Sunday, October 19, 2008

Role of low basal NO in capillary and vascular abnormalities

Vascular remodeling under low NO conditions.

I have really just skimmed the surface in what I have presented here. There are a lot more details that reinforce the chain of facts and logic that tie all of this together. This is already quite long and making it longer still isn't going to help you readers very much. Read what I have linked to, and if you have questions or want clarification of certain points, ask me.

There are many chronic degenerative diseases that are associated with what is termed vascular damage. The ultimate cause(s) of this vascular damage remains obscure. I suggest that much of what is perceived to be damage is not "damage" per se, but is simply the consequence of normal active remodeling of the vasculature under chronic conditions of low NO which results in the characteristic and dysfunctional morphology observed. In other words, the vascular state is the consequence of the low NO, and that vascular remodeling processes remain intact, active and essentially fully functional. These processes are simply operating in a low NO environment where the remodeling eventually result in dysfunction vascular morphology. Correcting the low NO environment should restore normal vascular remodeling and restore normal vascular function once there has been sufficient remodeling under conditions of normal NO levels.

In other words, to some extent the "damage" is not acute or even chronic "damage", rather it is simply a consequence of long term remodeling and is to a large extent reversible if caught early enough depending on age and the tissue compartment affected. This is an important point. I think it is not appropriate to call it "damage", if the normal remodeling pathways are working properly. Secondary damage to the tissues perfused by that dysfunctional vasculature is more problematic. There may be regeneration in some tissues such as muscle and liver, but less in others such as the brain or retina. Regeneration and regulation of vascularization is a critical aspect of wound healing. If the vasculature could not regenerate itself, wounds could not heal and death would occur fairly quickly. Even in the elderly, wounds do heal, demonstrating that the regulation of vascularization is intact even if it proceeds at a slow rate.

Vascularization is a complex process requiring many coupled and interacting pathways to function successfully. When vascularization "goes bad", how many pathways are affected and in how many different tissue compartments? A few, dozens, hundreds, thousands? Presumably each endothelial cell regulates itself from internal and external signals. For many endothelial cells to "go bad" simultaneously and in characteristic ways such that characteristic abnormalities develop over macroscopic spatial dimensions the regulation in millions of cells would have to simultaneously "go bad" in precisely "the same" way to achieve "the same" pathology over "the entire" tissue compartment (such as the retina) or systemically throughout the organism. That seems implausibly unlikely.

My hypothesis is that the observed vascular dysfunction is actually good regulation around a bad setpoint and that the bad setpoint is set external (to the endothelial cells) (the "setpoint" has to be external because what physiology is trying to "fix" is external levels of O2 and other nutrients in the local vicinity by modulating vascular perfusion) by the local NO level. Mostly the local level of NO is determined by the local NO production "source", eNOS in the endothelium and then taken up by the major "sink", hemoglobin in red blood cells. There is nothing simple or simplistic about this. The control system must have enough degrees of freedom to regulate the vasculature under each and every physiological condition that occurs, or vascular regulation fails and the organism dies. If the organism is still alive, then vascular regulation has not completely failed… yet.

My focus will be more on how low NO generates the characteristic morphology and what the long term consequences of those changes are rather than on other factors. I am not going to go into the detailed mechanisms of what regulates vascularization. I am going to more focus on the macroscopic control system which must exist even though the details are not very well understood. The experimental techniques necessary to measure and understand those details are quite challenging. They must involve gradients of diffusible (and so necessarily small) molecules over the smallest length scales of the vasculature. There are essentially no sensors to measure the small diffusible molecules that must be involved on the length, time and concentration scales involved. So long as those control systems use NO as a signaling molecule, a change in the basal level of NO will affect that control loop by skewing it in a characteristic direction.

The characteristic vascular remodeling I am most concerned about occurs "at rest", that is under the physiological state that organisms are in most of the time, and during sleep. During non-rest there are other pathways that provide additional regulation of the vasculature but because remodeling at rest occurs, the pathways that are involved at rest must be sufficient to mediate the remodeling.

I spend a lot of time talking in generalities, talking of the philosophy of design and control and how that must be applied in physiology. This is not the standard "hypothesis-experiment-conclusion" that is the standard methodology in science. The reason I don't take that approach is that the systems are too complex; they consist of too many coupled non-linear parameters for which there are no techniques to measure, let alone measure on the length and time scales that must be important to regulate vascularization. If we limit ourselves to what is possible to measure, the vast majority of physiology is completely inaccessible. Even though we can't measure parameters, we know that they must have certain properties due to stability considerations; certain properties because they evolved; certain properties because mammals gestate in utero under very different O2 levels and so on. The degree of certainty from such inferences isn't as high as from actual measurement, but the point of this argument is not to "prove" that low NO causes vascular abnormalities, but rather that NO is involved in "enough" pathways that having the "right" basal NO level must be important. How important? That is a good question and one that can only be answered experimentally. My purpose is to demonstrate that there is sufficient a priori justification to test the hypothesis that raising basal NO will improve vascular abnormalities. If my hypothesis about basal NO is correct, then raising basal NO will have no adverse effects because it simply restores a more normal state where all regulatory pathways work better.

We don't have the ability to measure basal NO levels, and even if we did measuring them in vivo would be to invasive and cause too much injury to be done ethically. There are so many different tissue compartments and so many different pathways that utilize NO as a signaling molecule, that the idea of external control through artificial means is completely preposterous. Because there are so many NO mediated pathways, it is very likely that different individuals will have different and to some extent idiosyncratic thresholds for some of them. What this means is that while there may be some generic symptoms from the effects of low basal NO, there will very likely also be idiosyncratic symptoms. Eventually as basal NO gets lower and lower, more and more NO mediated physiological pathways become marginal and eventually will "fail". As more and more NO mediated pathways become disrupted the course of many degenerative diseases gets closer and more similar. For example, as end stage kidney failure gets worse, vascular disease gets worse too and kidney failure is a common complication of vascular disease. I think the commonality of vascular abnormalities with many degenerative diseases is due to low NO being one of the final common pathways involved in them all.

I am a chemical engineer, so I will talk about and outline my reasoning about this in engineering terms, sensors, control systems, feedback, and that sort of thing. This is not to imply any type of design as ID proponents consider it. That is simply how I think of and understand physiology, just the same as any other chemical plant, a very complex chemical plant with exquisite controls most of which we have no idea how they work, what the design parameters are, what parameter is being controlled to do what and under what circumstances. Hacking into the control system of a complex chemical plant and perhaps bypassing the safety systems (when you don't know what is/is not a safety system) is not something to be done casually. That is how I see a lot of medicine, the same as trying to hack into a chemical plant where none of the pipes or wires are labeled as to what they are carrying, where they are coming from or where they are going, you don't know what the control system is based on, or even what is being controlled. Is that high pressure gas line carrying substrate for a process, a pneumatic control line, a pneumatic power line, a heat transfer line, or does it carry pneumatic messages inside tubes or some of all of these? We know that there is a gigantic amount of redundancy, and that there are so many different levels and layers of control that disabling all of them is difficult. Difficult but not impossible. The most important thing to remember in all of this is that these control systems have one and only one goal, the survival and reproduction of the organism. If for some reason we think a control system looks like it is doing something else that is probably a mistake on our part. A mistake based on the arrogance of our virtually complete ignorance of physiology.

I make a big point of how complicated physiology is, and how little we know about it because most people don't appreciate how complex it really is. The human genome has been sequenced (for some individuals), but the function of the vast majority of the DNA remains completely unknown. We don't know the complete function of any single protein; let alone how it interacts with thousands of other proteins under diverse circumstances.

A lot of what I cite is animal and in vitro research. I appreciate that in vitro and animal studies are not sufficient to base treatment in humans on. However there are no clinical trials where NO levels have been measured (there are no techniques that are sufficiently non-injurious to do in humans in vivo). There are no clinical trials where NO levels have been increased systemically (there are no generally recognized techniques to do so). With no techniques to measure basal NO and no techniques to increase it, some might say it can not be important. That would be wrong. 100 years ago there were no techniques to measure insulin levels in blood and no techniques to increase insulin levels either. Diabetes had been known for millennia. Eventually it was determined to be caused by insufficient insulin and that it could be effectively treated by supplying insulin from an external source. Diabetes in ancient Greece was caused by the same lack of insulin that modern diabetes is caused by.

Most of what I am talking about is quite generic to mammals. The "details" might be different, but the broad brush regulatory pathways are essentially the same. Those details are important for developing therapies specific to humans, but that is not my goal here. My goal is to illustrate how improved regulation of basal NO would improve regulation of vascularization. I don’t need to know the precise basal NO setpoint for a particular individual to know that bringing it closer to "normal" will improve the regulation of pathways mediated by NO.

We know that vascularization has not completely "failed". Even in the worst of circumstances wounds still heal although very slowly. The vascularization pathways are still working, still trying to maintain function, but they are having a very difficult time doing so. What ever is causing that must be a systemic agent that is at the heart of the regulation of vascularization. That agent is basal NO. Increasing basal NO won't act as a drug; it will simply allow normal physiology to reassert the control it evolved to do.

Something as important as basal NO is regulated, it is highly regulated. The problem is that our modern lifestyle has broken a critical component of that regulation. That critical pathway in NO/NOx physiology is the generation of NO and nitrite on the skin by a commensal biofilm of ammonia oxidizing bacteria due to release of ammonia in sweat.

Types of vascular effects covered.

General regulation of the vasculature via NO: vascular tone, blood and lymph flow, hypoxia, anemia, vascularization
Capillary rarefaction: hypertension, Raynaud's Rheumatoid arthritis and systemic sclerosis, Fibromyalgia
Acute changes: triggering of ischemic preconditioning à long term remodeling Retinopathy: Diabetic, hypertensive, tortuous vessels
Brain: Migraine, White matter hyperintensities, vascular dementia, reduced perfusion secondary to migraine, brain atrophy secondary to reduced perfusion
Diabetic vasculopathy: Diabetes type 1, metabolic syndrome, cause of vascular damage, cause of peripheral injury, nerve, poor healing, infections.

General Regulation

I have covered white matter hyperintensities in an earlier blog. I see that as the shut down of long range axonal transport due to low ATP as regulated by low NO. I will write about fibromyalgia in a later post. I did write about Morgellons, which I see as very similar to fibromyalgia except more in the skin and with greater systemic effects (likely because the skin is a big organ, and when a big organ is subjected to low NO stress, it affects a lot of things). I plan to write a future blog focused on fibromyalgia, so I won't spend as much effort on it as it deserves.

The engineering truism, "Good, Fast, Cheap; pick any two" is how evolution has configured physiology. Good only means good enough to survive and reproduce. Fast relates to the time scale of the organism's needs, when running from a bear, speed is of the essence. Cheap relates to the overhead in terms of metabolic cost and forgone reproduction. Every aspect of physiology has evolved as a compromise and trade-off between immediate costs and how that affects ultimate reproductive capacity. Regulation of blood flow is no exception. Every different aspect of vascular regulation is part of a unified physiology. The regulation of blood flow occurs over many different time scales (from seconds to months), under each and every different physiological state that a living organism can achieve (if regulation of blood flow ever "failed", the organism would die).

It is wrong to try and think about different aspects of that regulation in isolation, but only as part of the entire system. We know that the regulatory system evolved, and that every ancestor with a vasculature had a viable regulatory system (that would be every vertebrate ancestor). That constrains its properties considerably. This evolution probably explains a lot of the redundancy and robustness of physiological systems. When a new and improved pathway develops, the DNA encoding it becomes common in the gene pool because it provides improved survival and reproduction. The old pathway doesn't get removed; it stays there and is turned off, turned down, or inhibited, or relegated to a secondary, tertiary or later role. The "cost" of maintaining the DNA associated with a pathway now made obsolete is very small, the metabolic cost of maintaining DNA is that of the organic molecules that make it up, the metabolic cost of the enzymes that keep it repaired, the risk that a mutation in it will lead to something bad. Tiny risks compared to the risk that the newly evolved pathway won't always work in every situation that the old pathways worked in and which might be needed in some extreme circumstance, just in case. This is one of the difficulties in studying systems such as the vasculature. They are highly redundant and can tolerate quite large perturbations until they "break". When they do "break" the organisms dies. The transition between seemingly normal function and dysfunction leading to death can appear to be quite abrupt. It may be difficult to tell just how abrupt those changes actually are without knowing in detail exactly what is going on.

It is also wrong to try and apply any kind of "linear" model to physiology. Some times you can, but usually only for a small part of the actual dynamic range that physiology actually works in. Nothing in physiology is linear or continuous. Genes are expressed or not expressed, and discrete numbers of molecules of proteins are produced. Those proteins interact (or not) with discrete other proteins. Sometimes you can use a continuum model, but there are only 2 DNA molecules that the transcription factors can regulate. At the level of gene expression physiology is quite discrete.

The Good, Fast, Cheap tradeoff in human designed systems is somewhat different. Human labor in the design is often the largest part of the cost. Simplifying the design is done to minimize the design labor component. That always increases the cost and the response time. A typical heuristic for reducing design cost is to apply a "factor of safety". It is cheaper to simply use 3 times more structural material (as the ASME Boiler code calls for) than to calculate "exactly" what is needed, to make the system controls sufficiently precise and reliable such that a lower factor of safety is tolerable, or to tolerate occasional failure. Modular design is good engineering practice because it makes for easier design, easier debugging and easier coordination of human design efforts. None of those tradeoffs are important in living organisms, so what is perceived to be modular design is actually an artifact of systems that have evolved. Adopting a "physiology is modular" heuristic in examining evolved systems is fraught with potential error. There is no "evolutionary pressure" for organisms to evolve physiology in modules, only to evolve physiology that preserves the life and reproductive capacity of the organisms. Physiology may have the illusion of being modular because existing pathways can become duplicated and the now redundant pathways can diverge to accomplish different but similar tasks.

As human designed systems "evolve" down their "learning curve", more effort gets put into design. The design effort per unit may go down, but as the number of units goes up, the total design effort can become enormous. For a technology that is fully mature, the major cost is the raw materials. Evolved systems such as living organisms can in some ways be considered a "fully mature" design, that is where the engineering trade-off of "good, fast, cheap" has been "optimized" to a particular evolved value. That value may not be the value that we want, because it represents the optimization that occurred over many thousands of generations, mostly under conditions quite different than modern life. Our DNA didn't evolve for us to live happy lives. It evolved only because each and every one of our ancestors survived and reproduced, sometimes under quite horrific conditions.

General regulation of the vasculature via NO

There is an ok review that covers some of the basics.[1] There are a few misconceptions in this review. They make the very common error that too much NO is bad and that too much NO causes the formation of peroxynitrite (from NO and superoxide). This is simply incorrect. Peroxynitrite only occurs in vitro when there is near stoichiometric formation of NO and superoxide. [2] This is virtually certainly the case in vivo, but in vivo is considerably more complicated because superoxide (and peroxynitrite) is always confined by lipid membranes. They are both anions, and lipid membranes are impermeable to anions except through anion channels. Superoxide from mitochondria is confined to the mitochondrial inner matrix. Peroxynitrite is similarly confined. Peroxynitrite can decompose into NO2, and NO2 can diffuse through lipid membranes.

They are correct that it is a balance. They don't seem to appreciate how much feedback and crosstalk there is between NO and oxidative stress. NO and superoxide are very much complementary physiological principles. They are analogous to the conjugate variables of quantum mechanics, to the complementary principles of Yin and Yang, male and female, hot and cold, light and dark. These are only analogies, and shouldn't be taken literally. NO and superoxide react at near diffusion limited kinetics, as fast as it is possible for chemical species to react with each other. It is not possible to have both NO and superoxide present simultaneously. Which ever one is in excess will destroy the other. Because NO is lipid soluble (~10x over aqueous in isotropic lipid (lipid membranes are not isotropic, so that is more complicated still)) and superoxide is not, lipid membranes confine superoxide but not NO. This allows NO and superoxide to (nearly) co-exist in close proximity, provided the enzymes providing the NO and superoxide are kept separate (although nitric oxide synthase does make both NO and superoxide as discussed below).

A great many sources of both NO and superoxide are co-regulated by NO, superoxide and peroxynitrite. For example nitric oxide synthase generates both NO and superoxide. As the L-arginine level gets low, then NOS generates superoxide and forms peroxynitrite which has the effect of modifying NOS by oxidizing a critical zinc thiol complex so that NOS becomes uncoupled, and instead of making NO and superoxide makes only superoxide. This can be thought of as the "off" switch for NOS generating NO. A pulse of superoxide from another source can drop the NO level, accelerating the production of NO, locally depleting L-arginine, superoxide is formed by NOS, peroxynitrite is generated, this uncouples NOS which generates more superoxide until the NOS in the vicinity is all irreversibly switched to making superoxide instead of NO. This "switch" changes physiology from a low oxidative stress state dominated by NO to a high oxidative stress state dominated by superoxide. This is the generic "stress" response; lower NO levels switch physiology to respond to stress. This is how mitochondria respond, this is how ischemic preconditioning is triggered, this is how the respiratory burst is triggered, this is what mast cells do, release proteases to switch xanthine oxidoreductase to generate only superoxide. Low NO makes the threshold for all of these switches lower. In the limit, the threshold becomes so low the cells are only in the oxidative stress state. This can be sustained for considerable time, depending on the tissue compartment. It cannot be sustained indefinitely in all tissue compartments without adverse effects in multiple systems. Characteristic vascular remodeling is one of those adverse effects in the vasculature.

There is hysteresis when an organisms or tissue compartment enters an oxidative stress state. Usually stress states are conditions of high metabolic demand, it is advantageous to minimize the metabolic resources necessary to maintain the organism in that state, to free up those resources for productive use.

Peroxynitrite damage occurs due to slow turn off of oxidative stress

Peroxynitrite is a normal signaling compound. Peroxynitrite only occurs at near stoichiometric levels of NO and superoxide. Peroxynitrite effects are not observed in healthy individuals. Peroxynitrite damage doesn't occur in low NO states, it also doesn't occur in high NO states. Presumably peroxynitrite effects occur during the transitions of physiology, during the switching transients; from a superoxide dominated state to a NO dominated state and/or from a NO dominated state to a superoxide dominated state. We know the transition from a NO dominated state to a superoxide state is rapid and exhibits hysteresis. The main NO generating enzymes are turned off by peroxynitrite. The zinc thiolate couple in NOS becomes oxidized which decouples NOS so it produces only superoxide, similarly the Mo-thiol couple in xanthine oxidoreductase becomes oxidized so it no longer reduces nitrite to NO but only generates superoxide from O2 and reducing equivalents.

Presumably the damage observed and attributed to peroxynitrite occurs while physiology is attempting to switch from a superoxide dominated state to a NO dominated state. This requires sufficient NO to overcome the hysteresis of the low NO state. If there isn't enough NO, the transition cannot occur crisply, and physiology stays longer in the state where it generates the near stoichiometric levels of NO and superoxide that cause peroxynitrite damage. The solution to this ineffective and slow switching is to increase basal NO levels so that the transition can occur more rapidly and more robustly.

This is an extremely important point. The presence of peroxynitrite damage is not due to too much NO. Peroxynitrite damage is due to there being not enough NO (except under very rare those circumstances and the problem then isn't too much NO but not enough ATP see the blog on mitochondria damage).

The switching from the NO state to the superoxide state can occur very quickly. If you need to run from a bear, release of epinephrine causes acute oxidative stress. Revving up metabolism takes some time. Mitochondria need to disinhibit cytochrome c oxidase, the heart needs to start pumping blood at a high rate, get the liver putting out glucose at a high rate, the pancreas putting out insulin at a high rate and the lungs supplying O2 at a high rate. ATP cannot be stored. There is hysteresis in systems supplying ATP; ATP must be generated as fast as it is used. An analogy would be a pipeline which has inertia. You can't turn on and off a large pipeline instantaneously. The same is true of ATP. When it isn't needed, but might be in a few seconds, mitochondria get ready to generate it but dissipate the mitochondria potential as heat instead of generating ATP. This wastes substrate, but it is more important to be able to ramp up ATP production in a few seconds and escape with injury than ramp it up slowly and get caught. This readying of physiology to supply ATP at high rates is known as the "fight or flight" state.

When ATP is needed at high rates as in "fight or flight", an optimized organism would shut off ATP consuming pathways that are not needed during that time. If that time is brief, those longer term systems can be turned back on. If the time is prolonged, then what ever those pathways are supplying is lost until they are turned back on. Physiology can turn on the fight or flight state in a few seconds, it takes much longer to stand down from it.

A fundamental aspect of the damage that occurs from chronic low NO occurs because of the chronic activation of the "fight or flight" state. The fight or flight state evolved to be a temporary state. An emergency overload state where some necessary metabolic functions are put off until later to save ATP for immediate consumption. A state where damage is tolerable to save the life of the organism.

Modulating ATP demand over time is an extremely important physiological process, and one which is insufficiently appreciated because it is so automatic, so universal, and goes to such deep evolutionary time that all organisms exhibit it. The reason all organisms exhibit it is because is reduces the "overhead" associated with the production of ATP. That overhead includes the molecules that make up the ATP generating apparatus, the additional muscle to carry those extra muscles around. "Just in time" ATP generation allows those extra molecules to be used for reproduction instead. Over evolutionary time ATP allocation has evolved to be very efficient. This allocation of ATP is what occurs during fight or flight, it is what occurs during ischemic preconditioning.

Because peroxynitrite is a normal signaling compound, there will always be peroxynitrite effects, there will always be peroxynitrite "damage". Some amount of "damage" is unavoidable and physiology has evolved systems to deal with unavoidable amounts of damage. That damage isn't repaired during the low NO state because physiology is doing other things with the ATP, such as running from a bear. Repairing damage has too low a priority. The damage accumulates until there is a high NO state during which it can be repaired. Chronic low NO prevents the repair of peroxynitrite and other damage. When ever the damage rate exceeds the repair rate, damage will accumulate. The absolute rates don't matter, only that the damage exceeds the repair. The problem isn't too much damage, the problem is insufficient repair.

Vascular tone, blood flow, and lymph flow

The vasculature is active tissue. Arterial and venous blood is under pressure, the pressure drop between the heart outlet at the aorta and the heart inlet at the vena cava drives the flow of blood. The cross section of the vessels is regulated locally along their length, in (very) complex ways to regulate that flow. Red blood cells carry O2 from the lungs to the peripheral tissues and carry CO2 back to the lungs. Red blood cells are confined to the vasculature. O2 diffuses from red blood cells into the peripheral tissues. All tissues obtain O2 from the blood except for the external skin. The outer few hundred microns of the external skin derive O2 from the external air. All O2 diffusion is passive diffusion down a chemical potential gradient (more on this later).

The usual lack of blood flow in the skin is easily observed because non-pigmented skin is transparent and is not seen to be red except under conditions of hyperemia. Only a small fraction of the body is in direct contact with blood, only the endothelium. All other cells derive nutrients (other than O2) from extravascular fluid, that is from fluid that has "leaked out" of the vasculature (though it is not leakage per se, it is absolutely necessary extravascular flow). It is this extravascular fluid that carries glucose to the cells, other nutrients including protein (mostly as albumin), insulin, and all other nutrients and signaling components of blood. The extravascular fluid moves much slower than blood. Virtually all cells derive glucose from this extravascular fluid. Necessarily the glucose and insulin levels in plasma in contact with cells is lower than in bulk blood because intervening cells have consumed some of it. How much lower is a good question which is difficult to answer because getting samples to analyze is extremely difficult. The glucose level in the extravascular space next to the cells that are taking that glucose up is of course a much more important parameter than what the glucose level is in bulk blood remote from the cells that are using it.

Adequate flow of extravascular fluid is just as important as adequate flow of blood. The time constant for extravascular fluid flow is longer, but is obviously important and so obviously is actively regulated by physiology. If it were not actively regulated either there would be too much, or too little, or both in different tissue compartments.

The importance of extravascular flow of lymph is not always appreciated. Because it cannot be measured easily and is different in every tissue compartment (or even in the same tissue compartment due to gradients between capillaries), it is not as well mixed as blood is, it is not routinely measured and there are no clinical correlates with it. The fluid must "leak" out of capillaries at the proper rate, and then be transported along through the lymph vessels at the proper rate and then fed back into the circulation at the proper rate.

Accumulation of extravascular fluid is known as edema. This occurs for a variety of reasons, because the flow channels are blocked (as in filarial diseases such as elephantiasis), when there is too much fluid because the kidneys can't get rid of it and it has to go somewhere (the edema of congestive heart failure) and for things such as ascites (in the abdominal cavity).

CO2 must be carried back to the lungs also. CO2 transport is pretty complicated and won't be discussed in detail. CO2 as an uncharged gas diffuses pretty well. It is water soluble and forms carbonic acid, H2CO3. There are significant kinetic impediments to the formation of H2CO3, and so there are enzymes, carbonic anhydrase that catalyze it. H2CO3 disproportionates into H+, HCO3-, and CO3(2-) depending on pH. These are charged, and so cannot penetrate lipid membranes except through ion channels. Some of these are actively ported through cell membranes. Other ions must be co-ported to maintain ion neutrality. Chloride is the ion that does that in red blood cells, but nitrate and nitrate are similar to chloride ion in a lot of ways. They are not considered that important in ion channels, so the conductance of ion channels for nitrate and nitrite are not always measured along with other ions. CO2 can diffuse from tissue compartments containing carbonic anhydrase through intervening tissue compartments that don't, and into tissue compartments that do.

Regulation of vascular tone, blood flow, and lymph flow

The primary regulation of blood flow is via regulation of the cross section of vessels carrying that blood. The heart can pump more blood, and at a higher pressure, but for that blood to go where it is important for it to go the vessels have to modulate their cross section. Vessels are dilated where blood is being regulated to go, and constricted where blood is being regulated to not go. There is limited blood and also limited blood pumping capacity, so both types of control are needed, local to increase local blood flow, and non-local to decrease other blood flow.

The major regulator of vascular diameter and vascular tone is nitric oxide. NO is produced in the endothelium by eNOS. It is the NO that diffused into the vessel wall that regulates its tone. NO activates sGC which makes cGMP which relaxes smooth muscle. NO also diffuses into the blood and is taken up by red blood cells via kinetics that are first order in NO and first order in red blood cell concentration. The major passive sink for NO in the body is hemoglobin. Hemoglobin has a very high affinity for NO, and metabolizes it to either nitrite plus nitrate or to nitrosyl heme. Hemoglobin is normally confined to erythrocytes. Free hemoglobin destroys NO ~600 times faster than does Hb in erythrocytes. Free hemoglobin is responsible for the acute constriction and hypertension associated with hemolytic anemia as in sickle cell anemia.

How much NO diffuses into the blood, into red blood cells and is consumed and swept away and how much NO diffuses into the vessel wall and is consumed by superoxide and how much is left to activated sGC and cause vasodilation is a delicate balance between NO production, hematocrit, blood velocity, redox state, lipid vs. aqueous partitioning, ATP level, O2 level, L-arginine levels, asymmetric dimethyl arginine levels, nitrite, R-SNO thiols, NO from the extravascular space and other things. We know that all of those things are important, none of them can be measured on the length and times scales that we know are important in vivo. Neurogenic or receptor mediated production of superoxide can acutely consume NO and cause acute constriction. Superoxide can also be dismutated into H2O2 which can also cause vasodilation (but that is usually at high metabolic rate, not at rest).

When hematocrit is acutely decreased (taking out blood and replacing it with cell-free fluid, plasma or starch solution) as in isovolemic anemia, exhaled NO levels increase.[3] As Hct was decreased by dilution with hydroxyethyl starch (30, 23, 17, 11 %), cardiac output rose (0.52, 0.60, 0.70, 0.76 L/min), and exhaled NO levels rose (30, 34, 38, 43 nL/min). This demonstrates that NO levels in exhaled air are coupled to hemoglobin concentration in blood. This actually makes sense because the hormone that determines when more red blood cells need to be made is erythropoietin (Epo) and Epo is regulated by HIF-1-alpha which is regulated by low O2 (hypoxia) and also high NO. Both low O2 and high NO are signals of "not enough hemoglobin". HIF-1-alpha also causes expression of VEGF (vascular endothelial growth factor) which is one of the major factors that triggers angiogenesis.

There is starting to be some appreciation that the anemia observed in many chronic diseases may be an adaptive response and not solely something pathological. Anemia increases NO levels. High hemoglobin levels will decrease NO because hemoglobin is the sink for NO. It is the product of NO concentration and hemoglobin concentration that fixes the NO destruction rate. That destruction rate equals the production rate because there is no accumulation. The NO concentration (which is what NO sensors react to) then goes inversely with hemoglobin concentration. In a number of disorders associated with anemia (especially end stage kidney failure), increasing hemoglobin levels to "normal" causes increased death rates over increasing it to somewhat less than normal. Not enough hemoglobin is bad, but not enough NO (because a high hematocrit is destroying it) is worse. Increased hematocrit had the largest adverse effect on vascular disorders. The increased death rates are not concentrated in one or a few categories, but spread out over many. I see this as evidence of how many physiological systems are dependant on proper levels of NO, and how closely coupled that level is to hemoglobin levels in blood. Another example is systemic sclerosis, the death rate is ~2x that of standardized death rates after subtracting out deaths due to systemic sclerosis, but the causes of death are spread out over multiple causes. Presumably what ever is causing the systemic sclerosis is also causing the increased death rates.

When more flow is needed, NO levels are increased.

When greater blood flow is needed acutely through a particular vessel, the velocity goes up, that shear then activates eNOS and NO is generated which causes the vessel to dilate. When tissue becomes hypoxic, NO is generated via reduction of nitrite by deoxyhemoglobin and by other enzymes. When the vessel cannot supply sufficient oxygenated hemoglobin via blood, the increased NO level becomes chronic. Increased NO level would then be the ideal signal to trigger generation of more blood vessels through angiogenesis. It turns out that increased NO does trigger angiogenesis, and blocking NOS does inhibit angiogenesis. Supplemental IP nitrite substantially accelerates compensatory angiogenesis around a blocked artery in mice. The positive effects of nitrite were observed over a very broad dose range, something like a factor of 400.

Increased NO mediates increased blood flow over time scales from seconds to weeks. Presumably these multiple mechanisms for regulating blood flow evolved from an archetypal blood flow regulation mechanism which involved NO.

Acute Regulation of blood flow by NO, not by O2

Under conditions of isovolemic anemia, blood flow increases. The "conventional wisdom" is that it is "hypoxia" that causes the increased blood flow; however that cannot be correct because there actually is no hypoxia. There is no reduction in the O2 level in either the arterial blood, or the venous return blood. With no reduction in O2 level, there is no hypoxia. With no hypoxia, hypoxia cannot be a signal for the body to use to regulate blood flow.

At rest, acute isovolemic anemia is well tolerated. A 2/3 reduction in hematocrit has minimal effect on venous return PvO2, indicating no reduction in either O2 tension or delivery throughout the entire body. At 50% reduction (from 140 to 70g Hb/L), the average PvO2 (over 32 subjects) declined from about 77% to about 74% (of saturation). The reduction in O2 capacity of the blood is compensated for by vasodilatation and tachycardia with the heart rate increasing from 63 to 85 bpm. That the compensation is effective is readily apparent. The mechanism is not. The “obvious” explanation is that “hypoxia” sensors detected “hypoxia” and compensated with vasodilatation and tachycardia. However, there was no “hypoxia” to detect. There was a slight decrease in blood lactate (a marker for anaerobic respiration) from 0.77 to 0.62 mM/L perhaps indicating less anaerobic respiration and less “hypoxia” (though lactate production occurs under oxic conditions). The 3% reduction in venous return PvO2 is the same level of “hypoxia” one would get by ascending 300 meters in altitude (which from personal experience does not produce tachycardia). With the O2 concentration in the venous return staying the same, and the O2 consumption staying the same, there is no place in the body where there is a reduction in O2 concentration. Compensation during isovolemic anemia cannot occur because of O2 sensing.

When red blood cells of dogs are replaced with red blood cells that have been fully oxidized to methemoglobin (and so cannot carry O2), compensation for reduced O2 carrying capacity of blood is greatly reduced.[4] While maintaining the same hematocrit Hct (43%) and substituting (0, 26, 47%) fully metHb erythrocytes, the cardiac output (CO) declined (178, 171, 156 mL/m/kg) while the arterial PaO2 (93, 87, 84 mmHg) and PvO2 (55, 46, 38) also declined. In contrast, when acute isovolemic anemia (Hct 40, 30, 22) was induced using plasma, compensation was much better, CO (155, 177, 187), PaO2 (87, 88, 91), and PvO2 (51, 47, 42). When anemia was induced using dextran solution (Hct 41, 25, 15) cardiac output (143, 195, 243), PaO2 (89, 92, 93), PvO2 (56, 56, 51) compensation was better still. As part of their experiments with the metHb tests, a final dilution was done with dextran to lower the Hct to 26% while still maintaining 47% metHb. Compensation was much improved with CO (263 mL/m/kg), PaO2 (86 mmHg), and PvO2 (41 mmHg) all were increased, despite lower Hct, greater O2, and less “hypoxia”. The compensatory mechanisms to deal with this “hypoxia” cannot be due to reduced O2 levels because the O2 levels were not reduced, in fact, the O2 levels were increased. MetHb does bind NO, not quite as well as does Fe(II)Hb, but the presence of metHb erythrocytes clearly adversely effects compensation. The authors attributed the increased cardiac output to reduced blood viscosity in the case of reduced cell concentration. However when viscosity is increased, blood flow does increase.

When blood viscosity is increased during acute anemia, NO levels increase, flow mediated vasodilation increases and flow increases.

The optimum hemoglobin concentration for O2 delivery is as low as 15%. For O2 delivery to the brain it is about 30%. Normal hemoglobin levels are ~44%.

In summary, when the O2 carrying capacity of blood is reduced by removing erythrocytes, there is essentially complete compensation over a wide range by increased blood flow such that reduced O2 levels never occur. When the O2 capacity of blood is reduced by oxidizing hemoglobin to methemoglobin, there is much less compensation and reduced O2 levels do occur. When viscosity of blood is increased, there is increased shear, increased NO production and increased flow.

Long Term Regulation of vascularization

If acute regulation of flow of blood in the vasculature is not regulated by O2 levels, but is regulated by NO levels, should we expect that physiology uses a control system operating over a different time scale utilizing a different control scheme for other regulation?

Using a different control system presents potential difficulties when transitioning from one control scheme to the other. For the control to be stable, there can't be control regimes where one system is calling for more and the other system is calling for less. The control needs to be monotonic when averaged over a period that is long compared to the response time. We know that the control system evolved. In virtually all cases evolution takes an existing pathway or structure and modifies it for improved or different functionality.

If we know that acute increases in blood flow are mediated through increased NO, and we know that some instances of angiogenesis are increased by increased NO, it is likely that increased NO is the generic control system used for regulating vascularization.

Regulation of vascularization is a critically important physiological effect, and it is regulated exquisitely well and exquisitely complexly. Some of the details are known, many (probably most) are not. The vasculature is "well formed", that is it is very closely matched to the physiological needs of the tissue compartment it is in. There is no great excess of vessels and no great deficiency either. Organisms grow from a single cell, and have a well formed vasculature at all sizes. Organs grow in size, organs also shrink.

For vascularization to be regulated over so many orders of magnitude in size in so many different tissue compartments as organs grown and regress, there must be at least two types of regulation. There must be a mechanism that senses when there is not enough perfusion in a tissue compartment and signals the generation of more vasculature. There must also be a mechanism that senses when there is excess vascularization in a tissue compartment and ablates that excess. We know that there must be at least those two mechanisms. No doubt there are others, but I will focus on those two. It may also be a single mechanism operating in different regimes. I think a single mechanism is the most likely, that mechanism being NO with high NO triggering angiogenesis and low NO triggering ablation of vessels.

When are those signals generated?

They must be generated "at rest". Most growth occurs "at rest". The vasculature of organisms remains well formed after long periods of rest. The blood flow through some tissue compartments doesn't change much between periods of activity and inactivity. In utero, the fetus is always "at rest", capillary spacing is and must be regulated equally well in utero. Actually it must be regulated better in utero than after birth. A fertilized egg increases in size by many orders of magnitude very rapidly. An infant increases in size only about 1 order of magnitude as it becomes an adult and over a much longer time period. At rest would be the ideal time to fix the basal capillary spacing. Metabolic demands are low and constant. The appropriate level of vascularization could be established with the proper excess safety factor. At rest is a good time to remodel important physiological systems because metabolic need for those systems is at a minimum. While running from a bear is a bad time to divert resources into remodeling active systems. There may be additional signals that occur at other times (such as during exercise), but I will focus on the one(s) "at rest" which presumably are involved in all tissue compartments and so is likely to be the archetypal signaling system.

We know that hypoxia is involved in regulation of vascularization via HIF-alpha. Cells not getting enough O2 could generate a signal to generate more vasculature to bring more oxygenated blood to that tissue compartment. How can excess vasculature be measured? It cannot be measured by O2 level. The O2 level in arterial blood is very close to the level in air. That is the level in tissues at rest. At rest, the O2 level is essentially independent of capillary density. O2 demand is low, there are no large gradients in O2 concentration. All arterial blood is at near the saturation level in air. Venous blood is also regulated to a fairly constant O2 level. Gradients in O2 concentration between blood and mitochondria (where O2 is consumed) are in the extravascular space, not in the vasculature.

It also needs to be remembered that when embryos regulate capillary density they do so with quite different O2 partial pressures than do ex-utero organisms.

Physiology needs to generate a signal that measures how diffusively close red blood cells are to cells that require O2 and other nutrients in blood, that is, is a particular cell close enough to red blood cells such that it can obtain enough O2. If there are enough red blood cells close to a cell, that cell can indicate when it has sufficient vascularization and when there is not enough.

I suggest that an important component of that signal is NO. NO has physical properties very close to that of O2, the diffusion of NO through tissues is virtually identical to that of O2. O2Hb is the sink of NO, so the vasculature has the lowest NO level in the body (neglecting formation of superoxide (which is generated in mitochondria and microsomes) for the moment). If there was a volume source of NO, the basal NO level would be higher the farther from a capillary that tissue compartment was.

In summary, there must be a signal by which insufficient vascularization triggers angiogenesis, and also a mechanism by which excess vascularization is ablated. NO can signal both instances due to O2Hb acting as the sink for NO and with the extravascular space acting as a volume source.

Hypoxia and NO activate HIF-alpha which causes the expression of VEGF which is important in angiogenesis. NO is known to be important in angiogenesis, expression of iNOS is important in angiogenesis surrounding vascular infarcts. Neurogenic release of NO is what causes vasodilation by activating sGC. If the neurogenic NO were not sufficiently swept away by enough O2Hb, it would be a good signal for angiogenesis.

Regulation Oxygen delivery, Oxygen extraction, Ischemia reperfusion injury

Physiology can only use what are called intensive properties, properties that are proportional to the concentration or chemical potential of a substance, and not extensive properties, properties that are dependant on the amount of substance available. O2 partial pressure is the same as O2 chemical potential. O2 partial pressure is not proportional to the O2 content of blood because hemoglobin has a non-linear O2 dissociation functionality. O2 partial pressure is proportional to O2 content of plasma because plasma does have a linear O2 dissociation functionality (actually it is simple solubility via Henry's Law).

There are numerous misconceptions about this regarding O2 delivery, O2 extraction and the blood. O2 only moves by passive diffusion down a gradient in chemical potential. In homogeneous media this is down a concentration gradient via Fick's law of diffusion. By homogenous media I mean media where the chemical potential is strictly proportional to the concentration. In non-homogenous media such as blood or mixtures of lipid and aqueous phases one has to be careful. The "concentration" of O2 in red blood cells (mL/L), is not the same as the "concentration" of O2 in plasma in equilibrium with those red blood cells. The chemical potential of O2 in red blood cells and in plasma in a sample of blood is the same (and is the same in all fluids in mutual equilibrium that is the definition of equilibrium). In hemoglobin there is a non-linear relationship between O2 partial pressure and O2 concentration. Physiology can't measure O2 concentration in blood, all it can measure is O2 partial pressure, or more precisely the O2 chemical potential of the O2 sensors in equilibrium with that blood. Lipids have ~10x higher solubility of O2 and NO than do aqueous fluids. This can affect rates of chemical reactions a lot, as can the lower dielectric constant inside lipids. Ions can't enter lipids. Highly polar compounds like water or H2O2 can diffuse through lipid, but not as quickly as something nonpolar like NO or O2.

Some of the physiology literature talks about "oxygen extraction" from blood as if that is a real parameter. It isn't. Oxygen only moves by diffusion. There is no active transport. Tissues don't "extract" O2 from blood, O2 diffuses out of blood if the tissue the blood is flowing through has a lower O2 chemical potential than the blood flowing through it. If tissues are at the same O2 partial pressure as the blood, then they do not extract O2. If the tissues are at a higher O2 partial pressure, O2 diffuses out of the tissues and into the blood. There are no barriers to O2 diffusion. There is nothing that can block O2 diffusion. The vasculature can regulate where blood flows, and bypass less important organs to divert blood to more important organs. Tissues can only regulate O2 consumption by regulating the affinity for O2 of enzymes that consume O2.

Mitochondria are the ultimate sinks of O2; cytochrome c oxidase is the enzyme that reduces one O2 to two H2O's. The binding coefficient (Km) of cytochrome c oxidase for O2 is a sensitive function of the NO level. NO binds to cytochrome c oxidase and inhibits the binding of O2. This is an extremely important regulatory system for mitochondria. It is by regulating the NO level that the affinity of mitochondria for O2 is regulated. High NO, low O2 affinity. Low NO, high O2 affinity. The generation of superoxide by mitochondria under conditions of hypoxia is then seen as a necessary regulatory function. When cells become hypoxic, their mitochondria generate superoxide, that superoxide (confined to the inner matrix!) pulls down the NO level, cytochrome c oxidase is disinhibited, binds O2 at a lower O2 partial pressure, O2 is consumed to a lower partial pressure, the partial pressure gradient between the blood vessel (where it is nearly constant) and the mitochondria (where it is consumed by mitochondria) increases and so the flux of O2 (moles O2 per second) diffusing to the space where the mitochondria is now increases, relieving the "hypoxia". The problem of insufficient "oxygen extraction" is too much NO on the mitochondria. But is that really a problem? Cells don't need "oxygen extraction", they need ATP. If cells have enough ATP, they don't need anything else. Mitochondria are not the only source of ATP. Cells can make ATP via glycolysis which does not consume O2.

It is the attempt to make ATP using O2 under conditions of very high NO during sepsis that causes the mitochondrial damage and the multiple organ failure.

Under conditions of hypoxia, mitochondria first generate superoxide, and pull the NO level down to extract as much O2 as possible. Once that O2 is exhausted, mitochondria have a different need, to prevent the production of a massive amount of superoxide if and when O2 levels are restored. Most of the damage that occurs during ischemia-reperfusion occurs during the reperfusion, not the ischemia. When all the enzymes in mitochondria are primed to make superoxide, it is a bad time to supply a large bolus of O2 because much of it can get turned into superoxide.

Preventing damage following reperfusion is I think where nitrite comes in. There are many different enzymes that reduce nitrite to NO in the cytosol, in mitochondria, in microsomes. The nitrite reductase activity of these enzymes is O2 level dependant. O2 inhibits the production of NO from nitrite by them. When the O2 level drops, they become active nitrite reductases and produce large quantities of NO. This NO binds to heme enzymes and blocks their take-up of O2. This NO blocks the formation of superoxide following reperfusion.

As mentioned earlier, damage due to peroxynitrite from NO and superoxide occurs only when both are generated at near equimolar fluxes. This occurs during the transitions from a state dominated by one to a state dominated by the other.

Regulation of nutritive blood flow

In fMRI BOLD testing, it has been observed that the quantity of blood that flows in the region activated by the neurogenic NO release exceeds the nutritive quantity needed to supply the metabolic activity of that activated region. If the normal mechanism produces blood flow in excess of nutritive needs, there is a "factor of safety" and other mechanisms regulating blood flow are not needed. This is an important point. Blood contains many factors that are needed at different levels, O2, glucose, albumin, insulin, various proteins, hormones, immune cells, cytokines, etc. The levels of many of these factors in blood change week to week, day to day, even minute to minute. The needs for each of them in a particular tissue compartment also change. How many of them can the local regulation of blood vessel tone be determined by? In principle many of them, however if there is more than one control parameter, the control system may become over specified and instabilities may occur. It is much easier for evolution to modify and elaborate on a primitive ancestral trait than to evolve one de novo. The shortest time scale need is for O2. The time scale for O2 need is seconds, the time scale for glucose is minutes, the time scale for angiogenesis is days. If NO is both the shortest time scale control parameter and also the longest time scale, it seems implausible that a different control parameter and/or control system would have evolved to handle an intermediate time scale.

If blood flow in the brain is not acutely regulated to provide for acute nutritive needs, how is it that nutritive needs are met long term? The signals that do regulate acute blood flow either have some dependence on nutritive needs, or those nutritive needs are met by always providing an excess, or the tissue remodels to reduce demand when there is not sufficient excess.

If blood flow to the brain is reduced, how will the brain remodel itself to accommodate? Presumably brain cells self-regulate and reduce their metabolic load until it matches the supply of substrates provided by the blood. Presumably this involves pruning of lesser used cells. Pruning of brain cells is observed following a stroke. As cells necrose, the inhibitory signals they produce are lost, down stream cells become disinhibited, cells become over excited and excitotoxic death occurs. Excitotoxic death strikes cells with compromised metabolic capacity. Compromised due to insufficient blood supply, compromised due to mitochondrial damage, compromised due to other damage.

Capillary rarefaction

I suggest that the capillary rarefaction observed in many disorders, systemic sclerosis, Raynaud's, hypertension, dilative cardiomyopathy is ultimately caused by normal vascular remodeling via the same mechanism that leads to reduced blood flow, that of low NO. Reduced blood flow is observed in many neurological disorders, many even before there are overt signs of neurodegeneration. If blood flow is regulated to be chronically low, presumably efficient regulation of vascularization would ablate the seemingly excess vasculature that is seemingly present, resulting in capillary rarefaction. It needs to be appreciated that this is the completely normal response to a reduced perfusion setpoint. Physiology can't "compensate" because it is the compensatory pathways that are actually doing it.

If there is capillary rarefaction in an organ such as the heart, how will it respond? There isn't sufficient blood supply to maintain normal cell density, the cells "too far" from a capillary become stressed, die, and are cleared. If they are replaced, the replacement cells are insufficiently supplied also. The space could become filled with non-metabolically active fibrotic tissue. The heart still needs to pump sufficient blood, so it gets bigger, but weaker as fibrotic tissue replaces muscle. I think this is what eventually leads to dilative cardiomyopathy.

If the liver doesn’t have enough mitochondria to dispose of reducing equivalents, what does it do with them? Usually it makes fat. I think that is the source of fatty liver from chronic alcohol consumption. Alcohol is metabolized by alcohol dehydrogenase which makes NADH which can only be disposed of in complex I in mitochondria, or by making lipid. Ectopic lipid is an end stage symptom of many degenerative diseases associated with vascular abnormalities, liver failure, kidney failure, dilative cardiomyopathy. Excess NADH makes superoxide, and that superoxide lowers NO levels. Acutely that is adaptive, in that it disinhibits cytochrome c oxidase and allows for more O2 reduction to dispose of the reducing equivalents. In the long term, insufficient NO reduces mitochondria biogenesis resulting in systemic excess reducing equivalents which can only be disposed of by generating lipid. I think this is how the extreme obesity of hundreds of kg occurs. Individuals without enough mitochondria generate ATP via glycolysis; this generates lactate which is disposed of by generating lipid.

Hypertension

Hypertension occurs via increased vascular tone, the stiffness of vessels increases requiring higher pressure to drive the same volume of blood through the vessel bed.

Hypertension is associated with capillary rarefaction, with capillaries getting farther apart then is considered "normal". Capillaries farther apart means fewer capillaries in a tissue compartment and so blood flow through the remaining capillaries must be increased if the same blood delivery is going to occur.

Flow through a capillary and pressure drop across that capillary can be regulated independently. There is no need for a higher pressure drop to drive more blood through a capillary, the capillary could increase in cross section and accomplish the same thing. However bulk flow of blood through a capillary is not the only requirement. As mentioned before, extravascular flow of plasma is equally important (but on a different time scale, minutes as opposed to seconds). Extravascular cells derive nutrients only from local flow of plasma through the extravascular space. This plasma "leaks" out of the capillaries (however it is not "leakage" per se, it is a required flow). That "leakage" is likely proportional to the surface area of capillaries in that tissue compartment and also to the pressure drop from the inside to the outside of the capillaries. As capillary rarefaction reduces the number of capillaries, the total cross section goes down, to supply the same flow of extravascular fluid the pressure would have to go up.

I suspect that increasing extravascular flow is the physiological reason that blood pressure increases. Increased pressure drop through capillaries increases flow through the extravascular space that bypasses those capillaries. I suspect that increased extravascular flow is required to compensate for capillary rarefaction, both to supply the same extravascular flow, but also to increase extravascular flow to compensate for fewer mitochondria and greater ATP from glycolysis (which requires 19x more glucose for the same ATP production). NO is what triggers mitochondria biogenesis, so low basal NO will result in fewer mitochondria and more ATP from glycolysis necessitating increased extravascular fluid through fewer capillaries requiring higher pressure.

Raynaud's Syndrome

Raynaud's occurs when exposure to cold causes acute constriction of blood vessels in the skin, leading to pain, and in some cases necrosis. It is sometimes one of the first symptoms of capillary disorders, and usually accompanies all the others.

Because the output of the heart is limited, control of blood flow to peripheral tissues requires the regulation of both the pressure drop through the specific tissue being perfused, but also the pressure drop in the rest of the vasculature. The skin is a large organ, and constituting the outside surface, the skin is the only place where heat can be dissipated. Heat is brought to the surface by the blood and is easily observed as flushing. The external few hundred microns of skin derive O2 from the external air, they receive all other nutrients from the extravascular flow of plasma.

Conserving heat by constricting blood vessels in the skin when the skin is too cold is an essential part of maintaining the proper internal body temperature. Constricting blood vessels is usually done by generating superoxide, destroying NO and causing a reverse of the vasodilation that NO is producing.

Tortuous vessels

It is the take up of NO by hemoglobin in blood cells that results in the particular morphology of low NO damaged vessels, what is called "tortuous" vessels. There are some cases of familial tortuosity. This tortuosity is produced by essentially the same mechanism that stream meander is produced by. At high velocity there is erosion of the stream bank, and deposition of material at regions of low velocity. The same characteristic mechanism occurs in vessels but via different mechanisms. The crucially important similarity is that the flow of fluid inside the vessel affects the morphology of the flow pattern. In a stream the flow is within the stream banks. In blood vessels the flow is within the vessel.

Red blood cells are denser than plasma, so when there is accelerated flow there is segregation to the outside of the curved flow. This reduces the thickness of the boundary layer along the endothelium and increases the removal of NO at the high velocity outside region by the hemoglobin in the red blood cells that are now closer to the wall (just like in a stream meander). Removal of NO reduces the NO level on the inside and outside of the vessel, and this causes regression of that tissue via apoptosis due to low NO. This regression at high velocity allows for a vessel with an isotropic high velocity to enlarge in diameter. The tissue outside the vessel must regress so as to allow space for the vessel to enlarge in diameter. A series of images that just scream "low NO induced apoptosis" is here.[5] The images are of brain sections at autopsy of an individual with white matter hyperintensities. The vessels show a characteristic "tortuous vessel inside a cavity". The vessel is highly tortuous and the surrounding tissue has regressed leaving a cavity. Since there is no scaring, and there are markers characteristic of apoptosis, presumably the vessel is either a source of a pro-apoptotic diffusible factor, or a sink of an anti-apoptotic factor. It turns out that vessels are sinks for NO, which is an anti-apoptotic signal. The tortuousness of the vessel relates to how the flow changes the local source-sink properties of the vessel.

These blood vessels are often tortuous and appear as a tortuous arteriole in a cavity. These images are quite striking. The vessel is quite corkscrew-like, very tortuous and is in an empty cavity, devoid of white matter. These are sites of apoptosis. Low NO causes the tissues outside the vessel to regress (via apoptosis) and so the vessel migrates in that direction. Tortuous vessels like this are easily seen in retinopathy (where they accompany WMH) where they are caused by the same mechanism. In retinopathy, often when vessels cross there is observed to be "nicking", that is, a reduction in the diameter of the vessels. This is due to the decreased NO at the site of crossing (my hypothesis) where there is more hemoglobin to act as a sink of NO. This migration and remodeling of blood vessels by local NO levels is part of the normal regulation of capillary spacing (my hypothesis).

As cardiovascular risk factors increase there is decreased vascular reactivity; [6] that is there is reduced responsiveness shear induced increased blood flow, of exercise induced hyperemia, and even of NO induced hyperemia. This is what would be expected if basal NO levels are reduced because it is generation of NO by the endothelium that activates sGC and generates cGMP which relaxes the vascular smooth muscle. With a lower background level of NO, it takes more neurogenic NO, more shear generated NO, or more NO donor to achieve the same cGMP level and the same level of vasodilation.

Reductions in NO mediated vasodilation are observed in aged rats.[7]

These observation of long term remodeling of vascular morphology suggest that the remodeling is coupled to NO physiology, and that the basal level of NO is important in that regulation.

White matter hyperintensities

The tortuous vessels and cavities apparent on autopsy around those tortuous vessels indicate loss of neuronal tissue. There is also generalized reduction in capillary density observed in white matter hyperintensities,[8] which can be considered to be capillary rarefaction in the brain.

WMH are also observed during seizures. On acute occlusion of cerebral arteries, WMH occur very rapidly, in 2.7 minutes in the rat. It has been suggested that edema is the cause of WMH, however edema does not occurs this quickly, but ATP depletion does.

There may be other changes that result in WMH too. WMH are associated with markers for hypoxia. In any case, the relevance of this diversion into WMH is only to connect WMH to the ATP status of the brain. The association of WMH with low brain ATP is pretty well established, even if the mechanism(s) for that association is not.

NO and superoxide from iNOS are protective against excitotoxic injury, and this protection can be induced via LPS treatment.

Subjects with WMH have reduced density of blood vessels in regions which show hyperintensity. The reduced blood vessel density is likely due to a remodeling of the vasculature due to chronic low NO. With O2Hb being the sink for NO, if the level of NO is lower, then less hemoglobin is needed to act as a sink. I think this remodeling of the vasculature is the mechanism behind the lower brain blood flow observed in all of the neurodegenerative disorders characterized by WMH. I think it is also the mechanism for capillary rarefaction in non-neuronal tissues observed in hypertension and other disorders.

Ischemic preconditioning in the Brain

Ischemic precondition is a lower ATP state, but more importantly is a lower ATP consumption state. Some aspects of ischemic preconditioning are the same as the fight or flight state. Not enough is known about both to know if they are identical. They might be in some tissue compartments and not in others. All mammalian cells are aerobic and require continuous supply of O2 and substrate for continued ATP generation by mitochondria (except for red blood cells). When cells are deprived of O2 and substrate, they undergo ischemia and become damaged and eventually will die depending on the severity and length of the ischemia. This is the source of the injury when a vessel is occluded in the brain or heart for example. Ischemic preconditioning occurs when a tissue compartment is exposed to brief periods of ischemia prior to a prolonged severe ischemia. In an ischemic preconditioned state, tissues can survive ischemia that would otherwise cause necrosis. It only takes a few brief instances of ischemia to trigger the ischemic preconditioned state, which then persists for variable lengths of time, but it can be as much as a day or longer. The standing down from the ischemic preconditioned state takes longer.

Ischemic preconditioning can be triggered in a few minutes, and persists for hours to days. In the ischemic preconditioned state cells use less ATP. Presumably if cells could survive/reproduce while in the ischemic preconditioned state they would have evolved to do so because it would then free up more ATP for reproduction. Cells did not, so there is something incompatible with long term survival/reproduction with being in the ischemic preconditioned state too long. Presumably the time period that is "too long" depends on the tissue compartment and is probably longer than the normal duration of the normal ischemic preconditioned state.

Migraine

Migraine is a characteristic episodic type of headache that is often localized to a portion of the head, is sometimes preceded by visual hallucinations called aura or pro droma, and is sometimes triggered by a number of different environmental and/or physiological circumstances. The details of the physiology behind migraine are not well understood.

There has been considerable work on migraine using nitroglycerine because nitroglycerine does reliably induce migraine in susceptible patients. It is unfortunate that the effects of nitroglycerine on migraine have been attributed to nitroglycerine being a "NO donor". Nitroglycerine is not a "NO donor" in the classic sense. The chemistry and physiology behind the effects of nitroglycerine are complex and are not well understood. It can be a source of NO and nitrite via chemistry which is not fully understood, and which is subject to significant changes in fairly brief periods of time (few hours). Nitroglycerine exhibits what is termed "nitrate tolerance", where the dose of nitroglycerine must be increased; other NO donors such as sodium nitroprusside or authentic NO does not cause nitrate tolerance.

Nitroglycerine does irreversibly inhibit aldehyde dehydrogenase which appears to be the main enzyme responsible for generation of NO. This irreversible inhibition is exacerbated by oxidative stress. Nitroglycerine does induce late ischemic preconditioning. Ischemic preconditioning is a state where ATP concentration and consumption is reduced; it is a state that is protective in the short term, but (my hypothesis) detrimental in the long term. Long term treatment with organic nitrates increases cardiac events in patients with healed myocardial infarctions. I suspect that the therapeutic mechanism of nitroglycerine may be to induce ischemic preconditioning pharmacologically. This may be useful at reducing pain, and in reducing acute injury, but may not be helpful in the long term. That may be why some groups see increased cardiac events in long term treatment with nitroglycerine.

Migraine induced by nitroglycerine is not associated with changes in brain perfusion. This article is quite interesting and goes against a lot of conventional thinking and assumptions. It is consistent with the idea that migraine is not caused by vasodilation associated with NO. They did observe the prompt vasodilation associated with acute infusion of nitroglycerine, however there was no vasodilation associated with migraine following the nitroglycerine.

Migraine is pretty reliably triggered by sildenafil (Viagra). Sildenafil inhibits the phosphodiesterase 5 that is the main esterase that removes the cGMP produced by sGC after it is activated by NO. This is the mechanism by which sildenafil potentiates the action of NO through the cGMP pathway. However because there is feedback inhibition of NO production through the cGMP pathway, potentiating the level of cGMP will reduce the level of NO that is produced. Sildenafil thus will reduce the effects of NO mediated through non-cGMP pathways. This is apparent in men with obstructive sleep apnea, where a single dose of sildenafil significantly increases desaturation events. One of the triggers for breathing is S-nitrosothiols and is not mediated through cGMP.

When migraine is visually triggered, there is increased O2 levels by fMRI BOLD. This has been generally interpreted as being due to vasodilation, however the nitroglycerine study shows no vasodilation. I think it is more likely that the increase in O2 levels may be due to reduced O2 consumption due to triggering of ischemic preconditioning. Reduced O2 consumption is also consistent with reduced metabolism. The dynamic range of O2 level is substantially reduced, that is the level between activated and deactivated brain regions.

Migraine has been hypothesized to be associated with spreading depression. Spreading depression is a depolarization of neuronal tissue that propagates at up to a few mm/minute. It is not propagated by axons, but by some other type of signaling. It has many characteristics one would expect of ischemic preconditioning, and likely is related. This review article is interesting because it discusses both spreading depression (SD), and also hypoxia spreading depression like depolarization (HSD). I like the discussion of how they are different and how they are the same and the adherence to precision in naming and discussing the phenomena.

I suspect that they are even more similar than the author suggests, and perhaps are even indistinguishable. The major difference, that SD occurs in normal O2 environments and HSD occurs in low O2 environments simply means that O2 is not the causal factor. I think they are both simply ischemic preconditioning that has been turned on abruptly and hard. Under normal O2 levels, ischemic preconditioning turns off some pathways of ATP consumption, which reduces O2 consumption, so O2 levels go up. Under hypoxic conditions ATP production is reduced, so ATP consumption gets turned off. If the hypoxia is too severe, ATP cannot be produced and cells eventually die during HSD. SD can be tolerated many times with (apparently) little or no damage. I suspect that there is damage, that there is a "pruning" of a few neurons during each instance of SD to reduce the metabolic load and so bring it into better balance with what can be supplied by the vasculature. This "pruning" occurs during any type of seizure or excitotoxic damage. Cells that are firing too much and exceed their metabolic capacity are the ones that succumb to excitotoxic death. The cells that are the "weakest link" in the neural network of the brain. This pruning may not have apparent consequences with each episode, simply due to the redundancy and reserve of neurons present. When that reserve is exhausted, increased dysfunction will occur with each occurrence.

There are reports that people with migraine are at higher risk for lesions of various types visible on MRI. The increased risk due to migraines is additive to other risk factors and is somewhat higher in migraine with aura. Males who experience migraine are at slightly higher risk for cardiovascular disease. Women with migraine are at somewhat greater risk. I see the association of migraine with cardiovascular disease as both being due to and exacerbated by low NO.

Patients with migraine show subtle reductions in grey matter diffusivity compared to controls via high field MRI. Diffusivity relates to ATP levels as discussed above. These same patients also had reductions in grey matter density. The grey matter is where the cell bodies of neurons are, where protein synthesis and mitochondria biogenesis occurs.
The confinement of SD to the gray matter may be the attempt by physiology to spare the cell bodies of neurons. The white matter is mostly axons, which in principle can be replaced if the cell body of that axon remains in tact.

A number of conditions that are associated with mast cell activation are also associated with migraine including allergies, asthma, and irritable bowel syndrome. Elevated levels of histamine are sometimes associated with migraine, suggesting that mast cells in tissues associated with neurons in the brain may be involved in migraine. Agents that sensitize and activate mast cells also increase the sensitivity of intracranial meningeal pain receptors. These are thought to be the source of much of the pain felt during migraine.

When mast cells degranulate they release histamine as well as other agents that cause the production of ROS. ROS destroys NO, and this increases the sensitivity of mast cells to degranulation. Mast cells are responsible for release of ROS that is the inflammatory response to hypoxia.

There is another type of headache that is associated with excessive numbers of immune cells in the CSF, termed pseudomigraine lymphocytic pleocytosis. I see this as the production of superoxide by larger numbers of lymphocytes in the CSF, this superoxide reduces NO levels and triggers ischemic preconditioning and the low NO state.

Migraine is observed more frequently in people with other capillary/connective tissue disorders such as Sjögren's syndrome, Raynaud's and other rheumatic disorders. I think this relates to low NO being the final common pathway in all of these.

In conclusion, migraine is the triggering of ischemic preconditioning in the brain.

Reductions in Brain Blood flow associated with neurodegenerative diseases

Essentially all of the neurodegenerative diseases are characterized by reductions in blood flow, reductions in metabolism, accumulation of damaged proteins, and atrophy and shrinkage of the brain. These changes are not acute, but are progressive sometimes over many years and involve the whole brain.

I see this characteristic decline as the inevitable consequence of low basal NO. Once the NO level gets low enough that basal blood flow is affected, then basal blood flow is reduced, and tissues remodel themselves to accommodate to the now reduced nutritive blood flow. The reduced metabolic demands then set up another round of ablation of excess vasculature, reduced basal blood flow and still more remodeling. This progressive atrophy of tissue due to low NO is what I term the "low NO death spiral". The fundamental problem is the shifted setpoint brought about by reduced basal NO levels. Physiology is still regulating vascularization appropriately, it is simply to the wrong setpoint. The only way to fix the vascular remodeling is by restoring the correct setpoint. The only way to restore the correct setpoint is by restoring the appropriate basal NO level. This level is local to the tissue under consideration, and cannot be measured in vivo. It is on the order of nM/L.

In some ways the "low NO death spiral" is similar to the cell death that occurs during seizures or spreading depression. Those are acute episodes where cells are "tested" and the weakest cells ablated. We know there must be mechanisms for ablating cells because organs can and do shrink. Acute infarcts cause necrosis and scarring, less acute infarcts cause apoptosis and cell removal without scarring.

Diabetic vasculopathy

Vascular abnormalities leading to tissue damage are a common outcome in diabetes type 1 and diabetes type 2. I prefer the term metabolic syndrome over diabetes type 2 because there is a lot more going on than simply high blood sugar, and it is fundamentally different than diabetes type 1. One can have both diabetes type 1 and the metabolic syndrome simultaneously. I won't go into a lot of detail because there is quite an extensive literature on it. Diabetic vasculopathy is a chronic condition, it is not caused acutely. A serious complication is the very slow healing of even minor wounds which then become infected and if not treated adequately that healing occurs, amputation is not infrequently necessary.

There is a lot of thought that it is simply the high blood sugar that causes the damage. This is not strictly correct, but it is certainly related. There have been two recent large trials on standard blood glucose regulation vs. intensive glucose regulation, and with conflicting results. In one trial increased regulation of blood sugar doesn't reduced the death rate, it actually increases it. My interpretation is that in some cases trying to prevent hyperglycemia can be counterproductive. There is another study with a different conclusion, that intensive blood glucose control is beneficial. However in this study the incidence of hypoglycemic events requiring assistance and medical assistance was much higher in the intensive control group.

A recent (1999) "review points out that there is no compartment of glucose in the body at which all glucose is at the same concentration, and that models of glucose metabolism, including effects of insulin on glucose metabolism based on assumptions of concentration homogeneity, cannot be entirely correct." I would be more blunt; such models are wrong.

All cells in all tissue compartments need sufficient glucose. The only place where glucose can easily be measured is in bulk blood which is well mixed and essentially uniform in composition. Most cells derive glucose not from blood, but from plasma in the extravascular space. This plasma has a lower glucose level than bulk blood because cells have removed glucose from it before it reaches the sampling point. Physiology can't regulate extravascular glucose independently of blood glucose because it is plasma from the blood that makes up that extravascular plasma.

Preventing hyperglycemia will be counterproductive if it causes pathologically low glucose levels in the extravascular space (where it cannot be measured). Too much glucose is bad, but not enough glucose is worse. Not enough glucose in the extravascular space can occur even when there is pathologically high glucose in the blood stream. I suspect that to some extent that is the reason that physiology causes hyperglycemia in the first place (in the case of the metabolic syndrome, not diabetes type 1). NO is the signal for mitochondria biogenesis. With low NO, there ends up being not enough mitochondria. This shifts ATP production more to glycolysis, which takes 19 times more glucose per ATP molecule. If 5% of ATP production is shifted from mitochondria to glycolysis, that cell needs twice as much glucose to accommodate it. How can the vasculature deliver twice as much glucose? Only by increasing glucose concentrations in blood. If blood levels of glucose are not allowed to go up, then cells too far from a capillary become starved for glucose.

I suspect that if the groups were stratified for on the basis of capillary density that intensive glucose control would be beneficial for those with high capillary densities and the adverse events occur more in the group with low capillary densities, but it is probably more complicated than that.

In the intensive trial that was stopped, patients averaged 4 years younger and started out ~15 kg heavier and some exhibited larger weight gain since baseline (27.8% gained 10 kg or more compared to 14.1% in the standard group) (the averages are not provided). The starting weight in that trial was 93.5 and 93.6 kg. In the other trial, the starting weights were 78.2 and 78.0 and weight change was smaller, the ending weights were 78.1 and 77.0 kg. The standard treatment leg actually lost weight.

I suspect that weight and weight gain is a marker for degree of ATP production from glycolysis. When ATP is produced by glycolysis, lactate is produced and that lactate must be disposed of. Without enough mitochondria in the liver to recycle lactate into glucose via the Cori cycle, I think the excess lactate gets disposed of as fat. Since mitochondria biogenesis is triggered by NO, low NO will cause fewer mitochondria.

Diabetic vasculopathy is somewhat more complicated than just hyperglycemia. Low NO is a major final common pathway, but the cause is somewhat different. Acute hyperglycemia causes acute production of superoxide which reduces NO mediated regulation of vascular tone. What is interesting in this paper is that a transient elevation of glucose caused a sustained reduction in NO mediated vasodilation. This makes sense from a physiological control sense. When does blood glucose go up? When the body calls for more glucose to deal with an acute event such as running from a bear. The glucose is needed not in the bulk blood, but in the peripheral tissues, in the extravascular space. The only way that pulse of glucose can get to the extravascular space is to increase the pressure drop through the capillary bed and so transiently increase the extravascular flow and the flow velocity in the extravascular tissue compartment.

In obese Zucker rats, flow induced remodeling is characterized by low NO. Treatments that reduce NO decrease vasodilation due to shear, treatments that decrease superoxide (and so increase NO) increase vasodilation.

There have been suggestions that individuals with recurrent diabetic wounds have increased blood NO. This is incorrect. A paper which purports to have found this didn't actually measure NO, they measured the sum of nitrate plus nitrite. This is a common and fundamental error. NO has a very short lifetime in blood (less than 1 second) and is present at only nM/L levels. It is converted into nitrite and nitrate by oxyhemoglobin. Nitrite and nitrate are present at tens of microM/L. NO is extremely difficult to measure, nitrite and nitrate are easy to measure. Nitrite and nitrate are the terminal metabolites of NO, so there is some relationship between NO and nitrite and nitrate levels. Precisely what that relationship is remains largely unknown (and is likely very different in different tissue compartments). NOx levels in blood are more related to NO production rate than to NO concentration. The effects of NO as a signaling molecule are local and are related to the local NO concentration, not the NO production rate averaged over long times and multiple tissue compartments.

NO is one of the cytokines that has major regulatory effects on the immune system. NO attracts immune cells to the site of infection and regulates their function once they are there. This regulation is complex, and is affected by such things as temperature (NO being increased by fever range temperatures). NO causes vasodilation, bringing increased flow of blood. NO inhibits biofilm formation by Pseudomonas and Nitrite inhibited the formation of biofilms by Staphylococcus aureus and Staphylococcus epidermidis, and caused dissociation of biofilms already formed. Biofilm formation is a major virulence factor in infection. Suppression of virulence factor production renders even infectious strains of bacteria non-infectious. This is a point that is not always appreciated. Bacterial strains are infectious only because they produce toxins, proteases, and other virulence factors. Bacteria that do not produce virulence factors are non-virulent. Expression of virulence factors is regulated by bacteria, and until their expression is triggered, bacteria are non-virulent. Raising NO levels locally and systemically will improve the healing of diabetic wounds. Improving vascularization by increasing NO will prevent them from happening in the first place.

Summary

NO and NOx physiology is intimately connected with the regulation of vascularization. Capillary spacing is regulated not by gradients of O2, but by gradients of NO. Low NO causes physiology to decrease capillary spacing because low NO mimics the local signal of oxyhemoglobin being diffusively close. Physiology can't compensate because it is the compensatory pathways that are affected.

Reference:

1 Pechánová O, Simko F. The role of nitric oxide in the maintenance of vasoactive balance. Physiol Res. 2007;56 Suppl 2:S7-S16. Epub 2007 Sep 5. Review.

2 Espey MG, Thomas DD, Miranda KM, Wink DA. Focusing of nitric oxide mediated nitrosation and oxidative nitrosylation as a consequence of reaction with superoxide. Proc Natl Acad Sci U S A. 2002 Aug 20;99(17):11127-32. Epub 2002 Aug 12.

3 Deem, Steven, Richard G. Hedges, Steven McKinney, Nayak L. Polissar, Michael K. Alberts, and Erik R.
Swenson. Mechanisms of improvement in pulmonary gas exchange during isovolemic hemodilution. J. Appl. Physiol. 87(1): 132–141, 1999.

4 MURRAY,JOHN F., AND EDGARDO ESCOBAR. Circulatory effects of blood viscosity: comparison
of methemoglobinemia and anemia. JOURNAL OF APPLIED PHYSIOLOGY Vol. 25, No. 5, 594-599
November 1968.

5 Brown WR, Moody DM, Challa VR, Thore CR, Anstrom JA. Venous collagenosis and arteriolar tortuosity in leukoaraiosis. J Neurol Sci. 2002 Nov 15;203-204:159-63.

6 Silber HA, Lima JA, Bluemke DA, Astor BC, Gupta SN, Foo TK, Ouyang P. Arterial reactivity in lower extremities is progressively reduced as cardiovascular risk factors increase: comparison with upper extremities using magnetic resonance imaging. J Am Coll Cardiol. 2007 Mar 6;49(9):939-45. Epub 2007 Feb 16.

7 Sun D, Huang A, Yan EH, Wu Z, Yan C, Kaminski PM, Oury TD, Wolin MS, Kaley G. Reduced release of nitric oxide to shear stress in mesenteric arteries of aged rats. Am J Physiol Heart Circ Physiol. 2004 Jun;286(6):H2249-56. Epub 2004 Jan 29.
8 Brown WR, Moody DM, Thore CR, Challa VR, Anstrom JA. Vascular dementia in leukoaraiosis may be a consequence of capillary loss not only in the lesions, but in normal-appearing white matter and cortex as well. J Neurol Sci. 2007 Jun 15;257(1-2):62-6. Epub 2007 Feb

17 comments:

Oscar said...

A lot of good stuff in there.

I only managed to get partway thru. I will try and visit back and read it all.

Just wanted to post to let you know to keep up the good work.

Oscar said...

I've been trying to understand the NO pathway and how it pertains to sleep.

You might want to check out this paper on nNOS gene and RLS:

http://www.ncbi.nlm.nih.gov/pubmed/18058820

Also, a follow-up genome-wide analysis was done and a lot of genes were much more significant than the nNOS gene. And all those genes were involved in in-utero development I believe.

http://www.springerlink.com/content/jlv464808r03t030/

I just saw a presentation by her, and it's kinda bizarre that so many genes in early development are involved with RLS, when symptoms of RLS usually don't appear usually until 50 years old or so.

Thought you might find this interesting.

daedalus2u said...

A great many effects mediated through NO have nothing to do with nitric oxide synthase genes. NOS is not the only way to make NO, many enzymes reduce nitrite to NO. Low pH (less than 6 or so) will cause nitrite to make NO spontaneously; more if there are reducing equivalents available (such as ascorbic acid).

The basal level of NO is not set by nitric oxide synthase. Disorders of low basal NO are not due to polymorphisms in NOS (for the most part).

Nitric oxide synthase makes the NO that is used for signaling. That NO diffuses a distance, then adds to the basal NO level and when the sum exceeds the action level of the NO sensor (often sGC), then the NO sensor is triggered and the NO pathway is activated. All NO sensors only sense the sum of NO from all sources, including the background level. A change in the background NO level affects all NO pathways with no threshold.

I haven’t read much on RLS, but from these two abstracts, it looks like a great many disorders of low basal NO level. They are polygenetic with partial penetrance. This focus on genetics is (I think) quite misguided. It is focusing on genetics only because there are new and powerful tools that can be used to analyze DNA with.

I suspect that RLS is actually a “feature”, that when there is insufficient circulation, and insufficient NO to support proper vasodilation and produce sufficient circulation, that there is a compulsion to move so as to restore circulation via movement. It happens when people age because there is a decline in NO levels.

RLS is common in people with fibromyalgia. I suspect it is associated with other circulatory problems, and they all have the same final common pathway, low basal NO levels.

Oscar said...

Doesn't shear stress in the endothelium produce NO?

I generallly agree about genetics often being misguided, but sometimes it is fruitful.

Do you have any thoughts on using NOS Knockout mice for experiments? Is it valid?

And what about detecting NO bound to hemoglobin. We have oximeters that can detect Oxyhemoglobin, deoxyhemoglobin, carboxyhemoglobin, and methemoglobin.

Is there a specific light spectrum absorption pattern for NO bound hemoglobin?

Cause I totally agree that having a good non-invasive measure of NO bioactivity will crack this problem wide open.

daedalus2u said...

The data from the NOS ko mice is valid, it depends on what you are trying to use it for. The most important message from the NOS ko mice (to me) is how robust NO physiology is that whole sections of it can be removed (by ko) and the animals still survive. They have problems, but that they are able to stay alive is quite amazing.

The levels of nitrosylated heme are pretty small, and the spectrum isn't that different than that of oxygenated heme. The red in nitrite cured meat is due to nitrosylated heme.

The problem is that there are so many different NO species and there is lots of cross talk between them, NO, nitrite, nitrate, S-nitrosothiols (thousands), nitrosyl heme (hundreds), nitrotyrosine (thousands). A major NO reservoir is iron-NO-glutathione complexes. NO is one of the things that regulates metallization of zinc finger proteins (the largest class of transcription factors ~900). There can be cross talk without the intermediate formation of NO.

The problem is extremely complex. I am reminded of a quote from a book about the early development of computers, "Soul of a new machine". It was something to the effect that "oscilloscopes were what cavemen used to study fire." You couldn't look at digital signals with the analog oscilloscopes that were available at the time for studying analog repetitive AC signals.

Computers are run by electricity, trying to figure out how a computer works using a volt meter with an analog scale is going to be extremely slow and difficult. If your instruments don't have the resolution to measure something, you can't measure it. That is about where analytical technology is with NO. We know that we don't have the techniques to measure what is important on any scale that we know it is important on, length, time or concentration. We know that all of those are important, and also that many other things are coupled to NO physiology and we can't measure them either. Superoxide for example is extremely critical to NO physiology but it is mostly confined by lipid membranes and so all the little vesicles inside cells might have superoxide in them but unless we break them open we can't know, and once they are broken open, so is the cell.

Oscar said...

But didn't you say that you thought hemoglobin was the major sink for NO?

So, if you could determine NO bound Hb, then that's probably a pretty good measure. And, I'm confused when I read about Hb bound NO. Doesn't NO bind directly to the heme group, and also to the B-Cysteine 93 group? And, if so, what is the corresponding term for those?

I still think we need some measure of NO that provides some decent correlation to NO bioactivity.

daedalus2u said...

Oxygenated hemoglobin is the major sink for NO, the NO gets oxidized to nitrite and the ferrous heme gets oxidized to ferric heme (methemoglobin). Nitrosyl heme forms when NO bonds to ferrous hemoglobin that does not have O2 bonded to it. NO doesn’t bond to methemoglobin. Normal levels of methemoglobin are quite variable, on the order of a few percent. Nitrosyl heme levels are variable too, but smaller. S-nitrosyl hemoglobin is small and variable too. None of these hemoglobin species are probably important in delivery of actual NO to tissues (but that is controversial). Hemoglobin is only found inside the vasculature which is only a few percent of the body’s volume. Most everything related to NO happens outside the vasculature (because hemoglobin is the major sink for NO).

Oscar said...

I'm still a bit unclear.

So Ferrous Heme (deoxyhemeglobin) binds easily with NO. After NO binds (cause it has much higher affinity), then O2 also binds?

And that's the major NO store in hemoglobin? Some speculate that in areas of hypoxemia, the NO is released (causing vasodilation and thus increase Perfusion), and the heme bound O2 becomes more unstable so that the O2 can dissociate more quickly.

I've looked at methemoglobin measures during sleep via the Masimo CO Oximeter. Almost no change.

What do you think of the CO/NO crosstalk literature?

I'm inclined to think that NO in the vasculature is important. Wouldn't NO be binding & unbinding continually along with O2 delivery. Hemoglobin is a major sink for O2, but that doesn't mean that nothing happens with O2 in the vasculature. It's getting delivered.

daedalus2u said...

Hemoglobin has heme with ferrous iron. O2 can bind to that ferrous heme. So can NO and CO. The binding constants for NO are higher than for CO which are higher than O2. That is the reason that CO is poisonous because it binds to ferrous heme so that O2 can't.

Methemoglobin is hemoglobin with ferric iron. It doesn't bind anything.

Hemoglobin with O2 bound to it reacts with NO to form methemoglobin and nitrate. This reaction is quite fast. Since most hemoglobin is present as HbO2, NO has a very short lifetime in whole blood.

The amount of NO that binds to hemoglobin is small, too small to affect the bulk O2 carrying properties of hemoglobin.

I don't think there is a major store of NO in hemoglobin. I understand that some people do, but I don't think that production of NO by hemoglobin is important. The destruction of NO by hemoglobin is critically important. I think the idea that delivery of NO by hemoglobin is important is simply mistaken.

For NO from hemoglobin to be useful in regulating flow under conditions of hypoxia, the NO would have to be released where the constriction in the blood vessel is, which is upstream of where the vessel becomes hypoxic. Think about it. If the blood was hypoxic, it wouldn't have any O2 to deliver. It is only non-hypoxic blood that has O2 to deliver and the constriction in the vessels that regulates flow is upstream of the capillary bed where O2 is taken out. Blood isn't hypoxic where its flow is regulated.

I think there is some cross-talk between CO and NO, but only for a few NO pathways. CO does bind to heme, so there is cross-talk in the heme pathways but not in the S-nitrosothiol pathways (for example).

There are a lot of enzymes that reduce nitrite to NO, and these are inhibited by O2. Hemoglobin acts that way. Deoxyhemoglobin reduces nitrite to NO where O2Hb does not.

NO in the vasculature is critically important. It is what causes vasodilation. The NO that does that is produced in the endothelium, and in the extravascular space. The hemoglobin in the blood is the sink for NO, so the NO concentration is lowest at that sink in the blood. It is higher everywhere else (unless there is a specific thing pulling the NO level down).

Oscar said...

It's been awhile. I'd love to spend more time getting your perspective on NO & physiology, but am much too busy ATM.

Does the release of NO do anything at the level of the capillary bed where O2 is delivered?

What I've read about hemoglobin & NO, is that NO can be bound to oxyhemoglobin. And that when hypoxia is encountered, the NO is unbound first. This vasodilates the vascular in the vicinity and the loss of NO from hemoglobin makes the bound O2 more unstable, thus aiding again O2 delivery.

It's been awhile since I read that, so I could be wrong but that's what stuck in my head.

It seems to me, there is almost always quite a bit of O2 bound to hemoglobin, even when there is hypoxia. And, then there's hemoglobin bound O2, and then there's O2 in the blood overall. So, NO should be able to aid O2 delivery wherever it is, I would think.

And wouldn't NO released from hemoglobin vasodilate the endothelium? That should aid flow to the immediate area that is hypoxic.

I saw some research awhile back where a group was trying to treat tumors with inhaled NO, due to this effect. Since tumors become anerobic, the healthy cells could not survive in those conditions. To give the healthy cells a chance, they introduced NO to vasodilate the hypoxic areas and improve O2 delivery.

I think it's interesting what you say about nitrites and how deoxyhemoglobin breaks it down to NO. I was reading a couple of papers awhile back that was suggesting that nitrites was the mechanism of NO bio-transport and not hemoglobin.

David/Dad/Doc said...

daedalus2u

I am excited to see the research that you have done. I have come to realize and understand what you are researching through a back door approach. I am a dentist and my wife has Behcet's disease. It is an autoimmune disease that affects mainly capillaries in multiple parts of the body. Because of my awareness and study of the disease I have had referrals from patients with autoimmune diseases that end up affecting the gums and teeth.

In treating them, they have a large amount of pain in many places and their teeth and gums are more sensitive than the average person, so I quite often use nitrous oxide while treating them. Many say that the nitrous oxide gives them more relief of pain than almost anything else. It has (only temporarily) improved eyesight, hearing, relieved pain of headaches, joint pain, carpal tunnel pain, other pinched nerve pain, peripheral neuropathy in feet and hands, and the list goes on in these patients.

I have studied Behcet's extensively because of what my wife has been through. The nitrous oxide, I have just stumbled upon due to my clinical experiences. We have tried to duplicate the help with Arginine and/or OKG supplements to enhance the nitric oxide, but it has had little effect.

I will read your blog more in depth and get back to you more. You have done some wonderful work and I am excited to explore it more.

I saw that you have a way for me to send you my email without it getting to spammers. I will look more closely at that tomorrow and get my email to you.

Out of time for now. Thanks for your work.

David

daedalus2u said...

I am talking about nitric oxide, NO, not nitrous oxide N2O. They are very different. There might be some NO in the N2O, or NO might be made via unknown pathways from N2O, but they should not be confused and N2O is not a substitute for NO.

You might try increased nitrate consumption, lettuce a couple hundred grams several times a day. Nitrate is well absorbed, is concentrated ~10x in the saliva over plasma and is reduced to nitrite on the tongue. This nitrite is extremely important in oral health.

David/Dad/Doc said...

I realize that N2O and NO are two different entities. I was not sure of how much correlation there is between the two. The increase in vascularization by NO is very interesting to me because of the problems associated with Behcet's disease.

In briefly looking over your blog, I am assuming that you have someone dear to you that has Autism. Your study on the subject is amazing. I have done similar studies on Behcet's disease in efforts to help my wife's situation. I will take some time and digest your blog. Thanks for being willing to share your information.

David

Hip said...

QUOTING David/Dad/Doc FROM ABOVE: "Many [with autoimmune diseases] say that the nitrous oxide gives them more relief of pain than almost anything else. It has (only temporarily) improved eyesight, hearing, relieved pain of headaches, joint pain, carpal tunnel pain, other pinched nerve pain, peripheral neuropathy in feet and hands, and the list goes on in these patients".

It is possible that these above-mentioned benefits of nitrous oxide (N2O) on people with autoimmune diseases are due to the NMDA-receptor blocking effect of nitrous oxide.

NMDA (as well as AMPA and kainate) receptors can be overstimulated by the constant output of glutamate, which gets churned out by activated microglia. Chronic microglial activation is a characteristic of many neuroinflammatory and neurodegenerative conditions. This includes autism and Gulf War syndrome.

Once triggered by an infectious agent or a toxic chemical like rotenone, microglia sometimes stay permanently activated.

As a substitute for N2O, there are other useful and safe NMDA antagonists, such as high-dose transdermal magnesium. Transdermal magnesium has long been used in autism, chronic fatigue syndrome and fibromyalgia, and often gives mild benefit.

(Note that oral supplementation with magnesium is not really sufficient on its own, because bowel tolerance is reached at around 500 mg of oral magnesium.)

Other NMDA antagonists include: zinc, progesterone, L-taurine, L-huperzine A, agmatine, amantadine, ketamine, riluzole, memantine, dextromethorphan, ibogaine, and xenon gas.

Jcgilbert said...

Hi daedalus2u, is it possible I could contact you via email? You are clearly a very knowledgeable and learned man and I would to hear your thoughts on some issues im having. All the best , Jack.

daedalus2u said...

Jack, sure, send me an email. the address is on my corporate site, nitroceutic dot com.

joy said...

wow. never before have i wished that i had taken chemistry! thanks so much for all of this, even though i understood so little.

one question i have is about how muscle contraction plays into the vasodilation. would the contraction cause vasodilation even with low basal NO? could sufficient NO cause vasodilation in the absence of contraction?

as far as i understood, from a biomechanical standpoint, it's the muscle contracting/relaxing that causes the vasodilation. which is why it's so important to move all your muscles throughout the day.