Nitric oxide and the cardiovascular system.

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Nitric Oxide and the Cardiovascular System Harold Glenn Bohlen*1 ABSTRACT Nitric oxide (NO) generated by endothelial cells to relax vascular smooth muscle is one of the most intensely studied molecules in the past 25 years. Much of what is known about NO regulation of NO is based on blockade of its generation and analysis of changes in vascular regulation. This approach has been useful to demonstrate the importance of NO in large scale forms of regulation but provides less information on the nuances of NO regulation. However, there is a growing body of studies on multiple types of in vivo measurement of NO in normal and pathological conditions. This discussion will focus on in vivo studies and how they are reshaping the understanding of NO’s role in vascular resistance regulation and the pathologies of hypertension and diabetes mellitus. The role of microelectrode measurements in the measurement of [NO] will be considered because much of the controversy about what NO does and at what concentration depends upon the measurement methodology. For those studies where the technology has been tested and found to be well founded, the concept evolving is that the stresses imposed on the vasculature in the form of flow-mediated stimulation, chemicals within the tissue, and oxygen tension can cause rapid and large changes in the NO concentration to affect vascular regulation. All these functions are compromised in both animal and human forms of hypertension and diabetes mellitus due to altered regulation of endothelial cells and formation of oxidants that both damage endothelial cells and change the regulation of endothelial nitric oxide synthase. © 2015 American Physiological Society. Compr Physiol 5:803-828, 2015.

Introduction Nitric oxide has captured the interest and imagination of research and clinical scientists since its recognition as an endogenous vasodilator. The Nobel Prize for Murad, Furchgott, and Ignarro in 1996 ignited an explosive increase in interest in NO both for its normal physiology and complications during diseases such as hypertension and diabetes mellitus. So much has been discovered and reviewed about the vascular actions of NO that it is difficult to know what may best serve the interests of the Reader. One area that interestingly has received the least attention is the actual in vivo actions of NO on a moment to moment basis in health and as importantly, disease. It is fascinating that NO had not been measured in vivo at the time of the Nobel Prize, about the best estimate was something below 1 μmol/L seemed reasonable based on reactions to NO donors (120, 183). Very early measurements of in vivo perivascular [NO] with microelectrodes were consistent with a concentration range in the nanomolar range (51, 164, 249). One of the discussion items will be the current state of the art in NO measurement and the controversial nature of these measurements. This is an important consideration for future research as well as interpretation of the current literature—what is not measured correctly cannot be understood correctly. For those who have studied in vivo [NO] using microelectrodes, perhaps the most interesting aspect is that NO production is very dynamically influenced by many different physical and chemical events. For example, when the retina

Volume 5, April 2015

was stimulated by flickering light, Buerk et al. (51) measured a rapid increase in blood flow and NO in the optic nerve head. During occlusion of the middle cerebral artery, Malinski et al. (164) demonstrated an increase in [NO] that began within a few seconds after the occlusion occurred and was at a highly elevated steady state in about three minutes. Bauser-Heaton et al. (16) found a similar time course for increased periarteriolar [NO] in the brain when the arterial blood pressure was lowered. One of the more rapid increases in [NO] occurs when an increase in blood flow velocity at the endothelial surface causes a rapid increase in the [NO], as shown in Figure 1 for the arterioles of the intestinal wall (33). This process was termed flow mediated vasodilation based on the early studies of Bevan et al. (23, 24) and has been observed from major arteries to virtually all microvessels in multiple organ systems. It is very likely that flow dependent regulation of NO production is one of the dominant mechanisms for vascular resistance regulation through NO. However, increased endothelial production of NO in response to acetylcholine, bradykinin, serotonin, altered oxygen tension, pH, phosphorylated adenosine derivatives, and a growing list of tissue derived substances all play a role in tissue communicating to * Correspondence

to [email protected] of Cellular and Integrative Physiology, Indiana University Medical School, Indianapolis, Indiana, USA Published online, April 2015 (comprehensivephysiology.com) DOI: 10.1002/cphy.c140052 Copyright © American Physiological Society. 1 Department

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Nitric Oxide and the Cardiovascular System

Comprehensive Physiology

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Figure 1 (Panel A) Blood flow in an intestinal arteriole was increased by occlusion of parallel arterioles as [NO] was measured on the surface of the arteriole. The data at 3-s intervals (mean data straight lines) demonstrate a rapid increase in [NO] with increased flow and an equally rapid decline in [NO] when flow was returned to normal. Spikes represent artifacts of tissue movement which bend the microelectrode. (Panel B) A downstream section of an arteriole was occluded to rapidly lower flow and the mean [NO] decreased quickly. Upon release of the occlusion, flow increased to normal and [NO] was elevated. Adapted, with permission, from Figure 4 of (33).

vessels to alter blood flow through NO production. Evaluation of various mechanisms to increase NO production during in vivo conditions in the brain, intestine, heart, skeletal muscle, and kidney has revealed that unique chemical situations in each organ system are used as part of the specialized regulation of NO for that organ. For example, the brain uses NO from neurons containing neuronal nitric oxide synthase as a routine part of perivascular NO control but this mechanism is of much lower (8) consequence in other organ systems (16,182). In the small intestine, NaCl hyperosmolarity during nutrient

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absorption is a potent mechanism to increase microvascular and lymphatic endothelial NO production (272). The NaCl hyperosmolarity of the kidney may also be a factor in NO production (130). It is very unlikely that the NaCl hyperosmolarity mechanism is of much importance in other organ systems because NaCl osmolarity is so tightly regulated in the body. As with all regulated systems, the [NO] about arterioles and arteries tends to remain in a narrow range for each type of vessel and organ system. In all of the mechanisms to be


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discussed in this review, in every case when the natural stimulus to raise or lower NO production ceases, within several minutes or less the resting [NO] is restored to within 10% and often less of the original resting concentration. In larger vessels, such as small arteries, the [NO] is much higher than in progressively smaller resistance vessels (38,277) and while venules do generate NO (132), they do so at a slightly lower concentration compared to equivalent resistance vessels in the vascular tree. Just as in all other physiological regulatory systems, a feedback system of significant complexity and a set point range of the [NO] for routine life are both present in the in vivo regulation of NO. Perhaps the most important issue about resting NO production is that suppression by design or disease in all mammalian species studied thus far, including humans, causes a sustained increase in total vascular resistance which elevates arterial blood pressure (88, 131, 205). Consequently, abnormalities of the cardiovascular system that involved various forms of hypertension have received a great deal of interest in how NO is impacted. As will be subsequently discussed, both in vivo and in vitro studies of a variety of disease models related to inflammation, diabetes and hypertension have shown both activation and depression of normal NO function through endothelial nitric oxide synthase and in some studies, an increase in NO production by inducible nitric oxide synthase. In addition, increased oxygen radical production associated with diseases likely depresses the bioavailability of NO through increased destruction on NO as well as formation of the peroxynitrite radical which is quite damaging. The complexity of what happens to NO regulation in a given disease definitely complicates understanding what has been altered and by what mechanisms. To keep the discussion on point, the abnormalities of NO regulation in the three major vascular diseases of our time, hypertension, insulin-independent diabetes mellitus and obesity, will be considered. As these maladies are often interrelated, the overlap of abnormalities will be an important consideration. At this time, while abnormalities of NO regulation are known in these diseases, improving NO function through direct pharmacological intervention of specific NO mechanisms are quite limited, mostly involving improving the NO component of erectile dysfunction. However, there is compelling evidence of lifestyle improvement and pharmacological treatment of fundamental issues in each disease that do show some restoration of the overall NO set of mechanisms. Therefore, a review of how these diseases appear to compromise mechanisms of NO regulation, the current means to in part restore function, and opinions on what future directions to improve NO function might include will conclude the discussion.

What is the in vivo concentration of NO? The blood and NO It is vitally important to have reliable means to measure NO so that its dynamic regulation in health and disease can be


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understood. What cannot be measured accurately cannot be understood accurately. Quite surprisingly, the largest organ involved in NO regulation is the blood within the vascular system where measurement of dissolved NO and NO complexed with proteins and amino acids is particularly difficult. What is important is to what extent blood, principally the hemoglobin of red blood cells and the chemicals in plasma and red cell cytoplasm, carries NO through the vasculature to be released at vasoactive concentrations versus acting as a potentially huge mechanism to bind or even destroy NO in the case of hemoglobin. The classic work of the Ignarro (94) and Moncada (117) groups on identifying the chemical nature of endothelial derived relaxing factor as NO depended to a large extent on formation of nitrate in the presence of oxyhemoglobin. In effect, the huge amount of hemoglobin in resistance vessels should overwhelm local NO production and result in very low concentration of NO in the vessel wall, as multiple mathematical modeling studies have predicted (42,43,47,51,57-61). However, models have usually assumed a very high uptake or destruction of NO by hemoglobin when in the in vivo state, there may be a great deal of NO carried by arterial blood. The nondestructive interaction of NO with albumin, hemoglobin, cysteine, and glutathione to form S-nitrosylation products are well established as storage forms of NO to be transported by blood flow and potent vasodilators that may act directly or through release of NO. This area has been extensively studied by the Stamler group and their work has radically changed the understanding of the role of the hemoglobin-NO interaction (225, 228). Of interest to the current discussion is that the concentrations of S-nitrosylation forms in blood are not trivial and in the mid to high nanomolar, or even low micromolar range in arterialized blood. These concentrations are more than sufficient to cause activation of soluble guanylate cyclase in vascular smooth and initiate the cascade of events leading to relaxation and vascular dilation (58, 88, 106, 225). As importantly, in vivo measurements (16, 33) of [NO] on the outer wall of small arteries and throughout the microvasculature from multiple laboratories (not inclusive) (26,45,49,51,90,164,165,195,234,242,249,276) have found perivascular [NO] to be well into the nanomolar range with values of hundreds of nanomoles as routine concentrations. As will be discussed, measurement of [NO] in the in vivo state is quite difficult and depends upon microscopic NO sensors that do not consume so much NO as to lower the tissue concentration (28). When these technical caveats are properly adhered to, blood plasma and vessel wall concentrations of NO are sufficiently close that the concern that hemoglobin and to a lesser extent, albumin, cysteine, and glutathione, would deplete the vessel wall of NO is unlikely (38). There is a distinct possibility that exchange of blood-borne NO to and from the vessel wall could be an important form of vascular regulation (75, 155, 225). In future studies, the cooperative interaction of NO donation by blood reservoirs of NO to the vasculature when conditions warrant vasodilation, such as a decrease in tissue oxygen tension that lowers blood

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oxygenation, and a replenishment of NO to blood reservoirs under routine conditions needs to be considered. An alternative, perhaps adjunctive, form of NO in a reservoir form and available for NO generation is conversion of nitrite to NO by a variety of metal containing proteins when in a very low oxygen environment, including deoxyhemoglobin, myoglobin, neuroglobin, cytochrome c, cytochrome c oxidase, eNOS, xanthine oxidoreductase, aldehyde oxidase, and carbonic anhydrase, as reviewed by Bueno et al. (41). This is an interesting adjunct to eNOS because at very low oxygen tensions, formation of NO by eNOS is likely curtailed as molecular oxygen is essential to NO formation from arginine. The potential supply of nitrite is quite large as the typical plasma concentration in humans (72, 148) and rodents (3), is in the low micromolar range, typically 2 to 5 μmol/L. The typical vascular wall [NO] when measured correctly is of the order of 300 to 800 nmol/L, as will be explained in detail, such that nitrite even with inefficient conversion to NO in a low oxygen environment could be important for vascular regulation, Cosby et al. (72) have even proposed that nitrite is the largest potential reservoir for NO in the vasculature. However, two facts are very clear, typical resting nitrite concentrations do not appear to be particularly vasoactive at normal oxygenation conditions because to demonstrate vascular effects, the concentrations used must be high compared to the plasma nitrite concentration (3, 72, 148) and secondly, the conversion of nitrite to NO is best demonstrated in a low oxygen environment (41,72). A very good example of where nitrite might be very important for local NO generation is in the wall of damaged arteries where eNOS function is suppressed. In such a model, Alef et al. (3) have shown the conversion of nitrite to NO in damaged arteries is beneficial and improved by nitrite supplementation. In the peripheral circulation, nitrite infusion does increase NO generation under normal oxygen conditions, but the plasma concentrations reached are far in excess of normal (72) and accept at high concentrations, vasodilation is not always evident in peripheral vessels (148). In the pulmonary arterial circulation where deoxyhemoglobin is at a high concentration and oxygen tension is low, the Gladwin group has shown that providing exogenous nitrite lowers vascular resistance in pulmonary hypertension models and humans with the problem (41, 227). It is reasonable to assume that some version of this process occurs even under normal conditions in the pulmonary circulation before blood oxygenation. As a final thought, could nitrite conversion to NO in the lung be a potential source of NO to complex with newly formed oxyhemoglobin along with NO formed in the vessels of the lung? The Stamler group (209, 223, 270) has used gaseous ethyl nitrite to partially replenish hemoglobin bound NO and this process occurs at 20 to 50 ppm ethyl nitrite in in vitro and in vivo studies. If ideal conversion of nitrite to NO occurred at this concentration range, the [NO] would be of the order 25 to 70 nmol/L, compared to several hundred nanomoles per liter in typical arterial plasma (38). While certainly speculative, a relative low concentration of NO from vascular and nitrite sources in the lung could be important to loading the

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oxyhemoglobin with a storage form of NO about to enter the peripheral circulation. A potential exchange of NO from various regions of the overall cardiovascular system through blood transport (113, 206, 207, 228) that can interact with blood vessels requires that NO transit from vessel’s wall through plasma and into red blood cells as well as the reverse, movement from storage to act use at the vessel wall. The mathematical feasibility of such events has been shown by multiple models of the concept (61,146,250). The amount of NO in the plasma water would be of little issue because NO is very poorly soluble in water and has a very low content, which explains its very fast diffusion rate in water-based liquids (271). However, the significant concentrations of albumin and glutathione in plasma and the presence of nitroso compounds of each amino acid is likely a fast acting system for momentary equilibration of NO to and from the vessel wall, as proposed by Heuil et al. (113), Ng et al. (187), and Stamler et al. (228). It is likely that the NO exchange to and from hemoglobin in red blood cells is a comparatively slower process due in part to relatively slow diffusion of NO into the red blood cell (113). Circumstantial evidence of this is an observation by Liao et al. (154) that red blood cells perfused through isolated arterioles did not compromise endothelial-dependent vasodilation. If the red cells quickly removed NO, a diminution of dilation would be expected. Mixtures of NO into red blood cells in saline versus whole blood with plasma at similar hematocrit have very different time courses for the disappearance of NO. The half-life of NO in a simple RBC and saline solution is of the order of 4 s according to Liu et al. (157), compared to 1.8 ms in whole blood where albumin and glutathione could sequester NO before it subsequently moves to hemoglobin, which would be undetected with NO sensitive electrodes. These data clearly demonstrate that the NO reactive components of plasma have a rapid role in the immediate NO transport of blood and potentially can interact with the formidable reservoir capacity of hemoglobin to carry NO. Functional evidence from in vivo studies that NO provided at one point to blood in the vasculature can have vascular regulatory functions in another organ has been documented. Using inhalation of 80 ppm NO gas to increase local pulmonary gas [NO] of the order of 120 nmol/L, Fox-Robichaud et al. (89) demonstrated splanchnic vasodilation in cats. In a similar study using humans inhaling NO, Cannon et al. (54) demonstrated NO-induced peripheral vasodilation. Both of the studies were done when endothelial nitric oxide synthase was inhibited so that vasodilation would have to be due to some form of NO carried in blood that passed through the lung and was carried by arterial blood to peripheral resistance vessels. When a solution of NO in saline was injected into the brachial artery of normal intact humans, Rassaf et al. (206, 207) documented both local brachial artery dilation and increased forearm blood flow. These effects could be caused to a lesser extent by intravenous infusion of dissolved NO, indicating carriage of a vasoactive form of NO over a considerable distance through the lung vasculature and out to


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the peripheral vessels. As NO dissolved in saline is rapidly removed from blood by complexing with plasma amino acids and hemoglobin and some destruction, distal dilation would in part have to be caused by nitrosothiols in the blood in the opinion of these investigators. However, release of NO from storage forms at distal locations is also a potential contributor to the observed dilation. If blood can carry NO from the lungs to the peripheral tissues, is it also possible that transport of NO within the upstream to downstream locations from small arteries or larger arterioles to downstream vessels might occur? This question is of some interest because there is consistent finding that small arteries have a higher [NO] than large arterioles which in turn have higher [NO] than smaller arterioles and venules (32, 38, 118, 132, 277, 278). The transit time from small arteries to capillaries in most species is of the order of 2 s based on the velocities of blood and distances traveled as measured in in vivo microcirculatory studies. As mentioned earlier, NO mixed into whole blood disappears in a few milliseconds likely because of complexing first with plasma NO carriers, such as glutathione and albumin, before moving to hemoglobin (157). Therefore, NO can be rapidly protected from oxidation by dissolved oxygen in the plasma and potentially released at some point downstream. However, the oxidation of NO in arterioles may not be a particularly important because the PO2 declines to ∼60 mmHg in transit from large to small arterioles. The half-life of NO in oxygen increases exponentially with decreasing PO2 and approaches seconds at 60 mmHg (232) rather than less than 2 s based on mathematical simulations (145,236,237). Therefore, a combination of NO dissolved in plasma water equilibrium with the storage forms of NO in blood plasma and red blood cells might be an effective means to move NO from larger to small vessels. The potential role of this mechanism and the data available on the mechanism will be considered in the section on flow-mediated NO production.

Measurement of in vivo nitric oxide A great deal of what is known about NO in vivo physiology is based on suppression of eNOS by arginine analogs or damage of endothelial cells followed by comparison of vascular events before and after blockade. While this information sets the stage for how important NO may be in a given type of vascular regulation, the method so disrupts vascular regulation in general that intimate details of the regulation by specific mechanisms are difficult to appreciate. A very good example is the observation by Saito et al. (214) that suppression of eNOS with an arginine analog virtually eliminated functional hyperemia in a skeletal muscle during contractions. However, if the constriction at rest due to the loss of NO was avoided by adding a nitro vasodilator to the bathing media, functional hyperemia during muscle contractions was substantially restored yet NO could not have been produced. In this context, Nase and Bohlen (147) found that suppression of eNOS with hyperglycemia in the mouse


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cremaster muscle strongly suppressed the [NO] at rest and caused vasoconstriction plus eliminated the increased [NO] to acetylcholine. However, the vessels could still dilate quite well to acetylcholine due to a rapid improvement in response to an unknown hyperpolarizing factor. These studies show that interference with NO physiology has unintended consequences that go unappreciated without due diligence to find alternative mechanisms. Estimation of moment to moment production of NO has been focused on using microelectrode sensors and optical measurements to follow NO formation using the dye 4, 5diaminofluorescein (DAF). The DAF approach has been used in almost 1000 in vivo and in vitro studies since its introduction by Kojima et al. (135). This agent is useful to follow relative changes in [NO] about as well as NO sensitive microelectrodes (65). However, the chromophore is not able to predict specific concentrations of NO. One of the best and simultaneously most controversial methods to monitor in vivo NO at the vessel is the use of NO sensitive small electrodes to microelectrodes, which have evolved from the early work of Malinski et al. (166, 232), Buerk (51), and Friedemann et al. (90) as well as commercial electrodes made available by the World Precision Instruments company. The major advantages of electrodes are the ability to follow real time changes in concentration at a vascular site as well as approximately appreciate the actual [NO] involved at rest and during various mechanisms. The disadvantages are the difficulty of measuring the very low concentration of NO in the in vivo environment, the likely probability that the electrode reacts to multiple chemical species other than NO, and the possibility the electrode if too large consumes so much NO that the measurement is far below actual concentration. The question of whether NO measurements with electrodes is at all valid has been raised in reviews by Hall and Garthwaite in 2009 (106) and Chen et al. (58). The crux of the argument in these reviews is the very large range of [NO] in the literature from femptomolar to low micromolar concentrations. A possible explanation is that the higher concentrations are due to nonspecific sensing of chemicals in the in vivo environment other than NO. However, applying permeability barriers to electrode surfaces has yielded minimal sensitivity to physiological to supraphysiological concentrations of l-arginine (130,199), nitrite (4,90,166), ascorbic acid (4,26), cysteine (4), nitrate (4), H2 O2 (277), oxygen (15), glutathione (38), lysine (274), dopamine (90), norepinephrine (26), glucose (34, 147), and bradykinin (35). The opposing opinion is that when used correctly, electrodes do predict reliable concentrations of NO but the size of electrodes is a very serious concern. For example, in the review by Hall and Garthwaithe, they report the [NO] measured in 49 studies using a variety of different sized sensors. For electrodes with a sensor diameter of 30 μm or larger, the [NO] reported from tissues was consistently below 50 nmol/L. By comparison, for electrode diameters less than ∼15 μm, the [NO] was 100 to 1000 nmol/L. This disparity is so large as to predict the electrode size is a crucial issue in measurement of NO.

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All the NO electrode sensors used thus far in the literature are polarographic electrodes in which a polarizing positive current is chosen that best suits the measurement conditions. For NO, a voltage of ∼0.7 V or ∼0.9 V is usually chosen. In some cases, treatment of the electrode surface to improve sensing of NO has been used (90,166) while other laboratories have found “bare” electrodes with an appropriate semipermeable membrane made from Nafion are adequate (28, 51). The voltage at 0.7 V provides the greatest current difference from a 0 [NO] to whatever concentration of NO is present. The voltage at 0.9 V avoids false sensing of amino acids and their derivatives but there is a lower difference in measured current 1 from a 0 [NO] to whatever concentration of NO is present. Interesting, virtually all electrodes will yield a very linear current versus NO concentration relationship at both voltages. The higher range voltage requires a longer equilibration time to steady state current when the electrode is first used but a very stable current at 0 nmol/L NO thereafter for about 24 h of use, a higher absolute current at all concentrations of NO which is convenient for electrometer measurements but at the expense of a faster deterioration of the electrode surface due to greater current. As mentioned in a preceding paragraph, the physical size of electrodes may be the single most important issue in NO measurement. The first issue is that large electrode surfaces, that is diameters greater than about 30 μm, whether as a cylinder or a simple active face, require the fluid media about the electrode to be vigorously stirred to minimize an unstirred layer (28). In the early development of microscopic sized oxygen sensitive electrodes with diameters at or less than 10 μmol/L, Whalen et al. (259, 260) demonstrated how too large electrodes could lower the local oxygen tension environment in fluid. Modeling and tests of the unstirred layer thickness by Pohl (204) predict the size of the surface divided by the flow velocity determines the thickness of the unstirred layer. The concern is that if the unstirred layer is large in a relative sense, the electrode will consume the NO in the unstirred layer faster than diffusion can replace the consumed NO. Unstirred layers form at surfaces due to water molecules interacting at a molecular level, frictional forces limiting movement of the molecules near a stationary surface, and can be of the order of tens to hundreds of microns thick. The slower the fluid motion about the stationary object, the greater the thickness of the unstirred layer. The consequences are that the stirring conditions during calibration must be matched during measurements. It is important to appreciate that virtually any sized electrode will yield a linear [NO] versus current relationship in a properly stirred environment and then fail miserably in a situation where minimal stirring exists (28). The stirring requirement is virtually impossible if the electrode is pressed against a tissue surface, impossible if the electrode penetrates tissue, and quite difficult for electrodes measuring fluid conditions in a fluid bath with tissue. The end result is serious underestimation of the actual concentration of NO because the electrode consumes the NO within the unstirred layer faster than simple Fick diffusional forces supply the

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NO. To demonstrate this problem, one need simply equilibrate an overly large electrode in a well-stirred environment with NO and then stop the stirring, the current measured with large electrodes falls precipitously whereas the actual concentration measured with microscopic electrodes falls quite leisurely. The problems of unstirred layers are not unique to NO electrodes, as shown by Fatt (83) for polarographic oxygen electrodes and by Pohl et al. (204) for ion-selective microelectrodes. As the stirring was reduced in both studies, the measured concentration of the entity as judged by current generated by the electrode decreased dramatically when in fact the concentration was stable. Some of these problems can be avoided by using recessed tip microelectrodes such that a stable unstirred layer above the sensor element is established to minimize interactive effects of convective transport and diffusion. However, the electrode sensor diameter must be quite small, less than about 10 to 15 μm as mathematically modeled by Schneiderman and Goldstick (216) and experimentally determined by Crawford and Cole (73). The second major issue is that large electrode surfaces allow the electrode to consume a large amount of the sensed element such that if there are unstirred layer issues, the electrode will consume too much of the entity and report an erroneously low concentration. A very simple means to illustrate how physical size of electrodes interacts with the NO environment is to simply have very small electrodes, with tip diameters of less than 10 to 12 microns approach the surface of electrodes with diameters of 30 to 100 microns or larger in an NO environment (28). As shown in Figure 2 (28), the measured NO by the smaller electrode declined significantly as larger electrodes were approached but this did not happen with two small electrodes less than 10 to 12 μm in diameter. The open tip ∼10 μm microelectode is sufficiently small to enter the unstirred layer of larger electrodes and reveal the decline in [NO] when stirring is inadequate. The data demonstrate that unless used in a stirred environment equivalent to that used in calibration, larger NO electrodes of 30 microns in diameter or larger will grossly underestimate fluid concentrations of NO. Physical touch of the larger electrodes to a tissue surface where no stirring can occur is an even worse situation because the electrode can consume NO faster than tissue production and compound the problems. An interesting aspect of the study being discussed by Bohlen et al. (28) is that the diameter of the electrode, but not its length is the key issue in stirring and consumption artifacts. This is important because sub 10-μ-diameter tubular electrodes produced by World Precision Instruments as well as electrodes developed Friedemann et al. (90) and Malinski and Taha (166, 232) use very long but small diameter electrodes that work quite well in low flow velocity environs. The advantage of long but sub 15-μ-diameter sensor is that the currents measured are quite large compared to very tiny electrodes due to greater surface area and thus the currents are much easier to measure. The disadvantage when making tissue measurements is that the entire length of the electrode


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In Panels A and B, an open tip 7-μm-diameter carbon fiber microelectrode approached the surface of 2-mm metallic disk NO electrode: a similar process was done to 30-μm diameter by 2-mm-long carbon fiber WPI electrodes in Panel C and D in the presence of NO solution flowing through a tissue chamber. As the larger electrodes were approached in each panel, the NO current of the 7-μm-diameter carbon fiber microelectrode decreased as the electrode tip entered the unstirred layer about the larger electrode and NO within this layer had been consumed. The [NO] reported by larger electrodes was not shown because it did not change. Withdrawal of the open tip 7-μm microelectrode resulted in an increased [NO] at about 50 μm from the 2-mm WPI electrodes and at about 10 μm for 30 μm WPI electrodes, indicating the approximate distance of influence of the unstirred layer effects. When 7- and 10-μm microelectrodes were approached, the current of the exploring microelectrode did not change, indicating a minimal unstirred layer and diffusion interaction. Adapted, with permission, from Figure 4 of (28).

must be exposed to a physical tissue, such as a microvessel, otherwise part of the sensor will not monitor a correct tissue [NO] and the overall measurement underestimates the local [NO]. One of the recurring arguments against in vivo measurements of NO with microscopic sensors applied to the outer surface of arterioles or venules is that the flow of blood somehow influences the measurement. This could explain the mid to high nanomole per liter range in in vivo preparations of the eye (50, 51), small intestine (33, 35, 199), skeletal muscle (185), cheek pouch of hamsters (132), and cerebral cortex (15,16), unanesthetized hamster tissue (240,241) and in many more studies. In a strong magnetic field, flow of a salt solution


such as blood can induce a current and was the basis of the electromagnetic flow measurements. However, such physics cannot occur in the weak environmental magnetic field at the Earth’s surface. The potential stirring caused by the flow of blood is somewhat muted by the fact that the vessel wall acts as a gas semipermeable membrane added over the surface of the electrode tip and would minimize any stirring issue. When a very small electrode tip is put inside a vessel, stirring and motion artifacts become a worrisome issue and electrodes that have virtually no electronic response to fluid motion equivalent to that in flowing blood vessels have to be used. This has been tested (38), and can only be achieved with open tip microelectrodes with a tip diameter of about 10 μ and a recess

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of at least 5-μm filled with an appropriate semipermeable membrane. A secondary proof that very small NO microelectrodes reliably monitor the tissue concentration of NO is by using bioassay. If one accepts the premise that the in vivo [NO] required to cause arteriolar dilation is at least the order of 100 nmol/L or higher, then adding exogenous NO of this range and higher should be necessary to cause in vivo dilation. This has been tested by adding NO gas to nitrogen equilibrated physiological saline (protects the NO from oxidation) or NO derived from nitrosoglutathione (GSNO) added to the media (28). The results for both sources of NO are shown in Figure 3 as the change in nanomoles of [NO] versus dilation of the vessels for both sources of NO (28). The range of [NO] used was to simulate [NO] increases during intestinal arteriolar responses to absorption hyperemia (26), increased arteriolar flow velocity (33), and decreased perivascular oxygen tension (185). The tissue measurement of NO was on the surface of large arterioles before and during exogenous supply of NO in the overlying, flowing media. The data illustrate that to cause a 5% to 40% dilation, the background [NO] must be increased 30 to 200 nmol/L for both NO gas and NO from GSNO. In these studies, the resting [NO] were in the mid 300 nmol/L range and the highest exogenous NO increased the perivascular [NO] to the mid 500 nmol/L range. When the [NO] was elevated even higher, maximum dilation of intestinal vessels occurred in the 800 nmol/L range. The use

of GSNO in this study was to determine if a nitrosothiol would be more efficient as a vasodilator than NO gas. Dilation was not different for equivalent perivascular [NO] with NO gas or GSNO. S-nitrosothiol compounds in blood are typically reported in the 300 to 1000 nmol/L and higher for arterial hemoglobin, albumin, cysteine, and glutathione in plasma (113, 187, 206, 207, 225, 228). These arteriolar measurements along with the data on S-nitrosothiols in plasma predict that NO concentrations at the vessel wall have to be in the mid to higher nanomole per liter range to cause vasodilation. If the tissue concentrations of NO were very low, NO donated from plasma S-nitrosothiols in their reported range would cause overwhelming vasodilation. There are alternative means to predict that [NO] in the low to mid nanomole per liter are necessary to cause relaxation of vascular smooth muscle in the in vivo environment. Infusion studies into the human arm arteries have been used to address this issue. If one takes the face value infusion of single digit nanomoles per minute infusion, blood flow increased 50% to 100% (50, 83, 163). However, these studies did not take into account the concentration of S-nitrosothiols generated within the plasma of the flowing vessel. When corrected for plasma flow and the molar infusion rate, the milliliters per minute of plasma flow which received the nanomoles per min infusions would generate effective plasma concentrations of multiple hundreds of nanomolar concentration to cause well developed but not maximum dilation.

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Arteriolar inner diameter and perivascular [NO] were measured as either small volumes of 1200 nmol/L NO gas equilibrated solution or GSNO was added to the incoming bathing media over an in vivo intestinal preparation. The dilation to a given [NO] caused by either source of NO caused comparable dilation. After exposure of NO and the resulting dilation, when the NO source was removed, there was a rebound constriction and reduction in [NO] for both NO sources. With elevated or reduced [NO] from either source, the changes in diameter were equivalent, with similar regression statistics shown in the figure. Adapted, with permission, from Figure 6 of (28).

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As discussed in this section, when all aspects of NO measurement are considered from electrode physics and bioassay studies, the data point to higher concentrations of NO in at least the hundreds of nanomoles per liter around the resistance vessels. Very low measurements should be reconsidered in the light of using techniques that while quite reliable in wellstirred environments, do not translate to the reality of limited or no stirring for in vitro and in vivo measurements (58, 106). While true microelectrodes undoubtedly have their drawbacks in measurements of NO, until technology improves substantially, their measurements provide the best clues as the actual [NO] in the in vivo environment and have provided valuable insights into the real time physiology of NO.

Flow-Mediated Vasodilation Caused by Nitric Oxide When flow in resistance vessels is increased, there is a resulting vasodilation which is in part caused by release of NO, prostaglandins, and as yet undefined agents, such as hyperpolarizing factors. The increase in shear force by elevated flow velocity physically interacts with the endothelial surface to initiate these events and probably others as yet unknown. Reviews of this topic considering various mechanisms to cause or at least contribute to flow mediated dilation are available (99, 168, 208). For the scope of the current article, the probable role of NO in flow mediated events in the in vivo environment will be considered. Flow mediated vasodilation in the in vivo environment is an ongoing process that waxes and wanes with the blood flow demands of the host organ. Viewing flow-mediated vasodilation as just something that happens when flow increases or wanes limits the scope of this important mechanism. Flow mediated release of NO is ongoing process at all times, including at rest. However, to determine just how important flowmediated mechanisms are to resting vascular tone is very difficult because so many mechanisms influencing NO production occur plus multiple other mechanisms are influencing vascular tone. The general scenario for flow-mediated vasodilation to occur is that somewhere in the resistance vasculature, there is vasodilation which in turn increases flow in all up and downstream vessels. The distant vessels then respond by flow-mediated vasodilation to augment the ongoing increase in flow. The general thought in this field is that as arterioles are within the organ and subject to its chemical and physical environment, their dilation sets the stage for increased flow in small and large arteries which then can use increased NO release, as well as other mechanisms, to cause local dilation and somewhat additionally increase blood flow. For example as of this writing (April 2014), there are 1658 articles in PubMed using brachial artery flow-mediated dilation as a method to study various mechanisms of flow mediated dilation in humans. What is very important is that in most of these human studies, the quality of flow-mediated dilation of


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the brachial artery is used to evaluate how vascular diseases have suppressed the flow-mediated process and how pharmacological or lifestyle interventions improve the process. Studies of flow-mediated dilation by microscopic or small arterial vessels have predominately been through in vitro evaluation of isolated vessels where very controlled flow and physical properties can be maintained. Kuo et al. (141) in 1990 set a very good standard in this field by evaluation of how isolated coronary arterioles dilated in response to a change in flow at a constant internal pressure. The rapid onset of dilation as flow increased and constriction when flow was lowered was abolished after the endothelium of the vessel was removed. In a subsequent study (140), this group found that l-arginine, the precursor molecule of NO, restored flow-mediated dilation by coronary arterioles taken from atherosclerotic pigs. In aging pigs (83), there is also a loss of flow-mediated vasodilation by coronary arterioles which is due to a reduced vasodilatory signal from both NO and hydrogen peroxide. In unanesthetized hamsters using a skin/muscle flap preparation, Tsai et al. (240) used an innovative means to alter shear stress by hemodilution combined with either low or high viscosity plasma. The higher viscosity, which would elevate shear forces for a given hemodilution, did increase NO generation by large amounts whereas low viscosity plasma had no appreciable effects. Flow-mediated vasodilation secondary to changes in shear stress appears to be very widely distributed in the organs because isolated vessel studies of leg and diaphragm skeletal muscle (84, 129, 196), heart (128, 150), brain (188), retina (111, 112), and the mesentery (230) have all revealed flowmediated dilation that is at least in part caused by nitric oxide. In vivo studies of flow-mediated vasodilation using simultaneous measurements of NO with microelectrodes have shown very fast changes in vessel diameter and [NO] with both increases and decreases in blood flow. Hyre et al. (118) were the first to demonstrate actual increases in [NO] with elevated blood flow using the interconnected parallel arteries of the rat small intestine. For the parallel connected arterioles of the intestinal wall (33), selective occlusion of parallel arterioles or downstream portions of an arteriole could cause an increase or decrease in flow in a given vessel without compromising local blood flow in the capillary vasculature about the vessel of interest. In Figure 1 (33), the rapidity of increases in [NO] with increased flow and the decline in concentration when the flow increase was stopped can be appreciated. Equally fast reductions in [NO] occurred when a downstream section of an arteriole was temporarily occluded followed by recovery when the occlusion was ceased. Using both the increases in flow velocity, calculated shear rate, and measured [NO], the relationship of NO to velocity and shear rate for both large and intermediate diameter arterioles were similar and nearly linear as shown in Figure 4 (Fig. 9 (33). It is important to note that larger arterioles had a much higher resting [NO] than do intermediate diameter arterioles, perhaps due to higher shear rates and simple physical size of the endothelial cell pool. However, the relative changes in [NO] versus relative shear rate or flow velocity were quite similar, indicating a

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The relationships of [NO] to red blood cell velocity and shear rate for large (1A) and intermediate diameter (2A) in vivo intestinal arterioles as both were altered by either occlusion of a parallel arteriole or downstream occlusion of the arteriole studied. The change in [NO] was approximately linear for the range of velocity and shear rate studied and on a relative basis, about 1.3× larger for 1A than 2A. The linear regression equations were Velocity: 1A, 0.85× + 20.8, r2 = 0.53, P < 0.05; 2A, 0.52× + 50.7, r2 = 0.58. Shear rate: 1A, 0.75× + 33.6, r2 = 0.53, P < 0.05; 2A, 0.59× + 45.2, r2 = 0.56, P < 0.05. Adapted, with permission, from Figure 9 of (33).

common mechanism between these vessels to sense flow and respond with variable NO production. In this same context, the cerebral arterioles comparable in size to those of the small intestine have resting [NO] as much as 75% higher. However, studies of flow-mediated dilation of in vivo rat cerebral cortical arterioles by Bauser-Heaton et al. (15) in which only increases in blood flow could be caused have shown events quite similar to those in the small intestine. In these studies of both the small intestine and cerebral vasculatures, the increases in [NO] with elevated flow were always sustained for long periods of up to an hour with no indication of abatement. In this context, flow-mediated vasodilation should be viewed as a rapid responding but long-term controlled system that very likely is one of the major mechanisms setting the prevailing [NO] of arterioles along with other mechanisms that influence NO production. At the beginning of this section, the large body of studies on flow-mediation dilation of arteries, particularly those in humans, was mentioned. While the fact that large conduit arteries can be dilated by an NO mechanism to increased blood flow is interesting and useful, large arteries are not important resistance regulation vessels. However, the smaller generations of arteries are relevant to vascular resistance regulation and their flow-mediated vasodilation could be important to serving the needs of their host organ. A reduction in small artery resistance of significance when organ blood flow is increased has been established for cerebral (213), intestinal (26), and various skeletal muscles (124, 136, 218). Various

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means of communication to influence feed arteries preceding the organ from neural to electrical cell to cell communication from downstream vascular smooth muscle or endothelial cells has been proposed with the latter mechanism proposed by Duling and colleagues (77,85,218,219) receiving the greatest acceptance. Given that this type of communication would cause hyperpolarization of smooth muscle cells, relaxation should occur. Hyperpolarization of endothelial cells would also have vasoactive effects through the increased release of NO if additional calcium enters the cell. In vivo measurements of [NO] along mesenteric arteries have been done as blood flow was increased by forcing a small artery to perfuse more tissue when other parallel arteries were occluded (38). The [NO] did increase as the flow stimulus was increased and with the protocol used, there should have been little to no deficit of blood flow to the microvasculature and tissue, although some type of cell to cell communication influencing upstream NO should not be discounted. The key is, however, that NO was involved in resistance artery dilation under conditions that would mimic an increase in organ blood flow. As small arteries generate such large concentrations of NO when flow-mediated dilation occurs, it was questioned whether some of this NO could be carried to downstream arterioles within tissue. When this possibility was tested by increasing flow through intestinal arteries while monitoring downstream [NO] in intestinal arterioles, the arteriolar [NO] did increase in approximate proportion to the increase in large vessel [NO] (38). As discussed earlier, NO entering plasma is


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substantially protected from oxygen by rapid complexing with certain amino acids and hemoglobin and the transit time from small arteries to arterioles is less than 1 s. These observations raise the possibility that arteries, even those large vessels whose resistance changes are trivial to overall flow regulation, could be donating additional NO to downstream resistance vessels during periods of increased blood flow. The flow-mediated increase in NO formation in arteries and arterioles lead to questioning whether such a mechanism might have a role is the ebb and flow of lymph as lymphatic vessels go through cyclic contractions. Studies of the lymphatic vessels in the wall of the small intestine have shown that these vessels generated sufficient [NO] during the increased lymph flow during intestinal absorption that they could impact dilation of nearby arterioles (30). However, these lymphatics inside the wall of the intestine have very weak lymphatic contractions compared to those outside the organs. The very robust cyclic contractions of extra-organ lymphatics are similar to the cardiac cycle in terms of a diastolic and systolic volume phases, stroke volume, and contractility, as recognized by Granger et al. (96, 97) and McHale et al. (175). To accelerate the rate and volume of contraction in the in vivo state, hemodilution with simple saline has been used with rat mesenteric lymphatics by Benoit et al. (20). To achieve a similar state for in vitro isolated lymphatics from multiple species and organs (92,110,139,175,178,179,192,194), an increased transmural pressure with increased artificial lymph flow allows more precise regulation of physical conditions. The increase in the contraction phase in terms of stroke volume and active pressure developed is related to diastolic expansion (20) and constrictor prostaglandins (179). The relaxation or diastolic phase is very dependent upon nitric oxide, as judged by pharmacological studies (91, 92, 224, 243), and also hyperpolarization as evaluated by electrophysiological studies (253, 254), which might also lead to increased NO generation. Measurement of perilymphatic [NO] in the in vivo state has shown that shortly after the fast contraction of the lymphatic contraction cycle, the [NO] begins to increase as would be expected as lymph flow increases the shear rate and stress (37). Examples of actual recordings are shown in Figure 5. The resting and active [NO] were found to be much higher by almost a factor of 2× in the vicinity of lymphatic valve bulbs compared to the long tubular sections of lymphatic. These data are shown in Figure 6. Direct measurements of [NO] on the bulb valves indicated that these tissues generate the greatest [NO] during the contraction cycle (29,29), as would be expected in part by the high shear rates generated in the valve lumen during the bulb contraction. Immunocytochemistry for eNOS, shown in Figure 7, demonstrated well expressed eNOS at all locations but the greater population of endothelial cells in the expanded bulb region and associated valves may explain part of the increased [NO]. The use of what is basically flow-mediated vasodilation in the lymphatics during their contraction/ relaxation cyclic again points to the importance of the shear mechanism to activate NO production in the vascular system.


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The upper panel is a long-term [NO] recording along a valve structure of an in vivo rat mesenteric lymphatic before and after the animal was given intravenous saline to increase lymph production. The vertical gray marks are contractions. The vessel wall [NO] increased along with the frequency of contractions over a 30-min period. In the lower panel, the [NO] of a valve area and a downstream tubular area after saline infusion are shown: these are not simultaneous recordings but were recorded within about 10 min of each other. The valvular areas consistently have a higher [NO] than nearby tubular areas under all conditions. The higher frequency variations of NO are artifacts of mechanical ventilation moving the tissue preparation. The gray marks signal the start of contraction and within 1 to 2 s thereafter, the local [NO] began to rise. Adapted, with permission, from Figure 2 of (37).

Vascular Wall and Tissue Oxygen Tension Influence on NO Production The possible role of NO in responding to oxygen tension has been extensively reviewed by investigators whose expertise is in the area of quantitative microvascular studies (46, 58, 145). In the interaction of tissue oxygenation and nitric oxide regulation, there are two major areas to consider based on the expert opinions of these investigators and studies they have cited. First, reductions in tissue oxygenation due to increased metabolic rate, a decrease in blood flow, or systemic hypoxia require a rapid and sustained increased in NO formation. In

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Figure 6 Paired measurements of in vivo rat mesenteric lymphatic valve and tubular areas along with nearby (20-30 μm) adipose tissue and 500-μm distant in adipose tissue. The purpose of the measurements is to demonstrate that mesenteric tissue does have a background [NO] that under resting conditions is similar to the [NO] along tubular sections of the lymphatic. The tubular section [NO] did increase with flow stimulation as shown in Figure 5. The data were obtained for 12 lymphatics of 10 rats. The valve bulb area has a consistently high [NO] whereas the tubular region has a [NO] similar to that in tissue. However, both regions of the lymphatic can increase [NO] during increased contractions. Adapted, with permission, from Figure 3 of (37).

this type of scenario, it is very likely that the tissue is releasing many metabolically related chemicals that may ultimately influence eNOS through increased intracellular calcium and actions on the biochemical processes of the cell leading to the activation of eNOS. For example, potassium release from cells during reduced oxygen availability or increased metabolic rate could activate the various potassium channels to relax vascular smooth muscle cells and increase NO formation from endothelial cells (71, 162, 189, 190, 245). The second consideration is whether endothelial cells or other types of cells in the vicinity of arterioles are in some way directly capable of sensing oxygen availability and translating this capability into a rapid and sustained increase NO formation. This would be an ideal sensor to response situation because oxygen tension within and around arterioles can change in a few seconds, the NO has virtually immediate access to the smooth muscle for an appropriate change in vascular tone, and eNOS functions well down to quite low oxygen tensions. For example, cerebral periarteriolar [NO] during severe hypotension is dramatically increased by 50% even at perivascular PO2 of ∼10 mmHg and can be sustained for 30 to 60 min (15). The studies that have attempted to correlate in vivo oxygen tension to NO regulation are quite limited because they are very technically demanding. Buerk et al. (44, 45, 48, 51, 235)

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have clearly demonstrated in the eye and brain vasculatures, as well as the carotid body (48,144), that there is a link of oxygen tension to NO formation that is both fast and sustained. Mild to moderate reductions in in vivo tissue oxygen tension of the rat small intestine are possible by decreasing the oxygen content of physiological saline flowing over the tissue while simultaneously measuring perivascular [NO] and vascular diameter. This approach was developed by Duling et al. (79, 80, 201) in the 1970s and has been extensively used in microvascular studies by many laboratories. When such studies are done, the perivascular [NO] increased and vessels dilated as the oxygen tension was lowered (185) around both arterioles and venules, but the [NO] of general tissue away from intestinal vessels was unaffected. The vasodilation and increased [NO] to reduced oxygen tension was suppressed by eNOS inhibition with L-NAME but little effect was found with either a generalized cyclooxygenase blocker or potassium channel inhibitor. In subsequent studies by Zani et al. (274), suppression of arginine uptake into cells by adding l-lysine to the bathing media also suppressed the increase in [NO] and vasodilation. The l-lysine competes against l-arginine for transport through the cationic amino acid transporter-1 (CAT-1) (67, 100, 109), allows reversible suppression of NO production, and is highly reliable as a specific means to limit NOS activity in an acute experiment. The use of l-lysine as a transport competitor demonstrated that cationic CAT-1 must be acutely activated to provide arginine substrate to eNOS to increase NO production, as shown in Figure 8, although there are other interpretations (153). The use of excess arginine to acutely and very rapidly stimulate NO formation has been shown by Yukosavljevic et al. (255) and Pezzuto et al. (199) using NO sensitive microelectrodes. These studies indicate that extracellular arginine can enter endothelial cells to supplement internal cellular stores and at least acutely, influence the local generation of NO. As CAT-1 activity is calcium dependent within the caveoli complex (66, 68) and arginine transport appears essential to increased NO generation, this was a clue that calcium was entering the endothelial cells with even mild reductions of oxygen tension. By a process of elimination, it was determined that the sodium/calcium exchange (NCX) working in reverse mode to remove sodium from cells in exchange for calcium entry was a necessary component of the increased NO production to reductions in perivascular oxygen tension. However, for this exchanger to work in reverse mode, allowing calcium ions to enter the cell in exchange for sodium ions, the observations predicted that somehow sodium ions were entering endothelial cells as the oxygen tension was decreased or the NCX transporter was oxygen sensitive. Nase et al. (273) had shown that sodium chloride hyperosmolarity can cause increased NO production as sodium ions enter endothelial cells through the Na+ /K+ /2Cl− channel, which is routinely used by cells to balance intra- and extracellular osmolarity in the short term. Cellular studies have shown that this channel is oxygen sensitive and channel opening events increase as oxygen tension decreases (108, 265) such that sodium would


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Figure 7

Panel A presents the expression of eNOS measured by immunofluorescence histochemistry of isolated rat mesenteric lymphatic vessels using confocal microscopy. The left of panel A is a reconstruction of the three-dimensional view of a lymphatic vessel showing the valvular, sinus, valve leaflets, and tubular regions. The intensity of fluorescence is shown on the right side of panel A for 18 vessels with 100% relative intensity at a point as the valvular leaflet insert. The greatest intensity, eNOS expression, occurs in the valve bulb region and tapers off down the tubular region. Panel B is a reconstruction of the center one-fourth of the vessel to illustrate intensity of the wall of specific structures. The intensity data was color coded with hotter colors as greater intensity or expression relative to the valve insertion point area. The averaged data for six vessels on the left side of panel B show the highest expression in the valve wall and valve leaflet mid points. Adapted, with permission, from Figure 4 from (37).

enter the cell. When the channel is blocked with bumetanide, the increase in NO and vasodilation as oxygen tension is decreased is strongly suppressed. This would argue that the Na+ /K+ /2Cl− channel rather than NCX transporter was the oxygen sensitive link in the overall process. The interaction of all these processes is diagramed in Figure 9 (274) to illustrate that sodium entry into endothelial cells due to a variety of processes could be an important component of the regulation of eNOS and NO production. When studies of the type just discussed were extended to the cerebral circulation to determine if an oxygen tension link to NO production existed outside the small intestinal vasculature, the results were quite similar but the regulatory scheme was different. The brain vasculature has mechanisms to increase cerebral blood flow during mild to severe hypoxia even when normocapnia is preserved (6, 134, 276). The increase in blood flow was suppressed when NOS was suppressed, particularly when neuronal NOS (nNOS) was suppressed (116). However, in the earlier development of nNOS blockers, the agents were shown to be able to suppress nNOS, but the differential specificity for nNOS versus eNOS was


marginal at best (15). In effect, many studies in the literature in which nNOS blockade was the goal also caused eNOS blockade to some extent. A highly specific nNOS blocker, N-(4S)(4-amino-5-[aminoethyl]aminopentyl)-N’-nitroguanidine, is now available that has little capability to suppress eNOS (105). While the endothelial cells of the brain do express nNOS, the amounts are very limited in adult cerebral endothelial cells (158,269). However, there is a large pool of nNOS in neuronal and support cells, such as glial and astrocyte cells, around cerebral vessels (64, 116, 156, 210, 211, 267). The nNOS system is activated by the N-methyl-d-aspartate (NMDA) receptors which in term is stimulated by glutamate and leads to an increase in intracellular calcium concentration to start the actions of nNOS (182). As an aside, the glutamate concentration in plasma is so high that it would maximally stimulate cerebral NMDA receptors were it not for the selectivity of the blood brain barrier to exclude plasma glutamate. As shown in Figure 10, after nNOS blockade, neither vasodilation nor increased perivascular [NO] occurred when the local oxygen tension about vessels was lowered. After suppression

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Figure 9 A proposed scheme for NO generation by vascular endothelial cells during NaCl hyperosmolarity at point A and reduced oxygen tension at point C with both acting through the Na+ /K+ /2Cl− cotransporter followed by removal of sodium in exchange for calcium by the Na+ /Ca2+ exchanger (NCX). At B, increased flow shear increases calcium entry to activate eNOS. In all three scenarios, entry of L-arginine through the CAT-1 transporter is required to support the increased NO production, all of which fail if l-lysine is used to compete for transport. Adapted, with permission, from original Figure 7 of (274).

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L-Lysine was used to compete for transport against L-arginine by the CAT-1 transporter and in doing so, suppress eNOS formation of NO. Panel A for blood flow and Panel B for relative [NO] demonstrated that reduction in in vivo oxygen tension by lowering the bath oxygen percentage from 5% to 0% for intestinal arterioles increased blood flow and [NO]. During l-lysine exposure, flow and [NO] decreased at rest and neither responded appropriately to decreased oxygen tension. Data are means ± SE. ∗, P < 0.05 versus the control; #, P < 0.05 versus the natural paired condition. Adapted, with permission, from original Figure 7 of (274).

of eNOS with cavtratin (16) to bind to the caveoli domain of eNOS (21,98), the localized reduction of oxygen tension continued to cause both dilation and increased [NO]. Of interest, flow-dependent vasodilation by cerebral arterioles was not inhibited by nNOS blockade but was suppressed by blockade of eNOS with cavtratin. This indicated that nNOS is used as part of an oxygen-sensing mechanism in the brain arterioles whereas eNOS has an entirely independent role in flow sensing and undoubtedly many other mechanisms. This is quite different from the intestinal vasculature in which both flow and oxygen sensing are both part of the eNOS constellation of regulatory mechanisms. Having found that entry of sodium ions into endothelial cells of the small intestine arterioles was part of the eNOS system for oxygen sensing, it seemed reasonable to suspect that nNOS might have a sodium entry linkage in the brain microvasculature. Since the Na+ -K+ -2Cl− cotransporter was involved in intestinal arteriolar responses to oxygen-involving

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eNOS, it was a logical beginning choice for the nNOS system. Blockade of the channel with bumetanide did nearly completely suppress NO responses and dilation to reduced oxygen tension: suppression of the Na+ /H+ exchanger with amiloride had minor effects. Having established that sodium ions were involved in activation of nNOS, the Na+ /Ca2+ exchanger was suspected as being involved, particularly the NCX1 isoform (115,264). This isoform is highly expressed in the cerebral cortex and blocked by the highly selective agent KB-R7943 (14, 70). In an in vivo preparation, suppression of the sodium and calcium exchanger did all but eliminate increased [NO] and vasodilation to reduced oxygen tension. However, as was expected, blockade did cause some vasoconstriction and decreased [NO] at normal perivascular oxygen tension. These observations indicate this system is active during resting conditions and this function was impaired by pharmacological blockade. While the brain arterioles did have a different source of NO for oxygen sensing than the intestinal vasculature, the involvement of sodium entry into cells and subsequent replacement with calcium to activate a NOS is a common theme.

Consequences of Hypertension and Diabetes Mellitus on Vascular Nitric Oxide Regulation Hypertension While there is little doubt that NO function is compromised in essential hypertension, there are a large number of problems


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The data in panel A are in vivo cerebral periarteriolar [NO] responses and panel B is diameter responses to a decrease in cerebral oxygen availability before and after nNOS was blocked. Oxygen availability was reduced by lowering the bath oxygen tension. The nNOS blockade both caused constriction and reduced [NO], likely reflecting the loss of the NO generated by nNOS in normal conditions. The nNOS blockade eliminated both the dilation and increased [NO] to reduced oxygen availability. Tests of eNOS function during nNOS blockade demonstrated it was functional (15). The data are based on six rats and ∗ indicates statistical significance from control and, # is a significant difference from normal conditions at low oxygen tension. Adapted, with permission, from Figure 8 of (15).

to solve as to whether the compromise of NO biology is a contributing cause of hypertension or is itself compromised by the multiple cellular abnormalities that lead to hypertension, as Bernatova has questioned (22). In either event, the decreased ability of NO mechanisms to cause vasodilation would be part and parcel of the many mechanisms that contribute to peripheral vasoconstriction. Despite the extraordinary number of studies on probable causes of essential hypertension, isolation of a single cause of hypertension has been elusive but renal involvement appears essential (22,107). Fortunately, there are multiple pharmacological means to control the arterial blood pressure and generally they work through suppression of vascular smooth muscle contraction and limiting cardiac function, often by decreasing blood volume in part by renal excretion of sodium ions. It is tempting to


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conclude that if a pharmacological treatment works well to lower the arterial blood pressure, particularly if the method involves lowering vascular resistance, then the underlying mechanism suppressed is part of the mechanism in the cause of hypertension. In current clinical practice, a variety of therapeutic agents are used to control hypertension and include interfering with the conversion of angiotensin I to II or blockade of angiotensin receptors, a diuretic to increase sodium excretion, and the use of calcium channel blockers. The admonition to patients to lose weight, increase aerobic exercise, and decrease sodium chloride intake is usually necessary as many are overweight, sedentary, and have a diet rich in sodium. These are the least effective therapies in part due to lack of patient compliance and even with patient compliance; the blood pressure usually continues to rise with age despite the pharmacological interventions being used. The treatment of hypertension is necessary because it causes muscular hypertrophy of the heart and eventually heart failure, increases the progression of atherosclerosis throughout the body, and is a leading cause of coronary occlusion, strokes of all types, and peripheral vascular disease. In addition, the age-related decrease in glomerular filtration rate is increased and mild retinopathy may develop in hypertension and likely reflects damage of the microvascular structures over time. One of the enigmas of hypertension is that there is remarkably little compromise of microvascular beds in terms of healing of the organ after trauma, atrophy of tissues due to poor microvascular blood flow, direct loss of foot and lower leg tissue due specifically to microvascular disease, and peripheral or central nervous system neuropathy due to microvascular complications. Recent reviews of a large body of studies (169, 220) indicate aerobic exercise is generally well tolerated by humans with hypertension and helps control their hypertension to a limited extent. As exercise places high demands on the microvasculature throughout the body, the fact that exercise is so well tolerated by hypertensive humans demonstrates how functional the resistance vasculature is during the disease. Hypertensive people are capable of even the high-intensity interval exercise that seems one of the better approaches to improving aerobic exercise capability (258). The point to be made is that while the hypertensive vasculature undoubtedly undergoes compromises in hypertension in humans, it does perform reasonably well. A recurring theme in determining the role of NO in hypertension is to what extent is the production of NO compromised and whether oxygen radicals are destroying NO and thereby lowering the bioavailability of NO. Pharmacological studies of in vitro mesenteric artery reactivity to exogenous NO from sodium nitroprusside and S-nitroso-N-acetylpenicillamine by Chang et al. (55) of adult spontaneously hypertensive rats (SHRs) and Wistar Kyoto normotensive rats (WKY) indicated increased vasodilation to both NO donors. The suppression of NOS by L-NAME caused greater constriction in SHR than WKY. These data could either predict greater reactivity to NO in SHR than WKY or higher NO production in SHR. There are an enormous number of similar studies in the literature

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predicting how impaired NO production, excessive destruction, and compromised regulation might cause increased vascular resistance. Measurements of indices of NO production, such as nitrite/nitrate in plasma or in renal excretion in hypertensive humans have in the majority yielded decreased formation of NO (5, 25, 160, 161, 191), but studies finding increased or normal formation cannot be dismissed (176, 180). Endothelial cells can regenerate arginine from citrulline quite effectively (87, 152, 226), which further complicates understanding what the vascular [NO] might be. There are very limited measurements of the in vivo [NO] in hypertensive rats, but the results are startling. Direct measurements of perivascular [NO] along in vivo mesenteric vessels by Zhou et al. (277) revealed that the concentration in SHR was double that in WKY, however the [NO] did not increase during flow mediated dilation. Isolated arterioles from other vascular beds of the SHR demonstrate impaired flow-mediated dilation and reactivity to NO stimulation in general (102, 114, 137, 138, 170, 233). Humans with untreated essential hypertension generally have impaired flow-mediated vasodilation and reactivity to acetylcholine compared to matched normal patients (181,198,202,203,221) but can have relatively normal dilation to NO donors. Therefore, most of the human and animal studies would predict that NO physiology is compromised in hypertension in terms of making additional NO above the resting state with minor issues for vessels responding to NO. An area of major research interest in the past 20 years has been oxidant formation and their actions in hypertension as a means to compromise both vascular smooth muscle and endothelial function. There is overwhelming evidence that increased oxidant formation occurs during hypertension with over 1500 reviews as of 2014 of different aspects and mechanisms of oxidative stress in arterial hypertension. If one asks what mechanism causes increased oxidant formation in hypertension or whether the increase is a cause or consequence of hypertension, there are no particularly clear answers. Furthermore, even the most recent reviews indicate various types of antioxidative therapies whether with hypertensive drugs or antioxidants are generally less successful for hypertensive humans than in animal models (95, 212, 280). A general assumption is that increased oxygen radical formation during hypertension either destroys NO and perhaps forms the more toxic peroxynitrite radical in the process or harms the endothelial cells such that less NO is formed. Both processes would likely occur simultaneously. Zhou et al. (277) used a combined and simultaneous measurement of perivascular NO and H2 O2 to address how both are influenced by hypertension in the SHR. While the data are limited to the mesenteric arteries, the results revealed that both [H2 O2 ] and [NO] are increased at rest and NO responses to elevated blood flow were very limited whereas H2 O2 increased. The increase in relative blood flow by WKY and SHR intestinal preparations to equivalent stresses of flow-mediated stimulation were similar but by quite different mechanisms. Both reactive oxygen species (149, 177, 200) and NO (33, 118) have

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been implicated to increase during elevated flow in normal vessels and obese Zucker rats (27). After suppression of oxidants with apocynin as well as the NAD(P)H oxidase inhibitor gp91ds-tat, the basal [H2 O2 ] and [NO] in SHR decreased to near normal concentrations and the flow-mediated increase in [NO] was restored. In WKY rats, increased blood flow did not appreciably increase the [H2 O2 ] and blockade of NAD(P)H oxidase had little effect on NO responses. H2 O2 at 20 and 100 umol/L increased the [NO] in normal but not SHR, perhaps because the NO mechanism was already highly activated in SHR. When PEG-catalase was applied to SHR vessels to lower the [H2 O2 ] to normal, the [NO] was reduced to normal and flow-dependent increases in [NO] were restored. The data just described raise the issue that hypertension changes the regulation of at least flow-mediated vasodilation to an H2 O2 -dependent mechanism but the elevation of H2 O2 stimulates NO production at rest to perhaps a pathological concentration of about 2000 nmol/L. However, how these observations translate to chronic regulation of NO-dependent mechanisms and their consequences for the vasculature need to be addressed. A means to achieve this goal is evaluation of how long-term stimulation of the flow-mediated dilation system influences growth of normal mesenteric arteries over days to weeks. Within 1 week of a mesenteric artery forced to provide increased flow by occlusion of parallel arteries on either side, the vessel wall lumen expanded and hyperplasia of endothelial cells occurred, as shown by Unthank et al. (248). At 4 weeks, the expansion of the vessel lumen had restored shear rate to normal and the vessel wall had remodeled to increase cross sectional area. Subsequent studies indicate the remodeling process begins within one to 3 days (244) and was associated with increased expression of eNOS in the endothelial cells (246) by the first day of increased flow with a diminution, but still elevated expression at 1 week. Eventually at 4 weeks, the structure of the enlarged artery was similar to normal vessels in terms of vascular smooth muscle cell density (cells per cross sectional area) but much larger in cross-sectional area (82). While multiple mechanisms undoubtedly cause the vascular enlargement and cellular adaptation to increased shear forces (215, 247, 279), direct measurements of increased [NO] during acute increases in blood flow (38, 76, 103, 118, 277, 278) and elevation of eNOS expression on a chronic level as just mentioned predict that NO is certainly involved. Given that acute exposure of mesenteric arteries to H2 O2 caused increased NO production in normal rats and excessive production in SHR and mature normal rats inhibits further NO generation during flow stimuli (277,278), it is not surprising that oxidant formation has been implicated as a necessary step in the normal vascular remodeling process for elevated blood flow and a complicating factor to NO regulation when oxidant formation is excessively elevated in aging and hypertension (76, 103, 159, 247, 278). In summary, measurements of NO in vivo indicate an elevated concentration linked to elevated hydrogen peroxide formation, yet the vessels do not respond appropriately to NO so long as the peroxide concentration is elevated. A key


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issue here is that moderate increases in the concentration of peroxide, in the tens of micromolar as found by direct measurement in SHR, has these effects whether caused by hypertension in SHR or simulated with exogenous peroxide in normal rats. The often used peroxide concentrations in excess of 100 umol/L in many studies has only detrimental effects on eNOS activity and very likely only occurs in the most severe of pathological conditions. The unexpected activation of eNOS by peroxide at tens of micromolar concentrations which occur in the SHR arterioles appears to occur through AKT and MAPK activation (10, 40, 275).

Obesity, Diabetes Mellitus, and Nitric Oxide The destructive vascular consequences of type I diabetes in which insulin production is compromised have been described since antiquity. The development of obesity related diabetes, type II diabetes, and its associated health consequences that lead to premature death were well described by the 1800’s (Handbook of Physiology, Microcirculation, Chapter 19, 2008). Interestingly, while the cause of both types I and II diabetes were not known or even differentiated until the early 1900s, a low-calorie diet was appreciated to slow the progression of both diabetes in young people and mature obese people. The advent of insulin availability dramatically increased the life span of type I diabetics and slowed the progression of complications in both types I and II diabetes. However, even until the present time, accelerated atherosclerosis, microvascular disease, kidney disease, and peripheral neuropathy continue to have life-threatening consequences. The dramatic increase in obesity of the mature and now young population over the past 25 years has raised the incidence of type II diabetes. The Centers for Disease Control in the 2014 National Diabetes Statistics Report projects that about 9.3% of United States citizens have diabetes, most of which is caused by obesity issues. While accelerated atherosclerosis occurs almost as prevalently in obese as obese, diabetic populations (56, 93, 193), the incidence of lower body peripheral neuropathy and microvascular disease leading to loss of foot and leg tissue are extraordinarily different prior to atherosclerotic occlusion sufficient to impair blood flow. Development of the capillary overgrowth of retinal microvascular disease, the leading cause of blindness in adult populations in the United States and Europe, is rare in obese people prior to the development of hyperglycemia. The progression of kidney disease in the obese population is of concern but is far less of an issue than in people with type II diabetes (261). These epidemiological disparities for microvascular disease and peripheral neuropathy in obesity versus obesity associated with type II diabetes have increased the interest in whether hyperglycemia per se is a key issue in developing serious microvascular abnormalities. The current clinical guidelines to diagnose diabetes are fasting blood glucose of 129 mg/dL plasma and a glucose


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tolerance test concentration over 199 mg/dL in plasma. As a frame of reference for normal individuals, values of 99 and 139 mg/dL are used and so call prediabetics fall in between the normal and diabetic glucose concentrations. The blood glucose guidelines evolved from epidemiological studies of hyperglycemia and onset of typical diabetic microvascular disease. The time course of clinically relevant microvascular disease in diabetes is of the order of 5 to 10 years or more (186), but intensive therapy to control hyperglycemia has been shown to slow the progression (69). As mentioned, obese people without blood glucose in the diabetic range rarely have the significant microvascular disease of the forms common in the type II diabetic population. However, their vascular regulation is not normal, including NO physiology, as has been covered in recent reviews (not comprehensive) for obese humans, including children, and animal models of obesity (2, 18, 78, 101, 119, 167, 197, 239). At some point in the metabolic syndrome cascade both with aging or increased body mass, the development of hyperglycemia occurs and the risks for very serious microvascular disease increases. A fundamental mechanism that links obesity, the onset of hyperglycemia, and disturbed vascular regulation is increased activation of protein kinase C (PKC) in endothelial and vascular smooth muscle cells of the vasculature. Obesity and acute hyperglycemia are both associated with increased diacylglycerol in plasma that activates PKC forms in many different types of cells (74, 123, 263). In 1995 in one of the first studies of acute effects of hyperglycemia on endothelial function, Mayhan (173) demonstrated that in vivo cerebral arterioles of normal rats had suppressed endothelialdependent dilation to a variety of vasodilators beginning at d-glucose concentrations of 20 mmol/L, about four times higher than normal. This altered behavior could be both suppressed and substantially restored by suppression of PKC with the PKC blockers available at that time. During hyperglycemia, the metabolism of excessive glucose (121, 263) leads to increased diacylglycerol in the cell. As endothelial and vascular smooth muscle cells transport glucose through a noninsulin-dependent glucose carrier, GLUT1 (19, 127), they are particularly vulnerable to excessive glucose entry during hyperglycemia. Long-term studies of the effects of PKC inhibition by LY333531 in endothelial cells of diabetic mice by King’s group have shown very good improvement of the structural and biochemical properties of the retinal (266), heart (257), and renal microvascuatures (122). These improvements occurred even though the diabetic state was not changed. Clinical trials on LY333531 by the Eli Lilly Company using the intestinal absorbed version of the drug known as ruboxistaurin have included types I and II diabetic humans. This drug is particularly adept at blocking PKC-βII, the dominant isoform of PKC in endothelial cells. The available data from clinical trials have shown useful effects to slow the advance of microvascular disease in the eye, less protein leakage by the glomerular membranes, some improvement in neuropathy and nephropathy, and a decrease in mean arterial pressure (Eli Lilly Company) (1, 9, 222, 262). The diacylglycerol-PKC interaction is

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of interest in NO physiology because PKC inhibits eNOS and limits NO formation. LY333531 or ruboxistaurin treatment for one week in normal humans prevented the typical large decline in brachial blood flow vasodilation to intravascular methacholine during episodes of acute hyperglycemia (17). In later studies (63), type I diabetic patients exposed to systemic hyperglycemia experienced impaired flow-mediated dilation which was substantially reversed by ruboxistaurin. In normal rats, acute 300 mg/dL but not 200 mg/dL d-glucose exposed to in vivo intestinal and skeletal muscle arterioles for one hour greatly suppressed vascular dilation to acetylcholine applied directly to the vessel wall plus inhibited flowmediated vasodilation, and the associated [NO] responses (31, 34, 36, 125, 126, 147). In in vitro preparations of mouse kidney slices, Chu (65) demonstrated very similar data for NO production by glomerular capillaries activated by bradykinin. Glomerular tissue was studied because it is both a major target for problems in all forms of diabetes and illustrates that even capillaries are susceptible to hyperglycemic eNOS dysfunction. Both the resting and stimulated [NO] by the glomerular capillaries were suppressed by 20 mmol/L d-glucose in 10 to 30 min. In this study and the others mentioned above, ruboxistaurin pretreatment minimized the hyperglycemic suppression of NO mechanisms and when used after hyperglycemia, was able to recover some of the ongoing deficit of microvascular function in untreated tissue exposed to hyperglycemia. Given that PKC activates many enzymes that would compromise endothelial and vascular smooth muscle function, in particular NADPH oxidases, the benefits of ruboxistaurin likely are multifaceted and extend well beyond eNOS protection. For example, the Mayhan laboratory (171, 172) has shown that NADPH oxidase has a role in in vivo suppression of eNOS or at least NO bioavailability in type 1 diabetes. While hydrogen peroxide at physiological concentrations of a few micromolar can stimulate eNOS (52, 278), excessive production of oxygen radicals and hydrogen peroxide are well-established problems for endothelial function. Suppression of PKC in obese diabetic mice (231) and rats (184) also improves eNOS function by disinhibition of PKC’s effects on AKT which in turn would allow greater activity of eNOS. Given the broad range of enzymes influenced by excessive PKC activation and all of them involved at some level in eNOS function, as endothelial specific PKC inhibitors become available for clinical practice in the near future, substantial direct and indirect improvements in eNOS function may be possible in the diabetic population. An important issue to consider in humans with known type II diabetes is that the vast majority is under clinical care and while their blood glucose regulation may not be ideal, they are typically in the high normal to mild hyperglycemia range. However, this population is typically obese and would have the disturbed regulatory systems mentioned earlier. Does this situation make this population more susceptible to acute problems with bouts of more severe hyperglycemia? There are data from animal studies to predict this situation may exist. As shown in the Figure 11 (35), in Zucker Fatty rats just


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Figure 11 In Zucker obese rats prior to developing transient hyperglycemia, lowering the local intestinal vascular oxygen tension caused significantly less increased blood flow and elevated [NO] than in normal lean Zucker rats. After blockade of PKC β-11, lean animals were unaffected but obese animals experience increased blood flow at rest and during decreased oxygen availability. These data indicate PKC activation in obese animals limits NO physiology both at rest and when called upon to increase NO production at reduced oxygen tension. The data are based on five obese and six lean Zucker rats. An asterisk represents a significant event from 5% to 0% oxygen and two asterisks indicate a significant change after PKC blockade. Adapted, with permission, from Figure 1 of (27).

before they develop transient bouts of hyperglycemia after feeding, the resting [NO] was lower than normal in the presence of 5% oxygen in the tissue bath and did not increase as in normal rats when the local oxygen tension was reduced. In Figure 12, the obese animals are shown to have a mildly suppressed response of dilation and increased [NO] when cerebral arterioles are forced to undergo flow-mediated vasodilation as a parallel arteriole is occluded. When downstream occlusion of the same vessel was done, the [NO] decreased as flow declined but was less developed in obese animals. In both cases for oxygen and flow effects, suppression of PKC β-II restored much of the function in obese rats but had little effect in normal animals. The latter is not unexpected as this enzyme system should be of limited activity in normal rats at rest. The obese animals studied were about to enter the phase of life when postprandial hyperglycemia begins to develop. Therefore, it was of interest whether these animals would


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Figure 12 The percent of control changes in intestinal blood flow in panel A and perivascular [NO] in panel B as blood flow was increased by occlusion of parallel arterioles and decreased by downstream occlusion of the arteriole of interest. Both of these variables were suppressed in obese Zucker rats relative to responses in lean rats and these deficits were essentially restored to normal after acute blockade of PKC-βII. The data were obtained in seven obese and lean Zucker rats, one arteriole per animal. A single asterisk represents significant change within a rat type and two asterisks indicate a change with drug blockade. Adapted, with permission, from Figure 2 of (27).

be normally or excessively sensitive to acute hyperglycemia. As shown in Figure 13, obese animals are far more sensitive to the acute effects of hyperglycemia than are normal rats (35). Depression of NO with 200 mg/dL glucose occurred in obese rats, but to generate a similar deficit in lean rats, 300 mg/dL glucose was required: 200 mg/dL was benign in lean rats (35). In addition, deficits in dilation and increased [NO] to local bradykinin exposure was suppressed in intact obese animals and further compromised by 200 mg/dL d-glucose exposure: by comparison, 300 mg/dL was required to demonstrate a hyperglycemic effect in lean rats. In both lean and obese animals once hyperglycemia had compromised the NO mechanism, the problem persisted for at least three hours after local normoglycemia was restored. Studies of endothelial regulation in the peripheral vasculature of obese and normoglycemic diabetic humans typically indicate depressed NO mechanisms activated by


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Figure 13

The resting [NO] and diameter of large intestinal arterioles in obese, nonhyperglycemic Zucker rats was lower than normal in age matched lean animals. Both groups had suppressed NO function to topical acute 300 mg/dL hyperglycemia but 200 mg/dL d-glucose in obese rats caused as much compromise as 300 mg/dL in lean rats. The lean rats were insensitive to 200 mg/dL d-glucose for 1 h with a small deficit at 2 h of exposure. After the hyperglycemia of one hour duration was stopped, recovery was not evident in neither lean nor obese rats for at least two hours. Twelve normal vessels from 6 lean rats and 12 vessels from obese animals were studied. ∗ Significant change during hyperglycemia relative to control. Adapted, with permission, from Figure 3 of (35).

receptor mediated and flow-dependent endothelial processes, yet the vasculature typically has a near normal dilation to exogenous NO (53,151). In the obese but near normoglycemic Zucker rat, after blockade of PKC I and II beta isoforms, the resting [NO] in obese animals increased to be greater than in lean rats within 20 to 30 min and their ability to increase [NO] and blood flow in response to a low oxygen bath was dramatically improved (27,34). Normal arterioles did not demonstrate any response to PKC-βII blockade. These observations indicate that the endothelial cells of obese animals are capable of generating normal concentrations of NO with their eNOS but are likely having eNOS suppressed by excessive PKC activation. These data also support Erdos’ et al. (81), Schwaninger’s et al. (217), and Mayhan’s et al. (174) observation that eNOS expression in endothelial cells, including cerebral endothelial

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cells, of Zucker Obese and Zucker Fatty Diabetic Rats is at least normal and likely somewhat greater than normal. Bohlen et al. (27) found that suppression of NO formation secondary to PKC activation in Zucker obese rats had consequences for microvascular responses to changes in flow velocity during flow-mediated vasodilation. As shown in Figure 12, the vascular responses to increased and decreased shear rates caused by forcing arterioles to perfuse a larger or smaller than normal amount of tissue by temporary occlusions was substantially improved in obese rats after PKC blockade and to a lesser extent in lean rats. This improvement occurred within about 30 min of PKC blockade. Recently, Busija et al. (81) have shown that bioassay analysis of cerebral arterioles in Zucker Obese rats indicates improved dilation to acetylcholine after PKC blockade. They could not measure [NO] but assume that NO increased because the vessels responded normally to nitroprusside, an NO donor. The last issue to consider for NO and diabetes is that insulin is a vasoactive compound for the endothelial cells and stimulates increased release of NO. Endothelial cells internalize insulin at the blood interface and transport insulin to the tissue side of the cells (104, 133). At least 80% of the insulin survives intact during this process and is released to the tissue. Some of the insulin appears to activate the endothelial cells (39, 104) and NO appears to increase during insulin transport (256). Studies in predominately skeletal muscle vascular beds of both humans and animals demonstrate vasodilation at elevated but physiological insulin concentrations but dilation is suppressed by pharmacological blockade of eNOS (11, 12, 229). It has been questioned whether insulin has its vascular effects by acutely stimulating the endothelial cell NO generation system, activating NO release secondary to some insulin effect on tissue metabolism, or changing longer term expression of eNOS (62, 86). Studies of isolated arterioles have found both insulin causing and not causing endothelialdependent dilation linked to NO (62, 86). There is a recent concern that insulin stimulation increases oxidant formation by activating NADPH oxidase, which might cause odd NO responses if eNOS function is not ideal (252). However, the bulk of the literature supports an ability for insulin to increase release of NO from endothelial cells, at least in normal endothelial cells (11, 251). Both obesity and type II diabetes in humans suppressed insulin-mediated NO release in the leg vasculature (13, 142, 143, 238) as judged by vasodilation. The assumption is that fewer insulin receptors allow less of an insulin stimulation of eNOS through AKT activation. However, insulin concentrations in obese and obese diabetic humans and animals are very high so long as the pancreas is competent and maintained elevated or further increased in obese diabetics by the administration of insulin. Analysis of clinical trials (268) indicates that hyperinsulinemia per se was much less of a risk factor for complications than insulin resistance, in effect, lowered actions of insulin are more of a concern than the concentration of insulin. In this context, recent studies have correlated insulin resistance to increased activation of the renin-angiotensin-aldosterone system in

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diabetes as both a complication of the disease and factor in lowering the biological actions of insulin by inhibitory actions on the insulin receptor (167) and increasing oxidant formation to destroy additional NO formed in response to insulin (7).

Summary The in vivo actions of NO on the cardiovascular system and complications caused by hypertension, obesity, and diabetes have been and continue to be extraordinarily rich areas for basic science and clinical research. Nitric oxide has major roles in oxygen and blood flow shear sensing, responses to a myriad of biological compounds released by tissues and associated with vasodilation, remodeling of arteries when chronically exposed to increased blood flow, and the pumping cycle of lymphatics. In the areas of NO pathophysiology, interventions to improve endothelial function by pharmacology and lifestyle modifications are quite well established. While NO physiology appears compromised during essential hypertension, it does not appear that changing NO physiology is the initiating cause of increased vascular resistance. The endothelial cell compromise and damage associated with both types I and II diabetes has profound effects to limit NO formation and consequently the beneficial effects of NO are suppressed. This is unfortunate because the myriad studies of the physiology of NO in normal life have shown that this simple molecule and its vascular actions are among the most important forms of vascular regulation.

Acknowledgements The in vivo studies of [NO] measurements by the author were supported by NIH grants HL20605-29, HL-25824-23, and HL-70308-4. The author would like to acknowledge the special role of Dr. Donald Buerk for his many contributions to the measurement of NO with microelectrodes and development of multiple mathematical models of NO physiology in the microvasculature based on his unique perspective from in vivo studies.

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