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We developed a two-dimensional model of transport-dependent intracellular calcium signaling in endothelial cells (ECs). Our purpose was to evaluate the effects of spatial colocalization of endothelial nitric oxide synthase (eNOS) and capacitative calcium entry (CCE) channels in caveolae on eNOS activation in response to ATP. Caveolae are specialized microdomains of the plasma membrane that contain a variety of signaling molecules to optimize their interactions and regulate their activity. In ECs, these molecules include CCE channels and eNOS. To achieve a quantitative understanding of the mechanisms of microdomain calcium signaling and the preferential sensitivity of eNOS to calcium entering the cell through CCE channels, we constructed a mathematical model incorporating the cell morphology and cellular physiological processes. The model predicts that the spatial segregation of calcium channels in ECs can create transport-dependent sharp gradients in calcium concentration within the cell. The calcium concentration gradient is affected by channel density and cell geometry. This transport-dependent calcium signaling specificity effect is enhanced in ECs by increasing the spatial segregation of the caveolar signaling domains. Our simulation significantly advances the understanding of how [Ca.sup.2+], despite its many potential actions, can mediate selective activation of signaling pathways. We show that diffusion-limited calcium transport allows functional compartmentalization of signaling pathways based on the spatial arrangements of [Ca.sup.2+] sources and targets. capacitative calcium entry; caveolae; endothelial cells: endothelial nitric oxide synthase; mathematical model

Endothelial progenitor cells (EPCs) are essential in vasculogenesis and wound healing, but their circulating and wound level numbers are decreased in diabetes. This study aimed to determine mechanisms responsible for [...]

Nitric oxide (NO) is believed to play an important role in pancreatic islet physiology and pathophysiology. Research in this area has been hampered, however, by the use of indirect methods to measure islet NO. To investigate the role of NO in islet function, we positioned NOsensitive, recessed-tip microelectrodes in close proximity to individual islets and monitored oxidation current to detect subnanomolar NO in the bath. NO release from islets consisted of a series of rapid bursts lasting several seconds and/or slow oscillations with a period of 100-300 s. Average baseline NO near the islets in 2.8 mM glucose was 524 [+ or -] 59 nM (n = 12). Raising glucose from 2.8 to 11.1 mM augmented NO release by 429 [+ or -] 133 nM (n = 12, P < 0.05), an effect blocked by the NO synthase inhibitor L-NAME (n = 3). We also observed that glucose-stimulated increases in NO release were contemporaneous with changes in NAD(P)H and [O.sub.2] but occurred well before increases in calcium associated with glucose-stimulated insulin secretion. In summary, we demonstrate that NO release from islets is oscillatory and rapidly augmented by glucose, suggesting that NO release occurs early following an increase in glucose metabolism and may contribute to the stimulated insulin secretion triggered by suprathreshold glucose. calcium; oscillations; [N.sub.G]-nitro-L-arginine methyl ester; oxygen; reduced forms of islet nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide phosphate

We hypothesized that exposure to hyperbaric oxygen (HB[O.sub.2]) would mobilize stem/progenitor cells from the bone marrow by a nitric oxide (*NO) -dependent mechanism. The population of CD[34.sup.+] cells in the peripheral circulation of humans doubled in response to a single exposure to 2.0 atmospheres absolute (ATA) O2 for 2 h. Over a course of 20 treatments, circulating CD[34.sup.+] cells increased eightfold, although the overall circulating white cell count was not significantly increased. The number of colony-forming cells (CFCs) increased from 16 [+ or -] 2 to 26 [+ or -] 3 CFCs/100,000 monocytes plated. Elevations in CFCs were entirely due to the CD[34.sup.+] subpopulation, but increased cell growth only occurred in samples obtained immediately posttreatment. A high proportion of progeny cells express receptors for vascular endothelial growth factor-2 and for stromal-derived growth factor. In mice, HB[O.sub.2] increased circulating stem cell factor by 50%, increased the number of circulating cells expressing stem cell antigen-1 and CD34 by 3.4-fold, and doubled the number of CFCs. Bone marrow *NO concentration increased by 1,008 [+ or -] 255 nM in association with HB[O.sub.2]. Stem cell mobilization did not occur in knockout mice lacking genes for endothelial *NO synthase. Moreover, pretreatment of wild-type mice with a *NO synthase inhibitor prevented the HB[O.sub.2]-induced elevation in stem cell factor and circulating stem cells. We conclude that HB[O.sub.2] mobilizes stem/ progenitor cells by stimulating *NO synthesis. nitric oxide; CD34; CD133; CXCR4; cKit; colony-forming cells; progenitor cells

We tested the hypothesis that high-viscosity (HV) plasma in extreme hemodilution causes wall shear stress to be greater than low-viscosity (LV) plasma, leading to enhanced production of nitric oxide (NO). The perivascular concentration of NO was measured in arterioles and venules and the tissue of the hamster chamber window model, subjected to acute extreme hemodilution, with a hematocrit (Hct) of 11% using Dextran 500 (n = 6) or Dextran 70 (n = 5) with final plasma viscosities of 1.99 [+ or -] 0.11 and 1.33 [+ or -] 0.04 cp, respectively. HV plasma significantly increased the periarteriolar, perivenular, and tissue NO concentration by 2.0, 1.9, and 1.4 times the control (n = 7). The NO concentration with LV plasma was not statistically different from control. Arteriolar shear stress was significantly increased in HV plasma relative to LV plasma in arterioles but not in venules. Aortic endothelial NO synthase (eNOS) protein expression was increased with HV plasma but not with LV plasma. There was a weak correlation between perivascular NO concentration and the locally calculated shear stress induced by the procedures, when blood viscosity was corrected according to Hct values previously determined in studies of microvascular Hct distribution. The finding that the periarteriolar and venular NO concentration in HV plasma was the same although arteriolar shear stress was significantly greater than venular shear stress maybe be due to differences in vessel wall metabolism between arterioles and venules and the presence of NO transport through the blood stream in the microcirculation. Results support the concept that in extreme hemodilution HV plasma maintains functional capillary density through a NO-mediated vasodilatation. functional capillary density; microvascular flow; shear stress; plasma expanders; mechanotransduction

Abstract: Objectives: The effect of NO on smooth muscle cell contractility is crucial in regulating vascular tone, blood flow, and O2 delivery. Quantitative predictions for interactions between the NO production rate and the myogenic response for microcirculatory blood vessels are lacking. Methods: We developed a computational model of a branching microcirculatory network with four representative classes of resistance vessels to predict the effect of endothelium‐derived NO on the microvascular pressure‐flow response. Our model links vessel scale biotransport simulations of NO and O2 delivery to a mechanistic model of autoregulation and myogenic tone in a simplified microcirculatory network. Results: The model predicts that smooth muscle cell NO bioavailability significantly contributes to resting vascular tone of resistance vessels. Deficiencies in NO seen during hypoxia or ischemia lead to a decreased vessel diameter for all classes at a given intravascular pressure. At the network level, NO deficiencies lead to an increase in pressure drop across the vessels studied, a downward shift in the pressure‐flow curve, and a decrease in the effective range of the autoregulatory response. Conclusions: Our model predicts the steady state and transient behavior of resistance vessels to perturbations in blood pressure, including effects of NO bioavailability on vascular regulation. [ABSTRACT FROM AUTHOR]

Nitric oxide (NO) generated from nitrite through nitrite reductase activity in red blood cells has been proposed to play a major role in hypoxic vasodilation. However, we have previously predicted from mathematical modeling that much more NO can be derived from tissue nitrite reductase activity than from red blood cell nitrite reductase activity. Evidence in the literature suggests that tissue nitrite reductase activity is associated with xanthine oxidoreductase (XOR) and/or aldehyde oxidoreductase (AOR). We investigated the role of XOR and AOR in nitrite-mediated vasodilation from computer simulations and from in vivo exteriorized rat mesentery experiments. Vasodilation responses to nitrite in the superfusion medium bathing the mesentery equilibrated with 5% O2 (normoxia) or zero O2 (hypoxia) at either normal or acidic pH were quantified. Experiments were also conducted following intraperitoneal (IP) injection of nitrite before and after inhibiting XOR with allopurinol or inhibiting AOR with raloxifene. Computer simulations for NO and O2 transport using reaction parameters reported in the literature were also conducted to predict nitrite-dependent NO production from XOR and AOR activity as a function of nitrite concentration, PO2 and pH. Experimentally, the largest arteriolar responses were found with nitrite >10mM in the superfusate, but no statistically significant differences were found with hypoxic and acidic conditions in the superfusate. Nitrite-mediated vasodilation with IP nitrite injections was reduced or abolished after inhibiting XOR with allopurinol (p < 0.001). Responses to IP nitrite before and after inhibiting AOR with raloxifene were not as consistent. Our mathematical model predicts that under certain conditions, XOR and AOR nitrite reductase activity in tissue can significantly elevate smooth muscle cell NO and can serve as a compensatory pathway when endothelial NO production is limited by hypoxic conditions. Our theoretical and experimental results provide further evidence for a role of tissue nitrite reductases to contribute additional NO to compensate for reduced NO production by endothelial nitric oxide synthase during hypoxia. Our mathematical model demonstrates that under extreme hypoxic conditions with acidic pH, endogenous nitrite levels alone can be sufficient for a functionally significant increase in NO bioavailability. However, these conditions are difficult to achieve experimentally. [ABSTRACT FROM AUTHOR]

Inert gases diffuse into tissues in proportion to ambient pressure, and when pressure is reduced, gas efflux forms bubbles due to the presence of gas cavitation nuclei that are predicted based on theory but have never been characterized. Decompression stress triggers elevations in number and diameter of circulating annexin V-coated microparticles (MPs) derived from vascular cells. Here we show that ~10% MPs from wild-type (WT) but not inflammatory nitric oxide synthase-2 (iNOS) knockout (KO) mice increase in size when exposed to elevated air pressure ex vivo. This response is abrogated by a preceding exposure to hydrostatic pressure, demonstrating the presence of a preformed gas phase. These MPs have lower density than most particles, 10-fold enrichment in iNOS, and generate commensurately more reactive nitrogen species (RNS). Surprisingly, RNS only slowly diffuse from within MPs unless particles are subjected to osmotic stress or membrane cholesterol is removed. WT mice treated with iNOS inhibitor and KO mice exhibit less decompression-induced neutrophil activation and vascular leak. Contrary to injecting naïve mice with MPs from wild-type decompressed mice, injecting KO MPs triggers fewer proinflammatory events. We conclude that nitrogen dioxide is a nascent gas nucleation site synthesized in some MPs and is responsible for initiating postdecompression inflammatory injuries. [ABSTRACT FROM AUTHOR]

Endothelial progenitor cells (EPC) are known to contribute to wound healing, but the physiologic triggers for their mobilization are often insufficient to induce complete wound healing in the presence of severe ischemia. EPC trafficking is known to be regulated by hypoxic gradients and induced by vascular endothelial growth factor-mediated increases in bone marrow nitric oxide (NO). Hyperbaric oxygen (HBO) enhances wound healing, although the mechanisms for its therapeutic effects are incompletely understood. It is known that HBO increases nitric oxide levels in perivascular tissues via stimulation of nitric oxide synthase (NOS). Here we show that HBO increases bone marrow NO in vivo thereby increasing release of EPC into circulation. These effects are inhibited by pretreatment with the NOS inhibitor L-nitroarginine methyl ester (L-NAME). HBO-mediated mobilization of EPC is associated with increased lower limb spontaneous circulatory recovery after femoral ligation and enhanced closure of ischemic wounds, and these effects on limb perfusion and wound healing are also inhibited by L-NAME pretreatment. These data show that EPC mobilization into circulation is triggered by hyperoxia through induction of bone marrow NO with resulting enhancement in ischemic limb perfusion and wound healing. [ABSTRACT FROM AUTHOR]

The purpose of this investigation was to study the effect of the presence of red blood cells (RBCs) in the plasma layer near the arteriole wall on nitric oxide (NO) and oxygen (O2) transport. To this end, we extended a coupled NO and O2 diffusion-reaction model in the arteriole, developed by our group, to include the effect of the presence of RBCs in the plasma layer and the effect of convection. Two blood flow velocity profiles (plug and parabolic) were tested. The average hematocrit in the bloodstream was assumed to be constant in the central core and decreasing to zero in the boundary layer next to the endothelial surface layer. The effect of the presence or absence of RBCs near the endothelium was studied while varying the endothelial surface layer and boundary layer thickness. With RBCs present in the boundary layer, the model predicts that 1) NO decreases significantly in the endothelium and vascular wall; 2) there is a very small increase in endothelial and vascular wall Po2; 3) scavenging of NO by hemoglobin decreases with increasing thickness of the boundary layer; 4) the shape of the velocity profile influences both NO and Po2 gradients in the bloodstream; and 5) the presence of RBCs in the boundary layer near the endothelium has a much larger effect on NO than on O2 transport. [ABSTRACT FROM AUTHOR]

Nitric oxide (NO) and oxygen (02) transport in the microcirculation are coupled in a complex manner, since enzymatic production of NO depends on O2 availability, NO modulates vascular tone and O2 delivery, and tissue O2 consumption is reversibly inhibited by NO. The authors investigated whether NO bioavailability is influenced by the well-known Fåhraeus effect, which has been observed for over 70 years. This phenomenon occurs in small-diameter blood vessels, where the tube hematocrit is reduced below systemic hematocrit as a plasma boundary layer forms near the vascular wall when flowing red blood cells (rbcs) migrate toward the center of the bloodstream. Since hemoglobin iii the bloodstream is thought to be the primary scavenger of NO in vivo, this might have a significant impact on NO transport. To investigate this possibility, the authors developed a multilayered mathematical model for mass transport in arterioles using finite element numerical methods to simulate coupled NO and O2 transport in the blood vessel lumen, plasma layer, endothelium, vascular wall, and surrounding tissue. The Fahraeus effect was modeled by varying plasma layer thickness while increasing core hematocrit based on conservation of mass. Key findings from this study are that (1) despite an increase in the NO scavenging rate in the core with higher hematocrit, the model predicts enhanced vascular wall arid tissue NO bioavailability due to the relatively greater resistance for NO diffusion through the plasma layer; (2) increasing the plasma layer thickness also increases the resistance for O2 diffusion, causing a larger PO2 gradient near the vascular wall and decreasing tissue O2 availability, although this can be partially offset with inhibition of O2 consumption by higher tissue NO levels; (3) the Fåhraeus effect can become very significant in smaller blood vessels (diameters <30 µm); and (4) models that ignore the Fãhraeus effect may underestimate NO concentrations in blood and tissue. Objective: To examine whether prostaglandins are involved in endotheliurn-dependent vasodilatory responses of the skin microcirculation. Methods: Twenty-three young male volunteers were studied on 2 different days 1-3 weeks apart. On each experimental day the forearm skin blood flow response to iontophoretically applied acetylcholirie (Ach, an endothelium-dependent vasodilator) was determined with laser Doppler imaging following the intravenous administration of either the cyclo-oxygenase inhibitor lysine acetylsalicylate (L-AS), 900 mg, or the oral intake of indornethacin, 75 mg. Acetylcholine was iontophoresed both in presence and in absence of surface anesthesia. In some subjects, the effects of L-AS on skin reactive hyperemia were also assessed. Results: Acute cyclo-oxygenase inhibition with either drug influenced neither the skin blood flow response to 4 different doses of Ach (0.28, 1.4, 7, and 14 mC/cm²) nor reactive hyperemia. The peak vasodilatory response to Ach was significantly increased by skin anesthesia, regardless of whether the subjects received the cyclo-oxygenase inhibitor or not. For example, the mean response (+SD) to the largest dose of Ach (tested in 6 subjects, expressed in perfusion units) were as follows: in absence of anesthesia: L-AS 339 ± 105, placebo 344 ± 68; with anesthesia: L-AS 453 + 76, placebo 452 + 65 (p < .01 for effect of anesthesia). Conclusions: These data give no support for a contribution of prostaglandins to acetylcholine-induced vasodilation or to reactive hyperemia in the skin microcirculation. In this vascular bed, local anesthesia seems to amplify acetylcholine-induced vasodilation via a prostaglandin-independent mechanism. [ABSTRACT FROM AUTHOR]