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Endothelial dysfunction, characterised by impaired nitric oxide (NO) bioavailability, arises in response to a variety of cardiovascular risk factors and precedes atherosclerosis. NO is produced by tight regulation of endothelial nitric oxide synthase (eNOS) activity in response to vasodilatory stimuli. This regulation of eNOS is mediated in part by store-operated calcium entry (SOCE). We hypothesized that both ATP- and flow-induced eNOS activation are regulated by SOCE derived from Orai1 channels and members of the transient receptor potential canonical (TRPC) channel family. Bovine aortic endothelial cells (BAECs) were pre-treated with pharmacological inhibitors of TRPC channels and Orai1 to examine their effect on calcium signaling and eNOS activation in response to flow and ATP. The peak and sustained ATP-induced calcium signal and the resulting eNOS activation were attenuated by inhibition of TRPC3, which we found to be store operated. TRPC4 blockade reduced the transient peak in calcium concentration following ATP stimulation, but did not significantly reduce eNOS activity. Simultaneous TRPC3 & 4 inhibition reduced flow-induced NO production via alterations in phosphorylation-mediated eNOS activity. Inhibition of TRPC1/6 or Orai1 failed to lower ATP-induced calcium entry or eNOS activation. Our results suggest that TRPC3 is a store-operated channel in BAECs and is the key regulator of ATP-induced eNOS activation, whereas flow stimulation also recruits TRPC4 into the pathway for the synthesis of NO. [ABSTRACT FROM AUTHOR]

Interactions between cardiac myoglobin (Mb), nitrite, and nitric oxide (NO) are vital in regulating O 2 storage, transport, and NO homeostasis. Production of NO through the reduction of endogenous myocardial nitrite by deoxygenated myoglobin has been shown to significantly reduce myocardial infarction damage and ischemic injury. We developed a mathematical model for a cardiac arteriole and surrounding myocardium to examine the hypothesis that myoglobin switches functions from being a strong NO scavenger to an NO producer via the deoxymyoglobin nitrite reductase pathway. Our results predict that under ischemic conditions of flow, blood oxygen level, and tissue pH, deoxyMb nitrite reduction significantly elevates tissue and smooth muscle cell NO. The size of the effect is consistent at different flow rates, increases with decreasing blood oxygen and tissue pH and, in extreme pathophysiological conditions, NO can even be elevated above the normoxic levels. Our simulations suggest that cardiac deoxyMb nitrite reduction is a plausible mechanism for preserving or enhancing NO levels using endogenous nitrite despite the rate-limiting O 2 levels for endothelial NO production. This NO could then be responsible for mitigating deleterious effects under ischemic conditions. [ABSTRACT FROM AUTHOR]

Nitrite infusion into the bloodstream has been shown to elicit vasodilation and protect against ischemia-reperfusion injury through nitric oxide (NO) release in hypoxic conditions. However, the mechanism by which nitrite-derived NO escapes scavenging by hemoglobin in the erythrocyte has not been fully elucidated, owing in part to the difficulty in measuring the reactions and transport on NO in vivo . We developed a mathematical model for an arteriole and surrounding tissue to examine the hypothesis that dinitrogen trioxide (N 2 O 3 ) acts as a stable intermediate for preserving NO. Our simulations predict that with hypoxia and moderate nitrite concentrations, the N 2 O 3 pathway can significantly preserve the NO produced by hemoglobin nitrite reductase in the erythrocyte and elevate NO reaching the smooth muscle cells. Nitrite retains its ability to increase NO bioavailability even at varying flow conditions, but there is minimal effect under normoxia or very low nitrite concentrations. Our model demonstrates a viable pathway for reconciling experimental findings of potentially beneficial effects of nitrite infusions despite previous models showing negligible NO elevation associated with hemoglobin nitrite reductase. Our results suggest that additional mechanisms may be needed to explain the efficacy of nitrite-induced vasodilation at low infusion concentrations. [ABSTRACT FROM AUTHOR]

We developed a mass transport model for a parallel-plate flow chamber apparatus to predict the concentrations of nitric oxide (NO) and adenine nucleotides (ATP, ADP) produced by cultured endothelial cells (ECs) and investigated how the net rates of production, degradation, and mass transport for these three chemical species vary with changes in wall shear stress (τ w ). These simulations provide an improved understanding of experimental results obtained with parallel-plate flow chambers and allows quantitative analysis of the relationship between τ w , adenine nucleotide concentrations, and NO produced by ECs. Experimental data obtained after altering ATP and ADP concentrations with apyrase were analyzed to quantify changes in the rate of NO production (R NO ). The effects of different isoforms of apyrase on ATP and ADP concentrations and nucleotide-dependent changes in R NO could be predicted with the model. A decrease in ATP was predicted with apyrase, but an increase in ADP was simulated due to degradation of ATP. We found that a simple proportional relationship relating a component of R NO to the sum of ATP and ADP provided a close match to the fitted curve for experimentally measured changes in R NO with apyrase. Estimates for the proportionality constant ranged from 0.0067 to 0.0321 μM/s increase in R NO per nM nucleotide concentration, depending on which isoform of apyrase was modeled, with the largest effect of nucleotides on R NO at low τ w (<6 dyn/cm 2 ). [ABSTRACT FROM AUTHOR]

Biotransport of nitric oxide (NO) and of oxygen (O2) in the microcirculation are inherently interdependent, since all nitric oxide synthase (NOS) isoforms (eNOS, nNOS, and iNOS) require O2 to produce NO. Furthermore, tissue O2 consumption is reversibly inhibited by NO. To investigate these complex interactions, a mathematical model was developed for coupled mass transport of NO and O2 around a cylindrical arteriole using finite element computational methods. Steady-state radial NO and O2 gradients in the bloodstream, plasma layer, endothelium, vascular wall, and surrounding tissue were simulated for different conditions. Special cases of the model were solved, including O2-dependent NO production from eNOS alone, and with additional NO production from either nNOS or iNOS at specified locations.The model predicts that (a) concentration changes in one species can have significant effects on transport of the other species with the degree of interaction dependent on spatial gradients; (b) eNOS NO production rates required to maintain the concentration of NO in the vascular wall are more dependent on NO scavenging in blood than in tissue; (c) relatively low rates of NO production in tissue from either nNOS or iNOS can elevate vascular wall NO, compensating for possible reductions in NO production from eNOS; (d) depending on their physical location, nNOS and iNOS can be very sensitive to O2; and (e) increased tissue NO can increase O2 delivery to more distal regions by inhibiting O2 consumption in other regions. [Copyright &y& Elsevier]

We report the first dynamic measurements of tissue nitric oxide (NO) during functional activation of rat somatosensory cortex by electrical forepaw stimulation. Cortical tissue NO was measured electrochemically with rapid-responding recessed microelectrodes (tips <10 μm). Simultaneous blood flow measurements were made by laser–Doppler flowmetry (LDF). NO immediately increased, reaching a peak 125.5 ± 32.8 (SE) nM above baseline (P < 0.05) within 400 ms after stimulus onset, preceding any LDF changes, and then returned close to prestimulus levels after 2 s (123 signal-averaged trials, 12 rats). Blood flow began rising after a 1-s delay, reaching a peak just before electrical stimulation was ended at t = 4 s. A consistent poststimulus NO undershoot was observed as LDF returned to baseline. These findings complement our previous study (B. M. Ances et al., 2001, Neurosci. Lett. 306, 106–110) in which a transient decrease in rat somatosensory cortex tissue oxygen partial pressure was found to precede blood flow increases during functional activation. [Copyright &y& Elsevier]

Blood flow studies in the brain, heart, and other organs suggest that there could be interaction between nitric oxide (NO) and adenosine. This possibility was investigated in the optic nerve head (ONH) during photic stimulation of the dark-adapted cat eye. Functional activation of ONH blood flow was measured by laser Doppler flowmetry, simultaneously with NO and PO2 using double-barrel recessed electrochemical sensors. Photic stimulation (diffuse luminance flickering light at 30 Hz) increased ONH blood flow to 127.4 ± 4.7% (mean ± SEM) of baseline with a transient increase in NO by 79.8 ± 12.8 nM, while PO2 decreased from 24.5 ± 2.7 to 22.7 ± 2.4 Torr (control responses, 15 trials, 10 cats). Adenosine (3 mg/kg iv) increased baseline ONH blood flow to 113.8 ± 8.4% of control within 5 min. Functional activation of ONH blood flow was enhanced during photic stimulation, reaching a maximum of 155.8 ± 8.1% within 5 min, and remained enhanced for 30 to 45 min. NO responses during photic stimulation were not different from control responses. Treatment with a nonspecific NO synthase inhibitor (NG-nitro-l-arginine methyl ester, 40 mg/kg iv, 5 cats) did not alter the increase in resting ONH blood flow or the enhanced functional activation after adenosine. We conclude that there is no interaction between NO and adenosine during functional activation of cat ONH blood flow by photic stimulation. [Copyright &y& Elsevier]

Abstract: Nitric oxide (NO) produced by the endothelium is involved in the regulation of vascular tone. Decreased NO production or availability has been linked to endothelial dysfunction in hypercholesterolemia and hypertension. Shear stress-induced NO release is a well-established phenomenon, yet the cellular mechanisms of this response are not completely understood. Experimental limitations have hindered direct, real-time measurements of NO under flow conditions. We have overcome these challenges with a new design for a parallel-plate flow chamber. The chamber consists of two compartments, separated by a Transwell® membrane, which isolates a NO recording electrode located in the upper compartment from flow effects. Endothelial cells are grown on the bottom of the membrane, which is inserted into the chamber flush with the upper plate. We demonstrate for the first time direct real-time NO measurements from endothelial cells with controlled variations in shear stress. Step changes in shear stress from 0.1dyn/cm2 to 6, 10, or 20dyn/cm2 elicited a transient decrease in NO followed by an increase to a new steady state. An analysis of NO transport suggests that the initial decrease is due to the increased removal rate by convection as flow increases. Furthermore, the rate at which the NO concentration approaches the new steady state is related to the time-dependent cellular response rather than transport limitations of the measurement configuration. Our design offers a method for studying the kinetics of the signaling mechanisms linking NO production with shear stress as well as pathological conditions involving changes in NO production or availability. [ABSTRACT FROM AUTHOR]

Abstract: NO and PO2 microelectrodes were used to quantify the effects of increased availability of l-arginine in an exteriorized rat mesentery and small intestine microcirculatory preparation in n = 16 rats. During short periods of elevated l-arginine added to the superfusion bath, transient changes in perivascular NO or PO2 were measured at 171 perivascular sites near intestinal arterioles and venules, simultaneously with tissue perfusion using laser Doppler flowmetry (LDF). Excess l-arginine increased perivascular NO over twofold, by 411 ± 42 nM above the baseline of 329 ± 30 nM (P < 0.0001), and increased tissue perfusion by 35.5 ± 7.5% (P < 0.0001). No difference between arterioles and venules was observed in the magnitude or time course of the NO responses. Both increases and decreases in perivascular PO2 were observed after excess l-arginine, with a similar increase in tissue perfusion by 42.0 ± 12.3% (P < 0.0001). Our NO measurements confirm that increased bioavailability of l-arginine causes a significant increase in NO production throughout the microcirculation of this preparation, with increased tissue perfusion, and provides direct in vivo evidence for the l-arginine paradox. [Copyright &y& Elsevier]

We measured changes in nitric oxide (⋅NO) concentration in the cerebral cortex during experimental carbon monoxide (CO) poisoning and assessed the role for N-methyl-d-aspartate receptors (NMDARs), a glutamate receptor subtype, with progression of CO-mediated oxidative stress. Using microelectrodes, ⋅NO concentration was found to nearly double to 280 nM due to CO exposure, and elevations in cerebral blood flow, monitored as laser Doppler flow (LDF), were found to loosely correlate with ⋅NO concentration. Neuronal nitric oxide synthase (nNOS) activity was the cause of the ⋅NO elevation based on the effects of specific NOS inhibitors and observations in nNOS knockout mice. Activation of nNOS was inhibited by the NMDARs inhibitor, MK 801, and by the calcium channel blocker, nimodipine, thus demonstrating a link to excitatory amino acids. Cortical cyclic GMP concentration was increased due to CO poisoning and shown to be related to ⋅NO, versus CO, mediated guanylate cyclase activation. Elevations of ⋅NO were inhibited when rats were infused with superoxide dismutase and in rats depleted of platelets or neutrophils. When injected with MK 801 or 7-nitroindazole, a selective nNOS inhibitor, rats did not exhibit CO-mediated nitrotyrosine formation, myeloperoxidase (MPO) elevation (indicative of neutrophil sequestration), or impaired learning. Similarly, whereas CO-poisoned wild-type mice exhibited elevations in nitrotyrosine and myeloperoxidase, these changes did not occur in nNOS knockout mice. We conclude that CO exposure initiates perivascular processes including oxidative stress that triggers activation of NMDA neuronal nNOS, and these events are necessary for the progression of CO-mediated neuropathology. [Copyright &y& Elsevier]