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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]

We developed a two-dimensional model of transport-dependent intracellular calcium signaling in endothelial cells (Esc). Our purpose was to evaluate the effects of spatial colocalization of endothelial nitric oxide syntheses (enol) and capacitative calcium entry (CCE) channels in caveolae on enol 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 Esc, these molecules include CCE channels and enol. To achieve a quantitative understanding of the mechanisms of microdomain calcium signaling and the preferential sensitivity of enol 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 Esc 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 Esc by increasing the spatial segregation of the caveolar signaling domains. Our simulation significantly advances the understanding of how Ca2+, 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 Ca2+ sources and targets. [ABSTRACT FROM AUTHOR]

We hypothesized that elevated partial pressures of O[sub 2] would increase perivascular nitric oxide (·NO) synthesis. Rodents with O[sub 2-] and ·NO-specific microelectrodes implanted adjacent to the abdominal aorta were exposed to O[sub 2] at partial pressures from 0.2 to 2.8 atmospheres absolute (ATA). Exposures to 2.0 and 2.8 ATA O[sub 2] stimulated neuronal (type I) NO synthase (nNOS) and significantly increased steady-state · NO concentration, but the mechanism for enzyme activation differed at each partial pressure. At both pressures, elevations in ·NO concentration were inhibited by the nNOS inhibitor 7-nitroindazole and the calcium channel blocker nimodipine. Enzyme activation at 2.0 ATA O[sub 2] appeared to be due to an altered cellular redox state. Exposure to 2.8 ATA O[sub 2], but not 2.0 ATA O[sub 2], increased nNOS activity by enhancing nNOS association with calmodulin, and an inhibitory effect of geldanamycin indicated that the association was facilitated by heat shock protein 90. Infusion of superoxide dismutase inhibited ·NO elevation at 2.8 but not 2.0 ATA O[sub 2]. Hyperoxia increased the concentration of .NO associated with hemoglobin. These findings highlight the complexity of oxidative stress responses and may help explain some of the dose responses associated with therapeutic applications of hyperbaric oxygen. [ABSTRACT FROM AUTHOR]

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]

Protein Kinase C (PKC) is a promiscuous serine/threonine kinase regulating vasodilatory responses in vascular endothelial cells. Calcium-dependent PKCbeta (PKCβ) and calcium-independent PKCeta (PKCη) have both been implicated in the regulation and dysfunction of endothelial responses to shear stress and agonists. We hypothesized that PKCβ and PKCη differentially modulate shear stress-induced nitric oxide (NO) production by regulating the transduced calcium signals and the resultant eNOS activation. As such, this study sought to characterize the contribution of PKCη and PKCβ in regulating calcium signaling and endothelial nitric oxide synthase (eNOS) activation after exposure of endothelial cells to ATP or shear stress. Bovine aortic endothelial cells were stimulated in vitro under pharmacological inhibition of PKCβ with LY333531 or PKCη targeting with a pseudosubstrate inhibitor. The participation of PKC isozymes in calcium flux, eNOS phosphorylation and NO production was assessed following stimulation with ATP or shear stress. PKCη proved to be a robust regulator of agonist- and shear stress-induced eNOS activation, modulating calcium fluxes and tuning eNOS activity by multi-site phosphorylation. PKCβ showed modest influence in this pathway, promoting eNOS activation basally and in response to shear stress. Both PKC isozymes contributed to the constitutive and induced phosphorylation of eNOS. The observed PKC signaling architecture is intricate, recruiting Src to mediate a portion of PKCη's control on calcium entry and eNOS phosphorylation. Elucidation of the importance of PKCη in this pathway was tempered by evidence of a single stimulus producing concurrent phosphorylation at ser1179 and thr497 which are antagonistic to eNOS activity. We have, for the first time, shown in a single species in vitro that shear stress- and ATP-stimulated NO production are differentially regulated by classical and novel PKCs. This study furthers our understanding of the PKC isozyme interplay that optimizes NO production. These considerations will inform the ongoing design of drugs for the treatment of PKC-sensitive cardiovascular pathologies. • Flow-induced eNOS activation maintains vascular health via regulation by PKCs. • Classical PKCβ and novel PKCη both regulate basal eNOS phosphorylation. • Calcium-dependent PKCβ promotes flow-stimulated nitric oxide production. • PKCη mediates responses to agonists and flow, in part by src activation. • PKC regulates this pathway by multisite phosphorylation of calcium channels & eNOS. [ABSTRACT FROM AUTHOR]

NO has been shown to mediate angiogenesis; however, its role in vessel morphogenesis and maturation is not known. Using intravital microscopy, histological analysis, alpha-smooth muscle actin and chondroitin sulfate proteoglycan 4 staining, microsensor NO measurements, and an NO synthase (NOS) inhibitor, we found that NO mediates mural cell coverage as well as vessel branching and longitudinal extension but not the circumferential growth of blood vessels in B16 murine melanomas. NO-sensitive fluorescent probe 4,5-diaminofluorescein imaging, NOS immunostaining, and the use of NOS-deficient mice revealed that eNOS in vascular endothelial cells is the predominant source of NO and induces these effects. To further dissect the role of NO in mural cell recruitment and vascular morphogenesis, we performed a series of independent analyses. Transwell and under-agarose migration assays demonstrated that endothelial cell-derived NO induces directional migration of mural cell precursors toward endothelial cells. An in vivo tissue-engineered blood vessel model revealed that NO mediates endothelial-mural cell interaction prior to vessel perfusion and also induces recruitment of mural cells to angiogenic vessels, vessel branching, and longitudinal extension and subsequent stabilization of the vessels. These data indicate that endothelial cell-derived NO induces mural cell recruitment as well as subsequent morphogenesis and stabilization of angiogenic vessels. [ABSTRACT FROM AUTHOR]