Pressure-induced smooth muscle cell depolarization in pulmonary arteries from control and chronically hypoxic rats does not cause myogenic vasoconstriction.

Chronic obstructive pulmonary diseases, as well as prolonged residence at high altitude, can result in generalized airway hypoxia, eliciting an increase in pulmonary vascular resistance. We hypothesized that a portion of the elevated pulmonary vascular resistance following chronic hypoxia (CH) is due to the development of myogenic tone. Isolated, pressurized small pulmonary arteries from control (barometric pressure congruent with 630 Torr) and CH (4 wk, barometric pressure = 380 Torr) rats were loaded with fura 2-AM and perfused with warm (37 degrees C), aerated (21% O(2)-6% CO(2)-balance N(2)) physiological saline solution. Vascular smooth muscle (VSM) intracellular Ca(2+) concentration ([Ca(2+)](i)) and diameter responses to increasing intraluminal pressure were determined. Diameter and VSM cell [Ca(2+)](i) responses to KCl were also determined. In a separate set of experiments, VSM cell membrane potential responses to increasing luminal pressure were determined in arteries from control and CH rats. VSM cell membrane potential in arteries from CH animals was depolarized relative to control at each pressure step. VSM cells from both groups exhibited a further depolarization in response to step increases in intraluminal pressure. However, arteries from both control and CH rats distended passively to increasing intraluminal pressure, and VSM cell [Ca(2+)](i) was not affected. KCl elicited a dose-dependent vasoconstriction that was nearly identical between control and CH groups. Whereas KCl administration resulted in a dose-dependent increase in VSM cell [Ca(2+)](i) in arteries taken from control animals, this stimulus elicited only a slight increase in VSM cell [Ca(2+)](i) in arteries from CH animals. We conclude that the pulmonary circulation of the rat does not demonstrate pressure-induced vasoconstriction.     

Endothelium-dependent blunting of myogenic responsiveness after chronic hypoxia

Blunted agonist-induced vasoconstriction after chronic hypoxia is associated with endothelium-dependent vascular smooth muscle (VSM) cell hyperpolarization and decreased vessel-wall Ca(2+) concentration ([Ca(2+)]). We hypothesized that myogenic vasoconstriction and pressure-induced Ca(2+) influx would also be attenuated in vessels from chronically hypoxic (CH) rats. Mesenteric resistance arteries isolated from CH [barometric pressure (BP), 380 Torr for 48 h] or normoxic control (BP, 630 Torr) rats were cannulated and pressurized. VSM cell resting membrane potential was recorded at intraluminal pressures of 40-120 Torr under normoxic conditions. VSM cells in vessels from CH rats were hyperpolarized compared with control rats at all pressures. Inner diameter was maintained for vessels from control rats, whereas vessels from CH rats developed less tone as pressure was increased. Pressure-induced increases in vessel-wall [Ca(2+)] were also attenuated for arteries from CH rats. Endothelium removal restored myogenic constriction to vessels from CH rats and normalized VSM cell resting membrane potential and pressure-induced Ca(2+) responses to control levels. Myogenic constriction and pressure-induced vessel-wall [Ca(2+)] increases remained blunted in the presence of nitric oxide (NO) synthase inhibition for arteries from CH rats. We conclude that blunted myogenic reactivity after chronic hypoxia results from a non-NO, endothelium-dependent VSM cell hyperpolarizing influence.     

Cytochrome p-450 epoxygenase products contribute to attenuated vasoconstriction after chronic hypoxia

The systemic vasculature exhibits attenuated vasoconstriction following chronic hypoxia (CH) that is associated with endothelium-dependent vascular smooth muscle (VSM) cell hyperpolarization. We hypothesized that increased production of arachidonic acid metabolites such as the cyclooxygenase product prostacyclin or cytochrome p-450 (CYP) epoxygenase-derived epoxyeicosatrienoic acids (EETs) contributes to VSM cell hyperpolarization following CH. VSM cell resting membrane potential (Em) was measured in superior mesenteric artery strips isolated from rats with control barometric pressure (Pb, congruent with 630 Torr) and CH (Pb, 380 Torr for 48 h). VSM cell Em was normalized between groups following administration of the CYP inhibitors 17-octadecynoic acid and SKF-525A. VSM cell hyperpolarization after CH was not altered by cyclooxygenase inhibition, whereas the selective CYP2C9 inhibitor sulfaphenazole normalized VSM cell Em between groups. Iberiotoxin also normalized VSM cell Em, which suggests that large-conductance, Ca2+-activated K+ (BKCa) channel activity is increased after CH. Sulfaphenazole administration restored phenylephrine-induced and myogenic vasoconstriction and Ca2+ responses of mesenteric resistance arteries isolated from CH rats to control levels. Western blot experiments demonstrated that CYP2C9 protein levels were greater in mesenteric arteries from CH rats. In addition, 11,12-EET levels were elevated in endothelial cells from CH rats compared with controls. We conclude that enhanced CYP2C9 expression and 11,12-EET production following CH contributes to BKCa channel-dependent VSM cell hyperpolarization and attenuated vasoreactivity.     

48-h Hypoxic exposure results in endothelium-dependent systemic vascular smooth muscle cell hyperpolarization

Chronic hypoxia (CH) results in reduced sensitivity to vasoconstrictors in conscious rats that persists upon restoration of normoxia. We hypothesized that this effect is due to endothelium-dependent hyperpolarization of vascular smooth muscle (VSM) cells after CH. VSM cell resting membrane potential was determined for superior mesenteric artery strips isolated from CH rats (PB = 380 Torr for 48 h) and normoxic controls. VSM cells from CH rats studied under normoxia were hyperpolarized compared with controls. Resting vessel wall intracellular Ca(2+) concentration ([Ca(2+)](i)) and pressure-induced vasoconstriction were reduced in vessels isolated from CH rats compared with controls. Vasoconstriction and increases in vessel wall [Ca(2+)](i) in response to the alpha(1)-adrenergic agonist phenylephrine (PE) were also blunted in resistance arteries from CH rats. Removal of the endothelium normalized resting membrane potential, resting vessel wall [Ca(2+)](i), pressure-induced vasoconstrictor responses, and PE-induced constrictor and Ca(2+) responses between groups. Whereas VSM cell hyperpolarization persisted in the presence of nitric oxide synthase inhibition, heme oxygenase inhibition restored VSM cell resting membrane potential in vessels from CH rats to control levels. We conclude that endothelial derived CO accounts for persistent VSM cell hyperpolarization and vasoconstrictor hyporeactivity after CH.     

Chronic hypoxia augments protein kinase G-mediated Ca2+ desensitization in pulmonary vascular smooth muscle through inhibition of RhoA/Rho kinase signaling

Pulmonary vascular smooth muscle (VSM) sensitivity to nitric oxide (NO) is enhanced in pulmonary arteries from rats exposed to chronic hypoxia (CH) compared with controls. Furthermore, in contrast to control arteries, relaxation to NO following CH is not reliant on a decrease in VSM intracellular free calcium ([Ca(2+)](i)). We hypothesized that enhanced NO-dependent pulmonary vasodilation following CH is a function of VSM myofilament Ca(2+) desensitization via inhibition of the RhoA/Rho kinase (ROK) pathway. To test this hypothesis, we compared the ability of the NO donor, spermine NONOate, to reverse VSM tone generated by UTP, the ROK agonist sphingosylphosphorylcholine, or the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate in Ca(2+)-permeabilized, endothelium-denuded pulmonary arteries (150- to 300-microm inner diameter) from control and CH (4 wk at 0.5 atm) rats. Arteries were loaded with fura-2 AM to continuously monitor VSM [Ca(2+)](i). We further examined effects of NO on levels of GTP-bound RhoA and ROK membrane translocation as indexes of enzyme activity in arteries from each group. We found that spermine NONOate reversed Y-27632-sensitive Ca(2+) sensitization and inhibited both RhoA and ROK activity in vessels from CH rats but not control animals. In contrast, spermine NONOate was without effect on PKC-mediated vasoconstriction in either group. We conclude that CH mediates a shift in NO signaling to promote pulmonary VSM Ca(2+) desensitization through inhibition of RhoA/ROK.     

Reduced store-operated Ca2+ entry in pulmonary endothelial cells from chronically hypoxic rats

Chronic hypoxia (CH)-induced pulmonary hypertension may influence basal endothelial cell (EC) intracellular Ca(2+) concentration ([Ca(2+)](i)). We hypothesized that CH decreases EC [Ca(2+)](i) associated with membrane depolarization and reduced Ca(2+) entry. To test this hypothesis, we assessed 1) basal endothelial Ca(2+) in pressurized pulmonary arteries and freshly isolated ECs, 2) EC membrane potential (E(m)), 3) store-operated Ca(2+) current (I(SOC)), and 4) store-operated Ca(2+) (SOC) entry in arteries from control and CH rats. We found that basal EC Ca(2+) was significantly lower in pressurized pulmonary arteries and freshly isolated ECs from CH rats compared with controls. Similarly, ECs in intact arteries from CH rats were depolarized compared with controls, although no differences were observed between groups in isolated cells. I(SOC) activation by 1 muM thapsigargin displayed diminished inward current and a reversal potential closer to 0 mV in cells from CH rats compared with controls. In addition, SOC entry determined by fura 2 fluorescence and Mn(2+) quenching revealed a parallel reduction in Ca(2+) entry following CH. We conclude that differences in the magnitude of SOC entry exist between freshly dispersed ECs from CH and control rats and correlates with the decrease in basal EC [Ca(2+)](i). In contrast, basal EC Ca(2+) influx is unaffected and membrane depolarization is limited to intact arteries, suggesting that E(m) may not play a major role in determining basal EC [Ca(2+)](i) following CH.     

Chronic hypoxia upregulates pulmonary arterial ASIC1: a novel mechanism of enhanced store-operated Ca2+ entry and receptor-dependent vasoconstriction

Acid-sensing ion channel 1 (ASIC1) is a newly characterized contributor to store-operated Ca(2+) entry (SOCE) in pulmonary vascular smooth muscle (VSM). Since SOCE is implicated in elevated basal VSM intracellular Ca(2+) concentration ([Ca(2+)](i)) and augmented vasoconstriction in chronic hypoxia (CH)-induced pulmonary hypertension, we hypothesized that ASIC1 contributes to these responses. To test this hypothesis, we examined effects of the specific pharmacologic ASIC1a inhibitor, psalmotoxin 1 (PcTX1), on vasoconstrictor and vessel wall [Ca(2+)](i) responses to UTP and KCl (depolarizing stimulus) in fura-2-loaded, pressurized small pulmonary arteries from control and CH (4 wk at 0.5 atm) Wistar rats. PcTX1 had no effect on basal vessel wall [Ca(2+)](i), but attenuated vasoconstriction and increases in vessel wall [Ca(2+)](i) to UTP in arteries from control and CH rats; normalizing responses between groups. In contrast, responses to the depolarizing stimulus, KCl, were unaffected by CH exposure or PcTX1. Upon examining potential Ca(2+) influx mechanisms, we found that PcTX1 prevented augmented SOCE following CH. Exposure to CH resulted in a significant increase in pulmonary arterial ASIC1 protein. This study supports a novel role of ASIC1 in elevated receptor-stimulated vasoconstriction following CH which is likely mediated through increased ASIC1 expression and SOCE.     

Eicosapentaenoic acid (EPA) induces Ca(2+)-independent activation and translocation of endothelial nitric oxide synthase and endothelium-dependent vasorelaxation

Eicosapentaenoic acid (EPA), but not its metabolites (docosapentaenoic acid and docosahexaenoic acid), stimulated nitric oxide (NO) production in endothelial cells in situ and induced endothelium-dependent relaxation of bovine coronary arteries precontracted with U46619. EPA induced a greater production of NO, but a much smaller and more transient elevation of intracellular Ca(2+) concentration ([Ca(2+)]i), than did a Ca(2+) ionophore (ionomycin). EPA stimulated NO production even in endothelial cells in situ loaded with a cytosolic Ca(2+) chelator 1,2-bis-o-aminophenoxythamine-N',N',N'-tetraacetic acid, which abolished the [Ca(2+)]i elevations induced by ATP and EPA. The EPA-induced vasorelaxation was inhibited by N(omega)-nitro-L-arginine methyl ester. Immunostaining analysis of endothelial NO synthase (eNOS) and caveolin-1 in cultured endothelial cells revealed eNOS to be colocalized with caveolin in the cell membrane at a resting state, while EPA stimulated the translocation of eNOS to the cytosol and its dissociation from caveolin, to an extent comparable to that of the eNOS translocation induced by a [Ca(2+)]i-elevating agonist (10 microM bradykinin). Thus, EPA induces Ca(2+)-independent activation and translocation of eNOS and endothelium-dependent vasorelaxation.     

Altered membrane lipid domains limit pulmonary endothelial calcium entry following chronic hypoxia

Agonist-induced Ca(2+) entry into the pulmonary endothelium depends on activation of both store-operated Ca(2+) (SOC) entry and receptor-operated Ca(2+) (ROC) entry. We previously reported that pulmonary endothelial cell SOC entry and ROC entry are reduced in chronic hypoxia (CH)-induced pulmonary hypertension. We hypothesized that diminished endothelial Ca(2+) entry following CH is due to derangement of caveolin-1 (cav-1) containing cholesterol-enriched membrane domains important in agonist-induced Ca(2+) entry. To test this hypothesis, we measured Ca(2+) influx by fura-2 fluorescence following application of ATP (20 μM) in freshly isolated endothelial cells pretreated with the caveolar-disrupting agent methyl-β-cyclodextrin (mβCD; 10 mM). Cholesterol depletion with mβCD attenuated agonist-induced Ca(2+) entry in control endothelial cells to the level of that from CH rats. Interestingly, endothelial membrane cholesterol was lower in cells isolated from CH rats compared with controls although the density of caveolae did not differ between groups. Cholesterol repletion with a cholesterol:mβCD mixture or the introduction of the cav-1 scaffolding peptide (AP-cav; 10 μM) rescued ATP-induced Ca(2+) entry in endothelia from CH arteries. Agonist-induced Ca(2+) entry assessed by Mn(2+) quenching of fura-2 fluorescence was also significantly elevated by luminal AP-cav in pressurized intrapulmonary arteries from CH rats to levels of controls. Similarly, patch-clamp experiments revealed diminished inward current in response to ATP in cells from CH rats compared with controls that was restored by AP-cav. These data suggest that CH-induced pulmonary hypertension leads to reduced membrane cholesterol that limits the activity of ion channels necessary for agonist-activated Ca(2+) entry.     

Impaired NO-dependent inhibition of store- and receptor-operated calcium entry in pulmonary vascular smooth muscle after chronic hypoxia

We have recently demonstrated that chronic hypoxia (CH) attenuates nitric oxide (NO)-mediated decreases in pulmonary vascular smooth muscle (VSM) intracellular free calcium concentration ([Ca2+]i) and promotes NO-dependent VSM Ca2+ desensitization. The objective of the current study was to identify potential mechanisms by which CH interferes with regulation of [Ca2+]i by NO. We hypothesized that CH impairs NO-mediated inhibition of store-operated (capacitative) Ca2+ entry (SOCE) or receptor-operated Ca2+ entry (ROCE) in pulmonary VSM. To test this hypothesis, we examined effects of the NO donor, spermine NONOate, on SOCE resulting from depletion of intracellular Ca2+ stores with cyclopiazonic acid, and on UTP-induced ROCE in isolated, endothelium-denuded, pressurized pulmonary arteries (213 +/- 8 microm inner diameter) from control and CH (4 wk at 0.5 atm) rats. Arteries were loaded with fura-2 AM to continuously monitor VSM [Ca2+]i. We found that the change in [Ca2+]i associated with SOCE and ROCE was significantly reduced in vessels from CH animals. Furthermore, spermine NONOate diminished SOCE and ROCE in vessels from control, but not CH animals. We conclude that NO-mediated inhibition of SOCE and ROCE is impaired after CH-induced pulmonary hypertension.     

Disruption of smooth muscle gap junctions attenuates myogenic vasoconstriction of mesenteric resistance arteries

Communication between vascular smooth muscle (VSM) cells via low-resistance gap junctions may facilitate vascular function by synchronizing the contractile state of individual cells within the vessel wall. We hypothesized that inhibition of gap junctional communication would impair constrictor responses of mesenteric resistance arteries. Immunohistochemical experiments revealed positive staining for connexin 37 (Cx37) in both endothelium and smooth muscle of rat mesenteric arterioles, whereas connexin 43 (Cx43) immunoreactivity was not detected in the mesenteric vasculature. Administration of the gap junction inhibitory peptide Gap27, which targets Cx37 and Cx43, significantly diminished myogenic vasoconstriction (8.6 +/- 3.8% of passive diameter at 100 Torr) and changes in vessel wall intracellular [Ca2+] of mesenteric resistance arteries compared with vessels treated with either vehicle (physiological saline solution) (33.5 +/- 6.1%) or a control peptide (32.1 +/- 6.5%). Administration of 18alpha-glycyrrhetinic acid, structurally distinct from Gap27, also significantly attenuated myogenic constriction compared with its vehicle control (DMSO) (9.6 +/- 3.2% vs. 23.8 +/- 4.6%). In contrast, phenylephrine-induced vasoconstriction was not altered by gap junction blockers. Attenuated myogenic vasoconstriction resulting from inhibition of gap junctions persisted after disruption of the endothelium. In additional experiments, VSM cell membrane potential was recorded in mesenteric resistance arteries pressurized to 20 or 100 Torr. VSM membrane potential was depolarized at 100 Torr compared with 20 Torr. However, VSM cells in arteries treated with Gap27 were significantly hyperpolarized (-48.6 +/- 1.4 mV) at the higher pressure compared with vehicle (-41.4 +/- 1.5 mV) and Gap20-treated (-38.4 +/- 0.7 mV) vessels. Our findings suggest that inhibition of smooth muscle gap junctions attenuates pressure-induced VSM cell depolarization and myogenic vasoconstriction.     

Redistribution of the tight junction protein ZO-1 during physiological shedding of mouse intestinal epithelial cells

A novel vasodilatory influence of endothelial cell (EC) large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels is present following in vivo exposure to chronic hypoxia (CH) and may exist in other pathological states. However, the mechanism of channel activation that results in altered vasoreactivity is unknown. We tested the hypothesis that CH removes an inhibitory effect of the scaffolding domain of caveolin-1 (Cav-1) on EC BK(Ca) channels to permit activation, thereby affecting vasoreactivity. Experiments were performed on gracilis resistance arteries and ECs from control and CH-exposed (380 mmHg barometric pressure for 48 h) rats. EC membrane potential was hyperpolarized in arteries from CH-exposed rats and arteries treated with the cholesterol-depleting agent methyl-β-cyclodextrin (MBCD) compared with controls. Hyperpolarization was reversed by the BK(Ca) channel antagonist iberiotoxin (IBTX) or by a scaffolding domain peptide of Cav-1 (AP-CAV). Patch-clamp experiments documented an IBTX-sensitive current in ECs from CH-exposed rats and in MBCD-treated cells that was not present in controls. This current was enhanced by the BK(Ca) channel activator NS-1619 and blocked by AP-CAV or cholesterol supplementation. EC BK(Ca) channels displayed similar unitary conductance but greater Ca(2+) sensitivity than BK(Ca) channels from vascular smooth muscle. Immunofluorescence imaging demonstrated greater association of BK(Ca) α-subunits with Cav-1 in control arteries than in arteries from CH-exposed rats, although fluorescence intensity for each protein did not differ between groups. Finally, AP-CAV restored myogenic and phenylephrine-induced constriction in arteries from CH-exposed rats without affecting controls. AP-CAV similarly restored diminished reactivity to phenylephrine in control arteries pretreated with MBCD. We conclude that CH unmasks EC BK(Ca) channel activity by removing an inhibitory action of the Cav-1 scaffolding domain that may depend on cellular cholesterol levels.     

Chronic hypoxia induces Rho kinase-dependent myogenic tone in small pulmonary arteries

Myogenic tone in the pulmonary vasculature of normoxic adult animals is minimal or nonexistent. Whereas chronic hypoxia (CH) increases basal tone in pulmonary arteries, it is unclear if a portion of this elevated tone is due to development of myogenicity. Since basal arterial RhoA activity and Rho kinase (ROK) expression are augmented by CH, we hypothesized that CH elicits myogenic reactivity in pulmonary arteries through ROK-dependent vascular smooth muscle (VSM) Ca(2+) sensitization. To test this hypothesis, we assessed the contribution of ROK to basal tone and pressure-induced vasoconstriction in endothelium-disrupted pulmonary arteries [50-300 microm inner diameter (ID)] from control and CH [4 wk at 0.5 atmosphere (atm)] rats. Arteries were loaded with fura-2 AM to continuously monitor VSM intracellular Ca(2+) concentration ([Ca(2+)](i)). Basal VSM [Ca(2+)](i) was not different between groups. The ROK inhibitor, HA-1077 (100 nM to 30 microM), caused a concentration-dependent reduction of basal tone in CH arteries but had no effect in control vessels. In contrast, PKC inhibition with GF109203X (1 microM) did not alter basal tone. Furthermore, significant vasoconstriction in response to stepwise increases in intraluminal pressure (5-45 mmHg) was observed at 12, 15, 25, and 35 mmHg in arteries (50-200 microm ID) from CH rats. This myogenic reactivity was abolished by HA-1077 (10 microM) but not by GF109203X. VSM [Ca(2+)](i) was unaltered by HA-1077, GF109203X, or increases in pressure in either group. Myogenicity was not observed in larger vessels (200-300 microm ID). We conclude that CH induces myogenic tone in small pulmonary arteries through ROK-dependent myofilament Ca(2+) sensitization.     

Pregnancy-associated increase in rat systemic arteries endothelial nitric oxide production diminishes vasoconstrictor but does not enhance vasodilator responses

Late pregnancy in rats is characterized by a decrease in arterial pressure and in isolated arterial vessels response to vasoconstrictors. In uterine arteries the pregnancy-associated attenuation of the response to vasoconstrictors has been attributed to an increase in basal and agonist-induced endothelial NO production. However, the role of NO in pregnancy-associated changes of systemic arteries reactivity to vasoactive agents remains to be fully elucidated. We examined whether pregnancy influences the reactivity of systemic arteries to vasodilator or vasoconstrictor agents through NO-dependent mechanisms. Thoracic aortic rings and mesenteric arterial bed of late pregnant rats showed refractoriness to phenylephrine-induced vasoconstriction that was abolished by NO synthase inhibition. The potency of L-NNA to enhance tension of aortic rings preconstricted with phenylephrine (10–20% of their maximal response) was significantly lower in preparations from pregnant animals. In phenylephrine-contracted aortas and mesenteric bed, the effects of the endothelium-dependent vasodilators acetylcholine, A23187 and bradykinin, were not influenced by pregnancy. Similarly, pregnancy did not affect the vasodilator responses of adenosine, isoproterenol, capsaicin, nitroprusside, forskolin, and Hoe234 in the mesenteric bed. NO synthase activity measured by determining the conversion of L−[³H]-arginine to L−[³H]-citrulline in aorta and mesenteric arteries homogenates was not altered by pregnancy. These findings show that endothelial-dependent and -independent vasodilators action as well as NO synthase activity in systemic arteries is uninfluenced by pregnancy, whereas pregnancy-associated hyporeactivity of systemic arteries to vasoconstrictors is related to an enhanced endothelial NO production either spontaneous or elicited directly or indirectly by vasoconstrictor agents. This interpretation implies that the enhanced NO production observed in systemic arteries during late pregnancy involves cellular pathways other than the ones involved in the response to endothelium-dependent vasodilators such as acetylcholine.     

Platelet-activating factor increases endothelial [Ca2+]i and NO production in individually perfused intact microvessels

We have demonstrated that inhibition of NO synthase (NOS) in endothelial cells by either the NOS inhibitor N(omega)-monomethyl-l-arginine (l-NMMA) or the internalization of caveolin-1 scaffolding domain attenuated platelet-activating factor (PAF)-induced increases in microvessel permeability (Am J Physiol Heart Circ Physiol 286: H195-H201, 2004) indicating the involvement of an NO-dependent signaling pathway. To investigate whether an increase in endothelial cytoplasmic Ca(2+) concentration ([Ca(2+)](i)) is the initiating event and Ca(2+)-dependent NO production is crucial for permeability increases, PAF (10 nM)-induced changes in endothelial [Ca(2+)](i) and NO production were measured in individually perfused rat mesenteric venular microvessels via fluorescence microscopy. When venular microvessels were exposed to PAF, endothelial [Ca(2+)](i) increased from 69 +/- 8 nM to a peak value of 374 +/- 26 nM within 3 min and then declined to a sustained level at 190 +/- 12 nM after 15 min. Inhibition of NOS did not modify PAF-induced increases in endothelial [Ca(2+)](i). PAF-induced NO production was visualized and quantified at cellular levels in individually perfused microvessels using 4,5-diaminofluorescein diacetate and fluorescence imaging. Increased fluorescence intensity (FI), which is an indication of increased NO production, occurred in 75 +/- 7% of endothelial cells in each vessel. The mean maximum FI increase was 140 +/- 7% of baseline value. This increased FI was abolished by pretreatment of the vessel with l-NMMA and attenuated in the absence of extracellular Ca(2+). These results provide direct evidence from intact microvessels that increased endothelial [Ca(2+)](i) is the initial signal that activates endothelial NOS, and the subsequent increased NO production contributes to PAF-induced increases in microvessel permeability.     

Contribution of oxygen radicals to altered NO-dependent pulmonary vasodilation in acute and chronic hypoxia

Chronic hypoxia (CH) increases pulmonary arterial endothelial nitric oxide (NO) synthase (NOS) expression and augments endothelium-derived nitric oxide (EDNO)-dependent vasodilation, whereas vasodilatory responses to exogenous NO are attenuated in CH rat lungs. We hypothesized that reactive oxygen species (ROS) inhibit NO-dependent pulmonary vasodilation following CH. To test this hypothesis, we examined responses to the EDNO-dependent vasodilator endothelin-1 (ET-1) and the NO donor S-nitroso-N-acetyl penicillamine (SNAP) in isolated lungs from control and CH rats in the presence or absence of ROS scavengers under normoxic or hypoxic ventilation. NOS was inhibited in lungs used for SNAP experiments to eliminate influences of endogenously produced NO. Additionally, dichlorofluorescein (DCF) fluorescence was measured as an index of ROS levels in isolated pressurized small pulmonary arteries from each group. We found that acute hypoxia increased DCF fluorescence and attenuated vasodilatory responses to ET-1 in lungs from control rats. The addition of ROS scavengers augmented ET-1-induced vasodilation in lungs from both groups during hypoxic ventilation. In contrast, upon NOS inhibition, DCF fluorescence was elevated and SNAP-induced vasodilation diminished in arteries from CH rats during normoxia, whereas acute hypoxia decreased DCF fluorescence, which correlated with augmented reactivity to SNAP in both groups. ROS scavengers enhanced SNAP-induced vasodilation in normoxia-ventilated lungs from CH rats similar to effects of hypoxic ventilation. We conclude that inhibition of NOS during normoxia leads to greater ROS generation in lungs from both control and CH rats. Furthermore, NOS inhibition reveals an effect of acute hypoxia to diminish ROS levels and augment NO-mediated pulmonary vasodilation.     

Kinase-dependent activation of voltage-gated Ca2+ channels by ET-1 in pulmonary arterial myocytes during chronic hypoxia

Exposure to chronic hypoxia (CH) causes pulmonary hypertension. The vasoconstrictor endothelin-1 (ET-1) is thought to play a role in the development of hypoxic pulmonary hypertension. In pulmonary arterial smooth muscle cells (PASMCs) from chronically hypoxic rats, ET-1 signaling is altered, with the ET-1-induced change in intracellular calcium concentration (Δ[Ca(2+)](i)) occurring through activation of voltage-dependent Ca(2+) channels (VDCC) even though ET-1-induced depolarization via inhibition of K(+) channels is lost. The mechanism underlying this response is unclear. We hypothesized that activation of VDCCs by ET-1 following CH might be mediated by protein kinase C (PKC) and/or Rho kinase, both of which have been shown to phosphorylate and activate VDCCs. To test this hypothesis, we examined the effects of PKC and Rho kinase inhibitors on the ET-1-induced Δ[Ca(2+)](i) in PASMCs from rats exposed to CH (10% O(2), 3 wk) using the Ca(2+)-sensitive dye fura 2-AM and fluorescent microscopy techniques. We found that staurosporine and GF109203X, inhibitors of PKC, and Y-27632 and HA 1077, Rho kinase inhibitors, reduced the ET-1-induced Δ[Ca(2+)](i) by >70%. Inhibition of tyrosine kinases (TKs) with genistein or tyrphostin A23, or combined inhibition of PKC, TKs, and Rho kinase, reduced the Δ[Ca(2+)](i) to a similar extent as inhibition of either PKC or Rho kinase alone. The ability of PKC or Rho kinase to activate VDCCs in our cells was verified using phorbol 12-myristate 13-acetate and GTP-γ-S. These results suggest that following CH, the ET-1-induced Δ[Ca(2+)](i) in PASMCs occurs via Ca(2+) influx through VDCCs mediated primarily by PKC, TKs, and Rho kinase.     

Sphingomyelinase causes endothelium-dependent vasorelaxation through endothelial nitric oxide production without cytosolic Ca(2+) elevation

Neutral sphingomyelinase (N-SMase) elevated nitric oxide (NO) production without affecting intracellular Ca(2+) concentration ([Ca(2+)](i)) in endothelial cells in situ on aortic valves, and induced prominent endothelium-dependent relaxation of coronary arteries, which was blocked by N(omega)-monomethyl-L-arginine, a NO synthase (NOS) inhibitor. N-SMase induced translocation of endothelial NOS (eNOS) from plasma membrane caveolae to intracellular region, eNOS phosphorylation on serine 1179, and an increase of ceramide level in endothelial cells. Membrane-permeable ceramide (C(8)-ceramide) mimicked the responses to N-SMase. We propose the involvement of N-SMase and ceramide in Ca(2+)-independent eNOS activation and NO production in endothelial cells in situ, linking to endothelium-dependent vasorelaxation.     

Upregulation of endothelial cell Ca2+ signaling contributes to pregnancy-enhanced vasodilation of rat uteroplacental arteries

Normal pregnancy is characterized by an increased uterine blood flow due to growth and remodeling of the maternal uterine vasculature and enhanced vasodilation of the uterine arteries. The objective of the present study was to examine the role of endothelial cell Ca2+ signaling in augmented endothelium-mediated vasodilation of uteroplacental arteries in late pregnancy. We performed fura-2-based measurements of the intracellular Ca2+ concentration ([Ca2+]i) in the cytoplasm of endothelial cells simultaneously with diameter in pressurized uterine arteries from nonpregnant (NP) and late-pregnant (LP) rats. Basal levels of endothelial cell [Ca2+]i were higher in arteries from LP rats compared with NP controls. Withdrawal of extracellular Ca2+ resulted in a decrease in the level of basal [Ca2+]i that was significantly larger in arteries of LP than NP rats. The rate of Mn2+ -induced quenching of fura-2 fluorescence was significantly elevated in late pregnancy, implicating augmented Ca2+ influx as a cause of increased basal levels of [Ca2+]i in endothelial cells. Elevation of intraluminal pressure resulted in a transient increase in endothelial [Ca2+]i that was markedly potentiated in late gestation. ACh-induced [Ca2+]i and vasodilator responses were significantly augmented in arteries of LP compared with NP rats and were abolished by BAPTA treatment, demonstrating a critical role of [Ca2+]i elevation in the production of endothelium-derived vasodilators. Together, these results indicate that late pregnancy is a state of enhanced basal and stimulated Ca2+ signaling in endothelial cells of uterine vessels, which may represent an important underlying mechanism for augmented vasodilation in the maternal uterine circulation.     

Antihypertensive therapy increases tetrahydrobiopterin levels and NO/cGMP signaling in small arteries of angiotensin II-infused hypertensive rats

We previously reported that small mesenteric arteries from hypertensive rats have increased NOS-derived H(2)O(2) and reduced NO/cGMP signaling. We hypothesized that antihypertensive therapy lowers blood pressure through a tetrahydrobiopterin (BH(4))-dependent mechanism restoring NO/cGMP signaling and endothelial NOS (NOS3; eNOS) phosphorylation in small arteries. To test this hypothesis, small mesenteric arteries from normotensive rats (NORM), angiotensin II-infused rats (ANG), ANG rats with triple therapy (reserperine, hydrochlorothiazide, and hydralazine), or ANG rats with oral BH(4) therapy were studied. Both triple therapy and oral BH(4) therapy attenuated the rise in systolic blood pressure in ANG rats and restored NO/cGMP signaling in small arteries similarly. Triple therapy significantly increased vascular BH(4) levels and BH(4)-to-BH(2) ratio similar to ANG rats with BH(4) supplementation. Furthermore, triple therapy (but not oral BH(4) therapy) significantly increased GTP cyclohydrolase I (GTPCH I) activity in small arteries without a change in expression. NOS3 phosphorylation at Ser1177 was reduced in small arteries from ANG compared with NORM, while NOS3 phosphorylation at Ser633 and Thr495 were similar in ANG and NORM. NOS3 phosphorylation at Ser1177 was restored with triple therapy or oral BH(4) in ANG rats. In conclusion, antihypertensive therapy regulates NO/cGMP signaling in small arteries through increasing BH(4) levels and NOS3 phosphorylation at Ser1177.