Nitric oxide contributes to 20-HETE-induced relaxation of pulmonary arteries.

In contrast to its constrictor effects on peripheral arteries, 20-hydroxyeicosatetraenoic acid (20-HETE) is an endothelial-dependent dilator of pulmonary arteries (PAs). The present study examined the hypothesis that the vasodilator effects of 20-HETE in PAs are due to an elevation of intracellular calcium concentration ([Ca(2+)](i)) and the release of nitric oxide (NO) from bovine PA endothelial cells (BPAECs). BPAECs express cytochrome P-450 4A (CYP4A) protein and produce 20-HETE. 20-HETE dilated PAs preconstricted with U-46619 or norepinephrine and treated with the cytochrome P-450 inhibitor 17-octadecynoic acid and the cyclooxygenase inhibitor indomethacin. The dilator effect of 20-HETE was blocked by the NO synthase inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME) or by removal of endothelium. 20-HETE significantly increased [Ca(2+)](i) and NO production in BPAECs. 20-HETE-induced NO release was blunted by removal of extracellular calcium, as well as NO synthase inhibitors (L-NAME). These results suggest that 20-HETE dilates PAs at least in part by increasing [Ca(2+)](i) and NO release in BPAECs.     

Hypoxic regulation of the fetal cerebral circulation.

Fetal cerebrovascular responses to acute hypoxia are fundamentally different from those observed in the adult cerebral circulation. The magnitude of hypoxic vasodilatation in the fetal brain increases with postnatal age although fetal cerebrovascular responses to acute hypoxia can be complicated by age-dependent depressions of blood pressure and ventilation. Acute hypoxia promotes adenosine release, which depresses fetal cerebral oxygen consumption through action of adenosine on neuronal A1 receptors and vasodilatation through activation of A2 receptors on cerebral arteries. The vascular effect of adenosine can account for approximately half the vasodilatation observed in response to hypoxia. Hypoxia-induced release of nitric oxide and opioids can account for much of the adenosine-independent cerebral vasodilatation observed in response to hypoxia in the fetus. Direct effects of hypoxia on cerebral arteries account for the remaining fraction, although the vascular endothelium contributes relatively little to hypoxic vasodilatation in the immature cerebral circulation. In contrast to acute hypoxia, fetal cerebral blood flow tends to normalize during acclimatization to chronic hypoxia even though cardiac output is depressed. However, uncompensated chronic hypoxia in the fetus can produce significant changes in brain structure and function, alteration of respiratory drive and fluid balance, and increased incidence of intracranial hemorrhage and periventricular leukomalacia. At the level of the fetal cerebral arteries, chronic hypoxia increases protein content and depresses norepinephrine release, contractility, and receptor densities associated with contraction but also attenuates endothelial vasodilator capacity and decreases the ability of ATP-sensitive and calcium-sensitive potassium channels to promote vasorelaxation. Overall, fetal cerebrovascular adaptations to chronic hypoxia appear prioritized to conserve energy while preserving basic contractility. Many gaps remain in our understanding of how the effects of acute and chronic hypoxia are mediated in fetal cerebral arteries, but studies of adult cerebral arteries have produced many powerful pharmacological and molecular tools that are simply awaiting application in studies of fetal cerebral artery responses to hypoxia.     

Age-dependent modulation of endothelium-dependent vasodilatation by chronic hypoxia in ovine cranial arteries.

Although abundant evidence indicates that chronic hypoxia can induce pulmonary vascular remodeling, very little is known of the effects of chronic hypoxia on cerebrovascular structure and function, particularly in the fetus. Thus the present study explored the hypothesis that chronic hypoxemia also influences the size and shape of cerebrovascular smooth muscle and endothelial cells, with parallel changes in the reactivity of these cells to endothelium-dependent vasodilator stimuli. To test this hypothesis, measurements of endothelial and vascular smooth muscle cell size and density were made in silver-stained common carotid and middle cerebral arteries from term fetal and nonpregnant adult sheep maintained at an altitude of 3,820 m for 110 days. Chronic hypoxia induced an age-dependent remodeling that led to smooth muscle cells that were larger in fetal arteries but smaller in adult arteries. Chronic hypoxia also increased endothelial cell density in fetal arteries but reduced it in adult arteries. These combined effects resulted in an increased (adult carotid), decreased (adult middle cerebral), or unchanged (fetal arteries) per cell serosal volume of distribution for endothelial factors. Despite this heterogeneity, the magnitude of endothelium-dependent vasodilatation to A23187, measured in vitro, was largely preserved, although sensitivity to this relaxant was uniformly depressed. N(G)-nitro-L-arginine methyl ester, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, and endothelium denudation each independently blocked A23187-induced vasodilation without unmasking any residual vasoconstrictor effect. Indomethacin did not significantly attenuate A23187-induced relaxation except in the hypoxic adult middle cerebral, where a small contribution of prostanoids was evident. Vascular sensitivity to exogenous nitric oxide (NO) was uniformly increased by chronic hypoxia. From these results, we conclude that chronic hypoxia reduced endothelial NO release while also upregulating some component of the NO-cGMP-PKG vasodilator pathway. These offsetting effects appear to preserve endothelium-dependent vasodilation after adaptation to chronic hypoxia.     

High-salt diet depresses acetylcholine reactivity proximal to NOS activation in cerebral arteries

Rats were fed a low-salt (LS; 0.4% NaCl) or high-salt (HS; 4.0% NaCl) diet for 3 days, and the responses of isolated cerebral arteries to acetylcholine (ACh), the nitric oxide (NO)-dependent dilator bradykinin, and the NO donor 6-(2-hydroxy-1-methyl-2-nitrosohydrazino)-N-methyl-1-hex-anamine (NOC-9) were determined. ACh-induced vasodilation and NO release, assessed with the fluorescent NO indicator 4,5-diaminofluorescein (DAF-2) diacetate, were eliminated with the HS diet. Inhibition of cyclooxygenase, cytochrome P-450 epoxygenase, and acetylcholinesterase did not alter ACh responses. Bradykinin and NOC-9 caused a similar dilation in cerebral arteries of all groups. Arteries from animals on LS or HS diets exhibited similar levels of basal superoxide (O(2)(-)) production, assessed by dihydroethidine fluorescence, and ACh responses were unaffected by O(2)(-) scavengers. Muscarinic type 3 receptor expression was unaffected by dietary salt intake. These results indicate that 1) a HS diet attenuates ACh reactivity in cerebral arteries by inhibiting NO release, 2) this attenuation is not due to production of a cyclooxygenase-derived vasoconstrictor or elevated O(2)(-) levels, and 3) alteration(s) in ACh signaling are located upstream from NO synthase.     

Cholinergic reactivity of cerebral arteries in the developing fetal and newborn lamb.

Cerebral arteries of newborn pigs and baboons constrict to acetylcholine, suggesting that endothelium-dependent dilator mechanisms may be lacking in immature cerebral arteries. The present study tested this possibility in the immature sheep by examining the response of cerebral arterioles in fetal and newborn sheep to endothelium-dependent dilator, acetylcholine. Pial arteriolar diameter was measured in 9 anaesthetized foetuses in utero (4 preterm, 90-111 days gestation and 5 term, 128-143 days gestation) and in 5 anaesthetized, newborn lambs (14 days) using a closed cranial window with intravital microscopy. Application of acetylcholine to the pial surface induced dose-dependent increase in pial arteriolar diameter in all age groups; EC50 for acetylcholine was 0.10 +/- 0.03, 0.28 +/- 0.08 and 0.26 +/- 0.17 microM for preterm fetal, term fetal, and newborn lambs, respectively. The data demonstrate a sensitive dilator response to acetylcholine in immature fetuses as well as newborn lambs suggesting that cholinergic-mediated release of endothelium-dependent relaxing factor is functional early in gestation. The contractile response to acetylcholine observed in newborn pigs and premature baboons may reflect a species difference rather than maturational lack of endothelium-dependent dilator mechanisms.     

Gonadal hormones affect diameter of male rat cerebral arteries through endothelium-dependent mechanisms

Gender is known to influence the incidence and severity of cerebrovascular disease. In the present study, luminal diameter was measured in vitro in pressurized middle cerebral artery segments from male rats that were either untreated, orchiectomized (ORX), ORX with testosterone treatment (ORX+TEST), or ORX with estrogen treatment (ORX+EST). The maximal passive diameters (0 Ca(2+) + 3 mM EDTA) of arteries from all four groups were similar. In endothelium-intact arteries, myogenic tone was significantly greater in arteries from untreated and ORX+TEST compared with arteries from either ORX or ORX+EST. During exposure to N(G)-nitro-L-arginine-methyl ester (L-NAME), an NO synthase (NOS) inhibitor, myogenic tone significantly increased in all groups. The effect of L-NAME was significantly greater in arteries from untreated and ORX+EST compared with arteries from ORX and ORX+TEST rats. Differences in myogenic tone between ORX and ORX+TEST persisted after inhibition of NOS. After endothelium removal or inhibition of the cyclooxygenase pathway combined with K(+) channel blockers, myogenic tone differences between ORX and ORX+TEST were abolished. Wall thickness and forced dilation were not significantly different between arteries from ORX and ORX+TEST. Our data show that gonadal hormones affect myogenic tone in male rat cerebral arteries through NOS- and/or endothelium-dependent mechanisms.     

Erythropoietin for the Treatment of Subarachnoid Hemorrhage: A Feasible Ingredient for a Successful Medical Recipe

Subarachnoid hemorrhage (SAH) following aneurysm bleeding accounts for 6% to 8% of all cerebrovascular accidents. Although an aneurysm can be effectively managed by surgery or endovascular therapy, delayed cerebral ischemia is diagnosed in a high percentage of patients resulting in significant morbidity and mortality. Cerebral vasospasm occurs in more than half of all patients after aneurysm rupture and is recognized as the leading cause of delayed cerebral ischemia after SAH. Hemodynamic strategies and endovascular procedures may be considered for the treatment of cerebral vasospasm. In recent years, the mechanisms contributing to the development of vasospasm, abnormal reactivity of cerebral arteries and cerebral ischemia following SAH, have been investigated intensively. A number of pathological processes have been identified in the pathogenesis of vasospasm, including endothelial injury, smooth muscle cell contraction from spasmogenic substances produced by the subarachnoid blood clots, changes in vascular responsiveness and inflammatory response of the vascular endothelium. To date, the current therapeutic interventions remain ineffective as they are limited to the manipulation of systemic blood pressure, variation of blood volume and viscosity and control of arterial carbon dioxide tension. In this scenario, the hormone erythropoietin (EPO) has been found to exert neuroprotective action during experimental SAH when its recombinant form (rHuEPO) is administered systemically. However, recent translation of experimental data into clinical trials has suggested an unclear role of recombinant human EPO in the setting of SAH. In this context, the aim of the current review is to present current evidence on the potential role of EPO in cerebrovascular dysfunction following aneurysmal subarachnoid hemorrhage.     

Effect of endogenous and exogenous nitric oxide on calcium sparks as targets for vasodilation in rat cerebral artery

The potent vasodilator nitric oxide (NO), produced mainly by the endothelium, acts through a BK_Ca-dependent mechanism to increase the frequency of calcium sparks (Ca²⁺ sparks) in myocyte isolated from rat cerebral arteries. Our present aim has been to assess the role of endogenous and exogenous NO on the Ca²⁺ sparks through ryanodine-sensitive channels in the sarcoplasmic reticulum of an intact artery. Calcium sparks, detected with fluo-4 and laser scanning confocal microscopy, were examined in isolated pressurized rat posterior cerebral arteries with (intact) and without endothelium (denuded). Addition of the NO donor, DEA-NONOate (N-(2-aminoethyl)-N-(2-hydroxy-2-nitrosohydrazino)-1,2-ethylenediamine), did not change the amplitude and frequency of Ca²⁺ sparks in the intact artery. However, inhibition of nitric oxide synthase with N-ω-nitro-l-arginine or removal of endothelium reduced Ca²⁺ sparks frequency by about 50%. Under these conditions (i.e., absence of endogenous NO production), DEA-NONOate, increased Ca²⁺ spark frequency 3- to 4-fold. These results suggest that endothelial NO modulates local Ca²⁺ release events in the arterial smooth muscle and that this mechanism may contribute to the actions of nitrovasodilators.     

Permissive contributions of NO and prostacyclin in CO-induced cerebrovascular dilation in piglets

Endogenously produced CO is an important dilator in newborn cerebrovascular circulation. CO dilates cerebral arterioles by activating Ca2+-activated K+ channels, but modulatory actions of other effectors and second messenger inputs are unclear. Specifically, the mechanisms behind the obligatory permissive roles of prostacyclin and NO are uncertain. Therefore, the present study was performed using acutely implanted, closed cranial windows in newborn pigs to address the hypothesis that the permissive roles of NO and prostacyclin in cerebrovascular dilation in response to CO involve a common mechanism. The NO donor sodium nitroprusside restored dilation in response to CO after inhibition of that dilation with the prostaglandin cyclooxygenase inhibitor indomethacin. The stable prostacyclin analog iloprost restored CO-induced dilation blocked by the NO synthase inhibitor Nomega-nitro-L-arginine. Restoration of dilation in response to CO by the cGMP-dependent phosphodiesterase inhibitor zaprinast and blockade of CO dilation by the guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazole-[4,3-a]quinoxalin-1-one (ODQ) suggests involvement of the cGMP/PKG pathway. Iloprost or the cAMP-dependent dilator isoproterenol restored dilation in response to CO after ODQ administration. However, CO-induced dilation blocked by the cGMP-dependent PKG inhibitor Rp-8-[(4-chlorophenyl)thio]-cGMPS triethylamine could not be reversed by administration of sodium nitroprusside, iloprost, or isoproterenol. Conversely, PKA inhibition did not block dilation in response to CO. Overall, data indicate that activation of PKG is the predominant mechanism of the permissive actions of NO and prostacyclin for CO-induced pial arteriolar dilation.     

Elucidation of the temporal relationship between endothelial-derived NO and EDHF in mesenteric vessels

Although the endothelium co-generates both nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF), the relative contribution from each vasodilator is not clear. In studies where the endothelium is stimulated acutely, EDHF responses predominate in small arteries. However, the temporal relationship between endothelial-derived NO and EDHF over more prolonged periods is unclear but of major physiological importance. Here we have used a classical pharmacological approach to show that EDHF is released transiently compared with NO. Acetylcholine (3 x 10(-6) mol/l) dilated second- and/or third-order mesenteric arteries for prolonged periods of up to 1 h, an effect that was reversed fully and immediately by the subsequent addition of L-NAME (10(-3) mol/l) but not TRAM-34 (10(-6) mol/l) plus apamin (5 x 10(-7) mol/l). When vessels were pretreated with L-NAME, acetylcholine induced relatively transient dilator responses (declining over approximately 5 min), and vessels were sensitive to TRAM-34 plus apamin. When measured in parallel, the dilator effects of acetylcholine outlasted the smooth muscle hyperpolarization. However, in the presence of L-NAME, vasodilatation and hyperpolarization followed an identical time course. In vessels from NOSIII(-/-) mice, acetylcholine induced small but detectable dilator responses that were transient in duration and blocked by TRAM-34 plus apamin. EDHF responses in these mouse arteries were inhibited by an intracellular calcium blocker, TMB-8, and the phospholipase A(2) inhibitor AACOCF(3), suggesting a role for lipid metabolites. These data show for the first time that EDHF is released transiently, whereas endothelial-derived NO is released in a sustained manner.     

Estrogen reduces mouse cerebral artery tone through endothelial NOS- and cyclooxygenase-dependent mechanisms

Gender and estrogen status are known to influence the incidence and severity of cerebrovascular disease. The vasoprotective effects of estrogen are thought to include both nitric oxide-dependent and independent mechanisms. Therefore, using small, resistance-sized arteries pressurized in vitro, the present study determined the effect of gender and estrogen status on myogenic reactivity of mouse cerebral arteries. Luminal diameter was measured in middle cerebral artery segments from males and from females that were either untreated, ovariectomized (OVX), or OVX with estrogen replacement (OVX + EST). The maximal passive diameters of arteries from all four groups were similar. In response to increases in transmural pressure, diameters of arteries from males and OVX females were smaller compared with diameters of arteries from either untreated or OVX + EST females. In the presence of N(G)-nitro-L-arginine methyl ester, artery diameters decreased in all groups, but diameters remained significantly smaller in arteries from males and OVX females compared with untreated and OVX + EST females. After endothelium removal or when inhibition of nitric oxide synthase and cyclooxygenase were combined, differences in diameters of arteries from OVX and OVX + EST were abolished. These data suggest that chronic estrogen treatment modulates myogenic reactivity of mouse cerebral arteries through both endothelium-derived cyclooxygenase- and nitric oxide synthase-dependent mechanisms.     

How does ascorbic acid prevent endothelial dysfunction?

Human coronary and peripheral arteries show endothelial dysfunction in a variety of conditions, including atherosclerosis, hypercholesterolemia, smoking, and hypertension. This dysfunction manifests as a loss of endothelium-dependent vasodilation to acetylcholine infusion or sheer stress, and is typically associated with decreased generation of nitric oxide (NO) by the endothelium. Vitamin C, or ascorbic acid, when acutely infused or chronically ingested, improves the defective endothelium-dependent vasodilation present in these clinical conditions. The mechanism of the ascorbic acid effect is unknown, although it has been attributed to an antioxidant function of the vitamin to enhance the synthesis or prevent the breakdown of NO. In this review, multiple mechanisms are considered that might account for the ability of ascorbate to preserve NO. These include ascorbate-induced decreases in low-density lipoprotein (LDL) oxidation, scavenging of intracellular superoxide, release of NO from circulating or tissue S-nitrosothiols, direct reduction of nitrite to NO, and activation of either endothelial NO synthase or smooth muscle guanylate cyclase. The ability of ascorbic acid supplements to enhance defective endothelial function in human diseases provides a rationale for use of such supplements in these conditions. However, it is first necessary to determine which of the many plausible mechanisms account for the effect, and to ensure that undesirable toxic effects are not present.     

Selected contribution: Effects of aging on cerebrovascular tone and [Ca2+]i.

The lower limits of cerebral blood flow autoregulation shift toward high pressures in aged compared with young rats. Intraluminal pressure stimulates contractile mechanisms in cerebral arteries that might, in part, cause an age-dependent shift in autoregulation. The present project tested two hypotheses. First, cerebral artery tone is greater in isolated arteries from aged compared with mature adult rats. Second, aging decreases the modulatory effect of endothelium-derived nitric oxide (NO) and increases vascular smooth muscle Ca2+ sensitivity. Isolated segments of middle cerebral arteries from male 6-, 12-, 20-, and 24-mo-old Fischer 344 rats were cannulated and loaded with fura-2. Diameter and Ca2+ responses to increasing pressure were measured in HEPES, during NO synthase inhibition [NG-nitro-l-arginine methyl ester (l-NAME)], and after removal of the endothelium. Cerebral artery tone (with endothelium) increased with age. Only at the lowest pressure (20 and 40 mmHg) was intracellular Ca2+ concentration ([Ca2+]i) greater in arteries from 24-mo-old rats compared with the other age groups. l-NAME-sensitive constriction increased significantly in arteries from 6- to 20-mo-old rats but declined significantly thereafter in arteries from 24-mo-old rats. [Ca2+]i was less in arteries from 24-mo-old rats compared with the other groups after treatment with l-NAME. Another endothelial-derived factor, insensitive to l-NAME, also decreased significantly with age. For example, at 60 mmHg, the l-NAME-insensitive constriction decreased from 47 +/- 10, 42 +/- 5, 21 +/- 2, and 3 +/- 1 microm in 6-, 12-, 20-, and 24-mo-old rats, respectively. Our data suggest that aging alters cerebral artery tone and [Ca2+]i responses through endothelial-derived NO synthase-sensitive and -insensitive mechanisms. The combined effect of greater cerebral artery tone with less endothelium-dependent modulation may in part contribute to the age-dependent shift in cerebral blood flow autoregulation.     

Effects of bradykinin in the cerebral circulation

All components of an intracerebral kallikrein-kinin system have been described. Thus, bradykinin (BK) acting from the parenchymal side as well as from the blood side may influence cerebral microcirculation. BK is a potent dilator of extra- and intraparenchymal cerebral arteries when acting from the perivascular side. The vasomotor effect of BK is mediated by B2 receptors which appear to be located at the abluminal membrane of the endothelial cell. Signal transmission from the endothelial to the smooth muscle cell is mediated by NO, prostanoids, free radicals or H2O2 depending on the animal species and on the location of the artery. Selective opening of the blood-brain barrier for small tracers (Na+-fluorescein: MW, 376) has been found in cats during cortical superfusion or intraarterial application of BK. This leakage is mediated by B2 receptors located at the luminal and abluminal membrane of the endothelial cells and probably mediated by an opening of tight junctions. Formation of brain edema has been found after ventriculo-cisternal perfusion or interstitial infusion of BK. This can be explained by increase of vascular permeability and cerebral blood flow due to arterial dilatation thus enhancing driving forces for the extravasation. An increase of the BK concentration in the interstitial space of the brain up to concentrations which induce extravasation, dilatation and edema formation has been found under several pathological conditions. Thus, BK may be involved in edema and necrosis formation after cold lesion, concussive brain injury, traumatic spinal cord and ischemic brain injury.     

Rosuvastatin improves cerebrovascular function in Zucker obese rats by inhibiting NAD(P)H oxidase-dependent superoxide production

Insulin-resistance induces cerebrovascular dysfunction and increases the risk for stroke. We investigated whether rosuvastatin (RSV), a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, can reverse reduced cerebrovascular responsiveness in insulin-resistant rats. Dilator responses of the basilar artery (BA) were examined after 1-day or 4-wk RSV (2 mg.kg(-1).day(-1)) treatment in anesthetized 12-wk-old insulin-resistant Zucker obese (ZO) and lean (ZL) rats by using a cranial window preparation. Vehicle-treated ZO rats had significantly higher fasting insulin, total cholesterol (TC), and triglyceride (TG) levels compared with ZL rats. In addition, in the ZO rats, dilator responses of the BA to acetylcholine, iloprost, cromakalim, and potassium chloride were significantly reduced when compared with ZL rats. One-day RSV treatment improved dilator responses of the ZO BAs without altering lipid levels. Four-week RSV treatment lowered both TC and TG by 30% and also improved dilator responses of the ZO BAs, although without additional effects compared with the 1-day RSV treatment. NAD(P)H oxidase-dependent superoxide production was significantly higher in the cerebral arteries of vehicle-treated ZO rats compared with ZL rats, but both 1-day and 4-wk RSV treatments normalized elevated superoxide levels in the ZO arteries. These findings demonstrate that RSV improves cerebrovascular function in insulin-resistance independently from its lipid-lowering effect by the inhibition of NAD(P)H oxidase.     

Lack of prejunctional modulation of noradrenaline release by endogenous nitric oxide in guinea pig pulmonary artery

The regulation of noradrenaline (NA) release by endogenous endothelium-derived compounds was investigated in the isolated guinea pig pulmonary artery preloaded with [3H]NA. The radioactivity uptake, the basal and electrical field stimulation (10 Hz, 2 ms, 360 shocks) evoked release of [(3)H]NA was similar in arteries with intact endothelium and after removal of the endothelium. The wide selectivity nitric oxide (NO) synthase inhibitor N-omega-nitro-L-arginine (100 microM) did not affect significantly the basal and stimulation-evoked release of [(3)H]NA in control and endothelium-denuded preparations. These results indicate that neither endogenous NO nor other compounds derived from the endothelium have substantial influence on the NA outflow from sympathetic nerves innervating the pulmonary artery.     

17beta-estradiol decreases vascular tone in cerebral arteries by shifting COX-dependent vasoconstriction to vasodilation

We have previously shown that estrogen treatment increases cerebrovascular cyclooxygenase-1, prostacyclin synthase, and production of prostacyclin. Therefore, vascular tone and prostanoid production were measured to investigate functional consequences of estrogen exposure. Middle cerebral arteries were isolated from ovariectomized female Fischer-344 rats with or without chronic in vivo 17beta-estradiol treatment. In vivo 17beta-estradiol treatment increased cerebral artery diameter; functional endothelium was required for expression of these differences. The nonspecific cyclooxygenase inhibitor indomethacin constricted, whereas arachidonic acid dilated, cerebral arteries from estrogen-treated animals. Estrogen exposure increased production of prostacyclin by cerebral arteries. Conversely, in estrogen-deficient animals, indomethacin dilated and arachidonic acid constricted cerebral blood vessels. This correlated with vasorelaxation following inhibition of the thromboxane-endoperoxide receptor with SQ-29548 but not after selective blockade of thromboxane synthase with furegrelate, suggesting prostaglandin endoperoxide (i.e., PGH2) activity. Removal of the endothelium or selective blockade of cyclooxygenase-1 with SC-560 abolished estrogen-mediated differences in the effects of arachidonate on vessel diameter and on prostacyclin production by cerebral arteries. These data suggest 17beta-estradiol decreases cerebrovascular tone by shifting the primary end product of the endothelial cyclooxygenase-1 pathway from the constrictor prostaglandin PGH2 to the vasodilator prostacyclin. These effects of estrogen may contribute to the heightened thromboresistance and enhanced cerebral blood flow documented in pre-versus postmenopausal women.     

Nitric oxide and atherosclerosis.

Endothelial dysfunction has been shown in a wide range of vascular disorders including atherosclerosis and related diseases. Here, we examine and address the complex relationship among nitric oxide (NO)-mediated pathways and atherogenesis. In view of the numerous pathophysiological actions of NO, abnormalities could potentially occur at many sites: (a) impairment of membrane receptors in the arterial wall that interact with agonists or physiological stimuli capable of generating NO; (b) reduced concentrations or impaired utilization of l-arginine; (c) reduction in concentration or activity both of inducible and endothelial NO synthase; (d) impaired release of NO from the atherosclerotic damaged endothelium; (e) impaired NO diffusion from endothelium to vascular smooth muscle cells followed by decreased sensitivity to its vasodilator action; (f) local enhanced degradation of NO by increased generation of free radicals and/or oxidation-sensitive mechanisms; and (g) impaired interaction of NO with guanylate cyclase and consequent limitation of cyclic GMP production. Therefore, one target for new drugs should be the preservation or restoration of NO-mediated signaling pathways in arteries. Such novel therapeutic strategies may include administration of l-arginine/antioxidants and gene-transfer approaches.     

The relaxant effect of urocortin in rat pulmonary arteries

Urocortin is a potent vasodilator, which plays physiological or pathophysiological roles in systemic circulation. However, little is known about its action on pulmonary circulation. The present study was aimed to characterize some cellular mechanisms underlying the relaxant effect of urocortin in isolated rat pulmonary arteries. Changes in isometric tension were measured on small vessel myographs. Urocortin inhibited U46619-induced contraction with reduction of the maximal response. Urocortin-induced relaxation was independent of the presence of endothelium. Inhibitors of nitric oxide (NO)-dependent dilator, NG-nitro-L-arginine methyl ester or 1H-[1,2,4]oxadizolo[4,3-a]quinoxalin-1-one, did not affect the relaxation. Astressin (100-500 nM), a corticotropin-releasing factor (CRF) receptor antagonist and KT5720, a protein kinase A (PKA) inhibitor reduced urocortin-induced relaxation. Urocortin produced less relaxant effect in 30 mM K+- than U46619-contracted arterial rings. Urocortin did not reduce CaCl2-induced contraction in 60 mM K+-containing solution. Ba2+ (100-500 microM) but not other K+ channel blockers reduced the relaxant responses to urocortin. Urocortin also relaxed the rings preconstricted by phorbol 12,13-diacetae in normal Krebs solution while this relaxation was less in a Ca2+-free solution. Our results show that urocortin relaxed rat pulmonary arteries via CRF receptor-mediated and PKA-dependent but endothelium/NO or voltage-gated Ca2+ channel-independent mechanisms. Stimulation of Ba2+-sensitive K+ channel may contribute to urocortin-induced relaxation. Finally, urocortin relaxed pulmonary arteries partly via inhibition of a PKC-dependent contractile mechanism.     

Histamine induces intracellular Ca2+ oscillations and nitric oxide release in endothelial cells from brain microvascular circulation

The neuromodulator histamine is able to vasorelax in human cerebral, meningeal and temporal arteries via endothelial histamine 1 receptors (H₁Rs) which result in the downstream production of nitric oxide (NO), the most powerful vasodilator transmitter in the brain. Although endothelial Ca ²⁺ signals drive histamine-induced NO release throughout the peripheral circulation, the mechanism by which histamine evokes NO production in human cerebrovascular endothelial cells is still unknown. Herein, we exploited the human cerebral microvascular endothelial cell line, hCMEC/D3, to assess the role of intracellular Ca ²⁺ signaling in histamine-induced NO release. To achieve this goal, hCMEC/D3 cells were loaded with the Ca ²⁺- and NO-sensitive dyes, Fura-2/AM and DAF-FM/AM, respectively. Histamine elicited repetitive oscillations in intracellular Ca ²⁺ concentration in hCMEC/D3 cells throughout a concentration range spanning from 1 pM up to 300 μM. The oscillatory Ca ²⁺ response was suppressed by the inhibition of H ₁Rs with pyrilamine, whereas H ₁R was abundantly expressed at the protein level. We further found that histamine-induced intracellular Ca ²⁺ oscillations were initiated by endogenous Ca ²⁺ mobilization through inositol-1,4,5-trisphosphate- and nicotinic acid dinucleotide phosphate-sensitive channels and maintained over time by store-operated Ca ²⁺ entry. In addition, histamine evoked robust NO release that was prevented by interfering with the accompanying intracellular Ca ²⁺ oscillations, thereby confirming that the endothelial NO synthase is recruited by Ca ²⁺ spikes also in hCMEC/D3 cells. These data provide the first evidence that histamine evokes NO production from human cerebrovascular endothelial cells through intracellular Ca ²⁺ oscillations, thereby shedding novel light on the mechanisms by which this neuromodulator controls cerebral blood flow.