EDHF contributes to strain-related differences in pulmonary arterial relaxation in rats

Pulmonary arteries from the Madison (M) strain relax more in response to acetylcholine (ACh) than those from the Hilltop (H) strain of Sprague-Dawley rats. We hypothesized that differences in endothelial nitric oxide (NO) synthase (eNOS) expression and function, metabolism of ACh by cholinesterases, release of prostacyclin, or endothelium-derived hyperpolarizing factor(s) (EDHF) from the endothelium would explain the differences in the relaxation response to ACh in isolated pulmonary arteries. eNOS mRNA and protein levels as well as the NO-dependent relaxation responses to thapsigargin in phenylephrine (10(-6) M)-precontracted pulmonary arteries from the M and H strains were identical. The greater relaxation response to ACh in M compared with H rats was also observed with carbachol, a cholinesterase-resistant analog of ACh, a response that was not modified by pretreatment with meclofenamate (10(-5) M). N(omega)-nitro-L-arginine (10(-4) M) completely abolished carbachol-induced relaxation in H rat pulmonary arteries but not in M rat pulmonary arteries. Precontraction with KCl (20 mM) blunted the relaxation response to carbachol in M rat pulmonary arteries and eliminated differences between the M and H rat pulmonary arteries. NO-independent relaxation present in the M rat pulmonary arteries was significantly reduced by 17-octadecynoic acid (2 microM) and was completely abolished by charybdotoxin plus apamin (100 nM each). These findings suggest that EDHF, but not NO, contributes to the strain-related differences in pulmonary artery reactivity. Also, EDHF may be a metabolite of cytochrome P-450 that activates Ca(2+)-dependent K(+) channels.     

Hypoxic constriction of porcine distal pulmonary arteries: endothelium and endothelin dependence

To determine the role of endothelium in hypoxic pulmonary vasoconstriction (HPV), we measured vasomotor responses to hypoxia in isolated seventh-generation porcine pulmonary arteries < 300 microm in diameter with (E+) and without endothelium. In E+ pulmonary arteries, hypoxia decreased the vascular intraluminal diameter measured at a constant transmural pressure. These constrictions were complete in 30-40 min; maximum at PO(2) of 2 mm Hg; half-maximal at PO(2) of 40 mm Hg; blocked by exposure to Ca(2+)-free conditions, nifedipine, or ryanodine; and absent in E+ bronchial arteries of similar size. Hypoxic constrictions were unaltered by indomethacin, enhanced by indomethacin plus N(G)-nitro-L-arginine methyl ester, abolished by BQ-123 or endothelial denudation, and restored in endothelium-denuded pulmonary arteries pretreated with 10(-10) M endothelin-1 (ET-1). Given previous demonstrations that hypoxia caused contractions in isolated pulmonary arterial myocytes and that ET-1 receptor antagonists inhibited HPV in intact animals, our results suggest that full in vivo expression of HPV requires basal release of ET-1 from the endothelium to facilitate mechanisms of hypoxic reactivity in pulmonary arterial smooth muscle.     

Mechanisms of histamine-induced relaxation in bovine small adrenal cortical arteries

Adrenal steroidogenesis is closely correlated with increases in adrenal blood flow. Many reports have studied the regulation of adrenal blood flow in vivo and in perfused glands, but until recently few studies have been conducted on isolated adrenal arteries. The present study examined vasomotor responses of isolated bovine small adrenal cortical arteries to histamine, an endogenous vasoactive compound, and its mechanism of action. In U-46619-precontracted arteries, histamine (10(-9)-5 x 10(-6) M) elicited concentration-dependent relaxations. The relaxations were blocked by the H(1) receptor antagonists diphenhydramine (10 microM) or mepyramine (1 microM) (maximal relaxations of 18 +/- 6 and 22 +/- 6%, respectively, vs. 55 +/- 5% of control) but only partially inhibited by the H(2) receptor antagonist cimetidine (10 microM) and the H(3) receptor antagonist thioperamide (1 microM). Histamine-induced relaxations were also blocked by the nitric oxide synthase inhibitor N-nitro-L-arginine (L-NA, 30 microM; maximal relaxation of 13 +/- 7%) and eliminated by endothelial removal or L-NA combined with the cyclooxgenase inhibitor indomethacin (10 microM). In the presence of adrenal zona glomerulosa (ZG) cells, histamine did not induce further relaxations compared with histamine alone. Histamine (10(-7)-10(-5) M) concentration-dependently increased aldosterone production by adrenal ZG cells. Compound 48/80 (10 microg/ml), a mast cell degranulator, induced significant relaxations (93 +/- 0.6%), which were blocked by L-NA plus indomethacin or endothelium removal, partially inhibited by the combination of the H(1), H(2), and H(3) receptor antagonists, but not affected by the mast cell stabilizer sodium cromoglycate (1 mM). These results demonstrate that histamine causes direct relaxation of small adrenal cortical arteries, which is largely mediated by endothelial NO and prostaglandins via H(1) receptors. The potential role of histamine in linking adrenal vascular events and steroid secretion requires further investigation.     

NO and reactive oxygen species are involved in biphasic hypoxic vasoconstriction of isolated rabbit lungs

Hypoxic pulmonary vasoconstriction (HPV) matches lung perfusion with ventilation but may also result in chronic pulmonary hypertension. It has not been clarified whether acute HPV and the response to prolonged alveolar hypoxia are triggered by identical mechanisms. We characterized the vascular response to sustained hypoxic ventilation (3% O(2) for 120-180 min) in isolated rabbit lungs. Hypoxia provoked a biphasic increase in pulmonary arterial pressure (PAP). Persistent PAP elevation was observed after termination of hypoxia. Total blockage of lung nitric oxide (NO) formation by N(G)-monomethyl-L-arginine caused a two- to threefold amplification of acute HPV, the sustained pressor response, and the loss of posthypoxic relaxation. This amplification was only moderate when NO formation was partially blocked by the inducible NO synthase inhibitor S-methylisothiourea. The superoxide scavenger nitro blue tetrazolium and the superoxide dismutase inhibitor triethylenetetramine reduced the initial vasoconstrictor response, the prolonged PAP increase, and the loss of posthypoxic vasorelaxation to a similar extent. The NAD(P)H oxidase inhibitor diphenyleneiodonium nearly fully blocked the late vascular responses to hypoxia in a dose that effected a decrease to half of the acute HPV. In conclusion, as similarly suggested for acute HPV, lung NO synthesis and the superoxide-hydrogen peroxide axis appear to be implicated in the prolonged pressor response and the posthypoxic loss of vasorelaxation in perfused rabbit lungs undergoing 2-3 h of hypoxic ventilation.     

Insulin resistance in spontaneously hypertensive rats is associated with endothelial dysfunction characterized by imbalance between NO and ET-1 production

Insulin stimulates production of NO in vascular endothelium via activation of phosphatidylinositol (PI) 3-kinase, Akt, and endothelial NO synthase. We hypothesized that insulin resistance may cause imbalance between endothelial vasodilators and vasoconstrictors (e.g., NO and ET-1), leading to hypertension. Twelve-week-old male spontaneously hypertensive rats (SHR) were hypertensive and insulin resistant compared with control Wistar-Kyoto (WKY) rats (systolic blood pressure 202 +/- 11 vs. 132 +/- 10 mmHg; fasting plasma insulin 5 +/- 1 vs. 0.9 +/- 0.1 ng/ml; P < 0.001). In WKY rats, insulin stimulated dose-dependent relaxation of mesenteric arteries precontracted with norepinephrine (NE) ex vivo. This depended on intact endothelium and was blocked by genistein, wortmannin, or N(omega)-nitro-l-arginine methyl ester (inhibitors of tyrosine kinase, PI3-kinase, and NO synthases, respectively). Vasodilation in response to insulin (but not ACh) was impaired by 20% in SHR (vs. WKY, P < 0.005). Preincubation of arteries with insulin significantly reduced the contractile effect of NE by 20% in WKY but not SHR rats. In SHR, the effect of insulin to reduce NE-mediated vasoconstriction became evident when insulin pretreatment was accompanied by ET-1 receptor blockade (BQ-123, BQ-788). Similar results were observed during treatment with the MEK inhibitor PD-98059. In addition, insulin-stimulated secretion of ET-1 from primary endothelial cells was significantly reduced by pretreatment of cells with PD-98059 (but not wortmannin). We conclude that insulin resistance in SHR is accompanied by endothelial dysfunction in mesenteric vessels with impaired PI3-kinase-dependent NO production and enhanced MAPK-dependent ET-1 secretion. These results may reflect pathophysiology in other vascular beds that directly contribute to elevated peripheral vascular resistance and hypertension.     

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.     

Endothelium-derived reactive oxygen species and endothelin-1 attenuate NO-dependent pulmonary vasodilation following chronic hypoxia

Vasodilatory responses to exogenous nitric oxide (NO) are diminished following exposure to chronic hypoxia (CH) in isolated, perfused rat lungs. We hypothesized that both endothelium-derived reactive oxygen species (ROS) and endothelin-1 (ET-1) mediate this attenuated NO-dependent pulmonary vasodilation following CH. To test this hypothesis, we examined vasodilatory and vascular smooth muscle (VSM) Ca2+ responses to the NO donor spermine NONOate in UTP-constricted, isolated pressurized small pulmonary arteries from control and CH rats. Consistent with our previous findings in perfused lungs, we observed attenuated NO-dependent vasodilation following CH in endothelium-intact vessels. However, in endothelium-denuded vessels, responses to spermine NONOate were augmented in CH rats compared with controls, thus demonstrating an inhibitory influence of the endothelium on NO-dependent reactivity following CH. Whereas both the ROS scavenger tiron and the ETA receptor antagonist BQ-123 augmented NO-dependent reactivity in endothelium-intact vessels from CH rats, neither fully restored vasodilatory responses to those observed following endothelium denudation in vessels from CH rats. In contrast, the combination of tiron and BQ-123 or the nonselective ET receptor antagonist PD-145065 enhanced NO responsiveness in endothelium-intact vessels from CH rats similar to that observed following endothelium denudation. We conclude that both endothelium-derived ROS and ET-1 attenuate NO-dependent pulmonary vasodilation following CH. Furthermore, CH augments pulmonary VSM reactivity to NO.     

RhoA/Rho kinase and nitric oxide modulate the agonist-induced pulmonary artery diameter response time

We studied the amplitude and response time (RT; time to 50% of maximal response) of pulmonary vasoreactivity and investigated whether the characteristics of pulmonary vasoreactivity could be modulated by endothelium removal, nitric oxide (NO) synthase inhibition [N(G)-nitro-L-arginine (L-NNA)], RhoA activation [lysophosphatidic acid (LPA)] and Rho kinase inhibition (Y-27632). Slow acetylcholine-induced pulmonary vasodilation (262 +/- 5 s) was not due to the RT of endothelial NO release (45-55 s) and was always longer than RT in renal arteries (15 +/- 4 s). The rate-determining step is located in the smooth muscle cells. This was confirmed by the existing differences between the RT of the NO solution and KCl-induced renal and pulmonary vasoreactivity in endothelium-denuded arteries. We found that the pulmonary contractile amplitude increases and the RT decreases by L-NNA or LPA. In contrast, Y-27632 reduced the contractile amplitude and increased the RT in pulmonary arteries. These phenomena were dependent on the contractile stimulus (phenylephrine or KCl). In conclusion, slow pulmonary vasoreactivity is a smooth muscle cell characteristic that can be enhanced by RhoA and NO or endothelium removal. These effects were counteracted by Rho kinase inhibition. We show a role for RhoA/Rho kinase and NO in the modulation of pulmonary vascular reactivity.     

Correlation of acute with chronic hypoxic pulmonary hypertension in cattle.

We investigated acute and chronic hypoxic pulmonary pressor responses in two groups of calves, one bred to be susceptible, the other resistant to high-altitude pulmonary hypertension. Twelve 5-mo-old susceptible calves residing at 1,524 m increased their mean pulmonary arterial pressure from 26 +/- 2 (SE) to 55 +/- 4 mmHg during 2 h at a simulated altitude of 4,572 m. In 10 resistant calves pressure increased from 22 +/- 1 to 37 +/- 2 mmHg. Five calves were selected from each group for further study. When 9 mo old, the 5 susceptible calves again showed a greater pressor response to acute hypoxia (27 +/- 1 to 55 +/- 4 mmHg) than did 5 resistant calves (23 +/- 1 to 41 +/- 3 mmHg). When 12 mo old, the 5 susceptible calves also developed a greater increase in pulmonary arterial pressure (21 +/- 2 to 9 +/- 4 mmHg) during 18 days at 4,572 m than did the 5 resistant calves (21 +/- 1 to 64 +/- 4 mmHg). Acute and chronic hypoxic pulmonary pressor responses were highly correlated (r = 0.91; P less than 0.001) indicating that they were probably produced through a common mechanism.     

Chronic hypoxia attenuates nitric oxide-dependent hemodynamic responses to acute hypoxia

Alterations in the nitric oxide (NO) pathway have been implicated in the pathogenesis of chronic hypoxia-induced pulmonary hypertension. Chronic hypoxia can either suppress the NO pathway, causing pulmonary hypertension, or increase NO release in order to counteract elevated pulmonary arterial pressure. We determined the effect of NO synthase inhibitor on hemodynamic responses to acute hypoxia (10% O(2)) in anesthetized rats following chronic exposure to hypobaric hypoxia (0.5 atm, air). In rats raised under normoxic conditions, acute hypoxia caused profound systemic hypotension and slight pulmonary hypertension without altering cardiac output. The total systemic vascular resistance (SVR) decreased by 41 +/- 5%, whereas the pulmonary vascular resistance (PVR) increased by 25 +/- 6% during acute hypoxia. Pretreatment with N(omega)-nitro-L-arginine methyl ester (L-NAME; 25 mg/kg) attenuated systemic vasodilatation and enhanced pulmonary vasoconstriction. In rats with prior exposure to chronic hypobaric hypoxia, the baseline values of mean pulmonary and systemic arterial pressure were significantly higher than those in the normoxic group. Chronic hypoxia caused right ventricular hypertrophy, as evidenced by a greater weight ratio of the right ventricle to the left ventricle and the interventricular septum compared to the normoxic group (46 +/- 4 vs. 28 +/- 3%). In rats which were previously exposed to chronic hypoxia (half room air for 15 days), acute hypoxia reduced SVR by 14 +/- 6% and increased PVR by 17 +/- 4%. Pretreatment with L-NAME further inhibited the systemic vasodilatation effect of acute hypoxia, but did not enhance pulmonary vasoconstriction. Our results suggest that the release of NO counteracts pulmonary vasoconstriction but lowers systemic vasodilatation on exposure to acute hypoxia, and these responses are attenuated following adaptation to chronic hypoxia.     

High-altitude chronic hypoxia during gestation and after birth modifies cardiovascular responses in newborn sheep

Perinatal exposure to chronic hypoxia induces sustained pulmonary hypertension and structural and functional changes in both pulmonary and systemic vascular beds. The aim of this study was to analyze consequences of high-altitude chronic hypoxia during gestation and early after birth in pulmonary and femoral vascular responses in newborn sheep. Lowland (LLNB; 580 m) and highland (HLNB; 3,600 m) newborn lambs were cathetherized under general anesthesia and submitted to acute sustained or stepwise hypoxic episodes. Contractile and dilator responses of isolated pulmonary and femoral small arteries were analyzed in a wire myograph. Under basal conditions, HLNB had a higher pulmonary arterial pressure (PAP; 20.2 +/- 2.4 vs. 13.6 +/- 0.5 mmHg, P < 0.05) and cardiac output (342 +/- 23 vs. 279 +/- 13 ml x min(-1) x kg(-1), P < 0.05) compared with LLNB. In small pulmonary arteries, HLNB showed greater contractile capacity and higher sensitivity to nitric oxide. In small femoral arteries, HLNB had lower maximal contraction than LLNB with higher maximal response and sensitivity to noradrenaline and phenylephrine. In acute superimposed hypoxia, HLNB reached higher PAP and femoral vascular resistance than LLNB. Graded hypoxia showed that average PAP was always higher in HLNB compared with LLNB at any Po2. Newborn lambs from pregnancies at high altitude have stronger pulmonary vascular responses to acute hypoxia associated with higher arterial contractile status. In addition, systemic vascular response to acute hypoxia is increased in high-altitude newborns, associated with higher arterial adrenergic responses. These responses determined in intrauterine life and early after birth could be adaptive to chronic hypoxia in the Andean altiplano.     

In vivo evidence for endothelin-1-mediated attenuation of alpha1-adrenergic stimulation

Experiments were designed to determine the influence of endothelin A (ET(A)) receptors on the pressor response to acute environmental stress in Dahl salt-resistant (DR) and Dahl-sensitive (DS) rats. Mean arterial pressure (MAP) was chronically monitored by telemetry before and after treatment with the selective ET(A) receptor antagonist ABT-627. Rats were restrained and subjected to pulsatile air jet stress (3 min). In untreated animals, the total pressor response (area under the curve) to acute stress was not different between DR vs. DS rats (8.1 +/- 1.7 vs. 15.6 +/- 2.6 mmHg x 3 min, P = 0.10). Conversely, treatment with ABT-627 potentiated the total pressor response only in DR rats (36.3 +/- 6.2 vs. 22.6 +/- 5.9 mmHg x 3 min, DR vs. DS, P < 0.05). Treatment with ABT-627 allowed greater responses in anesthetized DR rats to exogenous phenylephrine (1-4 microg/kg) during ganglionic blockade (P < 0.05) and produced a significant increase in plasma norepinephrine at baseline and during stress in conscious DR rats compared with untreated animals (P < 0.05). ET(A) receptor blockade had no effect on these responses in DS rats. Our results suggest that endothelin-1 can inhibit alpha-adrenergic-mediated effects in DR, but not DS rats, consistent with the hypothesis that ET(A) receptor activation functions to reduce sympathetic nerve activity and responses in vascular smooth muscle to sympathetic stimulation.     

Hypoxia-induced inhibition of converting enzyme activity: role in vascular regulation

Systemic and pulmonary vascular reactivity to graded doses of angiotensin I (ANG I), angiotensin II (ANG II), and, as a control, phenylephrine were examined in 14- or 28-day hypoxia-exposed and air control rats. Hypoxic rats exhibited pulmonary hypertension that was reversible on return to room air, but systemic arterial pressure was not altered by hypoxia. Systemic pressor responses to ANG I and ANG II were significantly less in the hypoxic rats than in the control rats at 14 and 28 days but returned to control levels in hypoxic animals that were then returned to room air, demonstrating reversibility of the hypoxia-induced changes in vascular reactivity. Pulmonary pressor responses to ANG I were significantly less at 14 days, whereas responses to ANG II were significantly greater at 28 days, in hypoxic rats than in controls. There were no significant differences in systemic and pulmonary pressor responses to phenylephrine between the hypoxic and air control animals. The altered systemic and pulmonary pressor responsiveness to ANG I and ANG II in hypoxic rats is probably related to mechanisms specific to the renin-angiotensin system, such as inhibition of intrapulmonary angiotensin-converting enzyme activity and down regulation of ANG II receptors in the systemic circulation. Further study is needed to elucidate these mechanisms.     

Endogenous estrogen mediates vascular reactivity and distensibility in pregnant rat mesenteric arteries

The role of estrogen in the maternal systemic cardiovascular adaptations during pregnancy is still controversial. Female Sprague-Dawley rats were implanted at day 14 of pregnancy with either a 50-mg tamoxifen pellet (estrogen receptor blocker, n = 10) or placebo pellet (n = 10). Virgin female rats were a nonpregnant control (n = 7). At days 20-22 of pregnancy, resistance-sized mesenteric arteries were mounted onto a dual-chamber arteriograph system. Pregnancy significantly blunted the pressor response to phenylephrine [measurement of the effective concentration that yielded 50% maximum response (EC(50)) values were 1.5 +/- 0.22 vs. 0.69 +/- 0.16 microM (P < 0.05)] and enhanced vasodilation to ACh [EC(50) = 1.13 +/- 2.53 vs. 3.13 +/- 6.04 nM (P < 0.05)] compared with nonpregnant rats. However, tamoxifen treatment during pregnancy reversed these effects. Inhibition of nitric oxide (NO) synthase with N(G)-monomethyl-L-arginine (250 microM) shifted only the responses of the placebo-treated pregnant group to both phenylephrine and ACh. Arterial distensibility in the placebo-treated pregnant group was also significantly increased (P < 0.05) compared with nonpregnant and tamoxifen-treated pregnant animals. In summary, endogenous estrogen during pregnancy increases NO-dependent modulation of vessel tone and arterial distensibility.     

Epoxyeicosatrienoic acids constrict isolated pressurized rabbit pulmonary arteries

Little information is available regarding the vasoactive effects of epoxyeicosatrienoic acids (EETs) in the lung. We demonstrate that 5, 6-, 8,9-, 11,12-, and 14,15-EETs contract pressurized rabbit pulmonary arteries in a concentration-dependent manner. Constriction to 5,6-EET methyl ester or 14,15-EET is blocked by indomethacin or ibuprofen (10(-5) M), SQ-29548, endothelial denuding, or submaximal preconstriction with the thromboxane mimetic U-46619. Constriction of pulmonary artery rings to phenylephrine is blunted by treatment with the epoxygenase inhibitor N-methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide. Pulmonary arteries and peripheral lung microsomes metabolize arachidonate to products that comigrate on reverse-phrase HPLC with authentic regioisomers of 5,6-, 8,9-, 11,12-, and 14,15-EETs, but no cyclooxygenase products of EETs could be demonstrated. Proteins of the CYP2B, CYP2E, CYP2J, CYP1A, and CYP2C subfamilies are present in pulmonary artery and peripheral lung microsomes. Constriction of isolated rabbit pulmonary arteries to EETs is nonregioselective and depends on intact endothelium and cyclooxygenase, consistent with the formation of a pressor prostanoid compound. These data raise the possibility that EETs may contribute to regulation of pulmonary vascular tone.     

Selective estrogen receptor-alpha and estrogen receptor-beta agonists rapidly decrease pulmonary artery vasoconstriction by a nitric oxide-dependent mechanism

Both endogenous and exogenous estrogen decrease pulmonary artery (PA) vasoconstriction. Whether these effects are mediated via estrogen receptor (ER)-alpha or ER-beta, and whether the contribution of ERs is stimulus-dependent, remains unknown. We hypothesized that administration of the selective ER-alpha agonist propylpyrazole triol (PPT) and/or the selective ER-beta agonist diarylpropiolnitrile (DPN) rapidly decreases PA vasoconstriction induced by pharmacologic and hypoxic stimuli via a nitric oxide (NO)-dependent mechanism. PA rings (n = 3-10/group) from adult male Sprague-Dawley rats were suspended in physiologic organ baths. Force displacement was measured. Vasoconstrictor responses to phenylephrine (10(-8)M - 10(-5)M) and hypoxia (Po(2) 35-45 mmHg) were determined. Endothelium-dependent and -independent vasorelaxation were measured by generating dose-response curves to acetylcholine (10(-8)M - 10(-4)M) and sodium nitroprusside (10(-9)M - 10(-5)M). PPT or DPN (10(-9)M - 5 x 10(-5)M) were added to the organ bath in the presence and absence of the NO-synthase inhibitor N(omega)-nitro-l-arginine methyl ester (l-NAME) (10(-4)M). Selective ER-alpha activation (PPT, 5 x 10(-5)M) rapidly (<20 min) decreased phenylephrine-induced vasoconstriction. This effect, as well as PPT's effects on endothelium-dependent vasorelaxation, were neutralized by l-NAME. In contrast, selective ER-beta activation (DPN, 5 x 10(-5)M) rapidly decreased phase II of hypoxic pulmonary vasoconstriction (HPV). l-NAME eliminated this phenomenon. Lower PPT or DPN concentrations were less effective. We conclude that both ER-alpha and ER-beta decrease PA vasoconstriction. The immediate onset of effect suggests a nongenomic mechanism. The contribution of specific ERs appears to be stimulus specific, with ER-alpha primarily modulating phenylephrine-induced vasoconstriction, and ER-beta inhibiting HPV. NO inhibition eliminates these effects, suggesting a central role for NO in mediating the pulmonary vascular effects of both ER-alpha and ER-beta.     

Chronic administration of indomethacin increases role of nitric oxide in hypercapnic cerebrovasodilation in piglets.

Hypercapnia-induced cerebral vasodilation is associated with prostanoids in the piglet, but is a primarily nitric oxide (NO) associated response in many adult models. Hypercapnia-induced cerebral vasodilation is both NO and prostanoid associated in the juvenile pig. We hypothesized that with chronic administration of indomethacin the piglet would advance the role of the NO system in cerebrovascular responses. The closed cranial window technique was used in piglets to determine pial arteriolar response. Chronically indomethacin treated newborn animals dilated in response to CO2 similarly to control newborns (40.9+/-4.4% vs 48.4+/-4.1%). Topical n-nitro L-arginine (L-NA, 10(-3) M), attenuated CO2 induced dilation in the chronically indomethacin treated animals (11.7+/-3.3% vs 40.9+/-4.4%; p < 0.001), but had no effect on the response to hypercapnia of piglets not treated with indomethacin. Neither indomethacin nor L-NA altered response to topical isoproterenol (10(-6) M). We conclude that with chronic indomethacin administration there develops a significant hypercapnia-induced cerebral vasodilation in which NO has an important role. The chronic inhibition of the newborn's principal dilator system appears to increase the role of NO in newborn cerebral hemodynamics.     

Vasoconstrictor mechanisms in the cerebral circulation are unaffected by insulin resistance

Insulin-resistance (IR) impairs agonist-induced relaxation in cerebral arteries, but little is known about its effect on constrictor mechanisms. We examined the vascular responses of the basilar artery (BA) and its side branches in anesthetized Zucker lean (ZL) and IR Zucker obese (ZO) rats using a cranial window technique. Endothelin-1 (ET-1) constricted the BAs in both the ZL and ZO rats, but there was no significant difference between the two groups (ZL: 36 +/- 8%; ZO: 33 +/- 3% at 10(-8) M). Inhibition of the ET(A) receptors by BQ-123 slightly increased the diameters of the BAs, with no difference shown between the ZL (6 +/- 1%) and ZO (5 +/- 3%) rats. Expressions of the ET(A) receptors and ET-1 mRNA examined by immunoblot analysis and RT-PCR, respectively, were also similar in the ZL and ZO groups. Phorbol 12,13-dibutyrate (PDBu), an activator of protein kinase C (PKC), and the thromboxane A(2) (TxA(2)) mimetic U-46619 constricted the BAs, but similarly to ET-1, there was no significant difference between the ZL and ZO groups (10(-6) M PDBu: ZL: 33 +/- 2%; ZO: 32 +/- 4%; and 10(-7) M U-46619: ZL: 23 +/- 1%; ZO: 19 +/- 2%). Inhibition of Rho-kinase with Y-27632 induced dilation of the BAs, and these responses were also comparable in the ZL and ZO rats (ZL: 39 +/- 4%; ZO: 38 +/- 2% at 10(-5) M). In contrast, nitric oxide-dependent relaxation to bradykinin was significantly reduced in the ZO rats (10(-6) M: 10 +/- 3%) compared with ZLs (29 +/- 7%, P < 0.01). These findings indicate that vasoconstrictor responses of the BA mediated by ET-1, TxA(2), PKC, and Rho-kinase are not affected by IR.     

Pre- and postjunctional alpha(2)-adrenergic receptors in fetal and adult ovine cerebral arteries

In ovine cerebral arteries, adrenergic-mediated vasoconstrictor responses differ significantly with developmental age. We tested the hypothesis that, in part, these differences are a consequence of altered alpha(2)-adrenergic receptor (alpha(2)-AR) density and/or affinity. In fetal (approximately 140 days) and adult sheep, we measured alpha(2)-AR density and affinity with the antagonist [(3)H]idazoxan in main branch cerebral arteries and other vessels. We also quantified contractile responses in middle cerebral artery (MCA) to norepinephrine (NE) or phenylephrine in the presence of the alpha(2)-AR antagonists yohimbine and idazoxan and contractile responses to the alpha(2)-AR agonists clonidine and UK-14304. In fetal and adult cerebral artery homogenates, alpha(2)-AR density was 201 +/- 18 and 52 +/- 6 fmol/mg protein, respectively (P < 0.01); however, antagonist affinity values did not differ. In fetal, but not adult, MCA, 10(-7) M yohimbine significantly decreased the pD(2) for NE-induced tension in the presence of 3 x 10(-5) M cocaine, 10(-5) M deoxycorticosterone, and 10(-6) M tetrodotoxin. In fetal, but not adult, MCA, UK-14304 induced a significant decrease in pD(2) for the phenylephrine dose-response relation. In addition, stimulation-evoked fractional NE release was significantly greater in fetal than in adult cerebral arteries. In the presence of 10(-6) M idazoxan to block alpha(2)-AR-mediated inhibition of prejunctional NE release, the fractional NE release was significantly increased in both age groups. We conclude that in fetal and adult ovine cerebral arteries, alpha(2)-AR appear to be chiefly prejunctional. Nonetheless, the fetal cerebral arteries appear to have a significant component of postjunctional alpha(2)-AR.     

Red wine polyphenols induce vasorelaxation by increased nitric oxide bioactivity

The aim of the present study was to investigate the mechanism of vasorelaxant responses induced by red wine polyphenolic compounds (Provinol). Rings of rat femoral artery with or without functional endothelium were set up in a myograph for isometric recording and precontracted with phenylephrine (10(-5) M). Provinol in cumulative doses (10(-9) to 10(-3) mg/ml) elicited endothelium- and dose-dependent relaxation of the artery with maximal relaxation of 56 per cent at the concentration of 10(-5) mg/ml. The relaxant responses to Provinol correlated well with the increase of NO synthase activity in the vascular tissue after administration of cumulative doses of Provinol (10(-9) to 10(-3) mg/ml). N(G)-nitro-L-arginine methylester (L-NAME, 3x10(-4) M) significantly attenuated the endothelium-dependent relaxation produced by Provinol. Administration of L-arginine (3x10(-5) M) restored the relaxation inhibited by L-NAME. The relaxant responses of Provinol were abolished in the presence of Ca(2+)-entry blocker, verapamil (10(-6) M). Administration of hydrogen peroxide (H(2)O(2)) abolished acetylcholine (10(-5) M)-induced relaxation of the rat femoral artery, while administration of Provinol (10(2) mg/ml) together with H(2)O(2) helped to maintain the acetylcholine-induced relaxation. Provinol only partially affected the concentration-response curve for the NO donor sodium nitroprusside-induced relaxation in rings without endothelium. In conclusion, Provinol elicited endothelium-dependent relaxation of rat femoral artery by the Ca(2+)-induced increase of NO synthase activity and by protecting NO from degradation.