Analysis of tert-butyl hydroperoxide induced constrictions of perfused vascular beds in vitro

tert-Butyl hydroperoxide (t-BOOH), an inducer of oxidative stress in vitro, elicits constrictor responses of the isolated, rat kidney and mesenteric arteries perfused (5 ml/min) with physiological salt solutions (PSS) at 37 degrees C gassed with carbogen. We hypothesized that generation of superoxide anions (O(2)(-)) accounts for these responses. We assessed responses to t-BOOH in preparations with/without endothelium, and in the absence/presence of antioxidant compounds, catalase and tempol, scavengers of hydroxyl (OH(-)) radical and O(2)(-), respectively. t-BOOH (0.01-50 micromol) induced (expressed as % of 50 micromol KCl vasoconstriction) were abolished by endothelium denudation, perfusion with Ca(2+)-free PSS and by nifedipine, (1 nM). Infusion of t-BOOH (0.1 microM) did not significantly (P > 0.05) affect phenylepherine E(max) in the mesenteric arteries, however it reduced phenylepherine E(max) responses in the kidney from 94.9 +/- 3.9 % to 64.7 +/- 4.7 %. Nitroblue tetrazolium, as well as alpha-phenyl N-tert-butyl nitrone, at 100 microM, but not catalase (500 IU) significantly attenuated t-BOOH (10 micromol) vasoconstrictor responses. Tempol (100 microM), a membrane permeable antioxidant, also significantly reduced t-BOOH (10 micromol) responses from 17.0 +/- 1.9 % (control) to 9.6 +/- 0.5 % (+tempol) in the mesenteric arteries and from 40.4 +/- 4.2 % (control) to 20.7 +/- 1.5 % (+tempol) in the kidney. Our data suggest that t-BOOH elicits vasoconstriction via two distinct mechanisms: (i) increased influx of extracellular Ca(2+), and (ii) generation of free radicals including O(2)(-) anions.     

Pregnancy reduces RhoA/Rho kinase and protein kinase C signaling pathways downstream of thromboxane receptor activation in the rat uterine artery

During pregnancy, reduced vascular responses to constrictors contribute to decreased uterine and total vascular resistance. Thromboxane A(2) (TxA(2)) is a potent vasoconstrictor that exerts its actions via diverse signaling pathways, and its biosynthesis increases in preeclampsia. In this study, we hypothesized that maternal vascular responses to TxA(2) will be attenuated via Rho kinase, PKC, p38 MAPK, and ERK1/2 signaling pathways. Isolated ring segments of uterine and small mesenteric arteries from late pregnant (19-21 days) and virgin rats were suspended in a myograph, and isometric force was measured. Pregnancy did not affect uterine and mesenteric artery responses to the TxA(2) analog U-46619 (10(-9)-10(-5) M), but transduction signals associated with these contractions were different between pregnant and nonpregnant rats. Inhibition of Rho kinase (10(-6) M Y-27632) reduced sensitivity to U-46619 in virgin uterine vessels but did not inhibit these contractions in pregnant uterine arteries and had no effect on mesenteric vessels. Treatment of arterial segments with a PKC inhibitor (10(-6) M bisindolylmaleimide I) reduced U-46619-induced contractions in virgin uterine and mesenteric arteries and in pregnant mesenteric arteries. Pregnant uterine arteries, however, were unresponsive to PKC inhibition. Inhibition of ERK1/2 (10(-5) M PD-98059) and p38 MAPK (10(-5) M SB-203580) reduced U46619-induced contractions in nonpregnant vessels and in pregnant uterine and mesenteric vessels. These data suggest that normal pregnancy does not affect uterine and mesenteric contractile responses to TxA(2) but reduces the contribution of Rho kinase and PKC signaling pathways to these contractions in the uterine vasculature. In contrast, the role of ERK1/2 and p38 MAPK in U-46619-induced uterine contractions remains unchanged with pregnancy. TxA(2)-associated transduction signals and its regulators might present potential targets for the development of new treatments for preeclampsia and other pregnancy-associated vascular diseases.     

Increased H(2)O(2) counteracts the vasodilator and natriuretic effects of superoxide dismutation by tempol in renal medulla

A membrane-permeable SOD mimetic, 4-hydroxytetramethyl-piperidine-1-oxyl (tempol), has been used as an antioxidant to prevent hypertension. We recently found that this SOD mimetic could not prevent development of hypertension induced by inhibition of renal medullary SOD with diethyldithiocarbamic acid. The present study tested a hypothesis that increased H2O2 counteracts the effects of tempol on renal medullary blood flow (MBF) and Na+ excretion (UNaV), thereby restraining the antihypertensive effect of this SOD mimetic. By in vivo microdialysis and Amplex red H2O2 microassay, it was found that interstitial H2O2 levels in the renal cortex and medulla in anesthetized rats averaged 55.91 +/- 3.66 and 102.18 +/- 5.16 nM, respectively. Renal medullary interstitial infusion of tempol (30 micromol x min-1x kg-1) significantly increased medullary H2O2 levels by 46%, and coinfusion of catalase (10 mg x min-1x kg-1) completely abolished this increase. Functionally, removal of H2O2 by catalase enhanced the tempol-induced increase in MBF, urine flow, and UNaV by 28, 41, and 30%, respectively. Direct delivery of H2O2 by renal medullary interstitial infusion (7.5-30 nmol x min-1x kg-1) significantly decreased renal MBF, urine flow, and UNaV, and catalase reversed the effects of H2O2. We conclude that tempol produces a renal medullary vasodilator effect and results in diuresis and natriuresis. However, this SOD mimetic increases the formation of H2O2, which constricts medullary vessels and, thereby, counteracts its vasodilator actions. This counteracting effect of H2O2 may limit the use of tempol as an antihypertensive agent under exaggerated oxidative stress in the kidney.     

Increased superoxide leads to decreased flow-induced dilation in resistance arteries of Mn-SOD-deficient mice

The role of mitochondrial manganese-superoxide dismutase (Mn-SOD) in the maintenance of vascular function has not yet been studied. Thus we examined flow- and agonist-induced dilations in isolated mesenteric arteries (approximately 90 microm in diameter) of Mn-SOD heterozygous (Mn-SOD+/-) and wild-type (WT) mice. Increases in flow elicited dilations in all vessels, but the magnitude of the dilation was significantly less in vessels of Mn-SOD+/- mice than in those of WT mice (64 vs. 74% of passive diameter). N(omega)-nitro-L-arginine methyl ester inhibited the dilation in vessels of WT mice but had no effect on vessels of Mn-SOD+/- mice. Tempol or tiron (superoxide scavengers) increased flow-induced dilation in vessels of Mn-SOD+/- mice. Acetylcholine- and sodium nitroprusside-induced, but not adenosine-induced, dilations were also decreased in arteries of Mn-SOD+/- mice. Superoxide levels in the arteries of Mn-SOD+/- mice were significantly increased. Western blot analysis confirmed a 50% reduction of Mn-SOD protein in the vessels of Mn-SOD+/- mice. A 41% reduction in endothelial nitric oxide synthase (eNOS) protein and a 37% reduction in eNOS activity were also found in the vessels of Mn-SOD+/- mice. Whereas there was no difference in eNOS protein in kidney homogenates of WT and Mn-SOD+/- mice, a significant reduction of nitric oxide synthase activity was found in Mn-SOD+/- mice, which could be restored by the administration of tiron. We conclude that an increased concentration of superoxide due to reduced activity of Mn-SOD, which inactivates nitric oxide and inhibits eNOS activity, contributes to the impaired vasodilator function of isolated mesenteric arteries of Mn-SOD+/- mice. These results suggest that Mn-SOD contributes significantly to the regulation of vascular function.     

Characterization of thromboxane and prostacyclin effects on pulmonary vascular resistance.

Although thromboxane and prostacyclin (PGI2) have long been described as major controllers of pulmonary vascular resistance, little has been reported on the characteristics of the interactions between the two arachidonic acid products. The current study uses segmental vascular resistance and compliance measurements to evaluate the actions of thromboxane and PGI2 in isolated blood-perfused rat lung. The thromboxane analogue U-46619 increases pulmonary vascular resistance by increasing only small artery resistance and decreases pulmonary vascular compliance in the middle compartment. Among the vascular effects of U-46619 are a maximum increase in resistance (RmaxU-46619) of 60.3 +/- 15.6 cmH2O.l-1.min.100 g-1 and a concentration required for 50% of maximum increase (K0.5,U-46619) of 1.60 +/- 0.85 nM for small artery resistance, a minimum vascular compliance (CminU-46619) of -0.93 +/- 0.58 cmH2O, and a K0.5,U-46619 of 1.10 +/- 1.60 nM for middle compartment compliance. Similar results were obtained for total resistance and total compliance. The effects of PGI2 on thromboxane-induced resistance and compliance changes were evaluated using K0.5,PGI2, RmaxPGI2, and CmaxPGI2 at each dose of thromboxane. PGI2 was more effective in reversing the thromboxane constriction at higher concentrations of thromboxane. These data show that the absolute concentration of PGI2 and thromboxane and not a simple ratio of thromboxane to PGI2 determines vascular tone.     

Comparative responses to alpha,beta-methylene-ATP in cat pulmonary, mesenteric, and hindquarter vascular beds.

Responses to the P2X-purinoceptor agonist alpha,beta-methylene-ATP (alpha,beta-MeATP) were investigated in the pulmonary, hindquarter, and mesenteric vascular beds in the cat. Under constant-flow conditions, injections of alpha,beta-MeATP caused dose-related increases in perfusion pressure in the pulmonary and hindquarter beds and a biphasic response in the mesenteric circulation. In the pulmonary vascular bed, the order of potency was alpha,beta-MeATP > U-46619 > angiotensin II, whereas, in the hindquarters, the order of potency was angiotensin II > U-46619 > alpha,beta-MeATP. The order of potency was similar in the hindquarter and mesenteric beds when the pressor component of the response to alpha,beta-MeATP was compared with responses to angiotensin II and U-46619. The P2X-receptor antagonist pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid attenuated the pressor response to alpha,beta-MeATP in the hindquarter circulation and the pressor component in the mesenteric vascular bed. Pressor responses to alpha,beta-MeATP were not altered by cyclooxygenase, alpha-adrenergic, or angiotensin AT(1) antagonists. These data show that alpha,beta-MeATP has potent pressor activity in the pulmonary circulation, where it was 100-fold more potent than angiotensin II. In contrast, alpha,beta-MeATP had modest pressor activity in the systemic bed, where it was 1,000-fold less potent than angiotensin II. These data suggest that responses to alpha,beta-MeATP are dependent on the vascular bed studied and may be dependent on the density of P2X receptors in the vascular bed.     

Impaired flow-induced dilation in mesenteric resistance arteries from receptor protein tyrosine phosphatase-mu-deficient mice

The transmembrane receptor-like protein tyrosine phosphatase-mu (RPTPmu) is thought to play an important role in cell-cell adhesion-mediated processes. We recently showed that RPTPmu is predominantly expressed in the endothelium of arteries and not in veins. Its involvement in the regulation of endothelial adherens junctions and its specific arterial expression suggest that RPTPmu plays a role in controlling arterial endothelial cell function and vascular tone. To test this hypothesis, we analyzed myogenic responsiveness, flow-induced dilation, and functional integrity of mesenteric resistance arteries from RPTPmu-deficient (RPTPmu(-/-)) mice and from wild-type littermates. Here, we show that cannulated mesenteric arteries from RPTPmu(-/-) mice display significantly decreased flow-induced dilation. In contrast, mechanical properties, myogenic responsiveness, responsiveness to the vasoconstrictors phenylephrine or U-46619, and responsiveness to the endothelium-dependent vasodilators methacholine or bradykinin were similar in both groups. Our results imply that RPTPmu is involved in the mechanotransduction or accessory signaling pathways that control shear stress responses in mesenteric resistance arteries.     

Late-gestation betamethasone enhances coronary artery responsiveness to angiotensin II in fetal sheep

Antenatal glucocorticoids are used to promote the maturation of fetuses at risk for preterm delivery. While perinatal glucocorticoid exposure has clear immediate benefits to cardiorespiratory function, there is emerging evidence of adverse long-term effects. To determine if antenatal betamethasone alters vascular reactivity, we examined isometric contraction of endothelium-intact coronary and mesenteric arteries isolated from twin fetal sheep at 121-124 days gestation (term being 145 days). One twin received betamethasone (10 microg/h iv) while the second twin received vehicle (0.9% NaCl) for 48 h immediately before the final physiological measurements and tissue harvesting. Fetuses that received betamethasone had higher mean arterial blood pressures than the saline-treated twin controls (53 +/- 1 vs. 48 +/- 1 mmHg, P < 0.05). Coronary vessels from betamethasone-treated fetuses exhibited enhanced peak responses to ANG II (72 +/- 17 vs. 23 +/- 6% of the maximal response to 120 mM KCl, P < 0.05). There was no significant difference in response of the coronary arteries to other vasoactive compounds [KCl, U-46619, sodium nitroprusside, 8-bromo-cGMP (8-BrcGMP), isoproterenol, and forskolin]. Contractile responses to ANG II were similar in betamethasone and control mesenteric arteries (48 +/- 17 vs. 36 +/- 12% of the maximal response to 10-6 M U-46619). Western blot analysis revealed AT1 receptor protein expression was increased by betamethasone in coronary but not in mesenteric arteries. These findings demonstrate that antenatal betamethasone exposure enhances coronary but not mesenteric artery vasoconstriction to ANG II by selectively upregulating coronary artery AT1 receptor protein expression.     

NAD(P)H oxidase-derived reactive oxygen species contribute to age-related impairments of endothelium-dependent dilation in rat soleus feed arteries

We tested the hypothesis that age-related endothelial dysfunction in rat soleus muscle feed arteries (SFA) is mediated in part by NAD(P)H oxidase-derived reactive oxygen species (ROS). SFA from young (4 mo) and old (24 mo) Fischer 344 rats were isolated and cannulated for examination of vasodilator responses to flow and acetylcholine (ACh) in the absence or presence of a superoxide anion (O(2)(-)) scavenger (Tempol; 100 μM) or an NAD(P)H oxidase inhibitor (apocynin; 100 μM). In the absence of inhibitors, flow- and ACh-induced dilations were attenuated in SFA from old rats compared with young rats. Tempol and apocynin improved flow- and ACh-induced dilation in SFA from old rats. In SFA from young rats, Tempol and apocynin had no effect on flow-induced dilation, and apocynin attenuated ACh-induced dilation. To determine the role of hydrogen peroxide (H(2)O(2)), dilator responses were assessed in the absence and presence of catalase (100 U/ml) or PEG-catalase (200 U/ml). Neither H(2)O(2) scavenger altered flow-induced dilation, whereas both H(2)O(2) scavengers blunted ACh-induced dilation in SFA from young rats. In old SFA, catalase improved flow-induced dilation whereas PEG-catalase improved ACh-induced dilation. Compared with young SFA, in response to exogenous H(2)O(2) and NADPH, old rats exhibited blunted dilation and constriction, respectively. Immunoblot analysis revealed that the NAD(P)H oxidase subunit gp91phox protein content was greater in old SFA compared with young. These results suggest that NAD(P)H oxidase-derived reactive oxygen species contribute to impaired endothelium-dependent dilation in old SFA.     

Role of Cu,Zn-SOD in the synthesis of endogenous vasodilator hydrogen peroxide during reactive hyperemia in mouse mesenteric microcirculation in vivo

We have recently demonstrated that endothelium-derived hydrogen peroxide (H2O2) is an endothelium-derived hyperpolarizing factor and that endothelial Cu/Zn-superoxide dismutase (SOD) plays an important role in the synthesis of endogenous H2O2 in both animals and humans. We examined whether SOD plays a role in the synthesis of endogenous H2O2 during in vivo reactive hyperemia (RH), an important regulatory mechanism. Mesenteric arterioles from wild-type and Cu,Zn-SOD(-/-) mice were continuously observed by a pencil-type charge-coupled device (CCD) intravital microscope during RH (reperfusion after 20 and 60 s of mesenteric artery occlusion) in the cyclooxygenase blockade under the following four conditions: control, catalase alone, N(G)-monomethyl-L-arginine (L-NMMA) alone, and L-NMMA + catalase. Vasodilatation during RH was significantly decreased by catalase or L-NMMA alone and was almost completely inhibited by L-NMMA + catalase in wild-type mice, whereas it was inhibited by L-NMMA and L-NMMA + catalase in the Cu,Zn-SOD(-/-) mice. RH-induced increase in blood flow after L-NMMA was significantly increased in the wild-type mice, whereas it was significantly reduced in the Cu,Zn-SOD(-/-) mice. In mesenteric arterioles of the Cu,Zn-SOD(-/-) mice, Tempol, an SOD mimetic, significantly increased the ACh-induced vasodilatation, and the enhancing effect of Tempol was decreased by catalase. Vascular H(2)O(2) production by fluorescent microscopy in mesenteric arterioles after RH was significantly increased in response to ACh in wild-type mice but markedly impaired in Cu,Zn-SOD(-/-) mice. Endothelial Cu,Zn-SOD plays an important role in the synthesis of endogenous H(2)O(2) that contributes to RH in mouse mesenteric smaller arterioles.     

Selective cerebral vascular dysfunction in Mn-SOD-deficient mice.

We tested the hypothesis that the mitochondrial form of superoxide dismutase [manganese superoxide dismutase (Mn-SOD)] protects the cerebral vasculature. Basilar arteries (baseline diameter approximately 140 microm) from mice were isolated, cannulated, and pressurized to measure vessel diameter. In arteries from C57BL/6 mice preconstricted with U-46619, acetylcholine (ACh; an endothelium-dependent vasodilator) produced dilation that was similar in male and female mice and abolished by an inhibitor of nitric oxide synthase. Vasodilation to ACh was not altered in heterozygous male or female Mn-SOD-deficient (Mn-SOD+/-) mice compared with wild-type littermate controls (Mn-SOD+/+). Constriction of the basilar artery to arginine vasopressin, but not KCl or U-46619, was increased in Mn-SOD+/- mice (P<0.05), and this effect was prevented by tempol, a scavenger of superoxide. We also examined responses of cerebral (pial) arterioles (branches of the middle cerebral artery, control diameter approximately 30 microm) to ACh in anesthetized mice using a cranial window. Responses to ACh, but not nitroprusside (an endothelium-independent agonist), were reduced (P<0.05) in cerebral arterioles in Mn-SOD+/- mice, and this effect was prevented by tempol. Thus these are the first data on the role of Mn-SOD in cerebral circulation. In the basilar artery, ACh produced nitric oxide-mediated dilation that was similar in male and female mice. Under normal conditions in cerebral arteries, responses to ACh were not altered but constrictor responses were selectively enhanced in Mn-SOD+/- mice. In the cerebral microcirculation, there was superoxide-mediated impairment of responses to ACh.     

Role of hydrogen peroxide in ACh-induced dilation of human submucosal intestinal microvessels

The endothelium plays an important role in maintaining vascular homeostasis by synthesizing and releasing several mediators of vasodilation, which include prostacyclin (PGI(2)), nitric oxide, and endothelium-derived hyperpolarizing factor (EDHF). We have recently defined the role of nitric oxide and PGI(2) in the dilation of submucosal intestinal arterioles from patients with normal bowel function. However, significant endothelium-dependent dilator capacity to ACh remained after inhibiting both these mediators. The current study was designed to examine the potential role of EDHF in human intestinal submucosal arterioles. ACh elicited endothelium-dependent relaxation in the presence of inhibitors of nitric oxide synthase and cyclooxygenase (23 +/- 10%, n = 6). This ACh-induced relaxation was inhibited and converted to constriction by catalase (-53 +/- 10%, n = 6) or KCl (-30 +/- 3%, n = 7), whereas 17-octadecynoic acid and 6-(2-propargylloxyphenyl) hexanoic acid, two inhibitors of cytochrome P450 monooxygenase, had no significant effect (3 +/- 1% and 20 +/- 8%, n = 5, respectively). Exogenous H(2)O(2) elicited dose-dependent relaxation of intact microvessels (52 +/- 10%, n = 7) but caused frank vasoconstriction in arterioles denuded of endothelium (-73 +/- 8%, n = 7). ACh markedly increased the dichlorofluorescein fluorescence in intact arterioles in the presence of nitric oxide synthase and cyclooxygenase inhibitors compared with control and compared with catalase-treated microvessels (363.6 +/- 49, 218.8 +/- 10.6, 221.9 +/- 27.9, respectively, P < 0.05 ANOVA, n = 5 arbitrary units). No changes in the dichlorofluorescein fluorescence were recorded in vessels treated with ACh alone. These results indicate that endothelial production of H(2)O(2) occurs in response to ACh in human gut mucosal arterioles but that H(2)O(2) is not an EDHF in this tissue. Rather, we speculate that it stimulates the release of a chemically distinct EDHF.     

H2O2 increases production of constrictor prostaglandins in smooth muscle leading to enhanced arteriolar tone in Type 2 diabetic mice

Our previous study showed that arteriolar tone is enhanced in Type 2 diabetes mellitus (T2-DM) due to an increased level of constrictor prostaglandins. We hypothesized that, in mice with T2-DM, hydrogen peroxide (H(2)O(2)) is involved in the increased synthesis of constrictor prostaglandins, hence enhanced basal tone in skeletal muscle arterioles. Isolated, pressurized gracilis muscle arterioles ( approximately 100 microm in diameter) of mice with T2-DM (C57BL/KsJ-db(-)/db(-)) exhibited greater basal tone to increases in intraluminal pressure (20-120 mmHg) than that of control vessels (at 80 mmHg, control: 25 +/- 5%; db/db: 34 +/- 4%, P < 0.05), which was reduced back to control level by catalase (db/db: 24 +/- 4%). Correspondingly, in carotid arteries of db/db mice, the level of dichlorofluorescein-detectable and catalase-sensitive H(2)O(2) was significantly greater. In control arterioles, exogenous H(2)O(2) (0.1-100 micromol/l) elicited dilations (maximum, 58 +/- 10%), whereas in arterioles of db/db mice H(2)O(2) caused constrictions (-28 +/- 8%), which were converted to dilations (maximum, 16 +/- 5%) by the thromboxane A(2)/prostaglandin H(2) (TP) receptor antagonist SQ-29548. In addition, arteriolar constrictions in response to the TP receptor agonist U-46619 were not different between the two groups of vessels. Endothelium denudation did not significantly affect basal tone and H(2)O(2)-induced arteriolar responses in either control or db/db mice. Also, in arterioles of db/db mice, but not in controls, 3-nitrotyrosine staining was detected in the endothelial layer of vessels. Thus we propose that, in mice with T2-DM, arteriolar production of H(2)O(2) is enhanced, which leads to increased synthesis of the constrictor prostaglandins thromboxane A(2)/prostaglandin H(2) in the smooth muscle cells, which enhance basal arteriolar tone. These alterations may contribute to disturbed regulation of skeletal muscle blood flow in Type 2 diabetes mellitus.     

Aging and estrogen alter endothelial reactivity to reactive oxygen species in coronary arterioles

Endothelium-dependent, nitric oxide (NO)-mediated vasodilation can be impaired by reactive oxygen species (ROS), and this deleterious effect of ROS on NO availability may increase with aging. Endothelial function declines rapidly after menopause, possibly because of loss of circulating estrogen and its antioxidant effects. The purpose of the current study was to determine the role of O(2)(-) and H(2)O(2) in regulating flow-induced dilation in coronary arterioles of young (6-mo) and aged (24-mo) intact, ovariectomized (OVX), or OVX + estrogen-treated (OVE) female Fischer 344 rats. Both aging and OVX reduced flow-induced NO production, whereas flow-induced H(2)O(2) production was not altered by age or estrogen status. Flow-induced vasodilation was evaluated before and after treatment with the superoxide dismutase (SOD) mimetic Tempol (100 μM) or the H(2)O(2) scavenger catalase (100 U/ml). Removal of H(2)O(2) with catalase reduced flow-induced dilation in all groups, whereas Tempol diminished vasodilation in intact and OVE, but not OVX, rats. Immunoblot analysis revealed elevated nitrotyrosine with aging and OVX. In young rats, OVX reduced SOD protein while OVE increased SOD in aged rats; catalase protein did not differ in any group. Collectively, these studies suggest that O(2)(-) and H(2)O(2) are critical components of flow-induced vasodilation in coronary arterioles from female rats; however, a chronic deficiency of O(2)(-) buffering by SOD contributes to impaired flow-induced dilation with aging and loss of estrogen. Furthermore, these data indicate that estrogen replacement restores O(2)(-) homeostasis and flow-induced dilation of coronary arterioles, even at an advanced age.     

Nitric oxide-mediated arteriolar dilation after endothelial deformation

Previously, we frequently observed dilation of arterioles after agonist-induced constrictions. We hypothesized that deformation of the endothelium during decreases in diameter of isolated arterioles elicits the release of nitric oxide (NO). In isolated arterioles of rat mesentery, phenylephrine (PE, 10(-7) M)-, U-46619 (10(-7) M)-, and KCl (50 mM)-induced constrictions were followed by potent dilations. Inhibition of NO synthase with N(omega)-nitro-L-arginine (L-NNA, 2 x 10(-4) M) or removal of the endothelium significantly enhanced constriction and reduced the postconstriction dilation. In the presence of 80 mmHg of intraluminal pressure, an increase in extraluminal pressure (P(e)) to 75 mmHg for 20 s and 1 and 2 min decreased vessel diameter. After release of P(e), arterioles dilated as a function of the duration of diameter reduction by P(e). Removal of the endothelium or administration of L-NNA significantly diminished the post-P(e) dilations. In cultured mesenteric arteriolar endothelial cells (EC), PE, U-46619, or KCl did not increase, whereas ACh did increase, the production of NO, as measured by a fluorometric assay for nitrite. Furthermore, when EC, cultured on a stretched silicone membrane, were subjected to deformation by shortening the membrane to 50% of its original length, NO release increased significantly. Based on all of the above, we propose that deformation of EC per se elicits release of NO, a mechanism that modulates arteriolar constriction.     

Leaky intra-acinar arteries in rat lungs perfused with hydrogen peroxide

We have investigated the effects of H2O2 (150 or 300 microM) on the ultrastructure and permeability of the pulmonary endothelium in rat lungs perfused for 60 min with buffered Hanks' bovine serum albumin medium. In one group of experiments, we examined the effect of H2O2 on the uptake and transport of cationized ferritin (CF) by endothelial cells in intra-acinar arteries, alveolar capillaries, and interlobular veins. The influence of the oxidant on endothelial adsorptive endocytic processes was assessed by measuring the density of ferritin particles in luminal vesicles, multivesicular bodies, and basal lamina. In a second group of experiments, we examined the effects of H2O2 on the fine structure and permeability to electron-dense macromolecules of arterial, microvascular, and venous endothelium. For this purpose, at the end of the 60-min perfusion with H2O2, CF was perfused to identify leaky vessels. We found that H2O2 caused a dose-dependent inhibition of transcytosis of CF in all vascular segments. At the lower dose of H2O2, inhibition of transcytotic activity was not associated with structural injury to the vascular endothelium or with elevation of wet-to-dry ratios. At the higher oxidant dose, inhibition of transcytosis was associated with leaky arterial endothelium and elevation of wet-to-dry ratios (6.44 +/- 0.12 vs. 5.64 +/- 0.16, P less than 0.02). The effects of H2)2 were prevented by adding catalase to the perfusate. The selective loss of structural integrity and leakiness of the arterial endothelium were diminished but not completely abolished by perfusing the oxidant retrograde from the venous side.(ABSTRACT TRUNCATED AT 250 WORDS)     

Aging enhances pressure-induced arterial superoxide formation

The purpose of this study was to investigate the mechanisms that regulate superoxide (O(2)(*-)) production as a function of an acute elevation of intravascular pressure and age. Mesenteric arteries isolated from young (6 mo) and aged (24 mo) male Fischer 344 rats were used. O(2)(*-) production in vessels in response to 80 (normal pressure, NP) and 180 (high pressure, HP) mmHg was determined by the superoxide dismutase-inhibitable nitroblue tetrazolium (NBT) reduction assay. In vessels exposed to NP, O(2)(*-) production was significantly higher in aged than in young vessels (32.7 +/- 7.0 vs. 15.4 +/- 2.4 nmol.mg(-1).30 min(-1)). HP enhanced O(2)(*-) production in vessels of both groups, but the enhancement was significantly greater in aged than in young vessels (63.4 +/- 6.7 vs. 32.7 +/- 4.3 nmol.mg(-1).30 min(-1)). Apocynin (100 micromol/l) attenuated HP-induced increases in O(2)(*-) production in both groups, whereas allopurinol (100 micromol/l) and N(omega)-nitro-L-arginine methyl ester (100 mumol/l) inhibited the response only in aged vessels. Confocal microscopy showed increases in O(2)(*-) in response to HP in endothelial and smooth muscle layers of both groups, with much greater fluorescent staining in aged than in young rats and in the endothelium than in smooth muscle cells. No significant changes in NAD(P)H oxidase gene and protein expressions were observed in vessels of the two groups. Upregulation of protein expression of xanthine oxidase was detected in aged vessels. We conclude that NAD(P)H oxidase contributes importantly to HP-induced enhanced O(2)(*-) production in vessels of both young and aged rats, whereas xanthine oxidase and nitric oxide synthase-dependent O(2)(*-) production also contribute to the enhancement in mesenteric arteries of aged rats.     

Arachidonic acid- and prostaglandin E2-induced cerebral vasodilation is mediated by carbon monoxide, independent of reactive oxygen species in piglets

Arachidonic acid (AA) and prostaglandin (PG) E(2) stimulate carbon monoxide (CO) production, and AA metabolism is known to be associated with the generation of reactive oxygen species (ROS). This study was conducted to address the hypothesis that CO and/or ROS mediate cerebrovascular dilation in newborn pigs. Experiments were performed on anesthetized newborn pigs with closed cranial windows. Different concentrations of AA (10(-8)-10(-6) M), PGE(2) (10(-8)-10(-6) M), iloprost (10(-8)-10(-6) M), and their vehicle (artificial cerebrospinal fluid) were given. Piglets with PGE(2) and iloprost received indomethacin (5 mg/kg iv) to inhibit cyclooxygenase. AA, PGE(2), and iloprost caused concentration-dependent increases in pial arteriolar diameter. The effects of both AA and PGE(2) in producing cerebral vascular dilation and associated CO production were blocked by the heme oxygenase inhibitor chromium mesoporphyrin (2 × 10(-5) M), but not by the prostacyclin analog, iloprost. ROS inhibitor tempol (SOD mimetic) (1 × 10(-5) M) and the H(2)O(2) scavenger catalase (1,000 U/ml) also do not block these vasodilator effects of AA and PGE(2). Heme-L-lysinate-induced cerebrovascular dilation and CO production was blocked by chromium mesoporphyrin. Hypoxanthine plus xanthine oxidase, a combination that is known to generate ROS, caused pial arteriolar dilation and CO production that was inhibited by tempol and catalase. These data suggest that AA- and PGE(2)-induced cerebral vascular dilation is mediated by CO, independent of ROS.     

Selective effect of tempol on renal medullary hemodynamics in spontaneously hypertensive rats

The present study assessed the short- and long-term effect of tempol, a membrane-permeable mimetic of superoxide dismutase, on renal medullary hemodynamics in spontaneously hypertensive rats (SHR). Tempol was given in the drinking water (1 mM) for 4 days or 7 wk (4-11 wk of age), and medullary blood flow (MBF) was measured over a wide range of renal arterial pressure by means of laser-Doppler flowmetry in anesthetized rats. In addition, the response of the medullary circulation to angiotensin II (5-50 ng x kg(-1) x min(-1) iv) was determined in SHR treated for 4 days with tempol. Compared with control SHR, short- and long-term treatment with tempol decreased arterial pressure by approximately 20 mmHg and increased MBF by 35-50% without altering total renal blood flow (RBF) or autoregulation of RBF. Angiotensin II decreased RBF and MBF dose dependently (approximately 30% at the highest dose) in control SHR. In SHR treated with tempol, angiotensin II decreased RBF (approximately 30% at the highest dose) but did not alter MBF significantly. These data indicate that the antihypertensive effect of short- and long-term administration of tempol in SHR is associated with a selective increase in MBF. Tempol also reduced the sensitivity of MBF to angiotensin II. Taken together, these data support the idea that tempol enhances vasodilator mechanisms of the medullary circulation, possibly by interacting with the nitric oxide system. Increased MBF and reduced sensitivity of MBF to angiotensin II may contribute to the antihypertensive action of tempol in SHR.     

Progesterone regulation of vascular thromboxane A(2) receptors in rhesus monkeys

We hypothesized that progesterone regulates thromboxane A(2) receptor (TxA(2)R) density in primate vascular muscle and that TxA(2)R density correlates with coronary reactivity in vivo and in vitro. Reactivity to serotonin + U-46619 was determined by angiography in surgically postmenopausal [ovariectomized (Ovx)] rhesus monkeys without progesterone replacement and after 2-wk progesterone treatment (1-2 ng/ml). In untreated Ovx animals, 100 micromol/l serotonin + 1 micromol/l U-46619 (syringe concentrations) provoked vasospasm-like constrictions in six of six monkeys; zero of six progesterone-treated monkeys developed vasospasms. Sustained Ca(2+) responses in vascular muscle cells isolated from Ovx coronaries (208 +/- 63% of basal 20 min after stimulation) treated with serotonin + U-46619 contrasted with transient Ca(2+) responses (143 +/- 18% of basal and decreasing 5 min after stimulation) in progesterone-treated monkeys. The maximum density of [1S-(1I,2J(5Z),3I(1E,3R*),4I)]-7-[3-(3-hydroxy-4-(4'-[(125)I]iodophenoxy)- 1-butenyl)-7-oxabicyclo[2.2.1]heptan-2-yl]-5-heptenoic acid ([(125)I]-BOP) binding was greater (P < 0.01) in carotid arteries and aortic membranes from Ovx (109 +/- 11 fmol/mg) compared with progesterone-treated (43 +/- 15 fmol/mg) monkeys. TxA(2)R immunolabeling revealed greater coronary TxA(2)R labeling in Ovx compared with progesterone-treated monkeys. The results suggest that progesterone can decrease arterial TxA(2)R in Ovx monkeys.