Influence of the renal endothelin A system on the autoregulation of renal hemodynamics in SHRs and WKY rats

In this study, we investigated the influence of a short-term blockade of the renal endothelin A system on the autoregulation of total renal blood flow, cortical renal blood flow, and pressure-dependent plasma renin activity in spontaneously hypertensive rats (SHRs) and normotensive controls [Wistar-Kyoto (WKY) rats]. In anesthetized rats, renal blood flow was measured by a transit-time flow probe and cortical blood flow by a laser flow probe. Blood samples were taken for measurement of plasma renin activity. Renal perfusion pressure was reduced in 5-mm Hg steps by means of a servocontrolled electropneumatic device by an inflatable suprarenal cuff. During the experiments, the rats (n = 6, each group) received an intrarenal infusion of either the selective endothelin A-receptor antagonist BQ123 (3 mg/kg/h) or vehicle. We observed an improvement of total and cortical blood flow autoregulation as indicated by a shift of lower limits of autoregulation to lower threshold pressures [103 +/- 2 vs. 132 +/- 4 mm Hg compared with 98 +/- 3 vs. 120 +/- 4 mm Hg (mean +/- SEM); p < 0.01 resp. p < 0.05] in BQ123-treated SHRs, whereas BQ123 had no influence on breakpoints of autoregulation in WKY rats (p > 0.05). Pressure-dependent plasma renin activity in SHRs was not influenced by BQ123. Renal blood flow autoregulation is improved in SHRs after short-term blockade of the renal endothelin A system. This effect is independent of the renin-angiotensin system. The endothelin A system does not seem to play an important role in the autoregulation of renal blood flow in normotensive WKY rats.     

ETA and ETB receptors on vascular smooth muscle cells from mesenteric vessels of spontaneously hypertensive rats

1. To investigate the contribution of ETA and ETB receptors, calcium responses to the ETB agonist, IRL-1620, to endothelin-1 (ET-1) and to the ETA antagonist, BQ-123, were examined in primary cultured unpassaged vascular smooth muscle cells (VSMC) from mesenteric vessels of 3, 9 and 17 week old spontaneously hypertensive rats (SHR), Wistar and Wistar-Kyoto (WKY) rats using Fura-2 methodology. 2. IRL-1620 (10(-7) mol/L) and ET-1 (10(-9) mol/L) increased [Ca2+]i in all strains and ages. Responses to ET-1 and IRL-1620 were blunted in 17 week SHR. BQ-123 significantly reduced ET-1-stimulated [Ca2+]i. In endothelium-denuded mesenteric vessels, ET-1 and IRL-1620 induced significant [Ca2+]i responses. 3. Binding of ET-1 was significantly lower in mesenteric artery membranes from 17 week SHR compared to controls. 4. Thus, ETA and ETB receptors are present in rat mesenteric VSMC. In adult SHR, a reduced density of ET receptors results in decreased responses to IRL-1620 and to ET-1.     

Effects of timolol and betaxolol on choroidal blood flow in the rabbit

Endothelium-dependent hyperpolarization of vascular smooth muscle cells (VSMCs) plays a crucial role in regulating vascular tone, especially in resistance vessels. It has been proposed that metabolites of arachidonic acid (AA), formed by cytochrome P-450 monooxygenase (P450), are endothelium-derived hyperpolarizing factors (EDHFs). These metabolites have been reported to mediate dilation to endogenous vasoactive compounds, such as bradykinin and acetylcholine. However, it is not known whether these metabolites of AA contribute to dilation of human resistance vessels. This is important since it has been proposed that EDHF serves as a compensatory mechanism to maintain dilation in disease states. Therefore, we studied the effect of AA on vessel diameter and VSMC membrane potential in isolated human coronary microvessels. Arterioles (81+/-5 microm, n=70) were dissected from right atrial appendages at the time of cardiac surgery and cannulated at a distending pressure of 60 mm Hg and zero flow. Changes in internal diameter were recorded with videomicroscopy. Some vessels were impaled with glass microelectrodes to measure membrane potential of VSMCs while internal diameters were simultaneously recorded. After constriction (47+/-2%) with endothelin-1, AA (10(-10)to 10(-5)mol/L) induced substantial dilation of human coronary microvessels, which was abolished by removal of the endothelium. Treatment with 17-octadecynoic acid (17-ODYA, 10(-5) mol/L; a P450 inhibitor) attenuated maximal dilation to AA (49+/-9% versus 91+/-4% [control]; P<0.05 versus control), whereas indomethacin (INDO, 10(-5) mol/L; a cyclooxygenase inhibitor) and N omega-nitro-L-arginine methyl ester (L-NAME, 10(-4) mol/L; a NO synthase inhibitor) were without effect. Both 17-ODYA and miconazole (10(-5) mol/L, a chemically distinct P450 inhibitor) further reduced the dilation to AA in the presence of INDO. The presence of 40 mmol/L KCl or charybdotoxin (10(-8) mol/L, a blocker of large-conductance Ca2+-activated K+ channels) impaired dilation to AA (19+/-9% [KCI] versus 76+/-5% [control] and 47+/-6% [charybdotoxin] versus 91+/-3% [control]; P<0.05 for both). After depolarization with endothelin-1 (-26+/-1 mV from -48+/-3 mV [before endothelin]), AA (10(-5)mol/L) in the presence of INDO and L-NAME induced hyperpolarization of VSMCs (-57+/-5 mV). In the presence of 17-ODYA together with INDO and L-NAME, endothelin produced similar depolarization (-26+/-2 mV from - 48+/- 3 mV), but hyperpolarization to AA was reduced (-33+/-2 mV; P<0.05 versus absence of 17-ODYA). AA metabolites formed primarily by P450 produce potent endothelium-dependent dilation of human coronary arterioles via opening of Ca2+-activated K+ channels and hyperpolarization of VSMCs. These findings support an important role for P450 metabolites in the regulation of human coronary arteriolar tone.     

Superoxide released to high intra-arteriolar pressure reduces nitric oxide-mediated shear stress- and agonist-induced dilations

It is thought that elevated levels of reactive oxygen metabolites contribute to the dysfunction of vascular endothelium in hypertension. We hypothesized that high intravascular pressure itself elicits production of superoxide, which then interferes with nitric oxide (NO)-mediated responses of arterioles. Thus, isolated arterioles (approximately 80 microm in diameter) from gracilis muscle of normotensive Wistar rats were cannulated and exposed to 140 mm Hg perfusion pressure for 30 minutes (in the absence of perfusate flow). After high intravascular pressure treatment, dilations to increases in perfusate flow (0 to 30 microL/min) were significantly reduced (from 39+/-2.2 to 19+/-2.1 microm at 30 microL/min), eliciting an increase in wall shear stress from approximately 20 to approximately 60 dyne/cm2. Nomega-nitro-L-arginine (10(-4) mol/L) did not affect, whereas indomethacin eliminated, flow-induced dilations after pressure treatment. In control, substance P (SP, 10(-9) to 5x10(-8) mol/L), sodium nitroprusside (SNP, 10(-8) to 10(-6) mol/L), and adenosine (ADO, 10(-6) to 5x10(-5) mol/L) elicited dilations (SP: 31.5+/-1.9 microm, SNP: 45.6+/-4 microm, and ADO: 37.2+/-4.1 microm, at maximum concentrations, respectively). After pressure treatment, maximum dilations to SP and SNP were significantly reduced (by 49% and 39%, respectively), whereas responses to ADO were not affected. Presence of superoxide dismutase (120 U/mL) and catalase (80 U/mL), but not catalase alone, in the perfusate solution prevented the reduction in dilation of arterioles to flow and agonists after pressure treatment by restoring NO mediation. We conclude that high intravascular pressure per se elicits the release of superoxide, which then interferes with NO, a mechanism that contributes to the elevation of wall shear stress and peripheral resistance in hypertension.     

Characterization of endothelium-dependent vasodilation and vasoconstriction in coronary arteries from spontaneously hypertensive rats

Coronary artery disease often occurs in patients with hypertension. The present study was designed to evaluate coronary vascular function in isolated coronary arteries of spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats and to determine the effect of antihypertensive treatment on coronary vascular responsiveness. Male SHR and WKY rats (12 to 14 weeks old) were divided into control and hydralazine-treated (120 mg/L drinking water for 10 days) groups. After 10 days, arterial pressure and heart rate were recorded while rats were conscious and unrestrained. Left ventricular coronary arteries (200 to 300 microns diameter) were isolated and intraluminal diameter was continuously recorded while vessels were maintained at a constant intraluminal pressure of 40 mm Hg. Relaxation of coronary arteries to both acetylcholine and nitroprusside was slightly, but significantly, enhanced in vessels from SHR compared to WKY rats. The enhanced relaxation was a specific effect, since isoproterenol induced similar relaxation in coronary arteries from SHR and WKY rats. Contraction to phenylephrine, but not endothelin-1, was augmented in coronary arteries from SHR compared to WKY rats. Treatment with hydralazine significantly lowered arterial pressure in SHR and WKY rats, but did not alter the enhanced contraction to phenylephrine or the enhanced relaxation to acetylcholine and nitroprusside in coronary arteris from SHR. These results indicate that coronary arteries of 12 to 14 week-old SHR do not have impaired endothelium-dependent relaxation, but to exhibit enhanced alpha-adrenoceptor-mediated contraction that is not reduced by lowering arterial pressure.     

EndothelinA receptor blockade improves nitric oxide-mediated vasodilation in monocrotaline-induced pulmonary hypertension

BACKGROUND: Nitric oxide (NO) and endothelin (ET) have been implicated in the pathogenesis of pulmonary hypertension (PH). Chronic ETA antagonist therapy reduces PH in monocrotaline (MCT)-treated rats. Interactions between the L-arginine-NO pathway and the ET system have been described. We therefore studied the effect of long-term treatment with an oral ETA antagonist (LU 135252) on NO-related vasodilation in isolated lungs from control rats and rats with MCT-induced PH. METHODS AND RESULTS: Three weeks after MCT injection, PH was associated with an increase in right ventricular pressure (from 27.4 +/- 0.9 to 66.6 +/- 4.1 mm Hg) and a decrease in endothelium-independent vasodilation in response to sodium nitroprusside (10(-10) to 10(-5) mol/L; delta Emax, from 11.1 +/- 0.9 to 2.7 +/- 0.3 mm Hg). Endothelium-dependent vasodilation in response to acetylcholine (10(-9) to 10(-4) mol/L) and the calcium ionophore A23187 (10(-9) to 10(-7) mol/L) remained unaffected. Treatment with LU 135252 did not significantly affect the endothelium-dependent and -independent vasodilations in control rats. However, in MCT-treated rats, LU 135252 therapy significantly reduced right ventricular pressure (39.7 +/- 2.1 mm Hg), potentiated acetylcholine-induced vasodilatation (delta Emax, from 1.6 +/- 0.2 to 3.7 +/- 0.4 mm Hg), and improved the responses to sodium nitroprusside (delta Emax, from 2.7 +/- 0.3 to 5.6 +/- 0.6 mm Hg). LU 135252 did not significantly alter the non-receptor-mediated endothelium-dependent vasodilation to A23187 or pulmonary constitutive NO synthase activity. CONCLUSIONS: MCT PH is associated with a reduced smooth muscle responsiveness to NO but a maintained endothelium-dependent vasodilatory potency. Long-term ETA antagonist therapy not only restores smooth muscle responsiveness to NO but also increases endothelium-dependent dilation in response to acetylcholine. This mechanism may contribute to the therapeutic benefit of ETA antagonists in PH.     

Transradial approach for coronary procedures: initial experience and results

To characterize the myogenic response during the development of hypertension, we evaluated the myogenic response of small arteries isolated from the cremaster muscle of spontaneously hypertensive rats (SHR) aged 4-5 and 7-8 weeks, as compared with age-matched Wistar-Kyoto rats (WKY), using an in vitro system. The myogenic response of SHR aged 7-8 weeks (but not those aged 4-5 weeks) significantly exceeded that of WKY. Measurement of intracellular levels of free calcium ([Ca2+]i) in small arteries of the 7-8-week-old SHR and WKY loaded with a calcium-sensitive dye showed that the increase in [Ca2+]i in SHR was significantly greater than that in WKY during the myogenic response. The inhibitory effects of nitrendipine on the increased myogenic response and [Ca2+]i were greater in SHR. Thus, the myogenic response was enhanced in SHR and may be explained in part by an increase in Ca2+ entry through the voltage-dependent calcium channel (VDCC). The myogenic response and Ca2+ entry through the VDCC may be increased in association with the elevation of blood pressure.     

Central pathology review in clinical trials for patients with malignant glioma. A Report of Radiation Therapy Oncology Group 83-02

We previously described delayed pressor response (DPR) 3 h after endothelin (ET)-1 injection in normotensive rats. In the current study, we examined effects of the ETA receptor antagonist BQ123 (0.01 mumol/kg/min intravenously, i.v.), phosphoramidon (100 mumol/kg i.v.), the neutral endopeptidase inhibitor SQ28603 (112 mumol/kg + 0.04 mumol/kg/min i.v.), the angiotensin-converting enzyme inhibitor enalaprilat (10 mumol/kg i.v.), and the thromboxane receptor antagonist, SQ29548 (0.5 mumol/kg + 0.5 mumol/kg/h i.v.) on DPR. Vehicle and ET-1 (1.0 nmol/kg i.v.) were administered on day 1; vehicle or drug and ET-1 were administered on day 2. BQ123 inhibited DPR 36% (vehicle 44 +/- 5, BQ123 28 +/- 3 mm Hg); phosphoramidon inhibited DPR 56% (vehicle 45 +/- 4, and phosphoramidon 20 +/- 5 mm Hg). DPR was unchanged after SQ28603 (vehicle 39 +/- 2 and SQ28603 44 +/- 2 mm Hg), enalaprilat (vehicle 39 +/- 2 and enalaprilat 38 +/- 7 mm Hg), or SQ29548 (vehicle 46 +/- 6 and SQ29548 43 +/- 3 mm Hg). The results suggest that DPR 3 h after ET-1 injection in rats is mediated in part through ETA receptors. DPR does not appear to involve thromboxane or synthesis of angiotensin II (AII), but may be related to synthesis of ET-1.     

Regulation of capillary perfusion by small arterioles is spatially organized

To explore a mechanism for spatial recruitment of capillaries, this study determined whether the arterioles controlling capillary perfusion, which typically arise as sequential branches along a transverse arteriole, could respond differently from each other in situ in a spatially ordered way. Diameter changes were measured for these arterioles at a known location in the intact microvasculature in the cremaster muscle of anesthetized Golden hamsters (N = 67); each arteriole controls separate capillary groups. These arterioles all had the same concentration dependence to locally (by micropipette) applied norepinephrine (NE, 10(-9) to 10(-3) mol/L), and 10(-9) mol/L NE did not induce diameter changes when applied locally to individual vessels. However, 10(-9) mol/L NE added to the tissue superfusate, or 5% added superfusate oxygen (also locally subthreshold), each induced significant diameter changes (both constrictions and dilations), in different branches, that were presumably due to summation of individually subthreshold events that changed the prevailing conditions at the point of observation. These significant diameter changes were related to the maximal diameter or to initial tone of the branches, but these changes occurred in different ways for NE versus oxygen. With NE, the branch arterioles that constricted (versus dilated) were significantly larger (maximal diameter, 22.3 +/- 2.6 versus 15.9 +/- 2.1 microns) and had higher tone (fractional constriction, 0.53 +/- 0.05 versus 0.63 +/- 0.05); with oxygen, those that constricted were the same size as those that dilated (maximal diameter, 28.6 +/- 1.1 versus 30.5 +/- 2.7 microns), but constrictors had lower tone (fractional constriction, 0.49 +/- 0.04 versus 0.39 +/- 0.06).(ABSTRACT TRUNCATED AT 250 WORDS)     

Vasoconstrictor responses of coronary resistance arteries in exercise-trained pigs

Coronary resistance arteries isolated from exercise-trained pigs have been shown to exhibit enhanced myogenic reactivity (J. M. Muller, P. R. Myers, and M. Harold Laughlin. J. Appl. Physiol. 75: 2677-2682, 1993). The purpose of this study was to test the hypothesis that exercise training results in enhanced vasoconstrictor responses of these arteries to all vasoconstrictor stimuli [specifically acetylcholine (ACh), endothelin-1 (ET-1), KCl, and the Ca2+ channel-agonist Bay K 8644]. Female Yucatan miniature swine were trained (Trn) on a motor-driven treadmill (n = 16) or remained sedentary (Sed, n = 15) for 16-20 wk. Arteries 50-120 micron in diameter were isolated and cannulated with micropipettes, and intraluminal pressure was set at 60 cmH2O throughout experiments. Vasoreactivity was evaluated by examining constrictor responses to increasing concentrations of ACh (10(-9) to 10(-4) M), ET-1 (10(-10) to 10(-8) M), KCl (bath replacement with isotonic physiological saline solution containing 30 or 80 mM), and Bay K 8644 (10(-9) to 10(-6) M). Constricted diameters are expressed relative to the passive diameter observed after 100 microM SNP. All four constrictors produced similar decreases in diameter in arteries from both groups [ACh: 0.52 +/- 0.07 (Trn) and 0.54 +/- 0,06 (Sed); ET-1: 0.66 +/- 0.05 (Trn) and 0.70 +/- 0.07 (Sed); KCl: 0.66 +/- 0.05 (Trn) and 0.70 +/- 0.07 (Sed); Bay K 8644: 0.86 +/- 0.05 (Trn) and 0. 76 +/- 0.05 (Sed)]. Present results combined with previous observations indicate that exercise training does not alter vasoconstrictor responses of porcine coronary resistance arteries but specifically increases myogenic reactivity. Thus the underlying cellular mechanisms for myogenic tone are altered by training but not receptor-mediated mechanisms (ACh and ET-1) nor voltage-gated Ca2+ channels (KCl and Bay K 8644) in coronary resistance arteries.     

Endothelin-1 does not mediate the endothelium-dependent hypoxic contractions of small pulmonary arteries in rats

Various pulmonary artery preparations in vitro demonstrate sustained endothelium-dependent contractions upon hypoxia. To determine whether endothelin-1 could mediate this phenomenon, we examined the effect of bosentan, a new antagonist of both the ETA and ETB subtypes of the endothelin receptor. Small (300 pm) pulmonary arteries from rats were mounted on a myograph, precontracted with prostaglandin F2 alpha and exposed to hypoxia (PO2, 10 to 15 mm Hg, measured on-line) for 45 min. Endothelium-intact control rings exhibited a biphasic response, with a transient initial vasoconstriction (phase 1) followed by a second slowly developing sustained contraction (phase 2). Expressed in percent of the maximal response to 80 mmol/L KCl, the amplitudes of phase 1 (peak tension) and 2 (tension after 45 min of hypoxia) averaged 37 +/- 12% and 17 +/- 14%, respectively (n = 11). In endothelium-denuded rings, phase 1 persisted while the amplitude of phase 2 was reduced to 2 +/- 12% (p < 0.05, n = 8), showing the endothelium dependence of this contraction. Neither phase was significantly decreased in rings treated with 10(-5) mmol/L bosentan (38 +/- 15% and 17 +/- 12%, respectively, n = 6). The PO2 threshold for onset of hypoxic contraction was not significantly different among these three groups and averaged 32 +/- 24 mm Hg. In a separate experiment, we assessed the inhibitory effect of 10(-5) mol/L bosentan on the response to 10(-8) mol/L endothelin-I. Rings treated for 45 min with 10(-8) mol/L endothelin-1 alone exhibited a maximal contraction of 75 +/- 27% (n = 6). This was reduced to 4 +/- 17% (p < 0.01, n = 6) in rings treated with both 10(-8) mol/L endothelin-1 and 10(-5) mol/L bosentan. We conclude that complete blockade of all endothelin receptor subtypes has no effect on either endothelium-dependent or -independent hypoxic contractions in this preparation. This suggests that endothelial factors other than endothelin-I mediate the acute hypoxic contractions of small pulmonary arteries in the rat.     

Role of nitric oxide in regulation of brain stem circulation during hypotension

We tested the hypothesis that nitric oxide (NO) plays a role in CBF autoregulation in the brain stem during hypotension. In anesthetized rats, local CBF to the brain stem was determined with laser-Doppler flowmetry, and diameters of the basilar artery and its branches were measured through an open cranial window during stepwise hemorrhagic hypotension. During topical application of 10(-5) mol/L and 10(-4) mol/L N(omega)-nitro-L-arginine (L-NNA), a nonselective inhibitor of nitric oxide synthase (NOS), CBF started to decrease at higher steps of mean arterial blood pressure in proportion to the concentration of L-NNA in stepwise hypotension (45 to 60 mm Hg in the 10(-5) mol/L and 60 to 75 mm Hg in the 10(-4) mol/L L-NNA group versus 30 to 45 mm Hg in the control group). Dilator response of the basilar artery to severe hypotension was significantly attenuated by topical application of L-NNA (maximum dilatation at 30 mm Hg: 16 +/- 8% in the 10(-5) mol/L and 12 +/- 5% in the 10(-4) mol/L L-NNA group versus 34 +/- 4% in the control group), but that of the branches was similar between the control and L-NNA groups. Topical application of 10(-5) mol/L 7-nitro indazole, a selective inhibitor of neuronal NOS, did not affect changes in CBF or vessel diameter through the entire pressure range. Thus, endothelial but not neuronal NO seems to take part in the regulation of CBF to the the brain stem during hypotension around the lower limits of CBF autoregulation. The role of NO in mediating dilatation in response to hypotension appears to be greater in large arteries than in small ones.     

ATP-sensitive potassium channels in the basilar artery during chronic hypertension

We examined the hypothesis that dilatation of the basilar artery in response to activation of ATP-sensitive potassium channels is impaired in stroke-prone spontaneously hypertensive rats (SHRSP). Changes in basilar artery diameter in response to aprikalim, a direct activator of ATP-sensitive potassium channels, were measured in anesthetized SHRSP and normotensive Wistar-Kyoto (WKY) rats through a cranial window. Topical application of aprikalim increased basilar artery diameter in WKY rats. Glibenclamide, a selective inhibitor of ATP-sensitive potassium channels, abolished aprikalim-induced vasodilatation. Thus, ATP-sensitive potassium channels are functional in the basilar artery of WKY rats in vivo. Aprikalim (10(-6) mol/L) dilated the basilar artery by 31 +/- 5% (mean +/- SEM) in WKY rats but only 5 +/- 1% in SHRSP. The concentration-response curve to aprikalim in SHRSP was significantly shifted to the right, but the response to the highest concentration of aprikalim (10(-5.5) mol/L) was similar in SHRSP and WKY rats. Vasodilatation in response to norepinephrine was also impaired in SHRSP. Dilator responses of the basilar artery to forskolin, a direct activator of adenylate cyclase, and nitroprusside, a direct activator of guanylate cyclase, were normal in SHRSP. The findings suggest that dilatation of the basilar artery in response to direct activation of ATP-sensitive potassium channels is impaired in SHRSP compared with WKY rats in vivo.     

Microtiter plate immunoassay for the evaluation of platelet adhesion to fibronectin

This study shows the effects of a selective endothelin ET(B) receptor agonist, IRL 1720 {Ac-[Ala11,15]endothelin-1-(8-21)}, on cardiovascular responses in anesthetized spontaneously hypertensive rats and Wistar-Kyoto rats. Single intravenous bolus injection of IRL 1720 caused a dose-related short-lasting fall in blood pressure, left ventricular pressure and myocardial contractility. However, repeated intravenous bolus injection of 10(-5) mol/kg IRL 1720 produced a biphasic response consisting of an initial short-lasting decrease followed by a sustained increase in these parameters. The initial decrease was reduced, whereas the following increase was enhanced with the repeated injections of IRL 1720. The cardiovascular pressor response was not inhibited by the endothelin ET(A) receptor antagonist, FR139317 ((R)2-[(R)-2-[(S)-2-[[1-(hexahydro-1H-azepinyl)]carbonyl] amino-4-+methylpentanoyl] amino-3-[3-(1-methyl-1H-indolyl)]propionyl]amino -3- (2-pyridyl)propionic acid). The effects of IRL 1720 were qualitatively similar but more potent in spontaneously hypertensive rats than in Wistar-Kyoto rats. These results suggest the existence of two types of endothelin ET(B) receptor for IRL 1720: a tachyphylactic endothelin ET(B) receptor that mediates cardiovascular depressor responses and a less tachyphylactic endothelin ET(B) receptor that mediates pressor responses in the rat.     

Endothelin-1 does not contribute to ischemia/reperfusion-induced vasoconstriction in skeletal muscle

The experiment reported was designed to investigate whether endothelin-1 (ET-1) contributes to vasospasm and poor perfusion during the reperfusion after prolonged ischemia in skeletal muscle. Male Sprague-Dawley rats weighting 100 to 120 g were anesthetized with Nembutal. The vascular isolated rat cremaster muscle, coupled with local interarterial infusion, was the model used in this study. The diameters of feeding arterioles and terminal arterioles were measured utilizing intravital microscopy. The number of terminal arterioles with temporary cessation of flow were counted in each cremaster. Group 1: ET-dose response (8 rats)--various concentrations of ET-1 (from 10(-8) M to 10(-5) M) were infused into the cremaster to test whether this muscle was responsive to the agent in a dose-dependent manner. Group 2: ET-antagonist response (12 rats)--PD-142893, 10(-4) M (ETab receptor antagonist) plus ET-1 10(-7) M were infused into the cremaster to test whether vasospasm caused by exogenous ET-1 could be prevented by pretreatment with this specific ETab receptor antagonist. Group 3: ischemia/reperfusion response (12 rats)--PD-142893, 10(-4) M was infused into the cremaster before ischemia (4 hr warm ischemia) and during reperfusion to test whether ETab receptor antagonism was effective in preventing the vasospasm associated with ischemia/reperfusion injury. The results from this study show that a mixed ETab endothelin antagonist, PD-142893, infused before ischemia and during reperfusion at a dose which virtually abolished the vasoconstriction produced by a high concentration of exogenous endothelin-1, had no effect on ischemia/reperfusion-induced vasoconstriction in this model. These results suggest that ET-1 probably does not contribute to the ischemia/reperfusion-induced vasoconstriction and poor reflow in rat skeletal muscle.     

11 beta-Hydroxysteroid dehydrogenase activity in mesenteric arteries of spontaneously hypertensive rats

1. 11 beta-Hydroxysteroid dehydrogenase (11-HSD) activity in mesenteric arteries of spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats was determined and expressed as the percentage conversion of [3H]-corticosterone to [3H]-11-dehydrocorticosterone. 2. 11-HSD activity was significantly decreased in mesenteric arteries of both 4 and 9 week old SHR (8.4 +/- 0.8%, 5.0 +/- 1.5%, respectively) compared with WKY rats (12.4 +/- 0.6%, 15.8 +/- 0.7%, respectively; P < 0.05). 3. Total RNA from rat vascular smooth muscle cells (VSMC) and endothelial cells (EC) were prepared with selective precipitation in 3 mol/L LiCl/6 mol/L urea. The expression of 11-HSD mRNA was confirmed in the rat VSMC but its mRNA expression was not detected in EC, using northern blot analysis. 4. The results in this study indicate that 11-HSD in the vascular wall may play a role in the pathogenesis of hypertension in SHR.     

Immunological effects of an arginine side chain contaminating synthetically prepared peptides

We examined the effect of a nitric oxide (NO) quencher, stroma-free human hemoglobin A (HbA0; 0.01, 0.05, 0.1, 0.2 g/kg), on the blood flow measured using the Doppler flow technique, tumor oxygen pressure (pO2) and the diameter of the arterioles using R3230Ac mammary adenocarcinoma as the tumor model. In female Fischer 344 rats with 1-cm-diameter tumors implanted in the lateral aspect of the left quadriceps, intravenous infusion of 0.1 and 0.2 g/kg HbA0 decreased both central tumor and peripheral tumor blood flow by 20-30% (P < 0.05). Tumor pO2 decreased 28% with 0.2 g/kg HbA0, from 15 mm Hg (baseline) to 11 mm Hg at 10 min (P = 0.02). Although 0.2 g/kg HbA0 increased blood flow 55% in the left quadriceps muscle proximal to the implanted tumor (P < 0.05), HbA0 had little effect on blood flow in right quadriceps muscle with no tumor implanted, and increased right quadriceps pO2, from 21 mm Hg (baseline) to 23 mm Hg at 10 min (P = 0.03). HbA0 increased mean arterial pressure 5-10% in a manner that was dependent on dose while heart rate concurrently decreased 9-19%. The diameter of the arterioles supplying the tumor was rapidly reduced 10% by 0.2 g/kg HbA0 (P = 0.037) and remained stable through 60 min of observation (P = 0.005). HbA0 selectively reduces tumor blood flow and tumor pO2 through vasoconstriction of the arterioles supplying the tumor. Vascular NO quenching provides an alternative to NO synthase inhibition as a means to achieve the goal of selective tumor hypoxia.     

Role of nitric oxide in the regulation of vascular tone in pressurized and perfused resistance myometrial arteries from term pregnant women

OBJECTIVE: Our purpose was to evaluate flow-induced responses, myogenic tone, and norepinephrine-induced constriction in myometrial resistance arteries from normal term pregnant women and the role that nitric oxide and prostanoids may play in these responses. STUDY DESIGN: Arteries (approximately 200 microns, n = 14, at 40 mm Hg) were dissected from myometrial biopsy specimens from women undergoing cesarean section and then were mounted in a pressure arteriograph. Responses to intraluminal flow, pressure, and a constrictor agonist (norepinephrine 10(-6) mol/L) were studied in the absence and presence of N omega-nitro-L-arginine methyl ester (n = 7) or indomethacin (n = 5). Myogenic and norepinephrine-induced tone were calculated after the determination of artery diameter in the absence of extracellular calcium. RESULTS: Arteries developed myogenic tone (80 mm Hg) that was not modulated by nitric oxide or prostanoid release, whereas norepinephrine-induced tone was significantly enhanced by the nitric oxide inhibitor. An increase in intraluminal flow led to dilatation in physiologic salt solution and indomethacin, but to constriction in the presence of N omega-nitro-L-arginine methyl ester (percent increase in diameter at flow rate of 184.6 microliters/min, 24% +/- 8% in physiologic salt solution and 20% +/- 4% in the presence of indomethacin versus -27% +/- 12% in N omega-nitro-L-arginine methyl ester alone and -21% +/- 10% in indomethacin and N omega-nitro-L-arginine methyl ester, respectively, analysis of variance, p < 0.05). CONCLUSIONS: Flow-induced shear stress is a physiologic modulator of vascular tone in myometrial arteries from pregnant women. Nitric oxide, but not prostanoids, mediates this response and also blunts norepinephrine constriction. Nitric oxide may play a fundamental role in the maintenance of adequate blood supply to the fetus during human pregnancy.     

Endothelin converting enzyme (ECE) activity in human vascular smooth muscle

1. We have characterized the human smooth muscle endothelin converting enzyme (ECE) present in the media of the endothelium-denuded human umbilical vein preparation. 2. Endothelin-1 (ET-1) and ET-2 were potent constrictors of umbilical vein with EC50 values of 9.2 nM and 29.6 nM, respectively. ET-1 was at least 30 times more potent than ET-3 suggesting the presence of constrictor ETA receptors. Little or no response was obtained to the ETB-selective agonist sarafotoxin 6c. These data suggest that endothelin-mediated vasoconstriction is via ETA receptors in this preparation. 3. Autoradiographical visualization of endothelin receptors with subtype selective ligands confirmed the predominance of the ETA receptor in the media of umbilical vein. High density of binding was obtained with the ETA selective [125I]-PD151242, with much lower levels detected with the ETB selective [125I]-BQ3020. 4. Big ET-1 (EC50 = 42.7 nM) and big ET-2(1-38) (EC50 = 99.0 nM) were less potent than ET-1 and ET-2, respectively. Big ET-2(1-38) was more potent than its isoform big ET-2(1-37) with concentration-response curves to big ET-2(1-37) incomplete at 300 nM. No response was obtained to big ET-3 at concentrations up to 700 nM. The C-terminal fragments, big ET-1(22-38) and big ET-2(22-38) were inactive. 5. Responses to ET-1 were unaffected by either the neutral endopeptidase (NEP) inhibitor thiorphan (10(-5) M) or by the dual NEP/ECE inhibitor phosphoramidon (10(-5) M). Big ET-1 was also unaffected by thiorphan but antagonized in a concentration-dependent manner by phosphoramidon (10(-5) M and 10(-4) M). 6. Addition of all four big endothelin peptides to human umbilical vein preparations resulted in detectable amounts of ET-IR in the bathing medium. Therefore, although big ET-3 was functionally inactive this reflects the low potency of ET-3 at the ETA receptor rather than the lack of ability of this smooth muscle ECE to convert big ET-3 to ET-3. 7. To conclude we have demonstrated the presence of a phosphoramidon-sensitive ECE on the smooth muscle layer of the human umbilical vein which can convert big ET-1, big ET-2(1-37), big ET-2(1-38) and big ET-3 to their mature biologically active forms. The precise subcellular localization of this enzyme and its physiological relevance remains to be determined.     

Altered microvascular responses to C5a in rat striated muscle during two-kidney, one clip renovascular hypertension

OBJECTIVE: To determine how two-kidney, one clip (2-K,1C) renovascular hypertension alters microvascular responses in rat striated muscle to complement C5a, one of the most important inflammatory mediators. METHODS: 2-K,1C hypertension was induced in male Sprague-Dawley rats. Under anesthesia with pentobarbital (50 mg/kg, intraperitoneally) the cremaster muscle microcirculatory preparation with intact neurovascular connections was studied in vivo by closed-circuit videomicroscopy. Recombinant human C5a was applied topically in the tissue bath at concentrations of 10(-12), 10(-10) and 10(-8) mol/l, consecutively. Changes in the microvessel diameters in small arterioles, large arterioles and venules were measured. RESULTS: In normotensive rats complement C5a induces a significant dilation in small arterioles at low bath concentrations (10(-12) or 10(-10) mol/l), but the dilation is attenuated at a higher concentration (10(-8) mol/l). In contrast, in 2-K,1C hypertensive rats C5a constricts small arterioles at low concentrations (< 10(-10) mol/l) but dilates them at a higher concentration (10(-8) mol/l). Large arterioles and venules have minimal responses to C5a in either normotensive or 2-K,1C hypertensive rats. CONCLUSION: 2-K,1C hypertension dramatically alters C5a-induced microvascular responses in small arterioles. The alteration might be attributable to the enhanced vasoconstrictor mechanisms and impaired vasodilator mechanisms during 2-K,1C renovascular hypertension.