Adenosine- and hypoxia-induced dilation of human coronary resistance arteries: evidence against the involvement of K(ATP) channels

1. The ATP-sensitive potassium (K(ATP)) channel may be an important mediator of metabolic dilation in the human coronary circulation. As adenosine and hypoxia are considered to be major mediators of metabolic dilation in the coronary circulation, we investigated the effect of glibenclamide (a K(ATP) channel blocker) on adenosine and hypoxic dilation of human coronary resistance arteries, with myogenic tone, in vitro. 2. Vessels were dissected from the atrial appendage from consenting patients and studied in vitro using a pressure arteriograph system. Segments of coronary resistance artery were pressurized to 60 mmHg and the vessels studied developed spontaneous myogenic tone. 3. The K(ATP) opener pinacidil (final conc. 5 x 10(-6) M) resulted in dilation, which was completely reversed by 5 x 10(-6) glibenclamide (84+/-14 vs -10+/-9%, pinacidil and pinacidil plus glibenclamide, respectively, P=0.009, n=5). 4. Adenosine (final conc. 10(-5) M) resulted in dilation, glibenclamide (5 x 10(-6) and 10(-5) M) was without effect (118+/-12 vs 104+/-16% adenosine and adenosine plus 10(-5) glibenclamide, respectively, n.s., n=4). 5. Hypoxia (8+/-3 mmHg O2) resulted in a dilation that reversed when normoxic conditions were restored (60+/-9 vs 3+/-11% hypoxia and post-hypoxia, respectively, P=0.014, n=3). The hypoxic dilation was not affected by glibenclamide (63+/-14 vs 55+/-6% hypoxia and hypoxia plus glibenclamide, respectively, n.s., n=4). In a further series of experiments, vessels were incubated with glibenclamide (5 x 10(-6)) prior to a hypoxic challenge; again, glibenclamide was without effect on the hypoxic dilation (-0.008+/-2 vs 95+/-3% glibenclamide and glibenclamide plus hypoxia, respectively, P=0.0005, n=3). 6. These data demonstrate that glibenclamide is without effect on both adenosine and hypoxic dilation of human coronary resistance arteries with myogenic tone, from the right atrial appendage in vitro. Our findings suggest that the K(ATP) channel is unlikely to be a major mediator of metabolic dilation in these arteries.     

High NaCl intake decreases both flow-induced dilation and pressure-induced myogenic tone in resistance arteries from normotensive rats: involvement of cyclooxygenase-2

The effect of high NaCl diet on resistance arteries is not yet fully documented. In order to assess the effect of NaCl on myogenic tone and flow-induced dilation independent of arterial blood pressure change, we used normotensive rats which did not develop hypertension upon high NaCl intake. Normotensive Wistar Kyoto Rats received a high (8%) or a normal NaCl diet (0.4%). Mesenteric resistance arteries (150 microm, internal diameter) were cannulated in an arteriograph to allow perfusion of arteries under controlled pressure and flow. Pressure-induced myogenic tone was lower in the high NaCl group than in the control group. Cyclooxygenase inhibition with indomethacin and (N-(2-cyclohexyloxy)-4-nitro-phenyl)-methanesulphonamide, 1 micromol/l) NS 398 (specific cyclooxygenase-2 inhibitor) similarly decreased myogenic tone in rats fed high NaCl but had no effect in those fed a normal NaCl diet. Flow-induced dilation was decreased in the high NaCl group. Inhibition of nitric oxide synthesis with N(G)-nitro-L-arginine methyl ester decreased flow-induced dilation in both groups. Indomethacin and NS 398 did not change flow-induced dilation. As shown by immunofluorescence COX-2 was present in the endothelium of arteries from rats with a high NaCl diet but not in those fed a normal NaCl diet. Thus, chronic high NaCl intake decreased both flow-induced dilation and myogenic tone in resistance arteries. The chronic high NaCl did not affect the participation of nitric oxide on flow-induced dilation, but induced the expression of cyclooxygenase-2, which participates in myogenic tone. These results suggest that high NaCl changes flow and pressure mechanosensing processes and strengthen the hypothesis that sodium ions have an important role in both pressure and flow-mechanotransduction in vascular cells.     

Tissue angiotensin II and endothelin-1 modulate differently the response to flow in mesenteric resistance arteries of normotensive and spontaneously hypertensive rats

In resistance arteries pressure-induced (myogenic) tone (MT) and flow (shear stress)-induced dilation (FD) are potent determinant of vascular resistance. We investigated the role of angiotensin II and endothelin-1 in FD and MT in resistance arteries and their potential change in hypertension. Flow - diameter - pressure relationship was established in situ, under anaesthesia, in two daughter branches of a mesenteric resistance artery (180 microM, n=7 per group) from spontaneously hypertensive (SHR) or normotensive (WKY) rats. One artery was ligated distally, so that it was submitted to pressure only, while the other was submitted to pressure and flow. Drugs were added to the preparation and external diameter, pressure and flow measured continuously. External diameter (with flow) ranged from 150+/-3 to 191+/-7 microM in WKY (n=28) rats and from 168+/-6 to 186+/-6 microM in SHR (n=28). Flow induced a dilation of the non-ligated arteries which was lower in SHR (13+/-5 - 31+/-4 microM vs WKY: 5+/-5 - 44+/-4 microM). In the ligated artery, the diameter did not significantly change, due to MT. In the vessels submitted to flow angiotensin converting enzyme inhibition (perindopril, 10 micromol L(-1)) increased the diameter in SHR (+11+/-2 microM) significantly more than in WKY (+2+/-1 microM). Angiotensin type 1 receptor (AT(1)R) blockade (losartan, 10 micromol L(-1)) increased the diameter in the vessels with flow in SHR only (+6+/-1 microM). Angiotensin type 2 receptor (AT(2)R) blockade (PD 123319, 1 micromol L(-1)) decreased arterial diameter in WKY only (9+/-2). Endothelin-1 type A receptor (ET(A)R) blockade (LU135252, 0.1 micromol L(-1)) increased the diameter only in SHR in the artery submitted to flow (by 6+/-1 microM). Thus FD was counteracted by a flow-dependent AT(1) and ET(A) receptors-activation in SHR whereas in WKY FD AT(2)-dependent dilation is involved.     

Enhanced release of endothelium-derived hyperpolarizing factor in small coronary arteries from rats with congestive heart failure

1. Previous studies have suggested that the production of nitric oxide (NO) is reduced in coronary vessels of animals with congestive heart failure (CHF). However, the response to endothelium-derived hyperpolarizing factor (EDHF) in small coronary resistance arteries from CHF rats has not been investigated. The aim of the present study was to determine whether flow-induced dilation (FID) is altered in small coronary arteries from CHF rats and to characterize the role of EDHF in this process. 2. Small coronary arteries (97 +/- 6 microm) were isolated from control rats and from rats in which CHF was induced by left coronary artery ligation. The arteries were cannulated at 60 mmHg with flow. Changes in internal diameter were examined using videomicroscopy. 3. There was no significant difference in FID in small coronary arteries between control and CHF rats (68 +/- 6 vs 61 +/- 4% (expressed as a percentage of maximal dilation induced by nitroprusside (%MaxD(NP))), respectively). Flow-induced dilation in control rat vessels showed greater attenuation by N(G)-monomethyl-L-arginine (L-NMMA) than vessels from CHF rats (%NO-mediated FID 32 +/- 5 vs 16 +/- 3% (%MaxD(NP)), respectively). Pretreatment with indomethacin had no significant effect on FID in vessels from either rat group. Flow-induced dilation was attenuated by KCl (40 mmol/L) to a greater degree in vessels from CHF rats in the presence of L-NMMA and indomethacin compared with vessels from control rats (%EDHF-mediated FID: 36 +/- 4 vs 25 +/- 5% (%MaxD(NP)), respectively). Flow-induced dilation was abolished by removal of the endothelium and was significantly decreased in vessels from CHF rats in response to charybdotoxin plus apamin or tetrabutylammonium compared with control rat vessels. 17-Octadecynoic acid had no significant effect on FID in vessels from either control or CHF rats. 4. In conclusion, the FID of small coronary arteries is mediated by K+ channels, including the K(Ca) channels. Endothelium-derived hyperpolarizing factor-mediated dilation may compensate for the loss of NO-mediated dilation in CHF.     

Flow-dependent, endothelium-mediated dilation of epicardial coronary arteries in conscious dogs: effects of cyclooxygenase inhibition

Endothelial damage or removal abolishes the dilation of epicardial coronary arteries induced by increments in flow through these arteries in vitro. Therefore, we tested whether or not the release of a cyclooxygenase product from endothelial cells in vivo is the mechanism of this flow-dependent dilation. In eight conscious dogs, instrumented to register the external diameter of two epicardial branches--anterior descending and circumflex--of the left coronary artery, increments in coronary flow increased and reductions in coronary flow decreased the diameter of the left circumflex epicardial artery by 182 +/- 11 micron/100% change in flow. When mean coronary flow in one epicardial branch was kept constant by a distal, flow-limiting stenosis during the application of flow-augmenting stimuli (temporal coronary occlusion or 80-400 micrograms/kg adenosine i.v.), no dilation of this artery was observed. Cyclooxygenase inhibition (suppressing the bradykinin-induced elevation of plasma 6-keto-PGF1 alpha) by indomethacin (5 mg/kg) or by diclofenac (10 mg/kg) increased smooth muscle tone in both epicardial arteries, but did not modify the flow-diameter relation (181 +/- 10 and 179 +/- 9 microns/100% change in flow, respectively). It is concluded that a tonic, instantaneous influence of coronary flow on the smooth muscle tone of the epicardial coronary arteries exists in vivo. It is unlikely that prostacyclin or another prostanoid is a mediator of this endothelium-mediated influence of flow on smooth muscle tone.     

The influence of chronic inhibition of nitric oxide synthesis on contractile and relaxant properties of rat carotid and mesenteric arteries

Balloon denudation of the rat carotid artery leads to an immediate decrease in beta-adrenoceptor-medi-ated vasodilator response. However, this arterial function becomes significantly enhanced during subsequent formation of neointima with the endothelial cell lining still being absent. It was therefore hypothesized that chronic suppression of endothelium-dependent nitric oxide (NO) synthesis may eventually upregulate the beta-adrenoceptor system on vascular smooth muscle. To investigate this hypothesis, male Wistar rats were treated chronically with the L-arginine analogue NG-nitro-L-arginine methyl ester (L-NAME) to inhibit the synthesis of NO (i.e. 15 mg/kg per day or 0.06 mmol/kg per day for 6 weeks p.o.). Prior to the experiments the mean arterial blood pressure (MAP) was significantly elevated in the L-NAME-treated rats (i.e. 128.4+/-3.4 mmHg vs. 100.0+/-2.9 mmHg, L-NAME vs. control, n=4, P<0.05). The functional properties of the isolated vessel preparations were established by isometric force measurement in a myograph setup, in the absence of L-NAME. The maximal contractile responses to high potassium chloride solution (100 mM), to the alpha1-adrenoceptor agonist phenylephrine, and to the thromboxane A2-agonist U46619, were not influenced by chronic L-NAME-treatment in the carotid and mesenteric artery preparations. The vasodilator responses to the cholinergic agonist methacholine were significantly impaired in the carotid arteries of L-NAME-treated animals: 30.9+/-7.9% vs. 64.6+/-2.0%, P<0.05, L-NAME vs. control, n=10. However, these responses appeared not to be modulated in the mesenteric artery preparations after chronic L-NAME treatment. Separate experiments showed that these responses could be blocked both in the rat carotid and mesenteric artery with L-NAME (10 mM) in vitro. Addition of the NO synthase substrate L-arginine could partially but significantly reverse this blockade. Chronic inhibition of NO synthesis caused significant deterioration of beta-adrenoceptor-mediated vasodilator responses. For carotid arteries: Emax=36.1+/-9.4% vs. 65.9+/-6.0%, P<0.05, L-NAME vs. control, n=5; and pD2=6.7+/-0.2 and 7.4+/-0.1, respectively, P<0.05, n=5. For mesenteric arteries: pD2=7.7+/-0.0 and 8.0+/-0.0, respectively, P<0.05, n=5. From these data, it may be concluded that chronic L-NAME treatment results in a stable impairment of the endothelium-dependent NO system in the rat carotid but not mesenteric arteries. The stated hypothesis fails as the beta-adrenoceptor-induced vasorelaxation of carotid and mesenteric arteries became significantly impaired, rather than enhanced. Taken together, the beta-adrenoceptor function in the rat carotid artery is apparently more dependent on endothelial NO synthesis than that in the rat mesenteric artery.     

Interacting roles of nitric oxide and ATP in the pulmonary circulation of the rat.

1. The potentiating effect of NG-nitro-L-arginine methyl ester, (L-NAME) a nitric oxide synthesis inhibitor, on responses of the rat pulmonary vascular pressure (PVP) to purinoceptor agonists was examined. 2. At a constant flow of 23 ml min-1 the PVP was 22.4 +/- 2.5 mmHg (n = 15), and treatment with 100 microM L-NAME for 15 min was without effect on the PVP. After the tone was raised with 28 nmol 9,11-dideoxy-11 alpha, 9 alpha-epoxymethano-prostaglandin F2 alpha (U-46619), the PVP was 29.4 +/- 3.3 mmHg and treatment with 100 microM L-NAME was still without effect on the PVP. It appears that there is a graded release of nitric oxide in response to different levels of steady shear stress and in our experimental model the threshold for detection was not reached under basal conditions. 3. In contrast, when the circulation was challenged with 30 s step, additive increases in flow between 11 and 50 ml min-1 (n = 8), treatment with 100 microM L-NAME produced a significant (P < 0.05) increase in PVP suggesting that changes in flow-derived forces evoke the release of nitric oxide. This was evident for flow rates above 30 ml min-1. 4. In preparations in which tone was raised with U-46619, a dose of 1 x 10(-8) mol ATP or 2-meSATP evoked a drop in PVP while alpha,beta-meATP produced an increase in PVP under constant flow of 23 ml min-1. After treatment with 100 microM L-NAME, all three purinoceptor agonists evoked an increase in PVP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of the renal action of pranidipine in the rat

Although calcium antagonists elicit predominant dilation of afferent arterioles that might be associated with glomerular hypertension, there have been reported diverse observations demonstrating the effect of calcium antagonists on the progression of renal injury. The present study examined the effect of pranidipine (CAS 99522-79-9) on the renal microvascular tone in the isolated perfused hydronephrotic rat kidney, and the progression of renal insufficiency in subtotally nephrectomized spontaneously hypertensive rats. In the hydronephrotic kidney, angiotensin II caused marked constriction of both afferent and efferent arterioles. The subsequent addition of pranidipine (10 nmol/l, 100 nmol/l, 1 mumol/l) elicited dose-dependent afferent arteriolar dilation, with 97 +/- 3% reversal at 1 mumol/l. In contrast, efferent arterioles were resistant to pranidipine, with only 20 +/- 4% reversal at 1 mumol/l. In subtotally nephrectomized rats, 10-week treatment with pranidipine (3.0 mg/kg/day) markedly decreased blood pressure (from 270 +/- 6 to 158 +/- 8 mmHg) and improved renal histopathological changes, including glomerular and arteriolar sclerosis. Proteinuria was also less than than in the control rats (233 +/- 5 vs. 305 +/- 26 mg/day). Thus, although glomerular hypertension might develop as a consequence of preferential afferent arteriolar dilation, pranidipine actually improved the renal injury in subtotally nephrectomized SHR. These ostensibly discrepant observations could be attributed to the simultaneous reduction in blood pressure and the salutary actions of this agent mediated by non-hemodynamic mechanisms.     

Loss of endothelial KATP channel-dependent, NO-mediated dilation of endocardial resistance coronary arteries in pigs with left ventricular hypertrophy

The influence of left ventricular hypertrophy (LVH) on the endothelial function of resistance endocardial arteries is not well established. The aim of this study was to characterise the mechanisms responsible for UK-14,304 (alpha(2)-adrenoreceptor agonist)-induced endothelium-dependent dilation in pig endocardial arteries isolated from hearts with or without LVH. LVH was induced by aortic banding 2 months before determining endothelial function. Following euthanasia, hearts were harvested and endocardial resistance arteries were isolated and pressurised to 100 mmHg in no-flow conditions. Vessels were preconstricted with acetylcholine (ACh) or high external K(+) (40 mmol l(-1) KCl). Results are expressed as mean+/-s.e.m. UK-14,304 induced a maximal dilation representing 79+/-6% (n=8) of the maximal diameter. NO synthase (l-NNA, 10 micromol l(-1), n=7) or guanylate cyclase (ODQ, 10 micromol l(-1), n=4) inhibition reduced (P<0.05) UK-14,304-dependent dilation to 35+/-6 and 18+/-7%, respectively. Apamin and charybdotoxin reduced (P<0.05) to 39+/-8% (n=4) the dilation induced by UK-14,304. In depolarised conditions, however, this dilation was prevented (P<0.05). UK-14,304-induced dilation was reduced (P<0.05) by glibenclamide (Glib, 1 micromol l(-1)), a K(ATP) channel blocker, either alone (35+/-10%, n=5) or in combination with l-NNA (34+/-9%, n=4). In LVH, UK-14,304-induced maximal dilation was markedly reduced (25+/-4%, P<0.05) compared to control; it was insensitive to l-NNA (21+/-5%) but prevented either by the combination of l-NNA, apamin and charybdotoxin, or by 40 mmol l(-1) KCl. Activation of endothelial alpha(2)-adrenoreceptor induces an endothelium-dependent dilation of pig endocardial resistance arteries. This dilation is in part dependent on NO, the release of which appears to be dependent on the activation of endothelial K(ATP) channels. This mechanism is blunted in LVH, leading to a profound reduction in UK-14,304-dependent dilation.     

The effects of N omega-nitro-L-arginine methyl ester, sodium nitroprusside and noradrenaline on venous return in the anaesthetized cat.

1. The vascular actions of N omega-nitro-L-arginine methyl ester (L-NAME), sodium nitroprusside and noradrenaline were investigated in cats under chloralose anaesthesia with controlled vascular tone and ventilation. Cardiac output, heart rate, vascular pressures and mean circulatory filling pressure (MCFP) were measured. Total peripheral resistance (TPR) and resistance to venous return (Rvr) were calculated from steady-state readings. 2. L-NAME (37 mumol kg-1, i.v.) administered to ten cats receiving noradrenaline (6 nmol kg-1 min-1, i.v.) increased aortic pressure by 47.5 +/- 7.1 mmHg from 106 mmHg, and MCFP by 1.56 +/- 0.36 mmHg from 10.0 mmHg (means +/- s.e. means). Mean changes in portal venous pressure, RAP and heart rate were not significant. Cardiac output fell by 29.7 +/- 3.3% from 130 ml min-1 kg-1. TPR rose by 108 +/- 7.2% from 796 mmHg l-1 min kg and Rvr by 58.4 +/- 4.5% from 64 mmHg l-1 min kg. 3. Infusion of sodium nitroprusside into cats receiving noradrenaline evoked dose-related falls in aortic pressure, MCFP, TPR and Rvr. Changes in portal venous pressure, RAP and heart rate were not significant and cardiac output fell slightly. After L-NAME, sensitivity to nitroprusside was increased by 139 +/- 34% for MCFP, 176 +/- 19% for TPR and 351 +/- 39% for Rvr, and cardiac output rose slightly. The nitroprusside infusion required to restore TPR after L-NAME was estimated to be 5.8 x 10(+/- 0.41) nmol kg-1 min-1, which was approximately three times more than that required to restore MCFP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanism of butyrate-induced vasorelaxation of rat mesenteric resistance artery.

1. The vasorelaxant effect of the sodium salt of the short chain fatty acid, butyrate, on preconstricted rat small mesenteric arteries (mean inner diameter approximately 300 microns) was characterized. Isometric force development was measured with a myograph, and intracellular pH (pHi) was simultaneously monitored, in arteries loaded with the fluorescent dye BCECF in its acetomethoxy form. Sodium butyrate (substituted isosmotically for NaCl) was applied to arteries after noradrenaline (NA) or high K+ contractures were established. 2. Arteries preconstricted with a concentration of NA inducing an approximately half maximal contraction were relaxed by 91.5 +/- 6.3% by 50 mmol l-1 butyrate. This concentration of butyrate did not, however, cause a significant relaxation of contractures to a maximal (5 mumol l-1) NA concentration, and also failed to relax significantly contractures stimulated by high (45 and 90 mmol l-1) K+ solutions. Contractures elicited with a combination of NA (at a submaximal concentration) and 45 mmol l-1 K+ were, however, markedly relaxed by butyrate. 3. Investigation of the concentration-dependency of the butyrate-induced relaxation of the half maximal NA response revealed an EC50 for butyrate of approximately 22 mmol l-1. 4. Sodium butyrate (50 mmol l-1) caused pHi to decrease from 7.25 +/- 0.02 to 6.89 +/- 0.08 (n = 4, P < 0.001). However, the vasorelaxant effect of butyrate on the submaximal NA contracture was not significantly modified when this fall in intracellular pH was prevented by the simultaneous application of NH4Cl. 5. Butyrate-induced relaxation was also unaffected by endothelial denudation and inhibition of NO synthase with N omega-nitro-L-arginine methyl ester (100 mumol l-1). 6. The relaxation of the NA contracture by 50 mmol l-1 sodium butyrate was abolished in arteries pretreated with the cyclic AMP antagonist Rp-cAMPS (25 mumol l-1). 7. We conclude that the butyrate-induced relaxation of the NA contracture is independent of intracellular acidification. The ability of Rp-cAMPS to abolish the butyrate relaxation indicates that stimulation of the cyclic AMP second messenger system may play an important role in mediating this effect.     

Upregulated function of phosphatidylinositol-3-kinase in genetically hypertensive rats: a moderator of arterial hypercontractility

1. The growth enzyme phosphatidylinositol 3-kinase (PI3K) was recently implicated in the mediation of arterial spontaneous tone, an event observed in arteries from hypertensive, but not normotensive, subjects that contributes to changes in total peripheral resistance in the hypertensive state. We have shown this occurrence in experimentally induced models of hypertension. However, because the majority of hypertension is genetically based, it is important to demonstrate a similar change in genetically hypertensive animals. 2. Aorta from spontaneously hypertensive rats (SHR; systolic blood pressure = 183 +/- 4 mmHg) and Wistar Kyoto (WKY) rats (115 +/- 2 mmHg) were isolated for the measurement of isometric contractile force. Aorta from SHR displayed small increases (approximately 5% maximum phenylephrine (PE)-induced contraction) in spontaneous tone, whereas aorta from WKY rats displayed none. The non-selective PI3K inhibitor LY294002 (20 micromol/L) and the selective inhibitor of the p110delta catalytic subunit of PI3K IC87114 (20 micromol/L) caused a fall of basal tone in SHR aorta (20 +/- 7 and 24 +/- 6% of the initial PE contraction, respectively), but did not alter tone in arteries from WKY rats. LY294002, but not IC87114, normalized the increased potency of noradrenaline (NA) observed in aorta from SHR (-log EC50 values for NA in the presence of vehicle in WKY rats and SHR 7.5 +/- 0.1 and 7.8 +/- 0.1, respectively (P < 0.05); -log EC(50) values for NA in the presence of LY294002 in WKY rats and SHR 7.0 +/- 0.1 and 7.0 +/- 0.1, respectively). 3. Biochemical expression of the p110 catalytic and p85 regulator subunits of PI3K in western analyses revealed no difference in expression of the regulatory p85alpha or p110alpha protein subunits between WKY rats and SHR; p110gamma was not detected. In contrast, p110delta expression was increased greater than 30% in aorta from SHR compared with WKY rats (827.6 +/- 88.5 vs 576.8 +/- 53.4 arbitrary densitometry units, respectively). Immunohistochemical analyses revealed expression of the p110delta isoform in the smooth muscle of arteries. 4. These data underscore the relevance of an enzyme historically classified as one committed to growth/anti-apoptosis in modifying contractility and supports involvement of PI3K in genetically based hypertension.     

Mesenteric arterial function in the rat in pregnancy: role of sympathetic and sensory-motor perivascular nerves, endothelium, smooth muscle, nitric oxide and prostaglandins.

1. The effects of pregnancy on mesenteric arterial function were examined in constantly perfused (5 ml min-1) mesenteric arterial beds isolated from 21-day pregnant rats. The function of sympathetic and sensory-motor perivascular nerves, endothelium and smooth muscle was examined. The role of nitric oxide and prostaglandins in vasoconstrictor function was tested by use of NG-nitro-L-arginine methyl ester (L-NAME; 100 microM) and indomethacin (10 microM), respectively. 2. Electrical field stimulation (EFS; 4-32 Hz, 1 ms, 90V, 30s) at basal tone elicited frequency-dependent vasoconstriction which was markedly reduced in preparations from pregnant rats at all frequencies. Vasoconstrictor responses to vasopressin and endothelin were also reduced in pregnancy and there was a trend towards a reduction in maximal responses to noradrenaline (NA). In contrast, there was no difference in vasoconstrictor responses to ATP, 5-hydroxytryptamine (5-HT) or angiotension II. 3. L-NAME (100 microM) augmented responses to EFS, NA, ATP and vasopressin in control mesenteric arterial preparations. In contrast, L-NAME augmented responses only to EFS in pregnancy, having no significant effect on responses to NA, ATP and vasopressin. 4. Indomethacin (10 microM) attenuated responses to NA and vasopressin, but not to EFS, in controls and in pregnancy. Responses to ATP were attenuated by indomethacin in controls but not in pregnancy. 5. Mesenteric preparations from pregnant rats were resistant to having tone raised by continuous perfusion with methoxamine. Despite an approximately 10 fold greater concentration of methoxamine, there was a significantly smaller increase in tone in preparations from pregnant, 34.27 +/- 4.8 mmHg (n = 11) compared to control, 65.92 +/- 5.4 mmHg (n = 11), rats. EFS (4-12 Hz, 60 V, 0.1 ms, 30s) in the presence of guanethidine (5 microM) to block sympathetic neurotransmission elicited frequency-dependent vasodilatation due to activation of sensory-motor nerves. Percentage relaxations were similar in preparations from pregnant and non-pregnant rats. 6. Dose-dependent endothelium-dependent vasodilatations to acetylcholine and ATP were similar in preparations from pregnant and non-pregnant rats. Endothelium-independent vasodilatation to sodium nitroprusside and to calcitonin gene-related peptide were also similar between the two groups. 7. There was no significant difference in the basal perfusion pressure of mesenteric arterial beds from control (21.3 +/- 1.0 mmHg, n = 24) and pregnant (20.2 +/- 1.2 mmHg, n = 23) rats. However, a step-wise increase in perfusate flow from 5 to 10, 15, 20 and 24ml min-1 produced smaller increases in perfusion pressure in pregnancy compared to the controls. L-NAME (100 microM) or indomethacin (10 microM) had no significant effect on the relationship between flow and perfusion pressure. 8. The present results show that prejunctional changes are involved in blunted sympathetic vasoconstriction of rat mesenteric arteries in pregnancy. Non-specific postjunctional changes are implicated in the reduced constrictor responses to applied methoxamine, vasopressin and endothelin, but not to ATP. In contrast, sensory-motor nerves and endothelium-dependent and -independent vasodilatation was unchanged. The decrease in receptor-mediated mesenteric arterial constrictor responsiveness in pregnancy does not appear to be due to acute modulation by NO or prostaglandins, but may involve changes in the distensibility of the bed and/or changes in wall thickness.     

Effects of NG-nitro-L-arginine methyl ester on regional haemodynamic responses to MgSO4 in conscious rats.

1. We assessed regional haemodynamic responses to the vasodilator, MgSO4, in the absence and presence of the nitric oxide synthase inhibitor, NG-nitro-L-arginine methyl ester (L-NAME), in conscious chronically instrumented Long Evans rats (n = 9). 2. MgSO4 (loading dose 220 mumol kg-1 min-1 for 7 min, maintenance dose 56 mumol kg-1 min-1 for 7 min), alone, caused slight bradycardia and hypotension accompanied by reductions in renal and mesenteric flows, but a marked hyperaemic vasodilatation in the hindquarters (flow, delta 54 +/- 6%, vascular conductance, delta 77 +/- 5%). 3. L-NAME (183 nmol kg-1 min-1) caused hypertension (29 +/- 2 mmHg) accompanied by bradycardia (-51 +/- 6 beats min-1) and reductions in flow and vascular conductance in the renal (-18 +/- 4% and -35 +/- 3%, respectively), mesenteric (-35 +/- 3% and -49 +/- 3%, respectively), and hindquarters (-26 +/- 3% and -42 +/- 3%, respectively) vascular beds. In the presence of L-NAME, the hypotensive and bradycardic effects of MgSO4 were still apparent, but its hindquarters hyperaemic vasodilator effect was significantly attenuated. 4. In order to determine if the inhibitory action of L-NAME on the hindquarters hyperaemic vasodilator action of MgSO4 was a non-specific effect, due to the change in baseline conditions caused by L-NAME, we also examined responses to MgSO4 in the presence of endothelin-1 (12.5 pmol kg-1 min-1) or angiotensin II (50 pmol kg-1 min-1). In the presence of either peptide, the overall effects of MgSO4 on hindquarters flow and vascular conductance were unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of mibefradil and nifedipine on arteriolar myogenic responsiveness and intracellular Ca(2+)

1. Ca(2+) entry mechanisms underlying spontaneous arteriolar tone and acute myogenic reactivity remain uncertain. These studies aimed to compare the effects of nifedipine and the putative T-channel blocker, mibefradil, on arteriolar myogenic responsiveness and intracellular Ca(2+) (Ca(2+)(i)). 2. First order cremaster muscle arterioles (1A) were isolated from rats, cannulated, pressurized to 70 mmHg in the absence of intraluminal flow, and mechanical responses studied by video microscopy. The Ca(2+)(i) was measured using fluorescence imaging of Fura 2 loaded arterioles. 3. Both nifedipine and mibefradil showed dose-dependent inhibition of spontaneous myogenic tone (at 70 mmHg; pEC(50) 7.04+/-0.17 vs 6.65+/-0.20 respectively, n=6 for both, n.s.) and KCl-induced vasoconstriction (at 70 mmHg; pEC(50) 6.93+/-0. 38 vs 6.45+/-0.27 respectively, n=6 for both, n.s.). 4. In arterioles maintained at 50 mmHg, nifedipine (10(-7) and 10(-5) M) caused a concentration dependent reduction in Ca(2+)(i), however, mibefradil (10(-7) and 10(-5) M) had no effect. Furthermore nifedipine significantly attenuated the increase in Ca(2+)(i) associated with an acute pressure step (50 - 120 mmHg) whereas mibefradil was considerably less effective. 5. Mibefradil (10(-7) M) significantly attenuated contractile responses to 60 mM KCl without altering the KCl-induced increase in Ca(2+)(i), in contrast to nifedipine (10(-7) M) which reduced both Ca(2+)(i) and contraction. 6. Membrane potential of arterioles with spontaneous myogenic tone (70 mmHg) was -41.5+/-1. 0 mV. Nifedipine (10(-7) or 10(-5) M) had no effect on membrane potential, however mibefradil (10(-5) M) caused significant depolarization. 7. In summary, both mibefradil and nifedipine inhibit arteriolar spontaneous tone and acute myogenic reactivity. While there may be overlap in the mechanisms by which these agents inhibit tone, differences in effects on membrane potential and intracellular Ca(2+) levels suggest mibefradil exhibits actions other than blockade of Ca(2+) entry in skeletal muscle arterioles.     

Pressure modulates the reactivity of isolated rabbit epicardial arteries

1 The magnitude of responses to prostaglandin F_2α (PGF_2α) and adenosine (ADO) in pressurized rabbit epicardial coronary arteries (367 ± 44 μm o.d.) with intact endothelium was assessed when they developed spontaneous tone. 2 The arteries were cannulated and changes in arterial diameter registered with an automated video perfusion system. The vessels, in the stabilization period at 60 mmHg, were divided into two groups, one exposed to a longitudinal stretch of +20% of unstretched initial length (Lo), and the other to +35% Lo, which developed spontaneous tone. In both cases, diameter–pressure curves were obtained by changing the intravascular pressure from 30 to 120 mmHg in aleatory steps of 30 mmHg (dp/dt = 15 mmHg s^?1). 3 Diameter reduction of the arteries with stretch of +35% Lo in response to 1 μm PGF_2α was greater than that with stretch of +20% Lo. Likewise, increase of the arteries that had +35% Lo in response to 1 μm ADO was greater than with +20% Lo. 4 Intravascular flow (40 μl min^?1) increased the tone level of arteries. The addition of PGF_2α enhanced this tone which was similar to that obtained with a stretch of +35% Lo and no flow, whereas the effect of ADO was increased. 5 These data indicate that the vasomotor responses of PGF_2α and ADO are modulated by the degree of longitudinal stretch in epicardial arteries.     

Depletion of intracellular Ca2+ stores enhances flow-induced vascular dilatation in rat small mesenteric artery

The effect of depleting intracellular Ca2+ stores on flow-induced vascular dilatation and the mechanism responsible for the vasodilatation were examined in rat isolated small mesenteric arteries. The arteries were pressurized to 50 mmHg and preconstricted with phenylephrine. Intraluminal flow reversed the effect of phenylephrine, resulting in vasodilatation. Flow dilatation consisted of an initial transient peak followed by a sustained plateau phase. The magnitude of dilatation was markedly reduced by removing Ca2+ from the intraluminal flow medium.Depletion of intracellular Ca2+ stores with either cyclopiazonic acid (CPA, 2 microM) or 1,4-dihydroxy-2,5-di-tert-butylbenzene (BHQ, 10 microM) significantly augmented the magnitude of flow dilatation. Flow-induced endothelial cell Ca2+ influx was also markedly enhanced in arteries pretreated with CPA or BHQ.Flow-induced dilatation was insensitive to Nw-nitro-L-arginine methyl ester (100 microM) plus indomethacin (3 microM) or to oxyhemoglobin (3 microM), but was markedly reduced by 30 mM extracellular K+ or 2 mM tetrabutylammonium (TBA), suggesting an involvement of EDHF. Catalase at 1200 U ml-1 abolished the flow-induced dilatation, while the application of exogenous H2O2 (90-220 microM) induced relaxation in phenylephrine-preconstricted arteries. Relaxation to exogenous H2O2 was blocked in the presence of 30 mM extracellular K+, and H2O2 (90 microM) hyperpolarized the smooth muscle cells, indicating that H2O2 can act as an EDHF. In conclusion, flow-induced dilatation in rat mesenteric arteries can be markedly enhanced by prior depletion of intracellular Ca2+ stores. Furthermore, these data are consistent with a role for H2O2 as the vasodilator involved.     

Relaxation induced by histamine is not endothelium dependent in tail arteries of L-NAME-treated rats

The present study was carried out to evaluate the relaxation induced by histamine in tail arteries of rats after chronic inhibition of nitric oxide (NO) synthesis with the inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) compared to tail arteries of control rats. The maximum relaxation induced by histamine was greater in control (88.09% +/-5.50, n=6) than in L-NAME arteries (47.33% +/-6.40, n=6), although pD(2) values were not different between the two groups (control: 4.89+/-0.08; L-NAME: 4.81+/-0.10). After incubation with 100 microM L-NAME in vitro, the maximum relaxation induced by histamine was only reduced in the control arteries (44.93% +/-2.35, n=6), whereas it had no effect on aortas of rats pretreated with this inhibitor. The incubation with 100 microM L-NAME had the same effect as endothelium removal in both arterial groups. Furthermore, the relaxation induced by histamine was unaffected by indomethacin. The combination of L-NAME and the histamine antagonist cimetidine completely abolished the relaxation induced by histamine in both arterial groups. These results show that when NO synthesis is impaired, the relaxation induced by histamine is endothelium independent, and when NO-synthase is active, the relaxation involves both NO released from endothelial cells and an endothelium-independent mechanism that is sensitive to cimetidine.     

Sumatriptan elicits both constriction and dilation in human and bovine brain intracortical arterioles

1. Little is known about serotonin (5-HT) receptors present on brain microvessels that are innervated by brainstem serotonergic neurons. Using 5-HT, sumatriptan and subtype selective 5-HT(1) receptor agonists and/or the 5-HT(1) receptor antagonist GR127935, we characterized the 5-HT receptors involved in regulating microvascular tone of pressurized intracortical arterioles (approximately 40--50 microm) isolated from human and bovine cerebral cortex. The role of nitric oxide (NO) on these responses was assessed with the N(omega)-nitro-L-arginine (L-NNA, 10(-5) M), an inhibitor of NO synthesis. Bovine pial arteries were studied for comparative purposes. 2. At spontaneous tone, 5-HT induced a dose-dependent constriction of human and bovine microarteries (respective pD(2) values of 7.3+/-0.2 and 6.9+/-0.1); a response potently inhibited by GR127935 (pIC(50) value of 8.5+/-0.1) in bovine microvessels. 3. In both species, the 5-HT(1) receptor agonist sumatriptan induced a biphasic response consisting of a small but significant dilation at low concentrations (1 and/or 10 nM) followed by a constriction at higher doses (pD(2) for contraction of 6.9+/-0.1 and 6.6+/-0.2 in human and bovine vessels, respectively). Pre-incubation with L-NNA abolished the sumatriptan-induced dilation and significantly shifted the dose-response of the constriction curve to the left. In contrast, the selective 5-HT(1D) (PNU-109291) and 5-HT(1F) (LY344864) receptor agonists were devoid of any vasomotor effect. 4. In bovine pial vessels, 5-HT and sumatriptan elicited potent constrictions (respective pD(2) of 7.2+/-0.1 and 6.6+/-0.1), a weak dilation being occasionally observed at low sumatriptan concentrations. 5. A significant negative correlation was observed between pial and intracortical vessels diameter and the extent of the dilatory response to 10(-9) M sumatriptan. Together, these results indicate that sumatriptan, most likely via activation of distinctly localized microvascular 5-HT(1B) receptors, can induce a constriction and/or a dilation which is sensitive to inhibition of NO synthesis and dependent on the size and, possibly, the existing tone of the vessels.     

Differential effects of L-arginine on the inhibition by NG-nitro-L-arginine methyl ester of basal and agonist-stimulated EDRF activity.

1. An isolated, buffer-perfused rabbit ear preparation was used to investigate the influence of NG-nitro-L-arginine methyl ester (L-NAME) on endothelium-dependent vasodiltation and modulation of vasoconstrictor responses and vascular conductance. 2. Acetylcholine (0.55 pmol-1.6 nmol) caused dose-related vasodilatation of preparations constricted by the combination of 5-hydroxytryptamine and histamine (both 1 microM), with an ED50 = 31.1 +/- 7.8 pmol and a maximum dilatation of 69.9 +/- 4.3%. In the presence of 10 microM L-NAME the dose-response for vasodilator effects was shifted significantly (P less than 0.001) to the right (ED50 = 3.07 +/- 1.18 nmol) and there was a significant (P less than 0.01) depression of the maximum response (Rmax = 44.3 +/- 4.0%). The higher concentration of 100 microM L-NAME completely abolished vasodilatation to acetylcholine. L-Arginine (10 mM) did not reverse the inhibitory actions of L-NAME at either concentration. 3. L-NAME 100 microM, augmented vascular tone induced by 1 microM 5-hydroxytryptamine and 1 microM histamine, thus altering the characteristics of both pressure/flow and conductance/flow relationships such that conductance was reduced at all flow rates. The augmentation of constrictor tone was reversed in a concentration-dependent manner by L-arginine (10 microM-10 mM) and the effect of L-NAME on the conductance/flow relationships was similarly reversed by 10 mM L-arginine. The augmentation of tone was endothelium-dependent as it did not occur following functional destruction of the endothelium by perfusion of the vascular bed with the detergent CHAPS (0.3%) for 150s.(ABSTRACT TRUNCATED AT 250 WORDS)