Sensitivity Of Vascular Smooth Muscle Research Articles

Involvement of Src family protein tyrosine kinases in Ca(2+) sensitization of coronary artery contraction mediated by a sphingosylphosphorylcholine-Rho-kinase pathway.

We recently reported that sphingosylphosphorylcholine (SPC) is a novel messenger for Rho-kinase-mediated Ca(2+) sensitization of vascular smooth muscle (VSM) contraction. Subcellular localization and kinase activity of Src family protein kinases (SrcPTKs), except for c-Src, is controlled by a reversible S-palmitoylation, an event inhibited by eicosapentaenoic acid (EPA). We examined the possible involvement of SrcPTKs in SPC-induced Ca(2+) sensitization and effects of EPA. We used porcine coronary VSM and rat aortic VSM cells (VSMCs) in primary culture. An SrcPTKs inhibitor, PP1, and EPA inhibited SPC-induced contraction, concentration-dependently, without affecting [Ca(2+)](i) levels and the Ca(2+)-dependent contraction induced by high K(+) depolarization. A digitized immunocytochemical analysis in VSMCs revealed that SPC induced translocation of Fyn, but not of c-Src, from the cytosol to the cell membrane, an event abolished by EPA. Translocation of Rho-kinase from the cytosol to the cell membrane by SPC was also inhibited by EPA and PP1. The SPC-induced activation of SrcPTKs was blocked by EPA and PP1, but not by Y27632, an Rho-kinase inhibitor. Rho-kinase-dependent phosphorylation of myosin phosphatase induced by SPC was inhibited by EPA, PP1, and Y27632. Translocation and activation of SrcPTKs, including Fyn, play an important role in Ca(2+) sensitization of VSM contractions mediated by a SPC-Rho-kinase pathway.

Chronic Ethanol Consumption Alters Vascular Smooth Muscle Responses In Rats

1. The effect of chronic ethanol (10%) consumption for 5 months on vascular smooth muscle (VSM) using aortic rings of both sexes of Sprague-Dawley rats was studied. 2. Chronic ethanol consumption increased the sensitivity of VSM to noradrenaline (NA) in both male and female ethanol-treated rats. 3. There was no significant difference in the contractile response of male and female ethanol-treated rats to NA. Hence, the enhancement of vascular contractility to the agonist due to chronic ethanol consumption is independent of gender. 4. Vascular relaxation induced by acetylcholine showed similar responses in all experimental groups. Thus, chronic ethanol consumption seems to have no significant effect on the production of endothelium-dependent relaxing factor. 5. However, the VSM sensitivity to potassium chloride was increased in female ethanol-treated rats, whereas male ethanol-treated rats had similar responses as controls. 6. The results suggest that the effect of chronic ethanol consumption on VSM varies with gender. Impairment of VSM in male ethanol-treated rats is due partly to changes in the receptor-operated channel, whereas in females it is due to changes in both receptor- and potential-operated channels.

Chronic hypoxia attenuates cGMP-dependent pulmonary vasodilation.

Chronic hypoxia (CH) augments endothelium-derived nitric oxide (NO)-dependent pulmonary vasodilation; however, responses to exogenous NO are reduced following CH in female rats. We hypothesized that CH-induced attenuation of NO-dependent pulmonary vasodilation is mediated by downregulation of vascular smooth muscle (VSM) soluble guanylyl cyclase (sGC) expression and/or activity, increased cGMP degradation by phosphodiesterase type 5 (PDE5), or decreased VSM sensitivity to cGMP. Experiments demonstrated attenuated vasodilatory responsiveness to the NO donors S-nitroso-N-acetylpenicillamine and spermine NONOate and to arterial boluses of dissolved NO solutions in isolated, saline-perfused lungs from CH vs. normoxic female rats. In additional experiments, the sGC inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, blocked vasodilation to NO donors in lungs from each group. However, CH was not associated with decreased pulmonary sGC expression or activity as assessed by Western blotting and cGMP radioimmunoassay, respectively. Consistent with our hypothesis, the selective PDE5 inhibitors dipyridamole and T-1032 augmented NO-dependent reactivity in lungs from CH rats, while having little effect in lungs from normoxic rats. However, the attenuated vasodilatory response to NO in CH lungs persisted after PDE5 inhibition. Furthermore, CH similarly inhibited vasodilatory responses to 8-bromoguanosine 3'5'-cyclic monophosphate. We conclude that attenuated NO-dependent pulmonary vasodilation after CH is not likely mediated by decreased sGC expression, but rather by increased cGMP degradation by PDE5 and decreased pulmonary VSM reactivity to cGMP.

Nitric oxide induces dilation of rat aorta via inhibition of rho-kinase signaling.

NO induces vasodilation through cGMP-dependent protein kinase--dependent and --independent mechanisms. A recent study demonstrated that recombinant cGMP-dependent protein kinase can phosphorylate the small G protein, RhoA, thus inhibiting its activity. Additionally, sodium nitroprusside was found to reverse the phenylephrine-induced translocation of RhoA, which is further indicative of the inhibition of RhoA activity. RhoA is known to be involved in the Ca(2+) sensitization of vascular smooth muscle through the actions of one of its downstream effectors, Rho-kinase. This study examined whether NO endogenously induces the relaxation of intact rat aorta via the inhibition of the Rho-kinase--mediated Ca(2+)-sensitizing pathway. Endogenous Rho-kinase inhibitor activity was inhibited by the selective compound Y-27632. Treatment of endothelium-intact rat aorta with Y-27632 (1 micromol/L) resulted in an attenuation of maximal force generated in response to phenylephrine. In endothelium-denuded rings, however, 1 micromol/L Y-27632 was ineffective at inhibiting the phenylephrine-induced contraction. Additionally, 1 micromol/L Y-27632 was significantly less effective at inhibiting the phenylephrine-induced contraction of endothelium-intact rings in the presence of inhibitors of NO synthase or guanylate cyclase (N(omega)-nitro-L-arginine and 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one, respectively). Interestingly, sodium nitroprusside restored the ability of 1 micromol/L Y-27632 to attenuate phenylephrine-induced contraction. Rho-kinase inhibition was also found to increase the sensitivity of the endothelium-denuded aorta to sodium nitroprusside. These data demonstrate that NO inhibits Rho-kinase activity in the intact rat aorta, supporting the hypothesis that endogenous NO-mediated vasodilation occurs through the inhibition of Rho-kinase constrictor activity in the intact rat aorta.

Increased sensitivity of vascular smooth muscle to nitric oxide in dilated cardiomyopathy of Syrian hamsters (Bio TO-2 strain).

We assessed the evolution with time of the responsiveness of three vascular beds in dilated cardiomyopathic hamsters of the Bio TO-2 strain. Eight cardiomyopathic hamsters and 8 control hamsters were investigated at 180 and 300 days of age. Thoracic aorta and mesenteric and renal artery rings were studied in isolated organ baths. Cumulative concentration-response relations to phenylephrine, acetylcholine, sodium nitroprusside, and angiotensin II were established for each ring. Maximum effect (Emax) and concentration inducing 50% of Emax (EC50) were determined from each concentration-response curve and pD2 was calculated as -log(EC50). Compared with control hamsters, in cardiomyopathic hamsters, Emax of phenylephrine was not modified in aorta, whereas it was significantly lower in mesenteric (-6% and -33% at 180 and 300 days, respectively) and renal (-17% and -24%) arteries. Emax of acetylcholine was significantly higher in aorta (+57% and +30%), mesenteric (+42% and +34%), and renal (+168% and +70%) arteries. Emax of sodium nitroprusside was significantly higher in aorta (+26% and +16%) and tended to be higher in mesenteric (+25% and +23%) and renal (+27% and +10%) arteries. Emax of angiotensin II was not modified in aorta and tended to be lower in mesenteric artery at 300 days. The pD2 of phenylephrine was significantly increased in aorta and the pD2 of sodium nitroprusside was significantly increased in aorta and renal artery. In conclusion, in dilated cardiomyopathic hamsters, endothelium-dependent and -independent vasodilations are enhanced early, demonstrating increased sensitivity of vascular smooth muscle to nitric oxide. This abnormality may be involved in the decreased responsiveness to phenylephrine and angiotensin II.

Combined administration of Fasudil hydrochloride and nitroglycerin for treatment of cerebral vasospasm.

We examined the Ca2+ sensitivity of vascular smooth muscle in experimental cerebral vasospasm in canine basilar artery. From the results of our experiments we concluded that the increase in this Ca2+ sensitivity may be one of the most important factors in cerebral vasospasm [1, 2, 4]. Therefore, we considered that treatment to decrease Ca2+ sensitivity might be effective for the prevention of cerebral vasospasm. Based on this consideration, we examined a new therapy of combined administration of fasudil hydrochloride and nitroglycerin for cerebral vasospasm that decreases the Ca2+ sensitivity of vascular smooth muscle.KeywordsFasudil hydrochloridenitroglycerinvasospasm

Decreased sensitivity to nitric oxide in the aorta of severely hypercholesterolemic apolipoprotein E-deficient mice.

A normal response to nitric oxide donors has been cited as evidence that impaired endothelium-dependent vasodilation during hypercholesterolemia is due to decreased synthesis of nitric oxide. This tenet was examined by determining responses to nitric oxide gas as well as to acetylcholine and sodium nitroprusside in the isolated aorta of apolipoprotein E-deficient mice fed normal or Western-type cholesterol-rich diet until 21 or 35 weeks of age. In mice fed normal chow, relaxation to all agents remained comparable to that obtained in wild-type mice. In mice fed Western diet, the relaxation to acetylcholine as well as to nitric oxide was decreased at 35 weeks of age. At 21 weeks of age, decreased sensitivity to nitric oxide was observed despite a normal response to acetylcholine. The response to sodium nitroprusside was normal in all groups. A decrease in aortic superoxide dismutase activity as well as an increase in aortic superoxide anion generated in the presence of NADH as measured by lucigenin chemiluminescence was observed in the group fed Western diet at 35 weeks. This provides evidence that altered superoxide anion could contribute to the deterioration in nitric oxide sensitivity that underlies the impaired endothelium-dependent relaxation. These data indicate that decreased sensitivity to nitric oxide may contribute to the development of impaired endothelium-dependent relaxation in hypercholesterolemia. The response to sodium nitroprusside appears not to reflect the decreased sensitivity of vascular smooth muscle to authentic nitric oxide.

Effects of undernutrition in early pregnancy on systemic small artery function in late-gestation fetal sheep

Objective: The aim of this study was to investigate functional development of small arteries from the skeletal circulation of fetal sheep and to determine whether maternal undernutrition affects responses to vasoconstrictive and vasodilatory agonists in arteries from the late-gestation fetus. Study Design: We investigated vasoconstrictive and vasodilatory responses of isolated small (approximately 300 μm) arteries from the femoral vascular bed of fetal sheep and from late-gestation pregnant ewes. Ewes were fed either 100% of the nutritional requirement throughout pregnancy (control group) or a restricted diet of 85% or 50% of the nutritional requirement for the first 70 days of pregnancy. For the remainder of pregnancy all ewes were fed the complete diet. Results: Among control group animals vasoconstriction in response to norepinephrine was well developed in fetuses at 0.6 and 0.9 gestation with respect to that in the ewes. When expressed as a percentage of the response to 125-mmol/L potassium (to correct for differences in vessel size and muscle mass), maximum constriction in response to norepinephrine was greater in fetal vessels from 0.9 gestation than in either those at 0.6 gestation or those of the ewes. Endothelium-dependent vasorelaxation responses to acetylcholine and bradykinin were also well developed in fetuses at 0.6 and 0.9 gestation and were similar to those in the ewes. In fetuses at 0.9 gestation the 50% nutritional restriction of the ewe led to blunted endothelium-dependent vasodilatation in response to acetylcholine and blunted endothelium-independent vasodilatation in response to sodium nitroprusside. Responses in the fetuses at 0.9 gestation in which the ewes were fed a restricted diet of 85% were normal. Conclusion: This study shows that from midgestation onward small arteries from the skeletal circulation of the fetal sheep have the functional capacity to respond to norepinephrine and endothelium-dependent vasodilators (eg, acetylcholine and bradykinin). The blunted responses to acetylcholine and sodium nitroprusside in the fetuses at 0.9 gestation among the group of dietarily restricted ewes (restricted diet of 50% group) were indicative of impaired vascular smooth muscle sensitivity to nitric oxide. This defect may contribute to the development of hypertension in later life. (Am J Obstet Gynecol 2000;183:1301-7.)

Stimulated nitric oxide release and nitric oxide sensitivity in forearm arterial vasculature during normotensive and preeclamptic pregnancy

Objective: We sought to determine whether the enhanced forearm vascular activity of nitric oxide during pregnancy and preeclampsia is associated with altered smooth muscle sensitivity to nitric oxide or with stimulated nitric oxide release. Study Design: Forearm blood flow responses to brachial artery infusion of glyceryl trinitrate (a nitric oxide donor), serotonin (an endothelium-dependent nitric oxide–mediated agonist), and ritodrine (a β-adrenergic receptor agonist) were studied in 10 nonpregnant women, 12 pregnant women, and 7 women with preeclampsia by means of strain-gauge plethysmography. Responses to each drug (maximum dilator response and the sum of the percentage of dilator responses to each drug) were compared by analysis of variance. Results: Compared with nonpregnant women, pregnant subjects showed reduced responses to serotonin (summary response, 117 ± 19 vs 221 ± 30; P < .05). Responses to serotonin were reduced in the group with preeclampsia compared with those in the nonpregnant group (summary response, 71 ± 28; P < .05) but did not differ from the responses in pregnant women. There were no differences between responses to glyceryl trinitrate and responses to ritodrine in any of the groups. Conclusion: Vascular smooth muscle sensitivity to nitric oxide is not altered in normal pregnancy or preeclampsia, but dilator responses to serotonin appear blunted. Alterations in serotonin receptor coupling to nitric oxide synthase, or a limitation of availability of the substrate for nitric oxide synthase (L -arginine) during pregnancy, could account for the reduction in stimulated nitric oxide release. (Am J Obstet Gynecol 1999;181:1479-85.)

Impaired aerobic capacity in hypercholesterolemic mice: partial reversal by exercise training.

The present study assessed whether impaired aerobic capacity previously observed in hypercholesterolemic mice is reversible by exercise training. Seventy-two 8-wk-old female C57BL/6J wild-type (+, n = 42) and apolipoprotein E-deficient (-, n = 30) mice were assigned to the following eight interventions: normal chow, sedentary (E+, n = 17; E-, n = 8) or exercised (E+ex, n = 13; E-ex, n = 7) and high-fat chow, sedentary (E+chol, n = 6; E-chol, n = 8) or exercised (E+chol-ex, n = 6; E-chol-ex, n = 7). Mice were trained on a treadmill 2 x 1 h/day, 6 days/wk, for 4 wk. Cholesterol levels correlated inversely with maximum oxygen uptake (r = -0.35; P < 0. 02), which was blunted in all hypercholesterolemic sedentary groups (all P < 0.05). Maximum oxygen uptake improved in all training groups but failed to match E+ex (all P < 0.05). Vascular reactivity and nitric oxide (NO) synthesis correlated with anaerobic threshold (r = 0.36; P < 0.025) and maximal distance run (r = 0.59; P < 0.007). We conclude that genetically induced hypercholesterolemia impairs aerobic capacity. This adverse impact of hypercholesterolemia on aerobic capacity may be related to its impairment of vascular NO synthesis and/or vascular smooth muscle sensitivity to nitrovasodilators. Aerobic capacity is improved to the same degree by exercise training in normal and genetically hypercholesterolemic mice, although there remains a persistent difference between these groups after training.

Chronic nitric oxide inhibition model six years on.

The discovery in 1987 that endothelium-derived nitric oxide (NO) mediates the vasodilatory effect of certain endothelium-dependent agonists1 2 inaugurated the current huge field of NO biology. It is now recognized that NO plays essential roles in many diverse physiological processes and in some pathophysiologic events. Development of these concepts has been based largely on evidence obtained by limiting NO biosynthesis. This review is centered on the cardiovascular and particularly the renal functional and structural consequences of chronic pharmacologic NO inhibition by l-arginine analogues. We devoted special attention to the mechanisms of hypertension and organ injury that occur under these circumstances, while appreciating the inherent limitations surrounding interpretation of this data. NO is made by the enzymatic action of several widely distributed NO synthases (NOS). In the presence of the substrates l-arginine and oxygen, as well as a number of essential cofactors, NO is produced in response to appropriate stimuli. The constitutively expressed NOS play a major role in the physiological control of vascular tone and kidney function.3 4 Vascular endothelial NOS (eNOS) and brain-type NOS (bNOS) are widely distributed throughout the kidney5 as well as the cardiovascular system and in strategic locations in the peripheral and central nervous system (CNS).6 7 Both eNOS and particularly bNOS are abundant in the kidney, glomeruli, and vasculature as well as in most segments of the tubule,3 5 and NOS activity in medulla is considerably greater than in cortex.8 NO generated within the kidney controls the glomerular filtration rate (GFR), total renal and medullary blood flow, pressure natriuresis, epithelial sodium transport, and production of various vasoactive factors including renin.3 4 5 eNOS is distributed throughout most parts of the arterial and venous circulation, although there is considerable heterogeneity in the extent to which NO controls …

Evidence for the Involvement of Protein Kinase C in Depression of Endothelium-Dependent Vascular Responses in Spontaneously Hypertensive Rats

The goal of the present study was to evaluate the role of protein kinase C (PKC) in the depression of endothelium-dependent vacular response in spontaneously hypertensive Okamoto rats (SHR). Aortae from SHR demonstrated a decreased relaxant response to acetylcholine (Ach) as compared to aortae from normotensive Wistar-Kyoto (WKY) rats, while papaverine lowered the force of aorta to a similar degree in both strains of rats. PKC inhibitors, H-7 (5 × 10^–6 M) and chelerythrine chloride (10^–6 M), produced a greater decrease in the force developed by the aortae from SHR vs. WKY rats both in intact and chemically permeabilized tissues. In SHR aortae PKC inhibitors enhanced relaxation to Ach to a greater extent as compared to WKY aortae. Furthermore, in the presence of PKC inhibitors, the constrictor responses of SHR aortae to Ach were transformed into relaxant responses, and the concentration-response curve to Ach was shifted to the left. The sensitivity of aortae from SHR to authentic nitric oxide (NO) was lowere compared to WKY rats. EC₅₀s for authentic NO in SHR and WKY rat aortae were different: –2.9 ± 0.15 × 10^–6 M and 4.58 ± 0.1 × 10^–7 M (n = 15, p < 0.001), respectively. Bioassay experiments using SHR aortae showed that the addition of chelerythrine (10^–6 M) to the detector superfusate caused relaxation during treatment of the donor segment with Ach, indicating that the sensitivity of the aortae to NO had been restored. When SHR detector ring was substituted for denuded aortae from WKY rats and PKC inhibitors were not added to the detector superfusate, the relaxation of the detector aortae was also close to the normal Ach-induced relaxation. WKY aortae demonstrated a positive relationship between Ach-stimulated NO release and relaxant response amplitudes (correlation coefficient r = 0.905, p < 0.001, n = 10). In contrast, there was a significant negative correlation in SHR aortae (r = –0.712, p < 0.05, n = 10). Detection of NO release by chemiluminescence showed no significant difference in NO release in SHR and WKY aortae. Taken together, these data suggest that the blunted endothelium-dependent relaxations seen in SHR aortae are mainly due to a decreased sensitivity of vascular smooth muscle to EDRF/NO resulting from an increased PKC activity.

Calcium sensitivity and agonist-induced calcium sensitization in small arteries of young and adult spontaneously hypertensive rats.

The sensitivity of the myofilaments to Ca2+ is increased during agonist-induced contraction of vascular smooth muscle. Given the important contribution of vascular tone to the elevation of peripheral resistance observed in genetic hypertension, we have investigated whether alterations in myofilament Ca2+ sensitivity occur in small arteries from spontaneously hypertensive rats (SHR) and age-matched Wistar-Kyoto (WKY) controls during the developmental and established phases of hypertension. Segments of mesenteric, renal, and femoral artery with an average lumen diameter <300 microm from 5- or 20-week-old rats were mounted in a wire myograph. Morphological measurements were made and the vessels permeabilized with Staphylococcus aureus alpha-toxin. Dose-response curves to increasing concentrations of Ca2+ were obtained and the ability of 100 nmol/L endothelin-1 (ET-1) or 10 micromol/L norepinephrine (NE) in the presence of 10 micromol/L GTP to enhance tension in response to low Ca2+ (pCa6.7) was determined. Systolic, diastolic, and mean blood pressures were higher in SHR than in WKY at 5 and 20 weeks. The media thickness:lumen diameter ratio was increased in mesenteric and femoral arteries from SHR compared with WKY at 5 and 20 weeks. There was no difference in media thickness:lumen diameter ratio in renal arteries or between 5- and 20-week animals in any vascular bed. The pCa curves were not different in mesenteric, renal, or femoral arteries from hypertensive compared with normotensive rats or between age groups, except in femoral arteries at 20 weeks, which exhibited a greater sensitivity to Ca2+ in SHR. Tension developed in response to maximal Ca2+ (pCa5.0) was greater in permeabilized mesenteric arteries from SHR compared with WKY at 20 weeks of age only; media stress was again similar in both strains but increased in older animals compared with younger animals in mesenteric arteries from WKY. The submaximal contraction induced by pCa6.7 was greater in femoral and renal than mesenteric arteries. GTP (10 micromol/L) augmented the tension developed to pCa6.7 in mesenteric arteries at 5 and 20 weeks and in renal arteries at 20 weeks. Addition of 100 nmol/L ET-1 or 10 micromol/L NE in the continued presence of GTP markedly increased tension in mesenteric arteries at 5 and 20 weeks. In renal arteries, 10 micromol/L NE enhanced Ca2+ sensitivity in the presence of GTP in SHR at 5 and 20 weeks and WKY at 5 weeks. In femoral arteries, there was a tendency for ET-1 and NE to increase Ca2+ sensitivity, but this increase was significant in WKY at 20 weeks (ET-1) and SHR at 5 weeks (NE) only. We have demonstrated that the sensitivity of the myofilaments to Ca2+ and ET-1- or NE-induced Ca2+ sensitization is not different in permeabilized small mesenteric, renal, or femoral arteries from SHR compared with WKY controls. Only in SHR mesenteric arteries at 20 weeks of age was there evidence of increased active tension in response to maximal Ca2+, despite structural differences, consistent with increased muscle mass in femoral arteries from SHR. We conclude that it is unlikely that a ubiquitous abnormality of the sensitivity of the contractile apparatus to Ca2+ or agonist-induced Ca2+ sensitization in vascular smooth muscle underlies the elevated total peripheral resistance associated with hypertension.

Estrogen replacement attenuates resistance artery adrenergic sensitivity via endothelial vasodilators.

The objective of this study was to determine whether chronic estrogen replacement alters adrenergic constriction and endothelium-dependent dilation in resistance arteries from the rat. Resistance-sized (< 200 microns) mesenteric arteries from castrated female Sprague-Dawley rats with (E2; 21 day, 0.5-mg pellet) and without (OvX) estrogen replacement were removed for in vitro study on a pressurized arteriograph system. Sensitivity to alpha-adrenergic constriction and the role of the endothelium in its modulation and of agonist-provoked endothelium-dependent relaxation were determined. Estrogen-treated rats had decreased heart rate as well as systolic and diastolic blood pressure. Arteries from estrogen-replaced rats were fivefold less sensitive to alpha 1-adrenergic stimulation with phenylephrine (50% effective concentration: E2, 3.2 +/- 1.1 microM; OvX, 0.6 +/- 0.2 microM; P < 0.05). This difference was abolished by endothelial denudation, blockade of cyclooxygenase (1 microM ibuprofen), or nitric oxide synthase blockade (0.24 mM N omega-nitro-L-arginine). There was no difference in muscarinic agonist-provoked relaxation or vascular smooth muscle sensitivity to prostacyclin or sodium nitroprusside. These results indicate that estrogen replacement decreases resistance artery adrenergic sensitivity by increasing the basal release of relaxing factors from the endothelium. This effect on small artery function may produce dual cardioprotective effects by decreasing peripheral resistance, blood pressure, and the likelihood of thrombosis.

Gestation increases nitric oxide–mediated vasodilation in rat uterine arteries

OBJECTIVE: The purpose of this study was to determine the influence of endothelium-released nitric oxide on uterine arterial tone and reactivity during pregnancy. STUDY DESIGN: The effects of pregnancy on endothelial function were evaluated in isolated pressurized rat uterine arteries from late-pregnant rats (day 19 to 20) versus age-matched nonpregnant controls. The effects of nitric oxide synthase inhibition (Nω-nitro- l-arginine) on arterial tone and reactivity under basal and activated (phenylephrine) conditions were determined, as was arterial reactivity to endothelium-dependent (acetylcholine) and endothelium-independent (sodium nitroprusside) vasodilators, by evaluating changes in lumen diameter. RESULTS: (1) Maximal constriction to Nω-nitro- l-arginine was significantly enhanced under basal (nonstimulated) conditions in arteries from late-pregnant versus nonpregnant rats (changes in lumen diameter 37% ± 8% vs 9.3 ± 6.2%, respectively, p < 0.05). (2) Nitric oxide synthase blockade with 1 nmol/L Nω-nitro- l-arginine significantly increased phenylephrine sensitivity in arteries from late-pregnant animals (median effective concentration 115 ± 23 nmol/L vs 33 ± 8 nmol/L, control vs treated vessels, p < 0.05) but was without statistically significant effect on arteries from nonpregnant animals (control 255 ± 164 nmol/L, treated 250 ± 102 nmol/L, p > 0.05). (3) The threshold concentration of acetylcholine required to elicit endothelium-dependent dilation was significantly lower in late-pregnant versus nonpregnant arteries (1.4 ± 0.2 nmol/L vs 12.2 ± 3.8 nmol/L, p < 0.05). (4) Vascular smooth muscle sensitivity to an exogenous nitrodilator (sodium nitroprusside) was identical in late-pregnant versus nonpregnant vessels. CONCLUSION: Endothelial vasodilator influences are augmented during pregnancy under basal, activated (phenylephrine), and chemically provoked (acetylcholine) conditions in uterine arteries by enhanced release of nitric oxide. (Am J Obstet Gynecol 1997;176:856-64.)

Acetylcholine relaxation of renal artery and nitric oxide synthase activity of renal cortex increase with fetal and postnatal age.

Endothelium-derived nitric oxide (NO) regulates hemodynamics in the fetal kidney and modulates renal perfusion during postnatal maturation. We hypothesize that NO release by renal arteries increases with fetal maturation and contributes to the increased renal perfusion before and after birth. We tested the effect of maturation on relaxation to acetylcholine (ACh; 10(-9) M to 10(-5) M), the prototypic endothelium-dependent relaxing agent, and sodium nitroprusside (10(-9) M to 10(-5) M), an NO donor, on isolated main renal arteries obtained from anesthetized fetal guinea pigs of varying gestational age (0.5-0.8, 0.8-0.9, and 0.9-0.97 gestation), and neonatal (1-50 d) and reproductively mature adult guinea pigs. The effect of NO synthase inhibition by nitro-L-arginine (LNA; 10(-4) M) and cyclooxygenase inhibition by indomethacin (10(-5) M) on ACh relaxation was also measured. Ca(2+)-dependent NO synthase activity was measured in fetal (0.5-0.87 gestation), neonatal (1-10 d), and adult (mature) renal cortex by the conversion of [L-14C]arginine to [L-14C]citrulline and the time course compared with the relaxation responses. Sensitivity and maximal relaxation to ACh increased with fetal age. In neonatal renal arteries, maximal relaxation but not sensitivity to ACh increased relative to the fetal arteries. In adult renal arteries, both sensitivity and maximal relaxation increased compared with fetal arteries. Sensitivity but not maximal responses to sodium nitroprusside increased with age but exhibited a different maturational pattern than ACh relaxation. LNA inhibited ACh relaxation in arteries of all ages. Indomethacin reduced the sensitivity to ACh only in the fetal arteries. Ca(2+)-dependent NO synthase activity of the renal cortex increased during fetal development reaching levels at near term similar to those found in both the newborn and adult kidneys. These results suggest that endothelium-derived NO release by the renal artery and constitutive NO synthase activity in the renal microvasculature increases with fetal and postnatal maturation. Further, the sensitivity of vascular smooth muscle to NO also increases after birth. Thus, functional adaptations in both the endothelium and the vascular smooth muscle contribute to the maturational changes in mechanisms regulating renal hemodynamics before and after birth.

Chronic hypoxia selectively augments endothelium-dependent pulmonary arterial vasodilation.

We have previously demonstrated that chronic hypoxia (CH) augments pulmonary arterial dilation to the endothelium-derived nitric oxide (EDNO)-dependent pulmonary vasodilator arginine vasopressin (AVP). The present study examined 1) whether this enhanced vasoreactivity is observed with other agents that act by stimulating constitutive NO synthase (cNOS), 2) whether CH increases arterial vascular smooth muscle sensitivity to NO, and 3) whether endogenous endothelin (ET) or an endothelium-derived hyperpolarizing factor (EDHF) contributes to this altered arterial reactivity following CH. We examined responses to the receptor-mediated EDNO-dependent dilators histamine and ET-1, the nonreceptor-mediated EDNO-dependent dilator ionomycin, and the NO donors 1, 3-propanediamine, N-4-[1-(3-aminopropyl)-2-hydroxy-2-nitrosohydrazino] butyl (spermine NONOate) and S-nitroso-N-acetylpenicillamine (SNAP) in U-46619-constricted, isolated perfused lungs from control and CH rats. Additional experiments examined responses to AVP in the presence of the ET-receptor antagonist PD-145065 or the K+ channel blockers glibenclamide or tetraethylammonium (TEA) in lungs from each group. Microvascular pressure was assessed by double occlusion, allowing calculation of segmental resistances. Total and arterial vasodilatory responses to histamine, ET-1, and ionomycin were augmented in lungs from CH vs. control animals. However, CH did not alter the vasodilation to spermine NONOate or SNAP. PD-145065, glibenclamide, and TEA had no effect on responses to AVP in either group. We conclude that increased activity of arterial cNOS may be responsible for the augmented pulmonary arterial dilation to EDNO-dependent vasodilators following CH.

Endothelium-dependent relaxation in peripheral vasculature and kidney of non-insulin-dependent diabetes mellitus

Desmopressin (DDAVP), an AVP·V 2-receptor agonist, evokes endothelium-dependent relaxation (EDR) due to nitric oxide (NO), EDR factor (EDRF) in the systemic vasculature, and glomerular afferent arterioles via AVP receptor(s). Glyceryl trinitrate (GTN) causes endothelium-independent (nonreceptor-mediated) vasodilation. We elucidated the possible involvement of EDRF in early non-insulin-dependent diabetes mellitus (NIDDM) and glomerular hyperfiltration (GHF) by DDAVP and GTN infusions. Patients with advanced DM nephropathy (DM · Np) ( n = 7) were also examined. DDAVP and GTN decreased the mean blood pressure in DM with GHF (DM + GHF) and without GHF (DM-GHF) greater than that in normal subjects (N), without any difference in the heart rate changes in any group. Plasma levels of cGMP, a cellular messenger of NO, were significantly increased by DDAVP and GTN with a similar increment in each group. DDAVP caused a significant increase in urinary cGMP excretion in each group with a similar increment in each group. However, it caused a transient increase in creatinine clearance only in DM + GHF although GTN did not, and an exaggerated excretion of urinary albumin in early NIDDM, especially in DM + GHF, without a change in β 2-microglobulin excretion. In contrast, in DM · Np GTN caused a decrease in blood pressure and an increase in plasma cGMP levels, but DDAVP did not. In conclusion, in peripheral vasculature and kidney, an enhanced sensitivity of vascular smooth muscle to NO is present in early NIDDM. The exaggerated dilation of glomerular afferent arterioles by preferentially produced NO in in situ, which causes a rise in P GC, might be partly responsible for the glomerular hyperfiltration and subsequently the increase in the glomerular protein permeation of DM + GHF. However, in peripheral blood vessels of DM · Np EDR is impaired. Thus, EDR seems to change with the development of NIDDM.

Genistein, a tyrosine kinase inhibitor, reduces Ca2+ mobilization in swine carotid media

DiSalvo and colleagues (Biochem. Biophys. Res. Commun. 190: 968-974, 1993) found that tyrosine kinase inhibitors reduced force at constant Ca2+ concentrations in permeabilized mesenteric arterioles. These data suggest that tyrosine kinase activation could regulate Ca2+ sensitivity in intact vascular smooth muscle. We tested this hypothesis by examining the effects of the tyrosine kinase inhibitor genistein on intracellular Ca2+ concentration ([Ca2+]i), myosin regulatory light chain (MRLC) phosphorylation, and isometric stress in intact swine carotid media tissues. Pretreatment with 30 microM genistein attenuated histamine-induced increases in [Ca2+]i (estimated using the photoprotein aequorin), MRLC phosphorylation, and stress. The genistein-dependent decrease in [Ca2+]i quantitatively accounted for the decrease in MRLC phosphorylation and stress. There was no measurable change in the Ca2+ dependence of MRLC phosphorylation or the dependence of force on MRLC phosphorylation. Genistein inhibited contractions independently of the source of activator Ca2+. These data suggest that tyrosine kinase(s) may influence force development in the intact swine carotid media by altering [Ca2+]i rather than modulating the Ca2+ sensitivity of MRLC phosphorylation.

Coupling of the endothelin ET A and ET B receptors to Ca 2+ mobilization and Ca 2+ sensitization in vascular smooth muscle

Effects of endothelins on cytosolic Ca 2+ level ([Ca 2+]i) and contraction were examined in the swine pulmonary artery and vein. In the artery, endothelin-1 and endothelin-3, but not sarafotoxin S6c and IRL 1620 (300 nM each), induced transient increase followed by sustained increase in [Ca 2+]i and sustained contraction. These effects were inhibited by the ET A receptor antagonist, BQ-123. In the vein, endothelin-1 and endothelin-3 (300 nM each) induced sustained increase in [Ca 2+]i and sustained contraction whereas sarafotoxin S6c and IRL 1620 (300 nM each) transiently increased both [Ca 2+]i and contractile tension. The ET B receptor in the vein was desensitized by pretreatment with sarafotoxin S6c, abolishing the effects of sarafotoxin S6c and IRL 1620 without changing the effects of endothelin-1 and endothelin-3. In contrast, an ET B antagonist, RES-701-1, antagonized the effects of IRL 1620 without changing the effects of other stimulants. In both artery and vein, the maximum contraction induced by these stimulants was greater than that induced by KCl at a given [Ca 2+]i. In the absence of external Ca 2+, endothelin-1 and endothelin-3 induced transient increase in [Ca 2+]i and slow susained contraction in both artery and vein. In the vein, sarafotoxin S6c induced small sustained contraction without changing [Ca 2+]i. In the permeabilized artery and vein, endothelin-1 augmented the contraction induced by Ca 2+. These results suggest that the ET A receptors in the artery and vein are coupled to Ca 2+ release (which does not seem to trigger contraction), Ca 2+ influx and Ca 2+ sensitization. In contrast, the ET B receptor in the vein is coupled to Ca 2+ influx and Ca 2+ sensitization but not to Ca 2+ release.