Search

An insertion/deletion (ID) polymorphism of the angiotensin-converting enzyme (ACE) gene is associated with left ventricular hypertrophy. The present study examined polymorphisms of the ACE gene in patients with essential hypertension and left ventricular hypertrophy who were participants in a long-term trial of therapy with an ACE inhibitor.ACE inhibitor therapy was administered for2 years to 54 patients with hypertension who had moderate or severe left ventricular hypertrophy. Cardiac dimensions were monitored by echocardiography before the initiation of therapy and after 1 and 2 years of treatment. Serum ACE activity and plasma concentrations of brain natriuretic peptide, a marker for left ventricular hypertrophy, were also monitored.Eighteen patients had the II genotype for the angiotensin-converting enzyme gene, 19 had the ID genotype, and 17 had the DD genotype. Baseline (mean +/- SD) serum ACE activity was significantly greater (P0.05) in the DD (18 +/- 7 IU/L) group than in the II (7 +/- 4 IU/L) or ID (12 +/- 6 IU/L) groups. ACE inhibitor therapy was effective in controlling blood pressure, and it reduced posterior and septal wall thickness, left ventricular mass index, and plasma brain natriuretic peptide concentration in all three groups. Despite similar blood pressure reductions, after 2 years, mean (+/- SD) regression in posterior wall thickness was significantly less (P0.05) in the DD group (-9% +/- 5%) than in the ID (-21% +/- 7%) and II (-21% +/- 9%) groups. Similar results were seen for the reductions in brain natriuretic peptide levels. The magnitudes of regression of septal wall thickness and left ventricular mass index during therapy were less in the DD group than the II group (P0.05).Hypertensive patients with the DD genotype are less likely to have regression of left ventricular hypertrophy when treated with ACE inhibitors than are patients with other ACE genotypes.

Abstract The migration of medial smooth muscle cells (SMCs) into the intima is proposed to be an important process of intimal thickening in atherosclerotic lesions. The present study examined the possible effect of a novel endothelium-derived relaxing peptide, C-type natriuretic peptide (CNP), on oxidized low-density lipoprotein (LDL)–induced migration of cultured human coronary artery SMCs by the Boyden’s chamber method. The effect of CNP was compared with that of atrial and brain natriuretic peptides (ANP and BNP, respectively). Oxidized LDL stimulates SMC migration in a concentration-dependent manner between 20 and 200 μg/mL. This stimulation was chemotactic in nature but was not chemokinetic. By contrast, native LDL was without significant activity. CNP-22 clearly inhibited SMC migration stimulated with 200 μg/mL oxidized LDL in a concentration-dependent manner between 10 −9 and 10 −6 mol/L. ANP-(1-28) and BNP-32 also inhibited oxidized LDL–induced SMC migration at concentrations of 10 −7 and 10 −6 mol/L, but these effects were weaker than the effect of CNP-22. Such inhibition by these natriuretic peptides was paralleled by an increase in the cellular level of cGMP. Oxidized LDL–induced migration was significantly inhibited by a stable analogue of cGMP, 8-bromo-cGMP, or an activator of the cytosolic guanylate cyclase, sodium nitroprusside. These natriuretic peptides did not suppress the cell adhesion either in the absence or presence of oxidized LDL. These data indicate that oxidized LDL stimulates migration of human coronary artery SMCs and that natriuretic peptides, especially CNP, inhibit this stimulated SMC migration, at least in part, through a cGMP-dependent process. Taken together with the finding that oxidized LDL is present in the intima, CNP may play a role as a local antimigration factor during the process of intimal thickening in hypercholesterolemia-induced coronary atherosclerosis.

Abstract The migration of coronary artery medial smooth muscle cells (SMCs) into the intima is proposed to be an important process of intimal thickening in coronary atherosclerotic lesions. In the current study, we examined the possible interaction of adrenomedullin, a novel vasorelaxant peptide, and angiotensin II (Ang II) on human coronary artery SMC migration using Boyden’s chamber method. Ang II stimulated SMC migration in a concentration-dependent manner between 10 −6 and 10 −8 mol/L. This stimulation was clearly blocked by the Ang II type 1 receptor antagonist losartan but not by the type 2 receptor antagonist PD 123319. The migration stimulatory effect of Ang II was chemotactic in nature for cultured human coronary artery SMCs but was not chemokinetic. Human adrenomedullin clearly inhibited Ang II–induced migration in a concentration-dependent manner. Human adrenomedullin stimulated cAMP formation in these cells. Inhibition by adrenomedullin of Ang II–induced SMC migration was paralleled by an increase in the cellular level of cAMP. 8-Bromo-cAMP, a cAMP analogue, and forskolin, an activator of adenylate cyclase, inhibited the Ang II–induced SMC migration. These results suggest that Ang II stimulates SMC migration via type 1 receptors in human coronary artery and adrenomedullin inhibits Ang II–induced migration at least partly through a cAMP-dependent mechanism. Taken together with the finding that adrenomedullin is synthesized in and secreted from vascular endothelial cells, this peptide may play a role as a local antimigration factor in certain pathological conditions.

Abstract Adrenomedullin recently has been found to potently stimulate cAMP formation in cultured rat vascular smooth muscle cells (VSMCs). In the present study, we examined the effect of adrenomedullin on the production of a vasoconstrictive and growth-promoting peptide, endothelin-1, after stimulation with a clotting enzyme, thrombin, and a potent mitogen, platelet-derived growth factor (PDGF), in cultured rat VSMCs. Thrombin and PDGF stimulated endothelin-1 production in a dose-dependent manner. Rat adrenomedullin significantly inhibited thrombin- and PDGF-stimulated endothelin-1 production in a dose-dependent manner between 10 −7 and 10 −9 mol/L. Inhibition by rat adrenomedullin of thrombin- and PDGF-stimulated endothelin-1 production was paralleled by an increase in the cellular level of cAMP. Human adrenomedullin also inhibited thrombin- and PDGF-stimulated endothelin-1 production and increased cAMP levels. The addition of 8-bromo-cAMP, a cAMP analogue, reduced thrombin- and PDGF-induced endothelin-1 production. Furthermore, forskolin, a potent activator of adenylate cyclase, reduced thrombin- and PDGF-induced endothelin-1 production. In contrast, basal production of endothelin-1 was not altered by rat or human adrenomedullin. These results indicate that adrenomedullin inhibits not basal but thrombin- and PDGF-induced ET-1 production in cultured VSMCs probably through a cAMP-dependent process. Taken together with the finding that adrenomedullin is synthesized in and secreted from vascular endothelial cells, adrenomedullin may modulate vascular tone as a paracrine regulator partially through the inhibition of VSMC endothelin-1 production in some pathophysiological states.

Purpose Secretion of brain natriuretic peptide (BNP), a cardiac hormone, is accelerated via hypertrophied ventricles in experimental hypertension. The present study examined whether regression of left ventricular (LV) hypertrophy by long-term treatment with an angiotensin-converting enzyme inhibitor (ACEI) affects plasma BNP concentration in patients with essential hypertension. Patients and methods Thirty-one hypertensive patients with LV hypertrophy were treated with ACEI (16 with enalapril; 15 with lisinopril) for 1 year. Serial changes were recorded in LV mass index, LV systolic function, and plasma concentrations of BNP and atrial natriuretic peptide (ANP). Results ACEI therapy significantly reduced LV mass index at 6 months, and more so at 1 year. Septal and posterior wall thicknesses were also reduced. Plasma BNP and ANP were markedly elevated at study entry, but only BNP levels correlated with LV mass index. Both peptide levels declined after 6 months, and this decline was enhanced at 1 year. There was a close relation between BNP decline and LV mass index reduction overall and with enalapril and lisinopril separately. Changes in ANP and in LV mass index were not related. Conclusion Long-term ACEI therapy can reduce elevated plasma BNP. In this study, changes in BNP reflected the magnitude of regression of LVH. Plasma BNP may be a useful marker for LVH during antihypertensive therapy in patients with essential hypertension and LVH.

Heparin inhibits endothelin-1 production in cultured rat mesangial cells. The present study was designed to examine whether heparin inhibits basal or stimulated endothelin-1 production by arginine vasopressin (AVP) and platelet-derived growth factor (PDGF) in cultured rat mesangial cells. In addition, the reversibility of the heparin effect on mesangial cell endothelin-1 production was examined. AVP and PDGF stimulated endothelin-1 secretion in a concentration-dependent manner in these cells. Heparin (10 to 100 U/ml) exhibited concentration-related inhibition of AVP- and PDGF-stimulated endothelin-1 secretion. Heparin also had weak but significant inhibitory effects on basal endothelin-1 secretion in these cells. The protein kinase (PKC)-activating phorbor ester, phorbor myristate acetate (PMA), stimulated endothelin-1 secretion and heparin inhibited PMA-stimulated endothelin-1 secretion. In addition, the inhibitory effect of heparin was completely abolished in PKC-depleted mesangial cells. Mesangial cells which were exposed to a high concentration (100 U/ml) of heparin for 24 hours were capable of producing endothelin-1 after a short lag period of removal of heparin from the culture medium. These mesangial cells also showed recovery of responses to AVP and PDGF by secreting a significantly greater amount of endothelin-1 than the non-stimulated level. These results indicate that heparin potently inhibits mesangial cell endothelin-1 production, especially when stimulated by AVP or PDGF. This inhibitory effect of heparin is probably PKC dependent, and reversible.

Heparin shows blood pressure lowering effect in hypertensive patients and animal models. The present study examined the effect of heparin on vasoconstrictor endothelin-1 (ET-1) production in cultured human umbilical vein endothelial cells (ECs) to elucidate the mechanism of antihypertensive effect of heparin. Heparin suppressed both basal and thrombin-stimulated ET-1 mRNA expression paralleled with a decrease in ET-1 peptide release in a dose-dependent manner. Heparin concomitantly enhanced nitric oxide (NO) formation measured by NO2/NO3 levels and cGMP production in ECs. These enhancements were more marked when ECs were stimulated by thrombin. However, these heparin's effects were blunted in the presence of endothelium-derived nitric oxide (EDNO) synthesizing inhibitor NG-monomethyl L-arginine. Therefore, these results suggest that suppression of ET-1 production by heparin is EDNO mediated.

We examined the inhibitory effect of porcine C-type natriuretic peptide (CNP) on endothelin-1 secretion stimulated by thrombin and angiotensin II (Ang II) in cultured porcine endothelial cells. The results were compared with the effects of atrial (ANP) and brain (BNP) natriuretic peptides. Thrombin and Ang II produced a concentration-dependent stimulation of immunoreactive endothelin-1 secretion, and porcine CNP-22 potently inhibited this stimulated secretion in a concentration-dependent manner. CNP-22 had a stronger inhibitory effect than either porcine ANP(1-28) or porcine BNP-26. In addition, CNP potently increased the cellular level of cyclic guanosine 3',5'-monophosphate (GMP), with the inhibition of immunoreactive endothelin-1 secretion in response to thrombin and Ang II being paralleled by the increase in the cyclic GMP level. The increase of cyclic GMP produced by CNP was also greater than that due to porcine ANP(1-28) or porcine BNP-26. The immunoreactive endothelin-1 in the culture medium had two components on high-performance liquid chromatography; the major one corresponded to endothelin-1 (1-21) and the minor one to big endothelin-1 (porcine 1-39). Treatment with CNP did not affect this profile. Our results suggest that CNP probably inhibits the endothelin-1 secretion stimulated by thrombin and Ang II through a cyclic GMP-dependent process. The increase of cyclic GMP levels and the inhibition of immunoreactive endothelin-1 secretion produced by CNP appear to be greater than those produced by ANP or BNP.

The effect of glucocorticoids on the atrial natriuretic factor (ANF)-mediated formation of cyclic guanosine monophosphate (cGMP) by intact vascular smooth muscle cells (VSMC) was studied in rats. Cultured VSMC were obtained from the renal arteries of 14-week-old Wistar rats by the explant method. Micromolar concentrations of dexamethasone, given as pretreatment for 48 hours, suppressed the ANF-mediated response. The dexamethasone-induced suppression was detectable at 6 hours and reached a maximum 24 hours after administration in a dose-dependent manner. Inhibitors of protein synthesis blocked this effect of the glucocorticoid. The basal activity of guanylate cyclase in the dexamethasone-treated cells was lower than in the control cells. Other steroids having glucocorticoid action mimicked this suppression of the ANF-mediated response. This suppression was blocked by a glucocorticoid receptor antagonist. The results suggest that glucocorticoids suppress ANF-mediated cGMP formation by VSMC through glucocorticoid type II receptors and the induction of protein synthesis. Suppression of the ANF-mediated response may play a role in glucocorticoid-induced hypertension.

The present study was designed to examine the effect of antihypertensive therapy on plasma and atrial concentration of atrial natriuretic peptides (ANP) in spontaneously hypertensive rats (SHR) by using alpha-methyldopa and hydralazine. Methyldopa and hydralazine treatment reduced blood pressure (P less than .05, P less than .05, respectively); however, ventricular weight was reduced by methyldopa (P less than .05) but not by hydralazine. Plasma ANP concentration in untreated SHR was higher than that observed in Wistar-Kyoto rats (WKY). Methyldopa treatment decreased plasma ANP concentration, but hydralazine treatment did not. Moreover, plasma ANP concentration and ventricular weight were positively correlated in untreated and treated SHR. The left atrial ANP concentration in untreated SHR was lower than that observed in WKY. Methyldopa treatment increased left atrial ANP concentration, but hydralazine treatment did not. These results suggest that the ANP release from the left atrium is chronically stimulated in adult SHR, and that a decrease in plasma ANP concentration by methyldopa treatment is, in part, associated with the decline of ANP release from the heart due to the reductions of blood pressure and cardiac hypertrophy.

Heparin shows a blood pressure-lowering effect in various hypertensive rat models. This study was designed to examine the effect of heparin on vasoconstrictor endothelin-1 (ET-1) production by cultured human umbilical vein endothelial cells (HUVECs). ET-1 and cyclic GMP levels in the medium were determined by radioimmunoassay. ET-1 mRNA was quantified by densitometric Northern blot analysis. ET-1 was released into the medium in a time-dependent manner, and its release was augmented by thrombin (10 U/ml). Heparin suppressed both basal and thrombin-stimulated ET-1 secretion and its mRNA expression in a dose-dependent manner. Heparin suppressed ET-1 mRNA expression in a time-dependent fashion. Heparin did not suppress both basal and thrombin-stimulated ET-1 production in the presence of NG-monomethyl-L-arginine (L-NMMA) (10(-5) M). The production of cGMP stimulated by thrombin was significantly enhanced by heparin, but not in the presence of L-NMMA (10(-5) M). Heparin may suppress vasoconstrictor ET-1 production mediated by the enhancement of endothelium-derived nitric oxide in HUVECs.

Several mechanisms other than the inhibition of systemic and local formation of angiotensin II (Ang II) have been proposed to play a role in mediating the hypotensive effects of angiotensin-converting enzyme (ACE) inhibitors. In the present study, we measured plasma levels of nitric oxide (NO) and the related vasoactive factors bradykinin, 6-keto prostaglandin F1alpha (6-keto PGF1alpha) a stable metabolite of prostacyclin, and cyclic guanosine-3',5'-monophosphate (cGMP) before and after a 4-week treatment with the ACE inhibitor lisinopril in 17 patients with essential hypertension. Plasma NO levels were measured by the Griess method after conversion of nitrate to nitrite. Long-term lisinopril treatment significantly reduced blood pressure and increased plasma NO and 6-keto PGF1alpha. The treatment also tended to increase plasma levels of bradykinin and cGMP, but not to a significant extent. The posttreatment NO level was inversely correlated with posttreatment systolic, diastolic, and mean blood pressure (n = 17, r= -.68, P.01, n = 17, r= -.54, P.05, and n = 17, r= -.66, P.01, respectively). The posttreatment bradykinin level was also modestly correlated with posttreatment systolic and mean blood pressure (n = 17, r = -.51, P.05 and n = 17, r = -.55, P.05, respectively). In contrast, posttreatment 6-keto PGF1alpha and cGMP levels were not correlated with posttreatment systolic, diastolic, or mean blood pressure. These findings raise the possibility that increased formation of NO and bradykinin, as well as inhibition of the renin-angiotensin system, contribute to the hypotensive effect of the ACE inhibitor observed in our hypertensive patients.

Background —To explore the role of intracellular oxidative stress in high glucose–induced atherogenesis, we examined the effect of probucol and/or α-tocopherol on the migration and growth characteristics of cultured rabbit coronary vascular smooth muscle cells (VSMCs). Methods and Results —Chronic high-glucose-medium (22.2 mmol/L) treatment increased platelet-derived growth factor (PDGF)-BB–mediated VSMC migration, [ 3 H]thymidine incorporation, and cell number compared with VSMCs treated with normal-glucose medium (5.6 mmol/L+16.6 mmol/L mannose). Probucol and α-tocopherol significantly suppressed high glucose–induced increase in VSMC migration, cell number, and [ 3 H]thymidine incorporation. Probucol and α-tocopherol suppressed high glucose–induced elevation of the cytosolic ratio of NADH/NAD + , phospholipase D, and membrane-bound protein kinase C activation. Probucol, α-tocopherol, and calphostin C improved the high glucose–induced suppression of insulin-mediated [ 3 H]deoxyglucose uptake. Chronic high-glucose treatment increased the oxidative stress, which was significantly suppressed by probucol, α-tocopherol, suramin, and calphostin C. Conclusions —These findings suggest that probucol and α-tocopherol may suppress high glucose–induced VSMC migration and proliferation via suppression of increases in the cytosolic ratio of free NADH/NAD + , phospholipase D, and protein kinase C activation induced by high glucose, which result in reduction in intracellular oxidative stress.

Heparin, an anticoagulant, has been shown to reduce neointimal proliferation and restenosis following vascular injury in experimental studies, but the clinical trials of heparin in coronary balloon angioplasty have been negative. The current study, therefore, examined the effect of heparin on basal or stimulated migration by serum and platelet-derived growth factor (PDGF)-BB in cultured human coronary artery smooth muscle cells (SMCs) by Boyden's chamber method. In addition, the reversibility of the heparin effect on human coronary artery SMC migration was examined. Fetal calf serum (FCS) and PDGF-BB stimulated SMC migration in a concentration-dependent manner. Heparin in moderate to high concentration (10 to 100 U/mL) exhibited concentration-related inhibition of FCS- and PDGF-BB—stimulated SMC migration; however, a low concentration (1 U/mL) of heparin had no inhibitory effects. Heparin also had weak inhibitory effects on nonstimulated SMC migration. The SMCs that were exposed to a high concentration (100 U/mL) of heparin for 6 hours were capable of migrating after a short lag period of removal of heparin from the culture medium. These SMCs also showed recovery of responses to FCS and PDGF-BB by migrating significantly greater than the nonstimulated level. Furthermore, heparin-containing medium did not contain detached cells. These results indicate that heparin inhibits human coronary artery SMC migration, especially when stimulated by FCS or PDGF-BB, and that this inhibitory effect of hepari is reversible and not simply a function of killing cells.

Cyclosporine stimulates vasoconstrictor endothelin-1 (ET-1) synthesis. This study examined the effect of heparin on cyclosporine-induced ET-1 synthesis in Wistar rat aortic endothelial cells in culture. Cyclosporine (0.01-5 μmol/L) stimulated ET-1 mRNA expression in a dose-dependent manner. A nitric oxide synthesis inhibitor, N G -monomethyl-L-arginine (L-NMMA) (10 -5 mol/L), did not affect cyclosporine-induced ET-1 mRNA expression. Heparin (1-20 U/ml) suppressed cyclosporine-induced ET-1 mRNA expression in a dose-dependent manner. The inhibitory effect of heparin was blunted in the presence of either L-NMMA (10 -5 mol/L) or calmodulin inhibitors such as N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) (5 x 10 -5 mol/L) or calmidazolium (5 x 10 -5 mol/L) in the presence of the phosphodiesterase inhibitor 3-isobutyl 1-methylxanthine (0.1 mmol/L). These results suggest that heparin suppresses cyclosporine-induced ET-1 mRNA expression via both NO- and calmodulin-dependent pathways.

Abstract Recent findings suggest that high glucose levels may promote atherosclerosis in coronary vascular smooth muscle cells (VSMCs). To explore the intracellular mechanisms of action by which troglitazone affects this process, we examined the effect of troglitazone on the migration and growth characteristics of cultured rabbit coronary VSMCs. Treatment with chronic high glucose medium (22.2 mmol/L) for 5 days increased VSMC migration by 92%, [ 3 H]thymidine incorporation by 135%, and cell number by 32% compared with VSMCs treated with normal glucose (5.5 mmol/L glucose+16.6 mmol/L mannose) medium. Troglitazone at 100 nmol/L and 1 μmol/L significantly suppressed high glucose–induced VSMC migration by 34% and 42%, respectively, the proliferative effect (as measured by cell number) by 17% and 27%, and [ 3 H]thymidine incorporation by 45% and 60% (n=6, P 3 H]deoxyglucose uptake was blocked by a protein kinase C (PKC) inhibitor (calphostin C, 1 μmol/L) and was also improved by troglitazone without any change in insulin receptor number and affinity. The high glucose–induced insulin-mediated increase in cell number and in [ 3 H]thymidine incorporation was suppressed by troglitazone. Troglitazone (1 μmol/L) also suppressed high glucose–induced phospholipase D activation, elevation of the cytosolic NADH/NAD + ratio (as measured by the cytosolic ratio of lactate/pyruvate), and membrane-bound PKC activation. Flow cytometric DNA histogram analysis of cell cycle stage showed that high glucose–induced increase in the percentage of cells in the S phase was suppressed by 1 μmol/L troglitazone. These findings suggest that PKC may be a link between impairment of insulin-mediated glucose uptake and the increase in migration and proliferation induced by high glucose levels and that troglitazone may be clinically useful for the treatment of high glucose–induced coronary atherosclerosis.

We previously showed that plasma endothelin-1 (ET-1) concentration was increased in deoxycorticosterone acetate (DOCA)-salt-induced malignant hypertension in spontaneously hypertensive rats (SHR). In contrast, in normal SHR, this value is similar to that seen in Wistar-Kyoto (WKY) rats. The purpose of this study was to examine the effects of the new combined ET type A/type B (ETA/B) receptor antagonist, TAK-044, on the development of hypertension in this model of malignant hypertension. TAK-044 10 mg/kg, which effectively blocks both ETA and ETB receptors, was administered intraperitoneally once per day for 4 weeks in DOCA-salt SHR, and the effects on ET-1 and other parameters were compared with the same values in untreated WKY rats, untreated DOCA-salt SHR, and hydralazine-treated DOCA-salt SHR. DOCA-salt caused marked increases in blood pressure, blood urea nitrogen (BUN), serum creatinine, and plasma ET-1 concentrations in SHR. Both TAK-044 and hydralazine significantly suppressed the increase in blood pressure in DOCA-salt SHR to the same extent. Both treatments also suppressed the increase in BUN and serum creatinine, but this attenuation was less marked with hydralazine than with TAK-044. Neither TAK-044 nor hydralazine affected plasma ET-1 concentration in this model. TAK-044 significantly reduced kidney weight in DOCA-salt SHR, whereas the decrease seen with hydralazine was less marked. Prevention of DOCA-salt-induced renal structural injury (mesangial hypercellularity, glomerular sclerotic changes, and tubulointerstitial damage) in this model was clearly greater with TAK-044 treatment than with hydralazine treatment. These results suggest that endogenous ET-1 may, at least in part, contribute to renal functional and structural damage in malignant DOCA-salt SHR. Our results raise the possibility of renoprotective effects of ETA/B receptor blockers in certain forms of malignant hypertension.

Erythrocyte deformation is an important regulatory factor of the microcirculation. The present study was designed to examine whether erythrocyte deformability is altered in hypercholesterolemic patients and, if so, whether cholesterol-lowering therapy affects this parameter in these patients. The erythrocyte deformability of 37 hypercholesterolemic patients was evaluated before and after 1 year of therapy with pravastatin, an inhibitor of hepatic hydroxymethyl glutaryl coenzyme A reductase, under various shear stresses (4.7, 9.5, 23.6, 47.3, 118.1, and 236.2 dyne/cm2) using laser diffractometry. At study entry, erythrocyte deformability under 4.7 and 9.5 dyne/cm2 shear stress, which is actually observed in human vessels, was reduced compared with that in 20 age-matched normocholesterolemic subjects and was inversely correlated with serum cholesterol and low-density lipoprotein (LDL) cholesterol. Pravastatin therapy for 1 year, which reduced serum cholesterol from 288 ± 28 to 223 ± 20 mg/dL, significantly improved erythrocyte deformability by approximately 20%. There was a significant relation between the improvement of erythrocyte deformability and the reduction of serum cholesterol or LDL cholesterol. The results suggest that erythrocyte deformability is reduced in hypercholesterolemic patients, and that long-term cholesterol-lowering therapy can improve reduced erythrocyte deformability, which may contribute to the improvement of organ perfusion.

OBJECTIVE The present study was designed to examine whether adrenomedullin affects fetal calf serum (FCS)-stimulated proliferation in cultured rat vascular smooth muscle cells (VSMCs). METHODS Rat VSMCs were grown from explants of Sprague-Dawley rat aorta and were grown using the standard cell culture method. After incubation for 24 h with various concentrations of adrenomedullin in the presence of 5% FCS, trichloroacetic acid-insoluble tritiated thymidine was measured in a liquid scintillation counter. After incubation for 48 h, cell counts were performed. Cyclic adenosine 3',5'-monophosphate (AMP) levels were determined by radioimmunoassay. RESULTS Rat adrenomedullin exhibited concentration-dependent inhibition of the FCS-stimulated increase in thymidine incorporation between 10(-7) and 10(-9) mol/l and of cell number at 10(-7) mol/l. However, the calcitonin generelated peptide (CGRP) receptor antagonist human CGRP(8-37) abolished these antiproliferative effects of rat adrenomedullin. Inhibition by adrenomedullin of FCS-stimulated cellular proliferation was paralleled by an increase in the cellular level of cyclic AMP. 8-Bromocyclic AMP, a cyclic AMP analogue, and forskolin, an activator of adenylate cyclase, inhibited the FCS-stimulated increase in thymidine incorporation and cell number. CONCLUSIONS These results suggest that adrenomedullin inhibits FCS-stimulated proliferation in cultured rat VSMCs, probably through a cyclic AMP-dependent process. Taken together with the finding that adrenomedullin is synthesized in and secreted from vascular endothelial cells, adrenomedullin may play a role as an antiproliferative factor for VSMCs in a paracrine fashion.

Abstract Vascular remodeling is a key process in the pathophysiology of atherosclerosis. Recent evidence suggests that high glucose levels may function as a vascular smooth muscle growth and proliferation–promoting substance. To explore the role of the polyol pathway in this process, we examined the effect of an aldose reductase inhibitor (ARI), epalrestat, on the growth characteristics of cultured rat vascular smooth muscle cells (VSMCs). Epalrestat (10 nmol/L, 1 μmol/L) significantly suppressed the high glucose–induced proliferative effect as measured by [ 3 H]thymidine incorporation by 67% and 82% in cell number, suggesting ARI as an antimitogenic factor. In VSMCs, epalrestat (10 nmol/L, 1 μmol/L) significantly suppressed the high glucose–induced incorporation of [ 3 H]leucine by 45% and 58% with the concomitant reduction of the cell size estimated by flowcytometry. Epalrestat (1 μmol/L) also suppressed high glucose–induced intracellular NADH/NAD + increase and membrane-bound protein kinase C activation. These results indicate that this ARI possesses an antiproliferative and antihypertrophic action on VSMCs induced by high glucose possibly through protein kinase C suppression.

1. The present study examined the effect of heparin on nitric oxide (NO) formation and cyclic guanosine 3', 5'-monophosphate (cGMP) levels in cultured aortic endothelial cells (EC) from spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats. 2. Bradykinin (BK), adenosine diphosphate (ADP), Ca2+ ionophore A23187 (Io) and endothelin-3 (ET-3) stimulated the production of NO and cGMP. No significant difference was observed in both NO and cGMP production in EC between WKY and SHR. 3. Heparin enhanced BK-, ADP-, Io- and ET-3-stimulated NO and cGMP production. These enhancements by heparin in EC were significantly greater in SHR than in WKY. 4. Both NO formation and cGMP production stimulated by the agonists and/or heparin were blocked in the presence of NG-monomethyl-L-arginine (LNMMA, 10(-5) mol/L). 5. Increased sulphur level was observed on heparin-treated SHR EC surface compared with that on control SHR EC or on heparin-treated WKY EC surface. 6. These results suggest that heparin promotes agonist-induced NO-cGMP response in cultured EC from SHR.

The modulation of dopamine DA1 receptors of cultured rat renal arterial smooth muscle cells by phorbol ester, glucocorticoid and sodium chloride was studied. The extent of [3H]Sch-23390 binding to phorbol ester-treated cell was increased without any change in the dissociation constant (Kd). At a concentration of 10 nmol/l, the synthetic glucocorticoid dexamethasone increased maximum receptor binding (Bmax) but had no effect on the Kd. 100 mmol/l sodium chloride did not change Bmax, but increased the Kd for DA1 receptor. The production of cAMP in response to DA1 receptor stimulation was enhanced without any change of the adenylate cyclase activity. The glucocorticoid effect on DA1 of arterial smooth muscle cells became apparent after hours of incubation in the presence of the steroid and was significantly inhibited by cycloheximide (10 micrograms/ml) and by the glucocorticoid receptor antagonist RU-38486, indicating that the effect required protein synthesis through glucocorticoid receptors. Treatment of cells with 1 mumol/l dexamethasone for 24 h increased basal and DA1-stimulated adenylate cyclase activity. Basal adenylate cyclase was decreased by sodium chloride in a dose-dependent manner. These results suggest differential control of DA1 receptors on vascular smooth muscle cells by protein kinase C, glucocorticoid or sodium chloride.

The modulation of dopamine DA1 receptors of cultured rat renal arterial smooth muscle cells by glucocorticoid and sodium chloride was studied. At a concentration of 10 nM, the synthetic glucocorticoid dexamethasone increased maximum receptor binding but had no effect on the dissociation constant. However, the maximum binding of [3H]Sch-23390 in cells treated with 100 mM sodium chloride did not change. However, the dissociation constant for DA1 receptor was increased by adding sodium chloride. The glucocorticoid effect on DA1 of arterial smooth muscle cells became apparent after hours of incubation in the presence of the steroid and was significantly inhibited by cycloheximide (10 micrograms/ml) or by the glucocorticoid receptor antagonist RU-38486, indicating that the effect required protein synthesis through glucocorticoid receptors. Treatment of cells with 1 microM dexamethasone for 24 h increased basal and DA1-stimulated adenylate cyclase activity. Basal adenylate cyclase was decreased by sodium chloride in a dose-dependent manner. These results suggest differential control of DA1 receptors on vascular smooth muscle cells by glucocorticoid or sodium chloride.

Heparin given sc consistently lowers blood pressure in spontaneously hypertensive rats (SHR). This study was designed to examine heparin's effect on vasoconstrictor endothelin-1 production in cultured aortic endothelial cells (EC). Aortic EC from SHR and normotensive Wistar Kyoto rats (WKY) were cultured and incubated with or without different concentrations of heparin. Heparin suppressed endothelin-1 release and endothelin-1 mRNA expression in a dose- and a time-dependent fashion in both WKY and SHR. The suppressive effects were more augmented in SHR than in WKY: SHR versus WKY--endothelin-1 level at 6 h = 8 +/- 1.8 versus 14 +/- 2.2 pg/10(6) cells (P < 0.01) and mRNA expression--85 versus 52% maximal inhibition by heparin, 10 U/mL (=70 micrograms/mL) (P < 0.01). When heparin was added with transcriptional inhibitor actinomycin D and incubated for 30 min, no further inhibition of endothelin-1 mRNA level measured after another 30 minutes was observed compared with the endothelin-1 mRNA level in cultured EC of SHR treated with just actinomycin D at 30 min. These results suggest that heparin regulates endogenous endothelin-1 production by cultured EC, probably at the transcriptional level, and that this effect is more marked in SHR than in WKY.

The blood pressure was decreased after chronic treatment with enalapril, MK-954, and hydralazine in deoxycorticosterone acetate (DOCA)-salt-induced malignant hypertension of spontaneously hypertensive rats (SHR); however, ventricular weight and plasma brain natriuretic peptide (BNP) concentration were decreased after enalapril and MK-954 but not after hydralazine. The BNP secretory rates from the ventricle in enalapril- and MK-954-treated DOCA-salt SHR were decreased to approximately 50% of those in untreated DOCA-salt SHR. The BNP secretory rate from the ventricle was positively correlated with ventricular weight in untreated and treated DOCA-salt SHR. In contrast, acute administration of captopril or MK-954 did not decrease the BNP secretory rate from the heart. Results suggest that the decrease in plasma BNP after enalapril and MK-954 is attributed to a decline in the secretion from the ventricle but not from the atrium. The reduction in ventricular mass appeared to be related to this decline.

The interaction between dopamine DA1 receptors and a phorbol ester was studied to elucidate the role of protein kinase C in the response of this receptor. The in vitro binding of [3H]Sch 23390 to DA1 receptor sites on vascular smooth muscle cells was saturable. The extent of [3H]Sch 23390 binding to phorbol ester-treated cells was increased without any change in the dissociation constant. The production of adenosine 3',5'-cyclic monophosphate (cAMP) in response to DA1 receptor stimulation was enhanced by preincubation of vascular smooth muscle cells with the phorbol ester for 4 h. However, no enhancement was observed when the medium used for preincubation was supplemented with a protein kinase C inhibitor. Direct stimulation of stimulatory guanine nucleotide-binding regulatory protein with 5-guanylylimidodiphosphate and direct stimulation of adenylate cyclase with forskolin produced no significant differences in cyclase levels between phorbol ester-treated and untreated cells. These results suggest that activation of protein kinase C triggers an increase in the membrane expression of DA1 receptors, thereby enhancing receptor-coupled cAMP generation.

Previous studies have shown that chronic subcutaneous administration of heparin significantly reduces blood pressure in hypertensive rats. The intracellular mechanisms of how heparin prevents smooth muscle cell proliferation remain unclear. This study was designed to examine whether heparin affects endothelin-1 action and production, to further elucidate the mechanism of the antihypertensive effect of heparin. Four-week treatment with heparin (300 U/day sc) significantly decreased blood pressure in spontaneously hypertensive rats (SHR; 199 +/- 8 vs. 164 +/- 9 mmHg; P < 0.001) and completely blunted pressor response to endothelin-1 in SHR. Heparin treatment did not decrease blood pressure response nor did it attenuate blood pressure responses to endothelin-1 in Wistar-Kyoto rats (WKY). Heparin significantly suppressed endothelin-1-induced increase in intracellular calcium concentration and inositol 1,4,5-trisphosphate level in cultured vascular smooth muscle cells in a dose-dependent manner and endothelin-1 release from cultured endothelial cells. These inhibitions were significantly more pronounced in SHR than in WKY. In conclusion, our findings indicate that the antihypertensive effect of heparin is mediated, at least in part, by the inhibition of endothelin-1 action on vascular smooth muscle cells and endothelin-1 production from endothelial cells.

Angiotensin II stimulates endothelin-1 secretion in cultured rat mesangial cells. The present study was designed to test two hypotheses: (1) that angiotensin II (Ang II) stimulates endothelin-1 secretion in cultured rat mesangial cells and (2) that atrial and brain natriuretic peptides (ANP and BNP) inhibit the above-mentioned secretion in these cells. Ang II stimulated immunoreactive (ir) endothelin-1 secretion in a concentration-dependent manner between 10 -8 M and 10 -7 M. The protein kinase C (PKC) inhibitors from two chemical classes, H7 and staurosporine, inhibited secretion following such stimulation. The stimulatory effect of Ang II was also abolished in the PKC-depleted cells. Rat ANP(1-28) and rat BNP-45, which are the respective major circulating forms of ANP and BNP in rats, potently inhibited Ang II-stimulated endothelin-1 secretion in a concentration-dependent manner. Inhibition by ANP and BNP of Ang II-stimulated endothelin-1 secretion was paralleled by an increase in the cellular level of cyclic guanosine 5′-monophosphate (GMP). The addition of a cyclic GMP analogue, 8-bromo cyclic GMP, reduced the stimulated endothelin-1 secretion. Rat ANP(5-25) was less effective that rat ANP(1-28) with respect to inhibiting ir-endothelin-1 secretion and increasing cellular cyclic GMP. These findings indicate that Ang II stimulates endothelin-1 secretion in cultured rat mesangial cells by a mechanism probably involving activation of PKC, and that rat ANP and BNP inhibit this stimulated secretion through a cyclic GMP-dependent process.

Cultured porcine endothelial cells (EC) released immunoreactive endothelin-1 (ir-endothelin-1) and big endothelin-1 (ir-big endothelin-1) into the medium in a time-dependent way. Reverse-phase high-pressure liquid chromatography coupled with radioimmunoassay showed that the major component of ir-endothelin-1 corresponded to standard endothelin-1 (1-21) and that the major component of ir-big endothelin-1 corresponded to standard big endothelin-1 (porcine 1-39). This release was strongly inhibited by cycloheximide and was, therefore, related to de novo protein synthesis. The release of greater amounts was stimulated by thrombin. The protein kinase C (PKC) inhibitors from two chemical classes, H7 and staurosporine, inhibited release following such stimulation in a relatively dose-dependent way. Neither H7 nor staurosporine affected the basal release of both endothelin-1 and big endothelin-1. Phorbol myristate acetate, which activates PKC and the Ca2+ ionophore A23187, stimulated the release of ir-endothelin-1 and ir-big endothelin-1 in a dose-dependent way, respectively. In addition, the combination of both compounds had a synergistic effect. An inactive enantiomer of phorbol ester, 4 alpha-phorbol-12,13-didecanoate had no effect on the release of ir-endothelin-1 and ir-big endothelin-1. These results suggest that cultured EC release endothelin-1 and big endothelin-1 simultaneously, and that thrombin stimulates this release by a mechanism that probably involves intracellular Ca2+ mobilization and the activation of PKC.

Effects of endothelin-3 on the secretion of endothelin-1 and other endothelium-derived substances were investigated in cultured human umbilical vein endothelial cells. The present binding study showed two distinct subpopulations of binding sites for endothelin-3 with higher and lower affinities in cultured human endothelial cells. Endothelin-3 caused an increase in intracellular Ca2+ and inositol 1,4,5-trisphosphate levels and activated protein kinase C in a dose-dependent manner. Endothelin-3 also caused an increase in [3H]thymidine incorporation into cellular DNA and stimulated the production of cyclic guanosine 3',5'-monophosphate, 6-ketoprostaglandin F1 alpha, and immunoreactive endothelin-1 in cultured human endothelial cells. NG-Monomethyl L-arginine (3 x 10(-4) mol/l) and indomethacin (10(-5) mol/l) enhanced endothelin-3-induced endothelin-1 production. These results suggest that endothelin-3 bound to its specific receptors and then caused phosphoinositide breakdown, subsequently mobilizing intracellular Ca2+ and leading to protein kinase C activation and the initiation of DNA synthesis, resulting in the stimulation of endothelin-1 production by human endothelial cells. Furthermore, this endothelin-1 production may be suppressed by endothelium-derived relaxing factor and prostacyclin produced in response to endothelin-3 in cultured human endothelial cells.

We examined the inhibition by atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) of endothelin-1 secretion stimulated by angiotensin II (ANGII) and thrombin using cultured human umbilical-vein endothelial cells. ANGII and thrombin dose-dependently stimulated immunoreactive (ir) endothelin-1 secretion. Human ANP(1-28) and human BNP-32 both inhibited such secretion in a dose-dependent way. Inhibition of this secretion by ANP and BNP was paralleled by an increase in the level of cyclic guanosine 5'-monophosphate (GMP). The addition of a cyclic GMP analogue, 8-bromo cyclic GMP, reduced this stimulated secretion. Rat ANP(5-25) was weaker than human ANP(1-28) at inhibiting ir-endothelin-1 secretion and increasing cyclic GMP in the cells. ir-Endothelin-1 in the medium consisted of two components separated by high pressure liquid chromatography; the major one corresponded to endothelin-1(1-21) and the minor one corresponded to big endothelin-1(1-38). Treatment with ANP and BNP did not affect this profile. These findings suggest that human ANP and BNP inhibit endothelin-1 secretion stimulated by ANGII and thrombin in these cells through a cyclic GMP-dependent process. Taken together with endothelin stimulation of ANP and BNP secretion from the heart, our results suggest the existence of a cardiac-endothelium feedback.

In a randomized study in 26 elderly patients with mild essential hypertension, acute effects of alpha- and beta-adrenoceptor blockade on plasma ANP levels were examined at rest and during ergometric exercise. Plasma ANP level and LVEF were measured before and after administration of prazosin (an alpha 1-adrenergic blocker), atenolol (a cardioselective beta-adrenergic blocker), or carteolol (a nonselective beta-adrenergic blocker). Plasma ANP level was increased by exercise. Carteolol and atenolol increased plasma ANP levels at rest and during exercise, but the effect of atenolol was not statistically significant. Prazosin significantly suppressed the ANP values at rest and during exercise. The LVEF was increased by prazosin and decreased by beta-blockers, especially by carteolol. Multivariate regression analysis showed that LVEF was the most significant predictor of the plasma ANP level at maximal exercise; the resting blood pressure and heart rate were not predictors of this value. The results showed that single administrations of an alpha-blocker and a nonselective beta-blocker had opposite effects on the plasma ANP level both at rest and during exercise in elderly patients with mild essential hypertension. The observed difference in the ANP response seems to be related to changes in left ventricular function rather than changes in blood pressure or heart rate.

This study was designed to investigate the effect of propranolol on endothelin (ET)-1-induced increase in cytosolic Ca2+ [( CA2+]i) in cultured vascular smooth muscle cells (VSMCs) obtained from rat aorta. Propranolol (0.01 to 1 mmol/L) reduced the changes in the initial transient and the later sustained phase of [Ca2+]i induced by 100 nmol/L ET-1 as well as 10 mmol/L procaine. The inhibitory action of propranolol on the change in [Ca2+]i induced by ET-1 may be involved in one of the mechanisms of propranolol-induced reduction of vasospasms.

The present study was designed to examine the effect of thrombin on endothelin production by cultured human umbilical vein endothelial cells (HUVEC) by radioimmunoassay. Cultured HUVEC released immunoreactive endothelin-1 (ir-endothelin-1) into the medium in a time-dependent manner. This release was inhibited completely by 10 micrograms/mL cycloheximide and is, therefore, directly related to de novo protein synthesis. Further endothelin release was stimulated by the addition of thrombin to the culture medium. Thrombin-induced ir-endothelin-1 release also was inhibited completely by 10 micrograms/mL cycloheximide. These results suggest that HUVEC in culture produce ir-endothelin-1 slowly, but continuously, and that this production is stimulated by thrombin.

We recently demonstrated that porcine endothelin increases intracellular calcium ion concentration ([Ca2+]i) in monolayers of cultured vascular smooth muscle cells isolated from rat renal arteries. The present study was designed to examine the effect of porcine endothelin on [Ca2+]i in the platelets of rats. [Ca2+]i was serially measured with the fluorescent dye, fura 2, and the photoprotein, aequorin. Endothelin (10(-9) mol/L, 10(-8) mol/L, and 10(-7) mol/L) did not induce any change in [Ca2+]i in the platelets of rats while thrombin (2 U/mL) dramatically increased [Ca2+]i as measured by these methods. Our results indicate that [Ca2+]i in rat platelets is not influenced by porcine endothelin.

1. Basal endothelin-1 (ET-1) production in mesangial cells of spontaneously hypertensive rats (SHR) was not different from that of Wistar-Kyoto (WKY) rats, although a trend toward increased ET-1 production was observed in these cells of SHR. 2. Thrombin and platelet-derived growth factor (PDGF) stimulated ET-1 production in a concentration-dependent manner in these cells of both rat strains, but thrombin- and PDGF-induced stimulation of ET-1 production were clearly greater in cells of SHR than WKY rats. 3. The protein kinase C (PKC)-activating phorbol ester, phorbol myristate acetate, stimulated ET-1 production in cells of both rat strains, but this stimulation was significantly greater in cells of SHR than in cells of WKY rats. 4. An inactive enantiomer of phorbol ester, 4alpha-PDD, had no effect on the ET-1 production in these cells of both rat strains. 5. Neither thrombin nor PDGF stimulated ET-1 production in PKC-depleted cells of both rat strains.

To determine the involvement of nitric oxide production in hemodialysis-induced hypotension.Examination of nitric oxide synthesis, cyclic guanosine 3'5'-monophosphate (cGMP) levels, and endothelin-1 levels in plasma before and after hemodialysis.Veterans Affairs medical center.13 patients with end-stage renal failure who were receiving hemodialysis: Six patients had hypotensive episodes during dialysis and 7 did not.Patients received heparin at a bolus dose of 2000 U at the initiation of dialysis followed by 1000 U/h during 4-hour hemodialysis sessions.Nitric oxide production markedly increased during hemodialysis-induced hypotensive episodes; this increase was not seen in patients who did not have a hypotensive episode. In both groups, the plasma cGMP and endothelin-1 levels decreased after hemodialysis. According to multiple regression analysis, standard coefficients of nitric oxide production, plasma cGMP levels, and endothelin-1 levels with mean blood pressure after hemodialysis were -0.743, -0.07, and 0.31, respectively.Nitric oxide production increased in patients who had a hypotensive episode during hemodialysis but did not increase in those who did not have a hypotensive episode.

The effect of glucocorticoid on the prostaglandin E1 (PGE1)-mediated cyclic AMP (cAMP) formation by vascular smooth muscle cells (VSMC) from renal arteries (RA) was studied in rats. Dexamethasone (DEX) at concentrations ranging from 10(-10) to approximately 10(-8) mol/l dose-dependently potentiates the PGE1-mediated response. This facilitation began at 6 h and reached its maximum after 24 h of DEX administration. Aldosterone (10(-6) mol/l) did not affect the dose-response curve of PGE1. Inhibitors of protein and RNA synthesis blocked this glucocorticoid effect. The basal activity of adenylate cyclase in DEX-treated cells was twice as high as in control cells. Treatment of VSMC with DEX increased cholera toxin- and pertussis toxin-stimulated adenylate cyclase activity. DEX treatment also augments forskolin-stimulated adenylate cyclase activity. These results suggest that DEX increases PGE1-mediated cAMP formation of VSMC from RA through a mechanism that involves the induction of protein synthesis, and that the activation of the catalytic unit may play some role in this facilitating process.

The present study was designed to examine the possible effect of long-term treatment with diltiazem on plasma and atrial concentrations of atrial natriuretic peptides (ANP) in spontaneously hypertensive rats (SHR). Diltiazem treatment reduced blood pressure and ventricular weight in SHR. Plasma ANP concentration in untreated SHR was higher than Wistar-Kyoto rats (WKY). Diltiazem treatment decreased plasma ANP concentration in SHR near to the level of WKY; moreover, plasma ANP concentration was correlated with blood pressure and ventricular weight in treated and untreated SHR. Left atrial ANP concentration in untreated SHR was lower than WKY. Diltiazem treatment increased left atrial ANP concentration in SHR, but this effect was not noted in WKY. These results suggest that the ANP release from the left atrium is chronically stimulated in adult SHR, and that the prevention of an increase in plasma ANP by diltiazem treatment may be, in part, attributed to the improvement of cardiac overload induced by reductions in blood pressure and cardiac hypertrophy.

Heparin-induced endothelial cell cytoskeletal reorganization: A potential mechanism for vascular relaxation. We have previously shown that heparin given subcutaneously on a daily basis lowers blood pressure in hypertensive rat models, and that this blood pressure lowering effect is endothelium-dependent. The present study describes the effects of heparin on endothelial cell (EC) apical surface structures and cytoskeletal elements, namely, actin and vimentin as well as EC proliferative activity. The EC line (CRL 1998) was cultured, treated with different concentrations of heparin (0, 50, 100, 500 U/ml) for 4, 24 or 48 hours, and fixed for scanning electron microscopy (SEM), and immunofluorescence microscopy (IFM) studies. Enzyme-linked immunosorbent assays (ELISA) and flow cytometric analysis were performed on EC monolayers treated with different concentrations of heparin for quantitative detection of actin and vimentin. By SEM study the cell surface showed generalized smoothing as a result of blunting of surface microvilli with increasing time of exposure and dosage of heparin. By IFM study, the detectable actin signal within ECs became progressively reduced in both its cellular distribution and the apparent number of cells that remained reactive. By 48 hr/500 U heparin, the actin signal was almost undetectable. Vimentin showed a moderate reduction in the cellular distribution of labeling. Quantitatively, actin was significantly reduced after the 24 hour treatment with a higher dose of heparin (500 U/ml), from a baseline optical density (OD) of 1.12 ± 0.060 to 0.866 ± 0.008 (P < 0.0027). After 48 hours of treatment at both 100 U/ml and 500 Usol;ml heparin, actin was significantly reduced from a baseline OD of 1.347 ± 0.063 to 1.090 ± 0.039 (P < 0.0039) and 0.844 ± 0.074 (P < 0.008), respectively. However, vimentin was significantly reduced only after 48 hours of treatment with a high dose of heparin (500 U/ml), from baseline OD 1.82 ± 0.052 to 1.41 ± 0.004 (P < 0.002). The flow cytometric findings were virtually identical to the ELISA data for actin and vimentin. These qualitative and quantitative changes in actin and vimentin are consistent with apparent smoothing and relaxation of the EC's apical surface. Labeling with the cell cycle marker MIB-1 (monoclonal antibody Ki-67), showed a progressive reduction in the observed intensity in heparin treated cells with substantially fewer cells being positive. After a 48 hour treatment with heparin (500 U/ml), most ECs displayed only dim labeling of the nucleolus. This finding is consistent with an antiproliferative effect. Overall, these findings are additive to our previous observations, and demonstrate that heparin causes EC cytoskeletal reorganization which is a potential mechanism for vascular relaxation.

The effect of glucocorticoids on the dopamine (DA)-mediated cyclic adenosine monophosphate (cAMP) by intact vascular smooth muscle cells (VSMC) was studied in rats. Cultured VSMC were obtained from renal arteries of 14-week-old Wistar-Kyoto rats by explant method. Micromolar concentrations of dexamethasone (DEX) pretreatment for 48 hours potentiated DA-mediated response without any change of affinity constant. However, micromolar concentrations of aldosterone pretreatment for 48 hours had almost no effect on DA-mediated response. The DEX-induced facilitation began at 6 hours and reached maximum at 24 hours after DEX administration in a dose-dependent manner. Inhibitors of protein and RNA synthesis blocked this glucocorticoid effect. The basal activity of adenylate cyclase in DEX-treated cells was twofold higher than that in control cells. Treatment of VSMC with DEX increased cholera toxin-stimulated and forskolin-stimulated adenylate cyclase activity. However, pertussis toxin treatment did not augment or reduce the effect of DEX treatment. These results suggest that glucocorticoids increase DA-mediated cAMP formation by VSMC through glucocorticoid type II receptors and the induction of protein synthesis and that the activation of the catalytic unit may play some role in this facilitation.

In seven patients with mild to moderate essential hypertension, the effect of a new angiotensin converting enzyme (ACE) inhibitor, cilazapril, on atrial natriuretic factor (ANF) was examined at rest and during exercise. The exercise protocol consisted of three fixed workloads (25, 50 and 75W) every 4 min with the use of a sitting bicycle ergometer. Cilazapril reduced blood pressure without increasing the heart rate, both at rest and during exercise. The resting pre-exercise left ventricular ejection fraction was not altered. The plasma ANF level was increased by exercise, but at the 75-W workload it was decreased by cilazapril; the resting plasma ANF value was not altered. At the 75-W workload the plasma ANF level was inversely correlated to the left ventricular ejection fraction (n = 14, r = -0.64, P less than 0.01). These results suggest that the observed decrease in the plasma ANF level during exercise induced by cilazapril is, in part, related to an improvement in the cardiac overload, and that cilazapril may be effective in physically active hypertensive patients.

Cultured porcine endothelial cells derived from aortas spontaneously released immunoreactive endothelin into the medium in a time-dependent manner. This release was completely inhibited by cycloheximide and is, therefore, directly related to de novo protein synthesis. The endothlin-induced release was further stimulated by adrenaline. Adrenaline-induced stimulation was completely inhibited by the alpha-adrenergic blocker phentolamine and was not inhibited by the beta-adrenergic blocker propranolol. Cycloheximide completely prevented the adrenaline-stimulated as well as the basal release. These results suggest that cultured endothelial cells release endothelin slowly but continuously and that this release can be stimulated by adrenaline via alpha-adrenergic receptors. We speculate that the endothelium generates vasoconstrictor signals through endothelin production, thus contributing to the regulation of vascular tone.