The molecular heterogeneity of the smg-21/Krev-1/rap1 proteins, a GTP-binding protein having the same effector domain as ras p21s, in bovine aortic smooth muscle membranes.

smg p21 is a member of the ras p21/ras p21-like small GTP-binding protein (G protein) superfamily, having the same putative effector domain as ras p21s. In the preceding report, we showed that smg p21 was a major G protein in bovine aortic smooth muscle membranes. Recently, two different smg p21 cDNA clones, designated smg-21A and -B, were isolated from a bovine brain cDNA library. In the present studies, we resolved the bovine aortic smg p21 fraction into two distinct G protein fractions on hydroxyapatite column chromatography and purified them separately to near homogeneity (22K G1 and -2). Both 22K G1 and -2 were specifically recognized by an anti-smg p21 polyclonal antibody. 22K G1 and -2 were identified as smg p21B and -A, respectively, by peptide map and amino acid sequence analyses. Purified smg p21A and -B showed GDP/GTP-binding and GTPase activities similar to each other. The GTPase activities of smg p21A and -B were equally stimulated by smg p21 GTPase activating protein 1 and -2. Moreover, both smg p21A and -B were phosphorylated by cyclic AMP-dependent protein kinase with a stoichiometry of one phosphate/molecule of protein. These results indicate that smg p21A and -B coexist in bovine aortic smooth muscle membranes and suggest that smg p21A and -B may serve as intermediates for cyclic AMP actions.

Comparative effects of intracoronary vasodilators on restoring coronary perfusion during flow-reducing coronary stenosis in the dog.

The effects of intracoronary injection of nitroglycerin, adenosine, nifedipine and prostacyclin on restoring coronary perfusion during flow-reducing partial coronary obstruction in anesthetized dogs were studied. Coronary obstruction was obtained by inflation of an intraluminal balloon to decrease coronary blood flow and rate of rise in left ventricular pressure (dP/dt) by approximately 30 to 40 and 10%, respectively. Nitroglycerin (0.01 to 10 micrograms/kg per min) increased coronary blood flow and distal coronary pressure and decreased stenosis resistance associated with improved left ventricular dP/dt depending on its dose. In contrast, adenosine (0.3 to 1.0 micrograms/kg per min) decreased coronary blood flow and distal coronary pressure and intensified stenosis resistance associated with depression of left ventricular dP/dt. Nifedipine and prostacyclin caused divergent effects on the coronary circulation related to each dose. Nifedipine (0.01 and 0.1 micrograms/kg per min) and prostacyclin (0.01 micrograms/kg per min) increased coronary blood flow and distal coronary pressure and reduced stenosis resistance. Nifedipine (1.0 micrograms/kg per min) and prostacyclin (0.3 micrograms/kg per min) did not increase coronary blood flow, but reduced distal coronary pressure and intensified stenosis resistance. Thus, the vasodilators produced different effects on restoration of coronary perfusion during pliable severe coronary stenosis. Nitroglycerin and lower doses of nifedipine and prostacyclin improved coronary perfusion due to selective or preferential dilation of large coronary arteries. Adenosine and higher doses of nifedipine and prostacyclin had deleterious effects on the coronary circulation due to potent arteriolar vasodilation.

Endothelium-dependent vasodilating effect of substance P during flow-reducing coronary stenosis in the dog.

The effects of substance P, a putative central and peripheral neurotransmitter, on coronary vasculature and its mechanisms were studied in 31 anesthetized open chest dogs. Without coronary stenosis, intracoronary infusion of substance P (0.001 to 1 pmol/kg per min) for 40 s increased coronary blood flow up to 173 +/- 10.7% in dose-dependent fashion. Application of coronary stenosis created by an inflated intraluminal microballoon that preserved active vasomotion of the stenosed segment produced a pressure gradient of 34 +/- 2 mm Hg, a decrease in rest coronary blood flow of 21 +/- 1.6% and significant depression of the rate of rise in left ventricular pressure (dP/dt). During coronary stenosis, substance P increased coronary blood flow up to 150 +/- 9.4%, lowered mean distal coronary pressure and decreased stenosis resistance in dose-dependent fashion. After endothelial denudation of the proximal part of the coronary artery, the substance P-induced increments in coronary blood flow during coronary stenosis were abolished. In vitro measurements of isometric tension from both intact and denuded portions of coronary arteries confirmed a marked inhibition of substance P-induced relaxation in the denuded segments. These results show the obligatory role of the endothelium in substance P-induced coronary artery dilation. Furthermore, intracoronary infusion of substance P (1 pmol/kg per min) from the site distal to coronary stenosis that precluded the responsiveness of the large coronary artery decreased coronary blood flow by 24 +/- 4%, lowered mean distal coronary pressure by 15 +/- 1.9 mm Hg and intensified stenosis resistance by 77 +/- 7.2%. Thus, substance P exerts a direct potent dilating effect on both large and small coronary arteries. However, because of its strict endothelium-dependency, this peptide may play a detrimental role in the regulation of coronary blood flow when an atherosclerotic stenotic lesion with endothelial damage or dysfunction is present in the proximal part of the coronary artery.

Constriction of a large coronary artery contributes to serotonin-induced myocardial ischemia in the dog with pliable coronary stenosis.

To clarify the role of serotonin in eliciting myocardial ischemia, the effects of serotonin on coronary vasculature and cardiac performance were examined in anesthetized open chest dogs. Without coronary stenosis, intracoronary infusion of serotonin (0.001 to 1.0 micrograms/kg per min) for 40 s caused dose-dependent increases in coronary blood flow up to 175 +/- 8.3%. During fixed coronary stenosis produced by a metal constrictor that restricted active vasomotion of the stenosed segment, only the highest dose of serotonin increased coronary blood flow by 23 +/- 5.7% and decreased distal coronary pressure without any changes in stenosis resistance and systemic hemodynamic variables. During dynamic coronary stenosis created by inflation of an intraluminal microballoon that preserved active vasomotion of the stenosed segment, intracoronary serotonin (0.1 and 1.0 micrograms/kg per min) evoked marked decreases in coronary blood flow and distal coronary pressure and an increase in stenosis resistance, resulting in an elevation of left ventricular end-diastolic pressure and worsening of left ventricular dP/dt. These deleterious effects were reversed by additive intracoronary infusion of nitroglycerin (10 micrograms/min). The detrimental effects of serotonin were not attenuated by pretreatment with aspirin (10 mg/kg, intravenously), which suppressed adenosine disphosphate- and arachidonic acid-induced platelet aggregation in vitro. Furthermore, intracoronary infusion of serotonin (0.1 and 1.0 micrograms/kg per min) from the site distal to coronary stenosis failed to decrease coronary blood flow during dynamic coronary stenosis. Therefore, these detrimental effects of serotonin during dynamic coronary stenosis could be caused by constriction of a large coronary artery but not by platelet aggregation. Vascular effects of serotonin without coronary stenosis and with dynamic coronary stenosis were inhibited by pretreatment with methysergide (0.3 mg/kg, intravenously), but not with ketanserin (0.5 mg/kg, intravenously) which inhibited serotonin-induced platelet aggregation in vitro. Thus, serotonin produced opposing effects on coronary vasculature, that is, small coronary artery dilation and large coronary artery constriction. Both vascular effects were mediated by non-S2-serotonergic receptor activation of the coronary artery. The data obtained in this study suggest that serotonin-elicited constriction of a large coronary artery may play a crucial role in the pathogenesis of myocardial ischemia during pliable coronary stenosis.

Effects of left ventricular asynchrony on time constant and extrapolated pressure of left ventricular pressure decay in coronary artery disease.

To elucidate the effects of ventricular asynchrony with or without myocardial ischemia on the time constant of left ventricular pressure decay and asymptote, that is, the level to which pressure would decrease if isovolumic pressure decrease continued infinitely, left ventriculography and pressure measurements were investigated in 14 normal subjects and 25 patients with coronary artery disease. Ventricular asynchrony was quantitated by the segmental area-time curve. This study consisted of two parts. 1) After a right atrial pacing stress test, the time constant and asymptote remained unchanged in eight normal subjects. In 18 patients with coronary artery disease and pacing-induced angina, asynchrony increased, the time constant was prolonged (64 +/- 13 to 94 +/- 17 ms, p less than 0.01) and the asymptote decreased (-22 +/- 10 to -46 +/- 20 mm Hg, p less than 0.01) after the pacing. 2) During right ventricular pacing at 80, 100 and 120 beats/min in the patients, asynchrony increased and the time constant was prolonged (55 +/- 7 versus 70 +/- 10, 47 +/- 11 versus 66 +/- 19, 36 +/- 7 versus 53 +/- 13 ms, respectively, p less than 0.01 versus right atrial pacing), whereas the asymptote was unchanged in six normal subjects compared with the value during right atrial pacing at each pacing rate. In seven patients with coronary artery disease, right ventricular pacing at 80, 100 and 120 beats/min also produced an increase in the time constant, while the asymptote was unchanged. Thus, prolongation of the time constant of left ventricular pressure decay may result from ventricular asynchrony even in the absence of myocardial ischemia.(ABSTRACT TRUNCATED AT 250 WORDS)

Self suppression of phosphoinositide turnover and contraction by stimulating release of endogenous endothelium derived relaxing factor in vascular action of histamine.

STUDY OBJECTIVE: The aim of the study was to examine the relationship between phosphoinositide turnover and vascular contraction in response to histamine, and the role of endothelium derived relaxing factor (EDRF) in these activities. DESIGN: Thoracic aortic strips were studied under isometric tension in an organ bath. Phosphoinositide turnover was studied using [32P]Pi labelling. Cumulative concentration-response curves were obtained for histamine, and for histamine plus H1 and H2 antagonists. The effect of endothelial denudation was examined, as was that of removing Ca2+ from buffer medium, and of adding methylene blue, an inhibitor of EDRF. EXPERIMENTAL PREPARATIONS: Thoracic aortic rings were obtained from 81 male Japanese white rabbits, weight 2.0-2.4 kg. MEASUREMENTS AND MAIN RESULTS: Histamine (0.1 mumol-0.1 mmol.litre-1) caused concentration dependent contractions and increases of [32P]Pi incorporation into phosphatidylinositol (ED50 = 4.6 mumol.litre-1 and 4.0 mumol.litre-1 respectively). Diphenhydramine inhibited contractile response and phosphatidylinositol labelling by histamine. Cimetidine potentiated contractile response but had no effect on phosphatidylinositol labelling. Endothelial denudation potentiated maximum contraction by 15% and augmented phosphatidylinositol labelling at 0.1 mmol.litre-1 histamine by 30%. In intact aortic rings, methylene blue (10 mumol.litre-1), an inhibitor of EDRF, potentiated histamine induced phosphatidylinositol labelling, but had no influence on denuded rings. A-23187 (0.01 mumol.litre-1), which caused release of EDRF from intact endothelium, suppressed histamine induced phosphatidylinositol labelling in intact aortic rings. CONCLUSIONS: The results suggest that stimulation of the release of EDRF in response to histamine causes self suppression of phosphoinositide turnover and contraction.

Dynamic behaviour of coronary stenosis in response to increasing heart rate in the dog.

In order to assess the responses of coronary stenosis to an elevation of myocardial metabolic demand, the effects of pacing induced tachycardia on systemic and coronary haemodynamics were examined during four grades of compliant coronary stenosis which preserved stenosis vasomotility in eight open chest dogs. Grades of the coronary stenosis were defined as trifling, mild, moderate, and severe by pressure gradients of 12(0.5), 19(1.0), 28(1.4) and 37(1.6) mm Hg (mean [SEM]), respectively. Stepwise increases in heart rate (+30, +60 and +90 beats.min-1) decreased the resistance caused by trifling and mild coronary stenosis by 47(11.9) and 26(8.8)%, respectively, with an increase in coronary blood flow and a decrease in pressure gradient across the stenosis. By contrast, the resistance caused by moderate and severe stenosis increased by 96(24.2) and 148(63.2)%, respectively, with a decrease in coronary blood flow and an increase in pressure gradient. During lower grades of coronary stenosis, flow-dependent dilatation of large epicardial coronary arteries reduced the stenosis severity, whereas during higher grades of stenosis, passive narrowing of the stenosed arterial segment due to arteriolar vasodilatation outstripped the flow-dependent stenosis dilator effects and resulted in an intensification of the stenosis.

Myocardial ischaemia induced by endothelin in the intact rabbit: angiographic analysis.

STUDY OBJECTIVE: The aim was to clarify the effects of endothelin on coronary vasculature in the intact rabbit by coronary angiographic examination. DESIGN: By selective coronary angiography bolus injections of various amounts of endothelin (0.01-1 nmol) were given into the right coronary artery of anaesthetised rabbits. The preventive effects of nifedipine on endothelin induced myocardial ischaemia were examined. SUBJECTS: Adult male Japanese white rabbits (2.7-3.5 kg, n = 29) were used for in vivo and in vitro studies. MEASUREMENTS AND MAIN RESULTS: Intracoronary administration of 0.1 nmol endothelin led to the delayed filling of contrast medium into the distal coronary artery and the sustained retention of contrast medium in both large and small coronary arteries, associated with distinct ST elevation in leads II, III and aVF on the electrocardiogram. However, endothelin produced no evident constriction at any site in the large epicardial coronary arteries. Intracoronary administration of glyceryl trinitrate (50 micrograms) or nifedipine (5 micrograms) failed to reverse myocardial ischaemic changes induced by endothelin. Pretreatment with intravenous administration of nifedipine (3 micrograms.kg-1) partially inhibited the endothelin induced vasoconstrictor effect on small coronary arteries and subsequent ischaemic changes. In isolated coronary arterial preparation, endothelin produced long lasting contraction in a dose dependent manner [ED50 = 3.7 (SEM 1.8) nM]. Pretreatment with nifedipine inhibited the maximum contraction induced by endothelin, but did not affect the threshold dose or the half maximal effective dose (ED50). Endothelin (0.1 microM) per se did not produce aggregatory responses of rabbit platelets. CONCLUSIONS: Endothelin induced myocardial ischaemia cannot be ascribed to vasospastic constriction of large coronary arteries nor to platelet thrombi, but diffuse constriction of small coronary arteries occurs in the intact rabbit.

Myocardial ischaemia produced by ergonovine-induced vasoconstriction during preexisting coronary stenosis: experimental conditions for the geometric theory.

Experiments were designed to determine the contribution of active vasomotor tone of a large coronary artery during a preexisting coronary stenosis to the production of myocardial ischaemia. The quantitative relations between ergonovine dose and systemic and coronary haemodynamic and electrocardiographic responses during various degrees of coronary stenosis were evaluated in 55 anaesthetised open-chest dogs. In the absence of coronary stenosis, intracoronary infusion of ergonovine (0.04 to 4 micrograms X min-1) had no systemic or coronary haemodynamic effects. In dogs with coronary stenosis created with intraluminal microballoon occluder, ergonovine produced marked decreases in coronary blood flow and distal coronary pressure followed by a decline in left ventricular dP/dt and ST-elevation in epicardial electrogram in the presence of moderate (28 +/- 1.1 mmHg in pressure gradient) and severe (41 +/- 1.4 mmHg), but not mild (15 +/- 0.9 mmHg) stenosis. These detrimental effects of ergonovine were dependent on its dose as well as the severity of preexisting coronary stenosis. Interventions such as aspirin pretreatment or endothelial denudation did not attenuate the coronary vasomotor response or ergonovine, but pretreatment with nifedipine (3 micrograms X kg-1 iv) prevented this response. Intravenous injection of ergonovine (4 to 15 micrograms X kg-1) in doses relevant to clinical usage during intraluminal obstruction resulted in similar changes in coronary haemodynamics as those of intracoronary ergonovine. In contrast, in dogs with various degrees of coronary stenosis produced with an externally applied constrictor device, ergonovine did not affect systemic and coronary haemodynamics. These experiments demonstrate that normal vasomotion superimposed on moderate and severe pliable coronary stenosis can cause transient myocardial ischaemia, which helps to clarify the conditions to produce myocardial ischaemia according to geometric theory.

Oral magnesium supplementation in patients with essential hypertension.

To elucidate the effects of magnesium on high blood pressure, a 4-week study of oral magnesium supplementation (MgO 1 g/day) was conducted in 21 outpatients with uncomplicated essential hypertension. During the study, blood pressure and intraerythrocyte sodium concentration decreased significantly, and the erythrocyte ouabain-sensitive 22Na efflux rate constant (Kos) and intraerythrocyte magnesium concentration both increased. Serum triglyceride and free fatty acid concentrations were reduced. Furthermore, the elevation in Kos significantly and positively correlated with both the increase in intraerythrocyte magnesium concentration and the decrease in mean blood pressure. There was a significant inverse correlation between the prestudy Kos and the decrease in mean blood pressure. In addition, when patients were divided according to their overall decrease in mean blood pressure, the prestudy intraerythrocyte sodium concentration was significantly higher in patients with a mean blood pressure decrease of more than 7 mm Hg than that of patients whose mean blood pressure decrease was less than 7 mm Hg. These results suggest that oral magnesium supplementation may lower blood pressure through the activation of a cell membrane sodium pump and may reduce serum lipid concentration. It also suggests that the lower the prestudy Kos or the higher the prestudy intraerythrocyte sodium concentration, the more effective the oral magnesium treatment is in lowering blood pressure. Therefore, we concluded that appropriate oral magnesium intake might be effective as a nonpharmacological treatment for essential hypertension.

Effect of oral calcium on blood pressure response in salt-loaded borderline hypertensive patients.

To clarify the mechanism of the antihypertensive effect of oral calcium loading, we studied the effect of low versus high calcium intake on salt-induced blood pressure elevations in patients with borderline hypertension. After a 7-day period of dietary salt restriction (50 meq/day), 27 patients were placed on a high salt (300 meq/day), low calcium (250 mg/day) diet for 7 days; 14 of these patients were given 2,160 mg/day of supplementary calcium (Ca group), and 13 patients were given placebo (non-Ca group). With a high salt intake, the percent increase in mean blood pressure was smaller in the Ca group than in the non-Ca group (+2.85 +/- 1.22% vs. +8.63 +/- 1.66%, respectively, p less than 0.01). The Ca group showed a smaller weight gain (p less than 0.05) and a greater urinary excretion of sodium (p less than 0.005) than the non-Ca group. In the Ca group, but not in the non-Ca group, high salt intake resulted in an increase in intraerythrocyte magnesium content (p less than 0.01), which was correlated inversely with the salt-induced changes in mean blood pressure (r = -0.54, p less than 0.05). While the increase in cellular magnesium was greater in the Ca group, the changes in red blood cell sodium and sodium/potassium ratio were not different between the two groups. The results suggest that oral calcium supplementation may prevent a rise in blood pressure in patients on a high salt, low calcium diet by attenuating the sodium retention.(ABSTRACT TRUNCATED AT 250 WORDS)

Dietary linoleic acid prevents the development of deoxycorticosterone acetate-salt hypertension.

The aim of this study was to elucidate the effect of dietary variations of linoleic acid on the development of deoxycorticosterone acetate (DOCA)-salt hypertension in rats. All rats were divided into three groups and fed one of the following isocaloric diets with 8% NaCl: a high linoleic acid (HLA) (20% sunflower oil), a moderate linoleic acid (5% lard oil + 15% sunflower oil), or a low linoleic acid (DLA) (20% lard oil). After 4 weeks of feeding, we determined intraerythrocyte sodium, potassium, and magnesium concentrations, intra-aortic and lymphocyte magnesium content, and erythrocyte ouabain-sensitive 22Na efflux rate constant. Cytoplasmic free calcium concentration of lymphocytes from thymus was also determined with quin-2 as a fluorescent indicator. In the HLA group, the elevation of systolic blood pressure was significantly attenuated, and intraerythrocyte sodium concentration was significantly lower than in the DLA group. There were greater intraerythrocyte potassium and magnesium concentrations, intra-aortic and lymphocyte magnesium contents, and erythrocyte ouabain-sensitive 22Na efflux rate constant in the HLA group as compared with other groups. Cytoplasmic free calcium concentration in the HLA group was significantly lower than in other groups. Systolic blood pressure significantly correlated negatively with intraerythrocyte and intra-aortic magnesium concentrations and intraerythrocyte potassium concentration, and correlated positively with cytoplasmic free calcium concentration. Erythrocyte ouabain-sensitive 22Na efflux rate constant significantly correlated positively with intraerythrocyte magnesium concentration. These findings suggest that dietary linoleic acid can attenuate the development of DOCA-salt hypertension.(ABSTRACT TRUNCATED AT 250 WORDS)

Mass analysis of 1,2-diacylglycerol in cultured rabbit vascular smooth muscle cells. Comparison of stimulation by angiotensin II and endothelin.

We compared the effects of angiotensin II and endothelin on mass levels of 1,2-diacylglycerol, and endogenous activator of protein kinase C, in cultured rabbit vascular smooth muscle cells with the effects of these vasoconstrictors on contractile responses of rabbit aortic strips. At a high concentration (1 microM), both angiotensin II and endothelin induced a biphasic formation of 1,2-diacylglycerol with an early transient phase and a late sustained phase. At this high concentration, angiotensin II caused a transient contraction followed by a gradual relaxation, whereas endothelin caused a slowly developing and sustained contraction. At a low concentration (EC50 for the early phase of 1,2-diacylglycerol formation), angiotensin II also induced a biphasic formation of 1,2-diacylglycerol and caused a transient contraction, but endothelin induced a monophasic formation of 1,2-diacyglycerol with only an early peak. Despite a rapid decrease of 1,2-diacylglycerol, endothelin at this low concentration still caused a sustained contraction. At both the high and low concentrations, the 1,2-diacylglycerol level was sustained higher for angiotensin II, whereas the tension during the late tonic phase of contraction was greater for endothelin. These results suggest that the unique persistent nature of endothelin-induced contraction is not attributed simply to the stimulatory effect of this peptide on the 1,2-diacylglycerol/protein kinase C pathway.

Phosphatidylinositol turnover in human monocyte-derived macrophages by native and acetyl LDL.

Monocyte-derived macrophages play a key role in pathogenesis of atherosclerosis. However, the mechanism of activating macrophages in atheromatous lesions has not been fully investigated. This report describes the contribution of phosphatidylinositol turnover to the uptake of low density lipoproteins (LDL) by macrophages. Both native and acetyl LDL stimulated inositol 1,4,5-triphosphate (IP3) formation in a dose-dependent manner at concentrations of 0-70 micrograms/ml. The potency of IP3 formation by acetyl LDL (0.44 nmol/mg protein) was 2-fold higher than that by native LDL (0.21 nmol/mg protein). Time course studies showed that a maximal effect of IP3 formation by acetyl LDL at concentrations of 30 micrograms/ml was observed at 3 min. Longer incubation diminished IP3 formation. Oxidized LDL also stimulated IP3 formation with a similar efficiency to acetyl LDL. It was indicated that chemically modified LDL which were taken up through the scavenger receptor pathway activated the macrophages by mediating the phosphatidylinositol hydrolysis and IP3 formation.

Induction of protein kinase C activation and Ca2+ mobilization by fibroblast growth factor in Swiss 3T3 cells.

Addition of fibroblast growth factor (FGF) to quiescent cultures of Swiss 3T3 cells rapidly induced diacylglycerol formation, protein kinase C activation and Ca2+ mobilization. Protein kinase C-activating agents such as 12-O-tetradecanoylphorbol-13-acetate (TPA) and 1-oleoyl-2-acetylglycerol (OAG) mimicked the action of FGF and stimulated DNA synthesis in the presence of insulin. Prolonged treatment of the cells with phorbol-12,13-dibutyrate (PDBu) led to the down-regulation and complete disappearance of protein kinase C. In these cells, TPA and OAG did not induce DNA synthesis any more. FGF still elicited Ca2+ mobilization and DNA synthesis, but the magnitude of DNA synthesis was reduced to almost half as compared with that in the control cells. These results clearly indicate that both diacylglycerol and Ca2+ may serve as second messengers for FGF and suggest that these messengers may be involved in the mitogenic action of this growth factor.

Inhibition of DNA synthesis by phorbol esters through protein kinase C in cultured rabbit aortic smooth muscle cells.

In cultured rabbit aortic smooth muscle cells (SMC), 12-O-tetradecanoylphorbol-13-acetate (TPA) induced DNA synthesis in the presence of plasma-derived serum to a small extent, but inhibited markedly the rabbit whole blood serum (WBS)-, platelet-derived growth factor (PDGF)- and epidermal growth factor-induced DNA synthesis. Phorbol-12,13-dibutyrate (PDBu) mimicked this antiproliferative action of TPA, but 4 alpha-phorbol-12,13-didecanoate was inactive in this capacity. Prolonged treatment of the cells with PDBu caused the partial down-regulation of protein kinase C. In these protein kinase C-reduced cells, WBS still induced DNA synthesis, but TPA did not inhibit the WBS-induced DNA synthesis. We have previously shown that protein kinase C is involved at least partially in the PDGF-induced DNA synthesis in rabbit aortic SMC. The present results together with this earlier observation suggest that protein kinase C has not only a proliferative but also an antiproliferative action in rabbit aortic SMC.

Comparison of the modes of action of Ca2+ ionophore A23187 and thrombin in protein kinase C activation in human platelets.

In human platelets, the Ca2+ ionophore A23187 stimulated the phosphorylation of a 40 kDa protein and myosin light chain (MLC) to the same extents as those induced by thrombin, but the doses of A23187 for 40 kDa protein phosphorylation were higher than those for MLC phosphorylation, although the doses of thrombin for both reactions were nearly the same. Moreover, A23187 produced much less diacylglycerol than thrombin. However, the sites of the 40 kDa protein phosphorylated by the action of A23187 and thrombin were identical, and the 40 kDa protein phosphorylation induced by A23187 and thrombin was inhibited by tetracaine, an inhibitor for protein kinase C. Neither A23187 nor thrombin induced the production of a catalytic fragment of protein kinase C which might be generated by limited proteolysis with Ca2+-dependent protease. These results indicate that A23187 induces protein kinase C activation which phosphorylates the 40 kDa protein, but higher doses of A23187 are required for the activation of this enzyme than for the activation of MLC kinase.

Effect of hypoxia on cholesterol accumulation in cultured rabbit aortic smooth muscle cells.

We studied the effect of hypoxia on cholesterol accumulation in cultured rabbit aortic smooth muscle cells, which were incubated in a medium with normolipemic rabbit serum (NRS) or hyperlipemic rabbit serum (HRS). The cells were incubated in a humidified atmosphere of either 20% O2, 75% N2 and 5% CO2 (control cells) or 2% O2, 93% N2 and 5% CO2 (hypoxic cells). In a medium containing 20% NRS, the free cholesterol level of hypoxic cells was only a little higher than that of control cells, and there was no significant difference in esterified cholesterol content. On the other hand, in a medium containing 20% HRS, the free cholesterol level was slightly higher and the esterified cholesterol level was markedly higher in hypoxic cells compared with control cells. These results show that hypoxia promotes the accumulation of cholesterol, especially as ester, in smooth muscle cells cultured with hyperlipemic serum. These in vitro experiments indicate that hypoxia in the arterial wall associated with hyperlipidemia may play an important role in atherogenesis, although the precise mechanism remains unclear.

Serotonin plays a major role in serum-induced phospholipase C-mediated hydrolysis of phosphoinositides and DNA synthesis in vascular smooth muscle cells.

Whole blood serum (WBS) rapidly induced the phospholipase C-mediated hydrolysis of phosphoinositides and subsequently stimulated DNA synthesis in cultured rabbit vascular smooth muscle cells (VSMCs). Ketanserin, a serotonin (S2) receptor antagonist, markedly inhibited the WBS-induced phospholipase C reaction and DNA synthesis. Serotonin by itself had a weak mitogenic activity for VSMCs, but this vasoconstrictor markedly stimulated the platelet-derived growth factor- and epidermal growth factor-induced DNA synthesis. The stimulatory effect of serotonin on the growth factor-induced DNA synthesis was inhibited by ketanserin. The amount of serotonin contained in WBS was sufficient to induce the phospholipase C reaction and stimulate the growth factor-induced DNA synthesis. These results indicate that serotonin plays a major role in the WBS-induced phospholipase C-mediated hydrolysis of phosphoinositides and DNA synthesis in rabbit VSMCs and suggest that serotonin may act as an important growth regulator for VSMCs in addition to acting as a vasoconstrictor.

Antiproliferative action of cyclic GMP-elevating vasodilators in cultured rabbit aortic smooth muscle cells.

In cultured rabbit aortic smooth muscle cells (SMCs), sodium nitroprusside (SNP) (10(-7) to 10(-4) M), atrial natriuretic peptide (ANP) (10(-9) to 10(-6) M) and 8-bromo-cyclic GMP (10(-6) to 10(-3) M) inhibited the whole blood serum (WBS)-induced DNA synthesis by about 30%. The doses of SNP and ANP necessary for the inhibition of the WBS-induced DNA synthesis were similar to those necessary for the formation of cellular cyclic GMP (cGMP). These agents were effective even when added 6 h after stimulation of the cells with WBS. These results suggest that cGMP inhibits the proliferation of rabbit aortic SMCs by inhibiting the progression from the G1 into S phase of the cell cycle and raise the possibility that cGMP-elevating vasodilators may suppress the atherogenic process by inhibiting vascular SMC proliferation.