Hisashi Fukuzaki

Lysophosphatidylcholine: essential role in the inhibition of endothelium-dependent vasorelaxation by oxidized low density lipoprotein.

Endothelial cells are known to play an important role in the regulation of vascular tone. Here we demonstrate that modified low density lipoprotein (LDL) with copper oxidation or phospholipase A2 treatment elicits a potent inhibitory action on endothelium-dependent relaxations evoked by acetylcholine, although native LDL does not affect endothelium-dependent relaxations. Phosphatidylcholine of native LDL is converted to lysophosphatidylcholine during these modifications. Furthermore, lysophosphatidylcholine fraction separated from oxidized LDL (0.5mg.protein/ml) by thin layer chromatography abolished endothelium-dependent relaxations, although the remaining lipid fraction had little effects on endothelium-dependent relaxations. These results indicate that lysophosphatidylcholine is the principal substance for the impairment of endothelium-dependent relaxations by oxidized LDL and phospholipase A2 treated LDL.

Single-beat estimation of the slope of the end-systolic pressure-volume relation in the human left ventricle.

This study assessed a new method of estimating the slope (Ees) of the end-systolic pressurevolume relation (ESPVR) from a single beat of the human heart. Left ventricular pressure was recorded with a high-fidelity micromanometer in patients with heart disease during left ventriculography. Peak isovolumic pressure at the end-disastolic volume was estimated by a curve-fitting technique from an isovolumic left ventricular pressure curve. The ESPVR line was drawn from the estimated peak isovolumic pressure-volume point tangential to the left upper corner of the pressure-volume loop. The slope of this estimated ESPVR line from single-beat analysis was compared with the slope of the ESPVR line obtained from three pressure-volume loops in 16 patients given angiotensin II or nitroglycerin infusion. The estimated Ees was 5.0 ± 2.2 mm Hg/m1/m2, and the conventional Ees was 4.9 ± 2.7 mm Hg/mlm2. The estimated Ees showed a positive correlation with the conventional Ees (r = 0.91, p < 0.001, SEE= 1.2 mm Hg/ml/m2). In the other 13 patients, after dobutamine infusion (5, μg/kg/min i.v.) the estimated Ees increased significantly from 5.6 ± 1.4 to 7.4 ± 2.0 mm Hg/ml/m2 (p < 0.01). Thus, the estimated Ees approximated the conventional Ees and was sensitive to a positive inotropic intervention. We conclude that this single-beat analysis method facilitates assessment of the beat-by-beat ESPVR of the human heart.

Angiotensin II induces expression of the c-fos gene through protein kinase C activation and calcium ion mobilization in cultured vascular smooth muscle cells☆

Incubation of the serum-deprived cultures of rat vascular smooth muscle cells with angiotensin II, a potent vasoconstrictor, caused a rapid and transient increase in the c-fos mRNA level. The doses of this agonist necessary for the increase in the c-fos mRNA level coincided with those for the phospholipase C-mediated hydrolysis of phosphoinositides. Moreover, protein kinase C-activating 12-O-tetradecanoylphorbol-13-acetate and Ca2+-ionophore A23187 increased the c-fos mRNA level in an additive manner. These results suggest that angiotensin II induces expression of the c-fos gene through the activation of protein kinase C and Ca2+ mobilization in cultured vascular smooth muscle cells.

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.

Hyperreactivity of coronary arterial smooth muscles in response to ergonovine from rabbits with hereditary hyperlipidemia.

This study was undertaken to examine the response to ergonovine, an agent used to provoke spastic constriction of large epicardial coronary arteries, to elucidate the, responsible underlying mechanism, and to determine the impact of endogenous hyperlipidemia on contractile properties of isolated vessels from different beds. The isolated arteries from both control and Watanabe hereditary hyperlipidemic rabbits (WHHL rabbits) were suspended for recording isometric force in oxygenated Krebs buffer and exposed to agonists and antagonists. In athero- sclerotic aortas from WHHL rabbits, the concentration-response relations for ergonovine and serotonin exhibited a marked leftward shift with significantly depressed constrictor threshold concentration and lowered one-half maximally effective concentration values. In coronary arteries with no atherosclerotic lesions detectable macroscopically from WHHL rabbits, the concentration- response relations showed a leftward shift for ergonovine but not for serotonin. Coronary contraction evoked by ergonovine was remarkably inhibited by 0.1μm cyproheptadine and 0.3 μM methysergide, serotonergic antagonists, in both groups. α-Adrenergic blockade with 0.1 JZMprazosin was effective in inhibiting ergonovine-induced contraction of aortas from control rabbits, but not that of atherosclerotic ones. The constrictor response to ergonovine of atherosclerotic aortas was inhibited by cyproheptadine. The responsiveness to ergonovine of both carotid and femoral arteries from WHHL rabbits with no sclerotic lesions, which was suppressed by prazosin was not different from that of control rabbits. In contrast, the concentration-response relations for phenylephrine in the four different types of arteries did not differ appreciably between the two groups, and the constrictor responses to 20 ITIM KG were virtually identical. Thus, aortas and coronary arteries exposed to endogenous hyperlipidemia appear to be hyperreactive to ergonovine mediated by a serotonergic mechanism.

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.

Possible involvement of protein kinase C in platelet-derived growth factor-stimulated DNA synthesis in vascular smooth muscle cells☆

In cultured rabbit aortic vascular smooth muscle cells (VSMC), protein kinase C-activating phorbol esters such as 12-O-tetradecanoylphorbol-13-acetate (TPA) and phorbol-12,13-dibutyrate (PDBu) stimulated DNA synthesis in the presence of 10% cell-free plasma-derived serum. This stimulation was half that shown by PDGF. 4 alpha-Phorbol-12,13-didecanoate, known to be inactive for protein kinase C, was without effect in stimulating DNA synthesis. Prolonged treatment of the cells with PDBu led to a marked decrease in protein kinase C. In the pDBu-treated cells, the TPA-stimulated DNA synthesis was completely abolished whereas the PDGF-stimulated DNA synthesis was decreased to about half that in the control cells. These results suggest that protein kinase C is involved in PDGF-stimulated proliferation of VSMC.

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)

Effect of cyclic amp on cytoplasmic free calcium in human platelets stimulated by thrombin: Direct measurement with quiǹ2

With the fluorescent Ca2+ indicator quin2, we measured directly cytoplasmic free Ca2+ [( Ca2+]i) in washed human platelets stimulated by thrombin and examined the effect of cyclic AMP on [Ca2+]i levels and 14C-serotonin release. Thrombin (0.2 U/ml) evoked a rise in [Ca2+]i from the basal level of about 100 nM to integral of 3 microM which was fast enough to trigger serotonin release. This rise was inhibited in a dose-dependent manner by preincubation with prostaglandin E1 (PGE1) (0.1-10 microM) or dibutyryl cyclic AMP (0.01 -1 mM). Parallel to this, serotonin release was also inhibited by these drugs. When added to platelets after stimulation by thrombin, PGE1 caused the rapid decrease of elevated [Ca2+]i. These results provide direct evidence that [Ca2+]i levels in platelets are regulated by cyclic AMP.