Troglitazone and pioglitazone attenuate agonist-dependent Ca2+ mobilization and cell proliferation in vascular smooth muscle cells.

1. The effects of troglitazone and pioglitazone on agonist-induced Ca2+ mobilization and cell proliferation were studied using fluorescent Ca2+ indicator fura-2 AM and incorporation of [3H]-thymidine in rat aortic smooth muscle cells. The patch clamp techniques were also employed. 2. Vasopressin and platelet-derived growth factor-BB (PDGF) caused a transient elevation in [Ca2+]i by Ca2+ mobilization from intracellular stores, followed by a sustained rise due to Ca2+ entry. Nicardipine partly inhibited the sustained phase, but La3+ completely abolished it. 3. Troglitazone and pioglitazone did not significantly affect the transient rise elicited by these agonists, but preferentially inhibited the sustained phase of [Ca2+]i. 4. Under voltage clamp conditions, troglitazone and pioglitazone inhibited voltage-dependent L-type Ca2+ current (ICa.L). They also inhibited nonselective cation channels (Icat) elicited by vasopressin in a concentration-dependent manner. The half maximal inhibitory concentrations of troglitazone on ICa.L and Icat were 4.6 and 5.7 microM, respectively. On the other hand, nifedipine and nicardipine did not inhibit Icat. 5. Vasopressin and PDGF increased incorporation of [3H]-thymidine, and nifedipine and nicardipine partly suppressed it. However, the inhibitory effects of La3+ and exclusion of extracellular Ca2+ were more potent than the Ca2+ blocking agents. Troglitazone and pioglitazone also inhibited it concentration-dependently. 6. These results suggest that troglitazone and pioglitazone preferentially inhibited agonist (vasopressin and PDGF)-induced Ca2+ entry and proliferation in rat vascular smooth muscle cells, where the inhibitory effects of thiazolidinediones on ICa.L and Icat might be partly involved. Thus, thiazolidinediones may exert hypotensive and antiatherosclerotic effects.

Bile acids increase intracellular Ca(2+) concentration and nitric oxide production in vascular endothelial cells.

The effects of bile acids on intracellular Ca(2+) concentration [Ca(2+)](i) and nitric oxide production were investigated in vascular endothelial cells. Whole-cell patch clamp techniques and fluorescence measurements of [Ca(2+)](i) were applied in vascular endothelial cells obtained from human umbilical and calf aortic endothelial cells. Nitric oxide released was determined by measuring the concentration of NO(2)(-). Deoxycholic acid, chenodeoxycholic acid and the taurine conjugates increased [Ca(2+)](i) concentration-dependently, while cholic acid showed no significant effect. These effects resulted from the first mobilization of Ca(2+) from an inositol 1,4,5-triphosphate (IP(3))-sensitive store, which was released by ATP, then followed by Ca(2+) influx. Both bile acids and ATP induced the activation of Ca(2+)-dependent K(+) current. Oscillations of [Ca(2+)](i) were occasionally monitored with the Ca(2+)-dependent K(+) current in voltage-clamped cells and Ca(2+) measurements of single cells. The intracellular perfusion of heparin completely abolished the ATP effect, but failed to inhibit the bile acid effect. Deoxycholic acid and chenodeoxycholic acid enhanced NO(2)(-) production concentration-dependently, while cholic acid did not enhance it. The bile acids-induced nitric oxide production was suppressed by N(G)-nitro-L-arginine methyl ester, exclusion of extracellular Ca(2+) or N-(6-aminohexyl)-5-chloro-l-naphthalenesulphonamide hydrochloride (W-7) and calmidazolium, calmodulin inhibitors. These results provide novel evidence showing that bile acids increase [Ca(2+)](i) and subsequently nitric oxide production in vascular endothelial cells. The nitric oxide production induced by bile acids may be involved in the pathogenesis of circulatory abnormalities in liver diseases including cirrhosis.

Effects of Saiko-ka-ryukotsu-borei-to, a Japanese Kampo medicine, on tachycardia and central nervous system stimulation induced by theophylline in rats and mice.

Effects of Saiko-ka-ryukotsu-borei-to (SRBT) on theophylline-induced tachycardia in anesthetized rats and theophylline-induced locomotion and convulsions in mice were examined. An intraduodenal administration of SRBT (1 g/kg) prevented theophylline (5 mg/kg, i.v.)-induced tachycardia in rats. SRBT also attenuated an increase in arterial blood pressure with a slow reduction in heart rate of rats treated with theophylline, with no influence on the plasma level of theophylline. However, SRBT did not change the beating rate of right atrium isolated from rats in the absence or presence of theophylline or isoproterenol. The locomotor activity of theophylline in mice was reduced by the treatment with SRBT. Furthermore, the latency of convulsions in mice induced by administration of theophylline at a higher dose (240 mg/kg, i.p.) was prolonged by treatment with SRBT (1 g/kg, p.o.) and seven out of fifteen mice were saved from death due to convulsions. These results suggest that theophylline-induced tachycardia and central nervous stimulation are suppressed by SRBT and that SRBT may reduce the undesirable actions of theophylline on the cardiovascular and central nervous systems.

Effects of 9 Kampo medicines clinically used in hypertension on hemodynamic changes induced by theophylline in rats.

We examined the effects of 9 kinds of Kampo medicines, which are clinically used for the treatment of hypertension, on anesthetized rats with increases in arterial blood pressure, heart rate and peripheral blood flow induced by theophylline (5 mg/kg, i.v.) that were partially or completely mediated by endogenous catecholamines. Each Kampo medicine (1 g/kg) was intraduodenaly administered. Shinbu-to caused a severe disturbance of the arterial blood pressure. Saiko-ka-ryukotsu-borei-to, Oren-gedoku-to, San'o-shashin-to and Dai-jyoki-to had hypotensive effects, while Hachimi-jio-gan, Gosha-jinki-gan, Dai-saiko-to and Choto-san did not have such an effect. Moreover, Saiko-ka-ryukotsu-borei-to attenuated the heart rate. In Oren-gedoku-to, San'o-shashin-to and Dai-jyoki-to, a reduction in peripheral blood flow was observed. These results suggest that Saiko-ka-ryukotsuborei-to, Oren-gedoku-to, San'o-shashin-to and Dai-jyoki-to are ameliorative to the hypertension in sympathetic system dominance and Shinbu-to is occasionally dangerous to it.

Effects of San'o-shashin-to and the constituent herbal medicines on theophylline-induced increase in arterial blood pressure of rats.

San'o-shashin-to, composed of Scutellariae Radix, Coptidis Rhizoma and Rhei Rhizoma (volume ratio = 1:1:1), reduced an increase in arterial blood pressure of anesthetized rats induced by theophylline (5 mg/kg, i.v.). The hypotensive effect of San'o-shashin-to was produced in a dose dependent manner and was maximum at its 0.5 g/kg. Then the constituent herbal medicines were examined for their possible hypotensive effect. Scutellariae Radix of 0.2 g/kg slightly decreased in the blood presure. Rhei Rhizoma of 0.2 g/kg decreased in the blood pressure and the hypotensive effect was significantly produced even at the dose of 0.05 g/kg, while Coptidis Rhizoma had little effect. Among fractions of San'o-shashin-to separated by Diaion HP-20 column chromatography, the 50% methanol-eluted fraction had a large hypotensive effect. The 50% methanol-eluted fraction of Scutellariae Radix and Rhei Rhizoma were also effective and, especially, that of Rhei Rhizoma had a large hypotensive effect. In isometric tension study, Scutellariae Radix and Rhei Rhizoma (10-30 microg/ml) slightly exerted contractile and relaxant effects, respectively, on the phenylephrine-contracted endothelium-intact rat thoracic aorta. Coptidis Rhizoma (1-10 microg/ml) caused both endothelium-dependent and -independent relaxantion. These results suggest that the hypotensive effect of San'o-shashin-to is not mediated by the direct action on blood vessel but by other actions. Some components in Scutellariae Radix and Rhei Rhizoma, especially in the latter may play a main role in the hypotensive effect.

Enhancement of endothelial nitric oxide production by chenodeoxycholic acids in patients with hepatobiliary diseases.

The purpose of this study was to clarify whether physiological concentrations of bile acids could affect endothelial nitric oxide production. We investigated the relationships between clinical concentrations of individual bile acids observed in patients with hepatobiliary diseases and endothelial nitric oxide production induced by each bile acid. Fifteen serum bile acids were measured using high-performance liquid chromatography combined with enzymatic fluorometry in 8 patients with liver cirrhosis, obstructive jaundice, and 8 healthy subjects. The effects of individual bile acids on nitric oxide production were examined in human umbilical endothelial cells by measuring the concentration of NO2- in the cultured medium. NO release in the blood was also determined by measuring the NO2-/NO3- concentration in these patients. In patients with hepatobiliary diseases, the plasma concentrations of chenodeoxycholic acid, ursodeoxycholic acid and cholic acid (free acid, taurine and glycine conjugates) were markedly elevated. Incubation of cells with chenodeoxycholic acid and deoxycholic acid (free acid, taurine and glycine conjugates) enhanced NO2- production in a concentration-dependent manner, while cholic acid (free and its conjugates) did not. The effects of individual bile acids on nitric oxide production were additive. Patients with liver cirrhosis and obstructive jaundice had higher plasma levels of NO2-/NO3- levels than the control subjects. These results suggest that increased plasma concentrations of chenodeoxycholic acid (free, taurine and glycine conjugates) in patients with hepatobiliary diseases may induce endothelial nitric oxide production. Thus, nitric oxide production induced by bile acids may be involved in the pathogenesis of circulatory abnormalities in patients with liver diseases.