Hidehisa Masui

Effect of chromium administration on glucose tolerance in stroke-prone spontaneously hypertensive rats with streptozotocin-induced diabetes

The present study was conducted to assess the effect of chromium (Cr) administration on glucose tolerance in insulin-dependent diabetes that accompanies hypertension. Four rat groups were used: stroke-prone spontaneously hypertensive rats (SHRSP) and normotensive Wistar Kyoto rats (WKY) with and without streptozotocin (SZ, 40 mg/kg)-induced diabetes. Each group of rats was subdivided to the Cr-dose group and the control group. The Cr-dose group, which was intraperitoneally administered Cr solution (20 micrograms trivalent chromium/kg body weight/d for 4 weeks), and the control group (saline) were studied for plasma glucose and plasma insulin during intraperitoneal glucose tolerance test (IPGTT) and insulin action by isolated adipocytes. For diabetic SHRSP showing the highest plasma glucose and lowest plasma insulin among the four groups, Cr administration led to the greatest reduction in plasma glucose without a significant effect on plasma insulin during IPGTT. For each diabetic WKY and normal SHRSP and WKY, those given Cr showed lower levels of plasma glucose with lower levels of plasma insulin than the controls. For diabetic SHRSP, glucose uptake by isolated adipocytes in the Cr-dose group was higher than that in the control group. This effect of Cr administration involved enhancement of insulin responsiveness and sensitivity, attributed to enhanced affinity of the insulin receptor. A similar tendency was observed for diabetic WKY. However, for normal SHRSP and WKY, the increase in glucose uptake due to Cr administration coincided only with enhanced insulin responsiveness.(ABSTRACT TRUNCATED AT 250 WORDS)

Tyrosine phosphorylation increases Ca2+ sensitivity of vascular smooth muscle contraction.

In order to elucidate the role of tyrosine phosphorylation in vasoconstriction, we investigated the effects of inhibitors of tyrosine kinase (genistein, 30 microM) and phosphatase (sodium o-vanadate, 5 microM) on the contraction of aorta isolated from guinea pig. Genistein significantly inhibited norepinephrine-induced contraction, but it did not affect that induced by KCI. Thus, tyrosine phosphorylation may not be involved in the contractile response to KCI alone. The aortic contraction elicited by KCl was significantly augmented by sodium o-vanadate, which increased both the maximum force and pD2 values of KCl contraction. In the presence of verapamil, KCl-induced contraction was abolished even after pretreatment with sodium o-vanadate. Sodium o-vanadate also augmented Ca2+-induced contraction in the aortic strips depolarized with KCl, increasing both its maximum force and pD2 values. Neither basal 45Ca2+ uptake nor verapamil-sensitive 45Ca2+ uptake induced by KCl were affected by pretreatment with sodium o-vanadate. These results suggest that tyrosine phosphorylation is involved in the contraction of guinea-pig aorta not through transplasmalemmal Ca2+ entry but through increased Ca2+ sensitivity of the intracellular contractile pathway.

Effects of ethanol on contractile response of gall bladder isolated from guinea pig.

The effects of ethanol treatment in vitro and in vivo on gall bladder contraction were investigated using gall bladder strips isolated from guinea pigs. In vitro pretreatment of the strips with ethanol at a concentration of over 50 mM significantly attenuated the reactivity and sensitivity of contractile responses to KCl, acetylcholine and histamine in a concentration-dependent manner. Indomethacin treatment or removal of extracellular calcium remarkably reduced gall bladder contractile response to acetylcholine. The depressive effect of ethanol in vitro on gall bladder contraction was also noted in the presence of indomethacin or absence of calcium in the medium. The concentration-response curve of calcium-induced contraction in 40 mM KCl-depolarized gall bladder strip shifted to the right on pretreatment with ethanol. In the case of strips following the chronic administration of 3% ethanol solution ad libitum for 4 weeks, contractile responses to KCl, acetylcholine and histamine did not differ, compared to those in the pair-fed group. This chronic ethanol administration induced tolerance to the acute inhibitory effect of ethanol on gall bladder contractile responses to the agonists. Ethanol is thus shown to exert direct inhibitory action on gall bladder contraction by lowering the calcium sensitivity of the contractile apparatus of smooth muscle; it is unlikely that ethanol consumption would affect gall bladder motility in vivo, owing to the tolerance produced toward the acute inhibitory action of ethanol.

Extracellular Ca2+-dependent contractile action of phorbol 12,13-dibutyrate on gall bladder from guinea pig.

The effects of phorbol 12,13 dibutyrate (PDBu) on the isometric tone of gall bladder strips isolated from guinea pig were investigated. PDBu caused the development of a contractile force at concentrations of 5 nM to 1 microM, which was abolished by staurosporine (30 nM). The contraction was completely inhibited in Ca2+-free solution and by pretreatment with verapamil (1 microM) or nifedipine (1 microM). A lower concentration of PDBu (1 nM) potentiated the contractile response to KCl: it increased the sensitivity, but not the reactivity of KCl-induced contraction. Ca2+-induced contraction in the solution with 15 mM KCl was significantly augmented by the pretreatment with 1 nM PDBu. However, PDBu (1 nM) affected neither Ca2+-induced contraction in the presence of Ca2+ ionophore A23187 (0.7 microM) and verapamil (1 microM) nor Ba2+-induced contraction in the Ca2+-free solution. These results suggest that activation of protein kinase C induces completely extracellular Ca2+-dependent contraction by activating the voltage-dependent Ca2+ channels in gall bladder of guinea pig.

Effects of cadmiumin vitro on contractile and relaxant responses of isolated rat aortas

ObjectiveCadmium is known to affect the vascular tone of isolated blood vesselsin vitro and the arterial pressure of ratsin vivo. However, the mechanisms of cadmium actions on the vascular system have not been clarified. To elucidate the actions of cadmium on vascular tonus, effects of cadmium on vasocontractile and vasorelaxant responsesin vitro were investigated using aortic strips isolated from rats.MethodsAortic strips isolated from male Wistar rats were incubated with CdCl2 (10μM) for 24 hr, washed with fresh CdCl2-free medium, and then used for measurement of isometric tension and Western blot analysis of eNOS (endothelial nitric oxide synthase) and iNOS (inducible nitric oxide synthase).ResultsIn the aortas pretreated with cadmiumin vitro, the contractile response to phenylephrine was significantly higher than that in the control aortic strips pretreated with a vehicle. The sodium nitroprusside-induced relaxing response was significantly higher in the aortic strips pretreated with cadmium for 24 hr, compared with that in the control pretreated with a vehicle. The isoproterenol-induced relaxing response was also significantly higher in the cadmium-accumulated aortic strips.In vitro cadmium treatment slightly but not significantly increased the acetylcholine-induced relaxation of the aortic strips. Cadmium treatment induced expression of iNOS and significantly increased expression of eNOS in the aortic strips, while it did not affect expression of β-actin.ConclusionsCadmium treatmentin vitro augmented the α1 adrenoceptor-mediated contractile response, even though eNOS and iNOS were upregulated by cadmium treatment. NO-induced and β-adrenoceptor-mediated relaxing responses were also augmented by cadmium treatment. These results suggest that both vasocontractile and vasorelaxing responses are augmented in cadmium-accumulated aortas.

Potentiating effect of NH4Cl on vasoconstriction in rat aorta

The effect on the vasocontractile response of pretreatment with NH4Cl at a concentration (10 mM) that made almost no change in the resting tension was investigated using aortic strips from rats. NH4Cl pretreatment for 10 min significantly potentiated strip contractions induced by KCl (less than or equal to 30 mM), BAY K 8644 (0.1 microM) and phenylephrine (0.01 microM). This potentiating action of NH4Cl was eliminated in presence of nifedipine (1 microM). KCl (14.7 mM)-stimulated 45Ca uptake in rat aorta was significantly potentiated by pretreatment with NH4Cl (10 mM) for 10 min, but this NH4Cl effect was also eliminated in the presence of nifedipine. These results suggest that NH4Cl potentiates contractions induced by KCl and agonists in rat aorta by facilitating calcium influx through the nifedipine-sensitive calcium channel.

Intracellular alkalinization augments α1-adrenoceptor-mediated vasoconstriction by promotion of Ca2+ entry through the non-L-type Ca2+ channels

Modulation by intracellular pH of the vasoconstriction induced by alpha-adrenoceptor agonists was investigated in isolated guinea pig aorta. NH(4)Cl (15 mM) increased intracellular pH of aortic smooth muscle cells by about 0.2 pH unit and significantly augmented KCl-induced contraction of aortic strips, whereas simultaneous administration of NH(4)Cl (15 mM) plus Na(+) propionate (30 mM) failed to affect intracellular pH or contractility. NH(4)Cl (15 mM) potentiated contractions induced by alpha-adrenoceptor agonists, norepinephrine, phenylephrine and clonidine. Contraction induced by alpha(1)-selective adrenoceptor agonist, phenylephrine, but not that induced by norepinephrine or clonidine, was insensitive to inhibition by verapamil (1 microM). Phenylephrine-induced contraction was not affected by NH(4)Cl in Ca(2+)-free medium whereas extracellular Ca(2+)-induced contraction of phenylephrine-stimulated aorta was significantly augmented by NH(4)Cl. Consistently, 45Ca(2+)uptake into phenylephrine 1 microM)-stimulated aortic strips was increased by incubation with NH(4)Cl. The potentiating effects of NH(4)Cl on both phenylephrine-induced Ca(2+) entry and contraction were antagonized by Na(+) propionate. These results suggest that intracellular alkalinization facilitates alpha(1)-adrenoceptor-mediated vasoconstriction by facilitation of an agonist-induced Ca(2+) entry pathway that is independent of L-type Ca(2+) channels.

Diminished Inibitory Action of Ethanol on the Contraction of Gallbladder Isolated from Chronically Ethanol-Fed Guinea Pigs

Ethanol is known to decrease the gallbladder contractility. The purpose of this study was to clarify the mechanism of tolerance to the inhibitory action of ethanol on the gallbladder contractility. Male guinea pigs were fed ethanol (3%) or calorie-matched sucrose in the drinking water for 4 weeks. Then, the gallbladder was isolated, and its isometric tension was measured. The contractile responses to KCl, BAY K8644, histamine, and phorbol 12,13-dibutyrate in the normal medium were not different between the gallbladder strips from ethanol-fed and control guinea pigs. Ethanol at 25 mmol/l in vitro did not affect the contractile responses to KCl and BAY K8644 in the gallbladder strips from both ethanol-fed and control guinea pigs. On the other hand, ethanol at 25 mmol/l in vitro significantly inhibited the contractile responses to histamine and phorbol 12,13-dibutyrate in the gallbladder strips from the control guinea pigs, but it did not affect the contractile response to histamine and significantly augmented that to phorbol 12,13-dibutyrate in the strips from the ethanol-fed guinea pigs. Diphenhydramine, a selective H1 receptor antagonist, abolished the histamine contraction in gallbladder strips from both control and ethanol-fed guinea pigs, while cimetidine, a selective H2 receptor antagonist, did not affect histamine contraction, implying that histamine-induced contraction of guinea pig gallbladders is mediated only by H1 receptors. Verapamil (1 µmol/l) completely inhibited the phorbol 12,13-dibutyrate-induced contraction of the strips from both ethanol-fed and control guinea pigs. The histamine-induced contraction was partly inhibited in the absence of Ca2+ in the medium. In the gallbladder strips from both ethanol-fed and control guinea pigs, ethanol at 25 mmol/ in vitro did not affect the histamine-induced contraction in the absence of extracellular Ca2+. Tolerance to the inhibitory action of ethanol developed selectively on contractile responses to histamine and phorbol 12,13-dibutyrate. Chronic ethanol administration produces tolerance to in vitro gallbladder contractility mediated by the Ca2+ entry through L-type Ca2+ channels linked with protein kinase C activation.

Preferential inhibition by ethanol of phorbol ester-induced smooth muscle contraction of guinea pig gallbladder

In order to investigate how ethanol inhibits the contractility of gallbladder smooth muscle, the effects of different ethanol concentrations were examined on gallbladder contractile responses to stimulants which act on the smooth muscle by different mechanisms. A low concentration (25 mM) of ethanol significantly inhibited the contractile response to phorbol-12,13-dibutyrate (PDBu), but not that to KCl. It also did not significantly affect contractile response to Ca2+ ionophore A23187 in the presence of verapamil or that to Ba2+ in a medium without Ca2+. On the other hand, a higher concentration (200 mM) of ethanol significantly inhibited the contractile responses to PDBu, KCl, Ca2+ ionophore A23187 and BaCl2. These results together with our recent finding that PDBu-induced contraction of guinea pig gallbladder is completely dependent on activation of the voltage-dependent Ca2+ channel suggest that low concentrations of ethanol selectively inhibit the signal transduction of gallbladder smooth muscle cells in the pathway from activation of protein kinase C to that of the voltage-dependent Ca2+ channel, while high concentrations of ethanol inhibit the intracellular common pathway (probably in the cytoskeletal apparatus).

Effect of diethyldithiocarbamate on diabetogenic action of alloxan in rats.

To determine whether alloxan action is mediated by hydroxyl radicals in vivo, we assayed methane sulfinic acid (MSA), a product of the trapping reaction of dimethyl sulfoxide (DMSO) with hydroxyl radicals. In DMSO-treated rats, the plasma levels of MSA were increased after injection of alloxan (75 mg/kg). This supports the hypothesis that the diabetogenic action of alloxan is mediated by hydroxyl radicals in vivo. The role of cytosolic superoxide dismutase (SOD) in protecting B cells against chemically induced diabetes was studied in rats injected intraperitoneally with diethyldithiocarbamate (DDC). When rats were injected intraperitoneally with DDC (750 mg/kg), the SOD activity at 2.5 h was decreased by 44% in the whole pancreas. The decreased SOD activity was affected by DDC but not by alloxan. Intraperitoneal injection of rats with DDC (750 mg/kg) increased diabetogenic susceptibility to a nondiabetogenic dose of alloxan (20 mg/kg). Subcutaneous injection of vitamin E, prior to administration of both DDC and alloxan, provided partial protection to the rats against the diabetogenic action. These findings suggest that the susceptibility to diabetogenic action of alloxan in B cells is augmented when the cellular SOD activity is inhibited. Thus, cellular SOD may play an important role in the maintenance of B cell function.