Hideyo Ohshika

Membrane Viscosity Correlates with α1-Adrenergic Signal Transduction of the Aged Rat Cerebral Cortex

Abstract: We investigated, using adult (2‐month‐old) and senescent (12‐ and 24‐month‐old) rats, the effects of aging on the relationship between the α1‐adrenergic coupling system and the membrane viscosity of the cerebral cortex. There was no age‐related difference in the KD values of [3H]prazosin binding on the membranes. The Bmax values of [3H]prazosin binding were reduced with advanced age. Norepinephrine induced formation of 3H‐labeled inositol phosphates (3H‐IPs) in the slices increased with advanced age. The EC50 values for norepinephrine to stimulate the formation of 3H‐IPs at advanced age were lower than that at adult age. The cholesterol content in membranes increased with advanced age. No changes in the phospholipid content in membranes were observed with advanced age. Concomitantly, an increase of the molar ratio of cholesterol to phospholipids was observed with advanced age. The membrane viscosity as measured by 1,6 diphenyl‐1,3,5‐hexatriene increased with advanced age. These results indicate that the altered cholesterol content and/or viscosity in cortical membranes of the aged rat may account for the loss of α1‐adrenergic receptor density and/or compensatory changes in the receptor‐phospholipase C coupling system.

Effects of bilobalide on γ-aminobutyric acid levels and glutamic acid decarboxylase in mouse brain

We have previously demonstrated that bilobalide, a constituent of the Ginkgo biloba extract, possesses anticonvulsant activity, and suggested that the mechanism of its anticonvulsant action involves modulation of y-aminobutyric acid (GABA)-related neuronal transmission. This study examined the effects of bilobalide on the level of GABA and glutamate, the activity and the amount of glutamic acid decarboxylase (EC 4.1.1.15), and the function of GABA(A) receptors in the hippocampus, cerebral cortex and striatum of the mouse. GABA levels, glutamic acid decarboxylase activity, and the protein amount of 67 kDa glutamic acid decarboxylase in the hippocampus of mice treated with bilobalide (30 mg/kg, p.o., once a day for 4 days) were significantly higher than those in controls. However, there were no significant differences in glutamate levels or, the number and the dissociation constants of GABA(A) receptors in the hippocampus between control and bilobalide-treated mice. These results suggest that the anticonvulsant effect of bilobalide is due to elevation of GABA levels, possibly through potentiation of glutamic acid decarboxylase activity and enhancement of the protein amount of 67 kDa glutamic acid decarboxylase by bilobalide.

Ca Mobilizing Action of Sphingosine in Jurkat Human Leukemia T Cells EVIDENCE THAT SPHINGOSINE RELEASES Ca FROM INOSITOL TRISPHOSPHATE- AND PHOSPHATIDIC ACID-SENSITIVE INTRACELLULAR STORES THROUGH A MECHANISM INDEPENDENT OF INOSITOL TRISPHOSPHATE

Effects of sphingosine on Ca mobilization in the human Jurkat T cell line were examined. Sphingosine increased the cytoplasmic Ca concentration ([Ca]) in a dose-dependent manner with an ED of around 8 μM. Sphingosine and OKT3, a CD3 monoclonal antibody, transiently increased [Ca], which declined to the resting level in the absence of extracellular Ca. Under the same conditions, pretreatment with sphingosine inhibited but did not abolish an increase in [Ca] induced by the subsequent addition of OKT3 and vice versa. However, pretreatment with sphingosine did not affect an increase in [Ca] induced by OKT3 in the presence of Ca. OKT3 increased IP formation, but sphingosine did not affect the level of IP by itself nor did it cause IP formation induced by OKT3. In permeabilized Jurkat cells, the addition of IP released Ca from nonmitochondrial intracellular stores, but the addition of sphingosine did not. Sphingosine, stearylamine, and psychosine increased [Ca] and diacylglycerol (DG) kinase activation; however, ceramide did not, whereas sphingosine 1-phosphate slightly activated DG kinase without elevation of [Ca]. Pretreatment with R59022, a DG kinase inhibitor, abolished the peak but did not affect the sustained response of [Ca] to sphingosine. Phosphatidic acid (PA) elevated [Ca], after which it declined to a resting level even in the presence of extracellular Ca. In accordance with this, PA did not stimulate Ca uptake into cells, but sphingosine and OKT3 did. Pretreatment with PA partially inhibited a rise in [Ca] induced by the subsequent addition of sphingosine and vice versa in the absence of extracellular Ca. Under similar conditions, pretreatment with PA affected an elevation of [Ca] induced by OKT3 less, after which the subsequent addition of sphingosine did not increase [Ca]. In permeabilized Jurkat cells, the addition of IP did not release Ca, but PA did in the presence of heparin. Pretreatment with thapsigargin, a microsomal Ca-ATPase inhibitor, abolished the rises of [Ca] induced by the subsequent addition of sphingosine, OKT3, and PA in the absence of extracellular Ca. The present results suggest that at least two kinds of intracellular Ca stores exist in Jurkat cells, both of which are IP- and PA-sensitive, and that sphingosine mobilizes Ca from both stores in an IP-independent manner. Furthermore, the IP- but not the PA-sensitive intracellular Ca store seems to regulate Ca entry induced by sphingosine.

The relationship between serum nitrate and endothelin-1 concentrations in preeclampsia.

Recently it was suggested that abnormal endothelial function may contribute to the pathophysiological changes observed in preeclampsia (PE). Both nitric oxide (NO) and endothelin-1 (ET-1) are vasoactive substances produced by endothelial cells. NO is a vasodilator and has been believed to be decreased in PE. ET-1 is a vasoconstrictor and has been reported to be increased in PE. We simultaneously measured NO metabolites and ET-1 in sera from women with PE and investigated the correlation of NO and ET-1 concentrations. We obtained serum samples from 11 healthy nonpregnant (NP) women, 16 normotensive pregnant (NTP) women and 17 women with PE. In this study, the serum ET-1 level was assayed by the ET-1 RIA system, and serum NO metabolites were assayed by measuring nitrite (NO2-) and nitrate (NO3-) simultaneously in an HPLC-Griess reaction system. There was a significant correlation between NOx (nitrite + nitrate) and ET-1 in sera from all 44 women (NP, NTP and PE groups) (p<0.001). Nitrite and ET- in sera from each group were not significantly correlated. Nitrate and ET-1 in sera from the NP and NTP groups did not significantly correlate. However, there was a significant correlation between nitrate and ET-1 in sera from the PE group (p<0.05). The serum ET-1 and nitrate concentration in the PE group was significantly higher than in the NP and NTP groups (p<0.05 and p<0.001. respectively). These findings suggest that increased production of nitrate in PE may contribute to homeostatic vasodilation against vasoconstriction caused by a higher ET-1 concentration.

Impaired expression of Gsα protein mRNA in rat ventricular myocardium with aging

We have recently reported that the responsiveness of adrenoceptors is decreased with aging in rat ventricular myocardium. Thus, the current study determined aging-dependent changes in: (a) characteristics of myocardial G proteins as determined by Western blot analysis; (b) steady-state levels of G protein mRNA as determined by Northern blot analysis; and (c) the intropic response to isoproterenol, a beta-adrenoceptor agonist. Cardiac preparations were isolated from male Wistar rats of 6 (adult) and 24 (old) months old. Compared with adults, aging decreased the combined level of the three Gs alpha subunits (45, 47 and 52 kDa) by a total of 23% in ventricular membrane preparations. In contrast, levels of Gi alpha (40/41 kDa), Gq alpha (42 kDa), Go alpha (39 kDa) and G common beta (35/36 kDa) immunoreactivity were not affected by aging in the same membrane preparations. In ventricular myocardium, steady-state levels of Gs alpha mRNA (1.9 kb) decreased by 20-28% between 6 and 24 months of age with no change in Gi alpha mRNA (2.4 kb). An aging-associated decline in beta-adrenergic stimulation was observed in the maximum positive inotropic effect elicited by isoproterenol in the presence of prazosin in left papillary muscles, with no change in ED50 values. These results suggest that age-related changes in cardiac excitation and contraction coupling following beta-adrenoceptor stimulation are mediated, at least in part, by Gs alpha protein dysfunction.

Propofol-induced Depression of Cultured Rat Ventricular Myocytes Is Related to the M2-acetylcholine Receptor–NO–cGMP Signaling Pathway

BACKGROUND It is well-known that propofol sometimes causes bradycardia or asystole during anesthesia; however, the direct effect of propofol on the myocardium remains unclear. Previous reports showed the contribution of muscarinic acetylcholine receptors to propofol-induced bradycardia. Conversely, it was suggested recently that nitric oxide (NO) plays an important role in mediating the effect of vagal stimulation in the autonomic regulation of the heart. Therefore, the authors investigated the effects of propofol on spontaneous contraction and NO production in cultured rat ventricular myocytes. METHODS The authors measured chronotropic responses of cultured rat ventricular myocytes induced by propofol stimulation with a sensor, a fiber-optic displacement measurement instrument. The authors also quantitatively analyzed NO metabolite production in cultured myocytes by measuring the levels of nitrite and nitrate in a high-performance liquid chromatography reaction system. The influence of propofol on muscarinic acetylcholine receptors of myocyte membranes was also measured with a competitive binding assay using [3H]quinuclidinyl benzilate ([3H]QNB). RESULTS Propofol caused negative chronotropy in a dose-dependent manner. Propofol (IC50) also caused the enhancement of nitrite production in cultured myocytes. Eighty percent of the enhancement of nitrite production induced by propofol (IC50) stimulation was abolished by pretreatment with atropine, methoctramine, or N(G)-monomethyl-L-arginine acetate (L-NMMA). The negative chronotropy induced by propofol (IC50) stimulation was reduced to 40-50% by pretreatment with atropine, methoctramine, L-NMMA, or 1H[1,2,4]oxadiazolo[4,3-alpha]quanoxalin-1-one, a selective inhibitor of guanylyl cyclase. Propofol displaced [3H]QNB binding to the cell membrane of myocytes in a concentration-dependent manner. CONCLUSION These results suggest that the negative chronotropy induced by propofol is mediated in part by M2-acetylcholine receptor activation, which involves the enhancement of NO production in cultured rat ventricular myocytes.

TETRANDRINE AS A CALCIUM ANTAGONIST

1. The Ca2+‐antagonism of tetrandrine (TET) on the Ca2+ mobilization in various types of cells were reviewed. Inositol trisphosphate (IP3)‐generating drugs were used as Ca2+‐mobilizing agonists and the effects were compared with those produced by using the microsomal Ca2+‐ATPase inhibitor thapsigargin (TG), which is a tool for analysing Ca2+ which is a tool for analysing Ca2+ store ‐regulated Ca2+ entry (capacitative Ca2+ entry).

Participation of Tubulin in the Stimulatory Regulation of Adenylyl Cyclase in Rat Cerebral Cortex Membranes

Abstract: This study examined effects of tubulin on the activation of adenylyl cyclase in rat cerebral cortex membranes. Tubulin, prepared from rat brain by polymerization with the hydrolysis‐resistant GTP analogue 5′‐guanylylimidodiphosphate (GppNHp) caused significant activation of the enzyme by ∼156% under conditions in which stimulation rather than inhibition of the enzyme was favored. Tubulin‐GppNHp activated isoproterenol‐sensitive adenylyl cyclase, potentiated forskolin‐stimulated activity of the enzyme, and reduced agonist binding affinity for β‐adrenergic receptors. When tubulin, polymerized with the hydrolysis‐resistant photoaffinity GTP analogue [32P]P3(4‐azidoanilido)‐P1‐5′‐GTP ([32P]AAGTP), was incubated with cerebral cortex membranes, AAGTP was transferred from tubulin to Gsα as well as Giα. These results suggest that, in rat cerebral cortex membranes, the tubulin dimer participates in the stimulatory regulation of adenylyl cyclase by transferring guanine nucleotide to Gsα, as well as affecting the Gi‐mediated inhibitory pathway.

Down-regulation of β-adrenoceptors and loss of Gsα subunit levels in ventricular myocardium of rats treated with isoproterenol

Abstract We investigated the influence of chronic β-adrenergic stimulation on the β-adrenoceptor-G protein-adenylyl cyclase system in rat ventricular myocardium. The rats received twice-daily injections of 4 mg/kg isoproterenol (ISO) alone or with 8 mg/kg propranolol (PROP) for 4 days. The ISO (10 μM)-induced increase in tissue cAMP production was lower (50%) after chronic ISO treatment than in control myocardium. The β-adrenoceptor density decreased by 43% in ventricular membranes from ISO-treated rats. Immunoblotting techniques using specific antibodies against G proteins revealed that ventricular myocardium contains three Gsα subunit isoforms of 45, 47 and 52kDa. ISO treatment decreased levels of the three Gsα subunits by a total of 40%, while no change in Giα (40/41kDa) and Gcommonβ (35/36kDa) levels were found in the same membrane preparations. The antagonist PROP almost totally blocked the effects of ISO treatment on cAMP, β-adrenoceptors and Gsα subunits. These results suggest that chronic β-adrenergic stimulation causes not only down-regulation of β-adcenoceptors, but also loss of Gsα subunit levels in rat ventricular myocardium.

Bilobalide prevents reduction of γ-aminobutyric acid levels and glutamic acid decarboxylase activity induced by 4-O-methylpyridoxine in mouse hippocampus

We previously reported that bilobalide, a constituent of Ginkgo biloba L. leaves, protected mice against convulsions induced by 4-O-methylpyridoxine (MPN). To elucidate the mechanism of the anticonvulsant activity of bilobalide, this study examined the effect of bilobalide on MPN-induced changes in the levels of gamma-aminobutyric acid (GABA) and glutamate, and in the activity of glutamic acid decarboxylase (GAD) in the hippocampus, cerebral cortex and striatum of the mouse. GABA levels and GAD activity in the hippocampus and cerebral cortex were significantly enhanced by bilobalide treatment (30 mg/kg, p.o., for 4 days) alone. MPN significantly decreased GABA levels and GAD activity in the three brain regions tested compared with those in the control. Pretreatment with bilobalide effectively suppressed the MPN-induced reduction in GABA levels and GAD activity in the hippocampus and cerebral cortex. On the other hand, there were no significant differences in the glutamate levels in the three regions despite various treatments. These results suggested that bilobalide prevents MPN-induced reduction in GABA levels through potentiation by bilobalide of GAD activity, and this effect of bilobalide contributes to its anticonvulsant effect against MPN-induced convulsions.