Properties of the intracellular Ca store were studied using saponinskinned fiber bundles of guinea pig smooth muscles using a fluorescent Ca indicator method. There exist two Ca release mechanisms in the Ca store: Ca-induced Ca release (CICR) and inositol 1, 4, 5-trisphosphate (IP₃)-induced Ca release (IICR) mechanisms. The smooth muscle Ca store consists of two compartments: one (Sα) has both CICR and IICR, and the other (Sβ) has only IICR. The smooth muscle CICR is activated by >1 μM Ca²⁺, has essentially the same properties with that in striated muscles, and is open-locked by ryanodine. After ryanodine treatment, therefore, the Ca uptake capacity of Sα is selectively lost (‘functional removal’) with no effect on Sβ. The IICR is Ca²⁺-dependent: Ca²⁺ enhances the IICR below 300 nM, but has also an inhibitory effect above this concentration. Therefore, Ca²⁺ acts on the IICR in a positive feedback manner when muscle tension is about to rise, making IP₃ more effective, but this feedback is cut off as the tension approaches the maximum. ATP enhances the IICR as is the case in the CICR. 'Functional removal' of Sα in intact bundles by ryanodine was used to estimate the role of Ca release in agonist-induced contractions. Ca release from the Sα is important at least in the initial phase of contractions; and in the pulmonary artery, most of the activator Ca²⁺ originates from Sα.

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Effects of novel aldose reductase inhibitors, M16209 (1-(3-bromobenzo[b]furan-2-y!sulfonyl)hydantoin) and M16287 (1-(3-chlorobenzo[b]furan-2-ylsulfonyl)hydantoin), on galactose-induced cataract formation in rats were investigated. Rats fed a 30% galactose diet developed lenticular opacity in the peripheral region by the 6th day of galactose feeding and showed gradual progression of opacity from the equator to the center of lenses. Histological study on the 15th day showed apparent lens fiber swelling and vacuolation predominantly in the equatorial and anterior cortical regions. Biochemical changes such as accumulation of galactitol, depletion of myo-inositol and decrease in glutathione (GSH) content in lenses preceded the appearance of opacity. Remarkable increase in NADPH content and decrease in NADP⁺ content, in addition to elevation of the ratio of Na⁺/K⁺, in lenses were also observed on the 15th day. Both M16209 and M16287 (10, 30 and 100 mg/kg/day, p.o.) dose-dependently ameliorated these morphological and biochemical changes except that restoration of myo-inositol content was incomplete. These results indicate that M16209 and M16287 can prevent galactose-induced cataract formation through amelioration of metabolic disorders and thus have high potential for clinical use in the treatment of some diabetic complications.

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Catecholamine-induced stimulation of hepatic glycogenolysis in male and female rats was studied by detecting the cytosolic free Ca²⁺ concentration ( [Ca²⁺]₁), cAMP generation and adrenergic receptor function. Increase in α₁-adrenergic receptor-mediated [Ca²⁺]₁ and β-adrenergic receptor-mediated cAMP generation were examined using isolated hepatocytes. No difference was found in the α₁-adrenergic receptor-mediated response of [Ca²⁺]₁ in fura-2-loaded hepatocytes between males and females, while epinephrine-induced cAMP accumulation in hepatocytes was about 3-fold higher in females. The α₁ and β-adrenergic receptor properties of the plasma membrane were evaluated by ligand binding studies using[³H]prazosin (α₁-adrenergic antagonist) and [¹²⁵I] iodocyanopindolol (β-adrenergic antagonist); and little sex difference was found in either affinity or the number of binding sites of[³H]prazosin and [¹²⁵I] iodocyanopindolol. Activation of adenylate cyclase by forskolin and GTP-γ-S was also similar for both sexes. These results suggest that the sex difference of β-adrenergic response is due to a difference in the guanine nucleotide regulatory binding proteins (G proteins) and/or β-adrenergic receptor-Gs protein (the stimulatory G protein of adenylate cyclase) coupling ability.

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Effects of Kamikihi-To (KMK), a traditional Chinese medicine (Chinese name: Jia-Wei-Gui-Pi-Tang), on learning performance impairment caused by aging were evaluated in senescence accelerated mice (SAM). Normal diet containing 8% KMK extract was given to SAM-P/8, a senile-prone strain, and to SAM-R/1, a resistant strain, from 2 months old. Effects of KMK on learning performance were evaluated in 5 and 10 month old SAM using step through and step down type passive avoidance tests and shuttle box and lever press type conditioned avoidance tests. At 5 months old, KMK increased the retention rate in the step through test and decreased the number of errors in the step down test in SAM-P/8, though KMK had no effects in conditioned avoidance tests. KMK had no effects in any tests in SAM-R/1. At 10 months old, the decrease of the number of errors in the step down test and increase of the rate of the conditioned avoidance response in the shuttle box test were observed in SAM-P/8 treated with KMK. These results suggest that chronic administration of KMK can improve learning performance in the senescence model.

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The effects of 7% sodium bicarbonate on medial vestibular nucleus (MVN) neurons were examined to elucidate the mechanism underlying its antivertigo action, using α-chloralose-anesthetized cats. Intravenous injection of the drug at 1, 2 and 4 mI/kg every 10 min dose-dependently inhibited rotationand glutamate-induced firing of type I neurons, although a low dose of the drug enhanced firing in a few neurons. However, microiontophoretic application of bicarbonate ions did not inhibit rotation or glutamate-induced firing. After injection of the drug, the P0₂ level in arterial blood did not differ from previous levels, but the bicarbonate ion levels dose-dependently increased concomitantly with an increase in pH, as compared with previous levels. These results suggest that the intravenous injection of 7% sodium bicarbonate directly inhibits the neuronal activity of the MVN, although the lower dose may enhance neuronal activity by acting on the peripheral vestibule.

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Effects of high K⁺, acetylcholine (ACh), 9, 11 -epithio-1 1, 12-methano-thromboxane A₂ (STA₂), thrombin and endothelin were investigated on smooth muscles of the guinea pig coronary artery in intact and endothelium-denuded tissues. In intact tissues, ACh transiently inhibited but ATP produced maintained inhibition of the STA₂-induced contraction. However, in the endothelium-denuded tissue, ACh produced contraction and ATP inhibited the STA₂-induced contraction. In intact tissues, thrombin produced dual actions on the STA₂-induced contraction with an initial relaxation followed by contraction. In endothelium-denuded tissues, thrombin enlarged the STA₂-induced contraction without transient relaxation. In intact tissues prepared from both proximal and distal regions, endothelin showed the same dual action as observed with thrombin, whereas higher concentrations of endothelin showed only contraction. In endothelium-denuded tissues, endothelin consistently produced contraction. In intact tissues prepared from proximal and distal regions, ACh produced a biphasic response (initial hyperpolarization and subsequenty generated depolarization). The amplitudes of both potential changes occurred in a membrane potential-dependent manner. In endothelium-denuded tissues, ACh depolarized the membrane in both tissues. In intact and endothelium denuded tissues, ATP hyperpolarized the membrane in inverse proportion to the membrane potential level, whereas thrombin and endothelin consistently depolarized the membrane. The results indicate that ACh acts on endothelial and smooth muscle cells, and the former releases both EDRF and EDHF. ATP only acts on smooth muscle cells and hyperpolarizes the membrane. STA₂, thrombin and endothelin act on both endothelial and smooth muscle cells. STA₂ and endothelin may release EDRF but not EDHF, and thrombin may release EDRF and endothelin.

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Changes in monoamine levels during and after ischemia and effects of RS-8359, a type A monoamine oxidase (MAO-A) inhibitor, were studied in the cerebral cortex, hippocampus, and striatum of rats killed by microwave irradiation. The patterns of the changes in norepinephrine (NE), dopamine (DA), and serotonin (5HT) levels were similar during ischemia: All these monoamines decreased in all three regions. After reperfusion, however, the three monoamines showed different patterns of changes: NE, except in the striatum, decreased further; DA increased over the controls; 5HT remained suppressed in all three regions. With regard to the metabolites of the monoamines, the changes during and after reperfusion were almost similar in all regions: 0-methylated metabolites, normetanephrine and 3-methoxytyramine, markedly increased during ischemia; After reperfusion, the elevated levels of normetanephrine and 3-methoxytyramine returned to normal, while deaminated metabolites, 3, 4-dihydroxyphenylacetic acid (DOPAC) and 5hydroxyindoleacetic acid, homovanillic acid (HVA), and 3-methoxy-4-hydroxyphenylethyleneglycol clearly increased. RS-8359 pretreatment (30 mg/kg, p.o.) at an hour prior to ischemia elevated the levels of NE in the cortex and hippocampus during ischemia and inhibited the increases in DOPAC and HVA levels and the decrease in 3MT levels at 30 min after reperfusion. These results suggest that deamination of NE, DA, and 5HT is activated by the increases in the substrates for MAO in all three regions, except the noradrenergic system in the striatum, and that MAO-A participates in the activated deamination after reperfusion.

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Ethylcholine mustard aziridinium ion (AF64A), a synthesized cholinergic neurotoxin, was administered via intraperitoneal injection to the rat to study its effect on the central cholinergic nervous system. A single or consecutive daily injection of AF64A for 10 days resulted in a persistent reduction of acetylcholine (ACh) content in the several tested regions of the brain in the following order: hippocampus>cerebral cortex=striatum, the degree was the greatest in the hippocampus. Both resting and K⁺-stimulated release of ACh from the hippocampus were also significantly reduced 24 hr after a single injection of AF64A. Furthermore, daily injection of AF64A for 10 days induced a significant reduction of choline acetyltransferase (ChAT) activity in the homogenate obtained from the hippocampus but not from the cerebral cortex and striatum. ChAT activity in the crude synaptosomal fraction of the cerebral cortex was also significantly decreased. These results suggest that intraperitoneal administration of AF64A could induce cholinergic hypofunction more selectively in the nerve terminals. The high affinity choline uptake, which is located mainly on cholinergic nerve terminals, was not affected by the administration of AF64A. Any notable changes of ultrastructure in the cholinergic nerve terminals after the administration were not observed in all three regions examined. The present findings suggested that intraperitoneal administration of AF64A induces a specific damage of cholinergic nerve terminals by inhibiting ChAT activity. The cholinergic damage was most prominent in the hippocampus.

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The effect of Ba²⁺ on the catecholamine biosynthetic activity was studied by measuring the formation of [¹⁴C]catecholamines from L-[¹⁴C]tyrosine in cultured adrenal chromaffin cells. In the absence of Ca²⁺, [¹⁴C]catecholamine formation was markedly stimulated by Ba²⁺, and this stimulation was observed in a manner dependent on its concentration. The stimulation of [¹⁴C]catecholamine formation by relatively low concentrations of Ba²⁺ was suppressed by polymyxin B, a typical inhibitor of Ca²⁺/phosphol1pid-dependent protein kinase (protein kinase C); and this inhibitory action of polymyxin B was attenuated by increasing the Ba²⁺ concentration. On the other hand, a tendency toward the enhancement of Ba²⁺- stimulated [¹⁴C]catecholamine formation was observed by a protein kinase C activator, 12-0-tetradecanoylphorbol 13-acetate (TPA). In contrast to the acute effect of TPA, [¹⁴C]catecholamine formation stimulated by Ba²⁺ was reduced by long-term exposure of chromaffin cells to a high concentration of TPA, which has already been reported to cause the reduction of protein kinase C activity as a result of the down-regulation of this enzyme. These findings suggest that Ba²⁺ stimulates catecholamine biosynthesis, probably through its direct action on protein kinase C in adrenal chromaffin cells.

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Our purpose was to localize the intrarenal vascular sites of action of adenosine and glucagon. Renal blood flow (RBF) and glomerular filtration rate (GFR) were measured in anesthetized dogs, and renal perfusion pressure (RPP) was varied by an adjustable aortic clamp. At normal RPP, RBF was increased by all agents. In contrast, GFR was increased by glucagon, decreased by adenosine and unchanged by acetylcholine (ACh) or adenosine plus glucagon. The increases in RBF by glucagon occurred only at RPPs within the autoregulatory pressure range, and renal autoregulatory capability was attenuated during the infusion of glucagon. In contrast, adenosine increased RBF at RPPs both within and below the autoregulatory pressure range, and the autoregulatory capability was not perceptibly impaired. Superimposition of glucagon to adenosine caused further vasodilation, and the autoregulatory efficiency was completely attenuated. There was no difference between the RPP-RBF or RPP-GFR relations obtained during infusion of adenosine plus glucagon and ACh, which dilates both the afferent and efferent arterioles. It is generally accepted that afferent arteriolar resistance attains a minimum value at RPP near the lower limit of the autoregulatory range. Thus, our data indicate that glucagon and adenosine preferentially dilate the afferent arteriole and the efferent arteriole, respectively.

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Effect of oral and intraperitoneal administrations of [1, 3-di-n-butyl-7-(2-oxopropyl)-xanthine] (denbufylline) on acetylcholine (ACh) content and release in the rat striatum was investigated. Denbufylline (3, 10 and 30 mg/kg, p.o.) decreased striatal ACh contents in a dose-dependent manner. Denbufylline administration (30 mg/kg, i.p.) produced no significant change in the spontaneous release of ACh, while it increased a high potassium-evoked ACh release in the striatum. These results suggest that denbufylline may be a drug inducing the increased release of ACh in the brain.

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The immunomodulatory activity of ginsenoside Rg₁ from Panax ginseng was studied in mice using sheep red cells as the antigen. It was found that ginsenoside Rg₁ at a dose of 10 mg/kg administered for three consecutive days before immunization increased the number of spleen plaque-forming cells, the titers of sera hemagglutinins as well as the number of antigen-reactive T-cells. Ginsenoside Rg₁ also increased the number of T-helper cells with respect to the whole T-cell number and the splenocyte natural killer activity. Ginsenoside Rg₁ induced an augmentation of the production of IL-1 by macrophages and exerted a direct mitogenic effect on microcultured thymus cells. Ginsenoside Rg₁ also partly restored the impaired immune reactivity by cyclophosphamide treatment.

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In the course of investigating the mechanisms of the in vitro immunosuppressive effect of D-penicillamine in the presence of copper ion with murine spleen cells, we observed that copper ion, by itself, exhibited a strong immunosuppressive effect at a low concentration. Namely, the addition of CuSO₄ at a concentration of 0.1 to 2 μM markedly inhibited the development of SRBC-specific plaque forming cells (PFC) without causing cytotoxicity. CuSO₄, at the same concentration range, suppressed the lipopolysaccharide (LPS)-induced proliferative response, but not the response induced by concanavalin A (Con A). The suppressive effect of CuSO₄ on the antibody production was reversed by 500 U/ml of catalase, but that on the LPS-induced proliferative response was not. CuSO₄ also substantially suppressed the pokeweed mitogen-induced proliferative response. These findings suggest that CuSO₄ acts as an inhibitor of B cell proliferation and, through this effect, markedly inhibits the antibody production in murine spleen cells.

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Pressor effects of N^G-monomethyl-L-arginine (L-NMMA), a selective inhibitor of nitric oxide production from L-arginine, on mean blood pressure (MBP) were investigated in conscious Wistar Kyoto rats (WKY) and stroke-prone spontaneously hypertensive rats (SHRSP). L-NMMA (0.1-10 mg/kg, i.v.) elicited a dose-dependent increase in the MBP of WKY and SHRSP. The pressor response to L-NMMA was more marked in SHRSP than in WKY. These results suggest that nitric oxide may play an important role in the blood pressure regulation in the conscious SHRSP.

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Cytotoxic effect in vitro and antitumor effect in vivo of doxorubicin (DOX) combined with cepharanthine were investigated on DOX-resistant murine P388 leukemia (P388/R) cells. Cepharanthine was minimally cytotoxic in the cell line, but reversed DOX-resistance in a dose-related manner in P388/R cells. The administration of cepharanthine to mice bearing the P388 leukemia enhanced the antitumor activity of DOX. These results indicate that cepharanthine is an effective agent to reverse DOX-resistant cells.

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The effect of a non-protein fraction from an extract of inflamed skin of rabbits inoculated with vaccinia virus (Neurotropin, NSP) was studied on Meth A tumor-induced delayed type hypersensitivity (Meth A-DTH) in BALB/c mice. NSP enhanced the Meth A-DTH. NSP also enhanced the DTH suppressed with 5-fluorouracil (5-FU). Moreover, NSP inhibited the fatal effect of 5-FU and restored the decrease of body weight caused by 5-FU. However, NSP reduced partially but significantly the suppression of the tumor growth by 5-FU. NSP may be useful for cancer treatment in combination with chemotherapeutic agents, if NSP does not inhibit their antitumor activity.

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Effects of glibenclamide on the control membrane ionic currents, acetylcholine or adenosine-induced K⁺ current, and nicorandil-induced K⁺ current were examined in single atrial myocytes of guinea pig heart. The nystatin-whole cell clamp technique was used. Nicorandil evoked the time-independent K⁺ current which is probably the current through the ATP-sensitive K⁺ channel. Glibenclamide inhibited this current in a concentration-dependent fashion, although it had no effect on the other currents. We concluded that glibenclamide specifically inhibits the ATP-sensitive K⁺ channel current in cardiac myocytes.

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The present study employed various receptor-binding assays to clarify the biochemical characteristics of SM-9018. SM-9018 possessed very high affinity for 5-HT₂, D₂ and 5-HT_1A receptors (K₁=0.61, 1.4 and 2.9 nM, respectively), and it had moderate affinity for α₁, and D₁ receptors (K₁=17 and 41 nM, respectively). However, SM-9018 had only negligible affinity for α₂, opiate, glutamate, phencyclidine, benzodiazepine and GABA_A receptors. These results suggest that SM-9018 may be a novel antipsychotic agent with binding affinity for 5-HT₂ and 5-HT_1A receptors.

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In anesthetized rats, SKF 38393 (10 mg/kg, i.v.) did not facilitate urinary bladder motility, but bromocriptine (BR, 5 mg/kg, i.v.) alone and the combination of BR (1 mg/kg, i.v.) and SKF 38393 (1 mg/kg, i.v.) induced a hyperactive bladder response (HBR). Both HBR induced by BR alone or BR and SKF 38393 combined was suppressed by SCH 23390. sulpiride or haloperidol. These results indicate that HBR is mediated by the activation of D-2 receptors, and the effects of D-2 agonists on bladder motility are potentiated by the simultaneous stimulation of D-1 receptors.

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Higher plasma concentrations of 1-(tetrahydro-2-furanyl)-5-fluorouracil (FT) were obtained after administration of FT (100 mg/kg, i.v.) combined with Lcysteine (CySH, 500 mg/kg, p.o.). The volume of distribution (V_d) and body fluid volumes significantly decreased. These results suggest that the increase of the plasma concentrations of FT can be attributed to the decrease of the V_d of FT, which is considered to be based on the decrease of body fluid volumes by the combined administration of CySH.

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In order to clarify the mechanisms involved in the endothelium-dependent development of desensitization of norepinephrineinduced contraction in rat aorta, we have tested the effect of repeated generation of tension without receptor stimulation. Even when tension alone, with a magnitude almost equal to that generated by norepinephrine, was applied to the endothelium-intact ring without norepinephrine, the ring became desensitized. In the absence of endothelium, the development of desensitization did not occur. Furthermore, L-N^G-monomethyl arginine, which is an inhibitor of endothelium-dependent relaxing factor (EDRF) synthesis, prevented the occurrence of desensitization. It was even able to reestablish contractile force when added after the desensitization had developed, suggesting that an increased release of EDRF is necessary to produce the desensitization. Therefore, these results indicate that endothelium-dependent desensitization does not require adrenergic receptor stimulation, but rather that tension generation alone is sufficient to establish desensitization.

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