Genetically diabetic db/db mice and their normoglycemic littermates (+/+ mice) were studied to determine plasma levels of glucose, glucagon and insulin and hepatic gluconeogenic enzyme activities. Plasma glucose levels did not differ significantly between the 5-week-old db/db and +/+ mice, but increased with age in the former until the animals were 16-week-old. Similar age-associated changes were observed in the activities of the gluconeogenic enzymes, glucose-6-phosphatase (G-6-Pase) and fructose-1, 6-diphosphatase (F-1, 6-DPase). While the plasma levels of insulin and glucagon that peaked at 7 weeks of age did not parallel the hyperglycemia, the plasma glucagon/insulin (G/I) ratio roughly paralleled the hyperglycemia. Analysis of individual values for the db/db mice revealed statistically significant (P<0.001) correlations between plasma glucose levels and hepatic G-6-Pace (r=0.78) or F-1, 6-DPase (r=0.74) activity. There were also significant correlations between the G/I ratio and plasma glucose levels (P<0.001, r=0.66), hepatic G-6-Pase (P<0.01, r=0.48) or F-1, 6-DPase (P<0.01, r=0.57) activity. It is thus concluded that the relative predominance of glucagon over insulin action plays an important role in the ageassociated development of hyperglycemia in db/db mice. Glucagon presumably activates the hepatic gluconeogenic enzymes to enhance hepatic glucose output.

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We studied the effect of orally administered NIK-247 (9-amino-2, 3, 5, 6, 7, 8-hexahydro-1H-cyclopenta[b]quinoline monohydrochloride monohydrate) on basal extracellular acetylcholine (ACh) concentrations in the rat cerebral cortex using microdialysis without the addition of cholinesterase inhibitor to the perfusion fluid and radioimmunoassay for ACh. In addition, the effect of oral administration of NIK-247 on acetylcholinesterase (AChE) activity in rat cerebral cortex was determined. The mean basal ACh content in the perfusate from the cerebral cortex of freely moving rats was 123.2±21.8 fmol/30 min (n=7). NIK-247 (2.5 10.0 mg/kg, p.o.) increased the ACh content of the perfusate in a dose-dependent manner. NIK-247 at 10 mg/kg significantly increased the ACh content in the perfusate from 0.5 to 2.5 hr after administration, and the maximum increase was attained at 1 hr after administration. 9-Amino-1, 2, 3, 4-tetrahydroacridine (5 mg/kg, p.o.) and physostigmine (0.5 mg/kg, i.p.) significantly increased the ACh content in the perfusate from 1 to 2 hr and from 0.5 to 1.5 hr after administration, respectively. AChE activities in the cerebral cortex were about 32% and 12% below the control value at 1 hr and 3 hr after administration of NIK-247 at 10 mg/kg, respectively. These findings demonstrate that NIK-247 increases extracellular ACh concentration and inhibits AChE activity in the cerebral cortex after oral administration, and they suggest that NIK-247 facilitates central cholinergic transmission.

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We investigated the mechanism underlying stimulation of HCO₃^- secretion by the nitric oxide (NO) synthase inhibitor N^G-nitro-L-arginine methyl ester (L-NAME) in the gastroduodenal mucosa of anesthetized rats. A chambered stomach (in the presence of omeprazole) or a duodenal loop was perfused with saline, and HCO₃^- secretion was measured at pH 7.0 by a pH-stat method. Intravenous administration of L-NAME increased gastroduodenal HCO₃^- secretion with a concomitant rise in arterial blood pressure and a decrease in heart rate, and the changes were all antagonized by simultaneous administration of L-arginine. Vagotomy had no effect on the increased blood pressure response, but significantly inhibited the decrease of heart rate and increase of HCO₃^- secretion caused by L-NAME. The HCO₃^- stimulatory action of L-NAME was also inhibited by prior administration of yohimbine or prazosin. These agents alone lowered blood pressure and reduced the magnitude of the blood pressure response caused by L-NAME, leading to inhibition of heart rate changes. When ΔHCO₃^- output induced by L-NAME was plotted against Δblood pressure change (from basal values) under various conditions, a significant relationship was found between these two factors. These results suggest that L-NAME stimulates gastroduodenal HCO₃^- secretion in association with the inhibition of endogenous NO production, and this mechanism may be in part mediated by a neural reflex through the vagal efferent nerve, resulting from the pressor response to L-NAME.

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To develop a model to predict the efficacy and adversity of class I antiarrhythmic drugs, intraventricular conduction time (IVCT), coronary blood flow (CBF), developed tension of papillary muscle (DT) and idioventricular automaticity rate (VR) were measured following drug administration in an isolated canine papillary muscle preparation cross-circulated with the heparinized blood of a donor dog. Tetrodotoxin, the prototypic fast Na⁺ channel blocker, and class I drugs increased IVCT and CBF, but decreased DT and VR, in a dose-dependent manner. The profiles of known class I drugs, procainamide, disopyramide, lidocaine, mexiletine and flecainide were similar, but the potencies of each drug were different. Two new class I drugs, ME3202 and AN-132, were also tested and found to have effects that were similar to that of tetrodotoxin. There was a good correlation between the doses of drugs prolonging IVCT by 50% and the canine antiarrhythmic plasma concentrations in our previous study. This model can also be used to estimate the use-dependency and the kinetics of use-dependent sodium channel block; however, it is not suitable for extensive investigation of cellular and molecular mechanisms. Thus, the use of this model facilitates the comparison of multiple cardiac effects of class I drugs and may be an effective way to better assess new antiarrhythmic drugs.

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Itopride is a gastroprokinetic benzamide derivative. This agent inhibited both electric eel acetylcholinesterase (AChE) and horse serum butyrylcholinesterase (BuChE). The IC₅₀ of itopride with AChE (2.04±0.27 μM) was, however, 100-fold less than that with BuChE, whereas in the case of neostigmine with AChE (11.3±3.4 nM), it was 10-fold less. The recovery of AChE activity inhibited by 10^-7 M neostigmine was partial, but that inhibited by upto 3 × 10^-5 M itopride was complete when the reaction mixture was subjected to ultrafiltration. Double reciprocal plots of the experimental data showed that both K_m and V_max were affected by itopride, suggesting that the inhibition is a "mixed" type, although primarily being an uncompetitive one. The inhibitory effect of itopride on cholinesterase (ChE) activity in guinea pig gastrointestine was much weaker than that on pure AChE. However, in the presence of a low dose of diisopropyl fluorophosphate, just enough to inhibit BuChE but not AChE, the IC₅₀s of itopride against ChE activities were found to be about 0.5 μM. In conclusion, itopride exerts reversible and a "mixed" type of inhibition preferably against AChE. The IC₅₀ of itopride for electric eel and guinea pig gastrointestinal AChE inhibition was 200 times and 50 times as large as that of neostigmine, respectively.

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In cultured bovine adrenal chromaffin cells, pituitary adenylate cylase-activating polypeptide (PACAP) stimulated [¹⁴C]catecholamine synthesis from [¹⁴C]tyrosine (but not from [¹⁴C]DOPA) in a concentration-dependent manner, causing maximal stimulation at 10^-7 M. The stimulatory action of PACAP was not affected by staurosporine (an inhibitor of protein kinase C) or in the cells in which protein kinase C was down-regulated by prolonged exposure to TPA (an activator of protein kinase C), whereas it was partially attenuated in Ca²⁺-free medium. PACAP (10^-7M) increased the formation of [³H]inositol phosphates, [Ca²⁺]_i and ⁴⁵Ca2+ uptake as well as cAMP. The peptide also stimulated the phosphorylation of tyrosine hydroxylase, the enzyme catalyzing the rate-limiting step in catecholamine synthesis. Catecholamine synthesis and tyrosine hydroxylase phosphorylation stimulated by the maximal effective concentration of dibutyryl cAMP or high K⁺, which activates Ca²⁺ uptake, were further enhanced by PACAP, suggesting that both cAMP- and Ca²⁺-dependent protein kinases may be involved in the stimulation of tyrosine hydroxylase phosphorylation and catecholamine synthesis caused by PACAP.

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2, 5-Anhydro-D-mannitol (AM), a putative gluconeogenesis inhibitor, completely reversed the hyperglycemia in genetically diabetic (db/db) mice that exhibited hyperinsulinemia and enhanced hepatic gluconeogenic enzyme (glucose-6-phosphatase (G-6-Pase) and fructose l, 6-diphosphatase (F-1, 6-DPase)) activities compared with the control +/+ mice. In contrast, AM only partially reversed the hyperglycemia of streptozotocin (STZ)-treated +/+ mice in which the hepatic gluconeogenic enzyme activities were enhanced to the same degree as in the db/db mice, whereas the blood insulin level was depressed. In the db/db mice, the STZ-treatment attenuated the hyperinsulinemia and exaggerated the hyperglycemia as well as the hepatic gluconeogenic enzyme activities, and it greatly reduced the hypoglycemic action of AM. Not only the dose-response curve of AM but also the time-course of the blood glucose level (expressed as % of pre-treatment value) following 320 mg/kg of AM were almost identical between +/+, STZ-treated +/+ and STZ-treated db/db mice. In the STZ-treated +/+ mice, a combination treatment of insulin (320 μg/kg) with AM (320 mg/kg) caused hypoglycemia that was greater than that induced by AM or in sulin alone. On the other hand, in vitro studies with purified F-1, 6-DPase revealed that phosphorylated AM (AM-1, 6-diphosphate) but not AM itself inhibited the gluconeogenic enzyme activities. These results suggest that inhibition of gluconeogenesis is responsible, at least in part, for the hypoglycemic activity of AM. AM appears to inhibit hepatic gluconeogenic enzyme activities after being phosphorylated by an insulin-dependent mechanism.

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The effects of a mixture of three peptidase inhibitors (PIs), amastatin, captopril and phosphoramidon, on methionine-enkephalin (Met-enk)-, β-endorphin (β-end)-, dynorphin-(1-13) (Dyn)-and electroacupuncture (EA)-induced antinociception were compared in rats. EA was performed by passing electric pulses (3 Hz, 0.1-msec duration, for 45 min) through acupuncture needles inserted into the Hokupoint. The antinociceptive effect was estimated by the hind paw pressure test. The antinociceptive effects of Met-enk and β-end injected i.c.v. or Lt. and of Dyn injected i.t. were clearly potentiated by the PIs pretreated by the same administration routes as used for the injection of opioid peptides. The antinociceptive effects of Met-enk, β-end and Dyn injected i.c.v. were also potentiated significantly by i.t.-PIs. PIs injected into the periaqueductal gray (PAG) potentiated EA antinociception. However, the EA effect was not affected by i.t.-PIs and was rather attenuated by i.c.v.-PIs. These results suggest that: i) Met-enk hydrolyzing enzymes are involved in the degradation of not only Met-enk but also β-end and Dyn in the rat central nervous system; ii) Met-enk and β-end act on both supraspinal and spinal sites, while Dyn acts only on the spinal site; iii) EA antinociception is mediated by supraspinal Met-enk and/or β-end; and iv) an anti-opiate peptide system may be activated by EA stimulation, being susceptible to Met-enk hydrolyzing enzymes.

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The relationship between the morphological changes and vitamin A content during the development of acute toxicity induced by polychlorinated biphenyl (PCB) in mouse parotid glands was investigated. PCB was administered intraperitoneally at a single dose of 2 mg/kg. Ultrastructural studies revealed remarkable morphological changes in the rough endoplasmic reticulum, nucleus, Golgi apparatus and the secretory granules at 7 days after the administration of PCB. The activities of adenosine monophosphatase (AMPase) and alkaline phosphatase were increased 1 day after PCB administration. Then the activity of NADPH-cytochrome c reductase increased 4 days after PCB administration. Subsequently, the vitamin A content of the parotid glands significantly decreased at 7 days compared with the control. These sequential changes in enzyme activities implied that the decrease of vitamin A content in the parotid glands may be partly due to catabolism of vitamin A by increased activities of microsomal enzymes induced by PCB. In conclusion, it is suggested that PCB also induces drug metabolizing enzymes in the parotid gland cells and that the acute toxicity of PCB on this tissue may occur, at least partly, through the reduction of vitamin A not only by the secondary effect from liver impairment but also by the locally accelerated catabolism of vitamin A in the mouse parotid gland.

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Effects of cobalt chloride on liver glutathione S-transferase and protease activities were studied. When cobalt chloride (60 mg/kg) was given to rats, liver microsomal glutathione S-transferase and protease activities were significantly increased 24 hr after the injection, whereas glutathione peroxidase activity in microsomes was decreased. The increase in glutathione S-transferase activity caused by cobalt chloride treatment was not reversed by dithiothreitol, and the activation of the transferase by N-ethylmaleimide was similar to that of the control, indicating that the increase in the transferase activity by cobalt chloride is not due to a modification of the sulfhydryl group of the enzyme. Immunochemical analysis of the liver microsomes did not detect any proteolytic product of microsomal glutathione S-transferase. In puromycinor actinomycin D-treated rats, an increase in the transferase activity caused by cobalt chloride treatment was depressed. Thus it was suggested that liver microsomal glutathione S-transferase is induced by cobalt chloride treatment, but not activated by limited proteolysis via microsomal protease.

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Effects of 4-acylaminophenol derivatives, novel 5-lipoxygenase inhibitors, on the neutrophil infiltration in arachidonic acid (AA) (10 mg/site)-induced skin inflammation in rats were examined. Myeloperoxidase (MPO) activity in the skin lesion, used as an indicator of neutrophil infiltration, was significantly increased after intradermal injection of AA. Dual inhibitors of cyclooxygenase and 5-lipoxygenase, phenidone (100 mg/kg × 2, i.p. and p.o.) and BW-755C (50 mg/kg × 2, p.o.), and 5-lipoxygenase inhibitors, AA-861 (100 mg/kg × 2, i.p.) and the 4-acylaminophenol derivatives T-0799 and T-0757 (10-100 mg/kg × 2, p.o.), inhibited the increase in MPO activity 5 hr after AA-injection, but the cyclooxygenase inhibitor indomethacin (5 mg/kg, i.p.) showed no effect. These results suggest that products of lipoxygenase, but not of cyclooxygenase, are involved in the MPO activity increase (i.e., neutrophil infiltration), and that this model is useful for in vivo evaluation of 5-lipoxygenase inhibitors. It is suggested that the 4-acylaminophenol derivatives may be useful as orally active drugs for treatment of some leukotriene-mediated diseases.

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Butyrate at concentrations of 200-600 μM markedly enhanced the in vitro antibody productions against sheep red blood cells (SRBC) in murine splenocytes. However, other saturated short-chain fatty acids, including acetate, propionate and valeric acid, and 4-carbon compounds such as butanol, acetoacetate and β- and γ-hydroxybutyrate had no such effects. The presence of butyrate in the early phase of the cell culture was crucial for enhancement of the response. Butyrate also augmented the antibody production in T-cell-depleted splenocytes supplemented with the culture supernatant of concanavalin A (Con A)-stimulated lymphocytes. Interleukin (IL)-2 secreted from splenocytes in response to SRBC was increased by adding butyrate to the culture, but IL-1 secretion was not affected. On the other hand, Con A or lipopolysaccharide-stimulated proliferation of splenocytes was partly depressed by the addition of butyrate, while Con A-induced IL-2 production was not effected. These findings suggest that butyrate may act on T and B cells to promote their differentiation during the process of antibody production.

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The relationship between the influx of Ca²⁺ into cells or cytosolic Ca²⁺ concentration ([Ca²⁺]_i) and the histamine release following antigen stimulation in mouse bone marrow-derived mast cells (BMMC) was examined, and the results were compared with those from human lung mast cells (HLMC) and rat peritoneal mast cells (RPMC) in some experiments. Anaphylactic histamine release from BMMC as well as HLMC, but not that from RPMC, was dependent on the extracellular Ca²⁺. When BMMC were challenged by antigen following radioactive ⁴⁵Ca²⁺ addition, two phases of ⁴⁵Ca²⁺ influx into the cells were observed. The first phase, which was initiated and completed within 30 sec and 2 min, respectively, after antigen treatment, appeared to be related to anaphylactic histamine release. The second influx began 30 sec subsequent to the first one and lasted for at least 2 min, and this occurred after the completion of the histamine release; So far, it is not known how this second influx participates in the intracellular event(s). On the other hand, only one sustained elevation of [Ca²⁺]_i occurred that reached its maximum within 2 min after antigen stimulation. Following stimulation of BMMC with antigen in the absence of Ca²⁺, Ca²⁺ addition 1 to 5 min later time-dependently enhanced the histamine release, although the release was deteriorated by further extension of Ca²⁺ addition. In contrast, the releasability of HLMC was rapidly decreased. These results indicate that extracellular Ca²⁺ not only is prerequisite for anaphylactic histamine release from BMMC, but also may modulate the release and participate in some intracellular event(s) which has yet to be focused upon.

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Sex differences in the anticoagulant effects of warfarin were studied in rats. Warfarin was administered to rats from 7 days of gestation until 14 weeks of age. In male rats, the normal prothrombin level in the plasma was reduced, and the blood coagulation time was prolonged by treatment with warfarin at 4, 9 and 14 weeks of age. However, in female rats, the effects of warfarin on the prothrombin level and blood coagulation time were observed at 4 weeks to the same degree as in male rats, but these effects were reduced with aging, and at 14 weeks, no effect of warfarin was observed. Rats ovariectomized at 12 weeks of age and subsequently treated with warfarin for 2 weeks showed prolongation of blood coagulation time to the same level as in warfarin-treated male rats, which was inhibited by administration of 17β-estradiol (100 μg/kg /day for 4 days, i.m.). In male rats, treatment with 17β-estradiol also inhibited the anticoagulant effects of warfarin without changing the warfarin level in plasma. These results suggest that there is a sex difference in the anticoagulant effects of warfarin, and that this difference may be related to the estradiol level in plasma.

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We examined the possible existence of excitatory cholinergic innervation in isolated rabbit portal vein. Longitudinal strips of the vein in the presence of both phenoxybenzamine, an α-adrenoceptor antagonist, and N^G-nitro-L-arginine, an inhibitor of nitric oxide synthase, exhibited a small contraction in response to transmural electrical stimulation (ES). The contractile response to ES was augmented by physostigmine, an anticholinesterase agent, and inhibited by atropine. These findings indicate the existence of an excitatory cholinergic innervation in addition to the known adrenergic and non-adrenergic, non-cholinergic innervations in the rabbit portal vein.

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