We studied [3H]glutamate binding to human brain membrane preparations. We found that the binding assay of [3H]glutamate should be done at 4°C using glass-fiber filters pretreated with polyethyleneimine, sterile incubation buffer, and membrane preparations pretreated with a detergent. Under these assay conditions, we have characterized the three subtypes of glutamate receptors: N-methyl-D-aspartate-, quisqualate- and kainate-sensitive binding sites. Scatchard analysis revealed that the N-methyl-D-aspartate-sensitive [3H]glutamate binding sites consisted of a single component with a dissociation constant (Kd) of 28.2 ± 2.8 nM and total concentration (Bmax) of 36.2 ± 3.6 fmol/mg protein (N = 8). These data were much different from those presented in previous studies.
The present study was designed to elucidate pathophysiological changes in the brain energy metabolism after cerebral ischemia. Cerebral ischemia was induced in rats by administering microspheres into the right carotid canal, and the time course of changes in cerebral energy metabolism was examined up to the 7th day after the operation. Approximately 50% of the operated rats revealed typical symptoms of stroke. In the right hemisphere, cerebral ATP and creatine phosphate of the rat on the 1st to 7th day were greatly reduced by the microsphere-induced cerebral embolism (maximally 52 and 61%, respectively), whereas the tissue lactate level was increased on the 1st, 3rd and 5th day after the embolism (maximally 125%), suggesting an induction of microsphere-induced cerebral ischemia. These changes in the tissue metabolites were accompanied by a decrease in the mitochondrial oxidative phosphorylation measured in the presence of succinate. A similar trend in the changes of biochemical markers was observed in the left hemisphere, but to a lesser degree or to an insignificant degree. The pathophysiological alterations in behavior and cerebral metabolism of microsphere-injected rats tended to return toward the normal levels on the 7th day after the operation. The results provided information on a useful model for therapeutic studies of anti-ischemic agents in the brain.
The effect of famotidine on gastric lesions induced by the decrease in mucosal defensive resistance was investigated in rats and compared with those of cimetidine, pirenzepine and cetraxate. Famotidine (0.03, 0.1 and 0.3 mg/kg, p.o.) inhibited dose-dependently the development of gastric lesions produced by taurocholatehistamine in doses that suppressed histamine-induced acid secretion in pylorus-ligated rats. The H2-antagonist also prevented gastric mucosal lesions induced by taurocholate-serotonin, iodoacetamide, acidified aspirin and acidified ethanol. Cimetidine, pirenzepine and cetraxate showed the inhibitory effects on almost all types of the gastric lesions, but their inhibitory effects were much less potent than those of famotidine. On the other hand, famotidine inhibited the decreases of gastric mucosal blood flow induced by acidified ethanol and the mucosal contents of glycoprotein induced by water immersion restraint stress. In addition, famotidine increased the transgastric potential difference (PD) and promoted the recovery of decreased transgastric PD induced by acidified ethanol in rats. These results suggest that the preventive effect of famotidine on gastric lesions is attributable not only to suppression of acid secretion but to activation of the gastric mucosal defensive mechanisms.
The characteristics of [3H]choline transport with high affinity were investigated using primary cultured neurons obtained from the mouse cerebral cortex. [3H]Choline uptake was saturable as a function of extracellular [3H]choline concentration. Analysis by Lineweaver-Burk plot revealed that [3H]choline was transported into neurons by a high affinity transport system with a Km value of 19.8 ± 0.8μM and Vmax value of 0.334 plusmn; 0.022 nmol/mg protein/min. This high affinity transport of [3H]choline was significantly inhibited by the withdrawal of sodium from the incubation medium, incubation at low temperature (4°C) and addition of metabolic inhibitors such as monoiodoacetate. These results indicate that the high affinity [3H]choline uptake in primary cultured neurons is sodium- and energy-dependent. Hemicholinium-3 also showed a competitive inhibition on the [3H]choline transport. Depolarization by high K+ induced an enhancement of the [3H]choline uptake in the presence of Ca2+. The crude synaptosomal fraction obtained from primary cultured neurons possessed approximately forty-fold higher synthesizing activity of [3H]acetylcholine from [3H]choline than that found in the homogenate preparation of cultured neurons. The present results strongly suggest that the primary cultured neurons used in this study possess a sodium- and energy-dependent high-affinity choline uptake system as well as a synthesizing system for acetylcholine. Possible usefulness of these neurons for investigating neuronal uptake of choline and its functional role in the biosynthesis of acetylcholine are also suggested.
Effect of denbufylline, a low Km phosphodiesterase inhibitor, on cerebral cholinergic neurons was investigated using male Wistar rats. Single administrations of denbufylline (3, 10 and 30 mg/kg, p.o.) decreased cerebral cortical, hippocampal and striatal acetylcholine (ACh) contents in a dose-dependent manner. However, denbufylline (30 mg/kg, p.o.) had no effect on the activities of ACh synthesizing and degrading enzymes in these brain areas. In vitro addition of denbufylline (10-8 - 10-4M) produced no significant change in [3H]choline uptake in striatal slices, while denbufylline (10-4M) increased high (20 mM) potassium-evoked endogenous ACh release from striatal slices. Denbufylline (30 mg/kg, p.o.) increased cyclic AMP (cAMP) contents in the cerebral cortex, hippocampus and striatum. In vitro addition of dibutyryl cAMP (5 × 10-3M) also accentuated the high (20 mM) potassiumevoked endogenous ACh release from striatal slices. These results suggest that denbufylline may induce the facilitation of ACh turnover by enhancing endogenous ACh release via the increase of cAMP content in the brain.
The effects of a new TRH analog, TA-0910, orally administered, on experimental memory impairments for the one-trial passive avoidance response in anoxic mice (light-dark box), active avoidance response in basal forebrain (BF)-lesioned rats (shuttle box), and delayed alternation task in scopolamine-treated rats (T-maze) were studied. In mice, TA-0910 (3-30 mg) administered 60 min before the retention trial dose-dependently prolonged the passive avoidance response latency reduced by CO2-exposure that was given immediately after the acquisition trial, but not when it was given 60 min before the acquisition or just after the anoxic treatment. In rats, TA-0910 (0.3-3 mg/kg) administered 40-60 min before the test trial, dose-dependently prevented the reduction in mean avoidance rate caused by BF-lesioning and elevated the scopolamine (0.1 mg/kg, i.p.)-induced reduction in percent correct choice level in the alternation task. TRH (30-300 mg/kg), on the other hand, produced no improvements in any of the above tests. These results suggest that TA-0910 improves impaired memory by correcting the retrieval process of memory.
Effects of short-term treatment with basic fibroblast growth factor (FGF), epidermal growth factor (EGF) and nerve growth factor (NGF) on neurite outgrowth of superior cervical ganglia (SCG) in culture or on neuron survival and neovascularization of SCG in a transplantation system were examined in rats. SCG were preincubated with FGF, EGF and/or NGF for 30 min and cultured with drugfree medium for 2 days. FGF or EGF neither promoted neurite outgrowth of SCG nor potentiated the NGF-induced neurite elongation in culture. In the transplantation study, SCG were exposed to these factors for 30 min and grafted into the third ventricle of adult rats for 14 days. Although pretreatment of NGF, FGF, EGF or the combination of NGF and EGF were not effective, there was better neuron survival in SCG grafts pretreated with 1μg/ml NGF and 1μg/ml FGF together. In addition, density of capillaries in these grafts was significantly greater than in the other group tested. These results suggest that the synergistic interaction between NGF and FGF cause rapid neovascularization, which can prevent neuron death after transplantation.
Characteristics of the autophosphorylation of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) from the cytosol and in the postsynaptic densities (PSD) of rat brain were investigated. Several proteins were surveyed for their abilities to serve as a substrare for non-autophosphorylated and autophosphorylated CaM kinase Its from the cytosol and PSD. The tested substrates were separated into two groups. Autophosphorylation of the kinase slightly decreased or did not change its activities towards substrates of the first group: myosin light chain of chicken gizzard, synapsin I, tau factor and microtuble-associated protein 2. In contrast, autophosphorylation of the enzyme increased its activities towards substrates of the second group: syntide-2, histone H1, calcineurin and myelin basic protein. The Ca2+/calmodulin-independent kinase activity increased by autophosphorylation with any of substrates tested. Similar results were obtained with the cytosolic and PSD CaM kinase II. Trifluoperazine and mastoparan, calmodulin binding antagonists, inhibited the activity of the non-autophosphorylated CaM kinase II, but had no effect or only a slight inhibitory effect on the activity of the autophosphorylated CaM kinase II, indicating that the autophosphorylated kinase has no requirement for calmodulin for Ca2+dependent activity and/or a higher affinity for calmodulin The results suggest that the autophosphorylation of CaM kinase II is a subtle mechanism for regulating the interaction between the enzyme and substrate.
The effects of SA3443, a novel cyclic disulfide compound, on acute liver injuries induced by carbon tetrachloride (CCl4), D-galactosamine and DL-ethionine were studied in rats or mice. SA3443 (100-300 mg/kg, p.o.) significantly suppressed the increases of serum transaminase activity and liver triglyceride content in the CCl4 or DL-ethionine-induced model. Furthermore, SA3443 (300 mg/kg, p.o.) clearly reduced the formation of hepatic lipid peroxide in CCl4-treated rats. These results indicate that SA3443 protects the liver against acute liver injury.
The role of capsaicin-sensitive afferent nerves in gastroprotection by capsaicin was investigated in the absolute ethanol-induced gastric lesion model in rats. Capsaicin (0.1 and 0.5 mg/kg, p.o.) inhibited the lesion formation dose-dependently. The protective effect of capsaicin was attenuated by indomethacin-pretreatment and disappeared in capsaicin-sensitive nerve degenerated rats. Capsaicin did not induce the distension of gastric mucosal folds. These results suggested that stimulation of capsaicin-sensitive afferent nerves by capsaicin would enhance the prostaglandin formation, leading to an inhibition of gastric lesions.
When 17 macrocyclic lactone antibiotics were examined for their abilities to inhibit gastric H+, K+-ATPase, copiamycin A was found to have the strongest and relatively specific activity with IC50s of 15.7 μg/ml and > 100 μg/ml against the hog gastric H+, K+-ATPase and the dog kidney Na+, K+-APTase, respectively. Furthermore, this antibiotic inhibited the histamine-induced gastric acid secretion in the isolated gastric mucosal membrane of guinea pigs and the gastric ulcer formation in pylorus-ligated rats.
We prepared poly (A)+ mRNA from bovine adrenal medulla and electro physiologically examined the expression of H1-histaminergic receptors in Xenopus laevis oocytes. Histamine dose-dependently induced inward currents in oocytes injected with the mRNA, but not in those injected with water or the non-injected oocytes. This response was also induced by H1-agonists and antagonized by H1-antagonists, while both an H2-agonist and an antagonist had no effect. These results clearly demonstrate that H1-histaminergic receptors are functionally expressed in oocytes injected with exogenous mRNA.
Using the cannula inserting method, we investigated vascular responses to ACh and McN-A-343 (a M1-agonist) in isolated and perfused canine and simian facial veins in non-preconstricted conditions. ACh usually induced only a vasoconstriction, but McN-A-343 did not induce any significant vasoconstriction. It is concluded that canine and simian facial veins contain very few receptors of the muscarinic M1-subtype; and according to previous studies, these vessels have abundant M3-receptors.
Trichlormethiazide was given orally at 1200 hrs or 2400 hrs to rats. Its diuretic effects were greater at 1200 hrs than at 2400 hrs. There were significant correlations between urinary trichlormethiazide and its effects in both trials. The regression lines of two trials did differ. These findings indicate that the effects of trichlormethiazide vary with its administration time. Time-dependent variations in urinary trichlormethiazide and susceptibility to the agent might be involved in this phenomenon.
Effect of an intraperitoneal injection of cimetidine at a daily dose of 160 mg/kg for two weeks on biliary lipid secretion, fecal excretion of bile acids and intestinal bile acids was studied in rats. Bile flow, biliary lipid secretion and fecal excretion of bile acids remained unchanged, while the pool size of bile acids decreased by cimetidine treatment. Chenodeoxycholic acid increased with a concomitant decrease of β-muricholic acid in the biliary bile acid composition of cimetidine-treated rats.