Tissue levels of rat adrenal estrogens during the estrous cycle were determined by radioimmunoassay, and effect of ACTH on the adrenal estrogen levels was examined. The levels of estrone and estradiol in proestrus were 2.75±0.61 pg/mg tissue and 1.0± 0.19 pg/mg tissue (mean±SE), respectively. The higher level of estrone but not of estradiol was inclined to appear in proestrus rather than in other phases. Injection of 250 IU/kg of α1-24-ACTH significantly increased the estrone level at 15 and 30 min, and estradiol at 15 min. Administration of reserpine, which was used as the ACTH releaser, produced an increase in the estrone level but did not change the estradiol level. While dexamethasone injection decreased the estrone level significantly at 8 hours after the treatment, the level of estradiol remained unchanged. Hypophysectomy produced a considerable decrease in both steroid levels, particularly estrone. These results indicate that the major estrogen in the rat adrenal gland is estrone and that the level is regulated by hypophyseal ACTH.
Ionic mechanism of the resting membrane depolarization of frog sartorius muscle induced by dimorpholamine was studied in a variety of ionic conditions. The resting membrane was depolarized in a dose-dependent manner reaching a maximum with a dose of 6×10-5 M. The amount of the depolarization (1.4 mV) was suppressed with doses over 10-4 M. This dose-dependent response was accompanied by change in the membrane conductance. The chloride conductance was increased by 16 % and the potassium conductance by 7 %, however the sodium conductance did not significantly change with a dose of 2×10-5 M. The slight depolarization induced by the drug was well explained by the changes in the ionic conductance. The effects of these actions on the membrane action potentials were simulated, suggesting that this drug possesses the nature of a stimulant on excitable membranes, yet does not produce any appreciable depolarization blockade.
The developmental change of beta-adrenergic receptors in rat whole brain, except the cerebellum, was studied by binding assays with (-) 3H-dihydroalprenolol. The synaptic membrane fraction, prepared from 2-day-old and 6-week-old rat brains, had binding sites for (-) 3H-dihydroalprenolol, which seemed to represent physiological beta-adrenoceptors. The maximal binding capacity and the dissociation constant for (-) 3H-dihydroalprenolol of the synaptic membrane fraction did not vary with the age of the rats, but the yield of synaptic membrane fraction from the brain of 2-day-old rats was much less. Therefore, the total number of binding sites was less in 2-day-old rats. In addition, there was a significant difference in the inhibitory effects of 1-isoproterenol on binding of (-) 3H-dihydroalprenolol to the synaptic membrane fractions in the 2-day as compared to the tissues from the 6-week-old aminals. The developmental changes in the number and nature of beta-adrenoceptors may result in expression of catecholamine-sensitive adenylate cyclase activity.
Prolonged administration of insulin leads to the formation of insulin-binding antibodies due to contaminant peptides and the animal source of the insulin. It follows that quantitation and identification of these factors are of significant importance in pharmaceutical insulin preparations. The assay and test procedures stipulated in the current pharmacopoeia of various countries, nevertheless, cannot determine either of these effects. In the present study, the content of impurities in insulin preparations was measured by polyacrylamide gel disc electrophoresis and the animal source of insulin identified by amino acid analysis. Assays of 17 commercial insulin preparations by these techniques revealed diversity in purity and animal sources of insulin. The present results suggest potential usefulness of these assay methods and advisability of their adoption not only by the manufacturers but also by the official pharmacopoeia as well.
The influence of aphidicolin on cell multiplication and DNA synthesis was examined using synchronous mouse mastocytoma P-815 cells. Aphidicolin was cytotoxic specifically to the cells of the S phase of the cell cycle. This cytotoxicity was reversed by appropriately washing the drug-treated cells, but not by the addition of deoxyribonucleosides. Aphidicolin, a potent inhibitor of DNA synthesis, selectively inhibited the activity of partially purified DNA polymerase α from the nucleus and the cytosol of mastocytoma cells, but did not affect the activity of DNA polymerase β. Furthermore, aphidicolin had no effect on the synthesis of RNA and protein, and produced no changes in cell size at least for one generation.
The effect of the combination of aminopyrine and secobarbital at a molar ratio of 2:1, the mixture and molecular compound, on rabbit EEG activation was examined. Secobarbital 40 mg/kg p.o. elevated threshold voltages in the neocortical and hippocampal EEG activation by high frequency electrical stimulation of the midbrain reticular formation (MRF), nucleous centralis medialis (CM) of the thalamus, and posterior hypothalamus (PHA) by 40, 40 and 80 %, respectively. Aminopyrine 80 mg/kg p.o. alone did not affect the arousal responses. The molecular compound 120 mg/kg p.o. has more potent and long-lasting actions in inhibiting the arousal responses induced by the stimulation of MRF (80%) and CM (50%) than with secobarbital alone. The inhibitory action of the mixture 120 mg/kg p.o. on the PHA-arousal response (40 %) was significantly weaker than that of the molecular compound (80%). Secobarbital and the molecular compound slightly inhibited the neocortical augmenting and recruiting responses. Our findings suggest that although aminopyrine exerts a synergistic effect with secobarbital in inhibiting the EEG activation produced by MRF stimulation, in inhibiting the PHA-arousal response, there is no synergistic effect of the two drugs when they were given as the mixture. Moreover, the molecular compound rather than the mixture has a more potent inhibitory action on the EEG activation, particularly with PHA stimulation.
Effects of timepidium bromide (TB), hyoscine-N-butylbromide (HB) and atropine (Atr) were studied on the isolated guinea pig gallbladder and sphincter of Oddi. In the isolated gallbladder, the contraction induced by methacholine was competitively antagonized with TB, HB and Atr: pA2 values for TB, HB and Atr were 8.44, 7.55 and 9.11, respectively. Contraction of gallbladder after field stimulation at 5 Hz was also inhibited by TB, HB and Atr as well as by tetrodotoxin (TTX). On the other hand, TB had no significant influence on the electrically evoked (30 Hz) contraction which remained after treatment with TTX. It was estimated that the inhibitory activity of TB on the contraction of gallbladder was 1/5-1/6 that of Atr and 7 times as potent as HB. In the isolated sphincter of Oddi, TB, HB and Atr reduced the perfusion pressure increased by acetylcholine (ACh), the activity of TB being 1/4 that of Atr and 3 times of HB. The contractile responses to field stimulation (5 Hz) were also inhibited by TB, HB and Atr. The inhibitory activity of TB was 1/5 that of Atr and 12 times of HB. It was also demonstrated that TB produced no significant effect on the noradrenaline (Nor)-induced contraction and isoproterenol (Iso)-induced relaxation in the gallbladder or Nor-induced increase of the perfusion pressure in the sphincter of Oddi. It is suggested that TB acts on the gallbladder and sphincter of Oddi by virtue of its anticholinergic activity.
Two types of conditioned behaviors were investigated for the purpose of evaluating anxiolytic drugs. A conditioning procedure used was an active avoidance in poorly-performing mice. Chlordiazepoxide, diazepam, chlorazepate and meprobamate increased the avoidance rate, while chlorpromazine, haloperidol and nortriptyline did not produce such an effect. The effect of diazepam was potentiated by γ-aminobutyric acid (GABA) and aminoxyacetic acid (AOAA) and antagonized by picrotoxin and thiosemicarbazide, but was influenced little by spiroperidol, α-methyltyrosine, phenoxy-benzamine and levallorphan. In addition, the effect of other anxiolytics was potentiated by AOAA and antagonized by picrotoxin. Biperiden, methamphetamine, caffeine and morphine also induced an avoidance enhancement, which was not influenced by AOAA. A drug discrimination experiment was also performed using a milk-reinforced two-lever operant method. In the rats trained to discriminate phenobarbital from saline, diazepam produced a dose-related phenobarbital-lever selection, which was potentiated by AOAA and antagonized by picrotoxin. Chlordiazepoxide, chlorazepate and meprobamate also elicited responses on the phenobarbital-lever. On the other hand, haloperidol, nortriptyline, biperiden, methamphetamine, caffeine and morphine produced a saline-lever selection, at the doses tested. These results suggest that, among several drugs tested, the avoidance enhancement and discriminative response control by anxiolytics may be closely linked with the GABA system.
Properties of adenosine triphosphatases (ATPases) in the choroid plexus of rabbit cerebral ventricles were investigated using tissue homogenates and subcellular fractions. Na, K-ATPase or HCO3-ATPase activity of choroid plexus was significantly lower than that of the brain or the kidney, respectively. Among the homogenates of choroid plexus from the lateral, third and fourth ventricles, there were no differences in the activities of Na, K-, Mg- and HCO3-ATPases. In the choroid plexus, Na, K-ATPase activity was demonstrated to be highest in 10, 000 × g fraction, while the highest Mg- and HCO3-ATPase activities were observed in 8, 000 × g fraction, the so-called mitochondrial fraction. Ion requirement, pH optimum and ouabain sensitivity of Na, K-ATPase in 10, 000 × g fraction of choroid plexus were similar to findings in erythrocytes, brain and kidney. However, properties of Mg and HCO3-ATPases in 8, 000 × g fraction were considerably different from those found in the brain and gastric mucosa. In the choroid plexus, thiocyanate ion inhibited Mg-ATPase as well as HCO3-ATPase. These results indicate that characteristics of ATPase systems in the choroid plexus are to some extent different from those in tissues such as brain, kidney and gastric mucosa.
We devised a simple method for the separation of serotonin (5HT) and 5-hydroxyindoleacetic acid (5HIAA) using small P-cellulose and DEAE-Sephadex columns, respectively. Both substances were estimated fluorometrically by the reaction with o-phthalaldehyde (OPA). In this method, some known interfering substances in addition to many primary amines and amino acids were separated from 5HT and 5HIAA. The recoveries of 5HT and 5HIAA were 80 and 71 %, respectively. The fluorescence intensities of 30 pmol of 5HT and 15 pmol of 5HIAA were about twice over the reagent blank. The complete separation of 5-hydroxytryptophan (5HTP) from 5HT made it possible to determine 5HTP decarboxylase activity in rat brain areas without using an isotope-labeled substrate. The method allows for quantitation of the enzyme activity in mg amounts of the brain tissues. The activities in discrete areas were in good parallel with the 5HT contents, except for the striatum. The levels of 5HIAA were also much in parallel with those of 5HT.
Utilizing struggling to a repetitive stimulation of the tail, as a pain response, flexor reflex to a single stimulation of the sciatic nerve, as a spinal reflex activity, the analgesic activity and the muscle relaxant activity of certain analgesics were assessed alternately. Stable and reproducible response to a repetitive stimulation of the tail or a single stimulation of the sciatic nerve was obtained over a period of 5 hr, and quantitative measurement of struggling could be made. The 50% inhibitory doses of various drugs to struggling and flexor reflex were as follows (mg/kg, i.p.): morphine, 3.30 & 6.60, respectively; codeine, 12.80 & 24.00; pentazocine, 7.80 & 12.20; indomethacin, 4.60 & no effect; aspirin, 94.00 & no effect; baclofen, 1.04 & 2.20; chlorpromazine, 2.45 & 0.75; diazepam, 0.45 & 0.86; mephenesin, 36.00 & 32.00. Based on these results, it is suggested that the method is useful for quantitative measurement of analgesia and muscle relaxation.
Anaphylactic shock was induced by administration of ovalbumin to sensitized rats. Preventive effects of theophylline on anaphylactic shock were examined with regard to the relationship between cyclic AMP and prostaglandin (PG) E2 in lung tissue, and plasma histamine. During anaphylactic shock, levels of cyclic AMP content and PGE2 content in lung tissue decreased, while plasma histamine content increased. Theophylline increased levels of cyclic AMP content and PGE2 content in lung tissue, in a dose dependent manner, and pretreatment of animals with theophylline prevented the onset of anaphylactic shock. Pretreatment with indomethacin abolished the preventive effects of theophylline on anaphylactic shock, and the effect of theophylline on the cyclic AMP content. Dibutyryl cyclic AMP (DBcyclic AMP) increased PGE2 content in lung tissue, in a dose dependent manner, and also prevented the onset of anaphylactic shock. These effects of DBcyclic AMP were inhibited by the pretreatment with indomethacin, and here, cyclic AMP in lung tissue was maintained at a high level. In the group in which anaphylactic shock was prevented with these interventions, PGE2 content in lung tissue was significantly high in all cases. In addition PGE2 infusion prevented anaphylactic shock. These data suggest that theophylline increases cyclic AMP levels in lung tissue only in the presence of endogenous PG, that increased cyclic AMP content in lung tissue subsequently increases PGE2 content in lung tissue, and that the preventive effects of theophylline on anaphylactic shock result from increased PGE2 content in lung tissue.
The influence of various ulcerogenic treatments on healing gastric ulcers induced by thermocautery was studied in mice. A low dose of serotonin (5HT) which did not produce ulceration, was found to aggravate gastric ulcers at 15th or 30th day after thermocauterization, but other ulcerogenic treatments including histamine, norepinephrine, vasopressin, acetic acid ingestion and cold-restraint stress did not affect this induced gastric ulcer. Bleeding and ulceration, however, occurred in the gastric glandular portion in addition to thermocauterization ulcer by the treatment of acetic acid ingestion or cold-restraint stress. Histological sections of gastric ulcers (15th and 30th day) 24 hr after 5HT injection, showed severe necrosis of the regenerated mucosal layer. Microvessel structure in the gastric mucosa as revealed by the Indian-ink infusion, showed a local obstruction of blood flow on the edge of ulcers 1 or 3 hr after 5HT injection. Although acetic acid ingestion increased transmucosal fluxes of Na+ and K+, 5HT had no effect on the ion flux in normal mice. Thus the healed ulcer area was resistant to various ulcerogenic stimulants, except for 5HT, and the vasoactive factor of 5HT may be involved in the aggravating process of gastric ulcers induced by thermocautery.