Intracellular localization of histamine receptors in small intestinal smooth muscle of the cat was studied by investigating distribution of marker enzymes in the receptor rich fraction. Distribution of membrane markers coincided with that of the radiochemically labeled receptor fraction. Membrane fraction was further purified and it was concluded that the histamine receptor rich fraction is mainly composed of cell membrane. The chemical composition of the fraction lent support to this conclusion.

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Studies were made on tritiated histamine binding to the receptor-rich membrane fraction and solubilized sites and its displacement by various drugs. H₁-Agonists and antagonists displaced histamine most effectively. A H₂-agonist and atropine were less effective and propranolol, phentolamine and imidazole acetic acid had little effect. The solubilized binding sites showed the same specificity of binding as the membrane fraction. Membrane fragments had two binding constants, whereas solubilized sites had only one. Solubilized sites bound similar amounts of histamine and dibenamine: the latter was applied to intact tissue under conditions which would presumably cause specific binding to histamine receptors. These binding characteristics show that the method used was adequate for purification of histamine receptors from smooth muscle of cat small intestine.

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The effects of metiamide, a histamine H₂-receptor antagonist, and propranolol, a beta-adrenergic blocking agent, on gastric secretion were studied in anesthetized dogs. Metiamide, 1.45 mg/kg i.v., markedly inhibited the gastric secretion induced by a continuous i.v. infusion of tetragastrin (8 μg/kg-hr), histamine dihydrochloride (160 μg/kg-hr), or methacholine bromide (100 μg/kg-hr). Propranolol 0.5 or 1.0 mg/kg i.v. produced a significant potentiation of tetragastrin-induced gastric secretion but no influence on the secretion induced by methacholine. Propranolol at 5 or 10 mg/kg i.v. produced a slight reduction of the tetragastrin-induced secretion and a significant reduction of methacholine-induced secretion. Histamine-induced gastric secretion was not affected by propranolol at either 1 and 10 mg/kg i.v. These findings lend support to the hypothesis that interactions among histamine, gastrin and acetylcholine receptors do occur though the degree would not be the same in all directions.

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Slow depolarizations of dorsal root nerve terminals and motoneurons, which were produced by 5-hydroxytryptamine (5-HT) applied directly to isolated bullfrog spinal cords, were recorded by the sucrose-gap method. These depolarizations were eliminated in the Ca-deficient Ringer's solution containing Mg, suggesting that these 5-HT depolarizations were not caused by a direct action of 5-HT on dorsal root nerve terminals or motoneurons but rather by actions of transmitters released from interneurons. Indeed, mephenesin, which is a selective blocker of polysynaptic transmission in the spinal cord, inhibited more markedly the 5-HT depolarization than the L-glutamate or GABA depolarization. The transmitter directly responsible for the generation of the 5-HT depolarization of dorsal root nerve terminals was not considered to be GABA as the 5-HT depolarization was not antagonized by picrotoxin. It would thus appear that 5-HT stimulates interneurons in the amphibian spinal cord and unknown transmitters released from these interneurons depolarize the dorsal root nerve terminals.

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A comparative study was carried out on the effects of a soluble derivative of baicalein, disodium baicalein 6-phosphate (BPS) and disodium cromoglycate (DSCG) on the immediate type allergic reactions. BPS not only inhibited reaginic antibodymediated reactions including antigen-induced mediator release from monkey lung, homologous PCA in rats, and reaginic antibody-mediated degranulation of mast cell, but also non-reaginic antibody-mediated reactions such as mediator release from guinea pig lung sensitized with ovalbumin and that from human lung caused by antiIgE. The agent, however, did not affect the mediator release from lung of rats sensitized with dinitrophynylated ascaris extract plus Bordetella pertussis. On the other hand, DSCG showed characteristic properties as an inhibitor of reaginic antibody-mediated reaction. It is thus assumed that the functional site of reaginic antibody is well fixed with DSCG at a definite distance between the two-chromone-nuclei while that of IgG is readily fixed with the two molecules of baicalein or BPS.

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Effects of 1-methyl-5-chloroindoline methylbromide (S-6) on Renshaw cells were investigated in cats anesthetized with pentobarbital sodium. S-6 proved to have little effect on spike discharges of Renshaw cells when administered intravenously, but the agent electrophoretically applied revealed excitatory effects which resembled the action of methacholine and such effects were blocked by intravenous administration of atropine. It was concluded that (1) S-6 is impermeant to the blood brain barrier and (2) the excitatory action of S-6 on Renshaw cells is muscarinic in nature.

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Effects of clonidine on blood pressure, heart rate and rectal temperature in conscious rats were examined. Clonidine (0.1-1 mg/kg s.c.) caused a prevailing pressor response and dose-dependently a fall in heart rate and body temperature. The pressor response to clonidine (0.3 mg/kg s.c.) was completely reduced by phentolamine (10 mg/kg s.c.), chlorpromazine (10 mg/kg s.c.) but not by hexamethonium (30 mg/kg i.p.), guanethidine (30 mg/kg s.c.) or reserpine (5 mg/kg s.c. 18 hr + 1 mg/kg i.p. 4 hr prior to clonidine). Conversely, a remarkable potentiation of the pressor response to clonidine was observed after treatment with reserpine. The bradycardia with clonidine (0.3 mg/kg s.c.) was significantly reduced by phentolamine, chlorpromazine or atropine (5 mg/kg s.c.) but was potentiated by reserpine. The hypothermia with clonidine (0.3 mg/kg s.c.) was not influenced by phentolamine or atropine but was significantly potentiated by chlorpromazine. From the above results it is suggested that the prevailing pressor response to clonidine in conscious rats is due to a stimulation of peripheral α-adrenoceptors, the bradycardia with clonidine is exerted through the sympathetic pathway and the baroceptor-vagal reflex, and that the hypothermia with clonidine is mainly due to the central mechanism.

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The effects of local distension of the intestinal wall on the release of acetylcholine (ACh) from the adjacent non-distended part were studied with the segment of isolated guinea pig ileum. Local distension of the intestinal wall induced the increased release of ACh in the distended part and in its anal side but not in its oral side. Such aboral release of ACh by local distension was abolished by tetrodotoxin or atropine in the concentrations which did not block the release in the distended part. When hexamethonium was applied exclusively to the distending part, significant increase of ACh release was observed in both the regions oral to and anal to the distended part. It is suggested that distension stimuli applied to the myenteric plexus are transmitted aborally along the network of the Auerbach's plexus to the anal direction. The release of ACh from the intestine by nicotine or DMPP differed from that occurring during local distension in that the release was localized to the part of the intestine to which the drug was applied.

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Adult male rats were administered morphine twice a day for 45 days and the effects of morphine on the copulation rate, the weight of various organs, and on the hypothalamic-pituitary-gonadal axis were examined. Morphine administered rats showed a loss of weight, hypertrophy of the adrenals, decreased weight of accessory sex organs, low sperm count, and decreased copulation rate. The contents of the luteinizing hormone releasing hormone in the hypothalamus and the luteinizing hormone in the pituitary remained unchanged. Serum luteinizing hormone and testosterone levels decreased, but serum follicle-stimulating hormone levels increased. These results suggest that morphine inhibits the hypothalamic-pituitary-gonadal axis and causes a diminution in the number of fertilizations of the partner females.

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Mechanism of hypercholeretic effect of three polyoxybenzenes, 2, 4, 6-tri-hydroxypropiophenone (THPP), 4-methylumbelliferone (4-MU) and 3-(2', 4', 5'-tri-ethoxybenzoyl)propionic acid (AA-149), was studied in rats. Biliary clearance of ¹⁴C-labeled erythritol indicated that all the choleretics increased canalicular bile production. AA-149 excreted in the bile chiefly as the glucuronide provided no significant osmotic drive for bile formation. Both the biliary bile acid concentration and total biliary excretion of bile acids were lower in the rats treated with THPP, 4-MU or AA-149 than in control rats. These choleretics were also effective in the isolated rat liver perfusion system, in which concentration and output of biliary bile acids were low. Thus, it was unlikely that bile acids were involved in the hypercholeresis induced by the choleretics. THPP, 4-MU and AA-149 increased biliary excretion of sodium both in the biliary-cannulated rats and in the isolated rat liver perfusion system. It was concluded that the hypercholeresis induced by THPP, 4-MU and AA-149 was due to an enhanced formation of the bile acid-independent fraction of canalicular origin, probably mediated by the active transfer of sodium into the canaliculi.

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A technique was devised for the separation of rat renal tubules in order to study the transport of 5-hydroxytryptophan (5-HTP) and 5-hydroxytryptamine (5-HT). The separated tubules maintained activities of aromatic amino acid decarboxylase and monoamine oxidase, and these activities were markedly depressed by Ro 4-4602 and pargyline, specific inhibitors for the respective enzymes. In the tubule cells incubated with 0.001 mM of the substrates, 30.4% of ¹⁴C-5-HTP was converted to 5-HT and 5-hydroxyindoleacetic acid (5-HIAA), and 69.0% of ¹⁴C-5-HT was converted to 5-HIAA. This metabolic conversion was almost completely inhibited by Ro 4-4602 and pargyline. The uptake of ¹⁴C-5-HTP or ¹⁴C-5-HT by the tubule cells under the presence of an enzyme inhibitor such as Ro 4-4602 and pargyline, proceeded linearly until 20 min at 37°C and the equilibrium distribution ratio for ¹⁴C-5-HTP and ¹⁴C-5-HT at the medium concentration of 0.001 mM was 3.4 and 14.9, respectively. The ratio for ¹⁴C-5-HT was markedly higher than that for ¹⁴C-5-HTP. The uptake did not occur at 0°C. Kinetic analysis of the rate of uptake at various concentrations of ¹⁴-5-HTP and ¹⁴C-5-HT indicated the presence of two saturable transport systems with defferent affinities for each substrate (Km: 0.08 mM and 7.0 mM for 5-HTP, 0.14 mM and 1.7 mM for 5-HT). It is concluded that renal tubule cells have temperature Dependent, concentrating and saturable transport systemx for 5-HTP and 5-HT.

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Antihypertensive activity of orally administered methyl O-(4-hydroxy-3-methoxycinnamoyl)reserpate (CD-3400) was examined and compared with such activity of reserpine and its related agents in conscious hypertensive rats. CD-3400, 4-16 mg/kg p.o., produced a marked antihypertensive effect lasting 6 hr and a slight decrease in heart rate in spontaneously hypertensive rats (SHR). Reserpine, 1-4 mg/kg, rescinnamine, 4-16 mg/kg, and syrosingopine, 8-32 mg kg p.o., produced effects similar to those of CD-3400. The order of antihypertensive activities of the agents was as follows: reserpine>CD-3400=rescinnamine>syrosingopine. In renal hypertensive rats and deoxycorticosterone hypertensive rats, CD-3400 and rescinnamine, 4-16 mg/kg, and reserpine, 1-4 mg/kg p.o., also produced an antihypertensive effect and the order of potency was as follows: reserpine>rescinnamine≥CD-3400. Repeated administrations of CD-3400, 2-12 mg/kg/day p.o., for 14 days, to SHR produced the marked constant antihypertensive effects during the administration period. The order of the potency was as follows: reserpine> rescinnamine>CD-3400. CD-3400 possessing a sustained antihypertensive activity may be prescribed in cases when long-term therapy is required for hypertensive patients.

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The inhibitor constants of several inhibitors for cyclic AMP- and cyclic GMP-phosphodiesterase from various organs are compared. The inhibitors were classical theophylline, papaverine, and some of newly developed inhibitors: an imidazolidinone compound, R020-1724, and two phthalazinol compounds, EG 467 and EG 626. Among the inhibitors tested, papaverine and EG 626 were found to be the most potent. Both compounds were extremely inhibitory to platelet and arterial phosphodiesterases. EG 626 was much more inhibitory to cyclic AMP phosphodiesterase than to cyclic GMP phosphodiesterase in platelet and brain-extract and R020-1724 was inhibitory to cyclic AMP- but not cyclic GMP-phosphodiesterase in brainextract. When the skin adenyl cyclase was activated by AMP, the addition of theophylline blocked this activation, but EG 626 or EG 467 further potentiated the activation. These in vitro studies may serve as basic screening tests for the effectiveness of the specific phosphodiesterase inhibitors.

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Mechanical responses produced in isolated chicken rectum by nerve stimulation were subjected to a pharmacological analysis to determine the nerves involved. Stimulation of Remak's nerve caused biphasic responses, consisting of an initial, short-lasting contraction and a subsequent, long-lasting relaxation. A similar pattern of responses was observed following coaxial stimulation of intramural nerves. The contraction was unaffected by atropine and hyoscine, and also insensitive to α-adrenoceptor blockers. The relaxation was reduced in duration but not in amplitude by β-adrenoceptor blockers, and still observed in preparations from reserpinized animals. These results suggest the presence of non-cholinergic excitatory innervation and nonadrenergic inhibitory innervation in the chicken rectum. Changing the stimulating sites and sectioning some of the side branches of Remak's nerve extending to the gut revealed that the excitatory nerve fibers appear to reach the rectal wall preferentially via the lower side branches. The smooth muscle of the dorsocaudal region appears to receive more excitatory innervation, since the largest contraction was found in muscle strips taken from the rectal wall where the side branches terminated when field stimulation was applied to the anal end of these strips.

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Effects of central depressants—chlorpromazine, diazepam, pentobarbital and ethanol-on rota-rod and traction performances in mice were investigated. The walking technique of the animals on the rotating rod (3 cm in diameter, 24 r. p. m.) was established after about 15 trials, and was well maintained for one week thereafter. No training was required for the traction. Both the rota-rod and traction performances were inhibited by chlorpromazine, diazepam, pentobarbital and ethanol in fairly good parallel with the dosages. However, the sensitivities to one same drug markedly differed between the two performances. Chlorpromazine more than diazepam inhibited the rota-rod, while in the traction performance the inhibition with diazepam was greater than that with chlorpromazine. Pentobarbital and ethanol inhibited the two performances to nearly the same degree. On the basis of the present results, the rota-rod test is considered to be suitable for the estimation of the positive adaptability to forced motor activity, and the traction for that of muscle relaxation. By comparing the dose-effect relationships in the two performances, it may be possible to elucidate the characteristics of various central depressants, and to apply these procedures for the screening test in drug evaluations.

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Rat hepatic aminopyrine N-demethylase activity was measured by detecting the amount of formaldehyde produced from aminopyrine. Some optimal conditions for the N-demethylation were determined using both isolated microsomes and whole homogenates, and the standard assay method is described. Formaldehyde production from the substrate by microsomal enzyme system was linear to the amount of microsomes added during 3 min reaction time, whereas long-time incubation caused a decrease in the apparent activity of aminopyrine N-demethylation. The N-demethylase activity observed in normal rat liver homogenate was quite similar to that in microsomes when the activity was expressed on the basis of cytochrome P-450 as molecular activity. Pretreatment of animals with typical inducers, phenobarbital and 3-methylcholanthrene, resulted in alteration of the aminopyrine N-demethylase system, which was detectable in both microsomes and whole homogenates.

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Specific pressor activity of Asp¹-Val⁵-angiotensins I and II was determined by a 4-point assay in rats against various synthetic angiotensins. Specific pressor activity of various angiotensins was also obtained from the dose-blood pressure-response (DR) curve, using a single angiotensin sample per rat. Comparison of two values showed that two angiotensins interacted in the 4-point assay, yielding a potentiation factor of 0.465-1.373. Therefore, specific pressor activity from DR curve is more reliable, because two angiotensins are not able to interact. Potency ratio on a molar basis of Asp¹-IIe⁵-angiotensin I, Asp¹-Val⁵-angiotensins I and II, Asn¹-Val⁵-angiotensin II(Ciba Lot-094691), and Asp¹-Va1⁵-Ser⁹-angiotensin I against Asp¹-IIe⁵-angiotensin II were 0.76, 0.91, 2.02, 0.50, and 1.39, respectively. Asp¹-Va1⁵-angiotensin II had twice the pressor activity of Asp¹-IIe⁵-angiotensin II. Lyophylized Asp¹-IIe⁵-angio-tensins I and II kept desiccated at minus;20°C, were stable for 27 months. Solutions of 100, 10, and 0.8 μg/ml were stable for 12, 8, and 6 months at minus;20°C, respectively.

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Automutilation induced by a single large dose of clonidine was potentiated by pretreatment with methamphetamine, caffeine and theophylline, while it was inhibited by acute administration of reserpine, α-methyl-p-tyrosine, phenoxybenzamine, phentolamine and chlorpromazine. L-Dopa, 5-hydroxytryptophan and p-chlorophenylalanine had no effect on this abnormal behavior. Biochemical studies on brain monoamines revealed that noradrenaline was markedly increased and dopamine slightly so, but 5-hydroxytryptamine was never changed by clonidine. These results suggest that a central noradrenergic system may be involved in automutilation induced by clonidine in mice.

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Effects of biogenic amines and peptides on urine outflow and ADH release were studied using intracerebroventricular (i.c.v.) perfusion experiments and isolated neurohypophysis incubation studies. Decrease in the urine outflow was observed after norepinephrine, histamine and angiotensin II had been administered i.c.v.. The effect of norepinephrine was prevented by phentolamine. Phentolamine alone also acted as an antidiuretic. α-Adrenergic and histaminergic mechanisms may thus involved in ADH releasing system and this system might be also responsive to angiotensin II. When the isolated neurohypophysis was incubated in the presence of norepinephrine, histamine, angiotensin II amide 5-valine or bradykinin, release of ADH was increased, and the effects of norepinephrine and histamine were prevented by phentolamine and promethazine, respectively. Phentolamine but not promethazine alone increased ADH release. On the other hand, serotonin, dopamine and angiotensin II 5-isoleucine did not result in an ADH release from the isolated neural lobes. Our findings suggest that when the local concentration of norepinephrine, histamine or peptides is increased to the extent where the posterior lobe of the pituitary is stimulated directly, the ADH release is enhanced.

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