Appropriately substituted benzylamine (BZA) derivatives, fragmented derivatives of tetrahydroisoquinolines, were found to be directly acting adrenergic β-stimulants, exhibiting tracheal relaxing, positive chronotropic and free fatty acid (FFA) releasing activities. The chemical structures essential for manifestation of the β-action were i) 3, 4-dihydroxybenzylamine, ii) arylmethyl group at position α, iii) lower alkyl group on the N atom. The structure activity relationships of BZA-derivatives were almost similar to, but partly different from those of tetrahydroisoquinoline- and catecholamine-derivatives. The tracheal relaxing, positive chronotropic and FFA-releasing actions of α-(3, 4, 5-trimethoxybenzyl)-N-methyl-3, 4-dihydroxybenzylamine, the most active compound in the BZA-derivatives tested, were approximately one-hundred, thirty and fifty times less active than those of ISO, respectively. These results indicate that this compound is β₁-selective, while trimetoquinol is β₂-selective.

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Small tracking electrodes were inserted into the cat locus coeruleus (LC), and the effects of LC stimulation were determined on the transcallosal potential (TCP) evoked in cerebral cortex on the same side. LC stimulation at 8 volts inhibited TCP amplitude an average of 11% Rostral LC placements appeared most effective. LC stimulation, without drugs, did not affect the peak latency of the TCP. dl-Propranolol and FLA-63 blocked LC inhibition of the TCP and also increased TCP amplitude per se. Propranolol first increased the latency of the TCP during LC stimulation, then decreased it, while also prolonging the latency of the TCP per se. Phenoxybenzamine increased rather than blocked the LC inhibitory effect and also increased TCP amplitude. dl-Erythro-DOPS slightly increased the LC inhibitory effect, and substantially increased TCP amplitude. dl-Threo-DOPS produced somnolence in unanesthetized animals, led to increased norepinephrine levels in cortex and brain stem, and caused cortical potentials to fluctuate widely. A midbrain lesion of the dorsal ascending NE bundle blocked the LC inhibitory effect. LC stimulation alone, or in combination with evoked cortical stimulation, did not affect the interstimulus electrocorticogram or the heart rate. In the transcallosal system of the cortical regions studied, the LC appears to play mainly an inhibitory role.

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Effect of acetylcholine on hepatic circulation was investigated in 33 mongrel dogs. Mean arterial blood pressure, hepatic artery flow, portal vein pressure and portal vein flow were determined after laparotomy under nembutal anesthesia. In order to analyse the complicated responses in hepatic blood flows to 3 μg of acetylcholine hydrochloride, the peripheral resistance of each vascular system was observed in hypotensive dogs, in vascular obliteration dogs and in portal vein shunting dogs. Observation under a hypotensive condition produced by exsanguination revealed that hepatic artery resistance responded to decrease in hepatic artery pressure in different ways depending on whether the systemic blood pressure was above or below 90 mmHg. Experiments in which the hepatic artery or portal vein were obliterated disclosed that there was a certain regularity in the induction of resistance change in the non-injected one of the two hepatic vessels. The result of shunting the portal vein suggested that the complicated change above mentioned would be the expression of the overlapping two sequential responses. Since these results are difficult to explain merely by the direct response of vascular smooth muscle to acetycholine, the possibility that the hypothetical communication between the hepatic artery and portal vein might be a cholinergic system was discussed.

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Experiments were undertaken to substantiate the hypothesis that the mechanism of the direct effect of ouabain on the renal excretion of electrolytes is the result of inhibition of the transport enzyme, (Na, K)-ATPase. In dogs hydrated with saline, an injection of ³H-ouabain into the unilateral renal artery produced a continuing marked increase in excretion of water and sodium from the kidney, but not from the counter kidney. At maximal diuresis—90 min after ouabain injection, both kidneys were removed to assay microsomal ATPase activity and determine radioactivity distributed in subcellular structures. It was demonstrated that ³H-ouabain was deposited in the microsome fraction obtained from the injected kidney in concentrations ranging from 10^-7 to 10^-6 M/kg wet weight, and (Na, K)-ATPase activity of this fraction was inhibited as compared with that of the microsomal fraction obtained from control kidneys. Since (Na, K)-ATPase activity of renal microsomes was significantly inhibited in vitro by more than 10^-7 M of ouabain, ouabain concentration in microsomes obtained from the injected kidney was considered to be sufficient to inhibit ATPase activity. These findings indicate that ouabain diuresis under the present condition is closely related to direct inhibitory effect of ouabain on (Na, K)-ATPase activity of microsomes in tubular cells.

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Experiments were carried out on the isolated papillary muscle of the rabbit in order to further characterize the α-adrenoceptors mediating the positive inotropic effect. For this purpose dose-response relations of seven sympathomimetic amines were compared under the influence of α- and/or β-adrenolytic drugs. Phentolamine (10^-6 M) shifted the lower part of the dose-response curves for norfenephrine, synephrine and epinine as for phenylephrine and adrenaline to the right, while prindolol (10^-8 M) affected only the upper part of the curves. In the presence of both α- and β-adrenoceptor blocking agents the entire dose-response curves for sympathomimetic amines were shifted in a parallel manner. Noradrenaline affected preferentially β-adrenoceptors, whereas its effect on α-adrenoceptors was so weak that it could be detected only when the neuronal and extraneuronal uptake mechanism of amines were blocked by cocaine (3×10^-5 M) and corticosterone (4×10^-5 M), respectively. The effect of dopamine was not affected either by phentolamine or by prindolol, but was antagonized by the simultaneous application of both α- and β-adrenoceptor blocking agents. From the present results, it appears that the following relationships are present between the structure of amines and the α-adrenoceptor stimulating activity in the heart: (1) N-methylation increases the potency: (2) Absence of the hydroxyl group either in 3 or in 4 position decreases the intrinsic and β-adrenoceptor stimulating activities, but increases the α-adrenoceptor stimulating activity.

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When a given concentration of a catecholamine was applied to guinea pig tracheal preparation contracted by 20 μM histamine or by 30 mM-K⁺-Tyrode's solution, constant relaxations were observed, if the relaxation was submaximal. When a high concentration of catecholamine, 200 times the ED50, was once applied, subsequent responses to beta-stimulants (ED80) was reduced by about 30-40 %, in spite of repeated washings. The response was gradually recovered in 2 hr. Thus 45 μM epinephrine and 1 μM isoproterenol could cause desensitization to 0.65 μM epinephrine and 0.03 μM isoproterenol, respectively. Epinephrine and isoproterenol could cause desensitization to isoprophenamine, a non-catechol beta-stimulant. Epinephrine did not affect the response to cyclic AMP, dibutyryl cyclic AMP, aminophylline and prostaglandin E₁. This desensitization was not affected by phentolamine, normetanephrine nor by Ca^2- deprivation from the bathing solution. The mechanisms of the desensitization may relate to some step(s) between the receptor-drug interaction and cyclic AMP accumulation in the process of tracheal muscle relaxation induced by beta-stimulants.

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Effects of externally applied cyclic AMP and other adrenergic stimulants on the electrical and mechanical activities of the cat small intestine were observed by using pressure electrodes. The electrical and mechanical activities were suppressed by cyclic AMP and beta-stimulants. Those inhibitory actions of cyclic AMP and beta-stimulants were potentiated under the treatment with caffeine, theophylline and papaverine which inhibits the phosphodiesterase activity. On the other hand, the inhibitory action of cyclic AMP and beta-stimulants was decreased in imidazole, an agent that increases phosphodiesterase activity. Exogenous applied concanavalin A, an agent that inhibits the adenyl cyclase activity, showed no observable changes in both activities but the effects of beta-stimulants were decreased after treatment with concanavalin A. No obvious changes on both activities were obtained in cyclic GMP and dibutyryl cyclic GMP. These findings tentatively support the hypothesis that cyclic AMP is a second messenger in the inhibitory responses to beta-stimulants on the intestinal smooth muscle. However, it is also concluded that the inhibition of mechanical activity caused by cyclic AMP is partially due to suppression of the membrane activity.

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Relationship between pharmacological activities and blood levels of a new anticholinergic agent (SA-504). as well as excretion, and distribution in the gastro-intestinal tract were investigated in cats. The time courses of inhibitory effect of SA-504 on gastric motility were closely related to those of blood levels of SA-504 after intragastric administration of ³H-labelled compound. Effects of SA-504 on the pupil of the eye, heart rate and blood pressure were much less than its inhibitory effects on gastric motility. The contents of ³H-SA-504 in the gastric wall were extremely high as compared with those in the blood and heart. About 23 % of the radioactivity administered orally as ³H-SA-504 was excreted into the bile and 20% into the urine during a 24 hour period, suggesting that more than 4000 of the dose is absorbed from the alimentary tract of cats within 24 hours.

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The beta-receptor stimulating effect of isoprenaline on the spontaneous activities of the guinea-pig taenia coli was investigated in the presence of an alpha-receptor blocking agent, phentolamine (2 × 10^-6 M). Substitution of the external Na ion with Li, choline, tris-hydroxymethyl aminomethane or K ion greatly reduced the inhibitory effect of isoprenaline. A treatment with ouabain (4 × 10^-6 M) also had effects similar to those of Na removal. The spontaneous activity was transiently suppressed when 5.9 mM K ion was re-admitted into K-free solution, and this suppressing effect was blocked by ouabain and also by reducing Na concentration to 5.9 mM. However, isoprenaline retained its effects in K-free solution containing normal Na concentration or 5.9 mM Na. The relaxation by isoprenaline in these solutions was hardly reduced by ouabain (4 × 10^-6 M). These observations suggest that the beta-receptor stimulating effects of isoprenaline are partly mediated through an activation of the Na-pump and partly through some other mechanism which is inactivated when the preparation is deprived of Na or when the membrane is depolarized.

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Effects of lyophilization and storage of liver 9, 000 × g supernatant fraction on the activity of drug metabolizing enzymes in some species of animals were studied. Imipramine metabolizing enzyme activity and the content of cytochrome P-450 were stable for, at least, one month without any loss of the activity when lyophilized liver 9, 000×g supernatant fraction of some animal species, i.e., rat, mouse, guinea pig, rabbit and dog, was stored under reduced pressure in a deep freezer (-20°C). There were no differences of imipramine metabolism between lyophilized liver 9, 000 × g supernatant fraction which was kept in a deep freezer for one month and a fresh fraction which was prepared immediately before determinations of the enzyme activity and cytochrome P-450. The data presented here indicated that the lyophilized preparation of the liver 9, 000 × g supernatant as well as the fresh preparation can be used for the determination of the drug metabolizing enzyme activity.

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The activation of adenylate cyclase by NaF was dependent on the previous incubation time and the concentration of F^-. The activation by F^- was irreversible and Mg²⁺ was required for the maximum effect. Turbidity of microsome suspension was also greatly increased by F^- plus Mg²⁺ . These effects on adenylate cyclase and membrane turbidity were specific for F^- and F^--saturation curves for both were similar, though Mg²⁺-saturation curves for both were dissimilar. The increase in turbidity induced by F^- plus Mg²⁺ was rapidly reversed by ATP, GTP, ITP, UTP and CTP. However, ITP only, among all the triphospho-nucleotides tested, reversed the activity of adenylate cyclase previously activated by NaF plus MgCl₂. The activity of the enzyme reversed by ITP was not, however, re-enhanced by the presence of NaF in the assay medium. These results suggest the possibility that F^- induces a change in the membrane structure itself, and this change can be reversed by incubation with ITP. Consequently, adenylate cyclase may be conformed either to an activated or an unactivated state.

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Effects of parathion and di-isopropyl fluorophosphate (DFP) on acetylcholine (ACh) synthesis in the mouse brain were investigated. In addition to well known cholinesterase (ChE) inhibition, parathion showed inhibitory effects on the activity of synaptosomal choline acetyltransferase (ChAc), and on the uptake of [¹⁴C-methyl]-choline and ACh synthesis in subcellular fractions of the brain. DFP inhibited ChE activity, but had no significant effects on the choline uptake and ACh synthesis per se. Possible significance of these findings in the pharmacological actions of organophosphorus compounds is briefly discussed.

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An allometric method for estimating the volume change in inflamed paw of rats is described. The technique is effective and advantageous for long term observation of adjuvant arthritis which lacks a suitable reference criterion due to the simultaneous swelling of the control paw. In the present method, the inflammatory intensity (IF) of paw edema is estimated by means of the formula: IF(%)={2V_t/cX^d(I÷W_t/aX^b)-I}×100 where V_t is the paw volume, W_t is the body weight and X is the tail length of the inflamed rat. The constants a, b, c and d are obtained from the normal rats using the relative growth law, W=aX^b and V cX^d (where W is the body weight, V is the paw volume and X is the tail length).

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