Sensitivities of the stomach and duodenum to oral copper sulfate emesis were compared in dogs. 1) Dogs equipped with a stainless steel cannula in the middle of the duodenum were challenged to the oral threshold emetic dose of copper sulfate administered by a gastric tube. When the cannulas were opened, the oral thresholds were not effective to elicit vomiting in the most cases (1/13). Fairly rapid and high rate recoveries of copper through the open cannula were noted. With the closed cannulas, the thresholds were highly effective (16/16). 2) In the dogs with a cannula at the upper part of the jejunum, the oral threshold doses were always effective whether the canula was opened (9/9) or closed (11/11). Recovery rates of copper from the cannula were usually poor. 3) The oral thresholds administered into the proximal end or the middle of the duodenum through a PVC tubing were equally effective. 4) Although copper sulfate might irritate the stomach and upper duodenum to evoke vomiting, these results suggested a higher sensitivity of the lower duodenum.

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Morphine increased the serum prolactin (PRL) levels of male rats in a dose response manner. This effect was abolished by naloxone and apomorphine, but was not affected by diphenhydramine. The increase in the serum PRL levels by haloperidol was abolished by apomorphine, but not by naloxone. Repeated administrations of increasing doses of morphine attenuated the response of serum PRL to morphine. Naloxone did not alter the serum PRL levels of morphine-tolerant rats, while it precipitated full withdrawal signs on these rats. Although neither haloperidol nor morphine increased the release of PRL from the isolated anterior pituitary, haloperidol, but not morphine, reversed the inhibition by dopamine of the in vitro release of pituitary PRL. These results indicate that tolerance develops regarding the effect of morphine with a resulting increase in the serum PRL levels, abstinence precipitated by naloxone has no effect on the serum PRL levels, the mechanism of morphine involved in increase in the serum PRL is different from that of haloperidol as the effect of haloperidol is not antagonized by naloxone and morphine does not antagonize the effect of dopamine which inhibits the release of PRL from the anterior pituitary in vitro.

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The influence of a diuretic, clorexolone and a β-adrenoceptor blocking agent, alprenolol, alone and in combination, on blood pressure, heart rate and serum angiotensin II and aldosterone levels were examined in 5 conscious renal hypertensive dogs. Clorexolone (10 mg/kg p.o., twice a day) caused a slight increase in heart rate, and a gradual decrease in blood pressure which became significant after the second day of treatment (p<0.05). The mean reduction in systolic blood pressure by clorexolone alone was about 15 mm Hg. Addition of alprenolol (10 mg/kg p.o., twice a day) induced a further rapid decrease in arterial blood pressure and there was a tendency toward decrease in the increased heart rate. The mean decrease in systolic blood pressure was about 26 mm Hg and such was highly significant in comparison with that exerted by clorexolone alone (p<0.025). After withdrawal of clorexolone and final replacement of alprenolol with lactose, the decreased blood pressure gradually returned to the initial value. Increases in serum angiotensin II and aldosterone levels seen after clorexolone alone declined to the initial value when alprenolol was given in combination with clorexolone. The antihypertensive effect of this combination is attributed mainly to the inhibition of renin-angiotensin-aldosterone system, to decrease in circulating blood volume, and in part to decrease in the heart rate.

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Purified NADPH-cytochrome c reductase of rabbit liver microsomes was examined to determine whether or not the reported low lipid peroxidation activity of rabbit liver microsomes is due to the enzyme, NADPH-cytochrome c reductase. NADPH-cytochrome c reductase was purified from phenobarbital-treated rabbit liver microsomes to a specific activity of 14.9 to 21.4 unit per mg of protein with a yield of 15.2 to 16.4%. The purified sample (21.4 unit/mg of protein) was almost homogeneous as determined by sodium dodecylsulfate gel electrophoresis. This sample was used for determining lipid peroxidation activity. EDTA and ferrous ion but not ADP were essential requirements for the activity. FMN enhanced the activity when low concentrations of the NADPH-cytochrome c reductase were used for the assay. NADP and 2'-AMP, which are inhibitors of NADPH-cytochrome c reductase, inhibited the lipid peroxidation activity. α-Tocopherol and p-chloromercuribenzoate (PCMB) also inhibited the activity. From these results, we confirmed that rabbit liver microsomal enzyme NADPH-cytochrome c reductase plays a role in lipid peroxidation activity. The reported low lipid peroxidation activity in rabbit liver microsomes does not appear to be caused by the NADPH-cytochrome c reductase.

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In our studies with drug combinations, we searched for mixtures which would enhance the effectiveness of the related active substances. Ethenzamide was found to possess a specific suppressive effect on the gastric damage induced by aspirin. Such effect could not be demonstrated in analgesic agents such as salicylamide, bucetin, acetaminophen and phenacetin. The combination of aspirin with ethenzamide had a potentiating effect on analgesic activity and reduced motor incoordination and loss of righting reflex. We calculated the safety margins of various ratios of combinations and concluded that the best was aspirin and ethenzamide at a ratio of 2:3.

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A reconstituted lipid peroxidation system containing NADPH-cytochrome c reductase isolated from detergent-solubilized rat liver microsomes was used to determine the effects of several compounds, including drugs, on the lipid peroxidation activity. EDTA and ferrous ion were essential requirements for reconstitution of the activity. The addition of 1, 10-phenanthroline to the system containing both EDTA and ferrous ion further enhanced the activity. Pyrocatecol, thymol, p-aminophenol, imipramine, p-chloromercuribenzoate (PCMB) and α-tocopherol exhibited strong inhibition, aniline, N-monomethylaniline, aminopyrine, benzphetamine, SKF 525-A and NADP exhibited moderate inhibition, and phenol, benzoic acid, acetanilide and nicotinamide exhibited less or no inhibition at the concentrations lower than 1000 μM. Metal ions such as Hg⁺, Hg²⁺, Co²⁺, Cu²⁺, Mn²⁺ and U⁶⁺ inhibited lipid peroxidation strongly. In addition, Cd²⁺, St²⁺ and Ca²⁺ exhibited less potent to moderate inhibition, and Ba²⁺ and Mg²⁺ were without effects on the activity. Among sulfhydryl compounds tested, dithiothreitol inhibited lipid peroxidation to a greater extent than did the other three compounds, glutathione, cysteine and mercaptoethanol.

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Effects of estradiol on the release of prostaglandin-like substance (PG) from rat uterus was investigated. The amount of PG released into a medium that did not contain oxytocin was decreased by estradiol, but the release of PG was remarkably increased by the addition of oxytocin. No significant correlation was found in the relationship between PG release and uterine contraction in uterus of either the ovariectomized rat and that of the estradiol-injected rat. The amount of PG released from the uterus of ovariectomized rat and the uterus of 6 hours after estradiol injection was decreased by indomethacin. The PG release promoting action of oxytocin observed in estradiol-injected rat uterus disappeared completely with treatment of phospholipase C.

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Studies were made on the bindings of [³H]-acetylcholine and [³H]-atropine to synaptic plasma membranes from rat brain. Synaptic plasma membranes have reversible, high affinity binding sites for both ligands, the KD values for ACh and atropine being about 20 nM and 1 nM, respectively. The maximal binding capacities for ACh and atropine, respectively, are 0.8-1.2 pmoles and about 1.5 pmoles/mg protein of synaptic membranes. The specific binding of ACh is almost completely inhibited by oxotremorine and atropine.
5, 5'-Dithiobis (2-nitrobenzoic acid) (DTNB) increased the ACh-binding to about 1.5 pmole/mg protein. It also increased the inhibition of atropine-binding by ACh about 10-fold. Marked discrepancies were found in the inhibitions of atropine- and ACh-bindings by muscarinic agonists, but not in the inhibitions by antagonists. These findings support the hypothesis that muscarinic receptors have different sites for agonists and antagonists. The possibility that one receptor can be simultaneously occupied by both an agonist and an antagonist is also discussed.

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The variation of acetylcholine (ACh) sensitivity induced by development, denervation and glycerol treatment was studied using skeletal muscles of frogs and rats. In the course of metamorphosis from tadpole to adult, sensitivities (affinity) of rectus abdominis muscles of frogs to ACh and n-butyltrimethylammonium (BTMA) increased, as did the maximum responses (intrinsic activity) to BTMA, whereas the sensitivities to ACh-competitive antagonist decreased. The increasing phenomena were similar to the supersensitivity seen with denervation. The slope of dose-inhibitory response curve for curare-like agents changed from 1.0 to 1.5 with development of muscles. These results suggest changes in ACh receptors. With 4 or 10% glycerol treatment of chronically denervated diaphragm muscles, the responses to ACh decreased, whereas those to ATP increased. The maximum responses and sensitivity of ACh dose-response curves on glycerinated muscles decreased dose dependently with glycerol. ACh log dose-response curves of muscles treated with 2 % glycerol shifted in parallel with d-tubocurarine application and pA₂ values were observed to be similar to those of non-treated muscles. The responses of glycerinated muscles to ACh were potentiated by 1.5 μM neostigmine less than those seen in non-treated muscles. The increase in ACh sensitivity with development may be in close analogy to the supersensitivity seen with denervation, and the pattern of the increase in ACh sensitivity seen with development or denervation was the reverse of that seen with glycerol treatment. The sensitivity of ACh receptor is probably closely related with the glycerol treatment.

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Loperamide, an effective antidiarrheal agent, was investigated in attempts to determine the site of action which underlies the antiperistaltic and other antidiarrheal actions. In in vitro studies, this compound applied in a dose over 10^-8 g/ml, inhibited the release of both acetylcholine and prostaglandins during circumferential distension of the intestinal wall, in a dose dependent manner. The inhibited acetylcholine release, but not prostaglandin release, was reversed by naloxone. This suggests that loperamide inhibits acetylcholine release by interacting with opiate receptor sites in the myenteric plexus. The inhibition of prostaglandin release may be due to inhibition of prostaglandin synthesis in the intestine because loperamide prevented the biosynthesis of prostaglandin from arachidonic acid. Although a high concentration of loperamide (10^-6 g/ml) inhibited the contraction of the intestine to acetylcholine, this compound inhibited the contraction to nicotine and serotonin at a concentration which had no effect on the contraction to acetylcholine. Thus loperamide apparently inhibits the peristaltic movement principally by reducing the release of acetylcholine and prostaglandin, at least during circumferential distension of the intestinal wall in vitro. The finding that loperamide inhibited the biosynthesis of prostaglandin may lead to elucidation of the mechanism of its antidiarrheal activity.

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Contractile response of the isolated canine tracheal muscle to transmural nerve stimulation was considerably depressed by 10^-6 g/ml of prostaglandin Al (PGA₁), A₂ (PGA₂), B₂ (PGB₂), E₁ (PGE₁), E₂ (PGE₂) and F_2α (PGF_2α), while the response to exogenously administered ACh was only slightly affected by the pretreatment with prostaglandins (PGs). The order of the potency of the depressive effect was as follows; PGE₁>PGE₂>PGA₁>PGA₂≈PGB₂≈PGF2_2α. Therefore, if a negative feedback control mechanism through prostaglandins exists in the excitatory transmission in canine tracheal muscle, the PGE series may predominantly operate the mechanism. Indomethacin in a dose of 10^-6 g/ml produced a potentiation of the contractile responses to transmural nerve stimulation and to exogenously administered ACh.

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The membrane protein of rat erythrocytes, solubilized by 1 % sodium dodecyl sulfate, was analyzed by polyacrylamide gel electrophoresis. Six major protein bands (Coomassie brilliant blue positive, I-VI, molecular weight: 200, 000, 170, 000, 105, 000, 96, 000, 80, 000 and 54, 000) or three glycoprotein bands (PAS positive, 1-3, molecular weight: 108, 000, 96, 000 and 54, 000) were detected in the membrane produced by hypotonic hemolysis. Several additional protein bands (between 90, 000-40, 000 molecular weight) were detected in the membrane produced by mercuric chloride, methylmercuric chloride and N-ethylmaleimide hemolysis. A smaller number of bands (90, 000-40, 000 molecular weight) were detected in the membrane produced by p-chloromercuribenzoic acid hemolysis than the membrane hemolyzed by other mercurials. However, those additional bands did not appear when membranes were produced by hypotonic hemolysis then treated with mercurials. A band under band IV appeared when p-chloromercuribenzoic acid was used for the treatment. The profile of PAS positive bands of membrane produced by mercurial hemolysis was similar to that of membrane produced by hypotonic hemolysis. The degree of free sulfhydryl groups in membrane treated with mercurials was not closely related to the induction of hemolysis.

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The myelin fragments of rat brain stem were treated with butanol-water mixtures, and the butanol extracts (total extract=TE) were incubated with 5×10^-7 M of C¹⁴•5-HT. After incubation, protein, lipid phosphorus and radioactivity were analyzed by Sephadex LH₂₀ column chromatography. Two peaks of components eluted in chloroform-methanol 4:1 (peak I and II) showed the binding capacity for C¹⁴•5-HT. The displacement studies with unlabeled 5-HT (5×10^-4 M) suggested that peak II was the saturable binding component to 5-HT. On the other hand, butanol extracts from the synaptic membranes of rat brain stem did not show binding for C¹⁴•5-HT. Various compounds were studied to determine their inhibitory effects on the saturable binding of 5-HT to TE. The results indicated that acetylcholine, dopamine and tryptamine inhibited the 5-HT binding but LSD, reserpine, colchicine, vinblastine, 5-hydroxytryptophan (5-HTP) and 5-hydroxy-3-indole acetic acid (5-HIAA) had no effect.

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The anti-fertility activity of prostaglandin synthesis inhibitors namely the non-steroidal anti-inflammatory drugs: acetyl salicylic acid, indomethacin and oxyphenbutazone were investigated in male and female albino rats and female albino rabbits. Oxyphenbutazone and indomethacin affected the reproductive process in male rats. Indomethacin 3 mg/kg. and acetyl salicylic acid 300 mg/kg produced significant anti-ovulatory activity in the rabbit. In female rats, all three drugs given in high doses over a period of two oestrous cycles reduced mating significantly, while only indomethacin given in a low dose of 0.8 mg/kg over a period of six oestrous cycles could reduce mating significantly. Anti-implantation activity was seen with indomethacin 4 mg/kg. alone, and though post-implantation activity was seen with all three drugs, it was associated with maternal deaths. It appears that non-steroidal anti-inflammatory drugs affect reproduction in female animals.

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