The effects of exoenzyme C3 of Clostridium botulinum on Ca²⁺ -and drug-induced tension developments were investigated in β-escin skinned smooth muscle of guinea pig ileum to test the involvement of a small G-protein in the regulation of myofilament Ca²⁺ sensitivity. C3 is known to ADP-ribosylate the rho p21 family of small G-proteins. Treatment with C3 (0.35 μg/ml, for 30 min) shifted the pCa-tension curve rightward along the Ca²⁺ concentration axis, indicating a decrease in Ca²⁺ sensitivity of the contractile elements. The inhibitory effect of C3 was not preserved after treatment with GDPβS (1 mM), an antagonist of GTP for the binding to G-proteins. Stimulation of muscarinic receptors with carbachol (CCh, 100 μM) shifted the pCa-tension curve leftward, indicating Ca²⁺ sensitization of tension development. The Ca^2--sensitizing effect of CCh was not observed after C3 treatment. When GTPγS (10 μM), an activator of G-proteins, was applied at a plateau of tension development produced by a moderate concentration of Ca²⁺, further increase in tension was elicited and the effect of GTPγS was inhibited by C3 treatment. The results suggest the possible involvement of a rho p21-like small G-protein in the regulation of Ca²⁺ sensitivity of smooth muscle myofilaments.

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The mechanisms underlying the enhanced contractile response to phorbol 12, 13-dibutyrate (PDBu) were examined in de-endothelialized thoracic aortas isolated from rats with dietary magnesium (Mg) deficiency. PDBu (1.0 nM)-induced contractions were significantly larger in Mg-deficient rats than in the controls. The contraction was completely inhibited by nifedipine, removal of external Ca²⁺ or by l-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7). PDBu (1.0 nM) and phorbol 12-myristate 13-acetate (1.0μM) significantly decreased the K_D value and increased the B_max for the binding of [³H]PN200-110 to the aortas. The degree of the decrease in the K_D value was significantly greater in Mg-deficient rats than in the controls. The PDBu-induced decrease in the K_D value was abolished by H7. These results suggest that activation of protein kinase C by phorbol esters may participate in the activation of L-type Ca²⁺ channels, which increases both the affinity of [³H]PN200-110 binding and the magnitude of the external Ca²⁺-dependent contraction. Dietary Mg-deficiency may enhance these processes.

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We investigated the effects of the salivary peptide P-C (P-C), a saliva-derived peptide, on glucose (8.3 mM)- and arginine (10 mM)-induced insulin release and arginine (10 mM)-induced glucagon release using the perfused pancreas of spontaneously diabetic GK rats. Both its potentiating effect on insulinrelease and its inhibitory effect on glucagon release were concentration-dependent in diabetic GK rats. The ratio of insulin release obtained with P-C (194 nM) to that without P-C in GK rats was the same as ratio in normal Wistar rats. The ratio (0.40) of glucagon release obtained with P-C (194 nM) to that without P-C was smaller in diabetic GK rats than that (0.75) in normal Wistar rats. These results indicate that P-C inhibits arginine-induced glucagon release in diabetic GK rat pancreas more effectively than in normal Wistar rat pancreas.

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To understand the effect of oxygen free radicals on Ca²⁺-ATPase, we used sarcoplasmic reticulum (SR) microsomes of canine masseter muscle as a model system in which to explore the effects of oxidation on a biological membrane, and we investigated the effect of hydroxyl radicals (·OH) generated from Fenton''s reagent (H₂0₂/FeSO₄). H₂0₂(10 mM) alone had no effect on Ca²⁺ -ATPase activity; in the presence of FeSO₄ (0.2 mM), H₂0₂ inhibited the enzyme activity. Oxygen free radical species generated from H₂0₂/FeSO₄ under the conditions employed in the Ca²⁺ -ATPase assay were verified by highly sensitive electron spin resonance spectroscopy and the spin-trap 5, 5-dimethyl-1-pyrroline-N-oxide (DMPO) in the absence of SR vesicles; the 1:2:2:1 quartet (A_N= A_H=1.49 mT), characteristic of the DMPO-OH spin adduct, was observed. The Ca²⁺ -ATPase activity was inversely correlated with the calculated signal intensity of DMPO-OH, which is indicative of the amount of ·OH radical generated. The effect of Fenton''s reagent was effectively inhibited by catalase, dimethylsulfoxide, and dimethylthiourea; the effect was also inhibited by sulfhydryl (SH) group reducing agents, cysteine and dithiothreitol. The SH group modifying agents, p-chloromercuric benzoate and 5, 5'' -dithiobis(2-nitrobenzoic acid) depressed Ca²⁺-ATPase activity; the effects of the SH group modifying agents used were potentiated in the presence of Fenton''s reagent. It is suggested that ·OH radical-induced oxidant injury may be caused primarily by modification of the key SHgroup(s) on the ATPase molecule of masseter muscle SR vesicles.

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Effects of taurine on the spontaneous action potentials in rabbit sino-atrial nodal cells were examined at different extracellular Ca²⁺ concentrations ([Ca]_o). Experiments were performed at 36°C. The firing rate of spontaneous activity was 132.5± 12.1 beats/min (n= 18) in normal Tyrode''s solution ([Ca]_o = 1.8 mM). Increasing [Ca]_o level from 0.9 to 10.8 mM significantly changed the maximum rate of depolarization. Other parameters of the action potentials were unaffected. When [Ca]_o was 0.9 mM, application of taurine (1 to 20 mM) tended to cause a positive chronotropic effect and hyperpolarized the maximum diastolic potential. In the normal solution (at 1.8 mM [Ca]_o), taurine significantly enhanced only the maximum rate of depolarization. In contrast, under high [Ca]_o (5.4 and 10.8 mM), taurine at 1 and 5 mM had a negative chronotropic effect, but 20 mM taurine had a positive chronotropic effect. Also, taurine shortened the action potential duration and hyperpolarized the maximum diastolic potential. The maximum rate of depolarization was inhibited. In 10.8 mM [Ca]_o solution, irregular spontaneous activity (dysrhythmias) occurred in 4 of 6 preparations, and addition of taurine (1 to 20 mM) abolished it. These results indicate that taurine modulates the action potential configuration in the sino-atrial nodal cells dependent on [Ca]_o.

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The effects of K⁺ channel openers (PCO_s), NIP-121, levcromakalim and nicorandil, and the blockers of the specific binding sites for [³H]glibenclamide, ATP-sensitive K⁺ channel blocker, were investigated in rat brain and cardiac ventricle membrane preparations. When the microsomes were incubated with [³H]glibenclamide, the specific glibenclamide binding was fully inhibited by unlabeled glibenclamide (1 μM) and apamin (100 μM). However, the specific glibenclamide binding was not influenced by excess NIP-121, levcromakalim and nicorandil, although glibenclamide antagonized the increase in the ⁸⁶Rb⁺ efflux by PCOs. On the other hand, the binding of [³H]glibenclamide after a long pre-incubation (60 min) at 37°C with NIP-121 and levcromakalim at pharmacological effective concentrations (10 nM to 1 μM) was significantly influenced. Both PCOs partially reduced both K_d and B_max values of the specific [³H]- glibenclamide binding in a concentration-dependent manner that was not regulated by GTPγS. The doseeffect relationships for the B_max''s of NIP-121 and levcromakalim seemed similar to those for vasorelaxation. These findings indicate that the pharmacological effect of PCO may be caused by the binding to its own specific sites but not to the specific sulfonylurea sites. The binding of PCOs may inhibit, in a negative allosteric manner the binding of sulfonylureas.

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KW-5092 ({1- [2-[[[5-(piperidinomethyl)-2-furanyl]methyl]amino]ethyl]-2-imidazolidinylidene} propanedinitrile fumarate) is a novel gastroprokinetic agent with acetylcholinesterase (AChE) inhibitory activity and acetylcholine (ACh) release facilitatory activity. The present study examined the effects of KW-5092 on gastrointestinal (GI) propulsion in rats. KW-5092 at I to 30 mg/kg, p.o. dose-dependently enhanced the gastric emptying, small intestinal propulsion and the proximal and distal colonic propulsion. Metoclopramide, a dopamine D₂-receptor antagonist with ACh release facilitatory activity, dose-dependently enhanced the gastric emptying at 0.03 to 1 mg/kg, p.o., whereas this drug did not affect the small intestinal propulsion, or the proximal and distal colonic propulsion. Neostigmine, an AChE inhibitor, dosedependently enhanced the small intestinal propulsion and the proximal and the distal colonic propulsion at 0.3 to 10 mg/kg, p.o., whereas it delayed the gastric emptying at 10 mg/kg, p.o. The present results demonstrate that KW-5092 enhances the GI propulsion from the stomach to the colon and that metoclopramide or neostigmine enhances only the upper or the lower GI propulsion, respectively. Thus, KW-5092 may be a gastroprokinetic drug of a novel type for the treatment of GI motility dysfunctions in a wide range from the stomach to the colon.

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The photochemical reaction between rose bengal and light (540 nm) produces thrombotic occlusion in rat coronary artery. We have now developed an experimental myocardial infarction (MI) model by photochemically induced thrombosis (PIT) in rats and investigated the mechanisms responsible for the induction of MI. PIT in the coronary artery induced myocardial ischemia, which was determined by tissue oxygen tension (tpO₂), and resulted in MI. Pretreatment with a thromboxane (TX) A₂-receptor antagonist, vapiprost, prevented a decrease in myocardial tpO₂ and markedly reduced the MI area, although vapiprost inhibited collagen-induced platelet aggregation by 30% ex vivo. An ADP-induced platelet aggregation inhibitor, clopidogrel, also reduced the MI area. In contrast to vapiprost, clopidogrel inhibited collagen-induced platelet aggregation by 90% ex vivo. Pretreatment with a 5-HT₂-receptor antagonist, ketanserin, which did not inhibit collagen-induced platelet aggregation ex vivo, prevented the decrease in myocardial tpO₂ and reduced the MI area. These results suggest that TXA₂, 5-HT and ADP play a role in the induction of MI and that platelet aggregation and other factors induce ischemia in this model.

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We examined the effects of YM020 (3-cyanomethyl-2-methyl-8-[(3-methyl-2-butenyl)oxy]-imidazo[1, 2-α]pyridine), a novel H⁺, K⁺-ATPase inhibitor, on gastric acid secretion and experimental gastroduodenal lesions in rats and dogs. Intraduodenal, subcutaneous and oral YM020 inhibited basal gastric acid secretion in pylorus-ligated rats with ED₅₀ values of 9.1, 9.1 and 9.5 mg/kg, respectively. Oral pretreatment with YM020 5 hr before ligation still suppressed acid secretion, with a potency a little less than that of omeprazole. In anesthetized dogs, intravenous YM020 inhibited histamine-, methacholine- and pentagastrin-induced gastric acid secretion with ED₅₀ values of 0.05, 0.01 and 0.08 mg/kg, respectively. In Heidenhain pouch dogs, although oral YM020 (3 mg/kg) inhibited histamine-induced acid secretion, acid output returned to control levels faster than in dogs treated with omeprazole. Oral YM020 inhibited the formation of water-immersion restraint stress-, indomethacin-, absolute ethanol-, 0.7 N hydrochloric acid- and cysteamine-induced gastric or duodenal lesions with ED₅₀ values of 2.9, 4.3, 2.0, 11.7 and 8.4 mg/kg, respectively. Moreover, subcutaneous YM020 also suppressed the formation of ethanol- and HCl-induced gastric lesions. These results suggest that YM020 has an antisecretory effect almost the same as or 2 to 3 times weaker than those of omeprazole and that its duration is not as long as that of omeprazole in rats and dogs. Furthermore, YM020 possesses a cytoprotective effect and the mechanism of YM020 may be different to that of omeprazole.

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The effects of nomifensine (NOM) and methamphetamine (MA) on ambulation and rearing of rats in the open-field test were investigated. NOM and MA were injected i.p. into intact rats and nucleus accumbens (ACC)- and caudate-putamen (CP)-lesioned rats and infused into the ACC and CP. NOM (1-10 mg/kg, i.p.) and MA (1-5 mg/kg, i.p.) produced hyperactivity. However, NOM at 20 mg/kg, i.p. decreased the activity and induced repetitive head bobbing and squatting. Lesions of the ACC and CP increased open-field activity. However, lesion of the CP increased rearing more than lesion of the ACC. The increase in ambulation induced by NOM was inhibited by lesion of the ACC, whereas that induced by MA was inhibited by lesion of the CP. Although NOM (1-10 μg/2 μl) and MA (0.5 - 10 μg/2 μl) injections into the ACC and CP induced hyperactivity, the effect of NOM was greater after injection into the ACC, whereas the effect of MA was greater following injection into the CP. These results suggest that the ACC has a greater role in ambulation, while CP has a greater one in rearing. The present results, while they verified the significance of the ACC and CP in NOM- and MA-hyperactivity, also revealed a differential role of the ACC in NOM-hyperactivity and the CP in MA-hyperactivity.

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The amino acid domains in salivary peptide P-C (1-44 peptide fragments, P-C) that are essential to potentiate insulin release and inhibit glucagon release were investigated using isolated perfused rat pancreas. P-C significantly potentiated not only glucose (8.3 mM)-induced insulin release, but also arginine (10 mM)-induced insulin release. The essential domain responsible for potentiation of insulin release was the P-C-(23-29) fragment, ²³KPQGPPP²⁹, while that inhibiting glucagon release was the P-C-(12-18) fragment, ¹²HQQGPPP¹⁸. Since both domains share a common fragment, QGPP, these findings indicate that the functional amino acid sequences KP and HQ may potentiate insulin release and inhibit glucagon release, respectively.

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Injection of the muscarinic agonist oxotremorine into rat striatum for seven consecutive days resulted in a 44% in ³H-quinuclidinyl benzilate (³H-QNB) binding, and it caused decreases in binding affinity to agonists. Furthermore, it eliminated the shift from the high affinity site to the low affinity site that occurs in the presence of 5''-guanylyl imidodiphosphate (Gpp (NH)p). In addition, oxotremorine caused a 1.5-fold increase in the incorporation of ³H-leucine into the striatum, suggesting that it increased the syntheses of proteins other than the muscarinic receptor protein. The present results show that chronic treatment of the striatum with oxotremorine causes alterations in not only the quantity, but also the sensitivity of muscarinic receptors to guanine nucleotide.

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Nebracetam (10-100 μM) dose-dependently inhibited increases in intracellular Ca²⁺ concentrations evoked by various stimuli in cultured rat cerebellar granule cells. The magnitude of the nebracetam (100 μM)-induced inhibition of L-glutamate- and N-methyl-D-aspartate-evoked Ca²⁺ responses was 1.5-fold and 1.7-fold greater, respectively, than the inhibition of the high K⁺-evoked response. These findings suggest that in cultured cerebellar granule cells, nebracetam attenuates the external Ca²⁺ influx derived from the activation of N-methyl-D-aspartate receptor-gated rather than voltage-gated Ca²⁺ channels.

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The protective effect of nebracetam on ischemic neuronal damage was histologically examined in the pyramidal cell layer of the hippocampal CAI subfield 7 days after operation using stroke-prone spontaneously hypertensive rats (SHRSP) subjected to 10-min bilateral carotid occlusion. Nebracetam (50 and 100 mg/kg), given orally 10 min after reperfusion, dose-dependently protected against ischemic delayed neuronal damage in the SHRSP with occlusion; however, the blood pressure remained unchanged following nebracetam administration. These findings further support the notion that nebracetam protects against ischemic delayed neuronal cell death in the hippocampus.

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Angiotensin converting-enzyme inhibitors (alacepril and imidapril) or an AT₁, -receptor antagonist (SC-52458) was administered to 10-week-old spontaneously hypertensive rats (SHR) for 7 days, and cardiac mRNA levels for contractile proteins and atrial natriuretic polypeptide (ANP) were comprehensively measured. The expression of skeletal α-actin and ANP was selectively enhanced in the heart of vehicle-treated SHR compared with Wistar-Kyoto rats (WKY), thereby suggesting that the phenotypic modulation of myocytes occurred at the early stage of hypertension. The above-mentioned three drugs similarly suppressed these enhanced gene expressions, nearly to the control levels. In contrast, although the treatment with hydralazine lowered the blood pressure of SHR similarly, hydralazine did not suppress ANP expression at all and only partially suppressed skeletal α-actin. Moreover, alacepril did not affect these gene expressions in WKY. Thus, AT₁, receptor may be crucial for phenotypic modulation in the heart of SHR.

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