Imidazoline−binding sites are non−adrenergic receptors and classified into I₁/I₂ subtypes.There is strong evidence that I₁−binding sites, located in the rostro−ventrolateral medulla, are involved in regulation of blood pressure.However, less is known about the peripheral participation of I₁−binding sites in cardiovascular reactions.Therefore, the aim of this study was to investigate whether specific imidazoline derivatives influence myocardial contractility and whether imidazoline binding sites are expressed in rat heart.Agmatine, clonidine and idazoxan failed to alter inotropy in left atria within the whole concentration range tested(1 nM−100μM), whereas cirazoline(1−100μM)and moxonidine(100μM)increase inotropy by about 20−30%.After pre−incubation with the α₁−adrenoceptor antagonist prazosin, the cirazoline and moxonidine stimulated inotropy was antagonized, indicating more an α₁−adrenergic and less an imidazoline binding site mediated mechanism.Radioligand−binding studies in membranes of left ventricles using [³H]−clonidine to specify I₁−binding yielded K_D values of 12.7μM, confirming the functional results of an absence of I₁−binding sites in ventricles of rats.However, the existence of low affinity I₂−binding sites determined by [³H]−idazoxan labeling could not be excluded since a K_D of 0.5μM was calculated and since competition studies with guanabenz(K_i=0.1μM), clonidine(K_i=58.1μM)and moxonidine(K_i=129μM)confirmed the specificity of the I₂−binding.

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We describe the pharmacological characteristics of SM−19712 {4−chloro−N−[[(4−cyano−3−methyl−1−phenyl−1H−pyrazol−5−yl)amino]carbonyl]benzenesulfonamide, monosodium salt}.SM−19712 inhibited endothelin converting enzyme(ECE)solubilized from rat lung microsomes with an IC₅₀ value of 42 nM and, at 10−100μM, had no effect on other metalloproteases such as neutral endopeptidase 24.11 and angiotensin converting enzyme, showing a high specificity for ECE.In cultured porcine aortic endothelial cells, SM−19712 at 1−100μM concentration−dependently inhibited the endogenous conversion of big endothelin−1(ET−1)to ET−1 with an IC₅₀ value of 31μM.In anesthetized rats, either intravenous(1−30 mg/kg)or oral(10−30 mg/kg)administration of SM−19712 dose−dependently suppressed the pressor responses induced by big ET−1.In acute myocardial infarction of rabbits subjected to coronary occlusion and reperfusion, SM−19712 reduced the infarct size, the increase in serum concentration of ET−1 and the serum activity of creatinine phosphokinase.The present study demonstrates that SM−19712 is a structurally novel, nonpeptide, potent and selective inhibitor of ECE, and SM−19712 is a valuable new tool for elucidating the pathophysiological role of ECE.

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Effects of SM−19712 {4−chloro−N−[[(4−cyano−3−methyl−1−phenyl−1H−pyrazol−5−yl)amino]carbonyl] benzenesulfonamide, monosodium salt}, a novel endothelin converting enzyme(ECE)inhibitor, on ischemic acute renal failure(ARF)in rats were examined in comparison with those of phosphoramidon, a conventional ECE inhibitor.ARF was induced by occlusion of the left renal artery and vein for 45 min followed by reperfusion, 2 weeks after contralateral nephrectomy.Renal function in ARF rats markedly decreased at 24 h after reperfusion.Intravenous bolus injection of SM−19712(3, 10, 30 mg/kg)prior to the occlusion attenuated dose−dependently the ischemia/reperfusion−induced renal dysfunction.Histopathological examination of the kidney of ARF rats revealed severe renal damages such as tubular necrosis, proteinaceous casts in tubuli and medullary congestion, all of which were dose−dependently attenuated by SM−19712.Protective effects of phosphoramidon(10 mg/kg)on ARF−induced functional and tissue damages were less potent than that of the same dose of SM−19712.Endothelin−1(ET−1)content in the kidney after the ischemia/reperfusion was significantly increased, being the maximum level at 6 h after reperfusion, and this elevation was completely suppressed by the higher dose of SM−19712.Our findings support the view that renal ET−1 plays an important role in the development of ischemia/reperfusion−induced renal injury.SM−19712 may be useful in the treatment of ischemic ARF.

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The Ca²⁺ signaling mediated by activation of β−adrenoceptors was studied in a purified preparation of ducts from rat submandibular glands.At concentrations above 1 nM, isoproterenol(ISO)caused a small but significant increase in cytosolic Ca²⁺ concentration([Ca²⁺]_i).The ISO−induced increase in [Ca²⁺]_i was completely inhibited by the β−adrenoceptor antagonist propranolol but not by the α−adrenoceptor antagonist phentolamine.Forskolin was able to mimic the Ca²⁺ response to ISO.These results suggest that the ISO−induced increase in [Ca²⁺]_i in rat submandibular ducts is mediated by an accumulation of cAMP resulting from activation of β−adrenoceptors.In the absence of extracellular Ca²⁺, ISO or forskolin caused a transient increase in [Ca²⁺]_i, indicating Ca²⁺ mobilization from intracellular Ca²⁺ stores.Further, stimulation with ISO failed to mobilize Ca²⁺ after the depletion of intracellular Ca²⁺ stores by phenylephrine or carbachol, suggesting that the cAMP−mediated increase in [Ca²⁺]_i is due to a Ca²⁺ release from inositol trisphosphate(IP₃)−sensitive Ca²⁺ stores.As ISO did not stimulate a detectable production of IP₃, the cAMP−mediated Ca²⁺ mobilization may be evoked by a mechanism different from activation of phosphoinositide hydrolysis.

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In this study, [³H]hemicholinium−3([³H]HC−3)binding, which labels the presynaptic high affinity−choline transport sites, was examined in two brain regions, cerebral cortex and midbrain, of nicotine−treated and −untreated rat neonates.In nicotine−untreated neonates, [³H]HC−3 binding sites of cerebral cortex increased from 64 fmol/mg protein at postnatal day 7 to 142 fmol/mg protein at postnatal day 35.In nicotine−treated neonates, the development of [³H]HC−3 binding sites in cerebral cortex was significantly retarded, compared with control neonates on the 7th, 14th and 21st postnatal days.In parallel with this, the development of muscarinic receptor in cerebral cortex, which was detected by [³H]quinuclidinyl benzylate([³H]QNB)binding, was also retarded by nicotine treatment.However, in midbrain, neither [³H]HC−3 nor [³H]QNB binding sites at postnatal day 14 was affected by nicotine treatment.These results strongly suggest that perinatal treatment with nicotine inhibits presynaptic and postsynaptic development of the cholinergic pathway in cerebral cortex but not in midbrain of rat neonate.

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Since apomorphine actually reveals high efficacy in treatment of Parkinson’s disease but only has a very short half life, it is of only limited clinical significance.To overcome this substantial disadvantage, drug application by long term delivery systems could be one possibility.Based on this background, ethylene vinyl acetate polymeric delivery systems were manufactured that differed in size, with either coated or uncoated surfaces, but were similar in apomorphine loading.Release from uncoated polymeric delivery systems followed first order kinetics, whereas coated polymeric delivery systems showed within the first 40 days a period of first order kinetics release, in which the release rate is approximately half that of the uncoated polymeric delivery systems, followed by a zero order kinetics release for more than 130 days with a daily release rate of 3.1 ± 0.2 mg.In vivo release was investigated by determining plasma apomorphine concentrations after implanting polymeric delivery systems into the abdominal cavities of rats.Animals with uncoated polymeric delivery systems exhibited symptoms of an apomorphin overdosage within 20 days after surgery.Using coated polymeric delivery systems, a steady state plasma concentration of 15 ng/ml was observed, which was maintained over a period of 130 days after an initial period of high plasma concentrations.Based on our results, it is concluded that polymeric delivery systems might be an appropriate method for applying apomorphine for the treatment of Parkinson’s disease.

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We examined the influence of 2 gut hormones involved in the enhancement of pancreatic exocrine secretion, secretin and cholecystokinin(CCK), in the exacerbation of pancreatitis.We also examined the role of the vagal system, which was considered to be a transmission route for these hormones.Our model of pancreatitis in the rat was prepared by pancreatic bile duct ligation(PBDL), which simultaneously ligated the pancreatic duct and the common bile duct.Serum amylase activity and histopathological changes in the pancreas were used as indices of pancreatitis.We also measured the volume of pancreatic juice, as well as the amylase activity and protein level of the pancreatic juice, as indices of increased pancreatic exocrine secretion.Two gut hormones were given 6 times at 1−h intervals.Administration of secretin(1−3 μg/kg, s.c.)did not influence serum amylase activity in rats with PBDL−induced pancreatitis.However, food stimulation and administration of CCK−8(1 μg/kg, s.c.)increased serum amylase activity and promoted vacuolation of the pancreatic acinar cells in rats with PBDL−induced pancreatitis.Administration of atropine(3 mg/kg, s.c.)or a CCK₁−receptor antagonist, Z−203(0.1 mg/kg, i.v.), inhibited food−stimulated or CCK−8−induced(1 μg/kg, s.c.)enhancement of pancreatic exocrine secretion and exacerbation after the development of PBDL−induced pancreatitis.These results suggest that not secretin, which regulates the volume of pancreatic juice, but CCK, which regulates the secretion of pancreatic enzymes via the vagal system, plays an essential role in food−stimulated exacerbation after the development of pancreatitis.

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Fasting causes gastric mucosal damage in streptozotocin(STZ)−induced diabetic rats, but its pathogenic mechanism remains to be elucidated.The aim of the present study was to investigate the alteration of gastric mucosal mucin, one of the gastric defensive factors against the developmet of such damage.Diabetes was induced in rats by intravenous injection of STZ(65 mg/kg).The experiments were performed using 4−week STZ−diabetic rats with blood glucose levels above 350 mg/dl.The amount of gastric mucus glycoprotein was determined by gel filtration, and the distribution of neutral and acidic mucins in the stomach epithelium was examined by histochemical analysis.In normal rats, 24−h fasting neither affected the gastric mucin content nor caused any macroscopic gastric mucosal injury.In contrast, starvation significantly reduced the amount of total gastric mucus glycoprotein prior to the formation of mucosal lesions in the STZ−diabetic rats.Nine hours after food deprivation, the gastric damage developed in about 70% of the diabetic rats, the amount of mucus glycoprotein markedly decreased, and both the neutral and acidic mucins diminished in the epithelium.Taken together, in STZ−diabetic rats, fasting by itself depletes gastric mucus glycoprotein, and this depletion may be involved in the pathogenic mechanism of the formation of gastric mucosal lesions.

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We determined possible protective effects of benidipine hydrochloride(benidipine), a dihydropyridine calcium antagonist, on cerebrovascular lesions in salt−loaded stroke−prone spontaneously hypertensive rats(SHRSP).The animals were orally treated with benidipine at 1, 3 and 10 mg/kg daily for 7 weeks, and their neurological symptoms, body weight changes, systolic blood pressure and cerebrovascular lesions on magnetic resonance imaging(MRI)were determined at various time points of treatment.Moreover, the brains of the rats that showed cerebrovascular lesions on MRI in the course of treatment or completed 7−week treatment were examined histopathologically.Control rats presented such symptoms as sedation, ataxia and aggressiveness, while their MRI analysis revealed high signals over wide areas from the occipital to frontal cortex and from the corpus callosum to external capsule.These high signal areas corresponded in location to edematous or softening lesions revealed by the histopathological observation.Treatment with benidipine at 3 and 10 mg/kg ameliorated neurological symptoms, significantly suppressing cerebrovascular damages on MRI.Benidipine at 3 mg/kg significantly decreased blood pressure for the first four weeks but it did not thereafter.These findings demonstrate that benidipine can protect salt−loaded SHRSP from cerebrovascular injury as assessed by MRI.

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Polaprezinc [N−(3−aminopropionyl)−L−histidinato zinc](PZ), an anti−ulcer drug, is a chelate compound consisting of zinc and L−carnosine.PZ has been shown to prevent gastric mucosal injury.In the present study, we investigated the inhibitory effect of PZ on indomethacin(IND)−induced apoptosis in a rat gastric mucosal cell line, RGM1.Pretreatment with PZ suppressed caspase−3 activation and subsequent apoptosis in the cells exposed to 500 μM IND in a dose−dependent manner, and 50 μM PZ exhibited the maximum inhibitory effect.Among PZ subcomponents, zinc but not L−carnosine played a pivotal role in this anti−apoptotic function.PZ did not affect mitochondrial cytochrome c release upstream of caspase−3 activation in the IND−induced apoptotic signal pathway.Treatment with 500 μM IND evidently produced reactive oxygen species(ROS)in RGM1 cells.However, PZ did not scavenge ROS in IND−treated cells.Moreover, N−acetyl−L−cysteine, a potent antioxidant, inhibited ROS generation but did not suppress apoptosis in RGM1 cells exposed to IND.These observations demonstrate a novel pharmacological action of PZ;i.e., that PZ, and in particular its zinc subcomponent, inhibits apoptosis via inhibition of caspase−3 activation but not antioxidant activity.

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Serotonin−induced neuronal cell death has been implicated as a possible cause of neurodegenerative and neuropsychiatric disorders.To investigate the involvement of serotonin−induced apoptosis as a potential mechanism in the pathophysiology of serotonin−related diseases, MTT(3−(4, 5−dimethylthiazol−2−yl)−2, 5−diphenyltetrazolium bromide)assay, DAPI(4’, 6−diamidino−2−phenylindole)staining, TUNEL(terminal deoxynucleotidyl transferase−mediated dUTP−biotin nick end labeling)assay and flow cytometric analysis were performed using the immortalized pineal cell line PGT−β.Through morphological and biochemical analyses, it was demonstrated that cell death induced by serotonin in PGT−β cells shows classic apoptotic features.These data suggest that serotonin induces apoptosis in PGT−β cells.

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We studied the effect of chronic(7 days)angiotensin II infusion in a subpressor(200ng/kg per minute)dose on angiotensin II receptors in the left ventricle in rats.Infusion of angiotensin II caused an elevation in systolic blood pressure after 3 days, usually to values of about 150 mmHg, and the increase continued during the drug administration.The number of angiotensin II type 1 and angiotensin II type 2 receptors was significantly decreased by 20−30% in the angiotensin II−infused left ventricular membranes without affecting the affinity.Thus, these data suggest that angiotensin II may regulate the number of its own receptors in rat left ventricles.

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Adenosine was intraperitoneally(i.p.)injected to Wistar rats every 3 days with a total of 5 administrations.After a 7−day withdrawal, the animals were challengedwith methamphetamine(0.5 mg/kg, i.p.).The effect of methamphetamine on locomotor activity was significantly potentiated by repeated adenosine pre−treatment.Moreover, methamphetamine−induced dopamine release was also increased in the striatum.Methamphetamine−induced hyperactivity and dopamine release were significantly potentiated by repeated pre−treatment of an adenosine A₁ agonist, N⁶−cyclohexyladenosine(0.5 mg/kg, i.p.).These results suggest that the acute effect of methamphetamine is potentiated by repeated pre−treatment of adenosine via adenosine A₁ receptors.

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In a radioligand binding study using bovine coronary artery endothelial cell membranes, captopril changed a single bradykinin(BK)binding site(K_d=1.77 nM, B_max=60.2 fmol/mg protein)to high−(K_d=0.68 pM, B_max=17.7 fmol/mg protein)and low−(K_d1.00 nM, B_max=72.5 fmol/mg protein)affinity binding sites.This effect was reversed by GppNHp.Captopril also enhanced BK−induced endothelium−dependent relaxation in saponin−treated coronary rings, and GppNHp partially suppressed this enhancement.These results suggest that captopril may affect BK receptors that couple to G−proteins.

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The effect of intracerebroventricularly administered soybean lecithin transphosphatidylated phosphatidylserine(SB−tPS)on memory impairment was evaluated by a passive avoidance task.SB−tPS significantly prolonged the step−through latency induced by scopolamine treatment as in our previous report where SB−tPS was orally administered.The same doses of soybean phosphatidylcholine were ineffective.This result indicates that SB−tPS can act on the brain without any peripheral modification.

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The antinociceptive effect of mexiletine in diabetic mice was examined.Tail−flick latencies at heat intensity of 35 and 50 V in diabetic mice were shorter than those in non−diabetic mice.In diabetic mice, mexiletine increased the tail−flick latency at 35 V to the level observed in non−diabetic mice.The tail−flick latency at 50 V in diabetic mice, but not in non−diabetic mice, was increased by pretreatment with capsaicin(0.56 nmol, i.t., 24 h).The antinociceptive effect of mexiletine in diabetic mice was reduced by capsaicin.These results suggest that the mexiletine−induced antinociception in diabetic mice involves the inhibition of the nociceptive transmission of capsaicin−sensitive primary afferent fibers.

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The effects of soybean transphosphatidylated phosphatidylserine(SB−tPS)on cholinergic synaptic functions were investigated using cerebral cortical synaptosomes from mice.Treatment of the synaptosomes with SB−tPS increased high K⁺−induced acetylcholine(ACh)release in a bell−shaped, dose−dependent manner without affecting ACh synthesis.SB−tPS(10 and 50μM)also enhanced synaptosomal synthesis of sn−glycero−3−phosphocholine, but did not affect phosphorylcholine synthesis.In contrast, the choline synthesis was significantly reduced as SB−tPS concentration increased.The present result that SB−tPS modified the cholinergic pathway can partly explain its nootropic functions.

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The single channel current of intermediate conductance Ca²⁺−activated K⁺ channel(IK channel)was measured in mouse urinary bladder myocytes(MBM), and the molecular basis of the channel was suggested to be the SK4 subtype by RT−PCR.Among Ca²⁺−activated K⁺ channel subtypes(SK2, SK3, SK4 and BK), the mRNAs of SK4 and BK were predominantly expressed in MBM.IK channel currents recorded from MBM showed:38.7 pS slope conductance under symmetrical 140 mM K⁺ conditions, Ca²⁺−dependent activation, and blockade by charybdotoxin and econazole.These results strongly suggest that SK4 encodes IK channels in MBM.

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