The effects of inositol 1, 4, 5-trisphosphate (InsP₃) released from caged InsP₃ by flash photolysis on free intracellular Ca²⁺ concentration, [Ca²⁺]_i, and outward K⁺ current were simultaneously examined in a single smooth muscle cell of guinea pig small intestine using a patch pipette solution containing Indo-1 (0.1 mM), caged InsP₃ (50 μM) and KCl (130 mM). At a holding potential of −50 mV, a depolarizing pulse to + 10 mV for 200 msec caused a transient Ca²⁺ current and an increase in [Ca²⁺]_i. The amplitude of the Ca²⁺-transient was positively correlated with the peak Ca²⁺ current and negatively correlated with resting [Ca²⁺]_i. InsP₃ produced increases in [Ca²⁺]_i and outward K⁺ current in most of the cells at −50 and −30 mV. The outward K⁺ current response reached a peak sooner and decayed more quickly than the Indo-1 signal. Both responses to InsP₃ were resistant to the removal of extracellular Ca²⁺. The Ca²⁺-transient and outward K⁺ current responses to InsP₃ at −30 mV were larger than those at −50 mV. The InsP₃-induced Ca²⁺-transient was increased by increasing resting [Ca²⁺]_i at −30 mV but not at −50 mV. These results suggest that InsP₃-induced Ca²⁺ release from stores is potentiated by slight increases in [Ca²⁺]_i via membrane depolarization.

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Urinary kallikrein excretion during oxytocin (OT) infusion were studied in anesthetized (sodium pentobarbital, 50 mg/kg, i.p.) young (4-weeks-old) spontaneously hypertensive rats (SHR) and Wistar Kyoto rats (WKY). OT-infusion (30 nmol/kg/30 min) to WKY significantly increased urinary excretion of the active kallikrein from the basal levels (25.4±5.6 10^-2 × AU/15 min, n=5) to 37.3±5.0 10^-2 × AU/ 15 min (P<0.05, n=5) and 50.7±17.1 10^-2× AU/15 min (P<0.05, n=5) 15 and 30 min after the start of OT-infusion, respectively. In SHR, OT-infusion did not increase the urinary excretion of active kallikrein, but did decrease the urine volume and sodium excretion. The concentration of the active kallikrein in the kidney of WKY was not changed by OT-infusion, but that of SHR was slightly increased. The OTinfusion resulted in significantly higher concentrations of the active kallikrein in SHR kidney than in WKY kidney. These results suggest that less excretion of urinary kallikrein in SHR during OT-infusion may be attributable to a lower response in the secretion of kallikrein from the kidney.

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With flow cytometry, we investigated the effect of basic fibroblast growth factor (bFGF)-induced competence in subcultured endothelial cells (EC) (4—9-passage) of rat thoracic aorta. The cell population in each phase of the cell cycle was determined by a double staining technique with fluorescein isothiocyanate-conjugated mouse monoclonal antibody against the proliferation-associated nucleus antigen Ki-67 and propidium iodide for total DNA content. EC were cultured in medium containing 5% fetal bovine serum (FBS) for 6 days. After serum-starvation for 2 days, the treatment with bFGF (3—10 ng/ml) for 12 hr promoted the entry of cells into the G₁ phase from the G₀ phase concentration-dependently. bFGF (10 ng/ml) increased the cell population in the G₁ phase by 50% of the total EC, compared with the control culture without bFGF. A further 12—15-hr culture with 1% FBS after bFGF treatment promoted the entry of the cell into the S phase. Thus flow cytometric analysis demonstrates that bFGF stimulates the entry of EC into the G₁ phase from the G₀ phase.

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The motor-stimulating action of de(N-methyl)-N-isopropyl-8, 9-anhydroerythromycin A 6, 9-hemiacetal (EM574) on the upper gastrointestinal tract was studied in fasted conscious dogs using chronically implanted force transducers and compared with those of porcine motilin and cisapride. EM574 induced gastric phase III-like migrating contractions and increased the plasma motilin levels slightly. The gastric motility induced by low doses of EM574 and motilin was abolished by a 5HT₃-receptor antagonist ondansetron and acute vagal blockade, whereas under these conditions, high doses of both agents induced contractions, which were abolished by atropine. Cisapride-induced gastric motility was inhibited by atropine and acute vagal blockade, but not by ondansetron. EM574 did not stimulate gastric secretion in the basal state. These results indicate that EM574 and motilin-induced gastrointestinal motility is attributable mainly to motor-stimulating vagal cholinergic neurons, and 5HT₃-receptors are probably involved in the process. At high doses, EM574 and motilin also appear to stimulate cholinergic neurons in a non-vagal pathway, probably the enteric nervous system.

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The protective effects of ME3221, 3-met hoxy-2, 6-dimethyl-4-[[2''-(1H-tetrazol-5-yl)-1, 1''-biphenyl-4-yl]methoxy] pyridine, on aged (32-week-old) stroke-prone spontaneously hypertensive rats (SHRSP) were studied following long-term (for 8 months) oral administration. At a dose of 10 mg/kg/day, ME3221 suppressed the mortality and the hypertensive complications observed in control SHRSP: cerebral apoplexy (hemorrhage, and spongeform and malacia in the cerebral cortex), increased proteinuria, and total N-acetyl-β-_D-glucosaminidase activity, and cardiac hypertrophy and pleural effusion. The protective activity of ME3221, a surmountable angiotensin AT₁-receptor antagonist, was comparable to losartan, an insurmountable AT₁-antagonist, and also to enalapril, an angiotensin-converting enzyme inhibitor. In addition, ME3221 reduced the systolic blood pressure more effectively than the two reference drugs.

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Effects of quinacrine on voltage-dependent Ca²⁺ channel current (I_Ca) were examined using whole cell voltage clamp in single smooth muscle cells isolated from vas deferens and urinary bladder and single cardiac myocytes from ventricle of the guinea pig. When I_Ca was elicited by depolarization from a holding potential of −60 to 0 mV for 150 msec every 15 sec in vas deferens myocytes, external application of quinacrine reduced the amplitude of I_Ca in a concentration-dependent manner in a range of 0.1 ?? 30 μM, and the IC₅₀ of quinacrine was 1.3 μM. The block was at least partly removed by washout. The block of I_Ca by 1 μM quinacrine in vas deferens myocytes greatly depended upon the activation potentials but only slightly on the holding potentials. Use-dependent development of the block was also observed. Addition of 300 μM quinacrine to the pipette-filling solution did not significantly affect I_Ca. The IC₅₀ of quinacrine for I_Ca block in urinary bladder myocytes was 1.1 μM and comparable to that in vas deferens. On the other hand, IC₅₀ for the block of I_Ca elicited by depolarization from −45 to 0 mV in cardiac ventricular myocytes was 5.6 μM. It is concluded that quinacrine is a potent blocker of L-type Ca²⁺ channels in two types of smooth muscle myocytes and that the potency appeared to be approximately five times higher than that in cardiac myocytes. The action of quinacrine may be due to the direct block of Ca²⁺ channels from outside of the cell membrane.

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We examined the effects on urine outflow rate after microinjections of thiorphan (a carboxypeptidase inhibitor) and bestatin (an aminopeptidase inhibitor) into the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei of anesthetized hydrated rats to determine the possible role of neuropeptides in the regulation of urine production. After individual microinjection of the peptidase inhibitors into the nuclei, only thiorphan at 100 nmol administered into the PVN significantly decreased the urine outflow rate. Two consecutive microinjections of the peptidase inhibitors at 100 nmol each into the nuclei induced potent antidiuresis. These effects after microinjections of the peptidase inhibitors into the PVN and SON were diminished by pretreatment with [Sar¹, Ile⁸]angiotensin (ANG) II (an ANG II receptor antagonist) and naloxone (an opioid receptor antagonist) in the PVN and with [Sar¹, Ile⁸]ANG II in the SON, respectively. A vasopressin (AVP) receptor antagonist, d(CH₂)₅-_D-Tyr(Et)VAVP (i.v.), completely blocked the antidiuresis by microinjections of the peptidase inhibitors into both the nuclei. Urinary osmotic pressure was significantly increased by consecutive microinjections of the peptidase inhibitors into the PVN and SON. These results suggest that endogenously-released ANG II and opioid peptides in the PVN and ANG II in the SON regulate urine production mediated through increased AVP release.

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Our previous study has demonstrated that unilateral microinjection of norepinephrine (NE) into the right supraoptic nucleus (SON) of anesthetized hydrated rats elicited dose-dependent decreases in the urine outflow rate. This was antagonized by pretreatment with phenoxybenzamine (an α-antagonist) and timolol (a β-antagonist) in the same SON. In the present study, we examined the effects of NE, microinjected into the right, left and bilateral SON, on the urine outflow rate in order to investigate neural connections between the bilateral SON. NE administered by those three routes dose-dependently decreased the urine outflow rate. The order for the antidiuretic potency was as follows: the effect elicited by the intrabilateral-SON microinjection > the intra-left-SON microinjection = the intra-right-SON microinjection. The antidiuresis of NE microinjected into the right SON was inhibited by an electrolytic left-SON lesion and by pretreatment with phenoxybenzamine (20 nmol) and timolol (100 nmol), but not by atropine (300 nmol) in the left SON. These findings suggest adrenergic neural connections from the right to left SON, contributing to the regulation of urine production. Furthermore, there is a possibility that stimulation of endogenously-released NE in the bilateral SON is amplified through these neurons and elicits more potent effects than those produced in either the right or left nucleus.

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Effects of histamine H₁-receptor antagonists on L-dopa-induced behavioral excitement were examined in mice to confirm behaviorally the inhibition of dopamine uptake by these compounds. L-Dopa (100-300 mg/kg, s.c.) combined with pargyline hydrochloride (80 mg/kg, i.p.) caused a dose-dependent behavioral excitement. The marked excitement induced by L-dopa (300 mg/kg) plus pargyline was significantly inhibited by pimozide (0.1-1 mg/kg, s.c.), a selective dopamine antagonist. Tripelennamine (10 mg/kg, s.c.), d-chlorpheniramine (1 and 2 mg/kg, s.c.), homochlorcyclizine (2 and 5 mg/kg, s.c.), diphenhydramine (2 and 5 mg/kg, s.c.) and mepyramine (2 and 5 mg/kg, s.c.) each markedly enhanced the moderate behavioral excitement induced by L-dopa (150 mg/kg) plus pargyline. These findings are behavioral evidence for inhibition of dopamine uptake by H₁ antagonists, which has been suggested by neurochemical studies.

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We investigated prostanoid-induced intracellular Ca²⁺ mobilization in Ca²⁺-sensitive dye fura-2-loaded cultured astrocytes. The thromboxane (TX) A₂ analog STA₂ (9, 11-epithio-11, 12-methanoTXA₂) and/or prostaglandin (PG) F_2α (each used at 1 μM) stimulated intracellular Ca²⁺ mobilization in single cultured rat type 1 astrocytes. Three response patterns were observed: only STA₂-sensitive, only PGF_2α-sensitive, and both prostanoids-sensitive cells. The Ca²⁺ response was prostanoid-dose-dependent (0.1-1 μM) and showed a rapid spike-like Ca²⁺ rise that peaked within 30 sec after the stimulation by the ligand. These observations suggest that type 1 astrocytes are heterogeneous with respect to the expression of receptors for TXA₂ and PGF_2α, which are linked to Ca²⁺ mobilization.

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