The present study was undertaken to examine whether the natriuretic effect of spironolactone, a competitive antagonist of mineralocorticoid, varies with its time of administration. Wistar rat maintained under the condition of light from 7 hr to 19 hr were divided into two groups. The first group had a bilateral adrenalectomy and received a 50-mg deoxycorticosterone acetate (DOCA) tablet intraperitoneally (DOCA group). The second group had a sham operation (control group). Spironolactone (50 mg/kg) was given orally at 12 hr or 24 hr, and the 8-hr urine was collected. At the end of the experiment, the blood sample for measurement of aldosterone was obtained at 12 hr and 24 hr in the control group. The natriuretic effect of spironolactone in the 24 hr-trial was significantly greater than that of the 12 hr-trial in the control group. However, such a time-dependent difference was diminished and did not reach statistical significance in the DOCA group. The plasma aldosterone concentration at 24 hr was significantly higher than that at 12 hr in the control group. These results suggest that the natriuretic effect of spironolactone varies with its time of administration. Daily variation in mineralocorticoid activity might be involved in this chronopharmacological phenomenon of spironolactone.
The tert-butyl hydroperoxide (BHP)-induced release of oxidized glutathione (GSSG) and K+ was studied in relation to lipid peroxidation and cell death using isolated perfused rat livers. Infusion of BHP into the perfused liver resulted in an early and simultaneous release of GSSG and K+ and a sustained release of thiobarbituric-acid-reactive substances (TBARS) into the effluent perfusate, which was followed by further prenecrotic leakage of K+ followed by lactic dehydrogenase (LDH). These actions of BHP were not significantly affected by cutting or ligating the bile duct, and they were potentiated by omitting Ca2+ from the perfusion medium. Co-infusion of desferrioxamine, propyl gallate and diethyldithiocarbamate suppressed TBARS release as well as the later leakage of K+ and LDH. Desferrioxamine was also effective under Ca2+-free conditions. N, N''-diphenyl-p-phenylenediamine inhibited TBARS release, but it was not protective against cell death, although there was some delay. The action of dithiothreitol was only moderate. On the other hand, leakage of TBARS, K+ (prenecrotic) and LDH was enhanced by cysteamine and β-mercaptoethanol and most markedly enhanced by ferrous iron. However, none of these agents markedly affected the early release of GSSG and K+. These observations, which support our previous findings, suggest that the early and coupled sinusoidal efflux of GSSG and K+ caused by BHP is independent of lipid peroxidation and cell death and that they represent a physiological mechanism of GSSG release. The results also suggest that lipid peroxidation is not the sole cause of BHP-induced cell death.
We studied the antiarrhythmic effects of NS-2 (4-diisobutylamino-1, 1-diphenyl-l-butanol maleate) and AFD-19 (active metabolite of NS-2) on early stage ventricular arrhythmias induced by coronary artery occlusion and reperfusion in anesthetized male rats. These effects were compared with those of disopyramide and mexiletine. Drugs were intravenously administered either before or after coronary occlusion. When administered 5 min before occlusion, 3 mg/kg of NS-2 and AFD-19 exhibited equivalent anti-arrhythmic activity to that of 5 mg/kg of disopyramide and mexiletine, as assessed by reductions in the number of premature ventricular complexes and in the incidences of ventricular tachycardia and ventricular fibrillation. In a dose of 5 mg/kg, the antiarrhythmic effects of NS-2 and AFD-19 were more pronounced. When administered 5 min after coronary artery occlusion, only NS-2 and AFD-19 (in doses of 5 mg/kg) had significant antiarrhythmic effects. None of the drugs influenced the severe ventricular arrhythmias induced by reperfusion when administered 1 min before reperfusion. In conclusion, NS-2 might be effective in reducing the severity of the life-threatening ventricular arrhythmias that occur during acute myocardial infarction.
The stimulatory actions of veratridine (VTD) on catecholamine secretion and Na+ influx in cultured bovine adrenal chromaffin cells were studied in the presence of high concentrations of Ca2+ in the incubation mixture. Catecholamine secretion evoked by VTD was reduced by elevating the external Ca2+ concentration to higher than 2 mM. Under the same conditions, VTD-stimulated 22Na+ uptake into the cells was also reduced by elevating the extracellular Ca2+ concentration. In contrast, the secretory action of VTD was not significantly suppressed by preloading Ca2+ to the cells. Furthermore, the effects of extracellular Ca2+ on the sensitivities of the cells to VTD and tetrodotoxin (TTX) were examined, and neither their sensitivities to VTD nor those to TTX were shown to be significantly altered by elevating the Ca2+ concentration in the incubation mixture. These results seem to indicate that the elevation of extracellular Ca2+ concentration may cause the inhibition of VTD-induced catecholamine secretion as a consequence of the inactivation of voltage-dependent Na+ channels, and suggest that Ca2+ may directly act on the cell surface, and the site of Ca2+ action is presumably distinct from the sites of both VTD and TTX actions in the plasma membranes of adrenal chromaffin cells.
Antioxidative effects of the nitrovasodilator nicorandil (SG-75) and denitrated SG-75 (SG-86) were examined in vivo and in vitro. When the isolated rat liver was reperfused with Krebs-Henseleit solution after a 90-min ischemia, microsomal GSH S-transferase activity was increased significantly by oxidative modification of the sulfhydryl group of the enzyme. The increase in the transferase activity after ischemia/reperfusion was depressed by SG-75 but not by SG-86. Furthermore, only SG-75 significantly inhibited lipid peroxidation and the activation of microsomal GSH S-transferase induced by hydrogen peroxide treatment of liver microsomes. These data indicate that SG-75 has an antioxidative action and the nitro group of SG-75 may play a critical role for this action.
We investigated the effects of an ATP-sensitive K+ channel blocker, glibenclamide, on the negative chronotropic and inotropic responses to intracardiac parasympathetic nerve stimulation, acetylcholine (ACh, a muscarinic receptor agonist), ATP (a P2-purinergic receptor agonist), adenosine (a P1-purinergic receptor agonist) and cromakalim (a potassium channel opener) in the isolated, blood-perfused canine right atrium or left ventricle. A high dose of glibenclamide (3 μmol) did not affect the negative chronotropic and inotropic responses to parasympathetic stimulation (frequencies of 1-30 Hz), although it slightly but significantly attenuated the negative cardiac responses to exogenous ACh (0.3-10 nmol). Furthermore, adenosine (0.03-0.3 μmol)-induced negative chronotropic and inotropic responses were significantly inhibited by glibenclamide (3 μmol), but ATP (0.01-1 μmol)-induced negative cardiac responses were not affected. A cumulative administration of cromakalim (0.01-1 μmol) dose-dependently caused much greater decreases in the contractile force of atrial and ventricular muscles than in sinus rate. Glibenclamide (0.3-3 μmol) similarly blocked the negative chronotropic and inotropic responses to cromakalim in a dose-depend ent manner. These results suggest that glibenclamide modifies the negative cardiac responses to parasympathetic activation both in pre and postjunctional sites and the responses to adenosine but not to ATP at K+ channels in the dog heart, although the modifications are minor under physiological conditions.
A new experimental model of acute congestive heart failure was established in open-chest dogs, and it was employed to examine the effects of dobutamine, propranolol and nitroglycerin. The model was induced by intracoronary administration of saponin, volume loading and intravenous infusion of methoxamine. Left ventricular end-diastolic pressure (LVEDP) increased from 7.9±0.6 to 24.2±:1.4 mmHg, and aortic blood flow (AoF) decreased from 0.89±0.06 to 0.53±0.041/min. Systemic vascular resistance (SVR) increased from 9618±585 to 16492±1213 dynes·sec/cm5 and right atrial pressure (RAP) increased from 2.5±0.2 to 4.2±0.4 mmHg. Furthermore, Vmax decreased from 71.6±5.1 to 45.8±2.9 1/sec, and the time constant of left ventricular pressure decay (T) increased from 40.0±2.6 to 90.2±7.9 msec. These hemodynamic changes were stable for up to 80 min. Dobutamine improved cardiac function by increasing Vmax and by decreasing T. Consequently, dobutamine increased AoF and decreased LVEDP, while there was no change in SVR. Nitroglycerin reduced LVEDP, SVR and T; increased AoF; and did not change Vmax. Propranolol produced no improvement in the hemodynamics or cardiac function. These results indicate that the present congestive heart failure model is characterized by global left ventricular dysfunction with lowered cardiac output and increased peripheral vascular tone, and it is beneficial for evaluating the pharmacological properties of drugs for acute congestive heart failure.
Actions of substance P (SP, 10-9 to 10-6 M), neurokinin A (NKA, 10-9 to 10-6 M) and neurokinin B (NKB, 10-10 to 10-6 M) in the circular muscle of guinea pig ileum were investigated in segment and strip preparations, in which methacholine produced similar contractions. In the segment preparations, three tachykinins produced repeatedly occurring twitch-like contractions. Their efficacies were similar with the same maximal contractions, but their potencies were different (NKB > NKA = SP). Latency (38 sec) was observed before the initiation of contractions in response to NKA, but not to SP or NKB. Atropine (10-6 M) and tetrodotoxin (3×10-7M) did not affect NKA-induced contractions, but inhibited SP and NKB-induced contractions; the dose-response curves for SP and NKB were rightwardly shifted by atropine. The treatment with atropine brought out latency in the responses for NKB. In the strip preparations, SP did not substantially induce contractions, but NKA and NKB produced twitch-like contractions after latent periods of 28 and 36 sec, respectively. The efficacy of NKA was similar to that in segment preparations, while that of NKB was much lower in strip preparations. Unlike in segment preparations, atropine did not inhibit contractions induced by the two tachykinins in strip preparations. These results suggest that tachykinins induce contractions through myogenic and neurogenic mechanisms, the latter of which may be inoperative in strip preparations.
We investigated the mode of the 5-HT3-receptor antagonism of 4, 5, 6, 7-tetrahydrobenz-imidazole derivatives, YM060, YM114 (KAE-393), YM-26103-2 and YM-26308-2, against 5-HT-induced transient bradycardia in anesthetized rats. Results were compared with those of ondansetron and granisetron. YM060 (0.03-0.1 μg/kg, i.v.), YM114 (0.03-0.3 μg/kg, i.v.), YM-26103-2 (0.01-0.03 μg/kg, i.v.), YM-26308-2(0.01-0.03 μg/kg, i.v.) and granisetron (0.3-3 μg/kg, i.v.) displaced the 5-HT dose-response curve to the right, with apparent DR2 values of 0.068, 0.068, 0.019, 0.011 and 0.69 μg/kg, i.v., respectively. Higher doses of these compounds inhibited 5-HT-induced bradycardia with a reduced maximal response. In contrast, ondansetron displaced the 5-HT dose-response curve to the right without affecting the maximal response. Judged by the apparent DR2 values, YM060, YM114, YM-26103-2 and YM-26308-2 were approximately 13, 13, 50 and 79 times more potent than ondansetron, respectively, whereas granisetron was equipotent to ondansetron. Single i.v. doses of YM060 and granisetron inhibited 5-HT-induced bradycardia significantly longer than ondansetron. Moreover, inhibitory effects of p.o. doses of YM060(3 μg/kg), YM 114(80 μg/kg), YM-26103-2(12 μg/kg), YM-26308-2(5 μg/kg) and granisetron (250 μg/kg) on the von Bezold-Jarisch reflex lasted for 3-6 hr, whereas ondansetron (700 μg/kg, p.o.) antagonized 5-HT3 receptors for only 1 hr. In isolated guinea pig colon, the inhibitory effect of YM-compounds on 5-HT-induced contraction persisted significantly longer than those of ondansetron and granisetron after washout of the bath containing compounds. These results suggest that YM-compounds are highly potent 5-HT3-receptor antagonists. Furthermore, non-competitive 5-HT3-receptor antagonism of YM-compounds against the von Bezold-Jarisch reflex at higher doses may be reflected in their slow dissociation from the 5-HT3 receptor, and that of granisetron may be reflected in its slow metabolism in anesthetized rats.
The effects of trimebutine on Ca2+ release and modulation of Ca2+ sensitivity of contractile elements induced by carbachol (CCh) were investigated using a tension measuring method in β-escin-treated skinned smooth muscle of the longitudinal muscle layer of guinea pig ileum. Trimebutine (10-100 μM) concentration-dependently inhibited tension development brought about by Ca2+ release from intracellular stores induced by CCh (10 μM), but did not affect those induced by inositol 1, 4, 5-trisphosphate (IP3, 25 μM) or caffeine (5 mM). The inhibitory effect was reversible. Trimebutine (100 μM) neither altered the Ca2+ sensitivity of the contractile elements nor affected the effects of GTPγS (50 2+M) and CCh (100 2+M) in potentiating Ca2+ sensitivity of the contractile elements after the Ca2+ storage function had been eliminated by A23187. These results suggest that trimebutine inhibits CCh-induced Ca2+ release by acting at some point during the coupling of muscarinic receptors through a G-protein to phospholipase C and thus reducing the accumulation of IP3.
The thrombolytic activity of a novel modified tissue-type plasminogen activator (t-PA) (E6010) was examined in a canine model with copper coil-induced femoral artery thrombus. This model, in which thrombolytic activity can be easily and directly quantified by determining changes in thrombus weight, should be useful for comparing the activities of various thrombolytic agents. Using this model, the present study showed that the thrombolytic activity of bolus intravenous injection of E6010 was identical to that of continuous intravenous infusion of recombinant t-PA at the same dose. This thrombolytic activity can be explained by changes in blood concentrations of the administered thrombolytic agents. On the other hand, administration of the thrombolytic agents dose-dependently caused significant changes in the levels of hemostatic and fibrinolytic factors. These changes were not so marked with administration of E6010, and therefore we concluded that E6010 is unlikely to cause bleeding complications after administration
5-Hydroxytryptamine (5-HT)-receptor subtypes on pig basilar arteries were investigated by measuring the contractile responses to 5-HT agonists, the effects of antagonists on the responses and by carrying out a radioligand binding assay with [3H]5-HT. The rank order of contractile agonist potency (according to the pEC50 values) was 5-carboxamidotryptamine ≤ 5-HT > α-methyl-5-HT > (±)-8-hydroxy-dipropylaminotetralin. The contractile responses were not affected by endothelial denudation, and the 5-HT-induced contractions were antagonized competitively by ketanserin. Methiothepin shifted the 5-HT concentration-response curves to the right and downwards in a concentration-dependent manner. In the presence of ketanserin (10-6 M), however, methiothepin antagonized the 5-HT-induced contractions competitively. Specific [3H] 5-HT binding to 5-HT receptors was saturable, reversible and showed high (Kd, 2.5 nM) and low (Kd, 710 nM) affinities, with respective Bmax values of 29.5 and 1950 fmol/mg protein. These results indicate that both 5-HT1 and 5-HT2 receptors are present on pig basilar arterial smooth muscle cells, and their stimulation results in contraction.
Muscarinic agonists and guanylyl-5''-imidodiphosphate (Gpp(NH)p) stimulated formation of inositol phosphates in permeabilized longitudinal smooth muscle of guinea pig ileum. Gpp(NH)p markedly potentiated the formation of inositol bisphosphate (IP2) and inositol trisphosphate (IP3) stimulated by carbachol, but increased inositol monophosphate formation (IP1) only slightly. Gpp(NH)p enhanced the formation of IP2 + IP3 induced by either acetylcholine or carbachol about fourfold in a synergistic manner, but enhanced the effects of oxotremorine and pilocarpine less than twofold in an additive manner. Elevation of Ca2+ concentration resulted in increases of the inositol phosphate levels stimulated by both carbachol and Gpp(NH)p. The optimal concentration of Ca2+ for carbachol-stimulated formations of IP2 + IP3 was shifted to a lower Ca2+ concentration in the presence of Gpp(NH)p. These findings suggest that muscarinic receptor-stimulated polyphosphoinositide hydrolysis in ileal smooth muscle results in inositol polyphosphate formation via GTP binding protein (G-protein). The muscarinic receptor-activated G-protein decreases the Ca2+ requirement of polyphosphoinositide hydrolysis. Muscarinic agonists stimulate inositol polyphosphate formation by interaction of the G-protein activation of a phosphoinositide specific phospholipase C with Ca2+ influx.
The effect of activation of tachykinin NK2-receptors on gastrointestinal propulsion was studied in vivo in conscious rats. The selective NK2-receptor agonist [βAla8]NKA-(4-10) produced an atropine-resistant specific increase of the small intestinal transit measured by the charcoal method. This effect was restricted to the small intestine since gastric emptying was not affected by [βAla8]NKA-(4-10). The newly developed polycyclic peptide NK2-receptor antagonist MEN 10, 627 produced a dose-dependent inhibition of this stimulated transit. The spasmolytic effect of MEN 10, 627 was highly selective because it did not affect stimulated intestinal transit induced by equieffective doses of carbachol and reserpine. These findings indicate that MEN 10, 627 is a valuable tool for assessing the role of NK2-receptors in intestinal propulsive activity.
The effects of cerebral metabolism-improving drugs on NADPH diaphorase activity in the mouse brain were studied, and we found that diaphorase activity in the post-mitochondrial fraction of brain homogenate was enhanced by idebenone in a concentration-dependent manner. Histochemical studies also indicated that diaphorase staining was intensified by idebenone at the same concentration. These results suggest that idebenone may stimulate the production of nitric oxide, probably through its direct action on nitric oxide synthase, thus producing its protective action on neurological disorders due to cerebral hypoxia or ischemia as a consequence of dilating the cerebral blood vessels.
We investigated the antitumor effects of adriamycin (ADR)-loaded hydroxyapatite (HAP) beads in a Swarm rat chondrosarcoma model. When one ADR-loaded HAP bead (ADR: 0.8 mg/bead) was implanted into the central portion of a rat bearing tumor, the ADR-loaded HAP beads showed good therapeutic effects, increasing the life span (ILS) by 90%. Significantly, leukopenia and diarrhea were not observed. These results suggest that HAP delivery offers an interesting and a potentially effective method.
Effects of 7-hydroxy-2-(N, N-di-n-propylamino)tetralin (7-OH-DPAT), a dopamine D3-receptor agonist, on neuronal activities of the nucleus accumbens (Acc) were examined in chloral hydrate-anesthetized rats. Spikes elicited by stimulation of the parafascicular nucleus were dose-dependently inhibited by microiontophoretic application of dopamine and 7-OH-DPAT. Glutamate-induced firing was also inhibited by these drugs. These findings suggest that D3 receptors are also involved in the postsynaptic inhibition of Acc neuron by dopamine.