Renal excretion of rhodamine 123, a P-glycoprotein substrate, in rats with glycerol-induced acute renal failure.

To clarify renal handling of rhodamine 123, a substrate for P-glycoprotein, in normal and diseased states, in-vivo clearance studies were performed with normal rats and rats with glycerol-induced acute renal failure. For normal rats the excretion ratio of unbound rhodamine 123-to-inulin was 3.25, indicating the presence of the renal tubular secretion of rhodamine 123. Co-administration of cyclosporin, a P-glycoprotein inhibitor, significantly reduced tubular secretion of rhodamine 123. Administration of glycerol induced both an increase in blood urea nitrogen and a reduction in the glomerular filtration rate, confirming the induction of acute renal failure. Total plasma, renal, and tubular secretory clearances of rhodamine 123 were significantly lower for rats with acute renal failure than for control rats. There was no difference between the ATP content of the renal cortex in control rats and those with acute renal failure. In addition to the decrease in renal clearance, a decrease in the biliary clearance of rhodamine 123 was also observed in rats with acute renal failure. These results imply that rhodamine 123 is secreted via P-glycoprotein in renal tubules and that the renal secretory clearance of rhodamine 123 was reduced after acute renal failure, probably because of impairment of P-glycoprotein.

Pharmacological characterization of the nonpeptide angiotensin II receptor antagonist, U-97018.

We examined the pharmacological properties of U-97018, a novel nonpeptide angiotensin II (AII) receptor antagonist, in various in vitro and in vivo studies. U-97018 selectively displaced 125I-AII specific binding in the membrane fraction derived from the rat mesenteric artery and adrenal cortex (AT1 subtype) with IC50 of 1.3 +/- 0.2 and 7.7 +/- 1.3 nM, respectively, without altering the AII binding of the rat adrenal medulla (AT2 subtype). In rat adrenal cortical cells, U-97018 inhibited 1 nM AII-induced aldosterone secretion with an IC50 of 0.48 nM; it shifted concentration-secretion response curve for AII to the right and inhibited the maximal response to AII, yielding a pKB of 9.8. Similarly, U-97018 showed insurmountable antagonism with a pKB of 10.6 against the AII-induced contraction in the isolated rabbit aorta. U-97018 had no direct effect on the activities of renin and angiotensin converting enzyme in vitro. In pithed rats, U-97018 inhibited the AII-induced pressor response with an ED50 of 0.28 mg/kg, i.v. without any partial agonistic activity. In anesthetized rats and dogs, intraduodenal administration of U-97018 at a dose of 1 mg/kg inhibited the AII-induced pressor response by about 60%. In spontaneously hypertensive rats, U-97018 at 10 mg/kg p.o. produced antihypertensive effects which lasted for 24 hr after administration. Thus, U-97018 is an orally active, insurmountable AII receptor antagonist without any agonistic activity.

Effects of U-97018 on pressor responses to intracerebroventricularly administered angiotensin II in conscious normotensive rats.

We examined the effects of U-97018, an AT1 receptor antagonist, on the pressor response to intracerebroventricularly (i.c.v.) administered angiotensin II (AII) in conscious normotensive rats in comparison to losartan, EXP 3174, EXP 655, and saralasin. In an i.c.v. study, U-97018, losartan, and EXP 3174 reduced the pressor response. EXP 655, an AT2 selective antagonist, also inhibited the pressor response to i.c.v. AII. U-97018 combined with EXP 655 did not fully eliminate the pressor response to i.c.v. AII. Moreover, saralasin, a nonselective peptide AII antagonist, also failed to abolish the pressor response to i.c.v. AII. Therefore, both AT1- and AT2-receptors probably are functional in inhibiting the pressor response to i.c.v. AII and that a part of the i.c.v. AII-induced pressor response occurs through non-AT1- and non-AT2-receptors. In an intravenous (i.v.) study, U-97018, losartan, and EXP 3174 reduced the pressor response to i.c.v. AII. At 10 mg/kg orally (p.o.), which is an antihypertensive dose in spontaneously hypertensive rats (SHR), neither U-97018 nor losartan reduced the pressor response to i.c.v. AII even at 180 min after administration. This result indicates that neither U-97018 nor losartan, at the oral antihypertensive dose, reaches the brain in sufficient amount to affect the pressor response to i.c.v. AII.

Ca(2+)- and Cl(-)-dependent, NMDA receptor-mediated neuronal death induced by depolarization in rat hippocampal organotypic cultures.

The neurotoxicity induced by depolarization with high-K+ was investigated in rat hippocampal organotypic slice cultures. The exposure of cultures to 90 mM K+ solution for 30 min caused a severe neuronal injury in CA1 region while less damage was observed in CA3 and dentate gyrus over the following day. This neurotoxicity was prevented in a concentration dependent manner by NMDA antagonist MK-801 or CPP. Non-NMDA antagonist, DNQX, had no protective effect. Omission of Ca2+ from the exposure solution prevented the neurotoxicity. Voltage-dependent Ca2+ channel blockers, nifedipine and flunarizine, failed to prevent the neurotoxicity. These results suggest that the Ca2+ influx through the NMDA receptor is predominantly involved in this neurotoxicity. Apparent tissue swelling was observed immediately after the depolarization. This swelling was completely inhibited by omission of Cl- from the exposure solution, accompanied with complete protection against neurotoxicity. This suggests that Cl(-)-dependent tissue swelling also largely contributes to the neurotoxicity. Depolarization with application of MK-801 (10 microM) or omission of Ca2+ from the solution still caused apparent swelling, despite these treatment protected neuronal death. We hypothesize that Cl(-)-dependent tissue swelling may be involved in the release of the excitatory amino acid, which activates the NMDA receptor.

U-92032, a T-type Ca2+ channel blocker and antioxidant, reduces neuronal ischemic injuries.

Several diphenylmethylpiperazine derivatives are potential therapeutic agents for prevention of ischemic injuries in the heart and brain, because of their ability to block Ca2+ currents and their antioxidant activity. In this study, the current lead compound, U-92032 ((7-((bis-4-fluorophenyl)methyl)-1-piperazinyl)-2-(2-hydroxyethylamin o)- 4-(1-methylethyl)-2,4,6-cycloheptatrien-1-one), has been compared with flunarizine and nifedipine (well-known T- and L-type Ca2+ channel antagonists, respectively) for their effects on Ca2+ channels in a mouse neuronal cell line, N1E-115 cells, and their ability to preserve the phenomenon of long-term potentiation and to improve neurological symptoms in gerbil ischemic models. U-92032, like flunarizine, blocked transient Ba2+ currents (IBa) through T-type Ca2+ channels with no effect on nifedipine-sensitive non-inactivating currents. Transient IBa was reduced by U-92032 at a constant rate, the magnitude of which depended on the drug concentration, probably because of a time-dependent accumulation of the lipophilic drug in the membrane phase. For instance, the drug at 6 microM reduced IBa by 21% per min and abolished it in less than 5 min, about 3 times faster than flunarizine at the same concentration. Otherwise, U-92032 behaved like flunarizine, showing a use-dependent block without noticeable effects on the current-voltage relationship for transient IBa. Oral administration of U-92032 (1 and 25 mg/kg) or flunarizine (25 mg/kg), but not nifedipine (50 mg/kg), to gerbils 1 h prior to bilateral carotid artery occlusion, preserved long-term potentiation in hippocampal CA1 neurons, which were largely abolished by ischemia without the drug treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of spiradoline mesylate, a selective kappa-opioid-receptor agonist, on the central dopamine system with relation to mouse locomotor activity and analgesia.

Neurochemical and behavioral investigations were made to assess the role of central dopaminergic systems in mouse locomotor activity and analgesia by spiradoline mesylate. Analgesic activities of the kappa-opioid-receptor agonists spiradoline and U-50488H were not altered by haloperidol or L-dopa, whereas morphine analgesia was enhanced by haloperidol but attenuated by L-dopa. Spiradoline decreased spontaneous locomotor activity in mice and inhibited methamphetamine- or morphine-induced locomotor activity. In contrast, morphine given alone increased locomotor activity and enhanced methamphetamine-induced locomotor activity. In a neurochemical study, spiradoline decreased the amounts of dopamine metabolites in the striatum, but did not alter them in the brainstem and cerebral cortex. Morphine increased the dopamine metabolite contents in all three brain regions tested. These results suggest that inhibition of the dopaminergic pathway in the brain by spiradoline may be involved in its suppression of locomotor activity, but not in its analgesia; whereas, stimulation of the dopaminergic pathway by morphine seems to function in both behaviors: enhancement of locomotor activity and inhibition of analgesia.

Central monoaminergic mechanisms in mice and analgesic activity of spiradoline mesylate, a selective kappa-opioid receptor agonist.

We assessed the roles of brain monoaminergic systems in the analgesic action of spiradoline, a novel kappa-opioid agonist, behaviorally and biochemically by using noradrenaline (NE) and serotonin (5-HT) uptake inhibitors. Analgesic activity was evaluated by measuring latency time in the mouse tail-pinch test. Spiradoline at intramuscular doses of 0.3 mg/kg or more elicited a significant analgesic effect and the metabolism of both NE and 5-HT was significantly increased in brainstem and cortex. Pretreatment of the mice with imipramine, desipramine or clomipramine caused marked potentiation of spiradoline analgesia, whereas reserpine and phenoxybenzamine inhibited it. Morphine analgesia was enhanced by clomipramine but not by imipramine, desipramine or phenoxybenzamine. These results suggest that excitation of noradrenergic and serotonergic pathways in the brain appears to be involved in spiradoline analgesia, and that, as regards tail-pinch nociception, the kappa-opioid agonist acts on the noradrenergic pathway more potently than morphine.

Analgesic activity of spiradoline mesylate (U-62,066E), a kappa opioid agonist in mice.

The present study was undertaken to evaluate the analgesic potency of spiradoline mesylate, a k(kappa) opioid agonist, in comparison with that of morphine, by hot plate, tail-pinch and acetic acid-induced writhing assay. The ED50 values of spiradoline in hot plate, tail-pinch and acetic acid-induced writhing assay were 0.46, 0.26 and 0.20 mg/kg, respectively. The analgesic potency of spiradoline was 1.5-7.0 times higher than that of morphine. Repeated treatment with spiradoline as well as morphine developed tolerance to the analgesic effect in hot plate assay. In mice developed tolerance to one analgesic, response to the other analgesic did not alter compared to saline-treated mice. Single administration of spiradoline (1.5 and 3 mg/kg, s.c.) did not inhibit morphine-induced analgesia. These results suggest that spiradoline has more potent analgesic activity than morphine, presumably mediated through stimulation of receptors different from morphine.

Enhancement of ambulation-increasing effect produced by repeated administration of methamphetamine in rats and neurochemical changes in catecholaminergic neurons.

Male rats of the Wistar strain were repeatedly given methamphetamine (MAP) at 0.5 mg/kg, s.c., 10 times at a fixed interval of 4 days in different environmental situations. The effects of MAP administration on ambulatory activity and neurochemical parameters in the brain were investigated. MAP (0.5 mg/kg) markedly increased ambulatory activity. The ambulation-increasing effect of MAP was progressively enhanced without accompanying stereotyped behaviors when the drug was repeatedly given in the activity cage. The once enhanced effect was considered to be irreversible. In addition, the pretreatment with MAP in the activity cage produced a decrease in the maximum density of 3H-spiperone binding sites in the striatum and that of 3H-WB4101 binding sites in the cortex and hippocampus, with decreases in catecholamine and increases in its metabolites. However, the enhancement of the ambulation-increasing effect of MAP and changes in the binding sites or catecholamine metabolism were scarcely observed when the drug was repeatedly given to the rats in the narrow cage where horizontal ambulation of the animals was strongly impaired. These results suggest that the enhancing effect of MAP is closely related to the environmental situations to which the animals have been exposed to under the drug effect. Moreover, it is indicated that there is a correlation between the enhancement of the ambulation-increasing effect of MAP and changes in the neurochemical parameters. The enhancing effect of MAP is supposed to have been partially caused by changes in cerebral catecholaminergic systems, in particular an increase in catecholamine turnover.

Behavioral and neurochemical changes produced by postnatal pretreatments with methamphetamine in rats.

Male neonates of Wistar strain rats were given s.c. 1-4 mg/kg/day of methamphetamine (MAP) for 7 successive days from days 6 to 12 after birth. The acquisition processes of the discriminative lever-press avoidance response were investigated from day 60 after birth. Effects of the postnatal pretreatments with MAP on saturation constants for specific bindings of 3H-spiperone (SPP) and 3H-WB4101, respectively, in 8 brain regions were also investigated at 100-120 days after birth. In addition, dopamine, noradrenaline and the levels of their metabolites were measured in the brain. No significant difference was detected in body weight, gross behaviors and avoidance learning between saline- and MAP-pretreated groups. However, effects of MAP and apomorphine on the locomotor activity significantly increased in the MAP-pretreated group. Significant decreases in Bmax and Kd values of 3H-SPP binding sites in the striatum were detected in the MAP-pretreated group, while significant decreases in Bmax values of 3H-WB4101 binding sites in the cortex and hippocampus as well as those in Kd values in the hippocampus were found in the treated group. Dopamine and noradrenaline levels significantly decreased in the MAP-pretreated group, but on the contrary, their metabolites levels significantly increased. These results suggest that postnatal pretreatments with MAP produce long-lasting behavioral changes associated with an impaired development of catecholaminergic neurons in the rat brain after maturity.

Changes in 3H-spiperone, 3H-WB 4101 and 3H-dihydroalprenolol bindings to brain membranes produced by postnatal pretreatment with chlorpromazine in adult rats.

In order to elucidate possible mechanisms of the learning deficit produced by postnatal pretreatment with chlorpromazine (CPZ), changes in catecholamine receptors in the rat brain were investigated. Male neonates of Wistar strain rats were given s.c. 2 mg/kg/day of CPZ for 7 successive days from days 6 to 12 after birth. Effect of the postnatal pretreatment with CPZ on saturation constants for specific bindings of 3H-spiperone, 3H-WB 4101 and 3H-dihydroalprenolol, respectively, in 8 brain regions was investigated at 60 days after birth. Significant decreases in Bmax values of 3H-WB 4101 binding sites in the cortex, thalamus, hypothalamus, mid brain and medulla oblongata/pons and decreases in Kd values of the binding sites in thalamus, hypothalamus and mid brain were observed in CPZ-pretreated rats when compared with corresponding Bmax and Kd values obtained in saline-pretreated rats. Furthermore, significant decreases in both Bmax and Kd values of 3H-DHA binding sites in the thalamus were detected in CPZ-pretreated rats when compared with those obtained in saline-pretreated rats. However, no alterations in 3H-spiperone binding sites in all brain regions were found between CPZ- and saline-pretreated rats. These results suggest that the learning deficit observed in CPZ-pretreated rats may be produced by a functional disorder of catecholaminergic, in particular alpha 1-noradrenergic neurons in the brain.

Adrenal function affects morphine-induced feeding during dark period, but not during light period in rats.

The present study was undertaken to investigate the relationships between morphine-induced feeding and the adrenal functions. Morphine (5 mg/kg) was intraperitoneally administered at 10:45 (light period) or 18:45 (dark period). The orectic effects of morphine during the light period in normal rats were not influenced by adrenalectomy; however, the anorectic effects during the dark period in normal rats were attenuated by both adrenalectomy and adrenodemedullation. Corticosterone (10 mg/kg) itself had no effects on feeding during the light and dark period. Morphine did not alter blood insulin levels during the light period, but markedly decreased it during the dark period independently of feeding. These results show that morphine has two different effects on feeding by administration time, and they suggest that the adrenal affects morphine-induced feeding only during the dark period (hungry state), presumably through insulin release, but not during the light period (satiated state).

Opposite effects of morphine on feeding and drinking in rats relative to administration time.

The present study was undertaken to examine how morphine changes food and water intake in non-fasted or fasted rats with different administration times. Morphine (1, 3 and 10 mg/kg) was intraperitoneally administered at 10:45 (light period) or 18:45 (dark period). Morphine increased food and water intake in non-fasted rats 2 hr after the administration during the light period, whereas the total daily intakes were decreased. In contrast, morphine decreased food and water intake in non-fasted rats during the dark period and in fasted rats during both the light and dark period. These results suggest that morphine disorders the baseline levels of feeding and water drinking of naive rats.

Effects of epinephrine on plasma cholesterol levels in rats.

The present study was undertaken to evaluate whether epinephrine increases plasma cholesterol in rats. Epinephrine suspended in sesame oil was subcutaneously administered at 21:00 hr (9 PM). Blood was drawn 12 hr later, and plasma cholesterol was shown to be increased by epinephrine in a dose-dependent manner (0.5-2.0 mg/kg). This epinephrine-induced hypercholesterolemia was enhanced by phentolamine (25 mg/kg) and inhibited by propranolol (25 mg/kg). Although the effect of epinephrine in normal rats was abolished by adrenalectomy, corticosterone (10 mg/kg) increased plasma cholesterol in both normal and adrenalectomized rats. These results demonstrate that epinephrine increases plasma cholesterol levels in rats, and that the effect of epinephrine appears to be mediated by the beta-adrenergic receptors, depending upon adequate amounts of corticosteroids.

Determination of picomole amounts of choline and acetylcholine in blood by gas chromatography-mass spectrometry equipped with a newly improved pyrolyzer.

Blood levels of choline (Ch) and acetylcholine (ACh) have previously not been confirmed because of the difficulty in measuring these compounds. In this paper a recently developed method for the assay of Ch and ACh, which employs a chemical ionization (isobutane) quadrupole mass spectrometer (JEOL QH 100) equipped with a new type of pyrolyzer is reported. The correlation between the mass fragmentogram peak ratios and the amounts of compounds was good from 0.5 to 4 pmol. The assay limit for quantitation was ca. 0.3 pmol for Ch and ACh. The values for Ch from rat whole blood, serum and red blood cells were 10.88 +/- 1.46, 6.72 +/- 1.02 and 4.56 +/- 0.53 nmol/ml, respectively, and for ACh the respective values were 3.56 +/- 0.86, 1.69 +/- 0.16 and 1.87 +/- 0.83 nmol/ml. In human whole blood the levels of Ch and ACh were 22.55 +/- 3.97 and 3.23 +/- 0.23, respectively.

Measurement of gastrointestinal transit of solid food using a colestipol-phenol red complex as a marker.

The present study was undertaken to determine whether colestipol-phenol red complexes can be used as markers for gastrointestinal transit of solid food. Colestipol (1.0g) bound completely with phenol red (10 mg) to form a stable complex in water (pH 7.0) Phenol red was more readily eluted from colestipol with 20 ml of 1 N HCl than 1 N NaOH, but was not eluted with 0.1 N HCL (pH 1.0). It was therefore assumed that the complex would remain stable in the gastrointestinal tract. Also, phenol red in the gastrointestinal tract was quantitatively eluted from the complex with 20 ml of 1N HCl. In a study on gastric emptying, phenol red solution (liquid food) was excreted from the stomach in a zero-order manner, and the colestipol-phenol red complex (solid food) in a first-order manner. It was concluded that colestipol-phenol red complexes can be used as markers of gastrointestinal transit.