Antagonistic action of pitrazepin on human and rat GABA(A) receptors.
Pitrazepin, 3-(piperazinyl-1)-9H-dibenz(c,f) triazolo(4,5-a)azepin is a piperazine antagonist of GABA in a variety of electrophysiological and in vitro binding studies involving GABA and glycine receptors. In the present study we have investigated the effects of pitrazepin, and the GABA(A) antagonist bicuculline, on membrane currents elicited by GABA in Xenopus oocytes injected with rat cerebral cortex mRNA or cDNAs encoding alpha1beta2 or alpha1beta2gamma2s human GABA(A) receptor subunits. The three types of GABA(A) receptors expressed were reversibly antagonized by bicuculline and pitrazepin in a concentration-dependent manner. GABA dose-current response curves for the three types of receptors were shifted to the right, in a parallel manner, by increasing concentrations of pitrazepin. Schild analyses gave pA2 values of 6.42+/-0.62, n = 4, 6.41+/-1.2, n = 5 and 6.21+/-1.24, n = 6, in oocytes expressing rat cerebral cortex, alpha1beta2 or alpha1beta2gamma2s human GABA(A) receptors respectively (values are given as means +/- s.e. mean), and the Hill coefficients were all close to unity. All this is consistent with the notion that pitrazepin acts as a competitive antagonist of these GABA(A) receptors; and that their antagonism by pitrazepin is not strongly dependent on the subunit composition of the receptors here studied. Since pitrazepin has been reported to act also at the benzodiazepine binding site, we studied the effect of the benzodiazepine antagonist Ro 15-1788 (flumazenil) on the inhibition of alpha1beta2gamma2s receptors by pitrazepin. Co-application of Ro 15-1788 did not alter the inhibiting effect of pitrazepin. Moreover, pitrazepin did not antagonize the potentiation of GABA-currents by flunitrazepam. All this suggests that pitrazepin does not affect the GABA receptor-chloride channel by interacting with the benzodiazepine receptor site. (+info)
Epinastine (WAL 801CL) inhibits the electrical field stimulation-induced cholinergic contraction in guinea pig and human airways in vitro.
Epinastine is an antihistamine drug with binding affinities at 5-hydroxytryptamine (5-HT) receptors. The current study was performed to investigate whether epinastine could modulate the cholinergic contraction in guinea pig and human airways in vitro. Isolated guinea pig and human airway preparations were suspended in organ baths containing modified Krebs-Henseleit solution. Electrical field stimulation was applied to elicit cholinergic contractions. Epinastine produced a concentration-dependent inhibition of the cholinergic contraction in guinea pig airways and pretreatment with methysergide (5-HT1/2/7 antagonist) significantly attenuated these inhibitory effects of epinastine. Pretreatment with tropisetron (5-HT3/4 antagonist), ketanserin (5-HT2 antagonist), SDZ216-525 (5-HT1A antagonist) or phentolamine (alpha-adrenergic antagonist) had no effect. Epinastine did not displace the concentration-response curve to acetylcholine. These results suggest that epinastine inhibits the cholinergic contraction in guinea pig airways through stimulation of prejunctional 5-hydroxytryptamine receptors, located to postganglionic cholinergic nerves. Inhibitory effects of epinastine on the cholinergic contraction in human airways in vitro were also demonstrated, which suggests that a similar mechanism might be present in human airways. The pharmacological profile of epinastine, which shows binding affinity at the 5-hydroxytryptamine7 receptor but not at the 5-hydroxytryptamine1 receptor subtypes corroborates the hypothesis that the inhibitory prejunctional 5-hydroxytryptamine receptor on cholinergic nerves is of the 5-hydroxytryptamine7 subtype. (+info)
Bioactivation and covalent binding of hydroxyfluperlapine in human neutrophils: implications for fluperlapine-induced agranulocytosis.
The use of fluperlapine and the structurally related clozapine has been associated with the induction of agranulocytosis in humans. Unlike clozapine, fluperlapine is relatively resistant to chemical and biochemical oxidations. In this study we demonstrated that 7-hydroxyfluperlapine, the major metabolite of fluperlapine in humans, is oxidized to a reactive intermediate by HOCl and by myeloperoxidase in the presence of H(2)O(2) and Cl(-). This reactive intermediate was identified as an iminoquinone species with a M + 1 ion at m/z 324 by mass spectrometry. The iminoquinone intermediate was trapped by N-acetyl-L-cysteine (NAC) as well as GSH. NMR spectra of the NAC adducts indicated that the NAC was bound to the 6 and 9 positions of the aromatic ring. This is the same orientation as the binding of nucleophiles to the reactive metabolite of clozapine. We were able to use an antibody against clozapine to demonstrate that 7-hydroxyfluperlapine, but not fluperlapine itself, covalently modifies human myeloperoxidase. Furthermore, we demonstrated that 7-hydroxyfluperlapine is metabolized by activated neutrophils to a reactive intermediate that covalently binds to neutrophils. In the presence of NAC or GSH, such covalent binding was inhibited and the NAC or GSH adducts were formed. Thus, the reactivity and even the orientation of the binding of the reactive metabolite of 7-hydroxyfluperlapine is very similar to that of clozapine. These results provide a mechanism for the formation of a reactive metabolite of fluperlapine similar to clozapine that may explain its ability to induce agranulocytosis. (+info)
Suppressive effects of co-stimulatory molecule expressions on mouse splenocytes by anti-allergic agents in vitro.
The influence of anti-allergic drugs, epinastine hydrochloride (EP) and disodium cromoglycate (DSCG), on the co-stimulatory molecule expression was examined using in vitro cell culture technique. Spleen cells obtained from BALB/c mice 10 days after immunization with haemocyanin absorbed to aluminium hydroxide were cultured in the presence of 100.0 microg/ml haemocyanin and various concentrations of the agents. Low concentrations (<1.5 x 10(-4)M) of EP and DSCG did not influence spleen cell blastic activity induced by antigenic stimulation, whereas these agents caused significant inhibition of spleen cell activation when 2 x 10(-4) M of the agents were added to cell cultures. EP and DSCG also did not affect blastic activity of sensitized splenic T cells by anti-CD3 monoclonal antibody stimulation even when these cells were cultured in the presence of 2 x 10(-4) M of the agents. We next examined the influence of EP and DSCG on the expression of co-stimulatory molecules on spleen cells in response to antigenic stimulation. Sensitized spleen cells were cultured in the presence of 2 x 10(-4)M of the agents and the expression of molecules were examined by flow cytometer 24h later. EP and DSCG suppressed the expression of costimulatory molecules, CD40 and CD80, but not CD86, on splenic B cells which were enhanced by antigenic stimulation in vitro. (+info)
Pharmacokinetics of epinastine and a possible mechanism for double peaks in oral plasma concentration profiles.
The pharmacokinetics of epinastine (EPN), an anti-allergic agent, was investigated in rats. The plasma concentration-time profile of EPN after intravenous (i.v.) administration was triexponential. After oral administration of EPN (7.5 and 20 mg/kg), the drug was rapidly absorbed, and Cmax was reached 2 h after dosing. A minor secondary peak was observed in EPN plasma concentration-time profiles at both doses. The bioavailability of EPN after oral dosing was 41 and 40%. The kinetic parameters (T 1/2, AUC and MRT) for unlabeled EPN were much smaller than those for 14C-EPN, which has already been reported. The total biliary excretion of EPN at a 7.5 mg/kg dose was 15.5% of the dose, but the percentage of conjugates in bile was extremely low and about 11% of the total biliary excretion. The increase in the plasma concentration in bile duct-linked rats after oral administration of EPN (20 mg/kg) was not observed, indicating that a secondary increase in drug concentration based on enterohepatic circulation was ruled out. When the gastrointestinal (GI)-transit of phenol red (PR) after oral administration of EPN (20 mg/kg) was estimated, the GI-transit of PR was significantly delayed, and at 3-4 h after dosing half of the PR dose reached the jejunum. The remaining EPN in the small intestine after oral administration (7.5 mg/kg) reached peak levels 2 h after dosing, but then partly increased again at 4 h. As a result, it was clarified that the double peaks observed after oral doses are mainly due to the delayed absorption of a part of EPN, based on the reduction in gastric motility caused by the drug. (+info)
Acetylcholine mediates the estrogen-induced increase in NMDA receptor binding in CA1 of the hippocampus and the associated improvement in working memory.
Elevated levels of circulating estrogen in female rats result in increased spine and synapse density and parallel increases in NMDA receptor binding in area CA1 of the hippocampus. Estrogen also influences cholinergic neurochemistry in the basal forebrain and hippocampus. The objectives of the present study were to determine the role of acetylcholine in the estrogen-induced increase in NMDA receptor binding in CA1 of the hippocampus and to investigate the relationship between increased NMDA receptor binding in CA1 and performance on a task of working memory. In the current experiments, elevating endogenous levels of acetylcholine in ovariectomized rats by 3 d of continuous administration of physostigmine, an acetylcholinesterase inhibitor, increased NMDA receptor binding in CA1 as measured by quantitative autoradiography. This increase was comparable with the increase in NMDA receptor binding induced by injections of estradiol benzoate 72 and 48 hr before death. Additionally, the administration of 5,11-dihydro-8-chloro-11-[[4-[3-[(2,2-dimethyl-1-oxopentyl)ethylamino]propyl]-1-p iperidinyl]acetyl]-6H-pyrido[2,3-b][1,4]benzodiazepin-6-one (BIBN 99), an M2 receptor antagonist, blocked the ability of both estrogen and physostigmine to increase NMDA receptor binding. The regimen of estradiol replacement that was demonstrated to increase NMDA receptor binding in CA1 of ovariectomized rats also improved arm-choice accuracy in a working memory task in an eight-arm radial maze. The estrogen-induced improvement in working memory performance was blocked by BIBN 99, which also blocked the increase in NMDA receptor binding. These results indicate that acetylcholine acts at M2 muscarinic receptors to mediate the estrogen-induced increase in NMDA receptor binding in CA1 of the hippocampus as well as the associated improvement in working memory. (+info)
Effect of H1-antagonists on spatial memory deficit evaluated by 8-arm radial maze in rats.
AIM: To evaluate effects of certain H1-antagonists on spatial memory with 8-arm radial maze performance of rats. METHODS: Eight-arm radial maze performance was used to measure spatial memory in rats. RESULTS: Chronic treatments of classical H1-antagonists, diphenhydramine (5 mg/kg) and pyrilamine (20 mg/kg) impaired acquisition memory process regarding both parameters of radial maze performance. In addition, the memory retrieval process was also impaired significantly by a single administration of diphenhydramine (5, 10 mg/kg) and pyrilamine (50 mg/kg). However, the newly developed H1-antagonist, epinastine caused no appreciable effect on both acquisition and retrieval memory even at a high dose of 50 mg/kg. The memory deficit induced by diphenhydramine (10 mg/kg) or pyrilamine (50 mg/kg) was reversed by tacrine (1 mg/kg). CONCLUSION: Histamine H(1)-receptors plays a certain role in spatial cognition, and its action may be due to both histaminergic and cholinergic neurons. (+info)
Theta-frequency synaptic potentiation in CA1 in vitro distinguishes cognitively impaired from unimpaired aged Fischer 344 rats.
Hippocampal-dependent learning and memory deficits have been well documented in aging rodents. The results of several recent studies have suggested that these deficits arise from weakened synaptic plasticity within the hippocampus. In the present study, we examined the relationship between hippocampal long-term potentiation (LTP) in vitro and spatial learning in aged (24-26 months) Fischer 344 rats. We found that LTP induced in the CA1 region using theta-frequency stimulation (5 Hz) is selectively impaired in slices from a subpopulation of aged rats that had shown poor spatial learning in the Morris water maze. LTP at 5 Hz in aged rats that did not show learning deficits was similar to that seen in young (4-6 months) controls. We also found that 5 Hz LTP amplitude strongly correlated with individual learning performance among aged rats. The difference in 5 Hz LTP magnitude among aged rats was not attributable to an altered response to 5 Hz stimulation or to differences in the NMDA receptor-mediated field EPSP. In addition, no performance-related differences in LTP were seen when LTP was induced with 30 or 70 Hz stimulation protocols. Finally, both 5 Hz LTP and spatial learning in learning-impaired rats were enhanced with the selective muscarinic M2 antagonist BIBN-99 (5,11-dihydro-8-chloro-11-[[4-[3-[(2,2-dimethyl-1-oxopentyl)ethylamino]propyl]-1- piperidinyl]acetyl]-6H-pyrido[2,3-b][1,4]benzodiazepin-6-one). These findings reinforce the idea that distinct types of hippocampal LTP offer mechanistic insight into age-associated cognitive decline. (+info)