The effect of amixin and agmatine on cytochrome C release from isolated mitochondria

Mitochondrial nicotinic acetylcholine receptors (nAChRs) control permeability transition pore formation and cytochrome c release in the presence of apoptogenic factors. This study demonstrates that pharmacological agents amixin and agmatine affect mitochondrial nAChR functioning: they slightly suppress cytochrome c release from mouse brain and liver mitochondria stimulated with apoptogenic dose of Са2+ and prevent the effect of α7 nAChR agonist PNU282987. We conclude that mitochondria may be one of therapeutic targets of amixin and agmatine.

Activation of the α7 nicotinic ACh receptor induces anxiogenic effects in rats which is blocked by a 5-HT1a receptor antagonist.

The α7 nicotinic acetylcholine receptor (nAChR) is highly expressed in different regions of the brain and is associated with cognitive function as well as anxiety. Agonists and positive allosteric modulators (PAMs) of the α7 subtype of nAChRs have been shown to improve cognition. Previously nicotine, which activates both α7 and non-α7 subtypes of nAChRs, has been shown to have an anxiogenic effect in behavioral tests. In this study, we compared the effects of the α7-selective agonist (PNU-282987) and PAM (PNU-120596) in a variety of behavioral tests in Sprague Dawley rats to look at their effects on learning and memory as well as anxiety. We found that neither PNU-282987 nor PNU-120596 improved spatial-learning or episodic memory by themselves. However when cognitive impairment was induced in the rats with scopolamine (1 mg/kg), both PNU-120596 and PNU-282987 were able to reverse this memory impairment and restore it back to normal levels. While PNU-120596 reversed the scopolamine-induced cognitive impairment, it did not have any adverse effect on anxiety. PNU-282987 on the other hand displayed an increase in anxiety-like behavior at a higher dose (10 mg/kg) that was significantly reduced by the serotonin 5-HT1a receptor antagonist WAY-100135. However the α7 receptor antagonist methyllycaconitine was unable to reverse these anxiety-like effects seen with PNU-282987. These results suggest that α7 nAChR PAMs are pharmacologically advantageous over agonists, and should be considered for further development as therapeutic drugs targeting the α7 receptors.

Hyperforin--a key constituent of St. John's wort specifically activates TRPC6 channels.

Hyperforin, a bicyclic polyprenylated acylphloroglucinol derivative, is the main active principle of St. John's wort extract responsible for its antidepressive profile. Hyperforin inhibits the neuronal serotonin and norepinephrine uptake comparable to synthetic antidepressants. In contrast to synthetic antidepressants directly blocking neuronal amine uptake, hyperforin increases synaptic serotonin and norepinephrine concentrations by an indirect and yet unknown mechanism. Our attempts to identify the molecular target of hyperforin resulted in the identification of TRPC6. Hyperforin induced sodium and calcium entry as well as currents in TRPC6-expressing cells. Sodium currents and the subsequent breakdown of the membrane sodium gradients may be the rationale for the inhibition of neuronal amine uptake. The hyperforin-induced cation entry was highly specific and related to TRPC6 and was suppressed in cells expressing a dominant negative mutant of TRPC6, whereas phylogenetically related channels, i.e., TRPC3 remained unaffected. Furthermore, hyperforin induces neuronal axonal sprouting like nerve growth factor in a TRPC6-dependent manner. These findings support the role of TRPC channels in neurite extension and identify hyperforin as the first selective pharmacological tool to study TRPC6 function. Hyperforin integrates inhibition of neurotransmitter uptake and neurotrophic property by specific activation of TRPC6 and represents an interesting lead-structure for a new class of antidepressants.

Block of HERG-carried K+ currents by the new repolarization delaying agent H 345/52.

INTRODUCTION: The aim of this study was to analyze the block of HERG-carried membrane currents caused by H 345/52, a new antiarrhythmic compound with low proarrhythmic activity, in transfected mouse fibroblasts. METHODS AND RESULTS: Using the whole-cell configuration of the voltage patch clamp technique, it was demonstrated that H 345/52 concentration-dependently blocked HERG-carried currents with an IC50 of 230 nM. H 345/52 preferentially bound to the open channel with unusually rapid kinetics and was trapped by channel closure. Voltage-independent behavior of H 345/52 was observed during both square-pulse and action potential clamp protocols. In contrast, the Class III agents dofetilide (10 nM) and almokalant (250 nM) demonstrated significant membrane potential-dependent effects during square-pulse clamp protocols. When using action potential clamp protocols, voltage dependence was seen with dofetilide but not with almokalant. Mathematical simulations of human ventricular action potentials predicted that the different voltage-dependent behaviors would not produce marked variations in action potential duration prolongation patterns. CONCLUSION: We propose that block of IKr is the principal mechanism by which H 345/52 delays repolarization in human myocardium. The voltage independence of HERG/IKr block is unlikely to underlie the low proarrhythmic potential, and ancillary effects on other membrane currents must be considered.

Antinociceptive action of DBO 17 and DBO 11 in mice: two 3,8 diazabicyclo (3.2.1.) octane derivates with selective mu opioid receptor affinity.

Two 3,8 diazabicyclo (3.2.1.) octane derivates, namely DBO 17 and DBO 11, were studied for the opioid-like activity. In the rat brain membrane preparation binding studies, DBO 17 and DBO 11 showed a high affinity and selectivity for the mu opioid receptor (Ki's: 5.1 and 25 nM, respectively). DBO 17 and DBO 11 inhibited the nociceptive response in the hot-plate test of mice with ED50 values of 0.16 mg/kg and 0.44 mg/kg, respectively. The antinociceptive action of both DBO 17 and DBO 11 was blocked by naloxone. Tolerance to the antinociceptive action of DBO 17 and DBO 11 was present after 13 and 7 days of repeated treatment, respectively. Both DBO 17 and DBO 11 were ineffective in morphine-tolerant mice and vice versa. Chronic treatments (three times daily for seven consecutive days) of DBO 17 and DBO 11 induced a naloxone-precipitated withdrawal syndrome in DBO 17 treated mice similar to that in morphine treated mice, whereas in DBO 11 treated mice abstinence signs were virtually absent. These results indicate an interesting pharmacological profile that suggests these compounds as possible new candidates for the clinical treatment of pain.

Is epibatidine really analgesic? Dissociation of the activity, temperature, and analgesic effects of (+/-)-epibatidine.

The experiments in the present study were designed to determine if the activity, temperature, and analgesic effects of (+/-)-epibatidine treatment could be dissociated. Initially (i.e., 15 min) (+/-)-epibatidine treatment (0.1 mumol/kg = 28 micrograms/kg, IP) impaired rotorod performance, decreased activity, decreased temperature, and increased jump latency (e.g., analgesic effect). For the remaining time points measured (i.e., 30, 60, and 120 min), activity and temperature remained significantly reduced. In contrast, by 120 min (+/-)-epibatidine's effects on rotorod performance and analgesia (jump latency) were not observed. When administered after (+/-)-epibatidine (0.05 mumol/kg, IP), mecamylamine treatment (5 mumol/kg = 1 mg/kg, IP) produced a potentiation of analgesia. This potentiation effect was not observed on activity and temperature measures. The effect of (+/-)-epibatidine treatment (0.1 mumol/kg, IP) was also determined in mice with central nicotinic receptor blockade induced by treatment with chlorisondamine (23 mumol/kg = 10 mg/kg, IP). An (+/-)-epibatidine-induced reduction in activity was not attenuated in chlorisondamine-treated mice and only a minimal effect was observed on (+/-)-epibatidine-induced hypothermia in chlorisondamine-treated mice. In contrast, in chlorisondamine-treated mice (+/-)-epibatidine's analgesic effect was attenuated. Taken together, these data suggest that various centrally mediated effects of (+/-)-epibatidine can be dissociated.

Effects of 1-amino-5-bromouracil on the benzodiazepine-GABAA receptor complex.

We investigated the effects of 1-amino-5-bromouracil on the benzodiazepine-gamma-aminobutyric acid (GABA)A receptor complex to elucidate its central action. 1-Amino-5-bromouracil neither displaced nor enhanced [3H]muscimol, [35S]t-butylbicyclophosphorothionate (TBPS), or [3H]dehydroepiandrosterone sulfate binding to the rat brain synaptosomal membranes. The anesthesia induced by 1-amino-5-bromouracil was potentiated by diazepam, pentobarbital, and muscimol, and was antagonized by picrotoxin but not by bicuculline. 1-Amino-5-bromouracil protected mice from picrotoxin-induced seizure and slightly ameliorated TBPS-induced seizure, but did not antagonize bicuculline-induced seizure. Diazepam antagonized both the bicuculline- and the picrotoxin-induced seizure, and pentobarbital antagonized the picrotoxin- and the TBPS-induced seizure. Our in vivo studies suggest that part of the central action of 1-amino-5-bromouracil is concerned with the benzodiazepine-GABAA receptor complex including the chloride channel.

5-HT4 receptor stimulation facilitates acetylcholine release in rat frontal cortex.

The effect of the serotonergic 5-HT4 receptor agonists BIMU 1 and BIMU 8 on in vivo acetylcholine (ACh) release in brain hemispheric regions of freely moving rats was investigated using the microdialysis technique. Both agonists, applied intracerebroventricularly, facilitated the release of ACh selectively in the frontal cortex and were ineffective in the striatum or dorsal hippocampus. The facilitatory effect of BIMU 1 in frontal cortex was prevented by the selective 5-HT4 receptor antagonists GR 125487 and GR 113808 which by themselves did not alter basal release. the results provide the first evidence that serotonin facilitates ACh release in frontal cortex through stimulation of 5-HT4 receptors which are not tonically activated. 5-HT4 receptor agonists might thus offer a novel means of boosting central cholinergic function to overcome the cholinergic deficit in memory disorders.

Imidazenil, a new partial agonist of benzodiazepine receptors, reverses the inhibitory action of isoniazid and stress on gamma-aminobutyric acidA receptor function.

The imidazobenzodiazepine 6-(2-bromophenyl)-8-fluoro-4H-imidazo-[1,5-a] [1,4]benzodiazepine-3-carboxamide (imidazenil) is a new anxiolytic and anticonvulsant ligand of the benzodiazepine recognition site that possesses the characteristics of a partial allosteric modulator of the gamma-aminobutyric acid (GABA) type A receptor. The effects of imidazenil on GABAA receptor function were examined both in vitro and in vivo. Imidazenil inhibited [3H] flumazenil binding to mouse cerebral cortical membranes in vitro with an IC50 of 0.9 nM, showing that this compound binds with high affinity to benzodiazepine receptors. However, imidazenil failed to modify t-[35S]butylbicyclophosphorothionate ([35S]TBPS) binding to washed or unwashed mouse cortical membrane preparations. Furthermore, imidazenil injected i.p. into mice failed to affect [35S]TBPS binding subsequently measured in unwashed cortical membranes. In contrast, imidazenil reduced in a dose-dependent manner the increase in [35S]TBPS binding elicited by isoniazid (200 mg/kg s.c.), an effect mimicked by lorazepam and abecarnil++ but not by bretazenil. As expected, i.p. administration of lorazepam or abecarnil induced within 30 min a marked reduction in [35S]TBPS binding subsequently measured in unwashed cortical membranes of control mice. Moreover, imidazenil at a dose as low as 0.05 mg/kg (i.p.) delayed the onset of convulsions and death elicited by isoniazid and reduced significantly the number of mice exhibiting seizures. Accordingly, imidazenil also showed great potency in antagonizing the convulsions induced by pentylenetetrazole in rats. Imidazenil also completely abolished the increase in [35S]TBPS binding induced by foot-shock or exposure to carbon dioxide. Finally, imidazenil antagonized both in vitro and in vivo the effects of bretazenil or lorazepam on GABAA receptor function.

Effect of rociverine on P450-dependent monooxygenases and its N-deethylation metabolism in rat liver microsomes.

Rociverine [2-(diethylamino)-1-methylethyl cis-1-hydroxy [bicyclohexyl]-2-carboxylate] citrate (ROC) is an antispasmodic agent therapeutically active in humans at doses of 0.5-1 mg/kg. This study investigated the effect of acute administration of the drug on hepatic microsomal cytochrome P450 (P450)-catalysed drug metabolism. Only high doses (> or = 100 mg/kg) of ROC were able to induce in rats the hepatic microsomal pentoxyresorufin O-depenthylase (PROD) and 16 beta-testosterone hydroxylase activities both associated with P4502B1/2 and the erythromycin N-dimethylase (ErD) and 2 beta-testosterone hydroxylase activities both dependent on P4503A1/2. However, at 100 and 200 mg/kg of ROC, the 16 beta-testosterone hydroxylase and PROD were the most induced activities, suggesting that P4502B1/2 are the isoforms most sensitive to ROC induction. Accordingly, ROC treatment enhanced, in a dose-dependent manner, the amount of P4502B1/2 and 3A1/2 in microsomes as assayed by western blotting. The northern blot analysis of ROC-treated rat liver showed that the P4502B1/2 induction appears to be regulated at the mRNA level as in the induction by phenobarbital (PB). The oxidative metabolism of ROC with hepatic microsomes from control or PB- and ROC-induced rats resulted in a N-deethyl ROC derivative (major metabolite) and an unknown minor ROC derivative. The kinetic parameters for the N-deethylation of ROC were studied with purified P4502B1 and with microsomes from control or rats treated with various inducers (phenobarbital, ethanol, beta-naphthoflavone, dexamethasone and rociverine). It was found that phenobarbital-, dexamethasone- and rociverine-induced microsomes deethylated ROC with a Vmax about five times higher than that (0.9 nmol/min/mg protein) of control microsomes, although with a similar affinity (Km approximately 0.3 mM). In a reconstituted system, the purified P4502B1 metabolized ROC with a high deethylation rate (22 nmol/min/nmol P450). Moreover, the ROC deethylation was inhibited by compounds such as hexobarbital, metyrapone and triacetyloleandomicin, selective inhibitors for P4502B and/or P4503A enzymes. On the other hand ROC, when added in vitro, inhibited the 16 beta- and 2 beta-testosterone hydroxylases and the PROD and ErD activities. Taken together, these results indicate that the ROC-inducible P4502B and P4503A are involved in ROC deethylation. In conclusion, it has been demonstrated that ROC is a weak phenobarbital-like inducer of P450, probably able at high and reiterated doses to alter its own metabolism, at least in the rat liver.

The GABAA receptor complex in relation to epilepsy. Reversal of [3H]TBOB inhibition: a prediction of proconvulsive properties?

[3H]-t-Butylbicycloorthobenzoate ([3H]TBOB), a convulsant, is known to label a binding site on the GABAA receptor complex. Bicuculline methochloride (bicuculline MCl), folic acid, pentazocine, naloxone, ethyl-beta-carboline-3-carboxylate (beta CCE) and Ro 5-4864 have (pro)convulsive properties in vivo. In the present study, we determined the extent to which these compounds modify the binding of [3H]TBOB in the presence of IC50 amounts of GABA (5 microM) or diazepam (50 microM). We found that the GABA antagonist bicuculline MCl reversed the inhibitory effect of GABA on [3H]TBOB binding completely, as was expected. Folic acid, pentazocine and naloxone also reversed the inhibitory effect of GABA on [3H]TBOB binding. This finding is compatible with the view that the proconvulsive effects of these compounds can be credited to a reduction of GABAergic action at the GABAA receptor complex. We suggest that the reversal of GABA's inhibition of [3H]TBOB binding is a sufficient (but not a necessary) condition to predict proconvulsive (side) effects of drugs. beta CCE and Ro 5-4864 modified [3H]TBOB binding in the presence of GABA in a biphasic fashion. A unique relation between beta CCE, Ro 5-4864 and the GABAA complex might exist. Bicuculline MCl reversed the inhibitory effect of diazepam on [3H]TBOB binding only partly. beta CCE did not reverse the inhibitory effect of diazepam on [3H]TBOB binding, neither did Ro 5-4864. The presence of a GABA-independent interaction between a low affinity benzodiazepine recognition site and the TBOB site is proposed.

A specific B2-bradykinin receptor antagonist HOE 140 abolishes the antihypertrophic effect of ramipril.

To evaluate the role of bradykinin in the antihypertrophic effect of the angiotensin-converting enzyme (ACE) inhibitor, ramipril, we investigated the influence of HOE 140, a specific B2-receptor antagonist, on the effects of ramipril on left ventricular hypertrophy (LVH) in rats with aortic banding. Ramipril at a dose of 1 mg kg-1 day-1 for 6 weeks prevented the increase in blood pressure and development of LVH after aortic banding; plasma ACE activity was significantly inhibited. A lower dose of ramipril (10 micrograms kg-1 day-1 for 6 weeks) had no effect on the increase in blood pressure or on plasma ACE activity, but prevented LVH after aortic banding. The antihypertrophic effects of the higher and the lower dose ramipril, as well as the antihypertensive action of the higher dose of ramipril were abolished by the coadministration of HOE 140 (500 micrograms kg-1 day-1). The present data show for the first time that the beneficial effects of an ACE-inhibitor on LVH in rats with hypertension caused by aortic banding can be prevented by a specific B2-receptor antagonist.

Involvement of tryptophan residue(s) in the specific binding of agonists/antagonists to 5-HT3 receptors in NG108-15 clonal cells.

Chemical modification of the 5-HT3 receptors in membranes from NG108-15 hybridoma cells was achieved using protein modifying reagents specific for various amino acid residues: N-bromosuccinimide for tryptophan, dithiothreitol for cystine, sodium tetrathionate for cysteine, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline for aspartic and glutamic acids, diethylpyrocarbonate for histidine, tetranitromethane for tyrosine and 2,3-butanedione for arginine. Among all the reagents tested, N-bromosuccinimide produced the largest alteration in the specific binding of [3H]zacopride onto 5-HT3 receptors. A significant reduction in Bmax (approximately 50%) with no change in Kd were noted on [3H]zacopride specific binding to membranes which were incubated with 40 microM N-bromosuccinimide for 60 min at 25 degrees. The occupancy of 5-HT3 receptor binding sites by various 5-HT3 agonists and antagonists (phenylbiguanide, ondansetron, granisetron, MDL 72222) prevented, at least partially, any subsequent reduction in [3H]zacopride specific binding by N-bromosuccinimide treatment. However, neither m-chloro-phenylbiguanide, among the agonists, nor zacopride, among the antagonists, were able to prevent the effect of N-bromosuccinimide, suggesting that variations might exist in the molecular mechanisms implicated in the binding of 5-HT3 ligands to the recognition site on 5-HT3 receptors. Nevertheless, these data support the suggestion that tryptophan residue(s) are probably involved in the binding of agonists and antagonists onto 5-HT3 receptors in NG108-15 cell membranes.

Salt blocks the renal benefits of ramipril in diabetic hypertensive rats.

To establish if the benefit of angiotensin converting enzyme inhibitor therapy in retarding progressive diabetic renal injury is due to a specific intrarenal effect of the systemic hypotensive effect, we studied the effect of long-term ramipril treatment on blood pressure, glomerular filtration rate, and urinary protein excretion in streptozotocin-diabetic spontaneously hypertensive rats. The hypotensive effect of ramipril was prevented by a high salt diet, which did not alter the degree of renal angiotensin converting enzyme inhibition. Three weeks after uninephrectomy and induction of diabetes, rats were allocated to three groups. Groups 1 and 2 were given 1% NaCl, whereas group 3 was given water as drinking solution. One week later, groups 2 and 3 received 0.4 mg/kg/day ramipril in their drinking solution, which was continued over a 2-month period. Ramipril produced a blood pressure fall only in water-drinking rats (group 3) despite a similar reduction in plasma and renal angiotensin converting enzyme activity in groups 2 and 3. Salt-loaded rats had a progressive increase in urinary protein excretion over the duration of study. Ramipril treatment prevented an increase in protein excretion only in animals given water and with a reduced systolic blood pressure. Glomerular filtration rate was similar in all three groups. Ramipril treatment improved animal survival independently of a reduction in blood pressure or an effect on proteinuria. Although it is possible that angiotensin converting enzyme inhibitors have specific intrarenal effects reducing progression of diabetic proteinuria, concomitant control of systemic blood pressure appears to be necessary to demonstrate a benefit.

Regulation of 35S-TBPS binding by bicuculline is region specific in rat brain.

The allosteric regulation of specific 35S-TBPS binding to the convulsant site on the GABAA receptor/chloride (Cl-) ionophore complex was studied in various brain regions in an attempt to characterize regional heterogeneity of the protein subunits forming the complex. Bicuculline methiodide (BIC), a GABAA antagonist, enhanced binding in cortex (CTX), substantia nigra (SN) and cerebellum (CBL), inhibited binding in inferior colliculus (IC) and did not affect binding in superior colliculus (SC). Similar results were found in CBL and IC using SR-95531, another GABAA antagonist. The levels of endogenous GABA in the different tissue samples could not account for the regional differences in binding. When the functional regulation of these receptors was measured using 36Cl- uptake in microsomes, muscimol-stimulated uptake was completely blocked by BIC in CBL and IC but was not affected by BIC in SC. Additionally, picrotoxin completely blocked muscimol-stimulated uptake in CBL but had no effect in IC or SC. These findings provide a functional basis for regional heterogeneity of GABAA receptor.

Characterization of a thromboxane A2/prostaglandin H2 receptor in guinea pig lung membranes using a radioiodinated thromboxane mimetic.

Thromboxane A2 (TXA2) and prostaglandin H2 (PGH2) are potent constrictors of airway smooth muscle and may mediate some of the pulmonary effects of leukotrienes. To date, the TXA2/PGH2 receptor in lung has not been well characterized. In this report, we describe the evaluation of the TXA2/PGH2 receptor in guinea pig lung membranes using the new radiolabeled TXA2 mimetic [1S(1 alpha,2 beta(5Z),3 alpha(1E,3S*),4 alpha)]-7-[3-(3-hydroxy-4-(4'- iodophenoxy)-1-butenyl)-7-oxabicyclo-[2.2.1]heptan-2-yl]-5-h eptenoic acid (IBOP). IBOP elicited a dose-dependent contraction of guinea pig lung parenchymal strips (EC50 = 3.03 +/- 0.97 nM, three experiments), which was blocked by the TXA2/PGH2 antagonists SQ29548 (pKB = 7.44 +/- 0.2, three experiments), BM13505 (pKB = 6.29 +/- 0.26, three experiments), and I-PTA-OH (pKB = 5.82 +/- 0.36, three experiments). In radioligand binding studies, the binding of [125I]IBOP to guinea pig lung membranes prepared from perfused lungs was saturable, displaceable, and dependent upon protein concentration. Binding was optimal at pH 6.5 and was enhanced by the addition of mono- and divalent cations. The standard assay buffer was 25 mM 3-(N-morpholino)propanesulfonic acid, pH 6.5, 100 mM NaCl, 5 mM MgCl2. Binding was inhibited by pretreatment with dithiothreitol, N-ethylmaleimide, or beta-mercaptoethanol. Binding was unaffected by the addition of guanine nucleotide analogs at concentrations up to 300 microM. Analysis of the time course of binding of [125]IBOP at 30 degrees yielded k-1 = 0.0447 min-1, k1 = 2.49 x 10(8) M-1 min-1, and Kd = k-1/k1 = 180 pM. Computer analysis of equilibrium binding studies using nonlinear methods (LUNDON-1) revealed a single class of noninteracting binding sites with a Kd of 86.9 +/- 11.9 pM and a Bmax of 81.8 +/- 7.7 fmol/mg of protein (three experiments). [125I]IBOP binding to guinea pig lung membranes was inhibited by a series of TXA2/PGH2 receptor agonists and antagonists, with a rank order different from that previously determined for washed guinea pig platelets (Spearman's r = 0.686, p greater than 0.05). [125I]IBOP binding to guinea pig lung membranes was also inhibited by the prostanoids prostaglandin D2, prostaglandin E2, prostaglandin F2 alpha, and 9 alpha,11 beta-prostaglandin F2, all of which have been proposed to act at the TXA2/PGH2 receptor in lung.

The general anesthetic propofol enhances the function of gamma-aminobutyric acid-coupled chloride channel in the rat cerebral cortex.

The effect of the general anesthetic propofol on t-[35S]butylbicyclophosphorothionate ([35S]TBPS) binding to unwashed membrane preparations from rat cerebral cortex was studied and compared to that of other general anesthetics (pentobarbital, alphaxalone) which are known to enhance GABAergic transmission. Propofol produced a concentration-dependent complete inhibition of [35S]TBPS binding, an effect similar to that induced by pentobarbital and alphaxalone, although these agents differ markedly in potency (alphaxalone greater than propofol greater than pentobarbital). The concomitant addition of propofol either with alphaxalone or pentobarbital produced an additive inhibition of [35S]TBPS binding, suggesting separate sites of action or different mechanisms of these drugs. Moreover, although bicuculline (0.1 microM) completely antagonized the propofol-induced inhibition of [35S]TBPS binding, the effect of this anesthetic was not due to a direct interaction with the gamma-aminobutyric acidA (GABAA) recognition site. In fact, propofol, like alphaxalone and pentobarbital, markedly enhanced [3H]GABA binding in the rat cerebral cortex. Finally, propofol was able to enhance [3H]GABA binding in membranes previously incubated with the specific chloride channel blocker picrotoxin. Taken together these data strongly suggest that propofol, like other anesthetics and positive modulators of GABAergic transmission, might exert its pharmacological effects by enhancing the function of the GABA-activated chloride channel.

Zacopride and BRL 24924 induce an increase in EEG-energy in rats.

1. The substituted benzamides, zacopride and BRL 24924 induced dose-dependent increases of the total EEG-energy of rats when applied intracerebroventricularly (i.c.v.) with ED50 values of 8.0 +/- 0.6 and 3.6 +/- 0.9 micrograms, respectively. Not only the energy of the low frequency hippocampal theta rhythm but also that of the other frequency bands was increased. 2. In contrast to i.c.v. application intraperitoneal administration of zacopride or BRL 24924 (1 and 10 mg kg-1) did not lead to an increase in EEG-energy. 3. The increase in EEG-energy induced by zacopride (10 micrograms, i.c.v.) was blocked by ICS 205-930 (1 microgram, i.c.v.). Neither the 5-HT3 receptor agonist 2-methyl-5-hydroxytryptamine (30 micrograms, i.c.v.) nor the selective 5-HT3 receptor antagonist MDL 72222 (30 micrograms, i.c.v.) had any effect upon rat EEG. 4. Scopolamine (0.01 micrograms and 0.1 micrograms, i.c.v.) dose-dependently antagonized the effect of zacopride (10 micrograms, i.c.v.). 5. An agonist action of zacopride and BRL 24924 and inhibition of these effects by ICS 205-930 but not by MDL 72222 was recently described in isolated colliculi neurones from neonatal mice. The receptor involved was described as '5-HT4'. The present results indicate that the central effects of zacopride and BRL 24924 may be due to activation of such a 5-HT receptor.

gamma-Aminobutyric acid antagonism produced by an organophosphate-containing combustion product.

Results from [35S]t-butylbicyclophosphorothionate (TBPS) binding and gamma-aminobutyric acid (GABA)-dependent 36Cl- uptake indicated that the pyrolysis product of liquid trimethylol propane (TMP) containing organic phosphates interacts with the GABA/benzodiazepine receptor chloride ionophore complex (GBRC) producing a receptor-mediated reduction in 36Cl- flux. The pyrolysate-ligand interaction was competitive and directly proportional to the trimethylol propane phosphate (TMPP) content of the combustion product. TBPS displacement demonstrated that TMPP had less affinity than did TBPS and picrotoxin, but greater affinity than GABA, racemic dimethylbutyl barbituric acid (DMBB), clonazepam, and phenobarbital for their respective recognition sites. Addition of exogenous GABA was a necessary condition for demonstrating clonazepam-induced ligand displacement, thus being indicative of an allosteric interaction. The same rank order held for altering GABA-dependent 36Cl- uptake with the caveat of inhibitors vs enhancers, and noting that pentobarbital was less potent than DMBB. Concentration-dependent ligand displacement by GBRC agonists and antagonists quantitatively correlated with alterations in GABA-dependent 36Cl- uptake. The median convulsant dose (CD50) of pyrolysate TMPP was higher than that of synthetic TMPP. The presence of combustion by-products altering the dispositional kinetics of the former is felt to be the reason for this difference. Under our experimental conditions, phenobarbital was the most effective antidote for pyrolysate (TMPP) toxicity. The inhalant convulsant, flurothyl, was found not to displace [35S]TBPS binding, not to affect GABA-dependent 36Cl- uptake, and not to respond to the anticonvulsants in a manner consistent with a strict GABAA receptor phenomenon.

Emesis and defecations induced by the 5-hydroxytryptamine (5-HT3) receptor antagonist zacopride in the ferret.

Three antiemetic compounds (zacopride, batanopride, granisetron [BRL43694]) were evaluated for the production of gastrointestinal side effects in the conscious ferret after i.v. or p.o. administration. Zacopride evoked multiple emetic and defecatory responses at clinically relevant doses (0.003-0.3 mg/kg) and in a dose-dependent manner. The oral route was the more potent one for eliciting emesis (ED50 0.033 mg/kg). At 0.3 mg/kg p.o., zacopride reliably evoked an 80 to 100% incidence of emesis and a 40 to 80% incidence of defecation. In contrast, batanopride and BRL43694 i.v. evoked a small (10%) incidence of these side effects, but only at 0.1 to 10 mg/kg doses. When given p.o. (0.00003-10 mg/kg), these latter compounds never evoked emesis and significantly reduced (P less than .05) the incidence of defecation below that of vehicle. Responses to zacopride (0.3 mg/kg p.o.) were challenged by i.p. pretreatment with the 5-hydroxytryptamine receptor agonist 2-methyl serotonin, the 5-hydroxytryptamine receptor antagonist BRL43694, the quaternary atropine derivative glycopyrrolate, the dopamine receptor antagonist domperidone or selective abdominal vagotomies. All compounds and either bilateral or dorsal vagotomy significantly reduced the incidence of emesis, but did not abolish it. Latency to first emesis was delayed by BRL43694, domperidone or dorsal vagotomy. The data suggest that the emetic response to p.o. zacopride is mediated in part by 5-hydroxytryptamine receptors residing on either enteric neurons or vagal afferents. However, the underlying pharmacology of the response may also include activation of cholinergic and dopaminergic pathways.