Amperometric measurement of glutamate release modulation by gabapentin and pregabalin in rat neocortical slices: role of voltage-sensitive Ca2+ α2δ-1 subunit

Gabapentin (GBP; Neurontin) and pregabalin (PGB; Lyrica, S-(+)-3-isobutylgaba) are used clinically to treat several disorders associated with excessive or inappropriate excitability, including epilepsy; pain from diabetic neuropathy, postherpetic neuralgia, and fibromyalgia; and generalized anxiety disorder. The molecular basis for these drugs' therapeutic effects are believed to involve the interaction with the auxiliary α(2)δ subunit of voltage-sensitive Ca(2+) channel (VSCC) translating into a modulation of pathological neurotransmitter release. Glutamate as the primary excitatory neurotransmitter in the mammalian central nervous system contributes, under conditions of excessive glutamate release, to neurological and psychiatric disorders. This study used enzyme-based microelectrode arrays to directly measure extracellular glutamate release in rat neocortical slices and determine the modulation of this release by GBP and PGB. Both drugs attenuated K(+)-evoked glutamate release without affecting basal glutamate levels. PGB (0.1-100 μM) exhibited concentration-dependent inhibition of K(+)-evoked glutamate release with an IC(50) value of 5.3 μM. R-(-)-3-Isobutylgaba, the enantiomer of PGB, did not significantly reduce K(+)-evoked glutamate release. The decrease of K(+)-evoked glutamate release by PGB was blocked by the l-amino acid l-isoleucine, a potential endogenous ligand of the α(2)δ subunit. In neocortical slices from transgenic mice having a point mutation (i.e., R217A) of the α(2)δ-1 (subtype) subunit of VSCC, PGB did not affect K(+)-evoked glutamate release yet inhibited this release in wild-type mice. The results show that GBP and PGB attenuated stimulus-evoked glutamate release in rodent neocortical slices and that the α(2)δ-1 subunit of VSCC appears to mediate this effect.     

Stimulus-dependent modulation of [(3)H]norepinephrine release from rat neocortical slices by gabapentin and pregabalin

Gabapentin (GBP; Neurontin) has proven efficacy in several neurological and psychiatric disorders yet its mechanism of action remains elusive. This drug, and the related compounds pregabalin [PGB; CI-1008, S-(+)-3-isobutylgaba] and its enantiomer R-(-)-3-isobutylgaba, were tested in an in vitro superfusion model of stimulation-evoked neurotransmitter release using rat neocortical slices prelabeled with [(3)H]norepinephrine ([(3)H]NE). The variables addressed were stimulus type (i.e., electrical, K(+), veratridine) and intensity, concentration dependence, onset and reversibility of action, and commonality of mechanism. Both GBP and PGB inhibited electrically and K(+)-evoked [(3)H]NE release, but not that induced by veratridine. Inhibition by these drugs was most pronounced with the K(+) stimulus, allowing determination of concentration-effect relationships (viz., 25 mM K(+) stimulus: GBP IC(50) = 8.9 microM, PGB IC(50) = 11.8 microM). R-(-)-3-Isobutylgaba was less effective than PGB to decrease stimulation-evoked [(3)H]NE release. Other experiments with GBP demonstrated the dependence of [(3)H]NE release inhibition on optimal stimulus intensity. The inhibitory effect of GBP increased with longer slice exposure time before stimulation, and reversed upon washout. Combination experiments with GBP and PGB indicated a similar mechanism of action to inhibit K(+)-evoked [(3)H]NE release. GBP and PGB are concluded to act in a comparable, if not identical, manner to preferentially attenuate [(3)H]NE release evoked by stimuli effecting mild and prolonged depolarizations. This type of modulation of neurotransmitter release may be integral to the clinical pharmacology of these drugs.     

Activation of presynaptic 5-hydroxytryptamine1-like receptors on glutamatergic terminals inhibits N-methyl-D-aspartate-induced cyclic GMP production in rat cerebellar slices.

In rat cerebellar slices, depolarization with 35 mM KCl caused increase of cyclic GMP (cGMP) production. This increase was Ca(++)-dependent, similar to the K(+)-evoked release of glutamate and aspartate in the same preparation. The K(+)-induced cGMP formation was inhibited in a concentration-dependent manner by D-(-)-2-amino-5-phosphonopentanoic acid (maximal inhibition 60-70%; IC50 = 0.019 microM) indicating the involvement of N-methyl-D-aspartate receptors probably activated by excitatory amino acids (EAAs) released by K(+)-depolarization. The cGMP production evoked by high-K+ was also potently inhibited by 5-hydroxytryptamine (5-HT; IC50 = 0.42 nM) or by 8-hydroxy-2-(di-N-propylamino)tetralin (8-OH-DPAT; IC50 = 1 nM). Methiothepin prevented the action of both 5-HT and 8-OH-DPAT. These data suggest the involvement of 5-HT1-like receptors. When added alone to the depolarized slices, methiothepin (0.03-3 microM) produced a concentration-dependent increase of cGMP suggesting that the 5-HT1-like receptors may be physiologically activated by the endogenous transmitter. Endogenous 5-HT released by (+)-fenfluramine (1 microM) or remaining in the biophase due to reuptake inhibition by citalopram (1 microM) caused reduction of cGMP compatible with a close apposition between 5-HT and EAA terminals. It can be concluded that activation (either direct or indirect) or blockade of presynaptic 5-HT1-like receptors previously found to be sited on EAA terminals in rat cerebellum where they mediate decrease of EAA release may profoundly affect the postsynaptic response elicited by EAA receptors coupled to guanylate cyclase.     

Ca2(+)-evoked [3H]dopamine release from synaptosomes is dependent on neuronal type Ca2+ channels and is not mediated by acetylcholine, glutamate or aspartate release

Elevation of potassium concentrations ([K+]) in the presence of Ca2+ is the most common method of evoking neurotransmitter release from synaptosomes. However, we have been investigating a method of releasing dopamine from synaptosomes that does not involve using elevated [K+]. In this paradigm of neurotransmitter release, dopamine is released from synaptosomes, previously exposed to micromolar or lower [Ca2+], by 1.25 mM Ca2+ in the presence of non-depolarizing [K+] (4.5 mM). The present experiments characterize the Ca2+ channel(s) involved in the Ca2(+)-evoked release of dopamine from synaptosomes, and determine whether the release is mediated by acetylcholine, glutamate or aspartate. omega-Conotoxin (10 nM), which blocks N-, L- and possibly T-type voltage-sensitive Ca2+ channels (VSCC), inhibited the Ca2(+)-evoked [3H]dopamine release from either striatal or olfactory tubercle synaptosomes to less than 50% of control. Neither 1 microM nifedipine nor 1 microM verapamil, which block L-type VSCC, affected Ca2(+)-evoked release. The N- and T-type VSCC blocker neomycin and the nonspecific Ca2+ antagonist, cobalt2+, inhibited release to a greater extent than omega-conotoxin. At 1 mM, both compounds inhibited release to approximately 30% of control. Neither the excitatory neurotransmitter glutamate nor aspartate (2mM) affected 1 microM LY-171555 (a dopamine D2 agonist) inhibition of Ca2(+)-evoked [3H]dopamine release. Also, the glutamate antagonist, glutamic acid diethyl ester, did not affect either Ca2(+)-evoked release or 1 microM LY-171555 inhibition thereof. The nicotinic antagonist hexamethonium (10 microM) and the muscarinic antagonist atropine (1 microM) were also ineffective in inhibiting Ca2(+)-evoked release or LY-171555 inhibition of release.(ABSTRACT TRUNCATED AT 250 WORDS)     

Serotonergic modulation of the release of endogenous norepinephrine from rat hypothalamic slices

Ca+(+)-dependent release of endogenous norepinephrine (NE) and dopamine from superfused rat hypothalamic slices was stimulated by 40 mM K+. 20 mM K+ released only NE. Two consecutive exposures to 20 mM K+ (S1 and S2, respectively) produced NE release of similar magnitude (S2/S1 = 1.03 +/- 0.08). Serotonin (5-HT), 3 to 10 microM, in the presence of methylsergide or ritanserin (antagonists at 5-HT1-like and 5-HT2 receptors), caused a concentration-dependent decrease of K(+)-evoked NE release. 5-HT alone did not alter K(+)-evoked NE release. 2-Methyl-serotonin, 2-methyl-5-hydroxytryptamine, 3 to 10 microM (a selective 5-HT3 agonist), mimicked the 5-HT response in the presence and in the absence of ritanserin. A highly selective 5-HT3 antagonist, (3 alpha-tropanyl)1H-indole-3-carboxylic acid ester (ICS 205-930), 1 nM, inhibited the effect of both agonists. The isomers of another highly selective 5-HT3 antagonist, zacopride, inhibited the effect of 2-methyl-serotonin, 2-methyl-5-hydroxytryptamine, at a concentration range, 0.03 to 20 nM, characteristic of their interaction with 5-HT3 receptors. alpha-Methyl-serotonin, alpha-methyl-5-hydroxytryptamine, a selective 5-HT1-like/5-HT2 agonist, failed to affect the K(+)-evoked NE release, but antagonized the effect of 2-methyl-serotonin, 2-methyl-5-hydroxytryptamine. These observations provide direct evidence that, in rat hypothalamus, 5-HT modulates release of endogenous NE through activation of 5-HT3 and, possibly, 5-HT1C receptors.     

Functional evidence for multiple gamma-aminobutyric acidB receptor subtypes in the rat cerebral cortex.

The aim of this work was the identification of pharmacologically distinct subtypes of gamma-aminobutyric acidB (GABAB) receptors in the central nervous system. Inasmuch as GABAB receptors are often sited on axon terminals where they mediate inhibition of transmitter release, we chose as models the GABAB receptors mediating inhibition of release of 1) endogenous GABA; 2) endogenous glutamate; and 3) somatostatin-like immunoreactivity (SRIF-LI). The experimental set up consisted of rat cerebrocortical synaptosomes depolarized in superfusion with 12 or 15 mM KCl. Endogenous GABA and glutamate were measured by high-performance liquid chromatography and SRIF-LI by radioimmunoassay. The selective GABAB receptor agonist (-)-baclofen inhibited in a concentration-dependent manner the K(+)-evoked release of GABA, glutamate and SRIF-Ll with similar potencies and efficacies [EC50 values, 1.1-1.5 microM; maximal inhibition, 45-50% at about 10 microM (-)-baclofen]. The GABAB receptor antagonist phaclofen concentration-dependently reduced the effects of (-)-baclofen on the release of GABA and SRIF-Ll but not on the release of glutamate, where it was ineffective up to 1000 microM. The rank order of potency (Ki values are shown in parentheses) are: SRIF-Ll (7.8 microM); GABA (10.4 microM); and glutamate (greater than 115 microM). The novel GABAB receptor antagonist 3-aminopropyl(diethoxymethyl) phosphinic acid (CGP 35348) displayed a different pattern on the three release systems examined (Ki values are shown in parentheses): SRIF-Ll (0.38 microM); glutamate (0.48 microM); and endogenous GABA (greater than 115 microM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Opposite effects of enantiomers of clofibric acid derivative on rat skeletal muscle chloride conductance: antagonism studies and theoretical modeling of two different receptor site interactions.

The R-(+) enantiomer of 2-(p-chlorophenoxy) propionic acid (CPP) produces a biphasic effect on chloride channel conductance (GCl) of rat skeletal muscle, increasing GCl at low concentrations and decreasing it at concentrations greater than 10 microM; on the other hand, the S-(-) isomer mostly blocks GCl in a concentration-dependent manner. To explain the peculiar behavior of these compounds, a theoretical model based on the presence of two opposing receptor populations controlling chloride channel conductance has been used to fit the experimental data of the concentration-response curves of both S-(-) and R-(+) CPP. An analysis performed by means of the algebraic summation of two logistic terms suggests a reasonable merit of the proposed model and explains the resultant effect of each optical form as follows: S-(-) acts as a full agonist on an inhibitory sites, whereas R-(+) acts as a full agonist at both the inhibitory and excitatory sites. Antagonism studies appear to be consistent with the proposed model. Dose-response curves in which the block of GCl by the S-(-) isomer was evaluated in the presence of the R-(+) isomer (3-10 microM) clearly showed an antagonistic interaction between the two enantiomers, with an increase in the S-(-) concentration for half-maximal block. The antagonism was overcome by high concentrations of S-(-), and this might be consistent with the hypothesis that the block of GCl is modulated by an inhibitory site at which the two enantiomers compete.     

gamma-Aminobutyric acidB receptors mediate inhibition of somatostatin release from cerebrocortex nerve terminals.

The effects of gamma-aminobutyric acid (GABA) and of various GABA receptor agonists and antagonists on the calcium-dependent depolarization-evoked release of somatostatin (SRIF) from rat cerebrocortex synaptosomes have been studied by a superfusion technique. GABA (0.3-30 microM) decreased the K+ (15 mM)-evoked overflow of SRIF-like immunoreactivity (SRIF-LI) in a concentration-dependent manner (EC50 = 1.3 microM; maximal inhibition, 45% reached at 10 microM GABA). The effect of the amino acid was insensitive to the GABAA receptor antagonist bicuculline. Accordingly, the K(+)-evoked SRIF-LI release was not affected by muscimol, a GABAA receptor agonist, up to 100 microM. The effect of GABA was mimicked by the GABAB receptor agonist (-)-baclofen (EC50 = 1.2 microM; maximal effect, about 45% reached at 10 microM). The effect of baclofen was stereoselective, the (+)-enantiomer being inactive up to 100 microM. The inhibition of SRIF-LI release brought about by GABA was sensitive to the GABAB receptor antagonists 2-hydroxy-saclofen and CGP 35348 [3-aminopropyl(diethoxymethyl)phosphinic acid]. Also, the effect of (-)-baclofen was antagonized by CGP 35348 (IC50 = 4.8 microM). It is concluded that GABA can inhibit the depolarization-evoked release of SRIF by activating receptors which are located on SRIF-releasing nerve terminals and belong to the GABAB type.     

Muscarinic receptors mediating inhibition of gamma-aminobutyric acid release in rat corpus striatum and their pharmacological characterization

The effects of acetylcholine (ACh) and of cholinergic agonists on the release of tritiated gamma-aminobutyric acid ([3H]GABA) were studied in superfused synaptosomes prepared from rat corpus striatum and prelabeled with the radioactive amino acid. ACh, oxotremorine or (-)-nicotine, all tested at 100 microM had no effect on the spontaneous outflow of [3H]GABA. The depolarization-evoked overflow obtained by exposing the synaptosomes to 9 mM KCl was decreased in a concentration-dependent manner by ACh, oxotremorine, oxotremorine-M or carbachol. The maximal inhibition caused by ACh was 50%. The EC50 (agonist concentration causing half-maximal effect) amounted to 1 microM. Oxotremorine and oxotremorine-M were almost equipotent to ACh, whereas the concentration-response curve of carbachol was slightly (although not significantly) shifted to the right with respect to that of ACh. (-)-Nicotine (100 microM) did not affect the K(+)-evoked [3H]GABA overflow. ACh also inhibited the K(+)-evoked release of endogenous GABA. The inhibitory effect of 10 microM ACh on the release of [3H]GABA evoked by 9 mM KCl was insensitive to the nicotinic antagonist mecamylamine (10 microM) but it was potently blocked by the muscarinic antagonist atropine (IC50 = 5 nM) and weakly antagonized by pirenzepine, dicyclomine and AF-DX 116. The pharmacological profile of this receptor was very similar to that of the muscarinic autoreceptors regulating [3H]ACh release. The extent of [3H]GABA release inhibition caused by ACh did not differ between dorsal and ventral striatum. The inhibitory effect of ACh was much less pronounced in hippocampus and cortex than in the striatum.(ABSTRACT TRUNCATED AT 250 WORDS)     

Selective depression by general anesthetics of glutamate versus GABA release from isolated cortical nerve terminals

The role of presynaptic mechanisms in general anesthetic depression of excitatory glutamatergic neurotransmission and facilitation of GABA-mediated inhibitory neurotransmission is unclear. A dual isotope method allowed simultaneous comparisons of the effects of a representative volatile (isoflurane) and intravenous (propofol) anesthetic on the release of glutamate and GABA from isolated rat cerebrocortical nerve terminals (synaptosomes). Synaptosomes were prelabeled with L-[(3)H]glutamate and [(14)C]GABA, and release was determined by superfusion with pulses of 30 mM K(+) or 1 mM 4-aminopyridine (4AP) in the absence or presence of 1.9 mM free Ca(2+). Isoflurane maximally inhibited Ca(2+)-dependent 4AP-evoked L-[(3)H]glutamate release (99 +/- 8% inhibition) to a greater extent than [(14)C]GABA release (74 +/- 6% inhibition; P = 0.023). Greater inhibition of L-[(3)H]glutamate versus [(14)C]GABA release was also observed for the Na(+) channel antagonists tetrodotoxin (99 +/- 4 versus 63 +/- 5% inhibition; P < 0.001) and riluzole (84 +/- 5 versus 52 +/- 12% inhibition; P = 0.041). Propofol did not differ in its maximum inhibition of Ca(2+)-dependent 4AP-evoked L-[(3)H]glutamate release (76 +/- 12% inhibition) compared with [(14)C]GABA (84 +/- 31% inhibition; P = 0.99) release. Neither isoflurane (1 mM) nor propofol (15 microM) affected K(+)-evoked release, consistent with a molecular target upstream of the synaptic vesicle exocytotic machinery or voltage-gated Ca(2+) channels coupled to transmitter release. These findings support selective presynaptic depression of excitatory versus inhibitory neurotransmission by clinical concentrations of isoflurane, probably as a result of Na(+) channel blockade.     

Inhibition of [3H] γ-aminobutyric acid release from guinea-pig hippocampal synaptosomes by serotonergic agents

We studied the effects of (m-trifluoromethyl-phenyl)piperazine (TFMPP) and quipazine on the K(+)-evoked [3H]GABA release from guinea-pig hippocampal synaptosomes loaded with [3H]GABA.TFMPP and quipazine inhibited the K(+)-evoked release of [3H]GABA dose-dependently (IC50 = 153 and 123 microM, respectively). Serotonergic antagonists such as methiothepin (0.1, 0.3 and 1 microM), ketanserin (0.1, 0.3 and 1 microM), dihydroergotamine (0.1 microM), metergoline (0.1 and 0.3 microM), methysergide (0.3 microM), propranolol (1 microM) and yohimbine (1 microM) did not significantly alter the inhibitory effect of TFMPP on [3H]GABA release suggesting that neither 5-HT1 nor 5-HT2 receptors are involved in this process. By contrast, the effect of TFMPP was diminished by selective 5-HT3 receptor antagonist: MDL 72222 (0.3 microM), tropisetron (0.3 and 1 microM), ondansetron (0.3 microM) and metoclopramide (1 microM). Tropisetron (1 microM) and ondansetron (0.3 microM) also inhibited significantly the quipazine effect whereas methiothepin (1 microM), dihydroergotamine (0.1 microM), yohimbine (1 microM) and ketanserin (1 microM) were ineffective on the quipazine inhibition of [3H]GABA release. Our results show a serotonergic modulatory effect on the K(+)-evoked [3H]GABA release from guinea-pig hippocampal synaptosomes by receptors which are neither 5-HT1, 5-HT2 or 5-HT4. They appear to be pharmacologically related to the 5-HT3 type but different from the 5-HT3 ionic channel receptors.     

Involvement of adenosine in the spinal antinociceptive effects of morphine and noradrenaline

Intrathecal (i.t.) pretreatment of rats with either theophylline (50 micrograms) or 8-phenyltheophylline (3 micrograms) antagonized antinociception produced by i.t. injection of morphine (0.3-3 micrograms) in the tailflick and hotplate tests, but had no effect on antinociception produced by i.t. injection of noradrenaline (10-30 micrograms). In other experiments designed to test whether morphine released adenosine from the spinal cord, adenosine release from synaptosomes was measured by high-performance liquid chromatography with fluorescence detection of etheno-adenosine. Depolarization with 24 mM K+ or 50 microM veratridine released 3 times as much adenosine from dorsal than from ventral spinal cord synaptosomes. K+ released primarily adenosine whereas veratridine released both adenosine and nucleotide(s). Morphine (1-100 microM) produced a Ca++-dependent release of endogenous adenosine, comparable to K+-evoked adenosine release, which was blocked by 1 microM naltrexone. Noradrenaline (5-500 microM) produced a Ca++-dependent release of a nucleotide which was subsequently degraded extracellularly to adenosine by ecto-5'-nucleotidase. This release was antagonized by 10 microM phentolamine and by 1 microM yohimbine. These results suggest that, within the spinal cord, morphine may act on opioid receptors to release adenosine which subsequently acts at adenosine receptors to produce spinal analgesia. Spinal analgesia produced by noradrenaline does not appear to involve adenosine release.     

The novel N-type calcium channel blocker, AM336, produces potent dose-dependent antinociception after intrathecal dosing in rats and inhibits substance P release in rat spinal cord slices

N-type calcium channels modulate the release of key pro-nociceptive neurotransmitters such as glutamate and substance P (SP) in the central nervous system. Considerable research interest has focused on the therapeutic potential of the peptidic omega-conopeptides, GVIA and MVIIA as novel analgesic agents, due to their potent inhibition of N-type calcium channels. Recently, the novel peptidic N-type calcium channel blocker, AM336, was isolated from the venom of the cone snail, Conus catus. Thus, the aims of this study were to (i) document the antinociceptive effects of AM336 (also known as CVID) relative to MVIIA following intrathecal (i.t.) bolus dosing in rats with adjuvant-induced chronic inflammatory pain of the right hindpaw and to (ii) quantify the inhibitory effects of AM336 relative to MVIIA on K+-evoked SP release from slices of rat spinal cord. Both AM336 and MVIIA inhibited the K+-evoked release of the pro-nociceptive neurotransmitter, SP, from rat spinal cord slices in a concentration-dependent manner (EC50 values=21.1 and 62.9 nM, respectively), consistent with the antinociceptive actions of omega-conopeptides. Following acute i.t. dosing, AM336 evoked dose-dependent antinociception (ED50 approximately 0.110 nmol) but the doses required to produce side-effects were an order of magnitude larger than the doses required to produce antinociception. For i.t. doses of MVIIA0.07 nmol, produced a dose-dependent decrease in antinociception but the incidence and severity of the side-effects continued to increase for all doses of MVIIA investigated, suggesting that dose-titration with MVIIA in the clinical setting, may be difficult.     

Inhibitory presynaptic 5-hydroxytryptamine(2A) receptors regulate evoked glutamate release from rat cerebellar mossy fibers.

We studied the pharmacological characterization of the 5-hydroxytryptamine(2) (5-HT(2)) heteroreceptor located on glutamatergic cerebellar mossy fiber nerve terminals. Depolarization-evoked overflow of endogenous glutamate from rat cerebellar "giant" mossy fiber synaptosomes was inhibited by 5-HT or (+/-)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane [(+/-)-DOI], exhibiting pD(2) (= -log EC(50)) values of 7.37 and 7.29, respectively. Trazodone inhibited the depolarization-evoked glutamate overflow, exhibiting lower potency (pD(2) = 6.42) and lower efficacy with respect to 5-HT or (+/-)-DOI (maximal inhibition, 54%, compared with 70% for either 5-HT or (+/-)-DOI). Ketanserin, a 5-HT(2A)/5-HT(2C) receptor antagonist, counteracted the inhibitory effect of (+/-)-DOI or trazodone. Inhibition of glutamate overflow by 5-HT, (+/-)-DOI, or trazodone was prevented by the selective 5-HT(2A) receptor antagonist R-(+)-alpha-(2,3-dimethyoxyphenyl)-1-(2-(4-fluorophenyl)ethyl)-4-piperidine-methanol (MDL 100907), while the potent and selective 5-HT(2C) receptor antagonist 6-chloro-5-methyl-1-[6-(methylpyridin-3-yloxy)pyridin-3yl-carbamoyl] indoline (SB 242084) was ineffective. In cerebellar slices, MDL 100907 increased on its own the K(+)-evoked release of glutamate. It is concluded that the evoked release of glutamate from cerebellar mossy fibers can be controlled by inhibitory presynaptic 5-HT(2A) heteroreceptors, the receptors can be activated by endogenously released 5-HT, and trazodone behaves as a partial agonist at these receptors.     

A further evaluation of the effects of K+ depolarization on glutamate-evoked [3H]dopamine release from striatal slices.

Exogenous glutamate will evoke dopamine (DA) release from striatal slices in vitro. To further characterize glutamate-evoked DA release from striatal slices, experiments were designed to: 1) determine if sufficient endogenous glutamate can be released in vitro to presynaptically mediate [3H]DA release in the absence of Mg++ and 2) reevaluate how K+ depolarization affects glutamate-evoked [3H]DA release. Removal of Mg++ to potentiate N-methyl-D-aspartate (NMDA) receptor-mediated DA release increased 15 mM K(+)-evoked [3H]DA release to about 200% of control. The potentiation of this release was probably not mediated by NMDA receptors because it was not blocked by the glutamate receptor antagonists MK-801, 6,7-dinitroquinoxalinedione (DNQX) or kynurenate. Furthermore, the removal of Mg++ increased DA release substantially (200%) in the presence of 5 microM sulpiride and 10 microM nomifensine, indicating that DA reuptake and DA D2 autoreceptors are not primarily responsible for increased DA release. In the absence of Mg++, depolarization produced by 20 mM or greater [K+] inhibited DA released by exogenous glutamate, whereas a much higher [K+] was necessary to evoke endogenous glutamate release. In the presence of 1.5 mM Mg++, a reduction of the "Mg++ blockade" of NMDA receptors by 15 mM K+ depolarization during glutamate-evoked DA release was evaluated with and without the DA reuptake inhibitor nomifensine and the DA D2 antagonist sulpiride. DA released by K+ depolarization (Mg++ present) was markedly increased by 1 mM glutamate, but this effect was only partially reversed by kynurenate or high concentrations of either MK-801 (25 microM) or DNQX (100 microM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Enantioselective disposition of ofloxacin in humans.

The enantioselective disposition of ofloxacin (OFLX) was studied in healthy subjects after oral administration of (+/-)-OFLX at a dose of 200 mg. S-(-)-OFLX and R-(+)-OFLX concentrations in serum and urine were measured separately by high-performance liquid chromatography, and various pharmacokinetic parameters were calculated from the data. The ratio of S-(-) to R-(+) enantiomer concentrations in serum showed a increase with time, with S/R ratios of 1.01 at 2 h and 1.31 at 24 h. The terminal elimination half-life of S-(-)-OFLX was 6.9 h, which was significantly greater (P less than 0.05) than that of the R-(+) enantiomer (6.3 h). S-(-)-OFLX also revealed a significantly greater area under the concentration-time curve in serum, mean residence time, and total body clearance than the R-(+) enantiomer did. The renal clearance of S-(-)-OFLX (7.14 liters/h/1.73 m2) was significantly lower than that of the R-(+) enantiomer (7.53 liters/h/1.73 m2). Although the difference in the pharmacokinetic parameters of the enantiomers was small, their disposition in humans was found to be stereoselective. The difference between the enantiomers may be explained by the difference in their renal excretion.     

Endothelin (ET)-1-induced inhibition of ATP release from PC-12 cells is mediated by the ETB receptor: differential response to ET-1 on ATP, neuropeptide Y, and dopamine levels

During sympathetic neurotransmitter release, there is evidence for differential modulation of cotransmitter release by endothelin (ET)-1. Using nerve growth factor (NGF)-differentiated PC12 cells, the effects of ET-1 on K(+)-stimulated release of ATP, dopamine (DA), and neuropeptide Y (NPY) were quantified using high-pressure liquid chromatography or radioimmunoassay. ET-1, in a concentration-dependent manner, inhibited the release of ATP, but not DA and NPY. Preincubation with the ET(A/B) antagonist, PD 142893 (N-acetyl-beta-phenyl-D-Phe-Leu-Asp-Ile-Ile-Trp), reversed the inhibitory effect of ET-1 on ATP release, which remained unaffected in the presence of the ET(A)-specific antagonist BQ123 [cyclo(D-Asp-Pro-D-Val-Leu-D-Trp)]. The ET(B) agonists, sarafotoxin 6c (Cys-Thr-Cys-Asn-Asp-Met-Thr-Asp-Glu-Glu-Cys-Leu-Asn-Phe-Cys-His-Gln-Asp-Val-Ile-Trp), BQ 3020 (N-acetyl-[Ala(11,15)]-endothelin 1 fragment 6-21Ac-Leu-Met-Asp-Lys-Glu-Ala-Val-Tyr-Phe-Ala-His-Leu-Asp-IIe-IIe-Trp), and IRL 1620 (N-succinyl-[Glu(9), Ala(11,15)]-endothelin 1 fragment 8-21Suc-Asp-Glu-Glu-Ala-Val-Tyr-Phe-Ala-His-Leu-Asp-Ile-Ile-Trp), decreased K(+)-stimulated release of ATP in a dose-dependent manner, and this effect was reversed by the ET(B) antagonists RES 701-1 [cyclic (Gly1-Asp9) (Gly-Asn-Trp-His-Gly-Thr-Ala-Pro-Asp-Trp-Phe-Phe-Asn-Tyr-Tyr-Trp)] and BQ 788 (N-[N-[N-[(2,6-dimethyl-1-piperidinyl)carbonyl]-4-methyl-l-leucyl]-1-(methoxycarbonyl)-D-tryptophyl]-D-norleucine sodium salt). Preincubation of PC12 cells with pertussis toxin reversed the ET-1-induced inhibition of the K(+)-evoked ATP release. Real-time intracellular calcium level recordings were performed on PC-12 cell suspensions, and ET-1 induced a dose-dependent decrease in the K(+)-evoked calcium levels. Nifedipine, the L-type voltage-dependent Ca(2+) channel antagonist, caused inhibition of the K(+)-stimulated ATP release, but the N-type Ca(2+) channel antagonist, omega-conotoxin GVIA, did not reverse the effect on ATP release. These data suggest that ET-1 modulates the release of ATP via the ET(B) receptor and its associated G(i/o) G-protein through attenuation of the influx of extracellular Ca(2+) through L-type channels.     

Polymorphism in hydroxylation of mephenytoin and hexobarbital stereoisomers in relation to hepatic P-450 human-2

Stereoselective 4'-hydroxylations of R-(-)-mephenytoin and S-(+)-mephenytoin and 3'-hydroxylation of R-(-)-hexobarbital and S-(+)-hexobarbital were determined in liver microsomes of 14 Japanese subjects who were extensive metabolizers of mephenytoin and in five Japanese subjects who were poor metabolizers of mephenytoin. Content of P-450 human-2 assessed by Western blots was correlated to microsomal S-(+)-mephenytoin 4'-hydroxylation, R-(-)-hexobarbital 3' alpha-hydroxylation, and S-(+)-hexobarbital 3' beta-hydroxylation, and was less correlated to R-(-)mephenytoin 4'-hydroxylation, R-(-)-hexobarbital 3' beta-hydroxylation, and S-(+)-hexobarbital 3' alpha-hydroxylation. Antibodies raised against P-450 human-2 inhibited microsomal S-(+)-mephenytoin 4'-hydroxylation efficiently but was less efficient on R-(-)-mephenytoin 4'-hydroxylation in extensive metabolizers and on 4'-hydroxylation of mephenytoin enantiomers in poor metabolizers. The antibodies also inhibited R-(-)-hexobarbital 3' alpha-hydroxylation and S-(+)-hexobarbital 3' beta-hydroxylation but did not effectively inhibit the hydroxylation of the two other optical isomers of hexobarbital in extensive metabolizers and of four stereoisomers in poor metabolizers. These findings indicate the close relationship between polymorphic mephenytoin 4'-hydroxylation and two stereospecific hexobarbital hydroxylations, and they suggest that P-450 human-2 is a typical S-(+)-mephenytoin 4'-hydroxylase and a major hexobarbital 3'-hydroxylase in the livers of extensive metabolizers. The findings were further supported by the experiments that used P-450 human-2 complementary dexoyribonucleic acid-derived protein in yeast microsomes.     

Differential effects of diazoxide, cromakalim and pinacidil on adrenergic neurotransmission and 86Rb+ efflux in rat brain cortical slices.

The effects of diazoxide, cromakalim and pinacidil on depolarization-evoked tritium overflow from the rat brain cortical slices preloaded with [3H]noradrenaline were studied. Diazoxide inhibited both transmural nerve stimulation (TNS)- and 25 mM K(+)-evoked tritium overflows more potently than cromakalim. Diazoxide effects were only partially antagonized and cromakalim ones were totally reversed by 1 microM glibenclamide. Diazoxide, but not cromakalim, reduced the 45 mM K(+)-evoked tritium overflow, which was not antagonized by glibenclamide. Both diazoxide and cromakalim stimulated 86Rb+ efflux to a similar extent, the effects being completely abolished by glibenclamide. Glibenclamide (> or = 3 microM) by itself enhanced the TNS-evoked tritium overflow. Pinacidil increased both TNS- and K+ (25 and 45 mM)-evoked tritium overflows with little effect on 86Rb+ efflux. Pinacidil-induced increase in the TNS-evoked tritium overflow was still observed in the presence of cocaine or hydrocortisone. Pinacidil failed to affect the inhibitory action of xylazine on the TNS-evoked tritium overflow, whereas phentolamine attenuated it. These results indicate that ATP-sensitive K+ channels are present in the adrenergic nerve endings of rat brain. These channels seem to be pharmacologically different from those reported for vascular smooth muscles and pancreatic beta-cells.     

KW-7158 [(2S)-(+)-3,3,3-trifluoro-2-hydroxy-2-methyl-N-(5,5,10-trioxo-4,10-dihydrothieno[3,2-c][1]benzothiepin-9-yl)propanamide] enhances A-type K+ currents in neurons of the dorsal root ganglion of the adult rat

Recent studies revealed that a new compound, KW-7158 [(2S)-(+)-3,3,3-trifluoro-2-hydroxy-2-methyl-N-(5,5,10-trioxo-4,10-dihydrothieno[3,2-c][1]benzothiepin-9-yl)propanamide], can depress the excitability of afferent pathways from the urinary bladder and reduce bladder overactivity induced by chemical irritation of the urinary tract with xylene, an agent that sensitizes capsaicin-sensitive, C-fiber afferent nerves. In the present experiments, we examined the mechanisms that might underlie the depressant effect of KW-7158 on primary afferent neurons by studying the actions of the compound on ion channels and firing in dissociated dorsal root ganglion (DRG) cells from adult rats using whole cell patch-clamp techniques. KW-7158 increased transient, A-type K+ currents at concentrations ranging from 50 nM to 1 microM (20-50% increases). Similar effects were seen in fast blue identified bladder afferent neurons. Low concentrations of KW-7158 shortened the action potential duration, produced a 5- to 10-mV hyperpolarization, and inhibited repetitive firing induced by either 4-AP (50 microM) or substance P (0.5 microM) in phasic firing DRG neurons. Above 1 microM, KW-7158 elicited a smaller enhancement of A-type K+currents and in high concentrations inhibited the currents. Tetraethylammonium (5-60 mM) and verapamil (50 microM), which block noninactivating K+ currents, did not prevent the facilitatory effects of KW-7158. High concentrations of 4-AP (5 mM) inhibited A-type K+ currents and prevented the facilitatory effect of KW-7158 on the remaining currents. These data suggest that KW-7158 enhances A-type K+ currents in DRG neurons. Because A-type K+ channels regulate afferent neuron excitability and firing properties, KW-7158 is a promising new compound for treatment of hyper-reflexic bladder conditions.