Identification and characterization of a high-affinity peripheral-type benzodiazepine receptor in rabbit urinary bladder

The present study used radioligand binding and in vitro contractility experiments to identify and characterize a peripheral-type benzodiazepine receptor PBR in rabbit urinary bladder. [3H]PK11195 bound to bladder membranes with high-affinity and density (Kd = 5.2 nM., Bmax = 268 fmol./mg. protein), indicating the presence of a PBR. [3H]flunitrazepam bound with high-affinity and density (Kd = 1.2 nM., Bmax = 48 fmol./mg. protein). The rank order potency of various benzodiazepines and isoquinoline carboxamides in displacing the binding of [3H]PK11195 was Ro5-4864 > diazepam = flunitrazepam > Ro15-1788 = clonazepam. Ro5-4864 and PK11195 inhibited nerve-evoked contractions in a concentration-dependent manner (IC50 = 42 microM. and 56 microM., respectively). Carbachol- and KCl-induced contractions were also inhibited by Ro5-4864 and PK11195. KCl-induced contractions were inhibited to a greater extent than carbachol-induced or field-stimulated contractions with all the drugs tested. Both Ro5-4864 and PK11195 significantly increased the ED50 for calcium-induced contractions following a cholinergic stimulus compared with control. These data demonstrate the presence of a PBR in urinary bladder capable of altering contractility in vitro through modulation of calcium activity.     

Pharmacologic actions of the second-generation leukotriene B4 receptor antagonist LY293111: in vitro studies

The in vitro actions were investigated of LY293111, a potent and selective leukotriene B4 (LTB4) receptor antagonist, on human neutrophils, human blood fractions, guinea pig lung membranes, and guinea pig parenchymal and tracheal strips. The IC50 for inhibiting [3H]LTB4 binding to human neutrophils was 17.6 +/- 4.8 nM. LY293111 inhibited LTB4-induced human neutrophil aggregation (IC50 = 32 +/- 5 nM), luminol-dependent chemiluminescence (IC50 = 20 +/- 2 nM), chemotaxis (IC50 = 6.3 +/- 1.7 nM), and superoxide production by adherent cells (IC50 = 0.5 nM). Corresponding responses induced by N-formyl-L-methionyl-L-leucyl-L-phenylalanine were inhibited by 100-fold higher concentrations of LY293111. LTB4 binding to guinea pig tissues and subsequent activation were also inhibited. The Ki for inhibition of [3H]LTB4 binding to lung membranes was 7.1 +/- 0.8 nM; IC50 for preventing binding of [3H]LTB4 to spleen membranes was 65 nM. The compound inhibited LTB4-induced contraction of guinea pig lung parenchyma. At 10 nM, LY293111 caused a parallel rightward shift of the LTB4 concentration-response curve. At higher concentrations, plots were shifted in a nonparallel manner, and maximum responses were depressed. LY293111 did not prevent antigen-stimulated contraction of sensitized trachea strips. At micromolar concentrations, LY293111 inhibited production of LTB4 and thromboxane B2 by plasma-depleted human blood stimulated with N-formyl-L-methionyl-L-leucyl-L-phenylalanine and thrombin. In addition, at these higher concentrations, formation of LTB4 by A23187-activated whole blood and conversion of arachidonic acid to LTB4 by a human neutrophil cytosolic fraction were inhibited. In summary, LY293111 is a second-generation LTB4 receptor antagonist with much improved potency in a variety of functional assay systems.     

Pharmacological characterization of metabotropic glutamate receptors linked to the inhibition of adenylate cyclase activity in rat striatal slices

The pharmacological profile of mGlu receptors negatively linked to adenylyl cyclase was characterized in adult rat striatal slices. Among the mGlu agonists tested, (+)-2-aminobicyclo-[3.1.0]-hexane-2,6-di carboxylate (LY354740), was the most potent inhibitor of forskolin-stimulated cAMP formation (EC50 = 11 +/- 2 nM). Inhibition of forskolin stimulation by the group III agonist L-2-amino-4-phosphono-butanoate (L-AP4) was biphasic, the two parts of the concentration curve having EC50 values of 6 +/- 1 microM and 260 +/- 4 microM, suggesting a sequential recruitment of mGlu4/8 and mGlu7. The effects of several new phenylglycine derivative antagonists were tested on the inhibition of forskolin cAMP response by (2S,1'S,2'S)-2-(carboxy-cyclopropyl)-glycine (L-CCG I) and L-AP4. At 500 microM, (RS)-alpha-methyl-3-carboxy-methyl-pheny lglycine was unable to antagonize the effect of L-CCG I or L-AP4 but (S)-alpha-methyl-3-carboxy-phenylalanine inhibited the effect of L-AP4 with a low potency. Finally, (RS)-alpha-methyl-4-tetrazolylphenylglyc ine and particularly (RS)-alpha-methyl-4-phosphonophenylglyci ne, appeared to be the most potent and selective antagonists of L-AP4 induced inhibition of forskolin-stimulated cAMP production in adult rat striatal slices.     

Characterization of (2S,2'R,3'R)-2-(2',3'-[3H]-dicarboxycyclopropyl)glycine binding in rat brain

[(2S,2'R,3'R)-2-(2',3'-[3H]Dicarboxycyclopropyl)glycine ([3H]DCG IV) binding was characterized in vitro in rat brain cortex homogenates and rat brain sections. In cortex homogenates, the binding was saturable and the saturation isotherm indicated the presence of a single binding site with a K(D) value of 180 +/- 33 nM and a Bmax of 780 +/- 70 fmol/mg of protein. The nonspecific binding, measured using 100 microM LY354740, was <30%. NMDA, AMPA, kainate, L(-)-threo-3-hydroxyaspartic acid, and (S)-3,5-dihydroxyphenylglycine were all inactive in [3H]DCG IV binding up to 1 mM. However, several compounds inhibited [3H]DCG IV binding in a concentration-dependent manner with the following rank order of potency: LY341495 = LY354740 > DCG IV = (2S,1'S,2'S)-2-(2-carboxycyclopropyl)glycine > (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid > (2S,1'S,2'S)-2-methyl-2-(2-carboxycyclopropyl)glycine > L-glutamate = ibotenate > quisqualate > (RS)-alpha-methyl-4-phosphonophenylglycine = L(+)-2-amino-3-phosphonopropionic acid > (S)-alpha-methyl-4-carboxyphenylglycine > (2S)-alpha-ethylglutamic acid > L(+)-2-amino-4-phosphonobutyric acid. N-Acetyl-L-aspartyl-L-glutamic acid inhibited the binding in a biphasic manner with an IC50 of 0.2 microM for the high-affinity component. The binding was also affected by GTPgammaS, reducing agents, and CdCl2. In parasagittal sections of rat brain, a high density of specific binding was observed in the accessory olfactory bulb, cortical regions (layers 1, 3, and 4 > 2, 5, and 6), caudate putamen, molecular layers of the hippocampus and dentate gyrus, subiculum, presubiculum, retrosplenial cortex, anteroventral thalamic nuclei, and cerebellar granular layer, reflecting its preferential (perhaps not exclusive) affinity for pre- and postsynaptic metabotropic glutamate mGlu2 receptors. Thus, the pharmacology, tissue distribution, and sensitivity to GTPgammaS show that [3H]DCG IV binding is probably to group II metabotropic glutamate receptors in rat brain.     

Base and fog densities of fresh Ektaspeed Plus dental X-ray films

Binding of [3H]ethynylbicycloorthobenzoate ([3H]EBOB) to the gamma-aminobutyric acid type A (GABAA) receptor of cultured cerebellar granule neurons is inhibited by GABA, muscimol and 3-aminopropanesulfonic acid with IC50 values of 69-250 nM. Sensitivity to these GABA mimetics is lower by 3-4-fold for cerebellum and 10-20-fold for cerebral cortex, midbrain, and pons and medulla, a differential sensitivity by brain region and cell type consistent with earlier findings using tert-[35S]butylbicyclophosphorothionate and GABA. In contrast, the inhibitory potencies of two chloride channel blockers, alpha-endosulfan and picrotoxinin, do not differ in these assays. The hypothesis that this pharmacological profile is conferred by the alpha 6 subunit specific to cerebellar granule cells is supported by the finding that forskolin (which downregulates the alpha 6 subunit) but not the inactive dideoxyforskolin markedly decreases the sensitivity of [3H]EBOB binding to GABA without affecting inhibition by alpha-endosulfan.     

L-lysine is a barbiturate-like anticonvulsant and modulator of the benzodiazepine receptor

Our earlier observations showed that L-lysine enhanced the activity of diazepam against seizures induced by pentylenetetrazol (PTZ), and increased the affinity of benzodiazepine receptor binding in a manner additive to that caused by gamma-aminobutyric acid (GABA). The present paper provides additional evidence to show that L-lysine has central nervous system depressant-like characteristics. L-lysine enhanced [3H]flunitrazepam (FTZ) binding in brain membranes was dose-dependent and stimulated by chloride, bromide and iodide, but not fluoride. Enhancement of [3H]FTZ binding by L-lysine at a fixed concentration was increased by GABA but inhibited by pentobarbital between 10(-7) to 10(-3)M. While GABA enhancement of [3H]FTZ binding was inhibited by the GABA mimetics imidazole acetic acid and tetrahydroisoxazol pyridinol, the enhancement by pentobarbital and L-lysine of [3H]FTZ binding was dose-dependently increased by these two GABA mimetics. The above results suggest that L-lysine and pentobarbital acted at the same site of the GABA/benzodiazepine receptor complex which was different from the GABA binding site. The benzodiazepine receptor antagonist imidazodiazepine Ro15-1788 blocked the antiseizure activity of diazepam against PTZ. Similar to pentobarbital, the anti-PTZ effect of L-lysine was not blocked by Ro15-1788. Picrotoxinin and the GABA, receptor antagonist bicuculline partially inhibited L-lysine's enhancement of [3H]FTZ binding with the IC50s of 2 microM and 0.1 microM, respectively. The convulsant benzodiazepine Ro5-3663 dose-dependently inhibited the enhancement of [3H]FTZ binding by L-lysine. This article shows the basic amino acid L-lysine to have a central nervous system depressant characteristics with an anti-PTZ seizure activity and an enhancement of [3H]FTZ binding similar to that of barbiturates but different from GABA.     

Protein kinase C inhibitors block amphetamine-mediated dopamine release in rat striatal slices

The stimulant drug amphetamine is postulated to enhance dopamine release through the plasmalemmal dopamine transporter by an exchange diffusion with synaptosomal dopamine. Because protein kinase C has been shown to have an effect on dopamine transporter activity, we examined the effect of protein kinase C inhibitors on endogenous dopamine release stimulated by amphetamine in perfused rat striatal slices. At concentrations of 1 microM, the selective protein kinase C inhibitors chelerythrine, Ro31-8220 and calphostin C nearly completely inhibited endogenous dopamine release elicited by 1 microM amphetamine. The inactive analog bisindoylmaleimide V had no effect. Extracellular Ca++ was not required for the effect of the inhibitors. The importance of vesicular dopamine release was examined by determining inhibitor activity in reserpine-treated rats. Dopamine release elicited by 1 microM amphetamine was not significantly altered in reserpine-treated rats compared with control animals. Ro31-8220 at 1 microM completely blocked amphetamine-induced dopamine release in reserpine-treated rats. Activation of protein kinase C with 250 nM of the phorbol ester 12-O-tetradecanoylphorbol 13-acetate increased dopamine release, and the release was not additive with 1 microM amphetamine. Both chelerythrine and Ro31-8220 at 1 microM increased [3H]dopamine uptake by 17% and 30%, respectively, whereas a brief exposure to 12-O-tetradecanoylphorbol 13-acetate slightly inhibited [3H]dopamine uptake. Our results suggest that amphetamine-mediated dopamine release through the plasmalemmal transporter is highly dependent on protein kinase C activity.     

The specific binding of the platelet-activating factor (PAF) receptor antagonist WEB 2086 and the benzodiazepine flunitrazepam to rat hepatocytes

Thieno-triazolodiazepines WEB 2086 and BN 50739 have been described as the potent PAF receptor antagonists. Binding of radiolabeled [3H]WEB 2086 has been widely employed to characterize PAF receptors in different cells. In a search for a PAF receptor in isolated rat hepatocytes, we discovered that the binding of [3H]WEB to rat hepatocytes was highly specific but had a relatively low affinity with a Kd of 113 nM and Bmax of 0.65 pmol/10(6) cells in freshly isolated cell suspension and Kd of 1.65 muM and Bmax of 2.0 pmol/plate in cultured hepatocytes. No consistent specific binding of [3H]PAF itself was found in the same cell preparations. The binding of [3H]flunitrazepam in the presence of the peripheral type of benzodiazepine receptor antagonist Ro 5-4864 was saturated and exhibited a K(i) of 3.8 nM and Bmax of 3.5 pmol/plate. The central type of benzodiazepine receptor antagonist clonazepam was competed for the [3H]flunitrazepam binding, however with a much lower affinity. Various antagonists inhibited the binding of [3H]WEB 2086 with a rank order BN 50739>Ro 5-4864 > or = clonazepam. Interestingly, bicuculline, specific antagonist of GABA(A) recognition sites, also significantly reduced the binding of [3H]WEB 2086. The binding of [3H]flunitrazepam was inhibited with a rank potency BN 50739>WEB 2086. Taken together, these findings suggest that the specific binding of PAF receptor antagonists WEB 2086 and BN 50739 in rat hepatocytes does not involve PAF receptors and occurs via peripheral benzodiazepine and, possibly GABA(A) receptor sites.     

Mechanisms for facilitation of nitric oxide-evoked [3H]GABA release by removal of hydroxyl radical

We have investigated the mechanisms for enhancement of nitric oxide (NO)-evoked gamma-[3H]aminobutyric acid ([3H]GABA) release from mouse cerebrocortical neurons by hydroxyl radical (.OH) scavengers. .OH scavengers, such as N,N'-dimethylthiourea (DMTU), uric acid, and mannitol, dose-dependently facilitated NO-evoked [3H]GABA release evoked by NO liberated from S-nitroso-N-acetylpenicillamine. Ionomycin-evoked [3H]GABA release, which was significantly inhibited by hemoglobin and an NO synthase, N(G)-methyl-L-arginine, was also enhanced by DMTU. These results indicate that GABA release evoked by both endogenous and exogenous NO is facilitated by .OH scavengers. These enhancing actions of .OH scavengers were completely abolished by Ca2+ removal from incubation buffer and by an L-type voltage-dependent Ca2+ channel (VDCC) inhibitor, nifedipine, whereas each .OH scavenger showed no effects on [3H]GABA release in the absence of NO. Inhibitors for P/Q- and N-type VDCCs had no effects on the enhancement. NO-induced 45Ca2+ influx was also dose-dependently enhanced by .OH scavengers, although 45Ca2+ influx was not altered by .OH scavengers in the absence of NO. Nifedipine abolished this enhancement of the NO-induced 45Ca2+ influx by .OH scavengers. These results indicate that the removal of .OH by its scavengers facilitates the NO-evoked [3H]GABA release dependent on Ca2+ and that this enhancement is due to the increase in Ca2+ influx via L-type VDCCs.     

Effects of [3H]-BIDN, a novel bicyclic dinitrile radioligand for GABA-gated chloride channels of insects and vertebrates

1. The radiolabelled bicyclic dinitrile, [3H]-3,3-bis-trifluoromethyl-bicyclo[2.2.1]heptane-2,2-dicarbonitrile ([3H]-BIDN), exhibited, specific binding of high affinity to membranes of the southern corn rootworm (Diabrotica undecimpunctata howardi) and other insects. A variety of gamma-aminobutyric acid (GABA) receptor convulsants, including the insecticides heptachlor (IC50, 35 +/- 3 nM) and dieldrin (IC50, 93 +/- 7 nM), displaced [3H]-BIDN from rootworm membranes. When tested at 100 microM, 1-(4-ethynylphenyl)-4-n-propyl-2,6,7-trioxabicyclo[2.2.2]oct ane(EBOB), 4-t-butyl-2,6,7-trioxa-1-phosphabicy-clo[2.2.2]octane-1-thio ne (TBPS), 1-phenyl-4-t-butyl-2,6,7-trioxabicyclo[2.2.2]octane (TBOB) and picrotoxin failed to displace 50% of [3H]-BIDN binding to rootworm membranes indicating that the bicyclic dinitrile radioligand probes a site distinct from those identified by other convulsant radioligands. 2. Dissociation studies showed that dieldrin, ketoendrin, toxaphene, heptachlor epoxide and alpha and beta endosulphan displace bound [3H]-BIDN from rootworm membranes by a competitive mechanism. 3. Rat brain membranes were also shown to possess a population of saturable, specific [3H]-BIDN binding sites, though of lower affinity than in rootworm and with a different pharmacological profile. Of the insecticidal GABAergic convulsants that displaced [3H]-BIDN from rootworm, cockroach (Periplaneta americana) and rat brain membranes, many were more effective in rootworm. 4. Functional GABA-gated chloride channels of rootworm nervous system and of cockroach nerve and muscle were blocked by BIDN, whereas cockroach neuronal GABA(B) receptors were unaffected. 5. Expression in Xenopus oocytes of either rat brain mRNA, or cDNA-derived RNA encoding a GABA receptor subunit (Rdl) that is expressed widely in the nervous system of Drosophila melanogaster resulted in functional, homo-oligomeric GABA receptors that were blocked by BIDN. Thus, BIDN probes a novel site on GABA-gated Cl- channels to which a number of insecticidally-active molecules bind.     

Effects of the antiparkinsonian drug budipine on neurotransmitter release in central nervous system tissue in vitro

The effects of the antiparkinsonian drugs budipine and biperiden on spontaneous and electrically evoked release of dopamine (DA), acetylcholine (ACh), GABA or noradrenaline (NA) were studied in caudate nucleus or cortex slices, respectively, of the rabbit brain. Whereas both drugs (1-10 microM) strongly increased spontaneous [3H]outflow in caudate nucleus slices preincubated with [3H]DA, budipine inhibited but biperiden facilitated the evoked DA release. In the presence of the DA-reuptake inhibitor nomifensine, a significant part of the budipine-induced basal [3H] outflow consisted of unmetabolized DA. Synaptosomal high-affinity uptake of [3H]DA was only weakly affected by budipine and biperiden (IC50 values, 11 and 9 microM, respectively). Budipine enhanced also basal [3H]outflow from cortex slices prelabeled with [3H]NA, however this outflow consisted mainly of NA metabolites even in the presence of cocaine. The evoked release of [3H]ACh in rabbit caudate nucleus slices preincubated with [3H] choline was almost unaffected by budipine but enhanced by biperiden in the absence of further drugs. In the presence of nomifensine, however, budipine inhibited, but biperiden still enhanced, the evoked ACh release. Moreover, both drugs showed antimuscarinic properties in the presence of the ACh esterase inhibitor physostigmine, i.e., they facilitated the evoked ACh release, exhibiting pA2 values of about 6.9 (budipine) and 8.3 (biperiden). Addition of the D2 receptor antagonist domperidone diminished all inhibitory effects of budipine on the evoked ACh release. The evoked overflow of [3H] in caudate nucleus slices preincubated with [3H]GABA was reduced by both budipine and biperiden. It is concluded that both anticholinergic and indirect dopaminomimetic properties contribute to the antiparkinsonian effects of budipine, whereas biperiden exhibits mainly anticholinergic effects. Moreover, both drugs might disinhibit GABA controlled neurons in the central nervous system.     

Leukotriene B4 activates the NADPH oxidase in eosinophils by a pertussis toxin-sensitive mechanism that is largely independent of arachidonic acid mobilization

Experiments were designed to investigate whether leukotriene (LTB4) receptors can couple directly to phospholipase A2 (PLA2) in guinea pig eosinophils and the role of endogenous arachidonic acid (AA) in LTB4-induced activation of the NADPH oxidase. LTB4 (EC50 approximately 16 nM) and AA (EC50 approximately 6 microM) generated hydrogen peroxide (H2O2) in a concentration-dependent manner and at an equivalent maximum rate (5-6 nmol/min/10(6) cells). LTB4 stimulated PLA2 over a similar concentration range that activated the NADPH oxidase, although kinetic studies revealed that the release of [3H]AA (t1/2 approximately 2 s) preceded H2O2 generation (t1/2 > 30 s). Pretreatment of eosinophils with pertussis toxin abolished the increase in inositol(1,4,5)trisphosphate mass, [Ca2+]c, [3H]AA release, and H2O2 generation evoked by LTB4. Qualitatively identical results were obtained in eosinophils in which phospholipase C (PLC) was desensitized by 4beta-phorbol 12,13-dibutyrate with the exception that [3H]AA release was largely unaffected. Additional studies performed with the protein kinase C inhibitor, Ro 31-8220, and under conditions in which Ca2+ mobilization was abolished, provided further evidence that LTB4 released [3H]AA independently of signal molecules derived from the hydrolysis of phosphatidylinositol(4,5)bisphosphate by PLC. Pretreatment of eosinophils with the PLA2 inhibitor, mepacrine, abolished LTB4-induced [3H]AA release at a concentration that inhibited H2O2 by only 36%. Collectively, the results of this study indicate that agonism of LTB4 receptors on guinea pig eosinophils mobilizes AA by a mechanism that does not involve the activation of PLC. In addition, although LTB4 effectively stimulated PLA2, a central role for AA in the activation of the NADPH oxidase was excluded.     

Inhibition of nicotinic responses of bovine adrenal chromaffin cells by the protein kinase C inhibitor, Ro 31-8220

1. The effects of the protein kinase C inhibitor, Ro 31-8220, on the responses of cultured bovine adrenal chromaffin cells to nicotine, phorbol 12, 13-dibutyrate (PDBu) and K+ have been investigated. 2. Tyrosine hydroxylase activity was measured in situ in intact cells by measuring 14CO2 evolved following the hydroxylation and rapid decarboxylation of [14C]-tyrosine offered to the cells. Secretion of endogenous adrenaline and noradrenaline was measured by use of h.p.l.c. with electrochemical detection. Cyclic AMP levels were measured in cell extracts by RIA. 3. Ro 31-8220 produced a concentration-dependent inhibition of 300 nM PDBu-stimulated tyrosine hydroxylase activity with an IC50 of < 2 microM and complete inhibition at 10 microM. It had no effect on the responses to forskolin. 4. Ro 31-8220 produced a concentration-dependent inhibition of 5 microM nicotine-stimulated tyrosine hydroxylase activity, adrenaline and noradrenaline secretion and cellular cyclic AMP levels, with an IC50 of about 3 microM and complete inhibition by 10 microM. At concentrations up to 10 microM, Ro 31-8220 had little or no effect on the corresponding responses to 50 mm K+. 5. A structural analogue of Ro 31-8220, bisindolylmaleimide V, that lacks activity as a protein kinase C inhibitor, had no effect up to 10 microM on PDBu-stimulated tyrosine hydroxylase activity or on nicotine-stimulated cyclic AMP levels or noradrenaline secretion and only marginal inhibitory effects on nicotine-stimulated tyrosine hydroxylase activity and adrenaline secretion. 6. A structurally related protein kinase C inhibitor, bisindolylmaleimide I, inhibited PDBu-stimulated tyrosine hydroxylase activity with an IC50 of < 1 microM and complete inhibition by 3 microM, but had essentially no effect on nicotine stimulated tyrosine hydroxylase activity or catecholamine secretion. 7. The results suggest that Ro 31-8220 is not only a protein kinase C inhibitor but is also a potent inhibitor of nicotinic receptor responses in adrenal chromaffin cells by a mechanism unrelated to protein kinase C inhibition. The results are consistent with Ro 31-8220 being a nicotinic receptor antagonist.     

Kappa1- and kappa2-opioid receptors mediating presynaptic inhibition of dopamine and acetylcholine release in rat neostriatum

1. The effects of selective opioid receptor agonists and antagonists on N-methyl-D-aspartate (NMDA, 10 microM)-induced release of [3H]-dopamine and [14C]-acetylcholine (ACh) from superfused neostriatal slices were studied to investigate the possible occurrence of functional kappa-opioid receptor subtypes in rat brain. 2. The kappa receptor agonists (-)-ethylketocyclazocine ((-)-EKC), U69593 and the endogenous opioid peptide dynorphin A1-13 caused a naloxone-reversible inhibition of NMDA-induced [3H]-dopamine release, with pD2 values of about 9, 8.5 and 8.2, respectively, whereas both the mu agonist Tyr-D-Ala-Gly-(NMe)Phe-Gly-ol (DAMGO) and the delta agonist D-Pen2-D-Pen5-enkephalin (DPDPE) were ineffective in this respect. The inhibitory effect of submaximally effective concentrations of dynorphin A1-13, U69593 and (-)-EKC on NMDA-induced [3H]-dopamine release were not changed by the delta1/delta2-opioid receptor antagonist naltrindole (up to a concentration of 1 microM, but reversed by the kappa receptor antagonist nor-binaltorphimine (nor-BNI), with an IC50) as low as 0.02 nM, indicating the involvement of U69593-sensitive kappa1-opioid receptors. 3. NMDA-induced [14C]-ACh release was reduced in a naloxone-reversible manner by DPDPE (pD2 about 7.2), dynorphin A1-13 (pD2 6.7) and EKC (pD2 6.2), but not by U69593 and DAMGO. The inhibitory effect of a submaximally effective concentration of DPDPE, unlike those of dynorphin A1-13 and (-)-EKC, on NMDA-induced [14C]-ACh release was antagonized by naltrindole with an IC50 of 1 nM, indicating the involvement of delta-opioid receptors in the inhibitory effect of DPDPE. On the other hand, the inhibitory effects of dynorphin A1-13 and (-)-EKC on [14C]-ACh release were readily antagonized by nor-BNI with an IC50 of about 3 nM. A 100 fold higher concentration of nor-BNI also antagonized the inhibitory effect of DPDPE, indicating the involvement of U69593-insensitive kappa2-opioid receptors in the inhibitory effects of dynorphin A1-13 and (-)-EKC. 4. Although naloxone benzoylhydrazone (NalBzoH), displaying high affinity towards the putative kappa3-opioid receptor, antagonized the inhibitory effects of dynorphin A1-13 and (-)-EKC on [3H]-dopamine and [14C]-ACh release as well as that of U69593 on [3H]-dopamine release, it displayed a low apparent affinity (IC50 about 100 nM) in each case. 5. In conclusion, whereas activation of kappa1-opioid receptors causes presynaptic inhibition of NMDA-induced dopamine release, kappa2 receptor activation results in inhibition of ACh release in rat neostriatum. As such, this study is the first to provide unequivocal in vitro evidence for the existence of functionally distinct kappa-opioid receptor subtypes in the brain.     

Human brain cholecystokinin: release of cholecystokinin-like immunoreactivity (CCK-LI) from isolated cortical nerve endings and its modulation through GABA(B) receptors

The release of cholecystokinin-like immunoreactivity (CCK-LI) in human brain was investigated using synaptosomes prepared from neocortical specimens removed during neurosurgery. CCK-LI basal release from superfused synaptosomes was increased 3 to 4-fold during depolarization with 15 mM KCI. The K(+)-evoked overflow of CCK-LI was strictly Ca(++)-dependent. The gamma-aminobutyric acidB (GABA(B)) receptor agonist (-)baclofen (0.3-100 microM) inhibited CCK-LI overflow in a concentration-dependent manner (EC50 = 2.20 microM; maximal effect: 45%). The novel GABA(B) receptor ligand CGP 47656 mimicked (-)baclofen (EC50 = 2.45 microM; maximal effect: 50%), whereas the GABA(A) agonist muscimol was ineffective up to 100 microM. The inhibitory effect of 10 microM (-)baclofen on the CCK-LI overflow was concentration-dependently prevented by two selective GABA(B) receptor antagonists, CGP 35348 (IC50 = 13.91 microM) and CGP 52432 (IC50 = 0.08 microM). The effect of 10 microM CGP 47656 was abolished by 1 microM CGP 52432. In experiments on [3H]GABA release, CGP 47656 behaved as an antagonist at the GABA(B) autoreceptors: added at 10 microM, it prevented the inhibitory effect of 10 microM (-)baclofen on the K+ (15 mM)-evoked release of [3H]GABA from human synaptosomes. We conclude that 1) the release of CCK-LI evoked from human brain tissue appears of neuronal origin; 2) the CCK-releasing terminal possess inhibitory presynaptic GABA(B) receptors; 3) these receptors differ pharmacologically from human neocortex GABA(B) autoreceptors, which are CGP 35348-insensitive (Fassio et al., 1994) but can be blocked by CGP 47656; 4) because cholecystokinin has been implicated in anxiety, the GABA(B) receptors here characterized may represent targets for novel anxiolytic agents.     

Relation of exocytotic release of gamma-aminobutyric acid to Ca2+ entry through Ca2+ channels or by reversal of the Na+/Ca2+ exchanger in synaptosomes

The specific inhibitor of the gamma-aminobutyric acid (GABA) carrier, NNC-711, (1-[(2-diphenylmethylene)amino]oxyethyl)- 1,2,5,6-tetrahydro-3-pyridine-carboxylic acid hydrochloride, blocks the Ca(2+)-independent release of [3H]GABA from rat brain synaptosomes induced by 50 mM K+ depolarization. Thus, in the presence of this inhibitor, it was possible to study the Ca(2+)-dependent release of [3H]GABA in the total absence of carrier-mediated release. Reversal of the Na+/Ca2+ exchanger was used to increase the intracellular free Ca2+ concentration ([Ca2+]i) to test whether an increase in [Ca2+]i alone is sufficient to induce exocytosis in the absence of depolarization. We found that the [Ca2+]i may rise to values above 400 nM, as a result of Na+/Ca2+ exchange, without inducing release of [3H]GABA, but subsequent K+ depolarization immediately induced [3H]GABA release. Thus, a rise of only a few nanomolar Ca2+ in the cytoplasm induced by 50 mM K+ depolarization, after loading the synaptosomes with Ca2+ by Na+/Ca2+ exchange, induced exocytotic [3H]GABA release, whereas the rise in cytoplasmic [Ca2+] caused by reversal of the Na+/Ca2+ exchanger was insufficient to induce exocytosis, although the value for [Ca2+]i attained was higher than that required for exocytosis induced by K+ depolarization. The voltage-dependent Ca2+ entry due to K+ depolarization, after maximal Ca2+ loading of the synaptosomes by Na+/Ca2+ exchange, and the consequent [3H]GABA release could be blocked by 50 microM verapamil. Although preloading the synaptosomes with Ca2+ by Na+/Ca2+ exchange did not cause [3H]GABA release under any conditions studied, the rise in cytoplasmic [Ca2+] due to Na+/Ca2+ exchange increased the sensitivity to external Ca2+ of the exocytotic release of [3H]GABA induced by subsequent K+ depolarization. Thus, our results show that the vesicular release of [3H]GABA is rather insensitive to bulk cytoplasmic [Ca2+] and are compatible with the view that GABA exocytosis is triggered very effectively by Ca2+ entry through Ca2+ channels near the active zones.     

Halogenated mazindol analogs as potential inhibitors of the cocaine binding site at the dopamine transporter

A series of halogenated (F, Cl, Br, I), pyrimido and diazepino homologs of mazindol were prepared and evaluated for their ability to displace [3H]WIN 35,428 binding and to inhibit uptake of [3H]dopamine (DA) in rat striatal tissue. All of the compounds except for the 2'-chloro (6) and 2'-bromo (16) analogs of mazindol displaced [3H]WIN 35,428 binding and inhibited [3H]DA uptake more effectively than (R)-cocaine. Structure-activity studies indicated that best inhibition of [3H]WIN 35,428 binding occurred in the imidazo series with compounds containing one or two Cl or Br atoms in the 3'- or 4'-position of the free phenyl group. Replacement of the imidazo ring by a pyrimido or diazepino ring enhanced binding inhibition. The most potent inhibitors of [3H]WIN 35,428 binding and [3H]DA uptake were 6-(3'-chlorophenyl)-2,3,4,6-tetrahydropyrimido[2,1-alpha]isoind ol-6-ol (23; IC50 1.0 nM; 8 x mazindol) and 7-(3',4'-dichlorophenyl)-2,3,4,5-tetrahydro-7H-diazepino[2,1-alpha ]isoindol-7-ol (28; IC50 0.26 nM; 32 x mazindol), respectively. No significant differences was found between binding and uptake inhibition. Mazindol and the pyrimido and diazepino homologs 24 and 27 showed a selectivity for the DA uptake over the serotonin (5-HT) uptake site of 5-, 250-, and 465-fold, respectively, and displayed weak or no affinity for a variety of neurotransmitter receptor sites.     

Solvent and viscosity effects on the rate-limiting product release step of glucoamylase during maltose hydrolysis

The effects of 4-(4-fluorophenyl)-2-methyl-6-(5-piperidinopentyloxy) pyrimidine hydrochloride (NS-7), a novel neuroprotective compound, on the voltage-sensitive sodium channels (VSSC) were examined in the rat brain and cardiac myocytes. NS-7 inhibited [3H]batrachotoxinin A 20 alpha-benzoate (BTX) binding (neurotoxin receptor site 2) in brain membranes with a Ki value of 1 microM, while the compound was less effective in the cardiac myocytes (Ki = 13 microM). Aconitine, on the other hand, inhibited [3H]BTX binding to brain membranes and cardiac myocytes with the same potency. In contrast. NS-7 had no affinity for [3H]saxitoxin binding in brain (neurotoxin receptor site 1). In superfused slices of the rat cerebral cortex, NS-7 inhibited the veratridine (5 microM)-evoked glutamate release in a concentration-dependent manner, the IC50 value of which was 7.7 microM, whereas the compound showed a weak and not significant suppression of KCl-evoked glutamate release. The tissue concentrations of NS-7 in the rat cerebral cortex and heart were 89 and 28 nmole/g tissue, respectively, 5 min after its intravenous injection (8 mg/kg). Furthermore, in the cerebral cortex, NS-7 distributed preferentially to the membrane-enriched synaptosomal fraction. Since neurotoxin receptor site 2 is located in the transmembrane region of the VSSC moiety, the channel function may be substantially inhibited by a peripheral administration of NS-7. These results suggest that the blockade of neurotoxin receptor site 2 of VSSC in the brain contributes to the neuroprotective action of NS-7.     

Mechanisms for selective toxicity of fipronil insecticide and its sulfone metabolite and desulfinyl photoproduct

Fipronil, an N-phenylpyrazole with a trifluoromethylsulfinyl substituent, initiated the second generation of insecticides acting at the gamma-aminobutyric acid (GABA) receptor to block the chloride channel. The first generation includes the polychlorocycloalkanes alpha-endosulfan and lindane. In this study, we examine the mechanisms for selective toxicity of the sulfoxide fipronil and its sulfone metabolite and desulfinyl photoproduct relative to their target site interactions in vitro and ex vivo and the importance in fipronil action of biooxidation to the sulfone. Differences in GABA receptor sensitivity, assayed by displacement of 4'-ethynyl-4-n-[2, 3-3H2]propylbicycloorthobenzoate ([3H]EBOB) from the noncompetitive blocker site, appear to be a major factor in fipronil being much more toxic to the insects (housefly and fruit fly) than to the vertebrates (humans, dogs, mice, chickens, quail, and salmon) examined; in insects, the IC50s range from 3 to 12 nM for fipronil and its sulfone and desulfinyl derivatives, while in vertebrates, the IC50 average values are 1103, 175, and 129 nM for fipronil, fipronil sulfone, and desulfinyl fipronil, respectively. The insect relative to the vertebrate specificity decreases in the following order: fipronil > lindane > desulfinyl fipronil > fipronil sulfone > alpha-endosulfan. Ex vivo inhibition of [3H]EBOB binding in mouse brain is similar for fipronil and its sulfone and desulfinyl derivatives at the LD50 dose, but surprisingly, at higher doses fipronil can be lethal without detectably blocking the [3H]EBOB site. The P450 inhibitor piperonyl butoxide, acting in houseflies, increases the metabolic stability and effectiveness of fipronil and the sulfone but not those of the desulfinyl compound, and in mice it completely blocks the sulfoxide to sulfone conversion without altering the poisoning. Thus, the selective toxicity of fipronil and fipronil-derived residues is due in part to the higher potency of the parent compound at the insect versus the mammalian GABA receptor but is also dependent on the relative rates of conversion to the more persistent and less selective sulfone metabolite and desulfinyl photoproduct.     

Effects of intravenous general anesthetics on [3H]GABA release from rat cortical synaptosomes

BACKGROUND: Potentiation by general anesthetics of gamma-aminobutyric acid (GABA)-mediated inhibitory transmission in the central nervous system is attributed to GABA(A) receptor-mediated postsynaptic effects. However, the role of presynaptic mechanisms in general anesthetic action is not well characterized, and evidence for anesthetic effects on GABA release is controversial. The effects of several intravenous general anesthetics on [3H]GABA release from rat cerebrocortical synaptosomes (isolated nerve terminals) were investigated. METHODS: Purified synaptosomes were preloaded with [3H]GABA and superfused with buffer containing aminooxyacetic acid and nipecotic acid to inhibit GABA metabolism and reuptake, respectively. Spontaneous and elevated potassium chloride depolarization-evoked [3H]GABA release were evaluated in the superfusate in the absence or presence of various anesthetics, extracellular Ca2+, GABA receptor agonists and antagonists, and 2,4-diaminobutyric acid. RESULTS: Propofol, etomidate, pentobarbital, and alphaxalone, but not ketamine, potentiated potassium chloride-evoked [3H]GABA release (by 1.3 to 2.9 times) in a concentration-dependent manner, with median effective concentration values of 5.4 +/- 2.8 microM (mean +/- SEM), 10.1 +/- 2.1 microM, 18.8 +/- 5.8 microM, and 4.4 +/- 2.0 microM. Propofol also increased spontaneous [3H]GABA release by 1.7 times (median effective concentration = 7.1 +/- 3.4 microM). Propofol facilitation of [3H]GABA release was Ca2+ dependent and inhibited by bicuculline and picrotoxin, but was insensitive to pretreatment with 2,4-diaminobutyric acid, which depletes cytoplasmic GABA pools. CONCLUSIONS: Low concentrations of propofol, etomidate, pentobarbital, and alphaxalone facilitated [3H]GABA release from cortical nerve terminals. General anesthetics may facilitate inhibitory GABA-ergic synaptic transmission by a presynaptic mechanism in addition to their well-known postsynaptic actions.