Group I mGlu receptors potentiate synaptosomal [3H]glutamate release independently of exogenously applied arachidonic acid

In the current study, we have characterized group I metabotropic glutamate (mGlu) receptor enhancement of 4-aminopyridine (4AP)-evoked [3H]glutamate release from rat cerebrocortical synaptosomes. The broad spectrum mGlu receptor agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid ((1S,3R)-ACPD, 10 microM) increased 4AP-evoked [3H]glutamate release (143.32+/-2.73% control) only in the presence of exogenously applied arachidonic acid; an effect reversed by the inclusion of bovine serum albumin (BSA, fatty acid free). In contrast, the selective group I mGlu receptor agonist (S)-3,5-dihydroxyphenylglycine (DHPG) potentiated (EC50 = 1.60+/-0.25 microM; Emax = 147.61+/-10.96% control) 4AP-evoked [3H]glutamate release, in the absence of arachidonic acid. This potentiation could be abolished by either the selective mGlu1 receptor antagonist (R,S)-1-aminoindan-1,5-dicarboxylic acid (AIDA, 1 mM) or the selective PKC inhibitor (Ro 31-8220, 10 microM) and was BSA-insensitive. The selective mGlu5 receptor agonist (R,S)-2-chloro-5-hydroxyphenylglycine (CHPG, 300 microM) was without effect. DHPG (100 microM) also potentiated both 30 mM and 50 mM K+ -evoked [3H]glutamate release (121.60+/-12.77% and 121.50 +/-4.45% control, respectively). DHPG (100 microM) failed to influence both 4AP-stimulated 45Ca2+ influx and 50 mM K+ -induced changes in synaptosomal membrane potential. Possible group I mGlu receptor suppression of tonic adenosine A1 receptor, group II/III mGlu receptors or GABA(B) receptor activity is unlikely since 4AP-evoked [3H]glutamate release was insensitive to the selective inhibitory receptor antagonists 8-cyclopentyl-1,3-dimethylxanthine, (R,S)-alpha-cyclopropyl-4-phosphonophenylglycine or CGP55845A, respectively. These data suggest an 'mGlu1 receptor-like' receptor potentiates [3H]glutamate release from cerebrocortical synaptosomes in the absence of exogenously applied arachidonic acid. This PKC dependent effect is unlikely to be via modulation of synaptosomal membrane potential or voltage-activated Ca2+ channels and not via a suppression of tonically active inhibitory adenosine A1 receptor, group II/III mGlu receptors or GABA(B) receptors.     

Indirect modulation by alpha7 nicotinic acetylcholine receptors of noradrenaline release in rat hippocampal slices: interaction with glutamate and GABA systems and effect of nicotine withdrawal

Nicotinic acetylcholine receptors (nAChRs) can modulate transmitter release. Striatal [(3)H]dopamine ([(3)H]DA) release is regulated by presynaptic nAChR on dopaminergic terminals and alpha7 nAChR on neighboring glutamatergic afferents. Here, we explored the role of alpha7 nAChR in the modulation of [(3)H]noradrenaline ([(3)H]NA) release from rat hippocampal slices. The nicotinic agonist anatoxin-a (AnTx) evoked monophasic [(3)H]NA release (EC(50) = 1.2 microM) that was unaffected by alpha-conotoxin-MII or dihydro-beta-erythroidine, antagonists of alpha3/alpha6beta2* and beta2* nAChR, respectively. In contrast AnTx-evoked striatal [(3)H]DA release was biphasic (EC(50) = 138.9 nM; 7.1 microM) and blocked by these antagonists. At a high AnTx concentration (25 microM), alpha7 nAChR antagonists (methyllycaconitine, alpha-conotoxin-ImI) and glutamate receptor (GluR) antagonists [kynurenic acid, 6,7-dinitroquinoxaline-2,3-dione (DNQX)] partially inhibited [(3)H]NA release. The alpha7 nAChR-selective agonist choline evoked [(3)H]NA release (E(max) = 33% of that of AnTx) that was blocked by GluR antagonists, supporting a model in which alpha7 nAChRs trigger glutamate release that subsequently stimulates [(3)H]NA release. A GABAergic component was also revealed: choline-evoked [(3)H]NA release was partially blocked by the GABA(A) receptor antagonist bicuculline, and coapplication of bicuculline and DNQX fully abolished this response. These findings support alpha7 nAChR on GABAergic neurons that can promote GABA release which, in turn, leads to [(3)H]NA release, probably by disinhibition. To investigate the impact of long-term nicotine exposure on this model, rats were exposed for 14 days to nicotine (4 mg/kg/day) with or without 3 or 7 days of withdrawal. alpha7 nAChR responses were selectively and transiently up-regulated after 3 days of withdrawal. This functional up-regulation could contribute to the withdrawal effects of nicotine.     

Potentiation effects of (+/-)huprine X, a new acetylcholinesterase inhibitor, on nicotinic receptors in rat cortical synaptosomes

The present experiments were developed to analyze the direct and/or potentiation effect of (+/-)-12-amino-3-chloro-9-ethyl-6,7,10,11-tetrahydro-7,11-methanocycloocta[b]quinoline hydrochloride ((+/-)huprine X) on nicotinic receptors using a synaptosomal superfusion method. (+/-)Huprine X (1 microM, 10 microM) increased [(3)H]-ACh release only at 10 microM (46%; P < 0.001) in basal, but not in stimulated, conditions. This effect was completely reverted by mecamylamine (100 microM; MEC). Potentiation of evoked-[3H]-ACh release induced by ACh (1 microM) and by galantamine (GAL) 0.4 microM and physostigmine (PHY) 10 microM (55% and 50%, respectively; P < 0.001), two well-known allosteric compounds, corroborate that the present experimental approach is a suitable method to study potentiation effects on nicotinic receptors in the central nervous system nerve terminals. (+/-)Huprine X potentiated the evoked-[3H]-ACh release induced by ACh (1 microM) by 166% and 90% (P < 0.001) at 10 microM and 30 microM, respectively, and this effect was completely blocked by MEC (100 microM). In the presence of different ACh concentrations, (+/-)huprine X 10 microM potentiated evoked-[3H]-ACh release at low ACh concentrations, while a decrease in neurotransmitter release was observed at high ACh concentrations. The highest potentiation effect was obtained at the ACh/(+/-)huprine X concentration ratio of 1:10, and this potentiation was observed at as low a (+/-)huprine X concentration as 0.1 microM (P < 0.05). While the results suggest that huprine may enhance the potency or effectiveness of ACh by an effect involving nicotinic receptors we cannot completely discard that the results could be explained by acetylcholine esterase inhibition.     

Homologous and heterologous desensitization of histamine H1- and ATP-receptors in the smooth muscle cell line, DDT1MF-2: the role of protein kinase C.

1. The possible role of protein kinase C (PKC) in homologous and heterologous desensitization of histamine H1- and ATP-receptors has been studied in monolayers of cultured vas deferens smooth muscle cells (DDT1MF-2). Cells were loaded with the calcium-sensitive fluorescent dye fura-2 and increases in intracellular free Ca2+ concentration ([Ca2+]i) monitored in response to histamine H1- or ATP-receptor activation. 2. Histamine and ATP stimulated the release of Ca2+ from intracellular Ca2+ stores and Ca2+ influx across the plasma membrane. Activation of PKC with the phorbol ester beta-phorbol-12,13 dibutyrate (PDBu; 1 microM) attenuated histamine (100 microM) and ATP (10 microM)-induced release of intracellular Ca2+ and Ca2+ influx. 3. The selective PKC inhibitor, Ro 31-8220 (10 microM), reversed the PDBu-induced attenuation of histamine (100 microM)-stimulated Ca2+ responses. 4. Histamine H1- and ATP-receptors are readily susceptible to homologous desensitization since short-term exposure to histamine or ATP (450 s) attenuated the Ca2+ responses elicited by a second application of the same agonist. Furthermore, H1-receptor activation-induced heterologous desensitization of ATP stimulated Ca2+ responses and vice versa. 5. Homologous and heterologous desensitization of histamine and ATP Ca2+ responses still occurred in the presence of the PKC inhibitor, Ro 31-8220 (10 microM). 6. These data suggest that PKC activation can attenuate histamine H1- and ATP-receptor mediated Ca2+ responses. However, based on our experimental data, PKC-independent mechanisms appear to be involved in the homologous and heterologous desensitization of histamine H1- and ATP-receptor mediated Ca2+ responses in DDT1MF-2 cells.     

Role of protein kinase C in the regulation of histamine and bradykinin stimulated inositol polyphosphate turnover in adrenal chromaffin cells.

1. The possibility that bradykinin- or histamine-stimulated inositol polyphosphate accumulation may be regulated by protein kinase C (PKC) in bovine adrenal chromaffin cells has been addressed. 2. Initial experiments confirmed that the phorbol ester 12-O-tetradecanoyl-phorbol 13-acetate (TPA) dramatically inhibited agonist-stimulated [3H]-inositol phosphate accumulations in [3H]-inositol prelabelled cells. In contrast, the PKC inhibitor, Ro 31-8220, did not affect this response. 3. Histamine (100 microM) or bradykinin (100 nM) evoked rapid increases in inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) and inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4) mass accumulations (maximal accumulations within 10 s and 30 s, respectively) which declined towards basal values over a 10 min incubation period. TPA (1 microM) significantly attenuated the peak Ins(1,4,5)P3 response to bradykinin and histamine by 30% and 70% respectively. In contrast, TPA did not significantly affect agonist-stimulated Ins(1,3,4,5)P4 responses. 4. Ro 31-8220 (10 microM) significantly enhanced the maximal Ins(1,4,5)P3 accumulations elicited by both bradykinin and histamine. 5. The results indicate that the initial Ins(1,4,5)P3 response to either bradykinin or histamine in bovine adrenal chromaffin cells can be attenuated by PKC activation by phorbol ester and enhanced by PKC inhibition by Ro 31-8220. In contrast, agonist-stimulated Ins(1,3,4,5)P4 accumulation does not appear to be affected by these manipulations of PKC activity. Possible bases for differential modulation of Ins(1,4,5)P3 and Ins(1,3,4,5)P4 are discussed.     

Lobeline inhibits nicotine-evoked [(3)H]dopamine overflow from rat striatal slices and nicotine-evoked (86)Rb(+) efflux from thalamic synaptosomes

The present study evaluated the interaction of lobeline with neuronal nicotinic acetylcholine receptors using two in vitro assays, [(3)H] overflow from [(3)H]dopamine ([(3)H]DA)-preloaded rat striatal slices and (86)Rb(+) efflux from rat thalamic synaptosomes. To assess agonist interactions, the effect of lobeline was determined and compared to S(-)-nicotine. To assess antagonist interactions, the ability of lobeline to inhibit the effect of S(-)-nicotine was determined. Both S(-)-nicotine (0.1-1 microM) and lobeline (>1.0 microM) evoked [(3)H] overflow from superfused [(3)H]DA-preloaded striatal slices. However, lobeline-evoked [(3)H] overflow is mecamylamine-insensitive, indicating that this response is not mediated by nicotinic receptors. Moreover, at concentrations (<1.0 microM) which did not evoke [(3)H] overflow, lobeline inhibited S(-)-nicotine (0.1-10 microM)-evoked [(3)H] overflow, shifting the S(-)-nicotine concentration-response curve to the right. S(-)-Nicotine (30 nM-300 microM) increased (EC(50) value=0.2 microM) (86)Rb(+) efflux from thalamic synaptosomes. In contrast, lobeline (1 nM-10 microM) did not evoke (86)Rb(+) efflux, and the lack of intrinsic activity indicates that lobeline is not an agonist at this nicotinic receptor subtype. Lobeline completely inhibited (IC(50) value=0.7 microM) (86)Rb(+) efflux evoked by 1 microM S(-)-nicotine, a concentration which maximally stimulated (86)Rb(+) efflux. Thus, the results of these in vitro experiments demonstrate that lobeline inhibits the effects of S(-)-nicotine, and suggest that lobeline acts as a nicotinic receptor antagonist.     

Modulation of [3H]-dopamine released by different frequencies of stimulation from rabbit retina.

Rabbit retinal pieces were incubated with [3H]-dopamine and superfused with Krebs solution. Calcium-dependent tritium release was elicited twice within each experiment by electrical field stimulation (360 pulses, 2 ms, 20 mA) at frequencies of 1 Hz, 3 Hz, and 6 Hz. The evoked release of [3H]-dopamine for the first period of stimulation (S1) was 2.09 +/- 0.23% (n = 5) at 1 Hz and was of similar magnitude at all other frequencies of stimulation employed. The D2-dopamine receptor agonist, LY 171555 (quinpirole HCl; 0.01-1 microM) added to the superfusion medium before the second period of stimulation, inhibited the calcium-dependent release of [3H]-dopamine in a concentration-dependent manner, and was more potent the lower the stimulation frequency. The isomer of quinpirole, LY 181990 (0.01-1 microM) did not inhibit the stimulation-evoked overflow of tritium, regardless of concentration or stimulation frequency. The stereoisomers of the D2-dopamine receptor antagonist sulpiride (0.01-3 microM) increased the calcium-dependent release of [3H]-dopamine in a concentration-dependent manner, being more potent the higher the frequency of stimulation. S-sulpiride was more potent than R-sulpiride at all stimulation frequencies. The inhibitory effect of quinpirole was stereoselectively antagonized by sulpiride, but not by LY 181990. The alpha-adrenoceptor antagonist phentolamine (1 microM) did not modify the quinpirole-induced inhibition of [3H]-dopamine release. When the synaptic concentration of dopamine was increased by the presence of the dopamine uptake inhibitor nomifensine (3 microM), the potency of the agonist quinpirole in inhibiting the release of [3H]-dopamine elicited by field stimulation at 1 Hz (180 pulses) was decreased. In contrast, the potency of S-sulpiride in enhancing the evoked release of [3H]-dopamine was increased when nomifensine was present in the superfusion medium. Picomolar concentrations of the hormone melatonin (0.1-10 nM) inhibited the calcium-dependent release of [3H]-dopamine from rabbit retina with the same potency regardless of the frequency of stimulation applied (1 Hz, 3 Hz or 6 Hz). The potency of D2-dopamine receptor agonists and antagonists in modifying [3H]-dopamine release from rabbit retina appears to depend on the synaptic concentration of dopamine which is altered by the frequency of stimulation, and by the dopamine uptake inhibitor nomifensine.(ABSTRACT TRUNCATED AT 400 WORDS)     

Homologous and heterologous uncoupling of muscarinic M(3) and alpha(1B) adrenoceptors to Galpha(q/11) in SH-SY5Y human neuroblastoma cells

1. The present study employed a [(35)S]-GTPgammaS binding protocol in conjunction with immunoprecipitation (IP) of the Galpha subunits to investigate the desensitization of G(q/11)-coupled receptors at the level of the G-protein activation. Membranes from SH-SY5Y cells expressing the recombinant human alpha(1B)-adrenoceptor (alpha(1B)-AR) (and endogenously expressing the M(3) muscarinic acetylcholine receptor (M(3)-AChR)) exhibited G(q/11) activation in a concentration-dependent manner in response to noradrenaline or methacholine. 2. Pre-treatment of intact cells with agonist prior to membrane preparation and use in the [(35)S]-GTPgammaS IP assay demonstrated that both receptors were homologously desensitized by pre-treatment with agonist since the G(q/11) activation in response to a secondary challenge with agonist was markedly reduced. Stimulation of alpha(1B)-AR was effective at heterologously desensitizing the M(3)-AChR. The PKC inhibitor, Ro-31-8220 (10 microM) was ineffective at preventing the agonist-mediated receptor desensitization. 3. [(32)P]P(i)-labelled cells allowed the detection of increases in receptor phosphorylation. Phorbol 12,13 dibutyrate (PDBu) (1 microM) was effective at producing a Ro-31-8220 (10 microM)-sensitive, detectable increase in alpha(1B)-AR but not M(3)-AChR phosphorylation. Noradrenaline (30 microM) stimulated alpha(1B)-AR phosphorylation, which could be partially inhibited by Ro-31-8220 (10 microM). The phosphorylation of M(3)-AChR was increased by methacholine (100 microM) incubation and this effect appeared to be insensitive to Ro-31-8220 (10 microM). 4. These findings demonstrate that [(35)S]-GTPgammaS-Galpha-subunit IP can be used to estimate receptor desensitization as a decline in receptor-G-protein coupling. Both the alpha(1B)-AR and M(3)-AChR undergo rapid homologous desensitization that is associated with an increase in receptor phosphorylation. The heterologous desensitization of M(3)-AChR produced by alpha(1B)-AR stimulation is not associated with a detectable increase in M(3)-AChR phosphorylation, suggesting that receptor phosphorylation is not necessarily a prerequisite for desensitization.     

The structural requirements for phorbol esters to enhance noradrenaline and dopamine release from rat brain cortex.

1. The effects of various protein kinase C (PKC) activators on the stimulation-induced (S-I) release of noradrenaline and dopamine was studied in rat cortical slices pre-incubated with [3H]-noradrenaline or [3H]-dopamine. The aim was to investigate a possible structure-activity relationship for these agents on transmitter release. 2. 4 beta-Phorbol 12,13-dibutyrate (4 beta PDB, 0.1-3.0 microM), enhanced S-I noradrenaline and dopamine release in a concentration-dependent manner whereas the structurally related inactive isomer 4 alpha-phorbol 12, 13-dibutyrate (4 alpha PDB, 0.1-3.0 microM) and phorbol 13-acetate (PA, 0.1-3.0microM) were without effect on noradrednaline release. Another group of phorbol 12, 13-diesters containing a common 13-ester substituent (phorbol 12, 13-diacetate, PDA, 0.1-3.0 microM; phorbol 12-myristate 13-acetate, PMA, 0.1-3.0 microM; phorbol 12-methylaminobenzoate 13-acetate, PMBA, 0.03-3.0 microM) also enhanced S-I noradrenaline and dopamine release in a concentration-dependent manner with PMA being the least potent. 3. The 12-deoxyphorbol 13-substituted monoesters, 12-deoxyphorbol 13-acetate (dPA, 0.1-3.0 microM), 12-deoxyphorbol 13-angelate (dPAng, 0.1-3.0 microM), 12-deoxyphorbol 13-isobutyrate (dPiB, 0.03-3.0 microM) and 12-deoxyphorbol 13-phenylacetate (dPPhen, 0.1-3.0 microM) enhanced S-I noradrenaline and dopamine release in a concentration-dependent manner. In contrast, 12-deoxyphorbol 13-tetradecanoate (dPT, 0.1-3.0 microM) was without effect. 4. The involvement of PKC in mediating the effects of the various phorbol esters was further investigated. PKC was down-regulated by 20 h exposure of the cortical slices to 4 beta-phorbol 12,13-dibutyrate (1 microM). In this case the facilitatory effect of 4 beta PDB and dPA was abolished whilst that of dPAng was significantly attenuated. This indicates that these agents were acting selectively at PKC. In support of this the PKC inhibitors, polymyxin B (21 microM) and bisindolylmaleimide I (3 microM), attenuated the facilitatory effect of 4 beta PDB and dPAng although that of dPA was not significantly altered. 5. The effects of these agents on transmitter release were not correlated with their in vitro affinity and isozyme selectivity for PKC. Short chain substituted mono- and diesters of phorbol were more potent enhancers of action-potential evoked noradrenaline and dopamine release than the long chain esters. Interestingly, these former agents are the least potent or non effective (e.g. dPA, PDA) tumour promoters. We suggest that the reason for the poor effects of lipophilic long chain phorbol esters (PMA, dPT) on transmitter release is that they are sequestered in the plasmalemma and do not access the cell cytoplasm where the PKC may be located.     

Dual effects of adenosine on acetylcholine release from myenteric motoneurons are mediated by junctional facilitatory A(2A) and extrajunctional inhibitory A(1) receptors

1. The coexistence of both inhibitory A(1) and facilitatory A(2) adenosine receptors in the rat myenteric plexus prompted the question of how adenosine activates each receptor subtype to regulate cholinergic neurotransmission. 2. Exogenously applied adenosine (0.3-300 microm) decreased electrically evoked [(3)H]acetylcholine ([(3)H]ACh) release. Blocking A(1) receptors with 1,3-dipropyl-8-cyclopentylxanthine (10 nm) transformed the inhibitory action of adenosine into a facilitatory effect. Adenosine-induced inhibition was mimicked by the A(1) receptor agonist R-N(6)-phenylisopropyladenosine (0.3 microm), but the A(2A) agonist CGS 21680C (0.003 microm) produced a contrasting facilitatory effect. 3. Increasing endogenous adenosine levels, by the addition of (1) the adenosine precursor AMP (30-100 microm), (2) the adenosine kinase inhibitor 5'-iodotubercidin (10 microm) or (3) inhibitors of adenosine uptake (dipyridamole, 0.5 microm) and of deamination (erythro-9(2-hydroxy-3-nonyl)adenine, 50 microm), enhanced electrically evoked [(3)H]ACh release (5 Hz for 40 s). Release facilitation was prevented by adenosine deaminase (ADA, 0.5 U ml(-1)) and by the A(2A) receptor antagonist ZM 241385 (50 nm); these compounds decreased [(3)H]ACh release by 31+/-6% (n=7) and 37+/-10% (n=6), respectively. 4. Although inhibition of ecto-5'-nucleotidase by alpha,beta-methylene ADP (200 microm) or by concanavalin A (0.1 mg ml(-1)) attenuated endogenous adenosine formation from AMP, analysed by HPLC, the corresponding reduction in [(3)H]ACh release only became evident when stimulation of the myenteric plexus was prolonged to over 250 s. 5. In summary, we found that endogenously generated adenosine plays a predominantly tonic facilitatory effect mediated by prejunctional A(2A) receptors. Extracellular deamination and cellular uptake may restrict endogenous adenosine actions to the neuro-effector region near the release/production sites.     

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.     

Modulation of dopamine and noradrenaline release and of intracellular Ca2+ concentration by presynaptic glutamate receptors in hippocampus.

1. We studied the release of [3H]-dopamine and [3H]-noradrenaline (NA) from hippocampal synaptosomes induced by glutamate receptors and the associated Ca2+ influx through Ca2+ channels. The release of tritiated neurotransmitters was studied by use of superfusion system and the intracellular free Ca2+ concentration ([Ca2+]i) was determined by a fluorimetric assay with Indo-1 as a probe for Ca2+. 2. Presynaptic glutamate receptor activation induced Ca(2+)-dependent release of [3H]-dopamine and [3H]-NA from rat hippocampal synaptosomes. Thus, L-glutamate induced the release of both neurotransmitters in a dose-dependent manner (EC50 = 5.62 microM), and the effect of 100 microM L-glutamate was inhibited by 83.8% in the presence of 10 microM 6-cyano-7-nitroquinoxaline-2,3-dioxine (CNQX), but was not affected by 1 microM (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohepten-5,10-imine (MK-801). 3. Other glutamate receptor agonists also stimulated the Ca(2+)-dependent release of [3H]-dopamine and [3H]-NA as follows: N-methyl-D-aspartate (NMDA), at 200 microM, released 3.65 +/- 0.23% of the total 3H catecholamines, and this effect was inhibited by 81.2% in the presence of 1 microM MK-801; quisqualate (50 microM), S-alpha-amino-3-hydroxy-5-methyl-4-isoxazolopropionic acid (AMPA) (100 microM) or kainate (100 microM) released 1.57 +/- 0.26%, 1.93 +/- 0.17% and 2.09 +/- 0.22%, of the total 3H catecholamines, respectively. 4. The ionotropic glutamate receptor agonist, AMPA, induced an increase in the [Ca2+]i which was inhibited by 58.6% in the presence of 10 microM CNQX.(ABSTRACT TRUNCATED AT 250 WORDS)     

Release of [3H]-noradrenaline from rat hippocampal synaptosomes by nicotine: mediation by different nicotinic receptor subtypes from striatal [3H]-dopamine release.

1. The aim of the present experiment was to characterize nicotine-evoked [3H]-noradrenaline ([3H]-NA) release from rat superfused hippocampal synaptosomes, using striatal [3H]-dopamine release for comparison. 2. (-)-Nicotine, cytisine, DMPP and acetylcholine (ACh) (with esterase inhibitor and muscarinic receptor blocker) increased NA release in a concentration-dependent manner (EC50 6.5 microM, 8.2 microM, 9.3 microM, and 27 microM, respectively) with similar efficacy. 3. Nicotine released striatal dopamine more potently than hippocampal NA (EC50 0.16 microM vs. 6.5 microM). (+)-Anatoxin-a also increased dopamine more potently than NA (EC50 0.05 microM vs. 0.39 microM), and maximal effects were similar to those of nicotine. Isoarecolone (10-320 microM) released dopamine more effectively than NA but a maximal effect was not reached. (-)-Lobeline (10-320 microM) evoked dopamine release, but the effect was large and delayed with respect to nicotine; NA release was not increased but rather depressed at high concentrations of lobeline. High K+ (10 mM) released and NA to similar extents. 4. Addition of the 5-hydroxytryptamine (5-HT) reuptake blocker, citalopram (1 microM) to hippocampal synaptosomes affected neither basal NA release nor nicotine-evoked release. 5. The nicotinic antagonist, mecamylamine (10 microM), virtually abolished NA and dopamine release evoked by high concentrations of nicotine, ACh, cytisine, isoarecolone, and anatoxin-a. Although NA release evoked by DMPP (100 microM) was entirely mecamylamine-sensitive, DMPP-evoked dopamine release was only partially blocked. Dopamine release evoked by lobeline (320 microM) was completely mecamylamine-insensitive. 6. The nicotinic antagonists dihydro-beta-erythroidine and methyllycaconitine inhibited nicotine-evoked dopamine release approximately 30 fold more potently than NA release. In contrast, the antagonist chlorisondamine, displayed a reverse sensitivity, whereas trimetaphan and mecamylamine did not preferentially block either response. None of these antagonists, given at a high concentration, significantly altered release evoked by high K+. 7. Blockade of nicotine-evoked transmitter release by methyllycaconitine and dihydro-beta-erythroidine was surmounted by a high concentration of nicotine (100 microM), but blockade by mecamylamine, chlorisondamine, and trimetaphan was insurmountable. 8. Nicotine-evoked NA release was unaffected by tetrodotoxin, whereas veratridine-evoked NA release was virtually abolished. 9. We conclude that presynaptic nicotinic receptors associated with striatal dopamine and hippocampal NA terminals differ pharmacologically. In situ hybridization studies suggest that nigrostriatal dopaminergic neurones express mainly alpha 4, alpha 5, and beta 2 nicotinic cholinoceptor subunits, whereas hippocampal-projecting noradrenaline (NA) neurones express alpha 3, beta 2 and beta 4 subunits. Pharmacological comparisons of recombinant receptors suggest that release of hippocampal NA may be modulated by receptors containing alpha 3 and beta 4 subunits.     

Complex involvement of pertussis toxin-sensitive G proteins in the regulation of type 1alpha metabotropic glutamate receptor signaling in baby hamster kidney cells

Previously, we demonstrated that the coupling of the metabotropic glutamate receptor mGlu1alpha to phosphoinositide hydrolysis is enhanced by pertussis toxin (PTX) in stably transfected baby hamster kidney cells (BHK). Here, we show that the PTX effect on agonist-stimulated [(3)H]inositol phosphate accumulation can be resolved into two components: an immediate increase in agonist potency, and a more slowly developing increase in the magnitude of the response observed at maximally effective agonist concentrations. Using G(q/11)alpha- and G(i/o)alpha-selective antibodies to immunoprecipitate [(35)S]guanosine-5'-O-(3-thio)triphosphate-bound Galpha proteins, we also show that agonist stimulation of mGlu1alpha in BHK membranes increases specific [(35)S]guanosine-5'-O-(3-thio)triphosphate binding to both G(q/11) and G(i/o) proteins. Preincubation of BHK-mGlu1alpha with L-glutamate (300 microM) results in a progressive loss (60% in 30 min) of L-quisqualate-induced [(3)H]inositol phosphate accumulation (without a change in potency), providing evidence for agonist-induced receptor desensitization. Although such desensitization of mGlu receptor signaling was mimicked by a phorbol ester, agonist-induced phosphorylation of the receptor was not observed and protein kinase C inhibition by Ro 31-8220 did not prevent L-glutamate-mediated desensitization. In contrast, PTX treatment of the cells almost completely prevented L-glutamate-mediated desensitization. Together, these data provide evidence for a multifunctional coupling of mGlu1alpha to different types of G proteins, including PTX-sensitive G(i)-type G proteins. The latter are involved in the negative control of phospholipase C activity while also influencing the rate of desensitization of the mGlu1alpha receptor.     

Opioid receptor-mediated inhibition of dopamine and acetylcholine release from slices of rat nucleus accumbens, olfactory tubercle and frontal cortex

The modulation of the electrically evoked release of [3H]dopamine (DA) and [14C]acetylcholine (ACh) by opioid receptor activation was examined in superfused slices from rat nucleus accumbens, olfactory tubercle, and frontal cortex. In all brain areas examined, [3H]DA release was inhibited by the kappa agonist, U 50,488 (1-100 nM), and this inhibition was fully antagonized by the selective kappa antagonist, norbinaltorphimine (nor-BNI). In the frontal cortex, the mu agonist, [D-Ala2,MePhe4,Gly-ol5]enkephalin (DAGO, 0.01-1 microM), also inhibited the evoked release of tritium. However, further experiments (including the use of the D2-receptor agonist, LY 171555, and the alpha 2-adrenoceptor agonist, oxymetazoline) suggest strongly that in the frontal cortex DAGO only inhibits the release of [3H]catecholamine from noradrenergic nerve terminals, despite the use of desimipramine to prevent the uptake of [3H]DA into these terminals. [14C]ACh release from both the nucleus accumbens and olfactory tubercle, but not from the frontal cortex, was inhibited by DAGO (0.01-1 microM) and the delta agonist, [D-Pen2,D-Pen5]enkephalin (DPDPE, 0.01-1 microM). These inhibitory effects were antagonized by 0.1 microM naloxone but not by 3 nM nor-BNI. The irreversible delta ligand, fentanyl isothiocyanate (FIT, 1 microM), only antagonized the inhibition caused by DPDPE. The results indicate that the inhibitory effects of opioids on the in vitro release of DA from dopaminergic nerve fibres arising from the substantia nigra and the ventral tegmental area are mediated by presynaptic kappa receptors only. In those regions where ACh release is modulated by opioids, the type of opioid receptor involved may depend on the type of neuron, i.e. interneuron or afferent neuron.     

Ketamine induces hyperactivity in rats and hypersensitivity to nicotine in rat striatal slices

The symptoms of schizophrenia can be modeled in rats through blockade of ionotropic glutamate receptors, which induces changes in central dopamine circuits. These circuits also contain nicotinic acetylcholine receptors that are activated by nicotine. A role for nicotine in the etiology of schizophrenia is supported by clinical observations of high tobacco use rates in individuals experiencing the psychopathology. The present study investigated the effect of the ionotropic glutamate receptor antagonist ketamine on the function of striatal nicotinic acetylcholine receptors to understand better the potential role of these receptors in schizophrenia. Ketamine (0.1-300 microM) was ineffective to evoke [(3)H] overflow from rat striatal slices preloaded with [(3)H]dopamine. Application of psychotomimetic ketamine concentrations (1-10 microM) to striatal slices augmented nicotine-evoked [(3)H] overflow. Finally, rats received ketamine (30-50 mg/kg) injections for 30 days, to model the development of the disorder, and hyperactivity was observed, although repeated ketamine treatment did not significantly alter nicotine-evoked [(3)H]dopamine overflow. These data indicate that the function of nicotinic acetylcholine receptors that mediate dopamine release are altered by ketamine, and support a role for nicotinic acetylcholine receptors in schizophrenia pathology.     

Effects of riluzole on electrically evoked neurotransmitter release

1. The main purpose of the present study was to investigate the effects of the neuroprotective agent riluzole on the electrically evoked release of [(3)H]-glutamate ([(3)H]-Glu) in mouse neocortical slices. The reported selectivity of riluzole for excitatory amino acids was tested in release experiments with further neurotransmitters. Also distinct species, mouse, rat and man were compared. 2. [(3)H]-Glu was formed endogenously during incubation of slices with [(3)H]-glutamine ([(3)H]-Gln). Released [(3)H]-Glu and tissue [(3)H]-Glu was separated by anion exchange chromatography. Electrically evoked [(3)H]-Glu release was strongly diminished by tetrodotoxin (TTX) and Ca(2+)-withdrawal. 3. Riluzole (100 microM) depressed the release of [(3)H]-Glu up to 77% (IC(50)=19.5 microM). Riluzole was also able to inhibit strongly the electrically evoked release of [(3)H]-acetylcholine ([(3)H]-ACh) (at 100 microM by 92%, IC(50)=3.3 microM, and [(3)H]-dopamine ([(3)H]-DA) (at 32 microM by 72%, IC(50)=6.8 microM). However, the release of [(3)H]-serotonin ([(3)H]-5-HT) was less diminished (at 100 microM by 53%, IC(50)=39.8 microM). Riluzole up to 100 microM did not affect [(3)H]-noradrenaline ([(3)H]-NA) release. 4. Between species, i.e. in mouse, rat and human neocortex, no significant differences between the effects of riluzole could be observed. 5. The NMDA-receptor blocker MK-801 (1 microM) and the AMPA/Kainate-receptor blocker NBQX (1 microM) did neither affect the electrically evoked [(3)H]-ACh release nor its inhibition by riluzole, indicating that effects of riluzole on transmitter release were neither due to modulation of ionotropic Glu receptors, nor due to indirect inhibition of Glu release through these receptors. 6. Taken together, riluzole inhibits the release of distinct neurotransmitters differently, but is not selective for the excitatory amino acid Glu.     

Characterization of somatodendritic neuronal nicotinic receptors located on the myenteric plexus.

The effects of nicotine and dimethylphenylpiperazinium (DMPP) on resting and stimulation-evoked release of [3H]-acetylcholine ([3H]ACh) from cholinergic interneurons and neuro-effector neurons of the ileal longitudinal muscle and the responses of the smooth muscle to nicotinic agonists were studied. (-)-Nicotine was 15 times more effective than (+)-nicotine in releasing ACh. Since tetrodotoxin (1 microM) completely antagonized the effect of nicotinic agonists, the site of action of the nicotinic agonists studied was on the somatodendritic nicotinic receptors. The electrical field stimulation-evoked release was not affected by nicotinic agonists and antagonists, indicating that the axon terminals of cholinergic interneurons are not equipped with nicotinic receptors. This preparation proved to be useful to study the effect of nicotinic agonists on somatodendritic receptors, to determine the affinity constants of nicotinic antagonists, and to characterize these receptors. The rank order of antagonists was d-tubocurarine = mecamylamine greater than pipecuronium greater than pancuronium greater than vecuronium greater than hexamethonium; the apparent affinity constants (KD) were 1.15, 1.55, 3.06, 3.98, 13.59 and 32.88 microM, respectively. alpha-Bungarotoxin had no antagonistic activity at all. This finding indicates that nicotine and the endogenous ligand ACh act via a postsynaptic, somatodendritic nicotinic receptor that is pharmacologically similar to those located on the axon terminals of sympathetic neurons or in ganglions, but is dissimilar to those located at the postsynaptic site of the neuromuscular junction.     

Histamine H(3) receptor-mediated inhibition of depolarization-induced, dopamine D(1) receptor-dependent release of [(3)H]-gamma-aminobutryic acid from rat striatal slices

1. A study was made of the regulation of [(3)H]-gamma-aminobutyric acid ([(3)H]-GABA) release from slices of rat striatum by endogenous dopamine and exogenous histamine and a histamine H(3)-agonist. Depolarization-induced release of [(3)H]-GABA was Ca(2+)-dependent and was increased in the presence of the dopamine D(2) receptor family antagonist, sulpiride (10 microM). The sulpiride-potentiated release of [(3)H]-GABA was strongly inhibited by the dopamine D(1) receptor family antagonist, SCH 23390 (1 microM). Neither antagonist altered basal release. 2. The 15 mM K(+)-induced release of [(3)H]-GABA in the presence of sulpiride was inhibited by 100 microM histamine (mean inhibition 78+/-3%) and by the histamine H(3) receptor-selective agonist, immepip, 1 microM (mean inhibition 81+/-5%). The IC(50) values for histamine and immepip were 1.3+/-0.2 microM and 16+/-2 nM, respectively. The inhibitory effects of histamine and immepip were reversed by the H(3) receptor antagonist, thioperamide, 1 microM. 3. The inhibition of 15 mM K(+)-induced [(3)H]-GABA release by immepip was reversed by the H(3) receptor antagonist, clobenpropit, K(d) 0.11+/-0.04 nM. Clobenpropit alone had no effect on basal or stimulated release of [(3)H]-GABA. 4. Elevated K(+) caused little release of [(3)H]-GABA from striatal slices from reserpinized rats, unless the D(1) partial agonist, R(+)-SKF 38393, 1 microM, was also present. The stimulated release in the presence of SKF 38393 was reduced by 1 microM immepip to the level obtained in the absence of SKF 38393. 5. These observations demonstrate that histamine H(3) receptor activation strongly inhibits the dopamine D(1) receptor-dependent release of [(3)H]-GABA from rat striatum; primarily through an interaction at the terminals of GABA neurones.     

Effects of genistein and herbimycin, tyrosine kinase inhibitors, on catecholamine release in bovine adrenal chromaffin cells

1 The effects of genistein and herbimycin, tyrosine kinase inhibitors, on catecholamine (CA) release were examined in bovine adrenal chromaffin cells. 2 In intact cells, genistein (10-100 microm) and herbimycin (3-30 microm) inhibited CA release induced by acetylcholine (ACh; 100 microm) or the nicotinic receptor stimulant 1,1-dimethyl-4-phenyl-piperazinium (DMPP; 10 microm), but did not affect CA release induced by high K+ (40 mm). 3 Genistein and herbimycin inhibited (45)Ca2+ uptake induced by ACh (100 microm). 4 Neither genistein nor herbimycin affected [(3)H]nicotine binding with nicotinic receptors. 5 In beta-escin-permeabilized cells, neither genistein nor herbimycin affected CA release induced by Ca2+ (1 microm). 6 These results suggest that protein tyrosine kinase plays the facilitatory role in the regulation of CA release induced by nicotinic receptor stimulation in stimulus-secretion coupling of bovine adrenal chromaffin cells.