tetrodotoxin and 3-7-dimethyl-1-propargylxanthine

1. The modulation of voltage-dependent Ca2+ currents (ICa) by adenosine was investigated in magnocellular neurones acutely dissociated from the rat hypothalamic supraoptic nucleus (SON) by using the whole-cell patch-clamp technique. 2. Adenosine dose dependently and reversibly inhibited ICa elicited by depolarizing voltage steps from a holding potential of -80 mV to potentials ranging from -30 to +20 mV. The mean (+/- s.e.m.) maximum inhibition rate was 36.1 +/- 4.1 % (n = 6) at -20 mV and the EC50 was 9.8 x 10-7 M (n = 6). 3. The inhibition of ICa by adenosine was completely reversed by the selective A1 receptor antagonist 8-cyclopentyl theophylline (CPT), and was mimicked by the selective A1 receptor agonist N 6-cyclohexyladenosine (CHA). 4. The inhibition by CHA was strongly reduced when ICa was inhibited by omega-conotoxin GVIA, a blocker of N-type Ca2+ channels. 5. The adenosine-induced inhibition of ICa was largely reversed by a depolarizing prepulse to +150 mV for 100 ms, which is known to reverse the inhibition of Ca2+ channels mediated by G-protein betagamma subunits. 6. The adenosine receptor-mediated inhibition of ICa was not abolished by intracellularly applied preactivated pertussis toxin (PTX). 7. Using immunohistochemistry, Gzalpha-like immunoreactivity (a PTX-resistant inhibitory G-protein) was observed throughout the SON. 8. These results suggest that adenosine modulates the neuronal activity of SON neurones by inhibiting N-type voltage-dependent Ca2+ channels via A1 receptors which are coupled to PTX-resistant G-proteins.

Topics: Adenosine; Animals; Calcium Channels; Calcium Channels, N-Type; In Vitro Techniques; Male; Neurons; Patch-Clamp Techniques; Rats; Rats, Wistar; Receptor, Adenosine A2A; Receptor, Adenosine A2B; Receptor, Adenosine A3; Receptors, Purinergic P1; Reverse Transcriptase Polymerase Chain Reaction; Supraoptic Nucleus; Tetrodotoxin; Theobromine

In this study the regulation of cardiac sympathetic outflow by presynaptic P(2X) receptor-gated ion channels was examined. ATP (30 microM - 1 mM) and other P2-receptor agonists elicited [(3)H]-noradrenaline ([(3)H]-NA) outflow from the isolated guinea-pig right atrium with the potency order of ATP>2-methyl-thioATP>alpha,beta-methylene-ATP=ADP, whereas ss, gamma-methylene-L-ATP was inactive. Ca(2+)-free conditions abolished both electrical field stimulation (EFS)- and ATP-evoked release of tritium. Unlike from EFS-induced outflow, ATP-induced [(3)H]-NA outflow was not reduced by omega-Conotoxin-GVIA (100 nM), Cd(2+) (100 microM) and tetrodotoxin (1 microM). The rapid extracellular decomposition of ATP was revealed by HPLC analysis. However, the effect of ATP to promote [(3)H]-NA release was not prevented by 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 250 nM), 3, 7-dimethyl-1-propargylxanthine (DMPX, 250 nM), or by reactive blue 2 (RB2, 10 microM), antagonists of A(1)-, A(2)- and inhibitory P(2) receptors. Zn(2+) (50 microM), the P(2X)-receptor modulator potentiated, and P(2X) receptor antagonists, i.e. suramin (300 microM), pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS, 30 microM) and 2'-o-(trinitrophenyl)-adenosine 5'-triphosphate (TNP-ATP, 30 microM) antagonized the ATP (1 mM)-evoked response. RT - PCR study revealed the expression of P(2X2) and P(2X3) receptor mRNAs in guinea-pig superior cervical ganglion. PPADS (30 microM) significantly reduced the EFS-induced [(3)H]-NA outflow in the presence DPCPX (250 nM) and RB2 (10 microM). In summary a P(2X)-type purinoceptor regulates noradrenaline release from the isolated right atrium of the guinea-pig. The pharmacological profile of the receptor resemble to homo-oligomeric P(2X3) or hetero-oligomeric P(2X2)/P(2X3) complexes, and provide a new target to intervene on sympathetic neuroeffector transmission at the presynaptic site.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Animals; Cadmium; Dose-Response Relationship, Drug; Electric Stimulation; Gene Expression; Guinea Pigs; Heart Atria; Hippocampus; In Vitro Techniques; Male; Norepinephrine; omega-Conotoxin GVIA; Presynaptic Terminals; Purinergic P2 Receptor Agonists; Purinergic P2 Receptor Antagonists; Pyridoxal Phosphate; Receptors, Purinergic P2; RNA; Superior Cervical Ganglion; Tetrodotoxin; Theobromine; Thionucleotides; Time Factors; Tritium; Xanthines

This report describes the effects of midazolam and beta-carboline-3-carboxylate ethyl ester (beta-CCE) on extracellular concentrations of dopamine in the nucleus accumbens of freely moving rats measured by in vivo microdialysis. The two compounds had opposite effects, midazolam (0.075 and 0.15 mg/kg i.v.) dose dependently decreasing, and beta-CCE (3 and 10 mg/kg i.p.) dose dependently increasing, dialysate concentrations of dopamine. Flumazenil (6 micrograms/kg i.v.) did not affect the efflux of dopamine but it prevented the effects of both midazolam and beta-CCE on dopamine efflux. N6-Cyclohexyladenosine (0.1, and 1 mg/kg i.p.), a selective adenosine A1 agonist, dose dependently increased the efflux of dopamine. This effect was blocked by 8-cyclopentyl-1,3-dipropylxanthine (25 mg/kg i.p.), a selective adenosine A1 receptor antagonist, a dose which given alone did not affect dopamine efflux; responses to midazolam were not affected. 3,7-Dimethyl-1-propargylxanthine (1 and 3 mg/kg i.p.), a selective adenosine A2 receptor antagonist, did not mimic the effects of beta-CCE. The results suggest that midazolam and beta-CCE modulate dopamine release in the nucleus accumbens by an action at the benzodiazepine binding site associated with the GABAA receptor complex.

Topics: Adenosine; Animals; Carbolines; Dopamine; Dose-Response Relationship, Drug; Flumazenil; Ligands; Male; Microdialysis; Midazolam; Nucleus Accumbens; Purinergic P1 Receptor Antagonists; Rats; Rats, Sprague-Dawley; Receptors, GABA-A; Tetrodotoxin; Theobromine; Xanthines