bicuculline-methobromide and 5-(4-piperidyl)isoxazol-3-ol

Based on an unexpected high maximum response to piperidine-4-sulphonic acid (P4S) at human alpha1alpha6beta2gamma2 GABA(A) receptors expressed in Xenopus oocytes attempts to correlate this finding with the pharmacological profile of P4S and other GABA(A) receptor ligands in neuronal cultures from rat cerebellar granule cells and rat cerebral cortex were carried out. GABA and isoguvacine acted as full and piperidine-4-sulphonic acid (P4S) as partial agonists, respectively, at alpha1beta2gamma2, alpha6beta2gamma2 and alpha1alpha6beta2gamma2 GABA receptors expressed in Xenopus oocytes with differences in potency. Whole-cell patch-clamp recordings were used to investigate the pharmacological profile of the partial GABA(A) receptor agonists 4,5,6,7-tetrahydroisoxazolo-(5,4-c)pyridin-3-ol (THIP), P4S, 5-(4-piperidyl)isoxazol-3-ol (4-PIOL), and 3-(4-piperidyl)isoxazol-5-ol (iso-4-PIOL), and the competitive GABA(A) receptor antagonists Bicuculline Methbromide (BMB) and 2-(3-carboxypropyl)-3-amino-6-methoxyphenyl-pyridazinium bromide (SR95531) on cerebral cortical and cerebellar granule neurons. In agreement with findings in oocytes, GABA, isoguvacine and P4S showed similar pharmacological profiles in cultured cortical and cerebellar neurones, which are known to express mainly alpha1, alpha2, alpha3, and alpha5 containing receptors and alpha1, alpha6 and alpha1alpha6 containing receptors, respectively. 4-PIOL and iso-4-PIOL, which at GABA(A) receptors expressed in oocytes were weak antagonists, showed cell type dependent potency as inhibitors of GABA mediated responses. Thus, 4-PIOL was slightly more potent at cortical neurones than at granule neurones and iso-4-PIOL was more potent in inhibiting isoguvacine-evoked currents at cortical than at granule neurons. Furthermore the maximum response to 4-PIOL corresponded to that of a partial agonist, whereas that of iso-4-PIOL gave a maximum response close to zero. It is concluded that the pharmacological profile of partial agonists is highly dependent on the receptor composition, and that small structural changes of a ligand can alter the selectivity towards different subunit compositions. Moreover, this study shows that pharmacological actions determined in oocytes are generally in agreement with data obtained from cultured neurons.

Topics: Animals; Bicuculline; Cells, Cultured; Cerebellum; Cerebral Cortex; Electrophysiology; GABA Agonists; GABA-A Receptor Agonists; Isonicotinic Acids; Isoxazoles; Mice; Neurons; Oocytes; Patch-Clamp Techniques; Piperidines; Pyridazines; Receptors, GABA-A; Xenopus laevis

1. Whole-cell, patch-clamp recordings from cultured hippocampal neurones have been used to characterize the action of the GABAA ligand, 5-(4-piperidyl)isoxazol-3-ol (4-PIOL). The action of 4-PIOL was compared with that of the established GABAA agonist, isoguvacine. 2. With a symmetrical Cl- gradient across the membrane and a holding potential of -60mV, both isoguvacine and 4-PIOL evoked an inward current. The reversal potentials of the responses to both agents were identical (+8.8 mV, n = 4) and the current/voltage relationships showed outward-going rectification. 3. The response to 300 microM 4-PIOL was completely blocked by the GABAA antagonist, bicuculline methobromide (BMB, 10 microM). The pA2 of BMB was greater than 6.46. With 2 mM 4-PIOL about 15% of the response remained in the presence of 100 microM BMB. This may represent a non-specific component of the response to large concentrations of 4-PIOL. 4. 4-PIOL was about 200 times less potent as an agonist than isoguvacine. because of the rapid fade (desensitization) of isoguvacine-induced currents, the maximum response to this agonist was not determined. However, the response to 2 mM 4-PIOL was only a small fraction of that evoked by submaximal concentrations of isoguvacine. 5. Setting the response to 1 mM 4-PIOL as maximum, the EC50 for 4-PIOL was 91 microM (95% confidence limits:73-114 microM). 6. 4-PIOL antagonized the response to isoguvacine with a parallel shift to the right of the dose-response curve. The antagonist action of 4-PIOL was about 30 times weaker than that of BMB. When allowance was made for the intrinsic agonist action of 4-PIOL, the Ki was 116p microM (95% confidence limits: 102-130 microM). This was not significantly different from EC5, (P = 0.86; non-parametric Mann-Whitney test).7. It is concluded that 4-PIOL is a partial agonist at the GABAA receptor on cultured hippocampal neurones.

Topics: Animals; Bicuculline; Cells, Cultured; Female; Hippocampus; Isonicotinic Acids; Isoxazoles; Membrane Potentials; Neurons; Piperidines; Pregnancy; Rats; Receptors, GABA-A; Spinal Cord