piperidines and isoguvacine

The GABA(A) receptor system is implicated in a number of neurological diseases, making GABA(A) receptor ligands interesting as potential therapeutic agents. Only a few different classes of structures are currently known as ligands for the GABA recognition site on the GABA(A) receptor complex, reflecting the very strict structural requirements for GABA(A) receptor recognition and activation. Within the series of compounds showing agonist activity at the GABA(A) receptor site that have been developed, most of the ligands are structurally derived from the GABA(A) agonists muscimol, THIP or isoguvacine. Using recombinant GABA(A) receptors, functional selectivity has been shown for a number of compounds such as the GABA(A)agonists imidazole-4-acetic acid and THIP, showing highly subunit-dependent potency and maximal response. In the light of the interest in partial GABA(A) receptor agonists as potential therapeutics, structure-activity studies of a number of analogues of 4-PIOL, a low-efficacy partial GABA(A) agonist, have been performed. In this connection, a series of GABA(A) ligands has been developed showing pharmacological profiles from moderately potent low-efficacy partial GABA(A) agonist activity to potent and selective antagonist effect. Only little information about direct acting GABA(A) receptor agonists in clinical studies is available. Results from clinical studies on the effect of the GABA(A) agonist THIP on human sleep pattern shows that the functional consequences of a direct acting agonist are different from those seen after administration of GABA(A) receptor modulators.

Topics: Animals; Central Nervous System Diseases; GABA Agents; GABA Agonists; GABA Antagonists; Humans; Isonicotinic Acids; Isoxazoles; Ligands; Muscimol; Piperidines; Protein Subunits; Receptors, GABA-A

We wanted to ascertain whether the lateral parabrachial nucleus was involved in mediating the heart-rate response evoked during stimulation of somatic nociceptors. Reversible inactivation of the lateral parabrachial nucleus, using a GABA(A) agonist, reduced the reflex tachycardia evoked during noxious (mechanical) stimulation of the forelimb by approximately 50%. The same effect was observed after blockade of neurokinin 1 receptors within the lateral parabrachial nucleus, indicating a possible involvement for substance P as a neurotransmitter. Immunocytochemistry revealed a strong expression of substance P-immunoreactive fibers and boutons in all lateral subnuclei, but they were particularly dense in the lateral crescent subnucleus. Histological verification showed that the most effective injection sites for attenuating the noxious-evoked tachycardia were all placed in or near to the lateral crescent nucleus of the lateral parabrachial complex. Many single units recorded from this region were activated by high-intensity brachial nerve stimulation. The brachial nerve evoked firing responses of some of these neurons was reversibly reduced after local delivery of a neurokinin 1 receptor antagonist. However, only a minority of these neurons followed a paired-pulse stimulation protocol applied to the spinal cord, suggesting a predominance of indirect projections from the spinal cord to the parabrachial nucleus. We conclude that the cardiac component of the response to somatic nociception involves indirect spinal pathways that most likely excite neurons located in the lateral crescent nucleus of the parabrachial complex via activation of neurokinin 1 receptors.

Topics: Afferent Pathways; Animals; Brachial Plexus; Decerebrate State; Efferent Pathways; Female; GABA Agonists; GABA-A Receptor Agonists; Heart Rate; Hypertension; Isonicotinic Acids; Male; Neurokinin-1 Receptor Antagonists; Nociceptors; Pain; Piperidines; Pons; Posterior Horn Cells; Rats; Rats, Sprague-Dawley; Receptors, Neurokinin-1; Reflex; Spinal Cord; Substance P; Sympathetic Nervous System; Tachycardia

Somatic noxious stimulation can evoke profound cardiovascular responses by altering activity in the autonomic nervous system. This noxious stimulation attenuates the cardiac vagal baroreflex, a key cardiovascular homeostatic reflex. This attenuation is mediated via NK1 receptors expressed on GABAergic interneurones within the nucleus of the solitary tract (NTS). We have investigated the effect of noxious stimulation and exogenous substance P (SP) on the sympathetic component of the baroreflex. We recorded from the sympathetic chain in a decerebrate, artificially perfused rat preparation. Noxious hindlimb pinch was without effect on the sympathetic baroreflex although the cardiac vagal baroreflex gain was decreased (56 %, P < 0.01). Bilateral NTS microinjection of SP (500 fmol) produced a similar selective attenuation of the cardiac vagal baroreflex gain (62 %, P < 0.005) without effect on the sympathetic baroreflex. Recordings from the cardiac sympathetic and vagal nerves confirmed the selectivity of the SP inhibition. Control experiments using a GABAA receptor agonist, isoguvacine, indicated that both components of the baroreflex (parasympathetic and sympathetic) could be blocked from the NTS injection site. The NTS microinjection of a NK1 antagonist (CP-99,994) in vivo attenuated the tachycardic response to hindlimb pinch. Our data suggest that noxious pinch releases SP within the NTS to selectively attenuate the cardiac vagal, but not the sympathetic, component of the baroreflex. This selective withdrawal of the cardiac vagal baroreflex seems to underlie the pinch-evoked tachycardia seen in vivo. Further, these findings confirm that baroreflex sympathetic and parasympathetic pathways diverge, and can be independently controlled, within the NTS.

Topics: Animals; Baroreflex; GABA Agonists; Heart; Hindlimb; Isonicotinic Acids; Male; Microinjections; Nociceptors; Pain; Parasympathetic Nervous System; Physical Stimulation; Piperidines; Rats; Rats, Wistar; Receptors, Neurokinin-1; Solitary Nucleus; Substance P; Sympathetic Nervous System; Tachycardia; Vagus Nerve

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

We have previously characterized 5-(4-piperidyl)isoxazol-3-ol (4-PIOL) as a non-desensitizing partial agonist at GABAA receptors and shown that the responses are mediated by short-duration channel openings consonant with single-ligand gated openings of the Cl- channels. We presently investigate whether responses of cultured rat hippocampal neurones to 4-PIOL are modulated by benzodiazepine (BDZ) and barbiturate receptor ligands. Whole-cell patch-clamp recordings of maximal responses to 1 mM 4-PIOL were comparable in size to responses evoked by 10 microM of the full GABAA agonist, isoguvacine. The BDZ receptor inverse agonist, DMCM (1 microM) reduced responses to isoguvacine (to 65.7 +/- 11.0%) and 4-PIOL (to 69.3 +/- 3.5%) to a similar extent. The BDZ agonist, midazolam (0.1 microM) potentiated responses to both agonists, and resulted in responses with an early peak with later fading. Potentiation of the peak response to 4-PIOL (to 163 +/- 14%) was significantly less than for isoguvacine (215 +/- 11%). Pentobarbital (50 microM) caused a very marked, but variable, potentiation of the peak response to 4-PIOL (to 484 +/- 93%), which was significantly greater than the potentiation of the peak response to isoguvacine (to 304 +/- 46%), and induced fading. This suggests that a relatively larger number of the 4-PIOL-induced channel openings can be transformed to longer duration openings by pentobarbital. In conclusion, responses to 4-PIOL and isoguvacine are modulated by BDZ and barbiturate ligands in a qualitatively similar manner, but with a number of quantitative differences which cannot be readily explained by the kinetic model of Macdonald and Twyman (1992). Investigation of these responses at the single-channel level could provide further insight into the operation of the GABAA receptor-ionophore complex.

Topics: Animals; Barbiturates; Benzodiazepines; Carbolines; Cells, Cultured; Convulsants; Electrophysiology; GABA Agonists; GABA Modulators; GABA-A Receptor Agonists; Hippocampus; Hypnotics and Sedatives; Isonicotinic Acids; Isoxazoles; Membrane Potentials; Midazolam; Neurons; Patch-Clamp Techniques; Pentobarbital; Piperidines; Rats

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

GABAA receptor agonists modulate [3H]diazepam binding in rat cortical membranes with different efficacies. At 23 degrees C, the relative potencies for enhancement of [3H]diazepam binding by agonists parallel their potencies in inhibiting [3H]gamma-aminobutyric acid [( 3H]GABA) binding. The agonist concentrations needed for enhancement of [3H]diazepam binding are up to 35 times higher than for [3H]GABA binding and correspond closely to the concentrations required for displacement of [3H]bicuculline methochloride (BMC) binding. The maximum enhancement of [3H]diazepam varied among agonists: muscimol = GABA greater than isoguvacine greater than 3-aminopropane sulphonic acid (3APS) = imidazoleacetic acid (IAA) greater than 4,5,6,7-tetrahydroisoxazolo (4,5,6)-pyridin-3-ol (THIP) = taurine greater than piperidine 4-sulphonic acid (P4S). At 37 degrees C, the potencies of agonists remained unchanged, but isoguvacine, 3 APS, and THIP acquired efficacies similar to GABA, whereas IAA, taurine, and P4S maintained their partial agonist profiles. At both temperatures the agonist-induced enhancement of [3H]diazepam binding was reversible by bicuculline methobromide and by the steroid GABA antagonist RU 5135. These results stress the importance of studying receptor-receptor interaction under near-physiological conditions and offer an in vitro assay that may predict the agonist status of putative GABA receptor ligands.

Topics: Androstanes; Animals; Azasteroids; Bicuculline; Cell Membrane; Cerebral Cortex; Diazepam; GABA Antagonists; gamma-Aminobutyric Acid; Imidazoles; Isonicotinic Acids; Isoxazoles; Muscimol; Piperidines; Rats; Receptors, GABA-A; Taurine

THIP and piperidine-4-sulfonic acid (PSA) interact with [3H]GABA binding sites and have GABAmimetic efficacy in vivo, but fail to enhance benzodiazepine receptor binding performed at 0 degree C. However, when [3H]flunitrazepam binding is determined at elevated temperature (30 or 37 degrees C), THIP and PSA display potent chloride ion-dependent stimulatory effects. These results resolve apparent discrepancies between the properties of GABA receptors observed in vivo and in vitro, and they suggest that the modulation of benzodiazepine receptor binding investigated at physiological temperatures can be used as an experimental system for the characterization of GABA receptors.

Topics: Animals; Anti-Anxiety Agents; Chlorides; Flunitrazepam; gamma-Aminobutyric Acid; Isonicotinic Acids; Isoxazoles; Oxazoles; Piperidines; Rats; Stimulation, Chemical; Temperature; Tritium