piperidines and piperidine-4-sulfonic-acid

The GABA type A receptor (GABA(A)R) is expressed ubiquitously throughout the brain and is a target for many therapeutic agents, including general anesthetics and benzodiazepines, which enhance receptor function by increasing the open probability (P(o)) of the ion channel. It is commonplace for in vitro studies of receptor pharmacological characteristics to use negative membrane holding potentials to mimic the resting potential of neurons and symmetrical chloride to eliminate Goldman rectification, which results in chloride flow in the opposite direction, compared with in vivo conditions. This critical difference is usually overlooked because the GABA(A)R has been reported to behave as an ohmic pore, but our results show that the current-voltage relationship is nonlinear with respect to P(o). Specifically, we found that currents were outwardly rectifying at low P(o) and linear at high P(o). We confirmed the correlation between P(o) and rectification with a partial agonist, piperidine-4-sulfonic acid, and a gating-impaired mutation, α1(L277A); both exhibited enhanced outward rectification. Furthermore, this correlation was independent of Goldman rectification and persisted under altered chloride gradient conditions, which suggests that rectification is linked to the direction of chloride flux. Finally, our results showed that the degree of potentiation by general anesthetics (etomidate, propofol, and isoflurane) was greater at negative membrane potentials. Traditional in vitro experiments thus overestimate the action of positive allosteric modulators of the GABA(A)R. Our results show that the direction of the driving force on the permeant ion, as well as P(o), must be considered together for a complete understanding of drug actions on ligand-gated ion channels.

Topics: Allosteric Regulation; Chloride Channels; GABA Agonists; Humans; Ligand-Gated Ion Channels; Membrane Potentials; Piperidines; Probability; Protein Conformation; Receptors, GABA-A

GABA(A) receptors (GABA(A)Rs) composed of αβγ subunits are allosterically modulated by the benzodiazepines (BDZs). Agonists at the BDZ binding site potentiate submaximal GABA responses by increasing the apparent affinity of GABA(A)Rs for GABA. Although BDZs were initially thought to affect the binding of GABA agonists, recent studies suggest an effect on receptor gating; however, the involvement of preactivation steps in the modulation by BDZs has not been considered. Consequently, we examined whether BDZ agonists could exert their modulatory effect by displacing the equilibrium between resting and preactivated states of recombinant α1β2γ2 GABA(A)Rs expressed in Xenopus oocytes. For GABA and the partial agonists 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol and piperidine-4-sulfonic acid, we examined BDZ modulation using a simple three-step model incorporating agonist binding, receptor preactivation, and channel opening. The model accounted for diazepam modulation simply by increasing the preactivation constant by approximately fourfold. To assess whether BDZs preferentially affected a specific GABA binding site, pentameric concatamers were used. This demonstrated that single GABA-binding site mutant receptors were equally sensitive to modulation by BDZs compared with wild-type counterparts. Overall, our results suggest that BDZs affect the preactivation step to cause a global conformational rearrangement of GABA(A)Rs, thereby modulating receptor function.

Topics: Animals; Benzodiazepines; Binding Sites; Diazepam; Dose-Response Relationship, Drug; Drug Interactions; GABA Agonists; GABA Antagonists; GABA Modulators; gamma-Aminobutyric Acid; Ion Channel Gating; Isoxazoles; Larva; Membrane Potentials; Models, Biological; Mutation; Oocytes; Patch-Clamp Techniques; Piperidines; Protein Binding; Protein Subunits; Receptors, GABA-A; Xenopus

Neuroactive steroids are among the most efficacious modulators of the mammalian GABA-A receptor. Previous work has proposed that receptor potentiation is mediated by steroid interactions with a site defined by the residues alpha1Asn407/Tyr410 in the M4 transmembrane domain and residue alpha1Gln241 in the M1 domain. We examined the role of residues in the alpha1 subunit M1 domain in the modulation of the rat alpha1beta2gamma2L GABA-A receptor by neuroactive steroids. The data demonstrate that the region is critical to the actions of potentiating neuroactive steroids. Receptors containing the alpha1Q241W or alpha1Q241L mutations were insensitive to (3alpha,5alpha)-3-hydroxypregnan-20-one (3alpha5alphaP), albeit with different underlying mechanisms. The alpha1Q241S mutant was potentiated by 3alpha5alphaP, but the kinetic mode of potentiation was altered by the mutation. It is noteworthy that the alpha1Q241L mutation had no effect on channel potentiation by (3alpha,5alpha)-3-hydroxymethyl-pregnan-20-one, but mutation of the neighboring residue, alpha1Ser240, prevented channel modulation. A steroid lacking an H-bonding group on C3 (5alpha-pregnan-20-one) potentiated the wild-type receptor but not the alpha1Q241L mutant. The findings are consistent with a model in which the alpha1Ser240 and alpha1Gln241 residues shape the surface to which steroid molecules bind.

Topics: Amino Acid Substitution; Animals; Cell Line; gamma-Aminobutyric Acid; Humans; Ion Channel Gating; Mutant Proteins; Mutation; Piperidines; Pregnanetriol; Pregnanolone; Pregnenolone; Protein Structure, Secondary; Protein Structure, Tertiary; Protein Subunits; Rats; Receptors, GABA-A; Serine; Steroids; Tryptophan

The use of the antiepileptic drug, 4-aminobutyrate transaminase (GABA-T) inhibitor vigabatrin (VIGA), has been recently cautioned because it is associated to irreversible field defects from damage of the retina. Since novel GABA-T inhibitors might prove useful in epilepsy or other CNS pathologies as VIGA substitutes, the aim of the present investigation was to characterize the biochemical properties of some taurine analogues (TA) previously shown to act as GABA-T inhibitors. These include (+/-)piperidine-3-sulfonic acid (PSA), 2-aminoethylphosphonic acid (AEP), (+/-)2-acetylaminocyclohexane sulfonic acid (ATAHS) and 2-aminobenzenesulfonate (ANSA). Kinetic analysis of the activity of partially purified rabbit brain GABA-T in the presence of VIGA and TA showed that PSA and AEP caused a linear, mixed-type inhibition (Ki values 364 and 1010 microM, respectively), whereas VIGA, ANSA and ATAHS behaved like competitive inhibitors (Ki values 320, 434 and 598 microM, respectively). Among the compounds studied, only VIGA exerted a time-dependent, irreversible inhibition of the enzyme, with Ki and k(inact) values of 773 microM and 0.14 min(-1), respectively. Furthermore, the ability of VIGA and TA to enhance GABA-ergic transmission was assessed in rabbit brain cortical slices by NMR quantitative analysis. The results demonstrate that VIGA as well as all TA promoted a significant increase of GABA content. In conclusion, PSA, ANSA and ATAHS, reversible GABA-T inhibitors with Ki values close to that of VIGA, represent a new class of compounds, susceptible of therapeutic exploitation in many disorders associated with low levels of GABA in brain tissues.

Topics: 4-Aminobutyrate Transaminase; Aminoethylphosphonic Acid; Animals; Anticonvulsants; Brain; Enzyme Inhibitors; Male; Piperidines; Rabbits; Sulfanilic Acids; Taurine; Vigabatrin

The binding of the neurotransmitter GABA induces conformational changes in the GABAA receptor (GABAAR), leading to the opening of a gate that controls ion permeation through an integral transmembrane pore. A number of structural elements within each subunit, located near the membrane interface, are believed to undergo relative movements during this activation process. In this study, we explored the functional role of the beta-10 strand (pre-M1 segment), which connects the extracellular domain to the transmembrane domain. In alpha1beta2gamma2s GABAARs, analysis of the 12 residues of the beta-10 strand in the alpha1 subunit proximal to the first transmembrane domain identified two residues, alpha1V212 and alpha1K220, in which mutations produced rightward shifts in the GABA concentration-response relationship and also reduced the relative efficacy of the partial agonist, piperidine-4-sulphonic acid. Ultra-fast agonist techniques were applied to mutant alpha1(K220A)beta2gamma2s GABAARs and revealed that the macroscopic functional deficit in this mutant could be attributed to a slowing of the opening rate constant, from approximately 1500 s(-1) in wild-type (WT) channels to approximately 730 s(-1) in the mutant channels, and a reduction in the time spent in the active state for the mutant. These changes were accompanied by a decrease in agonist affinity, with half-maximal activation rates achieved at 0.77 mM GABA in WT and 1.4 mM GABA in the alpha1(K220A)beta2gamma2s channels. The beta-10 strand (pre-M1 segment) emerges, from this and other studies, as a key functional component in the activation of the GABAAR.

Topics: Cell Line; Computer Simulation; Dose-Response Relationship, Drug; GABA Agonists; GABA-A Receptor Agonists; gamma-Aminobutyric Acid; Humans; Ion Channel Gating; Kinetics; Membrane Potentials; Models, Biological; Mutation; Piperidines; Protein Conformation; Receptors, GABA-A; Transfection

Volatile anesthetics prolong inhibitory postsynaptic potentials in central neurons an allosteric action on the gamma-aminobutyric acid type A (GABA(A)) receptor, an effect that may underlie the hypnotic actions of these agents. Inhaled anesthetics such as isoflurane act to enhance responses to submaximal concentrations of GABA, but it is not clear whether their effect is mediated by an increase in the binding of the agonist or by changes in receptor gating behavior. To address this question, the authors studied the effects of isoflurane on a mutant GABA(A) receptor with a gating defect that decreases receptor sensitivity by lowering agonist efficacy. They then compared the effects of clinically relevant concentrations of isoflurane on the actions of GABA and piperidine-4-sulfonic acid (P4S), a partial agonist at the GABA(A) receptor.. The authors created a mutant of the GABA receptor alpha subunit (L277A) by site-directed mutagenesis. The mutant subunit was coexpressed with beta(2) and gamma(2S) subunits in HEK293 cells, and responses to GABA and P4S were recorded using the whole-cell patch clamp technique. EC values were determined for the full agonist GABA and the partial agonist P4S. The authors also determined the relative efficacy (epsilon) of P4S. These measurements were then repeated in the presence of isoflurane.. The concentration-response curve for GABA was shifted to the right (EC(50) = 278 microm) in the alpha(1)(L277A)beta(2)gamma(2S) mutant receptor, compared with the corresponding wild-type alpha(1)beta(2)gamma(2S) GABA(A) receptor (EC(50) = 16 microm). P4S is a partial agonist at both receptors, with a dramatically decreased relative efficacy at the mutant receptor (epsilon = 0.24). When the mutant receptor was studied in the presence of isoflurane, the concentration-response curves for both GABA and P4S were shifted to the left (EC(50) for GABA = 78 microm); the efficacy of P4S also increased significantly (epsilon = 0.40).. By studying a mutant GABA receptor with impaired gating, the authors were able to demonstrate clearly that isoflurane can increase the efficacy of a partial agonist, as well as increase agonist potency. These data suggest that the volatile anesthetic isoflurane exerts at least some of its effects on the GABA(A) receptor via alterations in gating rather than simply changing binding or unbinding of the agonist.

Topics: Anesthetics, Inhalation; Cell Line; Cells, Cultured; Dose-Response Relationship, Drug; Electrophysiology; GABA Agonists; gamma-Aminobutyric Acid; Humans; Ion Channel Gating; Isoflurane; Mutagenesis, Site-Directed; Mutation; Patch-Clamp Techniques; Piperidines; Receptors, GABA-A; Transfection

In order to study the correlation of the thermodynamic driving forces of binding with the efficacies of displacing ligands, the specific binding of [3H]SR 95531 [2-(3-carboxypropyl)3-amino-6-p-methoxyphenylpyridazinium bromide], a GABA(A) receptor antagonist, was studied in cell lines stably expressing human alpha(1)beta(3)gamma(2) and alpha(2)beta(3)gamma(2) GABA(A) receptors. Displacing potencies for the agonists with different efficacies (muscimol, 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP) and piperidine-4-sulfonic acid) and for antagonists (SR 95531 and 5-(4-piperidyl)isothiazol-3-ol) were determined at 0 degrees C, 20 degrees C and 37 degrees C. Displacing potencies were temperature-nearly independent for alpha(1)beta(3)gamma(2) receptors. At alpha(2)beta(3)gamma(2), receptor binding of the antagonists was exothermic, endothermic for the agonists THIP and piperidine-4-sulfonic acid and isothermic for muscimol. The free energy increments of displacement for the binding of the antagonist [3H]SR 95531 versus the agonist [3H]muscimol approach saturation as a function of the efficacies of the displacers only for alpha(1)beta(3)gamma(2) receptors. This suggests that, for binding to alpha(1)beta(3)gamma(2) GABA(A) receptors, displacement is an efficacy-dependent interaction predominantly driven by entropic increases.

Topics: Animals; Binding, Competitive; Dose-Response Relationship, Drug; Entropy; GABA Agonists; GABA Antagonists; Humans; Isoxazoles; L Cells; Membranes; Mice; Muscimol; Piperidines; Pyridazines; Receptors, GABA-A; Recombinant Proteins; Temperature; Tritium

Neurosteroids are produced in the brain, and can have rapid actions on membrane channels of neurons. Pregnenolone sulfate (PS) is a sulfated neurosteroid which reduces the responses of the [gamma]-aminobutyric acid A (GABA(A)) receptor. We analysed the actions of PS on single-channel currents from recombinant GABA(A) receptors formed from [alpha]1, [beta]2 and [gamma]2L subunits. Currents were elicited by a concentration of GABA eliciting a half-maximal response (50 microM) and a saturating concentration (1 mM). PS reduced the duration of clusters of single-channel activity at either concentration of GABA. PS had no discernable effect on rapid processes: no effects were apparent on channel opening and closing, nor on GABA affinity, and a rapidly recovering desensitised state was not affected. Instead, PS produced a slowly developing block which occurred at a similar rate for receptors with open or closed channels and with one or two bound GABA molecules. The rate of block was independent of membrane potential, implying that the charged sulfate moiety does not move through the membrane field. Change in a specific residue near the intracellular end of the channel lining portion of the [alpha]1 subunit had a major effect on the rate of block. Mutation of the residue [alpha]1 V256S reduced the rate of block by 30-fold. A mutation at the homologous position of the [beta]2 subunit ([beta]2 A252S) had no effect, nor did a complementary mutation in the [gamma]2L subunit ([gamma]2L S266A). It seems likely that this residue is involved in a conformational change underlying block by PS, instead of forming part of the binding site for PS.

Topics: Binding Sites; Cell Line; Estranes; GABA Agonists; GABA Modulators; gamma-Aminobutyric Acid; Humans; Ion Channel Gating; Kidney; Ligands; Membrane Potentials; Mutagenesis; Nitriles; Patch-Clamp Techniques; Pentobarbital; Piperidines; Pregnenolone; Receptors, GABA-A; Transfection

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. We have studied the kinetic properties of channel gating of recombinant alpha 1 beta 2 gamma 2L GABA(A) receptors transiently expressed in human embryonic kidney 293 cells, using the cell-attached, single-channel patch-clamp technique. The receptors were activated by GABA, beta-alanine or piperidine-4-sulfonic acid (P4S), and the effects of pentobarbital (PB) on single-channel activity were examined. 2. At relatively high concentrations of agonist, single-channel activity occurred in well-defined clusters. In global terms, PB increased the mean open time for events in clusters, without changing the mean closed time. The addition of PB shifted the curve relating the probability of being open in a cluster (P(o)) to lower agonist concentrations, and that shift could be accounted for by the changes in mean open time. 3. The intracluster closed-time histograms contained four components. The durations and relative frequencies of these closed-dwell components were not affected by the presence of 40 microM PB, at any agonist concentration. The duration of one component was dependent upon the concentration of agonist used to activate the receptor. Accordingly, the inverse of the mean duration of this component will be called the effective opening rate. 4. The channel-opening rate constant (beta) was determined from the value of the effective opening rate at a saturating agonist concentration. beta was about 1900 s(-1) when the receptors were activated by GABA, 1500 s(-1) when activated by beta-alanine, and too low to be determined when P4S was administered. In the presence of 40 microM PB, beta was about 1500 s(-1) when the receptors were activated by GABA, 1400 s(-1) when activated by beta-alanine, and 50 s(-1) when activated by P4S. Hence, the potentiating effect of PB is not mediated by a change in beta. The concentration of agonist producing a half-maximal effective opening rate also remained unaffected in the presence of PB, indicating that receptor affinity for agonists is not influenced by PB. 5. The distributions of the intracluster open durations elicited by GABA could be described by the sum of three exponentials, with mean durations of about 0.4, 2.4 and 6.3 ms. The duration and relative frequency of the components did not change with GABA concentration (20 microM to 1 mM). In the presence of 40 microM PB, however, the mean duration of the longest of the open times increased (mean durations of about 0.4, 2.0 and 13 ms). The intracluster open duration

Topics: beta-Alanine; Cell Line; Dose-Response Relationship, Drug; Drug Synergism; GABA Agonists; GABA Modulators; gamma-Aminobutyric Acid; Humans; Ion Channel Gating; Ion Channels; Osmolar Concentration; Pentobarbital; Piperidines; Protein Isoforms; Receptors, GABA-A; Recombinant Proteins

Using the whole-cell patch-clamp technique, we have determined that propofol, but not midazolam, increases the efficacy of piperidine-4-sulphonic acid (P4S), a partial agonist at alpha1beta1gamma2s, GABA(A) receptors expressed in HEK 293 cells. These findings are consistent with the idea that propofol facilitates receptor gating, while midazolam increases receptor occupancy by the agonist.

Topics: Allosteric Regulation; Anesthetics, Intravenous; Cells, Cultured; Drug Synergism; GABA Agonists; GABA Modulators; Humans; Ion Channel Gating; Kidney; Ligands; Midazolam; Patch-Clamp Techniques; Piperidines; Propofol; Receptors, GABA-A

Using human gamma-aminobutyric acid type A (GABAA) receptor subunit combinations, expressed in cell lines and Xenopus laevis oocytes, the pharmacology of a number of ligands interacting directly with the GABA recognition site has been studied in [3H]muscimol binding and electrophysiologically. The binding affinity of GABAA agonist and antagonist ligands showed small but statistically significant dependence on the subunit composition of receptors that include gamma 2 and different alpha and beta subunits. The potency of antagonist ligands was largely independent of receptor subunit composition, whereas the composition of receptors expressed in oocytes strongly influenced the EC50 value of agonists. An apparent reciprocal correlation between subunits favoring agonist binding and antagonist binding, respectively, was observed. Whereas antagonists showed comparable potencies in binding and functional studies, the potency of agonists in binding studies was generally two to three orders of magnitude higher than the agonist potencies measured electrophysiologically. 5-(4-Piperidyl)isothiazol-3-ol, which behaves as a low efficacy partial agonist at GABAA receptors in cultured cortical neurons, showed no efficacy in oocytes, but produced pure antagonist effects with a binding/functional affinity ratio between those observed for the agonists and antagonists. It is concluded that the GABAA receptor mechanisms transducing binding into physiological response, but not the binding per se, is dependent on the receptor subunit composition.

Topics: Animals; Binding, Competitive; DNA, Complementary; Electrophysiology; GABA Agonists; GABA Antagonists; Humans; Isoxazoles; Kinetics; Macromolecular Substances; Muscimol; Oocytes; Piperidines; Receptors, GABA-A; Transduction, Genetic; Transfection; Tritium; Xenopus laevis

Using systematic combination of alpha 1, alpha 3, and alpha 5 with beta 1, beta 2, and beta 3, together with gamma 1, gamma 2, and gamma 3, we have investigated the contributions of the various alpha, beta, and gamma subunits to the pharmacology of gamma-aminobutyric acid (GABA)A agonists. We have characterized GABA, (RS)-dihydromuscimol, piperidine-4-sulfonic acid, and 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol with recombinant human GABAA receptors expressed in Xenopus oocytes. Our observations indicate that the alpha subunit is the major determinant of efficacy for partial GABAA agonists. When alpha 1 and alpha 3 or alpha 1 and alpha 5 are coexpressed, the alpha 1 subunit determines the maximum efficacy, whereas the affinity is determined by the entire combination of subunits. Thus, the results of the present study demonstrate that the pharmacology of GABAA agonists is dependent on the subunit composition of the GABAA receptor complex. Functional GABAA receptors containing two different alpha subunits show pharmacological profiles distinctly different from those of receptors containing a single alpha subtype, indicating that two different alpha subunits can be coexpressed in one functional GABAA receptor complex.

Topics: Animals; GABA-A Receptor Agonists; gamma-Aminobutyric Acid; Humans; Isoxazoles; Muscimol; Oocytes; Oxazolidinones; Piperidines; Receptors, GABA-A; Recombinant Proteins; Structure-Activity Relationship; Xenopus

This study determined the effects of discrete microinjections of GABA agonists in the cholinergic nuclei of the pontomesencephalic tegmentum on spontaneous behavior and seizures induced by intravenous pentylenetetrazol, bicuculline or strychnine, in the rat. Injections of both the GABAA agonist piperidine-4-sulfonic acid and the GABAB agonist (-)baclofen in the laterodorsal tegmental nucleus produced a dose-dependent suppression of behavioral arousal and a reduction in the threshold of myoclonic and clonic but not tonic seizures induced by bicuculline and pentylenetetrazol. There were no significant effects on any type of strychnine seizure. Injections in the surrounding brainstem structures, including the pedunculopontine tegmental nucleus, had little effect on spontaneous behavior and did not significantly alter the thresholds of pentylenetetrazol-induced seizures. We have previously demonstrated that injections of GABA agonists in the central medial intralaminar nucleus of the thalamus have similar effects on behavior and seizures. Since the central medial nucleus receives important direct cholinergic projections from the laterodorsal tegmental nucleus, these two nuclei form a discrete ascending system which regulates seizure threshold.

Topics: Alcian Blue; Animals; Baclofen; Bicuculline; Female; Histocytochemistry; In Vitro Techniques; Microinjections; Pentylenetetrazole; Piperidines; Rats; Rats, Inbred Strains; Seizures; Strychnine; Tegmentum Mesencephali; Thalamic Nuclei

This study characterized the role of GABA in the central medial intralaminar nucleus on seizures induced by pentylenetetrazol given systemically. Injections of the direct selective GABAA agonist, piperidine-4-sulfonic acid or the indirect GABAA agonists, flurazepam and pentobarbital, in this region depressed arousal and facilitated myoclonic and clonic seizures induced by pentylenetetrazol but only caused slight inhibition of tonic seizures. In contrast the GABAB agonist (-)baclofen facilitated all three types of seizures. Recording after injection of piperidine-4-sulfonic acid and (-)baclofen revealed marked suppression and slowing of thalamic and cortical electrical activity. Thalamic injections of the GABAA antagonist, bicuculline methiodide, had opposite behavioral effects, causing hyperactivity and episodes of violent running, not accompanied by EEG discharges. When pentylenetetrazol was infused concommitantly there was marked facilitation of the tonic seizures, which occurred without preceding myoclonic of clonic seizures, or EEG spikes. These results demonstrate that GABA-mediated neurotransmission in the central medial intralaminar nucleus can control the threshold of seizures and that GABA agonists and antagonists have opposite effects. It is suggested that the central medial intralaminar nucleus is not a site of origination or spread of seizures, but controls seizures indirectly by regulating the excitability of other structures and that different synaptic mechanisms and anatomical connections mediate effects on different types of seizures.

Topics: Animals; Baclofen; Bicuculline; Electroencephalography; Epilepsy; Female; Flurazepam; gamma-Aminobutyric Acid; Pentobarbital; Pentylenetetrazole; Piperidines; Rats; Rats, Inbred Strains; Receptors, GABA-A; Thalamic Nuclei

In the spinal cord of pentobarbitone anaesthetised cats, increases in the electrical threshold of the terminations of extensor muscle group Ia afferent fibres, produced by tetanic stimulation of either the appropriate peripheral nerve or the central termination, were associated with parallel changes in the bicuculline-sensitive reduction in electrical threshold of the termination produced synaptically by impulses in flexor muscle low threshold afferent fibres (primary afferent depolarization, PAD) or by microelectrophoretic piperidine-4-sulphonic acid (P4S), an analogue of GABA. Since this post-tetanic hyperpolarization (PTH) could be produced by tetanic stimulation of a single termination centrally, and not by peripheral stimulation of heteronymous nerves, it presumably resulted from changes intrinsic to the tetanized termination. Increases in PAD and the effectiveness of P4S were probably associated with post-tetanic activation of an electrogenic Na+/K+ pump as the predominant cause of PTH, whereas decreases may have been largely the consequence of post-tetanic increases in intracellular Ca2+ levels. These results provide further evidence that GABA is the depolarizing transmitter at axo-axonic synapses upon primary afferent terminals, and that the underlying membrane conductance increase has a reversal potential at a more depolarized level than the resting potential.

Topics: Action Potentials; Afferent Pathways; Animals; Cats; Electric Stimulation; gamma-Aminobutyric Acid; Muscles; Neurons, Afferent; Piperidines; Spinal Cord

Divalent metal cations, including zinc, cadmium, cobalt, nickel, strontium, manganese, magnesium and calcium, reduced the depolarization by microelectrophoretic gamma-aminobutyric acid (GABA) and piperidine-4-sulphonic acid of the central terminations of muscle group Ia primary afferent fibres in the cat spinal cord without affecting the inhibition by GABA of the firing of spinal interneurones. There thus appears to be a difference in either the interaction of GABA with recognition sites, or in the mechanism by which such interaction activates chloride ionophores, at GABA-mediated bicuculline-sensitive synapses on the central terminals of peripheral primary afferent neurones and those on neurones located within the central nervous system.

Topics: Action Potentials; Animals; Bicuculline; Cations, Divalent; Cats; Electric Stimulation; gamma-Aminobutyric Acid; Interneurons; Membrane Potentials; Muscles; Neurons, Afferent; Piperidines; Spinal Cord

When administered microelectrophoretically GABA and piperidine-4-sulphonic acid depolarized the central terminations of muscle group Ia and Ib afferent fibres in the lumbar intermediate nucleus and Clarke's column of cats anaesthetised with pentobarbitone sodium. Both this depolarization, and primary afferent depolarization, generated by impulses in other primary afferent fibres which produce prolonged bicuculline-sensitive inhibition of the firing of group I afferent fibre-excited interneurones in the intermediate nucleus and cells in Clarke's column, are reduced by microelectrophoretic bicuculline methochloride. Systemically administered (+/-)-baclofen hydrochloride (maximum dose 8 mg kg-1) depressed the monosynaptic excitation of Clarke's column neurones by impulses in muscle and cutaneous afferent fibres. Microelectrophoretically administered (-)-baclofen reduced the bicuculline-sensitive primary afferent depolarization of group I terminations without, however, reducing the depolarizing action of GABA or piperidine-4-sulphonic acid. The depression by (-)-baclofen of the group I monosynaptic excitation of intermediate nucleus neurones is not reduced by concentrations of bicuculline methochloride adequate to suppress prolonged inhibition of these neurones.

Topics: Animals; Baclofen; Bicuculline; Cats; gamma-Aminobutyric Acid; Interneurons; Muscles; Neurons, Afferent; Piperidines; Spinal Cord; Synaptic Transmission

When administered microelectrophoretically, GABA and the GABA-mimetic piperidine-4-sulphonic acid (P4S) appear to have no direct hyperpolarizing or depolarizing effect on the terminations of motor axon collaterals excited electrically in the ventral horn of the lumbar spinal cord of the cat. This lack of effect on axon terminals of motoneurones, which contrasts with the bicuculline-sensitive depolarization by P4S of the spinal terminals of primary afferent fibres, is consistent with previous reports of the probable absence of pharmacologically detectable GABA receptors on the spinal terminals of other central excitatory neurones, namely those of the red and lateral vestibular nuclei.

Topics: Animals; Anterior Horn Cells; Bicuculline; Cats; gamma-Aminobutyric Acid; Interneurons; Motor Neurons; Piperidines; Receptors, GABA-A; Synaptic Transmission

In barbiturate anaesthetized cats, tonic inhibition of the excitation of lumbar dorsal horn neurones by impulses in unmyelinated primary afferents was measured by reversibly cooling the spinal cord at the thoraco-lumbar junction. Tonic inhibition was reduced by microinjection of the GABA analogue, piperidine-4-sulphonic acid (2.5 nM in 0.5 microliter) mainly at AP -7, L 2-5 and V -8 to -10. This area in the ventrolateral medulla is just ventral to the facial nucleus and has been shown to be important in cardiovascular control, particularly in relation to fear-defence reactions. It is proposed that tonic inhibition of the nociceptive responses of spinal neurones is part of such a reaction in response to the trauma of surgery. Since previous experiments had shown that the ventrolateral medulla was important in spinal inhibition produced by PAG stimulation, these experiments support the proposal that analgesia does not occur in isolation but is part of a complex behavioural response of an animal in a potentially injurious environment.

Topics: Action Potentials; Animals; Blood Pressure; Cats; Medulla Oblongata; Microinjections; Neural Inhibition; Neurons, Afferent; Piperidines; 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

(RS)-5-(Aminomethyl)-2-isoxazolin-3-ol (dihydromuscimol, DHM) is a potent 4-aminobutyric acid (GABA) agonist, the inhibitory effects of which on neurons are sensitive to the antagonist bicuculline methochloride (BMC), and it also interacts with the GABA uptake system in vitro. (S)-(+)-DHM (4) and (R)-(-)-DHM (5) were obtained in optically pure forms via resolution of tert-butyloxycarbonyl-protected DHM (1) using cinchonidine as the only resolving agent. The optical purity and absolute stereochemistry of 4 and 5 were established by chemical correlation to the (S)-(+) enantiomer of 3-hydroxy-4-aminobutyric acid (GABOB). While 4 was a specific and potent BMC-sensitive GABA agonist in vivo and in vitro, possibly the most potent GABA agonist so far described, the inhibition of GABA uptake by DHM proved to reside exclusively in the (R)-(-) enantiomer (5). The affinity of 5 for BMC-sensitive GABA receptor sites in vitro was some 50 times lower than that of 4. Compounds 4 and 5 can be considered semirigid isosteres of the conformationally flexible GABA analogues (S)-(+)- and (R)-(-)-GABOB, respectively, which show a very low degree of enantioselectivity with respect to GABA synaptic mechanisms. This correlation between the degree of enantioselectivity and conformational mobility of chiral GABA analogues might be of importance for the design of new drugs with specific actions at synapses at which GABA is the transmitter.

Topics: Animals; Bicuculline; Brain; Cats; Chemical Phenomena; Chemistry; Diazepam; gamma-Aminobutyric Acid; Isoxazoles; Molecular Conformation; Muscimol; Neurons; Oxazolidinones; Piperidines; Rats; Receptors, GABA-A; Spinal Cord; Stereoisomerism; Structure-Activity Relationship; Synaptic Membranes

The relationship between structure, in vivo activity, and in vitro activity of some analogues of the gamma-aminobutyric acid (GABA) agonist piperidine-4-sulphonic acid (P4S) was studied. The syntheses of 1,2,3,6-tetrahydropyridine-4-sulphonic acid (DH-P4S) and (RS)-pyrrolidin-3-yl-methanesulphonamide (PMSA-amide) are described. Like P4S, its unsaturated analogue DH-P4S and the five-ring isomer (RS)-pyrrolidin-3-yl-methanesulphonic acid (PMSA) were bicuculline methochloride (BMC)-sensitive inhibitors of the firing of neurones in the cat spinal cord. Whereas isonipecotic acid was less potent than its unsaturated analogue isoguvacine as a GABA-mimetic and as an inhibitor of GABA binding, the opposite relative potencies of P4S and DH-P4S were observed, P4S being proportionally more potent than DH-P4S. In contrast with P4S and DH-P4S, PMSA, which is an analogue of the potent GABA uptake inhibitor and BMC-sensitive GABA-mimetic homo-beta-proline, was a relatively weak inhibitor of GABA uptake in vitro. PMSA-amide was more than two orders of magnitude weaker than PMSA as an inhibitor of GABA binding and did not significantly affect GABA uptake in vitro. The effects of 3-aminopropanesulphonic acid (3-APS), PMSA, P4S, and DH-P4S on the binding of [3H]diazepam in vitro at 30 degrees C, in the presence or absence of chloride ions, were studied and compared with those of the structurally related amino acids GABA, homo-beta-proline, isonipecotic acid, and isoguvacine. Under these conditions the aminosulphonic acids were weaker than the respective amino acids in enhancing [3H]diazepam binding, the difference being more pronounced in the absence of chloride.

Topics: Animals; Binding Sites; Brain; Diazepam; gamma-Aminobutyric Acid; Piperidines; Pyridines; Pyrrolidines; Rats; Structure-Activity Relationship; Synaptic Membranes

Electrophysiological recordings from mouse neurones in tissue culture have been used to investigate how agents which interact with the benzodiazepine receptor modulate neuronal responses to gamma-aminobutyric acid (GABA) and its mimetics, 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP) and piperidine-4-sulphonic acid (P4S). Experiments were performed in a physiological medium, pH 7.35 at 34-36 degrees C. gamma-Aminobutyric acid, THIP and P4S were applied by iontophoresis to neuronal somata. Responses were assessed by current-clamp or voltage-clamp recordings. Midazolam (an agonist at the benzodiazepine receptor) and the beta-carboline, methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM; an inverse agonist at the BZ receptor), were applied by pressure ejection from blunt pipettes. The potency order of the agonists was GABA greater than P4S greater than THIP. Midazolam (10(-7)-10(-5) M) potentiated responses to all three agonists to a similar extent with a shift to the left of the dose-response curve. The drug DMCM (10(-6)-10(-5) M) decreased the responses to all three agonists to a similar extent. The DMCM-induced depression was of a non-competitive nature. It has previously been proposed that THIP is a partial agonist and P4S an antagonist at the GABA receptor coupled to the benzodiazepine receptor, or that the benzodiazepine-receptor-coupled and electrophysiological GABA receptors are distinct. In the present study, responses to the three agonists were modulated to a comparable extent following manipulation of the benzodiazepine receptor. It is therefore unnecessary to invoke the above explanations to account for these results.

Topics: Animals; Benzodiazepines; Carbolines; Cells, Cultured; Convulsants; Electrophysiology; Female; gamma-Aminobutyric Acid; Iontophoresis; Isoxazoles; Mice; Midazolam; Neurons; Oxazoles; Piperidines; Receptors, GABA-A

Naloxone, morphine, Met5-enkephalinamide (MENKA) and procaine were administered microelectrophoretically near extracellularly stimulated extensor muscle group Ia afferent fibres and terminations in the lumbar spinal cord of cats anaesthetized with pentobarbitone sodium. Observations were made of effects on the electrical threshold, on the depolarizing action of GABA or piperidine-4-sulphonate (P4S), and on bicuculline-sensitive primary afferent depolarization (PAD) generated by tetanic stimulation of flexor muscle low threshold afferents. All 4 agents reversibly elevated the threshold of Ia fibres in the dorsal column and Ia terminations in the ventral horn. The depolarizations of terminations by GABA or P4S were also reduced, an effect, which for all except MENKA, probably accounted for a concomitant reduction in PAD. In the absence of a consistent effect on either threshold or depolarization by GABAmimetics, MENKA reversibly diminished PAD, an action blocked by naloxone. Intravenously administered naloxone, in doses known to enhance spinal monosynaptic excitation in the cat, had no effect on GABAergic PAD and little or no effect on Ia termination threshold. The results are discussed in relation to a naloxone-sensitive effect of MENKA which reduces transmitter release from GABAergic axo-axonic synapses on Ia terminals, but which does not account for the enhancement of spinal reflexes by naloxone.

Topics: Afferent Pathways; Animals; Bicuculline; Cats; Electric Stimulation; Enkephalin, Methionine; gamma-Aminobutyric Acid; Morphine; Muscles; Naloxone; Piperidines; Procaine; Reflex, Monosynaptic; Synapses

The effects of GABA agonists on [3H] flunitrazepam binding were examined in membranes from CNS areas proposed to contain different populations of benzodiazepine binding site subtypes (BZ1/BZ2). Since these effects were broadly similar in both cerebellar and hippocampal membranes, it seems unlikely that GABA receptors interacting with the proposed BZ1/BZ2 binding sites have markedly different properties. Sodium chloride clearly facilitated the potentiating effect of muscimol on [3H] flunitrazepam binding but produced a complex interaction with the effects of GABA itself and the partial agonist imidazoleacetic acid in the phosphate buffer system used.

Topics: Animals; Cerebellum; Flunitrazepam; gamma-Aminobutyric Acid; Hippocampus; Imidazoles; Male; Membranes; Muscimol; Piperidines; Rats; Rats, Inbred Strains; Receptors, Cell Surface; Receptors, GABA-A

The binding of radioactive piperidine-4-sulphonic acid ([3H]P4S) to thoroughly washed, frozen, and thawed membranes isolated from cow and rat brains has been studied. Quantitative computer analysis of the binding curves for four regions of bovine brain revealed the general presence of two binding sites. In these brain regions less satisfactory computer fits were obtained for receptor models showing one or three binding sites or negative cooperativity. With the use of Tris-citrate buffer at 0 degree C the two affinity classes for P4S in bovine cortex membranes revealed the following binding parameters: KD = 17 +/- 7 nM (Bmax = 0.15 +/- 0.07 pmol/mg protein) and KD = 237 +/- 100 nM (Bmax = 0.80 +/- 0.20 pmol/mg protein). Heterogeneity was also observed for association and dissociation rates of [3H]P4S. The slow binding component (kon = 5.6 X 10(7) or 8.8 X 10(7) M-1 min-1, koff = 0.83 min-1, and KD = 14.7 or 9.4 nM, determined by two different methods in phosphate buffer containing postassium chloride) corresponds to the high-affinity component of the equilibrium binding curve (KD = 11 nM, Bmax = 0.12 pmol/mg protein in the same buffer system). The association and dissociation rates for the subpopulation of rapidly of dissociating sites, apparently corresponding to the low-affinity sites, were too rapid to be measured accurately. The binding of [3H]P4S appears to involve the same two populations of sites with Bmax values similar to those for [3H]GABA binding to the same tissue, although the kinetic parameters for the two ligands are somewhat different. Furthermore, comparative studies on the inhibition of [3H]P4S and [3H]GABA binding by various GABA analogues, strongly suggest that P4S binds to the GABA receptors. The different effects of P4S and GABA on benzodiazepine binding are discussed.

Topics: Animals; Brain; Cattle; Cerebral Cortex; gamma-Aminobutyric Acid; Kinetics; Organ Specificity; Piperidines; Rats; Receptors, Cell Surface; Receptors, GABA-A; Structure-Activity Relationship; Synaptic Membranes

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

Topics: Animals; Benzodiazepines; Bicuculline; Brain; Diazepam; gamma-Aminobutyric Acid; Macromolecular Substances; Male; Muscimol; Piperidines; Rats; Receptors, Cell Surface; Receptors, Drug; Receptors, GABA-A; Receptors, Neurotransmitter; Structure-Activity Relationship; Synaptic Membranes

Topics: Animals; Astrocytes; Bicuculline; Cats; gamma-Aminobutyric Acid; Mice; Neurons; Piperidines; Receptors, Drug; Spinal Cord; Structure-Activity Relationship