Methylphosphinic Research Articles

Anxiolytic agent, dihydrohonokiol-B, recovers amyloid β protein-induced neurotoxicity in cultured rat hippocampal neurons

The effects of anxiolytic honokiol derivative, dihydrohonokiol-B (DHH-B), on amyloid β protein (Aβ 25–35, 10 nM)-induced changes in Cl −-ATPase activity, intracellular Cl − concentration ([Cl −] i) and glutamate neurotoxicity were examined in cultured rat hippocampal neurons. DHH-B (10 ng/ml) recovered Aβ-induced decrease in neuronal Cl −-ATPase activity without any changes in the activities of Na +/K +-ATPase and anion-insensitive Mg 2+-ATPase. A GABA C receptor antagonist (1,2,5,6,-tetrahydropyridin-4-yl) methyl-phosphinic acid (TPMPA, 15 μM), inhibited the protective effects of DHH-B on Cl −-ATPase activity. DHH-B reduced Aβ-induced elevation of [Cl −] i as assayed using a Cl −-sensitive fluorescent dye, and prevented Aβ-induced aggravation of glutamate neurotoxicity. These data suggest that DHH-B exerts the neuroprotective action against Aβ through GABA C receptor stimulation.

Effects of GABA Receptor Antagonists on Retinal Glycine Receptors and on Homomeric Glycine Receptor Alpha Subunits

Glycinergic and GABAergic inhibition are juxtaposed at one retinal synaptic layer yet likely perform different functions. These functions have usually been evaluated using receptor antagonists. In examining retinal glycine receptors, we were surprised to find that commonly used concentrations of GABA antagonists blocked significant fractions of the glycine current. In retinal amacrine and ganglion cells, the competitive GABAA receptor antagonists (bicuculline and SR95531) were also competitive GlyR antagonists. Picrotoxinin produced a noncompetitive inhibition of retinal GlyRs. [1,2,5,6-tetrahydropyridine-4-yl] methylphosphinic acid, the GABACR antagonist, did not inhibit glycine receptors. All three GABAA receptor antagonists were competitive inhibitors of homomeric alpha1 or alpha2 GlyRs expressed in human embryonic kidney cells (HEK293) cells. Interestingly, bicuculline was much more effective at alpha2 GlyRs and might be used to separate glycine receptor subtypes. Thus commonly used concentrations of GABA antagonists do not unambiguously differentiate GABA and glycine pathways. Picrotoxinin inhibition of GABAC receptors requires two amino acids in the second transmembrane region (TM2): 2' serine and 6' threonine. Although TM2 regions in GABA and glycine receptors are highly homologous, neither 2' serine nor 6' threonine is essential for picrotoxinin sensitivity in glycine receptors.

Developmental Changes of GABA Synaptic Transient in Cerebellar Granule Cells

The time course of synaptic currents is largely determined by the microscopic gating of the postsynaptic receptors and the temporal profile of the synaptic neurotransmitter concentration. Although several lines of evidence indicate that developmental changes of GABAergic synaptic current time course are clearly correlated with a switch in postsynaptic receptors, much less is known about the modification of GABA release during development. To address this issue, we studied the sensitivity of miniature inhibitory postsynaptic currents (mIPSCs) to a quickly dissociating competitive antagonist, 1,2,5,6-tetrahydropyridine-4-yl)methylphosphinic acid (TPMPA), in neurons cultured for 6 to 8 days in vitro (DIV) ("young") and for 12 to 14 DIV ("old"). mIPSCs recorded in young neurons were significantly more resistant to the block by TPMPA. This observation was interpreted as a consequence of a more efficient displacement of TPMPA from GABA(A) receptors caused by a stronger GABA release in young neurons. The change in mIPSC sensitivity to TPMPA during development was not affected by the deletion of alpha(1) subunit, supporting its presynaptic origin. The effects of a second quickly dissociating antagonist, SR-95103 [2-(carboxy-3'-propyl)-3-amino-4-methyl-6-phenylpyridazinium chloride], on young, old, and alpha(1) -/- neurons were qualitatively the same as those obtained with TPMPA. Moreover, the analysis of current responses to ultrafast GABA applications showed that the unbinding rates of TPMPA in DIV 6 to 8 and in DIV 12 to 14 neurons are not significantly different, ruling out the postsynaptic mechanism of differential TPMPA action. Thus, we provide evidence that presynaptic GABA uniquantal release is developmentally regulated.

Charged Residues at the 2′ Position of Human GABAC ρ1 Receptors Invert Ion Selectivity and Influence Open State Probability

The ability of members of the nicotinicoid superfamily of ligand-gated ion channels to selectively conduct anions or cations is critical to their function within the central nervous system. Recent work has demonstrated that residues at the intracellular end of the second transmembrane domain, between the -3' and 2' positions, form the ion selectivity filter of these receptors. In this study, the proline residue at the 2' position (Pro-2') at the intracellular end of the second transmembrane domain of the gamma-aminobutyric acid type C rho 1 subunit was mutated to glutamate (rho 1P2'E) and arginine (rho 1P2'R). Dilution potential experiments indicated that the charge selectivity of the rho 1P2'E receptor channels had been inverted, with the channels now becoming predominantly cation selective, indicating the ability of negatively charged residues at this 2' position to control charge selectivity. The mutation was also seen to have significantly decreased agonist potency and intrinsic efficacy. In contrast, the rho 1P2'R receptor channels were anion-selective but were now found to be constitutively open with high holding currents (inhibited by low gamma-aminobutyric acid doses and the competitive antagonist, 1,2,5,6-tetrahydropyridine-4-yl)methylphosphinic acid alone) and increased agonist activity. Hill coefficients of both mutants were decreased, but competitive antagonist studies indicated that their binding sites were not significantly affected.

Picrotoxin Accelerates Relaxation of GABACReceptors

Picrotoxin is a plant alkaloid that is often used to block the activity of neuronal GABA and glycine receptors. However, the mechanism by which picrotoxin inhibits these receptors is still in debate. In this study, we investigated the picrotoxin inhibition on perch-rho subunits expressed heterologously in Xenopus laevis oocytes, and on native GABA(C) receptors of perch bipolar cells. Both competitive and noncompetitive mechanisms were observed for picrotoxin inhibition of the GABA(C) receptor. In oocytes expressing the rho1A subunit, terminating simultaneously the coapplication of GABA and picrotoxin induced a large rebound of membrane current. In addition, picrotoxin significantly accelerated the kinetics of GABA responses, particularly in the relaxation (offset) phase of GABA currents. Both current rebound and the large acceleration of GABA relaxation were unique to picrotoxin inhibition and were not observed with the competitive antagonist (1,2,5,6-tetrahydropyridin-4-yl)-methylphosphinic acid or the allosteric modulator zinc. The change in kinetics induced by picrotoxin was also observed on receptors formed by other GABA rho subunits, as well as on the GABA(C) receptors of retinal bipolar cells. Based on these observations, we proposed a model in which picrotoxin binds to the GABA(C) receptor in both channel open and closed states. Overall, this model provides a remarkably good approximation of the experimental findings we observed for picrotoxin inhibition of GABA(C) receptors. These results support an allosteric mechanism of picrotoxin inhibition of ligand-gated chloride channels.

Evidence for inhibition mediated by coassembly of GABAA and GABAC receptor subunits in native central neurons.

Fast inhibition in the nervous system is commonly mediated by GABA(A) receptors comprised of 2alpha/2beta/1gamma subunits. In contrast, GABA(C) receptors containing only rho subunits (rho1-rho3) have been predominantly detected in the retina. However, here using reverse transcription-PCR and in situ hybridization we show that mRNA encoding the rho1 subunit is highly expressed in brainstem neurons. Immunohistochemistry localized the rho1 subunit to neurons at light and electron microscopic levels, where it was detected at synaptic junctions. Application of the GABA(C) receptor agonist cis-4-aminocrotonic acid (100-800 microM) requires the rho1 subunit to elicit responses, which surprisingly are blocked independently by antagonists to GABA(A) (bicuculline, 10 microM) and GABA(C) [(1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid (TPMPA); 40-160 microM] receptors. Responses to GABA(C) agonists were also enhanced by the GABA(A) receptor modulator pentobarbitone (300 microM). Spontaneous and evoked IPSPs were reduced in amplitude but never abolished by TPMPA, but were completely blocked by bicuculline. We therefore tested the hypothesis that GABA(A) and GABA(C) subunits formed a heteromeric receptor. Immunohistochemistry indicated that rho1 and alpha1 subunits were colocalized at light and electron microscopic levels. Electrophysiology revealed that responses to GABA(C) receptor agonists were enhanced by the GABA(A) receptor modulator zolpidem (500 nm), which acts on the alpha1 subunit when the gamma2 subunit is also present. Finally, coimmunoprecipitation indicated that the rho1 subunit formed complexes that also containedalpha1 and gamma2 subunits. Taken together these separate lines of evidence suggest that the effects of GABA in central neurons can be mediated by heteromeric complexes of GABA(A) and GABA(C) receptor subunits.

Ionotropic GABA receptors with mixed pharmacological properties of GABA A and GABA C receptors

Ionotropic γ-aminobutyric acid (GABA) receptors form a large family of molecular isoforms with distinct properties. We have characterized a distinct new type of GABA receptors in CA1 pyramidal cells in rat hippocampal slices. Somatic application of GABA induced currents which were partially suppressed by (1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid (TPMPA), a specific antagonist of GABA C receptors. This sensitivity was enhanced when we evoked the currents by the GABA C receptor agonist cis-4-aminocrotonic acid (CACA). However, both GABA- and CACA-evoked currents were sensitive towards bicuculline and thus lack the defining feature of GABA C receptors, which are insensitive towards this antagonist. Spontaneous miniature post-synaptic currents (mIPSCs) revealed a similar pharmacological behaviour. We conclude that juvenile CA1 pyramidal cells express a fraction of ionotropic GABA receptors with mixed pharmacological properties of both, GABA A and GABA C receptors.

Gabapentin activates presynaptic GABA B heteroreceptors in rat cortical slices

In electrically stimulated rat neocortical brain slices preloaded with [ 3H]γ-aminobutyric acid (GABA) or [ 3H]glutamic acid, the pharmacological actions of 1-(aminomethyl)-cyclohexaneacetic acid (gabapentin, Gp) were compared with the GABA B receptor agonists baclofen (Bac) and (3-amino-2-( S)-hydroxypropyl)-methylphosphinic acid (CGP 44532). Gabapentin, baclofen and CGP 44532 all reduced the electrically stimulated release of [ 3H]glutamic acid (IC 50=20 μM, 0.8 μM and 2 μM, respectively). These effects were sensitive to the GABA B receptor antagonists (+)-( S)-5,5 dimethylmorpholinyl-2-acetic acid (Sch 50911) or N-3-[[1-( S)-(3,4-dichlorophenyl)ethyl]amino]-2-( S)-hydroxypropyl- P-(cyclo-hexylmethyl)-phosphinic acid (CGP 54626). By contrast, gabapentin was without effect on the release of [ 3H]GABA, whilst baclofen (IC 50=8 μM) and CGP 44532 (IC 50=1 μM) inhibited [ 3H]GABA release. It is concluded that gabapentin selectively activates presynaptic GABA B heteroreceptors, but not GABA B autoreceptors, and may be a useful ligand to discriminate between presynaptic GABA B receptor subtypes.

The spinal GABAergic system is a strong modulator of burst frequency in the lamprey locomotor network.

The spinal network coordinating locomotion is comprised of a core of glutamate and glycine interneurons. This network is modulated by several transmitter systems including spinal GABA interneurons. The purpose of this study is to explore the contribution of GABAergic neurons to the regulation of locomotor burst frequency in the lamprey model. Using gabazine, a competitive GABAA antagonist more specific than bicuculline, the goal was to provide a detailed analysis of the influence of an endogenous activation of GABAA receptors on fictive locomotion, as well as their possible interaction with GABAB and involvement of GABAC receptors. During N-methyl-D-aspartate (NMDA)-induced fictive locomotion (ventral root recordings in the isolated spinal cord), gabazine (0.1-100 microM) significantly increased the burst rate up to twofold, without changes in regularity or "burst quality." Gabazine had a proportionately greater effect at higher initial burst rates. Picrotoxin (1-7.5 microM), a less selective GABAA antagonist, also produced a pronounced increase in frequency, but at higher concentrations, the rhythm deteriorated, likely due to the unspecific effects on glycine receptors. The selective GABAB antagonist CGP55845 also increased the frequency, and this effect was markedly enhanced when combined with the GABAA antagonist gabazine. The GABAC antagonist (1,2,5,6-tetrahydropyridine-4-yl)methylphosphinic acid (TPMPA) had no effect on locomotor bursting. Thus the spinal GABA system does play a prominent role in burst frequency regulation in that it reduces the burst frequency by < or =50%, presumably due to presynaptic and soma-dendritic effects documented previously. It is not required for burst generation, but acts as a powerful modulator.

Mutations of the 2′ proline in the M2 domain of the human GABA C ρ1 subunit alter agonist responses

Mutations of the proline residue at the 2′ position (P2′) within the second transmembrane (M2) domain of the γ-aminobutyric acid C (GABA C) ρ1 subunit are known to produce receptors with altered pharmacology. In the present study, P2′ was mutated to alanine (ρ1P2′A), phenylalanine (ρ1P2′F), glycine (ρ1P2′G) and serine (ρ1P2′S). Mutant receptors were characterized using a range of agonists, partial agonists and antagonists. ρ1P2′A, ρ1P2′G and ρ1P2′S receptors were less susceptible than wild-type receptors to agonist activation. Most notably, the partial agonists, (±)- trans-2-(aminomethyl)cyclopropanoic acid ((±)-TAMP) and imidazole-4-acetic acid (I4AA) were converted to antagonists at ρ1P2′G and ρ1P2′S receptors and the partial agonist CACA acted as an antagonist at ρ1P2′S receptors. In contrast, ρ1P2′F receptors were more prone to activation by agonists. A correlation was observed between the pharmacological properties of the mutant receptors and the hydrophobicity of each residue. Unlike the agonists or partial agonists, the affinity of competitive antagonists, (1,2,5,6-tetrahydropyridine-4-yl)methylphosphinic acid (TPMPA) and 4,5,6,7-tetrahydroisoxazole[4,5-c]pyridine-3-ol (THIP), did not change significantly between wild-type and mutant receptors. Thus, the results suggest that the agonist/competitive antagonist binding site(s) were not significantly affected by the mutations, but that receptor activation properties altered such that the more hydrophobic the residue at the 2′ position, the more prone the receptor is to agonist activation.

Functional expression in frog oocytes of human rho 1 receptors produced in Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae was engineered to express the rho 1 subunit of the human gamma-aminobutyric acid rho 1 (GABA rho 1) receptor. RNA that was isolated from several transformed yeast strains produced fully functional GABA receptors in Xenopus oocytes. The GABA currents elicited in the oocytes were fast, nondesensitizing chloride currents; and the order of agonist potency was GABA > beta-alanine > glycine. Moreover, the receptors were resistant to bicuculline, strongly antagonized by (1,2,5,6 tetrahydropyridine-4-yl)methylphosphinic acid, and modulated by zinc and lanthanum. Thus, the GABA receptors expressed by the yeast mRNA retained all of the principal characteristics of receptors expressed by cRNA or native retina mRNAs. Western blot assays showed immunoreactivity in yeast plasma membrane preparations, and a rho 1-GFP fusion gene showed mostly intracellular distribution with a faint fluorescence toward the plasma membrane. In situ immunodetection of rho 1 in yeast demonstrated that some receptors reach the plasma membrane. Furthermore, microtransplantation of yeast plasma membranes to frog oocytes resulted in the incorporation of a small number of functional yeast rho 1 receptors into the oocyte plasma membrane. These results show that yeast may be useful to produce complete functional ionotropic receptors suitable for structural analysis.

GABA(A) and GABA(C) receptor antagonists increase retinal cyclic GMP levels through nitric oxide synthase.

The nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) signal transduction pathway plays a role in every retinal cell type. Previous studies have shown that excitatory glutamatergic synaptic pathways can increase cGMP-like immunoreactivity (cGMP-LI) in retina through stimulation of NO production, but little is known about the role of synaptic inhibition in the modulation of cGMP-LI. Gamma-amino-n-butyric acid (GABA) plays critical roles in modulating excitatory synaptic pathways in the retina. Therefore, we used GABA receptor antagonists to explore the role of GABAergic inhibitory synaptic pathways on the modulation of the NO/cGMP signal-transduction system. Cyclic GMP immunocytochemistry was used to investigate the effects of the GABA receptor antagonists bicuculline, picrotoxin, and (1,2,5,6-tetrahyropyridin-4-yl) methylphosphinic acid (TPMPA) on levels of cGMP-LI. Cyclic GMP-LI was strongly increased in response to the GABA(A) receptor antagonist bicuculline, while the GABA(C) receptor antagonist TPMPA had little effect on cGMP-LI. The GABA(A)/GABA(C) receptor antagonist, picrotoxin, caused a moderate increase in cGMP-LI, which was mimicked by the combination of bicuculline and TPMPA. The nitric oxide synthase inhibitor, S-methyl-L-thiocitrulline (SMTC), blocked the increased cGMP-LI in response to stimulation with either bicuculline or picrotoxin. Treatments with either of the glutamate receptor antagonists (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801) or 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) partially blocked the increases in cGMP-LI seen in response to bicuculline, but a combination of MK-801 and CNQX completely eliminated these increases. These results suggest that inhibitory synaptic pathways involving both types of GABA receptors work through excitatory glutamatergic receptors to regulate the NO/cGMP signal-transduction pathway in retina.

GABAC receptor agonist suppressed ammonia-induced apoptosis in cultured rat hippocampal neurons by restoring phosphorylated BAD level.

Ammonia-induced apoptosis and its prevention by GABAC receptor stimulation were examined using primary cultured rat hippocampal neurons. Ammonia (0.5-5 mm NH4Cl) dose-dependently induced apoptosis in pyramidal cell-like neurons as assayed by double staining with Hoechst 33258 and anti-neurofilament antibody. A GABAC receptor agonist, cis-4-aminocrotonic acid (CACA, 200 microm), but not GABAA and GABAB receptor agonists, muscimol (10 micro m) and baclofen (50 microm), respectively, inhibited the ammonia (2 mm)-induced apoptosis, and this inhibition was abolished by a GABAC receptor antagonist (1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid (TPMPA, 15 microm). Expression of all three GABAC receptor subunits was demonstrated in the cultured neurons by RT-PCR. The ammonia-treatment also activated caspases-3 and -9 as observed in immunocytochemistry for PARP p85 and western blot. Such activation of the caspases was again inhibited by CACA in a TPMPA-sensitive manner. The anti-apoptotic effect of CACA was blocked by inhibitors for MAP kinase kinase and cAMP-dependent protein kinase, PD98059 (20 microm) and KT5720 (1 microm), suggesting possible involvement of an upstream pro-apoptotic protein, BAD. Levels of phospho-BAD (Ser112 and Ser155) were decreased by the ammonia-treatment and restored by coadministration of CACA. These findings suggest that GABAC receptor stimulation protects hippocampal pyramidal neurons from ammonia-induced apoptosis by restoring Ser112- and Ser155-phospho-BAD levels.

GABAC receptors in the rat superior colliculus and pretectum participate in synaptic neurotransmission.

In mammals, GABA(C) receptors seem to be specifically expressed in the retina and the subcortical visual system, with highest extraretinal expression levels in the superior colliculus (SC). Although its presence in the superficial SC has been demonstrated physiologically, a direct involvement of this receptor type in fast synaptic neurotransmission still awaits verification. We addressed the question of a possible synaptic localization of GABA(C) receptors by performing in vitro whole-cell patch-clamp recordings of inhibitory postsynaptic currents (IPSCs) in single neurons of the rat SC and the neighboring pretectal nuclear complex, where GABA(C) receptors are also expressed at significant levels. To increase the likelihood to record IPSCs we induced spontaneous activity by application of the potassium channel blocker 4-aminopyridine (4-AP) and blocked glutamate-mediated excitatory neurotransmission with kynurenic acid. All 4-AP-induced postsynaptic currents were of synaptic origin because they were completely suppressed by lidocaine or by substitution of extracellular calcium with cobalt. In 40% of the SC cells and in 60% of the pretectal neurons, IPSCs in the presence of 4-AP and kynurenic acid were only partly blocked by the selective GABA(A) receptor antagonist bicuculline. Inhibitory currents that were insensitive to bicuculline, however, could be blocked by coapplication of either the specific GABA(C) receptor antagonist 1,2,5,6-tetrahydropyridine-4-yl)methylphosphinic acid or picrotoxin, an unselective GABA(A) and GABA(C) receptor antagonist. We conclude that GABA(C) receptors are, at least partially, located synaptically in SC and pretectal neurons in the rat, which indicates a direct function of this receptor type for synaptic processing in both structures.

Sometimes you see them, sometimes you don't: IPSCs in the rat superficial superior colliculus.

Superficial superior colliculus (SSC) neurones were voltage-clamped and the current-voltage relationship of synaptically evoked currents analyzed in vitro. A strong interplay between excitatory postsynaptic currents (EPSCs) and inhibitory postsynaptic currents (IPSCs) was identified. Glutamate receptor antagonists not only fully blocked EPSCs but IPSCs were also frequently reduced by the specific d,l,-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor antagonist (by 66.9%), indicative of glutamate-driven inhibitory projections. The GABA(A )receptor antagonist bicuculline enhanced EPSCs and either abolished or reduced (by 79.3%) IPSCs. The GABA(C) receptor antagonist 1,2,5,6-tetrahydro-(pyridin-4-yl)methylphosphinic acid decreased IPSCs in 80% of cells tested (by 24.1%), but had no effect on EPSCs. Varying the recording conditions influenced postsynaptic currents. At a holding potential of -60 mV, IPSCs were generally produced with intracellular chloride concentrations of both 5 and 10 mM (total n=24/30). However, with perforated-patch recordings using gramicidin, IPSCs were less frequently encountered (n=5/21), suggesting a higher intracellular chloride concentration in a large proportion of SSC neurones. Further assessment of experimental conditions revealed that two frequently used sodium channel blockers, QX-314 (bromide salt, intracellular) or tetrodotoxin (extracellular), shifted the IPSC reversal potential towards more positive values. Hence, IPSCs were not encountered at -60 mV in their presence. The level of stimulation intensity (minimal or maximal) did not influence IPSC production in these conditions. Thus, the current study describes the pharmacological properties of PSCs in the SSC and highlights the impact of experimental conditions on synaptic transmission, which requires consideration for past and present data reported in this preparation.

Phosphinic, phosphonic and seleninic acid bioisosteres of isonipecotic acid as novel and selective GABA C receptor antagonists

A number of amino acids bioisosterically derived from the specific GABA A agonist, isonipecotic acid, were electrophysiologically characterized as antagonists at GABA C ρ 1 receptors expressed in Xenopus oocytes. The phosphinic acid analogue of isonipecotic acid, piperidin-4-ylphosphinic acid ( 2), was comparable with the standard GABA C antagonist, (1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid (TPMPA), in terms of potency and GABA C versus GABA A receptor selectivity. Whereas the phosphonic acid analogue, piperidin-4-ylphosphonic acid ( 4), was at least an order of magnitude weaker than piperidin-4-ylphosphinic acid as a GABA C antagonist, the seleninic acid analogue, piperidin-4-ylseleninic acid (SEPI, 6), was the most potent and selective GABA C antagonist within the group of isonipecotic acid derived amino acids studied.

Gamma-aminobutyric acid-induced calcium signalling in rat superior collicular neurones

Ionotropic gamma-aminobutyric acid (GABA) receptors are known to mediate excitation in neonatal neurones as a crucial developmental factor. In the present study we employed calcium imaging techniques with the calcium indicator Fura-2-AM to study the pharmacology of GABA-induced calcium responses in cultures prepared from neonatal rat superficial superior colliculus (SC), after immunocytochemical labelling confirmed the presence of GABA C ρ 1 subunits in 35% of neurones. Rises in neuronal intracellular calcium were obtained in response to GABA and also to the subtype-specific GABA A agonist 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol. However, the GABA C agonist cis-4-aminocrotonic acid induced calcium response only at unspecifically high concentrations (500 μM). Co-application of GABA antagonists revealed that both GABA A&C agonists’ actions could be blocked by the GABA A antagonist bicuculline but not the GABA C antagonists 1,2,5,6-tetrahydro-(pyridin-4-yl) methylphosphinic acid. This suggests that activation of GABA A but not GABA C receptors contributes to excitatory GABA responses and related calcium signals in neonatal SC neurones.

Activation of mGluR5 modulates GABA(A) receptor function in retinal amacrine cells.

Amacrine cells in the vertebrate retina receive glutamatergic input from bipolar cells and make synapses onto bipolar cells, ganglion cells, and other amacrine cells. Recent studies indicate that amacrine cells express metabotropic glutamate receptors (mGluRs) and that signaling within the inner plexiform layer (IPL) of the retina might be modulated by mGluR activity. This study tests the hypothesis that activation of mGluR5 modulates GABA(A) receptor function in retinal amacrine cells. Whole cell voltage-clamp recordings were combined with pharmacology to establish the identity of the ionotropic GABA receptors expressed in primary cultures of chick amacrine cells and to determine how mGluR5 activity affected the behavior of those receptors. Application of GABA (20 microM) produced currents that reversed at -58.2 +/- 0.9 mV, near the predicted Cl(-) reversal potential of -59 mV. The GABA(A) receptor antagonist, bicuculline (50 microM), completely blocked the GABA-gated currents. cis-4-Aminocrotonic acid (CACA; 100 microM), a GABA(C) receptor agonist, produced small currents that were not blocked by the GABA(C) antagonist, (1,2,5,6-tetrahydropyridine-4-yl) methylphosphinic acid (TPMPA; 20 microM), but were completely blocked by bicuculline. These results indicate that cultured amacrine cells express GABA(A) receptors exclusively. Activating mGluR5 with (RS)-2-chloro-5-hydroxyphenylglycine (CHPG; 300 microM) enhanced GABA-gated currents by 10.0 +/- 1.5%. Buffering internal Ca(2+) with BAPTA (10 mM) blocked the CHPG-dependent enhancement. Activation of PKC with the cell-permeable PKC activators (-)-7-octylindolactam V, phorbol 12-myristate 13 acetate (PMA), or 1-oleoyl-2-acetyl-sn-glycerol (OAG) also enhanced GABA-gated currents in a dose-dependent manner. Preactivation of PKC occluded the mGluR5-dependent enhancement, and inhibition of Ca-dependent PKC isotypes with Gö6976 (35 nM) suppressed the effects of mGluR5 activation, suggesting that mGluR5 and PKC are part of the same pathway. To determine if mGluR5-dependent enhancement occurred at synaptic GABA(A) receptors, postsynaptic currents were recorded in the presence of CHPG. On average, the mean amplitudes of the quantal events were increased by about 18% when mGluR5 was activated. These results indicate that activation of mGluR5 enhances GABA-gated current in cultured amacrine cells in a manner that is both Ca(2+)- and PKC-dependent. These results support the possibility that glutamate released from bipolar cells can modulate the function of GABAergic amacrine cells and alter signaling in the inner plexiform layer.

Inhibitory effects of GABA B receptor agonists on swallowing in the dog

The effects of the GABA B receptor agonists baclofen (1.4 and 7 μmol/kg i.v.) and CGP 44532 ([(2 S)-3-amino-2-hydroxypropyl]methyl phosphinic acid], 0.2 and 0.7 μmol/kg i.v.) on transient lower esophageal sphincter relaxations and spontaneous and pharyngeally stimulated swallowing were investigated in conscious dogs. Both compounds inhibited transient lower esophageal sphincter relaxations dose-dependently, CGP 44532 being approximately fivefold more potent. In experiments designed to measure transient lower esophageal sphincter relaxations, spontaneous swallowing was suppressed by both compounds. When swallowing was evoked by intrapharyngeal water injection, both baclofen and CGP 44532 reduced the occurrence of primary peristalsis. It is concluded that centrally acting GABA B receptor agonists inhibit spontaneous and stimulated swallowing probably through an action in the central pattern generator for swallowing.

Palladium-catalyzed cross-coupling reaction of anilinium hypophosphite with alkenyl bromides and triflates: application to the synthesis of GABA analogs

Alkenyl bromides and triflates undergo palladium-catalyzed cross-coupling with anilinium hypophosphite to afford monosubstituted phosphinates (salts of alkenylphosphonous acids). The reaction is an extension of our previously reported methodology for the synthesis of aryl- and benzyl-phosphonous acids. Our preliminary results show that the best reaction conditions are observed with Pd(OAc) 2/dppp as a catalyst, in refluxing benzene or tetrahydrofuran. This novel PC bond forming reaction is applied to the synthesis of (1,2,3,6-tetrahydropyridin-4-yl)-methylphosphinic acid (TPMPA), a selective competitive antagonist for GABA C receptors. The divergent synthesis proceeds through protected (1,2,3,6-tetrahydropyridin-4-yl)-phosphinic acid, a previously unknown isoguvacine-like GABA analog. This synthetic intermediate is also an ideal precursor to other biologically interesting GABA analogs.