(+)- and (-)-cis-2-aminomethylcyclopropanecarboxylic acids show opposite pharmacology at recombinant rho(1) and rho(2) GABA(C) receptors

The effects of the enantiomers of (+/-)-CAMP and (+/-)-TAMP [(+/-)-cis- and (+/-)-trans-2-aminomethylcyclopropanecarboxylic acids, respectively], which are cyclopropane analogues of GABA, were tested on GABA(A) and GABA(C) receptors expressed in Xenopus laevis oocytes using two-electrode voltage clamp methods. (+)-CAMP was found to be a potent and full agonist at homooligomeric GABA(C) receptors (K:(D) approximately 40 microM: and I:(max) approximately 100% at rho(1); K:(D) approximately 17 microM: and I:(max) approximately 100% at rho(2)) but a very weak antagonist at alpha(1)beta(2)gamma(2L) GABA(A) receptors. In contrast, (-)-CAMP was a very weak antagonist at both alpha(1)beta(2)gamma(2L) GABA(A) receptors and homooligomeric GABA(C) receptors (IC(50) approximately 900 microM: at rho(1) and approximately 400 microM: at rho(2)). Furthermore, (+)-CAMP appears to be a superior agonist to the widely used GABA(C) receptor partial agonist cis-4-aminocrotonic acid (K:(D) approximately 74 microM: and I:(max) approximately 78% at rho(1); K:(D) approximately 70 microM: and I:(max) approximately 82% at rho(2)). (-)-TAMP was the most potent of the cyclopropane analogues on GABA(C) receptors (K:(D) approximately 9 microM: and I:(max) approximately 40% at rho(1); K:(D) approximately 3 microM: and I:(max) approximately 50-60% at rho(2)), but it was also a moderately potent GABA(A) receptor partial agonist (K:(D) approximately 50-60 microM: and I:(max) approximately 50% at alpha(1)beta(2)gamma(2L) GABA(A) receptors). (+)-TAMP was a less potent partial agonist at GABA(C) receptors (K:(D) approximately 60 microM: and I:(max) approximately 40% at rho(1); K:(D) approximately 30 microM: and I:(max) approximately 60% at rho(2)) and a weak partial agonist at alpha(1)beta(2)gamma(2L) GABA(A) receptors (K:(D) approximately 500 micro: and I:(max) approximately 50%). None of the isomers of (+/-)-CAMP and (+/-)-TAMP displayed any interaction with GABA transport at the concentrations tested. Molecular modeling based on the present results provided new insights into the chiral preferences for either agonism or antagonism at GABA(C) receptors.     

Synthesis of the nanomolar photoaffinity GABA(B) receptor ligand CGP 71872 reveals diversity in the tissue distribution of GABA(B) receptor forms

A radioiodinated probe, [125I]-CGP 71872, containing an azido group that can be photoactivated, was synthesized and used to characterize GABA(B) receptors. Photoaffinity labeling experiments using crude membranes prepared from rat brain revealed two predominant ligand binding species at approximately 130 and approximately 100 kDa believed to represent the long (GABA(B)R1a) and short (GABA(B)R1b) forms of the receptor. Indeed, these ligand binding proteins were immunoprecipitated using a GABA(B) receptor-specific antibody confirming the receptor specificity of the photoaffinity probe. Most convincingly, [125I]-CGP 71872 binding was competitively inhibited in a dose-dependent manner by cold CGP 71872, GABA, saclofen, (-)-baclofen, (+)-baclofen and (L)-glutamic acid with a rank order and stereospecificity characteristic of the GABA(B) receptor. Photoaffinity labeling experiments revealed that the recombinant GABA(B)R2 receptor does not bind [125I]-CGP 71872, providing surprising and direct evidence that CGP 71872 is a GABA(B)R1 selective antagonist. Photoaffinity labeling experiments using rat tissues showed that both GABA(B)R1a and GABA(B)R1b are co-expressed in the brain, spinal cord, stomach and testis, but only the short GABA(B)R1b receptor form was detected in kidney and liver whereas the long GABA(B)R1a form was selectively expressed in the adrenal gland, pituitary, spleen and prostate. We report herein the synthesis and biochemical characterization of the nanomolar affinity [125I]-CGP 71872 and CGP 71872 GABA(B)R1 ligands, and differential tissue expression of the long GABA(B)R1a and short GABA(B)R1b receptor forms in rat and dog.     

Enantiomers of cis-constrained and flexible 2-substituted GABA analogues exert opposite effects at recombinant GABA(C) receptors

The effects of the enantiomers of a number of flexible and cis-constrained GABA analogues were tested on GABA(C) receptors expressed in Xenopus laevis oocytes using two-electrode voltage-clamp electrophysiology. (1S,2R)-cis-2-Aminomethylcyclopropane-1-carboxylic acid ((+)-CAMP), a potent and full agonist at the rho1 (EC(50) approximately 40 microM, I(max) approximately 100%) and rho 2 (EC(50) approximately 17 microM, I(max) approximately 100%) receptor subtypes, was found to be a potent partial agonist at rho3 (EC(50) approximately 28 microM, I(max) approximately 70%). (1R,2S)-cis-2-Aminomethylcyclopropane-1-carboxylic acid ((-)-CAMP), a weak antagonist at human rho1 (IC(50) approximately 890 microM) and rho2 (IC(50) approximately 400 microM) receptor subtypes, was also found to be a moderately potent antagonist at rat rho3 (IC(50) approximately 180 microM). Similarly, (1R,4S)-4-aminocyclopent-2-ene-1-carboxylic acid ((+)-ACPECA) was a full agonist at rho1 (EC(50) approximately 135 microM, I(max) approximately 100%) and rho2 (EC(50) approximately 60 microM, I(max) approximately 100%), but only a partial agonist at rho3 (EC(50) approximately 112 microM, I(max) approximately 37%), while (1S,4R)-4-aminocyclopent-2-ene-1-carboxylic acid ((-)-ACPECA) was a weak antagonist at all three receptor subtypes (IC(50)>300 microM). 4-Amino-(S)-2-methylbutanoic acid ((S)-2MeGABA) and 4-amino-(R)-2-methylbutanoic acid ((R)-2MeGABA) followed the same trend, with (S)-2MeGABA acting as a full agonist at the rho1 (EC(50) approximately 65 microM, I(max) approximately 100%), and rho2 (EC(50) approximately 20 microM, I(max) approximately 100%) receptor subtypes, and a partial agonist at rho3 (EC(50) approximately 25 microM, I(max) approximately 90%). (R)-2MeGABA, however, was a moderately potent antagonist at all three receptor subtypes (IC(50) approximately 16 microM at rho1, 125 microM at rho2 and 35 microM at rho3). On the basis of these expanded biological activity data and the solution-phase molecular structures obtained at the MP2/6-31+G* level of ab initio theory, a rationale is proposed for the genesis of this stereoselectivity effect.     

Tax1-binding protein 1 is expressed in the retina and interacts with the GABA(C) receptor rho1 subunit

Macromolecular signalling complexes that link neurotransmitter receptors to functionally and structurally associated proteins play an important role in the regulation of neurotransmission. Thus the identification of proteins binding to neurotransmitter receptors describes molecular mechanisms of synaptic signal transduction. To identify interacting proteins of GABA(C) (where GABA is gamma-aminobutyric acid) receptors in the retina, we used antibodies specific for GABA(C) receptor rho1-3 subunits. Analysis of immunoprecipitated proteins by MALDI-TOF MS (matrix-assisted laser-desorption ionization-time-of-flight MS) identified the liver regeneration-related protein 2 that is identical with amino acids 253-813 of the Tax1BP1 (Tax1-binding protein 1). A C-terminal region of Tax1BP1 bound to an intracellular domain of the rho1 subunit, but not to other subunits of GABA(C), GABA(A) or glycine receptors. Confocal laser-scanning microscopy demonstrated co-localization of Tax1BP1 and rho1 in clusters at the cell membrane of transfected cells. Furthermore, Tax1BP1 and GABA(C) receptors were co-expressed in both synaptic layers of the retina, indicating that Tax1BP1 is a component of GABA(C) receptor-containing signal complexes.     

Phosphonic acid analogs of GABA through reductive dealkylation of phosphonic diesters with lithium trialkylborohydrides

Lithium trialkylborohydrides were found to effect rapid monodealkylation of phosphonic diesters, and this reaction was applied to the synthesis of alkylphosphonic acid 2-aminoethyl esters [H(2)N(CH(2))(2)OP(OH)R, 4], a little-explored class of analogs of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA). Compound 4a (R=Me) proved to be a potent antagonist at human rho1 GABA(C) receptors (expressed in Xenopus laevis oocytes), with an IC(50) of 11.1 microM, but is inactive at alpha(1)beta(2)gamma(2) GABA(A) receptors.     

Allosteric modulation of GABA(B) receptor function in human frontal cortex

In the present study, the effects of different allosteric modulators on the functional activity of gamma-aminobutyric acid (GABA)B receptors in membranes of post-mortem human frontal cortex were examined. Western blot analysis indicated that the tissue preparations expressed both GABA(B1) and GABA(B2) subunits of the GABA(B) receptor heterodimer. In [35S]-GTPgammaS binding assays, Ca2+ ion (1 mM) enhanced the potency of the agonists GABA and 3-aminopropylphosphinic acid (3-APA) and that of the antagonist CGP55845, but not that of the GABA(B) receptor agonist (-)-baclofen. CGP7930 (2,6-di-t-Bu-4-(3-hydroxy-2,2-dimethyl-propyl)-phenol), a positive allosteric modulator of GABA(B) receptors, potentiated both GABA(B) receptor-mediated stimulation of [35S]-GTPgammaS binding and inhibition of forskolin (FSK)-stimulated adenylyl cyclase activity. Chelation of Ca2+ ion by EGTA reduced the CGP7930 enhancement of GABA potency in stimulating [35S]-GTPgammaS binding by two-fold. Fendiline, also reported to act as a positive allosteric modulator of GABA(B) receptors, failed to enhance GABA stimulation of [35S]-GTPgammaS binding but inhibited the potentiating effect of CGP7930. The inhibitory effect was mimicked by the phenothiazine antipsychotic trifluoperazine (TFP), but not by other compounds, such as verapamil or diphenydramine (DPN). These data demonstrate that the function of GABA(B) receptors of human frontal cortex is positively modulated by Ca2+ ion and CGP7930, which interact synergistically. Conversely, fendiline and trifluoperazine negatively affect the allosteric regulation by CGP7930.     

Interactions between rho and gamma2 subunits of the GABA receptor

The gamma-aminobutyric acid type C (GABA(C)) receptor is a ligand-gated chloride channel with distinct physiological and pharmacological properties. Although the exact subunit composition of native GABA(C) receptors has yet to be firmly established, there is general agreement that GABA rho subunits participate in their formation. Recent studies on white perch suggest that certain GABA rho subunits can co-assemble with the GABA(A) receptor gamma2 subunit to form a heteromeric receptor with electrophysiological properties that correspond more closely to the native GABA(C) receptor on retinal neurons than any of the homomeric rho receptors. In the present study we examined the interactions among various perch GABA rho and gamma2 subunits. When co-expressed in Xenopus oocytes, the gamma2 subunit co-immunoprecipitated with Flag-tagged perch rho1A, rho1B, and rho2B subunits, but not with the Flag-tagged perch rho2A subunit. Immunocytochemical studies indicated that the membrane surface expression of the gamma2 subunit was detected only when it was co-expressed with perch rho1A, rho1B, or rho2B subunit, but not with the perch rho2A subunit or when expressed alone. In addition, co-immunoprecipitation of perch rho1B and gamma2 subunits was also detected in protein samples of the teleost retina. Taken together, these findings suggest that a heteromeric rho(gamma2) receptor could represent one form of GABA(C) receptor on retinal neurons.     

Identification of a tyrosine in the agonist binding site of the homomeric rho1 gamma-aminobutyric acid (GABA) receptor that,when mutated,produces spontaneous opening

Mutagenesis of recombinant rho1 gamma-aminobutyric acid (GABA) receptors has previously identified five residues in the amino terminal extracellular domain that play an important role in GABA binding. Here, we present evidence that the tyrosine at position 102 of the rho1 receptor is also associated with the agonist binding site. Wild-type and mutant rho1 receptors were expressed in Xenopus laevis oocytes and examined using the two-electrode voltage clamp. When Tyr-102 was mutated to cysteine, serine, tryptophan, or glycine the EC(50) increased 31-, 214-, 664-, and 8752-fold, respectively. An increase in the IC(50) was also observed for the competitive antagonist 3-APMPA, but not for the non-competitive antagonist picrotoxin. Y102C was accessible to modification by methanethiosulfonate, and this modification was prevented by both GABA and 3-APMPA. An interesting characteristic of the Y102S mutant receptor was that, in the absence of GABA, there was an unusually high oocyte resting conductance that was blocked by both 3-APMPA and picrotoxin, indicating spontaneously opening GABA receptors. It appears that mutation of Tyr-102 perturbs the binding site and gates the pore. We conclude that Tyr-102 is a component of the GABA binding domain and speculate that Tyr-102 might be important for coupling agonist binding to channel opening.     

Two pools of Triton X-100-insoluble GABA(A) receptors are present in the brain, one associated to lipid rafts and another one to the post-synaptic GABAergic complex

Rat forebrain synaptosomes were extracted with Triton X-100 at 4 degrees C and the insoluble material, which is enriched in post-synaptic densities (PSDs), was subjected to sedimentation on a continuous sucrose gradient. Two pools of Triton X-100-insoluble gamma-aminobutyric acid type-A receptors (GABA(A)Rs) were identified: (i) a higher-density pool (rho = 1.10-1.15 mg/mL) of GABA(A)Rs that contains the gamma2 subunit (plus alpha and beta subunits) and that is associated to gephyrin and the GABAergic post-synaptic complex and (ii) a lower-density pool (rho = 1.06-1.09 mg/mL) of GABA(A)Rs associated to detergent-resistant membranes (DRMs) that contain alpha and beta subunits but not the gamma2 subunit. Some of these GABA(A)Rs contain the delta subunit. Two pools of GABA(A)Rs insoluble in Triton X-100 at 4 degrees C were also identified in cultured hippocampal neurons: (i) a GABA(A)R pool that forms clusters that co-localize with gephyrin and remains Triton X-100-insoluble after cholesterol depletion and (ii) a GABA(A)R pool that is diffusely distributed at the neuronal surface that can be induced to form GABA(A)R clusters by capping with an anti-alpha1 GABA(A)R subunit antibody and that becomes solubilized in Triton X-100 at 4 degrees C after cholesterol depletion. Thus, there is a pool of GABA(A)Rs associated to lipid rafts that is non-synaptic and that has a subunit composition different from that of the synaptic GABA(A)Rs. Some of the lipid raft-associated GABA(A)Rs might be involved in tonic inhibition.     

A single amino acid in the second transmembrane domain of GABA rho subunits is a determinant of the response kinetics of GABAC receptors.

The rho subunits that constitute the gamma-aminobutyric acid (GABA)C receptors of retinal neurons form a unique subclass of ligand-gated chloride channels that give rise to sustained GABA-evoked currents that exhibit slow offset (deactivation) kinetics. We exploited this property to examine the molecular mechanisms that govern the disparate response kinetics and pharmacology of perch GABA rho1B and rho2A subunits expressed in Xenopus oocytes. Using a combination of domain swapping and site-directed mutagenesis, we identified the residues at amino acid position 320 in the second transmembrane domain as an important determinant of the receptor kinetics of GABAC receptors. When the site contains a proline residue, as in wild-type rho1 subunits, the receptor deactivates slowly; when serine occupies the site, as in wild-type rho2 subunits, the time course of deactivation is more rapid. In addition, we found that the same site also altered the pharmacology of GABA rho receptors, e.g., when the serine residue of the rho2A receptor was changed to proline, the response of the mutant receptor to imidazole-4-acetic acid (I4AA) mimicked that of the rho1B receptor. However, despite gross changes in receptor pharmacology, the apparent binding affinity for the drug was not significantly altered. These findings provide further evidence that the second transmembrane domain is involved in the gating mechanism that governs the response properties of the various rho receptor subunits. It is noteworthy that the proline residue in native rho1 subunits and the serine residue of rho2 subunits are well conserved in all species, a good indication that the presence of multiple GABA rho subunits serves to generate GABAC receptors that display the wide range of response kinetics observed on various types of retinal neurons.     

Phospho-dependent functional modulation of GABA(B) receptors by the metabolic sensor AMP-dependent protein kinase

GABA(B) receptors are heterodimeric G protein-coupled receptors composed of R1 and R2 subunits that mediate slow synaptic inhibition in the brain by activating inwardly rectifying K(+) channels (GIRKs) and inhibiting Ca(2+) channels. We demonstrate here that GABA(B) receptors are intimately associated with 5'AMP-dependent protein kinase (AMPK). AMPK acts as a metabolic sensor that is potently activated by increases in 5'AMP concentration that are caused by enhanced metabolic activity, anoxia, or ischemia. AMPK binds the R1 subunit and directly phosphorylates S783 in the R2 subunit to enhance GABA(B) receptor activation of GIRKs. Phosphorylation of S783 is evident in many brain regions, and is increased dramatically after ischemic injury. Finally, we also reveal that S783 plays a critical role in enhancing neuronal survival after ischemia. Together our results provide evidence of a neuroprotective mechanism, which, under conditions of metabolic stress or after ischemia, increases GABA(B) receptor function to reduce excitotoxicity and thereby promotes neuronal survival.     

GABA(A) receptor M2-M3 loop secondary structure and changes in accessibility during channel gating

The gamma-aminobutyric acid type A (GABA(A)) receptor M2-M3 loop structure and its role in gating were investigated using the substituted cysteine accessibility method. Residues from alpha(1)Arg-273 to alpha(1)Ile-289 were mutated to cysteine, one at a time. MTSET(+) or MTSES(-) reacted with all mutants from alpha(1)R273C to alpha(1)Y281C, except alpha(1)P277C, in the absence and presence of GABA. The MTSET(+) closed-state reaction rate was >1000 liters/mol-s at alpha(1)N274C, alpha(1)S275C, alpha(1)K278C, and alpha(1)Y281C and was <300 liters/mol-s at alpha(1)R273C, alpha(1)L276C, alpha(1)V279C, alpha(1)A280C, and alpha(1)A284C. These two groups of residues lie on opposite sides of an alpha-helix. The fast reacting group lies on a continuation of the M2 segment channel-lining helix face. This suggests that the M2 segment alpha-helix extends about two helical turns beyond alpha(1)N274 (20'), aligned with the extracellular ring of charge. At alpha(1)S275C, alpha(1)V279C, alpha(1)A280C, and alpha(1)A284C the reaction rate was faster in the presence of GABA. The reagents had no functional effect on the mutants from alpha(1)A282C to alpha(1)I289C, except alpha(1)A284C. Access may be sterically hindered possibly by close interaction with the extracellular domain. We suggest that the M2 segment alpha-helix extends beyond the predicted extracellular end of the M2 segment and that gating induces a conformational change in and/or around the N-terminal half of the M2-M3 loop. Implications for coupling ligand-evoked conformational changes in the extracellular domain to channel gating in the membrane-spanning domain are discussed.     

Sleep is differently modulated by basal forebrain GABA(A) and GABA(B) receptors

There is evidence that GABA plays a major role in sleep regulation. GABA(A) receptor agonists and different compounds interacting with the GABA(A) receptor complex, such as barbiturates and benzodiazepines, can interfere with the sleep/wake cycle. On the other hand, there is very little information about the possible role of GABA(B) receptors in sleep modulation. The nucleus basalis of Meynert (NBM), a cholinergic area in the basal forebrain, plays a pivotal role in the modulation of sleep and wakefulness, and both GABA(A) and GABA(B) receptors have been described within the NBM. This study used unilateral infusions in the NBM to determine the effects of 3-hydroxy-5-aminomethylisoxazole hydrobromide (muscimol hydrobromide, a GABA(A) receptor subtype agonist) and beta-(aminomethyl)-4-chlorobenzenepropanoic acid (baclofen, a GABA(B) receptor subtype agonist) on sleep parameters in freely moving rats by means of polygraphic recordings. Muscimol (0.5 nmol) and baclofen (0.7 nmol) induced an increase in slow-wave sleep and an inhibition of wakefulness. Muscimol, but not baclofen, also caused a decrease in desynchronized sleep parameters. The results reported here indicate that 1) the NBM activation of both GABA(A) and GABA(B) receptors influences the sleep/wake cycle, and 2) GABA(A) but not GABA(B) receptors are important for desynchronized sleep modulation, suggesting that the two GABAergic receptors play different roles in sleep modulation.     

Noncompetitive antagonist-binding sites of rat and housefly gamma-aminobutyric acid receptors display different enantiospecificities for tert-butyl(isopropyl)bicyclophosphorothionate

The enantiomers of 4-tert-butyl-3-isopropyl-2,6,7-trioxa-1-phosphabicyclo[2.2.2 ]octane 1-sulfide (TBIPPS) were prepared in nine steps from diethyl tert-butylmalonate, and their abilities to compete with [3H]1-(4-ethynylphenyl)-4-n-propyl-2,6,7-trioxabicyclo[2.2.2 ]octane (EBOB), a noncompetitive antagonist of ionotropic gamma-aminobutyric acid (GABA) receptors, at their binding site were investigated using rat brain and housefly head membranes. The (S)-(-)-isomer of TBIPPS (IC50 = 398 nM) was more potent than was the (R)-(+)-isomer of TBIPPS (IC50 = 1220 nM) in rat receptors, while the potencies of (S)-TBIPPS 104 nM) and (R)-TBIPPS (IC50 = 94.4 nM) in housefly receptors were almost the same. The different enantiospecificities of rat and housefly receptors indicate that the three-dimensional structure of the binding site might be different between these receptors. In a region of the rat binding site there might be a steric bulk that interacts less favorably with (R)-TBIPPS than with (S)-TBIPPS, while in the corresponding region of the housefly binding site there might not be such a steric bulk that leads to specificity for these compounds.     

Production of (R)-3-amino-3-phenylpropionic acid and (S)-3-amino-3-phenylpropionic acid from (R,S)-N-acetyl-3-amino-3-phenylpropionic acid using microorganisms having enantiomer-specific amidohydrolyzing activity

(R)-3-Amino-3-phenylpropionic acid ((R)-beta-Phe) and (S)-3-amino-3-phenylpropionic acid ((S)-beta-Phe) are key compounds on account of their use as intermediates in synthesizing pharmaceuticals. Enantiomerically pure non-natural amino acids are generally prepared by enzymatic resolution of the racemic N-acetyl form, but despite the intense efforts this method could not be used for preparing enantiomerically pure beta-Phe, because the effective enzyme had not been found. Therefore, screening for microorganisms capable of amidohydrolyzing (R,S)-N-acetyl-3-amino-3-phenylpropionic acid ((R,S)-N-Ac-beta-Phe) in an enantiomer-specific manner was performed. A microorganism having (R)-enantiomer-specific amidohydrolyzing activity and another having both (R)-enantiomer- and (S)-enantiomer-specific amidohydrolyzing activities were obtained from soil samples. Using 16S rDNA analysis, the former organism was identified as Variovorax sp., and the latter as Burkholderia sp. Using these organisms, enantiomerically pure (R)-beta-Phe (>99.5% ee) and (S)-beta-Phe (>99.5% ee) with a high molar conversion yield (67%-96%) were obtained from the racemic substrate.     

Structural modifications of (1S,3S)-3-amino-4-difluoromethylenecyclopentanecarboxylic acid, a potent irreversible inhibitor of GABA aminotransferase

Low brain levels of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) lead to convulsions. Inhibition of GABA aminotransferase increases the concentration of GABA and can terminate the convulsions. Earlier we reported the synthesis of (1S,3S)-3-amino-4-difluoromethylenecyclopentanecarboxylic acid (2), which is 186 times more potent an inactivator of GABA aminotransferase than the epilepsy drug S-vigabatrin. The corresponding dichloromethylene analogue of 2 (compound 3) has been made, but it shows only weak reversible inhibition of GABA aminotransferase. However, the tetrazole isostere of 2 (compound 4) has been found to be a time-dependent inactivator of GABA aminotransferase. Although it is 20 times less potent than carboxylic acid 2, it is 2.5 times more potent than S-vigabatrin. A calculation of the ClogP values indicates that 4 is the most lipophilic of the three, being 69 times more lipophilic than 2 and 55 times more lipophilic than S-vigabatrin, indicating potential for improved bioavailability.     

Reciprocal inhibition of G-protein signaling is induced by CB(1) cannabinoid and GABA(B) receptor interactions in rat hippocampal membranes

Cannabinoid CB(1) and the metabotropic GABA(B) receptors have been shown to display similar pharmacological effects and co-localization in certain brain regions. Previous studies have reported a functional link between the two systems. As a first step to investigate the underlying molecular mechanism, here we show cross-inhibition of G-protein signaling between GABA(B) and CB(1) receptors in rat hippocampal membranes. The CB(1) agonist R-Win55,212-2 displayed high potency and efficacy in stimulating guanosine-5'-O-(3-[(35)S]thio)triphosphate, [(35)S]GTPgammaS binding. Its effect was completely blocked by the specific CB(1) antagonist AM251 suggesting that the signaling was via CB(1) receptors. The GABA(B) agonists baclofen and SKF97541 also elevated [(35)S]GTPgammaS binding by about 60%, with potency values in the micromolar range. Phaclofen behaved as a low potency antagonist with an ED(50) approximately 1mM. However, phaclofen at low doses (1 and 10nM) slightly but significantly attenuated maximal stimulation of [(35)S]GTPgammaS binding by the CB(1) agonist R-Win55,212-2. The observation that higher concentrations of phaclofen had no such effect rule out the possibility of its direct action on CB(1) receptors. The pharmacologically inactive stereoisomer S-Win55,212-3 had no effect either alone or in combination with phaclofen establishing that the interaction is stereospecific in hippocampus. The specific CB(1) antagonist AM251 at a low dose (1 nM) also inhibited the efficacy of G-protein signaling of the GABA(B) receptor agonist SKF97541. Cross-talk of the two receptor systems was not detected in either spinal cord or cerebral cortex membranes. It is speculated that the interaction might occur via an allosteric interaction between a subset of GABA(B) and CB(1) receptors in rat hippocampal membranes. Although the exact molecular mechanism of the reciprocal inhibition between CB(1) and GABA(B) receptors will have to be explored by future studies it is intriguing that the cross-talk might be involved in balance tuning the endocannabinoid and GABAergic signaling in hippocampus.     

p-Hydroxybenzoylformic acid and (R)-(-)-p-hydroxymandelic acid, two antifungal compounds isolated from the liquid culture of the ectomycorrhizal fungus Pisolithus arhizus

Two antifungal compounds isolated from the liquid culture medium of Pisolithus arhizus were identified as p-hydroxybenzoylformic acid and (R)-(-)-p-hydroxymandelic acid and given the trivial names pisolithin A and pisolithin B, respectively. The efficacy of the compounds to inhibit the germination of conidia of Truncatella hartigii was compared with that of commercially available structural analogues, and a comparable range of effectiveness for 50% germination inhibition (GI50) of conidia was recorded. The commercially available synthetic compounds (R)-mandelic acid, benzoylformic acid, and racemic p-hydroxymandelic acid, had GI50 values of 82, 72, and 59 micrograms/mL, respectively, as compared with the natural compounds pisolithin A, 67 micrograms/mL, and pisolithin B, 71 micrograms/mL. Two synthetic S enantiomers of mandelic acid, (S)-mandelic acid and (S)-(+)-p-hydroxymandelic acid, were the most effective compounds, with GI50 values of 31 and 33 micrograms/mL, respectively. A sodium salt of mandelic acid had no activity below 500 micrograms/mL. Pisolithin A and pisolithin B were compared with polyoxin D for inhibition of hyphal growth, as measured by protein estimation. Both pisolithin A and B measured higher levels of putative extractable protein than polyoxin D, but less mycelial wet weight was measured. It is suggested that the pisolithins caused a disruption of cell turgor. A measurement of mycelial dry weights of phytopathogens, incubated with the commercially available analogues, benzoylformic acid and racemic p-hydroxymandelic acid, indicated that benzoylformic acid was either more effective than, or as effective as, racemic p-hydroxymandelic acid or nystatin in arresting fungal growth.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparative Stereostructure-Activity Studies on GABAA and GABAB Receptor Sites and GABA Uptake Using Rat Brain Membrane Preparations

The affinities of a number of analogues of gamma-aminobutyric acid (GABA) for GABAA and GABAB receptor sites and GABA uptake were studied using rat brain membrane preparations. Studies on the (S)-(+)- and (R)-(-)-isomers of baclofen, 3-hydroxy-4-aminobutyric acid (3-OH-GABA), and 4,5-dihydromuscimol (DHM) revealed different stereoselectivities of these synaptic mechanisms in vitro. Although (S)-3-OH-GABA and, in particular, (S)-DHM were more potent than the corresponding (R)-isomers as inhibitors of GABAA binding, the opposite stereoselectivity was demonstrated for the GABAB binding sites. Thus, (R)-3-OH-GABA and (R)-baclofen were more potent than the (S)-isomers as inhibitors of GABAB binding, (R)-baclofen being some five times more potent than (R)-3-OH-GABA. These two (R)-isomers actually have opposite orientation of the substituents on the GABA backbones, suggesting that the lipophilic substituent of (R)-baclofen interacts with a structural element of the GABAB receptor site different from that that binds the very polar hydroxy group of (R)-3-OH-GABA. The O-methylated analogue of 3-OH-GABA, 3-methoxy-4-aminobutyric acid (3-OCH3-GABA), did not interact significantly with GABAB sites. The homologues of GABA, trans-4-aminocrotonic acid (trans-ACA), muscimol, and 3-OH-GABA, that is, 5-aminovaleric acid (DAVA), trans-5-aminopent-2-enoic acid, homomuscimol, and 3-hydroxy-5-aminovaleric acid (3-OH-DAVA), respectively, were generally much weaker than the parent compounds, whereas 2-hydroxy-5-aminovaleric acid (2-OH-DAVA) showed a significantly higher affinity for GABAB sites than the corresponding GABA analogue.(ABSTRACT TRUNCATED AT 250 WORDS)