ABSTRACT
Antidepressants act at the GABA(A) receptor to inhibit GABA-stimulated 36Cl(-) influx and GABA reduction of [35S]TBPS binding. This study examined how selective knock-down (via antisense oligodeoxynucleotides, aODNs) of GABA(A) receptor subunits modified antidepressant activity. The specific aODNs used were for the alpha1, beta1, beta2 or gamma2 subunits of the GABA(A) receptor. The aODN microinjections reduced corresponding GABA(A) receptor subunit mRNA levels by 30-40% as assessed by RT-PCR. The inhibitory effect of the antidepressants amitriptyline and mianserin on GABA-stimulated 36Cl(-) influx was decreased after microinjections of alpha1, beta1, or beta2 subunit aODNs but potentiated after microinjections of gamma2 subunit aODNs. This pattern of aODNs effect on amitriptyline and mianserin modulation of GABA-stimulated 36Cl(-) influx was the same for both antidepressants and similar to GABA but different than that of diazepam and bicuculline. We conclude that multiple subunits of the GABA(A) receptor regulate the effect of amitriptyline and mianserin on the GABA(A) receptor chloride ionophore complex. However, the exact identity of the subunit mediating the direct or allosteric modulation of the antidepressant effect on GABA-stimulated 36Cl(-) influx remains unclear.
Subject(s)
Antidepressive Agents/pharmacology , Cerebral Cortex/drug effects , Chloride Channels/drug effects , Neurons/drug effects , Oligonucleotides, Antisense/pharmacology , Receptors, GABA-A/drug effects , Amitriptyline/pharmacology , Animals , Cerebral Cortex/cytology , Cerebral Cortex/metabolism , Chloride Channels/metabolism , Chlorides/metabolism , Male , Mianserin/pharmacology , Neurons/cytology , Neurons/metabolism , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , Receptors, GABA-A/genetics , Receptors, GABA-A/metabolismABSTRACT
GABA(A) receptor function was studied in cerebral cortical vesicles prepared from rats after intracerebroventricular microinjections of antisense oligodeoxynucleotides (aODNs) for alpha1, gamma2, beta1, beta2 subunits. GABA(A) receptor alpha1 subunit aODNs decreased alpha1 subunit mRNA by 59+/-10%. Specific [3H]GABA binding was decreased by alpha1 or beta2 subunit aODNs (to 63+/-3% and 64+/-9%, respectively) but not changed by gamma2 subunit aODNs (94+/-5%). Specific [3H]flunitrazepam binding was increased by alpha1 or beta2 subunit aODNs (122+/-8% and 126+/-11%, respectively) and decreased by gamma2 subunit aODNs (50+/-13%). The "knockdown" of specific subunits of the GABA(A )receptor significantly influenced GABA-stimulated 36Cl- influx. Injection of alpha1 subunit aODNs decreased basal 36Cl- influx and the GABA Emax; enhanced GABA modulation by diazepam; and decreased antagonism of GABA activity by bicuculline. Injection of gamma2 subunit aODNs increased the GABA Emax; reversed the modulatory efficacy of diazepam from enhancement to inhibition of GABA-stimulation; and reduced the antagonist effect of bicuculline. Injection of beta2 subunit aODNs reduced the effect of diazepam whereas treatment with beta1 subunit aODNs had no effect on the drugs studied. Conclusions from our studies are: (1) alpha1 subunits promote, beta2 subunits maintain, and gamma2 subunits suppress GABA stimulation of 36Cl- influx; (2) alpha1 subunits suppress, whereas beta2, and gamma2 subunits promote allosteric modulation by benzodiazepines; (3) diazepam can act as an agonist or inverse agonist depending on the relative composition of the receptor subunits: and (4) the mixed competitive/non-competitive effects of bicuculline result from activity at alpha1 and gamma2 subunits and the lack of activity at beta1 and beta2 subunits.
Subject(s)
Cerebral Cortex/metabolism , Chlorides/metabolism , Oligodeoxyribonucleotides, Antisense/pharmacology , Receptors, GABA-A/genetics , gamma-Aminobutyric Acid/pharmacology , Animals , Base Sequence , Bicuculline/pharmacology , Cerebral Cortex/drug effects , Flunitrazepam/pharmacokinetics , Male , Open Reading Frames , Rats , Rats, Sprague-Dawley , Receptors, GABA-A/physiology , Reverse Transcriptase Polymerase Chain Reaction , gamma-Aminobutyric Acid/metabolismABSTRACT
The clinically important antidepressant fluoxetine is established as a selective serotonin reuptake inhibitor. This study demonstrates that fluoxetine also interacts with the GABA(A) receptor complex. At concentrations above 10 microM fluoxetine inhibited the binding of both [3H]GABA (IC50 = 2 mM) and [3H]flunitrazepam (IC50 = 132 microM) to the GABA(A) receptor complex in brain cortical membranes. Low fluoxetine concentrations (1 nM) enhanced GABA-stimulated Cl- uptake by a rat cerebral cortical vesicular preparation. At higher concentrations (100 microM and 1 mM), however, fluoxetine inhibited GABA-stimulated Cl- uptake, an effect related to a reduction in Emax. These observations might assist in an explanation of the basis of the antidepressant action of fluoxetine.
Subject(s)
Fluoxetine/pharmacology , Receptors, GABA-A/drug effects , Selective Serotonin Reuptake Inhibitors/pharmacology , Animals , Chlorides/metabolism , Dose-Response Relationship, Drug , Flunitrazepam/metabolism , Ion Transport , Male , Rats , Rats, Sprague-Dawley , Tritium , gamma-Aminobutyric Acid/metabolismABSTRACT
The effects of three potential irreversible inhibitors of gamma-aminobutyrate aminotransferase from Pseudomonas fluorescens were studied in order to throw more light on the nature of the active site of the enzyme. The thiol group reagent mercuric chloride inactivated the enzyme in a concentration-dependent manner. Inhibition kinetics were consistent with a simple bimolecular reaction. The second-order rate constant was 4.2 x 10(3) +/- 0.61 M-1 sec-1. In contrast to either of the substrates, the cofactor pyridoxal 5'-phosphate could protect the enzyme from the inhibition, suggesting cysteinyl residues are important for cofactor binding at the active site. p-Chloromercuribenzoic acid produced a similar inactivation of the enzyme. 4-Amino-2-fluorobutanoic acid also inhibited enzymic activity but in this case the inhibition was reversible and competitive with respect to gamma-aminobutyric acid (GABA). The inhibitor constant (Ki) was 0.83 +/- 0.44 mM. We found no evidence that this fluorinated analogue of GABA could act as a substrate for the enzyme.
