ABSTRACT
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
Herpes simplex virus (HSV) is one of the most commonly identified viruses in the clinical laboratory. HSV is of clinical consequence because of its ability to produce such life-threatening infections as encephalitis and neonatal disease. A variety of cell lines are currently being used for detection of HSV in cell culture. This study compared several cell lines simultaneously in a 96-well cell culture system to determine which lines demonstrated viral CPE most quickly following infection. In considering speed at which viral CPE is demonstrated, our results showed that primary rabbit kidney cells surpassed all other cell lines at 48 h post-viral infection. Alternative cell lines demonstrated to be suitable by this study were human fetal foreskin, human embryonic lung, and Vero cells.
