Effects of treatment with GABA(A) receptor subunit antisense oligodeoxynucleotides on GABA-stimulated 36Cl- influx in the rat cerebral cortex.
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.
Malatynska
E
E
Department of Pharmacology and Toxicology, Indiana University, School of Medicine, Evansville 47712, USA. emalatyn@iupui.edu
Matheson
G K
GK
Goldenberg
R
R
Crites
G J
GJ
Schindler
N L
NL
Weinzapfel
D
D
Harrawood
D
D
Yochum
A
A
Tunnicliff
G
G
D000818:Animals; D001483:Base Sequence; D001640:Bicuculline / Q000494:pharmacology; D002540:Cerebral Cortex / Q000187:drug effects / Q000378:metabolism*; D002712:Chlorides / Q000378:metabolism*; D005445:Flunitrazepam / Q000493:pharmacokinetics; D008297:Male; D020319:Oligodeoxyribonucleotides, Antisense / Q000494:pharmacology*; D016366:Open Reading Frames; D051381:Rats; D017207:Rats, Sprague-Dawley; D011963:Receptors, GABA-A / Q000235:genetics* / Q000502:physiology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D005680:gamma-Aminobutyric Acid / Q000378:metabolism / Q000494:pharmacology*
D002712:Chlorides; D020319:Oligodeoxyribonucleotides, Antisense; D011963:Receptors, GABA-A; D005680:gamma-Aminobutyric Acid; D005445:Flunitrazepam; D001640:Bicuculline