ABSTRACT
Depolarization of cerebellar granule cells in culture leads to up-regulation of the GABA(A) receptor delta subunit expression. To determine the signaling molecules involved, we examined the effects of protein kinase inhibitors and cyclic AMP-elevating compounds on basal and AMPAR agonist-induced delta mRNA expression in cerebellar granule cells. Treatment with the c-Jun N-terminal kinase (JNK) inhibitor SP600125 or with pituitary adenylate activating polypeptide increased delta subunit expression by 70%. Selective activation of AMPA receptors with CPW-399 also increased delta mRNA expression (2-4-fold). CPW-399 induction of delta subunit mRNA was reduced by prior treatment with either the MEK1/2 inhibitor U0126 or protein kinase A (PKA) inhibitors KT 5720 and H89. These effects were additive and combined treatment with U0126 and H89 completely prevented induction of delta subunit expression above basal levels. These results suggest that the role of JNK and ERK1/2/PKA on maintainence of delta subunit expression is diammetrically opposite. While JNK activity negatively regulates delta subunit mRNA expression in unstimulated neurons, activity of ERK1/2 and PKA are required for full induction of GABA(A) receptor delta subunit expression in response to AMPA receptor stimulation.
Subject(s)
Receptors, AMPA/physiology , Receptors, GABA-A/physiology , Signal Transduction , Up-Regulation , Animals , Blotting, Western , Cells, Cultured , Mice , Mice, Inbred C57BLABSTRACT
Depolarization of cultured mouse cerebellar granule cells with potassium or kainate results in developmentally arrested state that includes down-regulation of GABA(A) receptor alpha1, alpha6 and beta2 subunit expression. These subunits are normally strongly expressed in cerebellar granule cells from second postnatal week throughout the adulthood. In the present study we demonstrate that selective activation of AMPA subtype of glutamate receptors down-regulates alpha1 and alpha6 subunit mRNA expression. Removal of AMPA agonist from culture medium restores expression of these subunits indicating reversibility of the down-regulation. In serum-free culture medium AMPA receptor activation did not down-regulate alpha1 or alpha6 subunit expression. Furthermore, the down-regulation was strongly attenuated when the cells were cultured in the presence of dialysed fetal calf serum. The results indicate that down-regulation of GABA(A) receptor alpha1 and alpha6 subunits by AMPA receptor activation is dependent on the presence of low molecular weight compounds present in fetal calf serum. In order to study mouse cerebellar granule cell maturation and/or regulation of GABA(A) receptor subunit expression in culture, the experiments should be performed in the absence of fetal calf serum.
Subject(s)
Cerebellum/metabolism , Neurons/metabolism , Receptors, AMPA/physiology , Receptors, GABA-A/biosynthesis , Alanine/analogs & derivatives , Alanine/pharmacology , Animals , Cells, Cultured , Cerebellum/cytology , Coloring Agents , Culture Media, Serum-Free , Down-Regulation , GABA Agonists/pharmacology , GABA Antagonists/pharmacology , GABA-A Receptor Antagonists , Kainic Acid/pharmacology , Mice , Neurons/drug effects , Protein Binding , Pyrimidinones/pharmacology , Quinoxalines/pharmacology , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Receptors, AMPA/antagonists & inhibitors , Receptors, AMPA/genetics , Receptors, GABA-A/genetics , Receptors, Kainic Acid/antagonists & inhibitors , Reverse Transcriptase Polymerase Chain Reaction , Tetrazolium Salts , Thiazoles , GluK2 Kainate ReceptorABSTRACT
We have studied the effects of AMPA/kainate receptor agonists on GABA(A) receptor subunit mRNA expression in vitro in cultured rat cerebellar granule cells (CGCs). Kainate (KA) (100 microM) and high K(+) (25 mM) dramatically up-regulated delta subunit mRNA expression to 500-700% of that in control cells grown in low K(+) (5 mM). KA or high K(+) had no effect on the expression of the other major GABA(A) receptor subunits alpha1, alpha6, beta2, beta3 or gamma2. Up-regulation of delta mRNA was also detected with the AMPA receptor-selective agonist CPW-399 and to a lesser extent with the KA receptor-selective agonist ATPA. AMPA/kainate receptor-selective antagonist DNQX completely inhibited KA-, CPW-399- and ATPA-induced delta mRNA up-regulation indicating that the effects were mediated via AMPA and KA receptor activation. NMDA receptor-selective antagonist MK-801 inhibited 76% of the KA- and 57% of the CPW-399-induced delta up-regulation suggesting that KA and CPW-399 treatments may induce glutamate release resulting in NMDA receptor activation, and subsequently to delta mRNA up-regulation. In CGCs, delta subunit is a component of extrasynaptic alpha6betadelta receptors that mediate tonic inhibition. Up-regulation of delta during prolonged glutamate receptor activation or cell membrane depolarization may be a mechanism to increase tonic inhibition to counteract excessive excitation.
