ABSTRACT
GABAA receptors of cerebellar granule cells obtained from neonatal rats and kept in culture were studied by labelled muscimol binding. The data show that, according to the maturational state of those cells in vivo, one or two binding components appear. The low affinity component seems to be the one appearing later. The expression of this component seems to be regulated by protein tyrosine phosphorylation. In fact, its expression is down regulated by the protein tyrosine kinase (PTK) inhibitor, genistein. Viceversa, its expression is upregulated by insulin like growth factor I (IGF-I), most probably via PTK activation. A possible interpretation of the data is that in vivo IGF-I is one of the endogenous messages leading to the expression of this component during development. Another endogenous factor involved may be GABA itself. Low affinity GABAA receptors appear to be the ones involved in inhibitory synaptic transmission at glomeruli. Whereas the high affinity ones probably correspond to extrasynaptic GABAA receptors mediating the tonic form of inhibition in cerebellar granules.
Subject(s)
Cerebellum/metabolism , Muscimol/metabolism , Neurons/metabolism , Receptors, GABA-A/metabolism , gamma-Aminobutyric Acid/metabolism , Animals , Animals, Newborn , Cells, Cultured , Cerebellum/cytology , Enzyme Inhibitors/pharmacology , GABA Agonists/metabolism , Genistein/pharmacology , Insulin-Like Growth Factor I/metabolism , Muscimol/chemistry , Neurons/drug effects , Protein Binding , Rats , Rats, Sprague-Dawley , Receptors, GABA-A/genetics , Tritium/chemistryABSTRACT
Gamma-aminobutyric acid (GABA) activated chloride currents were studied in rat cerebellum granule cells in culture by the whole cell patch-clamp technique. Both the peak and steady state currents were inhibited by 100 microM lanthanum. In the first case, inhibition is due to an increase of the EC50 for GABA. The inhibitory effect of lanthanum on the peak current at 3 microM GABA increased with the cation concentration. A tendency towards the same behavior was found also for the inhibition of the steady state current, at 3 microM GABA, as a function of lanthanum concentration, although inhibition in this case was lower. The comparison of the results with published data about the effects of lanthanum on recombinant GABA(A) receptors likely to occur in granule cells allows suggestions about the receptor types giving, respectively, the peak and the steady state component.
Subject(s)
Cerebellum/cytology , Lanthanum/pharmacology , Receptors, GABA-A/drug effects , Animals , Animals, Newborn , Cells, Cultured , Chloride Channels/drug effects , Chloride Channels/physiology , Membrane Potentials , Patch-Clamp Techniques , Rats , Rats, Wistar , Receptors, GABA-A/chemistry , Receptors, GABA-A/physiologyABSTRACT
Large conductance voltage-dependent and Ca(2+)-modulated K(+) channels play a crucial role in myometrium contractility. Western blots and immunocytochemistry of rat uterine sections or isolated cells show that MaxiK channel protein signals drastically decrease towards the end of pregnancy. Consistent with a transcriptional regulation of channel expression, mRNA levels quantified with the ribonuclease protection assay correlated well with MaxiK protein levels. As a control, Na(+)/K(+)-ATPase protein and RNA levels do not significantly change at different stages of pregnancy. The low numbers of MaxiK channels at the end of pregnancy may facilitate uterine contraction needed for parturition.
Subject(s)
Myometrium/metabolism , Placental Hormones/physiology , Potassium Channels, Calcium-Activated , Potassium Channels/biosynthesis , Potassium Channels/genetics , RNA, Messenger/biosynthesis , Animals , Blotting, Western , COS Cells , Cells, Cultured , Female , Large-Conductance Calcium-Activated Potassium Channel alpha Subunits , Large-Conductance Calcium-Activated Potassium Channels , Membrane Potentials , Myometrium/cytology , Potassium Channels/physiology , Pregnancy , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , Ribonucleases/metabolism , Sodium-Potassium-Exchanging ATPase/metabolism , Time FactorsABSTRACT
Interaction between GABAA and GABA(B) receptors was studied in rat cerebellar granule cells in culture, by the whole-cell patch-clamp approach. Our data show that the GABA(B) agonist (-)baclofen is not able, per se, to significantly change the muscimol-activated chloride current. However, (-)baclofen dose-dependently prevents the reduction of GABA(A) receptor function by forskolin, an activator of adenylate cyclase. The effect of baclofen is mediated by a pertussis toxin-sensitive G protein. In fact, in cells treated with pertussis toxin, baclofen and forskolin, the toxin is able to block baclofen action, allowing forskolin to act fully. The protective effect by GABA(B) receptor activation under these circumstances is most probably related to the prevention of cyclic AMP increases after forskolin treatment. In fact, in these neurons cyclic AMP and protein kinase A activation result in a down-regulation of GABA(A) receptor function. On the whole, the data indicate the presence of complex modulation of GABA(A) receptors by GABA(B) receptor types in cerebellum granule cells.
