ABSTRACT
We investigated whether the effect of phorbol-12-myristate-13-acetate (PMA) was altered by a kinase inhibitor and by down-regulation of protein kinase C (PKC) in order to determine if glycine receptors in mouse spinal neurons, unlike those in hippocampal and trigeminal neurons, can be inhibited by PKC. To examine the above, electrophysiological and immunofluorescence studies were carried out in mouse spinal neurons kept in culture for up to 3 weeks. The inhibition of the glycine activated current by PMA (1 microM) increased from 12+/-3% during week 1 to 27+/-6% during week 3. The effect of PMA was completely blocked by the PKC selective inhibitor RO 31-8220 (1 microM). After culturing the cells with 1 microM PMA for 24 h, the inhibitory effect of acute application of PMA disappeared altogether, suggesting that the effect of PMA was via PKC. Immunofluorescence studies showed that a short stimulation with PMA translocated the enzyme to the periphery whereas longer term stimulation (24 h) down regulated the PKC signal. These results indicate that activation of PKC by PMA inhibits the glycine receptor in cultured spinal neurons and that its sensitivity changes during neuronal development.
Subject(s)
Chloride Channels/drug effects , Chlorides/metabolism , Glycine/pharmacology , Indoles/pharmacology , Ion Transport/drug effects , Nerve Tissue Proteins/metabolism , Neurons/drug effects , Protein Kinase C/metabolism , Receptors, Glycine/drug effects , Spinal Cord/cytology , Tetradecanoylphorbol Acetate/pharmacology , Animals , Cells, Cultured , Chloride Channels/metabolism , Enzyme Activation , Enzyme Inhibitors/pharmacology , Female , Fluorescent Antibody Technique, Indirect , Ion Transport/physiology , Mice , Mice, Inbred C57BL , Nerve Tissue Proteins/antagonists & inhibitors , Neurons/metabolism , Patch-Clamp Techniques , Protein Kinase C/antagonists & inhibitors , Receptors, Glycine/metabolism , Signal Transduction/drug effectsABSTRACT
Five-day-old cultures of mouse glycinergic spinal interneurons were chronically treated with 100 mM ethanol and the glycine and gamma-aminobutyric acid (GABA)(A) receptors were assayed using whole-cell recordings and fluorescence-imaging techniques. Control neurons displayed a glycine(50) of 19 +/- 0.6 microM and a Hill coefficient of 3.1 +/- 0.3. Chronic ethanol treatment did not significantly change these parameters. The maximal responses were 310 +/- 80 pA/pF in control and 440 +/- 19 pA/pF in treated cells, and the fluorescence intensity associated to a monoclonal glycine receptor antibody was unchanged. Strychnine inhibited the glycine current with smaller potency (29%) in treated neurons, thus the IC(50) increased from 14 +/- 2 nM in control to 18 +/- 6 nM in treated neurons. Zn(2+) (10 microM) potentiated the glycine current by 43 +/- 33% in control, but only by 18 +/- 13% in treated neurons. Interestingly, no change on the inhibition produced by a high concentration of Zn(2+) was found in treated neurons. The inhibitory effect of picrotoxin on the glycine receptor, associated to a homomeric receptor, was eliminated with chronic ethanol, suggesting a faster switch to beta-subunit-containing receptors. Unlike glycine receptors, the sensitivity of GABA(A) receptors to GABA, pentobarbital, diazepam, and Zn(2+), as well as the fluorescence intensity associated to a high-affinity benzodiazepine analog was unchanged by chronic ethanol. In conclusion, we found that glycine receptors in spinal interneurons were altered by chronic ethanol treatment and this may reflect the expression of different subunits in control and treated neurons. GABA(A) receptors were resistant to the treatment.
