Phorbol 12-myristate 13-acetate potentiation of N-methyl-D-aspartate-induced currents in primary cultured cerebellar granule cells is mediated by protein kinase C alpha.
We have previously reported that activation of protein kinase C (PKC) by phorbol 12-myristate 13-acetate (PMA) results in potentiation of N-methyl-D-aspartate-induced currents (I(NMDA))of receptors contained in primary cultured cerebellar granule cells (CGCs). The purpose of this study was to identify which PKC isoform(s) was responsible for this effect by using the whole-cell patch-clamp technique. Experiments were conducted on CGCs that expressed both the NR2A and NR2B NMDA receptor subunits as well as the PMA-sensitive PKC isoforms alpha, betaI, betaII, delta, epsilon, gamma, and . As observed previously, N-methyl-D-aspartate-induced peak currents (I(Pk)) were enhanced by a 12.5-min, 100 nM PMA exposure at 37 degrees C under normal recording conditions. Potentiation of receptor function was not observed when extracellular Ca(2+) was removed and 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid was present inside the cell. PMA-induced potentiation of I(Pk) did not occur when PKCalpha-specific antibody was introduced into the cell via the recording electrode. However, in similar experiments with antibodies specific for PKCbetaII, delta, epsilon, gamma, and , PMA potentiation of I(Pk) was observed. Down-regulation of PMA-sensitive PKC isoforms by an overnight exposure of 100 nM PMA resulted in lack of potentiation by PMA that was rescued when catalytically active PKCalpha was introduced into the cell via the patch electrode. PMA potentiation of I(Pk) was not recovered when catalytically active PKCbetaI, PKCbetaII, or PKCgamma was introduced into the cell via the patch electrode. Collectively, our data provide strong evidence that PMA-enhanced function of native NMDA receptors expressed in primary cultured CGCs is mediated by activation of PKCalpha.
Reneau
Jason C
JC
Department of Pharmacology and Neuroscience, Texas Tech University Health Sciences Center, STOP 6592, Lubbock, Texas 79430, USA.
Reyland
Mary E
ME
Phillips
Jonathan
J
Kindy
Carissa
C
Popp
R Lisa
RL
D000818:Animals; D002478:Cells, Cultured; D002531:Cerebellum / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism; D004357:Drug Synergism; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D016202:N-Methylaspartate / Q000502:physiology*; D018408:Patch-Clamp Techniques; D051571:Protein Kinase C-alpha / Q000502:physiology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D016194:Receptors, N-Methyl-D-Aspartate / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000031:analogs & derivatives* / Q000494:pharmacology
D016194:Receptors, N-Methyl-D-Aspartate; C016658:phorbolol myristate acetate; D016202:N-Methylaspartate; D051571:Protein Kinase C-alpha; D013755:Tetradecanoylphorbol Acetate