Phorbol 12-Myristate 13-Acetate Potentiation ofN-Methyl-d-aspartate-Induced Currents in Primary Cultured Cerebellar Granule Cells Is Mediated by Protein Kinase Cα

Abstract

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 wholecell 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 ␣, ␤⌱, ␤⌱⌱, ␦, , ␥, 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°C under normal recording conditions. Potentiation of receptor function was not observed when extracellular Ca 2ϩ was removed and 1,2-bis(2-aminophenoxy)ethane-N,N,NЈ,NЈ-tetraacetic acid was present inside the cell. PMA-induced potentiation of I Pk did not occur when PKC␣-specific antibody was introduced into the cell via the recording electrode. However, in similar experiments with antibodies specific for PKC␤⌱⌱, ␦, , ␥, 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 PKC␣ was introduced into the cell via the patch electrode. PMA potentiation of I Pk was not recovered when catalytically active PKC␤I, PKC␤II, or PKC␥ 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 PKC␣.

Acute ethanol exposure prevents PMA-mediated augmentation of N-methyl-d-aspartate receptor function in primary cultured cerebellar granule cells

“…We also reported that similar results could be observed with briefer (12.5 min) PMA exposures, and that increases in I PK could be observed for up to 20 min with repeated agonist exposure clusters during whole-cell recordings (Reneau et al, 2009). In this current study, we corroborate our previous findings in that 100 nM PMA treatment at 37° C resulted in sustained augmentation of I Pk (Figure 1).…”

“…With this PMA exposure paradigm, small, significant increases in I Pk , with no significant increases in I SS were observed (Popp et al, 2008a). Thus, based on the results from Machu et al, 2006, we incorporated experiments in which CGCs were treated with PMA at physiological temperatures (37° C) and observed robust and significant increases in both I Pk and I SS (Popp et al, 2008a) and have continued to use this exposure paradigm to assess PMA-mediated alterations in I NMDA (Reneau et al, 2009). PMA was diluted and applied in conditioned feeding media.…”

“…Dishes were placed on the stage of a Nikon Eclipse TE2000-U inverted microscope (NIKON Inc., Melville, NY). Detailed methods have been previously published (Popp et al, 1999; Popp et al, 2008a; Reneau et al, 2009). Briefly, all currents were recorded with an Axopatch 200B amplifier (Molecular Devices, Sunnyvale, CA) at −60 mV holding potential.…”

“…Specifically, PMA-mediated alterations in NMDAR function of receptors expressed in mammalian cells (cultured CGCs), are significantly greater when PMA treatment occurs at a mammalian physiological temperature of 37° C (Popp et al, 2008a). Lastly, we have identified that augmentation of NMDAR function of receptors contained in primary cultured CGCs between 13 and 15 days in vitro (DIV), a time in culture in which the physiologically relevant NR2ANR2B NMDARs are present, is calcium dependent and is mediated by activation of PKCα and no other PKC isoform (Reneau et al, 2009). …”

Bryostatin-1 promotes long-term potentiation via activation of PKCα and PKCε in the hippocampus

“…In addition, PKC potentiates NMDA receptor function via phosphorylation (MacDonald et al, 2001). Activation of PKC by phorbol 12-myristate 13-acetate (PMA) shows that PMA-enhanced function of NMDA receptors expressed in primary cultured cerebellar granule cells is mediated by activation of PKCa (Reneau et al, 2009). Activation of PKCa by galantamine enhances NMDAR functionality via phosphorylation of Ser-896 of the NMDAR NR1 subunit (Tingley et al, 1997).…”