Characterization of protein kinase C isoforms in primary cultured cerebellar granule cells

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

“…This combination of NMDA receptor subunits is observed in the neonatal rat cerebellum (Farrant et al, 1994;Monyer et al, 1994). We have further confirmed through the use of PKC isoform-specific antibodies that nine of the 10 isoforms of this phospholipid-dependent kinase are expressed in CGCs during this time in vitro (Popp et al, 2006). PKC isoforms have been grouped according to sequence homology, substrate preference, and activators (Newton, 2001;Ohno and Nishizuka, 2002).…”

“…Stock PMA in dimethyl sulfoxide (DMSO) solutions was diluted to 100 nM (0.001% DMSO) and applied in conditioned cell media. We have reported previously that this concentration of DMSO does not result in translocation of PKC immunoreactivity for up to 18 to 20 h of exposure (overnight exposure) (Popp et al, 2006). NMDA-induced currents from receptors contained in 13 to 15 DIV CGCs when exposed to this concentration of DMSO (overnight) do not differ from NMDA-induced currents from control cells and are enhanced when challenged with 30-min, 100 nM PMA treatment at 37°C the next day (Popp et al, 2008a), indicating that increases in I NMDA are due to PMA and not the vehicle DMSO.…”

“…Antibodies directed against the ␣7-nicotinic acetylcholine (nACh) receptor have been introduced into the cell during whole-cell patch-clamp experiments to block acetylcholine-evoked currents in superior cervical ganglion neurons (Cuevas et al, 2000). We used a similar technique to confirm that PMA potentiation of I Pk was indeed mediated by a classical PKC isoform by introducing PKC isoform-specific antibodies (Popp et al, 2006) into the cell during whole-cell recordings. When antibodies directed against the novel, noncalcium-dependent PKC isoforms ␦, ε, and were introduced into the cell via the patch electrode, time-dependent potentiation of I Pk by PMA was still observed (Fig.…”

“…3, E and F). PKC␤I is a classical PKC isoform expressed in our CGCs during the first 2 weeks in culture (Popp et al, 2006). The method of intracellular dialysis with anti-PKC␤I to assess PMA potentiation of I NMDA could not be used because this antibody is not specific for PKC␤I and exhibits cross-reactivity with PKC␣ (Popp et al, 2006).…”

“…Dishes receiving an overnight PMA treatment were treated and returned to the incubator until the next day at which time they received a second 100 nM PMA dose for 30 min (ON PMA challenge). This treatment results in the down-regulation of PMA-sensitive PKC isoforms (Matthies et al, 1987;Popp et al, 2006Popp et al, , 2008a. Alternatively, dishes not receiving an overnight PMA treatment were treated once with 100 nM PMA and returned to the incubator for 12.5 min.…”