Related Experiment Videos
Phorbol ester stimulation of phosphatidylcholine synthesis in four cultured neural cell lines: correlations with
S A Sproull1, S C Morash, D M Byers
1Department of Pediatrics, Dalhousie University, Halifax, Nova Scotia, Canada.
Abstract:
Phosphatidylcholine (PtdCho) can provide lipid second messengers involved in signal transduction pathways. As a measure of phospholipid turnover in response to extracellular stimulation, we investigated differential enhancement of [3H]choline incorporation into PtdCho by phorbol esters. In C6 rat glioma and SK-N-SH human neuroblastoma cells, [3H]PtdCho synthesis was 2-4 fold stimulated by beta-12-O-tetradecanoylphorbol-13-acetate (beta-TPA) when [3H]choline was incubated simultaneously with, or 15 min prior to, beta-TPA treatment. By contrast, in N1E-115 mouse and SK-N-MC human neuroblastoma cells, phorbol esters had no appreciable effect on [3H]choline incorporation; however, in all cells, 200 microM oleic acid enhanced PtdCho synthesis, indicating a stimulable process. Alterations by thymeleatoxin (TMT), an activator of conventional PKC isoforms (alpha, beta and gamma), were similar to beta-TPA. We investigated whether expression of specific PKC isoforms might correlate with these effects of phorbol esters on PtdCho synthesis. All cell lines bound phorbol esters, had PKC activity that was translocated by phorbol esters and differentially expressed isoforms of PKC. Northern and western blot analyses, using specific cDNA and antibodies for PKC-alpha, -beta, -gamma, -delta, -epsilon, and -zeta, revealed that expression of alpha-isoform predominated in C6 and SK-N-SH cells. In contrast, TPA-responsive beta-isoform predominated in SK-N-MC cells. gamma-PKC was not detected in any cells and only in C6 cells was PKC-delta present and translocated by beta-TPA treatment. PKC-epsilon was not detected in SK-N-MC cell lines but translocated with TPA treatment in the other three cell lines. PKC-zeta was present in all cells but was unaltered by TPA treatment. Accordingly, stimulation of PtdCho turnover by phorbol esters correlated only with expression of PKC-alpha; presence of PKC-beta alone was insufficient for a TPA response.