Phorbol ester-mediated stimulation of phospholipase D activity in sciatic nerve from normal and diabetic rats.
Evidence for the presence of phospholipase D activity in sciatic nerve was obtained by incubation of 32P-prelabeled nerve segments in the presence of ethanol and measurement of [32P]phosphatidylethanol (PEth) formation expressed as a fraction of total phospholipid radioactivity. PEth synthesis was enhanced with increasing concentrations of ethanol (100 mM-2 M). 4-beta-Phorbol dibutyrate (100 nM-1 microM) stimulated PEth formation up to twofold in a time- and dose-dependent manner. The stimulatory effect evoked by 100 nM phorbol ester was completely abolished by Ro 31-8220 (compound 3), a selective protein kinase C inhibitor. Efforts to identify the phospholipid precursor of PEth were unsuccessful, suggesting this product arises from a small discrete precursor pool. On subcellular fractionation of nerve, the ratio of basal and 4-beta-phorbol dibutyrate-stimulated phospholipase D activity recovered in a myelin-enriched fraction, compared with a nonmyelin fraction, was 0.5 when results are expressed as a percentage of total phospholipid radioactivity. This ratio rises to 1.2 if the results are calculated assuming only phosphatidylcholine and phosphatidylethanolamine are potential precursors. The results suggest that myelin is a major locus of phospholipase D activity. Nerve from streptozotocin-induced diabetic and control animals displayed the same basal phospholipase D activity, but the enzyme in diabetic nerve was stimulated to a greater extent by a suboptimal concentration of 4-beta-phorbol dibutyrate. These results support the conclusion that protein kinase C modulates phospholipase D activity in nerve and suggest that in diabetic nerve the enzyme activation mechanism may possess increased sensitivity.
Eggen
B J
BJ
Department of Biochemical and Biophysical Sciences, University of Houston, Texas 77204-5934.
Eichberg
J
J
D000818:Animals; D003921:Diabetes Mellitus, Experimental / Q000201:enzymology*; D004305:Dose-Response Relationship, Drug; D020404:Glycerophospholipids; D007211:Indoles / Q000494:pharmacology; D008297:Male; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology*; D010712:Phosphatidic Acids / Q000378:metabolism; D010739:Phospholipase D / Q000378:metabolism*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D051381:Rats; D012016:Reference Values; D012584:Sciatic Nerve / Q000201:enzymology*; D013347:Subcellular Fractions / Q000201:enzymology
D020404:Glycerophospholipids; D007211:Indoles; D010712:Phosphatidic Acids; C051521:phosphatidylethanol; D015240:Phorbol 12,13-Dibutyrate; D011493:Protein Kinase C; D010739:Phospholipase D; C064758:Ro 31-8220