Growth-dependent accumulation of monoalkylglycerol in Madin-Darby canine kidney cells. Evidence for a role in the regulation of protein kinase C.
1-O-Alkyl-sn-glycerol (alkylglycerol) forms the backbone of complex ether-linked glycerolipids, including biologically active lipids such as platelet-activating factor. Synthetic alkylglycerol itself possesses several potent pharmacological activities and has been shown to inhibit protein kinase C (PKC) in vitro. In spite of these properties, free alkylglycerol has been regarded only as a potential product of the inflammatory degradation of complex ether lipids rather than a natural cell constituent. To explore the possibility that endogenous alkylglycerol functions as a physiological regulator in normal cells, we measured its content, along with related monoglycerides and diglycerides, by high performance liquid chromatography and gas-liquid chromatography in Madin-Darby canine kidney (MDCK) cells. The content of free alkylglycerol increased up to 20-fold during the growth of MDCK cell cultures to a confluent density. The increase was greatest during the log phase of growth, in which the content of alkylglycerol rose from 6.0 +/- 1.3 nmol/10(8) cells in preconfluent cultures to 23.6 +/- 3.4 nmol/10(8) cells in confluent cultures. Analysis of the molecular species of alkylglycerol showed that the higher content in quiescent MDCK cells was due primarily to an increase in 1-O-octadecyl-sn-glycerol. In contrast, the levels of monoacylglycerol and the PKC activator diacylglycerol were lower in confluent, quiescent cultures than in preconfluent, proliferating cultures. A similar pattern of changes in the monoglyceride and diglyceride content was observed in interleukin-3-dependent CFTL-12 mast cells when cell proliferation was blocked by growth factor withdrawal. Growth of MDCK cells to a confluent density resulted in a decrease in particulate PKC enzyme activity to a level that was only 6% of that in proliferating cells. To explore whether the accumulation of cellular alkylglycerol contributes to growth-dependent changes in PKC activity, we examined the effects of adding alkylglycerol to the activity and subcellular distribution of the enzyme in MDCK cells. Treatment of cells with 1-O-dodecyl-sn-glycerol resulted in a decrease in the activity of membrane-associated PKC activity and inhibited 12-O-tetradecanoylphorbol-13-acetate-stimulated translocation of PKC from the cytosol to the membrane fraction. Alkylglycerol was also shown to inhibit the activity of purified PKC in vitro when present at levels similar to that of the diacylglycerol activator. We propose that the accumulation of alkylglycerol during the growth of MDCK cells to a confluent density contributes to the decrease in PKC activity. The control of cellular alkylglycerol levels may be a novel mechanism for the regulation of cellular physiology.
Warne
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TR
Department of Biochemistry, James H. Quillen College of Medicine, East Tennessee State University, Johnson City 37614, USA.
Buchanan
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FG
Robinson
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M
D000215:Acylation; D000478:Alkylation; D000818:Animals; D002455:Cell Division; D002460:Cell Line; D002784:Cholesterol / Q000378:metabolism; D004075:Diglycerides / Q000302:isolation & purification / Q000378:metabolism; D004285:Dogs; D005989:Glycerides / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D006706:Homeostasis; D007668:Kidney; D007700:Kinetics; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*
D004075:Diglycerides; D005989:Glycerides; D002784:Cholesterol; D011493:Protein Kinase C