Activation and desensitization of phospholipase D in intact rat hepatocytes.

G Moehren,L Gustavsson,J.B Hoek

doi:10.1016/s0021-9258(17)42189-2

G Moehren, L Gustavsson + Show 1 more

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https://doi.org/10.1016/s0021-9258(17)42189-2

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Abstract

Activation of phospholipase D (PLD) by receptor-coupled stimuli (vasopressin, ATP), phorbol esters, and Ca2+ ionophores was studied in isolated rat hepatocytes, double labeled with [3H]arachidonate and [14C]stearate. Phosphatidylethanol (Peth) was formed when cells were stimulated in the presence of ethanol. The effect of combinations of agonists was not additive, indicating that the same PLD isozyme(s) were activated. With all agonists, the 3H- and 14C-specific radioactivity in Peth was higher than in any of the main phospholipid classes. The 3H/14C ratios of Peth and phosphatidylcholine (PC) were identical and differed from other phospholipid classes, indicating that the predominant PLD substrate was a PC pool labeled preferentially with radioactive fatty acids. Ethanol (50-300 mM) decreased the initial rate of phosphatidic acid (PA) formation, but did not affect total PLD activity. Agonist-induced changes in steady state accumulation of PA or 1,2-diacylglycerol were also unaffected. A slow degradation of Peth (apparent t1/2 > 60 min) occurred after ethanol removal from cells prestimulated with vasopressin. The rate of degradation was unaffected by agonists that stimulate PLD. Thus, Peth formation is a suitable cumulative indicator for PLD activation in intact hepatocytes. Peth accumulation declined over a period of 5-20 min, depending on the agonist. The decline was not due to increased Peth degradation, or limitations in substrate supply to PLD, or enzyme inhibition by accumulated Peth. Instead, a homologous desensitization of PLD occurs with all agonists. This desensitization may involve the action of selective protein kinase C isozymes.

Highlights

Phosphatidylethanol(Peth)was formed whencells were protein [5, 6]
The abbreviations used are: PLD, phospholipase D; [Ca2+l, cytoof PLD activation in isolated hepatocytes and address the issolic free Ca2+concentration; CPT-CAMP,8-(4-~hlorophenylthio)adeno- sues raisedby its reaction with ethanol
through activationof protein kinaseC (TPA) + va1s.8opressin TPA + ATP TPA + A23187 Vasopressin + ATP Vasopressin + A23187 ATP + A23187 nmol I 1 0 6 cells 15 min Isolation and Incubation of Liver Cells-Hepatocytes were isolated from male Sprague-Dawley rats (200-300byg)collagenase perfusiona s of ethanol (300 mM)

Summary

To whom correspondence should be addressed

Dept. of Pathology The experiments reported in this and theaccompanying paand Cell Biology, Thomas Jefferson University, 1020 Locust St., Phila- per [22]were designed to further characterize different modes delphia, PA 19107. The abbreviations used are: PLD, phospholipase D; [Ca2+l,,,, cytoof PLD activation in isolated hepatocytes and address the issolic free Ca2+concentration; CPT-CAMP,8-(4-~hlorophenylthio)adeno- sues raisedby its reaction with ethanol. Hepatocytes creased the initial rate ofPA formation, but it did not affect total PLD activity and had littleffect on the changes in steady state levels ofPA and 1,2-DAG induced by different agonists. Accumulated Peth was metabolized only slowly, even after removing either ethanolor the stimulifor PLD activation. These characteristics make Petha suitable indicator for PLD activity in intact cells. Hepatocytes were incubated with agonists for 5 min, with ethanol (300 m) added 5 minpriortotheagonists.Pethformationinthe.

EXPERIMENTAL PROCEDURES

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