Thyrotropin-releasing hormone and phorbol esters induce phosphatidylcholine synthesis in GH3 pituitary cells. Evidence for stimulation via protein kinase C.

R N Kolesnick

doi:10.1016/s0021-9258(18)47827-1

Abstract

Phorbol esters have been shown to stimulate phosphatidylcholine synthesis via the CDP-choline pathway. The present study compares the effects of phorbol esters and thyrotropin-releasing hormone (TRH) on phosphatidylcholine metabolism in GH3 pituitary cells. In a previous study (Kolesnick, R.N., and Paley, A.E. (1987) J. Biol. Chem. 262, 9204-9210), the potent phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA) induced time- and concentration-dependent incorporation of 32Pi and [3H]choline into phosphatidylcholine in short-term labeling experiments. In this study, TPA is shown to activate choline-phosphate cytidylyltransferase (EC 2.7.7.15), the regulatory enzyme of the CDP-choline pathway, by stimulating redistribution of the inactive cytosolic form of the enzyme to the membrane. Redistribution was quantitative. TPA reduced cytosolic activity from 3.5 +/- 0.4 to 1.5 +/- 0.3 nmol . min-1 x 10(7) cells-1 and enhanced particulate activity from 2.5 +/- 0.4 to 4.9 +/- 0.6 nmol . min-1 x 10(7) cells-1. TRH also stimulated time- and concentration-dependent 32Pi and [3H]choline incorporation into phosphatidylcholine. An increase was detectable after 5 min; and after 30 min, the levels were 164 +/- 9 and 150 +/- 11% of control, respectively; EC50 congruent to 2 X 10(-10) M TRH. These events correlated directly with TRH-induced 32Pi incorporation into phosphatidylcholine. TRH also stimulated redistribution of cytidylyl-transferase specific activity. TRH reduced cytosolic activity 45% and enhanced particulate activity 51%. Neither TRH nor TPA stimulated phosphatidylcholine degradation. In cells down-modulated for protein kinase C (Ca2+/phospholipid-dependent protein kinase), the effects of TPA and TRH on 32Pi incorporation into phosphatidylcholine were abolished. However, TRH-induced incorporation into phosphatidylinositol still occurred. These studies provide evidence that hormones may regulate phosphatidylcholine metabolism via the protein kinase C pathway.

Highlights

From the Division of Endocrinologyand Metabolism, Department of Medicine, MemorialSloan-Kettering Cancer Center, New York, New York 10021
The present study compares the eofffpechtosrbol cytidylyltransferase (EC 2.7.7.15), the regulatory enzyme for esters and thyrotropin-releasing hormone(TRH)on this pathway (I), and involved redistribution of an inactive phosphatidylcholine metabolism in GH3pituitary cells. cytosolic formof the enzyme to the membrane
Materials-Phorbol esters, TRH, and phospholipid and choline standards were purchased from Sigma.1,2-Diglyceridesobtained from egg lecithin were from Serdary Research Laboratories, Inc., London, Phorbol esters stimulate phosphatidylcholine metabolism in various cell types and this event correlates directly with other phorbol ester-induced biologic processes.The exact mechanismof stimulated phosphatidylcho

Summary

EVIDENCEFORSTIMULATION VIA PROTEINKINASE C*

The present study compares the eofffpechtosrbol cytidylyltransferase (EC 2.7.7.15), the regulatory enzyme for esters and thyrotropin-releasing hormone(TRH)on this pathway (I), and involved redistribution of an inactive phosphatidylcholine metabolism in GH3pituitary cells. In cells down-modulated for protein kinase C (lo),the effects of phorbol esters and TRH on phosphatidylcholine synthesis were abolished. These studiesprovide the firstevidence for receptor-mediated occurred.Thesestudiesprovideevidence thathor- activation of the CDP-choline pathwayby protein kinase C. mones may regulate phosphatidylcholine metabolism via the protein kinaseC pathway. Materials-Phorbol esters, TRH, and phospholipid and choline standards were purchased from Sigma.1,2-Diglyceridesobtained from egg lecithin were from Serdary Research Laboratories, Inc., London, Phorbol esters stimulate phosphatidylcholine metabolism in various cell types and this event correlates directly with other phorbol ester-induced biologic processes (for review see Ref. 1).The exact mechanismof stimulated phosphatidylcho-. Apparent K , and V, for enzymes of the major pathway for phosphatidylcholine biosynthesis

Choline phosphotransferase

RESULTS

Particulate measUurnambleeasurableCytosol

Choline phosphotransferased

DISCUSSION