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Phorbol ester and the actions of phosphatidylinositol 4,5-bisphosphate specific phospholipase C and protein kinase C in microsomes prepared from cultured cardiomyocytes

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Abstract

Microsomes were prepared from cultured neonatal rat cardiomyocytes. Incubation of microsomes in buffer containing 5µM CaCl2, 5 mM cholate and 100 nM [3H-]Phosphatidylinosito14,5-bisphosphate (PtdIns(4,5) P2) resulted in the formation of [3H-]InsP 3. GTP-gamma-S (125 µM) stimulated the production of [3H-]InsP 3. Microsomes prepared from phorbol ester-treated (100 nM phorbol 12-myristate 13-acetate, PMA) cardiomyocytes showed decreased activities of basal as well as GTP-gamma-S-stimulated [3H-]Ptdlns(4,5)P 2 hydrolysis. In the microsomes a 15 kD protein was demonstrated to be the major substrate phosphorylated by intrinsic protein kinase C, which was activated by 0.5 mM Ca2+. Addition of phorbol ester (100 nM PMA) enhanced the 32P-incorporation into the 15 kD protein. Protein kinase C, purified from rat brain, in the presence of Ca2+, diglyceride, and phosphatidylserine did not change the phosphorylation pattern any further. In conclusion, it was shown that phorbol ester pretreatment of neonatal rat cardiomyocytes reduces microsomel GTP-gamma-S-stimulated Ptdlns(4,5)P 2-specific phospholipase C activity, as estimated with exogenous substrate, and that in cardiomyocyte microsomes phorbol ester activates protein kinase C-induced 15 kD protein phosphorylation. The results indicate that phorbol ester may down-regulate α-adrenoceptor mediated Ptdlns(4,5)P 2 hydrolysis by activation of protein kinase C-induced 15 kD protein phosphorylation.

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Abbreviations

ATP:

Adenosine 5′-Trphosphate

CSU:

Catalytic Subunit of cyclic AMP-dependent protein kinase

DG:

Diacylglycerol

DMSO:

Dimethylsulfoxide

DTT:

DL-dithiothreitol

EDTA:

Ethylenedinitrilotetraacetic Acid

EGTA:

Ethyleneglycol-0,0′-bis(aminoethyl)-N,N,N′,N′,-tetraacetic acid

GTP-gamma-S:

Guanosine 5′-O-(3-thiotriphosphate)

HPTLC:

High Performance Thin Layer Chromatography

InsP 3 :

Inositol monophosphate

InsP 2 :

Inositol bisphosphate

InsP 3 :

Inositol trisphosphate

MES:

2-Morpholinoethanesulfonic acid

MOPS:

3-[N-Morpholino]Propanesulfonic acid

PAGE:

Polyacrylamide-gel Electrophoresis

PKC:

Protein Kinase C

PLase C:

Phospholipase C

PMA:

Phorbol 12-Myristate 13-Acetate

PMSF:

Phenylmethylsulfonyl Fluoride

PtdSer:

Phosphatidylserine

PtdIns:

Phosphatidyl inositol

PT:

Pertussis Toxin

Ptdlns(4)P :

Phosphatidylinositol 4-monophosphate

Ptdlns:

(4,5)PZ-Phosphatidylinositol4,5-bisphosphate

SDS-Sodium Dodecyl Sulfate:

Tris-Tris(hydroxymethyl) aminomethane

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Author notes

  1. Johanna T A. Meij

    Present address: Div. of Cardiovascular Sciences, St. Boniface G.H. Research Centre, and Dept. of Anatomy, University of Manitoba, Winnipeg, Canada

Authors and Affiliations

  1. Department of Biochemistry I, Erasmus University Rotterdam, Rotterdam, The Netherlands

    Johanna T A. Meij, Karel Bezstarosti & Jos M. J. Lamers

  2. Division of Cardiovascular Sciences, St. Boniface G. H. Research Centre, and Department of Anatomy, University of Manitoba, Winnipeg, Canada

    Vincenzo Panagia

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Meij, J.T.A., Bezstarosti, K., Panagia, V. et al. Phorbol ester and the actions of phosphatidylinositol 4,5-bisphosphate specific phospholipase C and protein kinase C in microsomes prepared from cultured cardiomyocytes. Mol Cell Biochem 105, 37–47 (1991). https://doi.org/10.1007/BF00230373

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