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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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Correspondence to Jos M. J. Lamers.

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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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Key words

  • cardiomyocytes
  • microsomes
  • phorbol ester
  • phosphatidylinositol 4,5-bisphosphate
  • phospholipase C
  • protein kinase C