Adenosine A1 stimulation activates delta-protein kinase C in rat ventricular myocytes.
By making use of immunoblotting and immunocytochemical analysis, we explored whether stimulation of adenosine A1 receptors would promote the activation of delta-protein kinase C (delta-PKC) immunolabeled with a polyclonal antibody. Immunoblot analysis of Triton X-100-soluble cell membrane and cytosolic fractions revealed the presence of a specific 75-kD band reactive to the delta-PKC polyclonal antibody. In freshly isolated rat cardiac myocytes, 28% of the total immunoreactive delta-PKC was associated with the membrane fraction, whereas 72% was associated with the soluble fraction. Under stimulation with the tumor-promoting phorbol 12-myristate 13-acetate (PMA, 50 nmol/L) used as a positive control, delta-PKC translocated to the cell membrane, with the membrane fraction representing 88% and the cytosolic fraction representing 12% of the total immunoreactive delta-PKC. Transverse optical sections performed with confocal laser microscopy showed that immunostaining with anti-delta-PKC antibody was distributed in the cytosol membrane under PMA stimulation. In the membrane fraction of cells pretreated with adenosine (100 mumol/L) or with the adenosine A1 agonist (--)-N6-(2-phenylisopropyl)-adenosine (R-PIA, 1 mumol/L), the 75-kD band corresponding to delta-PKC increased by 57% and 66%, respectively, when compared with nonstimulated cells processed under the same experimental conditions. In cells exposed to either of the purine agonists, specific fluorescence staining decorated the cell membrane, a pattern that was not observed in control cells. Activation of membrane delta-PKC produced either by adenosine itself or by its analogue R-PIA was fully antagonized by the specific A1 antagonist 8-cyclopentyl-1,3-dipropylxanthine (1 mumol/L). From these data, we conclude that adenosine A1 stimulation activates delta-PKC in freshly isolated rat ventricular myocytes.
Henry
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Laboratoire de Physiopathologie et de Pharmacologie, Cellulaires et Moléculaires, Hôpital G & R Laënnec, Nantes, France.
Demolombe
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S
Pucéat
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Escande
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D
D000241:Adenosine / Q000378:metabolism* / Q000494:pharmacology; D000818:Animals; D002462:Cell Membrane / Q000201:enzymology; D002478:Cells, Cultured; D004789:Enzyme Activation; D006352:Heart Ventricles / Q000378:metabolism*; D007527:Isoenzymes / Q000378:metabolism*; D018613:Microscopy, Confocal; D011493:Protein Kinase C / Q000378:metabolism*; D051745:Protein Kinase C-delta; D051381:Rats; D018047:Receptors, Purinergic P1 / Q000378:metabolism*
D007527:Isoenzymes; D018047:Receptors, Purinergic P1; C497260:Prkcd protein, rat; D011493:Protein Kinase C; D051745:Protein Kinase C-delta; D000241:Adenosine