Affiliation:
1. From the Laboratoire de Physiopathologie et de Pharmacologie Cellulaires et Moléculaires, Hôpital G & R Laënnec, Nantes, France; the Département de Cardiologie, Hôpital Broussais, Paris, France; and INSERM U-390, Hôpital Arnaud de Villeneuve, Montpellier, France.
Abstract
Abstract
By making use of immunoblotting and immunocytochemical analysis, we explored whether stimulation of adenosine A
1
receptors would promote the activation of δ-protein kinase C (δ-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 δ-PKC polyclonal antibody. In freshly isolated rat cardiac myocytes, 28% of the total immunoreactive δ-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, 500 nmol/L) used as a positive control, δ-PKC translocated to the cell membrane, with the membrane fraction representing 88% and the cytosolic fraction representing 12% of the total immunoreactive δ-PKC. Transverse optical sections performed with confocal laser microscopy showed that immunostaining with anti–δ-PKC antibody was distributed in the cytosol of unstimulated cells but accumulated in the cell membrane under PMA stimulation. In the membrane fraction of cells pretreated with adenosine (100 μmol/L) or with the adenosine A
1
agonist (−)-
N
6
-(2-phenylisopropyl)-adenosine (R-PIA, 1 μmol/L), the 75-kD band corresponding to δ-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 δ-PKC produced either by adenosine itself or by its analogue R-PIA was fully antagonized by the specific A
1
antagonist 8-cyclopentyl-1,3-dipropylxanthine (1 μmol/L). From these data, we conclude that adenosine A
1
stimulation activates δ-PKC in freshly isolated rat ventricular myocytes.
Publisher
Ovid Technologies (Wolters Kluwer Health)
Subject
Cardiology and Cardiovascular Medicine,Physiology
Reference28 articles.
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