Effect of P2Y agonists on adenosine transport in cultured chromaffin cells.
Adenosine transport in cultured chromaffin cells was inhibited by purinergic P2y-receptor agonists without significant changes in the affinity constant, the values being between 1 +/- 0.4 and 1.6 +/- 0.6 microM. The Vmax parameter was modified significantly, being 40 +/- 1.0, 26 +/- 5.0, 32 +/- 3.0, and 22 +/- 4.7 pmol/10(6) cells/min for control, adenosine-5'-O-(2-thiodiphosphate), 5'-adenylylimidodiphosphate, and P1,P4-di(adenosine-5'-) tetraphosphate (Ap4A) (100 microM for every effector), respectively. Ap4A, a physiological ligand for P2y receptors in chromaffin cells, showed the highest inhibitory effect (45%). This transport inhibition is explained by an increase in the cytosolic Ca2+ concentration ([Ca2+]i) and the activation of protein kinase C (PKC). Experiments of [Ca2+]i measurement with the fura-2 technique showed that P2y agonists, as well as bradykinin, were able to increase [Ca2+]i, this effect being independent of the presence of extracellular Ca2+. The peptide bradykinin, determined to be coupled to phosphatidylinositol hydrolysis and internal Ca2+ mobilization in chromaffin cells, exhibited a behavior similar to that of P2y agonists in adenosine transport inhibition (39%). P2y agonists and bradykinin increased PKC activity associated with the membrane fraction (about 50% increase in particulate PKC activity with respect to controls). The present studies suggest that adenosine transport is regulated by P2y-purinergic receptors mediated via Ca2+ mobilization and PKC activation.
Sen
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RP
Departamento de Bioquímica, Facultad de Veterinaria, Universidad Complutense, Madrid, Spain.
Delicado
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EG
Castro
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Miras-Portugal
M T
MT
D000241:Adenosine / Q000378:metabolism*; D000244:Adenosine Diphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology; D000266:Adenylyl Imidodiphosphate / Q000494:pharmacology*; D000313:Adrenal Medulla / Q000187:drug effects / Q000378:metabolism*; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D001920:Bradykinin / Q000494:pharmacology; D002217:Carbachol / Q000494:pharmacology; D002417:Cattle; D002478:Cells, Cultured; D015226:Dinucleoside Phosphates / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D007700:Kinetics; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism; D011983:Receptors, Purinergic / Q000187:drug effects / Q000502:physiology*; D013873:Thionucleotides / Q000494:pharmacology*
D015226:Dinucleoside Phosphates; D011983:Receptors, Purinergic; D013873:Thionucleotides; D000266:Adenylyl Imidodiphosphate; C030812:adenosine 5'-O-(2-thiodiphosphate); D015240:Phorbol 12,13-Dibutyrate; C020733:diadenosine tetraphosphate; D000244:Adenosine Diphosphate; D002217:Carbachol; D011493:Protein Kinase C; D000241:Adenosine; D001920:Bradykinin