Adenosine-sensitive phosphoinositide turnover in a newly established renal cell line

To aid in characterizing adenosine receptors in renal cells, primary cultures of rabbit cortical collecting tubule (RCCT) cells were infected with an adenovirus 12-simian virus 40 hybrid, resulting in a continuous cell line. The cells, designated RCCT-28A, retained their epithelial morphology and reacted with a monoclonal antibody specific for rabbit collecting tubule. Adenosine 3',5'-cyclic monophosphate (cAMP) accumulation was stimulated by vasopresin (AVP), isoproterenol, prostaglandin E₂ (PGE₂), calcitonin, parathyroid hormone, and a potent adenosine A₁- and A₂-receptor agonist, 5'-N-ethylcarboxamidoadenosine (NECA). A more selective adenosine A₁-receptor agonist, N⁶-cyclohexyladenosine (CHA) inhibited basal and AVP-stimulated cAMP accumulation. Cytosolic free calcium was transiently elevated by bradykinin, PGE₂, NECA, and CHA. To examine the mechanism by which adenosine analogues increase intracellular free calcium, phosphoinositide (PI) turnover was assessed in the 28A cells after labeling with myo-[³H]inositol. NECA and CHA increased [³H]inositol phosphate formation with an approximate half-maximal effective concentration of 0.1 μM for both analogues. The increase in PI turnover was blocked by the selective adenosine A₁-receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine and pretreatment of the 28A cells with pertussis toxin. These results suggest that adenosine analogues increase cytosolic free calcium by stimulating PI turnover.