CROSS-TALK BETWEEN MUSCARINIC-RECEPTOR AND ADENOSINE-RECEPTOR SIGNALING IN THE REGULATION OF CYTOSOLIC-FREE CA2-SECRETION( AND INSULIN)

The effects of A1-adenosine-receptor occupation on Ca2+ handling in th e insulin-secreting RINm5F cell line were investigated. The selective A1-agonist N6-cyclopentyladenosine (CPA) had no effect itself on the c ytosolic free Ca2+ concentration in cells loaded with Fura 2. However, CPA (1) attenuated the rise due to activation of voltage-gated Ca2+ c hannels with Bay K 8644, and (2) caused a secondary increase (EC50 app rox. 300 nM) if added after the primary Ca2+-mobilizing agonists vasop ressin or carbamoylcholine (carbachol). Prior addition of CPA (10 muM) also potentiated (by approx. 20%) the subsequent Ca2+ peak due to max imal (100 muM) carbachol, but did not alter the EC50 of the carbachol response. Detailed analysis of the secondary rise in Ca2+ revealed fur ther features. First, it was due to mobilization from intracellular st ores, since it persisted in the absence of extracellular Ca2+. Second, it was associated with a rapid (5-15 s) increase in phospholipase C ( PLC) activity, as measured by h.p.l.c. analysis of Ins(1,4,5)P3. This increase was only apparent after prior stimulation with carbachol. Thi rd, and unlike the response to carbachol, it was mediated by a pertuss is-toxin-sensitive G-protein. Fourth, it was not secondary to a decrea se in cyclic AMP. Fifth, it was absolutely dependent on continued occu pation of the primary receptor, since it was abolished if carbachol wa s displaced with the antagonist atropine. This implies a dynamic cross -talk between the two receptor coupling systems, rather than covalent modification as a result of the prior activation of PLC. Sixth, it was not associated with any desensitization of the ability of CPA to inhi bit forskolin-stimulated adenylate cyclase activity. Glyceraldehyde (1 0 mM)-induced insulin secretion was also potently inhibited by CPA > 1 0 nM, but the secretory response to 100 muM carbachol was unaffected u p to 10 muM. The results suggest that, in vivo, adenosine would inhibi t secretion due to carbohydrate nutrients much more effectively than t hat due to stimuli which activate PLC.