AJP - Cell Physiology, Vol 260, Issue 1 C143-C150, Copyright © 1991 by American Physiological Society

ARTICLES

Effects of adenosine on cAMP production and cytosolic Ca2+ in cultured rabbit medullary thick limb cells

M. A. Burnatowska-Hledin and W. S. Spielman
Department of Physiology, Michigan State University, East Lansing 48824.

The cellular signaling mechanism of adenosine action has been studied in highly purified populations of cultured cells from the rabbit medullary thick ascending limb of Henle's loop (MTAL). The effects of specific adenosine-receptor agonists 5'(N-ethylcarboxamido)adenosine (NECA; A2) and N6-cyclohexyladenosine (CHA; A1) on basal and hormone-stimulated adenosine 3',5'-cyclic monophosphate (cAMP) production, cytosolic free calcium concentration ([Ca2+]f), and formation of inositol phosphates were examined. Production of cAMP was stimulated by high doses of NECA and was inhibited by low doses of CHA. The inhibitory effect of CHA was observed in cells in which cAMP production was first stimulated with vasopressin, isoproterenol, prostaglandin E2 (10(-6) M), or calcitonin (100 ng/ml) and was abolished by pretreating the cells with pertussis toxin (PT) for 12-20 h. A highly selective adenosine A1 antagonist, 8-cyclopentyl-1,3-dipropylxanthine (CPX), also abolished the inhibitory effect of CHA. Both NECA and CHA induced a rapid (10 s) and transient increase in [Ca2+]f, and this was associated with an increased inositol trisphosphate (IP3) production. Single-cell [Ca2+]f measurements indicated that all MTAL cells responded to CHA. The removal of extracellular Ca2+ failed to inhibit these responses. Pretreatment with PT or administration of CPX abolished both the increase in [Ca2+]f and the formation of IP3 occurring in response to CHA and NECA. Our results suggest that both adenylate cyclase-coupled inhibitory (A1) and stimulatory (A2) adenosine receptors are present in pure populations of cultured MTAL cells. Moreover, activation of an adenosine receptor coupled to a PT substrate results in the increased production of inositol phosphate and elevation of [Ca2+]f.

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