THE EFFECTS OF ELEVATED CYCLIC-AMP LEVELS ON HISTAMINE-H1-RECEPTOR-STIMULATED INOSITOL PHOSPHOLIPID HYDROLYSIS AND CALCIUM MOBILIZATION IN THE SMOOTH-MUSCLE CELL-LINE DDT1MF-2

The effects of raising cyclic AMP levels, by forskolin stimulation, beta-adrcnoceptor activation or cyclic AMP phosphodiesterase inhibition, on inositol phospholipid hydrolysis and increases in intracellular free [Ca2+] ([Ca2+]i) elicited by a range of agonists have been investigated in the hamster vas deferens smooth-muscle cell line DDTMF-2. Isoprenaline {log [EC50 (M)] = -7.7+/-0.2}, forskolin and the type IV cyclic AMP phosphodiesterase inhibitor rolipram elicited significant increases in the accumulation of cyclic [H-3]AMP. Pretreatment with forskolin (10 muM) attenuated histamine (100 muM)- and N6-cyclopentyladenosine (CPA; 300 nM)-induced release of intracellular Ca2+, observed when cells are stimulated in Ca2+-free buffer containing 0.1 mM EGTA. Forskolin had no effect on ATP (100 muM)- or bradykinin (1 muM)-stimulated release of intracellular Ca2+. Histamine-induced intracellular Ca2+ release was also inhibited by pretreatment with rolipram (100 muM) or the membrane-permeant cyclic AMP analogue (S(p))-adenosine 3',5'-monophosphothioate (100 muM). Isoprenaline (1 muM) pretreatment (in the presence of 10 M rolipram, a concentration which on its own did not decrease the histamine response) attenuated histamine-induced intracellular Ca2+ release. Forskolin inhibited histamine (100 muM)- and CPA (100 nM) stimulated accumulation of [H-3]-inositol phosphates, but was without effect on ATP or bradykinin responses. Addition of forskolin (in the presence of 100 muM rolipram) after the cells had been stimulated with histamine (in experiments initiated in Ca2+-free buffer) inhibited the rise in [Ca2+], observed when extracellular Ca2+ (2 mM) was re-applied (owing to receptor-mediated Ca2+ influx). Finally, the refilling of intracellular Ca2+ stores (after receptor-mediated Ca2+ influx is blocked by mepyramine) can be demonstrated in the presence of raised cyclic AMP levels.