PITUITARY ADENYLATE CYCLASE-ACTIVATING POLYPEPTIDE ACTIVATES DIFFERENT SIGNAL-TRANSDUCING MECHANISMS IN CULTURED CEREBELLAR GRANULE CELLS

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a novel 38-residue neuropeptide which stimulates adenylate cyclase activity in rat pituitary cells as well as in other neuronal and non-neuronal tis sues. In this study we have investigated whether PACAP27 and PACAP38 m ay stimulate either cyclic AMP accumulation or phosphoinositide format ion in cultured cerebellar granule cells. In cultures at 8 days of mat uration in vitro (DIV) a 15-min exposure to PACAP27 or PACAP38 equally promoted a concentration-dependent increase in intracellular cAMP con tent: the effect was significant at 15 nM and maximal between 10 and 1 00 nM, while VIP was 1,000-fold less potent in elevating cAMP levels. In the presence of 3-isobutyl-1-methylxanthine (200 mu M), stimulation by PACAP was present already at 0.1 nM and was maximal (6-fold increa se) at 1 nM. A rapid elevation in intracellular cAMP (about 80%) was o bserved within a 30-second exposure to 10 mu M PACAP38 or PACAP27; the maximal activity of PACAP was present between 15 and 30 min and progr essively declined at 60 min without reaching basal values. PACAP27 and PACAP38, but not VIP, were also able to stimulate inositol phospholip id hydrolysis: PACAP38 (EC(50): 0.16 nM) was 10-fold more potent than PACAP27 (EC(50): 2.1 nM) in stimulating [H-3]inositol phosphate format ion. The effect of PACAP was rapid: fractionation of [H-3]inositol pho sphates revealed that inositol trisphosphate and inositol bisphosphate increased earlier (within 20 s) than inositol monophosphate (within 6 0 s). Stimulation of inositol phospholipid hydrolysis by PACAP was not mediated through the activation of the adenylate cyclase-cAMP system because neither 10 mu M forskolin nor VIP (1-10 mu M) were able to aff ect inositol phospholipid turnover. Interestingly, while the cAMP resp onse was already present (and maximal) at 2 DIV, PACAP increased phosp hoinositide hydrolysis only at 4 DIV. Our results provide clear eviden ce for the presence of PACAP receptors linked to both the adenylate cy clase-cAMP system and the phosphoinositide turnover in cerebellar gran ule cells.