Several evidence suggest that pituitary adenylate cyclase activating polypeptides (PACAP-38 and -27) could function as hypophysiotropic factors. Both peptides interact with either the type I receptor, which preferentially binds the two PACAPs and has a much lower affinity for vasoactive intestinal polypeptide (VIP) or the type II receptor, which binds the two PACAPs and VIP with a nearly equal affinity. In addition to the stimulation of adenylyl cyclase (AC) activity, in different cell types PACAP causes an increase of cytosolic calcium levels ([Ca2+]i), consequent to phospholipase-C activation. In the present study, we investigated the effect of PACAP on cAMP formation and [Ca2+]i levels in 16 human nonfunctioning pituitary adenomas (NFPA). PACAP-38 increased cAMP formation in all tumors; the peptide stimulated either AC activity in membrane preparations from 26 +/- 10 to 214 +/- 179 pmol/mg prot/min (P < 0.01) or cAMP efflux from 12 +/- 5.4 to 73.2 +/- 32 pmol/well (P < 0.01) in cultured cells. The effect, detectable at concentrations higher than 1-10 pM, was maximal at 0.1-10 nM. While PACAP-38 and PACAP-27 were nearly equally effective and potent, 100-fold higher concentrations of VIP were required to obtain similar AC activation. GHRH and CRH were ineffective in any NFPA. The PACAP effect was not antagonized by a VIP antagonist, while PACAP fragment 6-27 amide partially reduced the stimulatory effects of both PACAP-27 and VIP in 2 out of 3 tumors tested. PACAP-38 caused a [Ca2+]i rise in cells obtained from 7 NFPA (from 110 +/- 34 to 151 +/- 40 nM [Ca2+]i, P < 0.05) while in the remaining 7 the peptide was ineffective at any concentrations tested (from 1 nM to 10 microM). In the responsive tumors, PACAP-38 effect was not consequence of phospholipase-C activation since removal of extracellular Ca2+ as well as blockade of L-type Ca2+ channels by dihydropyridine antagonists abolished [Ca2+]i increase triggered by the peptide. These data indicate that PACAP is by far the most potent activator of cAMP formation in NFPA and suggest a possible modulatory action of this peptide on cell growth.