We have previously shown that both PACAP and VIP relax rat and guinea pig smooth muscle partly by formation of NO, generated in smooth muscle ceils. Furthermore we have provided evidence for the existence of at least two receptor-subtypes for PACAP. A common PACAP and VIP receptor, formerly designated as PACAP/VIP-2 receptor and a PACAP-specific apamin-sensitive receptor which represents probably the PACAP-1 receptor. The aim of the present study was to determine the signal-transduction pathways of PACAP and VIP mediating relaxation in human colonic muscle. Smooth muscle cells were isolated separately from circular (CM) and longitudinal (LM) layers of human colon. Relaxation of CCK-contracted ceils and formation of cAMP and cGMP induced by PACAP-38 and VIP were measured. PACAP-38 and VIP produced a concentration-dependent relaxation of CCK-contracted muscle ceils of both layers accompanied by an increase in cAMP (PACAP-38: CM 107.7-+25 %, LM 119.6-+29.8 %; VIP: CM 63.7-+9.8 %, LM 68.6 _+ 14.9 %; above basal levels) and cGMP (PACAP-38: CM 63.5 -+ 20 %, LM 60.7_+ 14.2 %; VIP: CM 54.1 _+8.9 %, LM 58.2_+ 11.5 % above basal levels). Relaxation and the increase in both cAMP and cGMP were not significantly modified by the NO-synthese inhibitor L-NNA (100 ~aM). Inhibition of guanylate cyclase by ODQ (! 0 laM) abolished the increase in cGMP and partly inhibited relaxation induced by both PACAP-38 and VIP. Relaxation elicited by PACAP-38 and VIP was partly inhibited by both the protein kinase A inhibitor H-89 (1 laM) and protein kinase G inhibitor KT-5823 (1 btM). The combination of the two types of inhibitors virtually abolished the peptide induced relaxation in both cell types. Apamin (1 laM) reduced significantly and exclusively the PACAP-induced relaxation and increase in cyclic nucleotides. Binding experiments using [125I]-VIP revealed 'identical displacement curves for PACAP-38 and VIP. In contrast, when using [I25I]-PACAP-27 as radioligand, the ability of VIP to inhibit binding of [125I]-PACAP-27 Was significantly diminished compared to PACAP-38. We conclude that PACAP-38 and VIP relax human longitudinal and circular colonic smooth muscle through stimulation of both cAMP and cGMP by NO independent pathways. The actions of PACAP and VIP are mediated through PACAP/VIP-I receptors. In contrast to VIP PACAP activates additionally distinct apamin-sensitive receptors that probably belong to the PACAP-1 receptor subtype.