In vitro activity: ACAP 6-38 is a novel and potent PACAP (pituitary adenylate cyclase-activating polypeptide) non-stimulating competitive antagonist with an IC50 value of 2 nM. It acts as a functional CARTp antagonist in vivo. It also blocks the CARTp-induced phosphorylation of ERK in differentiated cells. Intravesical administration of the PAC1 receptor antagonist, PACAP(6-38) (300 nM), significantly (p ≤ 0.01) increased intercontraction interval (2.0-fold) and void volume (2.5-fold) in NGF-OE mice. Intravesical instillation of PACAP(6-38) also decreased baseline bladder pressure in NGF-OE mice. PACAP(6-38) had no effects on bladder function in WT mice. Intravesical administration of PACAP(6-38) (300 nM) significantly (p ≤ 0.01) reduced pelvic sensitivity in NGF-OE mice but was without effect in WT mice. PACAP/receptor signaling contributes to the increased voiding frequency and pelvic sensitivity observed in NGF-OE mice.

Kinase Assay: PACAP (6-38), human, ovine, rat is a potent PACAP receptor antagonist with IC50s of 30, 600, and 40 nM for PACAP type I receptor, PACAP type II receptor VIP1, and PACAP type II receptor VIP2, respectively.

Cell Assay: Cells are incubated for 4 h with 300 nM PACAP-38 in the absence or presence of the PACAP antagonist PACAP (6-38), medium is collected, and proteins are separated by SDS/PAGE and blotted onto PVDF membranes. For APPsα detection, membranes are probed with the antibody 6E10 followed by an anti-mouse antibody labeled with HRP, detection is performed with the ECL plus system.