Figure 6.

PACAP is an autocrine factor. (a) At 24 h, 85% of precursors expressed intense PACAP immunoreactivty localized to the cytoplasm (arrow). Staining was blocked by antibody preincubation with PACAP but not related peptides, secretin and peptide–His–Ileu (not shown). (Arrowhead = negative cell) (5 expts). (b) At 24 h, 64% of cells exhibited immunoreactivity with a PACAP type I receptor antiserum (2 expts). (c and d) Detection of PACAP-responsive precursors by examining P-CREB immunoreactivity in 24 h cultures. Fifteen minutes after PACAP exposure, nuclear P-CREB staining was detected in 62 ± 3% of precursors (d) whereas only 2.3 ± 0.3% of cells exhibited signals in the vehicle-treated control (c) (5 expts). (e) Blockade of endogenous PACAP function increases [³H]dT incorporation. Cells were incubated for 24 h in control medium or in medium containing peptide antagonist PACAP_6–38 (100 nM). Antagonist-induced stimulation was reversed by coincubation with PACAP (3 expts); n = 11; ∗, P < 0.004. (f) PACAP-neutralizing antiserum increased [³H]dT incorporation. Precursors were incubated in control medium containing nonimmune rabbit serum or medium containing PACAP antiserum (1:3000) for 24 h (2 expts); n = 8; ∗, P < 0.016. (g) In serum containing medium, cell growth was observed at 24 h only in the presence of PACAP antagonist (100 nM) but not PACAP peptide (10 nM), suggesting that endogenous PACAP inhibits proliferation. Data are expressed as mean cells per dish ± SEM (2 expts); n = 6; ∗, P < 0.009. (Bar = 50 μm.) Con, control.