Vasoactive Intestinal Peptide Stimulates Hormone Release from Corticotropic Cells in Culture*

Abstract

The hypothalamic peptide vasoactive intestinal peptide (VIP) has been shown to stimulate the secretion of several pituitary hormones in vivo and PRL in vitro. We have examined the effects of VIP on ACTH and endorphin secretion by AtT20/D 16 v (D16) cells, a clonal strain of mouse pituitary tumor cells, and by primary cultures of rat pituitary cells. The addition of 100 nM VIP to D16 cells stimulated ACTH and endorphin secretion within 10 min, and stimulation continued for at least 24 h. However, treatment of cultures with VIP for18 h had no effect on the amount of [³⁵S]methionine incorporated into immunoprecipitable ACTH precursors during a 15- min pulse. Therefore, VIP acts primarily to stimulate the release of presynthesized intracellular hormones and not to alter proopiocortin synthesis. VIP stimulation of ACTH and endorphin release was dose dependent and biphasic. In the first phase, halfmaximal stimulation occurred with 1.6 ± 0.9 nM VIP and remained constant at 5.6-fold above control values with 10–100 nM VIP. In the second phase, half-maximal stimulation occurred with 160 ± 30 nM VIP, and a maximal effect of 9.4-fold over control values was observed at 1 μM peptide. The C-terminal VIP fragment VIP-(10–28) stimulated ACTH secretion only at concentrations greater than 0.3 μM, whereas structurally unrelated neuropeptides had no effect even at 1-μM concentrations. Dexamethasone inhibited VIP-stimulated ACTH secretion in a dose-dependent manner; the ED₅₀ occurred at 7 nM Dex, and a maximal inhibition of 65% was observed with 1 μM steroid. Somatostatin (SRIF), which inhibits ACTH release stimulated by K⁺ and hypothalamic extract, also inhibited VIP-stimulated ACTH secretion. The ED₅₀ for SRIF inhibition (1.9 nM) correlated well with the previously reported affinity of SRIF for its receptor in D16 cells (K_d = 1.7 nM), and maximal inhibition was 95–100%. In primary cultures of rat anterior pituitary cells, high concentrations of VIP (1μ3 μM) caused a 2-fold stimulation of ACTH release. In contrast, low doses of VIP (10–100 nM) did not reproducibly alter ACTH release, although stimulation of PRL release was usually observed at these concentrations. To conclude, low concentrations of VIP have a large stimulatory effect on ACTH and endorphin secretion by D16 corticotropic tumor cells. In addition, high doses of VIP stimulate ACTH secretion by primary pituitary cell cultures. These results suggest that VIP may regulate ACTH secretion in certain physiological or pathological states by direct action on the pituitary gland.