A Novel Hypothalamic Peptide, Pituitary Adenylate Cyclase-Activating Peptide, Regulates the Function of Rat Granulosa Cells in Vitro1

Abstract

Pituitary adenylate cyclase-activating peptide (PACAP) is a novel peptide that was isolated from ovine hypothalamic tissue on the basis of its ability to stimulate cAMP accumulation in cultured rat pituitary cells. Recently we demonstrated that PACAP can stimulate cAMP accumulation and secretory function in cultured rat Sertoli cells. Since ovarian granulosa cells share many properties with Sertoli cells, we have examined the effect of PACAP (consisting of 38 or 27 amino acid residues) on cultured granulosa cell function. Granulosa cells were obtained from the ovaries of 25-day-old rats implanted with a silastic capsule containing diethylstilbestrol 5 days prior to culture. PACAP 38 (0.1 microM-0.01 pM), both alone and in the presence of the phosphodiesterase inhibitor, methylisobutylxanthine, stimulated cAMP accumulation 4-8-fold with an ED50 of approximately 100 pM. Maximal PACAP 38 or PACAP 27 stimulation of granulosa cell cAMP was significantly greater than that produced by a maximally effective concentration of FSH. Because PACAP 38 and 27 have 68% sequence homology with vasoactive intestinal peptide (VIP), and since VIP stimulates granulosa cell cAMP accumulation and estradiol and progesterone secretion, we examined the possibility that PACAP could be acting via the VIP receptor. VIP stimulated cAMP only at concentrations of 10 nM or greater, whereas the PACAP stimulation was evident at 10 pM. Moreover, only one of three potent VIP antagonists inhibited VIP stimulation of cAMP accumulation, and only at 1 microM or greater. This VIP antagonist did not inhibit PACAP 38 action at 2000-fold excess concentration. Interestingly PACAP 38 was more effective than PACAP 27 with regard to steroid secretion and the ability to induce LH responsiveness. PACAP and VIP stimulation of granulosa cell cAMP accumulation or estradiol or progesterone secretion was not additive. Thus, these data support the hypothesis that granulosa cells have specific PACAP 38 receptors and that VIP acts via these receptors. In addition, PACAPs 38 and 27 are more potent stimulators of cAMP accumulation in luteinized granulosa cells than LH. These results both pre- and postovulation, along with previous data indicating that the PACAPs are found in the ovaries, suggest a role for PACAP in the regulation of ovarian function.

Pituitary adenylate cyclase-activating polypeptide acts synergistically with relaxin in modulating ovarian cell function in rats

“…The present study further demonstrates that PACAP, known to increase cAMP in target cells (Miyata et al 1989, 1990, Zhong & Kasson 1994, Heindel et al 1996, and cAMP signaling pathway mediators (8-Br-cAMP, forskolin and cholera toxin) alone did not stimulate the secretion of 71 kDa gelatinase in theca-interstitial cells, while PACAP and cAMP mediators augment this effect of relaxin. In addition, PACAP, follicle-stimulating hormone (FSH) or cAMP analog can stimulate steroidogenesis in granulosa cells , Richards et al 1987, Richards 1994, Zhong & Kasson 1994, Heindel et al 1996, whereas relaxin did not (Too et al 1982). Moreover, the present study demonstrates that cell-rounding phenomena occurred both in Cells were treated with cAMP signaling pathway mediators in the absence or presence of relaxin 10 g/ml for 24 h of culture.…”

“…We have previously shown that relaxin stimulates the secretion of 71 kDa gelatinase (MMP2) in theca-interstitial cells (Hwang et al 1996). The present study further demonstrates that PACAP, known to increase cAMP in target cells (Miyata et al 1989, 1990, Zhong & Kasson 1994, Heindel et al 1996, and cAMP signaling pathway mediators (8-Br-cAMP, forskolin and cholera toxin) alone did not stimulate the secretion of 71 kDa gelatinase in theca-interstitial cells, while PACAP and cAMP mediators augment this effect of relaxin. In addition, PACAP, follicle-stimulating hormone (FSH) or cAMP analog can stimulate steroidogenesis in granulosa cells , Richards et al 1987, Richards 1994, Zhong & Kasson 1994, Heindel et al 1996, whereas relaxin did not (Too et al 1982).…”

“…A recent study has shown the presence of PACAP type IA receptor in rat granulosa cells (Kotani et al 1998). In addition, the possible presence of PACAP type IB receptor in rat granulosa cells is supported by the work of Heindel et al (1996) who showed that PACAP38 was more effective than PACAP27 in stimulating steroidogenesis and cAMP accumulation in rat granulosa cells. The present study also shows that PACAPs at a sub-maximal dose of 10 8 M stimulate, synergistically with relaxin, the secretion of 63 kDa gelatinase in rat granulosa cells, while PACAPs at a maximal dose of 10 6 M alone or in combination with relaxin produced similar effects.…”

“…cells (Miyata 1989, 1990, Hart et al 1992, Zhong & Kasson 1994, Heindel et al 1996. In view of this, cAMP signaling pathway mediators (cholera toxin, activator of stimulatory G protein; forskolin, activator of adenylate cyclase; 8-Br-cAMP, cAMP analog) were used to determine whether these mediators could mimic PACAP effects on gelatinase secretion in rat ovarian cells.…”

“…Moreover, PACAP may play an autocrine/paracrine role in the ovary. First, PACAP stimulates steroidogenesis and increases cAMP levels in cultured rat granulosa cells (Zhong & Kasson 1994, Heindel et al 1996, Kotani et al 1998. Secondly, PACAP was recently shown to accelerate meiotic maturation in follicle-and cumulus-enclosed rat oocytes (Apa et al 1997).…”