PACAP Related Peptide (1-29) (rat)

VIP and PACAP38 are closely related peptides that are released in the adrenal gland and sympathetic ganglia and regulate catecholamine synthesis and release. We used PC12 cells as a model system to examine receptor and second messenger pathways by which each peptide stimulates transcriptional and post-transcriptional mechanisms that regulate the level of the mRNA for tyrosine hydroxylase (TH), the rate-limiting enzymatic step in catecholamine synthesis. Concentration-response studies revealed that PACAP38 had both greater efficacy and potency than VIP. The specific PAC1 receptor antagonist PACAP[6-38] blocked the effects of each peptide on TH mRNA content while the PACAP/VIP type II receptor antagonist (N-AC-Tyr(1)-D-Phe(2))-GRF-(1-29)-NH(2) was without effect. At equipotent concentrations, each peptide stimulated a transient increase in TH gene transcription lasting less than 3h. Continuous VIP treatment stimulated a transient increase in TH mRNA lasting less than 24h. In contrast, continuous exposure to PACAP38 stimulated a stable increase in TH mRNA that persisted for 2 days in the absence of elevated transcription, pointing to different post-transcriptional effects of the two peptides. PACAP38 alone had no effect on the magnitude of TH gene transcription or TH mRNA in A126-1B2 PKA-deficient PC12 cells. However, when combined with dexamethasone, PACAP38 produced a synergistic increase in TH mRNA in the absence of PACAP38-stimulated TH gene transcription. In contrast, VIP had no effect on either TH mRNA content or TH gene transcription in this model. PACAP38, but not VIP, stimulated PKC activity. Calphostin C antagonized the effect of PACAP38 on the persistent post-transcriptional elevation in TH mRNA. Thus, the results support the conclusion that VIP and PACAP38 each stimulate PAC1 receptors to increase TH gene transcription through a PKA-controlled pathway, but their divergent post-transcriptional effects result at least partly from differing abilities to stimulate PKC.

Although vasoactive intestinal polypeptide (VIP) is thought to be a prolactin releasing factor, in vivo studies on sheep suggest that it is inactive in this species. Recent studies, based primarily on the rat, suggest that the related pituitary adenylate cyclase-activating polypeptide (PACAP) is also a hypophysiotrophic factor but again in sheep, this peptide has no in vivo effects on hormone secretion despite being a potent activator of adenylate cyclase in vitro. This lack of response to either peptide in vivo in sheep could be due to the low concentration of peptide that reaches the pituitary gland following peripheral injection. In the present study we therefore adopted an alternative approach of evaluating in vitro effects of these peptides on GH, FSH, LH or prolactin secretion from dispersed sheep pituitary cells. In a time-course study, PACAP (1 mumol/l) increased GH concentrations in the culture medium between 1 and 4 h and again at 12 h but had no effect in the 6 and 24 h incubations. Prolactin, LH and FSH were not affected by PACAP. The response to various concentrations of PACAP (1 nmol/l-1 mumol/l) were then evaluated using a 3 h incubation. Again prolactin and LH were not affected by PACAP and there was a small increase in GH concentrations but only at high concentrations of PACAP (0.1 and 1 mumol/l; P < 0.05). PACAP also stimulated FSH secretion in cells from some animals although this effect was small. The GH response to PACAP was inhibited by PACAP(6-38), a putative PACAP antagonist, but not by (N-Ac-Tyr1, D-Arg2)-GHRH(1-29)-NH2, a GH-releasing hormone (GHRH) antagonist. The cAMP antagonist Rp-cAMPS was unable to block the GH response to PACAP suggesting that cAMP does not mediate the secretory response to this peptide. At incubation times from 1-24 h, VIP (1 mumol/l) had no effects on prolactin, LH or GH secretion and, in a further experiment based on a 3 h incubation, concentrations of VIP from 1 nmol/l-1 mumol/l were again without effect on prolactin concentrations. Interactions between PACAP and gonadotrophin releasing hormone (GnRH), GHRH and dopamine were also investigated. PACAP (1 nmol/l-1 mumol/l) did not affect the gonadotrophin or prolactin responses to GnRH or dopamine respectively. However, at a high concentration (1 mumol/l), PACAP inhibited the GH response to GHRH. In summary, these results show that PACAP causes a modest increase in FSH and GH secretion from sheep pituitary cells but only at concentrations of PACAP that are unlikely to be in the physiological range. The present study confirms that VIP is not a prolactin releasing factor in sheep.

The 27 amino acid peptide, pituitary adenylate cyclase-activating polypeptide (PACAP-27), and its 38 amino acid analogue, PACAP-38, stimulate serotonin-N-acetyltransferase (NAT) activity and N-acetylserotonin (NAS) and melatonin content of pineal glands from adult rats. Maximal stimulation of rat pineal NAT by PACAP-38 is not increased further significantly by concurrent stimulation with the two related peptides, vasoactive intestinal polypeptide (VIP) and/or peptide N-terminal histidine C-terminal isoleucine (PHI). Isoproterenol was a more potent inducer of NAT activity than any of these peptides alone or in combination. PACAP-38 also stimulates melatonin production by chicken pineal cells in culture as does VIP. Stimulation by both was not greater than after either alone. Prior stimulation of rat pineal NAT activity with VIP, PHI, or PACAP-38 reduces the magnitude of subsequent stimulation with PACAP-38 or forskolin. Concurrent stimulation of alpha-receptors or treatment with active phorbol ester augments rat pineal response to PACAP-38 stimulation just as it increases the response to VIP, PHI, and beta-receptor stimulation. Pineals from newborn rats respond to PACAP-38 with an increase in NAT activity and the increase is augmented by concomitant alpha 1-adrenergic stimulation. The putative PACAP inhibitor PACAP (6-38) and the putative VIP inhibitor (Ac-Tyr,D-Phe)-GRF 1-29 amide, in 100-1,000-fold excess, did not affect the stimulatory activity of any of the peptides. Pineal melatonin concentration parallels changes in pineal NAT activity.

Growth hormone (GH) is used or is being evaluated for efficacy in treatment of short stature, aspects of aging, cardiac disorders, Crohn's disease, and short bowel syndrome. Therefore, we synthesized several stable growth hormone-releasing factor (GRF) analogues that could be therapeutically useful. One potent analog, [D-Ala(2),Aib(8, 18,)Ala(9, 15, 16, 22, 24-26,)Gab(27)]hGRF(1-27)NH(2) (GRF-6), with prolonged infusion caused severe diarrhea in monkeys; however, it had no side-effects in rats. Because GRF has similarity to VIP/PACAP and VIPomas cause diarrhea, this study investigated the ability of this and other GRF analogues to interact with the VIP/PACAP receptors. Rat VPAC(1)-R (rVPAC(1)-R), human VPAC(1)-R (hVPAC(1)-R), rVPAC(2)-R and hVPAC(2)-R stably transfected CHO and PANC 1 cells were made and T47D breast cancer cells containing native human VPAC(1)-R and AR4-2J cells containing PAC(1)-R were used. hGRF(1-29)NH(2) had low affinity for both rVPAC(1)-R and rVPAC(2)-R while VIP had a high affinity for both receptors. GRF-6 had a low affinity for both rVPAC(1)-R and rVPAC(2)-R and very low affinity for the rPAC(1)-R. VIP had a high affinity, whereas hGRF(1-29)NH(2) had a low affinity for both hVPAC(1)-R and hVPAC(2)-R. In contrast GRF-6, while having a low affinity for hVPAC(2)-R, had relatively higher affinity for the hVPAC(1)-R. In guinea pig pancreatic acini, all GRF analogues were full agonists at the VPAC(1)-R causing enzyme secretion. These results demonstrate that in contrast to native hGRF(1-29)NH(2,) GRF-6 has a relatively high affinity for the human VPAC(1)-R but not for the human VPAC(2)-R, rat VPAC(1)-R, rat VPAC(2)-R or rat PAC(1)-R. These results suggest that the substituted GRF analog, GRF-6, likely causes the diarrheal side-effects in monkeys by interacting with the VPAC(1)-R. Furthermore, they demonstrate significant species differences can exist for possible therapeutic peptide agonists of the VIP/PACAP/GRF receptor family and that it is essential that receptor affinity assessments be performed in human cells or from a closely related species.