Pituitary adenylate cyclase-activating polypeptide (PACAP) and PACAP-receptor type 1 expression in rat and human placenta

Pituitary adenylate cyclase-activating polypeptide (PACAP), the new hypophy siotropic factor member of the vasoactive intestinal peptide (VIP)/secretin /glucagon/GHRH family of neuropeptides, exerts its biological action by int eracting with Tooth PACAP-selective type I receptors (PAC(1)) and type II r eceptors (VPAC(1)), which bind both PACAP and VIP. The placenta is a site o f production of hypophysiotropic factors that participate in the control of local hormone production, as well as the respective hypothalamic-pituitary neurohormones. In the present study, we show the expression of PACAP gene and irPACAP distribution within rat and human placental tissues, by means o f RT-PCR and immunohystochemical experiments. Tn both rat and human placent a, we evaluated the expression of PAC(1) gene by Northern hybridization ana lysis performed with a P-32-labeled 706 nt complementary DNA probe, derived from the full-length coding region of the rPAC(1) complementary DNA. The r esults of these experiments demonstrate the presence, in both human and rat placenta, of a 7.5-kb transcript similar in size to those detected in the ovary, brain, and hypothalamus. Alternative splicing of two exons occurs in human and rat PAC(1) gene generating splice variants with variable tissue- specific expression. To ascertain which of the splice variants were express ed in placental tissue we performed PT-nested PCR using primers flanking th e insertion sequence termed hip/hop cassette in rat or SV1/SV2 box in human gene. Electrophoretic analysis of the PCR products showed a different patt ern of expression of messenger RNA splicing variants in human and rat place nta. In particular, the rat placenta expresses the short PAC(1) receptor (P AC(1short)), the rPAC(1)-hip or hop (which are indistinguishable with the p rimers used), and the rPAC(1)-hip-hop, whereas the human placenta expresses only the PAC(1)SV(1) (or SV2) variant, structurally homologous to the rat PAC(1) hip (or hop). Sequence analysis of the human PCR-amplified PAC(1) va riant was therefore carried out and revealed that human placenta only expre sses the PAC(1)SV(2) isoform. The presence and characterization of PACAP bi nding sites was then investigated in human placenta by radioligand binding studies performed on crude membrane preparation using [I-126]PACAP27 as tra cer. Scatchard analysis of the binding results revealed the presence of two binding sites, one with high affinity and low capacity (K-d 0.33 +/- 0.04 nM; B-max 36.9 +/- 12.1 fmol/mg protein) and one with low affinity and high capacity (K-d 24 +/- 6.9 nM, B-max 9.3 +/- 0.19 pmol/mg protein). The rela tive potencies of PACAP-related peptides for inhibition of radioligand bind ing were: PACAP27 greater than or equal to PACAP38 > VIP, whereas GHRH and other unrelated peptides, such as CRH and beta-endorphin, did not inhibit [ I-125]PACAP27 binding. In conclusion, in this study, we provide evidence for the expression of PAC AP within rat and human placenta. We also demonstrate that both human and r at placenta express the PAC, gene and that the human tissue has binding sit es for PACAP. These findings may suggest a role for PACAP in the regulation of placental physiology through autocrine and/or paracrine mechanisms.