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Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) and PACAP-Receptor Type 1 Expression in Rat and Human Placenta¹

Department of Internal Medicine (M.L.S., G.F., C.M.), Department of Experimental Medicine and Biochemical Sciences (A.M., C.P.), and Department of Surgery (E.P.), University of TorVergata, 00133 Rome, Italy; Department of Medical Physiopathology (A.F.), University La Sapienza, 00174 Rome, Italy; and Department of Experimental Medicine (S.U.), University of L’Aquila, 67100 L’Aquila, Italy

Address all correspondence and requests for reprints to: Dr. Costanzo Moretti, M.D., Department of Internal Medicine, Chair of Endocrinology, Faculty of Medicine, University of Rome TorVergata, Via di TorVergata 135, 00133 Rome, Italy. E-mail: moretti{at}med.uniroma2.it

Pituitary adenylate cyclase-activating polypeptide (PACAP), the new hypophysiotropic factor member of the vasoactive intestinal peptide (VIP)/secretin/glucagon/GHRH family of neuropeptides, exerts its biological action by interacting with both PACAP-selective type I receptors (PAC₁) and type II receptors (VPAC₁), which bind both PACAP and VIP. The placenta is a site of 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 of RT-PCR and immunohystochemical experiments. In both rat and human placenta, we evaluated the expression of PAC₁ gene by Northern hybridization analysis performed with a ³²P-labeled 706 nt complementary DNA probe, derived from the full-length coding region of the rPAC₁ complementary DNA. The results 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 PAC₁ gene generating splice variants with variable tissue-specific expression. To ascertain which of the splice variants were expressed in placental tissue we performed RT-nested PCR using primers flanking the insertion sequence termed hip/hop cassette in rat or SV₁/SV₂ box in human gene. Electrophoretic analysis of the PCR products showed a different pattern of expression of messenger RNA splicing variants in human and rat placenta. In particular, the rat placenta expresses the short PAC₁ receptor (PAC_1short), the rPAC₁-hip or hop (which are indistinguishable with the primers used), and the rPAC₁-hip-hop, whereas the human placenta expresses only the PAC₁SV₁ (or SV₂) variant, structurally homologous to the rat PAC₁ hip (or hop). Sequence analysis of the human PCR-amplified PAC₁ variant was therefore carried out and revealed that human placenta only expresses the PAC₁SV₂ isoform. The presence and characterization of PACAP binding sites was then investigated in human placenta by radioligand binding studies performed on crude membrane preparation using [¹²⁵I]PACAP27 as tracer. 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 relative potencies of PACAP-related peptides for inhibition of radioligand binding were: PACAP27 PACAP38 > VIP, whereas GHRH and other unrelated peptides, such as CRH and ß-endorphin, did not inhibit [¹²⁵I]PACAP27 binding.

In conclusion, in this study, we provide evidence for the expression of PACAP within rat and human placenta. We also demonstrate that both human and rat placenta express the PAC₁ gene and that the human tissue has binding sites for PACAP. These findings may suggest a role for PACAP in the regulation of placental physiology through autocrine and/or paracrine mechanisms.

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