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Endocrinology Vol. 141, No. 3 1158-1167
Copyright © 2000 by The Endocrine Society

ARTICLES

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

Maria Lucia Scaldaferri, Andrea Modesti, Camilla Palumbo2, Salvatore Ulisse, Andrea Fabbri, Emilio Piccione, Gaetano Frajese and Costanzo Moretti

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


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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 (PAC1short), 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 (Kd 0.33 ± 0.04 nM; Bmax 36.9 ± 12.1 fmol/mg protein) and one with low affinity and high capacity (Kd 24 ± 6.9 nM, Bmax 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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PLACENTAL GROWTH AND functions are controlled by a complex local regulatory system that involves the interaction of neurohormones, neuropeptides, growth factors, and cytokines. In this network of signaling substances, many hypothalamic peptides exert a highly specialized role in the regulation of hormonogenesis, growth, and differentiation of the gestational unit (1). Pituitary adenylate cyclase-activating polypeptide (PACAP) is a newly identified hypothalamic peptide that was originally isolated from ovine hypothalamus by its potent activity in stimulating cAMP production in rat anterior pituitary cells (2, 3). It exists in two biologically active forms, PACAP38 and PACAP27, derived from the same precursor, pre-pro-PACAP, through posttranslational proteolysis and alternative {alpha}-amidation (4). PACAP is detectable and biologically active in many tissues, including pituitary, brain, adrenal, testis, and nerve fibers of both the gut and the lung (5, 6), where it is considered as a neurohormone, neurotransmitter, neuromodulator, and vasoregulator.

Several reports have demonstrated the presence of neuropeptides in the human placenta. CRH, calcitonin gene-related peptide, neuropeptide tyrosine, galanin, somatostatin, met-enkephaline, dynorphin A, and substance P-like immunoreactivities have been demonstrated within decidual cells and amniotic fluid (1, 7, 8, 9). Among the peptides structurally related to PACAP, which probably arose as a result of exon duplication and insertion of a common ancestral gene, vasoactive intestinal peptide (VIP) has been shown to regulate early postimplantation growth in rodents (10); and GHRH gene has been reported to be actively transcribed in human (11), rat (12), mouse (13, 14), and sheep (15) gestational intrauterine tissues. GHRH messenger RNA (mRNA) has been localized to the cytotrophoblast of rat and mouse placenta by in situ hybridization (12, 13, 14, 16). As far as PACAP is concerned, the two naturally occurring peptides, PACAP27 and PACAP38, have been detected by RIA in the human uteroplacental unit, where they have been shown to act as local vasoregulators (17). Interestingly, PACAP-38 has also been shown to positively regulate glycoprotein hormone {alpha}-gene expression in cultured placental cells, likely via a receptor-mediated mechanism (18).

PACAP and VIP share binding sites in a variety of rat and human tissues (19). Based on the relative potencies in ligand binding studies of PACAPs and natural and synthetic VIP analogues, the existence of at least 3 PACAP/VIP receptor subtypes has been suggested, namely PAC1, VPAC1, and VPAC2 (20). The genomic organization of rat PAC1 gene has been elucidated (21). It exists in 6 variant forms, generated by a tissue-specific different splicing, a short form and 5 variants having inserts at the C-terminal end of the third intracellular loop of the receptor (22). The isoforms include hip, a 28-amino acid insert; hop 1, a different 28-amino acid insert; hop 2, an insert similar to hop1, but lacking a single serine residue; and hip-hop 1, a combination of the hip and hop 1 inserts (21, 22, 23). The human PAC1 complementary DNA (cDNA) exhibits a nucleotide sequence identity of 93% with the rat homologue. Similarly to the rat gene, an alternative splicing of two exons allows for 4 major splice variants named: the PAC1 null form, encoding a 486-amino acid protein showing a 93% homology to the rPAC1short form; the hPAC1SV1, differing from its rat homologue (rPAC1-hip) by 2 aminoacids; the hPAC1 SV2, identical to the rPAC1 hop isoform; a sequence transcribed from both exons, showing the greatest homology to the rPAC1 hip/hop isoform, named hPAC1 SV3 (24). PAC1 and its isoforms are highly expressed in many areas of the CNS (25, 26) and moderately in the neurohypophysis (27) and the pituitary gland (28), adrenal medulla (29), and male reproductive tract (30); but there is no evidence, so far, on gene expression in placenta. All these findings prompted us to investigate the expression of PACAP and PACAP receptors in rat and human placenta. We report PACAP gene expression, the irPACAP distribution, and the expression of PAC1 receptors in both rat and human placenta. Thus, we postulate the existence of a new peptide-receptor local system that may act as a paracrine and/or autocrine regulator of placental function.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
PACAP27, PACAP38, VIP, GHRH, CRH, ß endorphin, and the PACAP38 antiserum were purchased from Peninsula Laboratories, Inc. (Belmont, CA). [125I]-PACAP27 (2200 Ci/mmol) was obtained from NEN Life Science Products (Boston, MA). Phenylmethylsulfonylfluoride, aprotinin, bacitracin, Tris, RIA grade BSA, and most of the other chemicals were purchased from Sigma (St. Louis, MO). M-MLV reverse transcriptase was purchased from Life Technologies (San Giuliano Milanese, Italy). Taq polymerase was purchased from Promega Corp. (Milano, Italy). The Bradford protein assay kit was purchased from Bio-Rad Laboratories, Inc. (Richmond, CA). Thermo Sequenase cycle sequencing kit and [{alpha}-35S] deoxy-ATP (dATP) were purchased from Amersham Pharmacia Biotech (Cleveland, OH). The random priming labeling kit was purchased from Boehringer Mannheim (Milano, Italy). [{alpha}-32P]dCTP (deoxycycidine triphosphate) was purchased from Amersham Pharmacia Biotech.

Tissue procurement
Pregnant (on day 20 of gestation) or adult male Sprague Dawley rats were obtained from Charles River Laboratories, Inc. Italia (Lecco, Italy) and were killed by decapitation to minimize stress and potential changes in hypothalamic function associated with anesthesia. Placenta, brain, and testis were then removed. Animal treatments were conducted in accordance with the guide for institutional approval for care and use of animals. Human placentas were obtained from women (n = 3) with uncomplicated term pregnancies after elective cesarean section. Human brain was obtained at autopsy from subjects who died of cardiac disease and who had no history of neurological or psychiatric disease. The study protocol has been approved by the University of TorVergata ethics committee. Tissues were immediately rinsed in ice-cold 0.9% saline, and the human decidua basalis and chorionic plate were dissected from the placental villous tissue. Fractions of all tissues (approximately 30–50 mg) were frozen in liquid nitrogen for subsequent RNA and membrane preparation or immunohistochemical analysis.

Membrane preparation
All the procedures were performed at 4 C and were slightly modified from the protocol of Gottshall et al. (31). Human placental tissue (500 mg) was placed in 10-fold-vol membrane isolation buffer (MIB): 50 mM Tris-HCl (pH 7.4), 5 mM MgCl2, 0.5 mg/ml bacitracin, 2 µg/ml phenylmethylsulfonylfluoride, and 200 U/ml aprotinin. The tissue was homogenized 3 times, 10 sec each time, at 10-sec intervals on ice with Polytron (Brinkmann Instruments, Inc., Westbury, NY) and centrifuged at 250 x g for 10 min. The supernatant was collected and centrifuged at 50,000 x g for 30 min. The pellet was resuspended in 6 ml MIB by passing the suspension several times through a 27-gauge needle. The suspension was centrifuged again at 50,000 x g for 30 min. The crude membrane pellet was resuspended into 5 ml MIB. After protein content determination by Bradford assay (32), the crude membranes were resuspended so as to provide the appropriate protein concentration per 200 µl MIB.

Receptor binding assay
Binding assays were performed on 100 µg of crude membranes, suspended in 200 µl MIB/1% BSA, with addition of 100 pM tracer, i.e. [125I] PACAP27 (2200 Ci/mmol), in a final incubation vol of 400 µl. The assay was conducted in 12 x 75 Sigma-Cote-pretreated borosilicate glass tubes, for 60 min at 15 C. The tubes were incubated in the presence of [125I] PACAP27 and indicated amounts of the structurally related peptides PACAP27, PACAP38, VIP, GHRH, or the unrelated peptides CRH and ß-endorphin. The reaction was terminated by transferring 250 µl incubation buffer into polyethylene microfuge tubes (Beckman Coulter, Inc., Fullerton, CA) followed by centrifugation for 4 min. To separate bound hormone from free hormone, the pellet was washed with 400 µl of binding buffer and centrifuged again for 4 min; moreover, after aspiration of the supernatants, the tip of each tube containing the membrane-bound tracer was severed, and the bound radioligand was quantified in a {gamma}-counter.

RNA preparation and Northern blot analysis
Total RNA from human and rat placenta and from rat hypothalamus was extracted from pools of frozen tissues using the single-step acid guanidinium thiocyanate-phenol-chloroform method (33). The purity and integrity of the RNA were checked spectroscopically and by gel electrophoresis before carrying out the analytical procedures. Total RNA (30 µg/lane) was fractionated by electrophoresis on a 1.5% (wt/vol) agarose gel containing 0.06 M formaldehyde in a 1-fold concentrated 3-morpholinopropanesulfonic acid buffer and blotted on nytran membranes (Shleicher & Schuell, Inc., Keene, NH) by capillary transfer in 20 x standard sodium citrate (3 M NaCl, 0.3 M SSC) followed by cross-linking under UV light for 3 min. Size markers were included as RNA ladders obtained from Life Technologies, Inc. (Gaithersburg, MD). Blots were prehybridized for 4 h at 42 C in a buffer containing 40% formamide, 6 x SSC, 1% SDS, 1 x Denhart’s, and 100 µg/ml sonicated salmon sperm DNA. Hybridization was performed at 42 C for 16 h in the same solution containing a 706-nt PAC1 probe obtained as previously described (34). The probe was labeled by random priming method with [{alpha}-32P]dCTP to a specific activity of 2 x 108 cpm/µg DNA. The filters were washed once in 2 x SSC/1% SDS at room temperature for 15 min, then twice in 2 x SSC/0.1% SDS at 42 C for 20 min. The blots were then exposed, at -70 C, to XAR-S film (Eastman Kodak Co.; Rochester, NY). The radioactive probe was stripped from the blot, which was then rehybridized with a [{alpha}-32P]dCTP-labeled cDNA of rat ßactin as RNA loading and transfer control.

Amplification of PACAP and PAC1 isoforms cDNA
First-strand cDNA synthesis was performed as follows: 1 µg total RNA from rat and human placenta was reverse transcribed by 200 U of Maloney-murine leukemia virus (M-MLV) reverse transcriptase using 2.5 µM random hexamers in the presence of 250 µM deoxynucleotides triphosphate in a final vol of 20 µl. Controls for DNA contamination or PCR carryover were performed omitting the M-MLV or the RNA during RT. The reaction mixture was incubated for 1 h at 37 C, then heat-denaturated for 5 min at 80 C. One-half of the cDNA obtained was used to amplify PAC1 or PACAP precursor. PAC1 cDNA was PCR- and nested-amplified as we have previously reported (34); briefly, the first round of PCR was carried out using primers designed for the amplification of PAC1 cDNA sequence spanning the region 609-1412 (22) [upstream 5'-CATCAAGGACTGGATCTTGTACGC-3'; downstream 5'-GGCTCAATGAATCACAGTAGG TGG-3', amplified product 803 bp]. The PCR product (0.1%) was nested-amplified using a set of primers flanking the receptor splicing site [upstream 5'-TTACTTTGTGCTTTTCATCGGGC-3'; downstream 5'-TCCCTCTTGCTGACGTTCTC-3'; multiple products are amplified related to the splice variants expressed (see Figs. 4Go and 5Go)]. The nested PCR was carried out for 20 (data not shown) or 30 cycles. PACAP PCR was performed using primers designed for the amplification of the fragment 502-1078 spanning exon 5 of the human PACAP gene [upstream hP1: 5'-AAACAAAGGACGACGCCGATAG-3; downstream hP2: 5' AGACTCACTGGGAAAGAATGC-3'; amplified product 576 bp] (35). To amplify the rat PACAP cDNA, primers spanning the region 675-1090 of the rat PACAP precursor cDNA sequence were designed [upstream rP1: 5' ATCAGACCAGAAGAGGC-3'; downstream rP2: 5' GCTATTCGGCGTCCTTTGTT-3'; amplified product 415 bp] (36). cDNAs were amplified using Taq polymerase (2 U per tube) with 15 pmol of both upstream and downstream primers, 1.5 mM magnesium chloride in a final vol of 50 µl that was overloaded with 25 µl mineral oil. Then, 30 cycles (94 C for 60 sec, 60 C for 60 sec, 72 C for 60 sec, with 10-min final extension for hP1/hP2 primers, and 94 C for 60 sec, 55 C for 45 sec, 72 C for 60 sec, with 10-min final extension for rP1/rP2) were applied. Finally, a 15-µl aliquot of all the amplified products was analyzed on 1.8% (wt/vol) agarose gel (NuSieve 3:1, FMC, Rockland, ME) and stained with ethidium bromide.



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  		Figure 4. Analysis of PAC1 and its splice variants by RT-nested PCR in rat placenta. Total RNA (1 µg) from rat placenta and hypothalamus was subjected to RT-nested PCR for 30 cycles, as indicated in Materials and Methods. PCR products (15 µl) were electrophoresed onto 3:1 Nusieve agarose gel and stained with ethidium bromide, HaeIII digested øx174 DNA was run to provide size markers. For either set of primers, no signal was detected in the negative control. PAC1-nested primers amplified, from both placenta and hypothalamus (positive control) cDNAs, three products corresponding to the size expected for PAC1 known splice variants, respectively: 352 bp for the hip-hop, 268 bp for the hip or hop, and 184 bp for the PACAP receptor short form.

 


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  		Figure 5. Analysis of PAC1 and its splice variants by RT-nested PCR in human placenta. Total RNA (1 µg) from human placenta and brain was subjected to RT-nested PCR for 30 cycles, as described in Materials and Methods. PCR products (15 µl) were electrophoresed onto 3:1 Nusieve agarose gel and stained with ethidium bromide, HaeIII digested øx174 DNA was run to provide size markers. PAC1-nested primers amplified three products from human brain cDNA corresponding to the size expected for PAC1 known splice variants, respectively: 352 bp for the SV3 268 bp for the SV1or SV2 and 184 bp for the PACAP receptor null form. From human placenta, cDNA was exclusively amplified the 268-bp product, corresponding to the SV1/SV2 receptor variant.

 
Direct SV1/SV2sequencing
SV1/SV2 PCR-nested amplified cDNA was prepared for direct sequencing as follows: mineral oil was removed by addition of 200 µl of chloroform, after a brief centrifugation, and the upper phase was transferred to a fresh tube, and the DNA was precipitated in the presence of ammonium acetate and ethanol. The pellet, obtained by centrifugation, was air-dried and resuspended in 50 µl DEPC water. The resuspended DNA (100 ng) was sequenced directly using Thermo Sequenase DNA polymerase (Amersham Pharmacia Biotech), [{alpha}-35S] dATP, and the Thermo Sequenase cycle sequencing kit (Amersham Pharmacia Biotech). The upstream primer (0.5 pmol) was labeled and extended by thermo sequenase in a reaction mixture containing the DNA template, the 7-deaza deoxy-GTP cycle mix, the dTTP cycle mix, and the labeled [{alpha}-35S] dATP, in a final vol of 17.5 µl. The reaction mix was thermally cycled (Gene Amp PCR system 2400) for 55 cycles as follows: 55 C for 30 sec, 95 C for 15 sec. Then, 3.5 µl of the labeling reactions were transferred to each dideoxynucleoside triphosphates and again thermally cycled for 55 times (95 C for 30 sec, 72 C 90 sec). Reaction products were resolved in a 6% polyacrylamide urea gel, dried ,and exposed to XAR film.

Immunohistochemistry
Immunohistochemistry was performed on the frozen sections obtained from snap-frozen specimens of human placenta using the Histostain SP kit (Zymed Laboratories, Inc.; San Francisco, CA). Briefly, human frozen sections were fixed in 4% neutral buffered formaldheyde, whereas the rat placenta specimens were fixed in 10% neutral buffer formaldheyde, and processed for paraffin embedding. Endogenous peroxidase activity was quenched using a 3% H2O2 solution for 10 min at RT, and the sections were incubated for 2 h at room temperature with a 1:1000 dilution of polyclonal anti-PACAP38 primary antibody (Peninsula Laboratories, Inc.). The reported cross-reactivity of the antiserum was less than 0.01 with PACAP27, and 0% with the human unrelated peptides CRH, neuropeptide tyrosine, and somatostatin. Ether rabbit preimmune serum (not shown) or primary antibody preadsorbed with a 1000-fold excess of synthetic human PACAP38 (Peninsula Laboratories, Inc.) were used, instead of the primary antibody, for control sections. Biotinylated goat antirabbit IgG and streptavidin-horseradish peroxidase were used according to the manufacturer’s protocol. Immunostaining was visualized using AEC substrate, and hematoxylin was used for nuclear counterstaining. The sections were finally observed under a Dialux 22 microscope (Leitz).

Data analysis and statistics
In the binding studies, each data point represents the mean ± SE of three replicates and the mean of three experiments. Data were analyzed by Student’s t test and ANOVA. Scatchard analyses were performed using the LIGAND computer program (37).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
PACAP expression and localization in placenta
The expression of PACAP38 at mRNA level in human and rat placenta was examined by RT-PCR employing primers spanning the exon 5 of the human PACAP gene that encode for PACAP38 and the region of rat precursor cDNA sequence encoding for the mature peptide (35, 36). A PACAP fragment was amplified from human (Fig. 1aGo) and rat (Fig. 1bGo) placenta cDNAs, representing the predicted size, based on primers location and corresponding to the products obtained from rat and human brain run as positive control.



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  		Figure 1. Expression of PACAP38 in human and rat placenta. Total RNA from (a) human and (b) rat tissues was subjected to RT-PCR using specific sets of primers (hP1-hP2; rP1-rP2), as indicated in Materials and Methods. An aliquot (15 µl) of each PCR product and DNA Molecular Weight VI (Boehringer Mannheim) were electrophoresed onto 1.5% agarose gel and stained with ethidium bromide. For either set of primers, no signal was detected in the negative control. a) PCR product of about 576 bp was amplified from human placenta and brain cDNAs; the latter was included as positive control. b) PCR product of about 415 bp was amplified from rat placenta, testis, and hypothalamus (included as positive controls) cDNAs. A representative negative control (nc) of PCR carryover is also shown.

 
PACAP localization was investigated in sections obtained from human and rat term placenta by an indirect immunoperoxidase technique with anti-PACAP 38 antiserum. No staining was observed in control slides (Fig. 2Go, a and f). In human sections, a specific immunostaining was found associated with stromal cells of both stem and terminal placental villi (Fig. 2Go, b–d). In the stem villi, strongly immunostained cells displayed a rather defined spatial distribution, being mainly restricted at the periphery of the villi in the stroma surrounding the blood vessels, whereas only a few lightly stained cells were occasionally found in blood vessel walls (Fig. 2bGo). In terminal villi, on the other hand, the stained cells seemed to lack a defined spatial distribution, being dispersed throughout the whole stroma (Fig. 2Go, c–d). In the sections obtained from rat term placenta, a specific immunostaining was observed both in the placental labyrinth and in the villous-like structures of the intraplacental yolk sac, resulting from the extension of the yolk sac into the placental disc during late pregnancy (38).



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  		Figure 2. Immnohistochemical detection of PACAP38 in sections from rat and human term placenta. a–d, Human placenta; a, negative control: the anti PACAP38 antibody has been preadsorbed with an excess of synthetic human PACAP38. b, Portion of stem villus; strongly immunostained cells are mainly present in the stroma surrounding the blood vessels (V), whereas few mildly stained cells are found in the vessel’s walls. c, Terminal villi; the staining is associated with stromal cells scattered within the villi, lacking a defined spatial distribution. d, High magnification of a terminal villus showing marked cytoplasmic staining in some stromal cells (arrow). e–f, Rat placenta. e, Portion of the rat placental labyrinth; several immunostained cells are present both in the labyrinth (L) and in the villous-like structures of the intraplacental yolk sac (IPY). f, Negative control. Scale bar represents 50 µm (a–c), 10 µm (d), and 160 µm (e, f).

 
PAC1 isoforms gene expression in placenta
To demonstrate the expression of PAC1 gene in placental tissue, we performed Northern blot analysis of total RNA extracted from rat and human placenta, as well as from rat hypothalamus (used as positive control). The results, shown in Fig. 3Go, revealed a 7.5-kb transcript, consistent with the size of the hypothalamic PAC1 mRNA, in both rat and human placental tissue. The expression of PAC1 was lower in human (compared with rat) placenta, as equal RNA loading and transfer was confirmed by ß-actin hybridization. To further characterize the PAC1 gene expression in the placenta, we used a sensitive RT nested PCR (RT-PCR). The results of these experiments demonstrated a different pattern of isoform expression in the rat (Fig. 4Go), compared with the human tissue (Fig. 5Go). Rat placenta expresses PAC1 splice variants either containing or lacking each of the two alternative exons. In fact, as shown in Fig. 4Go, the nested PCR revealed three products corresponding in size to the PAC1short, the hip or hop (indistinguishable with the primers used), and the hip-hop variant. Unlike the rat, human placenta uniquely expresses the SV1 or SV2 isoform of the PAC1 gene; whereas in the same experimental conditions, three PCR products [null, SV1 or SV2 and SV3 ] were amplified from human brain cDNA (Fig. 5Go). The characterization of the variant expressed in the human tissue was addressed by direct sequencing analysis, which demonstrated that the isoform expressed corresponded to the PAC1SV2, (Fig. 6Go).



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  		Figure 3. Northern blot analysis of PAC1 mRNA in rat and human placenta. Total RNA (30 µg) extracted from human or rat placenta and rat hypothalamus was resolved by formaldehyde agarose gel electrophoresis, transferred to nylon membrane, and probed with a 32P-labeled PAC1 cDNA. The blot was successively hybridized with a ß-actin probe as a control for RNA loading and transfer. Rat and human placenta RNA encode a 7.5-kb transcript, similar in size to the rat hypothalamus PAC1 (positive control).

 


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  		Figure 6. Human placenta SV1/SV2 sequence analysis. The PCR product SV1/SV2 was sequenced directly using Thermo Sequenase DNA polymerase, [{alpha}-35S] dATP, and a Thermo Sequenase cycle sequencing kit. The sequence autoradiography image is shown. The sequence obtained was aligned among the sequences SV1 and SV2 (accession numbers Z23373 and g404220). The analysis revealed the identity to SV2 PAC1 variant. Alignment of the SV1 and SV2 is partially shown. The square brings into evidence the restriction site Blp I (*), unique of PAC1SV2 variant.

 
Receptor binding assay
The binding sites expressed in human placenta were characterized by equilibrium binding studies performed on crude membrane preparations incubated for 60 min with 100 pM [125I]PACAP27 in the presence of increasing concentrations of the unlabeled peptide (Fig. 7Go). The specific binding of [125I]PACAP27, measured in the presence of 1 µM PACAP27 was 80% of the total binding to placental membranes. Scatchard analysis of the binding data (Fig 7Go, insert) showed the presence of two binding sites, one with high affinity and low capacity and another with low affinity and high capacity. The Kd values for the two sites were determined to be 0.3 ± 0.04 nM and 24 ± 6.9 nM; and their binding capacities were, respectively, 36.9 ± 12.2 fmol and 9.3 ± 0.19 pmol per mg of protein. The ligand specificity of the human placenta membrane binding sites for [125I] PACAP27 is also shown in Fig. 7Go. The unlabeled PACAP27 and PACAP38 were almost equipotent, with half-maximum inhibitory concentrations (IC50), respectively, of 0.34 nM and 0.65 nM; whereas VIP was 10 times less potent, with an IC50 of 50 nM. GHRH, as the unrelated peptides ß-endorphin and CRH, did not inhibit the binding of radioiodinated PACAP27.



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  		Figure 7. Inhibition of [125I] PACAP binding by PACAP27, PACAP38, VIP, and GHRH in human placenta membranes. Human placenta membranes (100 µg) were incubated with 100 pM [125I]PACAP27 and increasing concentrations of unlabeled PACAP38 ({blacktriangledown}), PACAP27 ({blacksquare}), VIP (*) GHRH (•), and the unrelated peptides ßendorphin ([circo) and CRH ({square}) at 16 C for 60 min. Binding is expressed as a percentage of the [125I]PACAP27 bound in absence of unlabeled peptide. The inset shows the Scatchard analysis of high- and low-affinity PACAP27 binding sites. Data are the mean ± SEM of three separate experiments performed in duplicate.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we have shown that PACAP and isoforms of its specific type I receptor (PAC1) are expressed in human and rat placenta, supporting the hypothesis of a novel system in the network of local regulatory neuropeptides.

Three receptors for PACAP have been cloned in mammals, two of which present also a high affinity for VIP (22, 24, 39, 40). Nucleotide and amino acid sequences of PACAP and PACAP receptors are highly conserved among mammals (41), birds (42), and fish (43). PACAP, in addition to its role as a hypophysiotropic hormone, neurotransmitter, and vasoregulator (44, 45), has also been shown to function as a growth and developmental factor. In fact, it promotes mitogenesis, survival, and neurite outgrowth on immature rat cerebellar granule cells (46) and stimulates male primordial germ cell proliferation in early gonadal development (47).

In this study, we focused on rat and human placenta because, in spite of some reports showing a biological role of the PACAP/VIP neuropeptide family, we were unaware of any data demonstrating the PACAP expression, its tissue distribution, or the expression of PACAP receptors within these tissues. Northern blot analysis showed, in both human and rat placenta, the presence of 7.5-kb PAC1 transcripts, consistent with those previously detected in other human and rat tissues.

Both in human and in rat, alternative splicing of two exons can occur, to generate the third intracellular loop splice variant forms of the PAC1 expressed in a tissue-specific manner and variably coupled to intracellular second messengers. In this report, we have demonstrated that rat placenta expresses at least three of the known rPAC1 variants, namely (from the shortest to the longest): the rPAC1short, the hip or hop, and the hip/hop; whereas the human placenta express only the PACAP receptor variant SV2. Previous studies have identified the null splice variant as the predominant form of PAC1 in human tissue (24). Our finding, of SV2 as a unique PAC1 variant in human placenta at term, may suggest the presence of a regulatory mechanism of the PAC1 splicing machinery leading to variable tissue expression. Moreover, the different pattern of splicing variants in the two species, within the same tissue, may imply an evolutionary regulation of such a mechanism. Because, in the present report, we have exclusively analyzed the placenta at term, we cannot exclude a different pattern of subtypes receptor expression during earlier gestational periods. These observations raise the question of PACAP biological effects in placenta physiology. The PACAP-PAC1 complex, through two genes (one encoding the hormone, the other encoding the receptor’s splice variants), allows a broader repertoire for regulation of intracellular signal transduction (44). Interestingly, it has been reported that in humans (unlike rats), each PAC1 splice variant has similar dose-response to PACAP for activation of both adenylate cyclase and phospholipase C, whereas the efficacy for coupling to phospholipase C is greater for the SV2, compared with the null and SV1 splice variants (24). These findings indicate that PACAP may generate a heterogeneous repertoire of signaling activation, depending on the predominant receptor isoform bound and on the network of signaling proteins expressed in each cell system. The different responses evoked by the PACAP/PAC1 receptor complex have, so far, been evaluated mainly by transfection studies (48, 49). Because, in our study, only a single isoform of the PACAP-preferring receptor was found in human placenta, we propose this tissue as a unique experimental system to further investigate the receptor pharmacology and signaling pathway activated in response to ligand binding.

In addition to the variable actions of PACAP via PAC1 receptor splice variants, PACAP has a further potential to induce a variety of biological responses interacting with VPAC1 and VPAC2 receptors. To investigate the binding properties of the PACAP receptors in placenta, we performed radioligand studies with [125I] PACAP27. The half-maximum inhibitory concentration (IC50) of PACAP38 and PACAP27 to inhibit the binding was almost 10 orders of magnitude higher than that of VIP, indicating the presence of a PACAP-preferring receptor on human placenta membranes. Moreover, Scatchard analysis evidenced a low affinity binding site, which may suggest the presence of either VPAC1 and/or VPAC2 receptors. Thus, we demonstrate that at least two classes of functional PACAP receptors exist in human placenta at term. The expression of VPAC2 in human placenta has been already reported (50) without mentioning the gestational period, whereas VIP binding sites have been demonstrated by autoradiographic studies on human placenta at term (51). These data and our present finding demonstrate that placenta serves as a site of PACAP production and receptor target.

The potential biological role of such receptors in the placenta could be suggested by the known actions of the peptides of the VIP/PACAP-family. These neuropeptides act via adenylate cyclase in the regional regulation of blood flow and hormone secretion (7, 17, 51) and are considered important trophic factors and developmental regulators (52). Increased levels of VIP have been described in preeclampsia in women with untreated gestational proteinuric hypertension, representing a powerful compensatory mechanism to restore vascular perfusion of the uterus and placenta (53). The evidence that PACAP displays an inhibitory effect on vascular and nonvascular smooth muscle activity in the nonpregnant rabbit female genital tract as well as in the nonpregnant human female genital tract (54), suggests a possible intragonadal role of the peptide in the local nervous control of blood flow and smooth muscle activity. PACAP has been detected by RIA at low concentrations throughout the uteroplacental unit, where it causes a concentration-dependent relaxation on stem villous and umbilical cord arteries (17). It has also been reported that PACAP positively regulates gene expression in JEG-3 choriocarcinoma cells that possess functional specific receptors for this peptide, the activation of which enhances cAMP formation, glycoprotein hormone {alpha}-subunit gene transcription, and interleukin-6 production (18).

Though it is well known that neuropeptides play a key role in human placenta physiology, only a few but confusing data are available on the localization and tissue distribution of these substances. To our knowledge, this is the first report showing the expression of PACAP in human placenta. Based upon the reported sequence of the human PACAP gene (35), we selected the primers for PACAP cDNA amplification to include the region of exon 5 that encodes the full length of PACAP38. In this respect, our data showing the expression of PACAP38 at both mRNA and protein levels in human placenta are in agreement with those of Steenstrup et al., who showed the presence, by RIA, of both PACAP38 and PACAP27 in the uteroplacental unit (17), PACAP38 being the dominant peptide expressed. In the same study, however, PACAP immunoreactivity was reported to be undetectable by immunohistochemistry. In contrast, our results showed the presence of PACAP-immunolabeling in human and rat term placenta. This discrepancy may be attributable to the different antibody used in the study. Our immunohistochemical data on human placenta revealed that the anti-PACAP38 immunostained cells were mainly confined to the periphery of the stem villi, whereas they were diffusely spread in the terminal ones. It will be interesting to investigate the meaning of this rather defined spatial distribution of immunoreactivity. In the rat tissue, we found anti-PACAP38 immunostaining associated with diffused cells belonging to both the components of the materno-fetal transport system, namely the chorioallantoic placental labirint and intraplacental york sac.

Although the physiological role of placental PACAP is still far from being elucidated, the evidence of the expression of both PACAP and different isoforms of the PAC1 receptor gene, together with the localization of the peptide, is intriguing and suggests the possibility that this new local system may play a role in the regulation of placental function in a paracrine and/or autocrine manner. Determination of the mechanisms controlling the PAC1 gene expression in the placenta may provide new insights into the biological significance of the PACAP/PAC1 system at this level.


   Acknowledgments
 
We would like to thank Mario Arizzi from the Department of Medical Physiopathology, University of Rome La Sapienza, for his assistance with rat immunohistochemistry.


   Footnotes
 
1 This work was supported by MURST 60% grants. Back

2 Supported by a Ph.D. fellowship in Experimental Medicine at the University of L’Aquila, L’Aquila, Italy. Back

Received July 15, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Pituitary Adenylate Cyclase-Activating Polypeptide and Its Receptors: From Structure to Functions
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