PAC1 is the neuropeptide pituitary adenylate cyclase activating polypeptide (PACAP) preferring receptor, which belongs to class B G protein-coupled receptors (GPCR) family. PAC1 mediates the most effects of PACAP as neurotransmitter, neuroregulator and neuroprotectant, while its high expression has close relationship with some physiological and pathological processes such as nerve-injury and tumor. To further understand the function of PAC1, a cell line that expressed inducible PAC1 was constructed to achieve Doxycycline (Dox) dependent expression of PAC1 in CHO (Chinese hamster ovary) cell using the improved Tet (tetracycline)-on Advanced System. First, the PAC1-EYFP fusion gene composed of PAC1 gene and gene encoding EYFP (enhanced yellow fluorescent protein) was sub-cloned to the tetracycline response element pTRE-Tight vector to construct the recombinant vector pEYFP-PAC1-EYFP by double enzyme digestion. Second, the tetracycline regulation components pTet-On advanced vector and the response element pTRE-PAC1-EYFP vector were both introduced into CHO cells successively and the positive clones were screened with G418 and hygromycin respectively. Third, the controlled expression of PAC1-EYFP in CHO was induced by tetracycline analogues Dox in different concentrations and the different levels of receptor PAC1-EYFP were detected. The results of fluorescence analysis and western blotting show that the cell strain with Dox dependent expression of PAC1-EYFP named PAC1-Tet-CHO was obtained. Moreover, in PAC1-Tet-CHO cells the expression of PAC1-EYFP was induced by Dox in a dose-dependent manner. The inducible expression of PAC1 still was stable after sub-culturing for more than 10 passages. It was also found by MTT assay that the higher expression level of PAC1 endowed the cells with higher proliferative viabilities. The construction of controlled expression system of PAC1 will lay a foundation for the further research on PAC1 profiles.
Animals ; Blotting, Western ; CHO Cells ; Cloning, Molecular ; Cricetinae ; Cricetulus ; Genetic Vectors ; Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide, Type I ; biosynthesis

Pituitary adenylate cyclase activating polypeptide (PACAP) was first isolated from ovine hypothalamus and was known to stimulate the release of growth factor in various cells. Recently, we reported the cellular localization of PACAP and its type I (PAC1 ) receptor in rat placenta during pregnancy. Placenta is a critical organ that synthesizes several growth factors and angiogenic factors for the fetal development and its own growth. However, there is little information regarding the cellular localization of PACAP and its receptor in human placenta at various gestations. The aim of the present study was to define the expression and distribution of PACAP and PAC1 receptor mRNAs in the human placenta during the pregnancy period. PACAP and PAC1 receptor mRNAs were expressed in stroma cells of stem villi and terminal villi. At the early stage, on 7 and 14 weeks, PACAP and PAC1 receptor genes were moderately expressed in stroma cells surrounding the blood vessels within stem villi. These genes were strongly expressed in stroma cells of stem villi and terminal villi on 24 and 38 weeks. The expression of these genes was increased as gestation advanced, and localized in the same areas. Localization of PACAP and PAC1 receptor demonstrate the evidence that PACAP may play an important role, as an autoregulator or pararegulator via its PAC1 receptor. In conclusion, our findings strongly suggest that PACAP may have a critical role in physiological function of the placenta for gestational maintenance and fetal growth.
Chorionic Villi/metabolism ; Female ; Gene Expression ; Humans ; Nerve Growth Factors/*biosynthesis ; Neuropeptides/*biosynthesis ; Neurotransmitter Agents/*biosynthesis ; Pituitary Adenylate Cyclase-Activating Polypeptide ; Placenta/*metabolism ; Pregnancy ; Pregnancy Trimester, First ; Pregnancy Trimester, Second ; RNA, Messenger ; Receptors, Cell Surface/*biosynthesis ; Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide ; Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide, Type I

VPAC2 is a co-receptor of pituitary adenylate cyclase activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) and mediates multiple bio-functions. In order to construct the CHO line expressing VPAC2 stably, pcDNA-VPAC2 was used to transfect CHO cells. The positive clones were selected by G418 and the clone VPAC2-CHO with high sensitivity to PACAP38 was picked out by its ability to promoting the concentration of cAMP. RT-PCR, Western blot and Immunofluorescenece assay were used to identify the express of VPACS. Binding competition with VPAC2 agonist and the bioactivity of mediating the ligand to promote the concentration of cAMP showed that VPAC2 was expressed effectively in VPAC2-CHO. The results of Scatchard analysis revealed that VAPC2-CHO expressed a receptor density of (1.1 +/- 0.2) pmol/mg protein, respectively, with Kd values of (0.55 +/- 0.10) nmol/L for PACAP38 used as a tracer. The construction of CHO cells expressing VPAC2 specially and functionally lays a foundation not only for the further research on the characters and functions of VPAC2 but also for the screening and characterization of novel agonists of antagonists for VPAC2.
Animals ; Binding, Competitive ; CHO Cells ; Cell Membrane ; drug effects ; metabolism ; Cricetinae ; Cricetulus ; Cyclic AMP ; metabolism ; Gene Expression ; Genetic Vectors ; genetics ; Iodine Radioisotopes ; chemistry ; Pituitary Adenylate Cyclase-Activating Polypeptide ; chemistry ; metabolism ; pharmacology ; Receptors, Vasoactive Intestinal Peptide, Type II ; agonists ; antagonists & inhibitors ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Transfection

BACKGROUND: Pituitary adenylate cyclase-activating polypeptide (PACAP) plays the role of a hypophysiotropic factor, which regulates the synthesis and secretion of pituitary hormones through the hypothalamo-hypophysial portal system. No clear evidence has yet been reported regarding the regulation of prolactin (PRL) by PACAP. In the present study, we tested a hypothesis that PACAP regulates the synthetic machinery of PRL during the estrus cycle and pubertal process using intracerebroventricular (i.c.v.) injection of an antisense oligodeoxynucleotide (ODN) against type I PACAP receptor (PAC1). METHODS: An RNase protection assay (RPA) was used to determine the pattern of hypothalamic PACAP and PAC1 mRNA expressions during the estrus cycle. Antisense PAC1 ODN was administered via i.c.v. injection to the female rats in normal estrus cycle of pubertal process. Northern blot analysis was used to determine the mRNA ievel of PRL in the pituitary gland. RESULTS: 1) PACAP mRNA in the medial basal hypothalamus was significantly increased at the diestrus I, while PAC1 mRNA showed no significant change. 2) PRL mRNA level of pituitary was increased by an injection of antisense PAC1 ODN at the proestrus and estrus stages. 3) PRL mRNA level of pituitary was significantly decreased by antisense PAC1 ODN injection at stage of prepuberty and initiate puberty, while its level was increased at stage of puberty. CONCLUSION: These data suggest that PACAP suppresses PRL mRNA synthesis through the PAC1 signaling pathway in the certain estrus cycle environments. It may be also involved in the regulation of pituitary PRL gene expression during the pubertal process
Adolescent ; Animals ; Blotting, Northern ; Diestrus ; Estrus ; Female* ; Gene Expression* ; Humans ; Hypothalamus ; Pituitary Adenylate Cyclase-Activating Polypeptide* ; Pituitary Gland* ; Pituitary Hormones ; Portal System ; Proestrus ; Prolactin* ; Puberty ; Rats* ; Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide ; Ribonucleases ; RNA, Messenger

PURPOSE: We analyzed the expression of vasoactive intestinal peptide (VIP), pituitary adenylate cyclase activating peptide (PACAP), VIP receptor 1 (VIPR1), VIP receptor 2 (VIPR 2) and PACAP receptor (PACAPR) genes in human neuroblastoma, neuroblastoma cell line, human stomach cancer, and human stomach cancer cell lines using RT-PCR and Sourthern hybridization. The results should permit identification of potential clinical applications for VIP and PACAP. METHODS: We isolated RNA from 1 neuroblastoma cell line, 8 stomach cancer cell lines, 13 neuroblastoma, and 10 stomach cancer tumor specimens. And then we performed RT-PCR, Sourthern hybridization, and sequencing. RESULTS: We detected the RNAs coding for VIP, VIPR1, VIPR2, PACAP, and PACAPR in 1, 11, 2, 12, and 13 out of 13 neuroblastoma tumor specimens, respectively. VIP and PACAPR RNA was expressed in SKNSH. VIPR1 RNA was expressed in 4 of 8 the stomach cancer cell lines and 6 of 10 stomach cancer tumor specimens. CONCLUSION: VIP/PACAP RNA and VIP/PACAP receptors RNA were expressed in SKNSH and neuroblastoma tumor specimens. VIPR1 was expressed in stomach cancer cell lines and tumor specimens. The present results suggested that VIP/PACAP analogues could be a candidate as the growth inhibitor of neuroblastoma and stomach cancer.
Adenylyl Cyclases* ; Cell Line ; Clinical Coding ; Humans ; Neuroblastoma* ; Pituitary Adenylate Cyclase-Activating Polypeptide ; Receptors, Peptide* ; Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide ; Receptors, Vasoactive Intestinal Peptide ; RNA ; Stomach Neoplasms* ; Stomach* ; Vasoactive Intestinal Peptide

AIMTo observe the protective role of pituitary adenylate cyclase activating polypeptide (PACAP) on hippocampal neuronal apoptosis induced by beta amyloid peptide in the culture.

METHODSHippocampal neurons were isolated from 1d old SD rat and neuronal survival and apoptosis were measured by MTT assay and DNA ladder.

RESULTS25 micromol/L Abeta could induce neuron apoptosis while co-treatment with PACAP could increase the survival of hippocampal neurons. The antagonist of PACAP receptor, P6-27, could reverse the effect of PACAP.

CONCLUSIONPACAP could protects cultured neurons from the neurotoxicity of Abeta through the activation of PACAP receptor and may have a bright use in treatment of neurodegenerative disease.

Amyloid beta-Peptides ; toxicity ; Animals ; Apoptosis ; drug effects ; Cells, Cultured ; Hippocampus ; cytology ; Neurons ; drug effects ; metabolism ; Rats ; Rats, Sprague-Dawley ; Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide ; metabolism

BACKGROUND: The pathogenesis of melasma has not yet been clearly demonstrated. But, clinical manifestations such as remarkable lesional symmetry and the distribution related to trigeminal nerves, suggest that the neural system could play a pathogenic role in melasma. OBJECTIVE: This study was carried out to examine the expression of some neuropeptides and their receptors, which are well known to be major contributors of neuroinflammation in many dermatoses, in melasma lesions. METHODS: Skin biopsies were obtained from the lesional and non-lesional facial skin of 6 Korean women with melasma. Immunofluorecence staining and confocal laser scanning microscopy were performed. RESULTS: In our results, no difference could be detected with regard to the intensity of immunoreactivity for vasoactive intestinal peptide (VIP), calcitonin gene-related peptide (CGRP), calcitonin gene-related peptide receptor (CGRPR), substance P (SP), substance P receptor (SPR), somatostatin (SOM), pituitary adenylate cyclase activating peptide (PACAP) and pituitary adenylate cyclase activating peptide receptor (PACAPR) in the lesional skins compared with the non-lesional skins. CONCLUSION: These results suggest that neuroinflammation induced by neuropeptides such as substance P, calcitonin gene-related peptide, vasoactive intestinal peptide, and somatostatin and their receptors included in this study, are not directly associated with melasma pathogenesis.
Adenylyl Cyclases ; Biopsy ; Calcitonin Gene-Related Peptide ; Female ; Humans ; Melanosis ; Microscopy, Confocal ; Neuropeptides ; Receptors, Neurokinin-1 ; Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide ; Skin ; Skin Diseases ; Somatostatin ; Substance P ; Trigeminal Nerve ; Vasoactive Intestinal Peptide

The present study was aimed to investigate the changes of vasoactive intestinal polypeptide (VIP) and VIP receptor 1 (VIPR1) in small intestinal and hepatic tissues during macaque development. The tissue samples of small intestine, liver and blood samples from peripheral and portal vein of 4 macaques of 6-month fetus, 2-day neonate, 45-day neonate and adult were obtained after anesthetization. The concentration of VIP in blood or tissues of macaques was measured by radioimmunoassay. The distribution of VIP in small intestinal or hepatic tissues was visualized by immunohistochemical staining. The expression of VIPR1 was detected by in situ hybridization. The results showed that: (1) VIP concentration in intestinal tissue of 6-month fetus was (20.7+/-14.3) ng/mg protein, and a few VIP-positive nerve fibers first appeared in intestinal villus root and submucosal layer but not in muscle layer. The intestinal concentration of VIP increased gradually with macaque development and reached (514.8+/- 49.2) ng/mg protein in adult, significantly higher than that in 6-month fetus (P<0.01). (2) In adult animal, VIP-positive nerve fibers became thicker and gradually extended into the mucosal crypt, submucosal layer nerve, myenteric nerve plexus of annular muscle and indulge muscle, and annular muscle. Correspondingly, the expression of VIPR1 in intestine was up-regulated during development. (3) On the contrary, the levels of VIP and VIPR1 in liver were gradually decreased during development. (4) VIP concentration in small intestinal tissue was higher than that in hepatic tissue during development. The VIP level in portal vein was also significantly higher than that in peripheral blood during development. In conclusion, the levels of VIP and VIPR1 in mucosal crypt, submucosal layer nerve, myenteric nerve plexus of annular muscle and indulge muscle increase rapidly after birth. Most of VIP from intestinal tract is degraded in portal vein before entering liver, suggesting that VIP does not metabolize and decompose in liver, and that VIPR1 is only present in embryo hepatic blood vessels.
Animals ; Animals, Newborn ; Fetus ; Intestine, Small ; metabolism ; Liver ; metabolism ; Macaca mulatta ; embryology ; growth & development ; metabolism ; Receptors, Vasoactive Intestinal Polypeptide, Type I ; metabolism ; Vasoactive Intestinal Peptide ; metabolism

It was hypothesized that the VPAC1 agonist may exert anti-obesity functions because VPAC1 is involved in the anorexigenic effects and the anti-inflammatory function of pituitary adenylate cyclase-activating polypeptide (PACAP)/vasoactive intestinal polypeptide (VIP). Furthermore, our in vitro test showed that the expression of VPAC1 increased significantly after the 3T3-L1 adipocytes were differentiated, and that incubation of adipocytes with VPAC1 agonist (10-1 000 nmol/L per 1x10(6) cells) resulted in stimulation of lipolysis. To test the effect of VPAC1 agonist [Lys15, Arg16, Leu27]-VIP (1-7) GRF (8-27) on diet-induced obesity (DIO), we further designed the following two in vivo experiments: (1) Mice were fed on high-fat diet (HFD) and intraperitoneally (i.p.) treated with VPAC1 agonist simultaneously for 28 d; (2) Mice were given HFD for 35 d, and subsequently fed on the same HFD and i.p. treated with VPAC1 agonist for the next 28 d. The physiological indices, including body weight, weight of white adipose tissue, plasma glucose and blood lipid, were collected. The results showed that treatment with VPAC1 agonist inhibited ingestion significantly and prevented the elevations in body weight and the weights of the white adipose tissues (epididymal and dorsal) induced by HFD. The increases in plasma glucose, cholesterol, triglycerides and LDL induced by HFD were also down-regulated in mice treated with VPAC1 agonist. VPAC1 agonist treatment also improved the glucose tolerance. Therefore, VPAC1 agonist treatment inhibits the development of the obesity induced by HFD and helps to improve the morbidities associated with DIO.
3T3-L1 Cells ; Adipocytes ; drug effects ; Animals ; Body Weight ; Diet, High-Fat ; Mice ; Obesity ; drug therapy ; Receptors, Vasoactive Intestinal Polypeptide, Type I ; agonists ; Vasoactive Intestinal Peptide ; pharmacology

OBJECTIVE: To determine whether chronic alcoholism alters the expression levels of Vasoactive intestinal polypeptide (VIP) and its receptor (VIPR1) in the cochlea of chronic alcoholism rats. METHOD: We measured their expression levels in 30 SD rats, in which we created models of different degrees of chronic alcoholism. We investigated the presence of the mRNA of VIP in the cochlea of chronic alcoholism rats and controls by reverse transcription-polymerase chain reaction (RT-PCR) method. We investigated the presence of proteins of VIPR1 in poisoned rats and controls by western blot. We also evaluated the local distribution of VIP cells by immunohistochemistry. RESULT: We found that the levels of VIP and VIPR1 were downregulated in the chronic alcoholism groups compared to the controls group. The differences in some expression levels were significant different between chronic alcoholism rats and control rats. Moreover, at different degrees of alcohol poisoning in rats, the contents of VIP and VIPR1 differed. Decreased levels of VIP and VIPR1 were detected in the deep chronic alcoholism group compared to the group with low-degree poisoning (P < 0.05). In spiral ganglion cell plasm the expression of VIP and VIPR1 had no significant difference in three groups (P > 0.05). CONCLUSION These results suggest that VIP and VIPR1 play an important role in the auditory function in rats with chronic alcoholism. Chronic alcoholism may cause a peptide hormone secretion imbalance in the auditory system, eventually leading to hearing loss.
Alcoholism ; metabolism ; Animals ; Cochlea ; metabolism ; Disease Models, Animal ; Down-Regulation ; RNA, Messenger ; Rats ; Rats, Sprague-Dawley ; Receptors, Vasoactive Intestinal Polypeptide, Type I ; metabolism ; Spiral Ganglion ; Vasoactive Intestinal Peptide ; metabolism