PACAP and its role in primary headaches.

Pituitary adenylate cyclase-activating peptide (PACAP) is a neuropeptide implicated in a wide range of functions, such as nociception and in primary headaches. Regarding its localization, PACAP has been observed in the sensory trigeminal ganglion (TG), in the parasympathetic sphenopalatine (SPG) and otic ganglia (OTG), and in the brainstem trigeminocervical complex. Immunohistochemistry has shown PACAP-38 in numerous cell bodies of SPG/OTG, co-stored with vasoactive intestinal peptide (VIP), nitric oxide synthase (NOS) and, to a minor degree, with choline acetyltransferase. PACAP has in addition been found in a subpopulation of calcitonin gene-related peptide (CGRP)-immunoreactive cells in the trigeminal system. The PACAP/VIP receptors (PAC1, VPAC1, and VPAC2) are present in sensory neurons and in vascular smooth muscle related to the trigeminovascular system. It is postulated that PACAP is involved in nociception. In support, abolishment of PACAP synthesis or reception leads to diminished pain responses, whereas systemic PACAP-38 infusion triggers pain behavior in animals and delayed migraine-like attacks in migraine patients without marked vasodilatory effects. In addition, increased plasma levels have been documented in acute migraine attacks and in cluster headache, in accordance with findings in experimental models of trigeminal activation. This suggest that the activation of the trigeminal system may result in elevated venous levels of PACAP, a change that can be reduced when headache is treated. The data presented in this review indicate that PACAP and its receptors may be promising targets for migraine therapeutics.

Development of a voided urine assay for detecting prostate cancer non-invasively: a pilot study.

OBJECTIVE: To validate a hypothesis that prostate cancer can be detected non-invasively by a simple and reliable assay by targeting genomic VPAC receptors expressed on malignant prostate cancer cells shed in voided urine. PATIENTS/SUBJECTS AND METHODS: VPAC receptors were targeted with a specific biomolecule, TP4303, developed in our laboratory. With an Institutional Review Board exempt approval of use of de-identified discarded samples, an aliquot of urine collected as a standard of care, from patients presenting to the urology clinic (207 patients, 176 men and 31 women, aged ≥21 years) was cytospun. The cells were fixed and treated with TP4303 and 4,6-diamidino-2-phenylindole (DAPI). The cells were then observed under a microscope and cells with TP4303 orange fluorescence around the blue (DAPI) nucleus were considered 'malignant' and those only with a blue nucleus were regarded as 'normal'. VPAC presence was validated using receptor blocking assay and cell malignancy was confirmed by prostate cancer gene profile examination. RESULTS: The urine specimens were labelled only with gender and presenting diagnosis, with no personal health identifiers or other clinical data. The assay detected VPAC positive cells in 98.6% of the men with a prostate cancer diagnosis (141), and none of the 10 men with benign prostatic hyperplasia. Of the 56 'normal' patients, 62.5% (35 patients, 10 men and 25 women) were negative for VPAC cells; 19.6% (11, 11 men and no women) had VPAC positive cells; and 17.8% (10, four men and six women) were uninterpretable due to excessive crystals in the urine. Although data are limited, the sensitivity of the assay was 99.3% with a confidence interval (CI) of 96.1-100% and the specificity was 100% with a CI of 69.2-100%. Receptor blocking assay and fluorescence-activated cell sorting (FACS) analyses demonstrated the presence of VPAC receptors and gene profiling examinations confirmed that the cells expressing VPAC receptors were malignant prostate cancer cells. CONCLUSION: These preliminary data are highly encouraging and warrant further evaluation of the assay to serve as a simple and reliable tool to detect prostate cancer non-invasively.

Atypical nuclear localization of VIP receptors in glioma cell lines and patients.

An increasing number of G protein-coupled receptors, like receptors for vasoactive intestinal peptide (VIP), are found in cell nucleus. As VIP receptors are involved in the regulation of glioma cell proliferation and migration, we investigated the expression and the nuclear localization of the VIP receptors VPAC1 and VPAC2 in this cancer. First, by applying Western blot and immunofluorescence detection in three human glioblastoma (GBM) cell lines, we observed a strong nuclear staining for the VPAC1 receptor and a weak nuclear VPAC2 receptor staining. Second, immunohistochemical staining of VPAC1 and VPAC2 on tissue microarrays (TMA) showed that the two receptors were expressed in normal brain and glioma tissues. Expression in the non-nuclear compartment of the two receptors significantly increased with the grade of the tumors. Analysis of nuclear staining revealed a significant increase of VPAC1 staining with glioma grade, with up to 50% of GBM displaying strong VPAC1 nuclear staining, whereas nuclear VPAC2 staining remained marginal. The increase in VPAC receptor expression with glioma grades and the enhanced nuclear localization of the VPAC1 receptors in GBM might be of importance for glioma progression.

Molecular cloning of VIP and distribution of VIP/VPACR system in the testis of Podarcis sicula.

Using molecular, biochemical, and cytological tools, we studied the nucleotide and the deduced amino acid sequence of PHI/VIP and the distribution of VIP/VPAC receptor system in the testis of the Italian wall lizard Podarcis sicula to evaluate the involvement of such a neuropeptide in the spermatogenesis control. We demonstrated that (1) Podarcis sicula VIP had a high identity with other vertebrate VIP sequences, (2) differently from mammals, VIP was synthesized directly in the testis, and (3) VIP and its receptor VPAC2 were widely distributed in germ and somatic cells, while the VPAC1 R had a distribution limited to Leydig cells. Our results demonstrated that in Podarcis sicula the VIP sequence is highly preserved and that this neuropeptide is involved in lizard spermatogenesis and steroidogenesis.

VIPoma with expression of both VIP and VPAC1 receptors in a patient with WDHA syndrome.

We report a case of VIPoma in a 72-year-old female patient who presented with excessive diarrhea, severe hypokalemia, and acidemia. She had been referred to our hospital three times because of severe diarrhea. No primary tumor site was found by conventional techniques, including contrast-enhanced CT and MRI, angiography, endoscopy, and positron emission tomography (PET), but a tumor was subsequently found in the head of the pancreas by octreotide scanning. Her diarrhea diminished dramatically after octreotide treatment, while her diarrhea has ceased without the therapy of octreotide at the first admission in the course of 2 years of her disease. Immunohistochemial analysis of the excised tumor tissue revealed the expression of both vasoactive intestinal peptide (VIP) and VIP and pituitary adenylate cyclase-activating peptide 1 (VPAC1) receptors. This is the first case report of a VIPoma that immunostains for VIP and VPAC1 receptors and indicates that abundant VIP produced by VIPoma might inhibit its growth and reduce VIP secretion via the VPAC1 receptor in vivo.

TCR signaling and environment affect vasoactive intestinal peptide receptor-1 (VPAC-1) expression in primary mouse CD4 T cells.

Strict regulation of T cell function is imperative to control adaptive immunity, and dysregulation of T cell activation can contribute to infectious and autoimmune diseases. Vasoactive intestinal peptide receptor-1 (VPAC-1), an anti-inflammatory G-protein coupled receptor, has been reported to be downregulated during T cell activation. However, the regulatory mechanisms controlling the expression of VPAC-1 in T cells are not well understood. Therefore, mouse splenic CD4 T cells were treated in complete media+/-anti-CD3 for 24h, total RNA isolated and VPAC-1 levels measured by qPCR. Surprisingly, we discovered that T cells incubated in complete media steadily upregulated VPAC-1 mRNA levels over time (24h). Importantly, CD4 T cells isolated from blood also showed elevated VPAC-1 expression compared to splenic T cells. Collectively, these data support that the vascular environment positively influences VPAC-1 mRNA expression that is negatively regulated by TCR signaling. This research was supported by a national service award (1KO1 DK064828) to G.D., the Center for Protease Research (2P20RR015566), and INBRE (P20 RR016741).

Expression localisation and functional activity of pituitary adenylate cyclase-activating polypeptide, vasoactive intestinal polypeptide and their receptors in mouse ovary.

Pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) positively affect several parameters correlated with the ovulatory process. PACAP is transiently expressed in rat preovulatory follicles, while VIP is present in nerve fibres at all stages of development. These two peptides act by interacting with three types of receptors: PACAP type I receptor (PAC1-R), which binds with higher affinity to PACAP, and two VIP receptors (VPAC1-R and VPAC2-R), which bind to PACAP and VIP with equal affinity. The aim of the present study was to characterise the PACAP/VIP/receptor system in the mouse ovary. Results obtained by RT-PCR, immunohistochemistry and in situ hybridisation showed that PACAP was transiently expressed in granulosa cells of preovulatory follicles after human chorionic gonadotrophin (hCG) stimulation, while VIP mRNA was never observed. All the receptors were present in 22-day-old untreated mice. In preovulatory follicles, PAC1-R was expressed both in granulosa cells and in residual ovarian tissue but was stimulated by hCG mainly in granulosa cells; VPAC2-R was present in both the cell compartments and was only mildly stimulated; VPAC1-R was present mainly in the residual ovarian tissue and was downregulated by hCG. PACAP and VIP were equipotent in inhibiting apoptosis in granulosa cells, confirming the presence of functional PACAP/VIP receptors. The contemporary induction by hCG of PACAP and PAC1-R in granulosa cells of preovulatory follicles suggests that, also in mouse ovary, PACAP may play a significant role around the time of ovulation. Moreover, the presence of PACAP/VIP receptors in the untreated ovary suggests a possible role for PACAP and VIP during follicle development.

Characterization and expression of different pituitary adenylate cyclase-activating polypeptide/vasoactive intestinal polypeptide receptors in rat ovarian follicles.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a bioactive peptide transiently expressed in preovulatory follicles. PACAP acts by interacting with three types of PACAP receptors. PACAP type I receptor (PAC(1)-R), which binds specifically to both PACAPs and vasoactive intestinal polypeptide (VIP), although with lower affinity, and two VIP receptors, VPAC(1)-R and VPAC(2)-R, which bind to PACAP and VIP with equal affinity. In the present study, we showed the expression of all three receptors in whole ovaries obtained from juvenile and gonadotropin-treated immature rats. A more detailed analysis on cells from preovulatory follicles showed that PAC(1)-R and VPAC(2)-R were expressed in granulosa cells, whereas only VIP receptors were expressed in theca/interstitial (TI) cells and fully grown oocytes presented only PAC(1)-R. The distribution of the VIP receptors was confirmed by immunofluorescence. HCG treatment induced stimulation of PAC(1)-R in granulosa cells and VPAC(2)-R in TI cells. The presence of functional PACAP/VIP receptors was also supported by metabolic studies. We further evaluated the presence of PACAP and VIP receptors by testing the effect of these peptides on apoptosis in granulosa cells cultured, isolated or in whole follicles. Treatment of follicles with PACAP and VIP dose-dependently inhibited apoptosis, while only PACAP significantly inhibited isolated granulosa cells. These results demonstrate a different expression of PACAP/VIP receptors in the various follicle compartments and suggest a possible role for PACAP and VIP on granulosa and TI cells, both during follicle development and ovulation.

Human plasmacytoid dendritic cell function: inhibition of IFN-alpha secretion and modulation of immune phenotype by vasoactive intestinal peptide.

Plasmacytoid dendritic cells (PDC) are considered the main sentinels against viral infections and play a major role in immune tolerance. Vasoactive intestinal peptide (VIP) is a potent immunomodulator, whose role in PDC function is unknown. The present study was designed to investigate whether human PDC express VIP receptors and whether VIP has immunological effects on PDC. Using real-time RT-PCR and immunofluorescence, we demonstrated that VIP receptors VPAC1 and VPAC2 are expressed on PDC. After culturing PDC with VIP and CpG oligodeoxynucleotides for 48 h, expression of surface molecules with significance for PDC-T cell interactions as well as IFN-alpha secretion were quantified using FACS analysis and ELISA, respectively. For functional assays, CFSE-stained CD4+ T cells were coincubated with differentially treated PDC. T cell proliferation and production of various cytokines were determined by FACS analysis and ELISA. VIP enhanced PDC expression of CD86, MHC II, and CCR7. In contrast, VIP inhibited PDC secretion of IFN-alpha and expression of Neuropilin-1 and MHC I. The potential of CpG oligodeoxynucleotide-activated PDC to induce proliferation of allogeneic CD4(+) T cells was impaired when VIP was present during activation. Furthermore, pretreatment of PDC with VIP resulted in a decrease of the IFN-gamma:IL-4 ratio in cocultured T cells, suggesting a modulation of the immune response toward Th2. Taken together, these results strongly suggest that VIP regulates the immunological function of human PDC. VIP may thus be involved in the modulation of immune responses to viral infections as well as in the maintenance of immune tolerance.