Vasoactive Intestinal Peptide Receptor Expression Research Articles

Effect of vasoactive intestinal peptide (VIP) on cytokine production and expression of VIP receptors in thymocyte subsets

Intrathymic T cell precursors undergo a programmed sequence of developmental changes resulting in the production of mature, self-MHC restricted, single positive T lymphocytes which migrate to the periphery. The intrathymic T cell development is controlled by various factors, including cytokines and possibly neuroendocrine hormones. Our previous studies indicate that vasoactive intestinal peptide (VIP) inhibits IL-2 and IL-4 production in thymocytes through different molecular mechanisms. Thymocytes acquire the competence to express IL-2 and IL-2R during thymic development in a maturation-dependent manner. In this study we investigate the effect of VIP on IL-2 production, and the expression of VIP-R1 and VIP-R2 mRNA in different thymocyte subsets in comparison to T cell lines. All thymocyte subsets and T cell lines tested express VIP-R2. In contrast, only single positive, CD4 +8 − and CD4 −8 + thymocytes express VIP-R1. VIP inhibits IL-2 production in CD4 +8 + and single positive CD4 +8 − and CD4 −8 + thymocytes and in TH1 cells stimulated through the TCR. No inhibition is observed in CD3 −4 −8 − and single positive CD4 +8 − and CD4 −8 + thymocytes, or in TH1 cells stimulated by a combination of calcium ionophores and phorbol esters. These findings suggest that VIP inhibits IL-2 production through VIP-R2, and that it interferes with a TCR-connected transduction pathway. We also investigate the expression of VIP mRNA in thymocyte subsets and T cell lines, and conclude that thymocytes as well as antigen-specific T cells may function as VIP sources within the lymphoid organs.

Ontogenic Development of the Adenylyl Cyclase Enzyme and the α s, α i1 and α i2 G-protein Regulatory Subunits from Rat Prostate

The expression of α s, α i1 and α i2 G-protein subunits measured by immunoblot increased in the rat prostate during sexual maturation, supporting their involvement in proliferation/differentiation. Northern blotting gave transcripts of 1.8 and 4 kb for α s, 1.4 and 4.5 kb (mainly) for α i1, and 2.4 kb for α i2 with levels suggesting a differential regulation (at transcription or post-transcription for α s, transcription for α i1, and translation for α i2). The stimulatory effects of forskolin, vasoactive intestinal peptide (VIP) and isoproterenol on adenylyl cyclase activity increased between 0.5–3 mo, remained constant up to 12 mo and decreased thereafter, conceivably following the expression of VIP and β-adrenergic receptors. However, G-protein activation of adenylyl cyclase (by GTP and Gpp[NH]p) was maximal at 0.5 mo and then decreased as it occurred with toxin-catalyzed ADP-ribose incorporation to α subunits suggesting that other factors are also involved in the regulation of G-protein activity during rat prostatic development.

Expression of vasoactive intestinal peptide in lymphocytes: a possible endogenous role in the regulation of the immune system

Experimental evidence is accumulating showing that vasoactive intestinal peptide (VIP) acts as an immunoregulatory peptide. Findings from our laboratory and others indicate that cells of the immune system are able to produce VIP. We have detected immunoreactivity for VIP in lymphocytes by immunohistochemical methods at specific locations of both central and peripheral lymphoid organs. Double immunofluorescence staining and flow cytometry analysis indicate that both T and B lymphocytes contain VIP that has been proved to be mostly VIP 1–28 by high-performance liquid chromatography and radioimmunoassay. VIP has been also demonstrated by ‘ in situ’ hybridization and reverse transcription followed by polymerase chain reaction. We have also detected induction of VIP in splenic lymphocytes after mitogenic stimulation. Lymphocytes should be sensitive to the endogenously produced VIP because we have also detected VIP receptor expression in different populations of lymphocytes. All this evidence indicates that VIP is an endogenous autocrine modulator of immune function.

The Autocrine Function of Vasoactive Intestinal Peptide on Human Neuroblastoma Cell Growth and Differentiation

Direct associations between serum concentrations and immunohistochemically detectable vasoactive intestinal peptide (VIP) and maturing neuroblastoma have been documented. Furthermore, VIP has been shown to induce both the growth inhibition and morphological differentiation of cultured human neuroblastoma cell lines. As such, it is hypothesized that VIP may be operative in the autocrine regulation of neuroblastic growth and differentiation. To test this hypothesis, VIP-induced differentiation of human neuroblastoma LA-N-5 cells was performed. Significant concomitant increases in both intracellular and extracellular VIP concentrations were observed. In addition, a marked increase in VIP receptor expression was demonstrated with VIP-induced cellular differentiation. Receptor function was maintained with enhanced expression, as evidenced by an increase in the generation of intracellular cyclic adenosine monophosphate in response to exogenous VIP stimulation. Concomitant enhancement of both intracellular and extracellular VIP expression, coupled with the induction of functional specific VIP receptors during VIP-induced differentiation, provides critical evidence for the autocrine regulation of neuroblastoma maturation by this peptide.

Insertion and internalization of vasoactive intestinal peptide (VIP) receptors in murine CD4 T lymphocytes

The specific binding of vasoactive intestinal peptide (VIP) to murine lymphocytes was investigated. CD4 T cells from mesenteric lymph nodes (MLN) bound more 125I-VIP than did unseparated MLN lymphocytes at 37°C, but not at 4°C. The differences between the amount of 125I-VIP bound by the CD4 T cells and unseparated MLN lymphocytes at 37°C depended upon a difference in the amount of the ligand that was internalized by the cells. The rate of insertion of unoccupied VIP receptors from the cytoplasm into the cell membrane (370 receptors/cell/min), the rate constants for internalization of ligand occupied VIP receptors (0.55 min −1) and unoccupied VIP receptors (0.11 min −1), and the rate constant for the elimination of internalized VIP (0.07 min −1) by CD4 T cells were evaluated. These results provide new understanding of the behaviour of VIP receptors on lymphocytes and indicate a mechanism by which CD4 T lymphocytes can homologously regulate their surface expression of VIP receptors in the presence of ambient VIP.

Autoregulation of neuroblastoma growth by vasoactive intestinal peptide

Elevated serum levels of vasoactive intestinal peptide (VIP) are associated with some cases of neuroblastoma and correlate with a favorable prognosis. VIP has previously been shown in our laboratory to cause the in vitro growth inhibition and morphological differentiation of the human neuroblastoma cell line, LA-N-5. It is now shown that LA-N-5 cells express immunoreactive VIP and bear specific VIP receptors. Antagonism of endogenous VIP, either by competitive inhibition or receptor blockade, increased cell proliferation, suggesting that VIP is operative in normal growth regulation. Intracellular and extracellular levels of VIP were also shown to increase significantly during the retinoic acid-induced differentiation of these cells. Furthermore, a concomitant marked increase in VIP receptor expression was demonstrated with cellular differentiation. These receptors remain functional as evidenced by a matching increase in the level of detectable cAMP generated in response to exogenous VIP. It is concluded that VIP is a normal autoregulator of neuroblastoma cell growth and differentiation, and that retinoic acid-mediated differentiation may be, in part, due to endogenous VIP.

VASOACTIVE-INTESTINAL-PEPTIDE (VIP) MODULATES THE GROWTH FRACTION OF EPITHELIAL SKIN CELLS

Using the human keratinocyte cell line HaCaT, modifications of the growth fraction due to vasoactive intestinal peptide (VIP) were determined by immunostaining with monoclonal antibody Ki67. In addition, the expression of VIP receptor and epidermal growth factor (EGF) receptor have been analysed. VIP (10-(7) to 10-(11) M) produced an almost doubling of the total number of Ki67-positive cells in cultures with 2% fetal calf serum (FCS), wheras it was ineffective in FCS-free and 10% FCS cultures. The nuclear Ki67-staining patterns were classified into four categories. In FCS-free cultures VIP induced a shift from type III (light nucleus, staining nuclei) to type II (multiple, intensely stained spots). In cultures with 2% FCS, VIP induced a shift from type II to type III. VIP receptor expression was facilitated by VIP, when cells were grown in a medium supplemented with 10% FCS. VIP increased EGF receptor expression in FCS-free cultures but decreased the number EGF receptor-positive cells in experiments with 2% FCS. In conclusion, VIP is capable to modulate the growth fraction and receptor expression of HaCaT cells in vitro. The effects are dependent on the concentration of FCS within the culture medium. The findings might be of interest for keratinocyte pathology in general and dermatooncology in particular.

Receptors for vasoactive intestinal peptide on murine lymphocytes turn over rapidly

The interaction of the neuropeptide vasoactive intestinal peptide (VIP) with its receptors on murine mesenteric lymph node lymphocytes (MLN) has been re-examined in detail. Intact MLN actively internalize surface bound VIP. the rate constants associated with the insertion of receptors at the MLN surface, the internalization of VIP occupied and unoccupied receptors and the elimination of the peptide were determined. At 37°C, MLN insert approximately 140 VIP receptors cell −1 min −1 at their surface, and the rate of internalization of occupied receptors (0.23 min −1) was much greater than that of the unoccupied receptors (0.02 min −1). Exposure of MLN to non-saturating concentrations of VIP markedly altered the expression of VIP receptors at the lymphocyte surface. The rapid turnover of VIP receptors combined with the differential clearance of occupied and unoccupied receptors from the cell surface provides a mechanism by which homologous regulation of VIP receptor expression can occur on these lymphocytes.

Vasoactive intestinal peptide receptor expression on human lymphoblasts.

This study was designed to determine which (if any) subtypes of leukemic blasts express a functional receptor for vasoactive intestinal peptide (VIP). Blasts harvested from bone marrow of 38 newly diagnosed patients were classified as acute lymphocytic leukemia (CALLA + pre-B-cell leukemia, CALLA-, pre-B-cell leukemia, T-cell leukemia) or acute myeloid leukemia based on cytochemical and histochemical markers. Of the 32 patients with lymphocytic leukemia, 22 expressed the VIP receptor as evidenced by VIP-mediated activation of adenylate cyclase in cell homogenates. Binding of 125I-VIP to ALL cells correlated with the ability of VIP to activate adenylate cyclase. The VIP receptor was not identified in myeloid blasts from any of six patients. Further correlation of 125I-VIP binding and VIP-mediated stimulation of adenylate cyclase was demonstrated in transformed cell lines: a pre-B-cell line (Nalm 6) and a T-cell line (Molt 4b) exhibited high-affinity binding of 125I-VIP and VIP-mediated activation of adenylate cyclase, whereas neither the histiocytic line (U937) nor the myelocytic line (HL60) appeared to express the VIP receptor. These observations suggest a role for VIP in the proliferation or differentiation of human T and B lymphocytes.

Modulation of the expression of the VIP receptor by serum factors on the human melanoma cell line IGR39

IGR39 cells, isolated from a human superficial melanoma, display at their surface high and low affinity receptors for the vasoactive intestinal peptide (VIP). When grown in DME medium supplemented with 10% fetal calf serum, cells display 1.6 x 10(5) high affinity (Kd 0.74 nM) and 5.6 x 10(5) low affinity (Kd 55 nM) VIP binding sites per cell. When cultured in a chemically defined medium containing EGF, transferrin, and selenium, IGR39 cells display many neurite-like extensions. Following these morphological changes, the specific [125I]VIP binding is increased four- to fivefold after 6 days in culture. This phenomenon is reversible and is the result of an increased number of VIP binding sites available at the cell surface, without modification of their affinities. The molecular mass of the binding sites is also unchanged whatever cell culture conditions. Increase in [125I]VIP binding is inversely correlated to the serum concentration in the culture medium. When added to the chemically defined medium, sera from various origins as well as some serum substitutes reduce [125I]VIP binding to the same extent as that of the serum. The total cAMP production by VIP-stimulated IGR39 cells is enhanced by a factor of six to seven when cells are cultured in serum-free medium, in good correlation with the increase of VIP binding capacity. These data suggest that factor(s) present in fetal calf serum inhibit(s) the expression of VIP receptor, thus demonstrating the importance of a strict control of cell culture conditions for in vitro studies.

Retinoic acid enhances VIP receptor expression and responsiveness in human neuroblastoma cell, SH-SY5Y

Retinoic acid (RA) induces partial differentiation of neuroblastoma (NB) cells in vitro. In the human NB line, SH-SY5Y (a neuroblastic subclone of SK-N-SH), RA was previously shown to enhance the stimulatory (PGE 1) and inhibitory (opioid) regulation of adenylyl cyclase. Since these cells are also sensitive to cAMP stimulation by vasoactive intestinal peptide (VIP), we have tested the effects of RA on VIP receptor expression and function. Pretreatment of SH-SY5Y cells with 10 μM RA over 6 days dramatically increased VIP receptor number from ∼3 000 to ∼70 000 sites per cell and enhanced threefold the cAMP accumulation after external VIP addition, while VIP immunoreactive content in the cells increased 2–3-fold. In the light of the recently proposed autocrine function of VIP in this cell lineage, the strong enhancement of the VIP system may contribute to the differentiation effects of RA.

Receptors for vasoactive intestinal peptide on isolated human sweat glands

Human sweat gland function is predominantly under cholinergic control. Sweat secretion may also be regulated by Vasoactive Intestinal Peptide (VIP), which coexists with acetylcholine in nerves to sweat gland acini and ducts. We have examined the expression of VIP receptors on isolated human eccrine sweat glands. Two classes of specific binding sites for VIP were demonstrated: one with high affinity (K d=0.58-2.4 nM), and another with low affinity (K d=175–288 nM). The data suggest a physiological regulatory role for VIP in sweat gland function.

In vitro alteration of receptors for vasoactive intestinal peptide changes the in vivo localization of mouse T cells.

The capacity of T lymphocytes exposed in vitro to the neuropeptide vasoactive intestinal peptide (VIP) to bind VIP in vitro and to migrate to different tissues in vivo has been studied. VIP treatment of T cells resulted in a time- and dose-dependent loss of the ability of T cells to specifically bind radioiodinated VIP. Altered binding was due to a decrease in the expression of cellular receptors for VIP on the treated cells rather than an alteration in the affinity of the cells for the neuropeptide. Alteration of VIP receptor expression was not associated with a change in the expression of Thy-1, Lyt-1, or Lyt-2 surface markers by the treated cells. VIP treatment of T cells in vitro resulted, however, in a dose-dependent decrease in the ability of the treated cells to localize in mesenteric lymph nodes (MLN) and Peyer's patches of recipient animals at early times after cell transfer, and this was due to a selective decrease in the rate of accumulation of the treated cells in these tissues. There was no alteration in the distribution of VIP-treated cells in the blood, spleen, liver, or other major organs of the recipient animals. It is concluded that the presence of VIP receptors on T cells facilitates the entry of T cells into MLN and Peyer's patches in vivo, and it is proposed that this effect is mediated by T cell-VIP interactions in the vicinity of the specialized endothelium of those tissues.