Activation of the CRF 2 receptor modulates skeletal muscle mass under physiological and pathological conditions

Two receptors activated by the corticotropin-releasing factor (CRF) family of peptides have been identified, the CRF 1 receptor (CRF1R) and the CRF 2 receptor (CRF2R). Of these, the CRF2R is expressed in skeletal muscle. To understand the role of the CRF2R in skeletal muscle, we utilized CRFR knockout mice and CRF2R-selective agonists to modulate nerve damage and corticosteroid- and disuse-induced skeletal muscle atrophy in mice. These analyses demonstrated that activation of the CRF2R decreased nerve damage and corticosteroid- and disuse-induced skeletal muscle mass and function loss. In addition, selective activation of the CRF2R increased nonatrophy skeletal muscle mass. Thus we describe for the first time a novel activity of the CRF2R, modulation of skeletal muscle mass.     

Epithelium modulates the potency of vasoactive intestinal peptide in the guinea pig.

The purpose of the study was to evaluate the importance of the epithelium in determining the potency of exogenous vasoactive intestinal peptide (VIP) in inhibiting responses of isolated guinea pig trachea to vagal stimulation. Isolated innervated tracheal preparations (n = 56) were mounted in glass organ baths in Krebs-Henseleit (K-H) solution at 37 degrees C and gassed with 95% O2-5% CO2. The inside of the trachea was separately perfused with K-H solution at 1 ml/min. The vagal nerve trunks were stimulated (20 V, 1-ms pulses, 10-s trains) at low (0.5 Hz) and high frequency (15 Hz) alternately, and the contractile responses were measured as increases in intratracheal pressures. VIP (10(-8)-10(-7) M) inhibited responses to both high- and low-frequency stimulation. VIP was more potent in inhibiting contractions when administered to the outside than the inside surface of the trachea, and disruptionon of the epithelium abolished this difference. The endopeptidase inhibitors phosphoramidon and thiorphan (5 x 10(-6) M) potentiated the action of VIP. These data indicate that the epithelium reduces the efficacy of VIP. We suggest that the epithelium is a site of degradation of VIP by endopeptidase and may also be a diffusion barrier.     

Structure of the human VIPR2 gene for vasoactive intestinal peptide receptor type 2.

The VPAC(2) (vasoactive intestinal peptide (VIP)(2)) receptor is a seven-transmembrane spanning G protein-coupled receptor which responds similarly to VIP and pituitary adenylate cyclase activating polypeptide (PACAP) in stimulating cAMP production. Recently, we reported the localisation of the human VPAC(2) receptor gene (VIPR2) to chromosome 7q36.3 (Mackay, M. et al. (1996) Genomics 37, 345-353). Here, we describe the characterisation of the VIPR2 gene structure and promoter region. The VIPR2 gene is encoded by 13 exons, the initiator codon of the 438 amino acid open reading frame is located in exon 1 and the termination signal and a poly-adenylation signal sequence are located in exon 13. The 5' untranslated region extends 187 bp upstream of the initiator codon and is extremely GC-rich (80%). The poly-adenylation signal is located 2416 bp downstream of the stop codon. Intron sizes range from 68 bp (intron 11) to 45 kb (intron 4) and the human gene spans 117 kb.     

Gender-dependent association of type 2 diabetes with the vasoactive intestinal peptide receptor 1

Type 2 diabetes is characterized by an inadequate pancreatic beta-cell response to the progressive insulin resistance. Its pathogenesis is complex and has been connected with a state of preclinical chronic inflammation. Vasoactive intestinal peptide (VIP) and its receptors play a relevant role in the homeostasis of insulin secretion as well as in the control of inflammation. In particular, VIP receptor 1 (VPAC1) has been found to be down-modulated during inflammation, and to be associated with several diseases. The objective of this study was to compare the distribution of SNPs mapping in the VIP receptor 1 gene in cases with type 2 diabetes and matched controls. Seven hundred cases with type 2 diabetes (423 males and 277 females) and 830 random controls (419 males and 411 females) were analyzed for the distribution of three common SNPs mapping in the VPAC1 gene. The results show a significantly different genotype distribution of the SNP rs9677 in the 3’-UTR of VPAC1 in female cases with type 2 diabetes compared to gender-matched controls (ptrend = 6 × 10^− 4). The rs9677 CC genotype confers the highest risk (OR: 2.1) and correlates with worse clinical parameters such as higher level of total cholesterol, higher LDL/HDL ratio and a higher HbA1c concentration. The genetic association reported here indicates that VIP/VPAC1 signaling can be a relevant pathway in the pathogenesis of type 2 diabetes in females suggesting that at least some aspects of the genetic predisposition to this disease can be gender-specific.     

Isolation of a low molecular mass vasoactive intestinal peptide binding protein.

A vasoactive intestinal peptide (VIP) binding protein was purified in active form by detergent solubilization of lung membranes, gel filtration, VIP-Sepharose affinity chromatography, reverse phase high performance liquid chromatography, and anion exchange chromatography. The mass of this protein was estimated at 18 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 17 kDa by gel filtration. The binding of VIP by this protein was inhibited by Mg2+, covalent cross-linking of [Tyr10-125I]VIP to the protein produced two radioactive bands at 22 and 26 kDa identified by electrophoresis, and the purified protein exhibited saturable and high affinity binding of VIP and the related neuropeptide, rat growth hormone releasing factor.     

Molecular properties of the vasoactive intestinal peptide receptor in aorta and other tissues.

The molecular weight of the vasoactive intestinal peptide (VIP) receptor was assessed in bovine aorta, and rat liver, lung, and brain by covalent cross-linking and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The receptor in all four tissues was found to be a single polypeptide of approximate M(r) 54,000, contradicting previous claims for substantial heterogeneity in the molecular weight of this receptor. Guanine nucleotides inhibit cross-linking of 125I-VIP to its receptor, and cross-linking with ethylene glycolbis(succinimidylsuccinate) provides further evidence for complex formation between VIP, its receptor and a guanine nucleotide-binding regulatory protein (G-protein). The precise mechanism of receptor-G-protein coupling may differ between the aorta and other tissues.     

Substance P but not vasoactive intestinal peptide modulates immunoglobulin secretion in murine schistosomiasis.

Neuropeptides including SP and VIP modulate Ig secretion by in vitro stimulated lymphocyte cultures. It is not known whether these neuropeptides effect the B cell directly, or if they significantly alter humoral immune responses to pathogens. We have previously shown that granulomas derived from schistosome-infected mice contain immunoglobulin secreting B cells (ISC) as well as eosinophils that secrete substance P (SP) and vasoactive intestinal peptide (VIP). It therefore seemed plausible that B cells derived from infected animals might respond to these neuropeptides, and that such responses might effect immunoregulatory signals. In this study, we addressed these issues in the murine Schistosoma mansoni model, at the level of immunoglobulin secretion in single B cells. Spontaneous ISC were observed in both splenic and granuloma cell preparations. The addition of SP resulted in a dose-dependent reduction in the number and size of plaques (a 50% reduction was observed at 10(-9) M). This effect was blocked with SP antagonists. Similar results were observed in T cell-depleted cell cultures. VIP had no effect on ISC number or plaque size. We conclude that SP, but not VIP, decreases spontaneous ISC number and Ig secretion in short-term cultures of spleen and granuloma cells. SP appears to exert its effects at the level of single B cells through a receptor-mediated mechanism and may thus play an immunoregulatory role in schistosomiasis.     

Characterization of vasoactive intestinal peptide receptors by a photoaffinity label. Site-specific modification of vasoactive intestinal peptide by derivatization of the receptor-bound peptide

The biological effects of vasoactive intestinal peptide (VIP) are mediated by binding to a membrane-bound receptor. Probes designed to trap this receptor by binding to it in a covalent way may suffer from a greatly reduced affinity. We report here, for the VIP receptor, the use of a photoaffinity probe obtained by derivatization of receptor-bound VIP with para-azidophenylglyoxal. This method protected the parts of the molecule essential for receptor binding. The VIP derivative thus obtained became covalently linked when irradiated. In the dark, however, it exhibited normal VIP-like behavior and retained its biological activity. This derivatization method might be generally applicable when hormone analogues have to be prepared without loss of receptor affinity. Receptor characterization studies on liver plasma membranes showed the presence of high- and low-affinity binding sites with KD = 0.1 and 5 nM, respectively. Treatment of membranes with dithiothreitol causes loss of high-affinity binding. The high-affinity site, trapped by the photoaffinity probe, resolved into two molecular mass forms, 50 and 200-250 kDa. Reduction of the receptor-probe complex left the 50-kDa form intact, whereas the amount of the 200-250-kDa form greatly diminished. We demonstrate the importance of the presence of disulfide bonds in one of the molecular forms involved in high-affinity binding.     

Regional distribution of vasoactive intestinal peptide in brains from normal and parkinsonian subjects

The distribution of vasoactive intestinal peptide (VIP) in the post-mortem human brain was determined by radioimmunoassay using a highly specific antiserum. The detection limit of the assay was 4 fmol/tube. The highest concentrations of VIP were found in the cerebral cortex, amygdala, hypothalamus and hippocampus. The lowest levels of peptide were detected in basal ganglia including caudate nucleus, external pallidum, putamen and substantia nigra. All dilution curves of acetic acid extracts from different brain areas were strictly parallel to the standard curve. Sephadex G-50 gel filtration of frontal cortex extract showed that VIP-like immunoreactivity (VIP-LI) eluted as a major peak comigrating with synthetic hVIP. Detailed mapping of VIP in the human cerebral cortex showed the existence of a rostro-caudal gradient of VIP-LI concentrations: the frontal cortex exhibited the highest VIP levels, the parietal and temporal cortex contained medium values and the occipital cortex contained the lowest VIP levels. The concentrations of VIP-LI were compared in various regions of the human brain from normal and parkinsonian subjects. No significant changes in VIP-LI levels occurred in the brains of patients dying with Parkinson's disease. No difference in VIP levels could be found either when the parkinsonian group was subdivided into nondemented and demented patients. These data indicate that VIP-containing neurons are not affected in parkinsonian patients. Our results also suggest that VIP neuronal systems are not involved in the course of dementing process in Parkinson's disease.     

Release of vasoactive intestinal peptide in the dumping syndrome.

To determine the effect of gastric surgery on the plasma vasoactive intestinal peptide (VIP) concentration, 13 patients with gastrectomy and seven controls were given an oral hypertonic load (200 ml 50% glucose solution). Blood was taken at intervals during the test for measurement of VIP and blood glucose concentrations and packed cell volume. At the same time observations were made on the occurrence of dumping symptoms and a record kept of the pulse rate. VIP values in the patients with gastrectomy were significantly increased by glucose ingestion, while these did not alter in controls (p less than 0.001). There was a highly significant correlation between the rate of rise in plasma VIP concentration and the rates of rise in packed cell volume (r = 0.85; p less than 0.001) and blood glucose concentration (r = 0.76; p less than 0.01) in patients with gastrectomy. Changes in packed cell volume and blood glucose values and the occurrence of dumping symptoms during the test were significantly different when postoperative patients were compared with controls (p less than 0.001, p less than 0.005, and p less than 0.001 respectively). Furthermore, when the patients with gastrectomy were divided into those without symptoms and those with dumping after meals the latter group showed a significantly greater rise of VIP (p less than 0.05). Despite the increased plasma VIP concentrations observed during dumping, VIP cannot be taken as the sole factor in the pathogenesis of the dumping syndrome.     

Immunohistochemical localization and vascular effects of vasoactive intestinal polypeptide in skeletal muscle of the cat

Summary Scattered vasoactive intestinal polypeptide (VIP) — immunoreactive nerves were found in the striated muscle of the hind limb of the cat, where they usually were associated with small blood vessels. VIP-immunoreactive nerves were also demonstrated in the sciatic nerve; after nerve ligation an abundance of intensely immunoreactive VIP fibres were seen proximal to the ligation. Intraarterial infusion of VIP into the isolated hind limb of the cat had dramatic effects on different sections of the vascular bed. Thus, VIP dilated the resistance vessels leading to a marked increment in muscle blood flow. VIP also relaxed the capacitance vessels causing regional pooling of blood; it increased the capillary surface area available for fluid exchange. Infusions of VIP at a dose of 8 g/min significantly inhibited the vasoconstriction induced by electrical stimulation of the regional sympathetic nerves. It is suggested that local nervous release of VIP may act as a modulator of vascular tone in skeletal muscle.     

Solubilization of the active vasoactive intestinal peptide receptor from human colonic adenocarcinoma cells

The non-ionic detergent n-octyl-beta-D-glucopyranoside was used to solubilize the VIP (vasoactive intestinal peptide) receptor from human colonic adenocarcinoma cell line HT29-D4. The binding of monoiodinated 125I-VIP to the solubilized receptor was specific, time-dependent, and reversible. Scatchard analysis of data obtained from competitive displacement of monoiodinated 125I-VIP by native VIP suggested the presence of two classes of VIP binding sites with Kd values of 0.32 and 46.7 nM. The binding capacities of these two classes were 1.7 x 10(10) and 30.2 x 10(10) sites/mg of proteins, respectively. The solubilized receptor retained the specificity of the human VIP receptor towards the peptides of the VIP/secretin/glucagon family. The order of potency in inhibiting monoiodinated 125I-VIP binding was VIP (IC50 = 1.0 x 10(-9) M) much greater than peptide histidine methionine amide (IC50 = 10(-7) M) greater than growth hormone-releasing factor (IC50 = 3 x 10(-7) M) greater than secretin (IC50 greater than 10(-6) M); glucagon had no effect on VIP binding. The reducing agent dithiothreitol inhibited in a dose-dependent manner the binding of 125I-VIP. Covalent cross-linking experiments between the solubilized receptor and 125I-VIP showed that after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography two major and one minor polypeptides of Mr 67,000, 72,000, and 83,000 were specifically labeled. When analyzed by gel filtration, the n-octyl-beta-D-glucopyranoside-solubilized 125I-VIP-receptor complex was resolved into two major peaks with molecular mass in the range of 60-70 and 270-300 kDa. Thus, the soluble form of the VIP receptor was probably a multimeric complex in which disulfide bonds may play an important role to hold the receptor in an active configuration.     

Activation of CFTR trafficking and gating by vasoactive intestinal peptide in human bronchial epithelial cells

Cystic fibrosis transmembrane conductance regulator (CFTR) is an apical membrane chloride channel critical to the regulation of fluid, chloride, and bicarbonate transport in epithelia and other cell types. The most common cause of cystic fibrosis (CF) is the abnormal trafficking of CFTR mutants. Therefore, understanding the cellular machineries that transit CFTR from the endoplasmic reticulum to the cell surface is important. Vasoactive intestinal polypeptide (VIP) plays an important role in CFTR-dependent chloride transport. The present study was designed to observe the affection of VIP on the trafficking of CFTR, and channel gating in human bronchial epithelium cells (HBEC). Confocal microscopy revealed CFTR immunofluorescence extending from the apical membrane deeply into the cell cytoplasm. After VIP treatment, apical extension of CFTR immunofluorescence into the cell was reduced and the peak intensity of CFTR fluorescence shifted towards the apical membrane. Western blot showed VIP increased cell surface and total CFTR. Compared with the augmented level of total CFTR, the surface CFTR increased more markedly. Immunoprecipitation founded that the mature form of CFTR had a marked increase in HBEC treated with VIP. VIP led to a threefold increase in Cl(-) efflux in HBEC. Glibenclamide-sensitive and DIDS-insensitive CFTR Cl(-) currents were consistently observed after stimulation with VIP (10(-8) mol/L). The augmentation of CFTR Cl(-) currents enhanced by VIP (10(-8) mol/L) was reversed, at least in part, by the protein kinase A (PKA) inhibitor, H-89 and the protein kinase C (PKC) inhibitor, H-7, suggesting PKA and PKC participate in the VIP-promoted CFTR Cl(-) currents.     

Generation of highly selective VPAC2 receptor agonists by high throughput mutagenesis of vasoactive intestinal peptide and pituitary adenylate cyclase-activating peptide

Pituitary adenylate cyclase-activating peptide (PACAP) has a specific receptor PAC1 and shares two receptors VPAC1 and VPAC2 with vasoactive intestinal peptide (VIP). VPAC2 activation enhances glucose-induced insulin release while VPAC1 activation elevates glucose output. To generate a large pool of VPAC2 selective agonists for the treatment of type 2 diabetes, structure-activity relationship studies were performed on PACAP, VIP, and a VPAC2 selective VIP analog. Chemical modifications on this analog that prevent recombinant expression were sequentially removed to show that a recombinant peptide would retain VPAC2 selectivity. An efficient recombinant expression system was then developed to produce and screen hundreds of mutant peptides. The 11 mutations found on the VIP analog were systematically replaced with VIP or PACAP sequences. Three of these mutations, V19A, L27K, and N28K, were sufficient to provide most of the VPAC2 selectivity. C-terminal extension with the KRY sequence from PACAP38 led to potent VPAC2 agonists with improved selectivity (100-1000-fold). Saturation mutagenesis at positions 19, 27, 29, and 30 of VIP and charge-scanning mutagenesis of PACAP27 generated additional VPAC2 selective agonists. We have generated the first set of recombinant VPAC2 selective agonists described, which exhibit activity profiles that suggest therapeutic utility in the treatment of diabetes.     

TRAF6 coordinates the activation of autophagy and ubiquitin-proteasome systems in atrophying skeletal muscle

Skeletal muscle wasting is a major reason for morbidity and mortality in many chronic disease states, disuse conditions and aging. The ubiquitin-proteasome and autophagy-lysosomal systems are the two major proteolytic pathways involved in regulation of both physiological and pathological muscle wasting. Tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF6) is an important adaptor protein involved in receptor-mediated activation of various signaling pathways in response to cytokines and bacterial products. TRAF6 also possesses E3 ubiquitin ligase activity causing lysine-63-linked polyubiquitination of target proteins. We have uncovered a novel role of TRAF6 in regulation of skeletal muscle mass. Muscle-wasting stimuli upregulate the expression, as well as the auto-ubiquitination, of TRAF6 leading to downstream activation of major catabolic pathways in skeletal muscle. Muscle-specific depletion of TRAF6 preserves skeletal muscle mass in a mouse model of cancer cachexia or denervation. Inhibition of TRAF6 also blocks the expression of the components of the ubiquitin-proteasome system (UPS) and autophagosome formation in atrophying skeletal muscle. While more investigations are required to understand its mechanisms of action in skeletal muscle, our results indicate that blocking TRAF6 activity can be used as a therapeutic approach to preserve skeletal muscle mass and function in different disease states and conditions.