Vasoactive intestinal peptide (VIP) is a naturally occurring 28-amino acid peptide with a wide range of biological activities. Recent reports suggest that VIP receptors are expressed on a variety of malignant tumor cells and that the receptor density is higher than for somatostatin. Our aims were to label VIP with 99mTc--a generator-produced, inexpensive radionuclide that possesses ideal characteristics for scintigraphic imaging--and to evaluate 99mTc-VIP for bioactivity and its ability to detect experimental tumors. METHODS: VIP28 was modified at the carboxy terminus by the addition of four amino acids that provided an N4 configuration for a strong chelation of 99mTc. To eliminate steric hindrance, 4-aminobutyric acid (Aba) was used as a spacer. VIP28 was labeled with 1251, which served as a control. Biological activity of the modified VIP28 agonist (TP3654) was examined in vitro using a cell-binding assay and an opossum internal anal sphincter (IAS) smooth muscle relaxivity assay. Tissue distribution studies were performed at 4 and 24 h after injection, and receptor-blocking assays were also performed in nude mice bearing human colorectal cancer LS174T. Blood clearance was examined in normal Sprague-Dawley rats. RESULTS: The yield of 99mTc-TP3654 was quantitative, and the yields of 125I-VIP and 1251-TP3654 were >90%. All in vitro data strongly suggested that the biological activity of 99mTc-TP3654 agonist was equivalent to that of VIP28. As the time after injection increased, radioactivity in all tissues decreased, except in the receptor-enriched tumor (P = 0.84) and in the lungs (P = 0.78). The tumor uptake (0.23 percentage injected dose per gram of tissue [%ID/g]) was several-fold higher than 125I-VIP (0.06 %ID/g) at 24 h after injection in the similar system. In mice treated with unlabeled VIP or TP3654, the uptake of 99mTc-TP3654 decreased in all VIP receptor-rich tissues except the kidneys. The blood clearance was biphasic; the alpha half-time was 5 min and the beta half-time was approximately 120 min. CONCLUSION: VIP28 was modified and successfully labeled with 99mTc. The results of all in vitro examinations indicated that the biological activity of TP3654 was equivalent to that of native VIP28 and tumor binding was receptor specific. (+info)
Neuroprotection of the developing brain by systemic administration of vasoactive intestinal peptide derivatives.
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Periventricular leukomalacia (PVL), a necrotic and often cystic lesion of the cerebral white matter occurring in very premature babies, is the leading cause of cerebral palsy in this population. Increased glutamate release and the excitotoxic cascade thus triggered may be critical factors in the development of PVL. The glutamatergic analog ibotenate injected intracerebrally into newborn mice produces white matter cysts that mimic human PVL. Concomitant injection of vasoactive intestinal peptide (VIP), a trophic factor, protects the white matter against excitotoxic lesions. The goal of the present study was to assess the protective properties of systemically injected VIP analogs against ibotenate-induced excitotoxic white matter lesions in newborn mice. VIP analogs were selected on the basis of their low susceptibility to endopeptidases and their potential ability to cross biological membranes. RO-25-1553, a long-lasting cyclic VIP analog, and stearyl-norleucine-VIP, a fatty derivative of VIP, reduced ibotenate-induced white matter cysts by up to 87% and 84%, respectively, when injected i.p. immediately after ibotenate. By comparison, i.p. coadministration of VIP and ibotenate was not protective against the excitotoxic insult. Furthermore, RO-25-1553 and stearyl-norleucine-VIP still induced significant neuroprotection of the developing white matter when injected systemically 8 and 12 h, respectively, after ibotenate, establishing these peptides as therapeutic agents in this murine model. VIP analogs may have therapeutic potential in human premature babies at high risk for PVL. (+info)
CFTR channel insertion to the apical surface in rat duodenal villus epithelial cells is upregulated by VIP in vivo.
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cAMP activated insertion of the cystic fibrosis transmembrane conductance regulator (CFTR) channels from endosomes to the apical plasma membrane has been hypothesized to regulate surface expression and CFTR function although the physiologic relevance of this remains unclear. We previously identified a subpopulation of small intestinal villus epithelial cells or CFTR high expressor (CHE) cells possessing very high levels of apical membrane CFTR in association with a prominent subapical vesicular pool of CFTR. We have examined the subcellular redistribution of CFTR in duodenal CHE cells in vivo in response to the cAMP activated secretagogue vasoactive intestinal peptide (VIP). Using anti-CFTR antibodies against the C terminus of rodent CFTR and indirect immunofluorescence, we show by quantitative confocal microscopy that CFTR rapidly redistributes from the cytoplasm to the apical surface upon cAMP stimulation by VIP and returns to the cytoplasm upon removal of VIP stimulation of intracellular cAMP levels. Using ultrastructural and confocal immunofluorescence examination in the presence or absence of cycloheximide, we also show that redistribution was not dependent on new protein synthesis, changes in endocytosis, or rearrangement of the apical cytoskeleton. These observations suggest that physiologic cAMP activated apical membrane insertion and recycling of CFTR channels in normal CFTR expressing epithelia contributes to the in vivo regulation of CFTR mediated anion transport. (+info)
Neuroregulation by vasoactive intestinal peptide (VIP) of mucus secretion in ferret trachea: activation of BK(Ca) channels and inhibition of neurotransmitter release.
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1. The aims of this study were to determine: (1) whether vasoactive intestinal peptide (VIP) regulates cholinergic and 'sensory-efferent' (tachykininergic) 35SO4 labelled mucus output in ferret trachea in vitro, using a VIP antibody, (2) the class of potassium (K+) channel involved in VIP-regulation of cholinergic neural secretion using glibenclamide (an ATP-sensitive K+ (K(ATP)) channel inhibitor), iberiotoxin (a large conductance calcium activated K+ (BK(ca)) channel blocker), and apamin (a small conductance K(ca) (SK(ca)) channel blocker), and (3) the effect of VIP on cholinergic neurotransmission using [3H]-choline overflow as a marker for acetylcholine (ACh) release. 2. Exogenous VIP (1 and 10 microM) alone increased 35SO4 output by up to 53% above baseline, but suppressed (by up to 80% at 1 microM) cholinergic and tachykininergic neural secretion without altering secretion induced by ACh or substance P (1 microM each). Endogenous VIP accounted for the minor increase in non-adrenergic, non-cholinergic (NANC), non-tachykininergic neural secretion, which was compatible with the secretory response of exogenous VIP. 3. Iberiotoxin (3 microM), but not apamin (1 microM) or glibenclamide (0.1 microM), reversed the inhibition by VIP (10 nM) of cholinergic neural secretion. 4. Both endogenous VIP (by use of the VIP antibody; 1:500 dilution) and exogenous VIP (0.1 microM), the latter by 34%, inhibited ACh release from cholinergic nerve terminals and this suppression was completely reversed by iberiotoxin (0.1 microM). 5. We conclude that, in ferret trachea in vitro, endogenous VIP has dual activity whereby its small direct stimulatory action on mucus secretion is secondary to its marked regulation of cholinergic and tachykininergic neurogenic mucus secretion. Regulation is via inhibition of neurotransmitter release, consequent upon opening of BK(Ca) channels. In the context of neurogenic mucus secretion, we propose that VIP joins NO as a neurotransmitter of i-NANC nerves in ferret trachea. (+info)
Muscarinic stimulation of calcium/calmodulin-dependent protein kinase II in isolated rat pancreatic acini.
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AIM: To study whether M3 receptor occupation would lead to activation of calcium/calmodulin-dependent protein kinase II (CaM kinase II). METHODS: In this study, we isolated rat pancreatic acini by collagenase digestion; measured the Ca2+/calmodulin-independent activity of autophosphorylated form of the CaM kinase II both before and after stimulation of the acini with muscarinic secretagogue bethanechol (Bet). RESULTS: Bet stimulated the activation of, or generation of Ca(2+)-independent activity of, this kinase, in a concentration (0.0001-1 mmol.L-1) and time (5-300 s)-dependent manner; with Bet of 100 mumol.L-1, Ca(2+)-independent activity increased from an unstimulated level of 4.5 +/- 0.3 (n = 4) to 8.9 +/- 1.3 (n = 4, P < 0.05) at 5 s. Another Ca2+ mobilizing secretagogue cholecystokinin (CCK) also activated the kinase; at 1 mumol.L-1, CCK increased Ca(2+)-independent kinase activity to 12.9 +/- 0.5 (n = 6, P < 0.05). Vasoactive intestinal peptide (VIP) at 1 mumol.L-1 did not produce significant Ca(2+)-independent kinase activity (from control 3.90 +/- 0.28 to 4.53 +/- 0.47, n = 6, P > 0.05). Atropine completely blocked Bet activation of the kinase. CONCLUSION: CaM kinase II plays a pivotal role in digestive enzyme secretion, especially during the initial phase of amylase secretion. (+info)
Depletion of cutaneous peptidergic innervation in HIV-associated xerosis.
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Severe xerosis occurs in approximately 20% of human immunodeficiency virus seropositive patients. Changes in cutaneous innervation have been found in various inflammatory skin diseases and in xerotic skin in familial amyloid. We have therefore carried out a quantitative examination of the cutaneous peptidergic innervation in human immunodeficiency virus-associated xerosis. Immunohistochemistry and image analysis quantitation were used to compare total cutaneous innervation (protein gene product 9.5), calcitonin gene-related peptide, substance P, and vasoactive intestinal peptide peptidergic fibers, at two sites in the skin of human immunodeficiency virus-associated xerosis patients (upper arm, n = 12; upper leg, n = 11) and site-matched seronegative controls (upper arm, n = 10; upper leg, n = 10). Measurement of lengths of fibers of each type was carried out for each subject in the epidermis and papillary dermis, and around the sweat glands. Immunostained mast cells in these areas were counted. Epidermal integrity and maturation were assessed by immunostaining for involucrin. There were significant (Mann-Whitney U test; p < 0.02) decreases in total lengths of protein gene product 9.5 fibers in both epidermis/papillary dermis and sweat gland fields; of calcitonin gene-related peptide innervation in the epidermis/papillary dermis; and of substance P innervation of the sweat glands. There were no differences in the distribution of mast cells, or in the epidermal expression of involucrin. Depletion of the calcitonin gene-related peptide innervation may affect the nutrient blood supply of the upper dermis, and the integrity and function of basal epidermis and Langerhans cells. Diminished substance P innervation of the sweat glands may affect their secretory activity. Both of these changes may be implicated in the development of xerosis. (+info)
Mapping the active site in vasoactive intestinal peptide to a core of four amino acids: neuroprotective drug design.
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The understanding of the molecular mechanisms leading to peptide action entails the identification of a core active site. The major 28-aa neuropeptide, vasoactive intestinal peptide (VIP), provides neuroprotection. A lipophilic derivative with a stearyl moiety at the N-terminal and norleucine residue replacing the Met-17 was 100-fold more potent than VIP in promoting neuronal survival, acting at femtomolar-picomolar concentration. To identify the active site in VIP, over 50 related fragments containing an N-terminal stearic acid attachment and an amidated C terminus were designed, synthesized, and tested for neuroprotective properties. Stearyl-Lys-Lys-Tyr-Leu-NH2 (derived from the C terminus of VIP and the related peptide, pituitary adenylate cyclase activating peptide) captured the neurotrophic effects offered by the entire 28-aa parent lipophilic derivative and protected against beta-amyloid toxicity in vitro. Furthermore, the 4-aa lipophilic peptide recognized VIP-binding sites and enhanced choline acetyltransferase activity as well as cognitive functions in Alzheimer's disease-related in vivo models. Biodistribution studies following intranasal administration of radiolabeled peptide demonstrated intact peptide in the brain 30 min after administration. Thus, lipophilic peptide fragments offer bioavailability and stability, providing lead compounds for drug design against neurodegenerative diseases. (+info)
Actions of vasoactive intestinal peptide on the rat adrenal zona glomerulosa.
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Previous studies, by this group and others, have shown that vasoactive intestinal peptide (VIP) stimulates aldosterone secretion, and that the actions of VIP on aldosterone secretion by the rat adrenal cortex are blocked by beta adrenergic antagonists, suggesting that VIP may act by the local release of catecholamines. The present studies were designed to test this hypothesis further, by measuring catecholamine release by adrenal capsular tissue in response to VIP stimulation. Using intact capsular tissue it was found that VIP caused a dose-dependent increase in aldosterone secretion, with a concomitant increase in both adrenaline and noradrenaline release. The effects of VIP on aldosterone secretion were inhibited by atenolol, a beta1 adrenergic antagonist, but not by ICI-118,551, a beta2 adrenergic antagonist. Binding studies were carried out to investigate VIP receptors. It was found that adrenal zona glomerulosa tissue from control rats contained specific VIP binding sites (Bmax 853+/-101 fmol/mg protein; Kd 2.26+/-0.45 nmol/l). VIP binding was not displaced by ACTH, angiotensin II or by either of the beta adrenergic antagonists. The response to VIP in adrenals obtained from rats fed a low sodium diet was also investigated. Previous studies have found that adrenals from animals on a low sodium diet exhibit increased responsiveness to VIP. Specific VIP binding sites were identified, although the concentration or affinity of binding sites in the low sodium group was not significantly different from the controls. In the low sodium group VIP was found to increase catecholamine release to the same extent as in the control group, however, in contrast to the control group, the adrenal response to VIP was not altered by adrenergic antagonists in the low sodium group. These data provide strong support for the hypothesis that VIP acts by the local release of catecholamines in adrenal zona glomerulosa tissue in normal animals. It does not appear that VIP acts through the same mechanism in animals maintained on a low sodium diet. The mechanism by which VIP stimulates aldosterone in this group remains to be determined. (+info)