vasoactive-intestinal-peptide and phosphoramidon

The functional effects of the intranasal application of exogenous vasoactive intestinal polypeptide (VIP), substance P (SP) and calcitonin gene-related peptide (CGRP) were evaluated in 12 healthy volunteers before and after neutral endopeptidase (NEP) inhibition with phosphoramidon (PA) and angiotensin-converting enzyme (ACE) inhibition with captopril. The three neuropeptides increased nasal airway resistance (NAR) measured by anterior rhinomanometry and superficial capillary blood flow measured by laser Doppler flowmetry (LDF). After pretreatment of the nasal mucosa with PA, the effects of VIP, SP and CGRP on the LDF signal, NAR and mucus production were potentiated, whereas local pretreatment with captopril did not modify these functional effects. These observations suggest that NEP, but not ACE, may participate in the catabolism of neuropeptides when applied directly to the human nasal mucosa. Furthermore, since these neuropeptides induced nasal obstruction, increased blood flow and rhinorrhea, a decreased activity of the enzymes involved in their degradation could be involved in the physiopathology of rhinitis symptoms.

Topics: Adult; Airway Resistance; Blood Flow Velocity; Calcitonin Gene-Related Peptide; Captopril; Drug Synergism; Endothelium, Vascular; Glycopeptides; Humans; Laser-Doppler Flowmetry; Middle Aged; Mucus; Nasal Mucosa; Neprilysin; Peptidyl-Dipeptidase A; Protease Inhibitors; Substance P; Time Factors; Vasoactive Intestinal Peptide

Vasoactive intestinal peptide (VIP) stimulates the neuroblastoma cell line (NMB) to proliferate. Neuropeptide activity can be inhibited by neutral endopeptidases that function intracellularly and in the extracellular milieu. NMB cells express neutral endopeptidase (NEP) activity that can be specifically inhibited by phosphoramidon (PA). Our data now show that phosphoramidon treatment increases the efficacy of VIP-stimulated neuroblastoma proliferation. These results suggest that membrane endopeptidases modulate VIP-associated cell proliferation and enhancement of endopeptidase activity may serve as a target for cancer therapy.

Topics: Cell Division; Enzyme Inhibitors; Glycopeptides; Humans; Neprilysin; Neuroblastoma; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

VIP-PACAP receptors were characterized in a human airway epithelial-like cell line (Calu-3), Pituitary adenylate cyclase activating polypeptide (PACAP) 1-27, PACAP 1-38, vasoactive intestinal polypeptide (VIP) and the beta 1- and beta 2-adrenoceptor agonist isoproterenol (3 nM-1 microM) increased cAMP concentration dependently. The peptides and isoproterenol displayed similar potencies (range of means pEC50[M]: 6.5-7.1). The maximum increase in cAMP (Emax in % of basal cAMP level) was similar for the peptides (range of means Emax: 2500-5100%). Pretreatment with the peptidase inhibitors captopril (10 microM) and phosphoramidon (1 microM) significantly increased the cAMP response to PACAP 1-38 (to 480% of control) only.

Topics: Captopril; Cyclic AMP; Epithelium; Glycopeptides; Humans; Isoproterenol; Lung; Neuropeptides; Neurotransmitter Agents; Pituitary Adenylate Cyclase-Activating Polypeptide; Protease Inhibitors; Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide; Receptors, Pituitary Hormone; Receptors, Vasoactive Intestinal Peptide; Receptors, Vasoactive Intestinal Polypeptide, Type I; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

The purpose of this study was to determine whether vasoactive intestinal peptide (VIP) dilates resistance arterioles in the in situ systemic circulation and whether inhibitors of neutral endopeptidase (NEP) and angiotensin I-converting enzyme (ACE), two membrane-bound metalloenzymes that are widely distributed in the microcirculation and cleave and inactive VIP, potentiate this response. Using intravital microscopy, we found that VIP (0.05 and 0.1 nmol) induced significant vasodilation in the hamster cheek pouch (13 +/- 1 and 20 +/- 2% increase from baseline, respectively; mean +/- SE; P < 0.05). These responses were significantly potentiated by topical application of phosphoramidon and thiorphan, two relatively selective NEP inhibitors, but not by captopril, a relatively selective ACE inhibitor. Furthermore, suffusion of a mixture of proteinase inhibitors consisting of leupeptin, Bestatin, and DL-2-mercaptomethyl-3-guanidinoethylthiopropanoic acid to inhibit serine proteinases, including mast cell tryptase, aminopeptidases, and carboxypeptidase N, respectively, had no significant effects on VIP-induced responses. These data indicate that VIP elicits vasodilation in the in situ systemic microcirculation and that NEP modulates this response.

Topics: Angiotensin-Converting Enzyme Inhibitors; Animals; Arterioles; Captopril; Cheek; Cricetinae; Drug Combinations; Glycopeptides; Male; Mesocricetus; Neprilysin; Protease Inhibitors; Thiorphan; Vasoactive Intestinal Peptide; Vasodilation

Topics: Albuterol; Animals; Bronchodilator Agents; Captopril; Glycopeptides; Guinea Pigs; In Vitro Techniques; Male; Muscle Relaxation; Muscle, Smooth; Neuropeptides; Pituitary Adenylate Cyclase-Activating Polypeptide; Protease Inhibitors; Theophylline; Trachea; Vasoactive Intestinal Peptide

An increase in subepithelial mesenchymal cells and associated connective tissue is a feature of bronchial asthma. We determined whether neuropeptides could modulate fibroblast activity, particularly with respect to proliferation and chemotaxis. Human lung fibroblasts were cultured with neurokinin A (NKA), substance P (SP), vasoactive intestinal peptide (VIP), and calcitonin-gene-related peptide (CGRP). After 48 h, fibroblast proliferation was measured by a colorimetric assay based on the uptake and subsequent release of methylene blue. The chemotactic response to neuropeptides was determined with the use of a modified Boyden chamber. Both NKA and SP (10(-7)-10(-4) M) stimulated human lung fibroblast proliferation in HFL1 and IMR-90 fibroblasts. VIP and CGRP had no effect on fibroblast proliferation. NKA alone stimulated fibroblast chemotaxis maximally at 10(-10) M. Neutral endopeptidase (NEP) activity of 0.52 and 5.2 pmol/10(6) cells was assayed in IMR-90 and Hs68 fibroblasts, respectively. Phosphoramidon (5 x 10(-6)-10(-5) M), an NEP inhibitor, enhanced fibroblast proliferation in a dose-dependent manner. Thus neuropeptides have the potential to cause activation of mesenchymal cells, and neuropeptide release may contribute to the structural abnormalities observed in asthmatic airways.

Topics: Calcitonin Gene-Related Peptide; Cell Division; Chemotaxis; Fibroblasts; Glycopeptides; Humans; Lung; Neprilysin; Neurokinin A; Neuropeptides; Substance P; Vasoactive Intestinal Peptide

1. The effect of passive sensitization on the mechanical activity of human isolated bronchial smooth muscle induced by the following neuropeptides substance P (SP), neurokinin A (NKA) and vasoactive intestinal peptide (VIP) was studied both in the absence and in the presence of the neutral endopeptidase (NEP) inhibitor, phosphoramidon. 2. Cumulative concentration-response curves (CCRC) to these neuropeptides were constructed in human passively sensitized isolated bronchial rings and compared to those in paired controls. Passively sensitized human isolated bronchial rings were tissues incubated overnight in serum from asthmatic patients atopic to Dermatophagoides pteronyssinus and paired controls were tissues originating from the same lung specimens but incubated overnight in serum from healthy donors. 3. In the absence of phosphoramidon, passive sensitization significantly increased the amplitude of the contractile responses to SP and NKA including that to the maximal concentration given from 50 +/- 5% to 76 +/- 6% (n = 5, P < 0.05) and from 70 +/- 7% to 101 +/- 6% (n = 5, P < 0.05) of the maximal response to acetylcholine, respectively. Passive sensitization significantly shifted to the left the CCRC for both tachykinins as measured by the geometric means dose-ratios which were 8.5 (95% confidence limits (CL): 3.1-13.9) and 7.3 (95% CL: 4.2-10.3) for SP and NKA, respectively. 4. In the presence of phosphoramidon (10 microM), passive sensitization still increased significantly the amplitude of the contractile responses to SP and NKA including that to the maximal concentration given from 74 +/- 4% to 115 +/- 7% (n = 5, P<0.05) and from 104 +/- 9% to 146 +/- 16% (n = 5, P<0.05)of the maximal response to acetylcholine, respectively. Passive sensitization still significantly shifted to the left the CCRC for both tachykinins as measured by the dose-ratios which were 9.0 (95% CL:4.3-13.6) and 5.4 (95% CL: 2.9-7.9) for SP and NKA, respectively.5. The relaxant response to the maximal concentration of VIP given in tissues precontracted with histamine (0.5 mM) was significantly reduced by passive sensitization from 41 +/- 4% to 25 +/- 3% (n = 5,P <0.05) of the amplitude of the precontraction in the absence of phosphoramidon and from 72 +/- 1%to 49 +/- 4% (n = 5, P<0.05) in the presence of phosphoramidon (10 microM). Passive sensitization significantly shifted to the right the CCRC for VIP as measured by the dose-ratios which were 10.4(95% CL: 6.6-14.1) and 6.4 (95%

Topics: Acetylcholine; Adult; Aged; Female; Glycopeptides; Histamine; Humans; Hypersensitivity; Immunization, Passive; In Vitro Techniques; Male; Middle Aged; Muscle Contraction; Muscle, Smooth; Neurokinin A; Phosphorylation; Substance P; Thermolysin; Vasoactive Intestinal Peptide

We studied the effect of vasoactive intestinal peptide (VIP) on ciliary activity in rabbit cultured tracheal epithelium by a photoelectric method in vitro. Administration of VIP (10(-7) M) elicited an increase in ciliary beat frequency (CBF) from the baseline values of 970 +/- 52 to 1139 +/- 75 beats/min (mean +/- S.E., P less than 0.01). This ciliostimulatory effect was dose-dependent, with the maximal increase and EC50 value being 17.4 +/- 1.0% (P less than 0.05) and 6.10(-11) M, respectively. The VIP-induced increase in CBF was abolished by pretreatment of cells with [4-Cl-D-Phe6, Leu17]-VIP, a VIP receptor antagonist. The neutral endopeptidase inhibitor phosphoramidon (10(-5) M) potentiated the effect of VIP, so that the CBF dose-response curve for VIP was shifted to lower concentrations by 0.5 log U. The administration of VIP increased cyclic AMP levels in epithelial cells, an effect that was also potentiated by phosphoramidon. These results suggest that VIP may interact with its specific receptors and stimulate airway ciliary activity probably through the activation of adenylate cyclase, and that neutral endopeptidase may play a role in modulating this effect of VIP.

Topics: Animals; Cells, Cultured; Cyclic AMP; Dose-Response Relationship, Drug; Epithelium; Glycopeptides; Indomethacin; Mucociliary Clearance; Neprilysin; Phosphorylation; Rabbits; Trachea; Vasoactive Intestinal Peptide

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.

Topics: Animals; Electric Stimulation; Epithelium; Female; Glycopeptides; Guinea Pigs; In Vitro Techniques; Male; Muscle Contraction; Thiorphan; Trachea; Vagus Nerve; Vasoactive Intestinal Peptide

Relaxations were induced in longitudinal muscle strips of the rat gastric fundus by exogenous administration of vasoactive intestinal polypeptide (VIP) and by transmural stimulation in the presence of atropine. These responses were not influenced by two neutral endopeptidase inhibitors, thiorphan (3 X 10(-5) M) and phosphoramidon (10(-5) M). This suggests that neutral endopeptidase is not involved in the breakdown of exogenous VIP and non-adrenergic non-cholinergic inhibitory transmitters of the rat gastric fundus.

Topics: Animals; Electric Stimulation; Female; Gallbladder; Gastric Fundus; Glycopeptides; Guinea Pigs; Male; Muscle Contraction; Muscle Relaxation; Neprilysin; Neurons; Rats; Thermolysin; Thiorphan; Vasoactive Intestinal Peptide

1. We have studied the effect of epithelium removal on relaxation of guinea-pig isolated tracheal smooth muscle induced by vasoactive intestinal peptide (VIP) or stimulation of non-adrenergic, non-cholinergic (NANC) inhibitory nerves. Also examined were the effects of inhibitors of neutral endopeptidase (NEP) and angiotensin-converting enzyme (ACE). 2. Epithelium removal produced a 3.6 +/- 0.4 fold leftward shift in the VIP concentration-response curve. The supersensitivity to VIP, following epithelium removal was abolished by phosphoramidon or thiorphan (NEP inhibitors), but unaffected by captopril (an ACE inhibitor). In intact trachea, the NEP inhibitors produced leftward shifts in the VIP curves similar to those produced by epithelium removal. 3. In contrast to responses to exogenous VIP, neurogenic NANC inhibitory responses to electrical field stimulation were affected neither by epithelial denudation nor by the peptidase inhibitors. 4. As in previous studies, epithelium removal increased tracheal sensitivity to isoprenaline. This was not altered by pretreatment with a cocktail of peptidase inhibitors. Thus, the effect of the NEP inhibitors on responses to VIP appears to be relatively specific. 5. These data indicate that exogenous VIP is a substrate for airway NEP, since inhibition of the enzyme potentiates the peptide. This is further evidence that the airway epithelium provides a source for the metabolism of mediators. 6. In guinea-pig trachea the NEP responsible for cleaving VIP may be located largely in the epithelial layer, since NEP inhibition was without effect on sensitivity to VIP in epithelium-denuded preparations. If VIP is a NANC inhibitory neurotransmitter in this tissue its degradation endogenously does not appear to involve epithelial NEP.

Topics: Animals; Atropine; Captopril; Electric Stimulation; Endopeptidases; Epithelium; Glycopeptides; Guinea Pigs; In Vitro Techniques; Male; Muscle Relaxation; Muscle, Smooth; Propranolol; Protease Inhibitors; Thiorphan; Trachea; Vasoactive Intestinal Peptide

Relaxation of guinea-pig trachea was induced by vasoactive intestinal peptide (VIP) or electrical field stimulation. Mechanical removal of airway epithelium potentiated responses to VIP and attenuated inhibitory non-adrenergic, non-cholinergic (i-NANC) responses to low stimulation frequencies. Phosphoramidon potentiated responses to VIP in intact but not de-epithelialised preparations, and had no effect on i-NANC responses. These findings suggest that neutral endopeptidase localised to the epithelium may modulate relaxation of guinea-pig trachea induced by VIP but not i-NANC nerve-stimulation.

Topics: Animals; Autonomic Nervous System; Electric Stimulation; Endopeptidases; Epithelium; Ferrets; Glycopeptides; Guinea Pigs; In Vitro Techniques; Male; Muscle Relaxation; Muscle, Smooth; Tetrodotoxin; Trachea; Vasoactive Intestinal Peptide

Human recombinant enkephalinase (neutral endopeptidase, EC 3.4.24.11) cleaved synthetic vasoactive intestinal peptide (VIP1-28) with time-and peptidase concentration-dependence, which left less than 30% intact after 30 micrograms was incubated at 37 degrees C with 0.1 micrograms and 10 micrograms of peptidase for 120 min and 15 min, respectively. The rank order of relative rates of peptidolysis amino-terminal to hydrophobic amino acids was Ala4 and Val5 greater than Tyr22 and Ile26 much greater than Leu13 and Met17. The many effects of VIP1-28 on epithelial cell and leukocyte functions thus may be influenced by degradation of the mediator by enkephalinase at the surface of target cells.

Topics: Glycopeptides; Humans; Kinetics; Neprilysin; Peptide Fragments; Recombinant Proteins; Substrate Specificity; Vasoactive Intestinal Peptide