vasoactive-intestinal-peptide and fenamic-acid

Chloride (Cl(-)) channels expressed in vascular smooth muscle cells (VSMC) are important to control membrane potential equilibrium, intracellular pH, cell volume maintenance, contraction, relaxation and proliferation. The present study was designed to compare the expression, regulation and function of CFTR Cl(-) channels in aortic VSMC from Cftr(+/+) and Cftr(-)(/)(-) mice. Using an iodide efflux assay we demonstrated stimulation of CFTR by VIP, isoproterenol, cAMP agonists and other pharmacological activators in cultured VSMC from Cftr(+/+). On the contrary, in cultured VSMC from Cftr(-)(/)(-) mice these agonists have no effect, showing that CFTR is the dominant Cl(-) channel involved in the response to cAMP mediators. Angiotensin II and the calcium ionophore A23187 stimulated Ca(2)(+)-dependent Cl(-) channels in VSMCs from both genotypes. CFTR was activated in myocytes maintained in medium containing either high potassium or 5-hydroxytryptamine (5-HT) and was inhibited by CFTR(inh)-172, glibenclamide and diphenylamine-2,2'-dicarboxylic acid (DPC). We also examined the mechanical properties of aortas. Arteries with or without endothelium from Cftr(-/-) mice became significantly more constricted (approximately 2-fold) than that of Cftr(+/+) mice in response to vasoactive agents. Moreover, in precontracted arteries of Cftr(+/+) mice, VIP and CFTR activators induced vasorelaxation that was altered in Cftr(-/-) mice. Our findings suggest a novel mechanism for regulation of the vascular tone by cAMP-dependent CFTR chloride channels in VSMC. To our knowledge this study is the first to report the phenotypic consequences of the loss of a Cl(-) channel on vascular reactivity.

Topics: Adrenergic beta-Agonists; Angiotensin II; Animals; Aorta, Thoracic; Benzoates; Cells, Cultured; Chlorides; Colforsin; Cystic Fibrosis Transmembrane Conductance Regulator; Genistein; Glyburide; In Vitro Techniques; Isoproterenol; Mice; Mice, Inbred CFTR; Muscle, Smooth, Vascular; ortho-Aminobenzoates; Quinolizines; Serotonin; Thiazoles; Thiazolidines; Vasoactive Intestinal Peptide; Vasoconstriction; Vasoconstrictor Agents; Vasodilation; Vasodilator Agents

Vasoactive intestinal polypeptide (VIP) plays an important role in water transport in the intestine. Several specialized channels termed aquaporins (AQP) facilitate water transport in the gastrointestinal tract. Aquaporin-3 localizes to epithelial cells in the human small intestine and colon. However, the regulatory mechanisms underlying the functions of AQP3 remain unclear. To characterize the regulation of AQP3 expression by VIP, we studied messenger (m)RNA expression, protein expression and DNA binding activity in a human colonic epithelial cell line, HT-29.. Human colonic epithelial cells, HT-29, were incubated with VIP (10-12-10-7 M). The cells were treated with protein kinase-A (PK-A) inhibitors (H-89, H-9) or chloride channel-blockers (diphenylamine-2-carboxylate (DPC), 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPD)). The expression of AQP3 mRNA and protein was determined by Northern blot and Western blot, respectively. The DNA-binding activities of cyclic adenosine monophosphate (cAMP) response elements/activating transcription factor (CRE/ATF)) in the nuclear extract were determined by electrophoretic mobility shift assay.. Aquaporin-3 mRNA was up-regulated at a concentration of 10-10 M VIP. The expression of AQP3 protein was enhanced at 3 h after addition of VIP. The PK-A inhibitors (H-89, H-9) inhibited the expression of AQP3 mRNA enhanced by VIP and cAMP. The gel shift assay of CRE/ATF in HT-29 cells revealed a single band.. These results indicate that VIP upregulated the expression of AQP3 mRNA and protein, and that a cAMP-dependent pathway mediated this effect in a human colonic epithelial cell line, HT-29.

Topics: Aquaporin 3; Aquaporins; Blotting, Northern; Blotting, Western; Chloride Channels; Colon; Cyclic AMP Response Element-Binding Protein; Cyclic AMP-Dependent Protein Kinases; Electrophoresis, Polyacrylamide Gel; Enzyme Inhibitors; Epithelial Cells; Humans; In Vitro Techniques; ortho-Aminobenzoates; Polymerase Chain Reaction; Receptors, Vasoactive Intestinal Peptide; RNA, Messenger; Up-Regulation; Vasoactive Intestinal Peptide

We have developed a cell culture of guinea pig gallbladder epithelial cells with which to study ion transport. When grown on permeable supports, the cultured epithelia developed a transepithelial resistance (R(t)) of approximately 500 Omega. cm(2). The epithelial cell origin of the cell culture was further confirmed by immunocytochemical localization of cytokeratin. Ionomycin and forskolin increased transepithelial voltage and short-circuit current (I(sc)) and decreased R(t). The response to ionomycin was transient, whereas that to forskolin was sustained. Both were attenuated by replacement of Cl(-) and/or HCO(3)(-). Mucosal addition of the anion transport inhibitors DIDS or diphenylamine-2-carboxylic acid (DPC) blocked the response to ionomycin. The response to forskolin was blocked by DPC but not by DIDS. Ionomycin, but not forskolin, increased intracellular Ca(2+) concentration in fura 2-loaded cells. PGE(2), histamine, vasoactive intestinal polypeptide, and secretin elicited a sustained increase in I(sc). Responses to ATP and CCK were transient. Thus cultured guinea pig gallbladder epithelia display the range of responses observed in the native tissue and are an appropriate model for studies of ion transport in gallbladder and intestinal epithelia.

Topics: 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Adenosine Triphosphate; Animals; Anions; Biological Transport; Calcium; Calcium Channel Blockers; Cells, Cultured; Cholecystokinin; Colforsin; Cyclic AMP; Dinoprostone; Epithelial Cells; Gallbladder; Gastrointestinal Agents; Guinea Pigs; Histamine; Ionomycin; Ionophores; Male; ortho-Aminobenzoates; Secretin; Vasoactive Intestinal Peptide

Previous studies demonstrated neurally mediated recurrent increases in short-circuit current (Isc) suggestive of anion secretion in guinea pig distal colon. To determine the neural pathways involved, segments of distal colon from guinea pigs were mounted in flux chambers. In muscle-stripped or whole thickness preparations, serosal addition of the histamine H2 receptor agonist, dimaprit, caused cyclical increases in Isc, which were reduced by the chloride channel blocker, N-phenylanthranilic acid, but not by the sodium channel blocker amiloride. Dimaprit stimulated release of [3H]acetylcholine and vasoactive intestinal polypeptide (VIP) from submucosal/mucosal sheets. Dimaprit caused recurrent increases in Isc, which were significantly decreased by mecamylamine, a nicotinic receptor antagonist, and nearly abolished by the muscarinic antagonist, atropine (M3 > M1 = M2). The muscarinic antagonist, 4-diphenylacetoxy-N-methyl-piperidine methiodide (4-DAMP, M3 > M1), was more potent than pirenzepine (M1 > M3) in reducing recurrent increases in Isc. Dimaprit- and electrically evoked secretion were inhibited by the VIP antagonists [4Cl-D-Phe6, Leu17]VIP and VIP hybrid. The results suggest the involvement of VIP-ergic and cholinergic neurons utilizing nicotinic and muscarinic synapses in mediating secretion.

Topics: Acetylcholine; Amiloride; Animals; Atropine; Choline; Colon; Dimaprit; Electric Conductivity; Guinea Pigs; Histamine Release; Intestinal Mucosa; Male; ortho-Aminobenzoates; Piperidines; Pirenzepine; Receptors, Histamine H2; Vasoactive Intestinal Peptide

In past studies we observed that the chloride channel blocker, diphenylamine-2-carboxylate (DPC) and chemically related drugs (Hoechst compounds 131, 143, 144) inhibited cAMP formation in mouse pituitary tumor cells. The object of this study was to determine whether these drugs inhibited chloride transport in human T-84 colonic carcinoma cells through an effect on cAMP metabolism. Chloride secretion (measured as 125I efflux from isotope-preloaded cells) was stimulated in a concentration-dependent manner by vasoactive intestinal polypeptide (VIP) (EC50 = 1.5 x 10(-10) M) which similarly increased cAMP synthesis (EC50 = 1.6 x 10(-8) M). The cAMP response to VIP was inhibited 17, 52, 55, and 78% maximally by DPC and compounds 144, 143, and 131, respectively. In untreated T-84 cells, 125I secretion fell by 66% after 3 min; VIP (10(-7) M) increased secretion about fivefold over the same period. Both basal and VIP-stimulated 125I secretion were inhibited up to 60% by compound 131. Pretreatment of cells with pertussis toxin did not attenuate the inhibitory effect of channel blockers on either VIP-stimulated cAMP synthesis or 125I secretion. The cationophore, A-23187, which had no effect on cAMP formation, and 8-Br-cAMP both stimulated 125I secretion from T-84 cells. These secretory responses were inhibited by compound 131. The mechanism by which phenylanthranilic acids antagonize cAMP synthesis and its significance is not known; however, the data suggest that this family of drugs may inhibit chloride transport by both cAMP-dependent and independent mechanisms.

Topics: Chloride Channels; Chlorides; Colonic Neoplasms; Cyclic AMP; Epithelium; Humans; Iodine; Iodine Radioisotopes; Membrane Proteins; ortho-Aminobenzoates; Pertussis Toxin; Stimulation, Chemical; Tumor Cells, Cultured; Vasoactive Intestinal Peptide; Virulence Factors, Bordetella

Somatostatin-14 (SS-14) and somatostatin-28 (SS-28) produce concentration dependent reductions in short-circuit current in rat colonic mucosa. EC50 values of 15.0 and 13.3 nM were obtained for SS-14 and SS-28 respectively while the N-terminal fragments of SS-28, namely somatostatin-(1-12) (SS1-12) and somatostatin-(1-14) (SS1-14) were inactive. Cyclo(Pro-Phe-D-Trp-Lys-Thr-Phe) and cyclo(Pro-Tyr-D-Trp-Lys-Thr-Phe) were potent antisecretory peptides, like SS-14 and SS-28; while the putative somatostatin antagonist, cyclo(7-aminoheptanoyl-Phe-D-Trp-Lys-Thr[Bzl]) exhibited neither agonist nor antagonist effects. Responses to SS-14 could be regulated by agents which affected the secretory state of the epithelium. Antisecretory effects of SS-14 were markedly attenuated by piroxicam and were restored following piroxicam plus either forskolin or vasoactive intestinal polypeptide (VIP). SS-14 also attenuated secretory responses produced by carbachol, substance P (SP), VIP and alpha- and beta-calcitonin gene related peptide (alpha-, beta-CGRP). Therefore, SS-14 exhibits broad spectrum antisecretory effects in rat descending colon mucosa.

Topics: Amino Acid Sequence; Animals; Calcitonin Gene-Related Peptide; Carbachol; Colforsin; Colon; In Vitro Techniques; Intestinal Mucosa; Male; Molecular Sequence Data; ortho-Aminobenzoates; Piroxicam; Protein Precursors; Rats; Rats, Inbred Strains; Somatostatin; Somatostatin-28; Substance P; Tetrodotoxin; Vasoactive Intestinal Peptide

The presently reported studies of factors involved in the regulation of acid-base excretory processes in isolated turtle urinary bladders have yielded the following data. (i) There exists in alkalotic and euhydric turtle bladders (but not in acidotic turtle bladders) a mechanism which drives a primary active electrogenic secretion of alkali; and the electrogenic output of this mechanism is up-regulated by exogenously added PDE inhibitors, such as theophylline or IBMX. (ii) VIP or cAMP up-regulates this alkali secretion in the presence of IBMX. (iii) Carbachol also initiates alkali secretion. But this action, independent of PDE activity, is probably associated with the phospho-inositol reaction cascade. (iv) Finally, low concentrations of mucosal DPC decreases the carbachol supported, but not the cAMP-supported alkali secretion.

Topics: 1-Methyl-3-isobutylxanthine; 3',5'-Cyclic-AMP Phosphodiesterases; Animals; Carbachol; Choline; Cyclic AMP; Hydrogen-Ion Concentration; ortho-Aminobenzoates; Phosphatidylinositols; Theophylline; Turtles; Urinary Bladder; Vasoactive Intestinal Peptide

This report describes a Cl- transport pathway in confluent monolayer cultures of the T84 human colonic carcinoma cell line which is: 1) activated by vasoactive intestinal polypeptide, or other agents which induce or mimic cAMP; 2) independent of extracellular Na+ or K+; 3) refractory to inhibition by 0.1 mM bumetanide and 1 mM 4-acetamido-4'-isothiocyanostilbene-2,-2'-disulfonic acid; 4) competitively inhibited by NO3-, I-, SCN-, and Br-; 5) inhibited in a noncompetitive-complex manner by the putative Cl- channel-blocking agent, N-phenylanthranilic acid; and 6) localized to the apical membrane of confluent monolayers. This Cl- transport system is, therefore, distinct from the bumetanide-sensitive, basolateral membrane-localized, Na+, K+, Cl- cotransport system previously described in these cells (Dharmsathaphorn, K., Mandel, K., Masui, H., and McRoberts, J.A. (1985) J. Clin. Invest. 75, 462-471). Kinetic studies revealed that Cl- transport by this pathway fit simple Michaelis-Menten kinetics with an apparent Km for Cl- of about 6 mM. Activation by vasoactive intestinal polypeptide increased the Vmax but did not alter the apparent Km. We discuss the possibility that this transport system is a Cl- channel which is intimately involved in hormonally mediated, electrogenic Cl- secretion across T84 cell monolayers.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; Anthracenes; Biological Transport, Active; Bromides; Bucladesine; Bumetanide; Calcimycin; Cell Line; Cell Membrane; Chlorides; Colon; Cyclic AMP; Dose-Response Relationship, Drug; Epithelium; Humans; Hydrogen-Ion Concentration; Kinetics; ortho-Aminobenzoates; Sodium; Valinomycin; Vasoactive Intestinal Peptide