NaCl and fluid secretion by the intestine of the teleost Fundulus heteroclitus: involvement of CFTR.
Sections of posterior intestine of the euryhaline killifish Fundulus heteroclitus adapted to sea water were stimulated by the calcium ionophore ionomycin (1 micromol l(-1)) in combination with agents to elevate intracellular cyclic AMP levels, 0.5 mmol l(-1) dibutyryl-cyclic AMP (db-cAMP) with 0.1 mmol l(-1) 3-isobutyl-1-methylxanthine (IBMX). Intestinal bag preparations from recently fed animals (but not from overnight unfed animals) changed from fluid absorption (+18.9+/-8.30 microl cm(-2) h(-1), N=8) in the untreated control period to net fluid secretion after stimulation (-7.43+/-1.30 microl cm(-2) h(-1), N=8, P<0.01; means +/- S.E.M.), indicative of the capacity of teleost intestine to undergo secretion. Posterior intestinal pieces mounted in vitro in Ussing-style membrane chambers showed net Cl(-) uptake (+2.245+/-0.633 microequiv cm(-2) h(-1), N=7) that turned to net secretion following stimulation by ionomycin + db-cAMP + IBMX (-3.809+/-1.22 microequiv cm(-2) h(-1), N=7, P<0.01). Mucosal application of the anion channel blocker 1 mmol l(-1) diphenylamine-2-carboxylate (DPC) after ionomycin + db-cAMP + IBMX treatment significantly reduced serosal-to-mucosal unidirectional Cl(-) flux (P<0.001), net Cl(-) flux (P<0.05), short-circuit current (I(sc), P<0.001) and tissue conductance (G(t), P<0.001), while 0.1 mmol l(-1) 4,4'-diisothiocyano-2,2'-stilbene-disulphonic acid (DIDS, a blocker of anion exchange) was without effect. Stimulation by db-cAMP + IBMX (no ionomycin) significantly increased unidirectional fluxes, I(sc) and G(t) but did not produce net Cl(-) secretion. Ionomycin alone produced a transient increase in I(sc) but had no effect on G(t) and caused no significant changes in unidirectional or net Cl(-) fluxes. Addition of db-cAMP + IBMX after ionomycin treatment produced net secretion of Cl(-) and large increases in unidirectional fluxes and G(t). Cystic fibrosis transmembrane conductance regulator (CFTR) was immunocytochemically localized with a monoclonal mouse antibody to the carboxy terminus and found to be present in the cytoplasm and basolateral membranes of all enterocytes and in the brush-border membrane of some cells, whereas NKCC immunofluorescence, demonstrating the presence of the Na(+)/K(+)/2Cl(-) cotransporter, was present in the cytoplasm and brush-border membrane. We conclude that the teleost intestine is capable of salt and fluid secretion only if intracellular Ca(2+) and cyclic AMP pathways are stimulated together and that this secretion appears to involve activation of CFTR ion channels in the apical membrane of a subpopulation of enterocytes.
Marshall
W S
WS
Department of Biology, St Francis Xavier University, Antigonish, Nova Scotia, Canada B2G 2W5. bmarshal@stfx.ca
Howard
J A
JA
Cozzi
R R F
RR
Lynch
E M
EM
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D000818:Animals; D001826:Body Fluids / Q000378:metabolism*; D003994:Bucladesine / Q000494:pharmacology; D002118:Calcium; D002121:Calcium Channel Blockers / Q000494:pharmacology; D002712:Chlorides / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000032:analysis / Q000502:physiology*; D004553:Electric Conductivity; D005260:Female; D005455:Fluorescent Antibody Technique; D023781:Fundulidae / Q000502:physiology*; D007150:Immunohistochemistry; D007413:Intestinal Mucosa / Q000187:drug effects / Q000378:metabolism; D007422:Intestines / Q000187:drug effects / Q000502:physiology*; D015759:Ionomycin / Q000494:pharmacology; D008297:Male; D012965:Sodium Chloride / Q000378:metabolism*; D028021:Sodium-Potassium-Chloride Symporters / Q000032:analysis; D062367:ortho-Aminobenzoates / Q000494:pharmacology
D002121:Calcium Channel Blockers; D002712:Chlorides; D028021:Sodium-Potassium-Chloride Symporters; D062367:ortho-Aminobenzoates; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D012965:Sodium Chloride; D015759:Ionomycin; D003994:Bucladesine; C001209:fenamic acid; D000242:Cyclic AMP; D002118:Calcium; D015056:1-Methyl-3-isobutylxanthine