Stilbenes stimulate T84 Cl- secretion by elevating Ca2+.
Basolateral but not apical application of 10-200 microM 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) or 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS) to T84 monolayers produced a transient increase in short-circuit current (Isc), followed by a sustained inhibition. 4,4'-Dinitrostilbene-2,2'-disulfonic acid (DNDS) had no effect. The increase in Isc produced by DIDS represents Cl- secretion and appears to result from Ca2+ elevation, because in all respects except time course the response to DIDS mimicked the response to the Ca(2+)-elevating agent thapsigargin. Fura-2 measurements established that thapsigargin elevates Ca2+ in T84 cells, but Ca2+ responses to DIDS could not be established directly because DIDS absorbs strongly at the critical wavelengths. Responses to DIDS and thapsigargin were 1) blocked by bumetanide; 2) not blocked by basolateral Ba2+; 3) completely nonadditive; 4) strongly synergistic with basal levels of Isc or with Isc increases produced by elevating adenosine 3',5'-cyclic monophosphate (cAMP; with forskolin) or guanosine 3',5'-cyclic monophosphate (with heat-stable enterotoxin); and 5) reversibly abolished by removal of basolateral Ca2+. Interactions between Ca2+ and cAMP-elevating agents strongly support a model of Cl- secretion in which apical Cl- conductance is activated by cyclic nucleotides but not by Ca2+ while basolateral K+ channels are activated by Ca2+. In contrast with this mechanism, whole cell patch-clamp recordings of nonconfluent T84 cells indicated that DIDS and other Ca(2+)-elevating agents stimulated an increase in Cl- conductance. Thus increases in cytosolic free Ca2+ in nonconfluent T84 cells activate conductances that differ from those in confluent monolayers.
Brayden
D J
DJ
Cystic Fibrosis Research Laboratory, Stanford University, California 94305-2130.
Krouse
M E
ME
Law
T
T
Wine
J J
JJ
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D002712:Chlorides / Q000378:metabolism*; D005576:Colforsin / Q000494:pharmacology; D003106:Colon / Q000166:cytology / Q000378:metabolism* / Q000502:physiology; D004357:Drug Synergism; D004594:Electrophysiology; D013267:Stilbenes / Q000494:pharmacology*; D013729:Terpenes / Q000494:pharmacology; D019284:Thapsigargin
D002712:Chlorides; D013267:Stilbenes; D013729:Terpenes; D005576:Colforsin; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; D019284:Thapsigargin; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D002118:Calcium