Cyclic AMP- and phorbol ester-regulated Cl- permeabilities in primary cultures of human and rabbit colonocytes.
Chloride transport in 24-h primary cultures of human and rabbit distal colonic crypt cells (90 +/- 5% viable) were characterized using the Cl(-)-sensitive fluorescent probe 6-methoxyquinolyl acetoethyl ester. To calculate the Cl- influx in millimolar per second, the Stern-Volmer quenching constant was determined to be 24.3 M-1 for human and 24.6 M-1 for rabbit colonocytes. Cl- influx was dependent on extracellular Cl- concentration ([Cl-]0), with maximal influx at [Cl-]0 > or = 20 mM. The adenosine 3',5'-cyclic monophosphate (cAMP)-dependent secretagogues forskolin (1 microM), prostaglandin E1 (1 microM), and 8-bromoadenosine 3',5'-cyclic monophosphate (100 microM) increased Cl- influx in human colonocytes from 0.35 +/- 0.08 to 2.14 +/- 0.65, 1.85 +/- 0.51, and 0.84 +/- 0.04 mM/s (n = 4), respectively, and in rabbit colonocytes from 0.22 +/- 0.03 to 1.04 +/- 0.11, 1.24 +/- 0.12, and 1.08 +/- 0.07 mM/s (n = 5), respectively. Depending on the secretagogue, this influx was inhibited 50-90% by the Cl- channel blocker diphenylamine-2-carboxylate (DPC; 50 microM) and > or = 65% by the Na-K-2Cl cotransport inhibitor furosemide (10 microM). Phorbol 12,13-dibutyrate, an activator of protein kinase C, increased Cl- permeability 3.8-fold in human and 2.4-fold in rabbit colonocytes. The phorbol 12,13-dibutyrate-stimulated Cl- permeabilities were sensitive to DPC and furosemide but not to indomethacin. These studies demonstrate DPC and furosemide-sensitive Cl- permeabilities in isolated cultured human and rabbit colonocytes, which can be activated by cAMP and protein kinase C stimulators.
Sahi
J
J
Department of Physiology and Biophysics, University of Illinois at Chicago 60612.
Goldstein
J L
JL
Layden
T J
TJ
Rao
M C
MC
D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000527:Alprostadil / Q000494:pharmacology; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002034:Bumetanide / Q000494:pharmacology; D002352:Carrier Proteins / Q000037:antagonists & inhibitors; D002463:Cell Membrane Permeability; D002469:Cell Separation; D002478:Cells, Cultured; D002712:Chlorides / Q000378:metabolism*; D005576:Colforsin / Q000494:pharmacology; D003106:Colon / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D000242:Cyclic AMP / Q000378:metabolism*; D005456:Fluorescent Dyes; D005665:Furosemide / Q000494:pharmacology; D006801:Humans; D007213:Indomethacin / Q000494:pharmacology; D007700:Kinetics; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology*; D011493:Protein Kinase C / Q000378:metabolism; D011817:Rabbits; D012007:Rectum / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D028021:Sodium-Potassium-Chloride Symporters; D013050:Spectrometry, Fluorescence; D013997:Time Factors
D002352:Carrier Proteins; D002712:Chlorides; D005456:Fluorescent Dyes; D028021:Sodium-Potassium-Chloride Symporters; D002034:Bumetanide; D005576:Colforsin; D015124:8-Bromo Cyclic Adenosine Monophosphate; D015240:Phorbol 12,13-Dibutyrate; D005665:Furosemide; D000242:Cyclic AMP; D011493:Protein Kinase C; D000527:Alprostadil; D007213:Indomethacin