AJP - Gastrointestinal and Liver Physiology, Vol 266, Issue 5 846-G855, Copyright © 1994 by American Physiological Society

ARTICLES

Cyclic AMP- and phorbol ester-regulated Cl- permeabilities in primary cultures of human and rabbit colonocytes

J. Sahi, J. L. Goldstein, T. J. Layden and M. C. Rao
Department of Physiology and Biophysics, University of Illinois at Chicago 60612.

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.

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