CYCLIC AMP-REGULATED 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) greater than or equal to 20 mM. The adenosine 3',5'-cyclic monophosphate (cAMP)-dependent secretagogues forskolin (1 mu M), prostaglandin E(1) (1 mu M), and 8-bromoadenosine 3',5'cyclic monophosphate (100 mu M) 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 mu M) and greater than or equal to 65% by the Na-K-2Cl cotransport inhibitor furosemide (10 mu M). 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.