Propranolol enhances cAMP and Ca2+-activated chloride secretion in T84 cells. Is PLD involved in the regulation of chloride secretion?

Abstract

Objective: Phospholipase D (PLD) is activated by cAmP and Ca2+agonists, but its role in the regulation of secretion in epithelial cells is unknown. Propranolol interferes with the conversion of PLD-generated phosphatidic acid to diacylglycerol. We studied the effect of propranolol on regulated C1secretion in T84 cells and examined the possible involvement of PLO in this secretory pathway. Material and methods: T84 cells grown on permeable supports were used for electrophysiological studies. Changes in PD were monitored with a voltage-current clamp. Short-circuit current (Isc in ~ . c m -2) was calculated by Ohm's law. Ise is equivalent to CI secretion in T84 cells. Forskofin (10 ~M) and carbachol (100 ~M) were used as cAMP and Ca2+agonists, respectively. Student's t test was used for statistical analysis. Statistical significance was defined as p<0.05. Results: Both apical and basulateral addition of proprannlol increased Isc in a dose-dependent fashion (500 riM-200 p,M). Apical propranolol (200 ~M) was more potent than basolateral propranolol for stimulation of secretion (peak Isc -65.4 -+ 1.8 vs. 39.6 -+ 0.8 for apical vs. basolateral propranolol after incubation for 150 minutes; n -3 for each condition; p<0.0001). Propranolol-elicited Isc was bumetanide-sensitive, suggesting that active Clsecretion is involved. Incubation with basolateral propranolol dose-dependently enhanced forskolin-stimulated Cl'secretion. (peak Isc = 137 -+ 8.9 vs. 76.4 -+ 1.7 for propranolol-treated vs. control samples; n = 3 for each condition; p<0.O01). Propranolol also showed a synergistic effect on Isc elicited by the Ca ~+ agonists(peak lsc = 275 -+ 40.2 vs. 112 8.4 for propranolol-treated vs. control samples; n = 6 for each condition; p<0.001). In T84 cells, carhachol pretreatment blunts the subsequent secretory response induced by forakolin. However, basolateral propranolol (200 p,M) overcame the inhibitory effect of carbachol pretreatment on forskolni-stimulated secretion (peak Isc = 236 -+ 11.9 vs. 59.4 -+ 1.8 for propranolol -+ carbachol vs. carhachol alone; n = 6 for each condition; p<0.0001). Conclusion: In T84 cells, propranolol dosedependently increases CI secretion and synergistically enhances cAMP and Ca2+-activated secretion. It also reverses the inhibitory effect of carbachol pretreatment on stimulated secretion. Our results suggest that PLD may be involved in the regulation of secretion in T84 cells and it may become a new therapeutic target.