The effect of phorbol esters on adenosine 3',5'-cyclic monophosphate (cAMP)-regulated epithelial Cl- transport was studied in T84 cells, a human colonic cell line that serves as a model for electrogenic Cl- secretion. Preincubation of T84 cell monolayers with phorbol 12-myristate 13-acetate (PMA) caused a time- and dose-dependent inhibition of the net transepithelial secretory response to 10 microM forskolin (half-maximal inhibition at a concentration of approximately 10 nM PMA and a time of 45 min). Similar inhibition was observed with phorbol 12,13-dibutyrate but not the inactive phorbol ester phorbol 12,13-diacetate. Na(+)-K(+)-2Cl- cotransporter activity, assessed by bumetanide-sensitive 86Rb+ uptake, and K+ conductance, assessed by 86Rb+ efflux, were both found to be markedly reduced by PMA with a time course that paralleled the loss of the cAMP-regulated Cl- secretory response. One- and four-hour treatment of T84 cells with 100 nM PMA caused a sustained increase in the membrane-bound fraction of protein kinase C (PKC) but a decrease in total cellular PKC. Although, at these time points, the Na(+)-K(+)-2Cl- cotransporter and K+ efflux pathways were markedly inhibited (associated with inhibition of the forskolin-stimulated transepithelial Cl- secretory response), the activity of the cAMP-regulated Cl- efflux pathway, assessed by 125I-labeled efflux, remained unaffected. With prolonged exposure to PMA (up to 10), the cAMP-regulated Cl- efflux pathway was also eventually inhibited, and transepithelial electrical resistance progressively declined.(ABSTRACT TRUNCATED AT 250 WORDS)
We previously reported that adenosine 3',5'-cyclic monophosphate-mediated stimulation of Cl- secretion in the human intestinal epithelial cell line T84 is accompanied by significant remodeling of F-actin and that both the secretory and cytoskeletal responses may be inhibited by phalloidin derivatives, agents that polymerize actin and prevent dynamic reorganization of microfilaments. In contrast, the carbachol-elicited Cl- secretory response (Ca2+ mediated) was not attenuated by phalloidin (J. Clin. Invest. 87: 1903-1909, 1991). In the present study, we examine the effect of phalloidin on the Cl- secretory response elicited by the heat-stable enterotoxin of Escherichia coli (STa), which induces elevations in intracellular guanosine 3',5'-cyclic monophosphate. We find that apical administration of 1 microM STa results in a regionally restricted redistribution of F-actin confined to the basal pole of the cells. In monolayers pretreated with phalloidin, the Cl- secretory response to STa was inhibited by > 60%. Sequential treatment of phalloidin-loaded monolayers with STa followed by carbachol resulted in a synergistic secretory response that was not different from control (unloaded) monolayers. Examination of efflux/uptake through specific membrane transport pathways involved in STa-stimulated Cl- secretion indicated normal activation of apical Cl- and basolateral K+ channels under phalloidin-loaded conditions. The ability of STa-treated monolayers to pump Na+ in an absorptive direction was also unaffected by phalloidin. der phalloidin-loaded conditions, STa-stimulated Na(+)-K(+)-2Cl- cotransporter activity was reduced by approximately 60%, sufficient to account for the observed inhibition of net Cl- secretory response.(ABSTRACT TRUNCATED AT 250 WORDS)
Adenosine release from mucosal sources during inflammation and ischemia activates intestinal epithelial Cl−secretion. Previous data suggest that A2b receptor-mediated Cl− secretory responses may be dampened by epithelial cell nucleoside scavenging. The present study utilizes isotopic flux analysis and nucleoside analog binding assays to directly characterize the nucleoside transport system of cultured T84 human intestinal epithelial cells and to explore whether adenosine transport is regulated by secretory agonists, metabolic inhibition, or phorbol ester. Uptake of adenosine across the apical membrane displayed characteristics of simple diffusion. Kinetic analysis of basolateral uptake revealed a Na+-independent, nitrobenzylthioinosine (NBTI)-sensitive facilitated-diffusion system with low affinity but high capacity for adenosine. NBTI binding studies indicated a single population of high-affinity binding sites basolaterally. Neither forskolin, 5′-( N-ethylcarboxamido)-adenosine, nor metabolic inhibition significantly altered adenosine transport. However, phorbol 12-myristate 13-acetate significantly reduced both adenosine transport and the number of specific NBTI binding sites, suggesting that transporter number may be decreased through activation of protein kinase C. This basolateral facilitated adenosine transporter may serve a conventional function in nucleoside salvage and a novel function as a regulator of adenosine-dependent Cl− secretory responses and hence diarrheal disorders.
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