PHARMACOLOGICAL EVIDENCE THAT CALCIUM IS NOT REQUIRED FOR P-2-RECEPTOR-STIMULATED CL- SECRETION IN HT29-CL.16E

Extracellular ATP at micro- to millimolar concentrations activates Cl- conductance and increases cytosolic calcium ([Ca](i)) in many epithel ial cells, including the colonic epithelial cell Line HT29-Cl.16E. The refore, [Ca](i) has been postulated to be the intracellular messenger for Cl- channel activation. HT29-Cl.16E is a highly differentiated cel l line that forms confluent monolayers and secretes mucins and Cl-. Th e involvement of [Ca](i) in the purinergically-stimulated Cl- secretio n was investigated pharmacologically in this cell line by whole-cell p atch-clamp and Ussing chamber techniques, as well as [Ca](i) measureme nts in fura-2 loaded cells. The calmodulin inhibitors W13 (5 mu M) and chlorpromazine (50 mu M) abolished increases in ATP-stimulated [Ca](i ) increases by 90% and 80%, respectively. However, these inhibitors ha d no effect on the ATP-stimulated Cl- conductance measured in either i ndividual cells or confluent monolayers. As controls, the effects of W 13 and chlorpromazine on Ca2+-ionophore stimulated Cl- conductance was measured. In this case, the two compounds inhibited whole cell Cl- co nductance and monolayer Isc by 90% and 100%, respectively. These data demonstrate: (1) The purinergically-stimulated increase in Cl- current does not require an increase in [Ca](i) suggesting the involvement of either another signaling pathway or direct activation of Cl- channels by purinergic receptors. (2) A calmodulin or a calmodulinlike binding site that is sensitive to W13 and chlorpromazine participates in the regulation of the [Ca](i) increase by purinergic receptors in HT29-Cl. 16E.