STIMULATION OF CL- SECRETION BY EXTRACELLULAR ATP DOES NOT DEPEND ON INCREASED CYTOSOLIC CA2-29.CL16E( IN HT)

Extracellular ATP and elevated cytosolic Ca2+ concentration ([Ca2+](i) ) are major secretagogues for Cl- in the goblet cell-like clone cl.16E derived from colcnic HT-29 cells. The involvement of [Ca2+]i as a mes senger for the purinergically stimulated Cl- secretion was investigate d using whole cell patch-clamp and Ussing chamber techniques, as well as [Ca2+](i) measurements using fura 2-loaded cells. Under voltage-cla mp conditions, the whole cell current at +50 mV was 3 +/- 1 pA/pF unde r unstimulated conditions. Stimulation of purinergic receptors with 20 0 mu M extracellular ATP increased the current at +50 mV to 41 +/- 10 pA/pF, with a half-maximal effective dose (ED(50)) Of similar to 3 mu M. The current was transient, usually lasting 1-2 min, and the current -voltage relationship was approximately linear between -70 and +50 mV. Evidence that the ATP-stimulated current was carried by Cl- included 1) the reversal potential of the current closely followed the Cl- equi librium potential, and 2) the stimulated current was absent when Cl- w as removed from both bath and pipette solutions. Exposure to ATP also increased [Ca2+](i), with an ED(50) of similar to 1 mu M and maximal c hanges (at 200 mu M) from baseline (71 +/- 3 nM) to 459 +/- 50 nM. The ATP-dependent Cl- conductance increase was not diminished when [Ca2+] (i) was clamped at 100 nM using a ,2-bis(2-aminophenoxy)ethane-N,N,N', N'-tetraacetic acid or Ca2+-ethylene glycol-bis(beta-aminoethyl ether) -N, N, N', N'-tetraacetic acid buffering system. However, the ATP effe ct did require some basal level of Ca2+ because clamping [Ca2+](i) at < 10 nM abolished activation of the Cl- conductance. The presence of t he protein kinase A inhibitor H-89 or the protein kinase C inhibitor s taurosprine did not change the ATP-activated Cl- conductance. These da ta demonstrate that the ATP-stimulated increase in Cl- current does no t require an increase in [Ca2+](i), suggesting the involvement of eith er another signaling pathway or direct activation of Cl- channels by p urinergic receptors.