Pharmacological evidence that calcium is not required for P2-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 epithelial cells, including the colonic epithelial cell line HT29-Cl. 16E. Therefore, [Ca]i has been postulated to be the intracellular messenger for Cl- channel activation. HT29-Cl.16E is a highly differentiated cell line that forms confluent monolayers and secretes mucins and Cl-. The involvement of [Ca]i in the purinergically-stimulated Cl- secretion was investigated pharmacologically in this cell line by whole-cell patch-clamp and Ussing chamber techniques, as well as [Ca]i measurements in fura-2 loaded cells. The calmodulin inhibitors W13 (5 microm) and chlorpromazine (50 microm) abolished increases in ATP-stimulated [Ca]i-increases by 90% and 80%, respectively. However, these inhibitors had no effect on the ATP-stimulated Cl- conductance measured in either individual cells or confluent monolayers. As controls, the effects of W13 and chlorpromazine on Ca2+-ionophore stimulated Cl- conductance was measured. In this case, the two compounds inhibited whole cell Cl- conductance 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.
Guo
X
X
Department of Physiology and Biophysics, School of Medicine, Case Western Reserve University, Cleveland, OH, 44106-4970, USA.
Merlin
D
D
Harvey
R D
RD
Laboisse
C
C
Hopfer
U
U
D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000037:antagonists & inhibitors; D002450:Cell Communication; D018118:Chloride Channels; D002712:Chlorides / Q000378:metabolism*; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D019073:HT29 Cells; D006801:Humans; D007476:Ionophores / Q000494:pharmacology; D018408:Patch-Clamp Techniques; D018048:Receptors, Purinergic P2 / Q000378:metabolism*; D015398:Signal Transduction
D002147:Calmodulin; D018118:Chloride Channels; D002712:Chlorides; D007476:Ionophores; D018048:Receptors, Purinergic P2; D002118:Calcium