Volume-sensitive chloride current in pigmented ciliary epithelial cells: the role of phospholipases. Mitchell, Claire H., Jin Jun Zhang, Liwei Wang, and Tim J. C. Jacob. Eye Research Lab, Physiology Unit, School of Molecular and Medical Bioscience, University of Wales, Cardiff, CF1 3US, UK
APStracts 3:0311C, 1996.
The whole cell recording technique was used to examine an outwardly rectifying chloride current activated by hypotonic shock in bovine pigmented ciliary epithelial (PCE) cells. Removal of internal and external Ca2+ did not affect the activation of these currents, but they were abolished by the phospholipase C inhibitor neomycin. The current was blocked by NPPB, SITS and DIDS in a voltage-dependent manner, but tamoxifen, dideoxyforskolin and quinidine did not affect it. This blocking profile differs from that of the volume sensitive chloride channel in neighbouring non-pigmented ciliary epithelial (NPCE) cells [40] and this difference implies that the volume responses of the two cell types are mediated by different chloride channels [20]. Intracellular administration of GTPS to PCE cells induced a transient, time-independent, outwardly rectifying chloride current which closely resembled the current activated by hypotonic shock. DIDS produced a voltage-dependent block of the GTPS-activated current similar to the block of the hypotonic-activated current. Intracellular neomycin completely prevented activation of this current as did incubation of the cells in calphostin C, an inhibitor of PKC. Removal of Ca2+ did not affect activation of the current by GTPS, but extended the duration of the response. Inhibition of phospholipase A2 (PLA2) with p-bromophenacyl bromide (pBPB) prevented the activation of the hypotonic-induced current and also inhibited the current once activated by hypotonic solution. The findings imply that the hypotonic response in PCE cells is mediated by both PLC and PLA2. Both phospholipases generate arachidonic acid and, in addition, the PLC pathway regulates the PLA2 pathway via a PKC-dependent phosphorylation of PLA2. 

Received 18 August 1995; accepted in final form 23 August 1996.
APS Manuscript Number C513-5.
Article publication pending Am. J. Physiol. (Cell Physiology).
ISSN 1080-4757 Copyright 1996 The American Physiological Society.
Published in APStracts on 5 November 1996