VOLUME-SENSITIVE CHLORIDE CURRENT IN PIGMENTED CILIARY EPITHELIAL-CELLS - ROLE OF PHOSPHOLIPASES

The whole cell recording technique was used to examine an outwardly re ctifying chloride current activated by hypotonic shock in bovine pigme nted ciliary epithelial (PCE) cells. Removal of internal and external Ca2+ did not affect the activation of these currents, but they were ab olished by the phospholipase C inhibitor neomycin. The current was blo cked by 5-nitro-2-(3-phenylpropylamino)benzoic acid, -acetamido-4'-iso thiocyanostilbene-2,2'-disulfonic acid, and 4,4'-diisothiocyanostilben e-2,2'-disulfonic acid (DIDS) in a voltage-dependent manner, but tamox ifen, dideoxyforskolin, and quinidine did not affect it. This blocking profile differs from that of the volume-sensitive chloride channel in neighboring nonpigmented ciliary epithelial cells (Wu, J., J. J. Zhan g, H. Koppel, and T. J. C. Jacob. J. Physiol. Lend. 491: 743-755, 1996 ), and this difference implies that the volume responses of the two ce ll types are mediated by different chloride channels (Jacob, T. J. C., and J. J. Zhang. J. Physiol. Lend. In press). Intracellular administr ation of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) to PCE cell s induced a transient, time-independent, outwardly rectifying chloride current that closely resembled the current activated by hypotonic sho ck. DIDS produced a voltage-dependent block of the GTP gamma S-activat ed current similar to the block of the hypotonically activated current . Intracellular neomycin completely prevented activation of this curre nt as did incubation of the cells in calphostin C, an inhibitor of pro tein kinase C (PKC). Removal of Ca2+ did not affect activation of the current by GTP gamma S but extended the duration of the response. Inhi bition of phospholipase A(2) (PLA(2)) with p-bromophenacyl bromide pre vented the activation of the hypotonically induced current and also in hibited the current once activated by hypotonic solution. The findings imply that the hypotonic response in PCE cells is mediated by both ph ospholipase C (PLC) and PLA(2). Both phospholipases generate arachidon ic acid, and, in addition, the PLC pathway regulates the PLA(2) pathwa y via a PKC-dependent phosphorylation of PLA(2).