The effects of haloalkene cysteine conjugates on cytosolic free calcium levels in LLC-PK(1) cells--studies utilising digital imaging fluorescence microscopy.

The aim of this study was to examine the effect of haloalkene S-cysteine conjugates on cytosolic free Ca(2+) levels in renal epithelial cells using digital imaging fluorescence microscopy (DIFM). S-(1,2,3,4,4-pentachloro-1,3,-butadienyl)-L-cysteine (PCBC) and S-(1,2-dichlorovinyl)-L-cysteine (DCVC) were both cytotoxic to LLC-PK(1) cells in culture. Prior treatment of the cells with aminooxyacetic acid (AOAA), an inhibitor of the enzyme cysteine conjugate beta-lyase, afforded complete protection against the toxicity at concentrations of PCBC up to 100 microM and DCVC up to 500 microM. The cytotoxicity produced by PCBC (100 microM) was time dependent with no loss of lactate dehydrogenase (LDH) into the medium being observed until 4 h after exposure, while removal of calcium from the medium prevented the toxicity. Addition of PCBC (100 microM) to LLC-PK(1) cells produced a small progressive increase in intracellular calcium ([Ca(2+)](i)) from 72+/-6 to 126+/-11 nM following 10 min of exposure. At this time there was a marked cellular heterogeneity in the calcium response with some cells showing marked increases in [Ca(2+)](i), while others cycled between low and high values and some just maintained basal levels. Exposure to PCBC (100 microM) for 1 h produced a more marked increase in [Ca(2+)](I), 469+/-46 nM, with all cells responding. The elevation in [Ca(2+)](i) was concentration-related with increases seen at concentrations of 5 microM PCBC and above. The increase in [Ca(2+)](i) produced by PCBC (100 microM) was prevented by treatment with AOAA, and markedly reduced by a nominally calcium free medium or the addition of the calcium chelator EGTA. DCVC (500 microM) also markedly elevated [Ca(2+)](i) following exposure for 1 h, this was also prevented by AOAA and a nominal calcium free medium. These findings indicate that elevation in [Ca(2+)](i) produced by PCBC in renal epithelial cells, is an early event in the cascade of signalling changes leading to renal cell death. The major source of calcium appears to be from increased influx although a small component is released from intracellular stores which my trigger a stress protein response.