We have previously demonstrated that [ATP]_o stimulates a K⁺ (⁸⁶Rb) efflux pathway in CYTS that is sensitive to charybdotoxin, suggesting that the response is via activation of Ca²⁺-activated K⁺ channels (Clarson et al. 2000). In this study we have examined the role of [Ca²⁺]_o on this efflux pathway and the effect of CYTS differentiation. CYTS isolated from human term placentas were maintained in culture for 18 or 66 h (the majority of cells are mono- or multinuclear, respectively) and ⁸⁶Rb efflux or intracellular Ca²⁺ concentration ([Ca²⁺]_i) measured.

Cells were loaded with 4 µCi ml^{-1 86}Rb in Hepes-efflux buffer for 2 h and then washed for 3 min with control or nominally 0 Ca²⁺ (no added Ca²⁺ with 0.5 mM EGTA) buffer. Efflux was measured by sequential addition and removal of buffer at 1 min intervals for 10 min in total, after which cells were lysed to determine intracellular ⁸⁶Rb content. ATP ± charybdotoxin was applied after 5 min and throughout the remaining experimental period. ⁸⁶Rb effluxed from the cells was expressed as % of the total ⁸⁶Rb available for efflux at the start of each 1 min collection. Cells were loaded with 1 µM fura-2 acetoxymethyl ester for measurement of [Ca²⁺]_i. Fluorescence was excited at 350 and 380 nm and observed through a Ω40 oil immersion objective. Cells were stimulated for 2 min with ATP ± nominally 0 Ca²⁺ buffer. 0 Ca²⁺ buffer was present for 8 min prior to treatment with ATP. The change in fluorescence ratio above control (▓Dgr│R) was measured for the peak response and following 2 min exposure.

ATP-stimulated ⁸⁶Rb efflux was significantly reduced in 0 Ca²⁺ buffer and the residual efflux was further decreased by charybdotoxin, demonstrating that ATP-stimulated K⁺ efflux from CYTS predominantly required [Ca²⁺]_o to raise [Ca²⁺]_i. Both the efflux and microfluorimetry data revealed a greater dependence on [Ca²⁺]_o in the 66 h CYTS than the 18 h CYTS. Therefore we postulate that the entry pathway for [Ca²⁺]_o may be altered during CYTS differentiation.

This work was supported by the MRC.

Clarson, L.H., Roberts, V.H.J. & Greenwood, S.L. (2000). J. Physiol. 528.P, 20P.