The role of the plasma-membrane Ca(2+)-ATPase in Ca (2+) homeostasis in Sinapis alba root hairs.

The regulation of cytosolic Ca(2+) has been investigated in growing root-hair cells of Sinapis alba L. with special emphasis on the role of the plasmamembrane Ca(2+)-ATPase. For this purpose, erythrosin B was used to inhibit the Ca(2+)-ATPase, and the Ca(2+) ionophore A23187 was applied to manipulate cytosolic free [Ca(2+)] which was then measured with Ca(2+)-selective microelectrodes. (i) At 0.01 μM, A23187 had no effect on the membrane potential but enhanced the Ca(2+) permeability of the plasma membrane. Higher concentrations of this ionophore strongly depolarized the cells, also in the presence of cyanide. (ii) Unexpectedly, A23187 first caused a decrease in cytosolic Ca(2+) by 0.2 to 0.3 pCa units and a cytosolic acidification by about 0.5 pH units, (iii) The depletion of cytosolic free Ca(2+) spontaneously reversed and became an increase, a process which strongly depended on the external Ca(2+) concentration, (iv) Upon removal of A23187, the cytosolic free [Ca(2+)] returned to its steady-state level, a process which was inhibited by erythrosin B. We suggest that the first reaction to the intruding Ca(2+) is an activation of Ca(2+) transporters (e.g. ATPases at the endoplasmic reticulum and the plasma membrane) which rapidly remove Ca(2+) from the cytosol. The two observations that after the addition of A23187, (i) Ca(2+) gradients as steep as-600 mV could be maintained and (ii) the cytosolic pH rapidly and immediately decreased without recovery indicate that the Ca(2+)-exporting plasma-membrane ATPase is physiologically connected to the electrochemical pH gradient, and probably works as an nH(+)/Ca(2+)-ATPase. Based on the finding that the Ca(2+)-ATPase inhibitor erythrosin B had no effect on cytosolic Ca(2+), but caused a strong Ca(2+) increase after the addion of A23187 we conclude that these cells, at least in the short term, have enough metabolic energy to balance the loss in transport activity caused by inhibition of the primary Ca(2+)-pump. We further conclude that this ATPase is a major Ca(2+) regulator in stress situations where the cytosolic Ca(2+) has been shifted from its steady-state level, as may be the case during processes of signal transduction.