Abstract:
The regulation of cytosolic Ca2+ has been investigated in growing root-hair cells of Sinapis alba L. with special emphasis on the role of the plasmamembrane Ca2+-ATPase. For this purpose, erythrosin B was used to inhibit the Ca2+-ATPase, and the Ca2+ ionophore A23187 was applied to manipulate cytosolic free [Ca2+] which was then measured with Ca2+-selective microelectrodes. (i) At 0.01 M, A23187 had no effect on the membrane potential but enhanced the Ca2+ 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 Ca2+ by 0.2 to 0.3 pCa units and a cytosolic acidification by about 0.5 pH units, (iii) The depletion of cytosolic free Ca2+ spontaneously reversed and became an increase, a process which strongly depended on the external Ca2+ concentration, (iv) Upon removal of A23187, the cytosolic free [Ca2+] returned to its steady-state level, a process which was inhibited by erythrosin B. We suggest that the first reaction to the intruding Ca2+ is an activation of Ca2+ transporters (e.g. ATPases at the endoplasmic reticulum and the plasma membrane) which rapidly remove Ca2+ from the cytosol. The two observations that after the addition of A23187, (i) Ca2+ gradients as steep as-600 mV could be maintained and (ii) the cytosolic pH rapidly and immediately decreased without recovery indicate that the Ca2+-exporting plasma-membrane ATPase is physiologically connected to the electrochemical pH gradient, and probably works as an nH+/Ca2+-ATPase. Based on the finding that the Ca2+-ATPase inhibitor erythrosin B had no effect on cytosolic Ca2+, but caused a strong Ca2+ 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 Ca2+-pump. We further conclude that this ATPase is a major Ca2+ regulator in stress situations where the cytosolic Ca2+ has been shifted from its steady-state level, as may be the case during processes of signal transduction.
