Calcium control of the hydraulic resistance in cells of Chara corallina.

The hydraulic resistance (the reciprocal of the hydraulic conductivity Lp) Lp-1 was measured in cells of Chara corallina by the method of transcellular osmosis. Treatment of cells with 100 mM KCl decreased Lp-1 significantly. Subsequent treatment of the cells with 70 mM CaCl2 recovered the decreased Lp-1 to the original value. To know whether K+ or Ca2+/Mg2+ acts on the cell wall and/or the membrane, the hydraulic resistances of the cell wall (Lpw-1) and that of the membrane (Lpm-1) were determined in one and the same cell. For this, a pair of cells (twin cells) were made from an internodal cell, one used for measurement of Lp-1 and the other used for the measurement of Lpw-1. From Lp-1 and Lpw-1, Lpm-1 was calculated. Both Lp-1 and Lpw-1 were decreased by K+, while Lpm-1 was not affected by K+. The same result was obtained with 5 mM EGTA. Lpw-1 was decreased more than it was by KCl but Lpm-1 remained constant after EGTA treatment. The recovery of the K+-decreased Lp-1 with Ca2+ can be explained exclusively by the recovery of Lpw-1 with Ca2+. The Ca2+ recovery of Lpw-1 was observed in the intact cell wall but not in the cell wall tube isolated from an internodal cell. The different response to Ca2+ between the intact cell wall and the isolated cell wall was discussed in relation to the tension in the cell wall which may be an important factor for the ionic regulation of hydraulic conductivity.

Age dependence of the hydraulic resistances of the plasma membrane and the tonoplast (vacuolar membrane) in cells of Chara corallina.

Hydraulic resistances (reciprocals of hydraulic conductivities) of the cell (Lp-1), the cell wall (Lpw-1), the membrane (Lpm-1), the plasma membrane (Lppm-1), and the tonoplast (Lptp-1) were determined in individual internodal cells of Chara corallina and their dependence on the cell age was studied. The thickness of the cell wall (d) was adopted as an index of the cell age, since the cell wall of spring-grown young cells (sg-cells) was found to be significantly thinner than that of winter-spent old cells (ws-cells). Both Lpw-1 and Lpm-1 were found to increase with cell age. Since Lpm-1 is the sum of Lppm-1 and Lptp-1, their dependence on the wall thickness was studied. It was found that both Lppm-1 and Lptp-1 increase with cell age using d as a proxy and that the former is distinctly higher than the latter. The ratio Lppm-1/Lptp-1 amounts to 30 for 5 µm of d, indicating that the tonoplast is a negligible barrier to osmotic water flow. The ratio decreases with the increase in d and amounts to 5.0 for 11 µm of d, showing that the tonoplast ages faster than the plasma membrane. The physiological meaning of the age dependence of hydraulic resistance of the tonoplast was discussed in terms of the role of the vacuole in the osmoregulation of the cytoplasm.

Chara myosin and the energy of cytoplasmic streaming.

Recently, it was found that myosin generating very fast cytoplasmic streaming in Chara corallina has very high ATPase activity. To estimate the energy consumed by this myosin, its concentration in the internodal cells of C. corallina was determined by quantitative immunoblot. It was found that the concentration of Chara myosin was considerably high (200 nM) and the amount of ATP consumed by this myosin would exceed that supplied by dark respiration if all myosin molecules were fully activated by the interaction with actin. These results and model calculations suggested that the energy required to generate cytoplasmic streaming is very small and only one-hundredth of the existing myosin is enough to maintain the force for the streaming in the Chara cell.

Cell physiological aspects of the plasma membrane electrogenic H+ pump.

This article deals with cell physiological aspects of the plasma membrane electrogenic proton (H+) pump and emphasizes the contribution of the giant algal cells of the Characeae in elucidating the mechanism of the pump. First, a history of the development of intracellular perfusion techniques in characean internodal cells is described, including preparation of tonoplast-free cells. Then, an outline of the hypothesis of the electrogenic H+ pump proposed by Kitasato is introduced, who prophesied the existence of an electric potential generated by an active H+ efflux. Subsequently, a history of finding ATP as the direct energy source of the electrogenic ion pump is presented. Quantitative agreement between the pump current and the ATP-dependent H+ efflux supports the notion that the ion carried by the electrogenic ion pump is H+. The role of the H+ pump in regulation of the cytosolic pH is discussed. Mechanisms of light-induced potential change through photosynthesis-controlled activation of the H+ pump are discussed in terms of changes in the levels of adenine nucleotides and in modulation of the Km value for the ATP of H+-ATPase. Recent progress in the molecular mechanism of the blue-light-induced activation of the H+-ATPase in guard cells is presented. However, there are cases where H+-ATPase activity is inhibited by blue light, indicating the flexibility of the control mechanisms of H+-ATPase activity. Finally, modulation of H+-pumping or H+-ATPase activities in response to environmental factors, such as anoxia, membrane excitation, osmotic and salt stresses, nutrient deficiencies and aluminum toxicity are described. Discussions are presented on the regulation of the electrogenic H+ pump.

Is Ca2+ release from internal stores involved in membrane excitation in characean cells?

An action potential in characean cells is accompanied by an increase in the cytosolic Ca(2+) concentration ([Ca(2+)](c)) which subsequently causes cessation of cytoplasmic streaming. Two Ca(2+ )origins are postulated for the increase in [Ca(2+)](c), extracellular and intracellular ones. For the extracellular origin, a Ca(2+) influx through voltage-dependent Ca(2+)-permeable channels is postulated. For the intracellular origin, a chain of reactions is assumed to occur, involving phosphoinositide-specific phospholipase C (PI-PLC) activation, production of inositol 1,4,5-trisphosphate (IP(3)) and IP(3)-dependent Ca(2+) release from internal stores [Biskup et al. (1999) FEBS Lett. 453: 72]. The hypothesis of the intracellular Ca(2+) origin was tested in three ways: injection of IP(3) into the streaming endoplasm, application of inhibitors of PI-PLC (U73122 and neomycin) and application of an inhibitor of IP(3)-receptor (2-aminoethoxydiphenyl borate; 2APB). Injection of 1 mM IP(3) into Chara cells did not change the rate of cytoplasmic streaming. Both U73122 (20 micro M) and neomycin (200 micro M) did not affect the generation of the action potential, cessation of cytoplasmic streaming and the increase in [Ca(2+)](c) caused by electric stimulus even 20-30 min after application. 2APB depolarized the membrane and inhibited the excitability of the plasma membrane. The results are not consistent with the data obtained by Biskup et al. (1999) who found inhibition of the excitatory inward current by neomycin and U73122. The hypotheses of internal and external Ca(2+) origins are discussed in the light of the present results.