Fluorescence ratio imaging was used for simultaneous measurement of cytosolic pH(c) and pCa(c) in Chara corallina, Nitella flexilis, and Eremosphaera viridis. In some experiments the electrical membrane potential was also recorded. The first hint of coupling between changes in pH(c) and pCa(c) was found in characean cells when the influence of butyrate on cytosolic streaming was studied by laser-Doppler-anemometry (LDA). The observed butyrate-induced cessation of cytosolic streaming supports the assumption that changes in pH(c) cause changes in pCa(c). This hypothesis was tested by simultaneously loading cells with Fura-2-dextran and BCECF-dextran. The addition of butyrate revealed strong coupling between pCa(c) and pH(c) although this only occurred when the difference between pH(c) and pCa(c) was less than 0.4 units (+/- 0.24, n = 7). The measured relationship between the changes in pCa(c) and pH(c) could be fitted by a cytoplasmic buffer exchange process. Protons imported by butyrate into the cytoplasm are able to displace Ca++ ions from cytoplasmic buffer sites. Three dissociation constants of the cytoplasmic buffer were pK(1) = 6.2, pK(2) = 7.1 for proton buffering, and pK(Ca) = 6.7 for Ca++ ion buffering. Other possible mechanisms, such as butyrate-induced Ca++ influx through the plasmalemma and Ca++ release from internal stores are discussed. They are not necessary to explain the observed coupling but cannot be excluded from the process. Using the butyrate technique, the cytosolic in vivo proton buffer capacities of N. flexilis, C. corallina, and E. viridis were determined as beta(i) = 30 mM . H+/pH-unit, beta(i) = 46 mM . H+/pH-unit, and beta(i) = 90 mM . H+/pH-unit, respectively. The values obtained in vivo are greater than those found previously using extraction methods. This can be explained in terms of pump activity and exchange with cell organelles, i.e., the vacuole. The high value of beta(i) found in Eremosphaera reflects adaptation to its habitat.
