Hypotonic Ca2+ signaling and volume regulation in proliferating and quiescent cells from multicellular spheroids

Hypotonicity-induced Ca²⁺ signals and volume regulation were studied in proliferating and quiescent subpopulations of multicellular prostate cancer spheroids. Enzymatic dissociation of multicellular spheroids 100 ± 19 μm in diameter, which are entirely proliferative, yielded a population of cells with a mean cell diameter of 17.5 ± 1.4 μm. After dissociation of spheroids in a size class of 200 ± 30, 300 ± 60, and 400 ± 65 μm in diameter, two subpopulations of cells with mean cell diameters corresponding to 12.9 ± 1.9 μm and 16.7 ± 2 μm were discriminated. The subpopulation of large cells was shown to be proliferative by positive Ki-67 antibody staining; the subpopulation of small cells was Ki-67 negative, indicating cell quiescence. In a spheroid size class of 100 ± 19 μm, a distinct subpopulation of quiescent cells was absent. Superfusion by hypotonic solutions revealed that only the proliferating cell fraction showed a regulatory volume decrease (RVD) and a Ca²⁺]_i transient. Both effects were absent in the quiescent cell population. The Ca²⁺]_i transient persisted in low (10 nM) Ca²⁺ solution and in the presence of 4 mM extracellular Ni²⁺ but was abolished in the presence of the endoplasmic reticulum Ca²⁺-ATPase blocker 2,5-di-tert-butylhydrochinone (t-BHQ). The t-BHQ likewise inhibited RVD, indicating that Ca²⁺ release from intracellular stores was necessary for RVD. Moreover, Ca²⁺]_i and RVD were dependent on an intact microfilament cytoskeleton because after 30 min of preincubation with cytochalasin B the Ca²⁺]_i transient was significantly reduced and RVD was abolished. The absence of RVD and Ca²⁺]_i transient in quiescent cells may be due to differences in the amount and the cytosolic arrangement of F-actin observed in quiescent cells. J. Cell. Physiol. 175:129–140, 1998. © 1998 Wiley-Liss, Inc.