Control of cell volume in the J774 macrophage by microtubule disassembly and cyclic AMP

Abstract

The possibilities that cell volume is regulated by the status of microtubule assembly and cAMP metabolism and may be coordinated with shape change were studied. Treatment of J774.2 mouse macrophages [subclone neoplastic cell line] with colchicine caused rapid microtubule disassembly and was associated with a striking increase (from 15-20 to > 90%) in the proportion of cells with a large protuberance at 1 pole. This provided a simple experimental system in which shape changes occurred in virtually an entire cell population in suspension. Parallel changes in cell volume could then be quantified by isotope dilution techniques. The shape change caused by colchicine was accompanied by a decrease in cell volume of .apprx. 20%. Nocodozole, but not lumicolchicine, caused identical changes in both cell shape and cell volume. The volume loss was not due to cell lysis nor to inhibition of pinocytosis. The mechanism of volume loss was examined. Colchicine induced a small but reproducible increase in activity of tht ouabain-sensitive Na+, K+-dependent ATPase. Inhibition of this enzyme/transport system by ouabain did not change cell volume nor did it block the colchicine-induced decrease in volume. SITS (4''-acetamido, 4-isothiocyano-2,2''-disulfonic acid stilbene), an inhibitor of anion transport, inhibited the effects of colchicine, thus suggesting a role for an anion transport system in cell volume regulation. Because colchicine is known to activate adenylate cyclase in several systems and because cell shape changes are often induced by hormones that elevate cAPM, the effects of cAMP on cell volume were examined. Agents that act to increase cAMP (cholera toxin, which activates adenylate cyclase; IBMX, an inhibitor of phosphodiesterase; and dibutyrl cAMP) all caused a volume decrease comparable to that of colchicine. To define the effective metabolic pathway, 2 mutants of J774.2 were studied, 1 deficient in adenylate cyclase and the other exhibiting markedly reduced activity of cAMP-dependent protein kinase. Cholera toxin did not produce a volume change in either mutant. CAMP produced a decrease in the cyclase-deficient line comparable to that in the wild type, but did not cause a volume change in the kinase-deficient line. This analysis established separate roles for cAMP and colchicine. The volume decrease induced by cAMP requires the action of a cAMP-dependent protein kinase. Colchicine induced a comparable volume change in both mutants and wild type and does not require the kinase.