Volume-dependent regulation of sodium and potassium fluxes in cultured vascular smooth muscle cells: Dependence on medium osmolality and regulation by signalling systems

Summary To identify ion transport systems involved in the maintenance of vascular smooth muscle cell volume the effects of incubation medium osmolality and ion transport inhibitors on the volume and ⁸⁶Rb and ²²Na transport in cultured smooth muscle cells from rat aorta (VSMC) have been studied. A decrease of medium osmolality from 605 to 180 mosm increased intracellular water volume from 0.6 to 1.3 l per 10⁶ cells. Under isosmotic conditions, cell volume was decreased by ouabain (by 10%, P< 0.005) but was not influenced by bumetanide, furosemide, EIPA and quinidine. These latter compounds were also ineffective in cell volume regulation under hypotonic buffer conditions. Under hyperosmotic conditions, cell volume was decreased by bumetanide (by 7%, P<0.05) and by ethylisopropyl amiloride (by 13%, P< 0.005). Ouabain-sensitive ⁸⁶Rb influx was decreased by 30–40% under hypoosmotic conditions. An increase in medium osmolality from 275 to 410 mosm resulted in an eightfold increase in bumetanide-inhibited ⁸⁶Rb influx and ⁸⁶Rb efflux. The (ouabain and bumetanide)-insensitive component of ⁸⁶Rb influx was not dependent on the osmolality of the incubation medium. However (ouabain and bumetanide)-insensitive ⁸⁶Rb efflux was increased by 1.5–2 fold in VSMC incubated in hypotonic medium. Ethylisopropyl amiloride-inhibited ²²Na influx was increased by sixfold following osmotic-shrinkage of VSMC. The data show that both Na⁺/H⁺ exchange and Na⁺/K⁺/2Cl^– cotransport may play a major role in the regulatory volume increase in VSMC. Basal and shrinkage-induced activities of Na⁺/K⁺/2Cl^– cotransport in VSMC were similarly sensitive to inhibition by either staurosporin, forskolin, R24571 or 2-nitro4-carboxyphenyl N,N-diphenylcarbomate (NCDC). In contrast basal and shrinkage-induced Na⁺/K⁺/2Cl^– cotransport were differentially inhibited by NaF (by 30 and 65%, respectively), suggesting an involvement of guanine nucleotide binding proteins in the volume-sensitive activity of this carrier. Neither staurosporin, forskolin, R24571 nor NCDC influenced shrinkage-induced Na⁺/H⁺ exchange activity. NaF increased Na⁺/H⁺ exchanger activity under both isosmotic and hyperosmotic conditions. These data demonstrate that different intracellular signalling mechanisms are involved in the volume-dependent activation of the Na⁺/K⁺/2Cl^– cotransporter and the Na⁺/H⁺ exchanger.The authors gratefully acknowledge the financial support of the Swiss National Foundation, grant No. 3.817.087. Bernadette Weber is thanked for preparing the figures.