Summary
Na+-K+-2Cl− cotransport in aortic endothelial cells is activated by cell shrinkage, inhibited by cell swelling, and is responsible for recovery of cell volume. The role of protein phosphorylation in the regulation of cotransport was examined with two inhibitors of protein phosphatases, okadaic acid and calyculin, and a protein kinase inhibitor, K252a. Both phosphatase inhibitors stimulated cotransport in isotonic medium, with calyculin, a more potent inhibitor of protein phosphatase I, being 50-fold more potent. Neither agent stimulated cotransport in hypertonic medium. Stimulation by calyculin was immediate and was complete by 5 min, with no change in cell Na + K content, indicating that the stimulation of cotransport was not secondary to cell shrinkage. The time required for calyculin to activate cotransport was longer in swollen cells than in normal cells, indicating that the phosphorylation step is affected by cell volume. Activation of cotransport when cells in isotonic medium were placed in hypertonic medium was more rapid than the inactivation of cotransport when cells in hypertonic medium were placed in isotonic medium, which is consistent with a shrinkage-activated kinase rather than a shrinkage-inhibited phosphatase. K252a, a nonspecific protein kinase inhibitor, reduced cotransport in both isotonic and hypertonic media. The rate of inactivation was the same in either medium, indicating that dephosphorylation is not regulated by cell volume. These results demonstrate that Na+-K+-2Cl− cotransport is activated by protein phosphorylation and is inactivated by a Type I protein phosphatase. The regulation of cotransport by cell volume is due to changes in the rate of phosphorylation rather than dephosphorylation, suggesting the existence of a volume-sensitive protein kinase. Both the kinase and the phosphatase are constitutively active, perhaps to allow for rapid changes in cotransport activity.
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Adragna, N.C., Perkins, C.M., Lauf, P.K. 1985. Furosemide-sensitive Na+-K− cotransport and cellular metabolism in human erythocytes. Biochim. Biophys. Acta 81:293–296
Altamirano, A.A., Breitwieser, G.E., Russell, J.M. 1988. Vanadate and fluoride effect on Na+-K+-Cl− cotransport in squid giant axon. Am. J. Physiol. 254:C582-C586
Altamirano, A.A., Russell, J.M. 1987. “Reverse” undirectional fluxes in squid giant axon. J. Gen. Physiol. 89:669–686
Bialojan, C., Takai, A. 1988. Inhibitory effect of marinesponge toxin, okadaic acid, on protein phosphatases. Biochem. J. 256:283–290
Bianchini, L., Woodside, M., Sardet, C., Pouyssegur, J., Takai, A., Grinstein, S. 1991. Okadaic acid, a phosphatase inhibitor, induces activation and phosphorylation of the Na+/H+ antiport. J. Biol. Chem. 266:15406–15413
Brock, T.A., Brugnara, C., Canessa, M., Gimbrone, M.A., Jr. 1986. Bradykinin and vasopressin stimulate in Na+, K+, Cl− cotransport in cultured endothelial cells. Am. J. Physiol. 250:C888-C895
Cooke, J.P., Stamler, J., Andon, N., Davies, P.F., McKinley, G., Lascalzo, J. 1990. Flow stimulates endothelial cells to release a nitrovasodilator that is potentiated by reduced thiol. Am. J. Physiol. 259:H804-H812
Dagher, G., Brugnara, C., Canessa, M. 1985. Effect of metabolic depletion on the furosemide-sensitive Na and K fluxes in human red cells. J. Membrane Biol. 86:145–155
Frangos, J.A., Eskin, S.G., McIntire, L.V., Ives, C.L. 1985. Flow effects on prostacyclin production by cultured human endothelial cells. Science 227:1477–1479
Grinstein, S., Rothstein, A., Cohen, S. 1985. Mechanism of osmotic activation of Na+/H+ exchange in rat thymic lymphocytes. J. Gen. Physiol. 85:765–787
Grinstein, S., Cohen, S., Goetz, J.D., Rothstein, A. 1985. Osmotic and phorbol ester-induced activation of Na+/H+ exchange: possible role of protein phosphorylation in lymphocyte volume regulation. J. Cell Biol. 101:269–276
Grinstein, S., Goetz-Smith, J.D., Stewart, D., Beresford, B.J., Mellors, A. 1986. Protein phosphorylation during activation of Na+/H+ exchange by phorbol esters and by osmotic shrinking. J. Biol. Chem. 261:8009–8016
Grinstein, S., Woodside, M., Sardet, C., Pouyssegur, J., Rotin, D. 1992. Activation of the Na+/H+ antiporter during cell volume regulation. J. Biol. Chem. 267:23823–23828
Haas, M. 1989. Properties and diversity of (Na-K-Cl) cotransports. Annu. Rev. Physiol. 51:443–457
Haystead, T.A.J., Sim, A.T.R., Carling, D., Honnor, R.C., Tsukitani, Y., Cohen, P., Hardie, D.G. 1989. Effects of the tumor promoter okadaic acid on intracellular protein phosphorylation and metabolism. Nature 337:78–81
Ishihara, H., Martin, B.L., Brautigan, D.L., Katraki, H., Ozaki, H., Kato, Y., Fusetani, N., Watabe, S., Hashimoto, K., Uemura, D., Hartshorne, D.J. 1989. Calyculin A and okadaic acid: Inhibitors of protein phosphatase activity. Biochem. Biophys. Res. Comm. 159:871–877
Ishihara, H., Ozaki, H., Sato, K., Hori, M., Karaki, H., Watabe, S., Kato, Y., Fusetani, N., Hashimoto, K., Uemura, D., Hartshorne, D.J. 1989. Calcium-independent activation of contractile apparatus in smooth muscle by calyculin-A. J. Pharm. Exp. Ther. 250:388–396
Jennings, M.L., Al-Rohil, N. 1990. Kinetics of activation and inactivation of swelling-stimulated K+/Cl− transport. J. Gen. Physiol. 95:1021–1040
Kaji, D. 1991. Regulation of Na-K-2Cl cotransport in medullary thick ascending limb (MTL) cells by phosphorylation and dephosphorylation. FASEB J. 5:A739
Killackey, J.J.F., Johnston, M.G., Movat, H.Z. 1986. Increased permeability of microcarrier-cultured endothelial monolayers in response to histamine and thrombin. Am. J. Pathol. 122:50–61
Klein, J.D., Bakir, S., O'Neill, W.C. 1991. Shrinkage of endothelial cells stimulates phosphorylation of myosin light chain. J. Am. Soc. Nephrol. 2:508
Klein, J.D., O'Neill, W.C. 1990. Effect of bradykinin on Na-K-2Cl cotransport and bumetanide binding in aortic endothelial cells. J. Biol. Chem. 265:22238–22242
Lamontagne, D., Pohl, U., Busse, R. 1992. Mechanical deformation of vessel wall and shear stress determine the basal release of endothelium derived relaxing factor in the intact rabbit coronary vascular bed. Circ. Res. 70:123–129
Lansman, J.B., Hallam, T.J., Rink, T.J. 1987. Single stretch activated ion channels in vascular endothelial cells as mechanotransducers? Nature 325:811–813
Levesque, M.J., Nerem, R.M. 1985. The elongation and orientation of cultured endothelial cells in response to shear stress. J. Biomech. Eng. 107:341–347
Ling, B.N., O'Neill, W.C. 1992. Ca2+-dependent and Ca2+-permeable ion channels in aortic endothelial cells. Am. J. Physiol. 263:H1827-H1838
Lytle, C., Forbush, B.III. 1990. The [Na-K-Cl] cotransport protein is activated and phosphorylated by cell shrinkage or cyclic AMP in a secretory epithelium. J. Cell Biol. 111:312a
Nakanishi, S., Yamada, K., Kase, H., Nakamura, S., Nonomura, Y. 1988. K-252a, a novel microbial product, inhibits smooth muscle myosin light chain kinase. J. Biol. Chem. 263:6215–6219
O'Donnell, M.E. 1989. Regulation of Na-K-Cl cotransport in endothelial cells by atrial natriuretic factor. Am. J. Physiol. 257:C36-C44
O'Donnell, M.E. 1991. Endothelial cell sodium potassium chloride cotransport. J. Biol. Chem. 266:11559–11566
Olesen, S-P., Clapham, D.E., Davies, P.F. 1988. Haemodynamic shear stress activates a K+ current in vascular endothelial cells. Nature 331:168–170
O'Neill, W.C., Kartsonis, N., Klein, J.D. 1991. Role of protein phosphorylation in the regulation of Na-K-2Cl cotransport by cell volume in aortic endothelial cells. J. Am. Soc. Nephrol. 2:747
O'Neill, W.C., Klein, J.D. 1992. Regulation of vascular endothelial cell volume by Na-K-2Cl cotransport. Am. J. Physiol. 262:C436-C444
O'Neill, W.C., Perry, P.B. Ling, B.N. 1992. Swelling-activated K transport and regulatory volume decrease (RVD) in vascular endothelial cells. J. Amer. Soc. Nephrol. 3:817
Paulais, M., Turner, R.J. 1992. Activation of the Na+-K+-2Cl− cotransporter in rat parotid acinar cells by aluminum fluoride and phosphatase inhibitors. J. Biol. Chem. 267:21558–21563
Perry, P.B., Klein, J.D., O'Neill, W.C. 1992. Na-K-2Cl Cotransport in vascular endothelial cells is regulated by protein phosphorylation. J. Gen. Physiol. 100:22a
Pewitt, E.B., Hegde, R.S., Haas, M., Palfrey, H.C. 1990. The regulation of Na-K-2Cl cotransport and bumetanide binding in avian erythrocytes by protein phosphorylation and dephosphorylation. J. Biol. Chem. 265:20747–20756
Rubanyi, G.M., Romero, J.C., Vanhoutte, P.M. 1986. Flowinduced release of endothelial derived relaxing factor. Am. J. Physiol. 250:H1145-H1149
Ruegg, U.T., Burgess, G.M. 1989. Staurosporine, K-252 and UNC-01: potent but nonspecific inhibitors of protein kinases. TIPS 10:218–220
Schmidt, W.F., III, McManus, T.J. 1977. Ouabain-insensitive salt and water movement in duck red cells. J. Gen. Physiol. 70:81–97
Shepard, J.M., Goderie, S.K., Brzyski, N., Del Vecchio, P.J., Malik, A.B., Kimelberg, H.K. 1987. Effects of alterations in endothelial cell volume on transendothelial albumin permeability. J. Cell. Physiol. 133:389–394
Shirinsky, V.P., Antonov, A.S., Konstantin, B.G., Sobolevsky, A.V., Romanov, Y.A., Kabaeva, N.V., Antonova, G.N., Smirnov, V.N. 1989. Mechano-chemical human endothelium orientation and size. J. Cell Biol. 109:331–339
Suganuma, M., Fujiki, H., Suguri, H., Yoshizawa, S., Hirota, M., Nakayasu, M., Ojika, M., Wakamatsu, K., Yamada, K., Sugimura, T. 1988. Okadaic acid: An additional non-phorbol-12-tetradecanoate-13-acetate-type tumor promoter. Proc. Natl. Acad. Sci. USA 85:1768–1771
Takai, A., Bialojan, C., Troschka, M., Ruegg, J.C. 1987. Smooth muscle myosin phosphate inhibition and force enhancement by black sponge toxin. FEBS Lett. 217:81–84
Torshia, J., Lytle, C., Forbush, B., III, Sen, A.K. 1992. Identification of the putative Na∶K∶Cl cotransporter in avian salt gland: Phosphorylation by VIP and carbachol. J. Gen. Physiol. 100:32a
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This work was supported by a Clinical Investigator Award DK01643 (to W.C.O) and a Grant-in-Aid from the American Heart Association of Georgia.
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Klein, J.D., Perry, P.B. & O'Neill, W.C. Regulation by cell volume of Na+-K+-2Cl− cotransport in vascular endothelial cells: role of protein phosphorylation. J. Membarin Biol. 132, 243–252 (1993). https://doi.org/10.1007/BF00235741
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DOI: https://doi.org/10.1007/BF00235741