Pflügers Archiv

, Volume 449, Issue 1, pp 42–55 | Cite as

Cell-volume-dependent vascular smooth muscle contraction: role of Na+, K+, 2Cl cotransport, intracellular Cl and L-type Ca2+ channels

  • Yana J. Anfinogenova
  • Mikhail B. Baskakov
  • Igor V. Kovalev
  • Alexander A. Kilin
  • Nickolai O. Dulin
  • Sergei N. OrlovEmail author
Cell and Molecular Physiology


This study elucidates the role of cell volume in contractions of endothelium-denuded vascular smooth muscle rings (VSMR) from the rat aorta. We observed that hyposmotic swelling as well as hyper- and isosmotic shrinkage led to VSMR contractions. Swelling-induced contractions were accompanied by activation of Ca2+ influx and were abolished by nifedipine and verapamil. In contrast, contractions of shrunken cells were insensitive to the presence of L-type channel inhibitors and occurred in the absence of Ca2+o. Thirty minutes preincubation with bumetanide, a potent Na+,K+,Cl cotransport (NKCC) inhibitor, decreased Cli content, nifedipine-sensitive 45Ca uptake and contractions triggered by modest depolarization ([K+]o=36 mM). Elevation of [K+]o to 66 mM completely abolished the effect of bumetanide on these parameters. Bumetanide almost completely abrogated phenylephrine-induced contraction, partially suppressed contractions triggered by hyperosmotic shrinkage, but potentiated contractions of isosmotically shrunken VSMR. Our results suggest that bumetanide suppresses contraction of modestly depolarized cells via NKCC inhibition and Cli-mediated membrane hyperpolarization, whereas augmented contraction of isosmotically shrunken VSMR by bumetanide is a consequence of suppression of NKCC-mediated regulatory volume increase. The mechanism of bumetanide inhibition of contraction of phenylephrine-treated and hyperosmotically shrunken VSMR should be examined further.


Ca2+ channels Cell volume Contraction Intracellular Cl Na+,K+,2Cl cotransport Smooth muscle 



This work was supported by grants from the Heart and Stroke Foundation of Canada, and the INTAS Young Scientist Fellowship (YSF 2001/2-0168). The editorial assistance help of Ovid Da Silva, Editor, Research Support Office, Research Centre, CHUM, is appreciated.


  1. 1.
    Adragna N, White RE, Orlov SN, Lauf PK (2000) K-Cl cotransport in vascular smooth muscle and erythrocytes: possible implication in vasodilation. Am J Physiol 278:C381–C390Google Scholar
  2. 2.
    Adragna N, Di Fulvio M, Lauf PK (2004) Regulation of K-Cl cotransport: from function to genes. J Membr Biol (in press)Google Scholar
  3. 3.
    Akar F, Skinner E, Klein JD, Jena M, Paul RJ, O’Neill WC (1999) Vasoconstrictors and nitrovasodilators reciprocally regulate the Na+-K+-2Cl cotransporter in rat aorta. Am J Physiol 276:C1383–C1390PubMedGoogle Scholar
  4. 4.
    Akar F, Jiang G, Paul RJ, O’Neill WC (2001) Contractile regulation of the Na+-K+-2Cl cotransporter in vascular smooth muscle. Am J Physiol 281:C579–C584Google Scholar
  5. 5.
    Alvarez-Guerra M, Nazaret C, Garay RP (1998) The erythrocyte Na,K,Cl cotransporter and its circulating inhibitor in Dahl salt-sensitive rats. J Hypertens 16:1499–1504CrossRefPubMedGoogle Scholar
  6. 6.
    Anfinogenova YJ, Rodriguez X, Grygorczyk R, Adragna N, Lauf PK, Hamet P, Orlov SN (2001) Swelling-induced K+ fluxes in vascular smooth muscle cells are mediated by charybdotoxin-sensitive K+ channels. Cell Physiol Biochem 11:295–310CrossRefPubMedGoogle Scholar
  7. 7.
    Anfinogenova YJ, Kilin AA, Kovalev IV, Baskakov MB, Orlov SN (2003) Vascular smooth muscle contraction in hyperosmotic medium: role of Ca2+, anion channels and cell volume-sensitive Na+,K+,Cl cotransport. J Hypertens 21:S101Google Scholar
  8. 8.
    Barandier C, Ming X-F, Yang Z (2003) Small G proteins as novel therapeutic targets in cardiovascular medicine. News Physiol Sci 18:18–22PubMedGoogle Scholar
  9. 9.
    Berk BC, Vallega G, Muslin AJ, Gordon HM, Canessa M, Alexander RW (1989) Spontaneously hypertensive rat vascular smooth muscle cells in culture exhibit increased growth and Na+/H+ exchange. J Clin Invest 83:822–829PubMedGoogle Scholar
  10. 10.
    Bianchi G, Ferrari P, Trizio P, Ferrandi M, Torielli L, Barber BR, Polli E (1985) Red blood cell abnormalities and spontaneous hypertension in rats. A genetically determined link. Hypertension 7:319–325PubMedGoogle Scholar
  11. 11.
    Brown RA, Chipperfield AR, Davis JPL, Harper AA (1999) Increased (Na+K+Cl) cotransport in rat arterial smooth muscle in deoxycorticosterone (DOCA)/salt-induced hypertension. J Vasc Res 36:492–501CrossRefPubMedGoogle Scholar
  12. 12.
    Chipperfield AR, Harper AA (2001) Chloride in smooth muscle. Prog Biophys Mol Biol 74:175–221CrossRefGoogle Scholar
  13. 13.
    Cuneo P, Margi E, Verzola A, Grazi E (1992) “Macromolecular crowding” is a primary factor in the organization of the cytoskeleton. Biochem J 281:507–512PubMedGoogle Scholar
  14. 14.
    Davis A, Hogarth K, Fernandes D, Solway J, Niu J, Kolenko V, Browning D, Miano JM, Orlov SN, Dulin NO (2003) Functional significance of protein kinase A (PKA) activation by endothelin-1 and ATP: negative regulation of SRF-dependent gene expression by PKA. Cell Signal 15:597–604CrossRefPubMedGoogle Scholar
  15. 15.
    Davis JPL, Chipperfield AR, Harper AA (1993) Accumulation of intracellular chloride by (Na-K-Cl) cotransport in rat arterial smooth muscle is enhanced in deoxycorticosterone acetate (DOCA)/salt hypertension. J Mol Cell Cardiol 25:233–237CrossRefPubMedGoogle Scholar
  16. 16.
    Flagella M, Clarke LL, Miller ML, Erway LC, Giannella RA, Andriga A, Gawenis LR, Kramer J, Duffy JJ, Doetschman T, Lorenz JN, Yamoah EN, Cardell EL, Shull GE (1999) Mice lacking the basolateral Na-K-2Cl cotransporter have impaired epithelial chloride secretion and are profoundly deaf. J Biol Chem 274:26946–26955CrossRefPubMedGoogle Scholar
  17. 17.
    Garay RP (1982) Inhibition of the Na+/K+ cotransport system by cyclic AMP and intracellular calcium in human red cells. Biochim Biophys Acta 688:786–792CrossRefPubMedGoogle Scholar
  18. 18.
    Glukhova MA, Frid MG, Koteliansky VE (1994) Phenotypic changes of human aortic smooth muscle cells during development and in adult. J Atheroscler Thromb 1 [Suppl 1]:S47–S49Google Scholar
  19. 19.
    Godfraind T (1994) Calcium antagonists and vasodilation. Pharmacol Ther 64:37–75CrossRefPubMedGoogle Scholar
  20. 20.
    Hoffmann EK, Simonsen LO (1989) Membrane mechanisms in volume and pH regulation in vertebrate cells. Physiol Rev 69:315–382PubMedGoogle Scholar
  21. 21.
    Isenring P, Forbush III B (1997) Ion and bumetanide binding by the Na-K-Cl cotransporter. Importance of transmembrane domains. J Biol Chem 272:24556–24562CrossRefPubMedGoogle Scholar
  22. 22.
    Isenring P, Jacoby SC, Payne JA, Forbush BI (1998) Comparison of Na-K-Cl cotransporters: NKCC1, NKCC2 and HEK cell Na-K-Cl cotransporter. J Biol Chem 273:11295–11301CrossRefPubMedGoogle Scholar
  23. 23.
    Janssen LJ, Lu-Chao H, Netherton S (2001) Excitation-contraction coupling in pulmonary vascular smooth muscle involves tyrosine kinase and Rho-kinase. Am J Physiol 280:L666–L674Google Scholar
  24. 24.
    Jiang G, Cobbs S, Klein JD, O’Neill WC (2003) Aldosterone regulates the Na-K-Cl cotransporter in vascular smooth muscle. Hypertension 41:1131–1135CrossRefPubMedGoogle Scholar
  25. 25.
    Jones AW (1973) Altered ion transport in vascular smooth muscle from spontaneously hypertensive rats. Influence of aldosterone, norepinephrine and angiotensin. Circ Res 33:563–572PubMedGoogle Scholar
  26. 26.
    Klein JD, O’Neill WC (1993) Myosin light chain phosphorylation in endothelial cells is regulated by cell volume and correlates with volume-regulatory Na-K-2Cl cotransport (abstract). J Gen Physiol 102:18aGoogle Scholar
  27. 27.
    Klein JD, O’Neill WC (1995) Volume-sensitive myosin phosphorylation in vascular endothelial cells: correlation with Na-K-2Cl cotransport. Am J Physiol 269:C1524–C1531PubMedGoogle Scholar
  28. 28.
    Kotelevtsev YuV, Orlov SN, Pokudin NI, Agnaev VM, Postnov YuV (1987) Genetic analysis of inheritance of Na+,K+ cotransport, calcium level in erythrocytes and blood pressure in F2 hybrids of spontaneously hypertensive and normotensive rats. Bull Exp Biol Med 103:456–458Google Scholar
  29. 29.
    Kovalev IV, Baskakov MB, Anfinogenova YJ, Borodin YL, Kilin AA, Minochenko IL, Popov AG, Kapilevich LV, Medvedev MA, Orlov SN (2003) Effect of Na+,K+,2Cl cotransport inhibitor bumetanide on electrical and contractile activity of smooth muscle cells in guinea pig ureter. Bull Exp Biol Med 136:145–149CrossRefPubMedGoogle Scholar
  30. 30.
    Kravtsov GM, Bruce IC, Wong TK, Kwan CY (2003) A new view of K+-induced contraction in rat aorta: the role of Ca2+ binding. Pflugers Arch 446:529–540CrossRefPubMedGoogle Scholar
  31. 31.
    Lang F, Busch G, Ritter M, Volkl H, Waldegger S, Gulbins E, Haussinger D (1998) Functional significance of cell volume regulatory mechanisms. Physiol Rev 78:247–306PubMedGoogle Scholar
  32. 32.
    Lauf PK, Adragna NC (2000) K-Cl cotransport: properties and molecular mechanism. Cell Physiol Biochem 10:341–354CrossRefPubMedGoogle Scholar
  33. 33.
    Madden TL, Herzfeld J (1993) Crowding-induced organization of cytoskeletal elements. I. Spontaneous demixing of cytoplasmic proteins and model filaments to form filament bundles. Biophys J 65:1147–1154PubMedGoogle Scholar
  34. 34.
    McDonald TF, Pelzer S, Trautwein W, Pelzer DJ (1994) Regulation and modulation of calcium channels in cardiac, skeletal, and smooth muscle cells. Physiol Rev 74:365–512PubMedGoogle Scholar
  35. 35.
    Meyer JW, Flagella M, Sutliff RL, Lorenz JN, Nieman ML, Weber GS, Paul RJ, Shull GE (2002) Decreased blood pressure and vascular smooth muscle tone in mice lacking basolateral Na+-K+-2Cl cotransporter. Am J Physiol 283:H1846–H1855Google Scholar
  36. 36.
    Mongin AA, Orlov SN (2001) Mechanisms of cell volume regulation and possible nature of the cell volume sensor. Pathophysiology 8:77–88CrossRefPubMedGoogle Scholar
  37. 37.
    Mount DB, Delpire E, Gamba G, Hall AE, Poch E, Hoover RS, Hebert SC (1999) The electroneutral cation-chloride cotransporters. J Exp Biol 201:2091–2102Google Scholar
  38. 38.
    Nakao F, Kobayashi S, Mogami K, Mizukami Y, Shirao S, Miwa S, Todoroki-Ikeda N, Ito M, Matsuzaki M (2002) Involvement of Src family protein tyrosine kinases in Ca2+ sensitization of coronary artery contraction mediated by a sphindosylphosphorylcholine-Rho-kinase pathway. Circ Res 91:953–960CrossRefPubMedGoogle Scholar
  39. 39.
    O’Neill WC (1999) Physiological significance of volume-regulated transporters. Am J Physiol 276:C995–C1011PubMedGoogle Scholar
  40. 40.
    O’Neill WC, Klein JD (1994) Regulation of vascular endothelial cell volume by Na-K-2Cl cotransport. Am J Physiol 262:C436–C444Google Scholar
  41. 41.
    Orlov SN (2003) Hypertension. In: Bernhardt I, Ellory JC (eds) Red cell membrane transport in health and disease. Springer, Berlin Heidelberg New York, pp 587–602Google Scholar
  42. 42.
    Orlov SN, Resink TJ, Bernhardt J, Buhler FR (1992) Na+-K+ pump and Na+-K+ co-transport in cultured vascular smooth muscle cells from spontaneously hypertensive rats: baseline activity and regulation. J Hypertens 10:733–740PubMedGoogle Scholar
  43. 43.
    Orlov SN, Resink TJ, Bernhardt J, Buhler FR (1992) Volume-dependent regulation of sodium and potassium fluxes in cultured vascular smooth muscle cells: dependence on medium osmolality and regulation by signalling systems. J Membr Biol 126:199–210Google Scholar
  44. 44.
    Orlov SN, Tremblay J, Hamet P (1996) Bumetanide-sensitive ion fluxes in vascular smooth muscle cells: lack of functional Na+,K+,2Cl cotransport. J Membr Biol 153:125–135CrossRefPubMedGoogle Scholar
  45. 45.
    Orlov SN, Tremblay J, Hamet P (1996) cAMP signaling inhibits dihydropyridine-sensitive Ca2+ influx in vascular smooth muscle cells. Hypertension 27:774–780PubMedGoogle Scholar
  46. 46.
    Orlov SN, Tremblay J, Hamet P (1996) Cell volume in vascular smooth muscle is regulated by bumetanide-sensitive ion transport Am J Physiol 270:C1388–C1397Google Scholar
  47. 47.
    Orlov SN, Adragna N, Adarichev VA, Hamet P (1999) Genetic and biochemical determinants of abnormal monovalent ion transport in primary hypertension. Am J Physiol 276:C511–C536PubMedGoogle Scholar
  48. 48.
    Orlov SN, Pchejetski D, Taurin S, Thorin-Trescases N, Maximov GV, Pshezhetsky AV, Rubin AB, Hamet P (2004) Apoptosis in serum-deprived vascular smooth muscle cells: evidence for cell volume-independent mechanism. Apoptosis 9:55–66CrossRefPubMedGoogle Scholar
  49. 49.
    Owen NE, Ridge KM (1989) Mechanism of angiotensin II stimulation of Na-K-Cl cotransport of vascular smooth muscle cells. Am J Physiol 257:C629–C636PubMedGoogle Scholar
  50. 50.
    Pedersen SF, Beisner KH, Hougaard C, Willumsen BM, Lambert IH, Hoffmann EK (2002) Rho family GTP binding proteins are involved in the regulatory volume decrease in NIH3T3 mouse fibroblasts. J Physiol (Lond) 541:779–796Google Scholar
  51. 51.
    Postnov YuV, Orlov SN, Gulak PV, Shevchenko AS (1976) Altered permeability of the erythrocyte membrane for sodium and potassium in spontaneously hypertensive rats. Pflugers Arch 365:257–263PubMedGoogle Scholar
  52. 52.
    Raat NJ, Hartog A, van Os CH, Bindels RJ (1994) Regulation of Na+-K+-2Cl cotransport in rabbit proximal tubule primary culture. Am J Physiol 267:F63–F69PubMedGoogle Scholar
  53. 53.
    Russell JM (2000) Sodium-potassium-chloride cotransport. Physiol Rev 80:212–276Google Scholar
  54. 54.
    Shrode LD, Klein JD, O’Neill WC, Putnam RW (1995) Shrinkage-induced activation of Na+/H+ exchange in primary rat astrocytes: role of myosin light-chain kinase. Am J Physiol 269:C257–C266PubMedGoogle Scholar
  55. 55.
    Smith JB, Smith L (1987) Na+/K+/Cl cotransport in cultured vascular smooth muscle cells: stimulation by angiotensin II and calcium ionophores, inhibition by cyclic AMP and calmodulin antagonists. J Membr Biol 99:51–63PubMedGoogle Scholar
  56. 56.
    Takeda M, Homma T, Breyer MD, Horiba N, Hoover RL, Kawamoto S, Ichikawa I, Kon V (1993) Volume and agonist-induced regulation of myosin light-chain phosphorylation in glomerular mesangial cells. Am J Physiol 264:F421–F426PubMedGoogle Scholar
  57. 57.
    Tseng H, Berk BC (1992) The Na/K/2Cl cotransporter is increased in hypertrophied vascular smooth muscle cells. J Biol Chem 267:8161–8167PubMedGoogle Scholar
  58. 58.
    Wagner CA, Huber SM, Warntges S, Zempel G, Kaba NK, Fux R, Orth N, Busch GL, Waldegger S, Lambert I, Nilius B, Heinle H, Lang F (2000) Effect of urea and osmotic cell shrinkage on Ca2+ entry and contraction of vascular smooth muscle cells. Pflugers Arch 440:295–301CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag  2004

Authors and Affiliations

  • Yana J. Anfinogenova
    • 1
  • Mikhail B. Baskakov
    • 1
  • Igor V. Kovalev
    • 1
  • Alexander A. Kilin
    • 1
  • Nickolai O. Dulin
    • 2
  • Sergei N. Orlov
    • 3
    • 4
    Email author
  1. 1.Department of Biophysics and Functional DiagnosticsSiberian State Medical UniversityTomskRussia
  2. 2.Department of MedicineUniversity of ChicagoChicagoUSA
  3. 3.Faculty of BiologyM.V. Lomonosov Moscow State UniversityMoscowRussia
  4. 4.Research CentreUniversity of Montreal Hospital (CHUM-Hôtel-Dieu)MontrealCanada

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