Role of ATP and Cl^– transporters in regulation of contractile activity of pulmonary artery smooth muscles in hyposmotic conditions

Objective. The role of Cl^– -transport in ATP-dependent regulation of contractile activity of rat pulmonary artery (PA) smooth muscle cells was studied. Design and methods. The study was performed on endotheliumdenuded ring segments of the PA of male Wistar rats. Mechanical tension was measured using organ bath technique. Contractions of the PA segments were induced by high-potassium solution (30 mM KCl), hyposmotic solution (40 mM NaCl), as well as restoration of the medium osmolarity (120 mM NaCl) after incubation in hyposmotic solution. Inhibitor of Na⁺ , K⁺ , 2Cl^– cotransport (NKCC) bumetanide (10 μM, 30 minutes preincubation), nonselective Cl^– -channel blocker SITS (100 μM) and Ca2⁺ -activated Cl^– channels blocker niflumic acid (NA, 10 μM) were used to modulate the Cl^– -transport. Results. ATP (10–500 μM) did not affect vascular tone of PA segments incubated in Krebs solution (120 mM NaCl), while 500 and 1000 μM ATP led to the development of transient contractions, the amplitude of which decreased in the presence of bumetanide, SITS and NA. Incubation of PA segments in a hyposmotic medium (40 mM NaCl) caused the development of transient contraction. Subsequent recovery of the medium osmolarity (120 mM NaCl) induced another transient contraction — isosmotic striction. ATP (500 μM) eliminated the relaxation phase of hyposmotic striction, and completely suppressed the development of isosmotic striction. Bumetanide did not affect the action of ATP during isosmotic striction, but restored the relaxation phase of hyposmotic striction. SITS and NA eliminated the effect of ATP on hypo- and isosmotic striction. Conclusions. The constrictive effect of ATP on the smooth muscle cells of the PA is associated with the activation of mechanisms of transmembrane Cl^– -redistribution, in which NKCC and Ca2⁺ -activated Cl^– channels are involved.

pulmonary artery, smooth muscle cells, Na⁺, K⁺, 2Cl^– cotransport, Cl^–-channels, ATP, hypoosmotic contraction

1. Pak O, Aldashev A, Welsh D, Peacock A. The effects of hypoxia on the cells of the pulmonary vasculature. Eur Respir J. 2007;30(2):364–372. doi:10.1183/09031936.00128706

2. Cahill E, Rowan SC, Sands M, Banahan M, Ryan D, Howell K et al. The pathophysiological basis of chronic hypoxic pulmonary hypertension in the mouse: vasoconstrictor and structural mechanisms contribute equally. Exp Physiol. 2012;97(6):796–806. doi:10.1113/expphysiol.2012.065474

3. Sweeney M, Jason XJY. Hypoxic pulmonary vasoconstriction: role of voltage-gated potassium channels. Respir Res. 2000;1(1):40–48. doi:10.1186/rr11

4. Sun H, Xia Y, Paudel O, Yang XR, Sham JS. Chronic hypoxia-induced upregulation of Ca2+-activated Cl-channel in pulmonary arterial myocytes: a mechanism contributing to enhanced vasoreactivity. J Physiol. 2012;590(15):3507–3521. doi:10.1113/jphysiol.2012.232520

5. Wang K, Chen C, Ma J, Lao J, Pang Y. Contribution of calcium-activated chloride channel to elevated pulmonary artery pressure in pulmonary arterial hypertension induced by high pulmonary blood flow. Int J Clin Exp Pathol. 2015;8(1):146–154.

6. Forrest AS, Joyce TC, Huebner ML, Ayon RJ, Wiwchar M, Joyce J et al. Increased TMEM16A-encoded calcium-activated chloride channel activity is associated with pulmonary hypertension. Am J Physiol Cell Physiol. 2012;303(12):C 1229–C 1243. doi:10.1152/ajpcell.00044.2012

7. Wang K, Ma J, Pang Y, Lao J, Pan X, Tang Q et al. Niflumic acid attenuated pulmonary artery tone and vascular structural remodeling of pulmonary arterial hypertension induced by high pulmonary blood flow in vivo. J Cardiovasc Pharmacol. 2015;66(4):383–391. doi:10.1097/FJC.0000000000000291

8. Orlov SN, Koltsova SV, Kapilevich LV, Gusakova SV, Dulin NO. NKCC 1 and NKCC 2: the pathogenetic role of cationchloride cotransporters in hypertension. Genes Dis. 2015;2(2):186–196. doi:10.1016/j.gendis.2015.02.007

9. Burnstock G, Ralevic V. Purinergic signaling and blood vessels in health and disease. Pharmacol Rev. 2014;66(1):102–192. doi:10.1124/pr.113.008029

10. Dietrich HH, Ellsworth ML, Sprague RS, Dacey RG. Red blood cell regulation of microvascular tone through adenosine triphosphate. Am J Physiol Heart Circ Physiol. 2000;278(4): H1294–H1298. doi:10.1152/ajpheart.2000.278.4.H1294

11. Ellsworth ML, Ellis CG, Goldman D, Stephenson AH, Dietrich HH, Sprague RS. Erythrocytes: oxygen sensors and modulators of vascular tone. Physiology. 2008;24:107–116. doi:10.1152/physiol.00038.2008

12. Locovei S, Wang J, Dahl G. Activation of pannexin 1 channels by ATP through P2Y receptors and by cytoplasmic calcium. FEBS Lett. 2006;580(1):239–244. doi:10.1016/j.febslet.2005.12.004

13. Sprague RS, Ellsworth ML. Erythrocyte derived ATP and perfusion distribution: role of intracellular and intracellular communication. Microcirculation. 2012;19(5):430–439. doi:10.1111/j.1549-8719.2011.00158.x

14. Sprague RS, Ellsworth ML, Stephenson AH, Lonigro AJ. Participation of cAMP in a signal-transduction pathway relating erythrocyte deformation to ATP release. Am J Physiol Cell Physiol. 2001;281(4): C1158–C1164. doi:10.1152/ajpcell.2001.281.4.C1158

15. Inglis SK, Olver RE, Wilson SM. Differential effects of UTP and ATP on ion transport in porcine tracheal epithelium. Br J Pharmacol. 2000;130(2):367–374. doi:10.1038/sj.bjp.0703324

16. Paradiso AM, Ribeiro CM, Boucher RC. Polarized signaling via purinoceptors in normal and cystic fibrosis airway epithelia. J Gen Physiol. 2001;117(1):53–67. doi:10.1085/jgp.117.1.53

17. Kunzelmann K, Schreiber R, Cook D. Mechanisms for the inhibition of amiloride-sensitive Na+ absorption by extracellular nucleotides in mouse trachea. Pflugers Arch. 2002;444(1–2):220–226. doi:10.1007/s00424-002-0796-y

18. Wong CH, Ko WH. Stimulation of Cl — secretion via membrane-restricted Ca2+ signaling mediated by P2Y receptors in polarized epithelia. J Biol Chem. 2002;277(11):9016–9021. doi:10.1074/jbc.M111917200

19. Mongin AA, Kimelberg HK. ATP regulates anion channelmediated organic osmolyte release from cultured rat astrocytes via multiple Ca2+-sensitive mechanisms. Am J Physiol Cell Physiol. 2005;288(1):C 204–C 213. doi:10.1152/ajpcell.00330.2004

20. Wang Y, Roman R, Lidofsky SD, Fitz JG. Autocrine signaling through ATP release represents a novel mechanism for cell volume regulation. Proc Natl Acad Sci USA. 1996;93(21):1202012025. doi:10.1073/pnas.93.21.12020

21. Akimova OA, Grygorzcyk A, Bundey RA, Bourcier N, Gekle M, Insel PA et al. Transient activation and delayed inhibition of Na+, K+, Cl– cotransport in ATP-treated C 11-MDCK cells involve distinct P2Y receptor subtypes and signaling mechanisms. J Biol Chem. 2006;281(42):31317–31325. doi:10.1074/jbc.m602117200

22. Burnstock G. Physiology and pathophysiology of purinergic neurotransmission. Physiol Rev. 2007;87(2):659–797. doi:10.1152/physrev.00043.2006

23. Fisher SK, Cheema TA, Foster DJ, Heacock AM. Volumedependent osmolyte efflux from neuronal tissues: regulation by G-protein-coupled receptors. J Neurochem. 2008;106(5):19982014. doi:10.1111/j.1471-4159.2008.05510.x

24. Franco R, Panayiotidis MI, de La Paz LD. Autocrine signaling involved in cell volume regulation: the role of released transmitters and plasma membrane receptors. J Cell Physiol. 2008;216(1):14–28. doi:10.1002/jcp.21406

25. Vazquez-Juarez E, Ramos-Mandujano G, HernandezBenitez R, Pasantes-Morales H. On the role of G-protein-coupled receptors in cell volume regulation. Cell Physiol Biochem. 2008;21(1–3):1–14. doi:10.1159/000113742

26. Sun XZ, Tian XY, Wang DW, Li J. Effects of fasudil on hypoxic pulmonary hypertension and pulmonary vascular remodeling in rats. Eur Rev Med Pharmacol Sci. 2014;18(7):959–964.

27. Roman RM, Feranchak AP, Salter KD, Wang Y, Fitz JG. Endogenous ATP release regulates Cl- secretion in cultured human and rat biliary epithelial cells. Am J Physiol. 1999;276(6):G1391G1400. doi:10.1152/ajpgi.1999.276.6.G1391

28. Feranchak AP, Fitz JG, Roman RM. Volume-sensitive purinergic signaling in human hepatocytes. J Hepatol. 2000;33(2):174–182. doi:10.1016/s0168-8278(00)80357-8

29. Sabirov RS, Okada Y. ATP release via anion channels. Purinergic Signaling. 2005;1(4):311–328. doi:10.1007/s11302005-1557-0

30. Gradilone SA, Masyuk AI, Splinter PL, Banales JM, Huang BQ, Tietz PS et al. Cholangiocyte cilia express TRPV4 and detect changes in luminal tonicity inducing bicarbonate secretion. Proc Natl Acad Sci USA. 2007;104(48):19138–19143. doi:10.1073/pnas.0705964104

31. Hoffmann EK, Lambert IH, Pedersen SF. Physiology of cell volume regulation in vertebrates. Physiol Rev. 2009;89(1):193–277. doi:10.1152/physrev.00037.2007

32. Bang L, Boesgaard S, Nielsen-Kudsk JE, Vejlstrup NG, Aldershvile J. Nitroglycerin-mediated vasorelaxation is modulated by endothelial calcium-activated potassium channels. Cardiovasc Res. 1999;43(3):772–778.

33. Gao YJ, Lee RMKW. Hydrogen peroxide is an endotheliumdependent contracting factor in rat renal artery. Br J Pharmacol. 2005;146(8):1061–1068. doi:10.1038/sj.bjp.0706423

34. Li JR, Zhao YS, Chang Y, Yang SC, Guo YJ, Ji ES. Fasudil improves endothelial dysfunction in rats exposed to chronic intermittent hypoxia through RhoA/ROCK/NFATc3 pathway. PLoS ONE. 2018;13(4): e019560. doi:10.1371/journal.pone.0195604

35. Anfinogenova YJ, Baskakov MB, Kovalev IV, Kilin AA, Dulin NO, Orlov SN. Cell-volume-dependent vascular smooth muscle contraction: role of Na+, K+, 2Cl – cotransport, intracellular Cl – and L-type Ca2+ channels. Pflügers Arch. 2004;449(1):42–55.

36. Koltsova SV, Gusakova SV, Anfinogenova YJ, Baskakov MB, Orlov SN. Vascular smooth muscle contraction evoked by cell volume modulation: role of the cytoskeleton network. Cell Physiol Biochem. 2008;21(1–3):29–36.

37. Orlov SN, Resink TJ, Bernhardt J, Buhler FR. Volumedependent regulation of sodium and potassium fluxes in cultured vascular smooth muscle cells: dependence on medium osmolality and regulation by signalling systems. J Membrane Biol. 1992;129(2): 199–210.

38. Burnstock G. Purinergic mechanisms. Ann N Y Acad Sci. 1990;603:1–17. doi:10.1111/j.1749-6632.1990.tb37657.x

39. Inoue T, Kannan MS. Nonadrenergic and noncholinergic excitatory neurotransmission in rat intrapulmonary artery. Am J Physiol. 1988;254(6Pt2):H1142–H114. doi:10.1152/ajpheart.1988.254.6.H1142

40. Katsuragi T, Tokunaga T, Ogawa S, Soejima O, Sato C, Furukawa T. Existence of ATP-evoked ATP release system in smooth muscles. J Pharmacol Exp Ther. 1991;259(2):513–518.

41. Pearson JD, Gordon JL. Vascular endothelial and smooth muscle cells in culture selectively release adenine nucleotides. Nature. 1979;281(5730):384–386.

42. Grygorczyk R, Orlov SN. Effects of hypoxia on erythrocyte membrane properties — implications for intravascular hemolysis and purinergic control of blood flow. Front Physiol. 2017;8:1110.

43. Hakim TS, Ferrario L, Freedman JC, Carlin RE, Camporesi EM. Segmental pulmonary vascular responses to ATP in rat lungs: role of nitric oxide. J Appl Physiol. 1997;82(3):852–858.

44. Boarder MR, Weisman GA, Turner JT, Wilkinson GF. G-protein-coupled P2purinoceptors: from molecular biology to functional responses. Trends Pharmacol Sci. 1995;16(4):133–139. doi:10.1016/S0165-6147(00)89001-X

45. Liu SF, McCormack DG, Evans TW, Barnes PJ. Characterization and distribution of P2-purinoceptor subtypes in rat pulmonary vessels. J Pharmacol Exp Ther. 1989;251(3):12041210.

46. Liu SF, McCormack DG, Evans TW, Barnes PJ. Evidence for two P2-purinoceptor subtypes in human small pulmonary arteries. Br J Pharmacol. 1989;98(3):1014–1020.

47. Neely CF, Haile DM, Cahill BE, Kadowitz PJ. Adenosine and ATP produce vasoconstriction in the feline pulmonary vascular bed by different mechanisms. J Pharmacol Exp Ther. 1991;258(3):753–761.

48. Neely CF, Kadowitz PJ, Lippton H, Neiman M, Hyman AL. Adenosine does not mediate the pulmonary vasodilator response of adenosine 5’-triphosphate in the feline pulmonary vascular bed. J Pharmacol Exp Ther. 1989;250:170–176.

49. Souza R, Amato MBP, Demarzo SE, Deheinzelin D, Barbas CSV, Schettino GPP et al. Pulmonary capillary pressure in pulmonary hypertension. Crit Care. 2005;9(2):R 132–R 138.

50. Rackow EC, Alan Fein I, Leppo J. Colloid osmotic pressure as a prognostic indicator of pulmonary edema and mortality in the critically ill. Chest. 1977;72:709–713. doi:10.1378/chest.72.6.709

51. Perry PB, O’Neill WC. Swelling-activated K fluxes in vascular endothelial cells: volume regulation via K-Cl cotransport and K channels. Am J Physiol. 1993;265(3Pt1): C 763–C 769. doi:10.1152/ajpcell.1993.265.3.C763