The Korean Journal of Physiology and Pharmacology

The Korean Society of Pharmacology (대한약리학회)
권호 표지
DOI QR코드

DOI QR Code

Differential expression of the enzymes regulating myosin light chain phosphorylation are responsible for the slower relaxation of pulmonary artery than mesenteric artery in rats

  • Seung Beom Oh (Department of Biomedical Sciences, Seoul National University College of Medicine) ;
  • Suhan Cho (Department of Biomedical Sciences, Seoul National University College of Medicine) ;
  • Hyun Jong Kim (Department of Physiology, Dongguk University College of Medicine) ;
  • Sung Joon Kim (Department of Biomedical Sciences, Seoul National University College of Medicine)
  • Received : 2023.09.26
  • Accepted : 2023.11.13
  • Published : 2024.01.01
Download PDF
⟨ Previous Next ⟩

Abstract

While arterial tone is generally determined by the phosphorylation of Ser19 in myosin light chain (p-MLC2), Thr18/Ser19 diphosphorylation of MLC2 (pp-MLC2) has been suggested to hinder the relaxation of smooth muscle. In a dual-wire myography of rodent pulmonary artery (PA) and mesenteric artery (MA), we noticed significantly slower relaxation in PA than in MA after 80 mM KCl-induced condition (80K-contraction). Thus, we investigated the MLC2 phosphorylation and the expression levels of its regulatory enzymes; soluble guanylate cyclase (sGC), Rho-A dependent kinase (ROCK) and myosin light chain phosphatase target regulatory subunit (MYPT1). Immunoblotting showed higher sGC-α and ROCK2 in PA than MA, while sGC-β and MYPT1 levels were higher in MA than in PA. Interestingly, the level of pp-MLC2 was higher in PA than in MA without stimulation. In the 80K-contraction state, the levels of p-MLC2 and pp-MLC2 were commonly increased. Treatment with the ROCK inhibitor (Y27632, 10 µM) reversed the higher pp-MLC2 in PA. In the myography study, pharmacological inhibition of sGC (ODQ, 10 µM) slowed relaxation during washout, which was more pronounced in PA than in MA. The simultaneous treatment of Y27632 and ODQ reversed the impaired relaxation in PA and MA. Although treatment of PA with Y27632 alone could increase the rate of relaxation, it was still slower than that of MA without Y27632 treatment. Taken together, we suggest that the higher ROCK and lower MYPT in PA would have induced the higher level of MLC2 phosphorylation, which is responsible for the characteristic slow relaxation in PA.

Keywords

Acknowledgement

This work was supported by the National Research Foundation of Korea funded by the Ministry of Science and ICT, Republic of Korea (grants NRF-2018R1D1A1B07048998 and NRF-2021R1A2C2007243), and also supported by 2023 Research Promoting program by Seoul National University Hospital.

References

  1. Guyton AC, Hall JE. Textbook of medical physiology. 10th ed. Saunders; 2000.
  2. Patton HD. Textbook of physiology: excitable cells and neurophysiology. W. B. Saunders; 1989.
  3. Townsley MI. Structure and composition of pulmonary arteries, capillaries, and veins. Compr Physiol. 2012;2:675-709. https://doi.org/10.1002/cphy.c100081
  4. Suresh K, Shimoda LA. Lung circulation. Compr Physiol. 2016;6:897-943. https://doi.org/10.1002/cphy.c140049
  5. Nelson MT, Patlak JB, Worley JF, Standen NB. Calcium channels, potassium channels, and voltage dependence of arterial smooth muscle tone. Am J Physiol. 1990;259:C3-C18. https://doi.org/10.1152/ajpcell.1990.259.1.C3
  6. Dorn GW 2nd, Becker MW. Thromboxane A2 stimulated signal transduction in vascular smooth muscle. J Pharmacol Exp Ther. 1993;265:447-456.
  7. Dupuis J, Jasmin JF, Prie S, Cernacek P. Importance of local production of endothelin-1 and of the ET(B)Receptor in the regulation of pulmonary vascular tone. Pulm Pharmacol Ther. 2000;13:135-140. https://doi.org/10.1006/pupt.2000.0242
  8. Muramatsu M, Rodman DM, Oka M, McMurtry IF. Endothelin-1 mediates nitro-L-arginine vasoconstriction of hypertensive rat lungs. Am J Physiol. 1997;272:L807-L812. https://doi.org/10.1152/ajplung.1997.272.5.L807
  9. Kim HJ, Yoo HY. Hypoxic pulmonary vasoconstriction and vascular contractility in monocrotaline-induced pulmonary arterial hypertensive rats. Korean J Physiol Pharmacol. 2016;20:641-647. https://doi.org/10.4196/kjpp.2016.20.6.641
  10. Boron WF, Boulpaep EL. Medical physiology. 3rd ed. Elsevier; 2016.
  11. Ogut O, Brozovich FV. Regulation of force in vascular smooth muscle. J Mol Cell Cardiol. 2003;35:347-355. https://doi.org/10.1016/S0022-2828(03)00045-2
  12. Walsh MP. Vascular smooth muscle myosin light chain diphosphorylation: mechanism, function, and pathological implications. IUBMB Life. 2011;63:987-1000. https://doi.org/10.1002/iub.527
  13. Takeya K, Wang X, Sutherland C, Kathol I, Loutzenhiser K, Loutzenhiser RD, Walsh MP. Involvement of myosin regulatory light chain diphosphorylation in sustained vasoconstriction under pathophysiological conditions. J Smooth Muscle Res. 2014;50:18-28. https://doi.org/10.1540/jsmr.50.18
  14. Katsumata N, Shimokawa H, Seto M, Kozai T, Yamawaki T, Kuwata K, Egashira K, Ikegaki I, Asano T, Sasaki Y, Takeshita A. Enhanced myosin light chain phosphorylations as a central mechanism for coronary artery spasm in a swine model with interleukin-1beta. Circulation. 1997;96:4357-4363. https://doi.org/10.1161/01.CIR.96.12.4357
  15. Shimokawa H, Seto M, Katsumata N, Amano M, Kozai T, Yamawaki T, Kuwata K, Kandabashi T, Egashira K, Ikegaki I, Asano T, Kaibuchi K, Takeshita A. Rho-kinase-mediated pathway induces enhanced myosin light chain phosphorylations in a swine model of coronary artery spasm. Cardiovasc Res. 1999;43:1029-1039. https://doi.org/10.1016/S0008-6363(99)00144-3
  16. Obara K, Nishizawa S, Koide M, Nozawa K, Mitate A, Ishikawa T, Nakayama K. Interactive role of protein kinase C-delta with rho-kinase in the development of cerebral vasospasm in a canine two-hemorrhage model. J Vasc Res. 2005;42:67-76. https://doi.org/10.1159/000083093
  17. Mam V, Tanbe AF, Vitali SH, Arons E, Christou HA, Khalil RA. Impaired vasoconstriction and nitric oxide-mediated relaxation in pulmonary arteries of hypoxia- and monocrotaline-induced pulmonary hypertensive rats. J Pharmacol Exp Ther. 2010;332:455-462. https://doi.org/10.1124/jpet.109.160119
  18. Cho S, Namgoong H, Kim HJ, Vorn R, Yoo HY, Kim SJ. Downregulation of soluble guanylate cyclase and protein kinase G with upregulated ROCK2 in the pulmonary artery leads to thromboxane A2 sensitization in monocrotaline-induced pulmonary hypertensive rats. Front Physiol. 2021;12:624967.
  19. Cho S, Oh SB, Kim HJ, Kim SJ. T18/S19 diphosphorylation of myosin regulatory light chain impairs pulmonary artery relaxation in monocrotaline-induced pulmonary hypertensive rats. Pflugers Arch. 2023;475:1097-1112. https://doi.org/10.1007/s00424-023-02836-6
  20. Schermuly RT, Stasch JP, Pullamsetti SS, Middendorff R, Muller D, Schluter KD, Dingendorf A, Hackemack S, Kolosionek E, Kaulen C, Dumitrascu R, Weissmann N, Mittendorf J, Klepetko W, Seeger W, Ghofrani HA, Grimminger F. Expression and function of soluble guanylate cyclase in pulmonary arterial hypertension. Eur Respir J. 2008;32:881-891. https://doi.org/10.1183/09031936.00114407
  21. Shimizu T, Fukumoto Y, Tanaka S, Satoh K, Ikeda S, Shimokawa H. Crucial role of ROCK2 in vascular smooth muscle cells for hypoxia-induced pulmonary hypertension in mice. Arterioscler Thromb Vasc Biol. 2013;33:2780-2791. https://doi.org/10.1161/ATVBAHA.113.301357
  22. Sutherland C, Walsh MP. Myosin regulatory light chain diphosphorylation slows relaxation of arterial smooth muscle. J Biol Chem. 2012;287:24064-24076. https://doi.org/10.1074/jbc.M112.371609
  23. Pfitzer G. Invited review: regulation of myosin phosphorylation in smooth muscle. J Appl Physiol (1985). 2001;91:497-503. https://doi.org/10.1152/jappl.2001.91.1.497
  24. Kimura K, Ito M, Amano M, Chihara K, Fukata Y, Nakafuku M, Yamamori B, Feng J, Nakano T, Okawa K, Iwamatsu A, Kaibuchi K. Regulation of myosin phosphatase by Rho and Rho-associated kinase (Rho-kinase). Science. 1996;273:245-248. https://doi.org/10.1126/science.273.5272.245
  25. Somlyo AP, Somlyo AV. Ca2+ sensitivity of smooth muscle and nonmuscle myosin II: modulated by G proteins, kinases, and myosin phosphatase. Physiol Rev. 2003;83:1325-1358. https://doi.org/10.1152/physrev.00023.2003
  26. Butler T, Paul J, Europe-Finner N, Smith R, Chan EC. Role of serine-threonine phosphoprotein phosphatases in smooth muscle contractility. Am J Physiol Cell Physiol. 2013;304:C485-C504. https://doi.org/10.1152/ajpcell.00161.2012
  27. Grassie ME, Sutherland C, Ulke-Lemee A, Chappellaz M, Kiss E, Walsh MP, MacDonald JA. Cross-talk between Rho-associated kinase and cyclic nucleotide-dependent kinase signaling pathways in the regulation of smooth muscle myosin light chain phosphatase. J Biol Chem. 2012;287:36356-36369. https://doi.org/10.1074/jbc.M112.398479
  28. Sakamoto K, Hori M, Izumi M, Oka T, Kohama K, Ozaki H, Karaki H. Inhibition of high K+-induced contraction by the ROCKs inhibitor Y-27632 in vascular smooth muscle: possible involvement of ROCKs in a signal transduction pathway. J Pharmacol Sci. 2003;92:56-69. https://doi.org/10.1254/jphs.92.56
  29. Mita M, Yanagihara H, Hishinuma S, Saito M, Walsh MP. Membrane depolarization-induced contraction of rat caudal arterial smooth muscle involves Rho-associated kinase. Biochem J. 2002;364:431-440. https://doi.org/10.1042/bj20020191
  30. Alessi D, MacDougall LK, Sola MM, Ikebe M, Cohen P. The control of protein phosphatase-1 by targetting subunits. The major myosin phosphatase in avian smooth muscle is a novel form of protein phosphatase-1. Eur J Biochem. 1992;210:1023-1035. https://doi.org/10.1111/j.1432-1033.1992.tb17508.x
  31. Shi F, Stewart RL Jr, Perez E, Chen JY, LaPolt PS. Cell-specific expression and regulation of soluble guanylyl cyclase alpha 1 and beta 1 subunits in the rat ovary. Biol Reprod. 2004;70:1552-1561. https://doi.org/10.1095/biolreprod.103.025510
  32. Stuehr DJ, Misra S, Dai Y, Ghosh A. Maturation, inactivation, and recovery mechanisms of soluble guanylyl cyclase. J Biol Chem. 2021;296:100336.
  33. Kostic TS, Andric SA, Stojilkovic SS. Receptor-controlled phosphorylation of alpha 1 soluble guanylyl cyclase enhances nitric oxide-dependent cyclic guanosine 5'-monophosphate production in pituitary cells. Mol Endocrinol. 2004;18:458-470. https://doi.org/10.1210/me.2003-0015
  34. Cabilla JP, Diaz Mdel C, Machiavelli LI, Poliandri AH, Quinteros FA, Lasaga M, Duvilanski BH. 17 beta-estradiol modifies nitric oxide-sensitive guanylyl cyclase expression and down-regulates its activity in rat anterior pituitary gland. Endocrinology. 2006;147:4311-4318. https://doi.org/10.1210/en.2006-0367
  35. Cabilla JP, Ronchetti SA, Nudler SI, Miler EA, Quinteros FA, Duvilanski BH. Nitric oxide sensitive-guanylyl cyclase subunit expression changes during estrous cycle in anterior pituitary glands. Am J Physiol Endocrinol Metab. 2009;296:E731-E737. https://doi.org/10.1152/ajpendo.90795.2008