Analysis of decay kinetics of the cytosolic calcium transient induced by oxytocin in rat myometrium smooth muscle cells

Journal of Muscle Research and Cell Motility volume 42, pages117–127(2021)Cite this article

Abstract

The method of kinetic analysis of the relaxation phase of the mechanical response of the smooth muscle previously proposed by Burdyga and Kosterin was applied to study the dynamics of the decay of oxytocin-induced calcium transients in cytosol of the rat myometrium smooth muscle cell detected by a fluorescence signal generated by a calcium-sensitive probe fluo-4 using a laser scanning confocal microscope. The experimental data were well linearized in the coordinates ln [(Fm – F)/F] vs lnt (F and Fm are the current fluorescence intensity of the calcium probe and the fluorescence intensity at the maximum of the calcium transient, respectively, while t is the time). The empirical parameters n and τ were determined by which the maximal normalized relaxation rate Vn was calculated for five different ROIs (regions of interest) in the myocyte cytosol. It proved to be almost the same for all ROIs. The maximal normalized relaxation rate calculated from the fluorescence intensity was always lower than that calculated from the corresponding calcium concentration, i.e. the cytosolic Ca2+ concentration in the relaxation phase decreases faster than the corresponding fluorescence intensity. The value of the maximal normalized relaxation rate calculated both from the fluorescence intensity and from the force of oxytocin-induced contractions of isolated rat uterus longitudinal smooth muscles (according to Tsymbalyuk and Kosterin) was exactly the same. This indicates that in the relaxation phase, the decreasing curves of both the fluorescence intensity and the contraction forces coincide.

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References

  1. Apati A, Paszty K, Erdei Z, Szebenyi K, Homolya L, Sarkadi B (2012) Calcium signalling in pluripotent stem cell. Mol Cell Endocrinol 353:57–67. https://doi.org/10.1016/j.mce.2011.08.038

    CAS  Article  PubMed  Google Scholar 

  2. Arrowsmith S (2020) Oxitocin and vasopressin signalling and myometrial contraction. Cur Opinion Physiol 13:62–70. https://doi.org/10.1016/j.cophys.2019.10.006

    Article  Google Scholar 

  3. Arrowsmith S, Wray S (2014) Oxitocin: its mechanism of action and receptor signalling in the myometrium. J Neuroendocrinol 26(6):356–369. https://doi.org/10.1111/jne.12154

    CAS  Article  PubMed  Google Scholar 

  4. Babich LG, Shlykov SG, Borisova LA, Kosterin SO (1994) Energy-dependent Ca2+-transport in intracellular smooth muscle structures. Biokhimiia 59(8):1218–1229 ((In Russian))

    CAS  PubMed  Google Scholar 

  5. Backx PH, Gao WD, Azan-Backx MD, Marban E (1995) The relationship between contractile force and intracellular [Ca2+] in intact rat cardiac trabeculae. J Gen Physiol 105(1):1–19. https://doi.org/10.1085/jgp.105.1.1

    CAS  Article  PubMed  Google Scholar 

  6. Baylor SM, Hollingworth S (2000) Measurement and interpretation of cytoplasmic [Ca2+] signals from calcium-indicator dyes. News Physiol Sci 15:19–26. https://doi.org/10.1152/physiologyonline.2000.15.1.19

    CAS  Article  PubMed  Google Scholar 

  7. Bootman MD, Rietdorf K, Collins T, Walker S, Sanderson M (2013) Converting fluorescence data into Ca2+ concentration. Cold Spring Harb Protoc. https://doi.org/10.1101/pdb.prot072827

    Article  PubMed  Google Scholar 

  8. Bootman MD, Rietdorf K, Collins T, Walker S, Sanderson M (2014) Ca2+-sensitive fluorescent dyes and intracellular Ca2+ imaging. In: Paris JB, Yule DI, Bootman MD, Bultynck G (eds) Calcium techniques: a laboratory manual.Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, p 25–49

  9. Brandt PW, Cox RN, Kawai M, Robinson T (1982) Effect of cross-bridge kinetics on apparent Ca2+ sensitivity. J Gen Physiol 79(6):997–1016. https://doi.org/10.1085/jgp.79.6.997

    CAS  Article  PubMed  Google Scholar 

  10. Bruschi G, Bruschi ME, Regolisti G, Borghetti A (1988) Mioplasmic Ca2+-force relationship studied with fura-2 during stimulation of rat aortic smooth muscle. Am J Physiol 254(5):H840–H854. https://doi.org/10.1152/ajpheart.1988.254.5.H840

    CAS  Article  PubMed  Google Scholar 

  11. Burdyga TV, Kosterin SA (1991) Kinetic analysis of smooth muscle relaxation. Gen Physiol Biophys 10(6):589–598

    CAS  PubMed  Google Scholar 

  12. Cannell MB, Cody SB (2006) Fluorescent ion measurement. In: Pawley JB (ed) Handbook of biological confocal microscopy, 3rd edition. SpringerScience+Business Media, New York, p 736–745. https://doi.org/10.1007/978-0-387-45524-2_42

  13. Diaz ME, Trafford AW, Eisner DA (2001) The effect of exogenous calcium buffers on the transient in rat venticular myocytes. Biophys J 80:1915–1925. https://doi.org/10.1016/S0006-3495(01)76161-9

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  14. Enyedi A, Brandt J, Minami J, Penniston JT (1989) Oxytocin regulates the plasma membrane Ca2+ transport in rat myometrium. Biochem J 261:23–28. https://doi.org/10.1042/bj2610023

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  15. Fluo calcium indicators. Product information (2011). https://assets.thermofisher.com/TFS-.Assets/LSG/manuals/mp01240.pdf

  16. Frank SA (2013) Input-output relations in biological systems: measurement, information and the Hill equation. Biol Direct 8:31. https://doi.org/10.1186/1745-6150-8-31

    Article  PubMed  PubMed Central  Google Scholar 

  17. Gee KR, Brown KA, Chen W-NU, Bishop-Stewart J, Gray D, Johnson I (2000) Chemical and physiological characterization of fluo-4 Ca2+-indicator dyes. Cell Calcium 27(2):97–106. https://doi.org/10.1054/ceca.1999.0095

    CAS  Article  PubMed  Google Scholar 

  18. Gesztelyi R, Zsuga J, Kemeny-Beke A, Varga B, Juhasz B, Tosaki A (2012) The Hill equation and the origin of quantitative pharmacology. Arch Hist Exact Sci 66:427–438. https://doi.org/10.1007/s00407-012-0098-5

    Article  Google Scholar 

  19. Gimpl G, Fahrengolz F (2001) The oxytocin receptor system: structure, function, and regulation. Physiol Rev 81(2):629–683. https://doi.org/10.1152/physrev.2001.81.2.629

    CAS  Article  PubMed  Google Scholar 

  20. Grynkiewicz G, Poenie M, Tsien RY (1985) A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 260(6):3440–3450

    CAS  Article  Google Scholar 

  21. Konhilas JP, Irving TC, de Tombe PP (2002) Length-dependent activation in three striated muscle types of the rat. J Physiol 544(1):225–236. https://doi.org/10.1113/jphysiol.2002.024505

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  22. Kosterin SO, Babich LG, Shlykov SG, Danylovych YV, Veklich TO, Mazur YY (2016) Biochemical properties and regulation of smooth muscle cell Ca2+-transporting systems. Kyiv, Naukova dumka (in Ukrainian)

    Google Scholar 

  23. Kosterin SA, Burdyga TV, Fomin VP, Grover AK (1994) Mechanisms of Ca2+ transport in myometrium. In: Garfield RE, Tabb TN (eds) Control of uterine contractility. CRC Press, Boka Raton, Ann Arbor, London, Tokyo, pp 129–153

    Google Scholar 

  24. Luckas MJM, Taggart MJ, Wray S (1999) Intracellular calcium stores and agonist-induced contractions in isolated human myometrium. Am J Obstet Gynecol 181(2):468–476. https://doi.org/10.1016/S0002-9378(99)70580-6

    CAS  Article  PubMed  Google Scholar 

  25. Maloney JA, Wheeler-Clark ES (1996) Reduction in sarcoplasmic reticulum Ca2+-ATPase activity contributes to age-related changes in the calcium content and relaxation rate of rabbit aortic smooth muscle. J Hypertens 14(1):65–74

    CAS  Article  Google Scholar 

  26. Mannhardt I, Breckwoldt K, Letuffe-Breniere D, Schaaf S, Schulz H, Neuber C, Benzin A, Werner T, Eder A, Schulze T, Klampe B, Christ T, Hirt MN, Huebner N, Moretti A, Eschenhagen T, Hansen A (2016) Human engineered heart tissue: analysis of contractile force. Stem Cell Reports 7:1–14. https://doi.org/10.1016/j.stemcr.2016.04.011

    CAS  Article  Google Scholar 

  27. McKillen K, Thornton S, Taylor CW (1999) Oxytocin increases the [Ca2+]i sensitivity of human myometrium during the falling phase of phasic contractions. Am J Physiol 276(2):E345–E351. https://doi.org/10.1152/ajpendo.1999.276.2.e345

    CAS  Article  PubMed  Google Scholar 

  28. Papp Z, van der Velden J, Borbely A, Edes I, Stienen GJM (2014) Altered myocardial force generation in end-stage human heart failure. ESC Heart Failure 1(2):160–165. https://doi.org/10.1002/ehf2.12020

    Article  PubMed  Google Scholar 

  29. Popescu LM, Nutu O, Panoiu C (1985) Oxytocin contracts the human uterus at term by inhibiting the myometrial Ca2+-extrusion pump. Biosci Rep 5(1):21–28. https://doi.org/10.1007/bf01117437

    CAS  Article  PubMed  Google Scholar 

  30. Rockwell PL, Storey BT (1999) Determination of the intracellular dissociation constant, KD, of the fluo-3.Ca2+ complex in mouse sperm for use in estimating intracellular Ca2+ concentrations. Mol Reprod Dev 54:418–428. https://doi.org/10.1002/(SICI)1098-2795(199912)54:4%3c418::AID-MRD13%3e3.0.CO;2-I

    CAS  Article  PubMed  Google Scholar 

  31. Ruzycky AL, Morgan KG (1989) Involvement of the protein kinase C system in calcium-force relationship in ferret aorta. Br J Pharmacol 97(2):391–400. https://doi.org/10.1111/j.1476-5381.1989.tb11966.x

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  32. Santillan M (2008) On the use of the Hill functions in mathematical models of gene regulatory networks. Math Model Nat Phenom 3(2):85–97. https://doi.org/10.1051/mmnp:2008056

    Article  Google Scholar 

  33. Shinozaki T, Wilkens JL, Yazawa T, Cavey MJ, ter Keurs HEDJ (2004) The steady-state force–Ca2+ relationship in intact lobster (Homarus americanus) cardiac muscle. J Comp Physiol B 174(5):407–414. https://doi.org/10.1007/s00360-004-0427-3

    CAS  Article  PubMed  Google Scholar 

  34. Shlykov SG (2010) Oxytocin and its role in the control of intracellular level of calcium ions in the myometrium. Ukr Biochem J 82(2):5–14 ((in Ukrainian))

    CAS  Google Scholar 

  35. Shlykov SG, Babich LG, Yevtushenko ME, Karakhim SO, Kosterin SO (2014) Modulation of myometrium mitochondrial membrane potential by calmodulin antagonists. Ukr Biokhim Zh 86(1):29–41 (In Ukrainian). https://doi.org/10.15407/ubj86.01.029

  36. Simpson AWM (2006) Fluorescent measurement of [Ca2+]c. In: Lambert DG (ed) Calcium signalling protocols, 2nd edn. Humana Press, Totowa, NJ, pp 3–36

    Google Scholar 

  37. Soloff MS, Sweet P (1982) Oxytocin inhibition of (Ca2+ + Mg2+)-ATPase activity in rat miometrial plasma membranes. J Biol Chem 257(18):10687–10693

    CAS  Article  Google Scholar 

  38. Taggart MJ, Menice CB, Morgan KG, Wray S (1997) Effect of metabolic inhibition on intracellular Ca2+, phosphorylation of myosin regulatory light chain and force in rat smooth muscle. J Physiol 499(2):485–496. https://doi.org/10.1113/jphysiol.1997.sp021943

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  39. Tahara A, Tsukada J, Tomura Y, Wada K, Kusayama T, Ishii N, Yatsu T, Uchida W, Tanaka A (2000) Pharmacologic characterization of the oxytocin receptor in human uterine smooth muscle cells. Br J Pharmacol 129:131–139. https://doi.org/10.1038/sj.bjp.0702996

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  40. Thomas D, Tovey SC, Collins TJ, Bootman MD, Berridge MJ, Lipp P (2000) A comparison of fluorescent Ca2+ indicator properties and their use in measuring elementary and global Ca2+ signals. Cell Calcium 28(4):213–223. https://doi.org/10.1054/ceca.2000.0152

    CAS  Article  PubMed  Google Scholar 

  41. Tsymbalyuk OV (2014) Comparative analysis of the mechano-kinetics of contractile activity of myometrium smooth muscles under calixarene C-99 and ouabain action. Studia Biologica 8(1):63–72 (in Ukrainian). https://doi.org/10.30970/sbi.0801.339

  42. Tsymbalyuk OV (2016) Influence of calixarene C-99 on contractile activity of rat large intestine smooth muscles. Studia Biologica 10(3–4):33–46 (in Ukrainian). https://doi.org/10.30970/sbi.1003.503

  43. Tsymbalyuk OV, Kosterin SO (2013) Influence of calixarene C-90 on contractile activity of rat myometrium smooth muscles. Studia Biologica 7(3):5–20 (in Ukrainian). https://doi.org/10.30970/sbi.0703.298

  44. Tsymbalyuk OV, Naumenko AM, Rohovtsov OO, Skoryk MA, Voiteshenko IS, Skryshevsky VA, Davydovska TL (2017) Titanium dioxide modulation of the contractibility of visceral smooth muscles in vivo. Nanoscale Res Lett 12:129. https://doi.org/10.1186/s11671-017-1865-7

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  45. Veklich TO, Mazur II, Kosterin SO (2015) Mg2+, ATP-dependent plasma membrane calcium pump of smooth muscle cells. II. Regulation of activity. Ukr Biochem J 87(2):5–25 (in Ukrainian). https://doi.org/10.15407/ubj87.02.005

  46. Virych PA, Shelyuk OV, Kabanova TA, Khalimova EI, Martynyuk VS, Pavlovsky VI, Andronati SA (2017) Effect of 3-substituted 1,4-benzodiazepin-2-ones on bradykinin-induced smooth muscle contraction. Ukr Biochem J 89(1):31–37. https://doi.org/10.15407/ubj89.01.031

  47. Walker JS, Li X, Buttrick PM (2010) Analysing force-pCa curves. J Muscle Res Cell Motil 31:59–69. https://doi.org/10.1007/s10974-010-9208-7

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  48. Williams DA (1990) Quantitative intracellular calcium imaging with laser-scanning confocal microscopy. Cell Calcium 11:589–597. https://doi.org/10.1016/0143-4160(90)90013-K

    CAS  Article  PubMed  Google Scholar 

  49. Yefimenko OYu, Savchenko YuO, Falalyeyeva TM, Beregova TV, Zholobak NM, Spivak MYa, Shcherbakov OB, Bubnov RV (2015) Nanocrystalline cerium dioxide efficacy for gastrointestinal motility: potential for prokinetic treatment and prevention in elderly. EPMA J 6:6. https://doi.org/10.1186/s13167-015-0029-z

    Article  PubMed  PubMed Central  Google Scholar 

  50. Yue DT, Marban E, Wier WG (1986) Relationship between force and intracellular [Ca2+] in tetanized mammalian heart muscle. J Gen Physiol 87(2):223–242. https://doi.org/10.1085/jgp.87.2.223

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  51. Zheng K, Jensen TP, Rusakov DA (2018) Monitoring intracellular nanomolar calcium using fluorescence lifetime imaging. Nat Protoc 13(3):581–597. https://doi.org/10.1038/nprot.2017.154

    CAS  Article  PubMed  Google Scholar 

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Acknowledgements

We would like to thank Prof. S.O.Kosterin, Member of NAS of Ukraine, for helpful discussion and comments on the manuscript.

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This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

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Affiliations

  1. Palladin Institute of Biochemistry of the National Academy of Sciences of Ukraine, 9 Leontovicha Street, Kyiv, 01054, Ukraine

    S. O. Karakhim, S. G. Shlykov & L. G. Babich

  2. Taras Shevchenko Kyiv National University, Educational-Scientific Center “Biology and Medicine Institute”, 64/13 Volodymyrska Street, Kyiv, 01601, Ukraine

    D. V. Sinko

Authors
  1. S. O. Karakhim
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  2. S. G. Shlykov
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  3. L. G. Babich
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Contributions

Karakhim S.O.—article concept, analysis and interpretation of data obtained, discussion of the results, writing the paper. Shlykov S.G.—attaching myocytes to the flow chamber for confocal microscopy and experiment with oxytocin, discussion of the results, writing the paper (Materials and methods). Babich L.G.—obtaining myometrium cells suspension, discussion of the results, writing the paper (Materials and methods). Sinko D.V.—calculation of kinetic parameters, analysis of data obtained, discussion of the results. All authors read and approved the final manuscript.

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Correspondence to S. O. Karakhim.

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All manipulations with animals were carried out according to “European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes” and law of Ukraine “On protection of animals from cruelty”.

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Karakhim, S., Shlykov, S., Babich, L. et al. Analysis of decay kinetics of the cytosolic calcium transient induced by oxytocin in rat myometrium smooth muscle cells. J Muscle Res Cell Motil 42, 117–127 (2021). https://doi.org/10.1007/s10974-021-09598-7

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Keywords

  • Smooth muscle
  • Contraction force
  • Fluorescence intensity
  • Intracellular calcium transient
  • Kinetic analysis of the relaxation phase
  • Oxytocin