Attenuation of calmodulin regulation evokes Ca2+ oscillations: evidence for the involvement of intracellular arachidonate-activated channels and connexons

  • Egor A. Turovsky
  • Valery P. Zinchenko
  • Nikolai P. KaimachnikovEmail author


Intracellular Са2+ controls its own level by regulation of Ca2+ transport across the plasma and organellar membranes, often acting via calmodulin (CaM). Drugs antagonizing CaM action induce an increase in cytosolic Ca2+ concentration in different cells. We have found persistent Са2+ oscillations in cultured white adipocytes in response to calmidazolium (CMZ). They appeared at [CMZ] > 1 μM as repetitive sharp spikes mainly superimposed on a transient or elevated baseline. Similar oscillations were observed when we used trifluoperazine. Oscillations evoked by 5 μM CMZ resulted from the release of stored Ca2+ and were supported by Са2+ entry. Inhibition of store-operated channels by YM-58483 or 2-APB did not change the responses. Phospholipase A2 inhibited by AACOCF3 was responsible for initial Ca2+ mobilization, but not for subsequent oscillations, whereas inhibition of iPLA2 by BEL had no effect. Phospholipase C was partially involved in both stages as revealed with U73122. Intracellular Са2+ stores engaged by CMZ were entirely dependent on thapsigargin. The oscillations existed in the presence of inhibitors of ryanodine or inositol 1,4,5-trisphosphate receptors, or antagonists of Ca2+ transport by lysosome-like acidic stores. Carbenoxolone or octanol, blockers of hemichannels (connexons), when applied for two hours, prevented oscillations but did not affect the initial Са2+ release. Incubation with La3+ for 2 or 24 h inhibited all responses to CMZ, retaining the thapsigargin-induced Ca2+ rise. These results suggest that Ca2+-CaM regulation suppresses La3+-sensitive channels in non-acidic organelles, of which arachidonate-activated channels initiate Ca2+ oscillations, and connexons are intimately implicated in their generation mechanism.


Ca2+ oscillations Calmodulin Arachidonic acid Connexons Calmidazolium Adipocytes 



This work was supported by a Grant of the President of the Russian Federation (Ref: МК-626.2018.4, EAT).

Author contributions

EAT performed all experiments, contributed to the experimental design and data analysis and prepared figures. VPZ contributed reagents and materials, participated in the discussion of results, and edited the manuscript. NPK conceived the study, designed the experiments, analyzed and interpreted the data, and wrote the manuscript.

Compliance with ethical standards

Conflict of interest

The authors state that they have no conflict of interest pertaining to this manuscript.


  1. 1.
    Carafoli E, Santella L, Brance D, Brini M (2001) Generation, control, and processing of cellular calcium signals. Crit Rev Biochem Mol Biol 36:107–260CrossRefGoogle Scholar
  2. 2.
    Berridge MJ (2012) Calcium signalling remodelling and disease. Biochem Soc Trans 40:297–309CrossRefGoogle Scholar
  3. 3.
    Berchtold MW, Villalobo A (2014) The many faces of calmodulin in cell proliferation, programmed cell death, autophagy, and cancer. Biochim Biophys Acta 1843:398–435CrossRefGoogle Scholar
  4. 4.
    Uhlén P, Fritz N (2010) Biochemistry of calcium oscillations. Biochem Biophys Res Commun 396:28–32CrossRefGoogle Scholar
  5. 5.
    Trebak M, Putney JW Jr (2017) ORAI calcium channels. Physiology (Bethesda) 32:332–342Google Scholar
  6. 6.
    Shuttleworth TJ (1999) What drives calcium entry during [Ca2+]i oscillations?—challenging the capacitative model. Cell Calcium 25:237–246CrossRefGoogle Scholar
  7. 7.
    Kawano S, Otsu K, Kuruma A, Shoji S, Yanagida E, Muto Y, Yoshikawa F, Hirayama Y, Mikoshiba K, Furuichi T (2006) ATP autocrine/paracrine signaling induces calcium oscillations and NFAT activation in human mesenchymal stem cells. Cell Calcium 39:313–324CrossRefGoogle Scholar
  8. 8.
    De Bock M, Wang N, Bol M, Decrock E, Ponsaerts R, Bultynck G, Dupont G, Leybaert L (2012) Connexin 43 hemichannels contribute to cytoplasmic Ca2+ oscillations by providing a bimodal Ca2+-dependent Ca2+ entry pathway. J Biol Chem 287:12250–12266CrossRefGoogle Scholar
  9. 9.
    Bol M, Wang N, De Bock M, Wacquier B, Decrock E, Gadicherla A, Decaluwé K, Vanheel B, van Rijen HV, Krysko DV, Bultynck G, Dupont G, Van de Voorde J, Leybaert L (2017) At the cross-point of connexins, calcium, and ATP: blocking hemichannels inhibits vasoconstriction of rat small mesenteric arteries. Cardiovasc Res 113:195–206CrossRefGoogle Scholar
  10. 10.
    Leybaert L, Lampe PD, Dhein S, Kwak BR, Ferdinandy P, Beyer EC, Laird DW, Naus CC, Green CR, Schulz R (2017) Connexins in cardiovascular and neurovascular health and disease: pharmacological implications. Pharmacol Rev 69:396–478CrossRefGoogle Scholar
  11. 11.
    Laird DW (2006) Life cycle of connexins in health and disease. Biochem J 394:527–543CrossRefGoogle Scholar
  12. 12.
    Bruzzone S, Guida L, Sturla L, Usai C, Zocchi E, De Flora A (2012) Subcellular and intercellular traffic of NAD+, NAD+ precursors and NAD+-derived signal metabolites and second messengers: old and new topological paradoxes. Messenger 1:34–52. CrossRefGoogle Scholar
  13. 13.
    D’hondt C, Ponsaerts R, De Smedt H, Bultynck G, Himpens B (2009) Pannexins, distant relatives of the connexin family with specific cellular functions? Bioessays 31:953–974CrossRefGoogle Scholar
  14. 14.
    Morgan AJ, Platt FM, Lloyd-Evans E, Galione A (2011) Molecular mechanisms of endolysosomal Ca2+ signalling in health and disease. Biochem J 439:349–374CrossRefGoogle Scholar
  15. 15.
    Tornquist K, Ekokoski E (1996) Inhibition of agonist-mediated calcium entry by calmodulin antagonists and by the Ca2+/calmodulin kinase II inhibitor KN-62. Studies with thyroid FRTL-5 cells. J Endocrinol 148:131–138CrossRefGoogle Scholar
  16. 16.
    Watanabe H, Takahashi R, Tran QK, Takeuchi K, Kosuge K, Satoh H, Uehara A, Terada H, Hayashi H, Ohno R, Ohashi K (1999) Increased cytosolic Ca2+ concentration in endothelial cells by calmodulin antagonists. Biochem Biophys Res Commun 265:697–702CrossRefGoogle Scholar
  17. 17.
    Harper JL, Daly JW (2000) Effect of calmidazolium analogs on calcium influx in HL-60 cells. Biochem Pharmacol 60:317–324CrossRefGoogle Scholar
  18. 18.
    Jan CR, Tseng CJ (2000) Calmidazolium-induced rises in cytosolic calcium concentrations in Madin-Darby canine kidney cells. Toxicol Appl Pharmacol 162:142–150CrossRefGoogle Scholar
  19. 19.
    Smani T, Zakharov SI, Csutora P, Leno E, Trepakova ES, Bolotina VM (2004) A novel mechanism for the store-operated calcium influx pathway. Nat Cell Biol 6:113–120CrossRefGoogle Scholar
  20. 20.
    Peppiatt CM, Holmes AM, Seo JT, Bootman MD, Collins TJ, McDonald F, Roderick HL (2004) Calmidazolium and arachidonate activate a calcium entry pathway that is distinct from store-operated calcium influx in HeLa cells. Biochem J 381:929–939CrossRefGoogle Scholar
  21. 21.
    Zinchenko VP, Kasymov VA, Li VV, Kaimachnikov NP (2005) The calmodulin inhibitor R24571 induces a short-term Ca2+ entry and a pulse-like secretion of ATP in Ehrlich ascites tumor cells. Biofizika 50:1055–1069Google Scholar
  22. 22.
    Liao WC, Huang CC, Cheng HH, Wang JL, Lin KL, Cheng JS, Chai KL, Hsu PT, Tsai JY, Fang YC, Lu YC, Chang HT, Huang JK, Chou CT, Jan CR (2009) Effect of calmidazolium on [Ca2+]i and viability in human hepatoma cells. Arch Toxicol 83:61–68CrossRefGoogle Scholar
  23. 23.
    Somogyi R, Stucki JW (1991) Hormone-induced calcium oscillations in liver cells can be explained by a simple one pool model. J Biol Chem 266:11068–11077Google Scholar
  24. 24.
    Uneyama H, Uneyama C, Akaike N (1993) Intracellular mechanisms of cytoplasmic Ca2+ oscillation in rat megakaryocyte. J Biol Chem 268:168–174Google Scholar
  25. 25.
    Turovsky EA, Kaimachnikov NP, Zinchenko VP (2014) Agonist-specific participation of SOC and ARC channels and iPLA2 in the regulation of Ca2+ entry during oscillatory responses in adipocytes. Biochem (Moscow) Suppl Ser A: Membr Cell Biol 8:136–143. Google Scholar
  26. 26.
    Veigl ML, Klevit RE, Sedwick WD (1989) The uses and limitations of calmodulin antagonists. Pharmacol Ther 44:181–239CrossRefGoogle Scholar
  27. 27.
    Turovsky EA, Kaimachnikov NP, Turovskaya MV, Berezhnov AV, Dynnik VV, Zinchenko VP (2012) Two mechanisms of calcium oscillations in adipocytes. Biochem (Moscow) Suppl Ser A: Membr Cell Biol 6:26–34.
  28. 28.
    Dolgacheva LP, Turovskaya MV, Dynnik VV, Zinchenko VP, Goncharov NV, Davletov B, Turovsky EA (2016) Angiotensin II activates different calcium signaling pathways in adipocytes. Arch Biochem Biophys 593:38–49CrossRefGoogle Scholar
  29. 29.
    Bogan SJ (2012) Regulation of glucose transporter translocation in health and diabetes. Annu Rev Biochem 81:507–532CrossRefGoogle Scholar
  30. 30.
    Park KH, Kim BJ, Shawl AI, Han MK, Lee HC, Kim UH (2013) Autocrine/paracrine function of nicotinic acid adenine dinucleotide phosphate (NAADP) for glucose homeostasis in pancreatic β-cells and adipocytes. J Biol Chem 288:35548–35558CrossRefGoogle Scholar
  31. 31.
    Zitt C, Strauss B, Schwarz EC, Spaeth N, Rast G, Hatzelmann A, Hoth M (2004) Potent inhibition of Ca2+ release-activated Ca2+ channels and T-lymphocyte activation by the pyrazole derivative BTP2. J Biol Chem 279:12427–12437CrossRefGoogle Scholar
  32. 32.
    Putney JW (2010) Pharmacology of store-operated calcium channels. Mol Interv 10:209–218CrossRefGoogle Scholar
  33. 33.
    Ackermann EJ, Conde-Frieboes K, Dennis EA (1995) Inhibition of macrophage Ca2+-independent phospholipase A2 by bromoenol lactone and trifluoromethyl ketones. J Biol Chem 270:445–450CrossRefGoogle Scholar
  34. 34.
    Duncan RE, Sarkadi-Nagy E, Jaworski K, Ahmadian M, Sul HS (2008) Identification and functional characterization of adipose-specific phospholipase A2 (AdPLA). J Biol Chem 283:25428–25436CrossRefGoogle Scholar
  35. 35.
    Jaworski K, Ahmadian M, Duncan RE, Sarkadi-Nagy E, Varady KA, Hellerstein MK, Lee HY, Samuel VT, Shulman GI, Kim KH, de Val S, Kang C, Sul HS (2009) AdPLA ablation increases lipolysis and prevents obesity induced by high-fat feeding or leptin deficiency. Nat Med 15:159–168CrossRefGoogle Scholar
  36. 36.
    Broad LM, Cannon TR, Taylor CW (1999) A non-capacitative pathway activated by arachidonic acid is the major Ca2+ entry mechanism in rat A7r5 smooth muscle cells stimulated with low concentrations of vasopressin. J Physiol 517:121–134CrossRefGoogle Scholar
  37. 37.
    Zimányi I, Buck E, Abramson JJ, Mack MM, Pessah IN (1992) Ryanodine induces persistent inactivation of the Ca2+ release channel from skeletal muscle sarcoplasmic reticulum. Mol Pharmacol 42:1049–1057Google Scholar
  38. 38.
    Gafni J, Munsch JA, Lam TH, Catlin MC, Costa LG, Molinski TF, Pessah IN (1997) Xestospongins: potent membrane permeable blockers of the inositol 1,4,5-trisphosphate receptor. Neuron 19:723–733CrossRefGoogle Scholar
  39. 39.
    Castonguay A, Robitaille R (2002) Xestospongin C is a potent inhibitor of SERCA at a vertebrate synapse. Cell Calcium 32:39–47CrossRefGoogle Scholar
  40. 40.
    Mignen O, Thompson JL, Shuttleworth TJ (2003) Ca2+ selectivity and fatty acid specificity of the noncapacitative, arachidonate-regulated Ca2+ (ARC) channels. J Biol Chem 278:10174–10181CrossRefGoogle Scholar
  41. 41.
    Naylor E, Arredouani A, Vasudevan SR, Lewis AM, Parkesh R, Mizote A, Rosen D, Thomas JM, Izumi M, Ganesan A, Galione A, Churchill GC (2009) Identification of a chemical probe for NAADP by virtual screening. Nat Chem Biol 5:220–226CrossRefGoogle Scholar
  42. 42.
    Lee H, Jun DJ, Suh BC, Choi BH, Lee JH, Do MS, Suh BS, Ha H, Kim KT (2005) Dual roles of P2 purinergic receptors in insulin-stimulated leptin production and lipolysis in differentiated rat white adipocytes. J Biol Chem 280:28556–28563CrossRefGoogle Scholar
  43. 43.
    Mignen O, Shuttleworth TJ (2000) IARC, a novel arachidonate-regulated, noncapacitative Ca2+ entry channel. J Biol Chem 275:9114–9119CrossRefGoogle Scholar
  44. 44.
    John SA, Kondo R, Wang SY, Goldhaber JI, Weiss JN (1999) Connexin-43 hemichannels opened by metabolic inhibition. J Biol Chem 274:236–240CrossRefGoogle Scholar
  45. 45.
    Kwan CY, Putney JW Jr (1990) Uptake and intracellular sequestration of divalent cations in resting and methacholine-stimulated mouse lacrimal acinar cells. Dissociation by Sr2+ and Ba2+ of agonist-stimulated divalent cation entry from the refilling of the agonist-sensitive intracellular pool. J Biol Chem 265:678–684Google Scholar
  46. 46.
    Sorkin A, von Zastrow M (2002) Signal transduction and endocytosis: close encounters of many kinds. Nat Rev Mol Cell Biol 3:600–614CrossRefGoogle Scholar
  47. 47.
    Weigel PH, Oka JA (1981) Temperature dependence of endocytosis mediated by the asialoglycoprotein receptor in isolated rat hepatocytes. Evidence for two potentially rate-limiting steps. J Biol Chem 256:2615–2617Google Scholar
  48. 48.
    Holmes AM, Roderick HL, McDonald F, Bootman MD (2007) Interaction between store-operated and arachidonate-activated calcium entry. Cell Calcium 41:1–12CrossRefGoogle Scholar
  49. 49.
    He LP, Hewavitharana T, Soboloff J, Spassova MA, Gill DL (2005) A functional link between store-operated and TRPC channels revealed by the 3,5-bis(trifluoromethyl)pyrazole derivative, BTP2. J Biol Chem 280:10997–11006CrossRefGoogle Scholar
  50. 50.
    Bodendiek SB, Raman G (2010) Connexin modulators and their potential targets under the magnifying glass. Curr Med Chem 17:4191–4230CrossRefGoogle Scholar
  51. 51.
    Beauvois MC, Arredouani A, Jonas JC, Rolland JF, Schuit F, Henquin JC, Gilon P (2004) Atypical Ca2+-induced Ca2+ release from a sarco-endoplasmic reticulum Ca2+-ATPase 3-dependent Ca2+ pool in mouse pancreatic beta-cells. J Physiol 559:141–156CrossRefGoogle Scholar
  52. 52.
    Yu F, Sun L, Machaca K (2010) Constitutive recycling of the store-operated Ca2+ channel Orai1 and its internalization during meiosis. J Cell Biol 191:523–535CrossRefGoogle Scholar
  53. 53.
    Tang Y, Stephenson JL, Othmer HG (1996) Simplification and analysis of models of calcium dynamics based on IP3-sensitive calcium channel kinetics. Biophys J 70:246–263CrossRefGoogle Scholar
  54. 54.
    Pizzo P, Lissandron V, Capitanio P, Pozzan T (2011) Ca2+ signalling in the Golgi apparatus. Cell Calcium 50:184–192CrossRefGoogle Scholar
  55. 55.
    Huotari J, Helenius A (2011) Endosome maturation. EMBO J 30:3481–3500CrossRefGoogle Scholar
  56. 56.
    Gerasimenko JV, Tepikin AV, Petersen OH, Gerasimenko OV (1998) Calcium uptake via endocytosis with rapid release from acidifying endosomes. Curr Biol 8:1335–1338CrossRefGoogle Scholar
  57. 57.
    Choi YO, Park JH, Song YS, Lee W, Moriyama Y, Choe H, Leem CH, Jang YJ (2007) Involvement of vesicular H+-ATPase in insulin-stimulated glucose transport in 3T3-F442A adipocytes. Endocr J 54:733–743CrossRefGoogle Scholar
  58. 58.
    Khan SZ, Longland CL, Michelangeli F (2000) The effects of phenothiazines and other calmodulin antagonists on the sarcoplasmic and endoplasmic reticulum Ca2+ pumps. Biochem Pharmacol 60:1797–1806CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Institute of Cell Biophysics of the Russian Academy of SciencesPushchino, Moscow RegionRussia

Personalised recommendations