Attenuation of calmodulin regulation evokes Ca2+ oscillations: evidence for the involvement of intracellular arachidonate-activated channels and connexons
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.
KeywordsCa2+ 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).
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.
- 5.Trebak M, Putney JW Jr (2017) ORAI calcium channels. Physiology (Bethesda) 32:332–342Google Scholar
- 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
- 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. https://doi.org/10.1166/msr.2012.1007 CrossRefGoogle Scholar
- 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
- 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.Uneyama H, Uneyama C, Akaike N (1993) Intracellular mechanisms of cytoplasmic Ca2+ oscillation in rat megakaryocyte. J Biol Chem 268:168–174Google Scholar
- 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. https://doi.org/10.1134/S1990747813050206 Google Scholar
- 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. https://doi.org/10.1134/S199074781106016X
- 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
- 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
- 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