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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
Article

Abstract

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.

Keywords

Ca2+ oscillations Calmodulin Arachidonic acid Connexons Calmidazolium Adipocytes 

Notes

Acknowledgements

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.

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Authors and Affiliations

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

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