Effects of Ca²⁺-ATPase Inhibitors, Ionomycin, and Pharmacological Modulators of Ryanodine Receptor on Calcium Release from Intracellular Pools and on Oscillatory Contractile Behavior in Physarum polycephalum

A. A. Kochegarov¹, S. I. Beylina^2*, N. B. Matveeva², G. A. Leont'eva¹, and V. P. Zinchenko¹

¹Institute of Cell Biophysics, Russian Academy of Sciences, Pushchino, Moscow Region, 142292 Russia; fax: (7-0967) 790-509; E-mail: akochegarov@mail.ru

²Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, Pushchino, Moscow Region, 142292 Russia; fax: (7-0967) 790-553; E-mail: beylina@venus.iteb.serpukhov.su

^* To whom correspondence should be addressed.

Received June 3, 1999; Revision received October 18, 1999

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Changes in calcium levels in organelles of the plasmodium of the myxomycete Physarum polycephalum were analyzed using the fluorescent calcium indicator chlortetracycline (CTC). Both the Ca²⁺-ATPase inhibitor 2,5´-di(tert-butyl)-1,4-benzohydroquinone (BHQ) (100 µM) and the calcium ionophore ionomycin (1 µM) induce a significant decrease in fluorescence level (by 30%) in CTC-stained microplasmodia; this is caused by release of calcium from intracellular storage compartments. An activator of ryanodine receptors, caffeine (10-50 mM), is less effective on Ca²⁺ release than BHQ or ionomycin, and their inhibitor, ryanodine (100 µM), almost completely blocks the response to caffeine, but only slightly decreases the effects of BHQ or ionomycin. Procaine, another inhibitor of ryanodine receptors, at 10 mM concentration completely abolishes both the BHQ and the ionomycin responses, but 50 mM is necessary to block the effect of 25 mM caffeine. These results suggest that both the BHQ- and the ionomycin-dependent Ca²⁺ releases occur through the ryanodine receptor and are to be considered as calcium-induced Ca²⁺ release (CICR). Both the ionomycin and the BHQ responses persist in the presence of Cd²⁺, which blocks Ca²⁺ channels of the plasmalemma. In most cases, Cd²⁺ itself induces release of Ca²⁺ from the CTC-stained calcium pool; the more effective Cd²⁺ is, the less the following ionomycin or BHQ responses occur. This indicates that Ca²⁺ entry through plasmalemma plays no significant role in the ionomycin- or BHQ-evoked initiation of CICR, and that the Cd²⁺- and BHQ/ionomycin-depleted Ca²⁺ stores overlap.

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KEY WORDS: Ca²⁺-ATPase, ryanodine receptor, calcium-induced Ca²⁺ release

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