When a Ca²⁺ signal is generated, the intracellular Ca²⁺ rise is provided by Ca²⁺ influx across the plasma membrane and Ca²⁺ release from organelles via IP₃ receptors (IP₃Rs) and ryanodine receptors (RyRs). The role of RyRs has been proved for pancreatic (Leite et al. 1999; Ashby et al. (2003) and parotid (Zhang et al. 1999) acinar cells but is not well defined. Previously we identified IP₃Rs in secretory cell ER membrane of Chironomus plumosus L. larvae salivary glands and assumed RyRs to be present. The aim of this study was to obtain direct confirmation of the presence of RyRs in these cells.

The Ca²⁺ content in gland tissue was measured with the help of arsenazo III. For cell permeabilization glands were incubated with saponin (0.1 mg ml^-1, 10 min), then (30 min) in solution that contained (mM): 15.3 NaCl, 129.9 KCl, 0.35 Na₂HPO₄, 0.44 KH₂PO₄, 5.55 glucose, pH 7. Ryanodine, cAMP and IP₃ were added to this solution as appropriate. Data are presented as means ± S.E.M. and were compared using Student’s t test.

(1) The ryanodine effect on the Ca²⁺ content of saponin-treated gland tissue depended on its concentration. It evoked a Ca²⁺ content decrease (by 37.95 ± 4.64% P < 0.01, n = 6) at a low concentration (5 nM), and an increase (by 40.72 ± 12.52% P < 0.05, n = 7) at a higher concentration (500 nM). This is possible only if RyRs are present. The effect of ryanodine at low concentration could be explained by RyR activation and its effect at high concentration by their suppression. (2) cAMP (10^-4 M) intensified the effect of ryanodine: ryanodine (500 nM) decreased Ca²⁺ content by 89.54 ± 30.96 % in the presence of cAMP. cAMP decreased Ca²⁺ content by 37.59 ± 10.18 % in the presence of heparin (500 µM) in the incubation medium. But cAMP did not cause Ca²⁺ content changes in saponin-treated gland tissue in the simultaneous presence of heparin and ryanodine. (3) Ryanodine (5 nM) evoked a Ca²⁺ content increase in gland tissue by 48.61 ± 11.00 % (P < 0.05, n = 6) in the presence of IP₃ (10 µM). Alone IP₃ caused the Ca²⁺ content to decrease by 45.24 ± 13.85 % (P < 0.01, n = 6). The effect of IP₃ in the presence of ryanodine (5 nM) was the opposite: the Ca²⁺ content of gland tissue increased by 30.94 ± 9.39 % (P < 0.01, n = 6). So, the simultaneous presence of ryanodine and IP₃ did not affect the Ca²⁺ content in gland tissue in comparison with control.

The effect of different ryanodine concentrations on the Ca²⁺ content of saponin-treated salivary gland tissue confirms the presence of RyRs in salivary gland secretory cells of Chironomus plumosus L. larvae. RyRs may be regulated directly or indirectly by cAMP. We suppose there to be tight connections between RyRs and IP₃Rs in secretory cells: possibly, inhibition of these channels in the presence of their stimulators is connected with mechanisms that limit extreme emptying of Ca²⁺ intracellular stores and/or a negative reverse effect of Ca²⁺ on these channels. But this idea requires further study and additional confirmation.