Velykopolska O. Purinergic receptors of salivary glands secretory cells of Chironomus plumosus L. larvae

the thesis P2X-and P2Y-receptors were identified and characterized. Their role in Ca2+ signaling of secretory cells of salivary glands of Chironomus plumosus larvae was investigated. P2-receptor agonists АТP or ADP in concentrations 0,1-2,0 mM caused decreasing in total Са2+ content of tissue (measured by arsenazo III), when glands were incubated in Ca2+ free medium, in contrast to mediums with normal and increased Са2+ concentration. Furthermore, it was established that extracellular ATP (100 mM) and ADP (100 mM) leads to the changes of stored Ca2+ content (measured with chlortetracycline fluorescence). The direction of these changes is determined by transmembrane Ca2+ gradient. Stored Ca2+ content increases upon 10 mM Са2+ concentration in medium, decreases upon Ca2+ free medium and does not change at 1,76 mM Са2+ concentration in medium. P2 receptor inhibitor suramin (100 µM) suppresses both purine-induced decrease and increase of stored Ca2+ content. This is consistent with presence of both P2X- and P2Y-receptors in plasma membrane of secretory cells. Role of InsP3Rs in intracellular signal transduction was investigated. Presence of InsP3Rs inhibitor 2-APB (10 ?mol/l) in nominally Ca2+-free medium abolished the ATP-induced release of Са2+ from stores, and did not influence ADP-induced decrease of stored Са2+ in the secretory cells. According to the obtained data, we assume the presence of two subtypes of Р2Y-receptors in secretory cells of Chironomus plumosus larvae salivary glands. Receptors of the first subtype (P2YATP) have higher affinity to ATP and are coupled with IP3-dependent Са2+-channels. Receptors of the second subtype (P2YADP) have higher affinity to ADP and exert their functions through other mechanism. Investigation of P2-receptors activation interaction with RyRs of salivary glands was performed. Ryanodine (10 nM) was shown to induce Ca2+ release from stores. Decrease of stored Ca2+ content caused by the activation of RyRs was more prominent than at purinergic receptors activation. Adding of ATP into Ca2+-free incubatory medium did not augment action of ryanodine, while at the presence of Ca2+ simultaneous ATP and ryanodine application potentiated Ca2+-release from store. We suggest that in case of P2YATP and RyRs activation potentiation of Ca2+-release from store occurs due to increase of endoplasmic Ca2+-functional unit activity by simultaneous activation of various its components - IP3Rs and RyRs. At simultaneous action of ADP and ryanodine in nominally Ca2+-free medium decrease of stored Ca2+ was not observed, and at the physiological [Ca2+] its content in stores even increased above control values. Thus, in cases of P2YADP and RyRs interaction inhibition of the unit is observed. Using Clark oxygen electrode, dependence of mitochondrial functions on Ca2+-release channels and P2 receptors activity was studied. It was found that adding of P2 receptor agonists (ATP and ADP) does not alter the rate of endogenous respiration of secretory cells, but promotes penetration of exogenous substrates (succinate, malate, pyruvate) and thus intensifies cells respiration. Ryanodine-induced activation of endoplasmic-mitochondrial Ca2+-functional unit leads to an intensification of endogenous respiration. Upon activation of P2 receptor ryanodine also increases rate of respiration stimulated with a mixture of malate and pyruvate (but not stimulated with succinate). Inhibition of InsP3Rs with 2-APB does not alter neither the rate of endogenous respiration nor the rate of respiration stimulated with a mixture of malate and pyruvate upon simultaneous activation of P2 receptors. However, succinate-stimulated respiration was increased under these conditions.