ИСТИНА

Cav1.2-channels are slow voltage-gated calcium channels present in terminals of mouse neuromuscular junctions (NMJs). Although they normally do not contribute to regulation of Ach release in this type of synapses, their activity is highly regulated by kinases and phosphotases. So, calcineurin (CaN) has been shown to constantly downregulate L-type calcium channel activity. Therefore, the aim of this study was to identify ways of L-type calcium channel upregulation in opposition to CaN activity. Experiments were held on «dissected» mouse diaphragm preparations. We registered both spontaneous and evoked (50 Hz) activity of NMJs in form of miniature end plate potentials (mEPP) and short trains of end plate potentials (EPP) respectively using standard microelectrode technique of biopotential registration. PKA is well known to phosphorylate L-type calcium channels and therefore enhance calcium influx through them in different types of tissue. But PKA inhibition by H-89 (1 μM) did not affect transmitter secretion in mouse NMJs. Nevertheless, H-89 prevented the expected rise of EPP quantal content (QC) caused by CaN inhibition by CsA (1 μM). PKA activity depends on intracellular cAMP levels and therefore on adenylate cyclase activity, which may be modulated through adenosine A2A-receptors (A2AR). A2AR agonist CGS-21680 1 μM application resulted in EPP QC increase by 20%. But preliminary H-89 or nitrendipine (Cav1.2-channel blocker) treatment fully abolished this rise. Thus activation of A2AR by CGS-21680 during short train activity may enhance PKA activity sufficiently enough to oppose CaN-dependent downregulation of L-type calcium channels. This CaN/PKA balance can also be shifted by altering the activity of an endogenous CaN regulator – calpain, since its inhibitor PD150 606 (100 mkM) caused a rise in EPP QC by approximately 32%. We showed that ACh secretion and L-type channel activity in mouse NMJs depends on the balance of CaN/PKA activity. Either by enhancing PKA or by inhibition of CaN activity in various ways makes it possible to upregulate L-type calcium channel activity and thereby increase ACh secretion.