Regulation of potential-dependent L-type Ca²⁺ currents by agmatine. Imidazoline receptors in isolated cardiomyocytes

The main goal of the present work was to study the mechanisms of voltage-gated L-type Ca²⁺ currents regulation by agmatine in isolated cardiomyocytes and to determine whether agmatine is involved in mediating the "arginine paradox". It was shown that agmatine at concentrations from 200 μM to 15 mM inhibited L-type Ca²⁺ currents in isolated cardiomyocytes in a dose-dependent manner. The selective antagonists of α₂-adrenoceptors (α₂-ARs), yohimbine and rauwolscine, did not modulate the effect of agmatine. In contrast, efaroxan and idazoxan known to antagonize both α₂-ARs and type 1 imidazoline receptors (I₁Rs) decreased the efficiency of agmatine almost twofold. The NO synthase inhibitor 7NI insignificantly influenced the suppressive action of agmatine on L-type Ca²⁺ currents, whereas the protein kinase C inhibitor, calphostin C, markedly reduced the effects of agmatine. Arginine did not affect L-type Ca²⁺ currents in the presence of agmatine and vice versa. These data suggest that agmatine is not involved in mediating the "arginine paradox" and that its effects are not due to the activation of endothelial NO synthase (eNOS) followed by cGMP-dependent inhibition of L-type Ca²⁺ current. Most likely, agmatine acts via I₁Rs coupled with the signaling pathway that involves the activation of protein kinase C. Previously nothing was known about possible localization of I₁Rs in isolated cardiomyocytes. Consistently, we have shown that single cardiomyocytes express the nischarin genes homologous to the IRAS gene, which is considered in the modern literature as the major candidate for the gene encoding I₁Rs. To the best our knowledge, this is the first demonstration of I₁Rs expression at the level of individual cells, including cardiomyocytes.