Convergence of Ionotropic and Metabotropic Signal Pathways upon Activation of P2X Receptors in Vascular Smooth Muscle Cells

Ionotropic P2X receptors (P2XRs) are involved in sympathetic control of the vascular tone; they mediate entry of Ca 2+ in smooth muscle cells (SMCs), which results in depolarization of the latter and activation of voltage-gated L-type calcium channels. In addition, Ca 2+ ions, after their entry into the cell, trigger Ca 2+ release from the sarcoplasmic reticulum (SR) of SMCs via ryanodine receptors (RyRs), and this amplifies calcium signals. We found earlier that Ca 2+ release mediated by inositol triphosphate (IP 3 ) receptors (IP 3 Rs) also provides a considerable contribution to P2XR-mediated calcium signaling. Thus, a metabotropic signal pathway is a component of the calcium signaling system triggered by ionotropic P2XRs. Using confocal detection of changes in the intracellular Ca 2+ concentration ([Ca 2+ ] i ) and applications of the inhibitors of calcium channels (nicardipine, 5 μM), sarco-endoplasmic Ca 2+ ATPase SERCA (CPA, 10 μM), IP 3 Rs (2-APB, 30 μM), RyRs (tetracaine, 100 μM), and phosphalipase C (PLC; U-73122, 2.5 μM), we estimated relative contributions of the above-mentioned four components to increase in the [Ca 2+ ] i induced by the action of an agonist of P2XRs, α,β-meATP. The contributions of transmembrane Ca 2+ entry via channels of P2XRs and calcium channels were comparable (11.0 ± 1.4 %, n = 14 and 8.0 ± 1.4 %, n = 14, respectively). The contribution of Ca 2+ release via IP 3 Rs was found to be three times greater than that via RyRs (41 ± 5 %, n = 26 and 14 ± 7 %, n = 16, respectively). Blocking of calcium channels resulted in a sevenfold decrease in the contribution of IP 3 R-mediated Ca 2+ release (from 41.0 to 5.6%); in this case, the contribution of RyR-mediated Ca 2+ release underwent no significant changes. This fact allows us to suppose that there is a functional relation between activation of calcium channels and functioning of a metabotropic PLC/IP 3 -mediated signal cascade. The efficiency of inhibition of α,β-meATP-induced calcium responses by the blocker of PLC, on the one hand, and by the IP 3 R blocker and nicardipine, on the other hand, is comparable, and this fact agrees with the above hypothesis. According to our data, P2XR activation-induced increase in [Ca 2+ ] I results not only from P2XR-mediated Ca 2+ entry that triggers Ca 2+ release via RyRs but also from Ca 2+ release via IP 3 Rs. The latter process is realized due to the functioning of the PLC-mediated pathway, is in close relation with activation of calcium channels, and provides a dominant contribution in Ca 2+ release from the stores after activation of the above ionotropic receptors.