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 Ca2+ in smooth muscle cells (SMCs), which results in depolarization of the latter and activation of voltage-gated L-type calcium channels. In addition, Ca2+ ions, after their entry into the cell, trigger Ca2+ release from the sarcoplasmic reticulum (SR) of SMCs via ryanodine receptors (RyRs), and this amplifies calcium signals. We found earlier that Ca2+ release mediated by inositol triphosphate (IP3) 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 Ca2+ concentration ([Ca2+](i)) and applications of the inhibitors of calcium channels (nicardipine, 5 mu M), sarco-endoplasmic Ca2+ ATPase SERCA (CPA, 10 mu M), IP(3)Rs (2-APB, 30 mu M), RyRs (tetracaine, 100 mu M), and phosphalipase C (PLC; U-73122, 2.5 mu M), we estimated relative contributions of the above-mentioned four components to increase in the [Ca2+] i induced by the action of an agonist of P2XRs, alpha, beta-meATP. The contributions of transmembrane Ca2+ 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 Ca2+ 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 IP3R-mediated Ca2+ release (from 41.0 to 5.6%); in this case, the contribution of RyR-mediated Ca2+ 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/IP3-mediated signal cascade. The efficiency of inhibition of alpha, beta-meATP-induced calcium responses by the blocker of PLC, on the one hand, and by the IP3R 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 [Ca2+] I results not only from P2XR-mediated Ca2+ entry that triggers Ca2+ release via RyRs but also from Ca2+ 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 Ca2+ release from the stores after activation of the above ionotropic receptors.