Original research article Mechanisms of the sarcoplasmic reticulum Ca 2+ release induced by P2X receptor activation in mesenteric artery myocytes Khrystyna Yu. Sukhanova a, *, Oleksandr M. Thugorka a , Vitali A. Bouryi a , Maksym I. Harhun b , Dmitri V. Gordienko a,c, * a Laboratory of Molecular Pharmacology and Biophysics of Cell Signalling, State Key Laboratory of Molecular and Cellular Biology, A.A. Bogomoletz Institute of Physiology, Kiev, Ukraine b Division of Biomedical Sciences, St. George’s, University of London, London, UK c Inserm U1003, Equipe labellise ´e par la Ligue Nationale Contre le Cancer, Universite ´ des Sciences et Technologies de Lille, Villeneuve d’Ascq, France Introduction Sympathetic nerve activity controls total peripheral vascular resistance and, hence, systemic blood pressure via regulation of the contractile activity of smooth muscle cells (SMCs) in the wall of small arteries. One of the principal sympathetic neurotransmitters, ATP, triggers contraction of arterial SMCs via activation of ionotropic P2X receptors (P2XRs) resulting in a robust increase of [Ca 2+ ] i . Both Ca 2+ entry across the plasma membrane through P2XRs themselves and Ca 2+ entry via voltage-gated Ca 2+ channels (VGCCs) activated by P2X-receptor-mediated membrane depolar- ization were found to contribute in the increase of [Ca 2+ ] i . We have recently demonstrated that net contribution of these two Ca 2+ Pharmacological Reports 66 (2014) 363–372 A R T I C L E I N F O Article history: Received 4 March 2013 Received in revised form 9 October 2013 Accepted 26 November 2013 Available online 13 April 2014 Keywords: Vascular smooth muscle cells P2X receptors Voltage-gated Ca 2+ channels Inositol 1,4,5-trisphosphate receptor Ryanodine receptor A B S T R A C T Background: ATP is one of the principal sympathetic neurotransmitters which contracts vascular smooth muscle cells (SMCs) via activation of ionotropic P2X receptors (P2XRs). We have recently demonstrated that contraction of the guinea pig small mesenteric arteries evoked by stimulation of P2XRs is sensitive to inhibitors of IP 3 receptors (IP 3 Rs). Here we analyzed contribution of IP 3 Rs and ryanodine receptors (RyRs) to [Ca 2+ ] i transients induced by P2XR agonist ab-meATP (10 mM) in single SMCs from these vessels. Methods: The effects of inhibition of L-type Ca 2+ channels (VGCCs), RyRs and IP 3 Rs (5 mM nicardipine, 100 mM tetracaine and 30 mM 2-APB, respectively) on ab-meATP-induced [Ca 2+ ] i transients were analyzed using fast x–y confocal Ca 2+ imaging. Results: The effect of IP 3 R inhibition on the [Ca 2+ ] i transient was significantly stronger (67  7%) than that of RyR inhibition (40  5%) and was attenuated by block of VGCCs. The latter indicates that activation of VGCCs is linked to IP 3 R-mediated Ca 2+ release. Immunostaining of RyRs and IP 3 Rs revealed that RyRs are located mainly in deeper sarcoplasmic reticulum (SR) while sub-plasma membrane (PM) SR elements are enriched with type 1 IP 3 Rs. This structural peculiarity makes IP 3 Rs more accessible to Ca 2+ entering the cell via VGCCs. Thus, IP 3 Rs may serve as an ‘‘intermediate amplifier’’ between voltage-gated Ca 2+ entry and RyR- mediated Ca 2+ release. Conclusions: P2X receptor activation in mesenteric artery SMCs recruits IP 3 Rs-mediated Ca 2+ release from sub-PM SR, which is facilitated by activation of VGCCs. Sensitivity of IP 3 R-mediated release to VGCC antagonists in vascular SMCs makes this mechanism of special therapeutic significance. ß 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved. Abbreviations: 2-APB, 2-aminoethoxydiphenyl borate; ab-meATP, ab-methylene- adenosine 5 0 -triphosphate; CICR, Ca 2+ -induced Ca 2+ release; CPA, cyclopiazonic acid; IP 3 , inositol 145-trisphosphate; [IP 3 ] i , intracellular concentration of IP 3 ; IP 3 R, inositol 145-trisphosphate receptor; [Ca 2+ ] i , intracellular concentration of ionized calcium; jSR, sub-plasmalemmal (‘‘junctional’’) sarcoplasmic reticulum; PLC, phospholipase C; RyR, ryanodine receptor; SERCA, sarco-/endoplasmic reticulum Ca 2+ -ATPase; SMC, smooth muscle cells; SPCU, sub-plasmalemmal [Ca 2+ ] i upstroke; SR, sarcoplasmic reticulum; VGCC, voltage-gated Ca 2+ channel. * Corresponding author. E-mail addresses: skhrist@biph.kiev.ua (K.Yu. Sukhanova), gordienkdv@googlemail.com (D.V. Gordienko). Contents lists available at ScienceDirect Pharmacological Reports jou r nal h o mep ag e: w ww .elsevier .co m /loc ate/p h arep http://dx.doi.org/10.1016/j.pharep.2013.11.005 1734-1140/ß 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.