Indian Journal of Biochemistry & Biophysics Vol. 50, October 2013, pp. 419-427 Effect of m-calpain in PKCα-mediated proliferation of pulmonary artery smooth muscle cells by low dose of ouabain Soni Shaikh, Jaganmay Sarkar, Asmita Pramanik, Kanchan Karmakar and Sajal Chakraborti* Department of Biochemistry and Biophysics, University of Kalyani, Kalyani 741235, West Bengal, India Received 26 June 2013; revised 28 August 2013 There is growing evidence that ouabain, a cardiotonic steroid may promote growth of cardiac and vascular myocytes, indicating its novel role in cell growth and proliferation, without appreciable inhibition of the sodium pump. The mechanism(s) by which low dose of ouabain produces pulmonary artery smooth muscle cell proliferation, a prerequisite for right ventricular hypertrophy, is currently unknown. Here, we analyzed the effects of low dose of ouabain (10 nM) on increase in [Ca 2+ ] i, m-calpain and protein kinase C (PKC) activities on pulmonary artery smooth muscle cell proliferation and determined their sequential involvement in this scenario. We treated bovine pulmonary artery smooth muscle cells with a low dose of ouabain (10 nM) and determined [Ca 2+ ] i in the cells by fluorometric assay using fura2-AM, m-calpain activity by fluorometric assay using SLLVY-AMC as the substrate, PKC activity using an assay kit and assay of Na + /K + ATPase activity spectrophotometrically. We purified m-calpain and PKCα by standard chromatographic procedure by HPLC and then studied cleavage of the purified PKCα by m-calpain using Western immunoblot method. Subsequently, we performed cell proliferation assay utilizing the redox dye resazunin. We used selective inhibitors of [Ca 2+ ] i (BAPTA-AM), m-calpain (MDL28170), PKCα (Go6976) and determined their involvement in ouabain (10 nM)-mediated smooth muscle cell proliferation. Our results suggested that treatment of bovine pulmonary artery smooth muscle cells with a low dose of ouabain (10 nM) increased [Ca 2+ ] i and subsequently stimulated m-calpain activity and proteolytically activated PKCα in caveolae (signaling microdomain also known as signalosomes) of the cells. Upon activation, PKCα increased the smooth muscle cell proliferation via Go/G1 to S/G2-M phase transition. Thus, [Ca 2+ ] i -mCalpain-PKCα signaling axis plays a crucial role during low dose of ouabain-mediated pulmonary artery smooth muscle cell proliferation. Keywords: Pulmonary artery, Smooth muscle cells, Caveolae, m-Calpain, Protein kinase Cα, Calcium, Ouabain, Cell proliferation. The caveolae are vesicular membrane structures that are found in almost all types of mammalian cells examined 1 . They are structurally and biochemically distinct cell membrane microdomains that contain a variety of transporters that regulate [Ca 2+ ] i and several important signaling components, such as Ca 2+ and Na + channels, Ca 2+ ATPase, Na + /K + ATPase, protein kinase C (PKC) and Ca 2+ sensing receptors 2-4 . Smooth muscle contractility depends on regulated changes in [Ca 2+ ] i . These changes could occur by localization of Ca 2+ handling components in the caveolae microdomain of plasma membrane, which have been suggested to play an important role in regulating [Ca 2+ ] i in the cells 5 . An increase in [Ca 2+ ] i has been shown to produce pulmonary hypertension with the involvement of PKC in isolated lung, which may subsequently cause pulmonary artery smooth muscle cell proliferation, leading to the right ventricular hypertrophy and failure 6-9 . Ouabain, a cardiotonic steroid (CTS) is capable of producing pulmonary hypertension 10 . Endogenous hormones identical to CTSs are present in nanomolar concentrations in plasma 11 . Ouabain has been shown to markedly inhibit the lungs ability to recover from edema, indicating that fluid clearance from lung tissue could result due to activation of ouabain-sensitive Na + pump 12 . The current concept for an increase in [Ca 2+ ] i during Na + /K + ATPase inhibition is centered —————— *Author for correspondence: Email: sajal_chakraborti@yahoo.com Fax: 0091-33-25828460 Tel: +91-9831228224 Abbreviations: BAPTA-AM, 1,2-Bis(2-aminophenoxy)ethane- N,N,N′ ,N′-tetraacetic acid tetrakis(acetoxymethyl ester); [Ca 2+ ] i , intracellular free Ca 2+ ; CTS, cardiotonic steroid; FCS, fetal calf serum; NKA, Na + /K + ATPase; PASMCs, pulmonary artery smooth muscle cells; PKC, protein kinase C; Go6976, [12-(2-Cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H- indolo(2,3-a)pyrrolo(3,4-c)-carbazole]; PMCA, plasma membrane Ca 2+ ATPase; SLLVY-AMC, N-Succinyl-Leu-Leu-Val-Tyr-7- amino-4-methylcoumarin.