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Effect of m-calpain in PKCα-mediated proliferation of pulmonary artery smooth muscle cells by low dose of ouabain

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Title Effect of m-calpain in PKCα-mediated proliferation of pulmonary artery smooth muscle cells by low dose of ouabain
 
Creator Shaikh, Soni
Sarkar, Jaganmay
Pramanik, Asmita
Karmakar, Kanchan
Chakraborti, Sajal
 
Subject Pulmonary artery
Smooth muscle cells
Caveolae
m-Calpain
Protein kinase Cα
Calcium
Ouabain
Cell proliferation
 
Description 419-427
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 [Ca2+]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 [Ca2+]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 [Ca2+]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 [Ca2+]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, [Ca2+]i-mCalpain-PKCα
signaling axis plays a crucial role during low dose of ouabain-mediated
pulmonary artery smooth muscle cell proliferation.
 
Date 2013-10-26T09:49:03Z
2013-10-26T09:49:03Z
2013-10
 
Type Article
 
Identifier 0975-0959 (Online); 0301-1208 (Print)
http://hdl.handle.net/123456789/22641
 
Language en_US
 
Rights CC Attribution-Noncommercial-No Derivative Works 2.5 India
 
Publisher NISCAIR-CSIR, India
 
Source IJBB Vol.50(5) [October 2013]