Pulak Kar

Mitochondrial calpain system: An overview

Calpain system is generally known to be comprised of three molecules: two Ca2+-dependent proteases: mu- and m-calpains, and their endogenous inhibitor, calpastatin. While calpains have previously been considered as the cytoplasmic enzymes, research in the recent past demonstrated that mu-calpain, m-calpain and calpain 10 are present in mitochondria, which play important roles in a variety of pathophysiological conditions including necrotic and apoptotic cell death phenomena. Although a number of original research articles on mitochondrial calpain system are available, yet to the best of our knowledge, a precise review article on mitochondrial calpain system has, however, not been available. This review outlines the key features of the mitochondrial calpain system, and its roles in several cellular and biochemical events under normal and some pathophysiological conditions.

μ-Calpain mediated cleavage of the Na+/Ca2+ exchanger in isolated mitochondria under A23187 induced Ca2+ stimulation

Treatment of bovine pulmonary artery smooth muscle mitochondria with the calcium ionophore, A23187 (0.2 microM) stimulates mu-calpain activity and subsequently cleaves Na(+)/Ca(2+) exchanger (NCX). Pretreatment of the A23187 treated mitochondria with the calpain inhibitors, calpeptin or MDL28170 or with Ca(2+) chelator, EGTA does not cleave NCX. Treatment of the mitochondria with A23187 increases Ca(2+) level in the mitochondria, which subsequently dissociates mu-calpain-calpastatin association leading to the activation of mu-calpain. Immunoblot study of the A23187 treated mitochondria with the NCX polyclonal antibody indicates the degradation of mitochondrial inner membrane NCX (110kDa) resulting in the doublet of approximately 54-56kDa NCX fragments. Moreover, in vitro cleavage of mitochondrial purified NCX by mitochondrial purified mu-calpain supports our conclusion. This cleavage of NCX may be interpreted as the main cause of Ca(2+) overload and could lay a key role in the activation of apoptotic process in pulmonary smooth muscle.

Calcium signaling phenomena in heart diseases: a perspective.

Ca2+ is a major intracellular messenger and nature has evolved multiple mechanisms to regulate free intracellular (Ca2+)i level in situ. The Ca2+ signal inducing contraction in cardiac muscle originates from two sources. Ca2+ enters the cell through voltage dependent Ca2+ channels. This Ca2+ binds to and activates Ca2+ release channels (ryanodine receptors) of the sarcoplasmic reticulum (SR) through a Ca2+ induced Ca2+ release (CICR) process. Entry of Ca2+ with each contraction requires an equal amount of Ca2+ extrusion within a single heartbeat to maintain Ca2+ homeostasis and to ensure relaxation. Cardiac Ca2+ extrusion mechanisms are mainly contributed by Na+/Ca2+ exchanger and ATP dependent Ca2+ pump (Ca2+-ATPase). These transport systems are important determinants of (Ca2+)i level and cardiac contractility. Altered intracellular Ca2+ handling importantly contributes to impaired contractility in heart failure. Chronic hyperactivity of the β-adrenergic signaling pathway results in PKA-hyperphosphorylation of the cardiac RyR/intracellular Ca2+ release channels. Numerous signaling molecules have been implicated in the development of hypertrophy and failure, including the β-adrenergic receptor, protein kinase C, Gq, and the down stream effectors such as mitogen activated protein kinases pathways, and the Ca2+ regulated phosphatase calcineurin. A number of signaling pathways have now been identified that may be key regulators of changes in myocardial structure and function in response to mutations in structural components of the cardiomyocytes. Myocardial structure and signal transduction are now merging into a common field of research that will lead to a more complete understanding of the molecular mechanisms that underlie heart diseases. Recent progress in molecular cardiology makes it possible to envision a new therapeutic approach to heart failure (HF), targeting key molecules involved in intracellular Ca2+ handling such as RyR, SERCA2a, and PLN. Controlling these molecular functions by different agents have been found to be beneficial in some experimental conditions.

Calcium-dependent cleavage of the Na+/Ca2+ exchanger by m-calpain in isolated endoplasmic reticulum

We have recently demonstrated the localization of associated m-calpain and calpastatin in the endoplasmic reticulum (ER) of bovine pulmonary artery smooth muscle. Herein, we sought to determine the role of m-calpain on calcium-dependent proteolytic cleavage of Na(+)/Ca(2+) exchanger (NCX) in the ER. Treatment of the ER with Ca(2+) (5 mM) dissociates m-calpain-calpastatin association leading to the activation of m-calpain, which subsequently cleaves the ER integral transmembrane protein NCX1 (116 kDa) to an 82 kDa fragment. Pre-treatment of the ER with calpain inhibitors, calpeptin (10 microM) or MDL28170 (10 microM), or Ca(2+) chelator, EGTA (10 mM) does not cleave NCX1. In vitro cleavage of the ER purified NCX1 by the ER purified m-calpain also supports our finding. Cleavage of NCX1 by m-calpain in the ER may be interpreted as the main cause of intracellular Ca(2+) overload in the smooth muscle, which could be important for the manifestation of pulmonary hypertension.

Role of protein kinase C in NADPH oxidase derived O2*(-)-mediated regulation of KV-LVOCC axis under U46619 induced increase in [Ca2+]i in pulmonary smooth muscle cells.

Treatment of bovine pulmonary smooth muscle cells with the TxA(2) mimetic, U46619 stimulated [Ca(2+)](i), which was inhibited upon pretreatment with apocynin (NADPH oxidase inhibitor). Pretreatment with cromakalim (K(V) channel opener) or nifedepine (L-VOCC inhibitor) inhibited U46619 induced increase in [Ca(2+)](i), indicating a role of K(V)-LVOCC axis in this scenario. Neither cromakalim nor nifedepine inhibited U46619 induced increase in NADPH oxidase activity, suggesting that the NADPH oxidase activation is proximal to the K(V)-LVOCC axis in the cells. Pretreatment with calphostin C (PKC inhibitor) markedly reduced U46619 induced increase in NADPH oxidase activity and [Ca(2+)](i) in the cells. Calphostin C pretreatment also markedly reduced p(47phox) phosphorylation and translocation to the membrane and association with p(22phox), a component of Cyt.b(558) of NADPH oxidase in the membrane. Overall, PKC plays an important role in NADPH oxidase derived O(2)(-)-mediated regulation of K(V)-LVOCC axis leading to an increase in [Ca(2+)](i) by U46619 in the cells.

Characteristic properties of endoplasmic reticulum membrane m-calpain, calpastatin and lumen m-calpain: a comparative study between membrane and lumen m-calpains

Previously, we reported that bovine pulmonary smooth muscle endoplasmic reticulum (ER) membrane possesses associated m-calpain and calpastatin and ER lumen contains only m-calpain. Herein, we report characteristic properties of ER membrane m-calpain (MCp), calpastatins and lumen m-calpain (LCp) and a brief comparative study between MCp and LCp. MCp containing 80 kDa large and 28 kDa small subunit is non-phosphorylated, whereas LCp containing only 80 kDa large subunit is phosphorylated. Optimum pH, Ca(2+) concentration and pI value of both MCp and LCp are 7.5, 5 mM and 4.5, respectively. MCp and LCp have similar kinetic parameters and circular dichroism (CD) spectra. Autolysis of MCp and LCp are different. Coimmunoprecipitation studies revealed that LCp is associated with ERp57 in the ER lumen, which suggests that the regulation of LCp differs from the regulation of MCp. In presence of Ca(2+), the activated LCp cleaves inositol 1,4,5-trisphosphate receptor-1 (IP(3)R1) in the ER lumen, whereas the activated MCp cleaves Na(+)/Ca(2+) exchanger-1 (NCX1) in the ER membrane. We have determined pI (4.6 and 4.7, respectively) and IC(50) (0.52 and 0.8 nM, respectively) values of 110 and 70 kDa calpastatins. For first time, we have determined the characteristic properties, regulation and functional activity of LCp in the ER lumen.

Identification, purification and partial characterization of a 70 kDa inhibitor protein of Na+/K+-ATPase from cytosol of pulmonary artery smooth muscle

AIMS We sought to identify, purify and partially characterize a protein inhibitor of Na(+)/K(+)-ATPase in cytosol of pulmonary artery smooth muscle. MAIN METHODS (i) By spectrophotometric assay, we identified an inhibitor of Na(+)/K(+)-ATPase in cytosolic fraction of pulmonary artery smooth muscle; (ii) the inhibitor was purified by a combination of ammonium sulfate precipitation, diethylaminoethyl (DEAE) cellulose chromatography, hydroxyapatite chromatography and gel filtration chromatography; (iii) additionally, we have also purified Na(+)/K(+)-ATPase alpha(2)beta(1) and alpha(1)beta(1) isozymes for determining some characteristics of the inhibitor. KEY FINDINGS We identified a novel endogenous protein inhibitor of Na(+)/K(+)-ATPase having an apparent mol mass of approximately 70kDa in the cytosolic fraction of the smooth muscle. The IC(50) value of the inhibitor towards the enzyme was determined to be in the nanomolar range. Important characteristics of the inhibitor are as follows: (i) it showed different affinities toward the alpha(2)beta(1) and alpha(1)beta(1) isozymes of the Na(+)/K(+)-ATPase; (ii) it interacted reversibly to the E(1) site of the enzyme; (iii) the inhibitor blocked the phosphorylated intermediate formation; and (iv) it competitively inhibited the enzyme with respect to ATP. CD studies indicated that the inhibitor causes an alteration of the conformation of the enzyme. The inhibition study also suggested that the DHPC solubilized Na(+)/K(+)-ATPase exists as (alphabeta)(2) diprotomer. SIGNIFICANCE The inhibitor binds to the Na(+)/K(+)-ATPase at a site different from the ouabain binding site. The novelty of the inhibitor is that it acts in an isoform specific manner on the enzyme, where alpha(2) is more sensitive than alpha(1).