m-Calpain-mediated cleavage of Na⁺/Ca²⁺ exchanger-1 in caveolae vesicles isolated from pulmonary artery smooth muscle

Using m-calpain antibody, we have identified two major bands corresponding to the 80 kDa large and the 28 kDa small subunit of m-calpain in caveolae vesicles isolated from bovine pulmonary artery smooth muscle plasma membrane. In addition, 78, 35, and 18 kDa immunoreactive bands of m-calpain have al...

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Veröffentlicht in:Molecular and cellular biochemistry 2010-08, Vol.341 (1-2), p.167-180
Hauptverfasser: Shaikh, Soni, Samanta, Krishna, Kar, Pulak, Roy, Soumitra, Chakraborti, Tapati, Chakraborti, Sajal
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Sprache:eng
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Zusammenfassung:Using m-calpain antibody, we have identified two major bands corresponding to the 80 kDa large and the 28 kDa small subunit of m-calpain in caveolae vesicles isolated from bovine pulmonary artery smooth muscle plasma membrane. In addition, 78, 35, and 18 kDa immunoreactive bands of m-calpain have also been detected. Casein zymogram studies also revealed the presence of m-calpain in the caveolae vesicles. We have also identified Na⁺/Ca²⁺ exchanger-1 (NCX1) in the caveolae vesicles. Purification and N-terminal sequence analyses of these two proteins confirmed their identities as m-calpain and NCX1, respectively. We further sought to determine the role of m-calpain on calcium-dependent proteolytic cleavage of NCX1 in the caveolae vesicles. Treatment of the caveolae vesicles with the calcium ionophore, A23187 (1 μM) in presence of CaCl₂ (1 mM) appears to cleave NCX1 (120 kDa) to an 82 kDa fragment as revealed by immunoblot study using NCX1 monoclonal antibody; while pretreatment with the calpain inhibitors, calpeptin or MDL28170; or the Ca²⁺ chelator, BAPTA-AM did not cause a discernible change in the NCX protein profile. In vitro cleavage of the purified NCX1 by the purified m-calpain supports this finding. The cleavage of NCX1 by m-calpain in the caveolae vesicles may be interpreted as an important mechanism of Ca²⁺ overload, which could arise due to inhibition of Ca²⁺ efflux by the forward-mode NCX and that could lead to sustained Ca²⁺ overload in the smooth muscle leading to pulmonary hypertension.
ISSN:0300-8177
1573-4919
DOI:10.1007/s11010-010-0448-z