Molecular and Cellular Biochemistry

, Volume 341, Issue 1, pp 167–180

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

  • Soni Shaikh
  • Krishna Samanta
  • Pulak Kar
  • Soumitra Roy
  • Tapati Chakraborti
  • Sajal Chakraborti
Article

DOI: 10.1007/s11010-010-0448-z

Cite this article as:
Shaikh, S., Samanta, K., Kar, P. et al. Mol Cell Biochem (2010) 341: 167. doi:10.1007/s11010-010-0448-z

Abstract

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.

Keywords

m-CalpainCalciumNCXCaveolaePulmonary arterySmooth muscle

Abbreviations

NCX1

Na+/Ca2+ exchanger-1

DHPC

1,2-Dihepatanoyl-sn-phosphatidylcholine

DOPC

Dioleoyl-phosphatidylcholine

SLLVY-AMC

Succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin

SEA0400

(2-[4-[(2,5-difluorophenyl)methoxy]phenoxy]-5-ethoxyaniline)

Copyright information

© Springer Science+Business Media, LLC. 2010

Authors and Affiliations

  • Soni Shaikh
    • 1
  • Krishna Samanta
    • 1
  • Pulak Kar
    • 1
  • Soumitra Roy
    • 1
  • Tapati Chakraborti
    • 1
  • Sajal Chakraborti
    • 1
  1. 1.Department of Biochemistry and BiophysicsUniversity of KalyaniKalyaniIndia