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Role of MMP-2 in PKCδ-mediated inhibition of Na+ dependent Ca2+ uptake in microsomes of pulmonary smooth muscle: Involvement of a pertussis toxin sensitive protein

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Abstract

Treatment of bovine pulmonary artery smooth muscle with the O2 •− generating system hypoxanthine plus xanthine oxidase stimulated MMP-2 activity and PKC activity; and inhibited Na+ dependent Ca2+ uptake in the microsomes. Pretreatment of the smooth muscle with SOD (the O2 •− scavenger) and TIMP-2 (MMP-2 inhibitor) prevented the increase in MMP-2 activity and PKC activity, and reversed the inhibition of Na+ dependent Ca2+ uptake in the microsomes. Pretreatment with calphostin C (a general PKC inhibitor) and rottlerin (a PKCδ inhibitor) prevented the increase in PKC activity and reversed O2 •− caused inhibition of Na+ dependent Ca2+ uptake without causing any change in MMP-2 activity in the microsomes of the smooth muscle. Treatment of the smooth muscle with the O2 •− generating system revealed, respectively, 36 kDa RACK-1 and 78 kDa PKCδ immunoreactive protein profile along with an additional 38 kDa immunoreactive fragment in the microsomes. The 38 kDa band appeared to be the proteolytic fragment of the 78 kDa PKCδ since pretreatment with TIMP-2 abolished the increase in the 38 kDa immunoreactive fragment. Co-immunoprecipitation of PKCδ and RACK-1 demonstrated O2 •− dependent increase in PKCδ-RACK-1 interaction in the microsomes. Immunoblot assay elicited an immunoreactive band of 41 kDa G i α in the microsomes. Treatment of the smooth muscle tissue with the O2 •− generating system causes phosphorylation of G i α in the microsomes and pretreatment with TIMP-2 and rottlerin prevented the phosphorylation. Pretreatment of the smooth muscle tissue with pertussis toxin reversed O2 •− caused inhibition of Na+ dependent Ca2+ uptake without affecting the protease activity and PKC activity in the microsomes. We suggest the existence of a pertussis toxin sensitive G protein mediated mechanism for inhibition of Na+ dependent Ca2+ uptake in microsomes of bovine pulmonary artery smooth muscle under O2 •− triggered condition, which is regulated by PKCδ dependent phosphorylation and sensitive to TIMP-2 for its inhibition. (Mol Cell Biochem xxx: 107–117, 2005)

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Abbreviations

O2 •− :

superoxide

SOD:

superoxide dismutase

ER:

endoplasmic reticulum

MMP-2:

matrix metalloprotease-2

TIMP-2:

tissue inhibitor of metalloprotease-2

PKC:

protein kinase C

RACK-1:

receptor for activated C kinase-1

E-64d:

trans-epoxysuccinyl-L-leucylamido-3-methylbutane ethyl ester

G i α:

α-subunit of the inhibitory G protein

HBPS:

Hank's buffered physiological saline

IAP:

islet activating protein (pertussis toxin)

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  1. Department of Biochemistry and Biophysics, University of Kalyani, Kalyani, 741235, West Bengal, India

    Sajal Chakraborti, Amritlal Mandal, Sudip Das & Tapati Chakraborti

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  1. Sajal Chakraborti
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Chakraborti, S., Mandal, A., Das, S. et al. Role of MMP-2 in PKCδ-mediated inhibition of Na+ dependent Ca2+ uptake in microsomes of pulmonary smooth muscle: Involvement of a pertussis toxin sensitive protein. Mol Cell Biochem 280, 107–117 (2005). https://doi.org/10.1007/s11010-005-8237-9

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