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  • Role of PKCζ-NADPH oxidase signaling axis in PKCα-mediated Giα2 phosphorylation for inhibition of adenylate cyclase activity by angiotensin II in pulmonary artery smooth muscle cells.

Role of PKCζ-NADPH oxidase signaling axis in PKCα-mediated Giα2 phosphorylation for inhibition of adenylate cyclase activity by angiotensin II in pulmonary artery smooth muscle cells.

Cell biology international (2020-01-23)
Animesh Chowdhury, Jaganmay Sarkar, Pijush Kanti Pramanik, Tapati Chakraborti, Sajal Chakraborti
ABSTRACT

We sought to determine the mechanism by which angiotensin II (AngII) inhibits isoproterenol induced increase in adenylate cyclase (AC) activity and cyclic adenosine monophosphate (cAMP) production in bovine pulmonary artery smooth muscle cells (BPASMCs). Treatment with AngII stimulates protein kinase C-ζ (PKC-ζ), nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, and PKC-α activities, and also inhibits isoproterenol induced increase in AC activity and cAMP production in the cells. Pertussis toxin pretreatment eliminates AngII caused inhibition of isoproterenol induced increase in AC activity without a discernible change in PKC-ζ, NADPH oxidase, and PKC-α activities. Treatment of the cells with AngII increases α2 isoform of Gi (Giα2) phosphorylation; while pretreatment with chemical and genetic inhibitors of PKC-ζ and NADPH oxidase attenuate AngII induced increase in PKC-α activity and Giα2 phosphorylation, and also reverse AngII caused inhibition of isoproterenol induced increase in AC activity. Pretreatment of the cells with chemical and genetic inhibitors of PKC-α attenuate AngII induced increase in Giα2 phosphorylation and inhibits isoproterenol induced increase in AC activity without a discernible change in PKC-ζ and NADPH oxidase activities. Overall, PKCζ-NADPH oxidase-PKCα signaling axis plays a crucial role in Giα2 phosphorylation resulting in AngII-mediated inhibition of isoproterenol induced increase in AC activity in BPASMCs.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Pertussis toxin from Bordetella pertussis, lyophilized powder
Sigma-Aldrich
Calphostin C from Cladosporium cladosporioides, ≥90% (HPLC), powder
Sigma-Aldrich
4-Chloro-1-naphthol solution, For HRP detection on Western Blots
Sigma-Aldrich
Adenosine 3′,5′-cyclic monophosphate tris salt, ≥97% (HPLC), powder