OBJECTIVE: Na/K-ATPase (Na,K pump) plays an important role in the regulation of intracellular ion composition. The purpose of this study is to determine whether Na+ regulates the levels of mRNA coding for Na/K-ATPase alpha and beta isoforms in cultured rat vascular smooth muscles cells. METHODS: Na/K-ATPase alpha and beta isoform mRNA expression was evaluated by northern blot analysis. The alpha 1 subunit content was determined by ELISA. A luciferase reporter gene assay was performed to study whether the alpha 1 promoter might contain Na+ responsive elements. RESULTS: Na/K-ATPase alpha 1 and beta 1 isoform mRNAs, but not alpha 2 and alpha 3 isoform mRNAs, were expressed in cultured rat vascular smooth muscle cells. Exposure of the cells to 10 microM veratridine, an Na+ channel activator, resulted in two- and threefold increases in alpha 1 and beta 1 mRNA accumulation, respectively, with a maximum at 60 min. The veratridine induced alpha 1 and beta 1 mRNA accumulation was still observed even in the absence of extracellular Ca2+. The increase in alpha 1 mRNA accumulation caused by 10 microM veratridine was associated with a significant increase in alpha 1 subunit protein accumulation. Transfection experiments with chimeric plasmids containing 5'-flanking sequences of alpha 1 isoform gene and luciferase reporter gene showed that 10 microM veratridine caused a threefold increase in luciferase activity. CONCLUSIONS: These results suggest that Na+ stimulates the transcription of Na/K-ATPase alpha 1 and beta 1 isoform genes in vascular smooth muscle cells. The transfection study further supports the premise that Na+ responsive elements are located within the 5'-flanking sequences of alpha 1 isoform gene.
OBJECTIVE: Na/K-ATPase (Na,K pump) plays an important role in the regulation of intracellular ion composition. The purpose of this study is to determine whether Na+ regulates the levels of mRNA coding for Na/K-ATPase alpha and beta isoforms in cultured rat vascular smooth muscles cells. METHODS: Na/K-ATPase alpha and beta isoform mRNA expression was evaluated by northern blot analysis. The alpha 1 subunit content was determined by ELISA. A luciferase reporter gene assay was performed to study whether the alpha 1 promoter might contain Na+ responsive elements. RESULTS: Na/K-ATPase alpha 1 and beta 1 isoform mRNAs, but not alpha 2 and alpha 3 isoform mRNAs, were expressed in cultured rat vascular smooth muscle cells. Exposure of the cells to 10 microM veratridine, an Na+ channel activator, resulted in two- and threefold increases in alpha 1 and beta 1 mRNA accumulation, respectively, with a maximum at 60 min. The veratridine induced alpha 1 and beta 1 mRNA accumulation was still observed even in the absence of extracellular Ca2+. The increase in alpha 1 mRNA accumulation caused by 10 microM veratridine was associated with a significant increase in alpha 1 subunit protein accumulation. Transfection experiments with chimeric plasmids containing 5'-flanking sequences of alpha 1 isoform gene and luciferase reporter gene showed that 10 microM veratridine caused a threefold increase in luciferase activity. CONCLUSIONS: These results suggest that Na+ stimulates the transcription of Na/K-ATPase alpha 1 and beta 1 isoform genes in vascular smooth muscle cells. The transfection study further supports the premise that Na+ responsive elements are located within the 5'-flanking sequences of alpha 1 isoform gene.
Authors: Sebastien Taurin; Nickolai O Dulin; Dimitri Pchejetski; Ryszard Grygorczyk; Johanne Tremblay; Pavel Hamet; Sergei N Orlov Journal: J Physiol Date: 2002-09-15 Impact factor: 5.182