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(Hypertension. 1997;30:191-198.)
© 1997 American Heart Association, Inc.

Articles

Characterization of a Sodium-Response Transcriptional Mechanism

Nelson Ruiz-Opazo; Jean-François Cloix; Maria-Grazia Melis; Xie Hou Xiang; ; Victoria L. M. Herrera

From the Section of Molecular Genetics, Whitaker Cardiovascular Institute, and Section of Cardiology (V.L.M.H.), Evans Department of Medicine, Boston (Mass) University School of Medicine.

Correspondence to Victoria L.M. Herrera, MD, Section of Molecular Genetics, W-609, Whitaker Cardiovascular Institute, Boston University School of Medicine, 80 E Concord St, Boston, MA 02118.

Abstract On the basis of paradigms in development wherein discrete transcriptional events are pivotal regulatory steps, we tested the hypothesis that transcriptional sodium (Na⁺)–response mechanisms are involved in in vivo Na⁺-induced responses relevant to normal (homeostatic) and pathophysiological (salt-sensitive hypertension) conditions. We used Na,K-ATPase -subunit genes as molecular probes and the Na⁺ ionophore monensin to induce a dose-specific incremental increase in [Na⁺]_i in rat A10 embryonic aortic smooth muscle cells. RNA blot analysis of rat A10 cells revealed a dose-specific (0.022 to 30 µmol/L monensin) upregulation of ₁-, ₂-, and ß₁-subunit Na,K-ATPase RNA levels. Control ß-actin and -tropomyosin RNA levels did not change. With the use of chloramphenicol acetyltransferase (CAT) as reporter gene, CAT assays of rat ₁[-1288]CAT and human ₂[-798]CAT promoter constructs exhibited induction of CAT activity in monensin (10 µmol/L)–treated A10 cells compared with untreated A10 cells. Promoter deletion constructs for rat ₁[-1288]CAT defined a positive Na⁺-response regulatory region within -358 to -169 that is distinct from the basal transcriptional activation region of -155 to -49 previously defined. Similarly, a positive Na⁺-response regulatory region is delimited to within -301 in the human ₂ Na,K-ATPase 5' flanking region. Analysis of transgenic TgH₂[-798]CAT rats demonstrated sodium activation of human ₂[-798]CAT transgene expression in aorta parallel to observations made in rat A10 aortic tissue culture cells. Southwestern blot analysis of nuclear extracts from monensin (10 µmol/L)–treated and control untreated A10 cells revealed a nuclear DNA binding protein (approximately 95 kD) that is upregulated by increased [Na⁺]_i. These data provide initial characterization of a transcriptional Na⁺-response mechanism delimiting a positive Na⁺-response regulatory region in two target genes (₁ and ₂ Na,K-ATPase) as well as detection of a Na⁺-response nuclear DNA binding protein. The in vitro data are corroborated by in vivo experimental and transgenic promoter expression studies, thus validating the biological relevance of the observations.

Key Words: monensin • gene regulation • muscle, smooth • rats, transgenic

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