CHARACTERIZATION OF A SODIUM-RESPONSE TRANSCRIPTIONAL MECHANISM

On the basis of paradigms in development wherein discrete transcriptio nal events are pivotal regulatory steps, we tested the hypothesis that transcriptional sodium (Na+)-response mechanisms are involved in in v ivo Na+-induced responses relevant to normal (homeostatic) and pathoph ysiological (salt-sensitive hypertension) conditions. We used Na,K-ATP ase cr-subunit genes as molecular probes and the Na+ ionophore monensi n to induce a dose-specific incremental increase in [Na+](i) in rat A1 0 embryonic aortic smooth muscle cells. RNA blot analysis of rat A10 c ells revealed a dose-specific (0.022 to 30 mu mol/L monensin) upregula tion of alpha(1)-, alpha(2)-, and beta(1)-subunit Na,K-ATPase RNA leve ls. Control beta-actin and alpha-tropomyosin RNA levels did not change . With the use of chloramphenicol acetyltransferase (CAT) as reporter gene, CAT assays of rat alpha(1)[-1288]CAT and human alpha(2)[-798]CAT promoter constructs exhibited induction of CAT activity in monensin ( 10 mu mol/L)-treated A10 cells compared with untreated A10 cells. Prom oter deletion constructs for rat alpha(1)[-1288]CAT defined a positive Na+-response regulatory region within -358 to -169 that is distinct f rom the basal transcriptional activation region of -155 to -49 previou sly defined. Similarly, a positive Na+-response regulatory region is d elimited to within -301 in the human alpha(2) Na,K-ATPase 5' flanking region. Analysis of transgenic TgH alpha(2)[-798]CAT rats demonstrated sodium activation of human alpha(2)[-798]CAT transgene expression in aorta parallel to observations made in rat A10 aortic tissue culture c ells. Southwestern blot analysis of nuclear extracts from monensin (10 mu mol/L)-treated and control untreated A10 cells revealed a nuclear DNA binding protein (approximately 95 kD) that is upregulated by incre ased [Na+](i). These data provide initial characterization of a transc riptional Na+-response mechanism delimiting a positive Na+-response re gulatory region in two target genes (alpha(1) and alpha(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 transge nic promoter expression studies, thus validating the biological releva nce of the observations.