Nelson Ruiz-Opazo

Characterization of a Sodium-Response Transcriptional Mechanism

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 alpha-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 micromol/L monensin) upregulation of alpha1-, alpha2-, and beta1-subunit Na,K-ATPase RNA levels. 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 alpha1[-1288]CAT and human alpha2[-798]CAT promoter constructs exhibited induction of CAT activity in monensin (10 micromol/L)-treated A10 cells compared with untreated A10 cells. Promoter deletion constructs for rat alpha1[-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 alpha2 Na,K-ATPase 5 flanking region. Analysis of transgenic TgH alpha2[-798]CAT rats demonstrated sodium activation of human alpha2[-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 micromol/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 (alpha1 and alpha2 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.

Renal immunocytochemical distribution and pharmacological properties of the dual angiotensin II/AVP receptor

We have recently characterized a novel angiotensin II/vasopressin (Ang II/AVP) dual receptor coupled to adenylate cyclase and responding with equal sensitivity to Ang II and AVP. To gain insight into putative renal physiological roles of the dual Ang II/AVP receptor, we determined its pharmacological binding properties and renal immunocytochemical distribution. The effective displacement of [3H]AVP by [1-deamino-Val14,D-Arg8]-vasopressin (DVDAVP), a specific antidiuretic AVP analogue, supports a V2-type AVP receptor characteristic of the Ang II/AVP receptor. Displacement of 125I-Ang II by losartan but not by PD 123319 defines the Ang II/AVP receptor as a novel AT1 receptor isoform coupled to adenylate cyclase, in contrast to prototype Ca(2+)-mobilizing AT1 receptors. Neither Ang II nor AVP displace each other, corroborating the predicted discrete binding domains for Ang II and AVP but presenting an enigma for the dissection of putative Ang II- and AVP-specific hierarchical roles of the dual Ang II/AVP receptor. The renal cytolocalization of the Ang II/AVP receptor to the outer medullary thick ascending limb tubules and inner medullary collecting ducts is consistent with the well-established AVP stimulation of sodium and water reabsorption in these tubules. These data suggest that the Ang II/AVP receptor might provide the molecular basis for the observed similar stimulatory effects of Ang II and AVP on renal tubular sodium and fluid reabsorption at physiological hormone concentrations.

Pressure-Overload Deinduction of Human α2 Na,K-ATPase Gene Expression in Transgenic Rats

The early and sustained deinduction of α 2 Na,K-ATPase gene expression in both cardiac left ventricle and aorta in various pressure-overload rat models and in hypertrophied human heart suggests a common transcriptional pressure response mechanism to pressure overload in both rats and humans. To test this hypothesis, we developed transgenic rat lines expressing the chloramphenicol acetyltransferase reporter gene regulated by the human α 2 Na,K-ATPase (−798 to +67) regulatory region, Hα 2 -CAT. Analysis of two homozygous transgenic rat lines revealed (1) parallel tissue-specific regulation of the Hα 2 -CAT transgene and rat α 2 Na,K-ATPase gene and (2) parallel load-induced deinduction of both cardiac and vascular (aortic) Hα 2 -CAT transgene and rat α 2 Na,K-ATPase gene expression in a 3-day model of induced pressure overload. Cardiac Hα 2 -CAT deinduction was detected at a systolic pressure greater than or equal to 150 mm Hg and correlated with the degree of systolic pressure elevation ( r =.82, P 2 Na,K-ATPase gene in both humans and rats.