-ACTH-inducedhypertension is commonly employed as a model of stress-related hypertension, and despite extensive investigation, the mechanisms underlying elevated blood pressure (BP) are not well understood. We have reported that ACTH treatment increases tail-cuff systolic pressure in wild-type mice but not in mutant mice expressing ouabainresistant ␣ 2-Na ϩ -K ϩ -ATPase subunits (␣2 R/R mice). Since tail-cuff measurements involve restraint stress, the present study used telemetry to distinguish between an effect of ACTH on resting BP vs. an ACTH-enhanced stress response. We also sought to explore the mechanisms underlying ACTH-induced BP changes in mutant ␣2 R/R mice vs. wild-type mice (ouabain-sensitive ␣ 2 -NaS/S mice). Baseline BP was not different between the two genotypes, but after 5 days of ACTH treatment, BP increased in ␣2 S/S (104.0 Ϯ 2.6 to 117.7 Ϯ 3.0 mmHg) but not in ␣2 R/R mice (108.2 Ϯ 3.2 to 111.5 Ϯ 4.0 mmHg). To test the hypothesis that ACTH hypertension is related to inhibition of ␣2-Na ϩ -K ϩ -ATPase on vascular smooth muscle by endogenous cardiotonic steroids, we measured BP and regional blood flow. Results suggest a differential sensitivity of renal, mesenteric, and cerebral circulations to ACTH and that the response depends on the ouabain sensitivity of the ␣ 2-Na ϩ -K ϩ -ATPase. Baseline cardiac performance was elevated in ␣2 S/S but not ␣2 R/R mice. Overall, the data establish that the ␣2-Na ϩ -K ϩ -ATPase ouabain-binding site is of central importance in the development of ACTH-induced hypertension. The mechanism appears to be related to alterations in cardiac performance, and perhaps vascular tone in specific circulations, presumably caused by elevated levels of circulating cardiotonic steroids.

Na,K-ATPase is ubiquitously expressed and is essential for maintaining electrochemical and osmotic gradients. The alpha subunit of Na,K-ATPase is the receptor for cardiotonic steroids, which act through the ouabain-binding site and are important in cardiovascular regulation. Interestingly, the presence of endogenous Na,K-ATPase ligands has been implicated in the natriuretic response to perturbations such as hypertension and salt loading; therefore, it is important to characterize the role of the ouabain-binding sites in this context. Because the alpha1 isoform of mice and rats is relatively ouabain resistant, gene-targeting strategies were used to produce mice with reversed responses of the alpha1 and/or alpha2 isoforms to ouabain to assess for altered natriuretic responses to acute salt loading. Regardless of the sensitivity of the alpha2 isoform to ouabain, conferring ouabain sensitivity to alpha1 augmented the natriuretic response to an acute salt load. In addition, when endogenous Na,K-ATPase inhibitors were sequestered with an anti-digoxin antibody fragment, the sodium excretion rates in the ouabain-sensitive alpha1 isoform mice were equivalent to the ouabain-resistant alpha1 isoform mice. These data suggest that the ouabain-binding site of the alpha1 Na,K-ATPase can participate in the natriuretic response to a salt load by responding to endogenous Na,K-ATPase ligands.

Recent studies have ascribed many non-pumping functions to the Na/K-ATPase. We show here that graded knockdown of cellular Na/K-ATPase ␣1 subunit produces a parallel decrease in both caveolin-1 and cholesterol in light fractions of LLC-PK1 cell lysates. This observation is further substantiated by imaging analyses, showing redistribution of cholesterol from the plasma membrane to intracellular compartments in the knockdown cells. Moreover, this regulation is confirmed in ␣1 ؉/؊ mouse liver. Functionally, the knockdown-induced redistribution appears to affect the cholesterol sensing in the endoplasmic reticulum, because it activates the sterol regulatory elementbinding protein pathway and increases expression of hydroxymethylglutaryl-CoA reductase and low density lipoprotein receptor in the liver. Consistently, we detect a modest increase in hepatic cholesterol as well as a reduction in the plasma cholesterol. Mechanistically, ␣1 ؉/؊ livers show increases in cellular Src and ERK activity and redistribution of caveolin-1. Although activation of Src is not required in Na/K-ATPase-mediated regulation of cholesterol distribution, the interaction between the Na/K-ATPase and caveolin-1 is important for this regulation. Taken together, our new findings demonstrate a novel function of the Na/K-ATPase in control of the plasma membrane cholesterol distribution. Moreover, the data also suggest that the plasma membrane Na/K-ATPase-caveolin-1 interaction may represent an important sensing mechanism by which the cells regulate the sterol regulatory element-binding protein pathway.

BACKGROUND We have shown that the ouabain-sensitive α2 Na,K-ATPase is required for adrenocorticotropic hormone (ACTH)-induced hypertension and gestational blood pressure regulation. It is therefore of interest to explore whether this binding site participates in the development of other forms of hypertension, such as deoxycorticosterone acetate (DOCA)-salt using mutant mice with altered sensitivity to ouabain. METHODS Wild-type (α1 ouabain-resistant, α2 ouabain-sensitive: αR/Rα2S/S), α1-resistant, α2-resistant (α1R/Rα2R/R) and α1 -sensitive, α2-resistant (α1S/Sα2R/R) mice were uninephrectomized and implanted with DOCA pellets. The animals were given either tap water or 1% NaCI, and blood pressure was measured before and after DOCA. RESULTS DOCA-salt-treated α1R/Rα2R/R mice developed hypertension to the same extent as α1R/Rα2S/S mice (wild type), and the α1S/Sα2R/R mice given DOCA-salt also showed no difference from the other two genotypes. The expression of the α1 isoform was not changed by DOCA-salt treatment in either α1R/Rα2S/S or α1 R/Rα2R/R mice. However, the α2 subunit was expressed at substantially higher levels in the hearts of α1R/Rα2R/R than α1R/Rα2S/S mice, regardless of treatment. Plasma levels of ouabain did not change consistently, but those of marinobufagenin were modestly higher in DOCA-salt treated mice relatively to those without salt. CONCLUSIONS The ouabain-binding site of either the α1 or α2 Na,K-ATPase subunit does not play an essential role in the development of DOCA-salt hypertension in this mouse model. These findings indicate that the underlying mechanisms of hypertension induced by DOCA-salt treatment are different from those of ACTH-induced hypertension.

Na,K‐ATPase plays a critical role in the absorption of water across the lung epithelial layer at birth and during lung injury. Until recently, it was accepted that the alpha 1 isoform was responsible for transporting Na+ out of Alveolar Type (AT) II cells. However, it has been shown that alpha 2, expressed in AT I cells, may in fact be the isoform that transports the majority of Na+ across the epithelium. While alpha 2 comprises only about 5% of the total Na,K‐ATPase expressed in the lung, there is evidence from isolated rat lung experiments suggesting that alpha 2 activity has a greater effect than alpha1. Ouabain‐induced inhibition of alpha 2 activity decreases alveolar fluid clearance by approximately 60%, while overexpression of alpha 2 increases fluid absorption by about 250%. Therefore, we hypothesize that the alpha 2 isoform is critical for alveolar fluid clearance. To investigate this, we have created a mouse model through gene targeting in which the alpha 2 gene is floxed. We will eliminate alpha 2 expression specifically in the lung epithelium by breeding these animals to mice that have doxycycline‐induced Cre recombinase expression driven by the lung‐specific SP‐C promoter. We will first determine if alpha 2 activity is critical for fluid clearance at birth. If not, we will determine if the absence of alpha 2 expression is detrimental during the resolution of lung injury. We will subject the animals to conditions that induce lung edema and look at the ability of the alpha 2 −/− lungs to clear alveolar fluid. We will also determine if beta‐adrenergic receptor agonists are capable of enhancing fluid clearance in the absence of alpha 2. This work will establish the role of the alpha 2 isoform of Na,K‐ATPase in alveolar fluid clearance.