The Na,K-ATPase contains a binding site for cardiac glycosides, such as ouabain, digoxin, and digitoxin, which is highly conserved among species ranging from Drosophila to humans. Although advantage has been taken of this site to treat congestive heart failure with drugs such as digoxin, it is unknown whether this site has a natural function in vivo. Here we show that this site plays an important role in the regulation of blood pressure, and it specifically mediates adrenocorticotropic hormone (ACTH)-induced hypertension in mice. We used genetically engineered mice in which the Na,K-ATPase ␣2 isoform, which is normally sensitive to cardiac glycosides, was made resistant to these compounds. Chronic administration of ACTH caused hypertension in WT mice but not in mice with an ouabain-resistant ␣2 isoform of Na,K-ATPase. This finding demonstrates that the cardiac glycoside binding site of the Na,K-ATPase plays an important role in blood pressure regulation, most likely by responding to a naturally occurring ligand. Because the ␣1 isoform is sensitive to cardiac glycosides in humans, we developed mice in which the naturally occurring ouabain-resistant ␣1 isoform was made ouabain-sensitive. Mice with the ouabainsensitive ''human-like'' ␣1 isoform and an ouabain-resistant ␣2 isoform developed ACTH-induced hypertension to greater extent than WT animals. This result indicates that the cardiac glycoside binding site of the ␣1 isoform can also mediate ACTH-induced hypertension. Taken together these results demonstrate that the cardiac glycoside binding site of the ␣ isoforms of the Na,K-ATPase have a physiological function and supports the hypothesis for a role of the endogenous cardiac glycosides. adrenocorticotropic hormone ͉ hypertension ͉ endogenous cardiac glycosides

An interesting feature of Na+-K+-ATPase is that it contains four isoforms of the catalytic alpha-subunit, each with a tissue-specific distribution. Our laboratory has used gene targeting to define the functional role of the alpha1- and alpha2-isoforms. While knockout mice demonstrated the importance of the alpha1- and alpha2-isoforms for survival, the knockin mice, in which each isoform can be individually inhibited by ouabain and its function determined, demonstrated that both isoforms are regulators of cardiac muscle contractility. Another intriguing aspect of the Na+-K+-ATPase is that it contains a binding site for cardiac glycosides, such as digoxin. Conservation of this site suggests that it may have an in vivo role and that a natural ligand must exist to interact with this site. In fact, cardiac glycoside-like compounds have been observed in mammals. Our recent study demonstrates that the cardiac glycoside binding site of the Na+-K+-ATPase plays a role in the regulation of blood pressure and that it mediates both ouabain-induced and ACTH-induced hypertension in mice. Whereas chronic administration of ouabain or ACTH caused hypertension in wild-type mice, it had no effect on blood pressure in mice with a ouabain-resistant alpha2-isoform of Na+-K+-ATPase. Interestingly, animals with the ouabain-sensitive alpha1-isoform and a ouabain-resistant alpha2-isoform develop ACTH-induced hypertension to a greater extent than wild-type animals. Taken together, these results demonstrate that the cardiac glycoside binding of the Na+-K+-ATPase has a physiological role and suggests a function for a naturally occurring ligand that is stimulated by administration of ACTH.

-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.

Idiopathic pulmonary arterial hypertension (IPAH) is associated with lower levels of the pulmonary vasodilator nitric oxide (NO) and its biochemical reaction products (nitrite , nitrate ), in part, due to the reduction in pulmonary endothelial NO synthesis. However, NO levels are also determined by consumptive reactions, such as with superoxide to form peroxynitrite, which subsequently may generate stable products of nitrotyrosine (Tyr-NO 2 ) and/or NO 3 -. In this context, superoxide dismutase (SOD) preserves NO in vivo by scavenging superoxide and preventing the consumptive reactions. Here, we hypothesized that reactive oxygen species (ROS) consumption of NO may contribute to the low NO level and development of pulmonary hypertension. To test this, nitrotyrosine and antioxidants glutathione (GSH), glutathione peroxidase (GPx), catalase, and SOD were evaluated in IPAH patients and healthy controls. SOD and GPx activities were decreased in IPAH lungs (all p < 0.05), while catalase and GSH activities were similar among the groups (all p > 0.2). SOD activity was directly related to exhaled NO (eNO) (R 2 = 0.72, p = 0.002), and inversely related to bronchoalveolar lavage (BAL) NO 3 -(R 2 = -0.73, p = 0.04). Pulmonary artery pressure (PAP) could be predicted by a regression model incorporating SOD, GPx, and NO 3 values (R 2 = 0.96, p = 0.01). These findings suggest that SOD and GPx are associated with alterations in NO and PAP in IPAH.

Hou X, Theriault SF, Dostanic-Larson I, Moseley AE, Lingrel JB, Wu H, Dean S, Van Huysse JW. Enhanced pressor response to increased CSF sodium concentration and to central ANG I in heterozygous ␣ 2 Na ϩ -K ϩ -ATPase knockout mice.

Background The cardiotonic steroid/ouabain-binding site of the α subunit of Na,K-ATPase is thought to play an important role in cardiovascular homeostasis. Previously, we demonstrated the cardiotonic steroid–binding site of the α2 Na,K-ATPase is involved in adrenocorticotropic hormone (ACTH)–induced hypertension by using gene-modified α2R/R mice in which the cardiotonic steroid–binding site is relatively resistant to ouabain compared to the ouabain-sensitive wild-type α2S/S mice. To further explore the importance of this site in the cardiovascular system, we investigated blood pressure regulation during pregnancy in mice with the α2R/R isoform. Methods The systolic blood pressure (SBP) of the α2S/S and α2R/R mice was measured before and during pregnancy by tail-cuff. The expression of the α isoforms of Na, K-ATPase in various tissues and plasma endogenous ouabain contents were assessed prior to pregnancy as well as days 7 and 17 of gestation. Results The α2S/S mice showed a gradual decrease in the SBP during the first two trimesters, followed by an increase above the preconceptional level in the third trimester. However, the α2R/R mice exhibited a lower blood pressure in the third trimester. The cardiac expression of the α2 Na,K-ATPase in the α2S/S mice was significantly less than that of the α2R/R mice throughout the pregnancy. The plasma endogenous ouabain concentration significantly increased by twofold at day 17 of pregnancy in the α2R/R mice but not in the α2S/S mice. Conclusions The cardiotonic steroid–binding site of the α2 Na,K-ATPase plays a role in maintaining normal SBP during pregnancy.

Initiation of tubuloglomerular feedback (TGF) depends on Na-K-2Cl co-transport in the macula densa (MD), but it is less clear whether Na,K-ATPase is responsible for establishing the inward Na ؉ gradient. It has been proposed that apical colonic H,K-ATPase, perhaps in concert with the Na/H exchanger (NHE2), may account for MD Na ؉ exit in these cells. This study evaluated TGF responses by micropuncture in mutant mice with altered ouabain sensitivity of the ␣1 and ␣2 Na,K-ATPase isoforms. TGF responses in ␣1-sensitive/␣2-resistant mice were inhibited by intravenous ouabain (control stop-flow pressure ‫؍‬ 9.7 ؎ 0.9 versus 1.6 ؎ 0.5 mmHg with intravenous ouabain). Subsequent inclusion of cyclohexyladenosine (10 M) in the tubule perfusate confirmed the ability of the afferent arteriole to contract in the presence of ouabain. In ␣1-resistant/␣2-resistant mice, ouabain infusion had no effect on TGF responses. In separate experiments, loop of Henle perfusion with 50 M ouabain decreased TGF responses (control stop-flow pressure) from 10.5 ؎ 1.1 to 3.9 ؎ 1.0 mmHg in ␣1-sensitive/␣2-resistant mice but had no effect in ␣1-resistant/␣2-resistant mice, and afferent arteriole responsiveness again was confirmed by cyclohexyladenosine. TGF responses in NHE2 and colonic H,K-ATPase knockout mice were not different from those of wild-type mice. These data indicate that TGF requires activity of the ␣1 Na,K-ATPase, presumably in the MD. Furthermore, the data show that neither NHE2 nor colonic H,K-ATPase is essential for initiation of TGF responses.

cannulation of the carotid artery can be performed on the right or left side in accordance with vascular pathology. 1 If the proper flow in the carotid artery cannot be restored by arch repair, which can occur occasionally when the repair of a dissected innominate artery bifurcation is not possible, an aorta-carotid bypass may be necessary and easily performed with a carotid side-graft used for cannulation. References1. Urbanski PP. Carotid artery cannulation in acute aortic dissection with malperfusion.