FN3-based monobodies selective for the receptor binding domain of the SARS-CoV-2 spike protein.

A phage library displaying 1010 variants of the fibronectin type III (FN3) domain was affinity selected with the biotinylated form of the receptor binding domain (RBD, residues 319-541) of the SARS-CoV-2 virus spike protein. Nine binding FN3 variants (i.e. monobodies) were recovered, representing four different primary structures. Soluble forms of the monobodies bound to several different preparations of the RBD and the S1 spike subunit, with affinities ranging from 3 to 14 nM as measured by bio-layer interferometry. Three of the four monobodies bound selectively to the RBD of SARS-CoV-2, with the fourth monobody showing slight cross-reactivity to the RBD of SARS-CoV-1 virus. Examination of binding to the spike fragments and its trimeric form revealed that the monobodies recognise at least three overlapping epitopes on the RBD of SARS-CoV-2. While pairwise tests failed to identify a monobody pair that could bind simultaneously to the RBD, one monobody could simultaneously bind to the RBD with the ectodomain of the cellular receptor angiotensin converting enzyme 2 (ACE2). All four monobodies successfully bound the RBD after overexpression in Chinese hamster ovary (CHO) cells as fusions to the Fc domain of human IgG1.

Salt-induced hypertension in a mouse model of Liddle syndrome is mediated by epithelial sodium channels in the brain.

Neural precursor cell expressed and developmentally downregulated 4-2 protein (Nedd4-2) facilitates the endocytosis of epithelial Na channels (ENaCs). Both mice and humans with a loss of regulation of ENaC by Nedd4-2 have salt-induced hypertension. ENaC is also expressed in the brain, where it is critical for hypertension on a high-salt diet in salt-sensitive rats. In the present studies we assessed whether Nedd4-2 knockout (-/-) mice have the following: (1) increased brain ENaC; (2) elevated cerebrospinal fluid (CSF) sodium on a high-salt diet; and (3) enhanced pressor responses to CSF sodium and hypertension on a high-salt diet, both mediated by brain ENaC. Prominent choroid plexus and neuronal ENaC staining was present in -/- but not in wild-type mice. In chronically instrumented mice, ICV infusion of Na-rich artificial CSF increased mean arterial pressure 3-fold higher in -/- than in wild-type mice. ICV infusion of the ENaC blocker benzamil abolished this enhancement. In telemetered -/- mice on a high-salt diet (8% NaCl), CSF [Na(+)], mean arterial pressure, and heart rate increased significantly, mean arterial pressure by 30 to 35 mmHg. These mean arterial pressure and heart rate responses were largely prevented by ICV benzamil but only to a minor extent by SC benzamil at the ICV rate. We conclude that increased ENaC expression in the brain of Nedd4-2 -/- mice mediates their hypertensive response to a high-salt diet by causing increased sodium levels in the CSF, as well as hyperresponsiveness to CSF sodium. These findings highlight the possible causative contribution of central nervous system ENaC in the etiology of salt-induced hypertension.

Low efficiency of functional translation of ouabain-resistant α2-Na(+),K(+)-ATPase mRNA in C7-MDCK epithelial cells.

Na(+),K(+)-ATPase is a heterodimer consisting of catalytic α1-α4 and regulatory ß1-ß3 subunits. Recently, we reported that transfection with ouabain-resistant α1R-Na(+),K(+)-ATPase rescues renal epithelial C7-MDCK cells exclusively expressing the ouabain-sensitive α1S-isoform from the cytotoxic action of ouabain. To explore the role of α2 subunit in ion transport and cytotoxic action of ouabain, we compared the effect of ouabain on K(+) ((86)Rb) influx and the survival of ouabain-treated C7-MDCK cells stably transfected with α1R- and α2R-Na(+),K(+)-ATPase. α2R mRNA in transfected cells was ∼8-fold more abundant than α1R mRNA, whereas immunoreactive α2R protein content was 5-fold lower than endogenous α1S protein. A concentration of 10 µmol/L ouabain led to complete inhibition of (86)Rb influx both in mock- and α2R-transfected cells, whereas maximal inhibition of (86)Rb influx in α1R-transfectd cells was observed at 1000 µmol/L ouabain. In contrast to the massive death of mock- and α2R-transfected cells exposed to 3 µmol/L ouabain , α1R-cells survived after 24 h incubation with 1000 µmol/L ouabain. Thus, our results show that unlike α1R, the presence of α2R-Na(+),K(+)-ATPase subunit mRNA and immunoreactive protein does not contribute to Na(+)/K(+) pump activity, and does not rescue C7-MDCK cells from the cytotoxic action of ouabain. Our results also suggest that the lack of impact of transfected α2-Na(+),K(+)-ATPase on Na(+)/K(+) pump activity and cell survival can be attributed to the low efficiency of its translation and (or) delivery to the plasma membrane of renal epithelial cells.

How NaCl raises blood pressure: a new paradigm for the pathogenesis of salt-dependent hypertension.

Excess dietary salt is a major cause of hypertension. Nevertheless, the specific mechanisms by which salt increases arterial constriction and peripheral vascular resistance, and thereby raises blood pressure (BP), are poorly understood. Here we summarize recent evidence that defines specific molecular links between Na(+) and the elevated vascular resistance that directly produces high BP. In this new paradigm, high dietary salt raises cerebrospinal fluid [Na(+)]. This leads, via the Na(+)-sensing circumventricular organs of the brain, to increased sympathetic nerve activity (SNA), a major trigger of vasoconstriction. Plasma levels of endogenous ouabain (EO), the Na(+) pump ligand, also become elevated. Remarkably, high cerebrospinal fluid [Na(+)]-evoked, locally secreted (hypothalamic) EO participates in a pathway that mediates the sustained increase in SNA. This hypothalamic signaling chain includes aldosterone, epithelial Na(+) channels, EO, ouabain-sensitive α(2) Na(+) pumps, and angiotensin II (ANG II). The EO increases (e.g.) hypothalamic ANG-II type-1 receptor and NADPH oxidase and decreases neuronal nitric oxide synthase protein expression. The aldosterone-epithelial Na(+) channel-EO-α(2) Na(+) pump-ANG-II pathway modulates the activity of brain cardiovascular control centers that regulate the BP set point and induce sustained changes in SNA. In the periphery, the EO secreted by the adrenal cortex directly enhances vasoconstriction via an EO-α(2) Na(+) pump-Na(+)/Ca(2+) exchanger-Ca(2+) signaling pathway. Circulating EO also activates an EO-α(2) Na(+) pump-Src kinase signaling cascade. This increases the expression of the Na(+)/Ca(2+) exchanger-transient receptor potential cation channel Ca(2+) signaling pathway in arterial smooth muscle but decreases the expression of endothelial vasodilator mechanisms. Additionally, EO is a growth factor and may directly participate in the arterial structural remodeling and lumen narrowing that is frequently observed in established hypertension. These several central and peripheral mechanisms are coordinated, in part by EO, to effect and maintain the salt-induced elevation of BP.

Hypertension from chronic central sodium chloride in mice is mediated by the ouabain-binding site on the Na,K-ATPase α2-isoform.

A chronic increase in the concentration of sodium chloride in the cerebrospinal fluid (CSF) (↑CSF [NaCl]) appears to be critically important for the development of salt-dependent hypertension. In agreement with this concept, increasing CSF [NaCl] chronically by intracerebroventricular (icv) infusion of NaCl-rich artificial CSF (aCSF-HiNaCl) in rats produces hypertension by the same mechanisms (i.e., aldosterone-ouabain pathway in the brain) as that produced by dietary sodium in salt-sensitive strains. We first demonstrate here that icv aCSF-HiNaCl for 10 days also causes hypertension in wild-type (WT) mice. We then used both WT and gene-targeted mice to explore the mechanisms. In WT mice with a ouabain-sensitive Na,K-ATPase α(2)-isoform (α2(S/S)), mean arterial pressure rose by ~25 mmHg within 2 days of starting aCSF-HiNaCl (0.6 nmol Na/min) and remained elevated throughout the study. Ouabain (171 pmol/day icv) increased blood pressure to a similar extent. aCSF-HiNaCl or ouabain given at the same rates subcutaneously instead of intracerebroventricularly had no effect on blood pressure. The pressor response to icv aCSF-HiNaCl was abolished by an anti-ouabain antibody given intracerebroventricularly but not subcutaneously, indicating that it is mediated by an endogenous ouabain-like substance in the brain. We compared the effects of icv aCSF-HiNaCl or icv ouabain on blood pressure in α2(S/S) versus knockout/knockin mice with a ouabain-resistant endogenous α(2)-subunit (α2(R/R)). In α2(R/R), there was no pressor response to icv aCSF-HiNaCl in contrast to WT mice. The α2(R/R) genotype also lacked a pressor response to icv ouabain. These data demonstrate that chronic ↑CSF [NaCl] causes hypertension in mice and that the blood pressure response is mediated by the ouabain-like substance in the brain, specifically by its binding to the α(2)-isoform of the Na,K-ATPase.

Cardiotonic steroid-resistant alpha1-Na+,K+-ATPase rescues renal epithelial cells from the cytotoxic action of ouabain: evidence for a Nai+,Ki+ -independent mechanism.

Mechanisms underlying the tissue-specific impact of cardiotonic steroids (CTS) on cell survival and death remain poorly understood. This study examines the role of Na(+),K(+)-ATPase alpha subunits in death of Madin-Darby canine kidney (MDCK) cells evoked by 24-h exposure to ouabain. MDCK cells expressing a variant of the alpha1 isoform, CTS-sensitive alpha1S, were stably transfected with a cDNA encoding CTS-resistant alpha1R-Na(+),K(+)-ATPase, whose expression was confirmed by RT-PCR. In mock-transfected and alpha1R-cells, maximal inhibition of (86)Rb influx was observed at 10 and 1000 muM ouabain, respectively, thus confirming high abundance of alpha1R-Na(+),K(+)-ATPase in these cells. Six-hour treatment of alpha1R-cells with 1000 muM ouabain led to the same elevation of the [Na(+)](i)/[K(+)](i) ratio that was detected in mock-transfected cells treated with 3 muM ouabain. However, in contrast to the massive death of mock-transfected cells exposed to 3 muM ouabain, alpha1R-cells survived after 24-h incubation with 1000 muM ouabain. Inversion of the [Na(+)](i)/[K(+)](i) ratio evoked by Na(+),K(+)-ATPase inhibition in K(+)-free medium did not affect survival of alpha1R-cells but increased their sensitivity to ouabain. Our results show that the alpha1R subunit rescues MDCK cells from the cytotoxic action of CTS independently of inhibition of Na(+),K(+)-ATPase-mediated Na(+) and K(+) fluxes and inversion of the [Na(+)](i)/[K(+)](i) ratio.

Enhanced pressor response to increased CSF sodium concentration and to central ANG I in heterozygous alpha2 Na+ -K+ -ATPase knockout mice.

Intracerebroventricular (ICV) infusion of NaCl mimics the effects of a high-salt diet in salt-sensitive hypertension, raising the sodium concentration in the cerebrospinal fluid (CSF [Na]) and subsequently increasing the concentration of an endogenous ouabain-like substance (OLS) in the brain. The OLS, in turn, inhibits the brain Na(+)-K(+)-ATPase, causing increases in the activity of the brain renin-angiotensin system (RAS) and blood pressure. The Na(+)-K(+)-ATPase alpha (catalytic)-isoform(s) that mediates the pressor response to increased CSF [Na] is unknown, but it is likely that one or more isoforms that bind ouabain with high affinity are involved (e.g., the Na(+)-K(+)-ATPase alpha(2)- and/or alpha(3)-subunits). We hypothesize that OLS-induced inhibition of the alpha(2)-subunit mediates this response. Therefore, a chronic reduction in alpha(2) expression via a heterozygous gene knockout (alpha(2) +/-) should enhance the pressor response to increased CSF [Na]. Intracerebroventricular (ICV) infusion of artificial CSF containing 0.225 M NaCl increased mean arterial pressure (MAP) in both wild-type (+/+) and alpha(2) +/- mice, but to a greater extent in alpha(2) +/-. Likewise, the pressor response to ICV ouabain was enhanced in alpha(2) +/- mice, demonstrating enhanced sensitivity to brain Na(+)-K(+)-ATPase inhibition per se. The pressor response to ICV ANG I but not ANG II was also enhanced in alpha(2) +/- vs. alpha(2)+/+ mice, suggesting an enhanced brain RAS activity that may be mediated by increased brain angiotensin converting enzyme (ACE). The latter hypothesis is supported by enhanced ACE ligand binding in the organum vasculosum laminae terminalis. These studies demonstrate that chronic downregulation of Na(+)-K(+)-ATPase alpha(2)-isoform expression by heterozygous knockout increases the pressor response to increased CSF [Na] and activates the brain RAS. Since these changes mimic those produced by the endogenous brain OLS, the brain alpha(2)-isoform may be a target for the brain OLS during increases in CSF [Na], such as in salt-dependent hypertension.

Endogenous brain Na pumps, brain ouabain-like substance and the alpha2 isoform in salt-dependent hypertension.

An endogenous ouabain-like substance (OLS) plays a critical role in the etiology of experimental models of human hypertension induced by a high salt diet. Early on, evidence for a role of this Na, K-ATPase inhibitor in blood pressure regulation was provided mainly by correlations of blood pressure with the levels of circulating Na, K-ATPase inhibitor. However, over the past decade, numerous studies have shown that endogenous Na pump inhibitors in the brain mediate salt-dependent hypertension in a variety of experimental models, including Dahl salt-sensitive (Dahl-S) and spontaneously hypertensive (SHR) rats on a high-salt diet. Other forms of hypertension that are known to be mediated by endogenous ouabain-like substances include steroid/salt- (e.g., DOCA-salt) and ACTH-induced hypertension. Even when exogenous ouabain is peripherally administered and/or the plasma ouabain/OLS level is increased in rats, the resulting hypertension is of CNS origin. After peripheral ouabain administration, ouabain levels increase in the plasma and the inhibitor subsequently accumulates in the brain. The ensuing hypertension is abolished by the intracerebroventricular (icv) administration of an anti-ouabain antibody (but not by the same antibody dose given iv), by discrete excitotoxic lesions in the brain or by ganglionic blockade, demonstrating that the response is neurally mediated. The pressor response to stimuli that increase the brain OLS (high salt diet, icv sodium) or to icv ouabain is abolished by icv losartan, demonstrating that the brain OLS activates the brain renin-angiotensin system (RAS) downstream. There are three isoforms of the catalytic alpha subunit of the Na, K-ATPase in the brain and cardiovascular system (alpha1, alpha2 and alpha3), but it is not known which brain isoform(s) mediate the hypertensive effects of circulating/CNS ouabain. Preliminary studies in gene-targeted mice suggest that the alpha2 isoform plays a critical role.

Physiological role of the alpha1- and alpha2-isoforms of the Na+-K+-ATPase and biological significance of their cardiac glycoside binding site.

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.

The highly conserved cardiac glycoside binding site of Na,K-ATPase plays a role in blood pressure regulation.

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 alpha2 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 alpha2 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 alpha1 isoform is sensitive to cardiac glycosides in humans, we developed mice in which the naturally occurring ouabain-resistant alpha1 isoform was made ouabain-sensitive. Mice with the ouabain-sensitive "human-like" alpha1 isoform and an ouabain-resistant alpha2 isoform developed ACTH-induced hypertension to greater extent than WT animals. This result indicates that the cardiac glycoside binding site of the alpha1 isoform can also mediate ACTH-induced hypertension. Taken together these results demonstrate that the cardiac glycoside binding site of the alpha isoforms of the Na,K-ATPase have a physiological function and supports the hypothesis for a role of the endogenous cardiac glycosides.

The alpha2-isoform of Na-K-ATPase mediates ouabain-induced hypertension in mice and increased vascular contractility in vitro.

Although ouabain is known to induce hypertension, the mechanism of how this cardiac glycoside affects blood pressure is uncertain. The present study demonstrates that the alpha2-isoform of the Na-K-ATPase mediates the pressor effects of ouabain in mice. To accomplish this, we analyzed the effect of ouabain on blood pressure in wild-type mice, where the alpha2-isoform is sensitive to ouabain, and genetically engineered mice expressing a ouabain-insensitive alpha2-isoform of the Na-K-ATPase. Thus differences in the response to ouabain between these two genotypes can only be attributed to the alpha2-isoform of Na-K-ATPase. As the alpha1-isoform is naturally resistant to ouabain in rodents, it will not be inhibited by ouabain in either genotype. Whereas prolonged administration of ouabain increased levels of ouabain in serum from both wild-type and targeted animals, hypertension developed only in wild-type mice. In addition, bolus intravenous infusion of ouabain increased the systolic, mean arterial, and left ventricular blood pressure in only wild-type anesthetized mice. In vitro, ouabain increased vascular tone and thereby phenylephrine-induced contraction of the aorta in intact and endothelium-denuded wild-type mice but in alpha2-resistant mice. Ouabain also increased the magnitude of the spontaneous contractions of portal vein and the basal tone of the intact aorta from only wild-type mice. The increase in aortic basal tone was dependent on the presence of endothelium. Our studies also demonstrate that the alpha2-isoform of Na-K-ATPase mediates the ouabain-induced increase in vascular contractility. This could play a role in the development and maintenance of ouabain-induced hypertension.

Pressor response to CSF sodium in mice: mediation by a ouabain-like substance and renin-angiotensin system in the brain.

Intracerebroventricular (i.c.v.) infusion of sodium in rats increases cerebrospinal fluid (CSF) [Na], mimicking the effects of a high salt diet in salt-sensitive strains and causing sympathetic hyperactivity and a pressor response that are mediated via both an endogenous brain ouabainlike substance (OLS) and the brain renin-angiotensin system (RAS). However, the concept that CSF sodium activates both the brain OLS and brain RAS to increase blood pressure has not been tested in any other species besides the rat. In the current study, it was established that continuous i.c.v. infusion of NaCl causes sustained increases in blood pressure and heart rate in both outbred (Swiss Webster, SW) and inbred (C57Bl/6) mouse strains. Subsequently, the mechanisms of the pressor effects were explored. In both SW and C57Bl/6, the i.c.v. administration of Fab fragments of an antibody with high affinity for ouabain and the OLS (Fab) abolished the pressor and tachycardic responses to i.c.v. sodium, as did the angiotensin II AT1 receptor antagonist losartan given i.c.v. In contrast, doses of NaCl, Fab and losartan that were effective i.c.v. were ineffective when given i.v. I.c.v. ouabain also caused the pressor and tachycardic responses, which were abolished by losartan (i.c.v.). In the reciprocal study, i.c.v. Fab had no effect on similar responses to i.c.v. angiotensin II. These studies demonstrate that the sustained blood pressure and heart rate responses caused by increases in CSF [Na] are mediated via both a brain OLS and the brain RAS. The RAS activation occurs downstream of the OLS effect.

Brain Na+,K+-ATPase isozyme activity and protein expression in ouabain-induced hypertension.

In normotensive rats, chronic infusion of exogenous ouabain causes hypertension involving central mechanisms. To determine whether ouabain-induced hypertension is associated with specific changes in brain Na+,K+-ATPase activity and expression, we assessed brain Na+,K+-ATPase isozyme activity and protein expression in rats treated with ouabain (50 microg/day s.c. or 10 microg/day i.c.v. for 14 days). Resting mean arterial pressure (MAP) was higher in s.c.- and i.c.v.-ouabain-treated animals vs. control (124+/-2 vs. 105+/-2 and 130+/-2 vs. 109+/-2, respectively, p<0.01). Ouabain infused s.c. or i.c.v. for 14 days had no effect on Na+,K+-ATPase isozyme activity in hypothalamic, pontine/medullary or cortical microsomes. However, the percent increase in total Na+,K+-ATPase activity produced in vitro by antibody Fab fragments that bind ouabain with high affinity (Digibind) was two-fold greater in s.c.- and i.c.v.-ouabain-treated rats vs. control, but only in hypothalamic microsomes. Thus, ouabain infused s.c. or i.c.v. does appear to directly inhibit Na+,K+-ATPase activity in the hypothalamus. On the other hand, in the hypothalamus, s.c.- and i.c.v.-ouabain infusions tended to increase alpha3 (by 30-44%), but had no effect on alpha1 or alpha2 Na+,K+-ATPase isozyme protein expression. In addition, ouabain was found to partially dissociate from the Na+,K+-ATPase enzyme following sample processing. Thus, the inability to detect a decrease in enzyme activity in the hypothalamus in response to ouabain may be due, in part, to an increase in enzyme expression and the dissociation of ouabain during sample processing.

Changes in brain Na, K-ATPase isoform expression and enzymatic activity after aortic constriction.

The objective of the present study was to test the hypothesis that brain Na, K-ATPase expression and/or activity is altered following an increase in blood pressure produced by constriction of the abdominal aorta just proximal to the renal arteries. A suprarenal constriction (SRC) was made to conform to the diameter of a 19-gauge (19-G) or 20-gauge (20-G) needle, while in a sham-operated group (Sham) the aorta was exposed surgically but not constricted. Within 1 week of SRC, mean arterial pressure was increased and remained elevated at 4 weeks post surgery. At 1 week, whole-brain Na, K-ATPase mRNA levels were depressed for all isoforms (alpha1 approximately beta1>alpha2>alpha3). No changes were observed in the hypothalamus. At 4 weeks, the mRNA levels of all alpha isoforms were significantly increased in the whole brain and these changes were paralleled by an increase of alpha2 and alpha3 transcript in the hypothalamus. beta1 mRNA expression was increased in the hypothalamus only. The alpha-isoform protein expression generally changed in the same direction as mRNA changes at both 1 and 4 weeks, as did alpha1 enzyme activity at 1 week and the combined alpha2/alpha3 enzyme activities at 4 weeks. Since inhibition of brain Na, K-ATPase increases sympathetic nervous system (SNS) activity and blood pressure, the decreases in brain Na, K-ATPase expression and activity at 1 week post SRC may contribute to the hypertension during its developmental phase, while the increase in the alpha2/alpha3 brain expression and activity at 4 weeks may be a compensatory response to established hypertension.