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.     

How does salt retention raise blood pressure?

A critical question in hypertension research is: How is long-term blood pressure controlled? Excessive NaCl ingestion or NaCl retention by the kidneys and the consequent tendency toward plasma volume expansion lead to hypertension. Nevertheless, the precise mechanisms linking salt to high blood pressure are unresolved. The discovery of endogenous ouabain, an adrenocortical hormone, provided an important clue. Ouabain, a selective Na+ pump inhibitor, has cardiotonic and vasotonic effects. Plasma endogenous ouabain levels are significantly elevated in approximately 40% of patients with essential hypertension and in animals with several forms of salt-dependent hypertension. Also, prolonged ouabain administration induces hypertension in rodents. Mice with mutant Na+ pumps or Na/Ca exchangers (NCX) and studies with a ouabain antagonist and an NCX blocker are revealing the missing molecular mechanisms. These data demonstrate that alpha2 Na+ pumps and NCX1 participate in long-term regulation of vascular tone and blood pressure. Pharmacological agents or mutations in the alpha2 Na+ pump that interfere with the action of ouabain on the pump, and reduced NCX1 expression or agents that block NCX all impede the development of salt-dependent or ouabain-induced hypertension. Conversely, nanomolar ouabain, reduced alpha2 Na+ pump expression, and smooth muscle-specific overexpression of NCX1 all induce hypertension. Furthermore, ouabain and reduced alpha2 Na+ pump expression increase myogenic tone in isolated mesenteric small arteries in vitro, thereby tying these effects directly to the elevation of blood pressure. Thus, endogenous ouabain, and vascular alpha2 Na+ pumps and NCX1, are critical links between salt and hypertension. New pharmacological agents that act on these molecular links have potential in the clinical management of hypertension.     

The alpha(1)- and alpha(2)-isoforms of Na-K-ATPase play different roles in skeletal muscle contractility

The Na-K-ATPase, which maintains the Na(+) and K(+) gradients across the plasma membrane, can play a major role in modulation of skeletal muscle contractility. Although both alpha(1)- and alpha(2)-isoforms of the Na-K-ATPase are expressed in skeletal muscle, the physiological significance of these isoforms in contractility is not known. Evaluation of the contractile parameters of mouse extensor digitorum longus (EDL) was carried out using gene-targeted mice lacking one copy of either the alpha(1)- or alpha(2)-isoform gene of the Na-K-ATPase. The EDL muscles from heterozygous mice contain approximately one-half of the alpha(1)- or alpha(2)-isoform, respectively, which permits differentiation of the functional roles of these isoforms. EDL from the alpha(1)(+/-) mouse shows lower force compared with wild type, whereas that from the alpha(2)(+/-) mouse shows greater force. The different functional roles of these two isoforms are further demonstrated because inhibition of the alpha(2)-isoform with ouabain increases contractility of alpha(1)(+/-) EDL. These results demonstrate that the Na-K-ATPase alpha(1)- and alpha(2)-isoforms may play different roles in skeletal muscle contraction.     

Transgenic mice expressing Na+-K+-ATPase in smooth muscle decreases blood pressure

The Na(+)-K(+)-ATPase (NKA) is a transmembrane protein that sets and maintains the electrochemical gradient by extruding three Na(+) in exchange for two K(+). An important physiological role proposed for vascular smooth muscle NKA is the regulation of blood pressure via modulation of vascular smooth muscle contractility (5). To investigate the relations between the level of NKA in smooth muscle and blood pressure, we developed mice carrying a transgene for either the NKA alpha(1)- or alpha(2)-isoform (alpha(1 sm+) or alpha(2 sm+) mice) driven by the smooth muscle-specific alpha-actin promoter SMP8. Interestingly, both alpha-isoforms, the one contained in the transgene and the one not contained, were increased to a similar degree at both protein and mRNA levels. The total alpha-isoform protein was increased from 1.5-fold (alpha(1 sm+) mice) to 7-fold (alpha(2 sm+) mice). The increase in total NKA alpha-isoform protein was accompanied by a 2.5-fold increase in NKA activity in alpha(2 sm+) gastric antrum. Immunocytochemistry of the alpha(1)- and alpha(2)-isoforms in alpha(2 sm+) aortic smooth muscle cells indicated that alpha-isoform distributions were similar to those shown in wild-type cells. alpha(2 sm+) Mice (high expression) were hypotensive (109.9 +/- 1.6 vs. 121.3 +/- 1.4 mmHg; n = 13 and 11, respectively), whereas alpha(1 sm+) mice (low expression) were normotensive (122.7 +/- 2.5 vs. 117.4 +/- 2.3; n = 11 or 12). alpha(2 sm+) Aorta, but not alpha(1 sm+) aorta, relaxed faster from a KCl-induced contraction than wild-type aorta. Our results show that smooth muscle displays unique coordinate expression of the alpha-isoforms. Increasing smooth muscle NKA decreases blood pressure and is dependent on the degree of increased alpha-isoform expression.     

Phenotypical features of long Q-T syndrome in transgenic mice expressing human Na-K-ATPase alpha(3)-isoform in hearts

To understand why the adult human heart expresses three isoforms of the sodium pump, we generated transgenic mice (TGM) with 2.3- to 5. 5-fold overexpression of the human alpha(3)-isoform of Na-K-ATPase in the heart. Hearts from the TGM had increased maximal Na-K-ATPase activity and ouabain affinity compared with control hearts, even though the density of Na-K-ATPase pump sites (of all isoforms) was similar to that of control mice. In perfused hearts, contractility both at baseline and in the presence of ouabain tended to be greater in TGM than in controls. Surface electrocardiograms in anesthetized TGM had a steeper dependence of Q-T on sinus cycle length, and Q-T intervals measured during atrial pacing were significantly longer in TGM. Q-T dispersion during sinus rhythm also tended to be longer in TGM. Thus TGM overexpressing human alpha(3)-isoform have several of the phenotypical features of human long Q-T syndrome, despite the absence of previously described mutations in Na(+) or K(+) channels.     

The alpha2 isoform of Na,K-ATPase mediates ouabain-induced cardiac inotropy in mice

Inhibition of Na,K-ATPase activity by cardiac glycosides is believed to be the major mechanism by which this class of drugs increases heart contractility. However, direct evidence demonstrating this is lacking. Furthermore it is unknown which specific alpha isoform of Na,K-ATPase is responsible for the effect of cardiac glycosides. Several studies also suggest that cardiac glycosides, such as ouabain, function by mechanisms other than inhibition of the Na,K-ATPase. To determine whether Na,K-ATPase, specifically the alpha2 Na,K-ATPase isozyme, mediates ouabain-induced cardiac inotropy, we developed animals expressing a ouabain-insensitive alpha2 isoform of the Na,K-ATPase using Cre-Lox technology and analyzed cardiac contractility after administration of ouabain. The homozygous knock-in animals were born in normal Mendelian ratio and developed normally to adulthood. Analysis of their cardiovascular function demonstrated normal heart function. Cardiac contractility analysis in isolated hearts and in intact animals demonstrated that ouabain-induced cardiac inotropy occurred in hearts from wild type but not from the targeted animals. These results clearly demonstrate that the Na,K-ATPase and specifically the alpha2 Na,K-ATPase isozyme mediates ouabain-induced cardiac contractility in mice.     

ACTH-induced hypertension is dependent on the ouabain-binding site of the alpha2-Na+-K+-ATPase subunit

ACTH-induced-hypertension 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 ouabain-resistant alpha(2)-Na(+)-K(+)-ATPase subunits (alpha2(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 alpha2(R/R) mice vs. wild-type mice (ouabain-sensitive alpha(2)-Na(+)-K(+)-ATPase, alpha2(S/S) mice). Baseline BP was not different between the two genotypes, but after 5 days of ACTH treatment, BP increased in alpha2(S/S) (104.0 +/- 2.6 to 117.7 +/- 3.0 mmHg) but not in alpha2(R/R) mice (108.2 +/- 3.2 to 111.5 +/- 4.0 mmHg). To test the hypothesis that ACTH hypertension is related to inhibition of alpha(2)-Na(+)-K(+)-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 alpha(2)-Na(+)-K(+)-ATPase. Baseline cardiac performance was elevated in alpha2(S/S) but not alpha2(R/R) mice. Overall, the data establish that the alpha(2)-Na(+)-K(+)-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.     

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.     

Knockout of the Na,K-ATPase α₂-isoform in the cardiovascular system does not alter basal blood pressure but prevents ACTH-induced hypertension

The α(2)-isoform of Na,K-ATPase (α(2)) is thought to play a role in blood pressure regulation, but the specific cell type(s) involved have not been identified. Therefore, it is important to study the role of the α(2) in individual cell types in the cardiovascular system. The present study demonstrates the role of vascular smooth muscle α(2) in the regulation of cardiovascular hemodynamics. To accomplish this, we developed a mouse model utilizing the Cre/LoxP system to generate a cell type-specific knockout of the α(2) in vascular smooth muscle cells using the SM22α Cre. We achieved a 90% reduction in the α(2)-expression in heart and vascular smooth muscle in the knockout mice. Interestingly, tail-cuff blood pressure analysis reveals that basal systolic blood pressure is unaffected by the knockout of α(2) in the knockout mice. However, knockout mice do fail to develop ACTH-induced hypertension, as seen in wild-type mice, following 5 days of treatment with ACTH (Cortrosyn; wild type = 119.0 ± 6.8 mmHg; knockout = 103.0 ± 2.0 mmHg). These results demonstrate that α(2)-expression in heart and vascular smooth muscle is not essential for regulation of basal systolic blood pressure, but α(2) is critical for blood pressure regulation under chronic stress such as ACTH-induced hypertension.     

Hypertrophy, increased ejection fraction, and reduced Na-K-ATPase activity in phospholemman-deficient mice

Phospholemman (FXYD1), a 72-amino acid transmembrane protein abundantly expressed in the heart and skeletal muscle, is a major substrate for phosphorylation in the cardiomyocyte sarcolemma. Biochemical, cellular, and electrophysiological studies have suggested a number of possible roles for this protein, including ion channel modulator, taurine-release channel, Na(+)/Ca(2+) exchanger modulator, and Na-K-ATPase-associated subunit. We have generated a phospholemman-deficient mouse. The adult null mice exhibited increased cardiac mass, larger cardiomyocytes, and ejection fractions that were 9% higher by magnetic resonance imaging compared with wild-type animals. Notably, this occurred in the absence of hypertension. Total Na-K-ATPase activity was 50% lower in the phospholemman-deficient hearts. Expression (per unit of membrane protein) of total Na-K-ATPase was only slightly diminished, but expression of the minor alpha(2)-isoform, which has been specifically implicated in the control of contractility, was reduced by 60%. The absence of phospholemman thus results in a complex response, including a surprisingly large reduction in intrinsic Na-K-ATPase activity, changes in Na-K-ATPase isoform expression, increase in ejection fraction, and increase in cardiac mass. We hypothesize that a primary effect of phospholemman is to modulate the Na-K-ATPase and that its reduced activity initiates compensatory responses.     

Reduced Na-K pump but increased Na-K-2Cl cotransporter in aorta of streptozotocin-induced diabetic rat

The activities of Na-K-ATPase and Na-K-2Cl cotransporter (NKCC1) were studied in the aorta, heart, and skeletal muscle of streptozotocin (STZ)-induced diabetic rats and control rats. In the aortic rings of STZ rats, the Na-K-ATPase-dependent (86)Rb/K uptake was reduced to 60.0 +/- 5.5% of the control value (P < 0.01). However, Na-K-ATPase activity in soleus skeletal muscle fibers of STZ rats and paired control rats was similar, showing that the reduction of Na-K-ATPase activity in aortas of STZ rats is tissue specific. To functionally distinguish the contributions of ouabain-resistant (alpha(1)) and ouabain-sensitive (alpha(2) and alpha(3)) isoforms to the Na-K-ATPase activity in aortic rings, we used either a high (10(-3) M) or a low (10(-5) M) ouabain concentration during (86)Rb/K uptake. We found that the reduction in total Na-K-ATPase activity resulted from a dramatic decrement in ouabain-sensitive mediated (86)Rb/K uptake (26.0 +/- 3.9% of control, P < 0.01). Western blot analysis of membrane fractions from aortas of STZ rats demonstrated a significant reduction in protein levels of alpha(1)- and alpha(2)-catalytic isoforms (alpha(1) = 71.3 +/- 9.8% of control values, P < 0.05; alpha(2) = 44.5 +/- 11.3% of control, P < 0.01). In contrast, aortic rings from the STZ rats demonstrated an increase in NKCC1 activity (172.5 +/- 9.5%, P < 0.01); however, in heart tissue no difference in NKCC1 activity was seen between control and diabetic animals. Transport studies of endothelium-denuded or intact aortic rings demonstrated that the endothelium stimulates both Na-K-ATPase and Na-K-2Cl dependent (86)Rb/K uptake. The endothelium-dependent stimulation of Na-K-ATPase and Na-K-2Cl was not hampered by diabetes. We conclude that abnormal vascular vessel tone and function, reported in STZ-induced diabetic rats, may be related to ion transport abnormalities caused by changes in Na-K-ATPase and Na-K-2Cl activities.     

Loss of alpha2B-adrenoceptors increases magnitude of hypertension following nitric oxide synthase inhibition

Vascular alpha(2B)-adrenoceptors (alpha(2B)-AR) may mediate vasoconstriction and contribute to the development of hypertension. Therefore, we hypothesized that blood pressure would not increase as much in mice with mutated alpha(2B)-AR as in wild-type (WT) mice following nitric oxide (NO) synthase (NOS) inhibition with N(omega)-nitro-l-arginine (l-NNA, 250 mg/l in drinking water). Mean arterial pressure (MAP) was recorded in heterozygous (HET) alpha(2B)-AR knockout mice and WT littermates using telemetry devices for 7 control and 14 l-NNA treatment days. MAP in HET mice was increased significantly on treatment days 1 and 4 to 14, whereas MAP did not change in WT mice (days 0 and 14 = 113 +/- 3 and 114 +/- 4 mmHg in WT, 108 +/- 0.3 and 135 +/- 13 mmHg in HET, P < 0.05). MAP was significantly higher in HET than in WT mice days 10 through 14 (P < 0.05). Thus blood pressure increased more rather than less in mice with decreased alpha(2B)-AR expression. We therefore examined constrictor responses to phenylephrine (PE, 10(-9) to 10(-4) M) with and without NOS inhibition to determine basal NO contributions to arterial tone. In small pressurized mesenteric arteries (inner diameter = 177 +/- 5 microm), PE constriction was decreased in untreated HET arteries compared with WT (P < 0.05). l-NNA (100 microM) augmented PE constriction more in HET arteries than in WT arteries, and responses were not different between groups in the presence of l-NNA. Acetylcholine dilated preconstricted arteries from HET mice more than arteries from WT mice. Endothelial NOS expression was increased in HET compared with WT mesenteric arteries by Western analysis. Griess assay showed increased NO(x) concentrations in HET plasma compared with those in WT plasma. These data demonstrate that diminished alpha(2B)-AR expression increases the dependence of arterial pressure and vascular tone on NO production and that vascular alpha(2B)-AR either directly or indirectly regulates vascular endothelial NOS function.     

Clearance of store-released Ca2+ by the Na+-Ca2+ exchanger is diminished in aortic smooth muscle from Na+-K+-ATPase alpha 2-isoform gene-ablated mice

Two alpha-isoforms of the Na+-K+-ATPase are expressed in vascular smooth muscle cells (VSMCs). The alpha 1-isoform is proposed to serve a cytosolic housekeeping role, whereas the alpha 2-isoform modulates Ca2+ storage via coupling to the Na+-Ca2+ exchanger (NCX) in a subsarcolemmal compartment. To evaluate the ramifications of this proposed interaction, Ca2+-store load and the contributions of the primary Ca2+ transporters to Ca2+ clearance were studied in aortic VSMCs from embryonic wild-type (WT) and Na+-K+-ATPase alpha 2-isoform gene-ablated, homozygous null knockout (alpha 2-KO) mice. Ca2+ stores were unloaded by inhibiting the sarco(endo)plasmic reticulum Ca2+-ATPase with cyclopiazonic acid (CPA) in Ca2+-free media to limit Ca2+ influx. Ca2+ clearance by the plasma membrane Ca2+-ATPase (PMCA), NCX, or mitochondria was selectively inhibited. In WT VSMCs, NCX accounted for 90% of the Ca2+ efflux. In alpha 2-KO VSMCs, preferential clearance of store-released Ca2+ by NCX was lost, whereas PMCA activity was increased. Selective inhibition of the alpha 2-isoform (0.5 microM ouabain for 20 min), before treatment with CPA enhanced the store load in VSMCs from WT, but not alpha 2-KO mice. A subsequent analysis of capacitative Ca2+ entry (CCE) indicated that the magnitude of Ca2+ influx was significantly greater in alpha 2-KO cells. Our findings support the concept of a subsarcolemmal space where the alpha 2-isoform coupled with NCX modulates Ca2+-store function and, thereby, CCE.     

Transgenic mice over-expressing ET-1 in the endothelial cells develop systemic hypertension with altered vascular reactivity

Endothelin-1 (ET-1) is a potent vasoconstrictor involved in the regulation of vascular tone and implicated in hypertension. However, the role of small blood vessels endothelial ET-1 in hypertension remains unclear. The present study investigated the effect of chronic over-expression of endothelial ET-1 on arterial blood pressure and vascular reactivity using transgenic mice approach. Transgenic mice (TET-1) with endothelial ET-1 over-expression showed increased in ET-1 level in the endothelial cells of small pulmonary blood vessels. Although TET-1 mice appeared normal, they developed mild hypertension which was normalized by the ET(A) receptor (BQ123) but not by ET(B) receptor (BQ788) antagonist. Tail-cuff measurements showed a significant elevation of systolic and mean blood pressure in conscious TET-1 mice. The mice also exhibited left ventricular hypertrophy and left axis deviation in electrocardiogram, suggesting an increased peripheral resistance. The ionic concentrations in the urine and serum were normal in 8-week old TET-1 mice, indicating that the systemic hypertension was independent of renal function, although, higher serum urea levels suggested the occurrence of kidney dysfunction. The vascular reactivity of the aorta and the mesenteric artery was altered in the TET-1 mice indicating that chronic endothelial ET-1 up-regulation leads to vascular tone imbalance in both conduit and resistance arteries. These findings provide evidence for the role of spatial expression of ET-1 in the endothelium contributing to mild hypertension was mediated by ET(A) receptors. The results also suggest that chronic endothelial ET-1 over-expression affects both cardiac and vascular functions, which, at least in part, causes blood pressure elevation.     

Functional characterization of Src-interacting Na/K-ATPase using RNA interference assay

We have shown that the Na/K-ATPase and Src form a signaling receptor complex. Here we determined how alterations in the amount and properties of the Na/K-ATPase affect basal Src activity and ouabain-induced signal transduction. Several alpha1 subunit knockdown cell lines were generated by transfecting LLC-PK1 cells with a vector expressing alpha1-specific small interference RNA. Although the alpha1 knockdown resulted in significant decreases in Na/K-ATPase activity, it increased the basal Src activity and tyrosine phosphorylation of focal adhesion kinase, a Src effector. Concomitantly it also abolished ouabain-induced activation of Src and ERK1/2. When the knockdown cells were rescued by a rat alpha1, both Na/K-ATPase activity and the basal Src activity were restored. In addition, ouabain was able to stimulate Src and ERK1/2 in the rescued cells at a much higher concentration, consistent with the established differences in ouabain sensitivity between pig and rat alpha1. Finally both fluorescence resonance energy transfer analysis and co-immunoprecipitation assay indicated that the pumping-null rat alpha1 (D371E) mutant could also bind Src. Expression of this mutant restored the basal Src activity and focal adhesion kinase tyrosine phosphorylation. Taken together, the new findings suggest that LLC-PK1 cells contain a pool of Src-interacting Na/K-ATPase that not only regulates Src activity but also serves as a receptor for ouabain to activate protein kinases.     

Decreased expression of both the alpha1- and alpha2-subunits of the Na-K-ATPase reduces maximal alveolar epithelial fluid clearance

Impaired epithelial sodium channel function predisposes to delayed resorption of pulmonary edema and more severe experimental lung injury, whereas even a small fraction of the normal Na-K-ATPase activity is thought to be sufficient to maintain normal ion transport. However, direct proof is lacking. Therefore, we studied baseline and cAMP stimulated alveolar fluid clearance (AFC) in mice with a 50% decrease in lung protein expression of the alpha(1)- and/or alpha(2)-subunit of the Na-K-ATPase. There was no difference in basal and stimulated AFC in alpha(1)(+/-) or alpha(2)(+/-) mice compared with wild-type littermates. Also, the compound heterozygous mice (alpha(1)(+/-)/alpha(2)(+/-)) had normal basal AFC. However, the combined alpha(1)(+/-)/alpha(2)(+/-) mice showed a significant decrease in cAMP-stimulated AFC compared with wild-type littermates (11.1 +/- 1.0 vs. 14.9 +/- 1.8%/30 min, P < 0.001). When exposed to 96 h of >95% hyperoxia, the decrease in stimulated AFC in the alpha(1)(+/-)/alpha(2)(+/-) mice was not associated with more lung edema compared with wild-type littermates (lung wet-to-dry weight ratio 6.6 +/- 0.9 vs. 5.9 +/- 1.1, respectively; P = not significant). Thus a 50% decrease in protein expression of the alpha(1)- or alpha(2)-subunits of the Na-K-ATPase does not impair basal or stimulated AFC. However, a 50% protein reduction in both the alpha(1)- and alpha(2)-subunits of the Na-K-ATPase produces a submaximal stimulated AFC, suggesting a synergistic role for alpha(1)- and alpha(2)-subunits in cAMP-dependent alveolar epithelial fluid clearance.     

Involvement of rho kinase in the ouabain-induced contractions of the rat renal arteries

Agonist and depolarization-induced vascular smooth muscle contractions include the activation of rho/rho kinase pathway. However, there are no reports addressing the question whether this pathway is involved in ouabain-induced vascular smooth muscle contractions. Therefore, in this study, the possible participation of the rho/rho kinase pathway in ouabain-induced contractions was evaluated in rat renal arteries. Effects of rho kinase inhibitors (fasudil and Y-27632) on ouabain-induced contractions, and phosphorylation of myosin binding subunits (MYPT/MBS85) of myosin phosphatase were determined using isolated tissue and Western blot experiments, respectively. Fasudil and Y-27632 inhibited ouabain-induced contractions in a concentration-dependent manner. The phosphorylation of MYPT was not altered by ouabain. However, ouabain significantly increased MBS85 phosphorylation of myosin phosphatase. The phosphorylation of both subunits of myosin phosphatase was inhibited by Y-27632. These results indicate that activation of rho kinase and the subsequent phosphorylation of MBS85 are involved in ouabain-induced contraction of rat renal arteries. This mechanism may be important in essential hypertension with elevated endogenous ouabain levels.     

Ouabain-induced hypertension is accompanied by increases in endothelial vasodilator factors

The involvement of nitric oxide (NO), prostaglandins, and calcium-dependent potassium channel (K(Ca)) activators on the negative modulation of phenylephrine-induced contractions was evaluated on the isolated aorta and caudal (CAU) artery obtained from rats treated with ouabain for 5 wk to induce hypertension. In ouabain-treated rats, the reactivity to phenylephrine was reduced in the endothelium-intact aorta but not the CAU segments. Endothelial modulation of phenylephrine contraction, as demonstrated by endothelium removal, NO synthase (NOS) inhibition with N(omega)-nitro-L-arginine methyl ester and aminoguanidine, as well as K(Ca) inhibition with tetraethylammonium, was more pronounced in segments from ouabain-treated animals, and here greater effects were seen in the aorta than in CAU. An increased expression of endothelial NOS and neuronal NOS was seen in the aorta after ouabain treatment. In CAU, only endothelial NOS was detected and ouabain treatment did not alter its expression. These results suggest that ouabain-induced hypertension is accompanied by increased NO release derived from endothelial NOS and neuronal NOS and increased release of an endothelial hyperpolarizing factor that presumably opens K(Ca), all of which contribute to the increased negative modulation of the phenylephrine contraction.     

Arterial remodeling and plasma volume expansion in caveolin-1-deficient mice

Caveolin-1 (Cav-1) is essential for the morphology of membrane caveolae and exerts a negative influence on a number of signaling systems, including nitric oxide (NO) production and activity of the MAP kinase cascade. In the vascular system, ablation of caveolin-1 may thus be expected to cause arterial dilatation and increased vessel wall mass (remodeling). This was tested in Cav-1 knockout (KO) mice by a detailed morphometric and functional analysis of mesenteric resistance arteries, shown to lack caveolae. Quantitative morphometry revealed increased media thickness and media-to-lumen ratio in KO. Pressure-induced myogenic tone and flow-induced dilatation were decreased in KO arteries, but both were increased toward wild-type (WT) levels following NO synthase (NOS) inhibition. Isometric force recordings following NOS inhibition showed rightward shifts of passive and active length-force relationships in KO, and the force response to alpha(1)-adrenergic stimulation was increased. In contrast, media thickness and force response of the aorta were unaltered in KO vs. WT, whereas lumen diameter was increased. Mean arterial blood pressure during isoflurane anesthesia was not different in KO vs. WT, but greater fluctuation in blood pressure over time was noted. Following NOS inhibition, fluctuations disappeared and pressure increased twice as much in KO (38 +/- 6%) compared with WT (17 +/- 3%). Tracer-dilution experiments showed increased plasma volume in KO. We conclude that NO affects blood pressure more in Cav-1 KO than in WT mice and that restructuring of resistance vessels and an increased responsiveness to adrenergic stimulation compensate for a decreased tone in Cav-1 KO mice.     

Role of myosin light chain kinase in regulation of basal blood pressure and maintenance of salt-induced hypertension

Vascular tone, an important determinant of systemic vascular resistance and thus blood pressure, is affected by vascular smooth muscle (VSM) contraction. Key signaling pathways for VSM contraction converge on phosphorylation of the regulatory light chain (RLC) of smooth muscle myosin. This phosphorylation is mediated by Ca(2+)/calmodulin-dependent myosin light chain kinase (MLCK) but Ca(2+)-independent kinases may also contribute, particularly in sustained contractions. Signaling through MLCK has been indirectly implicated in maintenance of basal blood pressure, whereas signaling through RhoA has been implicated in salt-induced hypertension. In this report, we analyzed mice with smooth muscle-specific knockout of MLCK. Mesenteric artery segments isolated from smooth muscle-specific MLCK knockout mice (MLCK(SMKO)) had a significantly reduced contractile response to KCl and vasoconstrictors. The kinase knockout also markedly reduced RLC phosphorylation and developed force. We suggest that MLCK and its phosphorylation of RLC are required for tonic VSM contraction. MLCK(SMKO) mice exhibit significantly lower basal blood pressure and weaker responses to vasopressors. The elevated blood pressure in salt-induced hypertension is reduced below normotensive levels after MLCK attenuation. These results suggest that MLCK is necessary for both physiological and pathological blood pressure. MLCK(SMKO) mice may be a useful model of vascular failure and hypotension.