Corresponding author M. P. Blaustein: Department of Physiology, University of Maryland School of Medicine, 655 W. Baltimore Street, Baltimore, MD 21201, USA. Email: firstname.lastname@example.org
A key question in hypertension is: How is long-term blood pressure controlled? A clue is that chronic salt retention elevates an endogenous ouabain-like compound (EOLC) and induces salt-dependent hypertension mediated by Na+/Ca2+ exchange (NCX). The precise mechanism, however, is unresolved. Here we study blood pressure and isolated small arteries of mice with reduced expression of Na+ pump α1 (α1+/−) or α2 (α2+/−) catalytic subunits. Both low-dose ouabain (1–100 nm; inhibits only α2) and high-dose ouabain (≥1 μm; inhibits α1) elevate myocyte Ca2+ and constrict arteries from α1+/−, as well as α2+/− and wild-type mice. Nevertheless, only mice with reduced α2 Na+ pump activity (α2+/−), and not α1 (α1+/−), have elevated blood pressure. Also, isolated, pressurized arteries from α2+/−, but not α1+/−, have increased myogenic tone. Ouabain antagonists (PST 2238 and canrenone) and NCX blockers (SEA0400 and KB-R7943) normalize myogenic tone in ouabain-treated arteries. Only the NCX blockers normalize the elevated myogenic tone in α2+/− arteries because this tone is ouabain independent. All four agents are known to lower blood pressure in salt-dependent and ouabain-induced hypertension. Thus, chronically reduced α2 activity (α2+/− or chronic ouabain) apparently regulates myogenic tone and long-term blood pressure whereas reduced α1 activity (α1+/−) plays no persistent role: the in vivo changes in blood pressure reflect the in vitro changes in myogenic tone. Accordingly, in salt-dependent hypertension, EOLC probably increases vascular resistance and blood pressure by reducing α2 Na+ pump activity and promoting Ca2+ entry via NCX in myocytes.
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Elevated blood pressure (BP), hypertension, is prevalent in developed societies, and is a major risk factor for disability and death (Kaplan, 2002; Chobanian et al. 2003). Salt (NaCl) retention by the kidneys typically leads to hypertension (Guyton, 1990; Kaplan, 2002; Johnson et al. 2005). Indeed, monogenic diseases of renal salt retention raise BP; in contrast, salt wasting syndromes lower BP (Lifton et al. 2001). Mutation, knockout or duplication of genes that affect BP induce either salt-dependent hypertension or unusual forms of salt-independent hypertension (Takahashi & Smithies, 1999). In essential hypertension, the primary defect may be an acquired renal injury rather than a genetic defect (Johnson et al. 2005). Nevertheless, none of those studies have addressed the question of precisely how salt retention leads to chronic hypertension (Kaplan, 2002; Johnson et al. 2005). In this paper we elucidate downstream molecular mechanisms and clarify the link between salt and hypertension.
Mean arterial BP depends primarily on cardiac output (CO) and total peripheral systemic vascular resistance (TPR) (Berne & Levy, 2001): at constant CO, mean BP ≈ CO × TPR. Acute plasma volume expansion elevates BP by increasing CO (Borst & Borst-de Geus, 1963; Guyton, 1990). With sustained volume expansion, however, TPR rises to maintain the elevated BP while CO declines (Borst & Borst-de Geus, 1963; Guyton, 1990). This condition of high TPR and near-normal CO is commonly observed in humans with essential hypertension (Cowley, 1992; Kaplan, 2002). Nevertheless, long-term control of BP is still poorly understood.
The shift from high CO to high TPR, called ‘whole-body autoregulation’, has been attributed to regulation of blood flow to meet metabolic demand (Guyton, 1990; Kaplan, 2002). This view is controversial (Julius, 1988), however, and the mechanisms are unresolved (Kaplan, 2002; Johnson et al. 2005). According to one hypothesis (Fig. 1) (Blaustein, 1977), salt retention promotes secretion of an endogenous cardiotonic (and vasotonic) steroid that inhibits Na+ pumps, including those in vascular smooth muscle. By raising the cytosolic Na+ concentration ([Na+]cyt), this agent would be expected to promote Na+/Ca2+ exchanger (NCX)-mediated Ca2+ entry into the myocytes. This should elevate the cytosolic Ca2+ concentration ([Ca2+]cyt), and thus increase TPR by enhancing myogenic tone, the intraluminal pressure-induced intrinsic arterial constriction that is prominent in small resistance arteries (Hill et al. 2001). Indeed, recent evidence reveals that NCX type-1 (NCX1) in arterial myocytes plays a central role in ouabain-induced hypertension and salt-dependent hypertension (Iwamoto et al. 2004b).
Here we show that exogenous ouabain, at low concentrations approaching circulating EOLC levels, elevates [Ca2+]cyt and augments vasoconstriction of pressurized small arteries. Moreover, mice heterozygous for α2 Na+ pumps (James et al. 1999) (α2+/−, which mimic the effects of nanomolar ouabain), but not mice heterozygous for α1 (α1+/−), have altered artery function and elevated BP. These data demonstrate, for the first time, that modulation of α2 Na+ pump activity, and not α1, regulates small artery contractility and exerts long-term control over BP (Fig. 1).
Wild-type (WT) C57/BL6 mice and mice with a null mutation in one Na+ pump α1 or α2 gene (α1+/− or α2+/−) were studied; the homozygous knockouts do not survive (James et al. 1999). Genomic DNA was obtained from tail biopsies for genotyping by PCR.
In some experiments, normal male Sprague-Dawley rats (150–240 g) were used. All rat and mouse protocols were approved by the Institutional Animal Care and Use Committee of the University of Maryland School of Medicine.
Mice (∼16 weeks old) were anaesthetized with isoflurane supplemented with 100% O2; core temperature was maintained at 37.5–38°C. The right femoral artery was surgically isolated and cannulated with a 1.4 F Mikro-tip pressure catheter (Millar Instruments, Houston, TX, USA). Blood pressure was acquired under 1.5% isoflurane anaesthesia (Janssen et al. 2004); data were calculated off-line (BioPac System, Santa Barbara, CA, USA). After the experiment the animal was killed by cervical dislocation following an isofluorane overdose. The data collection was performed ‘double-blind’: the individual who instrumented and measured the BP did not know the genotype. Animal code numbers were matched with genotype and BP after the data on all the mice had been collected.
Diameter and [Ca2+]cyt measurements
Mice and rats were killed by rapid cervical dislocation and decapitation. Mesenteric small arteries were isolated and pressurized to permit myogenic tone development (Zhang et al. 2002). Myogenic tone was generated at an intraluminal pressure of 70 mmHg unless otherwise noted. External diameter was monitored with a Nikon (Melville, NY, USA) TMS microscope (×10 objective) and a monochrome CCD camera operated by LabView software (National Instruments, Austin, TX, USA) (Zhang et al. 2002). Passive external diameter was measured in Ca2+-free solution (Zhang et al. 2002). Myogenic reactivity was determined by measuring the steady-state diameters following step changes in intraluminal pressure.
For [Ca2+]cyt measurement, pressurized arteries were loaded (room temperature) with 10 μm fura-2 at 20 mmHg for 45 min or 10 μm fluo-4 at 70 mmHg for 2 h in albumin-free dissection solution containing 1.0% DMSO (vol/vol) and 0.03% cremaphor EL (vol/vol).
Fluo-4 imaging Arteries were imaged in one of two scanning planes (Mauban et al. 2001; Zhang et al. 2002) with a Nipkow-Yokogawa spinning disc confocal imaging system (CSU10, Solomere Technology, Salt Lake City, UT, USA) connected to a Stanford XR-Mega 10 camera (Stanford Photonics, Palo Alto, CA, USA). The spinning disc was mounted on a Nikon Eclipse TE2000-U inverted microscope (×60, numerical aperture (NA) 1.2 water immersion objective). The confocal images shown here were obtained from an optical plane at the centre of the artery, parallel to the long axis. In this plane, the myocyte cross-sections in the artery walls remain in the plane of focus while the artery walls move horizontally during vasoconstriction and vasodilatation (Mauban et al. 2001). This permits simultaneous diameter determination and Ca2+ imaging (e.g. Fig. 2A); the vasoconstriction of dye-loaded arteries, however, is often somewhat attenuated (see Results section).
Fura-2 imaging Arteries were excited alternately at 340 and 380 nm by a Lambda DG-4 illumination system (Sutter Instruments, Novato, CA, USA) and were viewed with a TE2000-U inverted microscope (×40 oil objective). The myocytes in the artery walls were imaged in a focal plane tangential to the surface of the artery close to the floor of the tissue chamber (Mauban et al. 2001; Zhang et al. 2002). Images were acquired with an ORCA-ER camera (Hamamatsu Corp., Bridgewater, NJ, USA) using MetaFluor software (Universal Imaging, Chester, PA, USA). Individual myocyte [Ca2+]cyt was calculated using Grynkiewicz's equation:
with an apparent dissociation constant (Kd) of 282 (Knot & Nelson, 1998) after in situ calibration. Rmin, Rmax and β were determined for each region where R is the fluorescent emission ratio with excitation at 340 and 380 nm, Rmin and Rmax are the emission ratios under Ca2+- free (5mm EGTA and saturating (24 mm) Ca2+ conditions respectively, and β is the emission ratio with 380 nm excitation under Ca2+-free and saturating Ca2+ conditions.
Membrane potential measurements
Membrane potential was recorded in rat mesenteric small arteries (140–200 μm external diameter) using standard sharp microelectrodes. Pipettes were pulled on a Brown-Flaming electrode puller (Sutter Instruments) and filled with 3 m KCl (resistance, 70–100 MΩ). The preamplifier (M-707A, World Precision Instruments, Sarasota, FL, USA) output was digitized using a Digidata 1322A (Axon Instruments, Union City, CA, USA); data were analysed using pCLAMP software (Axon Instruments).
Mesenteric arteries, hearts and kidneys were minced and homogenized in NaCl/sucrose buffer and membrane fractions were prepared (Lencesova et al. 2004). Tissue extracts were analysed by Western blot with polyclonal α-isoform-specific and non-selective antibodies (Pressley, 1992); the antibodies were gifts from Drs T. Pressley (Texas Tech University, Lubbock, TX, USA) and A. McDonough (University of Southern California, Los Angeles, CA, USA). Band densities were quantified (Golovina et al. 2003) using Kodak ID image analysis software (Eastman Kodak, Rochester, NY, USA). For these analyses, we confirmed that the amounts of protein in the bands were within the linear range of signal intensities. Band densities were normalized with β-actin for arterial and renal membranes, and glyceraldehyde-3-phosphate dehydrogenase for cardiac membranes. The relative amounts of α1 and α2 in the tissue samples were calculated on the basis of evidence that 80–87% of the Na+ pumps in skeletal muscle have an α2 subunit, and the remainder is α1 (He et al. 2001; Golovina et al. 2003).
Reagents and solutions
Artery dissection solution (mm): NaCl, 145; KCl, 4.7; MgSO4·7H2O, 1.2; Mops, 2.0; EDTA, 0.02; NaH2PO4, 1.2; CaCl2·2H2O, 2.0; glucose, 5.0; pyruvate, 2.0; with 1% albumin (pH 7.4 at 5°C). Krebs perfusion solution (mm): NaCl, 112; NaHCO3, 26; KCl, 4.9; CaCl2, 2.5; MgSO4·7H2O, 1.2; KH2PO4, 1.2; glucose, 11.5; Hepes, 10 (pH adjusted to 7.3–7.4 with NaOH). Ca2+-free solution was made by omitting Ca2+ and adding 0.5 mm EGTA. Solutions were gassed with 5% O2, 5% CO2 and 90% N2. Solution for membrane potential measurement (mm): NaCl, 140; KCl, 5; NaH2PO4, 1.2; MgCl2, 1.4; Hepes, 10; NaHCO3, 5; CaCl2, 1.8; glucose, 11.5 (pH adjusted to 7.3 with NaOH).
Reagents and sources were as follows: ouabain, phenylepherine, phentolamine, acetylcholine and cremaphor EL (Sigma-Aldrich, St Louis, MO, USA); SEA0400 (Taisho, Tokyo, Japan); PST 2238 (Prassis/Sigma Tau, Milan, Italy); canrenone (Pharmacia Ltd, Morpeth, Northumberland, UK); KB-R7943 (Tocris, Ellisville, MO, USA); fluo-4 and fura-2 (Molecular Probes, Eugene, OR, USA). Other reagents were reagent grade or the highest grade available.
Data analysis and statistics
The data are expressed as means ±s.e.; n denotes the number of arteries studied (one per animal) unless otherwise stated. Comparisons of data were made using Student's paired or unpaired t test, as appropriate; one-way or two-way ANOVA was used where indicated (see figure legends). Differences were considered significant at P < 0.05. Images were analysed with customized Interactive Data Language software (IDL, Research Systems, Inc., Boulder, CO, USA).
Nanomolar ouabain augments myogenic tone
WT mouse mesenteric small arteries were loaded with the Ca2+ indicators, fluo-4 (Fig. 2A, C and D) or fura-2 (Fig. 2E). Fluo-4 fluorescence, a measure of [Ca2+]cyt, was relatively low in relaxed myocytes at 23°C (Fig. 2A). Cross-sections of individual myocytes are seen (arrows) in these confocal optical cross-sections of the artery wall (Zhang et al. 2002) that contains only a single layer of myocytes.
[Ca2+]cyt rose (i.e. fluorescence increased) after a 40–50 min delay when the arteries were pressurized to 70 mmHg and temperature was increased from 23 to 35°C. This was followed (Fig. 2Ab) by vasoconstriction (i.e. myogenic tone) (Hill et al. 2001). Under these conditions, but in the absence of Ca2+ indicator, the arteries constricted from 129 ± 1 μm (passive external diameter) to 99 ± 2 μm (n= 91); thus, myogenic tone at 70 mmHg in WT arteries was a 23 ± 1% constriction from passive diameter. Vasoconstriction in Ca2+ indicator-loaded arteries was usually attenuated, perhaps because of Ca2+ buffering by the indicator.
In arteries with myogenic tone, 100 nm ouabain induced a further, reversible constriction (Fig. 2B). On average, ouabain decreased external diameter from 101 ± 1 to 93 ± 1 μm; i.e. myogenic tone increased from 23 ± 1% of passive diameter to 29 ± 1% of passive diameter, a 25 ± 2% increase (n= 64; P < 0.001). This constriction corresponds to a 10 μm decrease in internal diameter, from 85 to 75 μm.
The arteries were exposed to ouabain for periods of only 2–10 min in these and most other experiments. In experiments in which the myogenic response to step increases in pressure was examined (see below), however, the exposure to ouabain was prolonged. In these arteries, myogenic tone at 70 mmHg was 27 ± 3% of passive diameter after 10 min, and 32 ± 3% (n= 5) after 50–60 min of treatment with 100 nm ouabain. Thus, this effect of ouabain on myogenic tone was maintained (or even slightly increased), in contrast to the transient response described in noradrenaline-contracted small arteries (Aalkjaer & Mulvany, 1985).
Ouabain did not constrict pressurized arteries before the generation of myogenic tone (Fig. 2B). Thus, the ouabain-induced vasoconstriction apparently depended upon some of the same cellular mechanisms that produce myogenic tone: elevation of [Ca2+]cyt (Fig. 2A) and/or enhanced Ca2+ sensitivity of the contractile apparatus (Hill et al. 2001). Ouabain (100 nm) elevated [Ca2+]cyt in pressurized arteries before (Fig. 2C) as well as after (Fig. 2D) the generation of myogenic tone but, in the latter case, the ouabain-induced effect was superimposed on a significantly higher prior [Ca2+]cyt (Fig. 2E). Even in two pressurized arteries treated with 0.3 μm nifedipine, which markedly reduces [Ca2+]cyt and myogenic tone (Zhang et al. 2002), 100 nm ouabain still constricted the arteries by 6 and 20% of the diameter with nifedipine alone. Therefore, both an appropriately high [Ca2+]cyt and a pressure-induced increase in Ca2+ sensitivity may contribute to the ouabain-induced constriction.
Fluo-4 is a non-ratiometric Ca2+ indicator and does not report absolute [Ca2+]cyt. The ratiometric Ca2+ sensor, fura-2, was therefore used to determine [Ca2+]cyt in individual myocytes imaged in a plane tangential to the surface of the artery (Fig. 2E). [Ca2+]cyt was 185 nm at 70 mmHg. Ouabain (100 nm) reversibly elevated [Ca2+]cyt by 22 ± 4 nm (n= 22 cells; P < 0.001); thus, the ouabain-induced constriction of internal diameter is ∼0.45 μm per 1 nm rise in [Ca2+]cyt. Therefore, when Ca2+ sensitivity is high and [Ca2+]cyt is already above contraction threshold in small arteries with myogenic tone, small changes in [Ca2+]cyt should significantly affect vessel diameter.
These findings are comparable with published data from somewhat larger (∼200 μm passive diameter) pressurized rat cerebral arteries with myogenic tone (Knot & Nelson, 1998): [Ca2+]cyt was ∼200 nm at 60 mmHg and 37°C, and K+ depolarization constricted the arteries by ∼1.05 μm per 1 nm increase in [Ca2+]cyt. Such small, but highly significant ouabain-induced increases in [Ca2+]cyt and myogenic tone (Figs 2B–E and 3A) have profound physiological implications. Blood flow through small arteries is governed by Poiseuille's law (Berne & Levy, 2001), and resistance to flow, R, is inversely proportional to the fourth power of the internal radius, r (i.e. R∝ 1/r4). For example, an ouabain-induced constriction from 85 to 75 μm internal diameter would be expected to increase R by 68% and markedly elevate BP.
Na+ pump α2 subunits are the low-dose ouabain receptor
To elucidate the mechanism of action of low-dose ouabain on myogenic tone, it is important to identify the high affinity ouabain receptor. A dose of 10 nm ouabain also raised [Ca2+]cyt (Fig. 3Aa), and the accompanying increase in myogenic tone (Fig. 3Ab and B) approached the maximal effect of 100 nm ouabain (Figs 2B and 3D). The relationship between the ouabain dose and the increase in myogenic tone was biphasic, with a plateau between 10 and 1000 nm (Fig. 3D). The apparent EC50 was ∼1.3 nm at the high affinity ouabain site. This effect must be mediated by Na+ pumps with high ouabain affinity α2 subunits. Even though the α1:α2 ratio is 4:1 in mesenteric arteries (Fig. 3E; the ratio is ∼2.3:1 in the aorta (Shelly et al. 2004; Staton et al. 2005)), in rodents, Na+ pumps with α1 subunits have ∼1000-fold lower affinity for ouabain (O'Brien et al. 1994; Blanco & Mercer, 1998).
The artery wall contains endothelial cells and neurones as well as myocytes, and all the cells have Na+ pumps/ouabain receptors. To determine whether endothelial cells play a major role in the response to ouabain, the endothelium was denuded. This is indicated by the loss of ACh-induced vasodilatation in arteries constricted with 5 μm phenylephrine (PE; Fig. 4A, red). Then, following development of myogenic tone, 100 nm ouabain still augmented myogenic tone by 21 ± 4% (n= 3; Fig. 4A, green). Thus, the endothelium had little influence on the response to nanomolar concentrations of ouabain.
The possible contribution of ouabain-induced catecholamine release from sympathetic nerve terminals to the ouabain-induced vasoconstriction (Bagrov et al. 1995) was tested by blocking myocyte α-adrenoceptors. Phentolamine (1 μm) blocked 90% of the vasoconstriction induced by 5 μm PE (not shown) but had no influence on the 100 nm ouabain-induced vasoconstriction (Fig. 4B, red line versus control blue and green lines). Even 10 μm phentolamine, which abolished the response to 10 μm PE, had no effect (not shown). Thus, catecholamine release by sympathetic nerves apparently contributed little to the ouabain-induced increase in myogenic tone.
Inhibition of Na+ pumps, which are electrogenic, might be expected to depolarize the myocytes and thereby trigger vasoconstriction (Haddy & Overbeck, 1976; but see Blaustein, 1981). However, 100 nm ouabain, which should block only about 20% of total Na+ pumps (i.e. only those with α2 subunits), had negligible effect on the membrane potential of myocytes within intact mesenteric arteries (Fig. 4C). On average, 100 nm ouabain depolarized the myocytes by only 0.1 ± 0.5 mV (n= 6). As these experiments were performed on rat arteries, it is important to note that 100 nm ouabain increased myogenic tone from a 21 ± 3% to a 27 ± 3% constriction relative to passive diameter in rat mesenteric small arteries (n= 5; P < 0.01).
The influence of the external K+ concentration ([K+]o) was tested in order to demonstrate that small, reversible changes in membrane potential could be detected. Unlike ouabain, elevating [K+]o from 4.9 to 10 mm depolarized the arteries by about 4–5 mV (Fig. 4C). Indeed, a hyperpolarization might have been expected (Weston et al. 2002) because such small increases in [K+]o often dilate small arteries (Emanuel et al. 1959), perhaps as a result of Na+ pump activation (Weston et al. 2002). Nevertheless, the subject is controversial because some other investigators also have observed that a 5 mm rise in [K+]o depolarizes small arteries (Quinn et al. 2000; Bratz et al. 2002).
Myogenic tone is increased in α2+/− mice
Figures 2–4 suggest that 10–100 nm ouabain raises [Ca2+]cyt and increases myogenic tone by inhibiting myocyte Na+ pumps with α2 subunits. Therefore we also studied myogenic tone in mesenteric small arteries from mice with a single null mutation in the gene that encodes either the α1 or α2 isoform of the Na+ pump α subunit (α1 or α2 heterozygotes: α1+/− or α2+/−; James et al. 1999). Arteries from the heterozygous mice expressed ∼50% of normal α1 or α2, respectively (Fig. 5A and B) (James et al. 1999; Shelly et al. 2004). Thus, despite up-regulation of α2, total Na+ pump expression was reduced by ∼40% in α1+/− arteries (Fig. 5A and B). Nevertheless, α1+/− arteries generated the same amount of myogenic tone at 70 mmHg pressure, and the same response to 100 nm ouabain as did WT mouse arteries (Fig. 6A).
In contrast, α2+/− mouse arteries, in which total Na+ pumps are reduced by only ∼10% (Fig. 5A and B), generated significantly more myogenic tone than did WT arteries, and the response to 100 nm ouabain was commensurately reduced (Fig. 6A). Indeed, 100 nm ouabain increased [Ca2+]cyt (measured with fura-2) by only 7 ± 2 nm in α2+/− artery myocytes (n= 28), versus 22 ± 5 nm in WT myocytes (n= 22; P= 0.011). Moreover, the myogenic responses to step increases in intraluminal pressure were augmented in both WT arteries treated with 100 nm ouabain and α2+/− arteries (without ouabain) relative to myogenic responses in control WT arteries (Fig. 6B). Thus, low-dose ouabain increases myogenic responses and myogenic tone by inhibiting, selectively, Na+ pumps with α2 subunits.
α2+/− mice have high blood pressure
Maintained low nanomolar plasma ouabain induces a sustained hypertension in rodents that requires α2 Na+ pumps with high affinity for ouabain (Dostanic et al. 2005). As reduced Na+ pump α2 subunit expression mimics the effects of nanomolar ouabain on small arteries (Fig. 6A and B), we reasoned that α2+/− mice might have elevated BP. Indeed, averaged mean BP (MBP) was significantly higher in α2+/− mice than in WT mice under isofluorane anaesthesia (Fig. 7).
In striking contrast, α1+/− mice, with far fewer α1 and total arterial Na+ pumps (Fig. 5A and B), have normal BP (Fig. 7) as well as normal myogenic tone (Fig. 6A). Nevertheless, acute inhibition by 10 μm ouabain markedly elevates arterial myocyte [Ca2+]cyt (not shown), constricts arteries (Fig. 6A) and has a large positive inotropic effect on hearts (James et al. 1999) from α1+/− mice. Comparable effects of 10 μm ouabain are observed in WT mouse arteries (Figs 3 and 6A) and hearts (James et al. 1999). What, then, is the explanation for this difference between the effects of congenitally reduced α1 expression and acutely reduced α1 activity on myogenic tone, and for the normal BP in α1+/− mice? The data imply that there is compensation for chronically reduced α1 activity. Up-regulation of α2 expression in the heart and arteries (Fig. 5A) of α1+/− mice may provide some compensation even though α1 and α2 are localized to different plasma membrane domains, are regulated differently and have different functions (Juhaszova & Blaustein, 1997). Other mechanisms also are likely to be involved, including reduced cell Na+ permeability. Thus, arterial myocytes from α1+/− mice may have relatively normal [Na+]cyt and [Ca2+]cyt despite the reduced α1 expression.
Alternatively, α1+/− mice might be normotensive because they lose salt due to reduced expression of α1 in the kidneys. This is not the case, however, because renal α1 expression is not reduced in the α1+/− mice, whereas cardiac and arterial α1 are reduced by ∼50% (Fig. 5B). As cardiac contractility is reduced in these mice (James et al. 1999), the BP might be normal because of a reduced CO. There is, however, no reason to expect a hypercontracted vasculature and an augmented TPR in α1+/− mice, as myogenic tone is normal in isolated α1+/− arteries (Fig. 6A).
Ouabain antagonists block ouabain's effect on tone
If the proposed sequence of events leading from ouabain to increased vascular tone (Fig. 1) is correct, it should be possible to interrupt this sequence with appropriate pharmacological tools. For example, PST 2238 and canrenone, known ouabain antagonists (Finotti & Palatini, 1981; Ferrari et al. 1998), should reduce ouabain's inhibition of Na+ pump α2 subunits and augmentation of myogenic tone. Indeed, 5 μm PST 2238, an antihypertensive agent (Ferrari et al. 1998) derived from digitoxigenin (Quadri et al. 1997), abolished the effect of 100 nm ouabain on myogenic tone (Fig. 8A and B); prior application of PST prevented ouabain from augmenting myogenic tone (not shown). Canrenone (5 μm), a spironolactone metabolite with antihypertensive activity (Semplicini et al. 1995; Mantero & Lucarelli, 2000), was a partial antagonist (Fig. 8B). Neither agent affected control myogenic tone. Also, neither agent affected the increase in myogenic tone in α2+/− arteries as, in this case, the reduced α2 activity was genetic and was not induced by ouabain.
NCX blockers inhibit effects of reduced α2 activity
In the present study, 1 μm SEA0400 reduced control myogenic tone by ∼10%, abolished the ouabain-induced increase in myogenic tone (Fig. 9A and C), and significantly attenuated the enhanced myogenic tone in arteries from α2+/− mice (Fig. 9B and C). The latter effect contrasts with the absence of responses to PST 2238 and canrenone in α2+/− mouse arteries (Fig. 8B). KB-R7943 (1 μm), another, less selective NCX inhibitor (Matsuda et al. 2001; Iwamoto et al. 2004a), had similar but less marked effects (Fig. 9C). These data cannot be explained by inhibition of L-type voltage-gated Ca2+ channels: Ca2+ entry through these channels accounts for most of control myogenic tone (Hill et al. 2001), but neither 1 μm SEA0400 nor KB-R7943 blocked the 75 mm K+-induced, nifedipine-sensitive vasoconstriction in these arteries (not shown). Rather, the in vitro data (Fig. 9C) indicate that ∼10% of control myogenic tone depends upon Ca2+ entry through NCX (presumably NCX1). Operation of a 3 Na+:1 Ca2+ exchanger in the Ca2+ entry mode is not surprising because the myocyte membrane potential in pressurized arteries (about −45 to −55 mV; Knot & Nelson, 1998) may be positive to the NCX reversal potential (Blaustein & Lederer, 1999). Reduction of myogenic tone by SEA0400 and KB-R7943, rather than augmentation, when myogenic tone was amplified by reduced α2 activity (Fig. 9A, B and C) also indicates that increased Ca2+ entry via NCX was responsible for the increased myogenic tone in ouabain-treated and α2+/− arteries. Thus, arterial myocyte NCX1 mediates the rise in myogenic tone and elevation of BP induced by nanomolar ouabain and by reduced Na+ pump α2 subunit expression.
α2 Na+pumps: long-term regulators of blood pressure in mice
This report reveals that low nanomolar ouabain augments myogenic reactivity and myogenic tone in small arteries by interacting specifically with arterial myocyte α2 Na+ pumps. These data are consistent with the recent report (Dostanic et al. 2005) that ouabain does not induce hypertension in mice with mutated, ouabain-resistant α2 Na+ pumps. The latter result demonstrates that interaction between ouabain and α2 is necessary for the induction of hypertension by ouabain, but this does not elucidate the mechanism.
Here, we employed ouabain concentrations approaching the EOLC levels in the circulation (∼0.1–3.0 nm; e.g. Rossi et al. 1995), and we addressed the mechanism of ouabain's action in intact, small arteries. The data demonstrate that low-dose ouabain-induced vasoconstriction is a result of its direct action on the arterial myocytes; it is not due to effects on the endo-thelium, catecholamine release from the sympathetic neuroeffector cells, or myocyte depolarization. In the isolated arteries, brief (e.g. 5–15 min) treatment with low-dose ouabain mimics the effects of genetically reduced α2 expression on myogenic reactivity and myogenic tone. Clearly, there is little adaptation or compensation for reduction of α2 activity even over the lifetime of the mice. Also, BP is elevated in α2+/− mice, as it is in ouabain-treated rodents (Yuan et al. 1993; Manunta et al. 1994; Iwamoto et al. 2004b; Dostanic et al. 2005). Thus, the ouabain-induced increase in myogenic reactivity and myogenic tone, as well as the elevated BP, are primarily due to reduction of arterial myocyte α2 activity, per se, and not to other reported actions of ouabain (Santana et al. 1998; Aizman et al. 2001; Gao et al. 2002; Xie & Askari, 2002; Saunders & Scheiner-Bobis, 2004). Moreover, a preliminary report that overexpression of α2, but not α1, in mouse smooth muscle induces hypotension (Staton et al. 2005) is consistent with this view of the central role of arterial myocyte α2 Na+ pump activity in regulating BP.
The evidence that BP is elevated in α2+/−, but not α1+/− mice implies that reduced α2 activity is both necessary and sufficient to induce hypertension, even without ouabain. Thus, arterial myocyte α2 Na+ pumps are a newly identified and long-sought long-term regulator of BP. The hypertension (and presumed increase in TPR) in the α2+/− mice are probably the consequence of the augmented myogenic reactivity and myogenic tone observed in the isolated α2+/− arteries. It is important to note, however, that these results do not preclude other, additional effects of EOLC in the pathogenesis of salt-dependent hypertension. For example, effects in the kidneys (in addition to vasoconstriction) may influence salt retention (Ferrandi et al. 2004), and in the kidneys and other tissues may contribute to target organ damage, possibly by promoting cell growth and proliferation (Aizman et al. 2001; Xie & Askari, 2002; Saunders & Scheiner-Bobis, 2004).
How does α2 activity influence blood pressure?
The increase in myogenic tone that results from reduced α2 Na+ pump activity can be explained by a rise in [Ca2+]cyt mediated by NCX1 (Iwamoto et al. 2004b), with which α2 Na+ pumps are coupled both geographically (Juhaszova & Blaustein, 1997; Lencesova et al. 2004) and functionally (Arnon et al. 2000; Golovina et al. 2003). This view is supported by the pharmacological evidence that the augmented myogenic tone in arteries from α2+/− mice as well as the increased myogenic tone induced by nanomolar ouabain are blocked by SEA0400 and KB-R7943.
Salt-dependent hypertension was not explored in the present study. Indeed, the observation that the ouabain antagonists, PST 2238 and canrenone, did not influence the augmented myogenic tone in arteries from α2+/− mice implies that the α2+/− hypertension model is ouabain (and salt) independent, presumably because salt and ouabain act upstream (Fig. 1). Nevertheless, a preliminary report indicates that the development of deoxycorticosterone acetate (DOCA)-salt hypertension is accelerated in α2+/− (versus WT) mice (Staton et al. 2005). This is expected if, when there are already a reduced number of α2 Na+ pumps, the DOCA and salt elevate plasma EOLC (Hamlyn et al. 1991; Rossi et al. 1995; Goto & Yamada, 2000).
We observed, however, both normal myogenic tone in isolated α1+/− arteries, and normal BP in α1+/− mice, despite the markedly reduced α1 activity in the arteries. Nevertheless, brief exposure to high-dose (≥1 μm) ouabain, which should inhibit rodent α1 Na+ pumps (O'Brien et al. 1994; Blanco & Mercer, 1998), induced profound vasoconstriction of the isolated, small arteries from α1+/− mice as well as those from WT and α2+/− mice. The implication is that the α1+/− mice compensate for the chronically (genetically) reduced α1 activity, and thereby avoid a rise in BP. This contrasts with the apparent absence of compensation for genetically reduced α2 activity, mentioned above.
Perhaps in humans, too, if there is compensation for chronically EOLC-inhibited α1 activity, functionally comparable with the reduced α1 expression in α1+/− mice, the dominant long-term effect of EOLC in humans may still be on arterial myocyte α2. This would reconcile the dilemma about the proposed role of EOLC in human hypertension (Fig. 1). Indeed, such compensation for α1 inhibition seems likely because this isoform is the ‘housekeeper’ that maintains the low global [Na+]cyt (Golovina et al. 2003). Complete inhibition or knockout of rodent α2 Na+ pumps (only ∼20% of total arterial Na+ pumps) has minimal effect on global [Na+]cyt (Golovina et al. 2003) and membrane potential (Fig. 4C). In contrast, acute inhibition of a significant fraction of the predominant α1 Na+ pumps (e.g. by micromolar ouabain concentrations) can be expected to elevate global [Na+]cyt substantially (Golovina et al. 2003) and markedly depolarize the myocytes (Aalkjaer & Mulvany, 1985); this should induce profound vasoconstriction.
Antagonism of ouabain's effect
An intriguing feature of cardiotonic steroid action is the fact that Strophanthus steroids such as ouabain and dihydro-ouabain induce hypertension in rodents, whereas Digitalis steroids such as digoxin and digitoxin do not (Kimura et al. 2000; Manunta et al. 2001). This seems surprising because both classes of steroids inhibit the Na+ pump and, as emphasized here, reduced α2 Na+ pump activity is necessary and sufficient for induction of hypertension in rodents. However, the fact that digoxin and digitoxin not only do not induce hypertension, but lower blood pressure in ouabain-hypertensive rats (Manunta et al. 2000) and even in human hypertensives (Abarquez, 1967), provides an important clue. This observation implies that the Digitalis steroids are partial Na+ pump agonists (i.e. they are pump inhibitors) and partial antagonists (i.e. they block ouabain's inhibition of the Na+ pump). Indeed, the digitoxigenin derivative, PST 2238, is a particularly interesting synthetic furane analogue of a Digitalis steroid because it has negligible agonist activity and strong ouabain-antagonist activity (Fig. 8) (Ferrari et al. 1998). The observation that PST 2238 antagonizes the low-dose ouabain-induced increase in myogenic tone, but has no effect on the augmented myogenic tone in arteries from α2+/− mice, is consistent with this mechanism of action.
We conclude that there is now compelling evidence for the sequence of events illustrated in Fig. 1. EOLC, Na+ pumps with α2 subunits and NCX1 are key downstream components in the regulation of myocyte [Ca2+]cyt and contractility, and long-term control of BP. This pathway (Fig. 1) provides several novel targets for antihypertensive therapy. These include the biosynthetic and secretory pathways of EOLC as well as Na+ pumps with α2 subunits and NCX1. Finally, by clarifying the mechanisms involved in salt-dependent hypertension, it should now be easier to elucidate the mechanisms that underlie the pathogenesis of other forms of essential hypertension.
This work was supported by NHLBI/NIH grants to C.W.B., M.P.B., J.B.L. and W.G.W., and postdoctoral fellowships from the American Heart Association Mid-Atlantic Affiliate (to J.Z.) and the Korea Science and Engineering Foundation, KOSEF (to M.Y.L.). α1+/− and α2+/− mice were originally generated by P. F. James and J. B. Lingrel (University of Cincinnati). We thank A. McDonough and T. Pressley for antibodies, S. Kinsey for mouse breeding and genotyping, and technical assistance, and V. Golovina for comments on the manuscript. We also thank Prassis-Sigma Tau (Milan, Italy) for supplying PST 2238 and the Taisho Pharmaceutical Co., Ltd. (Saitama, Japan) for SEA0400.
Author's present addresses
M. Cavalli: Dipartimento di Farmacologia ed Anestesiologia, Università degli Studi di Padova, Padua, Italy.
C. W. Balke: Departments of Medicine and Physiology and the Institute for Molecular Medicine, University of Kentucky College of Medicine, Lexington, KY, USA.