Chronic ouabain treatment induces vasa recta endothelial dysfunction in the rat.
ABSTRACT Descending vasa recta (DVR) are 15-microm vessels that perfuse the renal medulla. Ouabain has been shown to augment DVR endothelial cytoplasmic Ca(2+) ([Ca(2+)](CYT)) signaling. In this study, we examined the expression of the ouabain-sensitive Na-K-ATPase alpha2 subunit in the rat renal vasculature and tested effects of acute ouabain exposure and chronic ouabain treatment on DVR. Immunostaining with antibodies directed against the alpha2 subunit verified its expression in both DVR pericytes and endothelium. Acute application of ouabain (100 or 500 nM) augmented the DVR nitric oxide generation stimulated by acetylcholine (ACh; 10 microM). At a concentration of 1 mM, ouabain constricted microperfused DVR, whereas at 100 nM, it was without effect. Acute ouabain (100 nM) did not augment constriction by angiotensin II (0.5 or 10 nM), whereas l-nitroarginine methyl ester-induced contraction of DVR was slightly enhanced. Ouabain-hypertensive (OH) rats were generated by chronic ouabain treatment (30 microg.kg(-1).day(-1), 5 wk). The acute endothelial [Ca(2+)](CYT) elevation by ouabain (100 nM) was absent in DVR endothelia of OH rats. The [Ca(2+)](CYT) response to 10 nM ACh was also eliminated, whereas the response to 10 microM ACh was not. The endothelial [Ca(2+)](CYT) response to bradykinin (100 nM) was significantly attenuated. We conclude that endothelial responses may offset the ability of acute ouabain exposure to enhance DVR vasoconstriction. Chronic exposure to ouabain, in vivo, leads to hypertension and DVR endothelial dysfunction, manifested as reduced [Ca(2+)](CYT) responses to both ouabain- and endothelium-dependent vasodilators.
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ABSTRACT: The work of deWardener and colleagues stimulated longstanding interest in natriuretic hormones (NHs). In addition to the atrial peptides (APs), the circulation contains unidentified physiologically relevant NHs. One NH is controlled by the central nervous system (CNS) and likely secreted by the pituitary. Its circulating activity is modulated by salt intake and the prevailing sodium concentration of the blood and intracerebroventricular fluid, and contributes to postprandial and dehydration natriuresis. The other NH, mobilized by atrial stretch, promotes natriuresis by increasing the production of intrarenal dopamine and/or nitric oxide (NO). Both NHs have short (<35 min) circulating half lives, depress renotubular sodium transport, and neither requires the renal nerves. The search for NHs led to endogenous cardiotonic steroids (CTS) including ouabain-, digoxin-, and bufadienolide-like materials. These CTS, given acutely in high nanomole to micromole amounts into the general or renal circulations, inhibit sodium pumps and are natriuretic. Among these CTS, only bufalin is cleared sufficiently rapidly to qualify for an NH-like role. Ouabain-like CTS are cleared slowly, and when given chronically in low daily nanomole amounts, promote sodium retention, augment arterial myogenic tone, reduce renal blood flow and glomerular filtration, suppress NO in the renal vasa recta, and increase sympathetic nerve activity and blood pressure. Moreover, lowering total body sodium raises circulating endogenous ouabain. Thus, ouabain-like CTS have physiological actions that, like aldosterone, support renal sodium retention and blood pressure. In conclusion, the mammalian circulation contains two non-AP NHs. Identification of the CNS NH should be a priority.Frontiers in Endocrinology 12/2014; 5:199.
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ABSTRACT: Ouabain is a cardiac glycoside produced in the adrenal glands and hypothalamus. It affects the function of all cells by binding to Na+/K+-ATPase. Several lines of evidence suggest that endogenous ouabain could be involved in the pathogenesis of essential (particularly, salt-sensitive) hypertension. However, information regarding the postulated hypertensive effect of the long-term administration of low-dose exogenous ouabain is inconsistent. This study was designed to help settle this controversy through the use of telemetric monitoring of arterial blood pressure and to elucidate the ouabain-induced alterations that could either promote or prevent hypertension. Ouabain (63 and 324 µg/kg/day) was administered subcutaneously to male Wistar rats. Radiotelemetry was used to monitor blood pressure, heart rate and measures of cardiovascular variability and baroreflex sensitivity. The continuous administration of ouabain for 3 months did not elevate arterial blood pressure. The low-frequency power of systolic pressure variability, urinary excretion of catecholamines, and cardiovascular response to restraint stress and a high-salt diet as well as the responsiveness to α1-adrenergic stimulation were all unaltered by ouabain administration, suggesting that the activity of the sympathetic nervous system was not increased. However, surrogate indices of cardiac vagal nerve activity based on heart rate variability were elevated. Molecular remodeling in mesenteric arteries that could support the development of hypertension (increased expression of the genes for the Na+/Ca2+ exchanger and Na+/K+-ATPase α2 isoform) was not evident. Instead, the plasma level of vasodilatory calcitonin gene-related peptide (CGRP) significantly rose from 55 (11, SD) in the control group to 89 (20, SD) pg/ml in the ouabain-treated rats (PTukey's = 18.10-5). These data show that long-term administration of exogenous ouabain does not necessarily cause hypertension in rodents. The augmented parasympathetic activity and elevated plasma level of CGRP could be linked to the missing hypertensive effect of ouabain administration.PLoS ONE 10/2014; 9(10):e108909. · 3.53 Impact Factor
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Chronic ouabain treatment induces vasa recta endothelial dysfunction in the rat
Chunhua Cao1, Kristie Payne1, Whaseon Lee-Kwon1, Zhong Zhang1, Sun Woo Lim1, John
Hamlyn2, Mordecai P. Blaustein1,2, H. Moo Kwon1 and Thomas L. Pallone1
1Department of Medicine, and 2Department of Physiology, University of Maryland School of
Medicine, Baltimore, MD 21201
Running head: Ouabain induces endothelial dysfunction
Word count: manuscript (5,710) ; abstract (244)
Thomas L. Pallone
Division of Nephrology, N3W143
22 S. Greene St, UMMS
Baltimore, MD 21201
Ph: (410) 328-5720
FAX: (410) 328-5685
Articles in PresS. Am J Physiol Renal Physiol (October 22, 2008). doi:10.1152/ajprenal.90429.2008
Copyright © 2008 by the American Physiological Society.
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Descending vasa recta (DVR) are 15 μm vessels that perfuse the renal medulla.
Ouabain has been shown to augment DVR endothelial cytoplasmic Ca2+ ([Ca2+]CYT) signaling.
In this study we examined the expression of the ouabain sensitive NaKATPase α2 subunit in the
rat renal vasculature and tested effects of acute ouabain exposure and chronic ouabain
treatment on DVR. Immunostaining with antibodies directed against the α2 subunit verified its
expression in both DVR pericytes and endothelium. Acute application of ouabain (100 nM or
500 nM) augmented the DVR NO (nitric oxide) generation stimulated by acetylcholine (ACh, 10
μM). At a concentration of 1 mM, ouabain constricted microperfused DVR, whereas at 100 nM,
it was without effect. Acute ouabain (100 nM) did not augment constriction by angiotensin II
(0.5 or 10 nM), whereas L-NAME (L-nitroarginine methyl ester) induced contraction of DVR was
slightly enhanced. Ouabain hypertensive (OH) rats were generated by chronic ouabain
treatment (30 μg/Kg-day, 5 weeks). The acute endothelial [Ca2+]CYT elevation by ouabain (100
nM) was absent in DVR endothelia of OH rats. The [Ca2+]CYT response to 10 nM acetylcholine
was also eliminated whereas the response to 10 μM acetylcholine was not. The endothelial
[Ca2+]CYT response to bradykinin (100 nM) was significantly attenuated. We conclude that
endothelial responses may offset the ability of acute ouabain exposure to enhance DVR
vasoconstriction. Chronic exposure to ouabain, in vivo, leads to hypertension and DVR
endothelial dysfunction, manifest as reduced [Ca2+]CYT responses to both ouabain and
endothelium dependent vasodilators.
Key words: Rat, kidney, medulla, microcirculation, ouabain, nitric oxide, blood flow
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"Ouabain like factors" (OLF), synthesized by the adrenal gland and hypothalamus,
inhibit Na+ / K+ exchange (19; 46) and activate signaling cascades (6; 7; 44) by binding to
NaKATPase α subunits. In rodents, the α1 isoform of NaKATPase that maintains Na+ and K+
gradients across cell membranes has very low affinity (KD > 10 μM) for ouabain. In contrast,
the α2 and α3 isoforms have high affinity for ouabain (KD < 50 nM), but are less abundantly
expressed (3; 48). It has been proposed that targeting of the α2 isoform to cellular
microdomains where ER/SR protrusions abut the plasma membrane (4; 7) may modulate
intracellular Na+ and reduce Ca2+ extrusion via Na+ / Ca2+ exchange to enhance Ca2+
sequestration into ER/SR stores. Evidence favoring that hypothesis has been accumulating.
Low dose (10-100 nM) ouabain enhances Ca2+ release in smooth muscle and endothelium (2;
35), and increases resting cytoplasmic Ca2+ ([Ca2+]CYT) and myogenic tone (35; 48).
A role for OLF in hypertension is also well supported. Chronic administration of ouabain
into rodents induces hypertension (26; 28), and many patients with essential hypertension have
high plasma ouabain levels (43). Transgenic mice in which the α2 NaKATPase binding site for
ouabain has been mutated are resistant to both ouabain and ACTH induced hypertension,
supporting a causal role in hypertension for both ouabain and the α2 ouabain receptor (12; 13).
Detailed mechanisms by which ouabain induces hypertension remain to be elucidated.
Acute application of ouabain to a microvessel, ex vivo, can increase cytoplasmic Ca2+
concentrations ([Ca2+]CYT) and induce contraction (48; 49). In contrast, the chronic
administration of ouabain to rodents typically requires many 1-2 weeks to fully induce
hypertension (19; 28). At least one explanation for the time lag between onset of ouabain
exposure and generation of hypertension may be a delayed loss of compensatory mechanisms
that otherwise prevent a rise in vascular resistance. We recently demonstrated that the acute
exposure to ouabain increases endothelial [Ca2+]CYT signaling in renal medullary descending
vasa recta (35). Herein we describe a series of experiments designed to test whether ouabain
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enhances release of NO and whether chronic exposure of rats to ouabain inhibits DVR
endothelial signaling. The results show that the ouabain sensitive α2 NaKATPase isoform is
expressed in rat outer medullary vascular bundles and that the acute responses to ouabain and
endothelium dependent vasodilators are diminished by chronic exposure to ouabain in vivo.
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Isolation of DVR. Investigations involving animal use were performed according to protocols
approved by the Institutional Animal Use and Care Committee of the University of Maryland.
Sprague Dawley rats were anesthetized by intraperitoneal injection of ketamine (80 mg/kg) and
xylazine (10 mg/kg). The abdomen was opened and the kidneys excised, leading to euthanasia
by exsanguination. Small wedges of renal medulla were microdissected to isolate DVR. Acute
effects of ouabain were studied in DVR from male rats weighing 100 to150 grams. In those
young rats, DVR could be dissected without collagenase digestion of renal tissue.
Microdissection of DVR from older, ouabain hypertensive (OH) rats, (> 500 gram body weight)
required prior enzymatic digestion, which rendered explanted DVR unsuitable for microperfusion
and study of vasoactivity. Enzymatic digestion was accomplished by transferring wedges of
renal tissue to Blendzyme 1 (Roche, 0.27 mg/ml) in high-glucose DMEM media (Invitrogen), for
30 minutes at 37 OC. Tissue, whether enzymatically digested or undigested, was then
transferred to microperfusion buffer (in mM): NaCl 140, NaAcetate 10, KCl 5, MgCl2 1.2,
Na2HPO4 / NaH2PO4 2, CaCl2 1, alanine 5, glucose 5, HEPES 5, and albumin 0.5 g/dl, pH = 7.4
and stored at 4 OC. At intervals, DVR were isolated and either transferred to microscope slides
for fixation and immunostaining (23; 24) or to the stage of an inverted microscope for
microperfusion and fluorescence measurements (32; 35; 49).
Ouabain hypertensive rats. Male Sprague Dawley rats weighing ~ 250 g were obtained from
Charles River laboratories and acclimatized for several weeks prior to pellet insertions. Under
halothane anesthesia, a pellet containing or lacking ouabain in a proprietary matrix (Innovative
Research, Sarasota, FL) was implanted subcutaneously in the left flank. The ouabain pellet
was chosen to deliver a nominal dose of ~30 μg ouabain /(kg-day) for 60 days. Systolic blood
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pressures (SBPs) were measured weekly for 5 weeks after pellet insertions in awake, warmed
rats by automated tail cuff inflation and deflation (IITC model 29, Woodland Hills, CA).
Videomicroscopy and measurement of vessel diameters. To quantify changes in vessel
diameter, DVR were mounted on concentric pipettes, microperfused and recorded by
videomicroscopy. As previously described, vessel contraction was quantified by measuring
internal diameter at the site of maximal contraction by image analysis (32; 51).
Measurement of endothelial [Ca2+]CYT. DVR were loaded with fura2 (Molecular Probes) by
incubating them for 20 minutes in bath containing the fura2-AM ester (2 μmol/L). We have
previously shown that fura2 preferentially loads into endothelial cells (32; 33). Fura-2 was
excited at 350 / 380 nm wavelengths and the background-subtracted ratio of fluorescent
emission (R350/380) was calculated for conversion to [Ca2+]CYT assuming a dissociation constant
for fura2 at 37 oC of 224 nM. Rmax and Rmin were measured, as previously described, by
exposing vessels to buffer containing 5 mM CaCl2 or 0 CaCl2, 0.5 mM EGTA, respectively,
along with 10 μM calcium ionophore (32).
Fluorescent detection of NO with DAF-2. 4,5-diaminofluorescein diacetate (DAF-2DA,
Calbiochem) was loaded into DVR by incubating them for 20 minutes in bath containing DAF-
2DA AM ester (2 μmol/L) for 20 minutes. DAF-2 was excited at 485 nm (Photon Technology
International) and emission was measured at 530 nm, as previously described (35; 37). The
emission was quantified by photon counting with a photomultiplier assembly (D104B, PTI).
Immunoflourescent labeling of isolated DVR. Immunofluorescent labeling was performed to
localize NaKATPase α2 subunit expression using methods previously described (23; 24).
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Pericytes and endothelial cells were identified using anti- mouse α-smooth muscle actin (SMA)
antibody (Sigma, 1: 400 dilution) or anti-chicken aquaporin 1 (AQP1) antibody (1: 20 dilution),
respectively. Microdissected DVR on slides were fixed with 2% paraformaldehyde in 100
mmol/L cacodylate buffer, pH 7.4. The fixed vessels were preincubated in phosphate-buffered
saline (PBS) containing 5% BSA and 0.1% Triton X-100 followed by overnight incubation at 4oC
with purified polyclonal anti-α2 NaKATPase antibody (1:100 dilution, a generous gift from Dr.
Thomas Pressley, Department of Physiology, Texas Tech Univ. Health Sci Ctr, Lubbock, TX).
The primary antibodies were detected with goat anti-rabbit IgG labeled with Alexa Fluor 568,
goat anti-mouse or goat anti-chicken IgG labeled with Alexa Fluor 488. After several washes
with PBS/Triton, cover slips were mounted with Vectorshield (Vector Lab, Burlingame,CA).
Negative controls were performed in which the primary antibodies were omitted. Fluorescent
images were captured with a Zeiss LSM410 confocal microscope.
Light microscopic immunocytochemistry. As previously described (23), rat kidneys were
fixed by perfusing them for 2 min with PBS, 5 min in 2% paraformaldehyde, and 2 min in
cryoprotectant (10% EDTA, 0.1 mol/L Tris). The kidneys were post-fixed in 2%
paraformaldehyde in PBS and embedded in paraffin. De-paraffinized sections were
preincubated in PBS containing 5% BSA, 0.05% saponin, 0.2% gelatin (solution A). The tissue
sections were then incubated for overnight at 4oC with either purified polyclonal anti-α2
NaKATPase antibody (1: 100) or monoclonal anti-SMA antibody (1: 400) diluted in 1% BSA in
PBS (solution B). Control incubations were performed in solution B without the primary antibody.
After several washes with solution A, primary antibodies were detected with the indirect
immunoperoxidase method (DAKO Cytomation, Carpinteria, CA). Endogenous peroxidase was
blocked by 3% H2O2 for 30 min at room temperature. After rinsing three times for 10 minutes, the
sections were incubated in horseradish peroxidase-conjugated to goat anti-rabbit IgG or goat
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anti-mouse IgG (DAKO K609). To detect horseradish peroxidase, sections were incubated in
0.1% 3,3-diaminobenzidine. After wash in 0.05 M Tris buffer, sections were dehydrated in
graded ethanol solutions and embedded in permount (Sigma).
Reagents. DAF-2 DA (Calbiochem), was stored at 5 mmol/L in DMSO. Fura2 (Molecular
Probes) was stored at 1 mmol/L in DMSO. Angiotensin II (AngII), bradykinin (BK), and
acetylcholine (ACh) were stored in aliquots at 10 μmol/L, 100 μmol/L, and 10 mmol/L,
respectively, in water. Those reagents were diluted on the day of the experiment and the excess
discarded daily. The final concentration of DMSO during the loading of fluorescent probes was
1 - 2%.
Statistics. Data in the text and figures are reported as mean ± SE. The significance of
differences was evaluated with SigmaStat 3.11 (Systat Software, Inc., Point Richmond, CA)
using parametric or nonparametric tests as appropriate for the data. Comparisons between two
groups were performed with Student's t-test (paired or unpaired, as appropriate) or the Rank
Sum Test (nonparametric). Comparisons between multiple groups employed repeated
measures ANOVA, or repeated measures ANOVA on ranks (nonparametric). Post hoc
comparisons were performed using Tukey's or Holm-Sidak tests. P < 0.05 was used to reject
the null hypothesis.
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Immunolocalization of α α2 NaKATPase in DVR and vascular bundles of normal rats. We
verified expression of α2 NaKATPase in both DVR endothelia and pericytes of normal rats by
immunostaining. Smooth muscle actin (SMA) and aquaporin 1 (AQP1) are differentially
expressed in DVR pericytes and endothelia, respectively (30; 34). As expected, based on their
respective abluminal and luminal locations, pericytes (red, SMA) and endothelia (green, AQP1),
were labeled by antibodies directed to those targets (Figure 1A-1C). Endothelial expression of
α2 NaKATPase was demonstrated by its co-localization with AQP1 (Figure 1D-1F). Note that
both AQP1 positive cells (endothelia, arrowheads) and an AQP1 negative cell body (pericyte,
asterisk) show α2 staining.
In a separate series, α2 immunostaining was compared with distribution of SMA. SMA
(Figure 2A, 2D, green) identifies pericytes on the abluminal surface of DVR to include both cell
bodies, containing the nucleus, and the podocyte extensions that wrap around the vessel (31).
NaKATPase α2 isoform colocalized with SMA in a punctate pattern (Figure 2B, 2E). We also
examined α2 immunostaining in 50 micron tissue sections (Figure 3). Both renal cortical
vessels (Figure 3A, glomerular pole, arrowhead; interlobular arteriole, asterisk) and outer
medullary vascular bundles (Figure 3B) exhibited α2 expression. Structures surrounding
vascular bundles, wherein DVR reside, did not exhibit obvious expression of α2 or SMA
(Figures 3B, 3C).
NO generation by DVR from normal rats. Using DAF2 as a fluorescent probe, we tested
whether ouabain alone, or in combination with endothelium dependent vasodilators, can
enhance NO generation by DVR of normal rats. When compared to vehicle, exposure to
ouabain (500 nmol/L) for 30 minutes failed to significantly increase the conversion of DAF2 to its
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fluorescent form (data not shown). In contrast, ouabain increased the fluorescence of DVR
exposed to ACh (10 μmol/L). Fluorescence of DVR loaded with DAF2 was continuously
measured for 30 minutes during exposure to ACh or ACh + ouabain at concentrations of 0, 10,
100, or 500 nmol/L (n = 6, 8, 7, 6, respectively). The results are illustrated in Figure 4A and
summarized in Figure 4B where the data have been normalized, in the manner previously
described, by dividing the final accumulated fluorescence at 30 minutes by the mean of the
controls (37). The effects of ouabain were significant at 100 and 500 nmol/L concentrations.
The ability of ouabain (500 nmol/L) to increase bradykinin (BK, 100 nmol/L) induced NO
generation was also tested. Controls, exposed to BK alone (n = 9), showed a 4.3 ± 3.6 %
increase in DAF2 fluorescence over 30 minutes (not significant). In contrast, in the presence of
500 nM ouabain, BK induced a 21 ± 11 % rise in DAF2 fluorescence (n = 9, P < 0.05, data not
Effect of ouabain on vasoconstriction of DVR from normal rats. We have previously shown
that nanomolar ouabain raises both pericyte (49) and endothelial (35) [Ca2+]CYT. Given that
those effects might offset one another to favor either net vasoconstriction or dilation, we tested
the ability of ouabain to contract microperfused DVR from normal rats. As shown in Figure 5A,
compared to controls (n = 10), 100 nmol/L ouabain (n = 12) did not significantly alter luminal
diameter. At 100 nmol/L, ouabain is expected to saturate putative α2, α3 binding sites. In
contrast, 1 mmol/L ouabain, provides a positive control to induce significant, reversible
contraction. We also tested whether ouabain (100 nmol/L) would augment AngII induced
contraction of DVR (Figure 5B). At AngII concentrations of either 0.5 nmol/L (the EC50) or 10
nmol/L (maximal stimulation), there was no difference between control vessels (AngII alone, n =
8) and those exposed to the combination of AngII + ouabain (n = 10). Finally, we tested
whether L-NAME would eliminate offsetting stimulation of NO generation so that enhancement
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of pericyte contraction by ouabain might be uncovered. In those experiments, L-NAME alone
(100 μmol/L) constricted DVR, showing that despite the apparent insensitivity of DAF2 to detect
it (37), NO generation exists at a significant basal rate in isolated DVR. In the presence of L-
NAME (n = 10), ouabain (10, 100 nmol/L, n = 8) marginally intensified DVR contraction (Figure
Characteristics of OH rats. The above studies (Figures 1-5), as well as those previously
described (35) were focused on the presence of α2 and effects of ouabain stimulation in DVR of
normal rats. To facilitate dissection of DVR for those studies kidneys were harvested from
young rats (100 to 150 grams). In contrast, the prolonged pellet implantation times (5 weeks,
see methods) used to generate OH rats and their vehicle treated controls resulted in larger,
older animals from which dissection of DVR is not possible unless renal tissue is enzymatically
digested. The weights of the vehicle treated and OH rats at the time of sacrifice for the studies
that follow were 538.9 ± 19.6 and 540.0 ± 18.3 g, respectively. Tail cuff systolic blood pressures
(SBPs) in the vehicle treated rats remained stable, whereas OH rat blood pressures rose from
116 ± 6 to 149 ± 5 (P < 0.05 vs vehicle) over 5 weeks (Figure 6A). Immunostaining of DVR
isolated from OH rats showed persistent expression of α2 in pericytes and endothelia,
comparable to that of DVR of vehicle treated animals (Figure 6B).
[Ca2+]CYT responses of DVR from OH rats, ouabain stimulation. We tested whether DVR
isolated from vehicle vs OH rats have similar endothelial [Ca2+]CYT responses to acute
application of ouabain. Basal [Ca2+]CYT was similar, 70 ± 48 vs 91 ± 34 nmol/L, respectively (n =
6 each, not significant). As previously observed, ouabain (100 nmol/L) elicited an endothelial
[Ca2+]CYT response in vessels from vehicle treated rats, whereas the response was absent in
DVR from OH rats (Figure 7). This is not due to loss of expression of α2 NaKATPase
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expression which remains readily observable in immunostained DVR pericytes and endothelium
[Ca2+]CYT responses of DVR from OH rats, ACh and BK stimulation. To test for further
evidence of endothelial "dysfunction" in OH rats, we measured [Ca2+]CYT responses to acetyl
choline (ACh) and bradykinin (BK). In a first series, DVR from vehicle treated and OH rats (n =
6, each) were exposed sequentially to ACh at 10 nmol/L and 10 μmol/L. Responses to the
lower, threshold ACh concentration were eliminated by the prior, chronic ouabain exposure
(Figure 8A). There was also a tendency toward smaller responses to maximal ACh stimulation,
but the effect did not achieve significance, either by peak or "area under the curve" (AUC)
analysis (Figure 8B).
Similar experiments with BK also revealed diminished DVR endothelial [Ca2+]CYT
responses in the OH rats. The endothelial [Ca2+]CYT response was measured in fura2 loaded
DVR from control (n = 8) and OH rats (n = 9) at baseline (2 minutes) and during a subsequent
10 minute BK (100 nmol/L) exposure. A BK concentration of 100 nmol/L was chosen because it
has been previously shown to generate a maximal endothelial [Ca2+]CYT response (57). OH rat
DVR endothelial [Ca2+]CYT elevation in response to BK was significantly diminished (P < 0.05) for
all t > 4.5 minutes (Figure 9A). The peak [Ca2+]CYT response was not significantly different, but
the overall integrated (AUC) response was decreased (Figure 9B, P < 0.05, vehicle infused vs
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Ouabain interacts with a highly conserved site on the N-terminal H1-H2 extracellular
loop of the NaKATPase α subunit (3). Ouabain is secreted by the adrenal gland and circulates
in picomolar to nanomolar concentrations (17; 18; 27). Studies of its actions reveal modification
of vascular resistance, neural sympathetic activity, cellular hypertrophy and raised blood
pressure (7; 20; 44). Blaustein hypothesized that ouabain raises blood pressure acts by
inhibiting Na+ export via the arterial NaKATPase to elevate subplasmalemmal Na+
concentrations resulting in reduction of Ca2+ export (or enhancement of import) by the Na+/Ca2+
exchanger (NCX) (1). Those events favor an increase in the mass of Ca2+ that accumulates in
SR stores (5; 7). In rats and mice, the ubiquitous α1 isoform of NaKATPase that maintains
transcellular Na+ and K+ gradients is insensitive to physiological concentrations of ouabain. The
less abundant α2 and α3 isoforms retain high ouabain sensitivity (3) and possess an N-terminal
sorting motif that tethers them to cellular microdomains formed between abutments of the SR
and overlying plasma membrane (40). Co-localization of α2 Na+ pumps with those SR
protrusions has been verified (4; 16). Strong confirmation of a role for the α2 isoform in
hypertension has been obtained through studies in transgenic mice. Mutation of the α2 binding
site to eliminate ouabain interaction prevents murine hypertension due to chronic ouabain
treatment or ACTH infusion (12; 13). The importance of NCX in the overall scheme has been
strongly supported by the ability of SEA0400 blockade to reverse various forms of salt
dependent hypertension (21).
Not only does ouabain inhibit Na+ and K+ transport, Xie and colleagues have shown that
binding of ouabain to NaKATPase also stimulates tyrosine phosphorylation through Src kinase.
This leads to hypertrophy, reactive oxygen species generation and other events (44).
Stimulation of PLC-γ downstream of Src activation in LLC-PK1 cells results in generation of
inositol tris phosphate and [Ca2+]CYT elevation. Those events are dependent upon protein-
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protein interactions within caveolae and can involve NaKATPase that resides in a "nonpumping"
pool (25; 44; 47). A mathematical simulation of ouabain signaling supports offsetting roles for
the NCX and Src pathways to affect store Ca2+ (14).
Whatever the relative roles NCX and Src signaling might play in the development of
ouabain hypertension, there remains a need to explain why acute exposure of resistance
vessels to ouabain can cause vasoconstriction while chronic exposure, in vivo, takes many days
to induce hypertension (19; 28; 48). An inviting explanation is that compensatory mechanisms
that exist to offset ouabain-induced vasoconstriction are down-regulated during chronic ouabain
exposure. The potential sites of such interaction might include the central nervous system,
through effects on sympathetic tone, the kidney, through effects that favor sodium retention, and
the vascular endothelium, through local vasodilator release. The current study is consistent with
the latter possibility. We have previously shown that the microvascular DVR endothelium
acutely responds to ouabain by raising [Ca2+]CYT and increasing SR store Ca2+ (35). In this
study, we found that the aforementioned responses are eliminated by chronic in vivo exposure
to ouabain (Figure 7) and that this is not explained by loss of α2 expression (Figure 6B). The
loss of [Ca2+]CYT signaling to vasodilators also appears to be affected; chronic in vivo ouabain
administration blunted [Ca2+]CYT responses to ACh and BK (Figures 8, 9). In the case of ACh,
only responses at the threshold concentration (10 nmol/L) were affected. In contrast, responses
at the maximal stimulatory concentration (10 μmol/L) were not significantly reduced. Repeated
exposures to BK induce tachyphylaxis, so that we chose to study its effects at a concentration
(100 nmol/L) that yields maximal [Ca2+]CYT responses. Those responses were significantly
blunted (Figure 9). A potentially important experiment to verify that endothelial dysfunction
induced by chronic ouabain exposure augments DVR vasoconstriction could not be done.
Unfortunately, the enzymatic digestion required to explant DVR from those older OH rats
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renders them unsuitable for microperfusion because the walls of digested vessels rupture during
The acute effects of ouabain on cardiac and vascular myocytes have been well
documented (3; 6; 44). Although fewer studies have focused on its role to modulate signaling
and vasodilator release by the endothelium, evidence favoring such action has been obtained.
Rossoni and colleagues showed that interference with the endothelium enhanced the ability of
10 nM ouabain to increase phenylephrine induced contraction of rat tail vasculature (38). In
contrast to this study, which favors dysfunction of the microvascular DVR endothelium during
chronic ouabain exposure, an increased vasodilatory influence attributable to upregulation of
eNOS and nNOS and increased NO production was observed in the rat aorta (39). Ouabain
may augment release of both a diffusible vasodilator and vasoconstrictor from the
spontaneously hypertensive rat aortic endothelium (36). Most recently, a role for the
endothelium was uncovered by Dostanic et al; depending upon the presence or absence of the
endothelium, ouabain either attenuated or enhanced, respectively, phenylephrine induced
contraction of murine aortic rings (12).
Immunoblots probing NaKATPase α subunit expression in the kidney revealed the
overwhelming predominance of the α1 isoform (48). That is not surprising when one considers
the enormous task imposed upon Na+ transporting epithelia by glomerular filtration. In humans
with normal renal function, nearly 28,000 mEq / day is filtered and then reabsorbed through
several secondary active transport mechanisms, all of which are dependent upon the activity of
the NaKATPase α1 subunit. Such predominant α1 expression belies the possibility that
important signaling mechanisms in the renal vasculature, and possibly elsewhere, may involve
high affinity ouabain isoforms, particularly α2. Nevertheless our past studies and the present
data indicate the existence of ouabain mediated events at concentrations that are orders of
magnitudes below whose required to inhibit the α1 isoform. Accordingly, we suspected