Ouabain stimulates endothelin release and expression in humanendothelial cells without inhibiting the sodium pump Robert Saunders and Georgios Scheiner-Bobis Institut fu¨r Biochemie und
Trang 1Ouabain stimulates endothelin release and expression in human
endothelial cells without inhibiting the sodium pump
Robert Saunders and Georgios Scheiner-Bobis
Institut fu¨r Biochemie und Endokrinologie, Fachbereich Veterina¨rmedizin, Justus-Liebig-Universita¨t Giessen, Germany
Ouabain, a sodium pump (Na+/K+-ATPase) inhibitor, has
been shown to act as a hormone and is possibly involved in
the pathogenesis of hypertension The mechanism by which
ouabain may act was investigated using primary cultures of
human umbilical artery endothelial cells (HUAECs), which
are known to express and release the vasoconstrictive
hor-mone endothelin (ET-1) Five minutes after application, low
concentrations of ouabain induced Ca2+oscillations and
stimulated ET-1 release from endothelial cells into the
medium To investigate whether the observed effects were
due to inhibition of the sodium pump, the effects of ouabain
on the uptake of 86Rb+ by HUAECs were examined
Unexpectedly, ouabain concentrations below 10 nM
stimu-lated86Rb+uptake by 15–20%, and in some experiments
by 50%, results that are consistent with a stimulation of
the pump Within the concentration range 1–10 nM, ouabain
induced a 2.5-fold stimulation (phosphorylation) of mito-gen-activated protein kinase (MAP kinase) After incuba-tion of HUAECs with ouabain for 12 h, the glycoside stimulated cell growth by 49 ± 4%, as measured by cell number, with a maximum response at 5 nM At similar concentrations, ouabain also increased ET-1 mRNA
abun-dance by 19.5 ± 3.1% The results indicate that, by influ-encingET-1 expression and release, ouabain may contribute
to the regulation of vascular tone The data also confirm that it is not a global inhibition of the sodium pump that
is involved in the mechanism of action of this cardiac glycoside
Keywords: endothelin; human umbilical cord endothelial cell (HUAEC); mitogen-activated protein kinase (MAP kinase); sodium pump; ouabain
Na+/K+-ATPase (the sodium pump) is a
membrane-embedded protein in all animal cells that couples ATP
hydrolysis to a vectorial transport of Na+and K+ ions
against their electrochemical gradient For each ATP
hydrolyzed, three Na+ions are moved out of the cytosol
and two K+ ions are taken up from the environment,
resultingin the formation and maintenance of a negative
membrane potential
The sodium pump is specifically inhibited by a series of
naturally occurringsteroids, termed cardiac steroids or
cardiac glycosides, such as ouabain or digitalis glycosides
such as digoxin or digitoxin [1] Inhibition of the sodium
pump by cardiac steroids is of clinical use, as application of
these substances, especially digitalis and its congeners, helps
to increase muscular contractility of the failingheart [2]
In recent years, various research groups succeeded not
only in isolatingcirculatingfactors that interact with the
sodium pump and inhibit86Rb+uptake (Rb+is a surrogate
for K+) but also in identifyingseveral of them as ouabain or its isomer [3–6] or as its congeners, such as digoxin [7,8], proscillaridin A [9], 19-norbufalin [10] and marinobufagenin [11] In addition, evidence was provided in several investi-gations that the level of so-called endogenous ouabain increases in the plasma upon excessive work and is present
in higher concentrations in the serum of hypertensive patients [12]
All of these data indicate that ouabain may be directly or indirectly involved in the regulation of vascular tone and possibly also in the pathogenesis of hypertension To address mechanisms that may be involved in the generation
of hypertension, we investigated the effects of ouabain on human endothelial cells in culture
Experimental procedures
Isolation and culture of human umbilical cord endothelial cells (HUAECs)
Human umbilical cords were collected within 2 h of birth and kept on ice in wash buffer [Hanks buffered salt solution (HBSS) containing20 mMHepes] until ready for cell isolation An artery was cannulated, washed with the above solution, filled with collagenase (CLS 2; Worthing-ton) in Pucks saline solution (Seromed, Berlin, Germany) containing20 mM Hepes, and incubated at 37C for
20 min to detach the endothelium The cells were washed out using20–50 mL wash buffer containing10% fetal bovine serum and centrifuged at 50 g, 4C for 10 min The cell pellet was suspended in 10 mL endothelial cell
Correspondence to G Scheiner-Bobis, Institut fu¨r Biochemie und
Endokrinologie, Fachbereich Veterina¨rmedizin,
Justus-Liebig-Universita¨t Giessen, Frankfurter Str 100, D-35392 Giessen,
Germany Fax: + 49 641 9938189, Tel.: + 49 641 9938180,
E-mail: Georgios.Scheiner-Bobis@vetmed.uni-giessen.de
Abbreviations: ET-1, endothelin; HUAEC, human umbilical cord
endothelial cell; HBSS, Hanks buffered salt solution; ECGM,
endothelial cell growth medium; MAP kinase, mitogen-activated
protein kinase.
(Received 17 October 2003, revised 16 January 2004,
accepted 26 January 2004)
Trang 2growth medium (ECGM; Promocell, Heidelberg,
Ger-many) and transferred to a gelatin-coated, 94-mm cell
culture dish After 4 h, the medium and any nonadherent
cells were aspirated and the medium replaced During
culture, the medium was replaced every 48 h After
the first passage, ECGM was mixed with M199
(Gibco, Eggenstein, Germany) to give a 2 : 1 ratio (v/v),
with an additional 2% fetal bovine serum supplement
(ECGM-2)
Measurement of Ca2+oscillations induced by ouabain
Relative changes in intracellular Ca2+concentration were
measured usingthe Ca2+-sensitive fluorescent dye, fura-2
The cells were loaded as described below with the
membrane-permeable acetoxymethylester form of the dye
(fura-2 AM; Molecular Probes, Leiden, the Netherlands)
which is then converted into nonpermeable fura-2 by
intracellular esterase activity
Glass coverslips of 18-mm diameter were coated with
0.1 gÆL)1 poly(L-lysine) (Seromed) for 30 min at 4C in
12-well plates before a wash with phosphate-buffered saline
(NaCl/Pi) Thereafter, 1.5· 105HUAECs were pipetted on
to each coverslip in 300 lL ECGM-2 and allowed to adhere
for 1 h at 37C under 5% CO2 before the addition of a
further 300 lL medium The medium was changed every
48 h thereafter
After 4 days of culture on the coverslips, the cells were
incubated for 1 h in 600 lL ECGM-2 containing2.5 lM
fura-2 AM, 0.01% (w/v) Pluronic F-127 (Molecular
Probes) at 37C, 5% CO2 Then the incubation medium
was carefully removed by a pipette and cells were washed
once with HBSS/20 mM Hepes Duringmicroscopy, the
cells were maintained in fresh HBSS/20 mMHepes
Imaging was carried out on an inverted microscope
(Olympus IX-50) equipped with an epifluorescence set-up
and an image analysis system (Till Photonics, Martinsried,
Germany) The emission above 470 nm was measured from
several regions of interest, each approximately the size of
one cell The cells were excited alternately at 340 nm and
380 nm, and the ratio of the emission signal at the two
excitation wavelengths was calculated
86
Rb+uptake at various extracellular ouabain
concentrations
HUAECs were plated at a density of 2· 104cells per well
of a 12-well plate (Greiner, Frickenhausen, Germany)
precoated with 1% gelatin (Bio-Rad, Munich, Germany)
and grown to confluency The cells were washed three times
in a potassium-free uptake medium containing(in mM)
NaCl 150, Hepes 10, glucose 10, RbCl 5.0, MgCl25.0, and
CaCl20.5, pH 7.0, and equilibrated in uptake medium with
various concentrations of ouabain for 30 min at 37C
Then, 1 lCi86Rb was added to each well, and incubation
was continued for an additional 60 min Afterwards, the
medium was aspirated and the cells washed three times with
ice-cold 0.1M MgCl2 to stop pump activity and remove
excess 86Rb+ The washed cells were then disrupted by
treatment with 10% (w/v) trichloroacetic acid at 4C for
1 h to release intracellular86Rb+, and the radioactivity in
the lysate was measured by liquid-scintillation counting
The86Rb+activity was normalized against the amount of protein [13] per well
86Rb+uptake was also investigated after preincubating the cells for 15 min with 1 lM protein kinase C inhibitor Ro-31-8425, 5 lM Na+-channel inhibitor tetrodotoxin,
500 lM mitochondrial ATP-sensitive K+ [mitoK(ATP)] channel inhibitor 5-hydroxydecanoate, or with 50 lM
Na+/K+/2Cl–cotransporter inhibitor bumetanide
Binding of ouabain to sodium pumps on the surface
of the HUAEC plasma membrane This experiment was performed to investigate whether ouabain treatment influences the sodium pump number on the cell surface The experimental conditions were the same as for the Rb+-uptake experiment After incubation for 30 min
in the uptake medium containingeither no ouabain or 1 nM
or 5 nM[3H]ouabain (6.7·
Amersham-Pharmacia, Freiburg, Germany), [3H]ouabain was added instead of radioactive Rb+to a final concentration of 100 nM and incubation was continued for another 30 min After-wards, the medium was removed by aspiration, and cells were washed twice in 500 lL ice-cold water Then cells were dissolved by incubation for 20 min at 70C in 500 lL 1M NaOH Radioactivity was counted in a liquid-scintillation counter after neutralizing250 lL of the solution with 250 lL
1MHCl and adding3 mL liquid-scintillation fluid
Assay of mitogen-activated protein kinase (MAP kinase) activation
HUAECs were plated at a density of 1.5· 104cells per well
of a 24-well plate and grown to confluency The cells were then serum-starved (i.e 0.5% serum) in a 2 : 1M199/ ECGM (basal medium) mix (ECGM-SF) for 48 h before the start of the experiment Serum starvation was necessary because otherwise serum components might induce MAP kinase activation independently of ouabain
Ouabain was added to the cells at various concentra-tions in ECGM-SF, and the cells were further incubated
at 37C for 30 min The cells were then lysed usinga commercially available cell lysis buffer (Cell Signaling Technology, Frankfurt, Germany) The lysates were screened for MAP kinase activation usingSDS/PAGE (10% acrylamide, 0.3% N,N¢-methylenebisacrylamide) [14] followed by Western blottingwith an antibody against phospho-p44/42 (Cell Signaling Technology), and the resultingsignal was visualized usingthe luminescent ECL system (Amersham Pharmacia) In all cases, the protocols of the providers were followed A positive control derived from HEK cells after stimulation with serum (Cell Signaling Technology) was also run in parallel Pre-stained proteins (Cell Signaling Technology) were used
as molecular mass markers The bands were relatively quantified usinga digital documentation system (Biostep, Jahnsdorf, Germany) and Phoretix TotalLab gel image analysis software (Biostep)
To ensure that changes in phosphorylation are not due to changes in the overall content of MAP kinase, parallel samples were probed followingthe above protocols with the only exception that in this case an antibody against the overall (phosphorylated and nonphosphorylated) MAP
Trang 3kinase (Cell Signaling Technology) was used in place of the
antibody against the phosphorylated forms of MAP kinase
The same cell extracts were also used here as a positive
control
Effect of ouabain on cell number
HUAECs were plated at a density of 2· 104cells per well of
a 12-well plate precoated with 1% gelatin (Bio-Rad) and
grown to confluency The cells were then incubated for 12 h
in a 2 : 1M199/ECGM mix with 0.5% fetal bovine serum
(ECGM-0.5) to minimize serum-related growth signals
before the start of the ouabain treatment Ouabain was
added to the cells at various concentrations in fresh
ECGM-0.5, and the cells incubated for an additional 12 h For
counting, cells were detached and dispersed in trypsin/
EDTA (Gibco) The trypsin was then neutralized using
2 : 1 M199/ECGM with 50% fetal bovine serum and
placed on ice Suspended cells were counted usinga
Neubauer haemocytometer
Reverse transcription
Total cellular RNA was isolated from HUAECs usingthe
RNeasy kit (Qiagen, Hilden, Germany) Then, cDNA
synthesis with Moloney murine leukemia reverse
transcrip-tase was carried out by followingthe protocol of the enzyme
provider (Promega, Mannheim, Germany)
PCR amplification
Changes in the expression of endothelin (ET-1) mRNA
transcripts were measured usingPCR The 421-bp region
between bases 646 and 1067 of NM_001955.1 (GenBank;
NIH, Bethesda, MD, USA), the human prepro-endothelin-1
gene, was amplified with the primers: 5¢-GACCGTGA
GAATAGATGCCAATGTGCT-3¢ and 5¢-CTCCTGCT
CTGATCCCAGCCAG-3¢ The sequences of primers
used for the detection of the sodium pump a1-mRNA,
a2-mRNA and a3-mRNA have been published [15]
Normalization was performed by comparison with
amplif-icates of the housekeeper gene, glyceraldehyde-3-phosphate
dehydrogenase, which was amplified in parallel usingthe
primers 5¢-TGGGGAAGGTGAAGGTCGGAGTCAA-3¢
(ET-1 FORW) and 5¢-TAAGCAGTTGGTGGTGCAG
GAGGCA-3¢ (ET-1 REV) to amplify the 469-bp region
between bases 62 and 531 of NM_002046.1 (GenBank), the
human glyceraldehyde-3-phosphate dehydrogenase gene
PCR amplification was performed in a gradient PCR
Master Cycler (Eppendorf, Hamburg, Germany) following
published protocols [15] After 24 amplification cycles, the
amplified DNA was separated by electrophoresis on a 1%
agarose gel in 40 mM Tris/acetate/2 mM EDTA buffer,
pH 8.5, visualized by ethidium bromide, and quantified
using a digital gel documentation system and thePHORETIX
TOTALLABgel image analysis software
Dot-blot measurement of ET-1 protein
A total of 300 lL cell culture supernatant was blotted under
vacuum through a dot-blot apparatus on to a nitrocellulose
membrane presoaked in Tris/NaCl The membrane was
then blocked in Tris/NaCl containing5% (w/v) skimmed milk for 1 h before three washes in Tris/NaCl containing 0.1% (v/v) Tween 20 This was followed by incubation with
a mouse primary monoclonal antibody to ET-1 (MA3-005; Dianova, Hamburg, Germany) diluted 1 : 2000 in Tris/ NaCl containing0.1% (v/v) Tween 20 for 1 h before another wash as above The membrane was then incubated with the secondary antibody, an anti-mouse IgG (NIF-825; Amersham-Pharmacia) diluted 1 : 2500 in Tris/NaCl con-taining0.1% Tween 20 The membrane was washed four times in the same buffer before luminescent detection of ET-1 dots usingthe ECL system (Amersham-Pharmacia) Dots were relatively quantified usingthe Phoretix TotalLab array measurement
Under the conditions used here, the signals follow a linear dose/response relation in the range 0.1–32 ng ET-1 per vial This was established in experiments usingET-1 protein as
a standard (QBiogene-Alexis, Gru¨nberg, Germany), which was dissolved in cell culture medium before beinginvesti-gated under otherwise identical conditions
Statistical analysis Statistical analysis of the results was carried out by an unpaired, two-tailed t test P < 0.05 indicates that results are significantly different from each other
Results
Immediate, nongenomic effects Effect of ouabain on Ca2+ concentrations in endothelial cells As low concentrations of ouabain have been shown
to cause low frequency Ca2+ oscillations in rat tubule cells [16], we were interested in investigating the effects
of ouabain on cytosolic [Ca2+] of HUAECs As shown in Fig 1B, ouabain at the low concentration of 1 nMinduced clear Ca2+oscillations in these cells with a period of about 4–8 min (Fig 1B), sometimes even greater (Fig 1C) First oscillations were observed after 4 min (Fig 1C) The cells showingoscillations did not appear to be synchronized Nevertheless, not all cells investigated displayed such oscillations In the best case, four of the six cells observed displayed Ca2+oscillations The average over three inde-pendent experiments was, however, about 38% In the absence of ouabain, however, control cells never showed any calcium oscillations (Fig 1A), indicating that the observed slow Ca2+oscillations were specifically induced by ouabain Effects of ouabain on ET-1 release ET-1 is synthesized as
a prepro-hormone and is stored in vesicles As vesicular exocytosis is induced by a rise in intracellular [Ca2+], we were interested in determiningwhether ouabain added to endothelial cells in culture might induce such a response Various concentrations of ouabain were added to the cell culture wells, and, after 10 min of incubation, the super-natant was collected and analyzed for its ET-1 content using the dot-blot method The data in Fig 2A show a stimula-tion of ET-1 release even at 1 nMouabain The rise in ET-1 release follows a hyperbolic dose/response relation and reaches a more than twofold stimulation at 50 nMouabain (Fig 2A)
Trang 4When extracellular Ca2+was omitted by replacingthe
HBSS/20 mMHepes with NaCl/Pi, ET-1 in the supernatant
was considerably reduced (Fig 2B) The same was observed
when the Ca2+channel blockers NiCl2(1 mM) and CdCl2 (1 mM) and the Na+/Ca2+-exchanger-specific inhibitor 2¢,4¢-dichlorobenzamil (0.1 mM) were included in HBSS/
20 mM Hepes and allowed to act on the HUAECs for
20 min before the addition of ouabain (Fig 2B)
Under all these conditions, ET-1 in the supernatant was about half of the amount detected in the controls and only 25% of the ET-1 secreted after ouabain stimulation Effects of ouabain on 86Rb+ uptake To investigate whether the observations made thus far are based on a global inhibition of the sodium pump, the uptake of86Rb+ into endothelial cells was determined as a function of the ouabain concentration Rubidium is recognized by the sodium pump and its uptake can easily be inhibited by ouabain and several of its congeners This experiment, however, produced a most unexpected result Ouabain at low concentrations not only failed to inhibit86Rb+uptake
Fig 1 Ouabain-induced Ca2+oscillations in HUAECs (A) Without
ouabain, (B) with 1 n M ouabain (arrowhead), or (C) with 10 n M
ouabain Cells were cultured on coverslips and loaded with the Ca2+
-sensitive dye fura-2 Traces shown are for individual cells that were
representative of most of the cells observed in a field of view Ouabain
or control treatment started at the arrowhead See Experimental
procedures for details (D) False-color image of endothelial cells
loa-ded with fura-2 The picture was taken at an excitation of 340 nm The
scale on the right shows a relative loading condition with Ca 2+
Fig 2 Effects of ouabain and extracellular Ca 2+ on ET-1 release from HUAECs (A) Accumulation of ET-1 in the medium of HUAECs was measured by the dot-blot immunological method Medium was col-lected after 10 min of incubation with the various concentrations of ouabain shown (bars represent ± SEM; n ¼ 6) (B) In the absence of extracellular Ca 2+ , ET-1 in the supernatant is reduced by 50% when compared with the control and is only 25% of the amount secreted after stimulation by 10 n M ouabain A mixture of the Ca2+channel blockers NiCl 2 (1 m M ), CdCl 2 (1 m M ) and the Na + /Ca 2+ -exchanger inhibitor 2¢,4¢-dichlorobenzamil (0.1 m M ) added before ouabain have a similar effect.
Trang 5by the endothelial cells, but with all HUAEC preparations
from various umbilical cords a stimulation of86Rb+uptake
was observed at low ouabain concentrations Although in
most cases the stimulation observed was 15–20% above the
control without ouabain, in a series of measurements with
HUAECs prepared from umbilical cord number 4,
stimu-lation of 86Rb+ uptake reached 50 ± 22% at 0.1 nM
ouabain and 49 ± 2% at 1 nMouabain over the control
without ouabain (Fig 3A)
To investigate the mechanism for the observed
stimula-tion of86Rb+uptake by ouabain, the same experiment was
carried out after preincubation of the cells for 15 min with
various substances known to be specific inhibitors of cellular
components Thus, tetrodotoxin, which specifically inhibits
Na+channels, was used to determine whether the observed
stimulation was due to a secondary stimulation of the sodium pump by Na+cations that enter the cell via these channels The protein kinase C inhibitor Ro-31-8425 [17] was used to see whether the observed stimulation of the
86Rb+uptake was the result of Na+/K+-ATPase phos-phorylation by this kinase, which in the past has been repoted to activate or inactivate the sodium pump The 5-hydroxydecanoate inhibitor of the mitochondrial ATP-sensitive K+ [mitoK(ATP)] channels [18] was used to investigate whether the increased accumulation of86Rb+ was the result of increased transportation of the cation into the mitochondria, and finally, bumetanide, the specific inhibitor of Na+/K+/2Cl–cotransporters [19], was used to investigate whether the observed stimulation of Rb+uptake was due to the stimulation of this uptake system As shown
in Fig 3B, however, none of these substances had any effect
on the stimulation of86Rb+uptake by ouabain when used
at concentrations reported to affect the various channels and enzymes described above
Finally, ouabain-bindingexperiments with whole cells were carried out to investigate whether the observed stimu-lation of86Rb+uptake is due to a translocation of sodium pumps from cytosolic compartments to the surface of the plasma membrane The experiments, however, did not indicate any differences in [3H]ouabain bindingto the membrane surface after the cells were preincubated with either 1 or 5 nM[3H]ouabain Whereas cell membranes that were not preincubated with ouabain bound 685 ± 58 fmolÆmg)1protein, ouabain bindingafter preincubation with either 1 or 5 nM ouabain was 638 ± 68 or 702 ± 54 fmolÆmg)1, respectively (all values are mean ± SEM; n¼ 6) MAP kinase activation by ouabain
In rat cardiomyocytes, ouabain has been shown to stimulate the MAP kinase reaction cascade [20,21] To investigate a similar mechanism in endothelial cells, MAP kinase stimulation on ouabain exposure was investigated
by Western blottingusingan antibody against the phos-phorylated form of p44/p42 MAP kinase Usingthis method, MAP kinase activation was clearly detectable in HUAECs after 30 min of stimulation (Fig 4A) The increase in the phosphorylated MAP kinase forms is not due to an increase in overall MAP kinase in the various preparations, as shown by an antibody to MAP kinase that does not distinguish between phosphorylated and unphos-phorylated forms of the enzyme (Fig 4B) Notably, MAP kinase stimulation was detectable at the low ouabain concentration of 1 nM Measured over the ouabain concentration range 1 nM to 1 lM, stimulation was 2 to 2.5-fold over control (Fig 4C)
Delayed effects Cell proliferation in response to ouabain Ouabain has been described as a mitogen that induces cell proliferation in the upper micromolar and low millimolar range After 12 h
of incubation, the cell number of HUAECs increased linearly with increasingouabain concentrations (from 0.1 to
5 nM), peakingat 5 nM(Fig 5) At this concentration, the relative increase was 49 ± 4% (P < 0.05) above the proliferation observed in the absence of the glycoside Cell
Fig 3 Stimulation of Rb + uptake by ouabain and effects of various
inhibitors The accumulation of 86Rb+ during1 h of treatment of
HUAECs was measured as described under Experimental procedures.
(A) Cells were treated duringthe86Rb+incubation with the indicated
concentrations of ouabain or control buffer (B) Preincubation of
HUAECs with 5 l M tetrodotoxin (TTX), 1 l M Ro-31-8425, 500 l M
5-hydroxydecanoate (5-HDA)
6 , or 50 l M bumetanide was carried out
for 15 min before addition of control buffer or 1 or 10 n M ouabain.
The two experiments (A and B) were carried out with primary cell
cultures prepared from two different umbilical cords While the
stimulation of up to 50% above background was only observed in the
series of experiments shown here (A), most commonly the stimulation
of 86 Rb + uptake ranged between 15% and 20% above control, as
shown in B (bars represent ± SEM, n ¼ 3–6; *P < 0.05).
Trang 6numbers declined in response to higher ouabain
concentra-tions, such that 20 nM ouabain inhibited proliferation
relative to that of untreated cells (14%)
Effects of ouabain onET-1 mRNA The human ET-1 gene
promoter region contains two active transcription
regula-tory sites: an AP-1-bindingsite (TGACTAA) at)108 to
)102 bp from transcription initiation and a
GATA-2-bindingsite (TTATCT) at)136 to )131 bp [22] As
signal-transduction pathways that activate MAP kinase also
activate various genes through these promoters, it was
important to address the question of whether ouabain could
cause a long-term upregulation in ET-1 mRNA biosyn-thesis As shown in Fig 6, ouabain at 10 nM stimulated ET-1mRNA concentrations in HUAECs by 19.5 ± 3.1% (mean ± SEM; n¼ 8) after 12 h, as determined by semiquantitative RT-PCR
Discussion
Cardiac steroids have a positive inotropic effect on the heart muscle The mechanism is thought to involve inhibition of the sodium pump, which results in a reduction in the sodium gradient This in turn has an impact on the transport activity
of the Na+/Ca2+ exchanger, which does not transport
Ca2+ ions out of the cytosol as effectively Thus, the resultingincreased cellular concentration of Ca2+is thought
Fig 5 Effect of ouabain on HUAEC cell number Cells growing on 12-well plates were treated with the indicated concentrations of oua-bain for 12 h, after which cell number was assessed as described under Experimental procedures (bars represent ± SEM; n ¼ 6).
Fig 6 Effect of 12 h exposure to various ouabain concentrations on ET-1 mRNA in HUAECs HUAECs were incubated for 12 h with the various concentrations of ouabain shown Then, mRNA isolated from HUAECs was transcribed into cDNA by a reverse transcriptase step, and, by using ET-1-specific primers, the abundance of the latter was analyzed by semiquantitative PCR (bars represent ± SEM; n ¼ 8;
*P < 0.05).
Fig 4 MAP kinase activation of endothelial cells by ouabain (A) Cells
were incubated with the ouabain concentrations shown for 30 min as
described under Experimental procedures Thereafter, 20 lgprotein
was separated by SDS/PAGE and probed by an antibody to
phospho-p44/42 The resultingsignal shown was obtained by the luminescent
ECL system A positive control, commercially available phospho-p44/
42, was run in parallel (lane 5) Relative amounts of phosphoproteins
were analyzed with a digital documentation system and a gel image
analysis software (B) The conditions were the same as in (A) except an
antibody to the total (nonphosphorylated) MAP kinase was used.
(C) Relative amounts of MAP kinase activated by 1 n M ouabain Data
are derived from experiments similar to those described in (A) (bars
represent ± SEM; n ¼ 5).
Trang 7to stimulate the contractile elements of the heart or vascular
muscle and increase contractility
Although thus far plausible, the model implies that the
increase in contractility by cardiac steroids is associated with
inhibition of the pump and that the rise in cytosolic Na+
concentrations occurs before the rise in cytosolic Ca2+
Several investigations, however, do not appear to support
this mechanism Not only is inhibition of the pump not a
prerequisite for the positive inotropic effect [23], but
ouabain was shown to stimulate Ca2+transients in arterial
smooth muscle without raisingcytosolic [Na+] [24]
Our observations shown in Fig 3 are consistent with
these results.86Rb+uptake by HUAECs was not inhibited
by ouabain, but at 1 nM or 10 nM concentrations of the
steroid, the uptake was stimulated by at least 15% (Fig 3B)
and in some cases up to 50% over control uptake (Fig 3A)
Although the molecular basis for this observation is not yet
clear, effects of ouabain at low concentrations that do not
correlate with sodium pump inhibition have been described
in several investigations, including induction of positive
inotropic effects [23] or cytosolic [Ca2+] elevation [24] The
stimulation of Rb+uptake that we observed, however, was
not simply the result of cell swelling(data not shown) or
sodium pump recruitment to the plasma membrane
from cytosolic stores It was also not affected by the
Na+/K+/2Cl–cotransporter inhibitor bumetanide or by the
mitoK(ATP) channel-specific blocker 5-hydroxydecanoate
As these two major K+uptake systems are not involved in
the observed stimulation of Rb+uptake, the sodium pump
is the most likely route for86Rb+uptake Stimulation of the
sodium pump by protein kinase C [25,26], however, can be
excluded, as no effect was observed in the presence of the
protein kinase C-specific inhibitor Ro-31-8425 As a direct
activation of the sodium pump at very low concentrations
of ouabain has been shown in a recent investigation [27], this
possibility is currently the most likely explanation for the
ability of ouabain to stimulate86Rb+uptake
Several reports [20,21,28,29] show that ouabain induces
signalingcascades, resultingin both nongenomic and
genomic effects The most apparent of the nongenomic
effects described thus far are the rise in cytosolic Ca2+
concentration associated with slow Ca2+ oscillations,
activation of NF-jB, activation of ERK1/2 (MAP kinase
p42/p44) by interactions of the sodium pump with the
epidermal growth factor (EGF) receptor, or the release of
reactive oxygen species from mitochondria [28]
Consistent with these results, we determined that ouabain
at low concentrations stimulates MAP kinase p42/p44
phosphorylation (activation) by 2 to 2.5-fold Within the
same concentration and time range, it also induces Ca2+
oscillations of an approximate frequency of 8 min and a
duration lastingfor more than 40 min (until the experiment
was terminated; Fig 1) This phenomenon, which was first
described for rat renal proximal tubule cells [16], is clearly
confirmed here for human endothelial cells Whether such
Ca2+oscillations are also responsible for the induction of
the positive inotropic effect on the heart has still to be
investigated In smooth muscle cells of rat mesenteric
arteries, Ca2+transients were induced at low (3–100 nM)
concentrations of ouabain [24] Here, however, it was
thought that these effects are mediated by the a3 isoform of
the sodium pump a subunit, which binds ouabain with high
affinity Nevertheless, a3 and a2 mRNA were not detectable
in HUAECs by RT-PCR (not shown), leadingto the con-clusion that in the experiments described here the abundant a1 isoform is the most likely mediator of the effects of ouabain at low concentrations
HUAECs, like all endothelial cells, regulate the muscular tone of the underlyingarteries by releasingeither the vasoconstrictive ET-1 or the vasorelaxant NO Takinginto consideration the fact that ouabain in animal models applied over a longperiod of time causes hypertension [30], we investigated here its effects on ET-1 release and expression in HUAECs
ET-1, the only member of the endothelin peptide family produced by endothelial cells, is stored in vesicles before it is released towards the underlyingsmooth muscle cells of the artery As this substance is a potent vasoconstrictor and growth promoter of vascular smooth muscle cells via the
ETAreceptor, and because vesicular fusion and release has been shown in numerous cases to be triggered by Ca2+, we were interested in investigating whether ET-1 release from HUAECs to the medium might be stimulated by ouabain Indeed, a few minutes after application, ouabain causes ET-1 release into the medium (Fig 2A) This release was dependingon extracellular Ca2+, as its absence, or the presence of Ca2+channel inhibitors such as Ni2+or Cd2+ and the Na+/Ca2+-exchanger blocker 2¢,4¢-dichlorobenz-amil considerably reduced ET-1 release (Fig 2B) This findingis in good agreement with earlier reports showing that ET-1 release depends on extracellular Ca2+[31,32]
We do not know yet, however, whether or not ouabain stimulates ET-1 secretion also in vivo It is possible that ouabain – should it indeed be an endogenously produced hormone, as proposed by many – influences vascular tone
by such a mechanism and regulates blood pressure Elevated ET-1 production has been shown in human vessels subjec-ted to increased pressure and shear stress [33], and increased levels of circulatingET-1 are associated with pulmonary and essential hypertension [34,35]
Besides these immediate, nongenomic effects, ouabain has been shown previously to increase mitotic activity [36–38], a result complemented by the newer findings of ouabain-induced MAP kinase activation and induction of
Ca2+ oscillations followed by the translocation of the transcriptional factor NF-jB into the nucleus [16] In agreement with these results, we also showed that ouabain
at low concentrations stimulates the growth of HUAECs
by 49 ± 4% as measured by cell number (Fig 5) This stimulation was observed after 12 h of incubation At the same time, mRNA codingfor ET-1 is also increased, possibly because of ouabain stimulation of MAP kinase, which is known to stimulate the Fos and Jun transcription factors to form activator protein-1 (AP-1) heterodimers
As the human ET-1 gene promoter region contains an AP-1-bindingsite regulatingtranscription [22], this may be responsible for the apparent increased ET-1 gene transcrip-tion we observed In additranscrip-tion, the bovine ET-1 gene promoter region is also known to contain an NF-jB-responsive region [39] If this site were also to be present in the human ET-1 promoter region, the Ca2+oscillations we show to be induced by ouabain may also contribute to the observed ET-1 upregulation Although the increase in ET-1mRNA concentrations appears to be rather modest
Trang 8(19.5 ± 3.1%), ET-1 in the serum of hypertensive patients
is no more than 13% above normal [40] Thus, an increase
in ET-1 by this margin could be one of the reasons for the
observed induction of hypertension by ouabain in animal
experiments [30]
In conclusion, the results presented here clearly show that
ouabain can act in a hormone-like manner on endothelial
cells It becomes effective at concentrations that are within
the same range as the effective concentrations of other
hormones, includingthe steroid hormones, and, like the
latter, it also induces both genomic and nongenomic effects
that are independent of its ability to inhibit the sodium
pump at higher concentrations Assuming that ouabain
might be endogenously produced, as has been shown in
some instances [9,41], it could act as a hormone involved in
blood pressure regulation by fine-tuning and controlling
ET-1 release and expression
Acknowledgements
R S was supported through the Giessen Graduiertenkolleg Molekulare
Veterina¨rmedizin; G S B is supported through a grant from the
Deutsche Forschungsgemeinschaft (DFG), SCHE 307/5-1.
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