Cellular Biology |
From the Departments of Pharmacology and Toxicology (E.J.M., B.U.R.) and Anatomy and Cell Biology (G.W.W., J.J.B.), Indiana University School of Medicine, Evansville, Ind.
Correspondence to B. Raess, Department of Pharmacology and Toxicology, Indiana University School of Medicine, 8600 University Blvd, Evansville, IN 47712. E-mail braess{at}iupui.edu
| Abstract |
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100 compared with other unopposed calmodulin-stimulated
processes. Together, these findings support evidence for the presence
of a calmodulin-stimulated plasma membrane
(Ca2++Mg2+)-ATPase activity in cultured porcine
aortic endothelial cells.
Key Words: endothelium aorta (Ca2++Mg2+)-ATPase calmodulin calmidazolium
| Introduction |
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Much of the available information on endothelial and vascular smooth muscle Ca2+ regulation to date focuses on mechanisms of Ca2+-induced endothelial stimulation, agonist-induced stimulation of capacitative Ca2+ uptake, intracellular Ca2+ compartmentalization into endoplasmic reticulum (ER) and mitochondria, and the Na+/Ca2+ exchange mechanisms. Studies in these areas typically involve measurements of calcium-induced fluorescence or patch clamp current measurements, which show endpoint or temporal changes in levels of intracellular Ca2+ on a cell-to-cell basis, but are difficult to interpret in terms of exact compartmentalization mechanisms responsible for the observed Ca2+ change(s).6 7 8 9 10 11 Because of the relative difficulty in obtaining sufficiently large amounts of plasma membranes, and because of the abundance of ectonucleotide phosphohydrolase activities in endothelial cells12 13 14 15 and various other tissues,16 17 18 19 20 less attention has been paid to the regulation of the plasmalemmal Ca2+ extrusion pump in these cells. Recent inferential evidence, using relatively nonselective pharmacological inhibitors, suggests a functional role for both the Na+/Ca2+ exchanger and Ca2+ pump in intracellular Ca2+ removal.8 21 However, no information is available about the kinetic details of the (Ca2++Mg2+)-ATPase mechanism, its regulation by calmodulin, or its relative contribution to the overall nucleotide phosphohydrolase activity in endothelial cells.
Thus, the purpose of this study was to identify and measure the (Ca2++Mg2+)-ATPase activity of the plasma membrane Ca2+ pump of large vessel endothelial cells. Moreover, for the first time in endothelial cells, we were able to demonstrate calmodulin activation of the (Ca2++Mg2+)-ATPase. These findings should provide a basis for the future evaluation of Ca2+ transport regulation, which, together with the Na+/Ca2+ exchanger and intracellular Ca2+ regulatory systems (ER, mitochondria, nucleus), work to keep intracellular free Ca2+ at physiologically acceptable levels.
| Materials and Methods |
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Plasma membranes from porcine aortic endothelial cells (PAECs), grown in roller bottles, were isolated using a modification of a previously described method.23 The resulting plasma membranes were suspended in an equal volume of 0.25 mol/L sucrose, pH 7.5, and stored on ice or frozen at -80°C. Membrane protein was estimated using the Lowry method.24
Endothelial membrane ATPase activities were assessed at 37°C in a solution containing (in mmol/L) histidine 18, imidazole 18, NaCl 80, KCl 15, MgCl2 0.3, ouabain 0.1, CaCl2 0.2, EGTA 0.1, and ATP 3, as well as a membrane protein concentration of 5 µg/mL unless otherwise noted. Whereas (Ca2++Mg2+)-ATPase activity was assessed using the standard incubation solution, (Mg 2+)-ATPase activity was measured by the omission of Ca2+ from the incubation mixture, and (Na++K+)-ATPase activity was measured by omitting ouabain and Ca2+. Termination of reactions after 60 minutes was accomplished by addition of an equal volume of 2% SDS. Inorganic phosphate (Pi) hydrolyzed by the various ATPases was determined by a modification of the colorimetric method of Fiske and Subbarow25 described by Sadrzadeh et al.26
White membrane ghosts were prepared as described,27 and ATPase activities were assessed as in References 28 and 2928 29 , with variations to standard incubation conditions noted in figure legends.
Endothelial cells immunoreactive for (Ca2++Mg2+)-ATPase were stained using the 5F10 mouse monoclonal antibody and a modification of the avidin-biotin technique described by Borke et al.30 Aortic tissue was fixed in 4% buffered paraformaldehyde, embedded in polyethylene glycol, and sectioned in a cryostat.31 After permeabilizing and blocking steps, the sections were incubated for 1 hour in 5F10 antibody diluted 1:30, for 1 hour in biotin-conjugated goat anti-mouse IgG diluted 1:500, and for 30 minutes in streptavidin-peroxidase conjugate diluted 1:500. Finally, the sections were reacted with H2O2 and 3,3'-diaminobenzidine, counterstained with 0.3% methyl green, and mounted in Permount. This same procedure was used to stain (Ca2++Mg2+)-ATPase in the cultured PAECs and PAEC plasma membrane fractions fixed to glass slides.
Kinetic indices were derived from computer-fitted nonlinear regression logistic curves, as follows: y=y0+[a/1+(x/x0)b]. Probability of significant differences (probability value) were calculated using the 2-tailed Student t test distribution. Unless otherwise stated, all values for data listed are presented as mean±SEM of n independent experiments. Where missing, error bars were smaller than symbol size.
An expanded Materials and Methods section is available online at http://www.circresaha.org.
| Results |
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Figure 2
further depicts the
interrelationship of the Ca2+ and
Mg2+ activation of the ATPase. Full-spectrum
concentration-effect curves for MgCl2 in the
absence of Ca2+ and in the presence of ouabain
(10-4 mol/L) showed a specific
(Mg2+)-ATPase activity () with a
Vmax of 371.7±16.4 nmol
Pi/mg membrane protein ·
min1 at 3 mmol/L
MgCl2. When omitting ouabain, again in the
absence of Ca2+, the
Mg2+-dependent
(Na++K+)-ATPase activity
(
) with a Vmax of 131.4±5.9 nmol
Pi/mg membrane protein ·
min1 at 3 mmol/L
MgCl2 became apparent. Based on logistic
concentration-effect analysis, curve fitting yielded 2 apparent
dissociation constants of
0.04 mmol/L and 1.17±0.18
mmol/L for MgCl2.
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The addition of 0.2 mmol/L CaCl2 to the
incubation mixture (including 10-4 mol/L
ouabain) revealed a specific (Ca2+)-ATPase
activity (
) that was inhibited in a concentration-dependent manner
by Mg2+ with an IC50 of
0.152±0.004 mmol/L MgCl2 and nearly
complete inhibition at 10 mmol/L MgCl2. As
in Figure 1
, the presence of 10-8 mol/L
exogenous calmodulin (
) did not appear
to alter this relationship significantly. Larger amounts of exogenously
added calmodulin (up to 10-5 mol/L,
not shown) also had no stimulatory effects.
To further characterize the (Ca2+)-ATPase and
(Mg2+)-ATPase activities of PAEC plasma
membranes, full-spectrum concentration-effect curves for substrate
activation by ATP were assessed as shown in Figure 3
. In the presence of either
Ca2+ (
) or Mg2+ (
),
the preparations exhibited activity maxima in the 0.3 to 1 mmol/L
ATP range with apparent dissociation constants of
100 µmol/L
ATP. The presence of a combination of the 2 ions in the incubation
medium, ie, 0.3 mmol/L MgCl2 and 0.2
mmol/L CaCl2, produced both a noticeable decrease
in maximal enzyme velocity and a decrease in substrate affinity for
ATP. Again, in accordance with data shown in Figures 1
and 2
, the addition of exogenously added calmodulin did
not affect the
(Ca2++Mg2+)-ATPase
activity.
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Contributions of smooth endoplasmic reticular and mitochondrial
ATPase activities were ruled out on the grounds that sodium azide
(10-3 mol/L), oligomycin B (20 µg/mL),
thapsigargin (10-6 mol/L), and
cyclopiazonic acid (10-4 mol/L) did
not affect enzyme activity significantly (P>0.05).
(Na++K+)-ATPase activity
was inhibited by the presence of 10-4 mol/L
ouabain or 20 µg/mL oligomycin B and thus could not have contributed
to the overall ATPase activity. Sodium orthovanadate also inhibited the
(Na++K+)-ATPase activity
but did not affect the (Ca2+)- or
(Mg2+)-ATPase activity at the concentration of
0.5 mmol/L. Reactive blue 2 inhibited both
Mg2+- and
Ca2+-activated ATPase activities but not
the (Na++K+)-ATPase
activities (Table
).
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Because the
(Ca2++Mg2+)-ATPase of the
plasma membrane Ca2+ pump and the
(Ca2+)-ATPase and/or
(Mg2+)-ATPase activities associated with
ecto-ATPases have different substrate specificities, we compared human
red cell plasma membrane and PAEC plasma membrane preparations as to
activation by several other 5'-nucleotides. Figure 4
shows that, whereas the red cell
(Ca2++Mg2+)-ATPase activity
of the transport enzyme is relatively selective for ATP,
endothelial Mg2+- and
Ca2+-activated
ectonucleotide phosphohydrolase activities were capable
of hydrolyzing cytidine, guanosine, inosine, and uridine triphosphates,
and to a lesser extent adenosine diphosphate, but not
adenosine monophosphate. As expected, ATP was the only
substrate for the
(Na++K+)-ATPase activity in
both types of preparations.
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Without any apparent evidence of a calmodulin-sensitive
fraction of a PAEC plasma membrane
(Ca2++Mg2+)-ATPase activity
(Figures 1 through 4![]()
![]()
![]()
), and because cells in
culture often cease to express functional proteins with repeated
passage, it was necessary to establish that (1) calmodulin
was present in the preparations and (2) it could activate a
Ca2+-activated,
Mg2+-dependent ATPase activity. Therefore, a
simple, 3-pronged approach was used to establish that
calmodulin was present and that it was capable of
stimulating a Ca2+ transportrelated
(Ca2++Mg2+)-ATPase
activity.
First, by harvesting, separating, and dialyzing soluble intracellular PAEC contents, we affirmed the presence of calmodulin in a simple crossover experiment using the calmodulin-sensitive human red cell (Ca2++Mg2+)-ATPase preparation. The red cell enzyme could be fully and indistinguishably activated by the PAEC culture extract and/or commercially purified calmodulin from bovine testes (not shown).
Second, we used an immunohistochemical approach to identify the
physical presence of
(Ca2++Mg2+)-ATPase using
the F510 monoclonal antibody. As shown in Figure 5
, porcine endothelial
cells in the intact aorta and in culture for 7 days both displayed
immunoreactivity that was specific for the Ca2+
pump epitopes. This immunoreactivity was present in the isolated
plasma membrane fractions as well (not shown).
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Finally, to demonstrate the presence of a
calmodulin-sensitive
(Ca2++Mg2+)-ATPase activity
in PAEC plasma membrane, preparations were preincubated for 10 minutes
at 37°C with increasing concentrations of
1-(bis-[4-chlorophenyl]methyl)-3-(2-[2,4-dichlorophenyl]-2-[2,4-dichlorobenzyloxy]ethyl)-1H-imidazolium
chloride (calmidazolium, compound R24571). This
decreased (Ca2++
Mg2+)-ATPase activity in a
concentration-dependent manner from 138.3 nmol
Pi/mg membrane protein ·
min1 (the extrapolated maximal value from the
logistic concentration response curve fit; actual determined value was
143.0 nmol Pi/mg membrane protein ·
min1) by 96% to 5.8 nmol
Pi/mg membrane protein ·
min1 (Figure 6
, inset). The IC50 for this
calmidazolium effect was calculated to be
1.15±0.04 · 10-5 mol/L. On the basis of
these data, 10-5 mol/L
calmidazolium was chosen as the standard
preincubation addition to inhibit a
calmodulin-activated fraction of the
(Ca2++Mg2+)-ATPase
activity. Subsequent titration of exogenously added
calmodulin re-established maximal activity to 71.93±0.36
nmol Pi/mg membrane protein ·
min1 (Figure 6
, 100%) with a
half-maximally activating concentration of
3.17±0.36 ·
10-7mol/L calmodulin. To validate
this retitration approach in the presence of the calmodulin
antagonist, an analogous protocol with a red cell membrane
preparation prepared with high osmotic strength hemolysis was used.
Human red cells lysed in 300 mmol/L imidazole buffer yielded
"high-activity
(Ca2++Mg2+)-ATPase"
membranes (36.64±0.54 nmol Pi/mg membrane
protein · min1 [mean±SD; n=2]), which
were minimally responsive to exogenously added calmodulin
but, like the PAEC membranes, could be inhibited by the addition of
10-5 mol/L calmidazolium.
This inhibition, too, was overcome by retitration of
calmodulin similar to the data shown in Figure 6
with PAEC membranes (not shown). However, in the case of the red cell
membranes, maximal reactivation required 3 ·
10-6 mol/L calmodulin and the same
half-maximal stimulation of
3 · 10-7
mol/L as for PAEC membranes in Figure 6
.
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To ascertain that the reactivation of the
(Ca2++Mg2+)-ATPase by
calmodulin is specific for this enzyme and does not affect
the nonspecific Ca2+- or the
Mg2+-activated ecto-ATPases, reactivation
by calmodulin was attempted in the absence of any
Mg2+ addition
([Ca2+]-ATPase activity) and in the absence of
any Ca2+ addition
([Mg2+]-ATPase component). Figure 7
clearly indicates that, as expected for
a calmodulin-stimulated
(Ca2++Mg2+)-ATPase
activity, both cations are needed to see activation by
calmodulin in a dose-dependent manner.
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| Discussion |
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To build on these largely preliminary results and to examine these ion-transport mechanisms in a cardiovascular and atherogenesis-relevant tissue directly, large-vessel endothelial cells were cultured and scaled up to yield copious amounts of plasma membranes available for biochemical characterization. Although all plasma membranes of eukaryotic cells investigated are thought to possess an active, primary Ca2+ extrusion pump, little direct functional evidence exists for its existence in endothelial cell preparations. Hence, the purpose of the present study was first to establish the presence of a functional (Ca2++Mg2+)-ATPase in cultured PAECs, and second to delineate some of its biochemical properties. However, unlike the case with model single-membrane systems such as the red cell, the task in endothelial cells is complicated by the abundant presence of ectonucleotide phosphohydrolases associated with the extracellular degradation of 5'-nucleotides, which are largely activated by the same divalent cations that also activate p-type transport ATPases.12 13 14 15
In this communication, the term
(Ca2++Mg2+)-ATPase activity
is used either when referring to the Ca2+
translocating enzyme or when referring to an ATPase activity in the
presence of both ions, Ca2+ and
Mg2+. In case of the pump enzyme, the activity is
known to be Ca2+-activated and
Mg2+-dependent. In the case of the ecto-enzyme,
the 2 ions, Ca2+ and Mg2+,
are capable of activating the enzyme activity each in their own right
but appear antagonistic when the data are analyzed
as described. The overwhelming presence of nonpump ATPase activity is
best exemplified by the ion and substrate activation data in Figures 1 through 3![]()
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, which provide no evidence for the
functional presence of a calmodulin-stimulated
(Ca2++Mg2+)-ATPase
activity. This lack of calmodulin responsiveness is not
without precedence, because similar findings have been reported in
other tissues.39 40 41 42 Several explanations could account
for these findings. First, extensive proteolytic degradation of the
transport ATPase during membrane preparation could render the enzyme
inactive or, alternatively, partial proteolytic digestion of the
autoinhibitory calmodulin-binding domain could
yield a permanently activated enzyme.43 44
However, addition of protease inhibitors such as
phenylmethylsulfonyl fluoride, leupeptin, and aprotinin during
membrane isolation and preparation did not appear to make a difference
in terms of calmodulin activation (results not shown).
Besides proteolytic digestion of regulatory domains of the membrane
protein, highionic strength membrane preparation leading to possible
dimerization of ATPases could also render a fully activated
enzyme that would be unresponsive to added
calmodulin.45 46 47 Because it has been shown
that reconstitution of the purified red cell enzyme in
phosphatidylcholine and other acidic phospholipids yields a fully
activated conformational state that is unresponsive to
exogenously added calmodulin,48 49 highly
acidic phospholipid microenvironments could also be responsible for the
completely activated state of the enzyme. Moreover, the cell
harvesting and membrane preparation by several different
methods50 51 in the absence and the presence of
Ca2+ chelating agents also yield apparent
calmodulin-insensitive preparations. This suggests that the
particular preparation method used in the present work is not
responsible for the apparent lack of calmodulin regulation
of the ATPase activity. Furthermore, membrane preparations from 2
separate bovine pulmonary artery endothelial
cell lines prepared in a manner similar to that of those described here
yielded comparable quantitative and qualitative results with regard to
the lack of calmodulin activation of a
(Ca2++Mg2+)-ATPase
activity.
This prompted us to ask whether calmodulin was present in these cells in the first instance and whether it is functional in activating other known calmodulin-regulated processes. Furthermore, we felt it was important to show immunoreactivity with an antibody specific for calmodulin-regulated (Ca2++Mg2+)-ATPase activity, and if so, that we could antagonize the enzyme activity with a known, potent calmodulin antagonist.
As expected, isolation of soluble cellular PAEC contents, and their
subsequent addition to a calmodulin-sensitive red cell
(Ca2++Mg2+)-ATPase
preparation, indicated that calmodulin was present in
PAECs (results not shown). More importantly, immunohistochemical
evidence as seen in Figure 5
clearly indicated cross-reactivity
with a monoclonal antibody directed toward erythrocyte
calmodulin-stimulated
(Ca2++Mg2+)-ATPase in the
endothelial cell layer of intact aortic tissue, plasma
membranes of cultured PAECs, and isolated plasma membrane fractions.
However, none of these observations by themselves were deemed of
sufficient veracity to suggest the functional presence of a
calmodulin-stimulated
(Ca2++Mg2+)-ATPase activity
associated with the extrusion pump. Thus, using pharmacological
antagonism by treating the membrane preparation with
calmidazolium, we were able to partially decrease
the Ca2+-activated and
Mg2+-dependent fraction of the ATPase activity.
This decrease in activity could be reversed completely by the addition
of exogenous calmodulin (Figure 6
), although,
because of the presence of the antagonist, the apparent
Kd for calmodulin was about
3 · 10-7 mol/L. This is a roughly 50
to100 times higher concentration than that needed to activate
other calmodulin-sensitive processes in the absence of the
competitive calmodulin antagonist
calmidazolium (eg, Reference 2929 ). It is
interesting to note that it required only, relatively speaking,
10-6 mol/L calmodulin to completely
surmount inhibition by 10-5 mol/L
calmidazolium. A possible explanation for this may
be that the lipophilic inhibitor has a tendency to
intercalate in the lipid bilayer of the membrane, and thus reduces the
actual free concentration that can interact with
calmodulin. Alternatively or additionally,
calmodulin may bind >1 molecule of
calmidazolium. The former may also explain that the
antagonism of
(Ca2++Mg2+)-ATPase by
calmidazolium was not entirely selective, because,
at higher concentrations, calmidazolium also
inhibited (Mg2+)-ATPase and
(Na++K+)-ATPase activity.
Nevertheless, readdition of calmodulin and the consequent
restoration of full (Ca2++
Mg2+)-ATPase activity argues in favor of the
presence of a calmodulin-regulated,
Ca2+ transportassociated enzyme. This fact is
reinforced in Figure 7
by comparison of the replotted data from
Figure 6
(filled circles) to ATPase activities that are
activated by either Ca2+ or
Mg2+ independently (open symbols) but do not
respond to calmodulin stimulation after
calmidazolium exposure.
In summary, using both immunohistochemical and pharmacological antagonist evidence, this study demonstrates the presence of a calmodulin-stimulated (Ca2++Mg2+)-ATPase in PAECs. While the majority of the ATP hydrolyzing activity of PAECs is associated with Ca2+- and/or Mg2+-activated ecto-ATPases, a small but significant fraction can be shown to be inhibited by calmidazolium. In the presence of Ca2+ and Mg2+, this fractional inhibition can be reversed by exogenously added calmodulin in a concentration-dependent fashion, thus suggesting the presence of a calmodulin-stimulated (Ca2++Mg2+)-ATPase activity likely to be associated with the Ca2+ transport extrusion pump.
| Acknowledgments |
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Received May 12, 1999; accepted November 3, 1999.
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