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Circulation Research. 2000;86:191-197

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(Circulation Research. 2000;86:191.)
© 2000 American Heart Association, Inc.

Cellular Biology

Pharmacological and Immunohistochemical Characterization of Calmodulin-Stimulated (Ca2++Mg2+)-ATPase in Cultured Porcine Aortic Endothelial Cells

Elizabeth J. McConnell, Gary W. White, James J. Brokaw, Beat U. Raess

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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 *Abstract
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Abstract—Plasma membrane (Ca2++Mg2+)-ATPase and Ca2+ transport activities, best characterized in human erythrocytes, are stimulated by calmodulin and thought to play a crucial role in the termination of cellular Ca2+ signaling in all cells. In plasma membranes isolated from cultured porcine aortic endothelial cells, the (Ca2++Mg2+)-ATPase was not readily measured. This is in part because of an overabundance of nonspecific Ca2+- and/or Mg2+-activated ecto–5'-nucleotide phosphohydrolases. Moreover, addition of exogenous calmodulin (10-9 to 10-6 mol/L) produced no measurable stimulation of ATPase activities, suggesting a permanently activated state or, alternatively, a complete lack thereof. To establish and verify the presence of a calmodulin-regulated (Ca2++Mg2+)-ATPase activity in these endothelial cells, immunohistochemical localization using a monoclonal mouse anti–(Ca2++Mg2+)-ATPase antibody (clone 5F10) was applied to intact pig aorta endothelium, cultured endothelial monolayers, and isolated endothelial plasma membrane fractions. This approach clearly demonstrated Ca2+ pump immunoreactivity in each of these preparations. To confirm functional calmodulin stimulation of the (Ca2++Mg2+)-ATPase, 10-5 mol/L calmidazolium (R24571) was added to the isolated plasma membrane preparation, which lowered the (Ca2++Mg2+)-ATPase activity from 143.0 to 78.15 nmol Pi/mg protein · min–1. This calmidazolium-reduced activity could then be stimulated 113.1±0.8% in a concentration-dependent manner by the addition of exogenous calmodulin (10-7 to 2x10-6 mol/L) with an EC50 of 3.45±0.04x10-7 mol/L (n=4). This represents a competitive lowering of the apparent calmodulin affinity by {approx}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
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Mounting evidence in the biomedical literature suggests a pivotal role for the vascular endothelium in the pathophysiology of atherogenesis, blood pressure control, vasculitis, thromboembolic events, and angiogenesis. Intracellular Ca2+ is involved in all stages of the cell cycle and many signal transduction schemes and, eventually, orchestrates the events leading to apoptosis.1 In particular, Ca2+ compartmentalization and extrusion appear to be key regulatory processes in cellular Ca2+ signaling2 3 and are thought to play important roles in pathogenic processes such as atherogenesis (eg, see Reference 4 ). Today, much of the evidence regarding endothelial tissue function is derived from cells of various origins propagated in cell culture. This is an exceedingly valuable means of generating relatively large numbers of homogenous and harvestable cells. However, a potential drawback with this system is the apparent loss of some normal cell functions and physiological characteristics on repeated passages of cultures.5 Nonetheless, as long as it can be ascertained that the system under study is functionally expressed, cultured cells can serve as useful models for investigating ion-transport and regulatory mechanisms.

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 ecto–nucleotide 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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 *Materials and Methods
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 down arrowReferences
 
Endothelial cells from descending aorta of freshly slaughtered pigs were removed by scraping, collected in HBSS without Ca2+ and Mg2+, pelleted, resuspended in culture media, plated in 24-well tissue culture plates, and incubated in humidified 5% CO2 at 37°C. On confluency (6 to 7 days), cells were subcultured at a 1:5 ratio by conventional trypsinization. Cultures were identified by their typical cobblestone appearance and density-dependent inhibition after serial passage. Definitive verification was obtained using a modification of a previously reported method for the uptake of acetylated LDL.22

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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 up arrowMaterials and Methods
 *Results
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Porcine aortic apical and basolateral endothelial plasma membranes isolated by sucrose gradient centrifugation exhibit a nucleotide-triphosphohydrolase activity that is activated by Ca2+ and/or Mg2+. Figure 1Down shows the CaCl2 concentration-effect relationship in the absence and the presence of 0.3 mmol/L MgCl2 and 10-8 mol/L calmodulin. Increasing additions of CaCl2 to the incubation medium without Mg2+ produced a sigmoid concentration-effect curve with a maximal enzyme velocity (Vmax) of 796.6±0.7 nmol Pi/mg membrane protein · min–1 and an apparent dissociation constant of 248.0 µmol/L CaCl2 with a Hill coefficient of 1.51. In the presence of 0.3 mmol/L Mg2+, Vmax was reduced by 37.5% to 517.2±19.8 nmol Pi/mg membrane protein · min–1 with an approximate doubling of the apparent dissociation constant for Ca2+ to 422.3±31.5 µmol/L. The presence of exogenous calmodulin did not affect either situation significantly, in that it did not change Vmax (P>0.2) or the affinity (P>0.2) for Ca2+, as would be expected for a calmodulin-stimulated (Ca2++Mg2+)-ATPase activity.



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  		Figure 1. PAEC plasma membrane (Ca2+)-ATPase activity. Ca2+ concentration-effect relationship in the absence ({circ}, •) and the presence ({square}, {blacksquare}) of 0.3 mmol/L MgCl2. {circ} and {square} represent the absence of exogenously added calmodulin, and • and {blacksquare}, addition of 10-8 mol/L exogenous calmodulin. Activities of (Na++K+)-ATPase and (Mg2+)-ATPase activity have been subtracted as described in Materials and Methods. Data are mean±SEM of 4 independent experiments. Where missing, error bars are smaller than symbol size.

Figure 2Down 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 · min–1 at 3 mmol/L MgCl2. When omitting ouabain, again in the absence of Ca2+, the Mg2+-dependent (Na++K+)-ATPase activity ({circ}) with a Vmax of 131.4±5.9 nmol Pi/mg membrane protein · min–1 at 3 mmol/L MgCl2 became apparent. Based on logistic concentration-effect analysis, curve fitting yielded 2 apparent dissociation constants of {approx}0.04 mmol/L and 1.17±0.18 mmol/L for MgCl2.



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  		Figure 2. Concentration-effect relationship of magnesium. (Mg2+)-ATPase (•) and (Na++K+)-ATPase ({circ}) show concentration-dependent activation by Mg2+. (Ca2+)-ATPase in the absence of exogenously added calmodulin ({square}), and in the presence of 10-8 mol/L exogenously added calmodulin ({blacksquare}), are antagonized by increasing concentrations of Mg2+. (Ca2+)-ATPase activities were measured in the presence of 0.2 mmol/L CaCl2. Data are mean±SEM of 4 independent experiments. Where missing, error bars are smaller than symbol size.

The addition of 0.2 mmol/L CaCl2 to the incubation mixture (including 10-4 mol/L ouabain) revealed a specific (Ca2+)-ATPase activity ({square}) 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 1Up, the presence of 10-8 mol/L exogenous calmodulin ({blacksquare}) 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 3Down. In the presence of either Ca2+ ({circ}) or Mg2+ ({triangleup}), the preparations exhibited activity maxima in the 0.3 to 1 mmol/L ATP range with apparent dissociation constants of {approx}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 1Up and 2Up, the addition of exogenously added calmodulin did not affect the (Ca2++Mg2+)-ATPase activity.



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  		Figure 3. ATP substrate activation of PAEC plasma membrane ATPase activities. Shown are (Ca2+)-ATPase activities (0.2 mmol/L CaCl2 only) in the absence or presence of 10-8 mol/L exogenously added calmodulin ({circ} and •, respectively); (Ca2++Mg2+)-ATPase activity (0.2 mmol/L CaCl2 and 0.3 mmol/L MgCl2) in the absence of exogenously added calmodulin ({square}) and in the presence of 10-8mol/L exogenously added calmodulin ({blacksquare}); and (Mg2+)-ATPase activity in the presence of 0.3 mmol/L MgCl2 ({triangleup}) and its control (ie, no MgCl2, no Ca2+, and 0.10 mmol/L ouabain, {triangledown}). Data are mean±SD of 2 independent experiments. Where missing, error bars are smaller than symbol size.

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 (TableDown).


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  		Table 1. Effects of Variably Selective Enzyme Inhibitors on PAEC Plasma Membrane ATPase Activities

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 4Down shows that, whereas the red cell (Ca2++Mg2+)-ATPase activity of the transport enzyme is relatively selective for ATP, endothelial Mg2+- and Ca2+-activated ecto–nucleotide 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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  		Figure 4. Nucleotide substrate specificities of PAEC plasma membrane and human red blood cell (RBC) plasma membrane nucleotide phosphohydrolase activities. For PAEC and RBC activities, {square} indicates nucleotide phosphohydrolase activities in the presence of Mg2+ and ouabain; , presence of Na+, K+, and Mg2+; and {blacksquare}, presence of Ca2+, Mg2+, and ouabain. Nucleotide concentration of all substrates was 1 mmol/L. Specific activities for each membrane preparation are operationally defined and were calculated as outlined in Materials and Methods. Note the difference in the ordinate scale for the 2 plasma membrane ATPase activities. Data are mean±SD or SEM of 2 to 4 independent experiments. Where missing, error bars are smaller than symbol size.

Without any apparent evidence of a calmodulin-sensitive fraction of a PAEC plasma membrane (Ca2++Mg2+)-ATPase activity (Figures 1 through 4UpUpUpUp), 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+ transport–related (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 5Down, 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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  		Figure 5. Micrographs showing Ca2+-ATPase immunoreactivity in PAECs. A and B, Intimal layer (arrows) of porcine aorta immunostained in the absence (A) or presence (B) of 5F10 monoclonal antibody directed against the Ca2+ pump epitopes. C and D, Cultured aortic endothelial cells immunostained in the absence (C) or presence (D) of 5F10 antibody. Note the immunoreactive particles in panel D. Scale bar=50 µm.

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 · min–1 (the extrapolated maximal value from the logistic concentration response curve fit; actual determined value was 143.0 nmol Pi/mg membrane protein · min–1) by 96% to 5.8 nmol Pi/mg membrane protein · min–1 (Figure 6Down, 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 · min–1 (Figure 6Down, 100%) with a half-maximally activating concentration of {approx}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 · min–1 [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 6Down 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 {approx}3 · 10-7 mol/L as for PAEC membranes in Figure 6Down.



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  		Figure 6. Calmodulin reactivation of PAEC (Ca2++Mg2+)-ATPase activity; calmodulin concentration-effect relationship in the presence of 10-5 mol/L calmidazolium. Data are expressed as percentage activation, with 100% being equal to 71.9±13.9 nmol Pi/mg membrane protein · min–1 (5 mmol/L ATP and 10 µg/mL membrane protein). Inset, Calmidazolium inhibition of (Ca2++Mg2+)-ATPase. Ordinate units are nmol Pi/mg membrane protein · min–1. Data are mean±SD or SEM of 2 to 6 independent experiments. Where missing, error bars are smaller than symbol size.

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 7Down 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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  		Figure 7. Dependence of calmodulin reactivation of PAEC (Ca2++Mg2+)-ATPase activity on the simultaneous presence of Ca2+ and Mg2+. Shown is a comparison of calmidazolium-inhibited (Ca2++Mg2+)-ATPase reactivation data (•; n=4 to 6) from Figure 6Up with calmidazolium-inhibited ATPase activities in the absence of Mg2+ ({triangleup}, ie, [Ca2+]-ATPase, 0.2 mmol/L Ca2+, n=2) or in the absence of Ca2+ ({square}, ie, [Mg2+]-ATPase, 0.3 mmol/L Mg2+, n=4). Membrane, substrate, and calmidazolium (10-5 mol/L) concentrations for all activities as in Figure 6Up.


*    Discussion
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 up arrowResults
 *Discussion
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Endothelial cell dysfunction is now recognized as a pivotal culprit in various cardiovascular diseases such as atherogenesis, inflammatory tissue response, hypertensive disorders, and their interrelationship in these pathophysiologic processes.32 A number of pathophysiologic abnormalities such as dyslipidemias, excessive oxidatively modified lipids and lipoproteins, homocysteinemia, nitric oxide metabolism, and unopposed reactive oxygen metabolites are closely scrutinized as potential endothelial aggravating entities. Using various model systems, several of these factors have also been shown to have detrimental effects on Ca2+ transport and other ion-regulatory mechanisms of the plasma membrane.33 34 35 36 37 38

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 ecto–nucleotide 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 3UpUpUp, 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, high–ionic 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 5Up 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 6Up), 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+ transport–associated enzyme. This fact is reinforced in Figure 7Up by comparison of the replotted data from Figure 6Up (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
 
This work was supported in part by the American Medical Association Educational Research Foundation, Vanderburgh Medical Alliance of Evansville, a Research Enhancement Program award from Indiana University School of Medicine, and the Evansville Center for Medical Education. We are grateful for the generous help and advice of Dr D.R. Koritnik (Indiana University School of Medicine, Department of Pharmacology, Fort Wayne, Ind) in developing primary endothelial cultures. Moreover, we thank Dr R.B. Wysolmerski (St. Louis University School of Medicine, Department of Pathology, St. Louis, Mo) for the gift of bovine pulmonary endothelial cell culture, and Dewig Meats (Haubstadt, Indiana), for ample gifts of porcine tissues.

Received May 12, 1999; accepted November 3, 1999.


*    References
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 up arrowIntroduction
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 *References
 

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