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Am J Physiol Lung Cell Mol Physiol 277: L794-L801, 1999;
1040-0605/99 $5.00
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Vol. 277, Issue 4, L794-L801, October 1999

Increased expression of calreticulin is linked to ANG IV-mediated activation of lung endothelial NOS

Jawaharlal M. Patel1,2, Yong D. Li2, Jianliang Zhang2, Craig H. Gelband3, Mohan K. Raizada3, and Edward R. Block1,2

1 Research Service, Malcom Randall Department of Veterans Affairs Medical Center, and Departments of 2 Medicine and 3 Physiology, University of Florida College of Medicine, Gainesville, Florida 32608


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

This study demonstrates that ANG IV-induced activation of lung endothelial cell nitric oxide synthase (ecNOS) is mediated through mobilization of Ca2+ concentration and by increased expression and release of the Ca2+ binding protein calreticulin in pulmonary artery endothelial cells (PAEC). In Ca2+-free medium and in the presence of the ANG II AT1 and AT2 receptor antagonists losartan and PD-123319 (1 µM each), respectively, ANG IV (5, 50, and 500 nM) significantly increased intracellular Ca2+ release in PAEC (P < 0.05 for all concentrations). In contrast, ANG IV-mediated activation of ecNOS was abolished by the intracellular Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM. ANG IV stimulation resulted in significantly increased expression of calreticulin in cells as well as release of calreticulin into the medium of cells as early as 2 h after ANG IV stimulation (P < 0.05). Catalytic activity of purified ecNOS in the absence of calmodulin was increased in a concentration-dependent fashion by calreticulin. Immunocoprecipitation studies revealed that ecNOS and calreticulin were coprecipitated in ANG IV-stimulated PAEC. These results demonstrate that ANG IV-mediated activation of ecNOS is regulated by intracellular Ca2+ mobilization and by increased expression of calreticulin, which appears to involve interaction of ecNOS and calreticulin proteins in PAEC.

endothelial cell nitric oxide synthase; calcium; angiotensin IV; protein interaction; protein synthesis; protein-protein interaction


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

WE RECENTLY REPORTED that angiotensin IV (ANG IV) activates the constitutively expressed lung endothelial cell isoform of nitric oxide synthase (ecNOS) by a receptor-mediated pathway, leading to increases in nitric oxide (NO) release, production of cGMP, and NO-cGMP-mediated porcine pulmonary arterial vasodilation (14, 31). The catalytic activity of ecNOS is Ca2+ and calmodulin dependent and is transiently activated by agonist-mediated signaling pathways that increase mobilization of intracellular Ca2+ (7, 11, 15). These signaling mechanisms are associated with activation of several Ca2+-dependent enzymes responsible for mediating vascular endothelial cell function (3, 7, 16). Agonist-mediated intracellular Ca2+ mobilization also plays a critical role as a second messenger in the regulation of a variety of cell functions, including protein expression, cell proliferation, gene expression, and protein-protein interaction (4, 10, 17, 23, 25). Depletion of Ca2+ from the endoplasmic reticulum (ER) or sarcoplasmic reticulum, a known intracellular Ca2+ storage site, can facilitate a process that results in upregulation of a group of Ca2+ binding proteins, including calreticulin, located within the lumen of the ER (6, 22, 27).

Calreticulin is a 60-kDa, ubiquitous Ca2+ binding protein of the ER that consists of low- and high-affinity binding sites and is localized in various subcellular compartments, including the cytosol, the nucleus, and the cell surface membrane (1, 24, 37). Calreticulin has been reported to be secreted from cells (4, 28) and is present at low levels in human plasma (35). Calreticulin has also been recognized as a multifunctional protein involved in a wide variety of cellular processes, including its interaction with endothelium in canine coronary arteries, which results in stimulated NO production (9, 18). However, the mechanism by which calreticulin increases NO production by vascular endothelium is unknown. Because ANG IV-stimulated activation of ecNOS and release of NO are mediated by a posttranscriptional mechanism and because catalytic activity of ecNOS is elevated from 0.5 to 12 h after ANG IV stimulation (31), we examined whether ANG IV-mediated early and sustained activation of ecNOS is regulated through 1) intracellular Ca2+ release and 2) increased expression and release of calreticulin involving an ecNOS-calreticulin interaction in lung endothelial cells.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell culture and treatment. Endothelial cells were isolated from the main pulmonary artery of 6- to 7-mo-old pigs and propagated in monolayers, as previously described (30). Third- to fourth-passage cells in postconfluent monolayers maintained in RPMI 1640 medium (Life Technologies, Grand Island, NY) with 4% fetal bovine serum (HyClone Laboratories, Logan, UT) were used in all experiments. In each experiment, cell monolayers were studied 1 or 2 days after confluence and were matched for cell line, passage, and days after confluence.

To determine the potential effect of ANG IV on Ca2+ release from intracellular stores, cell monolayers in 35-mm culture dishes containing a round coverslip were incubated with 5 µM membrane-permeant fura 2-AM (fura 2-AM dissolved in 1 mM DMSO stock solution) in Tyrode solution (composition in mM: 134 NaCl, 5.4 KCl, 2.0 MgCl2, 0.3 NaH2PO4, 10.0 HEPES, and 10.0 dextrose, pH 7.4) for 30 min at 37°C and then washed to remove excess external fura 2-AM. The fura 2-loaded cells were then incubated (1 min) in the presence of 1 µM each losartan (an ANG II AT1-receptor antagonist) and PD-123319 (an ANG II AT2-receptor antagonist) and stimulated with ANG IV (5, 50, or 500 nM), and intracellular Ca2+ concentration ([Ca2+]i) was measured (see below).

To examine the role of Ca2+ in ANG IV-stimulated activation of ecNOS, cell monolayers were preincubated in Tyrode solution with or without 2 mM CaCl2 with and without 50 µM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-AM (dissolved in 1 mM DMSO stock solution) for 30 min at 37°C, then incubated 2 h at 37°C with or without 1 µM ANG IV. Controls were incubated in Tyrode solution alone under identical conditions. For studies involving an extracellular Ca2+-free environment, the Tyrode solution was supplemented with 5 mM EGTA. After incubation, the cells were used to measure total membrane fraction ecNOS activity.

To examine the effect of ANG IV on calreticulin expression and release of calreticulin into the medium, cell monolayers were incubated in RPMI 1640 with or without ANG IV (1 µM) for 2-12 h at 37°C. After incubation, calreticulin levels in the cells were determined by two-dimensional gel electrophoresis, microsequencing, and Western blot analysis. For estimation of released calreticulin in the medium, an equal volume (60 ml each) of medium from control cells and from cells incubated with ANG IV for 2 h was concentrated to 5 ml with use of a Centriprep-30 concentrator (Amicon, Beverly, MA) and analyzed by Western blot. To examine whether ANG IV-induced intracellular Ca2+ release is required for increased calreticulin expression, in some experiments, cell monolayers were preincubated in Tyrode solution without Ca2+ with or without 50 µM BAPTA-AM for 30 min at 37°C, then incubated for 2, 4, and 6 h at 37°C with or without 1 µM ANG IV. After incubation, calreticulin levels in the cell lysates were determined by Western blot analysis.

[Ca2+]i measurement. [Ca2+]i was measured using epifluorescence microscopy (36). Briefly, the fura 2-AM-loaded cells were alternately illuminated with ultraviolet light at 340- and 380-nm wavelengths with use of an IonOptix (Milton, MA) electronically controlled dual-excitation imaging fluorescence system. Cell fluorescence (emitted light) was collected through a 510-nm barrier filter before acquisition by a photomultiplier tube. The fluorescence signals at 340 and 380 nm (F340 and F380, respectively) were background subtracted, i.e., fluorescence signal from fura 2-AM-loaded ANG IV-unstimulated cells, during the experiment. The mean changes in F340 to F380 ratios were graphed to give a relative indication of the changes observed in [Ca2+]i. The percent intracellular Ca2+ release was determined from the F340 to F380 ratio (36).

Measurement of ecNOS activity. ecNOS activity was measured by monitoring the formation of L-[3H]citrulline from L-[3H]arginine in the total membrane fraction (31, 32). Total membranes (100-200 µg of protein) were incubated (total volume 0.4 ml) in buffer (50 mM Tris · HCl, 0.1 mM each EDTA and EGTA, 1 mM phenylmethylsulfonyl fluoride, and 1 mg/l leupeptin, pH 7.4) containing 1 mM NADPH, 100 nM calmodulin, 10 µM tetrahydrobiopterin, and 5 µM combined L-arginine and purified L-[3H]arginine for 30 min at 37°C. Purification of L-[3H]arginine and measurement of L-citrulline formation were carried out as previously described (29).

Two-dimensional gel electrophoresis and identification of calreticulin. Two-dimensional electrophoresis of control and ANG IV-stimulated cells was performed according to the method of O'Farrell (26) with use of a Bio-Rad protein II xi system (Bio-Rad, Richmond, CA), as previously described (20). Briefly, in the first dimension, the isoelectric focusing gel was loaded with equal amounts (180 µg) of TCA-precipitable cell lysate proteins. The second dimension (SDS-PAGE) was performed on a 7.5% separating Laemmli gel with a 3.9% stacking gel (20).

To identify calreticulin, immunoblot analyses of the two-dimensional polyacrylamide gels of the cell lysate proteins as well as of gels loaded with aliquots of concentrated (50 µl) medium from control and ANG IV-stimulated cells were performed. Proteins were electrophoretically transferred from the slab gels to polyvinylidine difluoride membranes, as described previously (20). To saturate nonspecific binding sites, the membranes were blocked by 1% blot-qualified BSA (Promega, Madison, WI) in 20 mM Tris · HCl, pH 7.5, 150 mM NaCl, and 0.05% Tween 20 for 1 h. The immunodetection was performed with a monoclonal human anti-calreticulin antibody and an anti-rabbit IgG horseradish peroxidase-linked whole antibody (Upstate Biotechnology, Lake Placid, NY). Immunoreactivity was detected by enhanced chemiluminescence (Amersham) (20). The blots were scanned using the Fluor-S MultImager system (Bio-Rad) to quantify protein content.

Expression and purification of ecNOS. Escherichia coli transformed with the plasmid Bov-eNOSpCW (kindly provided by Dr. B. S. S. Masters, University of Texas, San Antonio, TX) was incubated in 0.5 liter of modified TB (20 g yeast extract, 10 g bactotryptone, 2.65 g KH2PO4, 4.33 g Na2HPO4, and 4 ml glycerol per liter) containing ampicillin (50 µg/ml) and chloramphenicol (35 µg/ml). The cultures were grown in an orbital shaker (20 rpm; Forma Scientific) in the presence of 0.5 mM delta -aminolevulinic acid at 22°C. After 1 h of incubation, ecNOS gene expression was induced by adding 0.5 mM isopropyl beta -D-thiogalactopyranoside, 3 µM riboflavin, and 1 mM ATP. The flasks were kept on an orbital shaker in the dark at 22°C (200 rpm) for 48 h. After incubation, the cultures were centrifuged at 5,000 rpm for 10 min at 5°C, and the cell pellets were collected and used for purification of ecNOS.

Purification of ecNOS was carried out essentially as described previously with use of 2',5'-ADP-Sepharose 4B affinity chromatography (32). Briefly, the cell pellets were resuspended in buffer A (50 mM Tris · HCl, pH 7.4, 1 mM dithiothreitol, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 150 mM NaCl, 10% glycerol, and leupeptin and pepstatin at 1 µM each), lysed by pulsed sonication, and centrifuged at 100,000 g for 30 min at 4°C. The supernatants were applied to 2',5'-ADP-Sepharose 4B (Pharmacia Biotech) columns equilibrated in buffer B (50 mM Tris · HCl, pH 7.4, 0.1 mM EDTA, 0.1 mM dithiothreitol, 150 mM NaCl, and 10% glycerol). The columns were washed with 10 column volumes of buffer B, then with 10 column volumes of buffer B containing 600 mM NaCl, and then they were eluted with buffer B containing 600 mM NaCl and 5 mM adenosine 2'-monophosphate. The purity of the enzyme was determined by monitoring ecNOS activity as well as by Western blot analysis (39).

Calreticulin and catalytic activity of purified ecNOS. To determine whether the catalytic activity of ecNOS is supported by calreticulin in the absence of calmodulin, purified ecNOS (3 µg protein) was incubated with increasing amounts of calreticulin (10-70 nM) for 10 min at 37°C. After incubation, the catalytic activity of ecNOS was monitored as described in Measurement of ecNOS activity, except for the absence of calmodulin in the incubation mixture (31, 32). In some experiments, purified ecNOS (3 µg protein) was incubated with 50 nM calreticulin for 10 min at 37°C, and then the catalytic activity of ecNOS was monitored in the absence of calmodulin but in the presence of increasing concentrations (10-1,000 nM) of Ca2+. To determine the effects of the combination of calreticulin and calmodulin, purified ecNOS (3 µg of protein in each sample) was incubated as described in Measurement of ecNOS activity, except 100 nM calmodulin in the reaction mixture was replaced with various ratios of calreticulin to calmodulin (60:10-10:60 nM), with 60 nM calreticulin alone, or with 60 nM calmodulin alone. After 30 min of incubation at 37°C, the catalytic activity of ecNOS was measured by monitoring the formation of L-[3H]citrulline from L-[3H]argnine (31, 32).

Immunoprecipitation and analysis of ecNOS and calreticulin. Cell monolayers stimulated with or without ANG IV (1 µM) at 37°C for 4 h were lysed in buffer consisting of 20 mM Tris · HCl, pH 7.4, 2.5 mM EDTA, 100 mM NaCl, 10 mM sodium fluoride, 1 mM sodium vanadate, 1 mM Pefabloc, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, and 10 µg/ml each of pepstatin and leupeptin. The cell lysate proteins (50 µg) or buffer only (blanks) was incubated with 1 µg of anti-ecNOS monoclonal antibody (Transduction Laboratories, Lexington, KY) for 1 h at 4°C, and then 20 µl of Protein A/G Plus-Agarose (Santa Cruz Biotechnology, Santa Cruz, CA) were added and incubated with mixing overnight at 4°C. The reaction mixtures were centrifuged (2,500 rpm) for 15 min at 4°C, and the agarose pellets were collected, washed with PBS containing 1 M NaCl three times, and then boiled in 40 µl of loading buffer for 90 s. The samples were fractionated on a 7.5% SDS polyacrylamide gel and blotted onto polyvinylidine difluoride membranes (39). The blots were hybridized with monoclonal anti-ecNOS and anti-calreticulin antibodies, and the immunoreactive bands were visualized by enhanced chemiluminescence detection (39).

Statistical analysis. Statistical significance for the effect of ANG IV and BAPTA-AM on ecNOS activity, intracellular Ca2+ release, and calreticulin expression and for the effect of calreticulin on ecNOS activity was determined using ANOVA and Student's paired t-test (38). Values are means ± SE for n experiments. P < 0.05 was taken as significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

ANG IV stimulation increases [Ca2+]i. ANG IV stimulated an increase in [Ca2+]i in pulmonary artery endothelial cells (PAEC) as illustrated in Fig. 1. ANG IV caused rapid increases in [Ca2+]i in a dose-dependent manner, and the increases were significantly greater than the basal level of [Ca2+]i (P < 0.05 for all concentrations).


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  		Fig. 1.   ANG IV increases intracellular Ca2+ (Ca2+i) release in a dose-dependent manner. Pulmonary arterial endothelial cell (PAEC) monolayers loaded with fura 2-AM were preincubated in Tyrode solution without Ca2+ in presence of losartan and PD-123319 (1 µM each), then stimulated with 5, 50, and 500 nM ANG IV. Left: ANG IV concentration-dependent increases in fluorescence ratio [ratio of fluorescence at 340 nm to that at 380 nm (F340/F380)], representing transient intracellular Ca2+ release. Right: percent intracellular Ca2+ release from 4 independent samples. * P < 0.001 vs. basal level in presence of losartan and PD-123319.

ANG IV-mediated intracellular Ca2+ release is associated with ecNOS activation. To examine whether ANG IV-induced intracellular Ca2+ release is associated with activation of ecNOS, we examined the effect of the intracellular Ca2+ chelator BAPTA-AM on ecNOS activity. As shown in Fig. 2, incubation of PAEC in the presence of ANG IV but in the absence of extracellular Ca2+ significantly (P < 0.05) increased ecNOS activity compared with control. In contrast, preincubation of cell monolayers with BAPTA-AM in the absence of extracellular Ca2+ completely blocked ANG IV-stimulated activation of ecNOS. ecNOS activity was comparable to controls when cells were incubated with BAPTA-AM alone under identical conditions. Similar results were obtained if cell monolayers were stimulated by ANG IV in the presence of extracellular Ca2+ (data not shown).


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  		Fig. 2.   Effect of intracellular Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-AM on ANG IV-stimulated endothelial cell nitric oxide synthase (ecNOS) activation. Porcine PAEC were incubated in Tyrode solution without Ca2+ with or without 50 µM BAPTA-AM for 30 min, then incubated for 2 h at 37°C with or without 1 µM ANG IV. Respective controls were incubated in Tyrode solution alone or in Tyrode solution containing 50 µM BAPTA-AM under identical conditions. After incubation, total membrane fraction ecNOS activity was measured. Similar results were obtained when cell monolayers were incubated in RPMI 1640 (data not shown). Values are means ± SE (n = 4). * P < 0.05 vs. control.

ANG IV increases expression and release of calreticulin. Immunoblot analysis of two-dimensional gels of control and ANG IV-stimulated cells is shown in Fig. 3. A human monoclonal antibody for calreticulin reacted with a 60-kDa protein, and the intensity of this reaction was increased severalfold in cells exposed to ANG IV for 12 h compared with controls. To confirm the identity of calreticulin, NH2-terminal amino acid sequence analysis of the 60-kDa protein followed by protein database analysis of 25 residues (EPTIYFKEQFLDGDGWTDRWIESKH) matched 100% with rabbit uterine calreticulin and 96% with human placental calreticulin. The time course of the ANG IV-stimulated increased expression of calreticulin was determined and is shown in Fig. 4. ANG IV-mediated expression of calreticulin was significantly increased as early as 2 h and remained elevated for 6 h (P < 0.05 for all time points). In addition to increased expression of cellular calreticulin, similar immunoreactivity and increased levels of calreticulin were observed in medium from cells stimulated with ANG IV compared with medium from control cells (Fig. 5).


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  		Fig. 3.   Western blot analysis of calreticulin after ANG IV stimulation. PAEC monolayers in RPMI 1640 containing 1 µM ANG IV (B) or RPMI 1640 only (control, A) were incubated for 12 h at 37°C. Cell lysate proteins (180 µg) were separated by 2-dimensional electrophoresis and immunoanalyzed. pI, isoelectric point.



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  		Fig. 4.   Time-dependent effect of ANG IV on calreticulin expression. PAEC monolayers in RPMI 1640 containing 1 µM ANG IV or RPMI 1640 alone (Con) were incubated for 2, 4, and 6 h at 37°C. Cell lysate proteins (50 µg) were fractionated on a 7.5% SDS polyacrylamide gel, blotted onto polyvinylidine difluoride (PVDF) membranes and then hybridized with human anti-calreticulin monoclonal antibody. Blots were analyzed by densitometric analysis to quantify calreticulin protein content. A: representative data from 1 of 4 independent experiments. MW, molecular mass. B: results of densitometric analysis of blots from 4 independent experiments (means ± SE). * P < 0.05 vs. control (Con).



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  		Fig. 5.   ANG IV-stimulated expression of calreticulin increases its secretion into extracellular medium. PAEC monolayers in RPMI 1640 containing 1 µM ANG IV or RPMI 1640 only (Con) were incubated for 2 h at 37°C. After incubation, 60 ml of media were collected and concentrated, and calreticulin was identified by immunoblot analysis. A: representative data from 1 of 3 independent experiments. B: results of densitometric analysis of blots from 3 independent experiments (means ± SE). * P < 0.05 vs. control.

ANG IV-mediated level of intracellular Ca2+ release is critical for expression of calreticulin. To determine whether ANG IV-induced [Ca2+]i is critical for increased expression of calreticulin, we examined the effect of the intracellular Ca2+ chelator BAPTA-AM on calreticulin expression. As shown in Fig. 6, ANG IV-induced expression of calreticulin was blocked by BAPTA-AM.


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  		Fig. 6.   Effect of intracellular Ca2+ chelator on ANG IV-mediated calreticulin expression. PAEC monolayers were incubated in Tyrode solution without Ca2+ with or without 50 µM BAPTA-AM for 30 min, then incubated for 2, 4, or 6 h at 37°C with or without 1 µM ANG IV. After incubation, cell lysate proteins (50 µg) were fractionated on a 7.5% SDS polyacrylamide gel, blotted onto PVDF membranes, and then hydridized with human anti-calreticulin monoclonal antibody.

Calreticulin increases catalytic activity of ecNOS in the absence of calmodulin. Because the Ca2+ binding protein calmodulin is critical for the catalytic activity of ecNOS, we determined whether substitution of calreticulin for calmodulin can maintain catalytic activity of ecNOS. As shown in Fig. 7A, in the absence of calmodulin, calreticulin increased the catalytic activity of purified ecNOS in a dose-dependent manner. The effect of various concentrations of Ca2+ on calreticulin-mediated activation of ecNOS revealed that calreticulin can increase catalytic activity at physiologically relevant concentrations of Ca2+ (Fig. 7B).


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  		Fig. 7.   Calreticulin and Ca2+ concentration-dependent effects on catalytic activity of purified ecNOS in absence of calmodulin. Purified ecNOS (3 µg of protein) was incubated in presence of increasing concentrations (10-70 nM) of calreticulin for 10 min at 37°C (A) or in presence of 50 nM calreticulin and increasing concentrations (10-1,000 nM) of Ca2+ for 10 min at 37°C (B). After incubation, catalytic activity of ecNOS was measured in absence of calmodulin but in presence of other cofactors. Values are means ± SE; n = 3 for each data point.

Calreticulin-to-calmodulin ratio is critical for increased catalytic activity of ecNOS. To identify possible competitive effects between calreticulin and calmodulin on ecNOS activity, the catalytic activity of purified ecNOS was determined in the presence of increasing or decreasing concentrations of calmodulin and calreticulin. As shown in Fig. 8A, in the absence of calreticulin, catalytic activity of ecNOS was increased by calmodulin in a dose-dependent manner, with maximal activation observed at 50 nM calmodulin. In the absence of calmodulin, calreticulin (60 nM) caused only a limited increase in the catalytic activity of ecNOS (Fig. 8B). However, calreticulin-to-calmodulin ratios of 60:10, 50:20, 40:30, and 30:40 significantly increased the catalytic activity of ecNOS compared with ecNOS activities at 10, 20, 30, and 40 nM calmodulin alone (P < 0.05 for all; Fig. 8B), indicating that calreticulin can enhance the catalytic activity of ecNOS in the presence of calmodulin. Calreticulin or calmodulin alone at 60 nM increased the catalytic activity of ecNOS, but the calmodulin-mediated activation of ecNOS was severalfold greater than that observed with 60 nM calreticulin alone (Fig. 8B). Western blot analysis revealed that endogenous calmodulin levels in cells incubated for 2-12 h with ANG IV were comparable to controls (data not shown).


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  		Fig. 8.   Effect of calreticulin-to-calmodulin ratio on catalytic activity of ecNOS. A: purified ecNOS was incubated in absence of calreticulin but in presence of increasing concentrations (10-70 nM) of calmodulin. B: purified ecNOS was incubated with decreasing (60-10 nM) calreticulin and increasing (10-60 nM) calmodulin concentrations, with 60 nM calreticulin alone, or with 60 nM calmodulin alone for 10 min at 37°C. After incubation, catalytic activity of ecNOS was monitored. Values are means ± SE; n = 6 for each data point. * P < 0.05 vs. 10-40 nM calmodulin alone.

Calreticulin interacts with ecNOS protein. To determine whether calreticulin directly interacts with ecNOS in intact PAEC, ecNOS from control and ANG IV-stimulated cells was immunoprecipitated using anti-ecNOS monoclonal antibody, and the immunoprecipitates were analyzed for the presence of ecNOS and calreticulin proteins. As shown in Fig. 9, the presence of ecNOS and calreticulin proteins in the immunoprecipitates suggests that ecNOS exists in a complex with calreticulin in PAEC.


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  		Fig. 9.   Interaction between ecNOS and calreticulin. PAEC monolayers in RPMI 1640 containing 1 µM ANG IV or RPMI 1640 alone (control, C) were incubated for 4 h at 37°C. After incubation, cell lysate proteins (50 µg) or buffer only (blank, B) was immnoprecipitated with anti-ecNOS monoclonal antibody. Immunoprecipitated complex was denatured by boiling, fractionated, blotted on PVDF membranes, and hybridized with monoclonal ecNOS and calreticulin antibodies. Data are representative of 2 independent experiments with similar results.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We demonstrate here that ANG IV stimulation in the presence of ANG II AT1 and AT2 antagonists increases [Ca2+]i, which is associated with increased catalytic activity of ecNOS in PAEC. Our results further demonstrate for the first time that ANG IV stimulation results in increased expression and release of the Ca2+ binding protein calreticulin. The ANG IV-mediated activation of ecNOS and the ANG IV-mediated increase in expression of calreticulin are linked to release of intracellular Ca2+, and these responses were completely diminished by the intracellular Ca2+ chelator BAPTA-AM. The results of the present study are consistent with the well-established mechanism that agonist-induced transient release of intracellular Ca2+ can increase catalytic activity of ecNOS (7, 11, 15). However, ANG IV-mediated increased expression of calreticulin and its association with a sustained increase in ecNOS activity are of physiological relevance, because we previously reported that ANG IV-mediated increased catalytic activity of ecNOS is directly linked to increased NO production and pulmonary artery vasorelaxation through the NO-cGMP signaling mechanism (14, 31).

ANG IV-mediated activation of ecNOS and increased expression of calreticulin are clearly dependent on intracellular Ca2+ release, inasmuch as these responses were abrogated by the intracellular Ca2+ chelator BAPTA-AM. Agonist-mediated cytosolic Ca2+ release is associated with receptor-linked signaling pathways. One of the most ubiquitous pathways is activation of the phospholipase C-inositol 1,4,5-trisphosphate pathway, which releases Ca2+ from intracellular Ca2+ pools, namely, the ER (2). Although the precise signaling pathway involved in ANG IV-mediated intracellular Ca2+ release remains to be determined, two pieces of evidence indicate that the ER is the most likely source of the cytosolic Ca2+ elevation. First, ANG IV-stimulated increased intracellular Ca2+ release and activation of ecNOS were observed in the absence or presence of extracellular Ca2+, suggesting that elevation of cytosolic Ca2+ and activation of ecNOS were due to its release from intracellular stores and not as a result of increased influx. Second, our results demonstrated that the ANG IV-mediated increased expression of calreticulin was blocked by the intracellular Ca2+ chelator BAPTA-AM, and this is consistent with reports demonstrating an association between the depletion of ER Ca2+ stores and increased expression of a set of ER resident proteins, including calreticulin, in a variety of cells (5, 16, 27).

Our results also demonstrate that ANG IV-mediated increased expression of calreticulin resulted in release of this protein into the medium. Calreticulin secretion and the presence of calreticulin in human plasma have been previously reported (4, 35). Although the mechanism of secretion of calreticulin is unknown, it is suggested that perturbation of cellular Ca2+ enhances secretion of luminal ER proteins including calreticulin (4). The ANG IV-induced increased expression and secretion of calreticulin is particularly important, because a recent study by Kuwabara et al. (18) demonstrated that infusion of calreticulin increases endothelial cell surface binding in canine coronary artery and stimulates NO production. Although the mechanism of calreticulin-mediated NO generation was not examined by these authors, our results demonstrate that incubation of purified ecNOS with calreticulin increases catalytic activity of ecNOS, suggesting that the increased NO production is most likely associated with increased catalytic activity of ecNOS. Further examination of calreticulin-mediated activation of ecNOS revealed that, in the absence of calmodulin, calreticulin increases catalytic activity in a concentration-dependent manner. This calreticulin-mediated increase in ecNOS activity occurred in the presence of physiologically relevant concentrations of Ca2+. In addition, calreticulin enhanced the catalytic activity of ecNOS in the presence of calmodulin in vitro. We believe that a similar situation exists in cells stimulated with ANG IV, because calreticulin content was increased in these cells, but endogenous calmodulin expression was not changed.

Finally, our results suggest a protein-protein interaction between ecNOS and calreticulin in porcine PAEC. The existence of such a complex would provide a cellular construct for understanding how ANG IV-mediated increases in calreticulin result in ecNOS activation. Although the precise nature of the interaction between ecNOS and calreticulin remains to be determined, it is possible that the Ca2+ binding site of ecNOS protein may be conformationally changed by calreticulin. If this is so, it appears that such a molecular interaction might enhance the effect of calmodulin on the catalytic activity of ecNOS. Alternatively, an increased level of calreticulin may enhance the potential interactions between ecNOS and other proteins, such as heat shock protein 90, reported to be involved in ecNOS activation (12). This possibility is supported by reports that calreticulin can interact with other proteins such as nuclear hormone receptors, integrin alpha -subunit, and steroid hormone receptors, resulting in modulation of their function (5, 8, 19).

The physiological significance of the observations reported here relates not only to ANG IV-mediated sustained activation of ecNOS and vascular regulation through the NO-cGMP/signaling mechanism but to the multifunctional nature of calreticulin. For example, calreticulin has been shown to reduce intimal hyperplasia after arterial injury in the rat (9) and to protect lung and other tissues from a variety of pathophysiological conditions, including oxidant injury and heat shock (6, 21). Local release of calreticulin may alter Ca2+ homeostasis and generate excessive NO production, resulting in activation of inflammatory responses in the vasculature. In addition, vasostatin, a calreticulin fragment, has been reported to inhibit angiogenesis and to suppress tumor growth in mice (33). Similarly, NO was reported to play a role in suppression of angiogenesis and endothelial cell migration (33, 34). Our results emphasize the potential role of ANG IV/calreticulin-mediated responses in regulation of vascular function.


    ACKNOWLEDGEMENTS

We thank Bert Herrera for tissue culture assistance, Janet Wootten for excellent editorial help, Addy Heimer for secretarial assistance, and Weihong Han and Di-hau He for technical assistance.


    FOOTNOTES

This work was supported by the Medical Research Service of the Department of Veterans Affairs and by National Heart, Lung, and Blood Institute Grant HL-58679.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: J. M. Patel, Research Service (151), VA Medical Center, 1601 SW Archer Rd., Gainesville, FL 32608-1197 (E-mail: Pateljm{at}medicine.ufl.edu).

Received 30 March 1999; accepted in final form 14 May 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Am J Physiol Lung Cell Mol Physiol 277(4):L794-L801
0002-9513/99 $5.00 Copyright © 1999 the American Physiological Society


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