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CARDIOVASCULAR

Bradykinin Down-Regulates, Whereas Arginine Analogs Up-Regulates, Endothelial Nitric-Oxide Synthase Expression in Coronary Endothelial Cells

Division of Nephrology and Hypertension, Departments of Medicine (N.D.V., Z.N., C.H.B.) and Physiology and Biophysics (N.D.V.), University of California, Irvine, California; and Department of Pathology (Y.D.), Brown University, Providence, Rhode Island

Received August 20, 2004; accepted January 6, 2005.

	Abstract

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Bradykinin (BK) is an endogenous vasoactive peptide that promotes vasodilation by stimulating the release of nitric oxide (NO) from endothelial cells via activation of endothelial NO synthase (eNOS). Although the role of BK in modulation of eNOS activity is well understood, its possible effect on eNOS expression remains uncertain. Several studies have demonstrated negative feedback regulation of eNOS by NO. Therefore, we hypothesized that sustained stimulation with BK may down-regulate eNOS expression in endothelial cells. Human coronary endothelial cells were incubated for 24 h with either BK alone or BK plus BK receptor type 1 or type 2 blockers. NO production and eNOS abundance (Western analysis) were determined. In separate experiments, cells were incubated with either an NOS inhibitor alone or in combination with BK. Incubation with BK caused a concentration-dependent rise in NO production and a dose-dependent decline in eNOS protein expression. These effects were abrogated by BK-2 blockade but unaffected by BK-1 blockade. In contrast, NOS inhibitors lowered NO production and raised eNOS abundance in a dose-dependent fashion. The effects of BK on NO production and eNOS expression were abrogated by the NOS inhibitor. Thus, sustained activation of eNOS by BK results in a compensatory down-regulation of eNOS, whereas its sustained inhibition leads to a compensatory up-regulation of eNOS. The observed modulations of eNOS expression are mediated by NO and represent an adaptive physiologic response.

Stimulation of BK-2 receptor by BK on endothelial cells results in the activation of phospholipase C and A₂, which in turn triggers a rise in cytosolic Ca²⁺ concentration. This leads to activation of eNOS via calmodulin binding, followed by dissociation of the enzyme from its binding site to caveolin on the cell membrane and subsequent translocation to subcellular sites (Michel, 1999). Whereas the effect of BK on regulation of eNOS activity is well understood, the effect of BK on eNOS expression by endothelial cells is uncertain.

Earlier studies have demonstrated that the exogenous NO exerts a negative feedback influence on eNOS activity (Buga et al., 1993) in isolated vascular tissue and eNOS expression (Vaziri and Wang, 1999) in the cultured endothelial cells. Since BK augments NO production, we hypothesized that sustained stimulation with BK can lead to down-regulation of eNOS abundance in cultured endothelial cells. We further considered that such effect, if present, is mediated by BK-2 receptor and could be prevented by BK-2 receptor blockade or NOS inhibition. The present study was undertaken to test this hypothesis.

	Materials and Methods

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Cell Culture. Human coronary artery endothelial cells were purchased from Cambrex Bio Science Walkersville, Inc., (Walkersville, MD). The cells were cultured in a specific culture medium (Endothelial Cell Growth System) provided by Cambrex Bio Science Walkersville, Inc. and incubated in a humidified incubator at 37°C and 5% CO₂. The medium contained hydrocortisone, fibroblast growth factor, vascular endothelial growth factor, insulin-like growth factor, epidermal growth factor, ascorbic acid, gentamycin, and 5% fetal bovine serum. After 48 h, 10 ml of fresh medium were added, and incubation was continued for an additional 48 h. Once a monolayer was formed, the cells were subcultured. Cells obtained on passages 3 and 4 were used as described in our previous studies (Ding et al., 2000b).

Study Protocol. After reaching 90% confluence, the cultured coronary endothelial cells were incubated in a medium-containing bradykinin (Sigma-Aldrich, St. Louis, MO) at zero, 10^–7, 10^–6, or 10^–5 M concentrations. The incubation was conducted for 24 h.

In a second set of experiments, cells were incubated in a medium containing 10^–6 M bradykinin alone or in combination with a 10^–6 M concentration of either a BK-2 receptor blocker (HOE-140) or a BK-1 receptor blocker ([Des-Arg¹⁰] HOE-140) for 24 h. The BK receptor blockers were purchased from Sigma-Aldrich.

In an attempt to compare the effects of eNOS activation with those of eNOS inhibition, in a separate set of experiments the cultured endothelial cells were incubated with two different NOS inhibitors, N^G-monomethyl-L-arginine (LNMMA) and N-nitro-L-arginine-methylester (L-NAME) at zero, 10^–7, 10^–6, 10^–5, and 10^–4 M concentrations for 24 h. L-NMMA and L-NAME were purchased from Sigma-Aldrich. Finally, the interaction of BK and NOS inhibitor was tested in cells incubated with either BK alone, NOS inhibitor alone, or a combination thereof at 10^–6 M concentrations.

At the conclusion of each incubation period, cells were harvested for determination of eNOS protein abundance, and the medium was collected for measurement of nitrate plus nitrite (NO) as an index of NO production. On each occasion, four separate sets of parallel experiments were performed.

Measurement of NO Production. NO production was determined from the NO recovered in the culture medium. NO was quantified by use of the purge system of the model 270 B NOA Sievers NO Analyzer (Sievers Instruments, Boulder, CO). The amount of NO produced was normalized against total cellular protein. The procedures involved in this assay have been described in detail in our earlier studies (Vaziri and Wang, 1999; Wang and Vaziri, 1999).

Measurement of eNOS Protein. Endothelial cells were processed for determination of eNOS protein abundance by Western analysis, as described in our earlier studies (Ding and Vaziri, 2000a). Briefly, cells were washed with phosphate-buffered saline and extracted directly into the sample buffer (2% SDS, 10% glycerol, 0.0025% bromphenol, and 63 mM Tris·HCl, pH 6.8), and total protein was determined by using a bicinchoninic acid protein assay kit (Pierce, Rockford, IL). Fifty micrograms of cell lysate protein were size-fractionated on a 4 to 12% Tris-glycine gel at 130 V for 3 h. In preliminary experiments, the given protein concentrations were found to fall within the linear range of detection for our Western blot technique. After electrophoresis, proteins were transferred into Hybond enhanced chemiluminescence (ECL) membrane at 400 mA for 120 min by use of the Novex transfer system (Novex, San Diego, CA). The membrane was prehybridized in 10 ml of buffer A (10 mM Tris·HCl, pH 7.5, 100 mM NaCl, 0.1% Tween 20, and 10% nonfat mild powder) for 1 h and then hybridized for an additional 1 h in the same buffer containing 10 µl of the anti-eNOS monoclonal antibody (BD Biosciences Transduction Laboratories, Lexington, KY) at 1:1000 dilution. Thereafter, the membrane was washed for 30 min in a shaking bath, with the wash buffer (buffer A without nonfat milk) changed every 5 min before a 1-h incubation in buffer A plus goat anti-mouse IgG-horseradish peroxidase at the final titer of 1:1000. Experiments were carried out at room temperature. The washes were repeated before the membrane was developed with a light-emitting nonradioactive method using ECL reagent (Amersham Biosciences Inc., Piscataway, NJ). The membrane was then subjected to autoluminography for 1 to 5 min. The autoluminographs were scanned with a model PD 1211 laser densitometer (Amersham Biosciences Inc.) to determine the relative optical densities of the bands. In all instances, the membranes were stained with Ponceau stain before prehybridization to verify the uniformity of protein load and transfer efficiency across the test samples.

Data Presentation and Analysis. Data are presented as means ± S.E.M. Analysis of variance (two-way ANOVA; SigmaStat 2.0) and regression analysis (SigmaPlot 2000) were used in statistical evaluation of the data. P values 0.05 were considered significant.

	Results

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Effect of Bradykinin. Data are shown in Figs. 1 and 2. Incubation with BK at 10^–7 to 10^–5 M led to a dose-dependent rise in NO production by cultured human coronary endothelial cells. Similarly, short-term incubation (60 min) with BK resulted in a significant rise in NO production. In contrast, incubation with the given BK concentrations for 24 h caused a dose-dependent decline in eNOS protein abundance.

View larger version (19K): [in this window] [in a new window]   Fig. 1. Representative Western blot and group data depicting eNOS protein abundance and NO production in cultured human coronary endothelial cells incubated for 24 h in drug-free medium and media containing bradykinin at 10–7, 10–6, and 10–5 M concentrations. Data represent mean and S.E.M. of at least four separate experiments. *, P < 0.05 versus CTL (two-way ANOVA).

View larger version (18K): [in this window] [in a new window]   Fig. 2. NO production in cultured human coronary endothelial cells incubated in drug-free media and media containing different concentrations of bradykinin or NOS inhibitor (L-NNMA) for 1 h. Data represent mean and S.E.M. of at least four separated experiments. *, P < 0.05 versus CTL (two-way ANOVA).

Effect of BK-2 and BK-1 Receptor Blockade. Data are illustrated in Fig. 3. The addition of BK-2 receptor blocker completely abrogated the stimulatory action of BK on NO production by cultured coronary endothelial cells. Likewise, BK-2 receptor blockade prevented the BK-induced down-regulation of eNOS protein expression by coronary endothelial cells. In contrast, BK-1 receptor blocker failed to modify BK-mediated changes of NO production and eNOS abundance.

View larger version (20K): [in this window] [in a new window]   Fig. 3. Representative Western blot and group data depicting eNOS protein abundance and NO production in cultured human coronary endothelial cells incubated for 24 h in drug-free media (CTL) and media containing bradykinin (10–6 M) alone (BK) or in combination with BK-1 and BK-2 receptor blockers at 10–6 M concentrations. Data represent mean and S.E.M. of at least four separate experiments. *, P < 0.01 versus CTL and BK-2 group (two-way ANOVA).

Effect of NOS Inhibitors. Data are shown in Figs. 2 and 4. As expected, treatment with NOS inhibitors L-NMMA and L-NAME for 24 h resulted in a dose-dependent decline in NO production by coronary endothelial cells. Similarly, short-term incubation (60 min) with the NOS inhibitors led to a significant fall in NO production in this system. The reduction in NO production was coupled with a significant dose-dependent rise in eNOS abundance in cells treated with the NOS inhibitors for 24 h. The results obtained with L-NAME and L-NMMA were similar. Therefore, data from L-NAME are not shown.

View larger version (21K): [in this window] [in a new window]   Fig. 4. Representative Western blot and group data depicting eNOS protein abundance and NO production in cultured human coronary endothelial cells incubated for 24 h in drug-free media (CTL) and media containing the NOS inhibitor L-NMMA, at 10–7, 10–6, 10–5, and 10–4 M concentrations. Data represent mean and S.E.M. of at least four separate experiments. *, P < 0.05 versus CTL (two-way ANOVA).

Effect of BK Plus NOS Inhibitor. Data are shown in Fig. 5. In confirmation of the above experiments, BK-treated cells exhibited a significant increase in NO production, coupled with a significant down-regulation of eNOS protein abundance. The opposite responses were found with L-NNMA. These responses were completely abrogated when cells were treated with both BK and L-NNMA simultaneously.

View larger version (16K): [in this window] [in a new window]   Fig. 5. Representative Western blots and group data depicting eNOS protein abundance and NO production in cultured human coronary endothelial cells incubated for 24 h in the drug-free media (CTL) and media containing bradykinin alone (BK), L-NMMA alone (LN), and a combination thereof (LN + BK) at 10–5 M concentrations. Data represent mean and S.E.M. of at least four separate experiments. *, P < 0.05 versus CTL and LN + BK groups (two-way ANOVA).

Correlations. Data are depicted in Fig. 6. A significant inverse correlation was found between NO production and eNOS protein expression among cells incubated for 24 h in media containing different concentrations of BK and the NOS inhibitor.

View larger version (19K): [in this window] [in a new window]   Fig. 6. Correlation between eNOS protein abundance and NO production in coronary endothelial cells incubated for 24 h with different concentrations (a = 0, b = 10–7 M; c = 10–6 M; d = 10–5 M; and e = 10–4 M) of eNOS activator bradykinin (top panel) and NOS inhibitor L-NMMA (bottom panel).

	Discussion

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As expected, sustained stimulation with BK resulted in a significant dose-dependent rise in NO production by cultured human coronary endothelial cells. The BK-mediated increase in NO production was completely abolished by BK-2 receptor blockade but was unaffected by BK-1 blockade. These observations are consistent with the earlier findings in the endothelial cells (Regoli and Barbe, 1990; Pelc et al., 1991).

The rise in NO production in BK-stimulated endothelial cells was coupled with a significant concentration-dependent fall in immunodetectable eNOS protein abundance. Prevention of the BK-mediated rise in NO production by BK-2 receptor blocker completely abrogated the associated down-regulation of eNOS in cultured coronary endothelial cells. These observations suggested that BK-mediated down-regulation of eNOS may be mediated by the associated rise in NO production. If true, the opposite phenomenon should occur with inhibition of NO production by endothelial cells. In an attempt to explore this possibility, we treated cultured coronary endothelial cells with two different NOS inhibitors, LNMMA and LNAME. As expected, both NOS inhibitors significantly lowered NO production in this cell system. Inhibition of endothelial NO production by NOS inhibitors was accompanied by up-regulation of eNOS in cultured coronary endothelial cells. Moreover, concomitant treatment with NOS inhibitor and BK, which resulted in no change in NO production, was associated with no change in eNOS abundance.

The role of NO as the mediator of the observed negative feedback regulation of eNOS in response to BK and NOS inhibitors is supported by our earlier study, which showed dose-dependent down-regulation of eNOS in response to exogenous NO donors (nitroprusside or S-nitrosopenicillamine) and marked up-regulation of eNOS in response to an NO scavenger (oxyhemoglobin) in cultured human coronary artery endothelial cells (Vaziri and Wang, 1999). Moreover, NO inactivation by reactive oxygen species and the accompanying reduction of NO availability results in compensatory up-regulation of eNOS in spontaneously hypertensive rats, rats with lead-induced hypertension, and lead-exposed cultured endothelial cells (Vaziri et al., 2000, 2001; Vaziri and Ding, 2001). Finally, the compensatory up-regulation of eNOS in these models is reversed by antioxidant therapy, which restores NO availability (Vaziri et al., 2000, 2001; Vaziri and Ding, 2001). Thus, data obtained in the present study and those cited above demonstrate that despite their diversity, interventions that raise NO availability (BK-mediated eNOS activation and/or NO donor administration) down-regulate, whereas those limiting NO availability (BK-2 blockade, NOS inhibition, presence of NO scavengers, or NO inactivation by oxidative stress) up-regulate eNOS expression in coronary endothelial cells. These observations provide compelling evidence for the role of biologically active NO in the regulation of eNOS protein expression in coronary endothelial cells. This phenomenon does not appear to be limited to the coronary artery endothelial cells, since modulation of NO availability by NO donor administration in normal animals (Vaziri and Wang, 1999) or of endogenous NO availability by antioxidant therapy in hypertensive animals (Vaziri et al., 2000, 2001) causes directionally consistent changes in eNOS abundance in various other tissues, including aorta and kidney.

Together, the data demonstrate a reciprocal relationship between the level of eNOS activity and eNOS protein expression in the endothelial cells. Such a negative feedback regulation of the enzyme expression by its product appears to be a biologically appropriate adaptive response to chronic modifications of the enzyme activity. In this context, an exquisite negative feedback system is operative in short-term regulation of NO system as well (Michel, 1999). For instance, intracellular translocation of eNOS from plasma membrane in conjunction with its activation in response to the external agonists serves to uncouple the enzyme from the activating event at the cell membranes (Michel, 1999). Likewise, the decline in cytoplasmic Ca²⁺ in response to the rise in NO production contributes to dissociation of calmodulin and hence, inactivation of eNOS. These events provide a rapid and short-term counter-regulatory response by modifying the enzyme activity. The findings of this study elucidate the chronic adaptive response to conditions that may lead to persistent modifications of eNOS activity. It should be noted that changes in eNOS abundance in response to the stimulatory or inhibitory factors represent compensatory responses, and as such, serve to moderate, but not obviate, the effects of the inciting factor(s). Thus, the fall in eNOS abundance in response to sustained elevation of BK helps to moderate the rate of rise in NO production, as opposed to lowering it to or below the basal level. A similar argument can be made in reverse, concerning the effect of NOS inhibitors.

In conclusion, sustained BK-mediated activation results in compensatory down-regulation, whereas sustained inhibition leads to compensatory up-regulation of eNOS protein expression in cultured human coronary artery endothelial cells. The observed modulations of eNOS expression are mediated by NO and represent adaptive physiologic responses.

	Footnotes

 
doi:10.1124/jpet.104.076497.

Dr. Y. Ding is presently a resident in the Department of Pathology at Brown University, Providence, RI.

ABBREVIATIONS: BK, bradykinin; NO, nitric oxide; eNOS, endothelial nitric oxide synthase; HOE-140, H-D-Arg-Arg-Pro-Hyp-Gly-Thi-Ser-D-Tic-Oic-Arg-OH; L-NMMA, N^G-monomethyl-L-arginine; L-NAME, N-nitro-L-arginine-methylester; NO, nitrate plus nitrite; ECL, enhanced chemiluminescence; ANOVA, analysis of variance; CTL, control.

Address correspondence to: Dr. N. D. Vaziri, MACP, Division of Nephrology and Hypertension, UCI Medical Center, 101 The City Drive, Bldg. 53, Rm. 125, Rt. 81, Orange, CA 92868. E-mail: ndvaziri{at}uci.edu

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This Article Abstract Full Text (PDF) All Versions of this Article: jpet.104.076497v1 313/1/121    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Vaziri, N. D. Articles by Barton, C. H. Search for Related Content PubMed PubMed Citation Articles by Vaziri, N. D. Articles by Barton, C. H. Pubmed/NCBI databases Compound via MeSH Substance via MeSH Hazardous Substances DB (L)-ARGININE NITRIC OXIDE