JOURNAL OF CELLULAR PHYSIOLOGY 14321-25 (1990)

Bradykinin Induces Superoxide Anion Release From Human Endothelial Cells JAMES A. HOLLAND,' K I R K W O O D A. PRITCHARD, M I C U E L A. PAPPOLLA, MICHAEL S. W O L I N , NANCY J. ROGERS, AND MICHAEL B. STEMERMAN Departments of Medicine (I.A.H., K.A.P., N.I.R., M.B.S.), Pathology (M.A.P., M.B.S.), and Physiology (K.A.P., M.S.W., M.B.S.), New York Medical College, Valhalla New York 10595; Departments of Medicine (I.A.H.)and Pathology (M.A.P.), Veterans Administration Medical Center, Montrose, New York 10548

The time-dependent release of superoxide anion (0;) from bradykinin (Bk) -stimulated human umbilical vein endothelial cells (EC) was measured as the superoxide dismutase-inhibitable reduction of ferricytochrorne C employing a novel application of microspectrophotometry. In the absence of Bk, 0; release by EC was not detectable. EC exposure to Bk (1 O-' to 1 0-5 M) resulted in a rapid release of 0 ; . The release of 0; occurred within 5 minutes of exposure. 0; release was partially inhibited by indomethacin (63 5 6 % ) ,thus suggesting that arachidonic acid metabolism, through cyclooxygenase, contributes to EC 0; production. EC 0; release may be an important component in the pathophysiologic actions of Bk on vascular function.

Bradykinin (Bk) is a vasoactive peptide that is increased in plasma during numerous inflammatory diseases (Pisano and Austgen, 1976). The pathophysiologic effects of Bk on the blood vessel wall are diverse, including vasodilation, increased vascular permeability, margination and infiltration of leukocytes, and release of various endothelial-derived vasoactive substances (Erdos, 1979; Fujii et al., 1979). The precise mechanism(s) by which Bk causes these effects is not fully defined. One possible mechanism may be the induction of cellular superoxide anion (0,)production. In vivo studies by Kontos et al. (1984) have shown that exposure to Bk produces vasodilation through 0, generation in the cat cerebral microcirculation. However, the cellular source for 0; has not been determined with certainty. Studies by Rosenblum (1987) reveal that one likely 0; source in the cerebral microcirculation is vascular endothelium. Bk has been shown to modulate numerous endothelial functions such a s the release of endothelial-derived relaxing factor (EDRF) (Ignarro et al., 1986) and prostacyclin (PGI2) (McIntyre et al., 1985). In this report, we provide evidence that Bk also induces 0, release from endothelial cells and that cyclooxygenase is a potential source of the 0, produced.

MATERIALS AND METHODS

Reagents Medium 199 containing Earle's salts, HEPES buffer, heparin, cytochrome C (type 111: from horse heart), superoxide dismutase (SOD), and calcium ionophore A23187 were obtained from Sigma Chemical Company, St. Louis. Bradykinin was purchased from Calbiochem Biochemicals, San Diego. Collagenase was obtained from Cooper Biomedical, Malvern, PA. Antibiotidantimycotics and L-glutamine were from Gibco Labora0

1990 WILEY-LISS. INC.

tories, Grand Island, NY. [3H]-6-keto-PGF1, was obtained from Amersham, Arlington Heights, IL and 6keto-PGF,, antiserum was from Seragen, Inc., Boston. Scint A scintillation cocktail was purchased from Packard, Sterling, VA.

Endothelial cell culture EC were isolated from human umbilical veins by the method of Jaffe et al. (1973a). Briefly, the vein lumen was cannulated and washed with a HEPES buffer SOlution (pH 7.4). The vein was drained, filled with 0.05% collagenase, and incubated at 37°C for 10 minutes. The collagenase solution with cells was flushed from the vessel, then seeded in Primaria T 25 cm2 flasks (Falcon, Oxnard, CA) on a human fibronectin-coated surface (25 Fg/ml) (Maciag et al., 1981). Cells were cultured in media consisting of Medium 199 supplemented with 25% fresh pooled human serum, antibiotic/antimycotic, L-glutamine, HEPES buffer (21 mM), heparin (90 kg/ml), and endothelial cell growth factor (ECGF) (300 pg/ml), and cells were incubated in a n atmosphere of air containing 5% CO, at 37°C (Holland et al., 1988).The cultured cells were identified as EC by immunof luorescent staining for factor VIII antigen by the method of Jaffe et al. (1973b). Culture media was changed every 48 hours.

Measurement of 0, release The formation of 0, in the supernatant was monitored continuously using microspectrophotometry to measure the reduction of ferricytochrome C (cyt C) by 0;. Cells were passaged using trypsidEDTA onto Lab-

Received June 15, 1989; accepted November 20, 1989.

'To whom reprint requestskorrespondence should be addressed.

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HOLLAND ET AL

Tek tissue culture chamber/slides (#4812, Miles, Naperville, IL). These cell cultures were maintained at confluence, as determined by morphologic appearance, for 2-5 days prior to 0, measurements. 0, measurements were performed by washing cells 3 times with phosphate-buffered saline (pH 7.4) without calcium or magnesium (PBS). Medium 199 (pH 7.4) containing 0.14 mM cyt C was added to each chamber, and cells were incubated for 3 minutes a t room temperature in the presence or absence of 80 pg/ml SOD. The chambers were placed on a microscope stage (Labophot, Nikon, Japan), and the focus was adjusted on the cell surface. Reactions were started by withdrawal of the supernatant into a 1 ml syringe containing Bk (final M) or the calcium ionophore A23187 conc. lop6to (final conc. 1pM), mixed, and returned to the chamber. Transmittance measurements were recorded using microspectrophotometry with a Nikon P1 microphotometer equipped with a Hamamatsu Photonics photomultiplier tube (R1104HA, Hamamatsu, Japan) attached to a Nikon Labophot microscope. The microphotometer was adjusted to a maximum gain setting, and contained a 10 x objective (#85041, Nikon, Japan) with a 10 mm field diaphragm. Equipment calibration including determination of linearity for transmittance vs. reduced cyt C concentration was performed as previously described by Pappolla (1988). A 550 nm filter (bandwith 4.5nm) (Ditric Optics, Hudson, MA) was used with a 1mm slit between the filter and the microscope tungsten light source. Reduction ofcyt C in the supernatant was monitored continuously at room temperature by recording transmittance changes at 550 nm over a 5 minute period. The concentration of 0; (nanomoles/2 x lo5 cells) in the test media was calculated from transmission data as follows. First, transmittance values of 30 second time points were converted to their corresponding absorbence values. The 0; concentration at each time point was calculated by dividing the difference in absorbance of the samples with and without SOD by the extinction coefficient for ferrocytochrome (E 550 nm = 21.1 mM-’crn-’) and the light path (0.11 cm). The path length was calculated using the known medium volume (0.5 ml) and chambedslide dimensions (surface area = 4.26 cm2). After the completion of measurements, cells were harvested and counted with a Coulter Counter (Model ZF, Coulter Electronics, Hialeah, FL). For experiments employing indomethacin, cell cultures were incubated for 10 minutes in Medium 199 with or without 10 p-M indomethacin prior to stimulation. Measurement of prostacyclin accumulation Prostacyclin (PGI2) supernatant accumulation, following a 5 minute incubation of EC with Bk, was measured in the presence and absence of 10 pM indomethacin. Primary EC cultures were passaged onto 24 well plates. Cultures were maintained at confluence for 2-5 days prior to PG12 measurements. For measurements, EC were washed three times with PBS pH 7.4. An aliquot of 300 p1 of Medium 199 containing Bk (lo-’ to M) was added to each well for a 3 minute incubation or Medium 199 without Bk (control). In experiments employing indomethacin, cells were preincubated with 10 pM indomethacin for 10 minutes

A.

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Yo Trans I 0 oo/o

-3-2

-I

0

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BK

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+ SOD I00 i 1 I -3-2-1

T 0 BK

I I

I 2

I 3

I 4

I 5

I 6

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7

minutes

Fig. 1. This figure depicts a typical tracing from an experiment dernonstrating the change in transmittance produced by exposure of EC to M)in the absence (A) and presence (B) of SOD. Cells were Bk incubated with Medium 199 containing 0.14 mM cyt C for 3 minutes with or without 80 pgiml SOD. Reactions were started by withdrawal of the supernatant into a syringe containing Bk,mixing, and returning onto the cells. The decrease in transmittance at 550 nm was used to measure the generation of reduced cyt C (see “Materials and Methods”).

prior to Bk stimulation to loa5M). Subsequently, the supernatant was collected, and PGI2 accumulation was determined by radioimmunoassay of 6-ketoP GF la (Holland e t al., 1988).

Data analysis EC 0; generation was expressed as the mean standard error of the mean (SEM) in nanomoles of 0, per 2 x lo5 cells accumulated during a 5 minute incubation with varying Bk concentrations. PGIB accumulation in each well was reported as the mean & SEM in picograms of 6-keto-PGF1,/cells x lo3 formed per 5 minute incubation of EC at varying Bk concentrations, with or without indomethacin. The significance of differences in 0, data from varying time points was determined using a Student’s t-Test (Zar, 1974). Statistical analysis for 6-keto-PGF,, data was performed employing Analysis of Variance with Dunnett’s test for group comparisons.

*

RESULTS Measurement of 0; release f r o m EC 0, release by a confluent monolayer of EC was determined using microspectrophotometric measurement of cyt C reduction. This method compares the amount of cyt C reduced in the presence and absence of SOD, the difference corresponding to the amount of 0, generated. Typical recordings of transmittance data from Bk studies are illustrated in Figure lA,B. Figure 1

PRODUCTION OF SUPEROXIDE ANION BY THE ENDOTHELIUM

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0

1

Time (minutes) Fig. 2. Time course of the effect of Bk on endothelial 0;release. 0; release from EC incubated with M (open circles, n = 8) and 10 M (solid circles, n = 22) Bk for the 5 minute time course was measured as described in Figure 1.In the absence of Bk,0; formation was not detectable. Each time point is expressed as the mean 2 SEM. Group values for and 10 - 5 M Bk-stimulated cells a t each time point after 2 minutes were significantly different (P< .01).

shows transmittance changes following M Bk stimulation in the absence of SOD (A) and in the presence of SOD (B). Effect of bradykinin on EC 0; release Bk induces a rapid release of 0; from EC. Figure 2 shows the time course for 0, release during a 5 minute exposure of EC to Bk M (n = 22) and 1OP6M(n = 8). 0 irelease begins immediately with the addition of Bk. Similarly, and as previously reported by Matsubara and Ziff (1986a), calcium ionophore A23187 induces a continuous release of 0,by EC. Figure 3 shows the time course for 0; release from A23187-stimulated cells (n = 6). When EC are pre-incubated with 10 pM indomethacin, cellular 0; release in response to M Bk is decreased by 63 ? 6% (Fig. 4). Bradykinin and EC PGI2 generation The supernatant concentration of a stable metabolite of PGI2,6-keto-PGFla, following a 5 minute exposure of EC to Bk is measured as an index of EC cyclooxygenase activity. Bk a t M produces a stimulation in cyclooxygenase activity (n = 4) at a time period corresponding to an elevation in 0, generation (Table 1). Further, to demonstrate that indomethacin effectively blocked cyclooxygenase activity under the experimental conditions employed for the 0, studies, 6keto-PGF,, accumulation following Bk stimulation to lOP5M) is measured using indomethacin (10 FM)-pretreated cells (n = 4). Indomethacin reduced 6-keto-PGF,, accumulation by 84 1% and 84 f 5%, respectively. These responses are similar to those reported previously by Nawroth et al. (1984).

DISCUSSION This study demonstrates that EC generate 0; when stimulated with Bk. 0; release was rapid, occurring within minutes of Bk exposure. In addition, 0; release was partially blocked by indomethacin, an inhibitor of cyclooxygenase. Previous in vitro studies describe EC

2

3

5

4

Time (minutes) Fig. 3. Time course for calcium ionophore A23187-induced 0, release by EC. Cells were stimulated with 1 p M A23187 in the presence and absence of SOD,and 0, release was measured by microspectrophometry employing the reduction of cyt C as described for Figure 1. Each time point represents the mean 2 SEM from six individual experiments.

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04' 0

1

2

3

4

1

5

Time (minutes) Fig. 4. Inhibition by indomethacin of 0; release by EC. EC were preincubated for 10 minutes with (open diamonds) or without (solid diamonds) 10 pM indomethacin, and subsequently 0; release was measured during M Bk stimulation. Each time point represents the mean 2 SEM from 8 experiments. The group values for time points beyond 2 minutes were significantly different (P< .05).

0, release in response to various substances. Such

agents include phorbol myristate (Matsubara and Ziff, 1986a), calcium ionophore A23187 (Matsubara and Ziff, 1986a1, polystyrene microspheres (Gorog et al., 19871, low-density lipoprotein (Steinbrecher, 1988), interleukin 1 (Matsubara and Ziff, 1986b1, and gammainterferon (Matsubara and Ziff, 1986b). This report describes, for the first time, that EC are capable of releasing 0; when exposed to Bk.Studies by Kontos et al. (1984)suggest that such a release of reactive oxygen species is likely to contribute to Bk-mediated vascular responses. The microspectrophotometric technique employed in these studies was devised for direct and continuous monitoring of O,.generation by an in situ EC monolayer. In the original method by Babior et al. (1973), cyt C substrate was separated from the cells before cyt

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HOLLAND ET AL

TABLE 1. Bk-induced PGI2 release from human EC' Agent added Control Bk10 ' M Bk lo-' M Bk 10.' M

g-keto-PGF, (pg/i03 cells) 4.7 i 1.2 9.6 1.0 10.1 0.9 15.6 2 1.7

* *

'Concentration-dependenteffect of Bk on X I 2 release fmm human EC. EC cultured on 24 well plates were exposed to increasing concentrationsof Bk (lo-' to lo-' MI for 5 minutes. PGI2 production was quantified by radioimmunoassay of 6-keto-PGF1,,the stable metabolite of PGI2. The data represent the mean ? SEM of results obtainedfrom four experiments. Cells stimulated with M Bk produced a greater amount of 6-keto-PGF,, than those exposed to lower levels of Bk or vehicle (M199)(P< -01).

C reduction was measured spectrophotometrically. This method was not adequate to measure the timedependent O2 release by EC described in this study. The method used herein allows for the detection of rapid and brief 0; release with minimal perturbation or manipulation of the EC during the measurements. An unexpected finding was the concentration of Bk needed to elicit 0, release. Previous studies by Hong (1980)and Palmer et al. (1988)have demonstrated that PGIB and EDRF generation can be induced at Bk concentrations of lop8 to lop6 M. Ibe et al. (1989) have shown a concentration-dependent increase in PGIB release with Bk levels up to 10-4M. These differences may relate to the culture characteristics. In our experiments, first-passaged EC produced detectable 0, generation. Studies by Goldsmith et al. (1984) have shown that PG12 production by serial-passaged cells is highest in the early-passaged cells. In addition, they have demonstrated that angiotensin-converting enzyme activity, which is involved in Bk degradation, is highest in these early-passaged cells. Thus, increased Bk degradation by EC could account for this effect. Further, Kontos et al. (1985) required 1.9 x M Bk to elicit free radical production in the cat cerebral microcirculation. Studies examining EC 0, release have shown variability in the time course and amount of free radical generated (Matsubara and Ziff, 1986a,b; Rosen and Freeman, 1984).These differences may be explained by the culture conditions. Previous studies utilized cell suspensions and multi-passaged cultures. In the present study, fixed, confluent monolayers of first-passaged cultures were employed. Thus, cells were likely not perturbed prior to 0; measurements owing to detachment and suspension. Further, early-passaged cells are likely more metabolically active upon stimulation since studies show an inverse relationship between passage number and PG12 release (Goldsmith et al., 1984). Endothelial integrity is important for the maintenance of normal vascular physiology (Stemerman et al., 1984). Exposure t o reactive oxygen species may compromise endothelial vasoregulatory functions and potentially cause injury to the various vascular wall components. For example, studies by Egan et al. (1979) show that axygen free radicals can alter endothelial arachidonic acid metabolism by inactivating both cyclooxygenase and prostacyclin synthase. These enzymes are involved in PGIB formation, an important anti-platelet aggregating and vasodilating agent (Mon-

cada, 1983)). Further, Gryglewski et al. (1986) have shown that 0, inactivates EDRF, an endothelium-derived substance that mediates vascular relaxation responses to numerous vasoactive substances. Bk has numerous effects on EC function. This nonapeptide has been demonstrated to induce endothelial release of agents such as EDRF (Ignarro et al., 1986) and PGIB (McIntyre et al., 1985). Additionally, we now report that EC release 0; when stimulated with Bk. A paradox exists in that 0; degrades EDRF (Gryglewski et al., 1986) and may inhibit PGIB formation (Egan et al., 1979). Based on current knowledge, EC exposure to high Bk levels could result in generation of sufficient 0, to produce an inhibitory modulation of these other vasoregulatory systems, and 0, may also have a direct effect on vascular smooth muscle tone. These observations would be consistent with a vasoregulatory role for endothelial-derived oxygen free radicals. A role of oxygen species has been proposed by Kontos et al. (1984). They have demonstrated, using cat cerebral microcirculation, that SOD inhibits Bk-induced arteriolar vasodilation. Kontos et al. (1985) have also shown that cerebral Bk-induced microvascular vasodilation is partially inhibited by indomethacin. This finding corresponds t o our observation of a partial inhibition of endothelial 0, release by pretreatment with indomethacin. Indomethacin inhibition of PGIB release (83%)was substantially greater than 0; release (63%). This suggests that other cellular sources are also involved in 0; production (Freeman and Crapo, 1982). In vitro studies by Kukreja et al. (1986) have demonstrated that 0; is generated during arachidonic acid metabolism by cyclooxygenase. In these studies, purified PGH synthase (cyclooxygenase) co-metabolized NAD(P)H via the peroxidase reaction of this enzyme resulting in 0, generation. Recently, Rosenblum et al. (1987) have developed a method using light/dye injury to inactivate endothelium-dependent vascular regulation in vivo. This method demonstrated that the oxygen metabolite mediated vasodilation to Bk in the mouse cerebral circulation is endothelium-dependent. Thus, EC may be an important source of 0; in vivo. In conclusion, we report that Bk stimulation of cultured EC causes a SOD-inhibitable reduction of cyt C. This finding suggests that EC release 0; when exposed to Bk. Such endothelial release of 0, likely contributes to the numerous vascular actions of Bk.

ACKNOWLEDGMENTS We thank the Hudson Valley Blood Service for their support and Sue Murphy for assisting in the preparation of this manuscript. This research was supported by Grants HL43193 and HL33742 from the National Heart, Lung, and Blood Institute, National Institutes of Health and by a Grant-in-Aid No. 880907 from the American Heart Association. LITERATURE CITED Babior, B.M.,Kipnes, R.S.,and Curnette, J.T. (1973) Biological defense mechanisms. The production by leukocytes of superoxide, a potential bactericidal agent. J. Clin. Invest., 52:741-744. Egan, R.W., Gale, P.H., and Keuhl, F.A., Jr. (1979) Reduction of hydroperoxides in the prostaglandin biosynthetic pathway by a microsomal peroxidase. J. Biol. Chem., 254:3295-3302. Erdos, E. (ed.)(1979) Bradykinin, kallidin, and kallikrein (supple-

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Bradykinin induces superoxide anion release from human endothelial cells.

The time-dependent release of superoxide anion (O2-) from bradykinin (Bk)-stimulated human umbilical vein endothelial cells (EC) was measured as the s...
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