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. 2000 Nov;131(5):1019–1023. doi: 10.1038/sj.bjp.0703665

Endothelial nitric oxide synthase is a site of superoxide synthesis in endothelial cells treated with glyceryl trinitrate

Wayne H Kaesemeyer 1, Alison A Ogonowski 1, Liming Jin 1, Ruth B Caldwell 2, Robert W Caldwell 1,*
PMCID: PMC1572421  PMID: 11053225

Abstract

  1. Tolerance to glyceryl trinitrate (GTN) involves superoxide (O2.−) production by endothelial cells. Nitric oxide synthase (NOS) produces O2.− when L-arginine (L-arg) is limited. The purpose of this study was to test the hypothesis that GTN stimulates NOS to increase O2.− synthesis in endothelial cells when L-arg is limited.

  2. Production of O2.− by bovine aortic endothelial cells (BAEC, passages 3–5) was determined by spectrophotometrically measuring superoxide dismutase-inhibited reduction of ferricytochrome C to ferrocytochrome C. Cells were incubated in buffer without L-arg. O2.− production was measured using BAEC either untreated or treated with L-NAME or L-arg alone or following treatment with GTN (10−9 to 10−6 M) for 30 min or DPTA NONOate (10−7 and 10−6 M) alone or with GTN or DPTA NONOate after pretreatment with nitro-L-arginine methyl ester (L-NAME), L-arg or their inactive enantiomers, D-NAME or D-arg (all 5×10−4 M) (n=6–7/group).

  3. L-NAME alone produced a 69% reduction in O2.− levels. Treatment with L-arg alone had no effect. Cells treated with GTN alone exhibited an increase in O2.−. This effect was prevented by pretreatment with either L-NAME or L-arg, and was unaffected by D-NAME or D-arg. We observed a dose-response relationship in O2.− production to GTN over a range of 10−9 to 10−7 M.

  4. The NO donor, DPTA-NONOate, unlike GTN, did not have a significant effect on O2.− production.

  5. In conclusion, endothelial NOS is a site of O2.− synthesis in endothelial cells activated by GTN.

Keywords: Glyceryl trinitrate (nitroglycerin), endothelial nitric oxide synthase, L-arginine, nitrate tolerance, superoxide anion

Introduction

Previous studies have shown O2.− production by endothelial cells treated with a Ca2+-ionophore or bradykinin (Katusic & Vanhoutte, 1989; Holland et al., 1990) and also by GTN, a NO donor (Dikalov et al., 1998). In general, sites of O2.− synthesis include membrane-bound NAD(P)H oxidases (Münzel et al., 1995), xanthine oxidase (Katusic & Vanhoutte, 1989), mitochondrial electron transport chain (Jansson et al., 1993), cytochrome P450 oxidase (Jansson et al., 1993) and cyclo-oxygenase (Holland et al., 1990). In a recent study of rat aortic endothelial cells (Puey et al., 1998), O2.− production in response to angiotensin II was blocked by pretreatment with L-NMMA, an inhibitor of eNOS, indicating that eNOS is yet another site of O2.− production. When the supply of L-arginine to eNOS is limited, eNOS utilizes oxygen as its principal substrate and produces O2.− (Pritchard et al., 1995; Presta et al., 1997; Ogonowski et al., 2000). In this study we test and confirm the hypothesis that eNOS is a site of O2.− synthesis in endothelial cells activated by GTN when L-arginine is limited. The results of this study amplify our recent findings that agonists of eNOS increase superoxide production when L-arginine is restricted (Ogonowski et al., 2000) and that L-arginine is depleted by GTN, indicating a component of nitroglycerin tolerance that is L-arginine dependent (Abou-Mohamed et al., 2000).

Methods

Drugs and chemicals

Preservative free glyceryl trinitrate (Perlinganit®) was a kind gift of Dr Andreas Reimann of the Schwarz Pharma Company (Monheim, Germany). All other chemical reagents were purchased from the Sigma Chemical Company (St. Louis, MO, U.S.A.).

Cellular superoxide anion formation in the presence of glyceryl trinitrate

Primary cultures of bovine aortic endothelial cells (BAEC) were prepared from fresh blood vessels as described previously (Behzadian et al., 1995). The production of O2.− by BAEC was determined by spectrophotometrically measuring the superoxide dismutase-inhibitable reduction of ferricytochrome C according to Pritchard et al., (1995). BAEC (passages 3–5) were plated in 35 mm dishes containing 10.5×20 mm fibronectin-coated Thermanox coverslips. After reaching confluency in a M-199 medium containing 5×10−5 M L-arg, cells were washed three times (3 ml) with Dulbecco's phosphate buffered saline (DPBS) and two coverslips were placed in a disposable plastic cuvette facing each other. DPBS (1.8 ml) without L-arg was gently placed in a cuvette in addition to 200 μl of ferricytochrome C (final concentration, 5×10−6 M). The cuvette was inverted to mix the reagents. This was followed by a 30 min incubation period at 37°C to provide for the cells to equilibrate. After this period, the test substance or an equal volume of buffer (10 μl) was added and mixed. Absorbance was then recorded for 60 min using a spectrophotometer at 550 nm wavelength to monitor basal O2.− production or the effect of pretreatments. Production of O2.− was evident within minutes. The effects of pretreatment with L-NAME or L-arginine (L-arg, 5×10−4 M) on basal O2.− production were also determined. In order to determine whether addition of the L-arg analogue L-NAME or L-arg could prevent or reduce the formation of superoxide anion, experiments were performed in which L-NAME (5×10−4 M) or L-arg (5×10−4 M) were added prior to GTN treatment. Change in absorbance over time was recorded in the presence or absence of superoxide dismutase (400 u ml) to determine the portion of absorbance change due to O2.− production. The amount of superoxide anion [ε=2100 cm−1)/(mol l)−1] generated was determined over time and reported as pmoles O2.− min−1 (106 cells)−1. In order to evaluate the possible scavenging action of L-NAME and L-arg (Rehman et al., 1997), pretreatments of D-arg or D-NAME (both 5×10−4 m) were used as controls. In other experiments, varied concentrations of GTN (10−9 to 10−6 M) were added to the cuvette after the initial incubation period. We also examined the effects of the NO donor, DPTA-NONOate (at 10−7 and 10−6 M) on O2.− production.

Statistical analysis

Data are presented as mean±s.e.mean of the indicated number of observations (n) and the difference between groups was assessed using the paired t-test or analysis of variance, when appropriate. A probability value (P) less than 0.05 was considered to be statistically significant.

Results

Basal production of O2.− by BAEC in L-arg free media was 59 pmol min−1 (106 cells)−1 (Figure 1). Pretreatment of control cells with L-NAME suppressed basal production of O2.− by 70%. Pretreatment of these cells with L-arg did not alter basal O2.− production.

Figure 1.

Figure 1

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Basal production of O2.− by BAEC in L-arg free media. Effects of pretreatment of cells with L-NAME (5×10−4 M) or L-arginine (5×10−4 M) are also displayed. *Indicates difference from basal production of O2.− (P<0.01). n for each experiment ranged from 6–7.

Treating cells with GTN (10−6 M) increased O2.− production from 69 to 155 pmol min−1 (106 cells)−1, an increase of 125% over basal levels (Figure 2A). Experiments performed in parallel showed that treatment with L-NAME prior to GTN decreased O2.− production to 15 pmol min−1 (106 cells)−1, a decrease of 140 pmol min−1 (106 cells)−1, i.e. 90% of levels for GTN alone or 79% of control levels (Figure 2B vs 2A). Pretreatment of cells with L-arg also blocked the effect of GTN in increasing O2.− production (Figure 2B). L-arg pretreatment reduced O2.− levels to 88 pmol min−1 (106 cells)−1. This value is 43% of the response to GTN alone and indicates that L-arginine pretreatment reduced the O2.− response to treatment with GTN to basal (cells alone) levels (compare Figure 2A,B)].

Figure 2.

Figure 2

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Effect of GTN (10−6 M) on O2.− production by endothelial cells. (A) shows basal production of O2.− in the absence of L-arginine and the effect of treatment with GTN (10−6 M). (B) shows the effect on O2.− production of pretreating cells with L-NAME or L-arg followed by treatment with GTN. (C) shows the effect on O2.− production of pretreating cells with D-NAME or D-arginine followed by treatment with GTN. *Indicates difference from basal O2.− production, P<0.05, **indicates P<0.005, †indicates difference from cells treated with GTN alone, P<0.05, ‡indicates P<0.01. In A, there is a 125% increase in O2.− production when cells (in L-arginine free buffer) are treated with GTN alone. In B L-NAME pretreatments resulted in a decrease in O2.− which is different from both basal and GTN stimulated O2.− production, whereas L-arginine pretreatment is different from GTN stimulated O2.− production only. In C, both D-NAME and D-arginine pretreatments resulted in increased O2.− production over basal production; neither treatment resulted in changes in O2.− production which were different from cells treated with GTN alone. Δ Indicates difference from cells treated with L-arginine, P<0.05. n for each experiment ranged from 6–7.

In contrast, treating cells with either D-NAME or D-arg prior to the GTN treatment did not significantly change the responses to GTN which were increased by 150 and 86 per cent, respectively, over basal control (compare Figure 2A,C). Cells receiving pretreatments with D-NAME or D-arg and then treated with GTN were observed to have increases in O2.− production of 175 pmol min−1 (106 cells)−1 for D-NAME and 130 pmol min−1 (106 cells)−1 for D-arg (Figure 2C). Neither of these responses were different from cells treated with only GTN (compare Figure 2A,C); however, there is a difference between the effects of D-arg and L-arg (compare Figure 2B,C). All experiments displayed in Figure 2 were performed in parallel.

In another set of parallel experiments, we compared responses to GTN at concentrations from 10−9 M, to 10−6 M (Figure 3). This study revealed that treatment with 10−9 M GTN did not alter levels of O2.−. With 10−8 and 10−7 M GTN, O2.− production was significantly increased from 67 to 138 and 172 pmol min−1 (106 cells)−1), respectively. With 10−6 M GTN, O2.− production was elevated over basal levels (131 pmol min−1 (10−6 cells)−1) but was significantly lower than observed with the 10−7 M concentration of GTN. These elevations in O2.− production by GTN were also inhibited by prior treatment with either L-NAME or L-arg (Table 1).

Figure 3.

Figure 3

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Comparison of the effects of GTN at concentrations from 10−9 to 10−6 M on production of O2.−. *Indicates difference from basal O2.− production, P<0.01, **indicates P<0.005, †indicates difference from cells treated with GTN 10−7 M, P<0.05. n for each experiment ranged from 6–7.

Table 1.

Effect of pretreatment of BAEC with L-NAME (5×10−4 M) or L-arginine (5×10−4 M) on O2.− production in response to GTN

graphic file with name 131-0703665t1.jpg

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The NO donor, DPTA–NONOate did not have a significant action on superoxide production (Table 2). In comparison to GTN, addition of DPTA-NONOate resulted in O2.− production of 64 and 66 pmol min−1 (106 cells)−1 at 10−7 and 10−6 M, respectively, compared to a basal level of 59 pmol min−1 (106 cells)−1 for cells alone.

Table 2.

Effect of DPTA-NONOate (DPTA) on O2.− production in BAEC

graphic file with name 131-0703665t2.jpg

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Discussion

Our data confirm the previous studies that endothelial cell NOS is a site of O2.− synthesis (Huk et al., 1997; Münzel et al., 2000; Ogonowski et al., 2000; Puey et al., 1998). These data also demonstrate that GTN can stimulate NOS-mediated production of O2.− in endothelial cells when L-arginine is limited. This conclusion is supported by the reduced O2.− production to GTN in cells pretreated with L-NAME or L-arginine. In contrast, neither D-NAME nor D-arginine pretreatments prevented the GTN induced rise in O2.− production and neither yielded results different from cells treated with GTN alone. This finding, along with the finding of a difference between treatments with D- and L-arg, excludes a previously observed scavenging action of D-NAME and L-NAME and D-arginine and L-arginine on O2.− (Rehmann et al., 1997) as an explanation for the findings of our study.

Whereas L-arginine pretreatment of cells prior to GTN treatment resulted in O2.− values unchanged from control (cells alone), L-NAME pretreatment caused a significant decrease from control. Our present and previous data suggest that L-NAME inhibits both basal and stimulated O2.− production, while L-arginine inhibits only stimulated O2.− production (Ogonowski et al., 2000). We believe this difference is due to direct inhibition of eNOS activities in producing both NO and O2.− by L-NAME pretreatment. By contrast, we believe that addition of L-arginine supports NO production and diminishes O2.− production, i.e. shifts eNOS activity back into producing NO. Production of O2.−, which remains after L-NAME pretreatment, is felt to be from sites other than eNOS. Similar observations on the effect of NOS inhibitors have been made by others (Huk et al., 1997; Münzel et al., 2000; Ogonowski et al., 2000; Puey et al., 1998). It is interesting to note that basal O2.− production in our system is much less than basal O2.− levels observed by others in human umbilical vein endothelial cells in arginine-free buffer (Prichard et al., 1995).

Collectively, these data indicate that O2.− synthesis by endothelial cells treated with GTN involves eNOS activation. The findings of this study are in agreement with those of an earlier study by our group (Abou-Mohamed et al., 2000) which indicated that in addition to GTN's well known actions as an NO donor, it has an auxiliary action involving activation of eNOS.

Our finding that treatment with a 10−7 M concentration of GTN resulted in a higher level of O2.− production than with the 10−6 M concentration is consistent with the known avid reactivity of O2.− with NO to form peroxynitrite (ONOO−) (Huie & Padmaja, 1993; Amirmansour et al., 1999). Increasing concentrations of GTN will donate greater amounts of NO which will decrease levels of O2.− through the mechanism of ONOO− formation. Related interactions and a decrease in O2.− have been noted by others (Kooy & Royall, 1994; Amirmansour et al. 1999).

Control studies with the NO donor DPTA-NONOate demonstrated specificity of the GTN effect. We used a NONOate for these experiments in order to rule out the possible contribution of GTN's function as a NO donor in the O2.− response. Unlike the syndomomines, such as SIN-1, which are capable of generating O2.− as well as donating NO (Feelisch et al., 1989), NONOates have been shown to function as NO donors without generating O2.− (Wink et al., 1996; Rosenberg et al., 1999). In marked contrast with GTN, superoxide levels in the cells treated with NONOate at both 10−7 and 10−6 M were unchanged from basal values. We believe this indicates that the responses seen with GTN reflect its function as a NOS agonist function rather than as a NO donor.

It could be argued that the rate of release of NO from the two NO donors are different and at the concentrations used for DPTA-NONOate, little or no increase of NO may occur, resulting in no effect on NOS activity. However, 10−6 M DPTA-NONOate is chemically equivalent to 2×10−6 M of NO. DPTA-NONOate has a T1/2 of 180 min (Mooradian et al., 1995) and release of NO follows first order kinetics (Maragos et al., 1991). Therefore, 10−6 M DPTA-NONOate at 60 min will yield ∼0.33×10−6 M NO. We have previously determined that 10−6 M DPTA-NONOate decreases system y+ uptake of L-arginine in endothelial cells by about 22% at 60 min (Ogonowski et al., 2000). This observation strongly indicates that an adequate amount of NO is released at 1 h to affect cell function.

Although one might expect that NONOate would have reduced the measured O2.− levels due to formation of ONOO−, this did not occur in our study. The lack of O2.− reduction in the DPTA-NONOate-treated cultures may be explained by the failure of NO to reach critical levels required for ONOO− formation (Amirmansour et al., 1999).

We did not directly identify the NOS isoform stimulated by GTN in our experiments. The predominant isoform in BAEC is eNOS or NOS type III, but iNOS or NOS type II may be induced by cytokine treatment (Kaku et al., 1997). Therefore, O2.− production from iNOS might occur in some experimental conditions. However, basal levels of NO production in our endothelial cells are in line with only normal eNOS activity (Abou-Mohamed et al., 2000). As shown by Kaku et al., (1997), 8–16 h are required for a measurable increase in NO production due to induction of iNOS expression. In our study, the increased O2.− production resulting from GTN treatment occurred rapidly, within only 30 min of GTN addition. Therefore, any contribution of iNOS to O2.− production seen in this study is considered unlikely.

Our findings of eNOS as a site of O2.− production in response to GTN are in general agreement with those of another recent study (Münzel et al., 2000). Additionally, our experiments limiting the supply of L-arginine demonstrate directly for the first time that GTN-stimulated O2.− production occurs as a result of reduced L-arginine availability to eNOS. It has previously been reported that O2.− production occurs only with in vivo models of nitrate tolerance (Münzel et al., 1995). Our data plus those of (Dikalov et al., 1998) suggest that O2.− production also occurs in in vitro models of GTN tolerance. Our observation that addition of extracellular L-arginine prevents GTN-induced O2.− formation suggests that the prime source of this substrate for endothelial NOS is the extracellular fluid.

We believe these data shed new light on the long-standing problem of nitrate tolerance. Nitrate tolerance has been regarded as a problem with multiple etiologies (Parker & Parker, 1998). Recently, the oxidative hypothesis (Münzel et al., 1995) has gained considerable attention. According to this hypothesis, O2.− production results from activation of membrane-bound AND(P)H oxidase by angiotensin II, which is enhanced by GTN. This leads to scavenging of NO by O2.− to form peroxynitrite and results in reduced NO for vasorelaxation, i.e. tolerance. Recent work by Milone et al., (1999) raises questions about this hypothesis. In that study, an angiotensin II receptor antagonist, losartan, was ineffective in preventing tolerance to GTN. On the other hand, supplemental L-arginine seems to be beneficial in patients with angina treated with nitrates (Ceremuzynski et al., 1997; Kaesemeyer et al., 1997). These patients exhibited increased treadmill exercise time and reversal of tolerance to GTN.

In conclusion, endothelial cell NOS is a site of O2.− production in endothelial cells treated with GTN. Both L-NAME and L-arginine, but not their inactive enantiomers, reduced O2.− synthesis. This indicates that NOS activation by GTN, not scavenging by L-NAME and L-arginine, is involved. These findings suggest that supplemental L-arginine may be effective in reducing tolerance to GTN.

Acknowledgments

The excellent technical assistance of Suzanne Lee and Paula Jackson is gratefully acknowledged. The work was partially supported by NIH grants GM-40540 and EY-0468.

Abbreviations

BAEC

bovine aortic endothelial cells

D-arg

D-arginine

D-NAME

nitro-D-arginine methyl ester

DPTA NONOate

dipropylenetriamine NONOate

GTN

nitroglycerin (glyceryltrinitrate)

L-arg

L-arginine

L-NAME

nitro L-arginine methylester

L-NMMA

NG monomethyl L-arginine

NO

nitric oxide

eNOS

endothelial nitric oxide synthase

iNOS

inducible nitric oxide synthase

NOS

nitric oxide synthase

O2.−

superoxide anion

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