superoxide anions and hyperoxia inactivate endothelium...

Superoxide anions and hyperoxia inactivate endothelium-derived relaxing factor

G. M. RUBANYI AND P. M. VANHOUTTE Department of Physiology and Biophysics, Mayo Clinic and Mayo Foundation, Rochester, Minnesota 55905

RUBANYI, G. M., AND P. M. VANHOUTTE. Superoxide anions and hyperoxia inactivate endothelium-derived relaxing factor. Am. J. Physiol. 250 (Heart Circ. Physiol. 19): HSZZ-H827, 1986.-Experiments were designed to determine the effects of oxygen-derived free radicals on the production and biological activity of endothelium-derived relaxing factor or factors released by acetylcholine. Rings of canine coronary arteries without endothelium (bioassay rings) were superfused with solution passing through a canine femoral artery with endothelium. Superoxide dismutase caused maximal relaxation of the bioassay ring when infused upstream, but not downstream, of the femoral artery; this effect of superoxide dismutase was inhibited by catalase. Infusion of acetykholine relaxed the bioassay rings because it released a labile relaxing factor (or factors) from the endothelium. When infused below the femoral artery, superoxide dismutase and, to a lesser extent, catalase augmented the relaxations to acetylcholine. Superoxide dismutase, but not catalase, doubled the half-life of the endothelium-derived relaxing factor(s). This protective effect of the enzyme was augmented fivefold by lowering the oxygen content of the perfusate from 95 to 10%. These data demonstrate that: 1) superoxide anions inactivate the relaxing factor(s) released by acetylcholine from endothelial cells and 2) hyperoxia favors the inactivation of endothelium-derived relaxing factor(s).

acetylcholine; bioassay; catalase; coronary artery; dog; femoral artery; superoxide dismutase; vascular smooth muscle

ENDOTHELIAL CELLS release a diffusible relaxing substance (or substances) when exposed to acetylcholine (7, 9, 15, 16). The chemical nature of the endothelium- derived relaxing factor(s) is still unknown. Anoxia (3, 8) antioxidants and nonspecific radical scavengers (9, 15) inhibit endothelium-dependent relaxations evoked by acetylcholine, suggesting that oxidative mechanisms play an important role in the production or the release of endothelium-derived relaxing factor(s). Since oxidative processes in endothelial cells can generate oxygen-derived free radicals (13), early speculations suggested that the relaxing mediator may be a free radical (6,8). Exper- iments using isolated blood vessels of the rabbit (9, 19) and the dog (18) ruled out this possibility, since scavengers of oxygen-derived free radicals do not eliminate the endothelium-dependent relaxations to acetylcholine. However, generating or scavenging oxygen-derived free radicals can either augment or inhibit endothelium-dependent relaxations evoked by acetylcholine in canine coronary arteries (18). This study was designed to determine whether these effects of the scavengers are due to

interference with the production, the transit or the action on smooth muscle of endothelium-derived relaxing factor(s). The results demonstrate that under bioassay conditions, when the endothelium-derived relaxing factor is in prolonged contact with physiological salt solution, superoxide anions accelerate its destruction. As a consequence, superoxide dismutase (a specific scavenger of superoxide anions; 11) considerably prolongs the biological half-life of endothelium-derived relaxing factor(s), particularly when the partial pressure of oxygen is low- ered.

METHODS

Experiments were performed on femoral and left cir- cumflex coronary arteries taken from mongrel dogs of either sex (18-28 kg), anesthetized with pentobarbital sodium (30 mg/kg iv). The blood vessels were studied in modified Krebs-Ringer bicarbonate solution (control solution) of the following millimolar composition: NaCl 118.3, KC1 4.7, CaC12 2.5, MgSO, 1.2, KH2P0, 1.2, NaHC03 25.0, calcium disodium-EDTA 0.026, and glu- cose 11.1.

The bioassay apparatus designed in earlier work was used (15). Side branches of segments (3-3.5 cm long) of the left and right femoral artery were tied. The endothelium was kept intact as much as possible. The segments were fixed to stainless steel cannulas (1.5 mm ID) and placed into an organ chamber maintained at 37°C and filled with 12 ml of aerated (95% 02-5s COZ) control solution. The segments were perfused at constant flow (2 ml/min) by means of a multichannel roller pump (Gilson, Minipuls 2) with control solution maintained at 37°C and containing phentolamine ( 10B5 M), propranolol (5 x 10B6 M), and indomethacin (10B5 M), to inhibit CY- and ,&adrenoceptors and cyclooxygenase, respectively (1, 17). A stainless steel tube was also placed in the organ chamber through which control solution was pumped at the same rate. A ring of coronary artery, in which the endothelium had been removed (bioassay ring), was sus- pended directly below the organ chamber by means of two stainless steel stirrups passed through its lumen. One stirrup was connected to an isometric force transducer (Grass FT03C). Changes in isometric tension were recorded. The assembly of bioassay ring, stirrups, and force transducer could be moved freely below the organ chamber allowing the preparation to be superfused with the perfusate from either of the femoral segments with

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OXYGEN-DERIVED FREE RADICALS AND EDRF H823

endothelium (endothelial superfusion) or the stainless steel tube (direct superfusion) (Fig. 1).

In some experiments a stainless steel tube was fixed to the outflow cannula of the femoral artery with endothelium. With this setup, drugs could be infused sepa- rately into each perfusion line either above (site I; allowing contact with the inner surface of the femoral artery) or below the perfused femoral artery segment (sites 2 and 3). Infusion of drugs at site 2 avoided contact with the endothelium of the perfused vessel segment but allowed interaction with released vasoactive substances in transit. Infusion of drugs at site 3 (-0.5 s transit time) affected only the superfused bioassay tissue (15). To increase the transit time, polyethylene tubes of various lengths were placed into a heat exchanger (37°C). By connecting the appropriate tube to the outlet of the femoral artery and positioning the bioassay ring beneath the corresponding outlet from the heat exchanger, the transit time could be increased from 1 to 8,30,60, or 120 S.

Before the actual experiment, the bioassay ring was superfused directly with control solution for 60 min. During this interval it was stretched in a stepwise man- ner until the basal tension reached -10 g, the optimal tension for rings of isolated canine coronary arteries (1, 17). Drugs were infused by means of infusion pumps (Harvard model 901) at a rate of 0.1 ml/min or less.

Drugs

The following pharmacological agents were used: acetylcholine hydrochloride (Sigma); catalase (from bovine liver; 40,000 U/mg protein; Sigma); indomethacin (Sigma); meth 1 y ene blue (Eastman-Kodak); l-norepinephrine bitartrate (Sigma); phenidone (Sigma); phentolamine mesylate (Ciba-Geigy); dl-propranolol (Sigma); prostaglandin Fzn (Sigma); superoxide dismutase (from dog blood; 3,000 U/mg protein; Sigma). All concentrations are expressed as final concentrations (M or U/ml) in the superfusate.

Calculations and Statistics

The biological half-life of the relaxing substance(s) released by acetylcholine from the endothelium of perfused femoral arteries was calculated by relating the decrease in tension in the bioassay ring (expressed as percent) to the transit time imposed (9, 15). Unless otherwise noted, each experimental group consisted of at least six blood vessels taken from different dogs. The data are shown as means t standard error of the mean (SEM). Statistical analysis was performed using Stu- dent’s t test for paired and unpaired observations. Dif- ferences were considered to be statistically significant when P < 0.05.

RESULTS

All experiments were performed during contractions of the bioassay rings caused by prostaglandin FZCY (4 x 10m6 M) in the presence of indomethacin (10m5 M).

Site 1

These experiments were performed with a transit time of 1 s between the femoral artery and the bioassay ring.

All drugs were infused upstream of the femoral artery (Fig. 1).

During direct superfusion of the bioassay ring, the addition of superoxide dismutase (150 U/ml) to the perfusate caused a significant increase in tension (+20.2 t 5.4%). The same concentration of the enzyme caused relaxation (-98.3 t 5.0%; ~2 = 5) during endothelial superfusion (Fig. 2, upper tracing).

Catalase (860 U/ml) caused comparable, significant increases in tension during direct or endothelial superfusion (average increase in tension: +10.5 t 2.5 and +12.6 t 7.2%, respectively; n = 4) (Fig. 2, lower tracing).

During endothelial superfusion, acetylcholine ( 10B6 M) caused complete relaxation of the prostaglandin Fza- induced contraction. Catalase pretreatment (860 U/ml; 10 min) significantly and reversibly reduced the relaxations evoked by acetylcholine (to -31.7 t 9.0%; n = 5) and by superoxide dismutase during endothelial superfusion (to -15.2 t 4.3%, n = 3).

Site 2

In this series, acetylcholine (lo-” M) was infused upstream of the femoral artery, while all other drugs were given downstream from it. The transit time between the femoral artery and the bioassay ring was varied (Fig. 1).

Superoxide dismutase and catalase. When given at site 2 both superoxide dismutase and catalase caused small increases in tension (Fig. 3) that were not significantly affected by varying the transit time.

Acetylcholine. TRANSIT TIME, 8 s. Acetylcholine caused smaller relaxations (-51.2 t 3.6%) than with a transit time of 1 s. The response to acetylcholine was augmented by superoxide dismutase (150 U/ml) when it was added prior to or during acetylcholine infusion, and, to a lesser extent, by catalase (860 U/ml) (Fig. 3, upper).

TRANSIT TIME, 30 S. Acetylcholine caused an increase in tension that was not significantly affected by catalase; in the presence of superoxide dismutase (150 U/ml) acetylcholine caused relaxation (Fig. 3, lower).

In the presence of acetylcholine, but not in its absence, superoxide dismutase caused dose-dependent, reversible relaxations (Fig. 4). These relaxations were reversed by phenidone ( 10B5 M), norepinephrine ( 10d6 M), and methylene blue (low5 M), but not by catalase (860 U/ml) (Fig. 5) .

HALF-LIFE. Progressive lengthening (from 1 to 8, 30, 60, or 120 s) of the transit time between the femoral artery with endothelium and the bioassay ring caused a time-dependent reduction and eventually a reversal of the relaxations induced by acetylcholine (Fig. 6); the half-life of the response to acetylcholine was 8.1 t 1.2 s (n = 6). Superoxide dismutase (150 U/ml) potentiated the response to acetylcholine at 8 and 30 s; the enzyme significantly augmented the half-life (to 15.7 2 1.5 s; P < 0.05; n = 5) (Fig. 6). Catalase (860 U/ml) did not significantly affect the half-life of the relaxations induced by acetylcholine (9.2 t 1.6 s; n = 3).

LOW OXYGEN CONTENT. When the gas mixture aerating the endothelial perfusate was changed from 95% 02- 5% CO2 to 85% N2-10% 02-5% COZ, the contractions of the bioassay rings to prostaglandin Fza were augmented (Fig. 7) and with a transit time of 30 s, acetylcholine

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H824 OXYGEN-DERIVED FREE RADICALS AND EDRF

Multi-channel

of artery with endothelium

th

Diffusion

EDRF

I Action

muscle cell For;e

transducer Coronary artery ring without endothelium

Direct Endothelium

SOD i SOD

PG;pa Catalase ; Catalase -a-

.,/\

-i

I

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FIG. 2. Example of changes in tension in bioassay ring. Effect of superoxide dismutase (SOD, 150 U/ml; upper tracing) and catalase (860 U/ml; lower tracing) on contractions to prostaglandin Fz, (PGFs,; 4 x 10e6 M) of a canine coronary artery without endothelium during direct or endothelial superfusion. Rings relax after switching from direct to endothelial superfusion due to basal release of relaxing substance(s) from endothelium (16). Superoxide dismutase and catalase were infused at site 1 (see Fig. 1) during period indicated by horizontal bars.

caused a further increase in tension. Superoxide dismutase caused relaxations that decreased as the transit time increased (30 s: -29.0 + 9.2%, n = 4; 60 s: -10.7 f 1.2%, n = 3); it reversed the contraction evoked by acetylcholine to a relaxation (Fig. 7). The augmentation, by the enzyme of the relaxations induced by acetylcholine at transit time of 30 s was significantly larger, and started at lower concentrations in the presence of 10 than in 95% oxygen (Fig. 4). The half-life was not affected by the lower oxygen content alone (7.6 f 1.8 s; n = 4), but the combination of low oxygen content plus superoxide dismutase prolonged it to 81 + 5 s (P < 0.05; n = 4) (Fig. 6).

FIG. 1. Left: schematic illustration of 3 phases (production, diffusion, and action) of endothelium- dependent inhibitory responses to acetylcholine (ACh) in blood vessels. EDRF, endothelium-derived relaxing factor. Right: bioassay apparatus used for separate analysis of 3 phases (see left) of endothelium-dependent relaxations to ACh. Perfused segments of femoral artery with endothelium serve as “donor” of endothelium-derived relaxing factor, and a ring of canine coronary artery without endothelium serves as bioassay tissue. In this system, released endothelium-derived relaxing factor is transported to smooth muscle cells of bioassay ring by perfusate (transit). (Modified from Ref. 16).

Site 3. Superoxide dismutase (150 U/ml) and catalase (860 U/ml) given immediately upstream of the bioassay ring caused increases in tension, which were similar in the absence and presence of acetylcholine (10e6 M, infused at site 1) (Table 1). The contractions evoked by both enzymes were comparable to those that they caused during direct superfusion (Figs. 2 and 4) or when injected at site 2 during endothelial superfusion in the absence of acetylcholine (Figs. 3 and 5).

DISCUSSION

The present study confirms earlier observations that acetylcholine relaxes vascular smooth muscle cells by releasing a labile relaxing factor (or factors) from endothelial cells (2, 3, 6-9, 15, 16). It confirms that free oxygen radicals generated during univalent reduction of molecular oxygen are not the endothelium-derived relaxing factor(s) released by acetylcholine (9,X3,19). Indeed, in the bioassay-apparatus, infusion of superoxide dismutase and catalase downstream of the source of endothelial factors augmented rather than abolished the relaxations of the bioassay rings induced by acetylcholine.

In agreement with observations made on isolated rings of canine coronary arteries (18), the present experiments suggest that hydrogen peroxide facilitates the release of endothelium-derived relaxing factor(s). This conclusion is based on the finding that superoxide dismutase evokes catalase-sensitive endothelium-dependent relaxation in the organ chamber (18) and the release of endothelium- derived relaxing factor(s) in the bioassay apparatus. The data in the bioassay apparatus are consistent with a continuous generation of small amounts of superoxide anion, which does not trigger the release of endothelium- derived relaxing factor, except after its accelerated trans- formation to hydrogen peroxide by superoxide dismutase (11, 18). The facilitatory role of hydrogen peroxide on the release of endothelium-derived relaxing factor(s) by acetylcholine is demonstrated further by the inhibitory effect that catalase has an acetylcholine-induced relax-

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Tram 8sec.

it time,

Transit 3Osec.

time,

ACh, 1 O’%vl, site 1 ACh ACh ACh

Catalase, site2

ACh,lO’%l, site1

T /

SOD, site2

ACh

SOD, site2

ACh

b I

4min

Catalase, site2 SOD, site2

FIG. 3. Isometric tension recordings during endothelial superfusion of canine coronary arteries without endothelium contracted by prostaglandin Fza (PGFZ,, 4 X lo-” M). Effects of superoxide dismutase (SOD, 150 U/ml, infused at site 2) and catalase (860 U/ml, infused at site 2) on relaxations induced by acetylcholine (ACh, 10s6 M, infused at site I). Transit time, 8 s: for 6 experiments relaxations induced by ACh averaged -51.2 k 3.6, -99.4 2 1.2, and -65.2 k 3.1% in control solution, in presence of SOD dismutase and in that of catalase, respectively. Increases in tension caused by SOD and catalase averaged +9.2 k 2.3 and +10.5 2 3.1%, respectively (100% = 7.2 k 0.6 g). Transit time, 30 s: ACh caused contraction (+7.9 k 2.3%; n = 11) in control solution (left), which was not affected by catalase (middle) but reversed to relaxation (-47.3 k 7.2%; n = 5) by SOD (100% = 6.9 2 0.6 g).

% 02 + ACh

Soperoxide Dismutase, U/ml

FIG. 4. Effect of increasing concentrations of superoxide dismutase (infused at site 2) in presence of acetylcholine [ (ACh; 10e6 M) infused at site I] during endothelial superfusion of canine coronary arteries without endothelium contracted with prostaglandin Fga (4 x 10B6 M). Experiments were performed with a transit time of 30 s and at 2 levels of oxygenation (10 and 95% 02). For comparison, effects of enzyme during direct superfusion are also shown. Open triangles, direct superfusion + 95% 02; open squares, direct superfusion + 10% 02; fiZkd circles, endothelial superfusion + ACh ( 10m6 M) + 95% 02; filled squares, endothelial superfusion + ACh (10m6 M) + 10% 02. Data shown as means k SE (n = 4) and expressed as % of initial contraction to prostaglandin Fl,, (100% = 7.1 k 0.5 and 8.4 -t 0.5 g in 95 and 10% 02, respectively). * Difference between responses obtained at 95 and 10% O2 is statistically significant (P < 0.05).

ation when infused at site 1 in the bioassay apparatus; this must be due to an action of the enzyme involving the production of relaxing factor(s) by the endothelial cells, than

since catalase infused at site 2 inhibits the relaxation evoked by

augments acetylcholi

rather ne.

A major finding of the present study is that superoxide dismutase, infused downstream from the site of release of endothelium-derived factor(s), potentiates its effect and prolongs its half-life. One possibility is that the enzyme alters the vasodilator properties of substance(s) released by endothelial cells. However, the reversal by phenidone [inhibitor of the endothelial production of the relaxing factor(s)], norepinephrine [inactivator of the factor(s) in transit], and methylene blue [inhibitor of the action of the factor(s) on smooth muscle] of acetylcholine-induced relaxations both in the absence (8, 9, 15, 16) and in the presence of superoxide dismutase (present study) indicates that the known characteristics of the released relaxing factor are not changed by the enzyme. Superoxide dismutase did not augment the action of the endothelium-derived factor on the coronary smooth muscle, to judge from experiments where it was infused at site 3. Thus the most logical explanation for the poten- tiating effect of superoxide dismutase is that it prevents inactivation of the relaxing factor(s) in transit. The enzyme may protect endothelium-derived relaxing factor(s), either by decreasing the concentration of superoxide anion or by augmenting that of hydrogen peroxide (11, 12). The latter explanation is unlikely because catalase, infused at site 2, augments rather than inhibits acetylcholine-induced responses and does not reverse the facilitatory action of superoxide dismutase. The lack of effect of catalase per se on the half-life of endothelium- derived relaxing factor(s) implies that oxidizing free radicals (hydrogen peroxide, free hydroxyl radical) are not involved in the protection exerted by superoxide dismutase. Accelerated inactivation of endothelium-derived

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H826 OXYGEN-DERIVED FREE RADICALS AND EDRF

SOD, 150 U/ml, site2 I I I ACh,lO%, site1

95% 02

+25 r

QS% 02+ SOD

10w5M, site2

I(-[- Norepinephrine,

39 1.0 ;1 30 60

Transit time,sec.

120

FIG. 6. Effect of increasing transit times on relaxation induced by acetylcholine (10B6 M, infused at site I) during endothelial superfusion of canine coronary arteries (without endothelium) contracted with prostaglandin Fza (4 x 10m6 M) at 2 levels of 02 concentration (10 and 95% 02) in superfusate. Experiments were performed in absence and presence of superoxide dismutase (SOD, 150 U/ml) infused at site 2. Data shown as means k SE (numbers represent number of observations) and expressed as % of initial contraction to prostaglandin F,. * Effect of SOD is statistically significant.

PGF2a 1 Ow6M, site2

r I

4min

39

t PGF2Q

Catalase, site2 95% 02 1

1 10% 02

ACh, 10m6M, sits 1 FIG. 5. Isometric tension recording during endothelial superfusion of canine coronary arteries without endothelium contracted by prostaglandin Fg,(PGFz,, 4 x 10m6 M), with a transit time of 8 s. Superoxide dismutase (SOD, 150 U/ml) was infused at site 2, and acetylcholine (ACh, 10e6 M) at site 1. Phenidone (10B5 M), norepinephrine (10m6 M), and methylene blue ( 10B5 M) but not catalase (860 U/ml) (all infused at site 2) reversed inhibitory effect of ACh. In 3 experiments, reversal averaged 105 k 8.2, 92 + 6.5, and 101 -+ 4.5% for phenidone, norepinephrine, and methylene blue, respectively. Effect of these 3 substances SOD.150 U/ml. site2

was statistically significant (P C 0.05). pGF2~~y~ , ACh

T i

-

1 4min 2Q I I factor(s) by the superoxide anion would explain the augmented half-life due to superoxide dismutase. Super- oxide anions can reduce oxidized substances (12). Other reducing (antioxidant) agents, including cysteine, dithi- othreitol, phenidone, and norepinephrine inactivate endothelium-derived relaxing factor(s) in transit (9, 15). Thus it is likely that the inactivation of endothelium- derived relaxing factor is due to the reducing properties of the superoxide anion, which can be released from endothelial cells after its enzymatic intracellular production (13), or can be generated in salt solutions by pho- tolysis (4). This effect of superoxide anion probably becomes evident only with prolonged transit time, since under control conditions superoxide dismutase does not augment the activity of the endothelium-derived relaxing factor(s) at transit times of I (this study) or 4 (9) s. In rings with endothelium studied in organ chambers, the protecting effect of superoxide dismutase is not obvious (18, 19) because of the presumably short diffusion time between the endothelial cells and the smooth muscle in

Y I

PGF2a

FIG. 7. Isometric tension recording during endothelial superfusion (with a transit time of 30 s) of a canine coronary artery (without endothelium) contracted with prostaglandin FPa (PGFg,, 4 x lo* M). Switching gas mixture aerating superfusate from 95% O&I% COz (95% 02) to 85% &lo% 02-5% CO2 (10% 02) caused an increase in tension (mean increase 18.5 * 3.1%; n = 7); such increases in tension were not observed during direct superfusion (not shown). Acetylcholine (ACh, IOm6 M, infused at site I) caused contraction in absence (upper) but relaxation in presence (lower) of superoxide dismutase (SOD, 150 U/ ml, given at site 2). Note that in presence of low O2 SOD causes relaxation.

the intact blood vessel wall. If superoxide anions play a role in the inactivation of

endothelium-derived relaxing factor(s) in transit, the protective action of superoxide dismutase should depend on the extent of the generation of the free radical (which is the function of oxygen tension) and on the rate of its

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TABLE 1. Direct effects of superoxide dismutase and catalase on coronary arterial smooth muscle in bioassay system

Control Superoxide dismutase,

150 U/ml

Prostaglandin Fza, Prostaglandin Fza plus 4x 10*M Acetylcholine, lo4 M

7.5k1.5 3.9kl.l +0.69*0.10 +0.58&O. 12

Catalase, 860 U/ml +0.79*0.18 +0x&o. 15

Values are means & SE of 4 experiments. Results are expressed in absolute changes in tension (g); + = augmentation. Catalase and superoxide dismutase were infused at site 3 (see Fig. 1). Prostaglandin FzO and prostaglandin FZP plus acetylcholine were infused at site 1 (see Fig. 1).

elimination [which is determined by the concentration of the scavenger (5)]. In fact, the protective action of the enzyme was augmented by lowering the oxygen tension, and it was concentration-dependent. Reducing the oxygen content caused an increase in tension of the bioassay ring only during endothelial superfusion; this can be attributed to the release of vasconstrictor substances from the hypoxic endothelial cells (3, 16). Although reduction in oxygen content per se did not affect the half-life of the endothelium-dependent relaxing factor(s) released by acetylcholine, it reversed the contraction of the bioassay ring caused by superoxide dismutase, infused at site 2 in the absence of acetylcholine, into relaxation. This must reflect augmented protection by superoxide dismutase, at lower oxygen contents, of endothelium-derived factor released under basal conditions (9, 15). Likewise, the marked prolongation of the effect of acetylcholine in the combined presence of lower oxygen content and superoxide dismutase can only be ex- plained by reduced inactivation of endothelium-derived relaxing factor. Since a reduction in oxygen content per se did not affect the half-life of endothelium-derived relaxing factor(s) and since, under control conditions, maximally effective concentrations of superoxide dismutase restored the acetylcholine-induced relaxation only by -50% at a transit time of 30 s, the inactivation of the endothelium-derived factor by the higher oxygen content may not be due only to superoxide anions, which should be scavenged completely by the dose of superoxide dismutase used (S), but also to other oxygen-derived reducing radicals, which are not (or less) sensitive to the enzyme. One likely possibility is that the hyperoxic gas mixture used generates the formation of 0: as occurs in other biological systems (5). Thus the present data suggest that the short half-life of endothelium-derived relaxing factor, observed in this and previous studies (9, 15) may be the consequence of the artificial environment imposed.

The authors thank Helen Hendrickson for preparing the illustra- tions and (Janet Beckman for secretarial assistance.

This work is supported in part by National Heart, Lung, and Blood Institute research Grants HL-31183, HL-31547, and HL-34634.

Received 11 June 1985; accepted in final form 4 December 1985.

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