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(Circulation Research. 1995;77:174-181.)
© 1995 American Heart Association, Inc.

Articles

Dithionite Increases Radical Formation and Decreases Vasoconstriction in the Lung

Evidence That Dithionite Does Not Mimic Alveolar Hypoxia

Stephen L. Archer, Václav Hampl, Daniel P. Nelson, Erika Sidney, Douglas A. Peterson, E. Kenneth Weir

From the Minneapolis Veterans Affairs Medical Center and the University of Minnesota, Minneapolis.

Correspondence to Stephen Archer, MD (Associate Professor of Medicine), Minneapolis VA Medical Center, One Veterans Dr, Minneapolis, MN 55417.

	Abstract

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Abstract Dithionite is a powerful reducing agent used to deoxygenate hemoglobin and create anaerobic conditions in vitro. Recently, dithionite has been used as a convenient means of creating "hypoxia" in experiments studying the O₂ sensor in the pulmonary circulation and carotid body. We evaluated the hypothesis that hypoxia created by hypoxic ventilation and that created by dithionite have different effects on the pulmonary circulation. In vitro, dithionite (10^-5 to 10^-3 mol/L), added to oxygenated Krebs' solution, rapidly created superoxide anion in a dose-dependent manner. Dithionite consumed O₂ in parallel with the generation of superoxide radical, with both processes peaking within seconds. Anoxia was sustained only if resupply of O₂ was prevented. In isolated rat lungs (whether perfused with autologous blood or Krebs' solution), hypoxic ventilation alone lowered perfusate PO₂ from 140 to 40 mm Hg and decreased lung levels of activated oxygen species (AOS), measured by luminol-enhanced chemiluminescence, before the onset of hypoxic pulmonary vasoconstriction. Constrictor responses to angiotensin II and KCl were not impaired by intermittent hypoxic challenges, and lung weight did not increase. In contrast, dithionite impaired constrictor responses of the Krebs' solution–perfused lungs to all vasoconstrictors tested and increased lung weight. When given as a bolus (5x10^-3 mol/L) into the pulmonary artery during normoxic ventilation, dithionite caused no vasoconstriction and only briefly lowered PO₂ (because of constant resupply of O₂ from the alveoli). When superimposed on hypoxic ventilation, dithionite further lowered PO₂ from 40 to 0 mm Hg and caused additional constriction. Unlike hypoxic ventilation, dithionite increased AOS production. Antioxidant enzymes diminished dithionite-induced radical production and diminished the loss of vascular reactivity and lung edema. In conclusion, unlike hypoxic ventilation, dithionite causes edema and loss of vascular reactivity in the lung by generating superoxide anion and hydrogen peroxide. Hypoxia elicited by dithionite is not equivalent to authentic hypoxia because of the obligatory associated generation of AOS. Dithionite usage should not be substituted for authentic hypoxia in studies of O₂ sensing.

Key Words: dithionite • oxygen radicals • hypoxia • oxygen sensor • chemiluminescence

	Introduction

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Dithionite (Na₂S₂O₄) has been used to create hypoxia,¹ deoxygenate hemoglobin,² study the redox potential of mitochondrial cytochromes,³ evaluate the nitrogenase system,⁴ and create anoxia for anaerobic spectrophotometry.⁵ In the biochemical literature, it has been recognized that the reduction of O₂ by dithionite not only lowers PO₂ but also yields hydrogen peroxide⁵ and a highly reactive SO₂^- radical.⁴ For example, if dithionite or the milder reducing agent, dithiothreitol, is used to reduce hemoglobin, one must eliminate the concomitant hydrogen peroxide production by addition of catalase or horseradish peroxidase.¹ Dithionite can also reduce and thereby inactivate important enzymes, such as glutathione reductase.⁶

Dithionite has recently been used in experiments studying the mechanism of hypoxic pulmonary vasoconstriction (HPV) in cultured pulmonary artery (PA) myocytes,^{7 8} the sensing of oxygen in the carotid body,^{9 10} and the effects of anoxia on pH_i in cardiac cells.¹¹ Because of its convenience as a means of generating hypoxia and the difficulty of detecting radicals and peroxides, physiologists have paid little attention to the obligatory concomitant generation of these activated oxygen species (AOS). Instead, dithionite has been used as if it were equivalent to conventional hypoxia, often being referred to as "hypoxia"⁷ or "anoxia."⁹

In preliminary studies of isolated rat lungs, we found that unlike hypoxic ventilation, dithionite increased lung weight and impaired vascular reactivity to vasoconstrictors, including angiotensin II (Ang II), KCl, and alveolar hypoxia. This loss of vascular reactivity is reminiscent of the effects of radicals and peroxides but does not mimic the lung's response to hypoxic ventilation. To assess the hypothesis that hypoxia created by dithionite has different effects on the pulmonary circulation than does hypoxic ventilation, we compared the effects of alveolar hypoxia and dithionite on pulmonary vascular reactivity, lung production of AOS, and lung edema formation (as indicated by changes in lung weight) in the isolated perfused rat lung. Although hypoxic ventilation and dithionite both lower PO₂, only dithionite generates AOS, increases lung weight, and impairs vascular responsiveness to vasoconstrictors. We conclude that dithionite is not equivalent to authentic hypoxia in physiological studies.

	Materials and Methods

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Measurement of Radicals Created by Dithionite In Vitro
The effects of dithionite on PO₂ in vitro were determined by adding dithionite (3x10^-3 mol/L) to normoxic Krebs' solution while measuring PO₂ continuously for 15 minutes by use of an O₂ electrode. These experiments were performed in a glass beaker containing Krebs' solution, which was stirred continuously at room temperature. The beaker was open to room air and contained a submerged plastic tube to permit bubbling with normoxic gas. In some experiments, the Krebs' solution was bubbled with normoxic gas (20% O₂, 5% CO₂, and balance N₂, mimicking the effects of giving dithionite to a normoxia-ventilated lung); in others, the media were not resupplied with O₂ after the addition of dithionite. These two experimental conditions in vitro (with and without resupply of oxygen) were designed to correspond to the two conditions under which dithionite was given to the isolated lung (during normoxic and during hypoxic ventilation).

The effects of dithionite (10^-5 to 10^-3 mol/L, n=4 per dose) on superoxide radical production were measured by using the cytochrome c reduction assay.¹² Reactions were performed in normal saline at 25°C. Changes in cytochrome c reduction were measured as changes in absorbance multiplied by 10^-3 at 550 nm in a standard spectrophotometer. The contribution of superoxide anion to the net cytochrome c reduction was determined by measuring the proportion of reduction that was inhibited by bovine CuZn superoxide dismutase (SOD, 70 U/mL).

Radical production by dithionite was confirmed by measuring the effects of dithionite on luminol-enhanced chemiluminescence in the presence and absence of bovine CuZn SOD (4000 U). In these experiments the same glass beaker system was used, but dithionite was studied in saline at 25°C. The saline was bubbled with normoxic gas (20% O₂, 5% CO₂, and balance N₂).

Isolated Lungs
The isolated lung model was performed as described previously.^{13 14} Each rat was anesthetized with sodium pentobarbital (50 mg/kg IP) and mechanically ventilated (65 breaths per minute; tidal volume, 3 mL; room air; PEEP, 2.5 cm H₂O). The heart was exposed via a median sternotomy. The PA was cannulated with a double-lumen catheter used to measure perfusion pressure and deliver perfusate to the lungs. In protocols 1 and 3 (see below), the lungs were perfused with Krebs' solution (containing 4% albumin and 5 mg/mL meclofenamate); in protocol 2, the perfusate was autologous blood (hemoglobin, 13 g/dL). Perfusate was circulated by a roller pump through the lungs (0.04 mL/g rat weight per minute) to a left atrial cannula and then returned to a 40-mL reservoir (37°C). The left atrial cannula was suspended above the reservoir, thereby ensuring a left atrial pressure near 0 mm Hg. The lungs were ventilated with humidified gases: either normoxia (20% O₂, 5% CO₂, and balance N₂) or hypoxia (2.5% O₂, 5% CO₂, and balance N₂). The heart and lungs were dissected free, en bloc, and suspended over a heated water bath in a humidified chamber. O₂ was measured with an in-line O₂ electrode (Lazar Inc) that was placed in the circuit 25 cm distal to the lung. Lung weight was measured continuously with a force transducer (Radnotti Glass). Chemiluminescence was measured simultaneously with PA pressure, PO₂, and lung weight.

Chemiluminescence
Levels of AOS in the lung were measured by luminol-enhanced chemiluminescence, as previously described.^{13 14 15} The isolated lung was housed in a light-tight box with the lung within 2 mm of a foil-shielded Lucite rod (diameter, 2 in). The Lucite carried light to a red-sensitive photomultiplier tube (RCA C31034A) amplified at 1700 V by a high-voltage supply (EG & G, Princeton Applied Research, model 1121/99 HVS-1). The signal was processed by a discriminator (EG & G, Princeton Applied Research, model 1109) and recorded digitally with a MacLab A-D converter (AD Instruments) connected to a Macintosh computer. Chemiluminescence was expressed as counts per 0.1 second. Luminol was dissolved in warmed Krebs'-albumin solution and was added to the isolated lung at the beginning of each experiment. The dose of luminol was higher in blood-perfused lungs (3x10^-3 mol/L) than in Krebs'-perfused lungs (7x10^-5 mol/L) because of blood's effect of attenuating light transmission.

All vehicles were tested to establish their effects on chemiluminescence. Deoxygenated saline (the dithionite vehicle) and polyethylene glycol (PEG, 150 mL; the vehicle for PEG SOD) had no effect on chemiluminescence. Ammonium sulfate (the thymol buffer in which SOD is supplied) significantly decreased chemiluminescence and increased PA pressure (see "Results").

Protocols
Three protocols were used. In protocol 1, dithionite was given to Krebs'-perfused lungs during normoxia. Protocol 2 was identical to protocol 1 except that lungs were perfused with blood. In protocol 3, dithionite was given superimposed on hypoxic ventilation in Krebs'-perfused lungs. Protocols 1 and 2 evaluated the effects of dithionite under conditions in which O₂ was continuously resupplied by ventilation; thus, the fall in PO₂ was transient. In protocol 3, dithionite caused a more sustained fall in PO₂ because the hypoxic ventilation limited the resupply of O₂ after it was consumed by dithionite. Experiments in all protocols consisted of two to four 24-minute periods. In each period, 10 minutes of normoxia was followed by bolus injection of Ang II at 0.15 mg. Pressure was allowed to return to baseline over 8 minutes, and then 6 minutes of hypoxic ventilation was initiated.

Protocol 1
Dithionite (10^-5 to 5x10^-3 mol/L, n=35 lungs) was given as a bolus injection into the PA during the third normoxic control period. The goal of this part of the study was to establish the effects of dithionite on PA pressure, PO₂, and reactivity to subsequent challenges with Ang II, hypoxia, or KCl (40 mmol/L). Chemiluminescence and PO₂ were measured simultaneously in five additional lungs challenged with the highest dose of dithionite. Five lungs given vehicle (0.3 mL normal saline) instead of dithionite served as controls. In five other lungs, KCl was given 8 minutes after dithionite.

Protocol 2 (n=7 Lungs)
The lungs were treated in a manner identical to that in protocol 1, except the perfusate was blood (30 mL containing 600 U heparin), and a transfusion filter was placed proximal to the PA inflow cannula. Blood was harvested for each lung perfusion by ventricular puncture from four anesthetized rats by use of heparinized syringes. After the first two blood-perfused preparations (with no meclofenamate in the perfusate) were found to lack HPV, the perfusate for subsequent experiments was enriched with meclofenamate (5 mg/mL) to enhance hypoxic reactivity. One untreated lung and two of the meclofenamate-treated lungs received injections of normal saline (0.3 mL) rather than dithionite and served as controls.

Protocol 3 (n=41 Lungs)
Dithionite (5x10^-3 mol/L) was given as a bolus into the PA at the plateau phase of the third hypoxic challenge (after the third minute of hypoxic ventilation). The effects of pretreatment with bovine CuZn SOD (4000 U), catalase (100 000 U), CuZn SOD+catalase (4000 U and 100 000 U, respectively), or a cell-permeable SOD preparation (PEG SOD) plus catalase (4000 and 100 000 U, respectively) were determined. The antioxidants were given during the third normoxic period, 15 minutes before the dithionite. The doses of dithionite and O₂ radical scavengers were chosen because they either altered chemiluminescence and caused pulmonary vasoconstriction without overt producing pulmonary edema or were without effect at maximal doses.

Drugs and Statistics
The drugs were all reagent grade and were obtained from Sigma Chemical Co. Values were expressed as mean±SEM. Comparisons between two and three groups were by Student's t test and a factorial ANOVA, respectively. Fisher's PLSD test was performed for post hoc comparisons by using STATVIEW II (V4.0, Abacus Concepts). Vasoconstrictor reactivity before and after dithionite administration was compared by using the paired t test. A value of P<.05 was considered statistically significant.

	Results

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In Vitro
Dithionite lowered the PO₂ of Krebs' solution from 150 to near 0 mm Hg within seconds of being added (Fig 1A). If O₂ was resupplied, by bubbling the beaker with room air, the PO₂ recovered within 5 to 10 minutes. However, if the Krebs' solution was not bubbled, the PO₂ remained close to 0 mm Hg for 15 minutes and then gradually increased (because of the slow replenishment of O₂ from the room air). The results were the same with saline, water, or Krebs' solution containing 4% albumin (data not shown).

View larger version (34K): [in this window] [in a new window]   Figure 1. Dithionite generates superoxide radical in vitro. A, Example of a time course of dithionite-induced anoxia when a dose of dithionite is added to Krebs' solution in vitro is shown. Note that the anoxia is complete within 1 minute. If the solution is bubbled with room air, thereby resupplying oxygen, the PO2 recovers over 10 minutes. If the solution is not reoxygenated, the PO2 remains low. B, Time courses show that dithionite causes rapid dose-dependent reduction of cytochrome c by a mechanism that involves superoxide anion, as it is inhibited partially by superoxide dismutase (SOD) (n=4 measurements per group and dithionite dose). *P<.05 vs curve obtained with the same dose of dithionite in the absence of SOD; P<.01 vs curve obtained after the administration of dithionite (10-5 mol/L). C, Bar graph depicting luminol-enhanced chemiluminescence shows that radical formation induced in vitro by dithionite (3x10-3 mol/L) is virtually eliminated by 100 U/mL SOD (note the logarithmic scale) (n=3 per group). *P<.01 vs value obtained with dithionite alone.

Dithionite reduced cytochrome c within 15 seconds in a dose-dependent manner (Fig 1B). Both dithionite-induced cytochrome c reduction and luminol-enhanced chemiluminescence were inhibited by SOD, indicating that at least one of the reducing species is superoxide anion (Fig 1B and 1C). Desferrioxamine had no effect on radical generation by dithionite in vitro (data not shown).

Isolated Lungs
In all experiments, hypoxic ventilation alone caused a fall in PO₂ and a parallel rapid reduction in lung chemiluminescence (Fig 2A). The fall in chemiluminescence preceded the onset of HPV. When no dithionite was present in the perfusate, chemiluminescence, PA pressure, and PO₂ returned to baseline immediately after the end of a hypoxic challenge without an "overshoot" (Fig 2A). There was no change in lung weight during HPV or normoxia over the entire experiment in control lungs, which did not receive dithionite.

View larger version (29K): [in this window] [in a new window]   Figure 2. Effects on pulmonary artery (PA) pressure, chemiluminescence, and effluent PO2 in isolated lungs differ between dithionite and alveolar hypoxia. This is a computerized rendition of actual experimental tracings. PA pressure, radical production (enhanced chemiluminescence), and effluent PO2 were measured simultaneously. A, The response to alveolar hypoxia alone is from a lung studied under protocol 1. B, The response to dithionite in normoxically ventilated lungs is also from protocol 1. C, The response to dithionite in hypoxically ventilated lungs is from protocol 3.

Administration of Dithionite During Normoxic Ventilation: Protocol 1 (n=35 Lungs)
Dithionite (5x10^-3 mol/L) given during normoxic ventilation in Krebs' albumin–perfused lungs caused a brief, but intense, increase in luminol-enhanced chemiluminescence but did not change PA pressure (Fig 2B). Chemiluminescence had returned to baseline within 5 minutes (Fig 2B). Dithionite rapidly lowered PO₂ in the normoxically ventilated lungs, but the hypoxia was transient (Fig 3). PO₂ had returned to a normoxic level within 60 seconds (Fig 3). pH was 7.37±0.01 before and 7.35±0.01 5 minutes after dithionite. Vasoconstriction in response to a subsequent exposure to Ang II, hypoxic ventilation, or KCl was markedly reduced (10, 18, and 8 minutes after 5x10^-3 mol/L dithionite, respectively; Fig 4). This inhibition of vascular reactivity persisted for 30 to 40 minutes and then, in the case of Ang II, tended to recover (Fig 4). We did not detect any effect of dithionite doses lower than 5x10^-3 mol/L on PA pressure, effluent PO₂, or pulmonary vasoreactivity (chemiluminescence was measured only in experiments with 5x10^-3 mol/L dithionite).

View larger version (24K): [in this window] [in a new window]   Figure 3. Dithionite lowers PO2 in effluent from isolated lungs only briefly when O2 is resupplied by normoxic ventilation (protocols 1 and 2). Plotted values are the mean±SEM of eight experiments with Krebs'-perfused lungs (left) and four experiments with blood-perfused lungs (right). PO2 was measured continuously in the outflow line after the administration of 5x10-3 mol/L dithionite. Note that the fall in PO2 recovers within 60 to 90 seconds.

View larger version (20K): [in this window] [in a new window]   Figure 4. Administration of dithionite during normoxia inhibits subsequent constrictor response to angiotensin II, alveolar hypoxia, and KCl (protocol 1). Plotted values are the mean±SEM (n=5 per group) of the change in pulmonary artery (PA) perfusion pressure elicited by angiotensin II injection (0.15 µg), alveolar hypoxia (2.5% O2), or KCl (40 mmol/L). Responses to angiotensin II and hypoxia were measured both before and after dithionite administration (5x10-3 mol/L), whereas KCl was given only after the dithionite treatment. Baseline PA pressures before and after dithionite were 7.3±0.6 and 8.0±0.4 mm Hg, respectively, with no significant differences between the groups. Baseline PA pressure before KCl administration was 8.1±1.1 mm Hg. *P<.01 vs value obtained before dithionite administration; P<.01 vs corresponding value for the control group.

Administration of Dithionite During Normoxic Ventilation to Blood-Perfused Lungs: Protocol 2 (n=7 Lungs)
In blood-perfused lungs, basal chemiluminescence during normoxia was 105±44 counts per 0.1 second, and this fell rapidly and reversibly with hypoxic ventilation (Fig 5). As in the Krebs'-perfused lungs, dithionite (3x10^-5 mol/L) caused a rapid transient reduction in effluent blood PO₂ (Fig 3). Dithionite increased chemiluminescence transiently to 150±39 counts per 0.1 second, but after this initial "spike" in chemiluminescence, the basal chemiluminescence fell to 25±5 counts per 0.1 second (Fig 5). Lung weight was not changed significantly by dithionite injection in protocol 2. Dithionite caused constriction in the blood-perfused lungs whose perfusate contained meclofenamate (Fig 5). However, this was not a hypoxic vasoconstriction. The tone started to rise only after the PO₂ had returned to normal, and the vasoconstriction continued for several minutes while PO₂ was normal (Fig 5). KCl administered 10 minutes after vehicle or dithionite tended to cause more constriction in control than in dithionite-treated lungs (change in PA pressure with KCl, +24±8 and +16±5 mm Hg, respectively; P=.46).

View larger version (24K): [in this window] [in a new window]   Figure 5. Comparison of the effects of alveolar hypoxia and dithionite on pulmonary artery (PA) pressure, radical production (measured as luminol-enhanced chemiluminescence), and effluent PO2 in the blood-perfused meclofenamate-pretreated rat lung (protocol 2). This is a computerized rendition of actual tracings from a single experiment. AII indicates angiotension II. Note that dithionite-induced vasoconstriction starts after effluent PO2 returns to normoxia.

Administration of Dithionite During Hypoxic Ventilation: Protocol 3 (n=41 Lungs)
The initial event following bolus administration of dithionite during hypoxic ventilation was an increase in chemiluminescence (7.1±0.3 seconds after dithionite) (Fig 2C). This was followed by the virtually simultaneous peaking of PA pressure (12.1±0.4 seconds after dithionite) and fall in effluent PO₂ from 40 to 0 mm Hg (14.4±1.8 seconds after dithionite). This anoxia persisted until normoxic ventilation was resumed 3 minutes later (Fig 2C). The delay in the fall in PO₂ reflects the position of the O₂ sensor, 25 cm distal to the lung. The dithionite-induced increase in chemiluminescence was proportional to the ambient PO₂ of the perfusate and was smaller when dithionite was given during hypoxic ventilation (protocol 3; increase in chemiluminescence, +565±170 counts per 0.1 second) than during ventilation with normoxic gas (protocol 1; increase in chemiluminescence, +139 000±67 000 counts per 0.1 second).

Termination of the hypoxic challenge during which dithionite was given (reoxygenation) caused a second, even larger increase in radical production (Fig 2C). This reoxygenation increase in chemiluminescence was caused by the presence of dithionite, because switching from hypoxic to normoxic ventilation in the periods before the administration of dithionite caused chemiluminescence to return to baseline without any "reoxygenation spike" (Fig 2A).

The increases in chemiluminescence caused by dithionite administration during hypoxia (both initial and with reoxygenation) were obliterated by CuZn SOD or the combination of PEG SOD and catalase (Figs 6 and 7) but were not altered by ammonium sulfate, catalase alone, or desferrioxamine. The rise in chemiluminescence caused by dithionite during hypoxia was +565±170 counts per 0.1 second in control lungs, which did not differ significantly from the rise seen in lungs pretreated with desferrioxamine (+515±149 counts per 0.1 second), catalase (+740±197 counts per 0.1 second), or the CuZn SOD vehicle ammonium sulfate (+561±121 counts per 0.1 second). Similarly, these agents failed to reduce the magnitude of the reoxygenation spike in chemiluminescence (counts per 0.1 second: control +1078±391, desferrioxamine +873±85, catalase +1524±537, and ammonium sulfate +774±174; P>.05).

View larger version (27K): [in this window] [in a new window]   Figure 6. Bar graphs showing the effects of pretreatment with antioxidant enzymes on radical formation and vasoconstriction induced by dithionite (5x10-3 mol/L) given during hypoxia (protocol 3). Top, Dithionite-induced vasoconstriction is not inhibited by any antioxidant combination and in fact is enhanced by polyethylene glycol (PEG)–superoxide dismutase (SOD) plus catalase (CAT). Pulmonary artery (PA) pressure before dithionite injection was 21.1±1.2 mm Hg, with no difference among the groups. Middle, The rise in chemiluminescence caused by dithionite, administered during hypoxic ventilation, is virtually eliminated by SOD and PEG SOD plus CAT. Bottom, The rise in chemiluminescence caused by return to normoxic ventilation in dithionite-treated lungs is virtually eliminated by SOD and PEG SOD plus CAT. In lungs that do not receive dithionite, there is no reoxygenation increase in chemiluminescence. Dosage was as follows: SOD in thymol buffer, 4000 U (n=8); CAT, 100 000 U (n=6); and PEG SOD, 4000 U (n=5). Values are mean±SEM. The control group consisted of nine lungs. *P<.01 vs corresponding control value.

View larger version (31K): [in this window] [in a new window]   Figure 7. Polyethylene glycol (PEG)–superoxide dismutase (SOD) plus catalase inhibits dithionite-induced radical generation and partially protects angiotensin II (AII)–induced constriction. PA indicates pulmonary artery. This is a computerized rendition of an actual single experimental tracing.

Dithionite-induced increase in PA pressure was not prevented by pretreatment with any of the antioxidants/scavengers or their vehicles. PEG SOD plus catalase actually enhanced the dithionite-induced constriction despite eliminating the radical burst (Fig 6).

The baseline luminol-enhanced chemiluminescence (before hypoxic challenge during which dithionite was injected) was lowered by CuZn SOD suspended in ammonium sulfate (-79±2%), as previously reported.¹³ The same preparation of SOD plus catalase also lowered normoxic chemiluminescence -62±12%. In contrast, a membrane-permeable form of SOD, PEG–CuZn SOD, plus catalase had no effect on normoxic chemiluminescence (-5±10%) (Fig 7). Thus, it is the ammonium sulfate ("thymol") buffer in which CuZn SOD is suspended (rather than the enzyme) that is the cause of the normoxic reduction of chemiluminescence following CuZn SOD administration. Ammonium sulfate (in the same dose used to deliver 4000 U CuZn SOD) lowered normoxic chemiluminescence -65±2%, accounting for all the effects of SOD on normoxic chemiluminescence. Ammonium sulfate also accounts for vasoconstriction caused by CuZn SOD, because all preparations containing it (CuZn SOD+catalase, CuZn SOD alone, or ammonium sulfate alone) increased PA pressure (+2.2±0.6, +2.0±0.3, and +1.8±0.9 mm Hg, respectively; baseline PA pressure, 7.8±0.3 mm Hg). In contrast, PEG SOD plus catalase had no effect on PA pressure (Fig 7). This is an important technical point and indicates that CuZn SOD suspended in ammonium sulfate should not be used in studies of the effects of SOD on radical generation or vascular reactivity. However, ammonium sulfate did not have any effect on dithionite-induced chemiluminescence, whereas both CuZn SOD and PEG SOD (plus catalase) virtually eliminated the dithionite-induced chemiluminescence (Figs 6 and 7). This indicates that the dithionite chemiluminescence spike resulted from the production of superoxide radical.

The lung weights were increased (P<.05) in lungs that received dithionite (5.0±1.0 g) relative to control lungs (3.7±0.4 g), and this weight increase was prevented by pretreatment with catalase (3.3±0.9 g). As in protocol 1, the constrictor responses to hypoxic ventilation and Ang II were significantly impaired when tested within 20 minutes after dithionite (5x10^-3 mol/L) administration in protocol 3 (Fig 8). The loss of constrictor responsiveness was reduced by pretreatment with catalase or the combination of PEG SOD plus catalase but not by SOD alone (Fig 8).

View larger version (24K): [in this window] [in a new window]   Figure 8. Catalase (CAT) alone or in combination with superoxide dismutase (SOD) diminishes the inhibition of angiotensin II–induced constriction caused by dithionite (5x10-3 mol/L) (protocol 3). Values are the mean±SEM of the change in pulmonary artery (PA) perfusion pressure elicited by angiotensin II or alveolar hypoxia. Dosage was as follows: SOD in thymol buffer, 4000 U (n=5); CAT, 100 000 U (n=6); and polyethylene glycol (PEG) SOD, 4000 U (n=5). There were eight lungs in the control group. Baseline PA pressure was 8.7±0.3 mm Hg before and 9.5±0.6 after dithionite, with no differences among the groups. *P<.005, P<.05 vs corresponding values before dithionite (paired t test).

	Discussion

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The present study compared two different means of creating hypoxia: ventilation with hypoxic gas versus administration of the reducing agent dithionite in isolated lungs. As summarized in the Table and Fig 2, the two stimuli are not equivalent. Alveolar hypoxia reduces effluent PO₂ for the entire duration of the hypoxic challenge. Ventilation hypoxia also induces sustained pulmonary vasoconstriction, which neither increases lung weight nor impairs reactivity to subsequent vasoconstrictors. Hypoxic ventilation lowers production of AOS, consistent with previous studies showing that radical formation is directly proportional to PO₂ in lung mitochondria, lung homogenates, and the isolated lung.^{13 16 17 18 19}

View this table: [in this window] [in a new window]   Table 1. Effects of Hypoxia and Dithionite Compared

In marked contrast, dithionite causes only very brief reduction in effluent PO₂, unless supported by concurrent alveolar hypoxia. AOS production is remarkably elevated by dithionite administration. Dithionite does not cause vasoconstriction in normoxically ventilated Krebs'-perfused lungs, although they display HPV. Dithionite renders the lungs hyporesponsive to subsequent vasoconstrictor stimuli. Furthermore, unlike hypoxic ventilation, dithionite increases lung weight. The loss of vasoconstrictor responsiveness and increased lung weight after the administration of dithionite result, at least partially, from dithionite-induced AOS production. Dithionite-induced loss of vascular reactivity is diminished by catalase or PEG SOD and catalase but not by SOD alone. This suggests that it is largely due to the generation of hydrogen peroxide, similar to the "vascular paresis" caused by authentic hydrogen peroxide²⁰ or the hydrogen peroxide–generating enzyme-substrate pair, glucose–glucose oxidase.²¹ Consistent with this interpretation, the increased lung weight caused by dithionite was attenuated by catalase.

If AOS generation explains the loss of vascular reactivity, it may be unexpected that dithionite should initially cause vasoconstriction. There are two potential explanations: either the initial constriction does reflect true hypoxic constriction, or it is a form of radical-induced vasoconstriction, which is too intense to be reversed by the antioxidant enzymes used in the present study. Three pieces of data suggest the constriction induced by dithionite is not caused by its ability to generate radicals. First, when administered during normoxic ventilation, dithionite increases radical production but causes minimal vasoconstriction. Second, when given to lungs after antioxidant enzymes (eg, SOD), dithionite no longer increases radical levels but still causes vasoconstriction. Third, the biggest increase in radicals with dithionite occurs on return from hypoxic ventilation to normoxic ventilation. This reoxygenation-induced radical generation is not associated with any vasoconstriction. Thus, the radical generation accounts for the lung edema and loss of vascular reactivity but not for dithionite-induced vasoconstriction.

Arguing against the PO₂ dependence of the dithionite constriction are the data showing that anoxia does not produce greater constriction than does hypoxia in isolated lungs.¹³ Thus, it is surprising that a fall in PO₂ from 40 to 0 mm Hg, as occurred with dithionite, caused additional pulmonary vasoconstriction. Furthermore, the dithionite-induced constriction in normoxically ventilated blood-perfused lungs started only after PO₂ returned to normal (sometimes as much as several minutes later) and persisted long after the period of hypoxia (Fig 5). Thus, we cannot exclude the possibility that radical generation explains part of the dithionite-induced constriction. Large amounts of oxygen radical, as generated during reperfusion injury or with high doses of xanthine–xanthine oxidase, do initially cause vasoconstriction in many vascular beds.^{22 23 24 25 26 27} In contrast, lower doses of radicals cause pulmonary vasodilatation.²⁸ The mechanism of radical-induced vasoconstriction is probably multifactorial. Superoxide anion, generated by xanthine–xanthine oxidase, increases cytosolic calcium by stimulating calcium release from the sarcoplasmic reticulum.²⁹ Since dithionite generates large amounts of superoxide anion and thus presumably increases cytosolic calcium, it is not surprising that studies of hypoxia that use dithionite have reported that the initial event in response to "hypoxia" is a release of intracellular calcium stores.⁷ Such conclusions need to be reconsidered in light of the substantial confounding effect of radical generation that accompanies the dithionite-induced fall in PO₂.

The concomitant generation of AOS by dithionite during the induction of hypoxia makes it difficult to know whether to attribute an observed response to the fall in PO₂ or to the effects of excess radicals and peroxides. For example, HPV is associated with a decrease in AOS production,^{13 19} which in turn inhibits potassium channels in vascular smooth muscle.³⁰ This depolarizes the vascular smooth muscle, leading to vasoconstriction.³¹ Although dithionite also inhibits K⁺ channels and depolarizes vascular smooth muscle,⁸ it is unproved that these effects are solely due to the dithionite-induced fall in PO₂. Reducing agents (and oxidants) can directly alter the function of ion channels,^{32 33 34 35 36} independent of effects on PO₂,³⁷ by causing changes in the K⁺ channel that modulates its activity.³⁸

Conclusions
Dithionite is a powerful nonspecific reducing agent that causes anoxia by reducing oxygen with the associated generation of large amounts of AOS (including superoxide anion). This occurs in isolated lungs (whether perfused with blood or Krebs' solution) and in vitro. Although dithionite shares with alveolar hypoxia the ability to lower PO₂ and (under certain conditions) cause pulmonary vasoconstriction, it is in all other aspects the antithesis of hypoxia. Unlike HPV, the effects of dithionite on the pulmonary circulation include a radical-mediated loss of vascular reactivity and formation of edema. The effects of dithionite are somewhat analogous to those of hydrogen peroxide–generating enzyme-substrate pairs, such as glucose–glucose oxidase, and should not be considered to be equivalent to hypoxia.

	Acknowledgments

 
This study was supported by the Department of Veterans Affairs and National Institutes of Health grant R29-HL-45735.

Received October 12, 1994; accepted March 21, 1995.

	References

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