AMERICAN

JOURNAL

OF

PHYSIOLOGY

Vol. 230, No. 5, May 1976. Printed

in U.S.A.

Evaluation of redox perfused rat lung

state of isolated

ARON B. FISHER, LINDA FURIA, AND BRITTON CHANCE Departments of Physiology, Biophysics and Physical Biochemistry, and Medicine, Pennsylvania School of Medicine and Veterans Administration Hospital, Philadelphia, Pennsylvania 19174

METHODS

inhibitors; surface fluorescence; glycolytic inhibitor; nucleotides; anoxia; carbon monoxide; transaminase redox couples

STUDIES have shown that pulmonary parenchyma is an actively metabolic tissue that requires oxidative metabolism to carry out a variety of functions such as protein synthesis (l2), phagocytosis (14), phospholipid synthesis (I), and serotonin uptake (17). The purpose of this stcldy was to develop methods for the evaluation of lung oxidative metabolism and the effects of metabolic inhibitors on lung redox state. Two separate methods for evaluating redox state were investigated. The first method was based on measurement of fluorescence from the lung surface at wavelengths suitable for detection of reduced pyridine nucleotides (4, 7, 16). With this system, reduction of pyridine nucleotides results in an increased fluorescence from the lung surface, permitting continuous recording from a localized area of the lung parenchyma. The second method was based on measurement of the reduced and oxidized substrates from a pyridine nucleotide-dependent reaction in rapidly frozen lung tissue (19). A static value for redox RECENT

state could

be approximated

from

the ratio

of

olites. The results of the study define the redox response of the lung to metabolic inhibitors and provide a frame of reference for future studies of the relationship between oxidative metabolism and lung function.

FISHER,ARON B., LINDAFURIA,ANDBRITTON CHANCE. Eualuation of redox state of isoZated perfused rat lung. Am. J. Physiol. 230(5): 1198-1204. 1976. - The metabolic responsiveness of lung tissue to inhibition of oxidative metabolism was determined by measurement of the redox state of the isolated perfused and ventilated rat lung. Changes in redox state were evaluated by fluorescence from the lung surface at wavelengths suitable for reduced pyridine nucleotides and by measurement of the ratios of redox couples in rapidly frozen lung tissue. Maximal change of redox state was observed during ventilation with carbon monoxide; surface fluorescence increased 6.6%) lactate/pyruvate increased 5.8 times, glycerol-3P/dihydroxyacetone-P increased fourfold and glutamatela-ketoglutarate doubled. KCN infusion resulted in similar changes. Hypoxia produced with N, ventilation resulted in less than maximal changes in redox couple ratios until alveolar PO, was reduced below 0.1 mmHg. Redox changes observed during infusion of 0.5 mM aminoxyacetic acid suggested that maintenance of cytoplasmic redox state depended on functioning of a malate-aspartate “shuttle.” The isolated perfused lung appears suitable to study factors controlling pulmonary parenchymal oxidative metabolism. The results emphasize the need for ventilation with CO to establish intracellular anoxia. oxidative pyridine inhibitor;

University

of the metab-

Lung perfusion. Sprague-Dawley rats weighing 200250 g (Hilltop Lab Animals, Scottdale, Pa.) were anesthetized with intraperitoneal urethan (150 mg/kg body wt). The lungs were removed, placed in a water-jacketed incubation chamber maintained at 37”C, and perfused as previously described (I, 9). Lungs were ventilated with a Harvard rodent respirator at 100 cycles/min, 2 ml tidal volume, and 2-3 cm H,O end-expiratory pressure. The usual ventilating gas was 95% 0,:5% CO, that was humidified at room temperature. Gas with the same composition as the ventilating gas was continuously flushed through the incubation chamber. The perfusate was Krebs-Ringer bicarbonate solution plus 5 mM glucose, gassed with 95% 0,:5% CO,. The perfusate pH was adjusted to 7.4 with NaOH and the temperature was maintained at 37°C. The perfusate was infused at 10 ml/ min into the pulmonary artery with a peristaltic pump, flowed freely from the transected left atrium, and was not recirculated. Ventilation and perfusion pressures were monitored continuously with pressure transducers (Statham Instruments) and recorded with an oscillograph. PoZ in the perfusate was measured with a flow cuvette and Clark polarograph (Yellow Springs Instrument Co., Yellow Springs, Ohio). PO, in the expired gas was monitored with a rapidly responding electrochemical 0, analyzer (Applied Electrochemistry Inc., Sunnyvale, Calif.). The output of this cell is a logarithmic function of the difference between reference and sampled PO,, permitting precise measurement of low PO,. The analyzer was calibrated for each experiment with gases of known composition. Metabolic inhibitors were infused into a mixing chamber situated in the inflow line to the pulmonary artery. For studies of surface fluorescence, varying concentration of inhibitors was infused until maximal changes were produced. These concentrations of inhibitors were subsequently used in experiments designed to measure redox couples. To produce anoxia with NZ, a separate reservoir containing perfusate was gassed with 95% N,:5% CO, and the lungs were ventilated with the

1198

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LUNG

REDOX

1199

STATE

same gas mixture; in some experiments 95% He:5% CO, was used for ventilation since residual 0, in this mixture was slightly less. For CO-induced anoxia, lungs were ventilated with 95% CO:5% C02, while perfusate was equilibrated with 95% N,:5% CO,. For experiments with varying concentrations of CO and 0, in the ventilating gas, mixtures of CO and 0, (plus 5% CO,) were prepared “on line” with an accurate gas-mixing pump (Woesthoff I ns t ruments, Bochum, West Germany). Surface fhorescence. In order to measure surface fluorescence, a fiberoptic light guide, 5 mm in diameter, was passed through the wall of the incubation chamber and positioned against the pleural surface of the lung. The location chosen was generally the midportion of a lower lobe, although similar results were obtained when the probe was placed in other lung areas. The probe was placed gently against the lung to minimize distortion of the underlying parenchyma. The chamber was masked with black tape to exclude extraneous light. Fluorescence from the surface of the lung was measured by techniques similar to those used previously to study a variety of other organs (4, 5, 7, 13, 16). The lung surface was excited at 366 nm and the reflected light from the lung surface was passed through appropriate filters to a photomultiplier. The filters were contained in a timesharing device consisting of a rapidly spinning wheel, making it possible to monitor reflected light at both 366 and 450 nm (6). The 366-nm (reflectance) and 450-nm (fluorescence) signals were continuously recorded with an oscillograph (Bell and Howell, Datagraph). The fluorometer was calibrated under control conditions by setting the base-line signals equal to 1 V, and all changes were then recorded relative to the base-line condition. Perfusate and lung tissue metabolites and redox couples. To measure lactate and pyruvate in the perfusate, samples were collected in plastic vials over a 30-s period and frozen in liquid N, for analysis the subsequent day. At the conclusion of the experimental period, lungs were rapidly frozen by clamping between aluminum tongs precooled at the temperature of liquid N,. Ventilation and perfusion were continued during the clamping procedure which required only 1-2 s. The frozen lungs were immediately plunged into a flask of liquid N,. Subsequently, the frozen lung tissue was ground to a fine powder with mortar and pestle, and then extracted twice with cold ethanolic perchloric acid using a PotterElvehjem homogenizer (21). The remaining tissue was lyophilized for determination of dry weight. The neutralized tissue extracts were analyzed for lactate, pyruvate, glycerol3:phosphate, dihydroxyacetone phosphate, glutamate, a-ketoglutarate, NH,, NAD+, and NADP+. In additional experiments, alkaline extracts of lungs (21) were analyzed for NADH and NADPH. Metabolite concentrations were determined by fluorometric or spectrophotometric assay with standard enzyme methods (2, 21). a-Ketoglutarate was determined with glutamate-oxaloacetate transaminase (GOT) and malate dehydrogenase in the presence of Laspartate and NADH;l the commercial GOT was dil This assay utilizes the same reaction as described for measurement of L-aspartate (ref. 2, p. 381) with L-aspartate substituted for CYketoglutarate in the reaction vessel.

alyzed before use to remove contaminating cw-ketoglutarate. NH, was measured with glutamic dehydrogenase in the presence of a-ketoglutarate and NADH; the assay used the same reaction as described for cu-ketoglutarate (ref. 2, p. 324) with a-ketoglutarate substituted for NH,+ in the reaction vessel. All assays were run in duplicate and the results were averaged. Recovery of NADH introduced in the trachea before “freeze-clamping” exceeded 80%. Chemicals and standards were reagent grade obtained from Sigma Chemical Co., St. Louis, MO. Enzymes were obtained from Boehringer-Mannheim Corp., New York City. RESULTS

Measurement of surface fluorescence. Inflation and deflation of the lung introduced considerable noise into the fluorescence signal, but a satisfactory base-line signal could be achieved by using a 10-s time constant during recording. Changes in base-line signal were produced when the usual state of lung inflation was altered and were of sufficient magnitude to mask completely the fluorescence changes associated with metabolic perturbations. Several maneuvers were found to produce this change, including increased end-expiratory pressure, change in ventilatory rate, change in tidal volume, or changing gas cylinders.” To prevent base-line shifts, an attempt was made to maintain ventilatory parameters constant during each experiment. Circulatory changes, e.g., pulmonary edema or air embolism, also caused drift of the fluorescence base line (Fig. 1). When a fluorescence change was produced by ventilatory or circulatory factors, a change was also observed in the reflectance signal (Fig. 1). On the other hand, fluorescence changes due to metabolic alterations were generally associated with minimal reflectance change. Consequently, records with large changes in the reflectance signal were excluded from analysis of metabolic perturbations. With introduction of metabolic inhibitors into the lung, there was a rapid increase in surface fluorescence which was reversible. A typical effect for N, ventilation is illustrated in Fig. 2. During N, ventilation, the alveolar PO, was not reduced to zero but varied from 1.0 to 2.5 mm Hg. The presence of 0, in the alveoli was due to a small amount of 0, in the N, gas and to leaks in the gasdelivery system. In order to produce more complete anoxic changes, lungs were ventilated with CO. This resulted in a 80% greater increase of fluorescence compared with N, ventilation (Table 1). Changes in fluorescence similar to CO were observed with infusion of antimycin A and cyanide, while infusion of amytal produced a slightly lesser change. The combination of KCN infusion and N, ventilation did not result in greater fluorescence changes than KCN infusion alone. With these inhibitors, surface fluorescence increased by 56.7% (Table 1). The increased fluorescence observed with oxidative inhibitors was interpreted as a reduction of pyridine nucleotides. On the other hand, decreased 2 This was due to slight when the outDut Dressure

changes in output of the rodent respirator from the gas cvlinder was altered.

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1200

FISHER, 0

‘TR cm “20

10

366

nm

f

25 % P INCREASE 366-450

‘PA cm Hz0

I min W-L

t nm

30 20 IO

0

tl ml/min

R’

30



Air

cmbolus

FIG. 1. Effect of circulatory changes on reflectance (366 nm) and fluorescence (366 -+ 450 nm) signals from surface of isolated rat lung. PPA indicates perfusion pressure and PTR indicates ventilation pressure. Perfusate flow rate (Q) into pulmonary artery is indicated at bottom of figure. Abrupt changes in PPA are coincident with changes in Q. When perfusion flow rate was increased to 36 ml/min, ventilation pressure began to gradually increase suggesting development of pulmonary edema. Associated with this was a large decrease in reflectance and a smaller fluorescence decrease. Note low gain (compared with Figs. 2 and 4) of reflectance and fluorescence signals indicated by calibration mark. With intentional introduction of a bolus of air into pulmonary artery, reflectance and fluorescence both increased. As a consequence of air embolism, lungs became grossly edematous, ventilation pressure increased greatly, and there was a further decrease in reflectance and fluorescence signals. 0

PTR cmv

IO 366 nm

T

1

increase

2%

I min

f

-

1. Effect

fluorescence

nm .

.

1

t

t

T

t

N2

02

N2

02

FIG. 2. Effect of N, ventilation on fluorescence (366 + 450 nm) and reflectance (366 nm) from lung surface. Gas mixture used to ventilate lungs is indicated at bottom of the figure (all ventilating gases contained 5% CO,). Perfusate was gassed with same gas mixture used for lung ventilation. PTR indicates ventilation pressure; perfusion pressure was not recorded in this experiment. With N, ventilation, there was an increase in fluorescence which was reversible. A similar change was observed during a second period of N, ventilation. Small changes in reflectance with N, ventilation are possibly related to changes in internal alveolar geometry due to an influence of gas cylinder pressure on output of ventilator. Change in fluorescence and reflectance traces in middle of second N, cycle represents a shift in base line, possibly due to movement of tissue.

AND

CHANCE

fluorescence indicating oxidation of pyridine nucleotides was observed during infusion of pentachlorphenol, an uncoupler of oxidative phosphorylation (Table 1). Perfusate lactate and pyruvate. Further evidence concerning changes in redox state of the lung produced by metabolic inhibitors was obtained by measurement of lactate and pyruvate concentrations in the perfusate. The difference between inflow and outflow lactate and pyruvate multiplied by the perfusate flow rate indicated the rate of production of these metabolites. In eight control experiments, the rates of lactate and pyruvate production did not change during 20 min of perfusion. Lactate production was approximately 40 pmol x h-l x g dry wt-‘, a value similar to previous measurements in this laboratory using a recirculating perfusate system (1). The ratio of lactate to pyruvate production under control conditions was 5.2 t 0.9 (SE). The time course of response of perfusate lactate and pyruvate production was studied during ventilation with 95% CO:5% CO,. Perfusions were carried out with a lo-min control period followed by 10 min of CO ventilation. After the first minute of CO ventilation, lactate production had doubled, pyruvate production had decreased by a third, and the lactate-to-pyruvate ratio had increased fourfold (Fig. 3). After CO ventilation for 10 min, lactate production had increased by one and twothirds, and the lactate-to-pyruvate ratio was lo-fold greater than control. These results indicate a rapid and relatively brisk response of cytoplasmic redox state to inhibition of oxidative metabolism. Tissue redox couples. By measurement of the ratio of redox couples in the tissue, it was possible to evaluate changes in redox state of both the mitochondrial and cytoplasmic compartments. The ratios of lactate (L) to pyruvate (P) and glycerol-3-P (GP) to dihydroxyacetoneP (DHAP) were used as indexes of cytoplasmic redox state, while the ratio of glutamate (Glut) to cu-ketoglutarate (KG) was used to reflect the mitochondrial compartment. The use of the glutamate dehydrogenase reaction for calculation of redox state requires determination of tissue NH, content in addition to content of glutamate and cu-ketoglutarate. The mean value for lung tissue NH, in nanomoles per gram dry weight was 2.2 in two control experiments and 2.3 in two experiments with N, anoxia. Routine NH,, analysis was not carried out in this study because precise quantitation of TABLE

366-450

FURIA,

(366

of metabolic inhibitors on surface -+ 450 nm) of perfused rat lung

Inhibitor

Anoxia*

cot KCN, 1 mM Antimycin A, 1 pg/ml Amytal, 2 mM Pentachlorophenol, 1 mM Data percent regarded creased 95% He mixture.

N

35 5 11

3 5 3

Change in Fluorescence, %

3.6 6.6 6.7 6.2 5.0 -4.3

k 0.4 I? 0.7 + 0.7 + 0.7 ?I 1.2 + 0.5

are means + SE for N experiments. The results represent the change from base-line fluorescence. Increased fluorescence is as a positive change; a negative number indicates defluorescence. * Lungs were ventilated with 95% N, or plus 5% CO,. Perfusate was equilibrated with the same gas t Lungs were ventilated with 95% CO:5% CO,.

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LUNG

REDOX

STATE TABLE 2. Redox couples and metabolite content of freeze-clamped isolated, perfused rat lungs in presence of metabolic inhibitors -____

_ Lactate/Pyruvote

Lactate __..-__..Pyruvate

75

s

Control Amytal, 2 mM Anoxia* KCN, 2 mM cot AOA, 0.5 mM 2-DOG, 5 mM P1___

(,

IO

LACTATE

8

15 TIME

OF

PERFUSION

PYWVATE

20 (minutes)

3. Effect of 95% carbon monoxide: 5% CO, ventilation on lactate and pyruvate production by isolated perfused rat lung. During initial 10 min (control), lungs were ventilated with 95% 0,:5% CO,. Samples were taken at end of control perfusion and after 1, 5, and 10 min of CO ventilation. Perfusate was gassed with 95% 0,:5% CO, during control and 95% N,:5% CO, during CO ventilation. Results are means &SE for six experiments

Glycerol3-P/ Dihydroxyacetone-P 3.020.3 6.3 -eO.$

pmol/g Dry Wt 36k-4 5324

:,::1:::-

25 6 13

3223 6O-t14 $226

7.220.7 14.922.1 12.9k1.7

69510 78214 73212

7.9t0.7 9.92Io.9 10.6kl.O

4

2719

5.1-el.O

37k3

4.5t1.4

6

1012

1.220.3

32-tll

2.951.1

---

Results are means -+ SE for N observations. AOA, aminoxyacetic acid; 2-DOG, 2deoxyglucose. * Lungs were ventilated with 95% N, or 95% He plus 5% CO,. Perfusate was equilibrated with same gas mixture. Alveolar PO, was 0.1-2 mmHg. t Lungs were ventilated with 95% CO:5% CO,. 0

PTR cm H.$

IOI

r

“FREEZF_CCLAMP”

FIG.

NH, was dificult, the re ship between the tissue and mitochondrial was not known, a observed values for whole tissue in the small number of experiments did not change significantly between control and anoxia. Experiments for measurement tissue redox couples consisted of a 10-min control peri followed by a U.I-min infusion of inhibitor. Experiments were terminated b freeze-clamping the lung. 2 mM amytal sion, the L/P and ed 1.5 times an DHAP increased . I times (Table 2). With anoxia produced by ventilat Nv or He (plus 5% CO,), the changes in redox couples weie similar to amytal infusion (Table 29. However, with infusion, the changes and GP/DHAP increase doubled. An experiment indicating the relationship between fluorescence changes and tissue redox couples is shown in Fig. 4. Ventilation with 95% CO:5% CO, resulted in rapid increase in surface fluorescence. Redox couples measured in freeze-clamped lung tissue after 3 min of CO ventilation indicated reduction of both cytoplasmic and mitochondrial ne nucleotides. Relation ofnlveo to redox couples. The ratios of redox couples with N, anoxia (Table 2) were obtained in the presence of an alveolar PO, of 0.1-2 mmHg. Further insight into the relation between alveolar PO, and tissue redox state were obtained by plotting the L/P versus alveolar PO, (Fig. 5). these experiments the PO, was continuously monito at the tracheal cannula. On gas from control to 95% N,:5% changing the ventila COZYthere was a rapid decline of expired PO,, followe

I I 0.5 mm 366+

45Qnm

T 5% 4,

CO

ventilation on surface fluorescence (366 --+ 450 nm) and redox couples of freeze-clamped lung tissue. During control period, lungs were ventilated with 95% 0,:5% CO,. 95% CO:5% CO, ventilation was begun at time indicated by arrow. With CO, there was an increase of surface fluorescence; freeze-clamped tissue after 3 min of CO ventilation showed an increase in redox couple ratios compared with control lungs. Ventilation pressure (TTR), perfusion pressure (PPA), and reflectance (366 nm) did not change significantly with CO ventilation. In some experiments, N, or CO ventilation resulted in reversible increase in perfusion pressure (Le., hypoxic vasoconstriction), but effect was inconsistent.

radual decline that reached a plateau after ntilation with the hypoxic gas mixture was carried out for 10 min and terminated by freeze-clampchange was seen in the ing the lungs. No significant he alveolar PO, was decreased to maximal change in L/P (defined ng those obtained with KCN infuion) was not seen until alveolar PO, .I mmHg (Fig. 5). Similar results were obtaine when. alveolar PO, was lotted versus sate LIP. . The tissue 0 ventilation (Table 2) were o tained with 95% CO in the absence of O,? Since 0, a CO compete for cytochrome oxidase, the effect of CO --3 The actual alveolar PO, could not be measured in these experiments because CO is oxidized by the electrochemical cell, resulting in falsely low readings for PO,. Based on the N, ventilation experiments, alveolar PO, during CO anoxia was probably less than 2 mmHg.

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1202

FISHER,

60

0

0.5

1.0

Alveolar

1.5

20

-iTxl

PO, (mm Hg)

FIG. 5. Relationship between tissue lactate to pyruvate ratio and alveolar PO, at time of freeze-clamping of lung. Variation in alveolar PO, was produced through residual 0, in ventilating gas and by small additions of 0, to system. The plotted point for lactatelpyruvate at 700 mmHg is the mean -tSE for 20 control lungs (Table 2).

should be influenced presence gas. Therefore, the e t of mixt redox state was evaluated in a ser (Table 3). With 95% CO and zero 02, an increase was noted in all redox couples, although the change in cytoplasmic couples was slightly less observed in o%her experiments (Table 2). With 19% nd 76% CO in the ventilating gas giving a CO: of 4, perfusate/ lactate pyruvate did not than ung tissue redox state was similar to control. imilarly, no change in redox state was noted with CO:O, ratios of 1 or 0.125. These results indicate that vents the redox effects of C Infusion of aminoxyacetic acid. With oxidative inhibitors, changes in the same direction were noted for mitochondrial and cytoplasmic redox couples” This suggested the presence under control conditions of mechanisms for the transport of reducing equivalents between the two compartments, i.e., an H+ “shuttle’” mechanism. A major H+ shuttle in other tissues is related to influx of malate into the mitochondrial space and efIlux of aspartate (15, 22). Operation of this shuttle requires transamination in the cy%osolic and mitochondrial spaces. In other tissues (15, 22), t studied in the presence of aminoxyacetic acid (AOA), an inhibitor of transaminase reactions (IO). During infusion of AOA into t perfused lung, there was a slight increase in lacta oduction and a o- to threefold increase in llactat pyruvate ratio ( 6). Lung tissue redox couples showed a significant increase in L/P and GPIDHAP, but Glut/KG did not than (Table 2) . Reduction of pyr nucleotides in the %oplasmic space with AOA amination reaction is required cing equivalents between cytoplasmic and mitochondrial compartments of the lung. This data can be used as evidence for operation of a malate-asparate shuttle in lung tissue. Infusion of ,%-deQxy-D-&KQSe. Lung perfusions were generally carried out with 5 mM glucose in the perfusate. Preliminary perfusions in the absence of glucose demonstrated no significant change in lactate or pyruvate production during the 20-min experimental period. In order to more completely block glycolysis, glucose

FURIA,

AND

CHANCE

was omitted from the perfusate and 5 mM 2-deoxy-nglucose was infused. This inhibitor blocks glucose utilization through competitive inhibition of phosphoglucose isomerase (18). During the infusion of inhibitor, there was a significant decrease in both lactate and pyruvate production, but the perfusate lactate-to-pyruvate ratio did not change significantly (Fig. 7); tissue redox couples showed a slight change towards oxidation (Table 2) * Tissue pyridine nucleotides. Tissue pyridine nucleotide content was measured in control lungs and after infusion of 2 mM Na amytal in order to confirm the levels of reduced pyridine nucleotides that icted from measurement of surface fluoresredox couples. The results showed an 82% inTABLE 3. Effect of ventilation with varying @8 in alveolar gas on redox couples in perfusate and lung tissue _ll___l___ ~~__I_------ ___Ventilating

Gas,* %

Lactate/Pyruvate

Glutamate/ a-Ketoglutarate

GPDHAP

CO

02

Perfusate

Tissue

95 76 47.5 10 0

0 19 47.5 85 95

34 4 3 3 4

35 8 12 13 9

7.1 2.9 2.5 2.5

99 31 39 43

2.7

35

Lungs were ventilated and perfused under control conditions (95% 0,:5% CO,) for 15 min. The control perfusate lactate/pyruvate in each experiment was in the range of 3-6. Following the control for 15 min with CO-containing gas. f lungs were ventilated ate samples for lactate and pyruvate were taken at the end of CO period and then lungs were freeze-clamped and analyzed for tissue redox couples. Results are mean data for two experiments under each condition. * Ventilating gas contained 5% CO, in addition to CO and 0,. 20 Lactate

/Pyruvote

I)--*

LACTATE

+--o

PYRUVATE

g 400 \ z -5 ,E 300

.

20 k

I--

at2 il

>Q 2

0

0

IO

20 TlME

30

OF PERFUSION

40

2

50

(minutes)

FIG. 6. Effect of 0.5 mM AOA (aminoxyacetic acid) infusion on lactate and pyruvate production by isolated rat lung. Periods of AOA infusion are indicated by bars at bottom of figure. Results are means LSE for six experiments.

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LUNG

REDQX

1203

STATE

crease in reduced pyridine nucleotides (NADH plus NADPH) with infusion of amytal (Table 4). pyridine nucleotides in the lungs were NAD+. Control NAD+ content was 1,771 t 263 nmol/g dry wt and was 1,546 t 113 after amytal infusion.The reduced form (NADH) (Table 4) represented only a minor fraction of amytal infusion su cleotide in the lu bound) form simi The lung content o control perfusions an

tudy, the response of the redox state to ors was evaluated with two separate methods indicated reduction of tissue es in the presence of oxidative inhibiof redox state with both techwith CO ventilation and KCN lation with N., gave changes of lar results by both methods es as applied to the lung for Further validation was obincreased levels of re lung tissue following a

DISCUSSION

The isolated, perfused rat lung model was study to evaluate oxidative metabolism by Parameters of ventila approximate normal the physiological state was measurement of ventil have previously carri -

characteristics of the preparation (1,9, 17). In lungs that appear stable by the physiological monitors, weight gain represents less than 540% of wet weight, rates of production of lactate, pyruvate, and CO, from glucose14C are constant, and tissue ATP content and “energy charge” are maintained. Perfusion is evenly distributed to all portions of the lung by gross inspection after injection of Evans blue dye. Electron micrographs, after 2 h of perfusion, show no demonstrable abnormalities rsonal communication). The present study that responsiveness to oxidative inhibiined in the isolated lung. Therefore, this pears to be suitable for the study of factors ative metabolism by the lung paren-

La ctate/Pyruvate

ung, since s -

*--@

LACTATE

O--O

PYRUVATE

IO FIG. 7. Effect of pyruvate production ing 5 mM glucose Glucose was deleted case, indicated by lactate and pyruvate start of 2deoxyglucose experiments.

4. Reduced pyridine isolated perfused rat lung

TABLE

20

I5

TIME Of PERFUSION hinubs) 5 m&I Z-deoxyglucose infusion on lactate and by perfused rat lung. Usual perfusate containwas used during initial 10 min of perfusion. from perfusate during infusion of 2-deoxygluarrows. Samples of perfusion were taken for at end of control period and 5 and 10 min after infusion. Results are means *SE for six

nucleotide

content of

Control

NADH NADPH ZNAD(P)H Units for four

Amytal,

36.7 29.6 66.3

18.2 t 3.5 18.2 k 2.2 36.4 I!I 2.1

are nanomoles per gram dry lungs under each condition.

weight.

Data

2 mM

+ 6.1 AI 4.5 L 9.8

are means

+ SE

lung was measurement of perfusate lactate-to-pyruvate the ratio of tissue metabolite couples. Comfluorescen.ce, these techniques were ing and did not permit continuous “‘readout .” In addition, tissue metabolites could be measured only once for each lung perfusion so that considerably more experiments were required to obtain significant data. The signal-to-noise ratio of this technique was greater than with surface fluorescence, although changes in ratios of redox couples were relatively small compared with other tissues (20). The small change of pyridine nucleotides in the lung with oxidative inhibitors can possibly be attributed to a sizeable pool of cells with a relatively low rate of oxidative

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1204 metabolism. The relatively low mitochondrial density of membraneous epithelial and endothelial cells (11) suggests that these cells may comprise the poal of low oxidative activity. The ratios of redox couples can be used along with the equilibrium constants for the reactions to calculate the ratios of free oxidized to reduced pyridine nucleotides and the redox potential of the tissue (19). However, these calculations were not carried out with the present data for several reasons. First, the calculation assumes sufficient enzyme concentration to achieve equilibrium conditions and this has not been validated in lung tissue. In addition, the ratio of glycerol-3-P to dihydroxyace tone-P, alt*ho ugh reflecting chiefly the redox state of the cytoplasmic compartment, might be displaced from equilibrium by activity of the mitochondrial glycerol-3P-dehydrogenase. Second, the intracellu lar redox potential is a function of intracellular pH which w as not measured. Finally, compartmentation within cells and among various cell types in the lung diminishes the significance of calculated redox potentials. One manifestation of the compartmentation appears to be the difference between lactate-pyruvate ratios in perfusate and tissue, as has been noted with other perfused organs (3). For the se reasons, the metabolic couples were used to indicate direction of changes in overall redox state of the tissue without the attempt to give quantitative expression to redox potentials. A major finding of this study was that alveolar PO, must be reduced to extremely low levels in order to

FISHER,

FURIA,

AND

CHANCE

achieve maximal reduction of pyridine nucleotides. Only when alveolar PO, was less than 0.1 mmHg did perfusate and tissue redox couples approach values seen with carbon monoxide ventilation. In addition, there were lesser increases in surface fluorescence with N, ventilation compared with other oxidative inhibitors. These results suggest that some oxidative activity continued in the lung at extremely low driving gradients for oxygenation of lung cells. It seems likely that the persistence of oxidative activity at low PO, is related to the relatively short diffusion distances across the alveolar septa and the relatively large gas reservoir in the alveolar space. Of practical significance is that anoxia produced with N, ventilation may produce varying degrees of oxidative inhibition, resulting in difficulties with interpretation of results. Ventilation with carbon monoxide appears to be the best approach to produce maximal and consistent inhibition of sxidative activity in the lung. We thank Dr. Dana Jamieson and Brad Stuart for assistance with measurement of surface fluorescence and Dr. John R. Williamson and David Bassett for helpful advice. This study was supported by Public Health Service grants HL 15061 (SCOR), HL 15013, and the Veterans Administration Research Service. The results were presented in part at the meeting of the Federation of American Societies for Experimental Biology, April 1974 (Federation Proc. 33: 345, 1974) and at the Aspen Lung Conference, June 1974 (Chest 671 24S, 1974). Received

for

publication

2 June

1975.

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Evaluation of redox state of isolated perfused rat lung.

AMERICAN JOURNAL OF PHYSIOLOGY Vol. 230, No. 5, May 1976. Printed in U.S.A. Evaluation of redox perfused rat lung state of isolated ARON B. FI...
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