AMERICA
Vol.
230,
N JOURNAL
No.
3, March
OF PHYSIOLOGY
1976.
Printed
in U.S.A.
Metabolic response to carbon monoxide isolated rat lungs D. J. P. BASSETT Department Philadelphia,
AND
of Physiology, Pennsylvania
A. B. FISHER University
of Pennsylvania
lactate;
pyruvate;
C02, lipids;
School
of Medicine
19174
by nitrogen ventilation (2). This resulted in increased lactate production associated with decreased concentration of tissue adenine nucleotides, and an altered pattern of glucose carbon incorporation into lipids (2). However, total inhibition of oxidative metabolism is difficult to achieve by nitrogen ventilation (unpublished observations), so that the previous study did not evaluate the maximal metabolic response of the lung. Ventilation with carbon monoxide (CO) in the absence of oxygen produces more complete reduction of lung-tissue pyr-dine nucleotides (Am. J. Physiol., in press). These observations lead to the present study in which glucose metabolism and oxidation in lungs ventilated with oxygen (control), nitrogen (hypoxia), and CO are compared The results demonstrate the maximal glycolytic capacity of the perfused lung and the relationship of lung energy status and synthetic activity of the total inhibition of oxidative phosphorylation.
BASSETT, D. J. P., AND A. B. FISHER. Metabolic response to carbon monoxide by isolated rat lungs. Am. J. Physiol. 230(3): 658-663. 1976. - Glucose metabolism was studied in isolated rat lungs ventilated with 95% 0,:5% CO, (control), 95% N,: 5% CO, (hypoxia), and 95% CO:5% CO, (carbon monoxide) and perfused for loo-120 min with Krebs-Ringer-bicarbonate buffer, pH 7.4, containing [U-14C] and [3-3H]glucose. The production of 14C-labeled lactate plus pyruvate (L + P) and of 14C0, represented 48% and 22%, respectively, of the total [14C]glucose utilization. The lactate-to-pyruvate ratio (L/P) was 8.7. Tritium was recovered predominantly as 3H,0 in the perfusate. With carbon monoxide ventilation, L + P production was increased by 357% with an L/P of 52.9, and 14C0, production was markedly decreased. A 56% decrease in lung ATP content was associated with decreased incorporation of 14C into fatty acids. Compared with CO, changes with N, ventilation were less marked, indicating that ventilation with CO is a more effective method with which to study inhibition of oxidative metabolism. The lung exhibits a Pasteur effect, but glycolysis alone in the absence of oxidative metabolism is not sufficient to maintain ATP content or its supply for synthetic activity.
glucose;
by
ATP;
Pasteur
METHODS
Male Sprague-Dawley rats (HillPerfusion technique. top Lab Animals, Scottdale, Pa.) weighing‘ 180-220 g were kept in filtered air on a normal diet, feeding ad libitum. Rats were anesthetized with pentobarbital 50 mg/kg intraperitoneally, and lungs were isolated as previously reported (2) and placed in a water-jacketed incubation chamber held at 37°C (see Fig. 1). The perfusate was a Krebs-Ringer bicarbonate (KRB) solution, pH 7.35-7.40, containing 3% fatty-acid-free bovine serum albumin (dialyzed Pentex, Miles Laboratories), 5.5 mM n-glucose, and lo+ U/ml of insulin (U-40 Iletin regular, Lilly). All solutions were filtered through a Millipore filter of 0.45-p pore size. Lungs were perfused via the cannulated pulmonary artery at lo-12 ml/min with a peristaltic pump (Buchler) that maintained a constant output. Perfusate flowed freely from the cut left atrium into the chamber from which it was recycled at 30-35 ml/min to a water-jacketed aerator (pump 1 in Fig. 1). The perfusate was aerated by a steady flow of gas at 75 ml/min. The gas was drawn through the aerator, overflow circuit and incubation chamber and forced through two CO, traps in series (pump 2 in Fig. 1). Each trap contained 25 ml of 3N NaOH. The pH, Pco,, and PO, of the perfusate were continually monitored using a flow-through cuvette (Radiometer). Total recirculating volume in the perfusion circuit was 50 ml. Lungs were ventilated with the same gas mixture as
effect
GLUCOSE HAS BEEN SHOWN to be an important substrate for lung metabolism (2, 5, 12). Glycolysis and the subsequent oxidation of pyruvate to CO, by mitochondria provide a major source of energy for the metabolic processes of the lungs (6). Two- and three-carbon products derived from glucose provide intermediates for lipid metabolism (2, 8). These products are incorporated into both the glyceride-glycerol as well as the fatty acids of lung lipids (2, 8). Glucose carbon atoms are also incorporated into tissue glycoproteins (17). Glucose metabolized by the pentose cycle provides both ribose 5-phosphate for the synthesis of nucleic acids and generates NADPH for the synthesis of fatty acids. NADPH is also required to maintain the supply of reduced glutathione, which may be an important agent to protect the lung from the damage caused by oxidants (15). The incorporation of amino acids into tissue protein is also supported by the metabolism of glucose (11). The present study was designed to evaluate the pathways of glucose metabolism and the metabolic response to the inhibition of oxidative metabolism in the isolated perfused rat lung. In a previous study with this preparation, inhibition of oxidative metabolism was produced 658
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CO AND
LUNG
659
METABOLISM
Ii
Aer
PH
l
PO2
.
PCO*.
over
a-
i
Pump
lncubatlon
Chamber
2
C02Traps
End Explratar
y Pressure
FIG. 1. Schematic diagram of apparatus for isolated rat lung perfusion. Incubation chamber and aerator are heated by a water jacket to maintain a chamber temperature of 3’7°C.
for aeration by use of a Harvard rodent respirator. Tidal volume was 2-2.5 ml at 60 cycles/min with an endexpiratory pressure of 2-3 cmH,O. The gas used for ventilation was recycled from a 1.5-li ter reservoir. At the end of perfusion this reservoi .r was emptied through a NaOH solution to trap CO,. In the control experiments, 95% 0,:5% CO, was used for ventilation. For nitrogen-induced hypoxia, 95% N,:5% CO, was used. This resulted in a perfusate PO, of 3-5 mmHg. For experiments with carbon monoxide ventilation, 95% CO:5% CO, was used. Ventilation pressure and perfusion pressure were monitored with pressure transducers (Statham PM 131TC and P23V, respectively) and a direct-writing oscillograph (Gould Brush). Control and hypoxic perfusions were carried out for 120 min, while carbon monoxide perfusions were terminated after 100 min. Radioactive isotopes. Experiments were carried out with 5.5 mM [U-14C]glucose (sp act, 0.1 mCi/mmol) and [3-3H]glucose (sp act, 0.1 mCi/mmol) added to the perfusate. Isotopes were obtained from New England Nuclear. Radioactive analysis was done by scintillation counting (Packard Tri-Carb) with use of Aquasol (New England Nuclear) for aqueous samples and a toluene and Triton mixture containing 2,5-diphenyloxazole (PPO) and p-bis[2-(4-methyl-5-phenyloxazolyllbenzene (dimethyl POPOP) for nonaqueous samples. Counting efficiencies were determined routinely by methods based on internal standards. Perfusion analysis. Glucose, lactate, and pyruvate were measured in the perfusate by standard enzymatic techniques (3). 14C-labeled bicarbonate in the perfusate and the CO, traps was measured by radioactive scintillation counting after release with acid and concentration into 0.3 ml of Hyamine hydroxide. 3H,0 was measured after separation from 14C by evaporation of the sample under reduced pressure using Thunberg tubes. Lactate, pyruvate, 3H,0 and total 14C0, concentration was measured in samples taken at 20- to 30-min intervals during perfusion, and rates of production were estimated by least-mean-squares analysis. The specific activity of perfusate lactate was esti-
mated at the end of perfusion after separation from labeled glucose by ion-exchange chromatography with Dowex 1 acetate (Bio-Rad AGl-X4), or by extraction with ether at pH 3. The methods did not differentiate between lactate and other small anions that were potentially present but the extent of the latter was considered insignificant. The specific activity of pyruvate was estimated from the perfusate lactate/pyruvate ratio. Tissue anaZysis. For measurement of adenine nucleotides, the perfusions were terminated by freeze-clamping the lungs at liquid-nitrogen temperature, the major bronchi and large vessels were chipped away, and the tissue was extracted with perchloric acid in ethanol at -20” (16). ATP, ADP, and AMP were measured in the neutralized extracts by fluorometric assay with standard enzymatic methods (16). For measurements of other tissue components, lungs were trimmed before freezing, ground to a powder, and then lyophilizedl to a constant weight. The powdered lungs were weighed before and after lyophilization to determine dry weight-to-wet weight ratios. Lipids were extracted from the lyophilized material by homogenization in a chloroform-methanol(2: 1 vol/ vol) medium followed by incubation for 12-15 h at 60°C. The extracts were treated according to Folch et al. (7) to remove contaminants. The isolated lipids were saponified by incubation at 90°C for 2 h with 10% KOH in 90% ethanol. The liberated fatty acids were acidified and extracted with ether. The remaining portion represented a deacylated fraction consisting primarily of glyceride-glycerol but also possibly amines and amine alcohols. Both fractions were analyzed for radioactivity incorporation. The residue from the lipid extraction was washed 3 times with 95% ethanol. This ethanol-insoluble fraction was solubilized for radioactive analysis by heating in 0.2 N NaOH for 20 min at 85°C. Polysaccharides were precipitated from the alkali digest with ethanol (10). The results for 14C incorporation were expressed either as micromoles [U-14C]glucose or microatoms 14C incorporated per gram dry weight of tissue per hour of perfusion. Expression of results in this way is based on the assumption that the incorporation of the radioactive label into tissue components was linear. Estimation of glucose utilization. Total glucose metabolized was calculated from 14C recovered in C02, lactate, pyruvate, lipids, and the ethanol-insoluble fraction at the end of perfusion. An additional estimate was obtained from the recovery of 3H in perfusate H20, lactate, pyruvate, and tissue lipids and ethanol-insoluble material. These methods may underestimate total glucose utilization because of failure to account for tissue intermediates that might be removed during the ethanol washing step of the extraction procedure. Electron microscopy. Electron microscopy was carried out on lungs that were isolated and perfused in the usual manner. Lungs were fixed by infusion via the pulmonary artery of 4% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M Na-cacodylate buffer for approximately 10 min (pH 7.4, osmolarity 400 mosM). The fixed lungs were minced and small blocks were postfixed in 2% OsO,, dehydrated in ascending grades of alcohol,
Downloaded from www.physiology.org/journal/ajplegacy by ${individualUser.givenNames} ${individualUser.surname} (130.070.008.131) on January 16, 2019.
660
D. J. P. BASSETT
AND
A. B. FISHER
embedded in Epon 812, and stained with many1 acetatelead hydroxide. Thin sections were examined with a Siemens 101 electron microscope at 80 kV accelerating voltage and representative sections were photographed. Photographs were reviewed by an electron microscopist without knowledge of the lung exposure conditions. RESULTS
Rat lungs could generally be perfused for 120 min with no changes in ventilation or perfusion pressures. The pressure required for tidal ventilation was 5-7 cm H20, and the mean perfusion pressure was approximately 12-15 cm H20. The dry-to-wet weight ratio at the end of perfusion was 0.16-o. 18. The occasional lungs that developed gross alveolar edema during perfusion were discarded without further analysis. Experiments with CO ventilation were carried out for 100 min because perfusion beyond this time resulted in a higher incidence of gross alveolar edema. For electron microsFIG. 2. Electron micrograph of isolated rat lung fixed after 60 min of ventilation with 95% CO:5% CO,. Photograph illustrates a portion copy, two control lungs were studied after 60 and 90 min of an alveolar septum indistinguishable from that of rat lungs ventiof perfusion and two CO-ventilated lungs were fixed lated with 95% 0,:5% CO,. There is no evidence of interstitial or after 60 min. Electron microscopy demonstrated normal intracellular edema. Endothelial (J) and epithelial junctions (EJ) are ultrastructure at the level of the alveolar septum for all not widened and there is no swelling of endothelial (En) or type-1 pneumocyte (P.l) cytoplasm. A mitochondrion (Mt) in a type-II pneulungs (Fig. 2). mocyte (P.ll) appears normal. Symbols: Alv, alveolar space; Cap, Utilization of glucose during control perfusions. 14C0, capillary; Ic interstitial cell; LB, lamellar body. Bar indicates magproduction during the initial 0- to 30-min period ofpernification. fusions was alinear, suggesting that this time was required for the radioactive isotopes to equilibrate with ‘Or TSEM T endogeneous metabolic intermediates and substrates. 60 Between 30 and 120 min of perfusion, the production of lactate, pyruvate, 14C0, from [U-14Clglucose, and 3H,0 from [3-3Hlglucose was linear (Fig. 3). The lactate-topyruvate ratio (L/P) in the perfusate remained relatively constant with a mean value of 8.7 (Table 1). At the end of 120 min of perfusion, glucose utilization by the lung represented only 510% of the initial glucose added to the perfusate. Because of the small utilization, assay of the perfusate for glucose was considered to be an unsuitable method of determining total uptake. Therefore, metabolized glucose was calculated from the total recovery of 14C and 3H in the products of metabolism at the end of perfusion with [14C, 3H]glucose. Similar results were obtained with each isotope (Table 2), TIME OF FERFUSION (min) suggesting that most of the metabolized glucose was accounted for. The majority of 3H was recovered as 3H,0 FIG. 3. Production rates of lactate, pyruvate, 3H,0 from [33H]glucose and ‘*CO, from [U-‘Vlglucose in perfusate as a funcin the perfusate. This was derived from [3-3H]glucose by tion of perfusion time. WO, figures represent sum of HWO,in exchange with H,O at the triosephosphate isomerase perfusate plus NaHWO, in CO, traps. Results are mean 2 SE for 4 reaction of the glycolytic pathway (9). Varying amounts experiments. SE for pyruvate points was less than width of plotted of 3H were recovered in lactate, pyruvate, and tissue point. fractions, including 11% in tissue fatty acids. The likely source of 3H in fatty acids was from NADP3H generated * incorporated into both tissue lipids and the ethanolfrom [3-3Hlglucose in the reactions of the pentose cycle insoluble fraction (Table 3). Tissue fatty acids represented 60% of total incorpora(9). The distribution of 14C recovered from [U-14C]glucose tion into lipid with the remainder in the deacylated (Table 3) demonstrates that lactate and pyruvate were fraction. Polysaccharides were isolated from the the major metabolic products which together with carethanol-insoluble material and found to represent 22% bon dioxide production accounted for 70% of glucose of 14C recovered in this fraction (Fig. 4). The remaining uptake. The perfusate lactate at the end of perfusion 14C label in the ethanol-insoluble material was assumed had the same 14C specific activity as perfusate [Uto be other labeled-tissue components such as protein 14Clglucose indicating no dilution of the three-carbon and nucleic acids (17). pool by nonlabeled precursors. Glucose carbons were Glucose uptake during hypoxia. Ventilation with 95%
Downloaded from www.physiology.org/journal/ajplegacy by ${individualUser.givenNames} ${individualUser.surname} (130.070.008.131) on January 16, 2019.
CO AND
LUNG
661
METABOLISM
N,:5% CO, (hypoxia) resulted in a twofold increase in lactate production with no significant change in pyruvate production. The mean perfusate L/P with hypoxia was 15.7 (Table 1). Hypoxic conditions resulted in a 55% increase in [ 14C]glucose utilization compared with control (Table 2). This was accounted for by increased recovery of 14C in perfusate lactate (Table 3). The 14C specific activity of perfusate lactate was the same as for glucose. Measurements of 14C incorporation into synthesized products and 14C0, showed no significant diffferences when compared with the control (Table 3). Glucose uptake during ventilation with CO. In lungs ventilated with carbon monoxide (CO), lactate production increased and pyruvate production decreased giving a mean perfusate L/P of 52.9 (Table 1). There was a 151% increase in mean [14C]glucose utilization compared with control (Table 2). 14C recovered in the perfusate lactate plus pyruvate from [UJ4C]glucose was increased approximately fourfold, while the rate of 14C02 production was decreased 70% (Table 3). There was a TABLE
isolated
1. Lactate perfused
and pyruvate rat lung Lactate
Results are means tion. * Calculated Significantly different 0.05; $P < 0.001.
Pyruvate per
g dry
L/P*
Produced wt
29.7 * 2.1
3.5 + 0.3
8.7 f. 0.6
64.0 + 2.8$
4.2 f 0.2
15.7 t 0.6$
137.0 f 7.0$
2.8 * 0.2-l
52.9 + 4.9$
2. Glucose utilization determined of 14C and 3H in products of metabolism perfusion with [U- “C,3-3H]glucose [U-14ClGlucose
Utilized pmollh
by recovery during [3-3HlGlucose
per
g dry
3. Recovery
29.8 t 2.2
46.0 of: 5.1*
40.1 + 3.7
74.7 k 6.9-l
72.5 _+ 4.7-f
co2 39.2 & 1.3 (22.2%) 36.7 k 2.6 (13.5%) 11.6 + O.&i(2.6%)
q
Hypoxia
a
co
Fatty Acids
of glucose Lactate
metabolism plus
Deacylated Polysaccharide Fraction Fraction FIG. 4. Recovery of 14C in fatty acid and deacylated fractions of tissue lipids and polysaccharide fraction of ethanol-insoluble material isolated from lungs perfused with [UJ4C]glucose. Control and hypoxic lungs were perfused for 120 min, carbon monoxide lungs for 100 min, and results were represented as uptake of 14C per hour of perfusion. Results are means +SEM.
after perfusion
Pyruvate
Tissue wtoms
Control mew Hypoxia (N&O,) Carbon monoxide (CO:CO,)
Control
Utilized
29.6 -t 1.7
of 14C in products
0
wt
Results are means * SE of four lungs perfused under each condition. Control and hypoxic lungs were perfused for 120 min. Carbon monoxide perfusions were terminated after 100 min. Comparison with control significantly different: * (P < 0.025); UP < 0.001). TABLE
Characteristics of the preparation. Evaluation of the isolated perfused lung preparation suggests that it is suitable for metabolic studies. During the 2-h experimental period, the perfusion and ventilation pressures at constant perfusion rate and tidal volume remained unchanged, indicating that pulmonary vascular resistance and lung compliance were unaltered. Ultrastruc-
+ SE for 12 lungs perfused under each condias ratio of lactate to pyruvate production. from control by independent t test: tp
Vol.
230,
N JOURNAL
No.
3, March
OF PHYSIOLOGY
1976.
Printed
in U.S.A.
Metabolic response to carbon monoxide isolated rat lungs D. J. P. BASSETT Department Philadelphia,
AND
of Physiology, Pennsylvania
A. B. FISHER University
of Pennsylvania
lactate;
pyruvate;
C02, lipids;
School
of Medicine
19174
by nitrogen ventilation (2). This resulted in increased lactate production associated with decreased concentration of tissue adenine nucleotides, and an altered pattern of glucose carbon incorporation into lipids (2). However, total inhibition of oxidative metabolism is difficult to achieve by nitrogen ventilation (unpublished observations), so that the previous study did not evaluate the maximal metabolic response of the lung. Ventilation with carbon monoxide (CO) in the absence of oxygen produces more complete reduction of lung-tissue pyr-dine nucleotides (Am. J. Physiol., in press). These observations lead to the present study in which glucose metabolism and oxidation in lungs ventilated with oxygen (control), nitrogen (hypoxia), and CO are compared The results demonstrate the maximal glycolytic capacity of the perfused lung and the relationship of lung energy status and synthetic activity of the total inhibition of oxidative phosphorylation.
BASSETT, D. J. P., AND A. B. FISHER. Metabolic response to carbon monoxide by isolated rat lungs. Am. J. Physiol. 230(3): 658-663. 1976. - Glucose metabolism was studied in isolated rat lungs ventilated with 95% 0,:5% CO, (control), 95% N,: 5% CO, (hypoxia), and 95% CO:5% CO, (carbon monoxide) and perfused for loo-120 min with Krebs-Ringer-bicarbonate buffer, pH 7.4, containing [U-14C] and [3-3H]glucose. The production of 14C-labeled lactate plus pyruvate (L + P) and of 14C0, represented 48% and 22%, respectively, of the total [14C]glucose utilization. The lactate-to-pyruvate ratio (L/P) was 8.7. Tritium was recovered predominantly as 3H,0 in the perfusate. With carbon monoxide ventilation, L + P production was increased by 357% with an L/P of 52.9, and 14C0, production was markedly decreased. A 56% decrease in lung ATP content was associated with decreased incorporation of 14C into fatty acids. Compared with CO, changes with N, ventilation were less marked, indicating that ventilation with CO is a more effective method with which to study inhibition of oxidative metabolism. The lung exhibits a Pasteur effect, but glycolysis alone in the absence of oxidative metabolism is not sufficient to maintain ATP content or its supply for synthetic activity.
glucose;
by
ATP;
Pasteur
METHODS
Male Sprague-Dawley rats (HillPerfusion technique. top Lab Animals, Scottdale, Pa.) weighing‘ 180-220 g were kept in filtered air on a normal diet, feeding ad libitum. Rats were anesthetized with pentobarbital 50 mg/kg intraperitoneally, and lungs were isolated as previously reported (2) and placed in a water-jacketed incubation chamber held at 37°C (see Fig. 1). The perfusate was a Krebs-Ringer bicarbonate (KRB) solution, pH 7.35-7.40, containing 3% fatty-acid-free bovine serum albumin (dialyzed Pentex, Miles Laboratories), 5.5 mM n-glucose, and lo+ U/ml of insulin (U-40 Iletin regular, Lilly). All solutions were filtered through a Millipore filter of 0.45-p pore size. Lungs were perfused via the cannulated pulmonary artery at lo-12 ml/min with a peristaltic pump (Buchler) that maintained a constant output. Perfusate flowed freely from the cut left atrium into the chamber from which it was recycled at 30-35 ml/min to a water-jacketed aerator (pump 1 in Fig. 1). The perfusate was aerated by a steady flow of gas at 75 ml/min. The gas was drawn through the aerator, overflow circuit and incubation chamber and forced through two CO, traps in series (pump 2 in Fig. 1). Each trap contained 25 ml of 3N NaOH. The pH, Pco,, and PO, of the perfusate were continually monitored using a flow-through cuvette (Radiometer). Total recirculating volume in the perfusion circuit was 50 ml. Lungs were ventilated with the same gas mixture as
effect
GLUCOSE HAS BEEN SHOWN to be an important substrate for lung metabolism (2, 5, 12). Glycolysis and the subsequent oxidation of pyruvate to CO, by mitochondria provide a major source of energy for the metabolic processes of the lungs (6). Two- and three-carbon products derived from glucose provide intermediates for lipid metabolism (2, 8). These products are incorporated into both the glyceride-glycerol as well as the fatty acids of lung lipids (2, 8). Glucose carbon atoms are also incorporated into tissue glycoproteins (17). Glucose metabolized by the pentose cycle provides both ribose 5-phosphate for the synthesis of nucleic acids and generates NADPH for the synthesis of fatty acids. NADPH is also required to maintain the supply of reduced glutathione, which may be an important agent to protect the lung from the damage caused by oxidants (15). The incorporation of amino acids into tissue protein is also supported by the metabolism of glucose (11). The present study was designed to evaluate the pathways of glucose metabolism and the metabolic response to the inhibition of oxidative metabolism in the isolated perfused rat lung. In a previous study with this preparation, inhibition of oxidative metabolism was produced 658
Downloaded from www.physiology.org/journal/ajplegacy by ${individualUser.givenNames} ${individualUser.surname} (130.070.008.131) on January 16, 2019.
CO AND
LUNG
659
METABOLISM
Ii
Aer
PH
l
PO2
.
PCO*.
over
a-
i
Pump
lncubatlon
Chamber
2
C02Traps
End Explratar
y Pressure
FIG. 1. Schematic diagram of apparatus for isolated rat lung perfusion. Incubation chamber and aerator are heated by a water jacket to maintain a chamber temperature of 3’7°C.
for aeration by use of a Harvard rodent respirator. Tidal volume was 2-2.5 ml at 60 cycles/min with an endexpiratory pressure of 2-3 cmH,O. The gas used for ventilation was recycled from a 1.5-li ter reservoir. At the end of perfusion this reservoi .r was emptied through a NaOH solution to trap CO,. In the control experiments, 95% 0,:5% CO, was used for ventilation. For nitrogen-induced hypoxia, 95% N,:5% CO, was used. This resulted in a perfusate PO, of 3-5 mmHg. For experiments with carbon monoxide ventilation, 95% CO:5% CO, was used. Ventilation pressure and perfusion pressure were monitored with pressure transducers (Statham PM 131TC and P23V, respectively) and a direct-writing oscillograph (Gould Brush). Control and hypoxic perfusions were carried out for 120 min, while carbon monoxide perfusions were terminated after 100 min. Radioactive isotopes. Experiments were carried out with 5.5 mM [U-14C]glucose (sp act, 0.1 mCi/mmol) and [3-3H]glucose (sp act, 0.1 mCi/mmol) added to the perfusate. Isotopes were obtained from New England Nuclear. Radioactive analysis was done by scintillation counting (Packard Tri-Carb) with use of Aquasol (New England Nuclear) for aqueous samples and a toluene and Triton mixture containing 2,5-diphenyloxazole (PPO) and p-bis[2-(4-methyl-5-phenyloxazolyllbenzene (dimethyl POPOP) for nonaqueous samples. Counting efficiencies were determined routinely by methods based on internal standards. Perfusion analysis. Glucose, lactate, and pyruvate were measured in the perfusate by standard enzymatic techniques (3). 14C-labeled bicarbonate in the perfusate and the CO, traps was measured by radioactive scintillation counting after release with acid and concentration into 0.3 ml of Hyamine hydroxide. 3H,0 was measured after separation from 14C by evaporation of the sample under reduced pressure using Thunberg tubes. Lactate, pyruvate, 3H,0 and total 14C0, concentration was measured in samples taken at 20- to 30-min intervals during perfusion, and rates of production were estimated by least-mean-squares analysis. The specific activity of perfusate lactate was esti-
mated at the end of perfusion after separation from labeled glucose by ion-exchange chromatography with Dowex 1 acetate (Bio-Rad AGl-X4), or by extraction with ether at pH 3. The methods did not differentiate between lactate and other small anions that were potentially present but the extent of the latter was considered insignificant. The specific activity of pyruvate was estimated from the perfusate lactate/pyruvate ratio. Tissue anaZysis. For measurement of adenine nucleotides, the perfusions were terminated by freeze-clamping the lungs at liquid-nitrogen temperature, the major bronchi and large vessels were chipped away, and the tissue was extracted with perchloric acid in ethanol at -20” (16). ATP, ADP, and AMP were measured in the neutralized extracts by fluorometric assay with standard enzymatic methods (16). For measurements of other tissue components, lungs were trimmed before freezing, ground to a powder, and then lyophilizedl to a constant weight. The powdered lungs were weighed before and after lyophilization to determine dry weight-to-wet weight ratios. Lipids were extracted from the lyophilized material by homogenization in a chloroform-methanol(2: 1 vol/ vol) medium followed by incubation for 12-15 h at 60°C. The extracts were treated according to Folch et al. (7) to remove contaminants. The isolated lipids were saponified by incubation at 90°C for 2 h with 10% KOH in 90% ethanol. The liberated fatty acids were acidified and extracted with ether. The remaining portion represented a deacylated fraction consisting primarily of glyceride-glycerol but also possibly amines and amine alcohols. Both fractions were analyzed for radioactivity incorporation. The residue from the lipid extraction was washed 3 times with 95% ethanol. This ethanol-insoluble fraction was solubilized for radioactive analysis by heating in 0.2 N NaOH for 20 min at 85°C. Polysaccharides were precipitated from the alkali digest with ethanol (10). The results for 14C incorporation were expressed either as micromoles [U-14C]glucose or microatoms 14C incorporated per gram dry weight of tissue per hour of perfusion. Expression of results in this way is based on the assumption that the incorporation of the radioactive label into tissue components was linear. Estimation of glucose utilization. Total glucose metabolized was calculated from 14C recovered in C02, lactate, pyruvate, lipids, and the ethanol-insoluble fraction at the end of perfusion. An additional estimate was obtained from the recovery of 3H in perfusate H20, lactate, pyruvate, and tissue lipids and ethanol-insoluble material. These methods may underestimate total glucose utilization because of failure to account for tissue intermediates that might be removed during the ethanol washing step of the extraction procedure. Electron microscopy. Electron microscopy was carried out on lungs that were isolated and perfused in the usual manner. Lungs were fixed by infusion via the pulmonary artery of 4% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M Na-cacodylate buffer for approximately 10 min (pH 7.4, osmolarity 400 mosM). The fixed lungs were minced and small blocks were postfixed in 2% OsO,, dehydrated in ascending grades of alcohol,
Downloaded from www.physiology.org/journal/ajplegacy by ${individualUser.givenNames} ${individualUser.surname} (130.070.008.131) on January 16, 2019.
660
D. J. P. BASSETT
AND
A. B. FISHER
embedded in Epon 812, and stained with many1 acetatelead hydroxide. Thin sections were examined with a Siemens 101 electron microscope at 80 kV accelerating voltage and representative sections were photographed. Photographs were reviewed by an electron microscopist without knowledge of the lung exposure conditions. RESULTS
Rat lungs could generally be perfused for 120 min with no changes in ventilation or perfusion pressures. The pressure required for tidal ventilation was 5-7 cm H20, and the mean perfusion pressure was approximately 12-15 cm H20. The dry-to-wet weight ratio at the end of perfusion was 0.16-o. 18. The occasional lungs that developed gross alveolar edema during perfusion were discarded without further analysis. Experiments with CO ventilation were carried out for 100 min because perfusion beyond this time resulted in a higher incidence of gross alveolar edema. For electron microsFIG. 2. Electron micrograph of isolated rat lung fixed after 60 min of ventilation with 95% CO:5% CO,. Photograph illustrates a portion copy, two control lungs were studied after 60 and 90 min of an alveolar septum indistinguishable from that of rat lungs ventiof perfusion and two CO-ventilated lungs were fixed lated with 95% 0,:5% CO,. There is no evidence of interstitial or after 60 min. Electron microscopy demonstrated normal intracellular edema. Endothelial (J) and epithelial junctions (EJ) are ultrastructure at the level of the alveolar septum for all not widened and there is no swelling of endothelial (En) or type-1 pneumocyte (P.l) cytoplasm. A mitochondrion (Mt) in a type-II pneulungs (Fig. 2). mocyte (P.ll) appears normal. Symbols: Alv, alveolar space; Cap, Utilization of glucose during control perfusions. 14C0, capillary; Ic interstitial cell; LB, lamellar body. Bar indicates magproduction during the initial 0- to 30-min period ofpernification. fusions was alinear, suggesting that this time was required for the radioactive isotopes to equilibrate with ‘Or TSEM T endogeneous metabolic intermediates and substrates. 60 Between 30 and 120 min of perfusion, the production of lactate, pyruvate, 14C0, from [U-14Clglucose, and 3H,0 from [3-3Hlglucose was linear (Fig. 3). The lactate-topyruvate ratio (L/P) in the perfusate remained relatively constant with a mean value of 8.7 (Table 1). At the end of 120 min of perfusion, glucose utilization by the lung represented only 510% of the initial glucose added to the perfusate. Because of the small utilization, assay of the perfusate for glucose was considered to be an unsuitable method of determining total uptake. Therefore, metabolized glucose was calculated from the total recovery of 14C and 3H in the products of metabolism at the end of perfusion with [14C, 3H]glucose. Similar results were obtained with each isotope (Table 2), TIME OF FERFUSION (min) suggesting that most of the metabolized glucose was accounted for. The majority of 3H was recovered as 3H,0 FIG. 3. Production rates of lactate, pyruvate, 3H,0 from [33H]glucose and ‘*CO, from [U-‘Vlglucose in perfusate as a funcin the perfusate. This was derived from [3-3H]glucose by tion of perfusion time. WO, figures represent sum of HWO,in exchange with H,O at the triosephosphate isomerase perfusate plus NaHWO, in CO, traps. Results are mean 2 SE for 4 reaction of the glycolytic pathway (9). Varying amounts experiments. SE for pyruvate points was less than width of plotted of 3H were recovered in lactate, pyruvate, and tissue point. fractions, including 11% in tissue fatty acids. The likely source of 3H in fatty acids was from NADP3H generated * incorporated into both tissue lipids and the ethanolfrom [3-3Hlglucose in the reactions of the pentose cycle insoluble fraction (Table 3). Tissue fatty acids represented 60% of total incorpora(9). The distribution of 14C recovered from [U-14C]glucose tion into lipid with the remainder in the deacylated (Table 3) demonstrates that lactate and pyruvate were fraction. Polysaccharides were isolated from the the major metabolic products which together with carethanol-insoluble material and found to represent 22% bon dioxide production accounted for 70% of glucose of 14C recovered in this fraction (Fig. 4). The remaining uptake. The perfusate lactate at the end of perfusion 14C label in the ethanol-insoluble material was assumed had the same 14C specific activity as perfusate [Uto be other labeled-tissue components such as protein 14Clglucose indicating no dilution of the three-carbon and nucleic acids (17). pool by nonlabeled precursors. Glucose carbons were Glucose uptake during hypoxia. Ventilation with 95%
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CO AND
LUNG
661
METABOLISM
N,:5% CO, (hypoxia) resulted in a twofold increase in lactate production with no significant change in pyruvate production. The mean perfusate L/P with hypoxia was 15.7 (Table 1). Hypoxic conditions resulted in a 55% increase in [ 14C]glucose utilization compared with control (Table 2). This was accounted for by increased recovery of 14C in perfusate lactate (Table 3). The 14C specific activity of perfusate lactate was the same as for glucose. Measurements of 14C incorporation into synthesized products and 14C0, showed no significant diffferences when compared with the control (Table 3). Glucose uptake during ventilation with CO. In lungs ventilated with carbon monoxide (CO), lactate production increased and pyruvate production decreased giving a mean perfusate L/P of 52.9 (Table 1). There was a 151% increase in mean [14C]glucose utilization compared with control (Table 2). 14C recovered in the perfusate lactate plus pyruvate from [UJ4C]glucose was increased approximately fourfold, while the rate of 14C02 production was decreased 70% (Table 3). There was a TABLE
isolated
1. Lactate perfused
and pyruvate rat lung Lactate
Results are means tion. * Calculated Significantly different 0.05; $P < 0.001.
Pyruvate per
g dry
L/P*
Produced wt
29.7 * 2.1
3.5 + 0.3
8.7 f. 0.6
64.0 + 2.8$
4.2 f 0.2
15.7 t 0.6$
137.0 f 7.0$
2.8 * 0.2-l
52.9 + 4.9$
2. Glucose utilization determined of 14C and 3H in products of metabolism perfusion with [U- “C,3-3H]glucose [U-14ClGlucose
Utilized pmollh
by recovery during [3-3HlGlucose
per
g dry
3. Recovery
29.8 t 2.2
46.0 of: 5.1*
40.1 + 3.7
74.7 k 6.9-l
72.5 _+ 4.7-f
co2 39.2 & 1.3 (22.2%) 36.7 k 2.6 (13.5%) 11.6 + O.&i(2.6%)
q
Hypoxia
a
co
Fatty Acids
of glucose Lactate
metabolism plus
Deacylated Polysaccharide Fraction Fraction FIG. 4. Recovery of 14C in fatty acid and deacylated fractions of tissue lipids and polysaccharide fraction of ethanol-insoluble material isolated from lungs perfused with [UJ4C]glucose. Control and hypoxic lungs were perfused for 120 min, carbon monoxide lungs for 100 min, and results were represented as uptake of 14C per hour of perfusion. Results are means +SEM.
after perfusion
Pyruvate
Tissue wtoms
Control mew Hypoxia (N&O,) Carbon monoxide (CO:CO,)
Control
Utilized
29.6 -t 1.7
of 14C in products
0
wt
Results are means * SE of four lungs perfused under each condition. Control and hypoxic lungs were perfused for 120 min. Carbon monoxide perfusions were terminated after 100 min. Comparison with control significantly different: * (P < 0.025); UP < 0.001). TABLE
Characteristics of the preparation. Evaluation of the isolated perfused lung preparation suggests that it is suitable for metabolic studies. During the 2-h experimental period, the perfusion and ventilation pressures at constant perfusion rate and tidal volume remained unchanged, indicating that pulmonary vascular resistance and lung compliance were unaltered. Ultrastruc-
+ SE for 12 lungs perfused under each condias ratio of lactate to pyruvate production. from control by independent t test: tp
