Energetic response of coronary endothelial
cells to hypoxia
S. MERTENS, T. NOLL, R. SPAHR, A. KRUTZFELDT,AND H. M. PIPER Institut fiir Physiologie I, Universitkt Diisseldorf, D-4000 Diisseldorf, Federal Republic of Germany
MERTENS,~., T. NOLL, R. SPAHR,A. KR~TZFELDT,AND H. M. PIPER. Energetic response of coronary endothelial cells to hypoxia. Am. J. Physiol. 258 (Heart Circ. Physiol. 27): H689H694, 1990.-The response of endothelial energy metabolism to oxygen supply was studied in cultured coronary endothelial cells from the rat at defined PO:! levels between 0.1 and 100 Torr. In the presence of glucose (5 mM), endothelial respiration (4 nmol OZ.min-l l mg protein-‘) was independent of the exterior Pa > 3 Torr; oxygen consumption was half maximal at 0.8 Torr. At 100 Torr, lactate production was 26 nmol min-’ . mg protein-l; the decrease of the PO* to 0.1 Torr resulted in a %&fold increase in lactate production. The contents of ATP, ADP, and AMP were 21,4, and 2 nmol/mg protein, respectively; they remained constant for 2.5-h incubations at Po2 levels between 0.1 and 100 Torr. In the presence of palmitate (100 PM) plus glutamine (0.5 mM), oxygen consumption was 8 nmol . min-l . mg protein-l at Pop levels >3 Torr, and the halfmaximal rate was again observed at 0.8 Torr. Lactate production was negligible. At PO:! levels >3 Torr, the cells remained well energized. Below 3 Torr, however, the adenine nucleotide contents rapidly declined. These results demonstrate that the oxygen demand of coronary endothelial cells is low compared with the beating myocardium. In the presence of glucose, aerobic glycolysis is pronounced and the Pasteur effect small. In severe hypoxia (PO* < 0.1 Torr) the energetic state remained stable. In the absence of glucose, the energetic state of coronary endothelial cells is sensitive to the exterior Pop c 3 Torr, declining concomitantly with the decrease in respiration.
stimulate glycolytic energy production, and 2) in the presence of palmitate and glutamine to provide substrates that serve only oxidative energy production. A special technique was applied [“oxystat” (12)] that permitted continuous incubations of coronary endothelial cells at defined POT levels and the simultaneous monitoring of their oxygen consumption. The results demonstrate that the respiration of coronary endothelial cells is not affected by hypoxia unless the exterior PO:! drops to 0.05, n = 5)
*The addition
of 5 mM glucose to the medium
that
Pcells}
where [O&, is the O2 concentration of the reservoir equilibrated with calibrated OZ-NZ mixtures, [O,]i is the oxygen concentration in the incubation chamber, V is the volume per unit time being added into the cell suspension, Vi is the volume of the incubation chamber, and Pcellsis the protein concentration of the cell suspension. [O&, and [ O,]i were calculated according to Forster and Gnaiger (6). Analytical methods. For the analysis of acid soluble metabolites, samples of the cell suspension were taken up directly into 1.2 M HC104. The neutralized extracts were analyzed for lactate (according to Ref. 7) and for ATP, ADP, and AMP (according to Ref. 9). Samples of the effluent were also analyzed for lactate. In all samples withdrawn from the cell suspension, protein was determined, using bovine serum albumin as standard (11). Cell numbers were determined by counting trypsinized cells in a Neubauer chamber. Unless stated otherwise. all data are given as means t
Palmitate
0
+ Glutamine
ATP
i
i Time
6 ”
( h)
FIG. 2. Experiment on substrate effects on oxygen consumption, lactate formation, and adenine nucleotide (AN) concentrations of coronary endothelial cells. Cells were incubated at a constant PoQ of 10 Torr in modified Tyrode solution to which 1) 100 PM palmitate plus 0.5 mM glutamine and 2) 5 mM glucose were added as indicated.
Downloaded from www.physiology.org/journal/ajpheart by ${individualUser.givenNames} ${individualUser.surname} (130.070.008.131) on January 20, 2019.
ENDOTHELIAL
ENERGY
METABOLISM
contained palmitate plus glutamine caused a 50 t 5% decrease in the oxygen consumption rate and an increase of lactate production to 25.4 t 2.3 nmol lactate. min-’ . mg protein-l. The contents of the adenine nucleotides were not affected. Oxygen consumption, lactate production, and adenine nucleotide contents were the same as when glucose was supplied alone in the absence of palmitate plus glutamine (for all data: P > 0.05, n = 5). Oxygen dependence of energy metabolism inpresencz
of
glucose. Incubated at a PO:! of 10 Torr in the presence of 5 mM glucose, endothelial cells consumed oxygen at a rate of 4 nmol. min-’ mg protein-’ (Fig. 3). To vary the POT to >3 Torr left the oxygen consumption unchanged (Fig. 4). Below 3 Torr, the rate of oxygen consumption l
I O2
Uptake
H691
IN HYPOXIA
declined. It was half maximal at a Paz of 0.8 Torr. The depression of oxygen consumption at low POT levels was reversible; after a 2.5-h incubation 0.05, n = 5) ‘Oxygen dependence of energy metabolism in presence of
6 Lactate 3
Q) t;; 3 w
2 Time
3
4
n ”
(h)
3. Experiment on effect of hypoxia on oxygen uptake, lactate production, and adenine nucleotide (AN) content of coronary endothelial cells in presence of 5 mM glucose. Cells were incubated at PO:! levels of 10, 1, and 0.1 Torr in modified Tyrode solution. FIG.
palmitate plus glutamine. The importance of respiratory energy production was studied in experiments in which exogenous glucose was replaced by palmitate plus glutamine. With these substrates, oxygen consumption was 8 nmol . min-l gmg protein-l when incubations were performed at POT levels >3 Torr (Figs. 6 and 7). Thus the oxygen consumption was twice as high as in media containing glucose alone (Figs. 3 and 4) or glucose together with palmitate and glutamine (Fig. 2). The oxygen affinity of endothelial oxygen consumption was the same with palmitate plus glutamine as with glucose alone, i.e., the half-maximal rate of oxygen consumption was approached at 0.8 Torr (Fig. 7). In the presence of palmitate plus glutamine, endothelial lactate production was cl
+
Glucose
1
0
1
2
3 10 30 PO2 (torr)
50
70
90
FIG. 4. Rate of oxygen uptake of coronary endothelial cells as a function of Pop in presence of 5 mM glucose. Cells were incubated at various PO* levels in modified Tyrode solution. Symbols represent 3 exneriments using senarate culture nrenarations.
10
20
100
PO2 (torr) 5. Rate of lactate formation by coronary endothelial cells as a function of Paz in presence of 5 mM glucose. Cells were incubated at Pop levels of 100, 10, 1, and 0.1 Torr in modified Tyrode solution. Values at 1 and 0.1 Torr are significantly different from 100 Torr value (P < 0.01) (t SE. n = 5). FIG.
Downloaded from www.physiology.org/journal/ajpheart by ${individualUser.givenNames} ${individualUser.surname} (130.070.008.131) on January 20, 2019.
H692
ENDOTHELIAL
ENERGY
METABOLISM
IN HYPOXIA 25
10
t
h L L
0
.
I Lactate I)
l
ATP
=
l
PO2f ton) ATP
8. ATP content of coronary endothelial cells as a function of in presence of palmitate plus glutamine. Cells were incubated for 30 min at PO* levels of 100, 10, 1, and 0.1 Torr in modified Tyrode solution with 100 PM palmitate and 0.5 mM glutamine. Values at 1 and 0.1 Torr are significantly different from 100 Torr value (P < 0.005) (+ SE, n = 5). FIG.
Pop
0
1
2 Time
3 (h)
6. Experiment on effect of hypoxia on oxygen uptake, lactate production, and adenine nucleotide content of coronary endothelial cells in presence of palmitate plus glutamine. Cells were incubated at POT levels of 10, 1, and 0.1 Torr in modified Tyrode solution with 100 PM palmitate and 0.5 mM glutamine. FIG.
the Pop to 0.1 Torr resulted in a further decrease of the ATP content. After 2 h at POT levels 3 Torr. Below 3 Torr, the oxygen consumption gradually declined and was half maximal at 0.8 Torr. In isolated mitochondria, a POT of 0.2 Torr represents the & for oxygen consumption by cytochrome-c oxidase (2, 4). Whole cells generally have a reduced apparent affinity to oxygen due to intracellular gradients of oxygen. Therefore, the POT of the half-maximal oxygen consumption by coronary endothelial cells, i.e., 0.8 Torr, indicates that a gradient exists within the endothelial cell of 0.6 Torr toward the inner mitochondrial membrane. This value closely accords with estimates of intracellular oxygen gradients in other cell types (3). Interestingly, the sensitivity of individual endothelial cells to oxygen is comparable to that of cardiomyocytes (16). Oxygen tensions of 1 Torr and below are extremely low, since they are two orders of magnitude below the normal arterial oxygen tension. In the physiological range of conditions, such oxygen tensions are unlikely to occur in vivo in the coronary bed. But they occur in ischemic myocardium as indicated by high rates of lactate production by the cardiomyocytes (16). The aerobic production of lactate in the presence of glucose is unaffected by a reduction of medium Paz from 100 to 10 Torr. This shows that the high rates of lactate production are not because of partial hypoxia in the system. The maximal 2.2fold increase in lactate production, when Po2 was decreased to 0.1 Torr, shows that the Pasteur effect is small in coronary endothelial cells compared with the Pasteur effect in the cardiac muscle cell (13). This finding is consistent with the pronounced Crabtree effect under aerobic conditions (unpublished observations). Energetic response to hypoxia. In deep hypoxia (Po2 5 1 Torr), the adenine nucleotide contents of coronary endothelial cells remained stable when exogenous glucose was supplied. This could be because the increased glycolytic energy production meets the previous energy demand or because the energy demand becomes sufficiently reduced. From the lactate production at 0.1 Torr, a rate of glycolytic energy production equivalent to 55 nmol ATP min-’ . mg protein-’ is calculated. This corresponds closely to the aerobic energy production of 47 nmol ATP l
l
l
IN
H693
HYPOXIA
miril mg protein-l calculated above. This equivalence shows that enhanced glycolytic energy production is able to fully compensate for the lack of respiratory ATP generation and meet an energy demand as high as under aerobic conditions. The importance of glucose for hypoxic energy production in coronary endothelial cells was tested in experiments in which glucose was replaced by palmitate plus glutamine as exogenous substrates, both of which can be used only oxidatively. A lactate production ~5% of that in the presence of glucose indicates that glycolytic energy production from the mobilization of glycogen is indeed negligible. Under these conditions, the cellular ATP contents decreased at Paz levels
cells to hypoxia
S. MERTENS, T. NOLL, R. SPAHR, A. KRUTZFELDT,AND H. M. PIPER Institut fiir Physiologie I, Universitkt Diisseldorf, D-4000 Diisseldorf, Federal Republic of Germany
MERTENS,~., T. NOLL, R. SPAHR,A. KR~TZFELDT,AND H. M. PIPER. Energetic response of coronary endothelial cells to hypoxia. Am. J. Physiol. 258 (Heart Circ. Physiol. 27): H689H694, 1990.-The response of endothelial energy metabolism to oxygen supply was studied in cultured coronary endothelial cells from the rat at defined PO:! levels between 0.1 and 100 Torr. In the presence of glucose (5 mM), endothelial respiration (4 nmol OZ.min-l l mg protein-‘) was independent of the exterior Pa > 3 Torr; oxygen consumption was half maximal at 0.8 Torr. At 100 Torr, lactate production was 26 nmol min-’ . mg protein-l; the decrease of the PO* to 0.1 Torr resulted in a %&fold increase in lactate production. The contents of ATP, ADP, and AMP were 21,4, and 2 nmol/mg protein, respectively; they remained constant for 2.5-h incubations at Po2 levels between 0.1 and 100 Torr. In the presence of palmitate (100 PM) plus glutamine (0.5 mM), oxygen consumption was 8 nmol . min-l . mg protein-l at Pop levels >3 Torr, and the halfmaximal rate was again observed at 0.8 Torr. Lactate production was negligible. At PO:! levels >3 Torr, the cells remained well energized. Below 3 Torr, however, the adenine nucleotide contents rapidly declined. These results demonstrate that the oxygen demand of coronary endothelial cells is low compared with the beating myocardium. In the presence of glucose, aerobic glycolysis is pronounced and the Pasteur effect small. In severe hypoxia (PO* < 0.1 Torr) the energetic state remained stable. In the absence of glucose, the energetic state of coronary endothelial cells is sensitive to the exterior Pop c 3 Torr, declining concomitantly with the decrease in respiration.
stimulate glycolytic energy production, and 2) in the presence of palmitate and glutamine to provide substrates that serve only oxidative energy production. A special technique was applied [“oxystat” (12)] that permitted continuous incubations of coronary endothelial cells at defined POT levels and the simultaneous monitoring of their oxygen consumption. The results demonstrate that the respiration of coronary endothelial cells is not affected by hypoxia unless the exterior PO:! drops to 0.05, n = 5)
*The addition
of 5 mM glucose to the medium
that
Pcells}
where [O&, is the O2 concentration of the reservoir equilibrated with calibrated OZ-NZ mixtures, [O,]i is the oxygen concentration in the incubation chamber, V is the volume per unit time being added into the cell suspension, Vi is the volume of the incubation chamber, and Pcellsis the protein concentration of the cell suspension. [O&, and [ O,]i were calculated according to Forster and Gnaiger (6). Analytical methods. For the analysis of acid soluble metabolites, samples of the cell suspension were taken up directly into 1.2 M HC104. The neutralized extracts were analyzed for lactate (according to Ref. 7) and for ATP, ADP, and AMP (according to Ref. 9). Samples of the effluent were also analyzed for lactate. In all samples withdrawn from the cell suspension, protein was determined, using bovine serum albumin as standard (11). Cell numbers were determined by counting trypsinized cells in a Neubauer chamber. Unless stated otherwise. all data are given as means t
Palmitate
0
+ Glutamine
ATP
i
i Time
6 ”
( h)
FIG. 2. Experiment on substrate effects on oxygen consumption, lactate formation, and adenine nucleotide (AN) concentrations of coronary endothelial cells. Cells were incubated at a constant PoQ of 10 Torr in modified Tyrode solution to which 1) 100 PM palmitate plus 0.5 mM glutamine and 2) 5 mM glucose were added as indicated.
Downloaded from www.physiology.org/journal/ajpheart by ${individualUser.givenNames} ${individualUser.surname} (130.070.008.131) on January 20, 2019.
ENDOTHELIAL
ENERGY
METABOLISM
contained palmitate plus glutamine caused a 50 t 5% decrease in the oxygen consumption rate and an increase of lactate production to 25.4 t 2.3 nmol lactate. min-’ . mg protein-l. The contents of the adenine nucleotides were not affected. Oxygen consumption, lactate production, and adenine nucleotide contents were the same as when glucose was supplied alone in the absence of palmitate plus glutamine (for all data: P > 0.05, n = 5). Oxygen dependence of energy metabolism inpresencz
of
glucose. Incubated at a PO:! of 10 Torr in the presence of 5 mM glucose, endothelial cells consumed oxygen at a rate of 4 nmol. min-’ mg protein-’ (Fig. 3). To vary the POT to >3 Torr left the oxygen consumption unchanged (Fig. 4). Below 3 Torr, the rate of oxygen consumption l
I O2
Uptake
H691
IN HYPOXIA
declined. It was half maximal at a Paz of 0.8 Torr. The depression of oxygen consumption at low POT levels was reversible; after a 2.5-h incubation 0.05, n = 5) ‘Oxygen dependence of energy metabolism in presence of
6 Lactate 3
Q) t;; 3 w
2 Time
3
4
n ”
(h)
3. Experiment on effect of hypoxia on oxygen uptake, lactate production, and adenine nucleotide (AN) content of coronary endothelial cells in presence of 5 mM glucose. Cells were incubated at PO:! levels of 10, 1, and 0.1 Torr in modified Tyrode solution. FIG.
palmitate plus glutamine. The importance of respiratory energy production was studied in experiments in which exogenous glucose was replaced by palmitate plus glutamine. With these substrates, oxygen consumption was 8 nmol . min-l gmg protein-l when incubations were performed at POT levels >3 Torr (Figs. 6 and 7). Thus the oxygen consumption was twice as high as in media containing glucose alone (Figs. 3 and 4) or glucose together with palmitate and glutamine (Fig. 2). The oxygen affinity of endothelial oxygen consumption was the same with palmitate plus glutamine as with glucose alone, i.e., the half-maximal rate of oxygen consumption was approached at 0.8 Torr (Fig. 7). In the presence of palmitate plus glutamine, endothelial lactate production was cl
+
Glucose
1
0
1
2
3 10 30 PO2 (torr)
50
70
90
FIG. 4. Rate of oxygen uptake of coronary endothelial cells as a function of Pop in presence of 5 mM glucose. Cells were incubated at various PO* levels in modified Tyrode solution. Symbols represent 3 exneriments using senarate culture nrenarations.
10
20
100
PO2 (torr) 5. Rate of lactate formation by coronary endothelial cells as a function of Paz in presence of 5 mM glucose. Cells were incubated at Pop levels of 100, 10, 1, and 0.1 Torr in modified Tyrode solution. Values at 1 and 0.1 Torr are significantly different from 100 Torr value (P < 0.01) (t SE. n = 5). FIG.
Downloaded from www.physiology.org/journal/ajpheart by ${individualUser.givenNames} ${individualUser.surname} (130.070.008.131) on January 20, 2019.
H692
ENDOTHELIAL
ENERGY
METABOLISM
IN HYPOXIA 25
10
t
h L L
0
.
I Lactate I)
l
ATP
=
l
PO2f ton) ATP
8. ATP content of coronary endothelial cells as a function of in presence of palmitate plus glutamine. Cells were incubated for 30 min at PO* levels of 100, 10, 1, and 0.1 Torr in modified Tyrode solution with 100 PM palmitate and 0.5 mM glutamine. Values at 1 and 0.1 Torr are significantly different from 100 Torr value (P < 0.005) (+ SE, n = 5). FIG.
Pop
0
1
2 Time
3 (h)
6. Experiment on effect of hypoxia on oxygen uptake, lactate production, and adenine nucleotide content of coronary endothelial cells in presence of palmitate plus glutamine. Cells were incubated at POT levels of 10, 1, and 0.1 Torr in modified Tyrode solution with 100 PM palmitate and 0.5 mM glutamine. FIG.
the Pop to 0.1 Torr resulted in a further decrease of the ATP content. After 2 h at POT levels 3 Torr. Below 3 Torr, the oxygen consumption gradually declined and was half maximal at 0.8 Torr. In isolated mitochondria, a POT of 0.2 Torr represents the & for oxygen consumption by cytochrome-c oxidase (2, 4). Whole cells generally have a reduced apparent affinity to oxygen due to intracellular gradients of oxygen. Therefore, the POT of the half-maximal oxygen consumption by coronary endothelial cells, i.e., 0.8 Torr, indicates that a gradient exists within the endothelial cell of 0.6 Torr toward the inner mitochondrial membrane. This value closely accords with estimates of intracellular oxygen gradients in other cell types (3). Interestingly, the sensitivity of individual endothelial cells to oxygen is comparable to that of cardiomyocytes (16). Oxygen tensions of 1 Torr and below are extremely low, since they are two orders of magnitude below the normal arterial oxygen tension. In the physiological range of conditions, such oxygen tensions are unlikely to occur in vivo in the coronary bed. But they occur in ischemic myocardium as indicated by high rates of lactate production by the cardiomyocytes (16). The aerobic production of lactate in the presence of glucose is unaffected by a reduction of medium Paz from 100 to 10 Torr. This shows that the high rates of lactate production are not because of partial hypoxia in the system. The maximal 2.2fold increase in lactate production, when Po2 was decreased to 0.1 Torr, shows that the Pasteur effect is small in coronary endothelial cells compared with the Pasteur effect in the cardiac muscle cell (13). This finding is consistent with the pronounced Crabtree effect under aerobic conditions (unpublished observations). Energetic response to hypoxia. In deep hypoxia (Po2 5 1 Torr), the adenine nucleotide contents of coronary endothelial cells remained stable when exogenous glucose was supplied. This could be because the increased glycolytic energy production meets the previous energy demand or because the energy demand becomes sufficiently reduced. From the lactate production at 0.1 Torr, a rate of glycolytic energy production equivalent to 55 nmol ATP min-’ . mg protein-’ is calculated. This corresponds closely to the aerobic energy production of 47 nmol ATP l
l
l
IN
H693
HYPOXIA
miril mg protein-l calculated above. This equivalence shows that enhanced glycolytic energy production is able to fully compensate for the lack of respiratory ATP generation and meet an energy demand as high as under aerobic conditions. The importance of glucose for hypoxic energy production in coronary endothelial cells was tested in experiments in which glucose was replaced by palmitate plus glutamine as exogenous substrates, both of which can be used only oxidatively. A lactate production ~5% of that in the presence of glucose indicates that glycolytic energy production from the mobilization of glycogen is indeed negligible. Under these conditions, the cellular ATP contents decreased at Paz levels
