Intracellular NEWMAN Departme&
pH in hypoxic L. STEPHENS, of Physiology, G
EDWIN University
hypoxia
and glycolysis;
tracheal
smooth muscle
muscle
A. KROEGER, AND WILLIAM of Manitoba, Winnipeg, Canada
NEWMAN L., EDWIN A. KROEGER, AND WILLIAM Intracellular pH in hypoxic smooth muscle. Am. J. Physiol. 232(3): E330-E335, 1977 or Am. J. Physiol.: Endocrinol. Metab. Gastrointest. Physiol. l(3): E330-E335, 1977. Hypoxia impairs contractility in canine tracheal smooth muscle (TSM). This is attributed to intracellular lactacidosis. The present studies were undertaken t/o confirm this. Lactate was found to be significantly increased in hypoxic TSM (65.36 t 7.37 mg/lOO g wet tissue), compared to normoxic (29.83 t 5.05). Intracellular pH (pHi) was, however, significantly increased in hypoxic active TSM to 7.71 t 0.05 as compared to 7.30 * 0.03 in normoxic active muscle. pHi of resting normoxic muscle (7.20 t_ 0.04) was statistically not different from that of resting hypoxic muscle. The pHi’s of resting normoxic and active hypoxic muscles were significantly different. These results show that under in vitro, hypoxic conditions: 1) an increase in glycolysis in TSM is indicated by the increased lactate production, 2) there is a surprising, concomitant rise in pHi rather than a decrease as previously expected, and3) it is mechanical activity of the muscle which leads to this paradoxical result, inasmuch as pHi is unaltered in the resting hypoxic muscle. STEPHENS,
LOH.
smooth
LOH R3E OW3
the bath generally resulted in about a 30% impairment of contractile function (18, 19). Most of our studies of hypoxia have thus been conducted at this POz* From the above evidence, it is therefore evident that under hypoxic conditions, TSM partly compensates for the deficiency of energy supply by an increase in the rate of glycolysis. If such is the case, then we should expect a rise in the intracellular levels of the end products of glycolysis, namely lactate and/or pyruvate. The extent of the increase in intracellular concentration would depend on the membrane permeability to these end products. An increase in the levels of lactate and/or pyruvate may lower the p& of TSM. Because the enzymes in the glycolytic pathway have alkaline optimum pH’s, a lowering of pfi would be unfavorable to glycolysis and to the hypothesis that in hypoxia the rate of glycolysis is increased. Therefore, it seemed important to measure the levels of lactate and pyruvate and the pHi of TSM under hypoxic and normoxic conditions in order to differentitate between these possibilities. METHODS
MECHANICAL AND ELECTROPHYSIOLOGICAL properTrachealis muscle was obtained from the cervical traties of tracheal smooth muscle (TSM) have been studied cheae of mongrel dogs anesthetized with 30 mg/kg penin some detail under both normoxic and hypoxic condi- tobarbital. Muscle strips were dissected and equilitions (U-20, 21). A significant shift of the force-velocity brated in a lo-ml mammalian Krebs-Ringer bicarbonrelationships to the left has been observed under hy- ate solution of the following composition, in millimoles poxic conditions indicating a decrease in power producper liter: NaCl, 115; NaHCO:,, 25; NaH,PO,, 1.38; KC1, tion. This shift was found to be inversely proportional to 2.51; MgSO,*7 H20, 2.46; CaCl,, 1.91; dextrose, 5.56. both oxygen tension and dextrose concentration, with The solution was equilibrated w ,ith an OS-C0, gas mixthe sensitivity of the muscle to hypoxia increasing as ture so as to achieve a measured bath Po, of 600 mmHg, dextose concentration was progressively lowered. At Pco., of 45 mmHg, and pH of 7.40 at a temperature of $7 normal dextrose concentration, a decrease in PO, to less t 1°C. The bath PO, could be rapidly decreased to 50 mmHg while maintaining pH and Pco2 constant by than 10 mmHg resulted in an impairment of mechanical function to a level which was less than 50% of normal, equilibrating with a N,-CO,-0, gas mixture. whereas 10% of normal concentration of dextrose, with Measurement of effects of hypoxia on mechanical the same degree of hypoxia, resulted in a 97% impairproperties of TSM. The muscle was attached to an appament. Furthermore, with a substrate-free medium at ratus which is shown schematically in Fig. 1. The lower high PO,!, there was no reduction in mechanical function end of the trachealis muscle strip was attached firmly for 1 h (19). by 3-Obraided surgical silk to a hook at the bottom of the Further work (11) showed that glycogenolysis in TSM bath. The upper end was attached by a short length of is significantly increased under hypoxic conditions, and surgical silk to a Grass FT-03 transducer. The-force it has been reported that anaerobic glycolysis can sup- transducer was mounted on a rack-and-pinion enabling ply the energy requirement of a 70% maximal contracthe muscle to be stretched to any given length, at which tion of rabbit aortic smooth muscle (5). the muscle could be held isometrically. The output of Since a PO, of 0.05). Intracellular pH (n = 11). The intracellular pH’s of the muscles under the various incubation conditions are shown in Fig. 4. The unstimulated control experiments were conducted under resting n.ormoxic conditions, without electrical stimulation. The mean pH’s and standard errors of the muscle under various conditions are as follows: normoxic resting, 7.20 t .04; hypoxic resting, 7.19 & 0.04; normoxic active, 7.30 t 0.03; and hypoxic active, 7.71 t 0.05. The pHi was significantly higher under contracting hypoxic conditions (P < 0.05) as compared to normoxic contracting conditions, but there was no significant difference between the pHi’s of resting normoxic and resting hypoxic muscles, as assessedby the Student t test. There were 11 paired experiments for the hypoxia and normoxic muscles and 6 experiments for the unstimulated normoxic
Downloaded from www.physiology.org/journal/ajpendo by ${individualUser.givenNames} ${individualUser.surname} (150.216.068.200) on January 1, 2019.
SMOOTH
MUSCLE
LACTATE,
PYRUVATE,
AND
pHi
7.80
7.6 -
7.2 7.0c HA
FIG. moxie, smooth active; resting.
NA
NR
HR
4. Intracellular pH’s in stimulated active hypoxic and norand unstimulated resting normoxic and hypoxic tracheal muscles. Values indicate means with SE bars. HA, hypoxic NA, normoxic active; NR, normoxic resting; HR, hypoxic
controls, and the average weight of the muscle strips used in these experiments was 28.29 mg. Binding studies. Inasmuch as binding of DMO to cellular proteins would invalidate the use of this method in measuring pHi, the effect of adding unlabeled DMO to labeled DMO on determinations of p& was studied. The concentration of unlabeled DMO was 100 times that of the labeled DMO, and the active muscles were rendered hypoxic because it is in that condition we found the highest pH’s. Four experiments were carried out. Using only labeled DMO, a pH of 7.64 t .Ol was obtained. When unlabeled DMO was added, pH was 7.65 t .02. There was no statistically significant difference between the groups. No evidence of binding was detected when this study was conducted on normoxic resting tiuscles. Time studies. In comparing PHi’s under differing experimental conditions, it is important that the measurements be made at steady state. Data were obtained from resting muscles incubated for 1, 2, and 3 h, respectively. Mean values of 7.21, 7.25, and 7.22 were obtained with no statistically significant differences between these values. Hence, a steady state seems to have been established within 1 hr. Similar experiments conducted under normoxic contracting and hypoxic contracting conditions showed a steady state had developed within the same period of time. DISCUSSION
Effect of hypoxia on lactate and pyruvate contents of trachealis. The levels of lactate found in TSM are comparable to those reported for feline skeletal and bovine vascular smooth muscles (9, 13, 14) both in magnitude and direction of change under hypoxia. The total lactate production (tissue + medium) under hypoxic conditions is about 3 times that under normoxic conditions. This agrees with the hypothesis tested that in hypoxia, glycolysis in TSM is increased. The level of pyruvate in TSM under hypoxic conditions is slightly lowered as compared to normoxic TSM. This is in contradiction to findings in the gastrocnemius of the cat (9). But it was speculated that in this muscle the increase in pyruvate level was due to the
E333
loose-coupling of the oxidation of glyceraldehyde-3acid, to the reducphosphate to 1,3=diphosphoglyceric tion bf pyruvate to-lactate in the glycolytic pathway. Thus, the difference can be accounted for by 1) an absence of such loose coupling in TSM so that any amount of lactate could be formed without any net increase in the quantity of pyruvate present, and 2) a difference in the-activities of lactate dehydrogenases in the two muscles. The isozymes (H and M forms) of skeletal and smooth muscle lactic dehydrogenases tend to differ substantially (10) which could provide regulation of balance of the pyruvate-lactate ratio. Effect of hypoxia on intracellularpIT. The pI& of TSM was found to increase under hypoxic conditions rather than decrease as previously expected (19, 21). Two factors can most likely contribute to a falsely high pH, namely, the entry of inulin into intracellular space (15) and the binding of DMO to cellular proteins (2-4, 16). The mean TSM extracellular space (ECS) as determined by the inulin method in our experiments is 54% as compared to 33% in guinea pig taenia coli as determined by the Co-EDTA method (1). Inasmuch as the total TSM tissue water is only 78% fresh weight, a 54% ECS would strongly support a recent report that inulin enters intracellular space (15). The falsely high ECS, when substituted into the Waddell and Butler equation, will only increase the p& by approximately 1% as compared to an ECS of 33%. Consequently, the most important factor has to be the binding of DMO to cellular proteins. This can be elucidated by priming the tissue with unlabeled DMO and observing its effect on the scintillation counting of labeled DMO, but this remains to be done. Waddell and Butler (25) showed that the whole of the DMO present in homogenates of dog skeletal muscle was freely dialyzable and consequently not bound in any way to the tissue proteins. Izutsu (8) had adduced similar evidence for bullfrog toe striated muscle. Our own data confirm that significant binding does not occur in tracheal smooth muscle. The above discussion notwithstanding, because the same factors operate in both hypoxic and normoxic experiments, the observation that pa tends to increase in hypoxia over normoxia is still valid. This is strengthened by our finding that no significant binding was found in either nor-moxie resting or hypoxic muscles. Ifthe hypoxic insult does not create a leakage to DMO in the cell membranes in TSM as is the case in guinea taenia coli to sorbitol in anaerobic conditions (26), then the alkalosis in hypoxia would not be artifactual. The fact that the resting membrane potential of the TSM is unchanged under hypoxia (19) suggests that membrane damage does not exist. Finally, it may be argued that because of limitations in the sensitivity of the DMO technique as applied to tracheal smooth muscle we are unable to demonstrate any decrease in pH during hypoxia. However, we have shown (23) in studies of hypercapnic acidosis in TSM -that the DMO technique as used by us can indeed register decreases in PH. The following speculative explanations for the alkalosis are given: I) hydrogen ions are actively pumped out of the cells (7), and in hypoxia the activity of the
Downloaded from www.physiology.org/journal/ajpendo by ${individualUser.givenNames} ${individualUser.surname} (150.216.068.200) on January 1, 2019.
E334
STEPHENS,
hydrogen pump is enhanced. In this context, it should be pointed out that the size of the bath medium in these experiments was more than 300 times the size of the muscle strips used. This condition is unphysiological because the bath medium may constitute a reservoir of effectively infinite size into which H+, lactate, and pyruvate can be dumped. The calculated intracellular-extracellular lactate gradient in our hypoxia experiments is about 8O:l. 2) Previous work in this laboratory suggests that AMP is broken down in TSM under hypoxic conditions (11). The significance of the breakdown of AMP in muscle in the face of a strong ATP drain was discussed in detail by J. M. Lowenstein (12). The reaction involves adenylate deaminase and results in the production of IMP and ammonia as given below AMP
+ H,O
adenylate
dearninase
> IMP + NH,,
Alkalosis in TSM under hypoxic conditions could be attributed to the ammonia released in the above reaction. This line of reasoning is entirely speculative as we have no data at this time to support this hypothesis. 3) This laboratory has also reported a reduction of creatine phosphate levels in TSM in hypoxia to 16.0% of normals (11). The breakdown of CP is mediated by the enzyme creatine kinase and results in the formation of ATP and creatine, viz. creatine Creatine
phosphate has a high
+ ADP pK (-
--____) creatine
+ ATP 8.0) and hence is usually kinase
creatine
KROEGER,
AND
LOH
highly protonated in the normal range of pHi. Thus, the creatine released in TSM in hypoxia may contribute to the rise in pH,, at least transiently before glycolytic activity increases. In conclusion, it appears that in TSM under in vitro, hypoxic conditions which produce a reversible impairment of mechanical function, glycolysis is increased. However, instead of the expected acidosis an intracellular alkalosis develops. In this context it is to be noted that intracellular alkalosis would facilitate an increase in glycolysis, as the enzymes in the glycolytic pathway have alkaline optimal pH’s. Thus, though the mechanism of the production of the alkalosis is not understood at this time, its beneficial role is evident. To what extent this mechanism is operative in vivo is difficult to say, but it can be speculated that under in vivo conditions, intracellular alkalosis could be only a transient response to hypoxia because unlike in vitro experiments, the volume of the physiological extracellular fluid is less than that of the intracellular fluid. Hence, the extracellular lactate level will rise much more quickly, providing a substantial challenge to the cell in expelling the lactate and hydrogen ions. The invaluable technical assistance of Mr. Richard Mitchell is gratefully acknowledged. This investigation was supported by grants from the Medical Research Council of Canada, the Canadian Heart Foundation, and the Canadian Tuberculosis and Respiratory Disease Association.
Received
for publication
5 January
1976.
REFERENCES ,4. F., AND A. W. JONES. Distribution and kinetics of in smooth muscles and its use as an extracellular J. Physiol., London 200: 387-401, 1969. BUTLER, T. C., W. J. WADDELL, AND D. T. POOL. Intracellular pH based on the distribution of weak electrolytes. Federation Proc. 26: 1327, 1967. CAMPION, D. S., N. W. CARTER, F. C. RECTOR, JR., AND D. W. SELDIN. Intracellular distribution of I%-DMO. Clin. Res. 15: 353, 1967. CARTER, N. W., F. C. RECTOR, JR., D. S. CAMPION, AND D. W. SELDIN. Measurement of intracellular pH of skeletal muscle with pH-sensitive glass microelectrodes. J. Clin. Invest. 46: 920, 1967. FURCHGOT, R. F. Discussion on the biochemistry of the arterial wall. Bibliotheca Cardiol. 15: 20-23, 1964. HERBERG, R. J. Phosphorescence in liquid scintillation counting of proteins. Scierzce 128: 199-200, 1958. IRVINE, R. 0. H., S. J. SAUNDERS, M. D. MILNE, AND M. A. CRAWFORD. Gradients of potassium and hydrogen ion in potassium-deficient voluntary muscle. Clin. Sci. 20: 1-18, 1960. IZUTSU, K. T. Intracellular pH, H ion flux and H ion permeability coefficient in bullfrog toe muscle. J. PhysioZ., London 221: 1527, 1972. JACOB, S., AND J. H. MORTON. Lactic and pyruvic acid relations in contracting mammalian muscle. Am. J. Physiol. 186: 221-223, 1956. KAPLAN, N. O., AND J. EVERSE. Regulatory characteristics of In: Advances in Enzyme Regulation, lactate dehydrogenases. edited by G. Weber, New York: Pergamon, 1972, vol. 10, p. 323336. KROEGER, E., AND N. L. STEPHENS. Effect of hypoxia on energy and calcium metabolism in airway smooth muscle. Am. J. Physid. 220: 1199-1204, 1971.
1. BRADING,
Co-EDTA marker.
2.
3.
4.
5. 6. 7.
8.
9.
10.
11.
12. LOWENSTEIN, J. M. Ammonia production in muscle and other tissues: the purine nucleotide cycle. Physiol. Reu. 52: 382-414, 1972. 13. LUNDHOLM, L. L., AND E. MOHME-LUNDHOLM. Energetics of isotonic and isometric contraction in isolated vascular smooth muscle under anaerobic conditions. Acta Physiol. Stand. 64: 275-282, 1965. 14. LUNDHOLM, L. L., AND E. MOHME-LUNDHOLM. Contraction and glycogenolysis of smooth muscle. Acta PhysioZ. Stand. 57: 125129, 1963. 15. MCIVER, D. J. L., AND A. D. C. MCKNIGHT. Extracellular space in some isolated tissues. J. Physiol., London 239: 31-49, 1974. 16. MILLER, R. B., I. TYSON, AND A. S. RELMAN. pH of isolated resting skeletal muscle and its relation to potassium content. Am. J. Physiol. 204: 1048-1054, 1963. 17. MILNE, M. D., B. H. SCRIBNER, AND M. A. CRAWFORD. Non-ionic diffusion and the excretion of weak acids and bases. Am. J. Med. 24: 709-729, 1958. 18. STEPHENS, N. L., AND B. S. CHIU. Mechanical properties of tracheal smooth muscle and effect of 02, CO, and pH. Am. J. Physiol. 219: 1001-1008, 1970. 19. STEPHENS, N. L., AND E. KROEGER. Effect of hypoxia on airways smooth muscle mechanics and electrophysiology. J. Appl. Physiol. 28: 630-635, 1970. 20. STEPHENS, N. L., E. KROEGER, AND J. A. MEHTA. Force-velocity characteristics of respiratory airway smooth muscle. J. Appl. Physiol. 26: 685-692, 1969. 21. STEPHENS, N. L., J. L. MEYERS, AND R. M. CHERNIACK. Oxygen, carbon dioxide H+ ion, and bronchial length-tension relationship. J. AppZ. Physiol. 25: 1968. 22. STEPHENS, N. L., AND K. WROGEMANN. Oxidative phosphorylation in smooth muscle. Am. J. Physiol. 219: 1796-1801, 1970. 23. STEPHENS, N. L., AND R. W. MITCHELL. Effect of respiratory
Downloaded from www.physiology.org/journal/ajpendo by ${individualUser.givenNames} ${individualUser.surname} (150.216.068.200) on January 1, 2019.
SMOOTH
MUSCLE
LACTATE,
PYRUVATE,
AND
pHi
acidosis and activity on airway smooth muscle intracellular pH. Federation Proc. 35: 776, 1975. 24. VAUGHAN, M., D. STEINBERG, AND J. LOGAN. Liquid scintillation counting of Cl4 and H3-labelled amino acids and proteins. Science 126: 446-447, 1957. 25. WADDELL, W. J., AND T. C. BUTLER. Calculation of intracellular
E335 pH from the distribution of 5,5-dimethyl-2,4-oxazoldine dione (DMO). Application to skeletal muscle of the dog. J. Clin. Invest. 38: 720-729, 1959. 26. WUYTACK, F., AND R. CASTEELS. The energy-rich phosphates in smooth muscle of the guinea-pig taenia coli during metabolic depletion. Arch. Intern. Physiol. Biochem. 80: 820-845, 1972.
Downloaded from www.physiology.org/journal/ajpendo by ${individualUser.givenNames} ${individualUser.surname} (150.216.068.200) on January 1, 2019.
pH in hypoxic L. STEPHENS, of Physiology, G
EDWIN University
hypoxia
and glycolysis;
tracheal
smooth muscle
muscle
A. KROEGER, AND WILLIAM of Manitoba, Winnipeg, Canada
NEWMAN L., EDWIN A. KROEGER, AND WILLIAM Intracellular pH in hypoxic smooth muscle. Am. J. Physiol. 232(3): E330-E335, 1977 or Am. J. Physiol.: Endocrinol. Metab. Gastrointest. Physiol. l(3): E330-E335, 1977. Hypoxia impairs contractility in canine tracheal smooth muscle (TSM). This is attributed to intracellular lactacidosis. The present studies were undertaken t/o confirm this. Lactate was found to be significantly increased in hypoxic TSM (65.36 t 7.37 mg/lOO g wet tissue), compared to normoxic (29.83 t 5.05). Intracellular pH (pHi) was, however, significantly increased in hypoxic active TSM to 7.71 t 0.05 as compared to 7.30 * 0.03 in normoxic active muscle. pHi of resting normoxic muscle (7.20 t_ 0.04) was statistically not different from that of resting hypoxic muscle. The pHi’s of resting normoxic and active hypoxic muscles were significantly different. These results show that under in vitro, hypoxic conditions: 1) an increase in glycolysis in TSM is indicated by the increased lactate production, 2) there is a surprising, concomitant rise in pHi rather than a decrease as previously expected, and3) it is mechanical activity of the muscle which leads to this paradoxical result, inasmuch as pHi is unaltered in the resting hypoxic muscle. STEPHENS,
LOH.
smooth
LOH R3E OW3
the bath generally resulted in about a 30% impairment of contractile function (18, 19). Most of our studies of hypoxia have thus been conducted at this POz* From the above evidence, it is therefore evident that under hypoxic conditions, TSM partly compensates for the deficiency of energy supply by an increase in the rate of glycolysis. If such is the case, then we should expect a rise in the intracellular levels of the end products of glycolysis, namely lactate and/or pyruvate. The extent of the increase in intracellular concentration would depend on the membrane permeability to these end products. An increase in the levels of lactate and/or pyruvate may lower the p& of TSM. Because the enzymes in the glycolytic pathway have alkaline optimum pH’s, a lowering of pfi would be unfavorable to glycolysis and to the hypothesis that in hypoxia the rate of glycolysis is increased. Therefore, it seemed important to measure the levels of lactate and pyruvate and the pHi of TSM under hypoxic and normoxic conditions in order to differentitate between these possibilities. METHODS
MECHANICAL AND ELECTROPHYSIOLOGICAL properTrachealis muscle was obtained from the cervical traties of tracheal smooth muscle (TSM) have been studied cheae of mongrel dogs anesthetized with 30 mg/kg penin some detail under both normoxic and hypoxic condi- tobarbital. Muscle strips were dissected and equilitions (U-20, 21). A significant shift of the force-velocity brated in a lo-ml mammalian Krebs-Ringer bicarbonrelationships to the left has been observed under hy- ate solution of the following composition, in millimoles poxic conditions indicating a decrease in power producper liter: NaCl, 115; NaHCO:,, 25; NaH,PO,, 1.38; KC1, tion. This shift was found to be inversely proportional to 2.51; MgSO,*7 H20, 2.46; CaCl,, 1.91; dextrose, 5.56. both oxygen tension and dextrose concentration, with The solution was equilibrated w ,ith an OS-C0, gas mixthe sensitivity of the muscle to hypoxia increasing as ture so as to achieve a measured bath Po, of 600 mmHg, dextose concentration was progressively lowered. At Pco., of 45 mmHg, and pH of 7.40 at a temperature of $7 normal dextrose concentration, a decrease in PO, to less t 1°C. The bath PO, could be rapidly decreased to 50 mmHg while maintaining pH and Pco2 constant by than 10 mmHg resulted in an impairment of mechanical function to a level which was less than 50% of normal, equilibrating with a N,-CO,-0, gas mixture. whereas 10% of normal concentration of dextrose, with Measurement of effects of hypoxia on mechanical the same degree of hypoxia, resulted in a 97% impairproperties of TSM. The muscle was attached to an appament. Furthermore, with a substrate-free medium at ratus which is shown schematically in Fig. 1. The lower high PO,!, there was no reduction in mechanical function end of the trachealis muscle strip was attached firmly for 1 h (19). by 3-Obraided surgical silk to a hook at the bottom of the Further work (11) showed that glycogenolysis in TSM bath. The upper end was attached by a short length of is significantly increased under hypoxic conditions, and surgical silk to a Grass FT-03 transducer. The-force it has been reported that anaerobic glycolysis can sup- transducer was mounted on a rack-and-pinion enabling ply the energy requirement of a 70% maximal contracthe muscle to be stretched to any given length, at which tion of rabbit aortic smooth muscle (5). the muscle could be held isometrically. The output of Since a PO, of 0.05). Intracellular pH (n = 11). The intracellular pH’s of the muscles under the various incubation conditions are shown in Fig. 4. The unstimulated control experiments were conducted under resting n.ormoxic conditions, without electrical stimulation. The mean pH’s and standard errors of the muscle under various conditions are as follows: normoxic resting, 7.20 t .04; hypoxic resting, 7.19 & 0.04; normoxic active, 7.30 t 0.03; and hypoxic active, 7.71 t 0.05. The pHi was significantly higher under contracting hypoxic conditions (P < 0.05) as compared to normoxic contracting conditions, but there was no significant difference between the pHi’s of resting normoxic and resting hypoxic muscles, as assessedby the Student t test. There were 11 paired experiments for the hypoxia and normoxic muscles and 6 experiments for the unstimulated normoxic
Downloaded from www.physiology.org/journal/ajpendo by ${individualUser.givenNames} ${individualUser.surname} (150.216.068.200) on January 1, 2019.
SMOOTH
MUSCLE
LACTATE,
PYRUVATE,
AND
pHi
7.80
7.6 -
7.2 7.0c HA
FIG. moxie, smooth active; resting.
NA
NR
HR
4. Intracellular pH’s in stimulated active hypoxic and norand unstimulated resting normoxic and hypoxic tracheal muscles. Values indicate means with SE bars. HA, hypoxic NA, normoxic active; NR, normoxic resting; HR, hypoxic
controls, and the average weight of the muscle strips used in these experiments was 28.29 mg. Binding studies. Inasmuch as binding of DMO to cellular proteins would invalidate the use of this method in measuring pHi, the effect of adding unlabeled DMO to labeled DMO on determinations of p& was studied. The concentration of unlabeled DMO was 100 times that of the labeled DMO, and the active muscles were rendered hypoxic because it is in that condition we found the highest pH’s. Four experiments were carried out. Using only labeled DMO, a pH of 7.64 t .Ol was obtained. When unlabeled DMO was added, pH was 7.65 t .02. There was no statistically significant difference between the groups. No evidence of binding was detected when this study was conducted on normoxic resting tiuscles. Time studies. In comparing PHi’s under differing experimental conditions, it is important that the measurements be made at steady state. Data were obtained from resting muscles incubated for 1, 2, and 3 h, respectively. Mean values of 7.21, 7.25, and 7.22 were obtained with no statistically significant differences between these values. Hence, a steady state seems to have been established within 1 hr. Similar experiments conducted under normoxic contracting and hypoxic contracting conditions showed a steady state had developed within the same period of time. DISCUSSION
Effect of hypoxia on lactate and pyruvate contents of trachealis. The levels of lactate found in TSM are comparable to those reported for feline skeletal and bovine vascular smooth muscles (9, 13, 14) both in magnitude and direction of change under hypoxia. The total lactate production (tissue + medium) under hypoxic conditions is about 3 times that under normoxic conditions. This agrees with the hypothesis tested that in hypoxia, glycolysis in TSM is increased. The level of pyruvate in TSM under hypoxic conditions is slightly lowered as compared to normoxic TSM. This is in contradiction to findings in the gastrocnemius of the cat (9). But it was speculated that in this muscle the increase in pyruvate level was due to the
E333
loose-coupling of the oxidation of glyceraldehyde-3acid, to the reducphosphate to 1,3=diphosphoglyceric tion bf pyruvate to-lactate in the glycolytic pathway. Thus, the difference can be accounted for by 1) an absence of such loose coupling in TSM so that any amount of lactate could be formed without any net increase in the quantity of pyruvate present, and 2) a difference in the-activities of lactate dehydrogenases in the two muscles. The isozymes (H and M forms) of skeletal and smooth muscle lactic dehydrogenases tend to differ substantially (10) which could provide regulation of balance of the pyruvate-lactate ratio. Effect of hypoxia on intracellularpIT. The pI& of TSM was found to increase under hypoxic conditions rather than decrease as previously expected (19, 21). Two factors can most likely contribute to a falsely high pH, namely, the entry of inulin into intracellular space (15) and the binding of DMO to cellular proteins (2-4, 16). The mean TSM extracellular space (ECS) as determined by the inulin method in our experiments is 54% as compared to 33% in guinea pig taenia coli as determined by the Co-EDTA method (1). Inasmuch as the total TSM tissue water is only 78% fresh weight, a 54% ECS would strongly support a recent report that inulin enters intracellular space (15). The falsely high ECS, when substituted into the Waddell and Butler equation, will only increase the p& by approximately 1% as compared to an ECS of 33%. Consequently, the most important factor has to be the binding of DMO to cellular proteins. This can be elucidated by priming the tissue with unlabeled DMO and observing its effect on the scintillation counting of labeled DMO, but this remains to be done. Waddell and Butler (25) showed that the whole of the DMO present in homogenates of dog skeletal muscle was freely dialyzable and consequently not bound in any way to the tissue proteins. Izutsu (8) had adduced similar evidence for bullfrog toe striated muscle. Our own data confirm that significant binding does not occur in tracheal smooth muscle. The above discussion notwithstanding, because the same factors operate in both hypoxic and normoxic experiments, the observation that pa tends to increase in hypoxia over normoxia is still valid. This is strengthened by our finding that no significant binding was found in either nor-moxie resting or hypoxic muscles. Ifthe hypoxic insult does not create a leakage to DMO in the cell membranes in TSM as is the case in guinea taenia coli to sorbitol in anaerobic conditions (26), then the alkalosis in hypoxia would not be artifactual. The fact that the resting membrane potential of the TSM is unchanged under hypoxia (19) suggests that membrane damage does not exist. Finally, it may be argued that because of limitations in the sensitivity of the DMO technique as applied to tracheal smooth muscle we are unable to demonstrate any decrease in pH during hypoxia. However, we have shown (23) in studies of hypercapnic acidosis in TSM -that the DMO technique as used by us can indeed register decreases in PH. The following speculative explanations for the alkalosis are given: I) hydrogen ions are actively pumped out of the cells (7), and in hypoxia the activity of the
Downloaded from www.physiology.org/journal/ajpendo by ${individualUser.givenNames} ${individualUser.surname} (150.216.068.200) on January 1, 2019.
E334
STEPHENS,
hydrogen pump is enhanced. In this context, it should be pointed out that the size of the bath medium in these experiments was more than 300 times the size of the muscle strips used. This condition is unphysiological because the bath medium may constitute a reservoir of effectively infinite size into which H+, lactate, and pyruvate can be dumped. The calculated intracellular-extracellular lactate gradient in our hypoxia experiments is about 8O:l. 2) Previous work in this laboratory suggests that AMP is broken down in TSM under hypoxic conditions (11). The significance of the breakdown of AMP in muscle in the face of a strong ATP drain was discussed in detail by J. M. Lowenstein (12). The reaction involves adenylate deaminase and results in the production of IMP and ammonia as given below AMP
+ H,O
adenylate
dearninase
> IMP + NH,,
Alkalosis in TSM under hypoxic conditions could be attributed to the ammonia released in the above reaction. This line of reasoning is entirely speculative as we have no data at this time to support this hypothesis. 3) This laboratory has also reported a reduction of creatine phosphate levels in TSM in hypoxia to 16.0% of normals (11). The breakdown of CP is mediated by the enzyme creatine kinase and results in the formation of ATP and creatine, viz. creatine Creatine
phosphate has a high
+ ADP pK (-
--____) creatine
+ ATP 8.0) and hence is usually kinase
creatine
KROEGER,
AND
LOH
highly protonated in the normal range of pHi. Thus, the creatine released in TSM in hypoxia may contribute to the rise in pH,, at least transiently before glycolytic activity increases. In conclusion, it appears that in TSM under in vitro, hypoxic conditions which produce a reversible impairment of mechanical function, glycolysis is increased. However, instead of the expected acidosis an intracellular alkalosis develops. In this context it is to be noted that intracellular alkalosis would facilitate an increase in glycolysis, as the enzymes in the glycolytic pathway have alkaline optimal pH’s. Thus, though the mechanism of the production of the alkalosis is not understood at this time, its beneficial role is evident. To what extent this mechanism is operative in vivo is difficult to say, but it can be speculated that under in vivo conditions, intracellular alkalosis could be only a transient response to hypoxia because unlike in vitro experiments, the volume of the physiological extracellular fluid is less than that of the intracellular fluid. Hence, the extracellular lactate level will rise much more quickly, providing a substantial challenge to the cell in expelling the lactate and hydrogen ions. The invaluable technical assistance of Mr. Richard Mitchell is gratefully acknowledged. This investigation was supported by grants from the Medical Research Council of Canada, the Canadian Heart Foundation, and the Canadian Tuberculosis and Respiratory Disease Association.
Received
for publication
5 January
1976.
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