Acta Physiol Scand 1979, 106: 239-240
Observations on the localization of carbonic anhydrase in muscle YVONNE RIDDERSTRALE Department of Animul Physiology, Swedish University of Agricultural Sciences, Uppsala, Sweden
Moynihan (1977) has found carbonic anhydrase activity in rat skeletal muscle. M. soleus (which is predominantly red) contained a significantly higher concentration of the enzyme than the predominantly white m. extensor digitorum longus and m. vastus lateralis. It appears of interest to examine by a histochemical method the localization of the enzyme in the muscle and also to find out if there are differences in enzyme localization between red and white muscle fibers. Small pieces of m. soleus and m. vastus lateralis from rat were fixed by immersion in glutaraldehyde (2 % buffered with phosphate) and embedded in JB-4 plastic, sectioned and incubated for demonstration of carbonic anhydrase activity according to the modified Hansson method described by Ridderstrhle (1976).
The description given refers to what appears to be typical white fibers in m. vastus and to red fibers in m. soleus. In general the pattern of staining is similar in the two muscles. The strongest staining if found in the endothelium of the capillaries (Figs. 1, 2). The sarcolemma or its immediate neighbourhood is clearly stained. With equal incubation time and section thickness the staining is usually heavier in m. vastus lateralis than in m. soleus. The membrane-associated staining shows regular indentations (Fig. 1). Faint diffuse staining appears all over the muscle fibers. In two respects the staining pattern differs in the two muscles. Nuclei are stained in m. soleus. In m. vastus lateralis the connective tissue is stained. Acetazolamide in the concentration used (10 pM) abolishes all staining except weak diffuse staining in some muscle fibers and nuclei. Zborowska-Sluis et al. (1974) found indirect evidence for the presence of carbonic anhydrase in skeletal muscle. Moynihan (1977) determined the concentration of carbonic anhydrase in different muscles from rat. He found that m. soleus. m. vas-
tus lateralis and m. extensor digitorum longus had significant concentrations of the enzyme after correction for the enzyme content in the blood. M. soleus with mainly red fibers, had a significantly higher concentration than the other two, with predominantly white fibers. Lonnerholm (1974) shows a figure of dogfish muscle showing no staining of the muscle fibers but a capillary blackened by the carbonic anhydrase staining reaction. The present study shows the strongest staining to be localized to the endothelium of the capillaries. Weaker staining was found at the sarcolemma. M. soleus is more richly supplied with capillaries than m. vastus lateralis and this could contribute to the higher concentration of carbonic anhydrase found by Moynihan in m. soleus. It is impossible (with the light microscope) to ascertain if the superficial staining of the fibers is
Fig. 1 . M. vastus lateralis. Space between two fibers ( F ) with capillary (C). Arrows point to membrane indenta-
tions. Arttr Plr.vrio/ SctrnJ 106
240
Y. Ridderstrdle
carbonic acid. Rapid dehydration of this acid would diminish the resulting acidification. The carbon dioxide-bicarbonate system is a rapidly functioning buffer system only when carbonic anhydrase is available. This may be of importance for the damping of rapid acid-base changes e.g. during rhythmic contractions. Carbon dioxide formed during periods of capillary occlusion will mainly accumulate as bicarbonate (even without carbonic anhydrase). This bicarbonate may be more rapidly removed during the intermittent perfusion of the capillaries when the transformation of bicarbonate into carbon dioxide is catalyzed. However, when speculating about the function of carbonic anhydrase in muscle it may be appropriate to point out that carbonic anhydrase is found in the walls of capillaries in many organs e.g. mucosa of Fig. 2. M. soleus. Numerous stained capillaries surround- duodenum, cecum and colon in rabbits, mammary ing the muscle fibers. gland, cecum and colon in rats, rumen of the goat, the avian shell gland (unpublished observations by the author) where processes connected with conbound to the sarcolemma or located outside o r in- tractility are probably not important. side the membrane. The indentations of the staining into the muscle fibers at regular intervals may sugREFERENCES gest that the enzyme is very closely associated with the membrane and follows it at least some distance ENNS, T. 1967. Facilitation by carbonic anhydrase of carbon dioxide transport. Science 155: 44-47. along the transverse tubular system (T-system). LONNERHOLM. G . 1974. Carbonic anhydrase histoSpeculation with regard to the role of the chemistry, a critical study of Hansson's coboltcarbonic anhydrase in muscle may be premature. phosphate method. Acta Physiol Scand, Suppl. 418: 35. However, Zborowska-Sluis et al. suggested facilitaMOYNIHAN, J. B. 1977. Carbonic anhydrase activity in tion of carbon dioxide transport according to the mammalian skeletal and cardiac muscle. Biochem J scheme proposed by Enns (1967). Several other 168: 567-569. functions of carbonic anhydrase in muscle appear RIDDERSTRALE, Y. 1976. Intracellular localization of carbonic anhydrase in the frog nephron. Acta Physiol possible. Scand 98: 465-469. At least in some locations the enzyme may be ZBOROWSKA-SLUIS, D. T., L'ABBATE, A. & KLASinvolved in hydrogen ion transport. SEN, G . A. 1974. Evidence of carbonic anhydrase The production of lactic acid in the muscle fibers activity in skeletal muscle: a role for facilitative carbon during anaerobic work must lead to formation of dioxide transport. Respiration Physiology 2 1 : 341-350.
Observations on the localization of carbonic anhydrase in muscle YVONNE RIDDERSTRALE Department of Animul Physiology, Swedish University of Agricultural Sciences, Uppsala, Sweden
Moynihan (1977) has found carbonic anhydrase activity in rat skeletal muscle. M. soleus (which is predominantly red) contained a significantly higher concentration of the enzyme than the predominantly white m. extensor digitorum longus and m. vastus lateralis. It appears of interest to examine by a histochemical method the localization of the enzyme in the muscle and also to find out if there are differences in enzyme localization between red and white muscle fibers. Small pieces of m. soleus and m. vastus lateralis from rat were fixed by immersion in glutaraldehyde (2 % buffered with phosphate) and embedded in JB-4 plastic, sectioned and incubated for demonstration of carbonic anhydrase activity according to the modified Hansson method described by Ridderstrhle (1976).
The description given refers to what appears to be typical white fibers in m. vastus and to red fibers in m. soleus. In general the pattern of staining is similar in the two muscles. The strongest staining if found in the endothelium of the capillaries (Figs. 1, 2). The sarcolemma or its immediate neighbourhood is clearly stained. With equal incubation time and section thickness the staining is usually heavier in m. vastus lateralis than in m. soleus. The membrane-associated staining shows regular indentations (Fig. 1). Faint diffuse staining appears all over the muscle fibers. In two respects the staining pattern differs in the two muscles. Nuclei are stained in m. soleus. In m. vastus lateralis the connective tissue is stained. Acetazolamide in the concentration used (10 pM) abolishes all staining except weak diffuse staining in some muscle fibers and nuclei. Zborowska-Sluis et al. (1974) found indirect evidence for the presence of carbonic anhydrase in skeletal muscle. Moynihan (1977) determined the concentration of carbonic anhydrase in different muscles from rat. He found that m. soleus. m. vas-
tus lateralis and m. extensor digitorum longus had significant concentrations of the enzyme after correction for the enzyme content in the blood. M. soleus with mainly red fibers, had a significantly higher concentration than the other two, with predominantly white fibers. Lonnerholm (1974) shows a figure of dogfish muscle showing no staining of the muscle fibers but a capillary blackened by the carbonic anhydrase staining reaction. The present study shows the strongest staining to be localized to the endothelium of the capillaries. Weaker staining was found at the sarcolemma. M. soleus is more richly supplied with capillaries than m. vastus lateralis and this could contribute to the higher concentration of carbonic anhydrase found by Moynihan in m. soleus. It is impossible (with the light microscope) to ascertain if the superficial staining of the fibers is
Fig. 1 . M. vastus lateralis. Space between two fibers ( F ) with capillary (C). Arrows point to membrane indenta-
tions. Arttr Plr.vrio/ SctrnJ 106
240
Y. Ridderstrdle
carbonic acid. Rapid dehydration of this acid would diminish the resulting acidification. The carbon dioxide-bicarbonate system is a rapidly functioning buffer system only when carbonic anhydrase is available. This may be of importance for the damping of rapid acid-base changes e.g. during rhythmic contractions. Carbon dioxide formed during periods of capillary occlusion will mainly accumulate as bicarbonate (even without carbonic anhydrase). This bicarbonate may be more rapidly removed during the intermittent perfusion of the capillaries when the transformation of bicarbonate into carbon dioxide is catalyzed. However, when speculating about the function of carbonic anhydrase in muscle it may be appropriate to point out that carbonic anhydrase is found in the walls of capillaries in many organs e.g. mucosa of Fig. 2. M. soleus. Numerous stained capillaries surround- duodenum, cecum and colon in rabbits, mammary ing the muscle fibers. gland, cecum and colon in rats, rumen of the goat, the avian shell gland (unpublished observations by the author) where processes connected with conbound to the sarcolemma or located outside o r in- tractility are probably not important. side the membrane. The indentations of the staining into the muscle fibers at regular intervals may sugREFERENCES gest that the enzyme is very closely associated with the membrane and follows it at least some distance ENNS, T. 1967. Facilitation by carbonic anhydrase of carbon dioxide transport. Science 155: 44-47. along the transverse tubular system (T-system). LONNERHOLM. G . 1974. Carbonic anhydrase histoSpeculation with regard to the role of the chemistry, a critical study of Hansson's coboltcarbonic anhydrase in muscle may be premature. phosphate method. Acta Physiol Scand, Suppl. 418: 35. However, Zborowska-Sluis et al. suggested facilitaMOYNIHAN, J. B. 1977. Carbonic anhydrase activity in tion of carbon dioxide transport according to the mammalian skeletal and cardiac muscle. Biochem J scheme proposed by Enns (1967). Several other 168: 567-569. functions of carbonic anhydrase in muscle appear RIDDERSTRALE, Y. 1976. Intracellular localization of carbonic anhydrase in the frog nephron. Acta Physiol possible. Scand 98: 465-469. At least in some locations the enzyme may be ZBOROWSKA-SLUIS, D. T., L'ABBATE, A. & KLASinvolved in hydrogen ion transport. SEN, G . A. 1974. Evidence of carbonic anhydrase The production of lactic acid in the muscle fibers activity in skeletal muscle: a role for facilitative carbon during anaerobic work must lead to formation of dioxide transport. Respiration Physiology 2 1 : 341-350.
