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Peripheral nerve carbonic anhydrase activity and chronic acetazolamide treatment of rats
Brain Research, 406 (1987) 379-384 Elsevier
379
BRE 22113
Peripheral nerve carbonic anhydrase activity and chronic acetazolamide treatment of rats Terri Oswald and Danny A. Riley Department of Anatomy and Cellular Biology, Medical Collegeof Wisconsin, Milwaukee, W153226 (U.S.A.) (Accepted 18 November 1986) Key words: Carbonic anhydrase; Acetazolamide; Peripheral nerve
Examination of cranial nerves shows that the sensory infraorbital branch of the trigeminal nerve contains many carbonic anhydrasereactive axons whereas axons of the motor facial nerve are non-reactive. This motor/sensory axon staining difference holds for both cranial and spinal nerves. Chronic treatment with acetazolamide produced no apparent changes in carbonic anhydrase histochemical activity or the structure of peripheral nerve fibers.
Sulfonamides, such as acetazolamide, are potent inhibitors of carbonic anhydrase (CA) activity. They were first introduced clinically to treat cardiac edema because of their diuretic action 7. CA inhibitors'are also utilized in the treatment of edema, hydrocephaly, acute altitude sickness and gastric ulcers; the current major use is for treating glaucoma 16. Intraocular pressure is lowered when acetazolamide slows production of aqueous humor by blocking nearly 93% of the CA activity of the ciliary body 5. CA catalyzes the dehydration of H C O 3- to form CO 2 and H20. The CO z rapidly exits the cell through the plasma membrane. The diminution of intracellular H C O 3- decreases the rate of fluid secretion because movement of H20 out of the cell follows the excretion of Na + and H C O 3- (ref. 16). Chronic acetazolamide therapy can generate adverse neural side effects in humans, such as sensory paresthesias, tingling and numbness of the distal extremities and perioral region 9. Less common are complaints of shortness of breath on exertion, fatigue, ataxia, tremors and tinnitus 2,5. These symptoms suggest that acetazolamide disrupts peripheral nerve function. This is a reasonable assumption because sensory axons of peripheral spinal nerves in hu-
mans, monkeys, cats and rats have recently been shown to contain high levels of acetazolamide-inhibitable CA activity a2. However, it is unknown whether sensory axons of cranial nerves also possess CA activity. The present experiment examined the histochemical CA activity of cranial nerves and sought to determine whether chronic administration of acetazolamide altered peripheral nerve CA activity and/or disrupted axonal structure. The results of this study may provide insight into the function of neural CA. Two groups of 6 prepubertal Sprague-Dawley rats (130-190 g females) were gavaged twice daily with either 15 or 30 mg/kg b. wt. of sodium acetazolamide (Diamox; Lederle Parenterals) for 2 weeks. Controls (n = 12) of matched age, weight and sex received the vehicle, double distilled water, in otherwise identical treatment regimes. A 15 mg/kg dose can cause paresthesias in humans 4,5. Twice this clinical dosage was also tested because rodents may eliminate the drug more effectively. To insure that acetazolamide was absorbed into the blood following gastric administration, plasma acetazolamide was determined by a modified method of Maren 4,8. Aliquots of plasma were assayed for their ability to inhibit a standard
Correspondence: D.A. Riley, Department of Anatomy and Cellular Biology, 8701 Watertown Plank Road, Milwaukee, WI 53226, U.S.A. 0006-8993/87/$03.50 (~) 1987 Elsevier Science Publishers B.V. (Biomedical Division)
380 amount (2 W i l b e r - A n d e r s o n units of C A activity) of purified bovine erythrocyte C A (Sigma). A preliminary series of 4 rats were gavaged at the lower dose, and tail vein blood samples were collected immediately, 0.5, 1, 2 and 4 h later. The concentration of acetazolarnide in the test sample was calculated by comparing the percentage of inhibition obtained with the unknown plasma sample to a standard acetazolamide inhibition curve (Fig. 1). As previously found for humans, peak serum levels of the drug (50/~g/ml) occurred after 1 h, exceeding the level causing paresthesias in humans 4. Drug concentration decreased to 60% of the peak value by 4 h (Fig. 2). Based on the rate of loss of inhibitor from the blood, it was necessary to gavage the rats twice daily 10-12 h apart to maintain chronic exposure to the drug. In the chronically treated groups, tail vein blood were collected
nerves, L 4 spinal ganglia, facial motor nerves and infraorbital cutaneous nerves were removed, cryoprotected with 30% buffered sucrose at 5 °C and quick frozen in liquid nitrogen for cryostat microtomy as described previously 13. Sections (10 # m ) were cut
/
90
/
8O
09 E3
1 h following the final gavage. The mean acetazolamide concentration was 60 + 8/~g/ml for rats receiving 30 mg/kg/day. As expected, the plasma from control rats did not inhibit C A activity. No obvious abnormal behaviors or movement disorders were seen in rats chronically receiving the drug. Following chronic treatment, rats were anesthetized with chloral hydrate (400 mg/kg b. wt., i.p.) and perfused intracardially with 0.9% saline followed by 2.5% glutaraldehyde buffered with 0.1 M s o d i u m potassium phosphate at pH 7.4. Fixation was necessary to retain the soluble nerve C A 12-14. The sciatic
50
40
,J
70
"" 3 0 o9
Z
0 0
LU 03
A
r,r (.9 0 n- 2 0
60
./ 10
40-
0-~ i
o
i
' 5 J0
'
'
'
' 1"0'0
NANOGRAMS
Fig. 1. A standard curve showing inhibition of CA activity by acetazolamide. Each point represents the average of 4 measurements for a single inhibitor concentration.
i
i
i
i
1
2
3
4
HOURS
Fig. 2. Temporal changes in plasma acetazolamide levels after a single gavage of 15 mg/kg b. wt. Four rats were sampled per point. Bars represent S.E.M. Maximum concentration occurred at 1 h. Plasma from control rats did not inhibit CA activity.
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3
Fig. 3. C A activity of the l u m b a r (L4) dorsal sensory and ventral m o t o r roots and dorsal root ganglion sensory neurons in a control rat. Many darkly stained myelinated axons are present in the dorsal root (DR). Lightly to darkly staining sensory n e u r o n s are interspersed among the dorsal root axons. Serial sections revealed that at this level of sectioning the dorsal and ventral roots are partially merged. Most of the axons in the ventral root (VR) are non-reactive, x 156. Fig. 4. A sciatic nerve fascicle reacted for C A activity. T h e presence of stained and non-stained axons is consistent with the m o t o r and sensory composition of this nerve. ×156.
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5
6 Fig. 5. CA activity of the infraorbital sensory branch of the trigeminal cranial nerve. The majority of the axons are highly reactive as in the dorsal sensory root of the spinal nerves. × 156. Fig. 6. None of the motor axons in this cranial facial nerve branch show CA reactivity. This lack of staining is similar to that in ventral root motor axons, x 156.
383 The histochemical method may not be sensitive enough to detect changes; Sugai and Ito observed no visible differences in histochemical CA activity of fixed tissues in which enzymatic activity ranged from 4 to 32% of normal 15. In future studies, the CA activity of infraorbital nerve homogenates will be analyzed by the more sensitive biochemical method. Higher acetazolamide levels and longer exposure may be necessary to evoke a change. Alternatively, a temporal study may reveal alteration of enzyme activity with recovery by 2 weeks 1. In humans, Diamox administered at 7 mg/kg can produce paresthesia within 7 h (ref. 4). This suggests that acetazolamide affects sensory nerve function (conduction and/or generation of action potentials) rather than causing frank degeneration. The electrophysiological properties of axons in treated rats have not been studied. Acetazolamide functions as an anticonvulsant, and this effect is dependent on CA inhibition 1°. CA inhibition apparently increases CO 2 tension which indirectly decreases intracellular pH (ref. 3). A fall in pH can block voltage-sensitive inactivation of Na ÷ permeability resulting in sustained depolarization and loss of excitability n. Given the high levels of acetazolamide-inhibitable CA in oligodendrocytes and red blood cells, sensory disturbances may be obtained indirectly by multiple mechanisms 6'14. In conclusion, CA is present in both cranial and spinal sensory axons; acetazolamide does not appear neurotoxic at the dosages and duration administered in the present study. These findings indicate that the sensory nerve disturbances occurring in humans receiving acetazolamide may represent druginduced alteration of peripheral nerve excitability.
and reacted for CA activity as before 13, except for the following improvements: in the past, uneven wetting of sections by the incubation media produced spotty staining. To reduce hydrophobicity, sections were dipped 8 times over 30 s in 70% ethanol immediately before incubation. This maneuver produced even staining, although intensity was reduced somewhat. The black CoS reaction product was irreversibly oxidized by the mounting medium to a colorless product after 2 - 3 weeks when the sections were covered with Fisher Permount (manufactured after 1978). While adding 1% (w/v)butylated hydroxytoluene to the Permount and storing the slides in the refrigerator doubled the shelf life of the stain, a nonoxidizing replacement is being sought. When many tissues were sectioned, it was possible to store the slides in a humid chamber between section collection; this allowed multiple control and test sections to be placed and reacted on a single slide. At the light microscopic level, the structure and histochemical staining characteristics of the drugtreated tissues were indistinguishable from the controls. As reported previously 13A4, a subset of spinal sensory neurons were reactive for CA whereas most of the accompanying ventral root motor axons were unstained (Fig. 3). The sciatic nerve, which contains both motor and sensory axons, presented a mixture of reactive and non-reactive myelinated axons (Fig. 4). Consistent with sensory axon reactivity in spinal nerves, the axons of the infraorbital fifth cranial nerve, which are mostly sensory, were highly reactive (Fig. 5). Conversely, axons in the facial nerve, a primarily motor cranial nerve, were not stained (Fig. 6). Thus, CA reactivity of myelinated sensory axons is characteristic of both cranial and spinal nerves. Although the treated rats were exposed to plasma acetazolamide levels at or twice that seen clinically to produce paresthesia in humans, no morphological alterations or changes in CA activity of the myelinated axons were observed at the light microscopic level.
The Diamox was a generous gift from the Lederle Company. The authors wish to thank Mr. Jim Bain for his excellent technical support. This work was funded by NASA Grant NCC-2-266 to D.A.R.
1 Albrecht, J. and Hilgier, W., Brain carbonic anhydrase activity in rats in experimental hepatogenic encephalopathy, Neurosci. Lett., 45 (1984)7-10. 2 Bennett, D.R., AMA Drug Evaluations, Saunders, 5th ed., Philadelphia, 1983, p. 460. 3 Clark, M.A. and Eaton, D.C., Effect of CO2 on neurons of the house cricket Acheta domestica, J. Neurobiol., 14 (1983) 237-250.
4 Freidland, B.R., Mallonee, J. and Anderson, D.R., Shortterm dose response characteristics of acetazolamide in man, Arch. Ophthalmol., 95 (1977) 1809-1812. 5 Kupper, C., Lawrence, C. and Linner, E., Long-term administration of acetazolamide (Diamox) in the treatment of glaucoma, Am. J. Ophthalmol., 40 (1955) 673-680. 6 Lockwood, A.H., LaManna, J.C., Snyder, S. and Rosenthai, M., Effects of acetazolamide and electrical stimula-
384
7
8
9 10
11
tion cerebral oxidative metabolism as indicated by the cytochrome oxidase redox state, Brain Res., 308 (1984) 9-14. Maren, T . J , Carbonic anhydrase: the middle years, 1945-1960, an introduction to the pharmacology of sulfonamides, Ann. N. Y. Acad. Sci., 429 (1984) 10-17. Maren, T.H., Ash, V.I. and Bailey Jr., E.M., Carbonic anhydrasc inhibition. II. A method for determination of carbonic anhydrase inhibitors, particularly of Diamox, Bull. Johns Hopkins Hosp., 95 (1954) 244-255. Modell, W., Drugs of Choice, Mosby, St. Louis, 1983, p. 687. Millchap, J.G., Woodbury, D.M. and Goodman, L.S., Mechanism of the anticonvulsant action of acetazolamide, a carbonic anhydrase inhibitor, J. Pharmacol. Exp. Ther., 115 (1955) 251-258. Nonner, W., Spaulding, B.C. and Hille, B., Low intracellular pH and chemical agents slow inactivation gating in sodium channels of muscle, Nature (London), 284 (1980) 360-363.
12 Riley, D.A. and Lang, D.H., Carbonic anhydrase activity of human peripheral nerves: a possible histochemical aid to nerve repair, J. HandSurg., 9A (1984) 1809-1812. 13 Riley, D.A., Ellis, S. and Bain, J., Carbonic anhydrase activity in skeletal muscle fiber types, axons, spindles, and capillaries of rat soleus and extensor digitorum longus muscles, J. Histochem. Cytochem., 30 (1982) 1275-1288. 14 Riley, D.A., Ellis, S and Bain, J.L.W., Ultrastructural cytochemical localization carbonic anhydrase activity in rat peripheral sensory and motor nerves, dorsal root ganglia and dorsal column nuclei, Neuroscience. 13 (1984) 189-206. 15 Sugai, N. and Ito, S., Carbonic anhydrase, ultrastructural localization in the mouse gastric mucosa and improvements in the technique, J. Histochem. Cytochem., 28 (1980) 511-525. 16 Wistrand, P.J., The use of carbonic anhydrase inhibitors in ophthalmology and clinical medicine, Ann. N.Y. Acad. Sci., 429 (1984) 609-619.
379
BRE 22113
Peripheral nerve carbonic anhydrase activity and chronic acetazolamide treatment of rats Terri Oswald and Danny A. Riley Department of Anatomy and Cellular Biology, Medical Collegeof Wisconsin, Milwaukee, W153226 (U.S.A.) (Accepted 18 November 1986) Key words: Carbonic anhydrase; Acetazolamide; Peripheral nerve
Examination of cranial nerves shows that the sensory infraorbital branch of the trigeminal nerve contains many carbonic anhydrasereactive axons whereas axons of the motor facial nerve are non-reactive. This motor/sensory axon staining difference holds for both cranial and spinal nerves. Chronic treatment with acetazolamide produced no apparent changes in carbonic anhydrase histochemical activity or the structure of peripheral nerve fibers.
Sulfonamides, such as acetazolamide, are potent inhibitors of carbonic anhydrase (CA) activity. They were first introduced clinically to treat cardiac edema because of their diuretic action 7. CA inhibitors'are also utilized in the treatment of edema, hydrocephaly, acute altitude sickness and gastric ulcers; the current major use is for treating glaucoma 16. Intraocular pressure is lowered when acetazolamide slows production of aqueous humor by blocking nearly 93% of the CA activity of the ciliary body 5. CA catalyzes the dehydration of H C O 3- to form CO 2 and H20. The CO z rapidly exits the cell through the plasma membrane. The diminution of intracellular H C O 3- decreases the rate of fluid secretion because movement of H20 out of the cell follows the excretion of Na + and H C O 3- (ref. 16). Chronic acetazolamide therapy can generate adverse neural side effects in humans, such as sensory paresthesias, tingling and numbness of the distal extremities and perioral region 9. Less common are complaints of shortness of breath on exertion, fatigue, ataxia, tremors and tinnitus 2,5. These symptoms suggest that acetazolamide disrupts peripheral nerve function. This is a reasonable assumption because sensory axons of peripheral spinal nerves in hu-
mans, monkeys, cats and rats have recently been shown to contain high levels of acetazolamide-inhibitable CA activity a2. However, it is unknown whether sensory axons of cranial nerves also possess CA activity. The present experiment examined the histochemical CA activity of cranial nerves and sought to determine whether chronic administration of acetazolamide altered peripheral nerve CA activity and/or disrupted axonal structure. The results of this study may provide insight into the function of neural CA. Two groups of 6 prepubertal Sprague-Dawley rats (130-190 g females) were gavaged twice daily with either 15 or 30 mg/kg b. wt. of sodium acetazolamide (Diamox; Lederle Parenterals) for 2 weeks. Controls (n = 12) of matched age, weight and sex received the vehicle, double distilled water, in otherwise identical treatment regimes. A 15 mg/kg dose can cause paresthesias in humans 4,5. Twice this clinical dosage was also tested because rodents may eliminate the drug more effectively. To insure that acetazolamide was absorbed into the blood following gastric administration, plasma acetazolamide was determined by a modified method of Maren 4,8. Aliquots of plasma were assayed for their ability to inhibit a standard
Correspondence: D.A. Riley, Department of Anatomy and Cellular Biology, 8701 Watertown Plank Road, Milwaukee, WI 53226, U.S.A. 0006-8993/87/$03.50 (~) 1987 Elsevier Science Publishers B.V. (Biomedical Division)
380 amount (2 W i l b e r - A n d e r s o n units of C A activity) of purified bovine erythrocyte C A (Sigma). A preliminary series of 4 rats were gavaged at the lower dose, and tail vein blood samples were collected immediately, 0.5, 1, 2 and 4 h later. The concentration of acetazolarnide in the test sample was calculated by comparing the percentage of inhibition obtained with the unknown plasma sample to a standard acetazolamide inhibition curve (Fig. 1). As previously found for humans, peak serum levels of the drug (50/~g/ml) occurred after 1 h, exceeding the level causing paresthesias in humans 4. Drug concentration decreased to 60% of the peak value by 4 h (Fig. 2). Based on the rate of loss of inhibitor from the blood, it was necessary to gavage the rats twice daily 10-12 h apart to maintain chronic exposure to the drug. In the chronically treated groups, tail vein blood were collected
nerves, L 4 spinal ganglia, facial motor nerves and infraorbital cutaneous nerves were removed, cryoprotected with 30% buffered sucrose at 5 °C and quick frozen in liquid nitrogen for cryostat microtomy as described previously 13. Sections (10 # m ) were cut
/
90
/
8O
09 E3
1 h following the final gavage. The mean acetazolamide concentration was 60 + 8/~g/ml for rats receiving 30 mg/kg/day. As expected, the plasma from control rats did not inhibit C A activity. No obvious abnormal behaviors or movement disorders were seen in rats chronically receiving the drug. Following chronic treatment, rats were anesthetized with chloral hydrate (400 mg/kg b. wt., i.p.) and perfused intracardially with 0.9% saline followed by 2.5% glutaraldehyde buffered with 0.1 M s o d i u m potassium phosphate at pH 7.4. Fixation was necessary to retain the soluble nerve C A 12-14. The sciatic
50
40
,J
70
"" 3 0 o9
Z
0 0
LU 03
A
r,r (.9 0 n- 2 0
60
./ 10
40-
0-~ i
o
i
' 5 J0
'
'
'
' 1"0'0
NANOGRAMS
Fig. 1. A standard curve showing inhibition of CA activity by acetazolamide. Each point represents the average of 4 measurements for a single inhibitor concentration.
i
i
i
i
1
2
3
4
HOURS
Fig. 2. Temporal changes in plasma acetazolamide levels after a single gavage of 15 mg/kg b. wt. Four rats were sampled per point. Bars represent S.E.M. Maximum concentration occurred at 1 h. Plasma from control rats did not inhibit CA activity.
O! i¸i ~!
3
Fig. 3. C A activity of the l u m b a r (L4) dorsal sensory and ventral m o t o r roots and dorsal root ganglion sensory neurons in a control rat. Many darkly stained myelinated axons are present in the dorsal root (DR). Lightly to darkly staining sensory n e u r o n s are interspersed among the dorsal root axons. Serial sections revealed that at this level of sectioning the dorsal and ventral roots are partially merged. Most of the axons in the ventral root (VR) are non-reactive, x 156. Fig. 4. A sciatic nerve fascicle reacted for C A activity. T h e presence of stained and non-stained axons is consistent with the m o t o r and sensory composition of this nerve. ×156.
ii ~
;; :
5
6 Fig. 5. CA activity of the infraorbital sensory branch of the trigeminal cranial nerve. The majority of the axons are highly reactive as in the dorsal sensory root of the spinal nerves. × 156. Fig. 6. None of the motor axons in this cranial facial nerve branch show CA reactivity. This lack of staining is similar to that in ventral root motor axons, x 156.
383 The histochemical method may not be sensitive enough to detect changes; Sugai and Ito observed no visible differences in histochemical CA activity of fixed tissues in which enzymatic activity ranged from 4 to 32% of normal 15. In future studies, the CA activity of infraorbital nerve homogenates will be analyzed by the more sensitive biochemical method. Higher acetazolamide levels and longer exposure may be necessary to evoke a change. Alternatively, a temporal study may reveal alteration of enzyme activity with recovery by 2 weeks 1. In humans, Diamox administered at 7 mg/kg can produce paresthesia within 7 h (ref. 4). This suggests that acetazolamide affects sensory nerve function (conduction and/or generation of action potentials) rather than causing frank degeneration. The electrophysiological properties of axons in treated rats have not been studied. Acetazolamide functions as an anticonvulsant, and this effect is dependent on CA inhibition 1°. CA inhibition apparently increases CO 2 tension which indirectly decreases intracellular pH (ref. 3). A fall in pH can block voltage-sensitive inactivation of Na ÷ permeability resulting in sustained depolarization and loss of excitability n. Given the high levels of acetazolamide-inhibitable CA in oligodendrocytes and red blood cells, sensory disturbances may be obtained indirectly by multiple mechanisms 6'14. In conclusion, CA is present in both cranial and spinal sensory axons; acetazolamide does not appear neurotoxic at the dosages and duration administered in the present study. These findings indicate that the sensory nerve disturbances occurring in humans receiving acetazolamide may represent druginduced alteration of peripheral nerve excitability.
and reacted for CA activity as before 13, except for the following improvements: in the past, uneven wetting of sections by the incubation media produced spotty staining. To reduce hydrophobicity, sections were dipped 8 times over 30 s in 70% ethanol immediately before incubation. This maneuver produced even staining, although intensity was reduced somewhat. The black CoS reaction product was irreversibly oxidized by the mounting medium to a colorless product after 2 - 3 weeks when the sections were covered with Fisher Permount (manufactured after 1978). While adding 1% (w/v)butylated hydroxytoluene to the Permount and storing the slides in the refrigerator doubled the shelf life of the stain, a nonoxidizing replacement is being sought. When many tissues were sectioned, it was possible to store the slides in a humid chamber between section collection; this allowed multiple control and test sections to be placed and reacted on a single slide. At the light microscopic level, the structure and histochemical staining characteristics of the drugtreated tissues were indistinguishable from the controls. As reported previously 13A4, a subset of spinal sensory neurons were reactive for CA whereas most of the accompanying ventral root motor axons were unstained (Fig. 3). The sciatic nerve, which contains both motor and sensory axons, presented a mixture of reactive and non-reactive myelinated axons (Fig. 4). Consistent with sensory axon reactivity in spinal nerves, the axons of the infraorbital fifth cranial nerve, which are mostly sensory, were highly reactive (Fig. 5). Conversely, axons in the facial nerve, a primarily motor cranial nerve, were not stained (Fig. 6). Thus, CA reactivity of myelinated sensory axons is characteristic of both cranial and spinal nerves. Although the treated rats were exposed to plasma acetazolamide levels at or twice that seen clinically to produce paresthesia in humans, no morphological alterations or changes in CA activity of the myelinated axons were observed at the light microscopic level.
The Diamox was a generous gift from the Lederle Company. The authors wish to thank Mr. Jim Bain for his excellent technical support. This work was funded by NASA Grant NCC-2-266 to D.A.R.
1 Albrecht, J. and Hilgier, W., Brain carbonic anhydrase activity in rats in experimental hepatogenic encephalopathy, Neurosci. Lett., 45 (1984)7-10. 2 Bennett, D.R., AMA Drug Evaluations, Saunders, 5th ed., Philadelphia, 1983, p. 460. 3 Clark, M.A. and Eaton, D.C., Effect of CO2 on neurons of the house cricket Acheta domestica, J. Neurobiol., 14 (1983) 237-250.
4 Freidland, B.R., Mallonee, J. and Anderson, D.R., Shortterm dose response characteristics of acetazolamide in man, Arch. Ophthalmol., 95 (1977) 1809-1812. 5 Kupper, C., Lawrence, C. and Linner, E., Long-term administration of acetazolamide (Diamox) in the treatment of glaucoma, Am. J. Ophthalmol., 40 (1955) 673-680. 6 Lockwood, A.H., LaManna, J.C., Snyder, S. and Rosenthai, M., Effects of acetazolamide and electrical stimula-
384
7
8
9 10
11
tion cerebral oxidative metabolism as indicated by the cytochrome oxidase redox state, Brain Res., 308 (1984) 9-14. Maren, T . J , Carbonic anhydrase: the middle years, 1945-1960, an introduction to the pharmacology of sulfonamides, Ann. N. Y. Acad. Sci., 429 (1984) 10-17. Maren, T.H., Ash, V.I. and Bailey Jr., E.M., Carbonic anhydrasc inhibition. II. A method for determination of carbonic anhydrase inhibitors, particularly of Diamox, Bull. Johns Hopkins Hosp., 95 (1954) 244-255. Modell, W., Drugs of Choice, Mosby, St. Louis, 1983, p. 687. Millchap, J.G., Woodbury, D.M. and Goodman, L.S., Mechanism of the anticonvulsant action of acetazolamide, a carbonic anhydrase inhibitor, J. Pharmacol. Exp. Ther., 115 (1955) 251-258. Nonner, W., Spaulding, B.C. and Hille, B., Low intracellular pH and chemical agents slow inactivation gating in sodium channels of muscle, Nature (London), 284 (1980) 360-363.
12 Riley, D.A. and Lang, D.H., Carbonic anhydrase activity of human peripheral nerves: a possible histochemical aid to nerve repair, J. HandSurg., 9A (1984) 1809-1812. 13 Riley, D.A., Ellis, S. and Bain, J., Carbonic anhydrase activity in skeletal muscle fiber types, axons, spindles, and capillaries of rat soleus and extensor digitorum longus muscles, J. Histochem. Cytochem., 30 (1982) 1275-1288. 14 Riley, D.A., Ellis, S and Bain, J.L.W., Ultrastructural cytochemical localization carbonic anhydrase activity in rat peripheral sensory and motor nerves, dorsal root ganglia and dorsal column nuclei, Neuroscience. 13 (1984) 189-206. 15 Sugai, N. and Ito, S., Carbonic anhydrase, ultrastructural localization in the mouse gastric mucosa and improvements in the technique, J. Histochem. Cytochem., 28 (1980) 511-525. 16 Wistrand, P.J., The use of carbonic anhydrase inhibitors in ophthalmology and clinical medicine, Ann. N.Y. Acad. Sci., 429 (1984) 609-619.