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Histochemical demonstration of Cl−-ATPase in rat spinal motoneurons
Brain Research, 419 (1987)375-378 Elsevier
375
BRE 22464
Histochemical demonstration of Cl--ATPase in rat spinal motoneurons Chiyoko Inagaki, Wasako Oda, Kazuhiro Kondo and Masako Kusumi Department of Biology, Kyoto Pharmaceutical University, Yamashina, Kyoto (Japan) (Accepted 26 May 1987) Key words: CI--ATPase;Na+,K+-ATPase; Motoneuron; Spinal cord; Rat
Using an enzyme-histochemicaltechnique, rat spinal cords were stained for CI--ATPase, Na+,K+-ATPase and anion-insensitive Mg2+-ATPase. C1--ATPaseactivitywas demonstrated in plasma membranes of spinal motoneurons, Na÷,K+-ATPase activityand anion-insensitive Mg2+-ATPaseactivitywere detected in neuronal plasma membranes and blood vessels, respectively.
Inhibitory postsynaptic potentials of motoneurons are known to be generated by C1- ions 1'2,4, whose intracellular concentration is lower than that expected from passive distribution s. To account for such a CIgradient, the presence of a CI- extrusion mechanism has been proposed, but has not been demonstrated yet. Recently, we reported the presence of 'ethacrynic acid (EA) highly sensitive' and 'Cl--stimulated' Mg2+-ATPase (CI--ATPase) as a candidate for a CI-translocating system in the plasma membrane fractions of the brain 5-7,1°,11. This enzyme activity was selectively inhibited by 0.3 mM EA or replacement of medium CI- with NO~. In the present study, these biochemical characteristics were applied for enzymehistochemical demonstration of the localization of C1--ATPase in rat spinal motoneurons. Male Wistar rats weighing 180-200 g were anesthetized with ether, and peffused with a solution containing 333 mM formaldehyde, 155 mM NaC1, 0.5 mM ATP.2Na (Sigma) and 25 mM Tris-acetate (pH 7.4). The cervical segments of the spinal cord were removed and sectioned at 14/xm thickness in a cryostat at -20 °C. The sections were floated on ice-cold 10 mM Tris-acetate (pH 7.4) and stained for ATPase activities within 2 h. The staining procedures were carried out by the method of Wachstein and Meise112. The sections were preincubated for 5 min in freshly
prepared medium containing 3 mM MgCI2, 2.4 mM Pb(NO3)2, 100 mM NaC1, 10 mM KCI and 100 mM Tris-HC1 (pH 7.4) with or without 1 mM ouabain (Sigma) and/or E A (kindly supplied from Merck, Sharp & Dohme). When C1- in the medium was replaced with NO~, Mg(NO3)2, NaNO3, KNO 3 and Tris-HNO 3 (pH 7.4) were used instead of their chloride salts and buffer. Incubation was started by the addition of 3 mM ATP.2Na and carried out for 30 min at 37 °C. After the incubation, sections were treated with 147 mM ammonium sulfide solution for 4 min and then mounted in buffered glycerin for microscoPY. When the sections were incubated in the medium containing C1- without ouabain and EA, neuronal cells, nerve fibers and blood vessels were stained (Fig. 1A). In addition, nerve endings terminating on the motoneurons in the anterior horn were also well stained (Fig. 1B). As shown in Fig. 2, the effect of ouabain, EA or replacement of medium CI- with NO~ on the ATPase activities was evident in plasma membranes of anterior horn motoneurons. When the sections were incubated in C1- or NOj-medium without ouabain and EA, the total ATPase activity in plasma membranes was comparably high in both sections (Fig. 2A, D). By the addition of 1 mM ouabain, plasma membrane ATPase activity was reduced
Correspondence: C. Inagaki, Department of Biology, Kyoto PharmaceuticalUniversity,Yamashina-ku, Kyoto 607, Japan. 0006-8993/87/$03.50(~ 1987Elsevier SciencePublishers B.V. (BiomedicalDivision)
376 (Fig. 2B and E), suggesting that ouabain-sensitive ATPase (Na+,K+-ATPase) activity is located on the plasma membranes. In the presence of 1 mM ouabain, the remaining plasma membrane ATPase activity was much higher in Cl--medium than in NO~-medium (Fig. 2B, E). This suggests that the ouabain-insensitive and Cl--stimulated ATPase (CI--ATPase) activity is located in the plasma membranes. Further, 0.3 mM EA reduced the ouabain-insensitive activity in CI--medium (compare Fig. 2C with Fig. 2B), but not that in NO~-medium (compare Fig. 2F with Fig. 2E). The data also support the idea that C1--ATPase
is located in the plasma membranes. Since the anioninsensitive Mg2+-ATPase activity is not affected by EA at 0.3 mM or NOg (refs. 5-7), almost complete reduction by 1 mM ouabain of plasma membrane ATPase activity in NO~-medium may be due to the lower concentration of anion-insensitive Mg2+-ATPase in the plasma membranes, or to inactivation of the enzyme in the membranes during perfusion. Mitochondrial ATPase is known to be inhibited by NO~ (refs. 3, 7) but not by ouabain9 or E A 5"7, The difference between the intensity of ouabain-insensitive and EA-insensitive staining in the cytoplasmic area
Fig. 1. Enzyme-histochemical staining of ATPase activity in the rat spinal cord. A: the anterior horn of the spinal cord. Large arrows, motoneurons; small arrows, blood vessels; small double arrows, nerve fibers. Bar = 100/~m. B: nerve endings (small arrows) on motoneurons. Bar = 10/~m.
377
CI-
t'" m m
t~ t~ O .ib
< II! 4-
t" m n
t~ t-~
t~ O 4-
Fig. 2. Effects of ouabain (1 mM) and ethacrynic acid (EA, 0.3 mM) on the ATPase staining in the spinal motoneurons. Sections were incubated in C1--medium(A-C) or NO3-medium (D-F). Incubation medium without inhibitors (A,D), with 1 mM ouabain (B,E) or with 1 mM ouabain and 0.3 mM EA (C,F) was used. Large arrows, motoneurons; Small arrows, blood vessels. Bar = 20 b~m.
in Cl--medium and that in NO~-medium (Fig. 2C and F) is probably due to mitochondrial A T P a s e activity.
Findings on other structures are summarized in Table I. The staining pattern of other n e u r o n s in the spinal cord was similar to that of m o t o n e u r o n s , with
378 TABLE I Histochemical evaluation of A TPase activities in various structures of the rat spinal cord
Strong, mild, weak or no staining was scored as 3, 2, 1 or 0, respectively. Mean + S.E.M. of 40 scores from 8 experiments was calculated for each structure. ATPase activities were estimated as follows. Na+,K+-ATPase activity: difference between the scores for staining after the incubation in NO~-medium with and without 1 mM ouabain. CI--ATPase activity: difference between the scores for staining after the incubation in Cl--medium containing 1 mM ouabain with and without 0.3 mM ethacrynic acid (EA). Anion-insensitive Mg2+-ATPase activity: difference between the score for staining after the incubation in NO~-medium with 1 mM ouabain and 0.3 mM EA, and that after the incubation in ATP-free NO~-medium. The significance of difference was analyzed by Student's t-test. +: detectable, P < 0.05, +: faintly detectable, 0.05 < P < 0.10, - : not detectable, 0.10 < P.
Na +,K+-ATPase CI--ATPase Anion-insensitive Mg2+.ATPase
Motoneuron plasma mere branes
Nerve endings
Nerve fibers
Blood vessels
+ +
+ +
+ +
-
-
+
+
+
relatively low C I - - A T P a s e activity. T h e A T P a s e staining in nerve endings and nerve fibers was reduced by 1 m M o u a b a i n and 0.3 m M E A . H o w e v e r , a considerably high level of the activity r e m a i n e d after the t r e a t m e n t with both of the reagents and NO~. 1 Araki, T., Ito, M. and Oscarson, O., Anion permeability of the synaptic and non-synaptic motoneurone membrane, J. Physiol. (London), 159 (1961) 410-435. 2 Coombs, J.S., Eccles, J.C. and Fatt, P., The specific ionic conductions and the ionic movements across the motoneuronal membrane that produce the inhibitory post-synaptic potential, J. Physiol. (London), 130 (1955) 326-373. 3 Ebel, R.E. and Lardy, H.A., Stimulation of rat liver mitochondrial adenosine triphosphatase by anions, J. Biol. Chem., 250 (1975) 191-196. 4 Eccles, J.C., Eccles, R.M. and Ito, M., Effects produced on inhibitory postsynaptic potentials by the coupled injections of cations and anions into motoneurones, Proc. R. Soc. London Ser. B, 160 (1964) 197-210. 5 Hara, M., Miwa, S., Fujiwara, M. and Inagaki, C., Effects of several anions on ethacrynic acid high- and low-sensitive Mg-ATPase activities in microsomal fractions from rabbit cortical gray matter, Biochem. Pharmacol., 31 (1982) 877-879. 6 Hara, M., Fujiwara, M. and Inagaki, C., Non-mitochondrial origin of ethacrynic acid high-sensitive Mg2+-ATPase activity in microsomal fractions from rabbit cortical gray matter, Biochem. Pharmacol., 31 (1982) 4077-4079. 7 Inagaki, C., Tanaka, T., Hara, M. and Ishiko, J., Novel mi-
Thus, nerve endings and fibers s e e m e d to possess both Na + , K + - A T P a s e and C I - - A T P a s e , and a higher level of anion-insensitive Mg2+-ATPase. O n the other hand, A T P a s e activity in b l o o d vessels was unaffected by ouabain, E A and NO~ (Fig. 2), suggesting that anion-insensitive Mg2+-ATPase is p r e d o m i nantly located in b l o o d vessels. A o r t i c smooth muscles have been r e p o r t e d to contain both ouabain-sensitive and ouabain-insensitive A T P a s e using biochemical techniques Z3. T h e discrepancy between these and our data m a y be due to the difference between the distribution patterns of A T P a s e in the smaller and larger b l o o d vessels, or to differences in the other e x p e r i m e n t a l conditions. In the present study, the use of perfusion fluid with a lower concentration of f o r m a l d e h y d e than usual, A T P and NaCI e n a b l e d us to detect C F - A T P a s e activity in the spinal cord sections, though such mild fixation did not allow analysis of smaller neurons and glia cells. D e m o n s t r a t i o n of the localization, of C1-A T P a s e in the p l a s m a m e m b r a n e s of spinal m o t o n e u rons and nerve endings m a y contribute to the study of the mechanisms involved in the inwardly directed CIgradient across these m e m b r a n e s . This work was s u p p o r t e d by grants from the Ministry of Education, Science and Culture and the T a k e d a Science F o u n d a t i o n , Japan, and by the Science Research P r o m o t i o n F u n d of the J a p a n Private School P r o m o t i o n F o u n d a t i o n . crosomal anion-sensitive Mg2+-ATPase activity in rat brain, Biochem. Pharmacol., 34 (1985) 1705-1712. 8 Lux, H.D., Ammonium chloride extrusion: hyperpolarizing synaptic inhibition in spinal motoneurons. Science, 173 (1971) 509-517. 9 Schuurmans Stekhoven. F. and Bonting, L.. Transport adenosine triphospharase: properties and functions. Physiol. Rev., 61 (1981) 1-76. 10 Tanaka, T., Inagaki, C.. Kunugi, Y. and Takaori, S., SolubiUzation and separation of ethacrymc acid (EA) highly sensitive and EA less sensitive Mg2+-ATPase in the rat brain, Jpn. J. Pharmacol.. 43 (1987) 205-2t2. 11 Tanaka, T., Inagaki, C., Matsuda. K. and Takaori, S.. Characteristics of ethacrynic acid highly sensitive Mg2+ATPase in microsomal fractions of the rat brain: functional molecular size, inhibition by SITS and stimulation by CI-. Jpn. J. Pharmacol., 42 (1986) 351-359. 12 Wachstein, M. and Meisel, E.. Histochemistry of hepatic phosphatases at a physiologic pH, Am. J. Clin. Pathol., 27 (1957) 13-23. 13 Wolowyk, M.W., Kidwai. A.M. and Daniel, E.E., Sodium-potassium adenosine triphospbatase of vascular smooth muscle, Can. J. Biochem., 49 (1971) 376-384.
375
BRE 22464
Histochemical demonstration of Cl--ATPase in rat spinal motoneurons Chiyoko Inagaki, Wasako Oda, Kazuhiro Kondo and Masako Kusumi Department of Biology, Kyoto Pharmaceutical University, Yamashina, Kyoto (Japan) (Accepted 26 May 1987) Key words: CI--ATPase;Na+,K+-ATPase; Motoneuron; Spinal cord; Rat
Using an enzyme-histochemicaltechnique, rat spinal cords were stained for CI--ATPase, Na+,K+-ATPase and anion-insensitive Mg2+-ATPase. C1--ATPaseactivitywas demonstrated in plasma membranes of spinal motoneurons, Na÷,K+-ATPase activityand anion-insensitive Mg2+-ATPaseactivitywere detected in neuronal plasma membranes and blood vessels, respectively.
Inhibitory postsynaptic potentials of motoneurons are known to be generated by C1- ions 1'2,4, whose intracellular concentration is lower than that expected from passive distribution s. To account for such a CIgradient, the presence of a CI- extrusion mechanism has been proposed, but has not been demonstrated yet. Recently, we reported the presence of 'ethacrynic acid (EA) highly sensitive' and 'Cl--stimulated' Mg2+-ATPase (CI--ATPase) as a candidate for a CI-translocating system in the plasma membrane fractions of the brain 5-7,1°,11. This enzyme activity was selectively inhibited by 0.3 mM EA or replacement of medium CI- with NO~. In the present study, these biochemical characteristics were applied for enzymehistochemical demonstration of the localization of C1--ATPase in rat spinal motoneurons. Male Wistar rats weighing 180-200 g were anesthetized with ether, and peffused with a solution containing 333 mM formaldehyde, 155 mM NaC1, 0.5 mM ATP.2Na (Sigma) and 25 mM Tris-acetate (pH 7.4). The cervical segments of the spinal cord were removed and sectioned at 14/xm thickness in a cryostat at -20 °C. The sections were floated on ice-cold 10 mM Tris-acetate (pH 7.4) and stained for ATPase activities within 2 h. The staining procedures were carried out by the method of Wachstein and Meise112. The sections were preincubated for 5 min in freshly
prepared medium containing 3 mM MgCI2, 2.4 mM Pb(NO3)2, 100 mM NaC1, 10 mM KCI and 100 mM Tris-HC1 (pH 7.4) with or without 1 mM ouabain (Sigma) and/or E A (kindly supplied from Merck, Sharp & Dohme). When C1- in the medium was replaced with NO~, Mg(NO3)2, NaNO3, KNO 3 and Tris-HNO 3 (pH 7.4) were used instead of their chloride salts and buffer. Incubation was started by the addition of 3 mM ATP.2Na and carried out for 30 min at 37 °C. After the incubation, sections were treated with 147 mM ammonium sulfide solution for 4 min and then mounted in buffered glycerin for microscoPY. When the sections were incubated in the medium containing C1- without ouabain and EA, neuronal cells, nerve fibers and blood vessels were stained (Fig. 1A). In addition, nerve endings terminating on the motoneurons in the anterior horn were also well stained (Fig. 1B). As shown in Fig. 2, the effect of ouabain, EA or replacement of medium CI- with NO~ on the ATPase activities was evident in plasma membranes of anterior horn motoneurons. When the sections were incubated in C1- or NOj-medium without ouabain and EA, the total ATPase activity in plasma membranes was comparably high in both sections (Fig. 2A, D). By the addition of 1 mM ouabain, plasma membrane ATPase activity was reduced
Correspondence: C. Inagaki, Department of Biology, Kyoto PharmaceuticalUniversity,Yamashina-ku, Kyoto 607, Japan. 0006-8993/87/$03.50(~ 1987Elsevier SciencePublishers B.V. (BiomedicalDivision)
376 (Fig. 2B and E), suggesting that ouabain-sensitive ATPase (Na+,K+-ATPase) activity is located on the plasma membranes. In the presence of 1 mM ouabain, the remaining plasma membrane ATPase activity was much higher in Cl--medium than in NO~-medium (Fig. 2B, E). This suggests that the ouabain-insensitive and Cl--stimulated ATPase (CI--ATPase) activity is located in the plasma membranes. Further, 0.3 mM EA reduced the ouabain-insensitive activity in CI--medium (compare Fig. 2C with Fig. 2B), but not that in NO~-medium (compare Fig. 2F with Fig. 2E). The data also support the idea that C1--ATPase
is located in the plasma membranes. Since the anioninsensitive Mg2+-ATPase activity is not affected by EA at 0.3 mM or NOg (refs. 5-7), almost complete reduction by 1 mM ouabain of plasma membrane ATPase activity in NO~-medium may be due to the lower concentration of anion-insensitive Mg2+-ATPase in the plasma membranes, or to inactivation of the enzyme in the membranes during perfusion. Mitochondrial ATPase is known to be inhibited by NO~ (refs. 3, 7) but not by ouabain9 or E A 5"7, The difference between the intensity of ouabain-insensitive and EA-insensitive staining in the cytoplasmic area
Fig. 1. Enzyme-histochemical staining of ATPase activity in the rat spinal cord. A: the anterior horn of the spinal cord. Large arrows, motoneurons; small arrows, blood vessels; small double arrows, nerve fibers. Bar = 100/~m. B: nerve endings (small arrows) on motoneurons. Bar = 10/~m.
377
CI-
t'" m m
t~ t~ O .ib
< II! 4-
t" m n
t~ t-~
t~ O 4-
Fig. 2. Effects of ouabain (1 mM) and ethacrynic acid (EA, 0.3 mM) on the ATPase staining in the spinal motoneurons. Sections were incubated in C1--medium(A-C) or NO3-medium (D-F). Incubation medium without inhibitors (A,D), with 1 mM ouabain (B,E) or with 1 mM ouabain and 0.3 mM EA (C,F) was used. Large arrows, motoneurons; Small arrows, blood vessels. Bar = 20 b~m.
in Cl--medium and that in NO~-medium (Fig. 2C and F) is probably due to mitochondrial A T P a s e activity.
Findings on other structures are summarized in Table I. The staining pattern of other n e u r o n s in the spinal cord was similar to that of m o t o n e u r o n s , with
378 TABLE I Histochemical evaluation of A TPase activities in various structures of the rat spinal cord
Strong, mild, weak or no staining was scored as 3, 2, 1 or 0, respectively. Mean + S.E.M. of 40 scores from 8 experiments was calculated for each structure. ATPase activities were estimated as follows. Na+,K+-ATPase activity: difference between the scores for staining after the incubation in NO~-medium with and without 1 mM ouabain. CI--ATPase activity: difference between the scores for staining after the incubation in Cl--medium containing 1 mM ouabain with and without 0.3 mM ethacrynic acid (EA). Anion-insensitive Mg2+-ATPase activity: difference between the score for staining after the incubation in NO~-medium with 1 mM ouabain and 0.3 mM EA, and that after the incubation in ATP-free NO~-medium. The significance of difference was analyzed by Student's t-test. +: detectable, P < 0.05, +: faintly detectable, 0.05 < P < 0.10, - : not detectable, 0.10 < P.
Na +,K+-ATPase CI--ATPase Anion-insensitive Mg2+.ATPase
Motoneuron plasma mere branes
Nerve endings
Nerve fibers
Blood vessels
+ +
+ +
+ +
-
-
+
+
+
relatively low C I - - A T P a s e activity. T h e A T P a s e staining in nerve endings and nerve fibers was reduced by 1 m M o u a b a i n and 0.3 m M E A . H o w e v e r , a considerably high level of the activity r e m a i n e d after the t r e a t m e n t with both of the reagents and NO~. 1 Araki, T., Ito, M. and Oscarson, O., Anion permeability of the synaptic and non-synaptic motoneurone membrane, J. Physiol. (London), 159 (1961) 410-435. 2 Coombs, J.S., Eccles, J.C. and Fatt, P., The specific ionic conductions and the ionic movements across the motoneuronal membrane that produce the inhibitory post-synaptic potential, J. Physiol. (London), 130 (1955) 326-373. 3 Ebel, R.E. and Lardy, H.A., Stimulation of rat liver mitochondrial adenosine triphosphatase by anions, J. Biol. Chem., 250 (1975) 191-196. 4 Eccles, J.C., Eccles, R.M. and Ito, M., Effects produced on inhibitory postsynaptic potentials by the coupled injections of cations and anions into motoneurones, Proc. R. Soc. London Ser. B, 160 (1964) 197-210. 5 Hara, M., Miwa, S., Fujiwara, M. and Inagaki, C., Effects of several anions on ethacrynic acid high- and low-sensitive Mg-ATPase activities in microsomal fractions from rabbit cortical gray matter, Biochem. Pharmacol., 31 (1982) 877-879. 6 Hara, M., Fujiwara, M. and Inagaki, C., Non-mitochondrial origin of ethacrynic acid high-sensitive Mg2+-ATPase activity in microsomal fractions from rabbit cortical gray matter, Biochem. Pharmacol., 31 (1982) 4077-4079. 7 Inagaki, C., Tanaka, T., Hara, M. and Ishiko, J., Novel mi-
Thus, nerve endings and fibers s e e m e d to possess both Na + , K + - A T P a s e and C I - - A T P a s e , and a higher level of anion-insensitive Mg2+-ATPase. O n the other hand, A T P a s e activity in b l o o d vessels was unaffected by ouabain, E A and NO~ (Fig. 2), suggesting that anion-insensitive Mg2+-ATPase is p r e d o m i nantly located in b l o o d vessels. A o r t i c smooth muscles have been r e p o r t e d to contain both ouabain-sensitive and ouabain-insensitive A T P a s e using biochemical techniques Z3. T h e discrepancy between these and our data m a y be due to the difference between the distribution patterns of A T P a s e in the smaller and larger b l o o d vessels, or to differences in the other e x p e r i m e n t a l conditions. In the present study, the use of perfusion fluid with a lower concentration of f o r m a l d e h y d e than usual, A T P and NaCI e n a b l e d us to detect C F - A T P a s e activity in the spinal cord sections, though such mild fixation did not allow analysis of smaller neurons and glia cells. D e m o n s t r a t i o n of the localization, of C1-A T P a s e in the p l a s m a m e m b r a n e s of spinal m o t o n e u rons and nerve endings m a y contribute to the study of the mechanisms involved in the inwardly directed CIgradient across these m e m b r a n e s . This work was s u p p o r t e d by grants from the Ministry of Education, Science and Culture and the T a k e d a Science F o u n d a t i o n , Japan, and by the Science Research P r o m o t i o n F u n d of the J a p a n Private School P r o m o t i o n F o u n d a t i o n . crosomal anion-sensitive Mg2+-ATPase activity in rat brain, Biochem. Pharmacol., 34 (1985) 1705-1712. 8 Lux, H.D., Ammonium chloride extrusion: hyperpolarizing synaptic inhibition in spinal motoneurons. Science, 173 (1971) 509-517. 9 Schuurmans Stekhoven. F. and Bonting, L.. Transport adenosine triphospharase: properties and functions. Physiol. Rev., 61 (1981) 1-76. 10 Tanaka, T., Inagaki, C.. Kunugi, Y. and Takaori, S., SolubiUzation and separation of ethacrymc acid (EA) highly sensitive and EA less sensitive Mg2+-ATPase in the rat brain, Jpn. J. Pharmacol.. 43 (1987) 205-2t2. 11 Tanaka, T., Inagaki, C., Matsuda. K. and Takaori, S.. Characteristics of ethacrynic acid highly sensitive Mg2+ATPase in microsomal fractions of the rat brain: functional molecular size, inhibition by SITS and stimulation by CI-. Jpn. J. Pharmacol., 42 (1986) 351-359. 12 Wachstein, M. and Meisel, E.. Histochemistry of hepatic phosphatases at a physiologic pH, Am. J. Clin. Pathol., 27 (1957) 13-23. 13 Wolowyk, M.W., Kidwai. A.M. and Daniel, E.E., Sodium-potassium adenosine triphospbatase of vascular smooth muscle, Can. J. Biochem., 49 (1971) 376-384.