- Email: [email protected]
Effect of fibrillation on acetylcholinesterase mRNA in cultured embryonic rat myotubes
Effect of Fibrillation on Acetylcholinesterase in Cultured Embryonic Rat Myotubes
L. H. YOUNKIN,’ Departments
C. F. McTIERNAN,
mRNA
and S. G. YOUNKIN
of Pathology and Pharmacology, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106
Acetylcholinesterase (AChE) and AChE mRNA were evaluated in spontaneously tibrillating myotubes derived from 20-day-old rat fetuses and in matched cultures in which fibrillation was prevented by adding tetrodotoxin on the fourth day of culture. On the eighth day of culture, the AChE activity of fibrillating and nonfibrillating cultures was 5332 and 1861 pmol ACh hydrolyzed min-’ dish-‘, respectively (P
When adult mammalian muscle is denervated, there is a marked increase in the number of extrajunctional acetylcholine receptors [ 1, 21 and a decrease in extrajunctional acetylcholinesterase [2-4]. There is good evidence that the influence of innervation on these extrajunctional proteins is mediated by the electromechanical activity set up in musle by nerve [2, 5, 61. Cultured rat myotubes derived from 20-day embryos normally begin to fibrillate spontaneously on the fourth or fifth day in culture. Effects of this spontaneous electromechanical activity can be studied efficiently by comparing normal fibrillating cultures with those in which fibrillation has been inhibited with tetrodotoxin. In previous experiments, we used this approach to show that electromechanical activity increases the rate at which both globular and asymmetric forms of AChE are synthesized [7]. Soreq and her colleagues [S] have shown that microinjected Xenopus oocytes can be used to evaluate AChE mRNA in tissues where AChE comprises less than 0.01% of total protein. Meedel and Whittaker [9] used this system to show that translationally active AChE mRNA first appears during gastrulation in Ciona intestinalis. In this study, we used Xenopus oocytes to assess the effect of fibrillation on AChE mRNA in cultured embryonic rat myotubes. We could not evaluate AChE mRNA by Northern blot analysis because no cDNA clone for mammalian AChE has been isolated. Our comparison of matched fibrillating and nonfibrillating cultures showed that electromechanical activity significantly increases the level of translationally active AChE mRNA. Cultures were prepared in lOO-mm dishes and AChE was assayed as previously described [71. The culture medium was changed on Day 4. To inhibit fibrillation, tetrodotoxin (final concentration 1.0 @4) was added to half of the cultures in each set at the time that the medium was changed. AChE, ’ To whom reprint requests should be addressed. 279
Copyright @ 1988 by Academic Press, Inc. All rights of reproduction in any form reserved 0014-4827/88 $03.00
280 Short notes TABLE 1 AChE mRNA in fibrillating and nonfibrillating AChE/dish
mRNA/dish
myotubes AChEioocyte produced by myotube mRNA
Expt.
Fibrillating
Nonfibrillating
Fibrillating
Nontibrillating
Fibrillating
Nonfibrillating
1 2 3 4 5 6
3 550 2 965 4 923 6 116 6 801 7 634
1031 1 192 2 295 2 113 2 487 2047
34.8 25.2 41.6 20.8 22.1 19.2
23.5 29.8 40.0 18.9 22.6 17.6
0.47 0.70 0.83 0.27 0.27 0.24
0.05 0.08 0.39 0.00 0.06 0.05
Mean SE
5 332* 688
1 861 224
27.4 3.3
25.4 3.1
0.46* 0.09
0.10 0.05
Note. AChE activities are expressed as picomole ACh hydrolyzed/min. mRNA is expressed as ug poly(A)+ RNA. * P
total poly(A)+ RNA, and AChE mRNA were assessed on Day 8. RNA was extracted from fibrillating and nonfibrillating myotubes using the method of Chirgwin et al. [lo]; poly(A)+ RNA was isolated conventionally using one round of oligo(dT)-cellulose chromatography. Clumps of ovary were removed from anesthetized Xenopus laeuis (Nasco, WI), separated gently into small pieces, and incubated for 18 h on a rocker at 21°C in 5-10 ml of modified Barth’s buffer (88 mhf NaCl, 1.0 m&f KCl, 2.4 n-&f NaHCO,, 0.82 n&f MgS04, 0.33 mM Ca(NO,),.4 H20, 0.41 miV CaClr.2 HzO, 10 mM Hepes, penicillin/streptomycin 16 U/ml, pH 7.6) containing 0.5 mg/ml collagenase II (Cooper Biomedical). The separated oocytes were rinsed in fresh buffer and individual oocytes were selected for injection. Oocytes were injected with 50 nl of poly(A)+ RNA at a concentration of 2 mg/ml in 10 m&f Hepes buffer, pH 7.5. Control oocytes were injected with Hepes buffer alone. Groups of 20 oocytes that had been injected with mRNA from fibrillating cultures, mRNA from matched nontibrillating cultures, or Hepes alone were incubated in parallel in 0.3 ml of modified Barth’s medium at 20°C. After 22-24 h, the incubating medium was removed and each group of 20 oocytes was homogenized in 0.3 ml of 0.1 M phosphate buffer, pH 7.4, containing 1% Triton X-100. AChE was assayed in triplicate with [‘Hlacetylcholine after selectively inhibiting endogenous oocyte cholinesterase activity by adding p-chloromercuriphenylsulfonic acid to the homogenates at a final concentration of 1.O n&f [8]. The AChE produced by mRNA injection was calculated by subtracting the activity of Hepes-injected oocytes from the total activity of mRNA-injected oocytes.
In six separate experiments, we evaluated AChE activity, poly(A)+ RNA, and AChE mRNA in matched fibrillating and nonfibrillating cultures. The results of these experiments are shown in Table 1. As previously reported, fibrillating cultures had significantly more AChE than nonfibrillating cultures (P
Short notes
281
cal activity alters the transcription or post-transcriptional processing of AChE mRNA in a manner that increases the level of translationally active AChE mRNA. Merlie et al. [ 1l] used hybridization analysis with a cloned cDNA specific for AChR a-subunit to show that denervation markedly increases the level of AChR mRNA. Since neural control of extrajunctional AChR is mediated by electromechanical activity, they concluded that neurons are able to use conventional processes initiated by synaptic transmission to regulate gene expression in their synaptic targets. Our observation that electromechanical activity increases the level of translationally active AChE mRNA in cultured embryonic rat myotubes provides strong support for this concept. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
Axelsson, J., and Thesleff, S. (1959) J. Physiol. 147, 178. Fambrough, D. M. (1979) Physiol. Reu. 59, 165. Collins, P. L., and Younkin, S. G. (1982) J. Biol. Chem. 257, 13638. Guth, L., Albers, R. W., and Brown, W. C. (1964) Exp. Neural. 10, 236. Davey, B., and Younkin, S. (1978) Exp. Neurol. 59, 168. Drachman, D. B. (1972) J. Physio/. 226, 619. Brockman, S. K., Younkin, L. H., and Younkin, S. G. (1984) J. Neurosci. 4, 131. Soreq, H., Parvari, R., and Silman, I. (1982) Proc. Natl. Acad. Sci. USA 79, 830. Meedel, T. H., and Whittaker, J. R. (1983) Proc. Nutl. Acad. Sci. USA 80, 4761. Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J., and Rutter, W. J. (1979) Biochemistry
18,
5294.
11. Merlie, J. P., Isenberg, K. E., Russell, S. D., and Sanes, J. R. (1984) J. Cell Biol. 99, 332. Received April 13, 1987 Revised version received August 24, 1987
Printed
in Sweden
L. H. YOUNKIN,’ Departments
C. F. McTIERNAN,
mRNA
and S. G. YOUNKIN
of Pathology and Pharmacology, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106
Acetylcholinesterase (AChE) and AChE mRNA were evaluated in spontaneously tibrillating myotubes derived from 20-day-old rat fetuses and in matched cultures in which fibrillation was prevented by adding tetrodotoxin on the fourth day of culture. On the eighth day of culture, the AChE activity of fibrillating and nonfibrillating cultures was 5332 and 1861 pmol ACh hydrolyzed min-’ dish-‘, respectively (P
When adult mammalian muscle is denervated, there is a marked increase in the number of extrajunctional acetylcholine receptors [ 1, 21 and a decrease in extrajunctional acetylcholinesterase [2-4]. There is good evidence that the influence of innervation on these extrajunctional proteins is mediated by the electromechanical activity set up in musle by nerve [2, 5, 61. Cultured rat myotubes derived from 20-day embryos normally begin to fibrillate spontaneously on the fourth or fifth day in culture. Effects of this spontaneous electromechanical activity can be studied efficiently by comparing normal fibrillating cultures with those in which fibrillation has been inhibited with tetrodotoxin. In previous experiments, we used this approach to show that electromechanical activity increases the rate at which both globular and asymmetric forms of AChE are synthesized [7]. Soreq and her colleagues [S] have shown that microinjected Xenopus oocytes can be used to evaluate AChE mRNA in tissues where AChE comprises less than 0.01% of total protein. Meedel and Whittaker [9] used this system to show that translationally active AChE mRNA first appears during gastrulation in Ciona intestinalis. In this study, we used Xenopus oocytes to assess the effect of fibrillation on AChE mRNA in cultured embryonic rat myotubes. We could not evaluate AChE mRNA by Northern blot analysis because no cDNA clone for mammalian AChE has been isolated. Our comparison of matched fibrillating and nonfibrillating cultures showed that electromechanical activity significantly increases the level of translationally active AChE mRNA. Cultures were prepared in lOO-mm dishes and AChE was assayed as previously described [71. The culture medium was changed on Day 4. To inhibit fibrillation, tetrodotoxin (final concentration 1.0 @4) was added to half of the cultures in each set at the time that the medium was changed. AChE, ’ To whom reprint requests should be addressed. 279
Copyright @ 1988 by Academic Press, Inc. All rights of reproduction in any form reserved 0014-4827/88 $03.00
280 Short notes TABLE 1 AChE mRNA in fibrillating and nonfibrillating AChE/dish
mRNA/dish
myotubes AChEioocyte produced by myotube mRNA
Expt.
Fibrillating
Nonfibrillating
Fibrillating
Nontibrillating
Fibrillating
Nonfibrillating
1 2 3 4 5 6
3 550 2 965 4 923 6 116 6 801 7 634
1031 1 192 2 295 2 113 2 487 2047
34.8 25.2 41.6 20.8 22.1 19.2
23.5 29.8 40.0 18.9 22.6 17.6
0.47 0.70 0.83 0.27 0.27 0.24
0.05 0.08 0.39 0.00 0.06 0.05
Mean SE
5 332* 688
1 861 224
27.4 3.3
25.4 3.1
0.46* 0.09
0.10 0.05
Note. AChE activities are expressed as picomole ACh hydrolyzed/min. mRNA is expressed as ug poly(A)+ RNA. * P
total poly(A)+ RNA, and AChE mRNA were assessed on Day 8. RNA was extracted from fibrillating and nonfibrillating myotubes using the method of Chirgwin et al. [lo]; poly(A)+ RNA was isolated conventionally using one round of oligo(dT)-cellulose chromatography. Clumps of ovary were removed from anesthetized Xenopus laeuis (Nasco, WI), separated gently into small pieces, and incubated for 18 h on a rocker at 21°C in 5-10 ml of modified Barth’s buffer (88 mhf NaCl, 1.0 m&f KCl, 2.4 n-&f NaHCO,, 0.82 n&f MgS04, 0.33 mM Ca(NO,),.4 H20, 0.41 miV CaClr.2 HzO, 10 mM Hepes, penicillin/streptomycin 16 U/ml, pH 7.6) containing 0.5 mg/ml collagenase II (Cooper Biomedical). The separated oocytes were rinsed in fresh buffer and individual oocytes were selected for injection. Oocytes were injected with 50 nl of poly(A)+ RNA at a concentration of 2 mg/ml in 10 m&f Hepes buffer, pH 7.5. Control oocytes were injected with Hepes buffer alone. Groups of 20 oocytes that had been injected with mRNA from fibrillating cultures, mRNA from matched nontibrillating cultures, or Hepes alone were incubated in parallel in 0.3 ml of modified Barth’s medium at 20°C. After 22-24 h, the incubating medium was removed and each group of 20 oocytes was homogenized in 0.3 ml of 0.1 M phosphate buffer, pH 7.4, containing 1% Triton X-100. AChE was assayed in triplicate with [‘Hlacetylcholine after selectively inhibiting endogenous oocyte cholinesterase activity by adding p-chloromercuriphenylsulfonic acid to the homogenates at a final concentration of 1.O n&f [8]. The AChE produced by mRNA injection was calculated by subtracting the activity of Hepes-injected oocytes from the total activity of mRNA-injected oocytes.
In six separate experiments, we evaluated AChE activity, poly(A)+ RNA, and AChE mRNA in matched fibrillating and nonfibrillating cultures. The results of these experiments are shown in Table 1. As previously reported, fibrillating cultures had significantly more AChE than nonfibrillating cultures (P
Short notes
281
cal activity alters the transcription or post-transcriptional processing of AChE mRNA in a manner that increases the level of translationally active AChE mRNA. Merlie et al. [ 1l] used hybridization analysis with a cloned cDNA specific for AChR a-subunit to show that denervation markedly increases the level of AChR mRNA. Since neural control of extrajunctional AChR is mediated by electromechanical activity, they concluded that neurons are able to use conventional processes initiated by synaptic transmission to regulate gene expression in their synaptic targets. Our observation that electromechanical activity increases the level of translationally active AChE mRNA in cultured embryonic rat myotubes provides strong support for this concept. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
Axelsson, J., and Thesleff, S. (1959) J. Physiol. 147, 178. Fambrough, D. M. (1979) Physiol. Reu. 59, 165. Collins, P. L., and Younkin, S. G. (1982) J. Biol. Chem. 257, 13638. Guth, L., Albers, R. W., and Brown, W. C. (1964) Exp. Neural. 10, 236. Davey, B., and Younkin, S. (1978) Exp. Neurol. 59, 168. Drachman, D. B. (1972) J. Physio/. 226, 619. Brockman, S. K., Younkin, L. H., and Younkin, S. G. (1984) J. Neurosci. 4, 131. Soreq, H., Parvari, R., and Silman, I. (1982) Proc. Natl. Acad. Sci. USA 79, 830. Meedel, T. H., and Whittaker, J. R. (1983) Proc. Nutl. Acad. Sci. USA 80, 4761. Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J., and Rutter, W. J. (1979) Biochemistry
18,
5294.
11. Merlie, J. P., Isenberg, K. E., Russell, S. D., and Sanes, J. R. (1984) J. Cell Biol. 99, 332. Received April 13, 1987 Revised version received August 24, 1987
Printed
in Sweden