Serum regulation of acetylcholinesterase in cultured myotubes

EXPERIMENTAL

NEUROLOGY

(1986)

91,308-318

Serum Regulation of Acetylcholinesterase in Cultured Myotubes GARY T. PATTERSON

AND BARRY W. WILSON’

Department of Avian Sciences, University of California, Davis, Cali/otnia 95616 Received July 16, 1985 A large (20s) collagen-tailed form of acetylcholinesterase associated with the neuromuscular junction appears in cultures of chick embryo muscle cells when horse serum is withdrawn from the medium. In this report, I O-day-old cultures were incubated 2 days in serum-free medium or in medium containing either horse, bovine, fetal calf, chicken, heat-treated horse or chicken serum, low (< IOOK) or high (> 1OOK) molecular weight fractions of horse serum, or fibronectin. Total acctylcholinesterase activity and activity of the 20.5 form increased in medium without serum, with fetal calf serum and with the low-molecular-weight fraction of horse serum. The largest increase occurred with fibronectin. The results suggest that a factor(s) greater than 1OOK in adult set-a inhibits total acetylcholinesterase production and formation of the 20s form of the eIUyme.

@ 1986 Academic

F%s, Inc.

INTRODUCTION Acetylcholinesterase (AChE; EC 3.1.1.7) is a specialized protein of excitable tissue that has been extensively used to study mechanisms of development (23). Velocity sedimentation in sucrose density gradients separate small, globular forms (4 to 11s) and larger, collagen-tailed asymmetric forms of AChE [ 16s in rats; 20s in chickens and quail ( 17,18)]. At least four or five molecular forms of AChE with sedimentation coefficients of 2OS, 1lS, 7S, and 4 to 5s Abbreviations: AChE-acetylcholinesterase, MEM-Eagle’s minima1 essential medium. ’ The results herein were submitted by Dr. Patterson in partial completion of the Ph.D at the University of California, Davis, March, 1983; this present address is Dept. of Pharmacology, Vanderbilt University Medical Center, Nashville, TN 37232. The authors are grateful to Ms. P. S. Nieberg and Ms. Janelle Sonoda for their technical assistance in this study and to Dr. W. R. Randall, Dr. M. C. Kenney, and Dr. B. F. King for their useful discussions. The research was supported in part by grants to B.W.W. from the National Institutes of Health (ES 00202) and the Muscular Dystrophy Association. 308 0014-4886/86 $3.00 Copyright Q 1986 by Academic Press, Inc. AI1 rights of rcpmduction in any form reserved.

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are present in avian muscle (4, 18). The collagen-tailed forms have not been detected in ova until after the muscles become innervated (13, 2 1, 22,28). There have been several reports of the appearance of the 20s form of AChE in cultured myotubes after the addition of neurons or neural extracts (7, 16). Bulger et al. (2) showed that the activity of the 20s form dramatically increased after removal of horse serum from the culture medium of both chick and quail muscle cultures. The work reported here examines the effects of replacing the horse serum in the culture medium with other animal sera, heat-treated sera, molecular weight fractions of horse serum, and the serum protein, fibronectin. Some of the results were presented elsewhere in a preliminary form, (20, 28). MATERIALS

AND

METHODS

Primary cultures of embryonic pectoral muscle were prepared from 11 -day chick embryos of a White Leghorn line (03) maintained in the Department of Avian Sciences. Muscle cells were grown without antibiotics for 12 days at 375°C andpH 7.3 in a humidified atmosphere ofair and CO* in a medium of 88% Eagle’s minimal essential medium (MEM) with Earle’s salts (GIBCO, New York), 10% horse serum (K. C. Biological, Lenexa, Ka.), and 2% chick embryo extract (27). The medium was changed 72 h after plating the cells and every 2 days thereafter. The medium was removed on the 10th day in culture, the cultures rinsed with MEM, and the horse serum-containing medium was replaced for 48 h with serum-free medium or with medium containing bovine, newborn calf, fetal calf, chicken, heat-treated (55 to 60°C for 30 min) horse and chicken sera (10% v/v), high- and low-molecular-weight fractions of horse serum, or fibronectin. Embryo extract was maintained at 2% and MEM was increased to 98% of the medium when the serum was removed. Fractions of horse serum with weights above and below 1OOK were prepared using sterilized Diaflow Ultrafilters (Amicon, Danvers, Ma.). The percent of the fractions added to the medium was based on the total protein content of the fraction compared with whole horse serum and in no case exceeded 15% (v/v) of the medium. Fibronectin (provided by Dr. Edward G. Hayman, La Jolla Cancer Research Foundation, La Jolla, Ca.) was added to serum-free culture medium. The highest concentration of urea accompanying the fibronectin had no effect on the growth or AChE of the cells. After 48 h in the test media, the cultures were rinsed with physiologic saline and extracted with a solution of 0.5 M MgC12, 1% Na cholate, 0.05 M TrisHCl, pH 7.3. AChE velocity sedimentation profiles were determined with 5 to 20% sucrose gradients in Triton buffer (1 .O M NaCl, 0.2 mM EDTA, 0.05 A4 Tris-HCl, 0.5% Triton X-100, pH 7.3) with catalase ( 11.4s) and p-galac-

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tosidase (16.0s) as standards. The extracts were centrifuged 18 h at 150,000 g and 4°C and divided into 36 fractions, as described elsewhere (2). AChE activities were assayed radiometrically (10) using 0.2 mM acetylthiocholine iodide (Sigma, St. Louis, MO.) with 200 nCi [3H]acetylcholine iodide (New England Nuclear, Boston, Ma.) as the substrate. Nonspecific cholinesterase (ChE; EC 3.1.1.8) activity was selectively inhibited by the use of 0.1 mM tetraisopropyl pyrophosphoramide (iso-OMPA) in all AChE assays (27). One unit AChE activity represents 1 pmol substrate hydrolyzed per minute at 25 “C. The relative activities of the molecular forms of AChE were estimated by calculating the areas of the curves of AChE vs. fractions over the regions containing the peaks of activity (2). Protein synthesis was determined by measuring the incorporation of [3H]leucine in cultures incubated 1 to 2 h with 5 PCi [3H]leucine per dish. The cells were fixed 5 min on the culture dishes with ice-cold, 5% trichloracetic acid (TCA) rinsed three times with TCA, extracted 1 h in O.lN NaOH (I), and radiometrically counted in Triton-toluene fluor acidified with 10% TCA. Total protein content was determined by the method of Lowry et al. (15), using bovine serum albumin as the standard. AChE was localized at the light microscopic level by the method of Karnovsky and Roots (11) using isoOMPA to inhibit nonspecific ChE activity. RESULTS Cytochemical AChE activity was diffusely distributed in the myotubes of all cultures. Occasionally, clusters of intense AChE activity appeared near the sarcolemma. Intensity of the diffuse staining and the number of clusters of AChE activity associated with the sarcolemma (Fig. 1) were highest in cells incubated without serum, with fetal calf serum, the < 1OOK molecular weight fraction of horse serum, and fibronectin. Total AChE activity of muscle cells was 50% higher in serum-free medium than in horse serum-containing control medium. Activity of the 20s form was threefold greater (Table 1) and the activities of both the 7s and 11s forms were 23 and 27% less, respectively, than control values. Incorporation of [3H]leucine was 65% and total protein content was 74% that of control cultures (Table 2). The morphology, total AChE activity, relative activites of the molecular forms, [3H]leucine incorporation, and total protein of the cultures were not greatly affected when incubated with any of the following sera: chicken, heattreated chicken (55 to 60°C for 30 min), adult bovine, or newborn calf (Tables 1 and 2). However, cultures incubated with adult bovine serum had an incorporation rate of [3H]leucine that was 20% lower than that of controls (Table 2). Total AChE activity was lower and changes in the levels of activity of the 7 and 11s molecular forms of AChE were found in heat-treated horse

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FIG. 1. Localization of AChE activity in I2day chick muscle cultures incubated 2 days in media containing: A-horse serum, B-fetal calf serum, C-> IOOK fraction from horse Serum, D--
serum containing medium (Table 1). Representative profiles of the activities of the molecular forms of AChE from cultures incubated with several of the test media are shown in Fig. 2.

EXPERIMENTAL

NEUROLOGY

91,319-330

(1986)

Effects of Hypoxia on Rat Brain Metabolism: Unilateral in Vivo Carotid Infusion S. I. RAPOPORT, Laborator?, National

W. D. LUST, AND W. R. FREDERICKS’

of Neurosciences. National Institute on Aging and Laboratory ofNeurochemistry, Institute of Neurological and Communicative Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892 Received

August

12, 1985

An in vivo brain perfusion technique was used to examine effects of hypoxia on cerebral cortical metabolism in barbiturate-anesthetized rats. Dulbecco’s phosphatebuffered solution (PBS), or Dulbecco’s PBS + 6 mMghrcose, was infused into the right carotid circulation for 0 to 3 min, at a rate that reduced regional cerebral blood flow to the ipsilateral parietal lobe by more than 40% and O2 delivery by about 50%. The duration of infusion of either solution was correlated negatively with the ipsilateral parietal lobe concentrations of glucose, ATP, and phosphocreatine (PCr), and positively with parietal concentrations of lactate and CAMP. cGMP increased in relation to infusion duration of Dulbecco’s PBS. Statistically significant elevations of brain lactate occurred after 1 min of infusion of Dulbecco’s PBS; lactate was elevated and glucose was reduced after 2 min of infusion of either solution. Brain ATP, PCr, and glycogen concentrations decreased in relation to the elevation in brain lactate, and the [PCr]:[ATP] ratio declined. The results demonstrated that limited hypoxia stimulated cerebral glycolysis and produced a concurrent decrease in brain ATP and PCr. However, ATP was spared to a degree, at the expense of PCr. 0 1986 Academic PWS. I~C.

INTRODUCTION Cerebral metabolism following complete ischemia has been examined by occluding the middle cerebral artery in cats and monkeys, and by occluding Abbreviations: ATP-adenosine triphosphate; rCBF-regional cerebral blood flow; CAMP, cGMP-3’,5’-cyclic adenosine, guanosine monophosphate; GABA--y-aminobutyric acid; PBSphosphate-buffered solution; PCr-phosphocreatine. ’ We thank Dr. J. V. Passonneau for reading the manuscript and providing helpful suggestions. The experiments were conducted in accordance with the “Guide@ the Care and the Use of Laboratory Animals” (DHEW Publication No. NIH 78-23, revised 1978). The present address of Dr. Lust is Department of Neurosurgery, Case Western Reserve School of Medicine, Cleveland. OH 44106. 319 0014-4886/86 $3.00 CopyrigJs 0 1986 by Academic Press, Inc. All rights of reproduction in any form resewed

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FRACTION

FIG. 2. AChE sedimentation profiles from 12-day chick media containing: A-horse serum, B-no serum, C-adult E--> 1OOK fraction of horse serum, F--
muscle cultures incubated 48 h in bovine serum, D-fetal calf serum, hoerse serum, G-100 pg./ml fibroX 10e3. Arrows are locations of 11.4

Cells incubated in the presence of the high-molecular-weight (> IOOK) horse serum fraction were similar to unfractionated horse serum controls. AChE activity was almost 30% higher after incubation with the low-molecular-weight fraction of horse serum (< lOOK, Table 1); activity of the 20s form was 90% higher, activities of the 7s and 11s forms were less, total protein content was 82%, and the [3H]leucine incorporation rate was 78% that of control cultures (Table 2).

314

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TABLE 3 Effects of Fibronectin on Acetylcholinesterase’ Distribution Component

Total AChE

7s

11s

20s 9.9

N

Horse serum control

1.15

74.8

15.3

2

Fibronectin (&ml) 300 100 30 10

0.90 1.83** 1.90**q 1.80”

74.5 51.5 48.6** 51.1**

17.7 14.0 12.6; 14.1

9.8 34.6** 38.8***t 34.7**

2 2 2 2

Serum-free

1.73**

57.8*

10.7*

3 1.6**

2

’ AChE in units per dish X lo-‘; means of pooled samples of four dishes. Distribution: means of percentage activity in each region. N: number of experiments. * P i 0.05 compared with horse serum control.

** P < 0.01. t P < 0.05 compared with horse serum control.

Total AChE activity was higher in cultures treated with 10, 30, or 100 &ml fibronectin than in controls (Tables 3 and 4); the largest difference was in cultures treated with 30 &ml fibronectin; total AChE was 65% above that of controls (10% higher than that of serum-free cultures). However, total AChE was 20% less and total protein and [3H]leucine incorporation were 40% less than of controls in cultures treated with 300 pg/ml fibronectin. with 30

TABLE 4 Effect of Fibronectin on Protein Synthesis’

Component

Protein (mg/dish)

N

[“H]Leucine 6)

N

Horse serum control

2.36

2

100

8

Fibronectin (w/ml) 300 loo 30 10

1.40* 1.83 1.87 1.84

2 2 2 2

61.5 91.3 79.7 75.2

8 8 8 8

Serum-free

1.65*

2

68.7

8

u Protein: means of four dish sets.M number experiments. [3H]Ieucine: percentage of controls. * P < 0.05 compared with horse serum control.

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pg/ml fibronectin (Fig. 2), activity of the 7s form was 35% less, activity of the 11S form was 18% less, and activity of the 20s form was fourfold more than that of the controls. Activities of the 7 and 1 IS forms were less and that of the 20s form not as high as in cultures treated with 10 or 100 pg/ml fibronectin and were comparable to control cultures treated with 300 pg/ml. The total protein content of cultures treated with 10, 30, or 100 pg/ml fibronectin was approximately 22% less than in control cultures. However, this level was still approximately 13% higher than the total protein of serumfree cultures. [3H]leucine incorporation rate was dose-related; cultures treated with 100 pg/ml fibronectin had an incorporation rate similar to that of control cultures, those treated with 30 and 10 &ml had rates 20 and 25%, respectively, lower than that of controls. DISCUSSION Total AChE and the activities of the 7s and 20s forms increased; total protein content and protein synthesis decreased in serum-free cultures. Replacing horse serum with other adult sera (chicken, bovine, heat-treated horse, or heat-treated chicken) or neonatal serum (newborn calf) had no effect on total AChE activity or on the relative activities of its forms in cultured myotubes. Cells incubated in the presence of horse and chicken sera had more total protein and higher rates of protein synthesis than cells incubated in other adult animal sera. The study confirms the findings of Bulger et al. (2) that the 20s form of AChE associated with the motor end-plate preferentially increases in differentiated chick embryo muscle cells when horse serum is removed from the media. Fetal calf serum did not suppress AChE activity or the 20s AChE form. Activities of both total AChE and the 20s form were higher and the rate of protein synthesis was similar to that of control cultures. However, the total protein content of fetal calf serum cultures was also lower than that of control cultures. The activity of the 20s form in the cultures reported here was about eightfold higher than that found by Kato et al. ( 13) in cultures grown in fetal calf serum-containing medium. This may be due to differences in extraction procedures and culturing methods or to the fact that the cells in our study were grown in horse serum-containing medium and tested in other sera for only 2 days, whereas the cells in the study of Kate et al. (13) were grown from the outset in fetal calf serum-containing medium. Traditionally, horse serum, screened for cytotoxicity, has been the serum of choice for culturing muscle cells. In our laboratory, and in others (14) medium containing horse serum consistently provides better growth and more myotubes than any other, including media prepared with fetal calf serum or completely defined medium (5). In a sense, it is not surprising to find that nutrients that were pragmatically chosen for their ability to support morphogenesis are not the most appropriate for supporting later stages of maturation

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in culture. Removal of serum from culture media is known to stimulate a number of cell processes, including the AChE activity of neuroblastoma cells (12), intracellular proteolysis in muscle cultures (9), and protein synthesis and phosphorylation in quiescent Swiss 3T3 cells (24). Toutant et al. (25) found that addition of horse serum to homogenate of cultured muscle caused aggregation of AChE, resulting in broad bands of AChE activity at 15 to 17s and 25 to 30s. Such aggregations were not observed in the experiments presented here. As increases in the activity of low-molecular-weight AChE forms believed to be inside the cells did occur, it seems unlikely that the actions of serum studied here were due to an “aggregation” factor acting solely on extracellular AChE. Regardless, experiments examining this possibility would be of interest. Heating horse or chicken sera 30 min at 55 to 60°C did not destroy their ability to block the appearance of 20s AChE. The time-temperature combination used is one pragmatically applied by cell culturists to reduce “toxic” effects of serum ( 19) and is difficult, a priori, to correlate with the destruction of any particular classes of medium constituents beyond noting that such treatments denature some proteins and nucleic acids. Fibronectin is known to interact with AChE, as it does with many other proteins (29); the 20s form has been reported to be covalently crosslinked with fibronectin (6) and high densities of ACh receptors on the surface of myotubes have been associated with patches of fibronectin (3). It is interesting that the concentration of fibronectin that reduced AChE activity in our experiments (Table 3) was approximately the concentration (300 &ml) found in human plasma (29). Whether this decrease in AChE activity at high concentrations and the modest increase in AChE noted at a lower (30 &ml) concentration of fibronectin signify that this serum protein is one of the regulators of synthesis and assembly of AChE is a matter for future investigations. If fibronectin is necessary for the expression of the 20s form, then it is possible that a factor(s) in adult serum may be inhibiting the interaction of fibronectin with myotube surfaces, specifically inhibiting a domain of fibronectin that is sensitive to the AChE molecule. Fibronectin has been shown to be produced by muscle cultures, both by the myotubules themselves (8) and by mononucleated cells (26). Whether or not such syntheses would be influenced by the presence or absence of fibronectin-containing serum and the < 1OOK fraction are matters for future research. More research is needed to decide whether the effects of sera presented here and in earlier reports (2, 28) are important during muscle development and maturation. One possibility is that adult, but not embryo, tissue fluids contain factors that suppress assembly of the asymmetric forms of AChE everywhere but at nerve-muscle junctions where cells like the Schwann cells protect the synaptic clefts from contact with tissue fluids, providing for localization of the large forms of AChE at the junctions during maturation (2, 28).

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14. KONIGSBERG, I. R. 1979. Skeletal myobiasts in culture. Pages 51 l-527 in W. B. JAKOBY AND I. H. PASTAN, Eds., Methods in Enzymology, Vol. 58. Academic Press, New York. 15. LOWRY, 0. H., N. J. ROSEBROUGH,A. L. FARR, AND R. J. RANDALL. 195 1. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265-275. 16. MARKELONIS, G. J., V. F. KEMERER, AND T. H. OH. 1980. Sciatin: purification and characterization of a myotropic protein from chicken sciatic nerves. J. Biol. Chem. 255: 89678970.

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W. R., AND B. W. WILSON. 1982. Expression of AChE forms in interspecific muscle culture hybrids. In Vitro 18: 324. RIEGER, F., J. KOENIG, AND M. VIGNY. 1980. Spontaneous contractile activity and the presence of the 16s form of acetylcholinesterase in rat muscle cells in culture: reversible suppressive action of tetrodotoxin. Dev. Biol. 76: 358-365. ROSENBERRY,T. L. 1975. Acetylcholinesterase. Adv. Enzymol. 43: 103-218. THOMAS, G., AND M. SEIGMANN. 198 1. Effect of serum, EGF and insulin on S6 phosphorylation and the activation of protein synthesis. J. Cell Biol. 91(2): 3a. TOUTANT, J. P., M. TOUTANT, M. Y. FISZMAN, AND J. MASSOULIE. 1983. Expression of the A 12 form of acetylcholinesterase by developing avian leg muscle cells in vivo and during differentiation in primary cell cultures. Neurochem. Int. 5: 75 l-762. WALSH, F. S., S. E. MOORE, AND S. DHUT. 198 1. Monoclonal antibody to fibronectin: production and characterization using human muscle cultures. Dev. Biol. 84: 12 1- 132. WILSON, B. W., P. S. NIEBERG, C. R. WALKER, T. A. LINKHART, AND D. M. FRY. 1973. Production and release of acetylcholinesterase by cultured chick embryo muscle. Dev.

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E. CHOW, M. G. MCNAMEE, W. R. RANDALL, G. T. PATAND P. S. NIEBERG. 1984. Unifying mechanisms in AChE regulation and organophosphate induced neuropathy of the chicken. Pages 493-506 in M. BRZIN, E. A. BARNARD, AND D. SKET, Eds., Cholinesterases: Fundamental and Applied Aspects. deGruyter, Berlin. 29. YAMADA, K. M. 1983. Cell surface interactions with extracellular materials. Annu. Rev. Biochem. 52: 76 l-799.