butyryicholinesterase in nervous tissue of the cat - Europe PMC

Proc. Nati. Acad. Sci. USA

Vol. 84, pp. 7749-7752, November 1987 Neurobiology

Distributions of molecular forms of acetylcholinesterase and butyryicholinesterase in nervous tissue of the cat (Lsoenzymes)

GEORGE B. KOELLE*, JEAN MASSOULIu, DANIEL EUGtNEt, MARIAROSA A. B. MELONEt, GENEVIEVE BOULLA

AND

Laboratoire de Neurobiologie, Ecole Normale Sup~rieure, 46 rue d'Ulm, 75005 Paris, France

Contributed by George B. Koelle, June 29, 1987

We analyzed the activities of acetylcholinesABSTRACT terase and butyrylcholinesterase, and of the metabolic enzymes enolase and lactate dehydrogenase, in the superior cervical ganglion, ciliary ganglion, dorsal root ganglion, stellate ganglion, and caudate nucleus of the cat; we found that these tissues possess very different levels of enzymic activities. The proportions of the aa, ay, and yy enolase isozymes are also quite variable. We particularly studied the molecular forms of acetylcholinesterase and butyrylcholinesterase, in normal tissues and in preganglionically denervated SCG, in comparison with earlier histochemical fimdings. The results are consistent with the premise that the G, (globular monomer) forms of both enzymes are located in the cytoplasm, the G4 (globular tetramer) forms are at the plasma membranes, and the A12 (collagen-tailed, asymmetric dodecamer) form of acetylcholinesterase is at synaptic sites.

Acetylcholinesterase (EC 3.1.1.7; AcChoEase) and butyrylcholinesterase (EC 3.1.1.8; BtChoEase) have been shown to exist in a number of molecular forms, particularly the globular monomer (G1), dimer (G2), and tetramer (G4) and the collagen-tailed (or asymmetric) A4, A8, and A12 forms (1). In the superior cervical ganglion of the rat (2) and steer (3) the G1, G4, and A12 forms predominate. There is evidence (1) to suggest that the G1 form is associated with the cytoplasm or endoplasmic reticulum, the probable site of synthesis (4); the G4 form is probably located at cellular membranes, and the A12 form, by analogy with the motor endplate of skeletal muscle (5), at synaptic sites. Since the distributions of AcChoEase and BtChoEase have been studied extensively in the nervous tissue of the cat by light (6-8) and electron (9-11) microscopic histochemistry, this species appears to be ideal for correlation of biochemical and anatomical data. In this study, we describe the distributions of the molecular forms of AcChoEase and BtChoEase, determined by sucrose gradient sedimentation, in normal and preganglionically denervated superior cervical ganglion (SCG) and in normal ciliary ganglion (CG), dorsal root ganglion (DRG), stellate ganglion (StG), and caudate nucleus (CN). We also compare the levels of AcChoEase and BtChoEase with those of the metabolic enzymes lactate dehydrogenase (LDH) and enolase. Neurons produce a specific type of enolase subunit (y), while glial cells produce only the ubiquitous subunit (a) (12). These subunits form dimers, so that three different isozymes are found in the nervous tissue (13).

METHODS For determination of LDH and enolase activity, the tissues were extracted in 50 mM imidazole HCI buffer, pH 6.8/4 mM The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 7749

MgSO4/40 mM KCl. The supernatant obtained (50,000 x g, 30 min) was assayed for LDH activity with 20 mM (do-lactic acid and 10 mM NAD, in 10 mM Tris-HCI buffer (pH 8). The reaction was monitored at 340 nm. The total enolase activity was assayed by the method of Rider and Taylor (14), and the proportions of aa, ay, and yy isozymes were determined by DEAE-cellulose chromatography, according to the method of Keller et al. (15). The distributions of the molecular forms of AcChoEase and BtChoEase were examined in four different cats; similar results were obtained in all. General anesthesia was induced by the intramuscular injection of ketamine (15 mg/kg). A 1-cm segment was excised from the right vagosympathetic trunk to ensure complete preganglionic denervation of the corresponding SCG. The operative wound was sutured and Combiotic (0.5 ml) was given intramuscularly. Three or four days later, the cat was reanesthetized with sodium pentobarbital (50 mg/kg, intraperitoneally), and SCG, CG, StG, CN, and 20-30 DRG were excised and placed in ice-cold Dulbecco's modified Eagle's medium. The ganglia were trimmed of nerve fibers and connective tissue and weighed; for CG, the tissues obtained from several animals were stored at -70°C and pooled. The ganglia were minced with scissors and the tissues were homogenized in a motor-driven Teflon/ glass Potter homogenizer with extraction medium (1 M NaCI/1% Triton X-100/0.05 M MgCl2/0.01 M Tris-HCI, pH 7.4, containing bacitracin at 0.1 mg/ml) (16), at a volume/ weight (,A/mg) ratio of 6 (DRG), 10 (StG), 20 (CN), or 40 (SCG, CG). Homogenates were centrifuged at 10,000 x g for 15 min in a cold Eppendorf centrifuge; 200 Al of each supernatant was layered on sucrose density gradients (5-20%) containing the same ingredients as the extraction medium. Escherichia coli alkaline phosphatase (6.1. S) and ,B-galactosidase (16 S) were added to the enzyme samples as sedimentation markers. The samples were centrifuged for 18 hr at 39,000 rpm in a Beckman SW41 rotor at 4-5°C. Approximately 42 fractions were collected and assayed by a modification of the method of Ellman et al. (17). For AcChoEase, a final concentration of 0.1 mM Astra 1397 [10-(a-diethylaminopropionyl)phenothiazine] was included in the assay mixture; for BtChoEase, 3 ,uM BW 284C51 [1,5-bis(4-allyldimethylammoniumphenyl)pentan-3-one diAbbreviations: AcChoEase, acetylcholinesterase; BtChoEase, butyrylcholinesterase; LDH, lactate dehydrogenase; CG, ciliary ganglion (ganglia); CN, caudate nucleus; DRG, dorsal root ganglion (ganglia); SCG, superior cervical ganglion (ganglia); StG, stellate ganglion (ganglia). *Present address: Department of Pharmacology, Medical School, University of Pennsylvania, Philadelphia, PA 19104-6084. tPresent address: Departement de Cytologie, Institut des Neurosciences, Unite Associee Centre National de la Recherche Scientifique 1199, Universite Pierre et Marie Curie, 7 Quai Saint-Bernard, 75005 Paris, France. *Present address: Istituto di Scienze Neurologiche, Prima Facolta di Medicina e Chirurgia, Piazza Miraglia, 2-80100 Napoli, Italia.

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bromide] was employed. The reaction mixtures were held at room temperature for 45 min for all tissues excepting the CN (30 min) for AcChoEase, and for 180 min for BtChoEase, and absorbance was read at 412 nm with a Gilford spectrophotometer.

RESULTS AND DISCUSSION We determined the specific activities of AcChoEase and BtChoEase in SCG, CG, StG, DRG, and CN of cats, in comparison with the specific activities of the ubiquitous enzyme LDH. We also determined the total activity of enolase, and the proportions of its aa, ay, and yy isozymic forms. The results are presented in Table 1. It is apparent that each tissue possesses a distinctive pattern of AcChoEase, BtChoEase, LDH, and enolase activities. The proportions of the enolase aa, ay, and yy dimers are themselves variable: the level of the neuron-specific dimers is very high in the CN, high in the CG and DRG, and low or very low in the SCG and StG, respectively. The proportion of hybrid dimers is considerably smaller than would be expected if the association of monomers were random, in all cases except SCG, in agreement with an exclusively neuronal localization of the 'y subunits (12). The presence of ay dimers implies, however, that at least some neurons produce a as well as y subunits, as observed in the case of rat Purkinje cells (18). We analyzed the molecular forms of AcChoEase and BtChoEase by sedimentation in sucrose gradients. Fig. 1 presents the profiles obtained for AcChoEase (solid lines) and BtChoEase (broken lines). They are discussed below, in conjunction with earlier light and electron microscopic histochemical findings. AcChoEase. The cat is unique among common laboratory animals in that it shows, with certain exceptions, relative restriction of AcChoEase to cholinergic neurons (dendrites, perikarya, and axons) and their immediate postsynaptic sites (8). In the SCG, AcChoEase is present in high concentrations throughout the neuropil (the terminations of the preganglionic fibers and the dendrites of the ganglion cells) and only in trace amounts in the perikarya of the adrenergic (>99%) ganglion cells (6, 9, 19). Following preganglionic denervation, the AcChoEase content falls to 15-20% of the control value within a few days (20, 21); at this time it has virtually disappeared from the neuropil, but the low concentration remains in the ganglion cells (6, 10). These observations have led to the hypothesis that a neurotrophic factor, released by the preganglionic fibers, is essential for the maintenance of AcChoEase at postsynaptic dendritic sites (21), where nearly all of the synapses occur (22). To relate these earlier observations to the present findings, the low proportion of GO (at approximately 4 S) and G2 Table 1. Activities of AcChoEase, BtChoEase, LDH, and enolase in cat nervous tissue Enolase Tissue AcChoEase BtChoEase LDH Total y-y ay aa 600 100 0.5 0.9 15% 45% 38% SCG 900 80 CG 1.6 2.8 37% 22% 40% 30 6 0.8 0.9 29%. 24% 47% DRG StG 250 40 1.1 2.0 10% 9% 81% CN 10 0.6 5.0 46% 27% 26% 1800 AcChoEase and BtChoEase activities are expressed as nmol of substrate hydrolyzed per mg of tissue per hr. AcChoEase was assayed with acetylthiocholine as substrate in the presence of 0.1 mM Astra 1397, and BtChoEase with 3 1sM BW 284C51. LDH activity is expressed in OD units (340 nm) per mg of tissue per min, and enolase activity in OD units (240 nm) per mg of tissue per min. The proportions of the yy, ay, and aa enolase isozymes were determined as indicated in Methods.

Proc. Natl. Acad. Sci. USA 84 (1987)

(corresponding to a shoulder at about 7 S) in the normal SCG (Fig. 1A) is assumed to represent the AcChoEase of the cytoplasm, or more specifically at its site of synthesis in the rough endoplasmic reticulum (4). The high amount of G4 (11 S) probably represents the AcChoEase of the plasma membranes of the preganglionic terminals and of the dendrites of the ganglion cells, where most of the electron microscopic histochemical staining occurs (9). The trace amount of the A12 form (17 S) is assumed to represent the enzyme at synaptic sites, possibly only at axosomatic synapses (see below). Four days after denervation (Fig. 1B), there was a marked decrease in G4, consistent with the loss of AcChoEase from the neuropil at this time (6, 10), but no significant change in the level of the other forms. In the rat, in contrast, the perikarya of all neurons of the SCG exhibit variable but significant concentrations of AcChoEase (23, 24). Correspondingly, the G1 and G2 forms approximately equal the G4 form (figure 1 of ref. 2). Preganglionic denervation is followed by a temporary loss of half the G1 form, and a permanent loss of a somewhat greater proportion of G4 (figure 6 of ref. 2). The pattern of AcChoEase observed in the CG is quite different (Fig. 1C). Here the predominant form of AcChoEase was A12, and the next highest proportion was A8, at 13 S; G4 showed a somewhat lower peak, and G2 predominated over G1. The neurons of the parasympathetic CG are essentially all cholinergic, and in the cat the perikarya of all CG neurons exhibit significant staining for AcChoEase (6, 11); this is probably represented here by the G, and G2 forms. The anatomical arrangement is also markedly different from that of the SCG. Most of the synapses occur on large dendrites within the capsules surrounding the individual ganglion cells, and a significant proportion are axosomatic (25), in contrast to the SCG where this relationship is rare (22). Accordingly, the high proportions of A12 and its probable precursor, A8, suggest that these forms may be confined to axosomatic synapses or to synapses occurring on large dendrites. Significant concentrations of the asymmetric forms of AcChoEase have also been reported for the CG of the chicken (26, 27). The bipolar sensory neurons of the DRG exhibit histochemically light staining for AcChoEase in the perikarya and at the plasma membranes, probably represented here by G1 and G4, respectively (Fig. 1D). The relatively high proportion of the former, in comparison with the SCG, suggests that the rate of conversion of G1 to G4 is slower here than in the other ganglia (24). Although the polymerization of G1 into polymeric forms has been established (28), the relationship between these forms is a complex one because of the existence of multiple metabolic pools (29). The function of AcChoEase in the DRG neurons, if one exists, is elusive, since they are neither cholinergic nor cholinoceptive. The actual concentration of AcChoEase is considerably lower in the DRG than in the SCG, StG, and CG. The CN has perhaps the highest concentration of AcChoEase in the central nervous system (30). It is visualized histochemically as a dense mat of heavily stained nerve fibers interspersed with AcChoEase-staining neurons (31, 32). This distribution corresponds here to the high peak of G4 (membranes) and the small amount of G1 (perikarya) (Fig. 1F). The paucity of cholinergic synapses in the CN is suggested by the apparent lack of an A12 peak. BtChoEase. In the SCG of the cat, BtChoEase is completely absent from the cholinergic preganglionic terminals but is present at the dendritic and perikaryonal membranes of the ganglion cells (9). Its concentration in the perikaryonal cytoplasm of the latter cells is so low that it can be detected there only by a pharmacohistochemical technique (24). Following preganglionic denervation, the content of Bt-

Neurobiology: Koelle et al.

Proc. Natl. Acad. Sci. USA 84 (1987) 1100 -

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FIG. 1. Sedimentation profiles of AcChoEase and BtChoEase. Extracts of nervous tissues were prepared as described in Methods, under conditions that allow an essentially complete solubilization of cholinesterases. Samples were deposited on gradients, and 100-1ld aliquots of the fractions were assayed for AcChoEase and for BtChoEase, using acetylthiocholine in the presence of 0.1 mM Astra 1397 or 3 ,uM BW 284C51, respectively. The activities are expressed as nmol of substrate hydrolyzed per mg of tissue per hr in the gradient fraction. The left- and right-hand scales correspond respectively to AcChoEase (solid lines) and BtChoEase (broken lines), the latter scale being four times more expanded in all cases. (A) Normal SCG. (B) Preganglionically denervated (4 days previously) SCG. (C) CG. (D) DRG. (E) StG. (F) CN. AcChoEase activity is of the same order of magnitude in normal SCG, StG, and CG; much higher in CN; and much lower in DRG. The CG is characterized by high levels of A12 and A8 forms, while G4 is largely predominant in the other tissues. The activity of BtChoEase is much lower in the CN than in the other tissues. The major form of BtChoEase is G4 in all cases, together with less abundant G2 and G1 forms. Denervation of the SCG led to a dramatic reduction of G4 AcChoEase, without markedly modifying the levels of G2 or G1 AcChoEase or of BtChoEase.

ChoEase falls over the course of several days to approximately 60% of the control value (20, 21). These distributions are reflected in the high G4 peaks and relatively low G1 peaks in Fig. 1A (normal SCG) and B (denervated SCG). The nearly normal G4 peak in the latter is probably due to the slight

variation in AcChoEase and BtChoEase contents between the right and left ganglia (33). The CG shows, by electron microscopy, the same distribution of BtChoEase as the SCG (11). As in the case of AcChoEase, the CG showed a somewhat more elevated peak

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Neurobiology: Koelle et al.

for G2 and a barely perceptible peak for G1 BtChoEase (Fig. 1C). The neurons of the DRG exhibit histochemically a relatively high concentration of BtChoEase at the plasma membranes of the ganglion cells, and an extremely low concentration in the cytoplasm (6), reflected here by the high G4 peak and very low GI peak (Fig. 1D). The very low corresponding peaks for the CN (Fig. 1F) reflect the extremely small concentration of BtChoEase detected quantitatively (30) and histochemically (34) in the CN of various species. In no case did we find any significant A12 peak of BtChoEase. In conclusion, the present findings are entirely consistent with the hypothesis that, globally, the molecular forms of AcChoEase and BtChoEase correspond to the three types of location indicated in the introduction: G1 and G2 at intracellular sites, G4 at plasma membranes, and A12 at synaptic sites, perhaps mostly axosomatic. We express our deep appreciation to Prof. Jacques Taxi and Dr. Arlette Rougeul-Buser for providing additional laboratory facilities and to Dr. Isabel Llano and Dr. Lucienne Legault-Demare for helpful discussions. G.B.K. is indebted to the Centre National de la Recherche Scientifique (CNRS), France, for fellowship support and for the expenses incurred in this investigation, and M.A.B.M. is indebted to the Union des Myopathes de France for a fellowship. 1. Massoulid, J. & Bon, S. (1982) Annu. Rev. Neurosci. 5, 57-106. 2. Gisiger, V., Vigny, M., Gautron, J. & Rieger, F. (1978) J. Neurochem. 30, 501-516. 3. Bon, S., Vigny, M. & Massoulid, J. (1979) Proc. Nail. Acad. Sci. USA 76, 2546-2550. 4. Fukuda, T. & Koelle, G. B. (1959) J. Biophys. Biochem. Cytol. 5, 433-440. 5. Hall, Z. (1973) J. Neurobiol. 4, 343-361. 6. Koelle, G. B. (1955) J. Pharmacol. Exp. Ther. 114, 167-184. 7. Koelle, W. A. & Koelle, G. B. (1959) J. Pharmacol. Exp. Ther. 126, 1-8. 8. Koelle, G. B. (1963) in Cholinesterases and Anticholinesterase Agents, ed. Koelle, G. B. (Springer, New York), pp. 187-298. 9. Davis, R. & Koelle, G. B. (1979) J. Cell Biol. 78, 785-809. 10. Davis, R. & Koelle, G. B. (1981) J. Cell Biol. 88, 581-590.

Proc. Natl. Acad. Sci. USA 84 (1987) 11. Davis, R., Koelle, G. B. & Sanville, U. J. (1984) J. Histochem. Cytochem. 32, 849-861. 12. Zomzely-Neurath, C. E. (1983) Enzymes in the Nervous System, Handbook of Neurochemistry, ed. Lajtha, A. (Plenum, New York and London), Vol. 4, pp. 403-433. 13. Scarna, H., Keller, A., Pujol, J.-F., Legault-Demare, L., Zeitoun, Y., Lamandd, N., Lando, D. & Cousin, M. A. (1981) Neurochem. Int. 3, 295-301. 14. Rider, C. C. & Taylor, C. B. (1974) Biochim. Biophys. Acta 365, 285-300. 15. Keller, A., Scarna, H., Mermet, A. & Pujol, J.-F. (1981) J. Neurochem. 36, 1389-1397. 16. Bon, S. & Massoulie, J. (1980) Proc. Natl. Acad. Sci. USA 77, 4464-4468. 17. Ellman, G. L., Courtney, K. D., Andres, V., Jr., & Featherstone, R. M. (1961) Biochem. Pharmacol. 7, 88-95. 18. Kato, K., Suzuki, F. & Semba, R. (1981) J. Neurochem. 37, 998-1005. 19. Holmstedt, B. & Sjoqvist, F. (1959) Acta Physiol. Scand. 47, 284-296. 20. Sawyer, C. H. & Hollinshead, W. H. (1945) J. Neurophysiol. 8, 135-153. 21. Koelle, G. B. & Ruch, G. A. (1983) Proc. Natl. Acad. Sci. USA 80, 3106-3110. 22. Elfvin, L.-G. (1963) J. Ultrastruct. Res. 8, 403-440 and 441-476. 23. Eranko, L. (1972) Histochem. J. 4, 545-559. 24. Uchida, E. & Koelle, G. B. (1983) Proc. NatI. Acad. Sci. USA 80, 6723-6727. 25. Tobari, I. (1971) Acta Soc. Opthamol. Jpn. 75, Suppl. 719-738 and 739-747. 26. Vigny, M., Di Giamberardino, L., Couraud, J.-Y., Rieger, F. & Koenig, J. (1976) FEBS Lett. 69, 277-280. 27. Scarsella, G., Toschi, G., Chiappinelli, V. A. & Giacobini, E. (1978) Dev. Neurosci. 1, 133-141. 28. Brocktnan, S. K., Usiak, M. F. & Younkin, S. G. (1986) J. Biol. Chem. 261, 1201-1207. 29. Lazar, M., Salmeron, E., Vigny, M. & Massoulie, J. (1984) J. Biol. Chem. 259, 3703-3713. 30. Burgen, A. S. V. & Chipman, L. M. (1951) J. Physiol. (London) 114, 296-305. 31. Lynch, G. S., Lucas, P. A. & Deadwyler, S. A. (1972) Brain Res. 45, 617-621. 32. Tago, H., Kimura, H. & Maeda, T. (1986) J. Histochem. Cytochem. 34, 1431-1438. 33. Koelle, W. A., Smyrl, E. G., Ruch, G. A., Siddons, V. E. & Koelle, G. B. (1977) J. Neurochem. 28, 307-311. 34. Koelle, G. B. (1954) J. Comp. Neurol. 100, 211-235.