Phenylmethylsulfonyl fluoride inhibitory effects on acetylcholinesterase of brain and muscle

Neuropharmacology 38 (1999) 691 – 698

Phenylmethylsulfonyl fluoride inhibitory effects on acetylcholinesterase of brain and muscle Kenneth A. Skau *, Michael T. Shipley 1 Di6ision of Pharmaceutical Sciences, College of Pharmacy and Department of Anatomy and Cell Biology, College of Medicine, Uni6ersity of Cincinnati, 3223 Eden A6enue, Mail Location 4, Cincinnati, OH 45267 -0004, USA Accepted 7 October 1998

Abstract Differential inhibition of brain versus peripheral acetylcholinesterase (AChE) by phenylmethylsulfonyl fluoride (PMSF) suggested that PMSF might preferentially inhibit different AChE molecular forms. AChE inhibition was examined after systemic and in vitro PMSF treatment. Systemic administration resulted in no overt behavioral changes but produced a 71% reduction in brain AChE; hemidiaphragm, extensor digitorum longus and soleus muscles showed 65, 50 and 41% reductions. Muscle asymmetric AChE was reduced to the greatest extent (50 – 80%). The tetrameric form was inhibited in brain and hemidiaphragm (60–76%) but spared in other muscles (18–22%). Monomeric AChE was spared in all tissues. When PMSF was added to a muscle homogenate all forms were inhibited equally. Purified monomer and tetramer forms were inhibited equally in vitro. These results suggest that PMSF inhibition of AChE is a consequence of a selective inhibition of membrane-associated forms and that the apparent brain selectivity is related to the greater fraction of membrane-associated AChE in brain. © 1999 Elsevier Science Ltd. All rights reserved. Keywords: Acetylcholinesterase molecular forms; Phenylmethylsulfonyl fluoride; Alzheimer’s disease

1. Introduction The involvement of central cholinergic neurons in degenerative diseases such as Alzheimer’s dementia [AD] (Whitehouse et al., 1982) has led to clinical trials with anticholinesterase drugs in an attempt to enhance the effects of the dwindling amount of acetylcholine (reviewed by Becker and Giacobini, 1985). Such treatments have proved difficult for several reasons. First, commonly available anticholinesterase drugs, such as neostigmine, pyridostigmine, ambenonium and echothiophate, are quaternary ammonium compounds that penetrate the blood brain barrier poorly. Second, drugs that do penetrate the CNS, such as physostigmine, do not show significant selectivity for brain acetylcholinesterase (AChE; E.C. 3.1.1.7). Doses of * Corresponding author. Tel.: +1-513-5580741; fax: + 1-5135584372; e-mail: [email protected]. 1 Current address. Department of Anatomy, University of Maryland, School of Medicine, HSF Rm 22, 685 W. Baltimore St., Baltimore, MD 21201, USA.

physostigmine sufficient to inhibit brain AChE also produce peripheral cholinergic toxicity (Moss et al., 1985). Physostigmine has been tried in AD with mixed results (Becker and Giacobini, 1985) probably, in part, because of this lack of selectivity. Another reversible inhibitor of AChE, tacrine (tetrahydroaminoacridine) has been shown to concentrate in brain (Nielsen et al., 1989) and has been reported to produce remarkable alleviation of symptoms in AD patients (Summers et al., 1986). However, this effect has not been easily reproduced. More frequently, only modest results have been found with tacrine (Knapp et al., 1994). Also, tacrine can produce severe liver toxicity. A newer AChE inhibitor, donepezil, is also being used to treat AD symptomology and is more selective for AChE (Sugimoto et al., 1992). An alternative method of inhibiting AChE would be to use an irreversible inhibitor. Ideally, one would desire a drug that selectively inhibits brain AChE with less or no effects on peripheral tissue enzyme. Irreversible organophosphate inhibitors of AChE have been proposed for use in AD (Becker et al., 1996), but

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to date have not proven satisfactory. Moss et al. (1985, 1986) have observed that certain sulfonyl fluorides exhibit a selectivity for brain AChE. Their results in rats indicated that phenylmethylsulfonyl fluoride (PMSF, also called phenylmethanesulfonyl fluoride and a-toluenesulfonyl fluoride) or methanesulfonyl fluoride (MSF), when administered either intraperitoneally or orally, could inhibit 80% of brain AChE without causing peripheral cholinergic toxicity. They found that peripheral tissues exhibited less AChE inhibition as a result of a selectivity for brain enzyme as well as a more rapid turnover of peripheral AChE. They speculated that the differential AChE inhibition may be related to differential effects on molecular forms of the enzyme. Our study was initiated to examine the effects of PMSF on the AChE molecular forms of brain and muscle. Bon et al. (1979) classified the molecular forms of AChE into two broad categories of globular and asymmetric forms. The globular forms exist as monomers (G1), dimers (G2) and tetramers (G4) while the asymmetric forms are aggregates of tetramers. These aggregates exist as one (A4), two (A8) and three (A12) tetramers made asymmetric by attachment of a collagen tail. Our results indicate that these molecular forms, per se, show no differential inhibition by PMSF, but the subcellular location may influence the degree of inhibition. Some aspects of this study have been presented in abstract form (Skau and Shipley, 1988).

L3-50 ultracentrifuge and separated into 25 fractions (0.2 ml each). Marker proteins were included to calibrate each gradient. Fractions were assayed for AChE and marker proteins as described below. The separation of the AChE molecular forms has been described in detail (Skau, 1985).

2.2. Biochemical studies In some studies, hemidiaphragms from untreated rats were homogenized as described above. After low speed centrifugation, PMSF (dissolved in methanol) was added to one-half of the supernatant one hour before separation of the molecular forms. Methanol alone was added to the other half of the supernatants. The AChE molecular forms were then separated on sucrose density gradients as described above. In another series of studies, AChE was purified from rat serum (G4 form) and erythrocytes (G1 form) by affinity chromatography on a trimethyl-m-phenylenediamine affinity column (Berman and Young, 1971). The enzyme was eluted with 20 mM decamethonium directly onto a Concanavalin A Sepharose column. Methyl glucopyranoside (0.5 M) was used to elute the purified AChE. The sugar was removed by dialysis against sodium phosphate buffer (0.1 M, pH 7.4). Triton X-100 (1%) was included in all solutions during the purification of the erythrocyte AChE. The effects of PMSF on these purified forms was then assessed.

2.3. Assays 2. Materials and methods

2.1. Animal studies Sprague–Dawley rats (300 – 350 g) were lightly anesthetized with ether and a 1 ml sample of blood was withdrawn from a lateral tail vein. The animals were then injected i.p. with PMSF (85 mg/kg in 1 ml sesame oil) or vehicle. Twenty-four hours later the rats were sacrificed with carbon dioxide and a 1 ml blood sample was drawn by cardiac puncture. Forebrain, olfactory bulbs, left hemidiaphragm (HD), extensor digitorum longus (EDL) and soleus muscles (SOL) were removed to ice cold normal saline. The tissues were rinsed with saline and homogenized (10 ml/g) in buffer of the following composition: NaCl, 1.0 M; EDTA 0.2 mM; Tris –HCl (pH 7.424 ), 50 mM; Triton X-100, 1%. The homogenates were centrifuged at 100 000×g for 15 min (4°C) and the supernatants were assayed for total AChE activity. In addition, 0.1 ml of each supernatant was layered onto 5 – 20% linear sucrose density gradients prepared in homogenization buffer. The gradients were ultracentrifuged at 121 000×gav for 15 h at 4°C in a Beckman

For routine assays of AChE activity in tissue extracts the procedure of Ellman et al. (1961) was used. To assay AChE from gradient fractions the more sensitive radiometric assay of Potter (1967) was used. All samples, in both assays, were pretreated with 45 mM iso-OMPA (tetraisopropylpyrophosphoramide) to inhibit butyrylcholinesterase (E.C. 3.1.1.8). In the density gradient studies, the marker proteins were assayed as previously described (Skau, 1985). Bovine serum albumin (B; 4.4S) was estimated with the Bio-Rad dye binding protein assay (Bio-Rad Labs., Hercules, CA). Catalase (C; 11.2S) was assayed enzymatically through its action on hydrogen peroxide (Skau and Brimijoin, 1980). b-galactosidase (G; 16S) was assayed enzymatically by its action on o-nitrophenyl, b-D-galactopyranoside (Craven et al., 1965).

2.4. Data analysis Comparisons of PMSF treated rat tissues with control rat tissues were made with Student’s t-test. We used the techniques of Dreyfus et al. (1984) to quantitate the individual AChE molecular forms by resolving the peaks as gaussian curves.

K.A. Skau, M.T. Shipley / Neuropharmacology 38 (1999) 691–698 Table 1 PMSF inhibition of brain and muscle total AChE activity after systemic administration Tissue

Serum Day 1 Day 2 Forebrain Olfactory bulb Hemidiaphragm Extensor digitorum Longus Soleus

n

AChE activity* Control

PMSF

% Inhibited

7 7 6 6 7

378.19 68.3 382.4958.3 1233.59105.9 329.9939.9 122.89 22.0

313.59 46.1 137.0929.4§ 349.89 42.5† 96.0 9 17.1a 42.4 9 9.3b

N.A. 56.3 71.6 70.9 65.5

7 7

133.6916.6 92.29 13.5

66.59 16.0§ 54.6 9 11.0b

50.2 40.8

* Enzyme activity was measured by the Ellman assay in supernatants from homogenized tissues. Results are expressed as mU/0.1 g except for serum which is expressed as mU/ml and represent means 9 SEM. NA = not applicable. † PB0.001 versus control. a PB0.005 versus control. § PB0.01 versus control. b PB0.05 versus control.

3. Results

3.1. General obser6ations Administration of PMSF caused no gross alterations in the behavior of the rats in this study. We observed no muscle twitching, salivation, diarrhea nor any other sign of cholinergic overload within the time frame of the study. We did observe a significant inhibition of

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AChE activity in all tissues studied (Table 1). Rat serum, unlike human, has a high concentration of AChE (Fernandez et al., 1979; Skau, 1985) and thus served as a convenient measure of the inhibitory action of PMSF. In the 24 h period of these studies serum AChE was reduced about 55%. Brain AChE, both forebrain and olfactory bulb, was reduced by more than 70% when compared to controls. There was significant variation in the inhibition of skeletal muscle AChE. Over 65% of HD AChE was inhibited while the EDL enzyme was only 50% inhibited and SOL exhibited only 40% inhibition.

3.2. Systemic PMSF effects on AChE molecular forms Forebrain and olfactory bulb contained two forms of AChE activity (Fig. 1). Most of the enzyme (90%) was present as a tetramer that is believed to be primarily an externally facing membrane-bound enzyme (Bon et al., 1979). The remainder of the AChE corresponds to a monomer. These forms were differentially inhibited by systemically administered PMSF (Table 2). In the forebrain, more than 75% of the tetrameric form, but only 53% of the monomer, was inhibited. Similar ratios were evident in the olfactory bulb. Thus, the monomer accounts for a greater percentage of the total AChE in PMSF inhibited brains (11–19%) versus controls (8– 9%). The three skeletal muscles examined in this study showed differential distribution of the AChE forms (Fig. 2). In HD (Fig. 2A) four molecular forms could be resolved. Two asymmetric forms, A8 and A12, constituted 6.7 and 17.6% of total AChE in control tissues (Table 2). These forms were inhibited about 77% in

Fig. 1. PMSF inhibition of molecular forms of brain AChE. AChE was solubilized from forebrain (A) and olfactory bulbs (B) 24 h after i.p. injection of PMSF (85 mg/kg). The molecular forms were separated on 5 – 20% linear sucrose density gradients. AChE activity is expressed as nmoles of ACh hydrolyzed/h − 1 per fraction. Marker proteins used to calibrate the gradients were b-galactosidase (G; 16S), catalase (C; 11.2S) and bovine serum albumin (B; 4.4S) Each value is the mean9SEM of five control (open circles) and five PMSF injected (filled circles) rats.

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Table 2 PMSF inhibition of molecular forms of AChE from brain and muscle tissues after systemic administration Tissue

Forebrain Control Treated Olfactory bulb Control Treated Hemidiaphragm Control Treated SOL Control Treated EDL Control Treated

AChE activity* G1

G4

A8

A12

Total

195.39 17.93 (8.5) 69.99 4.75a (11.7)

2043.99 244.6 (88.7) 510.2 9 76.11† (85.6)

— —

— —

2304.1 9 25 596.3 988.43†

75.59 13.03 (9.2) 41.69 10.31 (19.0)

729.59 114.79 (88.8) 170.8 9 38.58a (78.0)

— —

— —

821.9 9126.3 218.9 9 49.83§

54.3912.3 (33.1) 28.497.94 (43.5)

64.99 6.61 (39.6) 25.79 6.36§ (39.4)

11.3 92.64 (6.9) 2.7 90.8§ (4.1)

28.5 9 4.0 (17.4) 6.5 91.9a (10.0)

164.0922.28 65.3 916.92§

41.49 4.88 (32.6) 33.59 5.89 (43.4)

21.69 1.40 (17.0) 16.89 3.03 (18.3)

28.4 9 1.29 (22.4) 14.1 9 2.11† (18.4)

34.7 9 2.15 (27.3) 13.0 92.25† (16.8)

127.095.87 77.29 11.17§

79.39 5.68 (48.3) 50.39 12.2 (47.2)

50.29 7.89 (30.6) 41.39 4.65 (38.7)

—

30.6 9 6.84 (18.6) 14.1 9 3.61a (13.2)

164.2 916.94 106.6 918.97

* Enzyme activity expressed as nmoles of AChE hydrolyzed/h−1 per peak. Numbers in parenthesis are percent of total. Each value is the mean9 SEM of five tissues. † PB0.005 versus control. a PB0.01 versus control. § PB0.05 versus control.

PMSF treated animals. The globular forms, G1 and G4, accounted for 31 and 41% of total AChE and were inhibited 48 and 60% by PMSF. SOL muscles were the least affected of the tissues examined. SOL had a greater concentration of the asymmetric A8 and A12 forms (22.5 and 27.5% of total AChE) which were less affected by PMSF (50.5 and 73% inhibited) than were asymmetric forms in HD and EDL. The globular G1 and G4 (32 and 17% of total) were even less inhibited by PMSF (19 and 22%). AChE activity in EDL muscles was somewhat more variable than in HD and SOL. In EDL only one asymmetric form (A12) was consistently observed. As with HD and SOL, this A12-AChe was most severely inhibited by PMSF. However, unlike the other tissues, the G4 form was inhibited to only a minor extent. The G1 form was inhibited to a greater extent than the G4, but less than the asymmetric form.

3.3. In 6itro inhibition by PMSF The apparent differential effects of PMSF on the molecular forms of AChE led us to examine, directly, whether or not the drug selectively inhibited the various molecular forms. In the first series of studies, AChE was solubilized from HD and PMSF was added directly to the solubilized enzyme, prior to separation of the molecular forms. Whereas systemic injection of PMSF exhibited differential inhibition of AChE molecular forms (Fig. 2, Table 2), when the

drug was added directly to solubilized enzyme this selectively was not evident (Fig. 3, Table 3). As previously demonstrated (Fernandez et al., 1979; Skau, 1985), rat plasma contains only G4-AChE and rat erythrocytes contain only G1 enzyme. We took advantage of these selective pools of enzyme forms to examine the effects of PMSF on individual forms without the need to separate the forms on density gradients. Rat whole blood was centrifuged to separate the erythrocytes and serum. The serum was dialyzed (MW cutoff: 12–14 000) for 12 h against 10 mM sodium phosphate (pH 7.4), centrifuged at 100 000× g for 30 min and the AChE was purified by affinity chromatography. Erythrocytes were washed three times with isotonic sodium phosphate (0.155 M), lysed by freeze-thawing in hypotonic phosphate buffer (0.015 M) and centrifuged at 10 000 ×g. The erythrocyte membranes were washed five times with hypotonic buffer, and AChE was solubilized in 10 mM phosphate buffer (pH 7.4) containing 1% Triton X-100. After centrifugation (100 000×g for 30 min) the solubilized AChE was purified by affinity chromatography. The partial purification of these molecular forms resulted in single peaks of enzyme (G4 for serum, G1 for erythrocytes) with no contaminating butyrylcholinesterase or hemoglobin to interfere with the spectrophotometric assay. There were no significant differences in the inhibitory activity of PMSF on these partially purified AChE molecular forms (Fig. 4).

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Fig. 2. PMSF inhibition of molecular forms of skeletal muscle AChE. Enzyme was solubilized from hemidiaphragm (A), soleus (B) and extensor digitorum longus (C) muscles. AChE activity and marker proteins are as described in Fig. 1. Each value is the mean 9SEM of five control (open circles) and five PMSF injected (filled circles) rats.

4. Discussion Since most mammalian tissues are generally considered to have an excess of AChE, it is not surprising that PMSF produced no gross signs of cholinergic overload. Moss et al. (1985) similarly reported no cholinergic symptoms. Our results show slightly less inhibition of brain AChE than did Moss. They also reported only about 35% inhibition of pectoral muscle AChE, which is similar to our results in SOL muscle. However, our results in HD and EDL show considerably more inhibition of AChE. Both forebrain and olfactory bulb of the rat exhibited patterns of AChE molecular forms similar to what has been reported for rat brain by others (Rieger and Vigny, 1976); forebrain had about three times as much enzyme activity, perhaps reflecting the cholinergic nature of this structure vs the cholinoceptive nature of

olfactory bulbs. Consistent with previous results, no significant asymmetric peaks were observed in either brain tissue. It has been estimated that asymmetric AChE constitutes less than 0.2% of total brain AChE (Rieger et al., 1980). Both structures had about 90% of the total AChE sedimenting in a peak that corresponds to a G4 form of the enzyme. Although studies on whole brain are not definitive, those with cultured neuronal cells suggest that 60–90% of this tetrameric enzyme is ectocellular (Taylor et al., 1981). By contrast, 90% of the monomeric AChE in neuronal cells appears to be intracellular. In our studies this intracellular enzyme appeared to be relatively spared from the inhibitory action of PMSF. Whereas the tetrameric form was inhibited 75% by PMSF, the monomer was only inhibited 45–64%. There appears to be only one mammalian gene for AChE, with the different molecular forms representing alternate exon expression and/or post-

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696

Fig. 3. PMSF inhibition of solubilized molecular forms of AChE from skeletal muscle. PMSF was added to solubilized samples of hemidiaphragm AChE in vitro 1 h prior to ultracentrifugation to separate the molecular forms. AChE activity and marker proteins are as described in Fig. 1. Values are mean 9 SEM of three samples of untreated (circles), PMSF = 0.05 mM (squares) and PMSF = 0.1 mM (triangles).

translational modifications of the protein (Li et al., 1991). The active sites for all of the molecular forms are identical. Thus, the differential inhibition of molecular forms cannot be attributed to differential inhibition at the active sites. Instead, the results suggest that PMSF may exert a selective inhibition of AChE molecular forms by acting primarily on those forms of the enzyme that are associated with external membranes. PMSF is a highly hydrophobic substance that may become concentrated in membrane lipid and partition poorly into the aqueous internal compartment of cells. Enz et al. (1993) reported on a brain-selective carbamylating AChE inhibitor, SDZ ENA 713 which appeared to preferentially inhibit the G1-AChE. They suggested that this effect may be desirable as it appeared to normalize the ratio of G4/G1 enzyme in Alzheimer’s disease. They, and others (Younkin et al., 1986) have found a selective reduction of G4-AChE in brains of Alzheimer’s patients. There are two difficulties Table 3 Effects of in vitro PMSF on hemidiaphragm AChE molecular forms [PMSF] mM

AChE (% of total)

G1 0 0.05 0.10

G4

33.790.47 36.59 3.55 30.7 91.49 41.89 4.95 25.0 92.34* 44.59 4.19

* PB0.05 versus no PMSF.

A8

A12

10.29 2.54 11.09 2.21 10.3 9 2.37

15.9 9 1.33 14.69 0.52 18.9 9 0.40

Fig. 4. PMSF inhibition of individual molecular forms of AChE in vitro. PMSF, 0.5 (open symbols) or 1.0 mM (closed symbols) was added to purified G4 AChE from plasma (circles) or G1 AChE from erythrocytes (triangles). At various times the samples were assayed for AChE activity and compared to untreated enzyme. Values are mean9 SEM of three determinations run in triplicate.

with these results. First, Enz et al. separated the two forms of AChE by extracting brain tissue without detergent followed by an extraction with 1% Triton X-100. They then treated the solubilized enzyme forms with inhibitor. We have found (manuscript in preparation) that certain carbamylating drugs are less effective in the presence of Triton X-100. Thus, the differential effect that they observed might be an artifact of the in vitro techniques that they used. Second, there is no indication that normalizing the G4/G1 ratio would be of any benefit. Since G1-AChE is found primarily in the cytoplasm of neuronal cells, it is unlikely that reversible inhibition of this form would have any significant effect on metabolism of acetylcholine. By contrast, the G4 form is primarily ectocellular and is the major form that metabolizes acetylcholine. Inhibition of this form would be expected to prolong the action of acetylcholine. The effects of PMSF on skeletal muscle were more complex. Inhibition of total AChE varied from 40% in SOL to 65% in HD. In all three muscles studied, the asymmetric forms were most severely affected by PMSF. Studies on the subcellular distribution of AChE in skeletal muscle have focused on HD because this relatively thin muscle allows penetration of inhibitors to all muscle fibers. The thicker EDL and SOL might result in exclusion of the deeper fibers from contact with inhibitors. The studies on HD suggest that asymmetric forms of AChE are concentrated at the endplate region where they are predominantly ectoproteins believed to be attached to the basal lamina of the muscle fiber by ionic interactions between the collagen tail and

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the heparan proteoglycans (Younkin et al., 1982; Torres and Inestrosa, 1983). However, although the greatest concentration of asymmetric AChE occurs at the endplate, a significant amount of asymmetric enzyme is present throughout the myofiber in intracellular sites (Younkin et al., 1982). This may represent asymmetric AChE that has been synthesized, but not yet externalized. The HD, by nature, has a greater area of endplates than does the EDL or SOL. Hence, the HD is likely to have a greater proportion of asymmetric enzyme external to the myofibers. If PMSF is relatively excluded from intracellular compartments, this may explain why a greater percent of asymmetric AChE was inhibited in HD. Two major peaks of globular AChE were present in all three muscles, although the tetrameric form in SOL was much less (as a percent of total) than in the other muscles. Younkin et al. (1982) have suggested that 70% of the G1-AChE is intracellular while less than 30% of G4-AChE is internal. This is consistent with our hypothesis that the molecular forms of AChE with active sites external to the cell are most susceptible to PMSF since the intracellular G1 was inhibited less than 50% but the membrane bound G4 was inhibited greater than 60%. Both globular forms of the enzyme in SOL and EDL were resistant to PMSF. However, there is little information on the subcellular location of the globular forms in these tissues. It is possible that these muscles have primarily intracellular pools of globular AChE. Further work is needed to establish the subcellular locations of the AChE molecular forms in these tissues. We considered the possibility that the apparent differential inhibition of AChE molecular forms by PMSF could be due to a differential effect on the individual molecular forms per se. To test this, we conducted two series of experiments. In the first of these, PMSF was added directly to solubilized muscle AChE prior to separation of the molecular forms on density gradients. In this case, all molecular forms were inhibited to approximately the same extent. The only significant difference was a greater inhibition of G1-AChE at the higher PMSF concentration (0.1 mM). This is the reverse of the effect of PMSF administered i.p. in which the G1 was most resistant. One explanation for the apparent sensitivity of the G1 form might be in the nature of the experiments. PMSF was added to solubilized AChE 1 h prior to initiation of density gradient centrifugation. PMSF, as a relatively small molecule, will penetrate the density gradient slowly, as will the G1 enzyme. Thus, the slow moving G1 was likely to be in contact with the inhibitor for a greater period of time than were the larger molecular forms. In support of this are the results of PMSF inhibition of purified AChE from plasma (G4) and erythrocytes (G1). There were no significant differences in the rate at which either 0.5 mM or 1.0 mM PMSF inhibited these forms of AChE.

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Although we favor the hypothesis of a differential penetration of PMSF to subcellular sites, we cannot eliminate the possibility that the differential effects observed might be due to different rates of synthesis of AChE. In the brain this would seem unlikely as brain AChE has a very slow turnover rate [approximately 11 days] (Moss et al., 1985). Skeletal muscle AChE is synthesized more rapidly and some of the effects that we observed might reflect this more rapid synthesis. In summary, these results show a differential inhibitory effect of PMSF on AChE molecular forms of brain and skeletal muscle. The differential inhibition appears to be related to a distributional effect in which enzyme associated with external membranes was most affected. The preponderance of ectoprotein AChE in the brain resulted in an overall greater inhibition of AChE in this tissue, whereas the greater amount of intracellular AChE in muscle confers some protection. Although it is unlikely that PMSF, itself, would be useful in degenerative diseases involving loss of cholinergic neurons, it is possible that derivatives of this compound might retain the differential brain AChE inhibitory actions without producing severe toxic effects. In fact, Moss et al. (1999) have presented preliminary data that MSF treatment improved cognitive performance in patients with Alzheimer’s dementia. However, it is necessary to establish that chronic treatment with sulfonyl fluorides continue to result in brain AChE inhibition without producing peripheral toxicity.

Acknowledgements The authors wish to thank Diane Hymel for technical assistance. This work was supported in part by the University of Cincinnati Research Council, the US Army Medical Research and Development Command DAMD 17-86-C-6005, and grant NINCD DC 00347.

References Becker, R.E., Colliver, J.A., Markwell, S.J., Moriearty, P.L., Unni, L.K., Vicari, S., 1996. Double-blind, placebo-controlled study of metrifonate, an acetylcholinesterase inhibitor, for Alzheimer Disease. Alz. Dis Assoc. Dis. 10, 124 – 131. Becker, R.E., Giacobini, E., 1985. Mechanisms of cholinesterase inhibition in senile dementia of the Alzheimer type: clinical, pharmacological and therapeutic aspects. Drug Dev. Res. 12, 163 – 195. Berman, J.D., Young, M., 1971. Rapid and complete purification of acetylcholinesterases of electric eel and erythrocyte by affinity chromatography. Proc. Natl. Acad. Sci. USA 68, 395 – 398. Bon, S., Vigny, M., Massoulie, J., 1979. Asymmetric and globular forms of acetylcholinesterase in mammals and birds. Proc. Natl. Acad. Sci. USA 76, 2546 – 2550. Craven, G.R., Steers, E., Anfinsen, C.B, 1965. Purification, composition, and molecular weight of the b-galactosidase of Escherichia coli K12. J. Biol. Chem. 240, 2468 – 2484.

698

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Dreyfus, P.A., Friboulet, A., Tran, L.H., Rieger, F., 1984. Polymorphism of acetylcholinesterase and identification of new molecular forms after sedimentation analysis. Biol. Cell 51, 35–41. Ellman, G.L., Courtney, K.D., Andres, V., Featherstone, R.M., 1961. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol. 7, 88–95. Enz, A., Amstutz, R., Boddeke, H., Gmelin, G., Malanowski, J., 1993. Brain selective inhibition of acetylcholinesterase: a novel approach to therapy for Alzheimer’s disease. Progr. Brain Res. 98, 431 – 438. Fernandez, H.L., Duell, M.J., Festoff, B.W., 1979. Neurotrophic control of 16S acetylcholinesterase at the vertebrate neuromuscular junction. J. Neurobiol. 10, 441–454. Knapp M.J., Knopman, D.S., Solomon P.R., Pendlebury W.W., Davis C.S. for the Tacrine Study Group, 1994. A 30-week randomized controlled trial of high-dose tacrine in patients with Alzheimer’s disease. J. Am. Med. Assoc. 271, 985–991. Li, Y., Camp, S., Rachinsky, T.L., Getman, D, Taylor, P., 1991. Gene structure of mammalian acetylcholinesterase. Alternative exons dictate tissue-specific expression. J. Biol. Chem. 266, 23083 – 23090. Moss D., Berlanga P., Hagan M., Sandoval H. Ishida C., 1999. Methanesulfonyl fluoride (MSF): a double blind, placebo controlled study of safety and efficacy in the treatment of senile dementia of the Alzheimer type. Alz. Dis. Assoc. Disord. (in press). Moss, D., Rodriguez, L., Selim, S., Ellett, S., Devine, J., Steger, R., 1985. The sulfonylfluorides: CNS-selective cholinesterase inhibitors with potential value in Alzheimer’s Disease? In: Hutton, J.T., Kenny, A.D. (Eds.), Proceedings of the Fifth Tarbox Parkinson’s Disease symposium: The Norman Rockwell Conference on Alzheimer’s Disease. Alan R. Liss, New York, pp. 337–350. Moss, D., Rodriguez, L., Herndon, W., Vincenti, S., Camarena, M., 1986. Sulfonyl fluorides as possible therapeutic agents in Alzheimer’s disease: structure/activity relationships as CNS selective cholinesterase inhibitors. In: Fisher, A., Hanin, I., Lachman, C. (Eds.), Alzheimer and Parkinson’s Diseases. Plenum Publ. Corp, New York, pp. 551–556. Nielsen, J., Mena, E., Williams, I., Nocerini, M., Liston, D., 1989. Correlation of brain levels of 9-amino-1,2,3,4-tetrahydroacridine (THA) with neurochemical and behavioral changes. Eur. J. Pharmacol. 173, 53 – 64.

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Potter, L., 1967. A radiometric microassay of acetylcholinesterase. J. Pharmacol. Exp. Ther. 156, 500 – 506. Rieger, R., Chetelat, R., Nicolet, M., Kamal, L., Poullet, M., 1980. Presence of tailed asymmetric forms of acetylcholinesterase in the central nervous system of vertebrates. FEBS Lett. 121, 169–173. Rieger, F., Vigny, M., 1976. Solubilization and physicochemical characterization of rat brain acetylcholinesterase: development and maturation of its molecular forms. J. Neurochem. 27, 121– 129. Skau, K.A., 1985. Acetylcholinesterase molecular forms in serum and erythrocytes of laboratory animals. Comp. Biochem. Physiol. 80C, 207 – 210. Skau, K.A., Brimijoin, S., 1980. Multiple molecular forms of acetylcholinesterase in rat vagus nerve, smooth muscle, and heart. J. Neurochem. 35, 1151 – 1154. Skau, K.A., Shipley, M.T., 1988. Phenylmethylsulfonyl fluoride (PMSF) inhibition of brain and muscle acetylcholinesterase. The Pharmacologist 30, A81. Sugimoto, H., Iimura, Y., Yamanishi, Y., Yamatsu, K., 1992. Synthesis and anti- acetylcholinesterase activity of 1-benzyl4-[(5,6-dimethoxy-1-indanon-2-yl)methyl]piperidine hydrochloride (E2020) and related compounds. Bioorg. Med. Chem. Lett. 2, 871 – 876. Summers, W., Majovski, L., Marsh, G., Tachiki, K., Kling, A., 1986. Oral tetrahydroaminoacridine in long-term treatment of senile dementia, Alzheimer type. New Engl. J. Med. 315, 1241–1245. Taylor, P., Rieger, F., Shelanski, M., Greene, L., 1981. Cellular localization of the multiple molecular forms of acetylcholinesterase in cultured neuronal cells. J. Biol. Chem. 256, 3827 – 3830. Torres, J.C., Inestrosa, N.C., 1983. Heparin solubilizes asymmetric acetylcholinesterase from rat neuromuscular junction. FEBS Lett. 154, 265 – 268. Whitehouse, P.H., Price, D.H., Struble, R.G., Clarke, A.W., Coyle, J.T., DeLong, M.R., 1982. Alzheimer’s disease and senile dementia: loss of neurones in the basal forebrain. Science 215, 1237– 1239. Younkin, S.G., Goodridge, B., Katz, J., Lockett, G., Nafziger, D., Usiak, M.F., Younkin, L.H., 1986. Molecular forms of acetylcholinesterases in Alzheimer’s disease. Fed. Proc. 45, 2982–2988. Younkin, S.G., Rosenstein, P.L., Collins, P.L., Rosenberry, T.L., 1982. Cellular localization of the molecular forms of AChE in rat diaphragm. J. Biol. Chem. 257, 13630 – 13637.