Multiple interactions of anticholinesterases withAplysia acetylcholine responses

Brain Research, 375 (1986) 407-412 Elsevier

407

BRE21598

Multiple interactions of anticholinesterases with Aplysla acetylcholine responses N.T. SLATER 1'2 M. FILBERT2 and D.O. CARPENTER 1

1Wadsworth Centerfor Laboratories and Research, New York State Department of Health, Albany, NY12201 and 2Neurotoxicology Branch, U.S.A.M.R.I. C.D., Aberdeen Proving Grounds, MD 21010 (U.S.A.) (Accepted February 25th, 1986)

Key words: neostigmine - - pyridostigmine- - acetylcholine receptor--Aplysia -- desensitization

The effects of the carbamate anticholinesterases neostigmine and pyridostigmine on the kinetics of desensitization of responses of isolated, voltage-clamped Aplysia neurons to microperfused acetylcholine (ACh) was examined. The peak ACh-induced current was potentiated at low carbamate doses and antagonized at higher doses (>10 -5 M); neostigmine was more potent than pyridostigmine in producing both effects. These effects suggest two mechanisms of action of these compounds: (a) inhibition of acetylcholinesterase at low doses, which increases the effective ACh dose, and (b) direct antagonism of the response at higher concentrations, which is associated with a slowing of both the activation and desensitization of the ACh response. These compounds may therefore have direct actions on the excitatory ACh receptor in Aplysia neurons which are similar to the effects of these drugs at the vertebrate endplate.

Acetylcholinesterase (ACHE) inhibitors m a y have direct actions on the nicotinic acetylcholine ( A C h ) receptor which lead to a b l o c k a d e of responses to A C h at somewhat higher doses than are required for the inhibition of A C h E 11A2. In addition to the proposed direct effects of A C h E inhibitors on the A C h receptor, c o m p l e m e n t a r y biochemical studies have d e m o n s t r a t e d a m o d u l a t o r y effect of nicotinic antagonists on A C h E 5'9'16'27-29, thus p r o m p t i n g the suggestion that the active sites of A C h E and the nicotinic A C h r e c e p t o r m a y be similar. A l t h o u g h several lines of evidence indicate that this view is a simplification 1'2'28, the cross interactions b e t w e e n these two classes of drugs are nevertheless of great interest. Most of these studies, however, have been confined to the interactions of A C h antagonists with ACHE, where they have b e e n p r o p o s e d to act as peripheral site ligands 5. By contrast, there have been few kinetic studies o f the actions of A C h E inhibitors on A C h receptors, although their cholinolytic effects are well known 11'12. Previous electrophysiologic studies of molluscan neurons have d e m o n s t r a t e d that A C h E inhibitors can antagonize responses to A C h or carbacho112'15'2°'26 and biochemical studies have

shown inhibition of a - b u n g a r o t o x i n binding to molluscan ganglionic h o m o g e n a t e s 4,12,17,21. A C h E is present in Aplysia neurons and can be inhibited by c a r b a m a t e A C h E inhibitors 4'12'13. In the present experiments we have c o m p a r e d the effects of two reversible c a r b a m a t e A C h E inhibitors, neostigmine and pyridostigmine, on the inward currents e v o k e d by the rapid micropeffusion of A C h on isolated, voltage-clamped Aplysia neurons. A l t e r a t i o n s of desensitization kinetics were used to derive information about the possible molecular mechanisms of the interactions of these drugs with the A C h receptor. A preliminary account of some of these experiments has been p r e s e n t e d elsewhere 25. The m e t h o d s used in this study were similar to those e m p l o y e d previously to study desensitization kinetics 24'25 and the effects of antagonists 22 in Aplysia neurons. Clusters of RB neurons from the a b d o m i n a l ganglia of Aplysia californica were dissected and secured to the base of a recording chamber. Artificial seawater ( A S W ) with o r without an inhibitor was microperfused over the cells through 0.86-mm (i.d.) tubing, which was directed at the cell u n d e r study by microscopic control. Perfusion solutions were sepa-

Correspondence: N.T. Slater, Department of Physiology, Northwestern University Medical School, 303 E. Chicago Avenue, Chicago, IL 60611, U.S.A. 0006-8993/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)

408 rated by air bubbles in the line, which escaped through a hole in the tubing 1-2 mm from the tip. This system allowed complete exchange of the bathing medium in less than 600 ms, as measured by ionsensitive microelectrodes placed behind the soma of the cell under study 22'24. Individual cell bodies were impaled with two shielded microelectrodes filled with 2 M potassium acetate (4-8 MQ) and voltage-clamped. The cells were perfused with 50 ~M ACh at 30-min intervals (to allow for recovery from each perfusion 24) in the presence or absence of a given dose of an AChE inhibitor. Each perfusion was continued for several minutes until the response attained a steady-state current amplitude. Then the cell was washed again for 30 min to allow for recovery from desensitization and to equilibrate in a new solution. The rapid microperfusion of ACh over these cells produces a large inward current, which peaks within 0.5-1.0 s and then declines in the continued presence of ACh towards a lower, steady-state value after several minutes due to desensitization. The rate of desensitization in these neurons can be described as the sum of two exponentials plus a constant 24. Thus the current at any given point in time after the onset of the response can be predicted by I = A e -at + B e -#t + C

(1)

where I is the current at time t, A and B are coefficients corresponding to the intercepts of the exponentials at time zero, a and fl are the corresponding rate constants, and C is the steady-state value to which the current declines. In molluscan neurons the time constants of the two exponentials, re (l/a) and r s (1//3) are sensitive to agonist dose but have differing pharmacologic properties and are not affected by membrane potential 3'24'25. To analyze the effects of each AChE inhibitor on desensitization kinetics, ACh was applied for several minutes until stable control responses were obtained in normal ASW. Then cells were exposed to a low concentration of the drug during the 30-min recovery period and tested again with an identical ACh concentration (50/~M). The dose of the drug was then increased, one response to ACh being obtained at each dose. When the data for each dose were fit to Eqn. 1 by a least-squares minimization routine 24, the effects

of the AChE inhibitor on the rate of desensitization could be quantitatively expressed in terms of the effects on rf and r s. The effects of the drugs were also examined on the peak ACh-induced current extrapolated to time zero, Ip (= A + B + C); on the fractional contributions of rf and r s, which can be given as A' (A/Ip) and B' (B/Ip), respectively; and on the plateau/peak current ratio, C' (C/Ip), which is a gross estimate of the overall degree of desensitization. The goodness of fit of Eqn. 1 to the data was always evaluated statistically by the F-test method, using the calculated values of the residuals 24. Neostigmine and pyridostigmine had multiple effects on the amplitude of responses to ACh in Aplysia neurons. At low doses Ip was enhanced by 1 × 10-8 M neostigmine or 1 × 10-5 M pyridostigmine. At higher concentrations this potentiation of ACh responses was reversed, and Ip was markedly depressed (Fig. 1B). Thus the relationship between the dose of AChE inhibitor and Ip was bell-shaped, low doses producing potentiation and higher doses antagonizing the peak response. Neostigmine was much more potent than pyridostigmine in potentiating the response, producing a maximal potentiation at only 1% of the dose required with pyridostigmine (Fig. 1A). If it is assumed that the potentiation of the ACh response by these compounds results from inhibition of AChE and that the blockade at higher doses results from direct action on the ACh receptor-ionophore complex, these results indicate that both sites are more sensitive to neostigmine than to pyridostigmine. Analysis of desensitization kinetics, similarly to other biophysical approaches, provides important clues to the mechanisms that may underlie these effects. It has previously been demonstrated that the onset of desensitization to micropeffused ACh is a biphasic process in the Aplysia neurons studied here 24 and in other molluscan neurons 3, as well as at the vertebrate neuromuscular junction 6-8'1°. In contrast to the biphasic effects of the AChE inhibitors on Ip, these compounds produced only an increase of the time constants of both exponential components of desensitization (rf, Vs; Fig. 2). An increase of the effective ACh dose resulting from inhibition of AChE would be expected to decrease the values of re, rs in the low carbamate dose range 24. This was not ob-

409

A f

B

CON T ROL

250

NEOSTIGMINE l x 1 0 -6 M

-

.-I

o

200-

fie Z

o

NEO

O

O. I-4

PYR

150NEOSTIGMINE 5 x i 0 "LM

/50,

100-

10s

0

8

6

7 -

5

t.

3

tog (M)

Fig. 1. Effects of carbamate anticholinesterases on responses of voltage-clamped Aplysia neurons to microperfused ACh. A: response to 50/zM ACh (continuously perfused from arrow) alone or in the presence of neostigmine. All 3 traces were obtained from the same cell. B: relationship between anticholinesterase dose and the peak ACh-induced inward current 'corrected' for desensitization (Ip; Eqn. 1). Data are mean values (___S.E.M.) obtained in 5 experiments with neostigmine (NEO; solid circles) and 4 experiments with pyridostigmine (PYR; open circles). Note that, in addition to dose-dependent effects on Ip (B), the anticholinesterases also produce a progressive increase in the time-to-peak of the response (A).

PYRIDOSTIGMINE NEOSTIGMINE

60-

A

B

14-

55-

12-

50u

.10-

®

45-

111

8--

"

6--

4035-

4--

a.

0

I 8

i 7

I 6

-tog

(M)

i 5

i L,

i 3

30-1

" 0

i

I

I

I

I

i

8

7

6

5

/,

3

-tog

(M)

Fig. 2. Effects of anticholinesterase dose on the time constants of the fast (A) and slow (B) exponential components of desensitization onset. Data represent mean values for 5 experiments with neostigmine and 4 experiments with pyridostigmine.

410 served with either compound, though there was a

sponse was increased (Fig. 1A), indicating that these drugs may slow the activation as well as desensitization of the response. We did not quantify this slowing of the time-to-peak because, at least at the low carbamate doses, it was observed when the A C h concentration was probably still equilibrating after the initial exposure to ACh. Measurements of the rate of rise of the response would thus be influenced by the discontinuous A C h concentration during the early phase of the perfusion. The effects of neostigmine (1 x 10 -7 M and 1 x 10-4 M) on responses to carbachol (200/~M) were studied in 4 cells. Only a reduction of Ip and a slowing of response kinetics was observed. In two cells the slopes of l o g - l o g plots of carbachol dose (50-500 ~M) vs Ip were 1.4-1.5 in normal A S W and in the presence of both neostigmine concentrations. The slowing of desensitization kinetics produced by these compounds may contribute to some extent to the potentiation of the responses at low carbamate doses, but it is more likely associated with the mechanism by which the response was antagonized. This antagonism was associated with 3 effects: a decrease of Ip at high doses, a slowing of desensitization kinetics over the whole dose range, and a slowing of the

lower slope for the relationship between r and drug dose at these concentrations, which likely reflects the opposing influences of A C h E inhibition (which would decrease r through an increase in effective ACh dose) and the antagonistic effects, which are associated with an increase of r. At higher doses, where A C h E was completely inhibited, the slope of the relationship between r and carbamate concentration steepened, as the effective A C h dose was constant and only the antagonistic effects were seen. Another sensitive measure of drug effects on A C h responses is the fractional contribution made by the two exponential components ( A ' , B ' ) , and the plateau/peak current ratio, C'. As the A C h dose is increased, these values shift in a characteristic manner 24. At low doses of the carbamates, which produce an overall potentiation of Ip, the relative contributions of rf (A'), r s (B'), and C' were shifted in a manner consistent with an increase in effective A C h dose (Fig. 3). No further shift was observed at the higher concentrations at which antagonism and a pronounced increase of the time constants of desensitization were observed. At the higher doses the time-to-peak of the re-

B

A NEOSTIGMINE I--

|.0

*" o tu or)

u

I.u

~ I1:

PYRIDOSTIGMINE

1.0-

A' 0.8-

0.8-

LU A'

0.6-

H

0.6--

z

Qt.-

a' z

0.4-

~

0.2-

o

o

o

u

O n,., u.

~ C'

,

00

8

~

,

,

;

,

,

7

6

5

,~

3

- log (M)

0.2-

~ 0-

I ~ 0

C' I 7

I 6

I 5

-log

(M)

i' 4

I 3

Fig. 3. Effects of carbamate anticholinesterases on the fractional contributions of the fast (A') and slow (B') exponential components and on the plateau/peak current ratio (C'). A: mean values for 5 experiments with neostigmine. B: mean values for 4 experiments with pyridostigmine. Note that the anticholinesterase dose at which no further significant shift in the fraetional contributions was observed (ca. 1 × 10-7 M neostigmine and 1 x 10-5 M pyridostigmine) correlates well with the maximal potentiation of Ip (Fig. 1B).

411 t i m e - t o - p e a k of the response. Such a direct action of the c a r b a m a t e s on the A C h r e c e p t o r - i o n o p h o r e complex cannot be explained by either competitive or channel block, as action at these sites would either have no effect or (in the case of channel block) accelerate the current decay after agonist perfusion. E i t h e r closed channel block or an allosteric site of action might be i n v o k e d to explain the effects, as these interactions could reduce the rate of transition of the agonist-bound, closed state of the receptor-ionophore complex to the open or desensitized state. T h e present e x p e r i m e n t s , however, do not distinguish between these two mechanisms of blockade. The bell-shaped d o s e - r e s p o n s e relations and the slowing of response kinetics in these experiments are similar to results o b t a i n e d at the v e r t e b r a t e neuromuscular junction, where a p r o l o n g a t i o n of channel lifetime and e n d p l a t e current decay was r e p o r t e d to be associated with the antagonistic effects of high doses of neostigmine 11 o r pyridostigmine 19. These c o m p o u n d s acted directly on the A C h r e c e p t o r to alter the o b s e r v e d kinetics of activation and desensitization. While the mechanism of action of these drugs

in Aplysia neurons and v e r t e b r a t e muscle is likely similar, the precise site of interaction remains to be demonstrated. The proposal that both the blocking effects and the slowing of desensitization kinetics m a y arise from the same direct action of A C h E inhibitors on the A C h receptor requires further e x p e r i m e n t a l evidence. Such alterations of response kinetics could also arise from indirect mechanisms, such as alteration of intracellular Ca 2÷ (refs. 18, 25) or p H t4.

1 Adams, P.R., Acetylcholine receptor kinetics, J. Membr. Biol., 58 (1981) 161-174. 2 Albuquerque, E.X., Adler, M., Spivak, C.E. and Aquayo, L., Mechanism of nicotinic channel activation and blockade, Ann. N. Y. Acad. Sci., 358 (1980) 204-328. 3 Andreev, A.A., Veprintsev, B.N. and Vulfius, C.A., Twocomponent desensitization of nicotinic receptors induced by acetylcholine agonists in Lymnaea stagnalis neurones, J. Physiol. (London), 353 (1984) 375-391. 4 Carpenter, D.O., Greene, L.A., Shain, W. and Vogel, Z., Effects of eserine and neostigmine on the interaction of abungarotoxin with Aplysia acetyicholine receptors, Mol. PharmacoL, 12 (1976) 999-1006. 5 Changeux, J.-P., Responses of acetylcholinesterase from Torpedo marmorata to salts and curarizing drugs, Mol. Pharmacol., 2 (1966) 369-392. 6 Chesnut, T.J., Two-component desensitization at the neuromuscular junction of the frog, J. Physiol. (London), 336 (1983) 229-241. 7 Clark, R.B. and Adams, P.R., Rapid flow measurements of desensitization of frog endplates, Biophys. J., 33 (1981) 16a. 8 Connor, E.A., Fiekers, J.F., Neel, D.S., Parsons, R.L. and Schnitzler, R.M., Comparison of cholinergic activation and desensitization at snake twitch and slow muscle fibre endplates, J. Physiol. (London), 351 (1984)657-674. 9 Dawson, R.M. and Poretski, M., Procaine as a substrate and possible allosteric effector of cholinesterases, Neurochem. Int., 5 (1983) 559-569.

10 Feltz, A. and Trautmann, A., Desensitization at the frog neuromuscular junction: a biphasic process, J. Physiol. (London), 322 (1982) 257-272. 11 Fiekers, J.F., Concentration-dependent effects of neostigmine on the endplate acetylcholine receptor channel complex, J. Neurosci., 5 (1985) 502-514. 12 Filbert, M.M.G., Cholinesterase-independenteffects ofanticholinesterase agents, Ph.D. Thesis, University of Maryland, 1984. 13 Giller, E. and Schwartz, J.H., Acetylcholinesterase in identified neurons of abdominal ganglion of Aplysia californica, J. Neurophysiol., 34 (1971) 108-115. 14 Goldberg, G. and Lass, Y., Evidence for acetylcholine receptor blockade by intracellular hydrogen ions in cultured chick myoballs, J. Physiol. (London), 343 (1983) 429-437. 15 Levitan, H. and Tauc, L., Acetylcholine receptors: topographic distribution and pharmacological properties of two receptor types on a single molluscan neurone, J. Physiol. (London), 222 (1972) 537-558. 16 Maayani, S., Weinstein, H., Ben-Zvi, N., Cohen, S. and Sokolovsky, M., Psychomimetics as anticholinergic agents. I. 1-cyclohexyipiperidine derivatives: anticholinesterase activity and antagonistic activity to acetylcholine, Biochem. Pharmacol., 23 (1974) 1263-1281. 17 Ono, J.K. and Salvaterra, P.M., Snake a-toxin effects on cholinergic and non-cholinergic responses of Aplysia californica neurons, J. Neurosci., 1 (1981) 259-270. 18 Parsons, R.L., Role of calcium in desensitization at the motor endplate of skeletal muscle. In G.B. Weiss (Ed.), Calci-

W e are grateful to A n d r e w F. Hall for participation in some experiments and to C. Sedlmeir and Drs. M. G a l v a n and D . L . Martin for assistance with c o m p u t e r programming. This w o r k was s u p p o r t e d by U . S . P . H . S . NS18435 and the Scientific Services Program of Battelle Columbus L a b o r a t o r i e s . The views, opinions, and/or findings contained in this report are those of the authors and should not be construed as an official D e p a r t m e n t of the A r m y position, policy, or decision, unless so designated by other documentation.

412

um and Drug Action, Plenum Press, New York, 1978, pp. 289-314. 19 Pascuzzo, G.J., Akaike, A., Maleque, M.A., Shaw, K.-P., Aronstam, R.S., Rickett, D.L. and Albuquerque, E.X., The nature of the interactions of pyridostigmine with the nicotinic acetylcholine receptor ionic channel complex. I. Agonist, desensitizing, and binding properties, Mol. Pharmacol., 25 (1984) 92-101. 20 Possier, P., Baux, G. and Tauc, L., Possible role of acetylcholinesterase in regulation of postsynaptic receptor efficacy at a central inhibitory synapse of Aplysia, Nature (London), 301 (1983) 710-712. 21 Shain, W., Greene, L.A., Carpenter, D.O., Sytkowski, A.J. and Vogel, Z., Aplysia acetylcholine receptors: blockade by and binding of a-bungarotoxin, Brain Research, 72 (1974) 225-240. 22 Slater, N.T. and Carpenter, D.O., A study of the cholinolytic actions of strychnine using the technique of concentration jump relaxation analysis, Cell. Mol. Neurobiol., 4 (1984) 263-272. 23 Slater, N.T. and Carpenter, D.O., Direct effects of neostigmine on acetylcholine receptor activation and desensitiza-

tion in Aplysia neurones, Pflagers Arch., 403 (1985) R50. 24 Slater, N.T., Hall, A.F. and Carpenter, D.O., Kinetic properties of cholinergic desensitization in Aplysia neurons, Proc. R. Soc. London Set. B, 223 (1984) 63-78. 25 Slater, N.T., Hall, A.F. and Carpenter, D.O., Trifluoperazinc and calcium antagonists accelerate cholinergic desensitization in Aplysia neurons, Brain Research, 329 (1985) 275-279. 26 Tauc, L. and Gerschenfeld, H.M., Cholinergic transmission mechanisms for both excitation and inhibition in molluscan central synapses, Nature (London), 192 (1962) 366-367. 27 Tomlinson, G., Mutus, B. and McLennan, I., Modulation of acetylcholinesterase activity by peripheral site ligands, Mol. PharmacoL, 18 (1980) 33-39. 28 Webb, G.D. and Johnson, R.L., Apparent dissociation constants for several inhibitors of acetylcholinesterase in intact electroplax of the electric eel, Biochem. Pharmacol., 18 (1969) 2153-2161. 29 Zupancic, A.O., Kinetic response of a membrane-bound acetyicholinesterase to cholinergic activating and blocking agents, FEBS Lett., 11 (1970) 277-280.