Cocaine and phencyclidine inhibition of the acetylcholine receptor:

Transcription

1 Proc. NatL Acad. Sci. USA Vol. 79, pp , April 1982 Biochemistry Cocaine and phencyclidine inhibition of the acetylcholine receptor: Analysis of the mechanisms of action based on measurements of ion flux in the millisecond-to-minute time region (Electrophorus electricus/torpedo spp./pc-12 cells/procaine/quench-flow techniques) JEFFREY W. KARPEN, HITOSHI AoSHIMA*, LEO G. ABOODt, AND GEORGE P. Section of Biochemistry, Division of Biological Sciences, 27 Clark Hall, Cornell University, Ithaca, New York Communicated by Choh Hao Li, January 11, 1982 ABSTRACT The effects of cocaine and of phencyclidine and procaine on acetylcholine receptor-controlled ion flux were measured in the millisecond-to-minute time region. Chemical kinetic measurements of ion flux were made in membrane vesicles prepared from the electric organ of Electrophorus electricus and in PC-12 cells, a sympathetic neuronal cell line. A quench-flow technique was used to measure ion flux in the millisecond-to-second range in membrane vesicles. Cocaine and phencyclidine both inhibit acetylcholine receptor-controlled ion flux, but by different mechanisms. Both compounds decrease the initial rate of ion flux, an effect observed with the local anesthetic procaine. This inhibition cannot be prevented by saturating concentrations of acetylcholine (1 mm). These results from chemical kinetic experiments are consistent with electrophysiological measurements which indicate that local anesthetics act by interfering with the movement of ions through receptor-formed channels. The chemical kinetic experiments, however, give additional information about the action of phencyclidine. They indicate that phencyclidine also increases the rate of receptor inactivation (desensitization) and changes the equilibrium between active and inactive receptor conformations, effects not observed in the presence of cocaine or procaine. The commonly abused drugs cocaine (8-azabicyclo[3.2. 1]octane- 2-carboxylic acid, 3-benzoyloxy-8-methyl methyl ester [1R- (exo-exo)]), and phencyclidine [PCP; 1-(1-phenylcyclohexyl)- piperidine] appear to have effects on multiple neurotransmitter systems (1, 2). Cocaine, for example, is known to be an effective inhibitor of norepinephrine uptake at peripheral and central sites (3). Because cocaine is a local anesthetic, it is of interest to study and compare its effects to those of other local anesthetics whose actions on the acetylcholine receptor have been extensively studied (4). Local anesthetics change the time course of neurally evoked end plate currents, causing them to decay as the sum of two exponentials (5, 6). Current pulses due to the opening ofsingle receptor channels are broken into shorter pulses in the presence of local anesthetics (7). The results of these and other studies have been accounted for by a simple mechanism in which the receptor exists in three states: closed, open, and blocked. In this model, local anesthetics bind to the open-channel form of the receptor, resulting in blockade of the receptor (4). PCP has been shown to have anticholinergic action which was originally attributed to inhibition of nicotinic acetylcholine receptors (8). Recently, this conclusion was confirmed, and the interaction of PCP with the ionic channel of the acetylcholine receptor has been investigated in frog sciatic nerve-sartorius 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 solely to indicate this fact. 259 HESSt muscle preparations by using electrophysiological techniques (9, 1) and in electroplax of Torpedo ocellata by measuring binding of PCP to the membranes (11). The conclusion from these studies was that PCP reacts with the ionic channel in both the closed and open conformations. Subsequently, the effect of PCP on nicotinic acetylcholine receptor function in nerve cells was demonstrated directly (unpublished data). Recently, techniques have been developed (for recent reviews see refs ) for measurement of acetylcholine receptor-controlled ion flux in membrane vesicles in the millisecondto-minute time region by using a quench-flow technique (14-16) and membrane vesicles isolated from the electric organ ofelectrophorus electricus. These techniques have proven useful in establishing the relationship between ligand binding and ion translocation processes (14, 17-21). The present study demonstrates that the mechanism ofaction of inhibitors of the acetylcholine receptor can be investigated by measuring the kinetics of ion flux. Four types of measurements in the presence and absence of inhibitors are reported here: (i) transmembrane ion flux, (ii) rate of inactivation (desensitization) of the receptor, (iii) ion flux mediated by an equilibrium mixture ofactive and inactive receptors, and (iv) ion flux in the second-to-minute time region in PC-12 cells, a sympathetic neuronal cell line. The results indicate that PCP and cocaine inhibit the acetylcholine receptor by different mechanisms. Cocaine, like the classical local anesthetic procaine, decreases the initial rate of receptor-controlled ion flux. This effect is also observed with PCP. The distinction, however, is that PCP also increases the rate of receptor inactivation (desensitization) and changes the equilibrium between active and inactive receptor forms, effects not observed in the presence of cocaine or procaine. MATERIALS AND METHODS The preparation of membrane vesicles from the electric organ of E. electricus has been described (22, 23). The quench-flow apparatus, the method of making 'Rb+ influx measurements, and the evaluation of rate coefficients have been described (16, 17, 2). 86Rb+ and 42K+ have been shown to give identical results in influx measurements (24). The PC-12 cell line is that derived by Greene and Tischler (25) and was obtained from The Salk Institute for Biological Studies. 22Na+ uptake, stimulated by acetylcholine, was measured at 22C and ph 7.4 as described (26-28). The concen- Abbreviation: PCP, 1-(1-phenylcyclohexyl)piperidine. * On leave from Yamaguchi University, Yamaguchi, Japan. t From the Center for Brain Research, University of Rochester Medical Center, Rochester, NY t To whom correspondence and reprint requests should be addressed.

2 251 Biochemistry: Karpen et al. tration of 22Na' was always 2,Ci/ml (1 Ci = 3.7 X 11 becquerels). Initial rates were determined by measuring the amplitude of ion flux in 15 sec and subtracting background ion flux in the absence of acetylcholine. Tetram (O,O-diethyl-5-diethylaminoethyl triphosphate), an inhibitor of acetylcholine esterase, was a gift from R. D. O'Brien. Carbamoylcholine-induced ion flux was found to be identical in the presence and absence of Tetram (21). Procaine was purchased from Schwarz/Mann. RESULTS Cocaine inhibited acetylcholine-mediated 22Na' influx in the PC-12 cell line in a dose-dependent fashion (Fig. 1). Because a fast phase of ion flux is not observed in PC-12 cells by quenchflow measurement (unpublished data), initial rates were determined by measuring the amplitude of the ion flux in 15 sec. 'Na' influx was measured at a nearly saturating acetylcholine concentration (1 mm) (18). The concentration of cocaine at which half-maximal inhibition occurred was approximately 1 AM, compared with 1,uM for the local anesthetic procaine (Fig. 1). PCP inhibits acetylcholine-mediated 22Na' influx in PC-12 cells with half-maximal inhibition occurring at.7 AuM (11). The PC-12 cell data have been included to show that, qualitatively, the inhibition and the relative order of affinity of cocaine, PCP, and procaine for receptor-rich membrane vesicles are not unique to membrane vesicles or the E. electricus receptor. Due to the extremely low number of receptors per unit internal volume in PC-12 cells, the effects of cocaine, procaine, and PCP on acetylcholine receptor-controlled ion translocation in membrane vesicles prepared from E. electricus electroplax can be treated more quantitatively. The minimum mechanism which accounts for the time-dependence of acetylcholine receptor-controlled ion flux over a 2-fold concentration range of carbamoylcholine (14, 17, 2, 29) and a 5,-fold concentration range of acetylcholine (18, 21) is as follows. L K1 L K1 (D I + _ + _- -- AL2 A A AL (closed) (open) kal 1 k12 k43 1lk34 K2 IL IL2 L M 1- exp (JA-_ JI) ( M,,,, a= 2K2k2l + k43l + L + 2K2 - C a JR' ion flux + lit] [1] 2Klkl2L + k34l2 L2(1 + () + 2K1FL + K1 4 The active forms of the receptor (A) and the inactive forms (I) bind ligand (L) in rapidly achieved equilibria denoted by microscopic equilibrium constants (K). Active receptor with two bound ligands (AL2) converts rapidly to an open channel (AL) with an equilibrium constant for channel opening (1/t'). AL2 permits ion flux with a first-order rate coefficient JMO, where J is the specific reaction rate (3 17M'-sec-1 at 1MC and ph 7.), X an intrinsic constant characteristic of the receptor. R' is characteristic of the vesicle preparation and represents the moles of receptor sites per liter of internal vesicle volume. The subscripts A and I represent fractions of the receptor and have Proc. Natl. Acad. Sci. USA 79 (1982) 25 X as ~~~ ~~~~~~~~~~~ C I~~~~ 5M6 51csm H 4,. 3 (M) [Inhibitor] FIG. 1. Inhibition of acetylcholine-induced "2Na' uptake by cocaine in PC-12 cells at ph 7.4 and 22 C. The buffer was 165 mm NaCl/ 5.4 mm KCI/2 mm CaCl2/5 mm glucose/25 mm Hepes, ph 7.4. The cells were plated on polylysine-coated 35-mm cell culture dishes as described (26-28). o, Dish was washed with buffer and then incubated with 5,uM Tetram (inhibitor of acetylcholinesterase) for 1 min. After removal of the solution, 22Na' uptake was measured in 1 mm acetylcholine/5 mm ouabain (an inhibitor of the Na',K' ATPase)/5 )M Tetram with cocaine at different concentrations. A, Procaine was used instead of cocaine under the same conditions. m, Activity was measured in the presence of.7,um PCP. The activity of the receptor is expressed as a percentage of the activity found in the absence of inhibitor. been defined previously in terms of ligand concentration and constants in the minimum mechanism (17). Conversion between the active and inactive receptor states occurs with firstorder rate constants (k). The integrated rate equation based on the above mechanism is given by Eq. 1. M represents metal ion concentration inside the vesicles, and t and refer to the times of measurement. The relationship between the rate coefficients JA and J. and the fraction of the receptor in the open channel form is given byja = JRO[AL2] and J, = JR'[AL2]L, where and denote the open-channel form ofthe receptor before the onset of inactivation and after inactivation is complete, respectively. a, the first-order rate constant of inactivation, is given by Eq. 2. The rate equation (Eq. 1) based on this model (17, 29) relates the ligand binding process to ion translocation. All the constants in Eq. 1 have been evaluated (17, 21). The rate equation was used in all subsequent measurements to evaluate the effects of cocaine and PCP on the receptor-controlled ion translocation process. Fig. 2 shows the time-dependence of acetylcholine (1 mm)-mediated 86Rb+ influx in membrane vesicles from E. electricus in the presence and absence of PCP (5 AuM) and cocaine (2 AtM). These concentrations were chosen because they both result in approximately 5% inhibition ofthe ion flux amplitude in 1 sec. The solid lines were obtained by a least squares fit of the data to the integrated rate equation (Eq. 1) based on the minimum mechanism. It is clear that both PCP and cocaine inhibit ion flux in the millisecond time region. A fit of the data to the integrated rate equation indicates that the inhibitors decrease JA, the initial rate constant of ion flux, by about 4% (PCP) and 6% (cocaine). However, PCP also increases a, the first-order rate coefficient of inactivation (desensitization), by a factor of 2, whereas cocaine has no effect on the rate of inactivation. Qualitatively, the influx curve in the presence of PCP rises more sharply and levels off more quickly than the influx curve in the presence of cocaine. When influx was measured at 8,uM acetylcholine [a concentration that is approximately half saturating (21)1, 2 glm cocaine and 5 AuM PCP decreased JA by the same factor as they did in the presence of saturating acetylcholine (data not shown). The degree of inhibition is therefore independent of acetylcholine concentration,

3 Biochemistry: Karpen et al. Proc. Natl. Acad. Sci. USA 79 (1982) ~~~~~~~~~~ ~~~~~.6 Mt MOa.4.2 A 1 A A 1-a d 5 \ F -I- I-! I Time (sec) FIG. 2. Acetylcholine receptor-mediated 86Rb+ influx in electroplax membrane vesicles in the presence and absence of cocaine (2 AuM) and PCP (5 MM). Membrane vesicles (8,ug of protein per ml treated with' 5 AM Tetram were mixed with an equal volume of 86Rb+ (1 ACi/ml) and acetylcholine (2 mm) solution without inhibitor (s), with 4 AM cocaine (o), or with 1,M PCP (A). The final concentrations of cocaine and PCP were 2 AM and 5 AM, respectively. The influx was quenched by d-tubocurarine (7 mm after mixing) at the times shown on the abscissa. After Millipore filtration of the vesicles and washing, the 86Rb+ content of the vesicles was measured by scintillation counting. The same procedure was followed in the absence of acetylcholine, and the difference between these measurements at each time is expressed as a fraction of the total ion exchange (M/MX) and plotted against time. The procedure was carried out in a quench-flow machine in E. electricus Ringer's solution at ph 7. and 1 O (16). Each point is a mean of three measurements. The coordinates of the curves and the values of the parameters (below) were obtained by fitting the data to Eq. 1 with a nonlinear least squares computer program. For (control): JA = sec-' (mean ± SD); a = 4.2 sec-'. For o (2,M cocaine): JA = sec'; a = 5.3 sec-'. For A (5 M PCP): JA = sec'; a = 1.6 sec1. Also shown are measurements of ion flux in 1 min obtained from a separate out-of-machine experiment. The conditions of influx were identical to quench-flow measurements. After incubation for 1 min,.5 ml of the solution was mixed with 1.7 ml of 5 mm d-tubocurarine. After exactly 15 min, two 1-ml aliquots were filtered and washed as before. Background values in the absence of acetylcholine were measured in the same fashion and subtracted. Values were expressed as a fraction of total ion exchange (M1/Mj) and plotted on the same graph. indicating that inhibition by cocaine and PCP is not competitive. Other differences between the effects of cocaine and PCP are evident from Fig. 2. In the presence of cocaine the vesicles fill (equilibrate with the external 86Rb+ solution) within 1 min, whereas they are only half-filled in the presence of PCP after 1 min. The significance of this result is treated in the Discussion. An independent verification of the results from influx is shown in Fig. 3. In these.experiments the membrane vesicles were preincubated with acetylcholine for varying periods of time before addition of radioactive tracer and measurement of influx for a constant period of time. It is possible by this method to measure receptor inactivation as a function of preincubation time (2, 3). Fig. 3 shows inactivation by 1 mm acetylcholine in the presence and absence of cocaine (2,M) and PCP (5 MM). The slopes of the lines in Fig. 3 represent a, the firstorder rate coefficient of inactivation. The results indicate that a is unchanged in the presence of 2,M cocaine (5 sec- 1) but is increased by a factor of 2 in the presence of 5 AM PCP. These values are in close agreement with the values obtained from analysis of the influx curves in Fig. 2. In Fig. 4, influx mediated by 1 mm acetylcholine after prein Preincubation Time (sec) FIG. 3. Effect of cocaine (2 AM) and PCP (5 AM) on the rate of receptor inactivation in the presence of 1 mm acetylcholine. Preincubation of vesicles with 1 mm acetylcholine for the times indicated on the abscissa was followed by incubation with 86Rb+ solution containing 1 mm acetylcholine for 1 sec to assay for flux activity. The other details of the experimental conditions were as in Fig. 2. e, Absence of inhibitor; o, 2,M cocaine in the final preincubation and incubation mixtures; A, A, 5 AuM PCP in the final preincubation and incubation mixtures. Each point shown is the mean of three determinations within one experiment. The curve in the presence of PCP represents a composite of three separate experiments (A represents points averaged over more than one experiment). Activity remaining is expressed as a percentage of activity observed in the absence of preincubation and is defined as ([AL2]t [AL2]L)/([AL2] [AL2D. The observed - - rate coefficient of inactivation, a, is obtained from the slope of the semilogarithmic plot. The solid lines were computed by a least squares program. For e (control), a 5.2 = sec-1; for o (2,uM cocaine), a 5.1 = sec-1; for A, (5 AM PCP), a 1.3 sec1. = cubation for 1 hr is shown. This time interval is more than sufficient to obtain an equilibrium mixture of active and inactive receptor forms (2, 3). PCP (5,uM) and cocaine (2 /im) were 1.~~~~~~~~ Mt.5- t X MtD Time (min) FIG. 4. Effect of cocaine (2 KM) and PCP (5,uM) on influx mediated by an equilibrium mixture of active and inactive receptors. Vesicle suspensions (8,ug of protein per ml) were equilibrated with 1 mm acetylcholine for 1 hr and mixed with an equal volume of a solution containing 86Rb+ (1 /.Ci/ml) and 1 mm acetylcholine (), 1 mm acetylcholine and 4 AM cocaine (), or 1 mm acetylcholine and 1,M PCP (A). Final concentrations of cocaine and PCP were 2 AM and 5,uM, respectively. Quenching and measurement of influx were as in Fig. 2. For e (control), J1 8.7 = x 1-3sec-1; for o (2,M cocaine), J, = 3.7 x 1-3sec-1; for A (5 AuM PCP), J1 = 2 x 1-3sec'-. J1 values are an estimate and are not corrected for a process occurring in the minute time region which reflects the properties of both the receptors and the vesicles (31).

4 2512 Biochemistry: Karpen et at l. Mt MOD.1.. A4/.2 ~~~~~~~~ ~~~~ o I. 2.. Time (sec) FIG. 5. Acetylcholine receptor-mediated 'Rb+ influx in electroplax membrane vesicles in the presence and absence of procaine (1 PM). The conditions of the experiment were as in Fig. 2. The control curve (in the absence of inhibitor) is the same one shown in Fig. 2. For * (control), JA = sec- (mean SD); a 4.2 sec'1; for o(1 jlm = procaine), JA 4.4 = ±.2 sec-1; a = 4.5 sec'1. added after preincubation. Concentrations ofthe two inhibitors that result in approximately the same influx amplitude in 1 sec, in the absence of preincubation (Fig. 2), have clearly different effects on slow influx, characterized by a rate coefficientji (17). JI is representative of ion translocation that occurs in E. electricus membrane vesicles after the inactivation process has gone to completion (2, 3). The ion flux shown in Fig. 4 is receptormediated because background ion flux in the absence of acetylcholine has been subtracted. The significance of the effects on J1, in terms of the equilibrium between active and inactive receptors, is treated in detail in the Discussion. Influx mediated by 1 mm acetylcholine in the presence and absence of 1 AM procaine is shown in Fig. 5. An analysis of the influx curve in the presence of 1 pim procaine indicates that, like cocaine, procaine decreasesja but has no effect on a. Therefore, based on the parameters that can be measured from influx, cocaine and procaine appear to act by similar mechanisms. DISCUSSION The present study demonstrates that cocaine inhibits acetylcholine receptor-controlled ion flux. In terms of the mechanism of inhibition, there is good agreement between the results obtained here on cocaine and procaine and those from studies, using electrophysiological techniques, on local anesthetics (4-7). The hypothesis from previous studies is that local anesthetics bind to the open-channel form of the acetylcholine receptor and prevent ion translocation. In terms of the kinetics of receptor-controlled ion flux, this effect would manifest itself as a decrease in the rate coefficientja which is directly proportional to the number of open receptor channels. This is, in fact, what is observed in the presence of cocaine or procaine. The primary significance of the present study is the finding that PCP inhibits the acetylcholine receptor by a mechanism different from that of cocaine or procaine. PCP also decreases the initial rate of ion flux, a result in agreement with results of previous studies (9, 1). However, PCP has the additional effect of increasing the rate of receptor inactivation (desensitization) (Fig. 3) and shifting the equilibrium to the inactive form of the receptor in the presence of acetylcholine. This last conclusion is most easily demonstrated by the experiments shown in Fig. 4. Incubation of acetylcholine receptor-rich vesicles with ace- Proc. Nad Acad. Sci. USA 79 (1982) tylcholine results in an equilibrium mixture of active and inactive receptor forms (17, 2, 3). The slow acetylcholine-dependent flux observed in E. electricus vesicles after prolonged preincubation with acetylcholine is proportional to the fraction of active receptor form in the equilibrium mixture (2). If the inhibitor interferes with the channel opening process and does not shift the equilibrium to the inactive form of the receptor (i.e., does not bind preferentially to inactive receptor but binds inactive receptor with the same affinity as receptor in the openchannel form), the slow flux measured by J. is expected to be inhibited to the same extent as the fast flux measured byja. Therefore, the ratioja/ji is expected to be the same in the presence and absence of inhibitor. This is observed when cocaine is the inhibitor:jjj1 is approximately13 in the absence and presence of cocaine. In contrast to this is the finding that PCP inhibitsjl to a much greater extent thanja.ja/j, in the presence of PCP is found to be 3 times larger than in the absence of PCP (Figs. 2 and 4), indicating that PCP does not merely interfere with the channel opening process but also changes the equilibrium between active and inactive receptors. PCP, therefore, binds inactive (desensitized) receptor with higher affinity than receptor in the open or closed channel conformations (AL2 and AL2). Fig. 2 lends further support to this conclusion. Although the amplitude of ion flux in the presence of both inhibitors is approximately the same after 1 sec, in the presence of PCP the vesicles fill only to the extent of 5% in 1 min. In the presence of cocaine, however, the vesicles fill completely within 1 min. It has been reported that the local anesthetic SKF-525A enhances the rate of receptor desensitization, based on 22Na' efflux measurements using membrane vesicles from Torpedo Recently, however, two inactivation processes marmorata (32). have been detected in receptors from T. californica with halftimes on the order of 3 msec and 6 sec at saturating carbamoylcholine concentrations (33, 34). Only a single fast inactivation process can be detected in receptors from E. electricus (2), and the present study is concerned with the effects of inhibitors on that process. SKF-525A appears to alter a much slower inactivation process (32). Studies of the effect of histrionicotoxin on the acetylcholine receptor in cultured chicken muscle cells (35) and the effect of substance P on the receptor in PC-12 cells (36) have been reported. These studies indicate that both inhibitors enhance desensitization. The states of receptor inactivation have not been characterized at all in these cell systems. The rates of conversion between active and inactive receptor states also are not known. As is the case with SKF-525A, these studies may be concerned with a slow inactivation process similar to the one observed in T. californica (33, 34). Therefore, it is difficult to compare the effects of PCP described here with the effects described in these other studies at this time. Analysis of acetylcholine-induced noise in muscle cells (37-39) allows one to measure elementary steps in the formation of ion channels through the cell membrane and to assess the effect of pharmacologically interesting compounds on these steps. The present study demonstrates that chemical kinetic measurements in a well-defined system gives information about the steps that are affected prior to channel opening as well as about the process ofreceptor inactivation. Use of this approach, therefore, allows one to elucidate the mode of action of important compounds such as cocaine and PCP. We are grateful to Lisa Lapish for making excellent membrane preparations. HiA. received a Senior Investigator's Fellowship from the Muscular Dystrophy Association. J.W.K. was supported by National Institutes of Health Training Grant 8-T2GM The work was supported by National Institutes of Health Grants EA464 (to L.G.A.) and DA2445 (G. P. H.).

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