Molecularand CellularBiochemistry95: 13-20, 1990. © 1990KluwerAcademic Publishers. Printedin the Netherlands. Original Article

The chlorpromazine inhibition of transport ATPase and acetylcholinesterase activities in the microsomal membranes of rat in vitro and in vivo Barsanjit Mazumder, Shyamali Mukherjee 1 and Parimal C. Sen Department of Chemistry, Bose Institute, Calcutta - 700 009, India; 1Present address: Department of Biochemistry, Meharry Medical College, School of Medicine, Nashville, TN37209, USA Received23 January 1989; accepted14 September 1989

Key words: Na+,K+-ATPase, Ca+2-ATPase, aceltylcholinesterase, chlorpromazine, different organs, microsomal membranes (rat) Abstract

Chlorpromazine, an antipsychotic drug, is found to inhibit Na',K---ATPase activity in rat brain microsomal membranes in vitro in concentration and time dependent manner but some inconsistency is observed when the effect was studied with respect to different temperatures. Various ligands and/or substrate affect the inhibition by chlorpromazine in different ways. The in vivo study with this drug shows that the activities of Na ÷,K÷-ATPase, Ca÷2-ATPase and acetylcholinesterase in the microsomal membranes of different organs are inhibit with increases in concentration or lengths of time of treatment and then levels off.

Introduction

The Na +,K+-ATPase is responsible for the coupled transport of Na + and K + ions and Ca+2-ATPase for the transport of Ca +2ion across the cell membranes at the expense of ATP hydrolysis [1]. Acetylcholinesterase is a synaptosomal membrane bound enzyme and is responsible for the restoration of the polarization of post synaptic membrane by hydrolyzing acetylcholine ester. All these enzyme activities have been found to be affected by inhibitors [1], drugs [24] and hormones [5, 6]. The inhibition of ATPase activities leads to a decrease of the uptake of K" and increase of intracellular concentration of Na + [7, 8] and so does Ca +2 concentration through the Na+/Ca+2-exchange [9]. Chlorpromazine, an antipsychotic drug, can antagonize dopamine receptors within the central nervous system and is known to affect various neurological and metabolic functions at cellular and molecular levels [10]. It can inhibit lipid peroxidation [11], alter

biosynthesis of acidic lipids [12] and induce intralysosomal accumulation of phospholipids and inhibit phospholipase activity [13]. It can displace membrane bound Ca +2 [14], alter membrane permeability [15] and readily accumulate inside the membrane vesicles [16]. Chlorpromazine can disrupt the membrane structure due to its high surface activity [17]. It has been reported previously [18, 19] that chlorpromazine free radicals produced by uv-irradiation could affect the transport enzymes and membrane functions. In the present study, we describe the effect of chlorpromazine on Na+,K +ATPase activity in rat brain microsomal membranes in vitro and for the first time on Na +,K +ATPase, p-nitrophenyl phosphatase activities in brain and kidney, acetylcholinesterase activity in brain and Mg+2-dependent Ca+2-ATPase activity in spleen and testes in vivo in order to explore in detail the mechanisms of alteration of these enzyme activities by chlorpromazine.

14 Materials and methods

Materials

ATP,[3-mercaptoethanol (I]ME), imidazole, phenylmethyl sulfonyl fluoride (PMSF) and chlorpromazine hydrochloride (CPZ) were purchased from Sigma Chemical Co., USA. Sucrose and other chemicals of analytical grade were obtained from Sisco Research Laboratories (SRL, India) or E. Merck (Germany). Double distilled deionized water was used throughout the study. Methods

Isolation of microsomal membranes - The study was carried out using male rats of Charls foster strain of 9 months old and weigh about 150-200 gm. Each animal was killed by anesthatization in chloroform and the tissues were collected on ice. The microsomal membranes from brain were isolated following the method of Robinson [20] and from kidney, spleen and testes as described previously [4]. The pellet in each case was suspended in 25 mM imidazole buffer containing l mM EDTA and 250 mM sucrose (pH 7.5) ans assayed for protein following the method of Lowry et al. [21] using bovine serum albumin as standard.

E n z y m e assay

The Na+,K+-ATPase activity was assayed as described by Robinson [20] and Sen et al. [22]. The reaction mixture in a total volume of i ml contained 30raM imidazole buffer (pH 7.5), 3mM MgC12, 3 mM ATP, 50-200 tzg membrane protein, 130 mM NaC1 and 20 mM KC1. When present, ouabain was used at a concentration of 1 mM. Na+,K+-ATPase activity was taken to be the ouabain sensitive component of the total ATPase activity and was about 95% of the total ATPase activity. The p-nitrophenyl phosphatase (pNNPase) activity was also assayed as described by Robinson [20]. The medium contained, in a total volume of 2 ml, 30 mM imidazole (pH 7.5), 2mM MgCI2, 3 mM p-nitrophenyl phosphate, 100-250/zg membrane protein with and

without 10 mM KC1. After 30 min incubation the reaction was stopped with 2 ml of 1.5 mM NaOH and the liberated p-nitrophenol was read at 410 nm against the blank. The MgZ2-dependent Ca ÷2ATPase activity was measured as described previously [23, 24]. The assay mixture in a total volume of 1 ml contained 50 mM histidine buffer in 25 mM sucrose and 0.5 mM EDTA (pH 7.5), i mM MgC12, 3 mM ATP with and without 3 mM CaC12. The difference between the activity with and without Ca +2 was considered as Ca+2-ATPase activity. The incubation time for ATPase assay was 30 min at 37° C. The reaction was terminated with 5% icecold TCA and the liberated Pi was estimated spectrophotometrically at 820 nm against a blank that contained all the ingredients except membrane protein. Acetylcholinesterase activity was assayed using acetylthiocholine iodide as substrate following the method of Ellman et al. [25]. The membrane protein (20-25/zg) was incubated with 5 mM acetylthiocholine iodide for 30 min at 37° C. It was then stopped with 12% perchloric acid and centrifuged at 2,000 r.p.m, for 5 min. To 0.1ml of the supernatant, 0.4 ml of 67 nm DTNB (5.5'-dithiobis-2-nitro-benzoic acid) was added, mixed and read at 400 nm in 3 min. The enzyme activity was calculated from a standard curve prepared with different concentrations of L-cysteine. In vitro treatment of rat brain microsomal membranes with chlorpromazine - For concentration effect study, the membranes in 25mM imidazole buffer containing 1 mM EDTA and 25 mM sucrose (pH 7.5) were treated with different concentrations of chlorpromzine at a fixed time (15 rain) and temperature (30 ° C). For time course study, the membranes were treated with a final concentration of 0.5 mM of drug at a fixed temperature (30° C) for different lengths of time. For temperature effect study, the membranes were treated with 0.5 mM drug at a fixed time (15 min) at different temperatures. For studying the effect of ligand(s) and substrate on inhibition by chlorpromazine, the membranes were treated with optimum concentration of drug (0.5 mM) in the presence of a particular concentration of ligand(s) and/or substrate and incubated for 15 min at 30° C. The excess or unbound drug was removed by centrifugation at

15 100,000 g for 1 h in each case and the pellet was resuspended in 25 m M imidazole buffer containing i mM E D T A , 25 m M sucrose (pH 7.5) and assayed for protein and Na+,K+-ATPase activity as described above. In all the cases, the control membranes without drug was run under identical conditions and percent inhibition was calculated taking the activity of the control membranes as 100. The specific activities of control membranes were in the following ranges: Na+,K+-ATPase (3.5-4.5 /xmoles/hr/mg protein), Ca+2-ATPase (1.5-2.0 /xmoles/hr/mg protein), pNPPase (2.0-3.5 t~moles/ hr/mg protein) and acetylcholinesterase (2.5-3.5 /zmoles/hr/mg protein). In vivo treatment of rat with chlorpromazine For dose dependence study, the rats were divided into six groups with five animals in each group. The first group of animals served as control and the animals of the 2nd, 3rd, 4th, 5th and 6th groups were intraperitoneally injected with 2, 4, 8, 16 and 32/xg of drug per gm body weight of the animal in physiological saline respectively. Each animal was sacrificed on the 16th day. For time course study except control animal in each group, the other animals of each group were treated with 32/xg chlorpromazine/gm body weight at different periods of time. Each set of animals was sacrificed on 8, 12, 16, 20, 24 and 28 days respectively. The control animal in each set was injected with an equal volume of saline. Isolation of membranes and subsequent assays were done as described before.

Statistical calculation - The results shown were the mean + SD of a few determinations as described in each table and figure. Student's 't' test was used to calculate the significance of differences between different data points.

Results The effect of different concentrations of chlorpromazine on Na+,K+-ATPase activity in vitro is shown in Table 1. A gradual increase in inhibition of enzyme activity is observed with increase in drug concentration and reaches a maximum inhibition of 82% with 0.75 mM chlorpromazine. The inhibition of enzyme activity by the drug in different lengths of time is also shown in Table 1. A n optimum inhibition of 45% by 0.5 mM drug is obtained in 10 min and remains in that level up to 60 min of incubation. Incubation beyond 60 rain leads to the loss of activity of the control membranes. The effect of temperature on the inhibition of enzyme activity by a fixed concentration of drug at a particular time (Table 1) shows that the inhibition increases slightly from 24 to 30 ° C, a sharp fall at 34 ° C and again increases at 37° C. The effect of drug on inhibition in the presence of different ligand(s) and/or substrate is shown in Table 2. Mg +2 and K ÷ do not have any significant effect on the inhibition but Na + and ATP increase the inhibition slightly.

Table 1. Effect of different concentrations of chlorpromazine, time and temperature of incubation on Na÷,K+-ATPaseactivityin the microsomal membranes of rat brain in vitro

Conc" (mM)

Percent inhibition

Timeb (min)

Percent inhibition

Temp° (0° C)

Percent inhibition

0 0.25 0.35 0.50 0.75 1.00

0 4.0+ 2.2 35.2+ 7.5* 53.2+ 7.2* 82.2 + 10.7" 85.7 + 10.6

0 5 10 20 30 60

0 30.0+ 7.8 45.8+ 8.3* 45.0+ 10.3 47.0___ 8.5 43.0 + 6.2

24 27 30 34 37 40

45.9 + 9.2 43.0+ 9.6 50.8+ 7.4 6.1 + 3.3* 37.4 + 6.7 35.5 + 10.0

The membranes were incubated with different concentrations of chloropromazine at a fixed temp (30° C) and time (20 min) [a] or different time at a fixed concentration (0.50mM) and temperature (30°C) [b] or different temperature at a fixed concentration (0.50mM) and time (30 min) [c]. The data shown was the mean + S.D. of 5-6 separate experiments. The other procedures were the same as described in Methods (n = 4). * The difference is significant from the preceding data point (P < 0.05).

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Chlorpromoz;ne(~tg ) Fig. 1. Effect of different concentrations of chlorpromazine on

Na+,K+-ATPase (0), pNPPase (O) and acetylcholinesterase (/~) activities in the microsomal membranes of rat brain in vivo. The animals were treated with different concentrations of the drug for 16 days. Each animal was sacrificed, brain collected and microsomal membranes were isolated for protein and enzyme activities assay as described in Methods. Each point was the mean + S.D. of 8-10 determinations obtained as duplicate assay of 4-5 experiments. The difference is significant from the control values in the case of Na+,K+-ATPase (P < 0.02) and AchE (P < 0.05) from 4/xg to 32/.~g of drug. Table 2. Effect of different ligands and/or substrate on the inhibition of Na ÷,K+-ATPase activity by chlorpromazine in the microsomal membranes of rat brain in vitro a Ligands/Substrate

% inhibition

None CPZ Mg +2 Na + K+ ATP Mg +z+ Na + + K + + A T P

0 48.0 -+ 8.3 44.0 + 7.5 58.0 -+ 9.9 45.0 _+ 6.0 60.0 -+ 6.7 51.4+7.6

"The membranes were incubated without (none) or with chlorpromazine alone (0.50 raM) or in the presence of Mg +2 (3 raM) or Na ÷ (130mM) or K ÷ (20raM) or ATP (3mM) or Mg +2 (3 mM) + Na ÷ (130 mM) + K ÷ (20 mM) + ATP (3 mM) for 20 min at 30° C. Each reaction mixture was centrifuged at 100,000 g for I hr and the pellet resuspended in sample buffer and assayed for enzyme activity as described in the text. The difference is not significant when the inhibition was compared between CPZ alone and CPZ in the presence of ligands and/or substrate (n = 4).

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Fig. 2. Effect of different concentrations of chlorpromazine on

Na ÷,K÷-ATPase (0) and pNPPase (O) activities in the microsomal membranes of rat kidney in vivo. The other experimental conditions were the same as in Fig. 1. P < 0.02 in Na+,K÷ATPase and < 0.05 in pNPPase from the control values from 8/zg to 32 ~g of drug used.

T h e effect o f d i f f e r e n t c o n c e n t r a t i o n s o f t h e d r u g in v i v o o n N a + , K + - A T P a s e , p N P P a s e a n d acetylc h o l i n e s t e r a s e activities in b r a i n a n d k i d n e y a r e s h o w n in Figs 1 a n d 2. T h e activities of N a + , K +ATPase, pNPPase and acetylcholinesterase have b e e n f o u n d to inhibit with r e s p e c t to d i f f e r e n t conc e n t r a t i o n s o f t h e d r u g a n d a l m o s t levels off at 16/zg o f C P Z p e r g m b o d y w e i g h t of t h e animals. N o effect o n Ca+2-ATPase activity o f t h e testes a n d s p l e e n is o b s e r v e d u n d e r t h e s e c o n d i t i o n s (results n o t s h o w n ) . H o w e v e r , if t h e a n i m a l s a r e t r e a t e d with t h e d r u g for l o n g e r p e r i o d s o f t i m e , a significant i n h i b i t i o n is o b t a i n e d (Fig. 3). T h e i n h i b i t i o n of N a + , K + - A T P a s e , p N P P a s e a n d a c e t y l c h o l i n e s t e r a s e activities of b r a i n a n d k i d n e y a r e f o u n d to i n c r e a s e with t i m e a n d a l m o s t levels off at 20 d a y s w h e n t r e a t e d w i t h 32 ~ g / g m b o d y w e i g h t o f t h e a n i m a l s (Figs 4 a n d 5).

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Fig. 3. Mg÷,Ca÷2-ATPaseactivityin the microsomalmembranes of rat testes (0) and spleen (C)) in vivo when the animals were treated

without or with 32 tzg of drug/gm body weight of the animal for different lengths of time. P < 0.05 in 24 and 28 days from the control values. The enzyme activitieswithout drug are shown at zero time. Discussion

It has been reported that the inhibition of Na+,K +ATPase by chlorpromazine occurred due to the formation of semiquinone free radicals of chlorpromazine by light [18, 19]. However, in contrast, we have found that chlorpromazine itself can inhibit the transport enzyme activities both in vitro and in vivo. The findings in the present study indicate that Na÷,K÷-ATPase in rat brain microsomal membranes is inhibited by chlorpromazine on dose dependent manner to a concentration of 0.75 mM and then levels off (Table 1). With respect to time, the inhibition reaches a plateau level when incubated for 10 min with 0 . 5 m M of the drug (Table 1). Chlorpromazine has been reported to react with - SH groups of the proteins [18] and the activity of Na+,K+-ATPase is believed to be dependent on - SH groups of the enzyme [1]. The increase in inhibition with respect to concentration and time of incubation may be due to the reaction of drug with - SH groups of proteins which reaches a maximum level in 10 min incubation and/or 0.75 m M concentration of the drug. The findings of the temperature effect though are interesting but strange in that the extent of inhibition is almost the same at 24, 27 and 30°C (approx. 48%, Table 1) and then falls to

about 6% at 34 ° C, again increases at 37 and 40 ° C to about 35 %. The nature of this enzyme inhibition may be due to the non-specific binding of chlorpromazine of the enzyme at different temperatures. The Na÷,K÷-ATPase has two binding sites for ATP, low affinity and high affinity binding sites [1]. Hence the other explanation of inconsistent inhibition by chlorpromazine with respect to temperature may be due to the binding of drug to either or both of these sites; thus affecting the binding of the substrate leading to variable level of inhibition. The different level of inhibition of Na÷,K+-AT Pase by the drug in the presence of ligand(s) and/or ATP (Table 2) (the difference is not significant) may be due to the different conformational states of the enzyme that reacts with the drug differently. It is pertinent to mention in this connection that various ligands like Na ÷, K ÷ and Mg ÷2 are capable of changing the conformation of the enzyme [1, 22]. Injection of any drug may produce several overlapping effects simultaneously, but still retain their functional individuality by retaining the chemical specificity of the synapses at the site of overlap [26]. The in vivo study with different concentrations of chlorpromazine (Figs 1-2) shows a depletion of the acetylcholinesterase activity in the microsomal membranes of brain, Na+,K+-ATPase, pNPPase

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D~,s Fig. 4. Na÷,K+-ATPase ( I ) , pNPPase (O) and acetylcholinesterase (A) activities of brain microsomal membranes in vivo when the animals were treated without or with 32/.Lg of drug/gin body weight of the animal for different lengths of time. The other experimental conditions were the same as in Fig. 1. P < 0.05 in 16 to 28 days from the control in all three cases.

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Days Fig. 5. Na+,K+-ATPase (O) and pNPPase (O) activities of kidney microsomal membranes in vivo when the animals were treated without or with 32/zg of drug/gin body weight of the animal for different lengths of time. The other experimental conditions were the same as in Fig. 1. P < 0.05 in Na ÷,K÷-ATPase and < 0.02 in pNPPase in 20 to 28 days from the control values.

19 activities in brain, kidney and reaches saturation level of inhibition at 16 ~g/gm body weight of the animal. However, the Ca+2-ATPase activity of the microsomal membranes of spleen and testes are found to be insensitive to chlorpromazine up to the dose of 32 tzg/gm body weight of the animal for 16 days (Fig. 3) and then inhibition is observed. The higher dose of the drug could not by used because of the high rate of mortality of the animal. A significant decrease of Na÷,K÷-ATPase, pNPPase and acetylcholinesterase activities have been observed when the animals were treated with 16 ~g drug/gm body weight of the animal (Figs 1 and 2). The activities of the enzymes reach almost a plateau level in 20 days of treatment (Figs 4 and 5). Except in Ca+2-ATPase of testes and spleen (Fig. 3) a significant inhibition of enzyme activities is observed in 20 days in all other cases. In case of testes and spleen, we have to treat the animal for 28 days to get a significant inhibition. All these enzymes reported here are known to be membrane bound and the activities of membrane bound enzymes are believed to depend on the phospholipid environment of the membranes. Thus any agent and/or drug which can alter the composition of phospholipid of the membranes will also alter the activities of the enzymes [1]. Chlorpromazine get readily accumulated inside the membrane vesicle [16], alter membrane permeability and fluidity [15] and phospholipid metabolism through the alteration of phospholipase activity [13], phospholipid transfer protein [27] and the biosynthesis of acidic lipids [12]. The changes in phospholipids and fatty acids composition as well as the fluidity of the membranes in CPZ treated animals compared to the control (unpublished observations) are expected to have definite contributions towards the inhibition of the enzyme activities by chlorpromazine in vivo. Furthermore, inhibition of these enzyme activities by CPZ on time and concentration dependent manner both in vitro and in vivo and the effect of substrate and some ligands on CPZ inhibition in vitro led us to believe that the CPZ inhibition may be specific. Our findings are in confirmation with a recent report by Van Dye et al. [28] who showed that the effect of chlorpromazine

on Na+,K+-pumping and solute transport in rat hepatocytes was specific.

Acknowledgements Authors are thankful to Professor N.K. Sinha, Chairman, Department of Chemistry and Professor B.B. Biswas, Director, Bose Institute for their interest in this work. A part of this work was supported from a grant of DST, Government of India [22(77)/84-STP-I].

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Address for offprints: P.C. Sen, Section of Biochemistry, Molecular & Cell Biology, Cornell University, Ithaca, NY 14853, USA