Differential acetylcholinesterase activity in rat cerebrum, cerebellum and hypothalamus

Indian Journal of Experimental Biology Vol. 44, May 2006, pp. 381-386 Differential acetylcholinesterase activity in rat cerebrum, cerebellum and hypothalamus Rini Roy (Pal) & Aditi Nag Chaudhuri* Department of Microbiology, Lady Brabourne College, P1/2 Suhrawardy Avenue, Kolkata 700 017, India Received 14 March 2005: revised 26 December 2005 Acetylcholinesterase (AChE) has been purified from three different regions of rat brain using Sephadex G 200 column. SDS PAGE (6%) showed single band for the purified AChE fractions. Purified and lyophilized AChE from different (NH 4 ) 2 SO 4 precipitated fractions of three brain parts were utilized for in vitro enzyme kinetics using Dimethoate (Dmt) as inhibitor. K m values for cerebellum and hypothalamus were almost similar whereas cerebrum showed a different K m value compared to other two regions. With the drug Rivastigmine it was found that % G 1 and G 4 forms of AChE in three different parts of brain are different. Keywords: Acetylcholinesterase activity, Cerebrum, Cerebellum, Hypothalamus, Rat Organophosphorus pesticides (OPs) are widely used in Indian agricultural fields. Most of the OP affected areas have shown the occurrence of Non-Hodgkin s Lymphoma (NHL). In pursuit of designing less toxic to non-targets organophosphorus pesticides experiments were performed on rat model with various OPs having different LD 50 values and different octanol water partition coefficient values (P ow ). OPs affect the enzyme acetylcholinesterase (AChE) in the mammalian brain. Effects of OPs like methyl parathion, malathion, phorate, dimethoate, chlorpyrifos, monocrotophos and dichlorvos (widely used in Indian agricultural fields), on AChE activity in three different parts of rat brain viz hypothalamus (H), cerebellum (CR) and cerebrum (C) showed a relationship with LD 50 values and P ow values of the pesticides 1. The results showed that not all parts of the brain are equally sensitive to the same pesticide. Cerebrum was totally different in behaviour compared to other regions of rat brain. The observations have been supported by multiple correlation coefficient calculation, which is the measure of efficacy for different predicting equations (formulated by Multiple Regression Analysis) involving AChE activity, level of LD 50 and P ow values 1,2. To find out the molecular level of interaction between AChE and particular pesticide in particular part of rat brain, AChE from different parts of the rat brain was purified and the *Correspondent author Phone: 91-33 24625927 E-mail: odditybio@yahoo.com interaction is reported in the present study. From statistical analysis also it has been observed that cerebrum was behaving differently from other brain regions. The present study has been undertaken with the aim to verify whether this different behaviour of cerebrum was the result of mathematical calculation or due to inherent property of it. Materials and Methods Healthy male Albino Charles Foster rats (~100g body weight) were provided with ad libitum food and water for 12-hr light-dark cycle for acclimatization in the laboratory atmosphere. Since from all of the pesticides dimethoate (30% EC, 30% w/w, Rallis India Ltd.) affected all the parts of the rat brain though not equally 1 this pesticide was used for in vitro interaction study. Common laboratory reagents used in the experiments were of analytical reagent grade and purchased either from E. Merck, Sigma or SRL. Chemicals like acetylthiocholine iodide, 5,5 - dithiobisnitrobenzoic acid, Folin-Ciacalteü, Perchloric acid, were obtained from Sigma Chemical Company. Sephadex G 200 (PHARMACIA, Uppsala Sweden) was used for gel filtration. One capsule, (4.5 mg) of Rivastigmine gifted from NOVARTIS was dissolved in 5ml ethanol and then diluted to 10 ml by adding distilled water and vigorously shaken before use. Following buffers were used: (a) 10 mm phosphate buffer, ph 7, (b) 1 mm phosphate buffer, ph 7, (c) 30

382 INDIAN J EXP BIOL, MAY 2006 mm phosphate buffer, ph 8, (d) 0.1 M phosphate buffer, ph 8, (e) 0.1 M phosphate buffer, ph 7.5. Purification of AChE Cerebrum, cerebellum and hypothalamus portions of rat brain were pooled from rats (number of rats = 6) and weighed individually. All the three tissues of brain were homogenized (15% w/v) with 0.32 M sucrose containing 1 mm EDTA and 1 mm phosphate buffer (ph 7). The crude homogenate was centrifuged at 5000 g for 10 min. Volume of supernatants (S 1 ; supernatant obtained from centrifugation of the crude homogenate) was measured, enzyme activity of three regions was assayed in duplicate and protein content was also estimated. (NH 4 ) 2 SO 4 (10%) curdling was done by noting the volume of the S 1. This was centrifuged at 5000 g. Pellet (P 1 ; 10%) was collected for further investigation. Now, taking the S 2 (supernatant separated from P 1 ), 30% (NH 4 ) 2 SO 4 curdling 3,4 was done by noting the volume of S 2. Centrifugation was done at 5000 g. Pellet (P 2 ; 30%) and supernatant (S 3 ; supernatant separated from P 2 ) were collected. P 1 and P 2 were washed with 10mM phosphate buffer. All the fractions S 3 and P 1 and P 2 were dialyzed overnight against 1 mm phosphate buffer ph 7 at 10 C. Purification of AChE is favoured by great stability of the enzyme as it may be kept in refrigerator for years 3. Preliminary chromatographic runs in the cold were compared with runs at room temperature. No significant difference in recovery of enzyme activity, protein content or chromatographic pattern was observed 5. Enzyme activity in S 2, S 3 and P 1 and P 2 were measured by Ellman method 6 using acetylthiocholine iodide as substrate. In all the cases protein was measured using Lowry s method 7. Sephadex G 200 equilibrated with 30 mm phosphate, buffer ph 8 was poured in 1 22.5 cm column to get 10 cm gel length. Gel was allowed to settle overnight. The dialysed protein [10 % (NH 4 ) 2 SO 4 precipitated pellet fractions and 30 % (NH 4 ) 2 SO 4 precipitated supernatant fractions of three regions of rat brain] was loaded on Sephadex G 200. Eluate 3.5 ml was collected in each tube at the rate of 14ml/hr. For each fraction for each eluate, AChE activity and protein were measured by the above mentioned methods. The fractions showing AChE activity and protein content were pooled together and lyophilized. Purified, lyophilized AChE was used from each part of the brain to see the AChE and pesticide interaction and was expressed in form of K m and V max.. SDS-PAGE of Brain AChE Sodium dodecyl sulphate-polyacrylamide gel (6%) electrophoresis (SDS-PAGE) of different parts of brain AChE were done following Chan et al. 8. Estimation of protein Protein was estimated as per Lowry et al. 7 at 660 nm. Kinetic study of purified enzyme K m and V max of purified enzyme were done using dimethoate as inhibitor 9. AChE activity was determined by measuring the rate of hydrolysis of acetylthiocholineiodide in phosphate buffer (ph 8.0, 0.1 M) following the method of Ellman et al. 6. Specific activity (Sp. activity) of the AChE in different fractions of brain was determined. Determination of K m and V max A Lineweaver- Burk curve was drawn by plotting 1/[substrate]/ (mm -1 ) on the X-axis and 1/[Sp. Activity] ( OD/mg pr/hr) on the Y-axis. Lineweaver-Burk plots showed that the compound tested was competitive with reference to the substrate acetylthiocholineiodide. Results and Discussion The pellet and supernatant portions of brain were labelled as Fraction I = 30%C supernatant, Fraction II = 30%H supernatant, Fraction III = 30%CR supernatant, Fraction IV = 10%C pellet, Fraction V = 10%H pellet, Fraction VI = 10%CR pellet, Fraction VII = 30%C pellet, Fraction VIII = 30%H pellet, Fraction IX = 30%CR pellet. The % recovery and the fold purification of the P 1, P 2 and S 3 portions of three regions of rat brain after dialysis of P 1, P 2 and S 3 obtained from (NH 4 ) 2 SO 4 saturation of the crude extract are given in Table 1. The elution profile of AChE (P 1, P 2 and S 3 portions of three regions) on Sephadex G 200 column showing the specific activity ( OD/mg pr/hr) and the protein content (in mg) against fraction number has been shown in Figs 1-3. The % recovery and the fold purification of the P 1 and S 3 portions of three regions of rat brain after performing Column Chromatography with Sephadex G 200 are given in Table 2. The % recovery of Fraction VII was too low to carry out further study. The % recovery of Fraction VIII and Fraction IX were lower than the other cut portions (Fraction III and Fraction VI for cerebellum and Fraction II and Fraction V for hypothalamus). Therefore, these two portions were not taken for further study.

ROY & NAG CHAUDHURI : ACETYLCHOLINESTERASE ACTIVITY IN RAT BRAIN 383 Table 1 Purification of AChE in (a) cerebrum, (b) hypothalamus and (c) cerebellum of rat brain after dialysis Fraction Total protein (mg) Total activity (units) Specific activity (unit/mg pr/hr) Recovery (%) Fold purification Cerebrum Crude Homogenate 18.645 153.746 8.246 100 1.0 Fraction I 2.910 58.724 20.176 38.195 2.45 Fraction IV 1.914 9.786 5.113 6.36 0.62 Hypothalamus Crude Homogenate 13.583 59.847 4.406 100 1.0 Fraction II 0.421 12.451 29.576 20.805 6.71 Fraction V 2.125 19.947 9.387 33.33 2.13 Cerebellum Crude Homogenate 9.148 69.293 7.000 100 1.0 Fraction III 3.557 45.375 12.755 65.48 1.82 Fraction VI 0.643 2.495 3.880 3.6 0.49 Fig. 1 Elution profile of (a) 10% and (b) 30% (NH 4 ) 2 SO 4 precipitated pellet and supernatant fraction, respectively of AChE in cerebrum after overnight dialysis against 1 mm phosphate buffer ph 7 on Sephadex G 200 equilibrated with 30 mm phosphate buffer ph 8 at the rate of 14 ml/ hr. [ - - Protein (mg), - - Specific activity (Δ OD/mg pr/hr)]. Fig. 2 Elution profile of (a) 10% and (b) 30% (NH 4 ) 2 SO 4 precipitated pellet and supernatant fraction, respectively of AChE in hypothalamus after overnight dialysis against 1 mm phosphate buffer ph 7 on Sephadex G 200 equilibrated with 30 mm phosphate buffer ph 8 at the rate of 14 ml/ hr. [ - - Protein (mg), - - Specific Activity (Δ OD/mg pr/hr)].

384 INDIAN J EXP BIOL, MAY 2006 Table 2 Purification of AChE in (a) cerebrum, (b) hypothalamus and (c) cerebellum of rat brain after Sephadex G 200 Fraction Total protein (mg) Total activity (units) Specific activity (unit/mg pr/hr) Recovery (%) Fold purification Cerebrum Crude Homogenate 18.645 153.746 8.246 100 1.0 Fraction I 0.099 7.31 73.863 4.5 8.96 Fraction IV 0.199 1.577 13.257 1.026 1.60 Hypothalamus Crude Homogenate 13.583 59.847 4.406 100 1.0 Fraction II 0.133 1.295 9.740 10.40 2.210 Fraction V 0.099 1.312 13.257 10.53 3.008 Cerebellum Crude Homogenate 9.148 69.293 7.000 100 1.0 Fraction III 0.058 4.519 77.921 6.52 10.287 Fraction VI 0.099 0.750 7.576 1.082 1.08 Fig. 3 Elution profile of (a) 10% and (b) 30% (NH 4 ) 2 SO 4 precipitated pellet and supernatant fraction, respectively of AChE in cerebellum after overnight dialysis against 1 mm phosphate buffer ph 7 on Sephadex G 200 equilibrated with 30 mm phosphate buffer ph 8 at the rate of 14 ml/ hr. [ - - Protein (mg), - -Specific Activity (Δ OD/mg pr/hr)]. The lyophilized portion after column chromatography showed a single band of ~ 67 kda on 6% SDS PAGE (Fig. 4) for Fractions I-V indicating homogeneity of the purified enzyme. The K m and V max were calculated using the Lineweaver Burk plot in presence and absence of inhibitor (dimethoate). The K m and V max of AChE from different fractions with or without in vitro use of dimethoate have been represented in Table 3. The K m values in both cerebellum and hypothalamus were almost similar but that obtained from cerebrum was different from the above two regions. It was earlier observed that nature of graph of AChE activity vs octanol-water partition coefficient (P OW ) values of different pesticides in cerebrum was totally different from other regions of the rat brain 1,2 (Figs 5, 6). To find out the molecular forms of AChE in cerebellum, cerebrum and hypothalamus Rivastigmine, a drug for Alzheimer s disease was employed in the present study. Among the inhibitors of AChE, only one inhibitor, i.e., Rivastigmine displayed preferential inhibition for G 1 form of AChE 10. In animals, Rivastigmine inhibits in the brain much more potently than in peripheral organs and consequently has minimal peripheral side effects. In addition, the drug is more potent in inhibiting AChE in cortex and hippocampus than in other brain regions such as striatum and pons/medulla. The existence of AChE in different molecular forms is well established. In human brain, the total AChE level and the distribution of its molecular forms vary regionally 11. Recently, G 1 form and G 4 form of AChE have been isolated from cerebral cortex of rat brain 12.

ROY & NAG CHAUDHURI : ACETYLCHOLINESTERASE ACTIVITY IN RAT BRAIN 385 Fractions Cerebrum Hypothalamus Cerebellum Table 3 K m and V max of AChE activity from different fractions without and with in vitro use of Dimethoate [Values are mean ± SD from 3 observations] K m (mm) V max (ΔOD/mg pr/hr) -dmt +dmt -dmt +dmt I 2.631±0.02 a 3.703±0.03 a 4.360±0.07 a 4.360±0.07 a IV 2.702±0.00 a 20.000±1.10 b 5.079±0.05 a 5.079±0.05 a II 4.545±0.08 b 9.090±0.23 b 6.695±0.08 b 6.695±0.08 b V 4.160±0.07 b 25.000±1.15 b 4.619±0.06 a 4.619±0.06 a III 5.000±0.01 a 7.692±0.17 b 8.619±0.12 b 8.619±0.12 b VI 6.660±0.12 b 14.280±0.80 b 9.735±0.28 c 9.735±0.28 c Mean affinity as well as V max of abovementioned regions of rat brain with different superscripts are significantly different. The F values in one-way ANOVA, with degrees of freedom 2 and 3 are significant at both 1 and 5% level of significances. Fig. 4 SDS-PAGE (6%) of different fractions of AChE [1- Fraction I, 2- Fraction II, 3- Fraction III, 4- Fraction IV, 5- Fraction V, 6- BSA]. Fig. 5 Acetylcholinesterase activity (Δ OD/ mg pr/hr) with increase in octanol-water partition coefficient (P ow ) in cerebellum of pesticide treated rat brain at (a) 1/20 LD 50, (b) 1/10 LD 50 and (c) 1/5 LD 50 after 30 days of treatment [Dimethoate: logp ow = 0.65; Methyl parathion: logp ow = 2.68; Malathion: logp ow = 2.89; Phorate: logp ow = 3.9]. Fig. 6 Acetylcholinesterase activity ( Δ OD/ mg pr/hr ) with increase in octanol-water partition coefficient (P ow ) in cerebrum of pesticide treated rat brain at (a) 1/20 LD 50, (b) 1/10 LD 50 and (c) 1/5 LD 50 after 30 days of treatment [Dimethoate: logp ow = 0.65; Methyl parathion: logp ow = 2.68; Malathion: logp ow = 2.89; Phorate: logp ow = 3.9].

386 INDIAN J EXP BIOL, MAY 2006 The most abundant form found in human brain is the tetrameric G 4, while the monomeric G 1 is present in smaller amounts. Rivastigmine is 4 to 5 times more potent in inhibiting G 1 form as compared to the G 4 form extracted from cortex and hippocampus in the patients of Alzheimer s disease 11. From the in vitro inhibition study with Rivastigmine in the three portions of the normal rat brain, it was observed that 55.38% inhibition occurred in cerebrum compared to 21.51 and 23.74% inhibition in cerebellum and hypothalamus, respectively. This suggests that G 1 form exists in cerebrum in higher amount than in cerebellum and hypothalamus, which is showing differential variation in total acetylcholinesterase activity in different parts. Acknowledgement Thanks are due to the University Grants Commission (UGC), New Delhi for financial assistance. References 1 Roy R, Das Gupta N & Nag Chaudhuri A, Designing of less toxic to non targets organophosphorus pesticide: Predicting equation of Acetylcholinesterase activity dependence on LD 50 values and octanol water partition coefficients of the pesticides, Indian J Biochem Biophys, 2004 (communicated). 2 Roy R, Das Gupta N& Nag Chaudhuri A, Designing of less toxic to non targets organophosphorus pesticide through multiple correlation analysis (MRA), a part of QSAR, paper presented to the Symposium on Fourth Indo-US Workshop on Mathematical Chemistry, University of Pune, Maharashtra, India, 8-12 January, O12 (2005) 17. 3 Colowick Sidney P & Kaplan, Purification of acetylcholinesterase, in Methods in enzymology (Academic Press Inc., New York) 1955, 645. 4 Chan S l, Shirachi D Y & Trevor J J, Purification & properties of brain acetylcholinesterase (EC 3.1.1.7), J Neurochem, 19 (1972) 437. 5 Ho I K & Ellman G L, Triton solubilized Acetylcholinesterase of brain, J Neurochem, 16 (1969) 1505. 6 Ellman G L, Courtney K D, Andres V, Jr & Featherstone R M, A new and rapid colorimetric determination of acetylcholinesterase activity, Biochem Pharmacol, 7 (1961) 88. 7 Lowry O H, Rosebrough N J, Farr A L & Randall R J, Protein measurement with folin-phenol reagent, J Biol Chem, 193 (1951) 265. 8 Chan S L, Schirachi D Y, Bhargava H N, Gardner E & Trevor J J, Purification and properties of multiple forms of brain acetylcholinesterase (EC 3.1.1.7), J Neurochem, 19 (1972) 2747. 9 Cohen S D, Williams R A, Killinger J M & Freudenthal R I, Comparative Sensitivity of Bovine and Rodent Acetylcholinesterase to in vitro Inhibition by Organophosphate Insecticides, Toxicol Appl Pharmacol, 81 (1985) 452. 10 Rakonczay Z, Potencies and Selectivities of inhibitors of acetylcholinesterase and its molecular forms in normal and Alzheimer s disease brain, Acta Biol Hung, 54 (2003) 183. 11 Enz A, First KUOPIO Symposium under the auspices of Department of Neuroscience & Neurobiology, University of KUOPIO, Proceedings of the Symposium, 28-30 January, 1999. 12 Qin Z & Xi-Can T, Isolation of acetylcholinesterase G 4 and G 1 molecular isoforms from rat cortex, Acta Pharmacol Sin, 23 (2002) 173.