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Changes of cholinesterase activities in the plasma and some tissues following administration of l -carnitine and galanthamine to rats
Neuroscience Letters 411 (2007) 212–216
Changes of cholinesterase activities in the plasma and some tissues following administration of l-carnitine and galanthamine to rats Jiri Bajgar a,∗ , Lucie Bartosova a , Josef Fusek a , Zdenek Svoboda b , Josef Herink b , Jaroslav Kvetina b , Vladimir Palicka c , Pavel Zivny c , Vaclav Blaha a a Faculty of Military Health Sciences, University of Defence, 500 01 Hradec Kralove, Czech Republic Institute of Experimental Biopharmaceutics, Heyrovskeho 1207, 500 03 Hradec Kralove, Czech Republic c Institute of Clinical Biochemistry and Diagnostic, Faculty Hospital, 500 05 Hradec Kralove, Czech Republic b
Received 21 July 2006; received in revised form 14 September 2006; accepted 14 September 2006
Abstract Changes of acetylcholinesterase (AChE) activities in the hypophysis and brain (frontal cortex, hippocampus, medial septum and basal ganglia), and butyrylcholinesterase in plasma and liver following galanthamine (GAL) administration were studied in rats pretreated with l-carnitine (CAR). Following only GAL administration (10 mg/kg, i.m.), both cholinesterases (without clinical symptoms of GAL overdosage) were significantly inhibited. Pretreatment with CAR (3 consecutive days, 250 mg/kg, p.o.) followed by GAL administration showed higher AChE inhibition in comparison with single GAL administration. However, a statistically significant difference was observed for AChE in the hippocampus only. The activity of peripheral cholinesterases was not influenced by CAR pretreatment. Thus, pretreatment with CAR enhanced AChE inhibition in some brain parts of the rat following GAL administration. © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Rat; Acetylcholinesterase; Brain parts; Butyrylcholinesterase; Plasma; Liver; l-carnitine; Galanthamine
Alzheimer disease (AD) is characterized by neuronal loss, synaptic damage, neurofibrillary tangles and neuritic and vascular plaques. At the cerebral level, AD is associated with reduced level of synaptic acetylcholine and other neurotransmitters. This has led to treatment attempts with cholinomimetics including cholinesterase inhibitors [5,7,16,15,23,31,32,38,44]. Most of AD treatments have been focused on the inhibition of acetylcholinesterase (AChE, EC 3.1.1.7) in order to raise the level of the neurotransmitter acetylcholine to augment cognitive functions of affected patients. Reversible inhibitors of cholinesterases belong to a group of drugs which were reported to be useful in the therapy of AD, as antidotes against intoxication with anticholinergics [12,13], as prophylactics against nerve agents [2,41], in tardive dyskinesias and others [12,13,10,11].
∗ Corresponding author at: Faculty of Military Health Sciences, University of Defence, Trebesska 1575, 500 01 Hradec Kralove, Czech Republic. Tel.: +420 973 251 507/511; fax: +420 495 518 094. E-mail address: [email protected] (J. Bajgar).
0304-3940/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2006.09.087
Galanthamine (GAL) is a cholinesterase inhibitor and allosteric modulation ligand at cholinergic receptors. It is valuable addition to agents available for pharmacological treatment of AD [34,37,42]. The natural component of the mammalian tissue l-carnitine (CAR) is known to increase penetration of some drugs or chemical groups through biological barriers [4,6,21,22,24,28,30]. Moreover, both drugs (inhibitors and CAR) enhance cholinergic transmission in the brain [33]. It was demonstrated previously that pretreatment with CAR in different doses (p.o.) increased AChE inhibition in the rat brain following administration of other reversible inhibitor, 7-methoxytacrine [19,20,39]. Highest effect for CAR dose of 250 mg/kg was observed [19,20,39]. Higher penetration of GAL through the blood-brain-barrier towards the target sites is connected with an assumed reduction of the therapeutic dose; a limitation of side effects from simultaneous administration of GAL and CAR can be expected. On the other hand, CAR has though weak cholinomimetic activity [33]. Some degree of protection of the cholinergic synapses could not be therefore excluded [4]. The influence of pretreatment with CAR on cholinesterase inhibition caused by GAL administration is the aim of this study.
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1. Material and Methods Male Wistar Han II rats (BioTest Ltd., Konarovice), 22 weeks old, weighing 270 ± 10 g, were used. The animals were maintained in an air conditioned room (22 ± 1 ◦ C and 50 ± 10% of relative humidity, with light from 07:00 to 19:00 h) and were allowed free access to standard chow and tap water. The directives of the Council of the European Communities (86/609/EEC) on animal care have been duly observed. Handling of experimental animals was performed under supervision of the local Ethic Committee. The animals were divided into groups with seven in each. Control group 1: Doses of 0.1 ml/100g of water (p.o.) were administered daily for 3 consecutive days. On the third day of experiments, saline was injected i.m., 30 min after the third water administration. Control group 2: The animals were treated with CAR (250 mg/kg, p.o.) daily for 3 consecutive days. On the third day of experiments, saline was injected i.m., 30 min after the third CAR adminstration. Experimental groups: The animals in groups were treated daily p.o. with CAR (250 mg/kg) for 3 consecutive days. On the third day, GAL in dose of 2.5 or 10 mg/kg was injected (i.m.). Used GAL doses did not cause symptoms of GAL over-dosage. The animals were killed by bleeding from a carotid artery 30 min after the GAL injection, and blood, liver, hypophysis and brain parts (frontal cortex-FC, basal ganglia-BG, medial septum-S, and hippocampus-HIPP) were collected and homogenates were prepared. CAR (l-carnitine hydrochloride), 5,5-dithio bis-2-nitrobenzoic acid were obtained from Sigma, GAL (galanthamine hydrochloride) from ICI Biomedicals Inc. Acetylthiocholine and butyrylthiocholine iodides were obtained from Lachema, Brno (Czech Republic). AChE activity in homogenates (water 1:10) of the brain parts and hypophysis was determined according to modified Ellmanˇıs method [9]. Butyrylcholinesterase activity (BuChE, EC 3.1.1.8) in plasma and liver homogenates was determined with butyrylthiocholine as substrate. The activities were expressed as cat/l or kg wet weight tissue. Regression analysis using last square method was used for transformation of the dependence enzyme activity versus GAL dose. The statistically significant differences in the slopes were determined and considered to be significant when p < 0.05. Regression was considered significant when the R2 (correlation coefficient) was higher than 0.835.
2. Results Normal BuChE activity in the liver (11.2 cat/kg) was higher than that observed in plasma (3.12 cat/l). Normal AChE activity in different parts decreased in order BG (1460.0 cat/kg), S (729.0 cat/kg), HIPP (420.7 cat/kg), and FC (224.2 cat/kg), respectively. Relative standard errors varied from ±13 to ±22%. CAR given p.o. in 3 consecutive days (control 2) did not influence either AChE activity in the brain parts studied or BuChE activity in plasma and liver in comparison with control group 1 (administration of saline only). Single administration of GAL (i.m.) has led to a decrease in cholinesterase activities in all tissues studied. Plasma BuChE changes for all experimental animals is given in Fig. 1. The slopes of the lines “BuChE activity versus GAL dose” are linear not differing in the slope for GAL with and without pretreatment with CAR. The same situation was observed for the enzyme activities in liver and hypophysis. The courses of cholinesterase (AChE and BuChE) inhibition caused by GAL in the hypophysis and liver were not influenced by CAR pretreatment. On the contrary, AChE activity decrease in HIPP was more expressed in animals pretreated with CAR (Fig. 2) and the slopes were significantly different. The inhibition of AChE activity in FC was enhanced by GAL pretreatment, however, the slopes of this decrease were not significantly differ-
Fig. 1. Changes in rat plasma butyrylcholinesterase activities in rats pretreated with l-carnitine. Plasma BuChE activity changes following GAL administration in rats with (+CAR) and without (−CAR) pretreatment with CAR. All experimental values are shown. Differences in the activities were not statistically significant.
Fig. 2. AChE activities in hippocampus following galanthamine administration in rats pretreated with l-carnitine. HIPP AChE activity changes following GAL administration in rats with (+CAR) and without (−CAR) pretreatment with CAR. All experimental values are shown. The differences in the slopes of the lines with (−6.62) and without (−1.53) carnitine were statistically (p < 0.05) significant.
ent. AChE inhibition was not significantly influenced by CAR pretreatment in BG or S. To compare relative inhibition, all data were transformed in per cents. Statistical differences in the slopes were observed for HIPP though some tendencies for FC AChE were registered (Fig. 3). When we compare relative changes of activities for
Fig. 3. AChE activities in hippocampus and frontal cortex following galanthamine administration in rats pretreated with l-carnitine. ChE activity changes following GAL administration with and without preatreatment with CAR. The results are expressed as means of percentage of control values. (1) Hippocampus without carnitine; (2) hippocampus with carnitine; (3) frontal cortex without carnitine; (4) frontal cortex with carnitine. The slopes of the lines 1, 3 ,4 were significantly different from the slope of the line 2 (p < 0.05).
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Fig. 4. Cholinesterase activity changes following GAL administration with (+CAR) and without (−CAR) pretreatment in the liver, hypophysis, BG and S. The results are expressed as means of percentage of control values. The numbers for each group indicate the slope of the line “activity vs. dose” and correlation coefficient (R2 ).
both cholinesterases (AChE and BuChE as % of control values), BuChE activity in liver and plasma and AChE activity in hypophysis were inhibited in a similar manner (Fig. 4). The enzyme inhibition of activities in peripheral tissues (hypophysis, liver, plasma) were stronger than those observed for the brain parts (FC and HIPP). Used GAL doses (2.5–10 mg/kg) did not cause symptoms of GAL over-dosage. Thus, pretreatment with oral CAR administration significantly enhanced anticholinesterase potency of GAL in HIPP only, though this tendency was observed also in FC. This pretreatment did not influence inhibition of BuChE in the plasma and liver or AChE in the hypophysis, FC, S and BG caused by GAL administration. 3. Discussion Highest AChE activity observed in BG and the lowest one in FC as well as BuChE activity in plasma (lower than that observed in liver) is in a good agreement with our previous [20,39] and literature [17] data. The use of compounds causing the inhibition of AChE allows a longer lasting presence of the neurotransmitter, acetylcholine, at the synaptic cleft compensating the cholinergic deficiency. The doses of GAL were chosen as doses without clinical symptoms of GAL over-dosage [19,20]. The inhibitory activity of GAL in vitro is somewhat lower than that of acridine derivatives [26] but it is very similar for cholinesterases from different sources [41,34]. On the other hand, inhibitory activity of these reversible inhibitors in vivo is not similar [2,40,14]. This discrepancy could be explained by non-uniform distribution of the drugs in tissues as it was demonstrated for tacrine, GAL, and 7-methoxytacrine [39,43,25]. However, the studies on distribution in the brain was limited to the whole brain only. It appears from our results that AChE inhibition is different also for various parts of the brain.
Changes of AChE activity following administration of GAL and reversible cholinesterase inhibitors of acridine group are in a good agreement with our previous and literature data [2,36,8]. The differences in the inhibition of AChE in the brain parts (especially FC and HIPP) could indicate various importance of them for the action of these drugs. The highest inhibition of AChE in the FC and HIPP would support a hypothesis on the importance of these structures for GAL effects. It was also described a role of GAL as an allosteric potentiating ligand probably enhancing function of both nicotinic and muscarinic excitatory neurotransmission on hippocampal pyramidal neurons [27]. More detailed study on cholinesterases in vivo and in vitro in connection with these results and especially with AD and other pathological states including poisonings can be recommended [1,35]. Higher AChE inhibition in the brain parts following GAL administration in animals pretreated with CAR suggest an influence of CAR on GAL-induced inhibition probably by enhanced GAL penetration into the brain. This potentiation of inhibition efficacy after CAR pretreatment was observed for other reversible inhibitor (7-methoxytacrine), too [19,20,18]. The permeation of different drugs such as clodronate through Caco-2 cells could be significantly promoted by the absorption enhancers including palmitoyl-CAR [30]. The enhanced absorption of simazine by carnitine was observed [6]. Moreover, acetyl-CAR and CAR are known to improve many aspects of the neural activity even its exact role in neurotransmission is still unknown [24]. Our results have not yet direct impact to clinical practice but they should be considered as possible new approach in the treatment of Alzheimer’s disease. In this connection, the effect of acetyl-CAR in association with donepezil or rivastigmin not responding to treatment with these inhibitors has been reported. The response rate was increased by 12% after the addition of acetyl-CAR. However, these data do not permit a conclusion as to the possible mechanism of action of the association of the two treatments [3]. Our results would suggest that this improvement could be caused by an influence
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of CAR on inhibition potency of GAL, either by direct effect (it can be excluded because this effect was not observed for peripheral cholinesterases) or by an influencing of GAL penetration into the brain. These inhibitory effects were different for AChE in various brain areas. A hypothetic reasons for this phenomenon should be discussed as different ratio of AChE/BuChE in these areas and different affinity of GAL to these two enzymes, dependence of AChE inhibition by GAL on the activity (i.e. its concentration) in the particular area, influence of the blood flow in various brain regions. A differentiation among these possibilities needs to be evaluated. The AChE inhibition in vivo was more expressed when the animals were pretreated with CAR. Theoretically, one of the possible mechanisms for increased AChE inhibition could be an enhancement of GAL penetration through the blood-brain barrier. It is also known that glial cells are the main sensors of neuronal function. Glial homeostasis of the extracellular milieu is circuit-specific, limiting the functional-metabolic coupling to discrete regions of the brain generating the classical pattern of the heterogenous activity in the different modules of the nervous tissue [29]. Thus, different action of CAR to various brain areas can not be excluded. Acknowledgement The authors express their appreciation to Mrs. M. Zechovska for her skilful technical assistance.Support by grant No. NR7935-3/2004 of the IGA MZ CR is gratefully acknowledged. References [1] J. Bajgar, Organophosphates/nerve agent poisoning: mechanism of action, diagnosis, prophylaxis, and treatment, Adv. Clin. Chem. 38 (2004) 151–216. [2] J. Bajgar, J. Fusek, F. Skopec, Changes of cholinesterases in the blood and some tissues following administration of Tacrin and its two derivatives to rats, Neurochem. Int. 24 (1994) 555–558. [3] A. Bianchetti, R. Rozzini, M. Trabucchi, Effects of acetyl-l-carnitine in Alzheimerˇıs disease patients unresponsive to acetylcholinesterase inhibitors, Curr. Med. Res. Opin. 19 (2003) 350–353. [4] J.J. Bohl, R. Brester, The pharmacology of carnitine, Ann. Rev. Pharmacol. Toxicol. 27 (1987) 257–277. [5] M. Colombres, J.P. Sagal, N.C. Inestrosa, An overview of the current and novel drugs for Alzheimerˇıs disease with particular reference to anticholinesterase compounds, Curr. Pharm. Res. 10 (2004) 3123–3132. [6] M. Cruz-Guzman, R. Celis, M.C. Hermosin, J. Cornejo, Adsorption of the herbicide simazine by montmorillonate modified with natural organic cations, Env. Sci. Technol. 38 (2004) 180–186. [7] S. Darvesh, R. Walsh, R. Kumar, A. Caines, S. Roberts, D. Magee, K. Rockwood, E. Martin, Inhibition of human cholinesterases by drugs used to treat Alzheimer disease, Alzheimer Dis. Assoc. Disord. 17 (2003) 117–126. [8] P. de Sarno, M. Pomponi, E. Giacobini, X.C. Tang, E. Williams, The effects of heptyl-physostigmine, a new cholinesterase inhibitor, on the central cholinergic system of the rat, Neurochem. Res. 14 (1989) 971–977. [9] G.L. Ellman, D.K. Courtney, V. Andres, R.M. Featherstone, A new rapid colorimetric determination of acetylcholinesterase activity, Biochem. Pharmacol. 7 (1961) 88–95. [10] V. Filip, J. Vachek, V. Albrecht, I. Dvorak, J. Dvorakova, J. Fusek, J. Havlu, Pharmacokinetics and tolerance of 7-methoxytacrine following the single dose administration in healthy volunteers, Int. J. Clin. Pharmacol. Ther. Toxicol. 29 (1991) 431–436.
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Changes of cholinesterase activities in the plasma and some tissues following administration of l-carnitine and galanthamine to rats Jiri Bajgar a,∗ , Lucie Bartosova a , Josef Fusek a , Zdenek Svoboda b , Josef Herink b , Jaroslav Kvetina b , Vladimir Palicka c , Pavel Zivny c , Vaclav Blaha a a Faculty of Military Health Sciences, University of Defence, 500 01 Hradec Kralove, Czech Republic Institute of Experimental Biopharmaceutics, Heyrovskeho 1207, 500 03 Hradec Kralove, Czech Republic c Institute of Clinical Biochemistry and Diagnostic, Faculty Hospital, 500 05 Hradec Kralove, Czech Republic b
Received 21 July 2006; received in revised form 14 September 2006; accepted 14 September 2006
Abstract Changes of acetylcholinesterase (AChE) activities in the hypophysis and brain (frontal cortex, hippocampus, medial septum and basal ganglia), and butyrylcholinesterase in plasma and liver following galanthamine (GAL) administration were studied in rats pretreated with l-carnitine (CAR). Following only GAL administration (10 mg/kg, i.m.), both cholinesterases (without clinical symptoms of GAL overdosage) were significantly inhibited. Pretreatment with CAR (3 consecutive days, 250 mg/kg, p.o.) followed by GAL administration showed higher AChE inhibition in comparison with single GAL administration. However, a statistically significant difference was observed for AChE in the hippocampus only. The activity of peripheral cholinesterases was not influenced by CAR pretreatment. Thus, pretreatment with CAR enhanced AChE inhibition in some brain parts of the rat following GAL administration. © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Rat; Acetylcholinesterase; Brain parts; Butyrylcholinesterase; Plasma; Liver; l-carnitine; Galanthamine
Alzheimer disease (AD) is characterized by neuronal loss, synaptic damage, neurofibrillary tangles and neuritic and vascular plaques. At the cerebral level, AD is associated with reduced level of synaptic acetylcholine and other neurotransmitters. This has led to treatment attempts with cholinomimetics including cholinesterase inhibitors [5,7,16,15,23,31,32,38,44]. Most of AD treatments have been focused on the inhibition of acetylcholinesterase (AChE, EC 3.1.1.7) in order to raise the level of the neurotransmitter acetylcholine to augment cognitive functions of affected patients. Reversible inhibitors of cholinesterases belong to a group of drugs which were reported to be useful in the therapy of AD, as antidotes against intoxication with anticholinergics [12,13], as prophylactics against nerve agents [2,41], in tardive dyskinesias and others [12,13,10,11].
∗ Corresponding author at: Faculty of Military Health Sciences, University of Defence, Trebesska 1575, 500 01 Hradec Kralove, Czech Republic. Tel.: +420 973 251 507/511; fax: +420 495 518 094. E-mail address: [email protected] (J. Bajgar).
0304-3940/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2006.09.087
Galanthamine (GAL) is a cholinesterase inhibitor and allosteric modulation ligand at cholinergic receptors. It is valuable addition to agents available for pharmacological treatment of AD [34,37,42]. The natural component of the mammalian tissue l-carnitine (CAR) is known to increase penetration of some drugs or chemical groups through biological barriers [4,6,21,22,24,28,30]. Moreover, both drugs (inhibitors and CAR) enhance cholinergic transmission in the brain [33]. It was demonstrated previously that pretreatment with CAR in different doses (p.o.) increased AChE inhibition in the rat brain following administration of other reversible inhibitor, 7-methoxytacrine [19,20,39]. Highest effect for CAR dose of 250 mg/kg was observed [19,20,39]. Higher penetration of GAL through the blood-brain-barrier towards the target sites is connected with an assumed reduction of the therapeutic dose; a limitation of side effects from simultaneous administration of GAL and CAR can be expected. On the other hand, CAR has though weak cholinomimetic activity [33]. Some degree of protection of the cholinergic synapses could not be therefore excluded [4]. The influence of pretreatment with CAR on cholinesterase inhibition caused by GAL administration is the aim of this study.
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1. Material and Methods Male Wistar Han II rats (BioTest Ltd., Konarovice), 22 weeks old, weighing 270 ± 10 g, were used. The animals were maintained in an air conditioned room (22 ± 1 ◦ C and 50 ± 10% of relative humidity, with light from 07:00 to 19:00 h) and were allowed free access to standard chow and tap water. The directives of the Council of the European Communities (86/609/EEC) on animal care have been duly observed. Handling of experimental animals was performed under supervision of the local Ethic Committee. The animals were divided into groups with seven in each. Control group 1: Doses of 0.1 ml/100g of water (p.o.) were administered daily for 3 consecutive days. On the third day of experiments, saline was injected i.m., 30 min after the third water administration. Control group 2: The animals were treated with CAR (250 mg/kg, p.o.) daily for 3 consecutive days. On the third day of experiments, saline was injected i.m., 30 min after the third CAR adminstration. Experimental groups: The animals in groups were treated daily p.o. with CAR (250 mg/kg) for 3 consecutive days. On the third day, GAL in dose of 2.5 or 10 mg/kg was injected (i.m.). Used GAL doses did not cause symptoms of GAL over-dosage. The animals were killed by bleeding from a carotid artery 30 min after the GAL injection, and blood, liver, hypophysis and brain parts (frontal cortex-FC, basal ganglia-BG, medial septum-S, and hippocampus-HIPP) were collected and homogenates were prepared. CAR (l-carnitine hydrochloride), 5,5-dithio bis-2-nitrobenzoic acid were obtained from Sigma, GAL (galanthamine hydrochloride) from ICI Biomedicals Inc. Acetylthiocholine and butyrylthiocholine iodides were obtained from Lachema, Brno (Czech Republic). AChE activity in homogenates (water 1:10) of the brain parts and hypophysis was determined according to modified Ellmanˇıs method [9]. Butyrylcholinesterase activity (BuChE, EC 3.1.1.8) in plasma and liver homogenates was determined with butyrylthiocholine as substrate. The activities were expressed as cat/l or kg wet weight tissue. Regression analysis using last square method was used for transformation of the dependence enzyme activity versus GAL dose. The statistically significant differences in the slopes were determined and considered to be significant when p < 0.05. Regression was considered significant when the R2 (correlation coefficient) was higher than 0.835.
2. Results Normal BuChE activity in the liver (11.2 cat/kg) was higher than that observed in plasma (3.12 cat/l). Normal AChE activity in different parts decreased in order BG (1460.0 cat/kg), S (729.0 cat/kg), HIPP (420.7 cat/kg), and FC (224.2 cat/kg), respectively. Relative standard errors varied from ±13 to ±22%. CAR given p.o. in 3 consecutive days (control 2) did not influence either AChE activity in the brain parts studied or BuChE activity in plasma and liver in comparison with control group 1 (administration of saline only). Single administration of GAL (i.m.) has led to a decrease in cholinesterase activities in all tissues studied. Plasma BuChE changes for all experimental animals is given in Fig. 1. The slopes of the lines “BuChE activity versus GAL dose” are linear not differing in the slope for GAL with and without pretreatment with CAR. The same situation was observed for the enzyme activities in liver and hypophysis. The courses of cholinesterase (AChE and BuChE) inhibition caused by GAL in the hypophysis and liver were not influenced by CAR pretreatment. On the contrary, AChE activity decrease in HIPP was more expressed in animals pretreated with CAR (Fig. 2) and the slopes were significantly different. The inhibition of AChE activity in FC was enhanced by GAL pretreatment, however, the slopes of this decrease were not significantly differ-
Fig. 1. Changes in rat plasma butyrylcholinesterase activities in rats pretreated with l-carnitine. Plasma BuChE activity changes following GAL administration in rats with (+CAR) and without (−CAR) pretreatment with CAR. All experimental values are shown. Differences in the activities were not statistically significant.
Fig. 2. AChE activities in hippocampus following galanthamine administration in rats pretreated with l-carnitine. HIPP AChE activity changes following GAL administration in rats with (+CAR) and without (−CAR) pretreatment with CAR. All experimental values are shown. The differences in the slopes of the lines with (−6.62) and without (−1.53) carnitine were statistically (p < 0.05) significant.
ent. AChE inhibition was not significantly influenced by CAR pretreatment in BG or S. To compare relative inhibition, all data were transformed in per cents. Statistical differences in the slopes were observed for HIPP though some tendencies for FC AChE were registered (Fig. 3). When we compare relative changes of activities for
Fig. 3. AChE activities in hippocampus and frontal cortex following galanthamine administration in rats pretreated with l-carnitine. ChE activity changes following GAL administration with and without preatreatment with CAR. The results are expressed as means of percentage of control values. (1) Hippocampus without carnitine; (2) hippocampus with carnitine; (3) frontal cortex without carnitine; (4) frontal cortex with carnitine. The slopes of the lines 1, 3 ,4 were significantly different from the slope of the line 2 (p < 0.05).
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Fig. 4. Cholinesterase activity changes following GAL administration with (+CAR) and without (−CAR) pretreatment in the liver, hypophysis, BG and S. The results are expressed as means of percentage of control values. The numbers for each group indicate the slope of the line “activity vs. dose” and correlation coefficient (R2 ).
both cholinesterases (AChE and BuChE as % of control values), BuChE activity in liver and plasma and AChE activity in hypophysis were inhibited in a similar manner (Fig. 4). The enzyme inhibition of activities in peripheral tissues (hypophysis, liver, plasma) were stronger than those observed for the brain parts (FC and HIPP). Used GAL doses (2.5–10 mg/kg) did not cause symptoms of GAL over-dosage. Thus, pretreatment with oral CAR administration significantly enhanced anticholinesterase potency of GAL in HIPP only, though this tendency was observed also in FC. This pretreatment did not influence inhibition of BuChE in the plasma and liver or AChE in the hypophysis, FC, S and BG caused by GAL administration. 3. Discussion Highest AChE activity observed in BG and the lowest one in FC as well as BuChE activity in plasma (lower than that observed in liver) is in a good agreement with our previous [20,39] and literature [17] data. The use of compounds causing the inhibition of AChE allows a longer lasting presence of the neurotransmitter, acetylcholine, at the synaptic cleft compensating the cholinergic deficiency. The doses of GAL were chosen as doses without clinical symptoms of GAL over-dosage [19,20]. The inhibitory activity of GAL in vitro is somewhat lower than that of acridine derivatives [26] but it is very similar for cholinesterases from different sources [41,34]. On the other hand, inhibitory activity of these reversible inhibitors in vivo is not similar [2,40,14]. This discrepancy could be explained by non-uniform distribution of the drugs in tissues as it was demonstrated for tacrine, GAL, and 7-methoxytacrine [39,43,25]. However, the studies on distribution in the brain was limited to the whole brain only. It appears from our results that AChE inhibition is different also for various parts of the brain.
Changes of AChE activity following administration of GAL and reversible cholinesterase inhibitors of acridine group are in a good agreement with our previous and literature data [2,36,8]. The differences in the inhibition of AChE in the brain parts (especially FC and HIPP) could indicate various importance of them for the action of these drugs. The highest inhibition of AChE in the FC and HIPP would support a hypothesis on the importance of these structures for GAL effects. It was also described a role of GAL as an allosteric potentiating ligand probably enhancing function of both nicotinic and muscarinic excitatory neurotransmission on hippocampal pyramidal neurons [27]. More detailed study on cholinesterases in vivo and in vitro in connection with these results and especially with AD and other pathological states including poisonings can be recommended [1,35]. Higher AChE inhibition in the brain parts following GAL administration in animals pretreated with CAR suggest an influence of CAR on GAL-induced inhibition probably by enhanced GAL penetration into the brain. This potentiation of inhibition efficacy after CAR pretreatment was observed for other reversible inhibitor (7-methoxytacrine), too [19,20,18]. The permeation of different drugs such as clodronate through Caco-2 cells could be significantly promoted by the absorption enhancers including palmitoyl-CAR [30]. The enhanced absorption of simazine by carnitine was observed [6]. Moreover, acetyl-CAR and CAR are known to improve many aspects of the neural activity even its exact role in neurotransmission is still unknown [24]. Our results have not yet direct impact to clinical practice but they should be considered as possible new approach in the treatment of Alzheimer’s disease. In this connection, the effect of acetyl-CAR in association with donepezil or rivastigmin not responding to treatment with these inhibitors has been reported. The response rate was increased by 12% after the addition of acetyl-CAR. However, these data do not permit a conclusion as to the possible mechanism of action of the association of the two treatments [3]. Our results would suggest that this improvement could be caused by an influence
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of CAR on inhibition potency of GAL, either by direct effect (it can be excluded because this effect was not observed for peripheral cholinesterases) or by an influencing of GAL penetration into the brain. These inhibitory effects were different for AChE in various brain areas. A hypothetic reasons for this phenomenon should be discussed as different ratio of AChE/BuChE in these areas and different affinity of GAL to these two enzymes, dependence of AChE inhibition by GAL on the activity (i.e. its concentration) in the particular area, influence of the blood flow in various brain regions. A differentiation among these possibilities needs to be evaluated. The AChE inhibition in vivo was more expressed when the animals were pretreated with CAR. Theoretically, one of the possible mechanisms for increased AChE inhibition could be an enhancement of GAL penetration through the blood-brain barrier. It is also known that glial cells are the main sensors of neuronal function. Glial homeostasis of the extracellular milieu is circuit-specific, limiting the functional-metabolic coupling to discrete regions of the brain generating the classical pattern of the heterogenous activity in the different modules of the nervous tissue [29]. Thus, different action of CAR to various brain areas can not be excluded. Acknowledgement The authors express their appreciation to Mrs. M. Zechovska for her skilful technical assistance.Support by grant No. NR7935-3/2004 of the IGA MZ CR is gratefully acknowledged. References [1] J. Bajgar, Organophosphates/nerve agent poisoning: mechanism of action, diagnosis, prophylaxis, and treatment, Adv. Clin. Chem. 38 (2004) 151–216. [2] J. Bajgar, J. Fusek, F. Skopec, Changes of cholinesterases in the blood and some tissues following administration of Tacrin and its two derivatives to rats, Neurochem. Int. 24 (1994) 555–558. [3] A. Bianchetti, R. Rozzini, M. Trabucchi, Effects of acetyl-l-carnitine in Alzheimerˇıs disease patients unresponsive to acetylcholinesterase inhibitors, Curr. Med. Res. Opin. 19 (2003) 350–353. [4] J.J. Bohl, R. Brester, The pharmacology of carnitine, Ann. Rev. Pharmacol. Toxicol. 27 (1987) 257–277. [5] M. Colombres, J.P. Sagal, N.C. Inestrosa, An overview of the current and novel drugs for Alzheimerˇıs disease with particular reference to anticholinesterase compounds, Curr. Pharm. Res. 10 (2004) 3123–3132. [6] M. Cruz-Guzman, R. Celis, M.C. Hermosin, J. Cornejo, Adsorption of the herbicide simazine by montmorillonate modified with natural organic cations, Env. Sci. Technol. 38 (2004) 180–186. [7] S. Darvesh, R. Walsh, R. Kumar, A. Caines, S. Roberts, D. Magee, K. Rockwood, E. Martin, Inhibition of human cholinesterases by drugs used to treat Alzheimer disease, Alzheimer Dis. Assoc. Disord. 17 (2003) 117–126. [8] P. de Sarno, M. Pomponi, E. Giacobini, X.C. Tang, E. Williams, The effects of heptyl-physostigmine, a new cholinesterase inhibitor, on the central cholinergic system of the rat, Neurochem. Res. 14 (1989) 971–977. [9] G.L. Ellman, D.K. Courtney, V. Andres, R.M. Featherstone, A new rapid colorimetric determination of acetylcholinesterase activity, Biochem. Pharmacol. 7 (1961) 88–95. [10] V. Filip, J. Vachek, V. Albrecht, I. Dvorak, J. Dvorakova, J. Fusek, J. Havlu, Pharmacokinetics and tolerance of 7-methoxytacrine following the single dose administration in healthy volunteers, Int. J. Clin. Pharmacol. Ther. Toxicol. 29 (1991) 431–436.
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