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Beta amyloid protein and plant cysteine proteinases influence the (3H)hemicholinium-3 binding site in vitro in rat hippocampus

Krištofiková Z., Klaschka J.

Prague Psychiatric Centre, Ústavní 91, Prague, Czech Republic

 2nd FEPS Congress, June 29 - July 4, 1999, Prague, Czech Republic 

 Physiological Research 1999: Vol. 48, Suppl. 1, S87.- poster 

463 KA (18-11-1)



Recent research suggests that some forms of beta amyloid peptides (Ab) inhibit the high-affinity choline uptake in rat hippocampus in vitro [1,2]. It seems that the effect could lead to autocannibalism of cholinergic neurons observed in Alzheimer disease. The specific binding of (3H)hemicholinium-3 is closely associated with substrate recognition site on the high-affinity choline carrier [3]. However, the in vitro effect of Ab on this binding site has not been evaluated yet.

 Our experiments on hippocampal synaptosomes of male and female 3-month old Wistar rats suggest that lipophilic Ab 1-40 eliminates depolarization effects and reminds actions of ethanol [4]. However, 100 nM concentration incubated 10-20 min has evoked no significant effects on the high-affinity transport and specific binding under basal conditions. Plant cysteine proteolytic enzymes bromelain and papain (100 mg/ml, short-term 10-20 min preincubation), two anti-inflammatory drugs [5] and perspective supportive agents for Alzheimer disease therapy, have decreased the uptake and specific binding under both basal and stimulated conditions. Membrane-localized carrier protein seems to be cleft by both proteases independently of its actual functional state, i.e. active as well as inactive carriers. It appears that papain influences rather external part of carrier and surrounding membrane in comparison with a more specific action of bromelain. The high-affinity transport measured on hippocampal synaptosomes of young rats and previously inhibited by Ab 1-40 is more sensitive to the effects of bromelain and papain in vitro.

[1] D.S.Auld et a., TINS, 21: 43-49, 1998.

 [2] S.Kar et al., J Neurochem, 70: 2179-2187, 1998.

 [3] S.S.G.Ferguson et al., J Neurochem, 63: 1328-1337, 1994.

 [4] Z.Krištofiková et al., Neurochem Res, 23: 923-929, 1998.

 [5] K.L.Lee et al., Biochem J, 327: 199-202, 1997.

 unpublished paper


Brains of patients with Alzheimer disease (AD) are characterized among others by the presence of amyloid deposits in selected brain regions (1). Recent research suggests important physiological functions for amyloid beta peptides (Abeta) proteolytically derived from a larger amyloid precursor protein (2). However, depending also on the fragment length and on the degree of aggregation, higher concentrations of Abeta can induce apoptotic or necrotic neuronal degeneration (1,2).

 Enhanced vulnerability of cholinergic forebrain system under normal and pathological aging (cholinergic hypothesis of AD) has been described for many times (e.g., 3). Recent research suggests direct actions of Abeta on cholinergic neurons leading probably to their autocannibalism under pathological conditions. While higher concentrations of Abeta (mM) incubated for a sufficiently long time (hours) increase generally the lipid peroxidation (1,2) and the leakage of choline across cell membranes (2,4,5), low concentrations (pM-nM) incubated for a short time (minutes) influence significantly cholinergic neurotransmission (2,6). Some fragments of Abeta inhibit among others the hippocampal and cortical high-affinity choline uptake (HACU) that is a rate-limiting step in acetylcholine synthesis (6). However, effects of Abeta on the specific binding of (3H)hemicholinium-3 ((3H)HC-3, specific and competitive inhibitor of HACU) have not been evaluated yet.

 At the present time, great attention concentrates on the therapy of AD and a possible administration of plant cysteine proteases as supportive agents is suggested. It seems that different endogenous proteases or their inhibitors play a role in the ethiology of senile plaques and neurofibrillary tangles. Some of these mechanisms could be influenced by exogenous proteases. Moreover, the proteolytic enzymes act as anti-inflammatory agents and increase the permeability of the blood-brain barrier. Our preliminary results of experiments in vitro indicate certain advantages for a perspective application in vivo to demented patients especially for bromelain (BRO) and papain (PAP).

Aim of the study

•   To evaluate in vitro effects of Abeta on the specific binding of (3H)HC-3 and to contribute to the more detailed elucidation of action mechanism on hippocampal presynaptic cholinergic nerve terminals.

•   To evaluate in vitro effects of BRO and PAP on HACU and (3H)HC-3 binding.

•   To compare these results with our preliminary data and to assess possible risks and advantages for a protease application in AD therapy.

Material and methods

•   Brain tissue: Hippocampal synaptosomes were isolated from 68 male and female 3-month old Wistar rats of the Konárovice breed.

•      Chemicals: Abeta (amyloid beta-protein fragment 1-40, Sigma), BRO (Ananas Comosus, Mucos Pharma), PAP (Carica papaya, Mucos Pharma), (3H)choline (NEN), (3H)HC-3 (NEN) and HC-3 (Sigma) were used.

•      Methods: HACU and (3H)HC-3 binding were measured in accordance with our previous work (7). Abeta and proteases were incubated for 10 min (HACU) or for 20 min ((3H)HC-3 binding) at 37°C before measurement. Concentration of proteins was estimated by a method of Bradford (8).

•   Data analysis: ANOVA and Student's t-test (separate variance) were applied. Data are presented as the means ± S.D.


•   100 nM Abeta influenced significantly the HACU and (3H)HC-3 specific binding only under stimulated conditions. More detailed analysis revealed that the decrease of transport is linked to the change in KM, the decrease of binding to the change in both Bmax and Kd.


•      Concentrations of proteases lower than 100 mg/ml did not significantly influence the HACU levels. The effect on HACU was not dependent on protein concentrations in the incubation mixture.


•   100 nM Abeta significantly decreased the HACU levels previously incubated with PAP but not with BRO.


•   100 mg/ml BRO decreased markedly the HACU levels and moderately the (3H)HC-3 binding on synaptosomes previously incubated with 100 nM Abeta. The effect of PAP was not significant.



•   The in vitro effect of Abeta on high-affinity choline carriers

Our results support the works where Abeta-mediated inhibiton of HACU transport in the hippocampus in the case of very low concentrations and especially under stimulated conditions has been reported (2,6). It seems that lipophilic Abeta eliminates depolarization effects and reminds in vitro actions of ethanol that reduces K+-stimulated increase in intracellular Ca2+ concentrations (7). Recent research indicate depolarization-dependent changes rather in the activity of membrane high-affinity carriers for choline than in their number (9). However, comparison of data from both HACU and (3H)HC-3 binding including Lineweaver-Burk and Scatchard analysis suggests that Abeta actions are not probably connected only with a simple repeated decrease in the activity of carriers (Table I and II). While the high-affinity carrier conformation seems to be returned to conditions before depolarization for choline transport, this conformation seems to be totally changed for (3H)HC-3 binding that is closely associated with choline recognition site (9). Therefore, it appears that more pools of the carriers (active and inactive membrane-localized as well as occult membrane pools) are involved in process of depolarization and in the following Abeta actions.

•   The in vitro effects of BRO and PAP on high-affinity choline carriers

Plant cysteine proteolytic enzymes have decreased the uptake and specific binding under both basal and stimulated conditions (Table V). Therefore, it seems that membrane-localized carrier protein is cleft by both proteases independently of its actual functional state, i.e. active as well as inactive carriers. In contrast to brain capillaries (our unpublished results), the effect of both proteases on synaptosomal uptake has been pronounced only for higher concentrations (Table III) and not dependent on the concentration ratio of enzymes and proteins in the incubation mixture (Table IV). Comparison of both HC-3 sensitive and HC-3 insensitive choline uptakes indicates that PAP influences rather external part of carrier and surrounding membrane in comparison with a more specific action of BRO (Table VI).

•   The evaluation of results from the point of view of AD therapy

Our previous experiments with Abeta and both proteases have indicated certain positive circumstances for the application of enzymes in AD therapy. Firstly, the effects of Abeta and of proteases on the lipid peroxidation in hippocampal homogenates after long-term preincubation (30-60 minutes) have been significantly age-dependent with antagonistic trends. Secondly, both proteases have been able to eliminate the negative effects of Abeta on the lipid peroxidation. Our experiments with HACU and (3H)HC-3 binding using in vitro test with Abeta suggest possible advantages rather for the application of BRO (Abeta is not able to decrease HACU previously affected by BRO -Table VI, BRO is able to cleave carrier protein previously affected by Abeta - Table VII) in contrast to PAP. However, more detailed experiments and especially the application in vivo to young and old rats must be further performed in our laboratory in future.

The research was performed under grant of GACR n. 305/99/1317.


1 Cotman CW and Pike CJ (1994): Alzheimer Disease (Terry RD, Katzman R and Bick KL, eds.). New York, Raven Press, pp. 305-315.

2 Auld DS, Kar S and Quirion R (1998): Trends Neurosci. 21, 43-49.

3 Hagan JJ (1994): Anti-dementia Agents (Nicholson CD, ed.). London, Academic Press, pp. 85-138.

4 Allen DD, Galdzicky Z, Brining SK, Fukuyama R, Rapoport S and Smith QR (1997): Neurosci. Lett. 234, 71-73.

5 Ehrenstein G, Galdzicky Z and Lange G.D. (1997): Biophys. J. 73, 1276-1280.

6 Kar S, Issa AM, Seto D, Auld DS, Collier B and Quirion R (1998): J. Neurochem. 70, 2179-2187.

7 Krištofiková Z, Klaschka J. and Tejkalová H (1998): Neurochem. Res. 23/7, 923-929.

8 Bradford MM (1976): Anal. Biochem. 72, 248-254.

9 Fersugon SSG, Rylett RJ and Collier B (1994): J. Neurochem. 63, 1328-1337.


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