Actions of ginsenoside Rb1 on choline uptake in central cholinergic nerve endings

Neurochem. Int. Vol. 21, No. 1, pp. l-5, 1992 Printed in Great Britain. All rights reserved

0197-0186/9255.00+0.00 Copyright © 1992Pergamon Press Ltd

ACTIONS OF GINSENOSIDE Rbl ON CHOLINE UPTAKE IN CENTRAL CHOLINERGIC NERVE ENDINGS CHRISTINA G. BENISHIN Department of Physiology, Faculty of Medicine, University of Alberta, Edmonton, Alberta, Canada (Received 21 August 1991; accepted 5 December 1991)

Abstract--The ginsenoside Rb~ has previously been reported to improve memory deficits induced by anticholinergic drug treatment, and to facilitate acetylcholine (ACh) release from rat brain hippocampal slices. The increase in ACh release was not associated with an increase in calcium uptake into nerve terminals, but was associated with an increase in uptake of the precursor choline. In the present studies, analysis of choline uptake kinetics indicated that Rb~ increased the maximum velocity of choline uptake, while the affinity of the choline uptake carrier for choline (Kin) was not significantly altered. Acute treatment with Rb~ did not alter the number of [3H]hemicholinium-3 (HC-3) binding sites in any of three cholinergic brain regions examined, suggesting that the increase in the maximum velocity of choline uptake was not associated with an increase in the number of choline carriers. However, chronic (3 day) administration of Rb~ did increase the number of choline uptake sites in the hippocampus, and to a lesser extent in the cortex.

Although a number of central neurotransmitters may be involved in memory and learning, some specific cholinergic drugs have been found to have profound effects on these behaviors. Therefore, the central cholinergic nervous systems are thought to play crucial roles in memory and learning (Perry, 1986). In particular, muscarinic cholinergic antagonists, such as scopolamine, were found to induce memory and learning impairment in laboratory animals, as well as humans (Rush, 1988). Drugs which increase central cholinergic function or act as cholinergic agonists have been found to improve memory dysfunctions. For example, acetylcholinesterase inhibitors have been found to reverse memory deficits induced by chemical (Yamazaki et al., 1991) or electrolytic lesions (Haroutunian et al., 1985 ; Sweeney et al., 1990). These drugs have also been used in humans with memory impairments o f various etiologies (Hollander et al., 1986; Summers et al., 1986), however, the clinical efficacy may be inconsistent (Goodnick and Gershon, 1984; G a m z u et al., 1990). These compounds have been the most extensively studied for potential therapeutic benefit in the alleviation of some of the symptoms of senile dementia of the Alzheimer's type (SDAT). Several recent studies have examined the ability of extracts and individual components of ginseng to reverse m e m o r y deficits (Saito, 1985 ; Benishin et al., 1991). The effects of the saponin components of

ginseng, the ginsenosides, on central cholinergic function have also been examined (Zhang, 1990; Benishin et al., 1991). Recently, we have found that one ginsenoside, Rb~, is capable of reversing the scopolamine-induced memory deficit in rats, and also facilitating the release of acetylcholine (ACh) from hippocampal slices. The increase in ACh release is not associated with an increase in calcium influx, but is associated with increased choline uptake (Benishin et al., 1991). The objective of the present study is to examine more closely the mechanism of the increase in choline uptake.

EXPERIMENTAL PROCEDURES

Choline uptake into rat brain synaptosomes was measured as described previously (Benishin et al., 1991) with some modifications. Rats were killed by decapitation and the forebrains removed into ice cold 0.32 M sucrose solution, buffered at pH 7.4 with 10 mM HEPES. The crude synaptosomal fraction (P2) was prepared by homogenization and centrifugation as described by Benishin et al. (1988). The uptake incubation was initiated by addition of an aliquot of the Pz synaptosome suspension containing 41~50 mg protein (determined by the method of Lowry et al., 1951) to the warmed uptake solution consisting of normal Kreb's solution containing 0.1 to I0/~M [3H]choline chloride (1/zCi/ml), and with or without Rb ~. The total volume of the incubation was 2 ml per tube and each concentration was run in duplicate. After 5 min the incubation was terminated by chilling the tubes in an ice bath followed by rapid filtration on Whatman GF/B filters. The filters were washed three times with

CHRISTINA G. 5 ml of ice cold Kreb's solution, and then the ~H retained on the filter counted by liquid scintillation spectrometry. Under these conditions, choline uptake is reduced by at least 98% by 10 pM hemicholinium-3. K,,, and Vm~,~for synaptosomal choline uptake were calculated with the analysis by the Enzfitter computer program. Hemicholinium-3 (HC-3) binding to synaptosomal membranes was determined as described previously (Sandberg and Coyle, 1985). Rats were killed by decapitation, and the brains removed into ice cold 0.32 M sucrose solution. The cortex, hippocampus and striatum were dissected out and the membrane fractions prepared by homogenization and centrifugation (Sandberg and Coyle, 1985). The final washed pellets were resuspended in glycylglycine NaC1 buffer (composition : glycylglycine, 50 mM : NaC1, 200 mM ; pH 7.8). Aliquots of the membrane fractions containing 50 1000 #g protein, depending on the brain region (assayed according to the method of Lowry et al., 1951) were incubated in glycylglycine NaCI buffer solution containing [3H]HC-3 (10.8 Ci/mmol), with (non-specific binding) or without (total binding) 10 #M unlabeled HC-3. The total volume was 500 #1. In order to quantitate total binding sites, the concentration of [3H]HC-3 used was l0 riM, as has been used previously (Saltarelli et al., 1988). Incubations were carried out for 30 min at 30C, and were terminated by removing the tubes to an ice bath, adding 5 ml of ice-cold glycylglycine NaCI buffer, followed by rapid filtration on Whatman GF/B filters which were previously treated with 0.1% polyethyleneimine. Filters were then washed three times with Tris NaC1 buffer (composition: Tris HCI, 50 raM; NaC1, 200 raM). The 3H retained on the filters was counted by liquid scintillation spectrometry. Specific binding of HC-3 to synaptosomal membranes was calculated as the difference between the amount bound in the absence (total) and the presence (non-specific) of unlabeled HC-3. Materials Rb, was prepared from North American ginseng (Panax quinqu!Jblium L.) as described previously (Shoji, 1981). [3H]choline chloride and [3H]hemicholinium-3 chloride were purchased from New England Nuclear (Boston, MA).

RESULTS

Previous studies have s h o w n that Rb, stimulates the uptake of 1 # M choline into rat brain nerve endings. Choline uptake under these conditions is essentially completely inhibited by 1 # m HC-3 (data not shown). The uptake of various c o n c e n t r a t i o n s o f choline ranging from 0.1 to 10.0 # M was also stimulated by Rb~. The results are presented as a double reciprocal plot in Fig. l, a n d suggest that the increase in uptake results from an increase in the V,.... of uptake. Table 1 presents the results of the kinetic analysis of choline uptake in the absence a n d the presence of 0.1 and 10 n M Rb,. The effects of Rb, on the K,,, were not consistent or significant, while Rb~ produced a significant, dose dependent increase in the Vn,~,x. Increase in the Vn,~,, o f an uptake process may be accounted for by either one o f two different mech-

BENISHIN 15

• Control V Rbl,lOnM /

10

/

/

E N ~

/

5

0

I

0

t~

I

2

I

4

t

/

I

6

[

8

I

10

12

1/[- Choline ],/.L M Fig. 1. Double reciprocal plot of the rate of choline uptake at different concentrations of choline in the absence ( 0 ) or presence (V) of 10 ~ M Rb,. Each point is the average of 13 ( 0 ) or 6 (V) determinations.

anisms: (1) an increase in the n u m b e r of t r a n s p o r t sites, or (2) an increase in the t u r n o v e r rate of each carrier. In order to discriminate between these two possibilities, the effect of Rb~ on the specific binding of HC-3, a specific ligand for the choline uptake site was determined. Rbl up to a concentration of 10 v M did not alter HC-3 binding to cortical and hippocampal m e m b r a n e s in vitro (data not shown). Results presented in Fig. 2 indicate that acute a d m i n i s t r a t i o n of Rb~ (5 m g / k g i.p., [ h prior to sacrifice) to rats tended to slightly, but insignificantly, increase the n u m b e r of HC-3 binding sites in three cholinergic brain regions. These results would suggest that, acutely, R b t does not increase the n u m b e r of choline carriers in synaptosomal membranes. Figure 3, however, presents the results of chronic a d m i n i s t r a t i o n of Rb~ to rats (5 m g / k g / d a y i.p. for 3 days). This dosage regimen was used in a previous study, a n d was shown to reverse

Table I. S u m m a r y o f the effects o f Rb, on the kinematic parameters of synaptosomal choline uptake

Control l0 I ° M Rbl 10 ~ M Rbl

K,,, app (itM)

V,,,,, (cpm/5 min)

N

8.11 ± 1.54 15.65+5.53 23.2±6.59

686.01 + 146.19 1326.98+241.56" 1903.33_+ 188.06"

13 8 6

* Indicates value is significantly different from control, P < I).05, N e w m a n - K e u l s test.

Ginsenoside Rb~ on choline uptake in central cholinergic nerve endings brain regions examined, the hippocampus, and to a lesser extent the cortex. Rbl had no significant effect on the HC-3 binding in the striatum.

200

2

~5o

g DISCUSSION ~

100

e-

0 I

5O

Cortex (8)

Hippo (6)

Striotum (6)

Fig. 2. The effects of acute (5 mg/kg i.p., l h prior to sacrifice) Rb, on the HC-3 binding in three cholinergic brain regions, cortex, hippocampus (hippo), and striatum. Results are expressed as a percent of control. Numbers in parentheses under each bar are N's for each region.

scopolamine-induced memory deficits in rats (Benishin et al., 1991). In this case the longer term administration of Rbj does increase the number of HC-3 binding sites in two of the three cholinergic 300 0

-

250

(~ 200 L.) 150 c

~5

100

]

L) I 50

Cortex (16)

Hippo (16)

m

Striotum (8)

Fig. 3. The effects of chronic (5 mg/kg/day i.p. for 3 days) Rb, on the HC-3 binding in three cholinergic brain regions, cortex, hippocampus (hippo), and striatum. Results are expressed as a percent of control. Numbers in parentheses under each bar are N's for each region. "indicates significantly greater than control (saline) group, P < 0.05, t-test.

Rb~ has been reported to improve memory and learning. On the basis of results presented here, as well as results presented earlier (Benishin et al., 1991), the ability of Rb, to reverse memory deficits may be related to the ability of this compound to also increase cholinergic neurotransmission centrally. In vitro, Rb, produces an increase in the maximal velocity (Vmax) of choline uptake into synaptosomes exposed to the drug, while it does not significantly alter the affinity of the carrier (Kin) for choline. This is similar to the results of Simon et al. (1976), who also noted that treatments which increase choline uptake do so by increasing the maximal velocity of uptake. The increase in //maxis associated with a minimal change in the number of HC-3 binding sites on synaptic membranes which are exposed to Rb, in vitro. This would be consistent with a generalized increase in the turnover of ACh in synaptosomes, as has been seen by others (Swann and Hewitt, 1988; Yamada et al., 1988). One would not expect Rb, to alter HC-3 binding to membranes in the absence of any change in ACh turnover, such as is the case in the synaptic membrane preparations used in the HC-3 binding studies. These data also suggest that Rb ~does not have a direct effect on the choline uptake carrier, such as increasing the number by unmasking carrier sites. In vitro, Rb~ increased ACh release following 20 min of exposure to hippocampal slices (Benishin et al., 1991), and increased choline uptake even at 5 rain. However, acute (1 h prior to sacrifice) administration of Rb~ produced only a minimal, but insignificant, increase in HC-3 binding in the hippocampus. Others have suggested that increased neuronal activity produces an increase in HC-3 binding sites (e.g. Swann and Hewitt, 1988), and therefore one would expect acute administration of Rb~ to increase HC-3 binding. This discrepancy might be explained by the very limited permeability of Rb, into the brain (Odani et al., 1983), and because of the relatively large molecular weight, this drug may take more than 1 h to reach its equilibrium concentration in the brain. In vivo, the behavioral effects of Rb, (improvement of scopolamine-induced amnesia) were noted after a more prolonged (3 day) administration (Benishin et al., 1991). This dosage regimen also produced an

4

CHRISTINA G. BENISHIN

increase in HC-3 binding sites. This was most pronounced in the hippocampus, and was also seen, to a lesser extent, in the cortex. Both of these regions are associated with memory and learning functions. The HC-3 binding sites in another cholinergic region, the striatum, were not significantly altered by the prolonged administration of Rb~. This brain region also is not as closely associated with memory and learning. The mechanism by which Rb, increases the number of HC-3 binding sites is not clear. Several groups have reported that drugs which stimulate ACh release also concomitantly increase high-affinity choline uptake (Murrin and Kuhar, 1976; Antonelli et al., 1981: Saltarelli et al., 1988; Lowenstein and Coyle, 1986). Furthermore, changes in high-affinity choline uptake are paralleled by similar changes in [~H]HC-3 binding to synaptic membranes (Swarm and Hewitt, 1988; Saltarelli et al., 1988). Such changes in [3H]HC-3 binding are reported to be due to changes in the number of binding sites (Bm~,0 and not due to changes in the affinity of the carrier for the ligand (Kd) (Saltarelli et al., 1987). The Kd for HC-3 binding to synaptic membranes is reported to be 2 3 nM (Saltarelli et al.. 1987); therefore, the 10 nM [3H]HC-3 used here is most appropriate for detecting changes in B,,,,~. In the present study, the effects on [3H]HC-3 binding correlate with in l, itro effects on ACh release and choline uptake, and also correlate with the behavioral actions. Non-specific depolarization of nerve tissue to stimulate release could account for the increase in choline uptake, as seen earlier (Murrin and Kuhar, 1976); however, our previous study has shown that the increase in release by Rb~ was not accompanied by an increase in calcium uptake, as would be predicted by tissue depolarization. Nootropic drugs are defined as those which improve memory and learning (Giurgea and Salama, 1977). The mechanism of neurochemical action of members of this class of drugs is not yet clear, but it is generally felt that these drugs act on brain cholinergic mechanisms (Pepeu and Spignoli, 1990). Pavlik et al. (1987) reported that administration for 6 days of oxiracetam and pramiracetam, but not piracetam, significantly increased hippocampal high-affinity choline uptake. Funk and Schmidt (1988) also found that a single dose of pramiracetam increased choline uptake in cortex and hippocampus, but this effect was not maintained with repeated doses. In striatum, oxiracetam had less pronounced an effect in normal (sham-operated) animals, but restored choline uptake (Consolo et al., 1990) and HC-3 binding (Forloni el al., 1990) which were depressed as a result of deafferentation. Rbp may also be classified as a nootropic

drug; the spectrum of actions seems similar, but not identical, to other nootropic drugs extensively studied. Several recent reports have suggested that phospholipase A_, (PLA~) activation may be involved in the up-regulation of high-affinity choline uptake. Treatment of tissue with P L A , increases choline uptake (Yamada et al., 1988). This increase is mediated by increases in both the B ..... and the affinity of the carrier for HC-3 (Yamada et al., 1989). Furthermore, the increase in HC-3 binding induced by atropine was not additive with the PLA2 action (Yamada et al., 1989) and the PLA~ antagonists reduced the increase in HC-3 binding by KCI depolarization (Saltarelli el al., 1990). Rb~ apparently increased the capacity of HC-3 binding in hippocampus and cortex; the conditions of the experiment might not have detected any change in receptor affinity. Therefore, a possible mechanism of action of Rb~ through a pathway involving PLA~ cannot be excluded at this time. Ackmm'h'd.qcment.~ The author would like to acknowledge the technical assistance of Brenda Matheson, Richard Liu and Lei Zhang. This work was supported by a research grant from Zenyaku Kogyo Co. tad., Japan.

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