The “antidementia drug” pantoyl-γ-aminobutyric acid increases high affinity uptake of choline by slices of rat brain

Neuropharmacology Vol. 25, No. 3, pp. 227-230, Printed in Great Britain. All rights reserved

1986 Copyright

0

002%3908/86 $3.00 + 0.00 1986 Pergamon Press Ltd

THE “ANTIDEMENTIA DRUG” PANTOYL-?, -AMINOBUTYRIC ACID INCREASES HIGH AFFINITY UPTAKE OF CHOLINE BY SLICES OF RAT BRAIN M. Department

of Pharmacology

and H.

NAKAHIRO

I, Osaka

YOSHIDA

University School Osaka 530, Japan

of Medicine,

Nakanoshima,

Kita-ku,

(Accepted 9 July 1985) Summary-Studies were made on the effects of an “antidementia drug”, pantoyl-y-aminobutyric acid (pantoyl-GABA), on high affinity uptake of choline by slices of rat brain. Depolarization caused by increasing the K+ concentration to 25 mM for 30 min before incubation with [‘HIcholine enhanced the uptake of radioactivity by slices of cerebral cortex, hippocampus and striatum during a 5 min incubation in the presence of 1 PM [‘HIcholine. Pantoyl-GABA (1 mM) increased the depolarization-induced uptake of radioactivity by the slices of cortex and hippocampus, but not by the slices of striatum; it had little effect on the uptake when the slices were not depolarized. It also had no effect when the slices were depolarized in the incubation medium without Ca*+, Since the high affinity uptake of choline is considered to be a regulatory step in the synthesis of acetylcholine (ACh), these results suggest that pantoyl-GABA increases the synthesis of ACh in cholinergic terminals in the cerebral cortex and hippocampus. This action may be involved in its effect as an antidementia drug, because cholinergic deficits are assumed to occur in Alzheimer’s disease. Key words:

antidementia

drug,

pantoyl-GABA,

high affinity

Several lines of evidence suggest that Alzheimer’s disease is associated with cholinergic dysfunction in the CNS, particularly in the cerebral cortex and hippocampus (Terry and Davies, 1980; Bartus, Dean, Beer and Lippa, 1982; Coyle, Price and DeLong, 1983). Neurochemical studies on patients with Alzheimer’s disease demonstrated a reduction in the activity of choline acetyltransferase (ChAT; Davies and Maloney, 1976; White, Hiley, Goldhardt, Carrasco, Keet, Williams and Bowen, 1977; Perry, Gibson, Blessed, Perry and Tomlinson, 1977). However, the rate-limiting step in the synthesis of acetylcholine (ACh) does not seem to be choline acetyltransferase but the supply of choline (Haubrich and Chippendale, 1977; Marchbanks, 1982). Thus, the high affinity uptake of choline, that is transmembrane passage of choline into the cholinergic terminals, was measured and was found to be decreased in patients with Alzheimer’s disease (Sims, Bowen, Allen, Smith, Neary, Thomas and Davison, 1983; Rylett, Ball and Colhoun, 1983). The calcium salt of pantoyl-y -aminobutyric acid (pantoyl-GABA) has been widely used in Japan for treatment of Alzheimer’s disease and has been found to prevent progressive loss of memory and cognition in some of these patients (Kaneda, Yagasaki, Kobayashi, Miyasaki, Hino, Nishimura, Tanino, Yamashita, Sugiyama, Kitajima, Tada, Hosaka, Takeda, Ozaki, Hariguchi and Nishimura, 1980). Little is known about its pharmacological mechanism of action, but it was recently found, in behavioral 227

choline

uptake,

Alzheimer’s

disease.

studies on mice, that pantoyl-GABA reversed the action of ACh antagonists and increased the release of ACh from slices of rat brain, suggesting that its effect as an “antidementia drug” may result from a facilitating effect on central cholinergic function (Nakahiro, Fujita, Fukuchi, Saito, Nishimura and Yoshida, 1985). In the present study, the effects of pantoyl-GABA on the high affinity uptake of choline by slices of rat brain were investigated. METHODS

Male Sprague-Dawley rats (Charles River Japan, Inc.) were decapitated and their brains were removed, chilled and sectioned sagittally at 350 pm thickness. Slices of the cerebral cortex, hippocampus and striatum were incubated for 1.5 hr in a modified Krebs-Ringer solution (composition in mM: NaCl 124, KC1 5, KH,PO, 1.24, MgSO, 1.3, CaCI, 2.4, NaHCO, 26 and glucose 10). The solution was bubbled with a mixture of 95% O2 and 5% CO, and maintained at 37°C throughout the assays. The slices were further incubated for 1 hr in the presence or absence of 1 mM pantoyl-GABA and were depolarized by increasing the K+ concentration to 25 mM in the last 30min of incubation. In some experiments, depolarization was omitted or a solution without Ca*+ was used for depolarization. Then the slices were incubated for 5 min in Ca*+-free normal-K+ solution containing 1 PM [3H]choline. Blank values were taken as those in the same solution supple-

M. NAKAHIRO and H. YOSHIDA

228

mented with 500 p M hemicholinium-3 (HC-3). The reaction was terminated by brief washing of the slices in ice-cold solution. The slices were then sonicated in 1 ml of 0.05% Triton X-100 and an aliquot was mixed with scintillation fluid (Amersham, ACS-II) for measurement of the radioactivity, using a liquid scintillation counter. Specific uptake was defined as the total counts minus those of the blank. Protein was determined by measuring the absorbance shift of Coomassie Brilliant Blue G-250 bound to protein (Bradford, 1976), using a Bio-Rad Protein Assay Kit. When the calcium salt hemihydrate of pantoylGABA was used, the total amount of Ca2+ was adjusted to the indicated concentration. In solution without Ca2+, pantoyl-GABA, not containing calcium was used: the potency of this preparation on the uptake of choline was the same as that of the calcium salt. These two forms of pantoyl-GABA, calcium salt hemihydrate and calcium free pantoyl-GABA, were gifts from Tanabe Pharmaceutical Co. Ltd. [3H]Choline chloride (80.0 Ci/mmol) was purchased from New England Nuclear and HC-3 was obtained from Aldrich Chemical Co.

physiological techniques. Since the electrical activity in these preparations is well accepted to be very susceptible to anoxic conditions, an anoxic effect on the slices, possibly due to the rather long incubation period, could be excluded in experiments on uptake. It is reported that choline, at a concentration of less than 1 PM, is taken up predominantly by cholinergic neurons and converted to ACh (Kuhar, Sethy, Roth and Aghajanian, 1973; Yamamura and Snyder, 1973). Hemicholinium is reported to inhibit this high affinity uptake of choline (Guyenet, Lefresne, Rossier, Beaujouan and Glowinski, 1973; Haga and (A)

Cerebral

DISCUSSION

Using slices prepared in a similar manner to those employed in the present study, good electrical responses were observed for at least 3 hr in hippocampal neurons studied by conventional electro-

E

COntrOl

(13)

(131

P- GABA

K+

(14)

I+

K+andP-GAEA

RESULTS

The uptake of radioactivity by the slices was linear for at least 5 min. A rather long preincubation period (i.e. 1.5 hr) was employed before application of the drug, as described in Methods, because the effects of the drug and of depolarization were not marked after a shorter preincubation period. As shown in Fig. lA, 1B and lC, when the slices were depolarized by increasing the K+ concentration to 25 mM for 30min before incubation with [3H]choline, the uptake of choline was significantly increased in the cerebral cortex, hippocampus and striatum. A further increase of the uptake was observed with slices of cortex and hippocampus when they were depolarized in the presence of 1 mM pantoyl-GABA. With slices of striatum, the effect of depolarization was much greater than with slices from the other two regions, but pantoyl-GABA had little effect on the depolarization-induced uptake. Pantoyl-GABA had no effect on uptake when the slices were not depolarized. Depolarization of the slices in the absence of Ca2+ resulted in a smaller increase in uptake and in this condition pantoyl-GABA had no enhancing effect on uptake by either of the regions of the brain examined (Fig. 2). Pantoyl-GABA (1 mM) also had no effect on the uptake when added to the solution during a 5 min incubation with [3H]choline (data not shown).

cortex

Choline

uptake

(pmol/mg

protein

5min)

( B 1 Hippocampus

k (13)

P-GABA

KC

(13)

K+ EI P-GABA

I

I 10

0 Choline

uptake

I

I

20

30

(pmol/mg

protein

5mln)

(ClStrlatum COlWOl

(7)

K+

(6)

K+B P-GABA

15)

0

k 0 Chohne

20 uptake

40 (pmol/mg

60 protein

5mln)

Fig. 1. Effects of 25 mM K+ and/or 1mM pantoyl-GABA on the uptake of choline by slices of cerebral cortex (A), hippocampus (B) and striatum (C). Slices were incubated in normal medium (control) or in medium containing I mM pantoyl-GABA (P-GABA), 25mM K+ (K+), 25mM K+ and 1 mM pantoyl-GABA (K+ and P-GABA), respectively, for 30 min and then the uptake of [‘HIcholine was measured in Ca*+-free normal-K+ medium. Values are means k SD for the numbers of determinations shown in parentheses. Significantly different from control at P -c0.001. b and Significantly different from 25 mM K+ alone at P < 0.001 and P < 0.01, respectively, by Student’s f-test.

Effect of pantoyl-GABA Cerebral

Cortex

1

Control KC K+EI P-GABA

H~ppo~ampus Control Kf K+ EL P-GA6A

Choline

uptake

(pmol/mg

prOteln

5mln)

Fig. 2. Effects of 25 mM K+ and/or 1 mM pantoyl-GABA on the uptake of choline in medium without Ca2+. Slices were incubated as described in the legend of Fig. 1 except that Ca*+ was omitted from the medium. Values are means k SD for the numbers of determinations shown in parentheses. a and bSignificantly different from the control at P < 0.01 and P < 0.05, respectively, by Student’s f-test.

Noda, 1973). Thus, it is concluded that the values obtained in this study represent the amount of choline taken up by cholinergic terminals and subsequently used to synthesize ACh. This study confirms other reports (Barker, 1976; Murrin and Kuhar, 1976; Polak, Molenaar and Van Gelder, 1977) that the uptake of choline is enhanced after a period of depolarization. The mechanism regulating the high affinity uptake of choline is unknown, but several workers have suggested that it is regulated by the content of ACh inside the cholinergic terminals (Jenden, Jope and Weiler, 1976; Roskoski, 1978; Marchbanks, Wannacott and Rubio, 1981). If so, the enhanced uptake of choline after a period of depolarization may be due to a decreased concentration of ACh in the terminals as the result of the release of ACh. This possibility is compatible with the present finding that the stimulatory effect of depolarization on the uptake was reduced in the absence of Ca’+. The finding that in the absence of Ca2+ the uptake was not reduced to control level may be ascribed to the fact that slices of brain contain abundant Ca’+. Similarly, the enhancement by pantoyl-GABA of the uptake by depolarized slices may be attributed to a further release of ACh than that caused by depolarization alone. This idea is consistent with a previous finding that pantoylGABA increased the release of ACh, stimulated by 25 mM K+, from slices of cerebral cortex and hippocampus (Nakahiro et al., 1985). It is also consistent with the present finding that pantoyl-GABA had no effect on the uptake of choline by the striatum, where it also did not increase the release of ACh. In these in vitro experiments, pantoyl-GABA was effective only when the slices were depolarized. It was recently observed that in vivo treatment with this drug also increased the high affinity uptake of choline into synaptosomes (unpublished observations). This

on choline

uptake

229

difference between in vitro and in vivo observations may be accounted for by the fact that the afferent fibers of cholinergic neurons are disrupted in slices of cerebral cortex and hippocampus, because these neurons are extrinsic to these areas of the brain (Fibiger, 1982). Accordingly, it seems likely that impulse-flow is necessary for the action of pantoyl-GABA in vivo. High affinity uptake of choline is considered to be a regulatory step in the synthesis of ACh (Haubrich and Chippendale, 1977; Marchbanks, 1982). Therefore, pantoyl-GABA, by enhancing the uptake of choline, may also increase the synthesis of ACh in cholinergic neurons. This effect may be involved in its action as an antidementia drug, since preparations from patients with Alzheimer’s disease are reported to show reduced high affinity uptake of choline (Sims et al., 1983; Rylett et al., 1983). Thus, the present results support a previous proposal that pantoylGABA facilitates cholinergic function in the CNS (Nakahiro et al., 1985). Acknowledgements-We thank Dr Kihachi Saito for reading the manuscript, Mrs Mieko Nakamura for secretarial assistance and Tanabe-Seiyaku Co. Ltd for a gift of pantoylGABA. This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan. REFERENCES

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