Effects of Nebracetam (WEB 1881 FU), a Novel Nootropic, As a Mi-Muscarinic Agonist

Short Communications Japan. J. Pharmacol. 55, 177 (1991)

Effects of Nebracetam (WEB 1881 FU), a Novel Nootropic, as a Mi-Muscarinic Agonist

Yoshihisa Kitamura, Toshio Kaneda and Yasuyuki Nomura *

Department of Pharmacology, Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo 060, Japan

Received August 28, 1990 Accepted November 21, 1990

ABSTRACT-We here investigated the effects of nebracetam (WEB 1881 FU, 4- aminomethyl-1-benzylpyrrolidin-2-one-hemifumarate), a novel pyrrolidinone nootropic, on the rise of intracellular Ca2+ concentration ([Ca 2+]i) in Jurkat cells, a human leukemic T cell line. Nebracetam induced a rise of [Ca2+]i in the medium with 1 mM Ca2+ and without Ca 2+ (plus 1 mM EGTA). The nebracetam-induced [Ca2+]i rise was blocked by atropine > pirenzepine > AF-DX 116. From these results, nebracetam seems to act as an agonist for human M1-muscarinic receptors.

Nebracetam (WEB 1881 FU, 4-aminomethyl- zepine and AF-DX 116 were generous gifts 1-benzylpyrrolidin-2-one-hemifumarate) has from Nippon Boehringer Ingelheim. Anirace- been developed as a novel nootropic drug (1). tam (Ro 13-5057) and calcium hopantenate Two pharmacological studies have showed that also were generous gifts from Roche and nebracetam ameliorates the impairment of Tanabe, respectively. Muscarine, nicotine and working memory induced by scopolamine and oxotremorine-M (Research Biochemicals by cerebral ischemia in rats (2) and facilitates Inc.); atropine, carbachol, phytohemagglutinin the muscarinic transmission via action on mus- (PHA) (Sigma) were used. carinic receptors in dogs (3). Recently, we re- Jurkat cells were maintained in Iscove's ported that nebracetam has binding activity modified Dulbecco's medium (IMDM) sup- for acetylcholine receptors in rat brain: the plemented with 10% (vol./vol.) heat-inacti- order of the potency of binding affinity was vated (56°C for 30 min) fetal calf serum M1-muscarinic > M2-muscarinic > nicotinic (FCS). They were kept at 37°C in humidified receptors and that long-term administration of 10% C02/90% air. The medium (containing nebracetam caused a decrease in the number 10% FCS) was supplied at the quantity of 5 of M1-muscarinic receptors in rat hippocampus volumes every 48 hr. (4). These findings led us to examine whether Jurkat cells were washed and resuspended nebracetam has action as an agonist or anta- in the IMDM to the final concentration of gonist of muscarinic receptors. We here ex- 1-5 X 107 cells/ml. Intracellularly loading amined the effect of nebracetam on the rise of with Fura-2 was carried out by a 10-min in- intracellular Ca 2+ concentration QCa 2+]i) in cubation with 5.0 ,uM Fura-2-AM at 37°C in a Jurkat cells, a human leukemic T cell line (5). water bath. The loaded Jurkat cells were then Fura-2-AM was purchased from Dojin diluted 1:10 with the IMDM and incubated for (Japan). Nebracetam (WEB 1881 FU), piren- a further 30 min at 37°C and washed by centri- fugation at 200 X g for 3 min at room temper- * To whom all correspondence should be addressed. ature. An aliquot of 0.5 -1 X 107 cells was used for autofluorescence measurements. The [Ca2+]i slowly and cholera toxin-sensitively. suspension was transferred to a fluorometer Thus the [Ca211i was raised by the Mi-agonist cuvette housed in a thermostatted holder. or PHA through different mechanisms in Jur- Fluorescence readings were taken in a Hitachi kat cells (7). The pretreatment with muscarine F-2000 fluorescent spectrophotometer, at ex- inhibited the oxotremorine-M-induced [Ca211i citation and emission wavelenghs of 340/380 rise. Reversibly, pretreatment with oxo- nm and 510 rim, respectively. At the end of tremorine-M inhibited the muscarine-induced each measurement, changes of Fura-2 rise completely (data not shown). The PHA- fluorescence intensities were calibrated in terms of intracellular Ca2+ concentration ([Ca2+];) as described by two wavelength pro- tocols similar to that reported by Grynkiewicz et al. (6). The KDof the Fura-2/Ca2+ interac- tion was taken to be 224 nM. We preliminarily examined whether several acetylcholine receptor agonists affect the [Ca2+]i rise in Jurkat cells. At 100 ,uM, M,- muscarinic agonists, e.g., carbachol, pilocar- pine, muscarine and oxotremorine-M, raised [Ca 2+]i immediately after injection of these drugs. Oxotremorine-M-induced [Ca 2+]; rise was more potently inhibited by pirenzepine than by AF-DX 116, indicating that M1-receptors are involved in the [Ca 2+]i rise (7). In addition, oxotremorine-M induced stimulation of 1,4,5-inositol trisphosphate (IP3) formation. The [Ca2+]; rise in response to oxotremorine- M could be due to release of stored Ca 22+by IP3. We also observed the [Ca2+]; rise induced by oxotremorine-M was insensitive to pertussis toxin (7). It is known that M1-muscarinic receptors mediate 1,4,5-IP3 formation which is not inhibited by pertussis toxin (8). Therefore, M1-muscarinic receptors seem to cause IP3 formation, followed by [Ca2+]i rise through pertussis toxin-insensitive GTP-binding proteins. We here examined the effect of nebracetam on [Ca2+]i in Jurkat cells, which may express M1-muscarinic receptors. Nebracetam at 1 mM also raised the Fig. 1. Effects of nootropics and muscarinic agonists [Ca2+]i, similar to a Mi-agonist, but anirace- on [Ca2+]i of Jurkat cells. A, effects of nebracetam (1 tam and calcium hopantenate (Ca-Hopate), mM), aniracetam (1 mM) and calcium hopantenate other nootropics, did not affect the [Ca2+]i (Ca-Hopate) (1 mM); B, effects of nebracetam (10 (Fig. 1A). In addition, nebracetam raised mM) and PHA (40,ug/ml) under Ca2+-free conditions (IMDM in the absence of Ca 2+ plus 1 mM EGTA); [Ca2+]i under the condition of extracellular C, effect of oxotremorine-M (0.1 mM) on [Ca2+]; rise Ca 2+ deficiency, in the absence of Ca2+ plus 200 sec after exposure to nebracetam (10 mM); D, 1 mM EGTA in IMDM (Fig. 1B). effect of nebracetam (10 mM) on [Ca2+]; rise 200 sec In contrast, PHA, a lectin, increased after exposure to oxotremorine-M (0.1 mM). Short Communications Japan. J. Pharmacol. 55, 179 (1991)

Table 1. Simultaneous measurement of [Ca 2+]i in Jurkat cells

The [Ca2±]i rise induced by 10 mM nebracetam (control) in the absence of antagonist was 259 ± 27 nM. Effects of muscarinic antagonists, AF-DX 116 (2 ,uM), pirenzepine (2 ,u M) and atropine (2 aM), on nebracetam-induced [Ca2+]i rise were examined as described in the text. Values are means ± standard errors of three determinations. Significantdifference by Student's t-test: *P < 0.05, **P < 0.01 vs. the value at the same concentration of nebracetam alone. induced [Ca 2+]i rise, however, was not gene-products of ml- and/or m3-receptor gene affected by pretreatment with muscarine or (8, 10). It will be interesting to determine if oxotremorine-M. Thus, MI-muscarinic recep- ml- and/or m3-receptor genes are expressed tors seem to be homologously desensitized in in Jurkat cells, and this will be elucidated by Jurkat cells. The oxotremorine-M-induced rise Northern blotting in the near future. was also inhibited by pretreatment with neb- It is well known that [Ca 2+], mobilization is racetam (Fig. 1C), and the nebracetam-in- activated by intracellular 1,4,5-IP3 production duced rise was completely blocked by pre- via activation of phospholipase C, with the treatment with oxotremorine-M (Fig. 1D), possible involvement of GTP-binding proteins while the PHA-induced rise occurred without (11-13). These observations suggest that neb- being changed by pretreatment with nebracetam activates human MI-muscarinic recep- racetam and oxotremorine-M (Fig. 1, C and tors and result in accumulation of 1,4,5-IP3 via D). GTP-binding proteins. It is reported that the Nebracetam elevated [Ca2+1i in a concen- intracerebroventricular injection of pirenze- tration-dependent manner (EC50 = 1.59 mM) pine, a specific MI-antagonist, causes the (Table 1). The concentration-dependent curve blockade of spatial learning (14), indicating of nebracetam was shifted rightward by the the importance of MI-muscarinic receptors in addition of 2,uM AF-DX 116 (M2-antagonist), learning and memory. Nebracetam ameliorates 2 ,uM pirenzepine (MI-antagonist) or 2 ,uM the impairment of working memory caused by atropine (MI/M2 full antagonist); the EC50 scopolamine and by cerebral ischemia (2). values were 2.38, 4.04, and 5.68 mM, respec- Thus it is presumed that nebracetam seems to tively (Table 1). Thus, the nebracetam-in- be a partial agonist for human MI-muscarinic duced rise was inhibited by Mi-antagonists, receptors, and this property could be involved with the order of inhibitory potency being in the nootropic effects of nebracetam. It has atropine > pirenzepine > AF-DX 116. been recently reported that an immunologic In addition, nebracetam induced the meta- dysfunction was also observed in Alzheimer's botropic current in Xenopus oocytes injected patients (15). The effect of nebracetam in the with rat brain mRNA (data not shown). It is present results was similar to the effects of known that MI-muscarinic receptors cause the PHA, indicating that nebracetam may have metabotropic current, which is different from improving effects on immunologic activity the M2-muscarinic receptor-evoked current (9, decreased during senile dementia and/or 10). Recently, it was reported that carbachol Alzheimer's disease. induces IP3 production by the activation of Acknowledgments 7 Kaneda, T. and Nomura, Y.: Effects of oxo- We would like to thank Dr. Isobe (Toyama Medical tremorine-M on the second-messenger generating and Pharmaceutical Univ.) for supplying Jurkat cells, system in Jurkat cells, cloned human T lympho- and we are also grateful to Dr. Kohno (Nippon cytes. Bull. Japan. Neurochem. Soc. 29, 58-59 Boehringer Ingelheim) and Mr. Yada for their perti- (1990) (in Japanese) nent help. This study was entrusted to Hokkaido Uni- 8 Ashkenazi, A., Peralta, E.G., Winslow, J.W., versity by the Science and Technology Agency of Ramachandran, J. and Capon, D.J.: Functional Japan using the Special Coordination Funds for Pro- role of muscarinic receptor subtype diversity. In moting Science and Technology. 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