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Ligand-binding characteristics of [3H]QNB, [3H]prazosin, [3H]rauwolscine, [3H]TCP and [3H]nitrendipine to cerebral cortical and hippocampal membranes of senescence accelerated mouse
334
Neuroscience Letters, 106 (1989) 334 33~ Elsevier Scientific Publishers Ireland Ltd
NSL 06456
Ligand-binding characteristics of [3H]QNB, [3H]prazosin, [3H]rauwolscine, [3H]TCP and [3H]nitrendipine to cerebral cortical and hippocampal membranes of senescence accelerated mouse Yoshihisa Kitamura, Xue-Hui Zhao, Toshio Ohnuki and Yasuyuki Nomura Department of Pharmacology, Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo (Japan) (Received 17 July 1989; Accepted 24 July 1989) Key words: Muscarinic acetylcholine receptor; a-Adrenoceptor, N-Methyl-D-aspartate receptor channel; L-type Ca 2+ channel; Cerebral cortex; Hippocampus; Senescence accelerated mouse (SAM) The senescence accelerated mouse (SAM) is known as a murine model of aging and memory dysfunction. In the cerebral cortical membranes of male 9-month-old SAM mice, the B~,~xvalues of [3H]rauwolscine and [3H]nitrendipine binding, and the values of both Kd and Bmaxof [3H]TCP binding in the accelerated aging strain SAM-P/8, were significantly increased compared with the values in the control strain SAM-R/1. In hippocampal membranes, however, the B ~ values of [3H]quinuclidinylbenzilate and [3H]nitrendipine binding were significantly decreased in SAM-P/8 compared with those in SAM-R/1. These resuits suggest that muscarinic acetylchotine receptors, ~-adrenoceptors, N-methyl-D-aspartate receptor channels and L-type Ca 2÷ channels are changed in cerebral cortex and hippocampus in SAM-P/8 at 9 months.
A g i n g is a s s o c i a t e d with several deficits in the C N S which have been c o r r e l a t e d with a n i m p a i r e d f u n c t i o n i n g o f neurons, involving synthesis, storage, u p t a k e a n d release, a n d r e c e p t o r f u n c t i o n s o f the n e u r o t r a n s m i t t e r s [17]. These n e u r o n a l dysfunctions s o m e t i m e s induce d i s t u r b a n c e s in l e a r n i n g a n d m e m o r y [1, 17]. T h e senescent a c c e l e r a t e d m o u s e ( S A M ) , a m u r i n e m o d e l o f a c c e l e r a t e d aging, s h o w s a n early onset a n d irreversible a d v a n c e m e n t o f senescence [18]. In a d d i t i o n , the senescence a c c e l e r a t e d p r o n e m o u s e ( S A M - P / 8 ) s h o w s a n a g e - r e l a t e d d e t e r i o r a t i o n o f the learning ability c o m p a r e d with the c o n t r o l m o u s e , the senescence-accelerated resistant m o u s e ( S A M - R / l ) [12, 19]. W e have r e p o r t e d the a g e - r e l a t e d changes o f cq-, ct2- a n d f l - a d r e n o c e p t o r s , a n d D 2 - d o p a m i n e r e c e p t o r s in the rat C N S [8, 13, 14, 15]. F u r t h e r m o r e , we d e t e r m i n e d the c h a n g e s o f b i o c h e m i c a l p a r a m e t e r s such as m a l o n d i a l d e Correspondence." Y. Nomura, Ph.D., Department of Pharmacology, Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-Ku, Kita 12-Nishi 6, Sapporo 060, Japan. 0304-3940/89/$ 03.50 © 1989 Elsevier Scientific Publishers Ireland Ltd.
335 hyde, superoxide dismutase and monoamine oxidase B in the SAM [16]. To clarify aging of neurotransmitter receptor/ion channel functions in the brain, we here investigated the ligand-binding characteristics of neurotransmitter receptors (~1, ~2-adrenoceptors and muscarinic acetylcholine (mACh) receptors) and ion channels (N-methyl-D-aspartate (NMDA) receptor channels and L-type Ca 2+ channels) in cerebral cortical and hippocampal membranes of SAM R/1 and P/8 of 9 months when both behavioral and biochemical agings were clearly observed. SAM-P/8 acquires severe forms of degeneration in its appearance accompanying pathological amyloidosis and memory dysfunctions at about 8 months after birth, while such senile signs do not emerge in the SAM-R/I at the same age [12, 18, 19]. We used male, 9-month-old SAM-R/i and P/8. The brains of SAMs were rapidly removed and dissected on ice. Cerebral cortex and hippocampus were homogenized with 10 vols. of 50 mM Tris-HCl buffer (pH 7.4) and the homogenate was centrifuged at 15,000 g for 10 rain at 4°C. The pellet thus obtained was washed 3 times with the same buffer. The sedimented membranes were suspended (2-5 mg/ml) in 50 mM TrisHCI buffer (pH 7.4) and stored at -80°C. The total volume of the reaction mixture was 100/A, and contained 50 mM Tris-HCl (pH 7.4), the membranes (20-100/~g protein) and radioligand: either [3H]prazosin (0.14 nM, for cq-adrenoceptor), [3H]rauwolscine (0.5-20 nM, for e2-adrenoceptor), [3H]quinuclidinyl benzilate (QNB) (0.24 nM, for mACh receptor), [3H]TCP (3-90 nM, for NMDA receptor channel) or [3H]nitrendipine (0.1-2 nM, for L-type Ca 2+ channel). After the reaction mixture was incubated at 30°C for 60 min, it was filtered under vacuum through a Whatman GF/
TABLE I BINDING CHARACTERISTICS OF [3H]QNB, [3HIPRAZOSIN, [3H]RAUWOLSCINE, [3H]TCP AND [3H]NITRENDIPINE TO CEREBRAL CORTICAL MEMBRANES OF SAM R/1 AND P/8 Values are means + S.E.M. of 3 independent experiments with samples in duplicate. Ligand (site)
Strain
Kd (nM)
Bma×(fmol/mg protein)
[~H]QNB (mACh receptor)
R/I P/8
0.18_+0.03 0.14 + 0.02
2,510_+ 150 2,200_+ 70
[3H]Prazosin (~ ~-adrenoceptor)
R/1 P/8
0.58 + 0.18 0.18 + 0.02
134 _+ 15 107 + 13
[~H]Rauwolscine (c~2-adrenoceptor)
R/1 P/8
6.0 -+2.1 9.6 -+2.1
115 + 9 221 +% . _37*
[3H]TCP (NMDA receptor channel)
R/1 P/8
40-+8 83 -+6*
211 +42 345 -+ 25*
[3H]Nitrendipine (L-Ca 2+ channel)
R/1 P/8
0.39 -+0.02 0.50_+ 0.08
148 _+3 184 + 12*
*P < 0.05, with respect to the value of SAM R/l.
336 C filter and washed 3 times with 1.5 ml of ice-cold buffer. Specific binding was defined as radioactivity bound after subtraction of the non-specific binding (in the presence of I ¢tM atropine for mACh receptor; 10/tM phentolamine for ~1- and ~2-adrenoceptor; 10/zM MK-801 for NMDA receptor channel; and 10/tM nifedipine for L-type Ca 2+ channel) from the total binding. Protein content was estimated by the method of Lowry et al. [9]. Student's t-test was used for statistical significance. In the cerebral cortex (Table I), the values of Kd and Bmaxof [3H]QNB and [3H]prazosin binding were not different between SAM-R/I and SAM-P/8. The Bm~ values of [3H]rauwolscine and [3H]nitrendipine binding were significantly (P<0.05) increased in SAM-P/8 vs SAM-R/I without a change in the Kd of both bindings. In [3H]TCP binding, both the Kd and Bmax values were significantly (P < 0.05) increased in SAM-P/8 compared to SAM-R/1. In the hippocampal membranes (Table II), neither Kd nor Bmax was different between SAM-R/1 and P/8 regarding the binding of [3H]prazosin, [3H]rauwolscine, and [3H]TCP. However, the Bmax values of [3H]QNB and [3H]nitrendipine binding were significantly (P < 0.01 and P < 0.05, respectively) decreased in SAM-P/8 compared to SAM-R/1 but the Kd values were not. It is possible that ~2-adrenoceptors, NMDA receptor channels and L-type Ca 2+ channels in cerebral cortex and mACh receptors and L-type Ca 2+ channels in hippocampus already change at 9 months in SAM-P/8 and have not changed in SAM-R/I of the same age. We previously demonstrated in the rat brain that the densities of ~1-, c~2- and fl-adrenoceptor are not changed [8] during normal aging until 24
TABLE II LIGAND-BINDING PARAMETERS OF MUSCARINIC RECEPTOR, ~q, ct:-ADRENOCEPTOR, NMDA RECEPTOR CHANNEL, AND L-TYPE CA 2÷ CHANNEL TO HIPPOCAMPAL MEMBRANES OF SAM R/1 AND P/8 Values are means _+ S.E.M. of 3 independent experiments with sample in duplicate. Ligand
Strain
Kd (nM)
Bm~ (fmol/mg protein)
[3H]QNB
R/l P/8
0.27 ± 0.04 0.19 ± 0.03
2,280 ___30 1,750 ± 70**
[3H]Prazosin
R/1 P/8
0.67 ± 0.23 0.72 _+0.32
103 + 19 69 __+20
[3H]Rauwolscine
R/1 P/8
21 __.5 21 +4
260 __+69 278+41
[3H]TCP
R/1 P/8
95 ± 22 80+ 18
504 ± 89 642±66
[3H]Nitrendipine
R/1 P/8
0.45 + 0.04 0.48+0.10
213 ___3 174_+ 11"
*P < 0.05 and **P< 0.01, with respect to the Bma×value of SAM R/I.
337
months but that the coupling, activities of G i and Gs with the catalytic units of adenylate cyclase are decreased [14, 15]. The density of mACh receptors in the rat brain is decreased during aging [6] and the decrease can be correlated with the disturbances of learning and memory in human [1] and rat [4]. Govoni et al. reported that the Kd value of [3H]nitrendipine binding [5] and the Bmaxvalue of [3H]verapamil binding [2] are increased during aging in the rat brain, indicative of a qualitative change in the L-type Ca 2+ channel in the senescent brain. L-type Ca 2+ channels are composed of a complex of ~l, ~2, fl, ~, and 6 subunits and dihydropyridines and phenylalkylamines bind to el-subunits (MW= 165-195 kDa) of L-type Ca 2+ channels in brain, heart and skeletal muscle [3]. It is known that NMDA receptor channels also induce to raise directly intracellular concentration of free Ca 2+ [10] and that the channels are correlated with long-term potentiation (LTP), synaptic plasticity and neurotoxity, although such a causal relationship has never been known. Recently, intracellular microinjection of protein kinase C [7], or its activation by phorbol ester [11] has been examined to produce a LTP-like effect. Therefore, the phenomena after transmembrane signalling, e.g. the production of second messengers (Ca 2+, cyclic AMP, inositol 1,4,5-trisphosphate (IP3), diacylglycerol etc.) and the intracellular signalling mechanism in SAM-P/8, remain an interesting subject to be elucidated. In conclusion, SAM seems to be a pertinent animal model for studying the neurotransmitter receptor/ion channel mechanisms with regard to the memory deficit seen in senile human. We thank Prof. T. Takeda (Kyoto Univ.) for the original provision of SAM. This study was supported in part by Grant-in-Aid from the Fujisawa Foundation. I Bartus, R.T., Dean III, R.L., Beer, B. and Lippa, A.S., The cholinergic hypothesis of geriatric memory dysfunction, Science, 217 (1982)408~417. 2 Battaini, F., Govoni, S., Rius, R.A. and Trabucchi, M., Age-dependent increase in [3H]verapamil binding to rat cortical membranes, Neurosci. Lett., 61 (1985) 67 71. 3 Catterall, W.A., Structure and function of voltage-sensitive ion channels, Science, 242 (1988) 50- 61. 4 Fukuchi, I., Kato, S., Nakahiro, M., Uchida, S., Ishida, R. and Yoshida, H., Blockade of cholinergic receptors by an irreversible antagonist, propylbenzilylcholine mustard (PrBCM), in the rat cerebral cortex causes deficits in passive avoidance learning, Brain Res., 400 (1987) 53 61. 5 Govoni, S., Rius, R.A., Battaini, F., Bianchi, A. and Trabucchi, M., Age-related reduced affinity in [3H]nitrendipine labeling of brain voltage-dependent calcium channels, Brain Res., 333 (1985) 374 377. 6 Gurwitz, D., Egozi, Y., Henis, Y.I., Kloog, Y. and Sokolocsky, M., Agonist and antagonist binding to rat brain muscarinic receptors: influence of aging, Neurobiol. Aging, 8 (1987) 115 122. 7 Hu, G.-Y., Hvalby, O., Walaas, S.I., Albert, K.A., Skjeflo, P., Andersen, P. and Greengard, Protein kinase C injection into hippocampal pyramidal cells elicits features of long term potentiation, Nature (Lond.), 328 (1987) 426429. 8 Kawai, M., Sakaue, T., Watanabe, C., Nomura, Y. and Segawa, T., Specific binding of [3H]WB 4101, [~H]clonidine and [3H]dihydroalprenolol in cerebral cortical membranes in developing, adult and old rats, Jpn. J. Pharmacol., 36 (1984) 265 -267. 9 Lowry, O.H., Rosebrough, N.J., Faro A.L. and Randall, R.J., Protein measurement with the Folinphenol reagent, J. Biol. Chem., 193 (1951) 265--275. I0 MacDermott, A.B., Mayer, M.L., Westbrook, G.L., Smith, S.J. and Barker, J.L., NMDA-receptor activation increases cytoplasmic calcium concentration in cultured spinal cord neurones, Nature (Lond.), 321 (1986) 519-522.
338 11 Malenka, R.C., Madison, D.V. and Nicoll, R.A., Potentiation ofsynaptic transmission in the hippocampus by phorbol esters, Nature (Lond.), 321 (1986) 175-177. 12 Miyamoto, M., Kiyota, Y., Yamazaki, N., Nagaoka, A., Matsuo, T., Nagawa, Y. and Takeda, T., Age-related changes in learning and memory in the senescence-accelerated mouse (SAM), Physiol. Behav., 38 (1986) 399-406. 13 Nomura, Y., Oki, K., Yotsumoto, I. and Segawa, T., Age-related change of binding regulation in dopamine and ~2-adrenergic receptors in the brain. In E. Giacobini et al. (Eds.), The Aging Brain: Cellular and Molecular Mechanisms of Aging in the Nervous System, Raven Press, New York, 1982, pp. 185-194. 14 Nomura, Y., Kitamura, Y., Kawai, M. and Segawa, T., 72-Adrenoceptor-GTP binding protein-adenylate cyclase system in cerebral cortical membranes of adult and senescent rats, Brain Res., 379 (1986) 118 124. 15 Nomura, Y., Kitamura, Y. and Kawata, K., Function and mechanism of the interaction of GTP-binding proteins with ~2-adrenoceptors in the brain. In S. Kito et al. (Eds.), Neuroreceptors and Signal Transduction, Plenum, 1988, pp. 301-311. 16 Nomura, Y., Wang, B.X., Qi, S.B., Namba, T. and Kaneko, S., Biochemical changes related to aging in the senescence-accelerated mouse, Exp. Gerontol., 24 (1989) 49-55. 17 Pradhan, S.N., Central neurotransmitters and aging, Life Sci., 26 (1980) 1643-1656. 18 Takeda, T., Hosokawa, M., Takeshita, S., Irino, M., Higuchi, K., Matsushita, T., Tomita, Y., Yasuhira, K., Hamamoto, H., Shimizu, K., Ishii, M. and Yamamuro, T., A new murine model of accelerated senescence, Mech. Ageing Dev., 17 (1981) 183-194. 19 Yagi, H., Katoh, S., Akiguchi, I. and Takeda, T., Age-related deterioration of ability of acquisition in memory and learning in senescence accelerated mouse: SAM-P/8 as an animal model of disturbances in recent memory, Brain Res., 474 (1988) 86-93.
Neuroscience Letters, 106 (1989) 334 33~ Elsevier Scientific Publishers Ireland Ltd
NSL 06456
Ligand-binding characteristics of [3H]QNB, [3H]prazosin, [3H]rauwolscine, [3H]TCP and [3H]nitrendipine to cerebral cortical and hippocampal membranes of senescence accelerated mouse Yoshihisa Kitamura, Xue-Hui Zhao, Toshio Ohnuki and Yasuyuki Nomura Department of Pharmacology, Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo (Japan) (Received 17 July 1989; Accepted 24 July 1989) Key words: Muscarinic acetylcholine receptor; a-Adrenoceptor, N-Methyl-D-aspartate receptor channel; L-type Ca 2+ channel; Cerebral cortex; Hippocampus; Senescence accelerated mouse (SAM) The senescence accelerated mouse (SAM) is known as a murine model of aging and memory dysfunction. In the cerebral cortical membranes of male 9-month-old SAM mice, the B~,~xvalues of [3H]rauwolscine and [3H]nitrendipine binding, and the values of both Kd and Bmaxof [3H]TCP binding in the accelerated aging strain SAM-P/8, were significantly increased compared with the values in the control strain SAM-R/1. In hippocampal membranes, however, the B ~ values of [3H]quinuclidinylbenzilate and [3H]nitrendipine binding were significantly decreased in SAM-P/8 compared with those in SAM-R/1. These resuits suggest that muscarinic acetylchotine receptors, ~-adrenoceptors, N-methyl-D-aspartate receptor channels and L-type Ca 2÷ channels are changed in cerebral cortex and hippocampus in SAM-P/8 at 9 months.
A g i n g is a s s o c i a t e d with several deficits in the C N S which have been c o r r e l a t e d with a n i m p a i r e d f u n c t i o n i n g o f neurons, involving synthesis, storage, u p t a k e a n d release, a n d r e c e p t o r f u n c t i o n s o f the n e u r o t r a n s m i t t e r s [17]. These n e u r o n a l dysfunctions s o m e t i m e s induce d i s t u r b a n c e s in l e a r n i n g a n d m e m o r y [1, 17]. T h e senescent a c c e l e r a t e d m o u s e ( S A M ) , a m u r i n e m o d e l o f a c c e l e r a t e d aging, s h o w s a n early onset a n d irreversible a d v a n c e m e n t o f senescence [18]. In a d d i t i o n , the senescence a c c e l e r a t e d p r o n e m o u s e ( S A M - P / 8 ) s h o w s a n a g e - r e l a t e d d e t e r i o r a t i o n o f the learning ability c o m p a r e d with the c o n t r o l m o u s e , the senescence-accelerated resistant m o u s e ( S A M - R / l ) [12, 19]. W e have r e p o r t e d the a g e - r e l a t e d changes o f cq-, ct2- a n d f l - a d r e n o c e p t o r s , a n d D 2 - d o p a m i n e r e c e p t o r s in the rat C N S [8, 13, 14, 15]. F u r t h e r m o r e , we d e t e r m i n e d the c h a n g e s o f b i o c h e m i c a l p a r a m e t e r s such as m a l o n d i a l d e Correspondence." Y. Nomura, Ph.D., Department of Pharmacology, Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-Ku, Kita 12-Nishi 6, Sapporo 060, Japan. 0304-3940/89/$ 03.50 © 1989 Elsevier Scientific Publishers Ireland Ltd.
335 hyde, superoxide dismutase and monoamine oxidase B in the SAM [16]. To clarify aging of neurotransmitter receptor/ion channel functions in the brain, we here investigated the ligand-binding characteristics of neurotransmitter receptors (~1, ~2-adrenoceptors and muscarinic acetylcholine (mACh) receptors) and ion channels (N-methyl-D-aspartate (NMDA) receptor channels and L-type Ca 2+ channels) in cerebral cortical and hippocampal membranes of SAM R/1 and P/8 of 9 months when both behavioral and biochemical agings were clearly observed. SAM-P/8 acquires severe forms of degeneration in its appearance accompanying pathological amyloidosis and memory dysfunctions at about 8 months after birth, while such senile signs do not emerge in the SAM-R/I at the same age [12, 18, 19]. We used male, 9-month-old SAM-R/i and P/8. The brains of SAMs were rapidly removed and dissected on ice. Cerebral cortex and hippocampus were homogenized with 10 vols. of 50 mM Tris-HCl buffer (pH 7.4) and the homogenate was centrifuged at 15,000 g for 10 rain at 4°C. The pellet thus obtained was washed 3 times with the same buffer. The sedimented membranes were suspended (2-5 mg/ml) in 50 mM TrisHCI buffer (pH 7.4) and stored at -80°C. The total volume of the reaction mixture was 100/A, and contained 50 mM Tris-HCl (pH 7.4), the membranes (20-100/~g protein) and radioligand: either [3H]prazosin (0.14 nM, for cq-adrenoceptor), [3H]rauwolscine (0.5-20 nM, for e2-adrenoceptor), [3H]quinuclidinyl benzilate (QNB) (0.24 nM, for mACh receptor), [3H]TCP (3-90 nM, for NMDA receptor channel) or [3H]nitrendipine (0.1-2 nM, for L-type Ca 2+ channel). After the reaction mixture was incubated at 30°C for 60 min, it was filtered under vacuum through a Whatman GF/
TABLE I BINDING CHARACTERISTICS OF [3H]QNB, [3HIPRAZOSIN, [3H]RAUWOLSCINE, [3H]TCP AND [3H]NITRENDIPINE TO CEREBRAL CORTICAL MEMBRANES OF SAM R/1 AND P/8 Values are means + S.E.M. of 3 independent experiments with samples in duplicate. Ligand (site)
Strain
Kd (nM)
Bma×(fmol/mg protein)
[~H]QNB (mACh receptor)
R/I P/8
0.18_+0.03 0.14 + 0.02
2,510_+ 150 2,200_+ 70
[3H]Prazosin (~ ~-adrenoceptor)
R/1 P/8
0.58 + 0.18 0.18 + 0.02
134 _+ 15 107 + 13
[~H]Rauwolscine (c~2-adrenoceptor)
R/1 P/8
6.0 -+2.1 9.6 -+2.1
115 + 9 221 +% . _37*
[3H]TCP (NMDA receptor channel)
R/1 P/8
40-+8 83 -+6*
211 +42 345 -+ 25*
[3H]Nitrendipine (L-Ca 2+ channel)
R/1 P/8
0.39 -+0.02 0.50_+ 0.08
148 _+3 184 + 12*
*P < 0.05, with respect to the value of SAM R/l.
336 C filter and washed 3 times with 1.5 ml of ice-cold buffer. Specific binding was defined as radioactivity bound after subtraction of the non-specific binding (in the presence of I ¢tM atropine for mACh receptor; 10/tM phentolamine for ~1- and ~2-adrenoceptor; 10/zM MK-801 for NMDA receptor channel; and 10/tM nifedipine for L-type Ca 2+ channel) from the total binding. Protein content was estimated by the method of Lowry et al. [9]. Student's t-test was used for statistical significance. In the cerebral cortex (Table I), the values of Kd and Bmaxof [3H]QNB and [3H]prazosin binding were not different between SAM-R/I and SAM-P/8. The Bm~ values of [3H]rauwolscine and [3H]nitrendipine binding were significantly (P<0.05) increased in SAM-P/8 vs SAM-R/I without a change in the Kd of both bindings. In [3H]TCP binding, both the Kd and Bmax values were significantly (P < 0.05) increased in SAM-P/8 compared to SAM-R/1. In the hippocampal membranes (Table II), neither Kd nor Bmax was different between SAM-R/1 and P/8 regarding the binding of [3H]prazosin, [3H]rauwolscine, and [3H]TCP. However, the Bmax values of [3H]QNB and [3H]nitrendipine binding were significantly (P < 0.01 and P < 0.05, respectively) decreased in SAM-P/8 compared to SAM-R/1 but the Kd values were not. It is possible that ~2-adrenoceptors, NMDA receptor channels and L-type Ca 2+ channels in cerebral cortex and mACh receptors and L-type Ca 2+ channels in hippocampus already change at 9 months in SAM-P/8 and have not changed in SAM-R/I of the same age. We previously demonstrated in the rat brain that the densities of ~1-, c~2- and fl-adrenoceptor are not changed [8] during normal aging until 24
TABLE II LIGAND-BINDING PARAMETERS OF MUSCARINIC RECEPTOR, ~q, ct:-ADRENOCEPTOR, NMDA RECEPTOR CHANNEL, AND L-TYPE CA 2÷ CHANNEL TO HIPPOCAMPAL MEMBRANES OF SAM R/1 AND P/8 Values are means _+ S.E.M. of 3 independent experiments with sample in duplicate. Ligand
Strain
Kd (nM)
Bm~ (fmol/mg protein)
[3H]QNB
R/l P/8
0.27 ± 0.04 0.19 ± 0.03
2,280 ___30 1,750 ± 70**
[3H]Prazosin
R/1 P/8
0.67 ± 0.23 0.72 _+0.32
103 + 19 69 __+20
[3H]Rauwolscine
R/1 P/8
21 __.5 21 +4
260 __+69 278+41
[3H]TCP
R/1 P/8
95 ± 22 80+ 18
504 ± 89 642±66
[3H]Nitrendipine
R/1 P/8
0.45 + 0.04 0.48+0.10
213 ___3 174_+ 11"
*P < 0.05 and **P< 0.01, with respect to the Bma×value of SAM R/I.
337
months but that the coupling, activities of G i and Gs with the catalytic units of adenylate cyclase are decreased [14, 15]. The density of mACh receptors in the rat brain is decreased during aging [6] and the decrease can be correlated with the disturbances of learning and memory in human [1] and rat [4]. Govoni et al. reported that the Kd value of [3H]nitrendipine binding [5] and the Bmaxvalue of [3H]verapamil binding [2] are increased during aging in the rat brain, indicative of a qualitative change in the L-type Ca 2+ channel in the senescent brain. L-type Ca 2+ channels are composed of a complex of ~l, ~2, fl, ~, and 6 subunits and dihydropyridines and phenylalkylamines bind to el-subunits (MW= 165-195 kDa) of L-type Ca 2+ channels in brain, heart and skeletal muscle [3]. It is known that NMDA receptor channels also induce to raise directly intracellular concentration of free Ca 2+ [10] and that the channels are correlated with long-term potentiation (LTP), synaptic plasticity and neurotoxity, although such a causal relationship has never been known. Recently, intracellular microinjection of protein kinase C [7], or its activation by phorbol ester [11] has been examined to produce a LTP-like effect. Therefore, the phenomena after transmembrane signalling, e.g. the production of second messengers (Ca 2+, cyclic AMP, inositol 1,4,5-trisphosphate (IP3), diacylglycerol etc.) and the intracellular signalling mechanism in SAM-P/8, remain an interesting subject to be elucidated. In conclusion, SAM seems to be a pertinent animal model for studying the neurotransmitter receptor/ion channel mechanisms with regard to the memory deficit seen in senile human. We thank Prof. T. Takeda (Kyoto Univ.) for the original provision of SAM. This study was supported in part by Grant-in-Aid from the Fujisawa Foundation. I Bartus, R.T., Dean III, R.L., Beer, B. and Lippa, A.S., The cholinergic hypothesis of geriatric memory dysfunction, Science, 217 (1982)408~417. 2 Battaini, F., Govoni, S., Rius, R.A. and Trabucchi, M., Age-dependent increase in [3H]verapamil binding to rat cortical membranes, Neurosci. Lett., 61 (1985) 67 71. 3 Catterall, W.A., Structure and function of voltage-sensitive ion channels, Science, 242 (1988) 50- 61. 4 Fukuchi, I., Kato, S., Nakahiro, M., Uchida, S., Ishida, R. and Yoshida, H., Blockade of cholinergic receptors by an irreversible antagonist, propylbenzilylcholine mustard (PrBCM), in the rat cerebral cortex causes deficits in passive avoidance learning, Brain Res., 400 (1987) 53 61. 5 Govoni, S., Rius, R.A., Battaini, F., Bianchi, A. and Trabucchi, M., Age-related reduced affinity in [3H]nitrendipine labeling of brain voltage-dependent calcium channels, Brain Res., 333 (1985) 374 377. 6 Gurwitz, D., Egozi, Y., Henis, Y.I., Kloog, Y. and Sokolocsky, M., Agonist and antagonist binding to rat brain muscarinic receptors: influence of aging, Neurobiol. Aging, 8 (1987) 115 122. 7 Hu, G.-Y., Hvalby, O., Walaas, S.I., Albert, K.A., Skjeflo, P., Andersen, P. and Greengard, Protein kinase C injection into hippocampal pyramidal cells elicits features of long term potentiation, Nature (Lond.), 328 (1987) 426429. 8 Kawai, M., Sakaue, T., Watanabe, C., Nomura, Y. and Segawa, T., Specific binding of [3H]WB 4101, [~H]clonidine and [3H]dihydroalprenolol in cerebral cortical membranes in developing, adult and old rats, Jpn. J. Pharmacol., 36 (1984) 265 -267. 9 Lowry, O.H., Rosebrough, N.J., Faro A.L. and Randall, R.J., Protein measurement with the Folinphenol reagent, J. Biol. Chem., 193 (1951) 265--275. I0 MacDermott, A.B., Mayer, M.L., Westbrook, G.L., Smith, S.J. and Barker, J.L., NMDA-receptor activation increases cytoplasmic calcium concentration in cultured spinal cord neurones, Nature (Lond.), 321 (1986) 519-522.
338 11 Malenka, R.C., Madison, D.V. and Nicoll, R.A., Potentiation ofsynaptic transmission in the hippocampus by phorbol esters, Nature (Lond.), 321 (1986) 175-177. 12 Miyamoto, M., Kiyota, Y., Yamazaki, N., Nagaoka, A., Matsuo, T., Nagawa, Y. and Takeda, T., Age-related changes in learning and memory in the senescence-accelerated mouse (SAM), Physiol. Behav., 38 (1986) 399-406. 13 Nomura, Y., Oki, K., Yotsumoto, I. and Segawa, T., Age-related change of binding regulation in dopamine and ~2-adrenergic receptors in the brain. In E. Giacobini et al. (Eds.), The Aging Brain: Cellular and Molecular Mechanisms of Aging in the Nervous System, Raven Press, New York, 1982, pp. 185-194. 14 Nomura, Y., Kitamura, Y., Kawai, M. and Segawa, T., 72-Adrenoceptor-GTP binding protein-adenylate cyclase system in cerebral cortical membranes of adult and senescent rats, Brain Res., 379 (1986) 118 124. 15 Nomura, Y., Kitamura, Y. and Kawata, K., Function and mechanism of the interaction of GTP-binding proteins with ~2-adrenoceptors in the brain. In S. Kito et al. (Eds.), Neuroreceptors and Signal Transduction, Plenum, 1988, pp. 301-311. 16 Nomura, Y., Wang, B.X., Qi, S.B., Namba, T. and Kaneko, S., Biochemical changes related to aging in the senescence-accelerated mouse, Exp. Gerontol., 24 (1989) 49-55. 17 Pradhan, S.N., Central neurotransmitters and aging, Life Sci., 26 (1980) 1643-1656. 18 Takeda, T., Hosokawa, M., Takeshita, S., Irino, M., Higuchi, K., Matsushita, T., Tomita, Y., Yasuhira, K., Hamamoto, H., Shimizu, K., Ishii, M. and Yamamuro, T., A new murine model of accelerated senescence, Mech. Ageing Dev., 17 (1981) 183-194. 19 Yagi, H., Katoh, S., Akiguchi, I. and Takeda, T., Age-related deterioration of ability of acquisition in memory and learning in senescence accelerated mouse: SAM-P/8 as an animal model of disturbances in recent memory, Brain Res., 474 (1988) 86-93.