Gerontology 1992;38(suppl 1):24—28
Department of Geriatric Medicine. Osaka University Medical School, Osaka, Japan
Key Words Senile dementia, Alzheimer type Muscarinic acetylcholine receptor Circulating suppressing factor
Circulating Suppressing Factor for the Muscarinic Acetylcholine Receptor in Patients with Senile Dementia of the Alzheimer Type
Abstract Circulating suppressing factor for the binding of quinuclidinyl benzilate (QNB), an antagonist for the muscarinic acetylcho line receptor, to the synaptic membranes was evaluated in 48 patients with senile dementia of the Alzheimer type (SDAT), in 17 patients with the vascular type dementia (VTD) and in 11 nondemented elderly subjects (NE). The mean suppression rate on the binding in the SDAT group was significantly greater than that in the NE group, although that in the VTD group was similar to that in the NE group. Moreover, the per cent QNB binding was significantly (p < 0.05) correlated with the score of the mini-mental state in the SDAT group. The circulating suppressing factor may participate in the pathogen esis of SDAT.
Senile dementia of the Alzheimer type (SDAT) is one of the most prevalent forms of senile dementia, although its pathogenesis is still unknown. Recent biochemical findings on SDAT have revealed a defect in the cholin ergic nerve activity, including decreases in the activities of the acetylcholine receptor of the cerebral cortex [1], in choline acetyl transfer ase [1] and in acetylcholine synthetase [1]. Degeneration in the nucleus basalis of Meynert, the main nucleus of the upper choliner-
gic neuron pathway, has been also reported [1]. The cholinergic neuron system in the brain was generally thought to participate in memory and recognition [2, 3]. Moreover, at least two types of receptors for acetylcholine have been observed in the brain, the musca rinic acetylcholine receptor and the nicotinic acetylcholine receptor. The muscarinic acetyl choline receptor has been postulated to regu late the production of cAMP by adenylate cyclase, and the nicotinic acetylcholine recep-
Shigeto Morimoto, MD Department of Geriatric Medicine Osaka University Medical School Fukushima-ku, Osaka 553 (Japan)
© 1992 S. Kargcr AG, Basel 0304-324X/92/ 0387-0024$2.75/0
Downloaded by: East Carolina University - Laupus Library 150.216.68.200 - 1/11/2019 10:08:25 PM
Shoichi Kitano Shigeto Morimoto Keisuke Fukuo Keiko Miya Osamu Yasuda Eio Koh Atsnshi Hirotani Tsuyoshi Nakahashi Michio Tamalani Toshio Ogihara
Subjects and Methods Subjects This study included 48 patients with SDAT, mean age ( ± SD) 79 ± 8 years, 17 patients with VTD (78 ± 8 years) and 11 NE subjects (78 ± 6 years). The clini cal diagnosis of SDAT or VTD was established by DSM III-R criteria [7] and the Hachinski ischemic score [8]. All fulfilled DSM III-R criteria for dementia and had a score of 20 or less on the mini-mental state (MMS) by Folstein et al. [9], All the NE subjects showed normal cognitive functions with MMS scores of more than 25. Patients in whom dementia was part of another neurological disorder, such as Parkinson’s disease, or with evidence of dementia of endocrine or electrolyte metabolism were not included. Blood sam ples obtained from these subjects after an overnight fast were incubated at room temperature for 1 h, and sera were separated after centrifugation of the samples at 3,000 g for 30 min. Materials [3H]QNB was purchased from Amersham (Amersham, UK). Glass fiber filter paper Whatman GF/C was from Whatman International (Maidstone, UK). Other chemicals were obtained either from Sigma (St. Louis, Mo., USA) or from Wako Pure Chemicals (Tokyo, Japan) with the highest grade available.
Preparation o f Synaptic Membranes o f Rat Brain Synaptosomes were prepared from mitochondrialsynaptosomal fraction (P2 fraction) of rat brain by the method of Whittaker et al. [ 10]. The synaptosomes were then resuspended in 10 ml per brain of low osmotic solution consisting of 1 mM EDTA and 5 mM Tris-Mes (pH 7.4), stirred gently for 60 min to remove proteolytic enzymes in the vesicles and centrifuged at 12,000# for 20 min. The protein content of the sam ples was determined by the Coomassie brilliant blue G protein-dye binding method [11] with bovine serum albumin as a standard. Radioreceptor Assay for the Muscarinic Acetylcholine Receptor The radioreceptor assay for the muscarinic acetyl choline receptor was carried out using the synaptic membranes and [3H]QNB as a ligand. Total volume of 1,000 pi consisting of 60 gg protein of the synaptic membranes, 0.025-0.4 mV/ [3H]QNB and assay buffer (120mM NaCl, 5 mM KC1, 2m M CaCl2, lm M MgCl2, 50 mA/ Tris-HCl, pH 7.4) with and without serum sample was incubated at 4 °C for 24 h. Bound/ free separation was carried out by the filter method using filter paper (Whatman GF/C), and the bound 3H count was measured. Nonspecific binding was mea sured by addition of 80 pM of unlabeled QNB. The suppressive activity of the serum on the binding of [3H]QNB to the synaptic membranes was determined with 0.2 nM [3H]QNB and 40 pi of serum. Statistical analysis was performed by Student’s t test, and Spear man’s rank correlation analysis.
Results The synaptic membranes were revealed to have a specific receptor activity for [3H]QNB with high affinity and low capacity with the dissociation constant (Kj) of 0.03 11M and the maximal displacement of 1.5pmol/mg pro tein (fig. 1). Addition of sera from SDAT patients sig nificantly inhibited the [3H]QNB binding, with the mean (± SD) value of 13.3 ± 6.1 % of the control, which was significantly higher than that of age-matched NE subjects, being 8.0 ± 3.8%. The mean inhibitory rate of the sera of the VTD patients, which was 10.0 ±
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tor to regulate ion transport of the membrane [4], Although the quantity of muscarinic ace tylcholine receptors has been reported to be much larger than that of the nicotinic acetyl choline receptors in the brain [5], the number of muscarinic acetylcholine receptors has been reported to be decreased in the cerebral cortex in patients with SDAT [6]. In this study, the circulating suppressing activity for the muscarinic acetylcholine receptor of syn aptic membranes was investigated in patients with SDAT in comparison with that in pa tients with the vascular type dementia (VTD) and in nondemented elderly (NE) subjects, using [3H]quinuclidinyl benzilate ([3H]QNB), a specific antagonist for the muscarinic ace tylcholine receptor.
b
Fig. 1. a Saturation curves of total (o), nonspecific ( a ) and spe cific binding (•) of [3H]QNB to the synaptic membrane. Specific bind ing was calculated by subtracting the nonspecific binding from total binding, b Scatchard plot of the specific binding.
Fig. 2. Inhibition of the [3H]QNB binding to the synaptic membranes by addition of serum samples from NE subjects (□ ), SDAT patients (|D and VTD patients ® . Data were shown as percent decrease in the binding of 0.2 nM of [3H]QNB from the control value without serum, using 40 pi of serum samples and 60 pg protein of synaptic membranes. Each point represents the mean of the percent inhibition by serum samples in triplicate assays. Columns and bars repre sent means ± SD.
26
Kitano/Mori moto/Fukuo/Miva/ Yasuda/Koh/Hirotani/Nakahashi/ Tamatani/Ogihara
Circulating Suppressing Factor for the Muscarinic Acteylcholine Receptor in SDAT
Downloaded by: East Carolina University - Laupus Library 150.216.68.200 - 1/11/2019 10:08:25 PM
3.5%, did not differ from that of age-matched NE subjects (fig. 2). There was a significantly positive correla tion between the percent binding of [3H]QNB to the synaptic membranes by addition of serum and the score for MMS in the SDAT patients (r = 0.462, p < 0.01; fig. 3) and in all the 76 subjects studied (r = 0.395, p < 0.01). However, there was no correlation between the two values in the groups of VTD patients or of NE subjects. The percent binding of [3H]QNB to the synaptic membrane was not significantly correlated with age, either in the SDAT patients or in the other subjects stud ied.
Fig. 3. Correlation between the percent binding of [3H]QNB to the synaptic membranes by addition of serum and the score for MMS in the SDAT patients.
In the present study, sera from the major ity of patients with SDAT suppressed the binding of [3H]QNB up to 20% or more of control. The suppressive rate in the SDAT patients was significantly greater than that of NE subjects, although there was no statistical significance in the suppressive rate between the VTD patients and the NE subjects. More over, there was a statistically significant posi tive correlation between the MMS score and binding of [3H]QNB to the membrane in the group of SDAT and in all the elderly subjects studied. Although the incidence of SDAT gen erally increases with age, the inhibitory activ ity of the serum did not significantly correlate with the age of the subjects in this study. These findings suggest the presence of a circu lating suppressive factor on the binding of [3H]QNB for the muscarinic acetylcholine re ceptor, which may participate particularly in the pathogeneses of SDAT. The nature of the circulating inhibitory factor to the muscarinic acetylcholine recep tor in the SDAT patients has not been fully elucidated in this study. However, possible circulating factor(s) which may affect the re ceptors for acetylcholine have been reported.
Tanaka et al. [12] showed the frequent pres ence of autoantibody to the nicotinic acetyl choline receptor for the neuromuscular junc tion in the serum of VTD. However, in the present study, a significant increase in the activity of autoantibody to the nicotinic ace tylcholine receptor in the sera of SDAT pa tients was observed (data not shown). More recently, Foley et al. [13] also reported the presence of antibody to cholinergic synaptic vesicles in the circulation of patients with Alz heimer disease, measuring the release of en zyme from the synaptosomes with the pres ence of complement factors. Further studies are necessary on the nature of the circulating suppressive factor for the muscarinic acetylcholine receptor, including the possibility of autoantibodies to the musca rinic acetylcholine receptor, to elucidate its pathophysiological role(s) in the development of SDAT.
Acknowledgments This work was partly supported by grants from the Sandoz Foundation for Gerontological Research and from the Ministry of Science and Culture of Japan. The authors thank Ms. Y. Mayumi and H. Shirai for their secretarial and technical helps.
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Discussion
1 Hardy J, Adolfsson R, Alafuzoff I, et al: Transmitter deficits in Alzhei mer's disease. Neurochem Int 1985; 7:545-563. 2 Drachman DA, Leavitt J: Human memory and the cholinergic system. Arch Neurol 1974;30:113-121. 3 Drachman DA: Memory and cogni tive function in man: Does the cho linergic system have a specific role? Neurology 1977;27:783-790. 4 Changeux JP, Rcvah F: The acetyl choline receptor molecule: Alloste ric sites and the ion channel. Trends Neurosci 1987;10:245-250. 5 Shimohama S, Taniguchi T, Fujiwara M, et al: Changes in nicotinic and muscarinic cholinergic recep tors in Alzheimer-type dementia. J Neurochem 1986;46:288-293.
6 Caulfield MP, Straughan DW. Cross AJ, et al: Cortical muscarinic recep tor subtypes and Alzheimer’s dis ease. Lancet 1982;ii: 1277. 7 American Psychiatric Association: Diagnostic and Statistical Manual of Mental Disorders (DSM-I1I-R). Washington, American Psychiatric Association, 1987, pp 103-109. 8 Hachinski VC, IlifF LD, Zilhka E, et al: Cerebral blood flow in dementia. Arch Neurol 1975;32:632-637. 9 Folstein MF, Folstein SE, McHugh PR: ‘Mini-mental state’. A practical method for grading the cognitive state of patients for the clinician. J Psychiatr Res 1975;12:189-198. 10 Whittaker VP, Michaelson 1A, Kirk land RJ: The separation of synaptic vesicles from nerve-ending particles (‘synaptosomes’). Biochem J 1964; 90:293-303.
11 Bradford MM: A rapid and sensitive method for the quantitation of mi crogram quantities o f protein utiliz ing the principle of protein-dye binding. Anal Biochem 1976:72: 248-254. 12 Tanaka M, Miyake T: Anti-acetylcholine receptor antibody in aged individuals and in patients with Down’s syndrome. J Neuroimmunol 1983;4:17-24. 13 Foley P. Bradford HF, Docherty M, et al: Evidence of the presence of antibodies to cholinergic neurons in the serum of patients with Alzhei mer’s disease. J Neurol 1988;235: 466-471.
28
Kitano/Morimoto/Fukuo/Miya/ Y asuda/Koh/H irotani/Nakahashi/ Tamatani/Ogihara
Circulating Suppressing Factor for the Muscarinic Acteylcholine Receptor in SDAT
Downloaded by: East Carolina University - Laupus Library 150.216.68.200 - 1/11/2019 10:08:25 PM
References
Department of Geriatric Medicine. Osaka University Medical School, Osaka, Japan
Key Words Senile dementia, Alzheimer type Muscarinic acetylcholine receptor Circulating suppressing factor
Circulating Suppressing Factor for the Muscarinic Acetylcholine Receptor in Patients with Senile Dementia of the Alzheimer Type
Abstract Circulating suppressing factor for the binding of quinuclidinyl benzilate (QNB), an antagonist for the muscarinic acetylcho line receptor, to the synaptic membranes was evaluated in 48 patients with senile dementia of the Alzheimer type (SDAT), in 17 patients with the vascular type dementia (VTD) and in 11 nondemented elderly subjects (NE). The mean suppression rate on the binding in the SDAT group was significantly greater than that in the NE group, although that in the VTD group was similar to that in the NE group. Moreover, the per cent QNB binding was significantly (p < 0.05) correlated with the score of the mini-mental state in the SDAT group. The circulating suppressing factor may participate in the pathogen esis of SDAT.
Senile dementia of the Alzheimer type (SDAT) is one of the most prevalent forms of senile dementia, although its pathogenesis is still unknown. Recent biochemical findings on SDAT have revealed a defect in the cholin ergic nerve activity, including decreases in the activities of the acetylcholine receptor of the cerebral cortex [1], in choline acetyl transfer ase [1] and in acetylcholine synthetase [1]. Degeneration in the nucleus basalis of Meynert, the main nucleus of the upper choliner-
gic neuron pathway, has been also reported [1]. The cholinergic neuron system in the brain was generally thought to participate in memory and recognition [2, 3]. Moreover, at least two types of receptors for acetylcholine have been observed in the brain, the musca rinic acetylcholine receptor and the nicotinic acetylcholine receptor. The muscarinic acetyl choline receptor has been postulated to regu late the production of cAMP by adenylate cyclase, and the nicotinic acetylcholine recep-
Shigeto Morimoto, MD Department of Geriatric Medicine Osaka University Medical School Fukushima-ku, Osaka 553 (Japan)
© 1992 S. Kargcr AG, Basel 0304-324X/92/ 0387-0024$2.75/0
Downloaded by: East Carolina University - Laupus Library 150.216.68.200 - 1/11/2019 10:08:25 PM
Shoichi Kitano Shigeto Morimoto Keisuke Fukuo Keiko Miya Osamu Yasuda Eio Koh Atsnshi Hirotani Tsuyoshi Nakahashi Michio Tamalani Toshio Ogihara
Subjects and Methods Subjects This study included 48 patients with SDAT, mean age ( ± SD) 79 ± 8 years, 17 patients with VTD (78 ± 8 years) and 11 NE subjects (78 ± 6 years). The clini cal diagnosis of SDAT or VTD was established by DSM III-R criteria [7] and the Hachinski ischemic score [8]. All fulfilled DSM III-R criteria for dementia and had a score of 20 or less on the mini-mental state (MMS) by Folstein et al. [9], All the NE subjects showed normal cognitive functions with MMS scores of more than 25. Patients in whom dementia was part of another neurological disorder, such as Parkinson’s disease, or with evidence of dementia of endocrine or electrolyte metabolism were not included. Blood sam ples obtained from these subjects after an overnight fast were incubated at room temperature for 1 h, and sera were separated after centrifugation of the samples at 3,000 g for 30 min. Materials [3H]QNB was purchased from Amersham (Amersham, UK). Glass fiber filter paper Whatman GF/C was from Whatman International (Maidstone, UK). Other chemicals were obtained either from Sigma (St. Louis, Mo., USA) or from Wako Pure Chemicals (Tokyo, Japan) with the highest grade available.
Preparation o f Synaptic Membranes o f Rat Brain Synaptosomes were prepared from mitochondrialsynaptosomal fraction (P2 fraction) of rat brain by the method of Whittaker et al. [ 10]. The synaptosomes were then resuspended in 10 ml per brain of low osmotic solution consisting of 1 mM EDTA and 5 mM Tris-Mes (pH 7.4), stirred gently for 60 min to remove proteolytic enzymes in the vesicles and centrifuged at 12,000# for 20 min. The protein content of the sam ples was determined by the Coomassie brilliant blue G protein-dye binding method [11] with bovine serum albumin as a standard. Radioreceptor Assay for the Muscarinic Acetylcholine Receptor The radioreceptor assay for the muscarinic acetyl choline receptor was carried out using the synaptic membranes and [3H]QNB as a ligand. Total volume of 1,000 pi consisting of 60 gg protein of the synaptic membranes, 0.025-0.4 mV/ [3H]QNB and assay buffer (120mM NaCl, 5 mM KC1, 2m M CaCl2, lm M MgCl2, 50 mA/ Tris-HCl, pH 7.4) with and without serum sample was incubated at 4 °C for 24 h. Bound/ free separation was carried out by the filter method using filter paper (Whatman GF/C), and the bound 3H count was measured. Nonspecific binding was mea sured by addition of 80 pM of unlabeled QNB. The suppressive activity of the serum on the binding of [3H]QNB to the synaptic membranes was determined with 0.2 nM [3H]QNB and 40 pi of serum. Statistical analysis was performed by Student’s t test, and Spear man’s rank correlation analysis.
Results The synaptic membranes were revealed to have a specific receptor activity for [3H]QNB with high affinity and low capacity with the dissociation constant (Kj) of 0.03 11M and the maximal displacement of 1.5pmol/mg pro tein (fig. 1). Addition of sera from SDAT patients sig nificantly inhibited the [3H]QNB binding, with the mean (± SD) value of 13.3 ± 6.1 % of the control, which was significantly higher than that of age-matched NE subjects, being 8.0 ± 3.8%. The mean inhibitory rate of the sera of the VTD patients, which was 10.0 ±
25
Downloaded by: East Carolina University - Laupus Library 150.216.68.200 - 1/11/2019 10:08:25 PM
tor to regulate ion transport of the membrane [4], Although the quantity of muscarinic ace tylcholine receptors has been reported to be much larger than that of the nicotinic acetyl choline receptors in the brain [5], the number of muscarinic acetylcholine receptors has been reported to be decreased in the cerebral cortex in patients with SDAT [6]. In this study, the circulating suppressing activity for the muscarinic acetylcholine receptor of syn aptic membranes was investigated in patients with SDAT in comparison with that in pa tients with the vascular type dementia (VTD) and in nondemented elderly (NE) subjects, using [3H]quinuclidinyl benzilate ([3H]QNB), a specific antagonist for the muscarinic ace tylcholine receptor.
b
Fig. 1. a Saturation curves of total (o), nonspecific ( a ) and spe cific binding (•) of [3H]QNB to the synaptic membrane. Specific bind ing was calculated by subtracting the nonspecific binding from total binding, b Scatchard plot of the specific binding.
Fig. 2. Inhibition of the [3H]QNB binding to the synaptic membranes by addition of serum samples from NE subjects (□ ), SDAT patients (|D and VTD patients ® . Data were shown as percent decrease in the binding of 0.2 nM of [3H]QNB from the control value without serum, using 40 pi of serum samples and 60 pg protein of synaptic membranes. Each point represents the mean of the percent inhibition by serum samples in triplicate assays. Columns and bars repre sent means ± SD.
26
Kitano/Mori moto/Fukuo/Miva/ Yasuda/Koh/Hirotani/Nakahashi/ Tamatani/Ogihara
Circulating Suppressing Factor for the Muscarinic Acteylcholine Receptor in SDAT
Downloaded by: East Carolina University - Laupus Library 150.216.68.200 - 1/11/2019 10:08:25 PM
3.5%, did not differ from that of age-matched NE subjects (fig. 2). There was a significantly positive correla tion between the percent binding of [3H]QNB to the synaptic membranes by addition of serum and the score for MMS in the SDAT patients (r = 0.462, p < 0.01; fig. 3) and in all the 76 subjects studied (r = 0.395, p < 0.01). However, there was no correlation between the two values in the groups of VTD patients or of NE subjects. The percent binding of [3H]QNB to the synaptic membrane was not significantly correlated with age, either in the SDAT patients or in the other subjects stud ied.
Fig. 3. Correlation between the percent binding of [3H]QNB to the synaptic membranes by addition of serum and the score for MMS in the SDAT patients.
In the present study, sera from the major ity of patients with SDAT suppressed the binding of [3H]QNB up to 20% or more of control. The suppressive rate in the SDAT patients was significantly greater than that of NE subjects, although there was no statistical significance in the suppressive rate between the VTD patients and the NE subjects. More over, there was a statistically significant posi tive correlation between the MMS score and binding of [3H]QNB to the membrane in the group of SDAT and in all the elderly subjects studied. Although the incidence of SDAT gen erally increases with age, the inhibitory activ ity of the serum did not significantly correlate with the age of the subjects in this study. These findings suggest the presence of a circu lating suppressive factor on the binding of [3H]QNB for the muscarinic acetylcholine re ceptor, which may participate particularly in the pathogeneses of SDAT. The nature of the circulating inhibitory factor to the muscarinic acetylcholine recep tor in the SDAT patients has not been fully elucidated in this study. However, possible circulating factor(s) which may affect the re ceptors for acetylcholine have been reported.
Tanaka et al. [12] showed the frequent pres ence of autoantibody to the nicotinic acetyl choline receptor for the neuromuscular junc tion in the serum of VTD. However, in the present study, a significant increase in the activity of autoantibody to the nicotinic ace tylcholine receptor in the sera of SDAT pa tients was observed (data not shown). More recently, Foley et al. [13] also reported the presence of antibody to cholinergic synaptic vesicles in the circulation of patients with Alz heimer disease, measuring the release of en zyme from the synaptosomes with the pres ence of complement factors. Further studies are necessary on the nature of the circulating suppressive factor for the muscarinic acetylcholine receptor, including the possibility of autoantibodies to the musca rinic acetylcholine receptor, to elucidate its pathophysiological role(s) in the development of SDAT.
Acknowledgments This work was partly supported by grants from the Sandoz Foundation for Gerontological Research and from the Ministry of Science and Culture of Japan. The authors thank Ms. Y. Mayumi and H. Shirai for their secretarial and technical helps.
27
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Discussion
1 Hardy J, Adolfsson R, Alafuzoff I, et al: Transmitter deficits in Alzhei mer's disease. Neurochem Int 1985; 7:545-563. 2 Drachman DA, Leavitt J: Human memory and the cholinergic system. Arch Neurol 1974;30:113-121. 3 Drachman DA: Memory and cogni tive function in man: Does the cho linergic system have a specific role? Neurology 1977;27:783-790. 4 Changeux JP, Rcvah F: The acetyl choline receptor molecule: Alloste ric sites and the ion channel. Trends Neurosci 1987;10:245-250. 5 Shimohama S, Taniguchi T, Fujiwara M, et al: Changes in nicotinic and muscarinic cholinergic recep tors in Alzheimer-type dementia. J Neurochem 1986;46:288-293.
6 Caulfield MP, Straughan DW. Cross AJ, et al: Cortical muscarinic recep tor subtypes and Alzheimer’s dis ease. Lancet 1982;ii: 1277. 7 American Psychiatric Association: Diagnostic and Statistical Manual of Mental Disorders (DSM-I1I-R). Washington, American Psychiatric Association, 1987, pp 103-109. 8 Hachinski VC, IlifF LD, Zilhka E, et al: Cerebral blood flow in dementia. Arch Neurol 1975;32:632-637. 9 Folstein MF, Folstein SE, McHugh PR: ‘Mini-mental state’. A practical method for grading the cognitive state of patients for the clinician. J Psychiatr Res 1975;12:189-198. 10 Whittaker VP, Michaelson 1A, Kirk land RJ: The separation of synaptic vesicles from nerve-ending particles (‘synaptosomes’). Biochem J 1964; 90:293-303.
11 Bradford MM: A rapid and sensitive method for the quantitation of mi crogram quantities o f protein utiliz ing the principle of protein-dye binding. Anal Biochem 1976:72: 248-254. 12 Tanaka M, Miyake T: Anti-acetylcholine receptor antibody in aged individuals and in patients with Down’s syndrome. J Neuroimmunol 1983;4:17-24. 13 Foley P. Bradford HF, Docherty M, et al: Evidence of the presence of antibodies to cholinergic neurons in the serum of patients with Alzhei mer’s disease. J Neurol 1988;235: 466-471.
28
Kitano/Morimoto/Fukuo/Miya/ Y asuda/Koh/H irotani/Nakahashi/ Tamatani/Ogihara
Circulating Suppressing Factor for the Muscarinic Acteylcholine Receptor in SDAT
Downloaded by: East Carolina University - Laupus Library 150.216.68.200 - 1/11/2019 10:08:25 PM
References
