GABA와 Benzodiazepine 수용체 및 그 기능

정영조,한기석 大韓神經精神醫學會 1991 신경정신의학 Vol.30 No.3

GABA is ubiquitously distributed throughout the CNS. It is probably the major central inhibitory aminoacid neurotransmitter which hyperpolarizes the postsynapic neurons and inhibits the release of neurotransmitters. Generally inhibition of GABA activity causes excitation leading to anxiety and convulsions, whereas activation of its activity causes antiexpressive. anticonvulsive activity and sedation. The GABA receptors have been divided into two distinct groups named GABA(A) and GABA(B) receptors by their pharmacological and physiological properties. GABA(A) receptors are coupled with Bz binding site and that in conjunction with a CI­ channel they form a supramolecular receptor complex which mediates rapid increasing of CI­ influx into postsynaptic neurons. thus contributing to the prompt inhibition of cellular excitability. On the contrary, the GABA(B) receptor does not contain an integral ion channel and is responsible for slow responses through receptor-G protein-effector complexes. GABA(B) receptors appear to be localized on presynaptic nerve terminals and stimulation of presynaptic GABA(B) receptors reduced Ca²+ influx, resulting in decreased release of neurotransmitters. Stimulation of presynaptic GABA(B) receptors increases K efflux, resulting in inhibition of cell firing. A division of function among the two types of GABA receptors appears to exist : GABA(A) receptor complex mediates anxiety, anticonvulsive activity and feeding, GABA(B) receptors, on the other hand, are involved in depression and analgesia. In those cases where GABA(A) and GABA(B) receptors mediate similar functions (e.g., cardiovascular regulation), they do so by affecting different transmitter systems and cellular mechanisms. The putative involvement of GABA(A) and GABA(B) receptors in various behavioral and physiological effects is summarized in Table 3. There are two types of Bz receptors in brain which are central and peripheral type Bz receptors. Central Bz receptors are in many regions of the brain, coupled with the receptors for GABA and they mediate the acute actions of Bz in CNS. Although there is general acceptance that Bz evert their major actions via the GABA(A) receptor, more recent studies suggest that other systems may be involved. In addition, endogenous anxiogenic ligands that interact with Bz receptors in several ways have been discovered, which of these,β-carbolines and DBI may produce anxiety and convulsion in human, The recent investigation of endogenous ligands and specific receptor agonist or antagonist and inverse agonist provide important new conceptual tools for the studies of anxiety and depression mechanisms. It is expected that a better understanding of GABA and Bz receptors will eventually help to demonstrate the biology of anxiety and depression.

IM-9 Lymphocyte에서 포도당과 인슐린이 인슐린 수용체 유전자의 발현에 미치는 영향

정수경 ( Jeong Su Gyeong ),김성운 ( Kim Seong Un ),양인명 ( Yang In Myeong ),김진우 ( Kim Jin U ),김영설 ( Kim Yeong Seol ),김광원 ( Kim Gwang Won ),최영길 ( Choe Yeong Gil ),정해원 ( Jeong Hae Won ) 대한내과학회 1992 대한내과학회지 Vol.42 No.5

연구배경 : 인슐린 수용체의 조절은 인슐린과 수용제의 복합체가 세포내로 내재화한 후 분해되는 비율과 합성된 수용체가 세포막으로 출현하는 비율의 조화에 의해 이루어진다. 세포막 수준의 인슐린 수용체수의 저하는 인슐린 수용체의 합성의 저하즉 인슐린 수용체 mRNA의 발현이 저하된 것으로 고려해 볼 수 있다. 인슐린 구용체의 발현에 영향을 미치는 요인을 분석하여 그중 인슐린과 포도당이 인슐린 수용체 유전자의 발현에 미치는 영향을 보고한다. 방법 : 인슐린 수용체의 조절기전을 연구하기 위하여 인슐린 수용체가 다량으로 세포막에 표현되는 세포인 IM-9 lymphocyte를 이용하여 인슐린과 포도당 농도의 변화가 인슐린 수용체의 변동과 그 유전자의 발현에 미치는 영향을 조사하였다. 포도당은 생체의 저혈당 농도인 2.BmM/L에서 고혈당 농도인 22mM/L까지, 그리고 인슐린은 인슐린이 없는 대조군에서부터 최고 10^(-6)M까지 노출하여 16시간까지 배양한 후 방사수용체 분석법으로 인슐린 수용체를 분석하였다. RNA를 추출하여 Northern blot을 시행하여 분자 수준의 변화인 mRNA의 전사양상을 관찰하였다. 결과 : 인슐린의 농도가 증가됨에 따라 인슐린 수용체의 최대 결합율은 대조군에서 13.6%였으나, 점차 감소하여 10^(-6)M에서 2%로 유의하게 감소하였다(p<0.01). 고친화성 인슐린 수용체의 수는 대조군이 5.14×10^(3)sites/cell 이었으며, 최고 인슐린 농도인 10^(-6)M에서 0.26×10^(3) sites/cell로 감소하였으나(p<0.01), 저친화성 인슐린 수용체는 변화하지 않았다. 포도당의 농도가 증가됨에 따라 인슐린 수용체의 최대 결합율은 2.8~5.5 mM/L까지는 23% 정도로 변화가 없었으나, 11-22mM/L에서는 약 15%로 유의하게 감소하었다 (p<0.05). 고친화성 인슐린 수용체의 수는 생체내 저혈당 농도인 2.8mM/L에서 5.08×10^(3) sites/cell 이었으나, 고혈당 농도인 22mM/L에서는 2.53×10^(-3) sites/cell로 감소하였으며(p<0.05), 저친화성 수용체 역시 감소하는 경향이었다(p<0.01). Northern blot을 시행한 결과 인슐린 수용체 mRNA는 11과 8.5 kb의 두 종류로 발현되었다. 인슐린 수용체 mRNA 발현양상은 인슐린 농도의 증가에도 유의한 변화가 없었다. 포도당의 농도가 증가됨에 따라 lIkb의 mRNA는 전사량이 증가하였으나, 8.5kb의 전사량은 감소하었다. 이 두 종류 mRNA의 비(8.5kb/11kn)는 인슐린 수용체 수와 정상관 관계에 있었다. 결론 : 이러한 연구결과를 토대로 인슐린 수용체의 조절은 전사과정에서부터 두 종류의 mRNA로 전사됨을 확인할 수 있었다. 수용체 조절의 첫단계인 전사와 마지막 단계인 수용체 결합을 측정한 결과 인슐린은 수용제의 전사단계에는 영향을 미치지 않고 세포막에서 수용체 단백의 표현에 영향을 주며. 포도당은 인슐린 수용체의 전사단계에 영향을 미치며 이를 통하여 세포막 수용체의 조절에 영향을 미치는 것으로 생각된다. 포도당의 농도가 증가됨에 따라 두 종류 mRNA의 전사비(8.5kb/11kb)가 감소하는 것으로 미루어 인슐린 수용체 mRNA는 전사량 뿐만 아니라 전사비의 변화도 세포막 인슐린 수용체의 증가나 감소에 관여하리라고 사료되었다. Backgroundhw level of cell surface insulin receptor seems to result either from a low level of insulin receptor gene expression or from structural changes in the receptor which interfere with proper processing of the primary gene product. The balance of insulin receptor degradation and de novo synthesis determines the final number of receptor on the plasma membrane. We have studied the factors influencing mRNA levels of the insulin receptor, and report on the effects of insulin and glucose on insulin receptor mRNA levels in IM-9 lymphoblastic cells. Methode:IM-9 cells were cultured in RPMI 1640 (glucose free) media containing various concentrations of glucose and insulin for 16 hours. mRNA levels was quantified by Northern blot analysis using a labled cDNA (phINSR-13.1) probe for the insulin receptor. And the number of cell surface insulin receptor was estimated by Scatchard plot simultaneously. Results:The number of insulin receptor was decreased with increasing glucose and insulin concentration in the culture media. Northern blot analysis of insulin receptor gene mRNA revealed two major size of 11 kb (band Ⅰ) and 8.5 kb (band Ⅱ). The concentration of insulin up to 1 pM had no effect on hybridizable insulin receptor mRNA levels. The level of transcripted mRNA band Ⅰ (11 kb) was increased, while band Ⅱ (8.5 kb) was decreased with increasing glucose concentration. The changes of band ratio (band Ⅱ/band Ⅰ) correlated well with the decreased number of insulin receptor. Conclusion: These results suggested that glucose had suppressive effect on the expression of insulin receptor mRNA, but insulin had no direct effect on the expression of insulin receptor mRNA. And it was suggested that the changes of band ratio as well as the amount of transcripted mRNA might play a role in regulaiton of the cell surface insulin receptor.

M₁과 M₂ 무스카린성 수용체에서 아미노산 Triplet Repeat의 Site-Mutagenesis가 수용체기능에 미치는 영향

이석용(Seok-Yong Lee),이상복(Sang-Bok Lee) 대한약리학회 1996 대한약리학잡지 Vol.32 No.3

M₁과 M₂ 무스카린성 수용체의 두 번째 transmembrane domain의 C-말단에는 leucine(L), tyrosine(Y), threonine(T)로 구성된 3중체(triplet)가 있다. 이 3중체는 M₂ 무스카린성 수용체에서는 두 번째 transmembrane domain과 첫 번째 세포외 고리사이의 연접부위에서 LYT-LYT의 반복구조로 존재하며 M₁ 무스카린성 수용체에서는 흥미롭게도 LYT-TYL의 역상구조로 존재한다. 본 연구에서는 site-directed mutagenesis방법을 사용하여 이와 같은 특이한 구조적차이가 두 subtype의 수용체의 기능상 차이와 관련한 역할을 가지고 있는지를 확인하고자 하였다. M₁ 수용체에서는 LYTTYL서열을 M₂ 수용체의 서열에 해당하는 LYTLYT로 mutation시켰으며 M₂ 수용체에서는 LYTLYT8서열을 M₁ 수용체의 서열에 해당하는 LYTTYL로 mutation시켰다. 이와같은 mutation은 M₁과 M₂ 수용체에서 효능제 carbachol의 수용체 결합친화력에 유의한 변화를 주지 않았다. 또한 M₁ 수용체에서의 mutation은 cyclic AMP 증가작용에 대한 coupling은 변화시키지 않고 phosphoinositides (PI) hydrolysis 촉진작용과 세포내 Ca²⁺ 농도 상승을 현저히 증가시켰다. 또한 M₂ 수용체에서의 mutation은 adenylate cyclase 억제에 대한 coupling은 변화시키지 않고 PI hydrolysis 촉진을 약간 증가 시켰다. 이상의 결과는 M₁과 M₂ 수용체에서 LYTTYL/LYTLYT 아미노산 서열의 차이는 두 수용체의 PI hydrolysis에 대한 coupling을 조절하는 역할을 하지만, 두 수용체 사이에서 ligand 결합과 신호전달계의 차이를 구분하는데 중요한 역할을 하지는 않는다. Both M₁ and M₂ muscarinic receptors contain a triplet of amino acid residues consisting of leucine (L), tyrosine (Y) and threonine (T) at C-terminus ends of the second putative transmembrane domains. This triplet is repeated as LYT-LYT in M₂ receptors at the interface between the second transmembrane domain and the first extracellular loop. Interestingly, however, it is repeated in a transposed fashion (LYT-TYL) in the sequence of M₁ receptors. In this work, we employed site-directed mutagenesis to investigate the possible significance of this unique sequence diversity for determining the distinct differential cellular function at the two receptor subtypes. Mutation of the LYTTYL sequence of M₁ receptors to the corresponding M₂ receptor LYTLYT sequence did not result in a significant change in the binding affinity of the agonist carbachol. The reverse mutation at the M₂ receptor also did not modify agonist affinity. Surprisingly, the LYTLYT M₁ receptor mutant demonstrated markedly enhanced coupling to activation of phospholipase C without a change in its coupling to increased cyclic AMP formation. There was also an enhanced receptor sensitivity in transducing elevation of intracellular Ca²⁺. On the other hand, the reverse LYTLYT{\rightarrow}LYTTYL mutation in the M₂ receptor did not alter its coupling to inhibition of adenylate cyclase, but slightly enhanced its coupling to stimulation of phosphoinositide (PI) hydrolysis. Our data suggest that the LYTTYL/LYTLYT sequence differences between M₁ and M₂ muscarinic receptors are not important for specifying ligand binding and coupling of various subtypes of muscarinic receptors to different cellular signaling pathways although they might play a role in the modulation of muscarinic reseptor coupling to PI hydrolysis.

Effects of Site-Mutagenesis of an Amino Acid Triplet Repeat at $M_1$ and $M_2$ Muscarinic Receptors on Receptor Function

이석용,이상복,Lee, Seok-Yong,Lee, Sang-Bok The Korean Society of Pharmacology 1996 대한약리학잡지 Vol.32 No.3

$M_1$과 $M_2$ 무스카린성 수용체의 두 번째 transmembrane domain의 C-말단에는 leucine(L), tyrosine(Y), threonine(T)로 구성된 3중체(triplet)가 있다. 이 3중체는 $M_2$ 무스카린성 수용체에서는 두 번째 transmembrane domain과 첫 번째 세포외 고리사이의 연접부위에서 LYT-LYT의 반복구조로 존재하며 $M_1$ 무스카린성 수용체에서는 흥미롭게도 LYT-TYL의 역상구조로 존재한다. 본 연구에서는 site-directed mutagenesis방법을 사용하여 이와 같은 특이한 구조적차이가 두 subtype의 수용체의 기능상 차이와 관련한 역할을 가지고 있는지를 확인하고자 하였다. $M_1$ 수용체에서는 LYTTYL서열을 $M_2$ 수용체의 서열에 해당하는 LYTLYT로 mutation시켰으며 $M_2$ 수용체에서는 LYTLYT8서열을 $M_1$ 수용체의 서열에 해당하는 LYTTYL로 mutation시켰다. 이와같은 mutation은 $M_1$과 $M_2$ 수용체에서 효능제 carbachol의 수용체 결합친화력에 유의한 변화를 주지 않았다. 또한 $M_1$ 수용체에서의 mutation은 cyclic AMP 증가작용에 대한 coupling은 변화시키지 않고 phosphoinositides (PI) hydrolysis 촉진작용과 세포내 $Ca^{2+}$ 농도 상승을 현저히 증가시켰다. 또한 $M_2$ 수용체에서의 mutation은 adenylate cyclase 억제에 대한 coupling은 변화시키지 않고 PI hydrolysis 촉진을 약간 증가 시켰다. 이상의 결과는 $M_1$과 $M_2$ 수용체에서 LYTTYL/LYTLYT 아미노산 서열의 차이는 두 수용체의 PI hydrolysis에 대한 coupling을 조절하는 역할을 하지만, 두 수용체 사이에서 ligand 결합과 신호전달계의 차이를 구분하는데 중요한 역할을 하지는 않는다. Both $M_1$ and $M_2$ muscarinic receptors contain a triplet of amino acid residues consisting of leucine (L), tyrosine (Y) and threonine (T) at C-terminus ends of the second putative transmembrane domains. This triplet is repeated as LYT-LYT in $M_2$ receptors at the interface between the second transmembrane domain and the first extracellular loop. Interestingly, however, it is repeated in a transposed fashion (LYT-TYL) in the sequence of $M_1$ receptors. In this work, we employed site-directed mutagenesis to investigate the possible significance of this unique sequence diversity for determining the distinct differential cellular function at the two receptor subtypes. Mutation of the LYTTYL sequence of $M_1$ receptors to the corresponding $M_2$ receptor LYTLYT sequence did not result in a significant change in the binding affinity of the agonist carbachol. The reverse mutation at the $M_2$ receptor also did not modify agonist affinity. Surprisingly, the LYTLYT $M_1$ receptor mutant demonstrated markedly enhanced coupling to activation of phospholipase C without a change in its coupling to increased cyclic AMP formation. There was also an enhanced receptor sensitivity in transducing elevation of intracellular $Ca^{2+}$. On the other hand, the reverse $LYTLYT{\rightarrow}LYTTYL$ mutation in the $M_2$ receptor did not alter its coupling to inhibition of adenylate cyclase, but slightly enhanced its coupling to stimulation of phosphoinositide (PI) hydrolysis. Our data suggest that the LYTTYL/LYTLYT sequence differences between $M_1$ and $M_2$ muscarinic receptors are not important for specifying ligand binding and coupling of various subtypes of muscarinic receptors to different cellular signaling pathways although they might play a role in the modulation of muscarinic reseptor coupling to PI hydrolysis.

Effects of $17{\beta}$-Estradiol and Estrogen Receptor Antagonists on the Proliferation of Gastric Cancer Cell Lines

Kim, Myung-Jin,Cho, Sung-Il,Lee, Kun-Ok,Han, Hyung-Joon,Song, Tae-Jin,Park, Seong-Heum The Korean Gastric Cancer Association 2013 Journal of gastric cancer Vol.13 No.3

Purpose: The aims of this study were as follow: 1) to de scribe the expression status of estrogen receptor-${\alpha}$ and -${\beta}$ mRNAs in five gastric carcinoma cell lines; 2) to evaluate in vitro the effects of $17{\beta}$-estradiol and estrogen receptor antagonists on the proliferation of the cell lines. Materials and Methods: Detection of estrogen receptor-${\alpha}$ and estrogen receptor-${\beta}$ mRNA in five human gastric cancer cell lines (AGS, KATO III, MKN28, MKN45 and MKN74) was made by the reverse transcription-polymerase chain reaction system. To evaluate the effect of $17{\beta}$-estradiol and estrogen receptor antagonists on the proliferation of gastric cancer cell line, the cell lines which expressed both es trogen receptors were chosen and treated with $17{\beta}$-estradiol and estrogen receptor antagonists (methyl-piperidino-pyrazole and pyrazolo [1,5-a] pyrimidine). Cell proliferation was assessed with the methylthiazol tetrazolium test. Results: Estrogen receptor-${\alpha}$ and estrogen receptor-${\beta}$ mRNAs were expressed in three (KATO III, MKN28 and MKN45) and all of the five gastric cancer cell lines, respectively. At higher concentrations, $17{\beta}$-estradiol inhibited cell growth of MKN28, MKN45 and KATO III cell lines. Neither estrogen receptor-${\alpha}$ nor estrogen receptor-${\beta}$ antagonist blocked the anti-proliferative effect of $17{\beta}$-estradiol. Conclusions: Our results indicate that estrogen receptor-${\beta}$ mRNAs are preferentially expressed in gastric cancers and also imply that hormone therapy rather than estrogen receptor blockers may be a useful strategy for the treatment of estrogen receptor-${\beta}$ positive gastric cancer. Its therapeutic significance in gastric cancer are, however, limited until more evidence of the roles of estrogen receptors in the gastric cancer are accumulated.

Functions of Eel Luteinizing Hormone Receptor Mutants (Activating and Inactivating Receptors)

Munkhzaya Byambaragchaa,Jeong-Soo Kim,Hun-Ki Seong,Kwan-Sik Min 한국동물생명공학회(구 한국동물번식학회) 2017 Reproductive & Developmental Biology(Supplement) Vol.41 No.2

Gonadotropin receptors are members of the seven transmembrane (TM) receptor families. Several point mutations in TM II, III, V and VI have been identified in the luteinizing hormone receptor (LHR) gene, leading to constitutive activation and inactivation of the receptor. In eelLHR, we generated 3 types of constitutive activating mutations (M410T, L469R and D590Y) and 2 types of constitutive inactivating mutations (D383N and Y546F) to investigate how they work on hormone-receptor interaction and receptor activation system on eel. To assess the functional effects of 5 receptor mutations directly, wt and mutant eel- LHRs were transiently expressed in CHO-K1 cells, and basal and recombinant eel LH-stimulated cAMP and IP-1 accumulations were measured. Rec-eelLH (0.076~1,200 ng/mL) produced a concentration-dependent increase in cAMP production in wt eelLHR expressing cells with an EC50 of 160 ng/mL and basal cAMP level of 2.6 nM. In contrast, the L469R activation mutant had most elevated (16.88 fold higher than wt) basal cAMP production (basal cAMP level=43.9 nM). Compared with the wt eelLHR, all the activation mutant receptors produced higher basal levels of cAMP (18.4 nM for D590Y and 7.9 nM for M410T). However, eelLH-stimulated (0.076~1,200 ng/mL) basal cAMP levels in the constitutive inactivation mutants D383N and Y546F did not obviously altered from that in wt eelLHR. D383N mutation increased the EC50 value to 185 ng/mL (inhibited receptor activity to 86%), while Y546F mutation increased that to 170 ng/mL which implies that receptor activity was inhibited to 94% only. As seen in IC50 values in IP-1 accumulation, activity for M410T mutant was 19% higher than that for wt receptor, but other activation mutants did not show any difference in IP-1 production. In case of inactivation mutants, there were no significant differences in IP-1 production and only 7% decreased activity was identified in D383N mutant. In summary, we have demonstrated 3 mutations that are responsible for constitutive activation of eelLHR. Although predicted 2 inactivation mutations led to slightly diminished activation of receptor, those could not impair signal transduction of eelLHR.

Drawing Phenomenon #19

이태희 ( Lee Tae Hee ) 한국기초조형학회 2019 한국기초조형학회 작품집 Vol.2019 No.2

Brain Receptor Mosaics and Their Intramembrane Receptor-Receptor Interactions: Molecular Integration in Transmission and Novel Targets for Drug Development

Kjell Fuxe,Daniel Marcellino,Diego Guidolin,Amina S. Woods,Luigi Agnati 사단법인약침학회 2009 Journal of Acupuncture & Meridian Studies Vol.2 No.1

The concept of intramembrane receptor-receptor interactions and evidence for their existence was introduced by Agnati and Fuxe in 1980/81 suggesting the existence of heteromerization of receptors. In 1982, they proposed the existence of aggregates of multiple receptors in the plasma membrane and coined the term receptor mosaics (RM). In this way, cell signaling becomes a branched process beginning at the level of receptor recognition at the plasma membrane where receptors can directly modify the ligand recognition and signaling capacity of the receptors within a RM. Receptorreceptor interactions in RM are classified as operating either with classical cooperativity, when consisting of homomers or heteromers of similar receptor subtypes having the same transmitter, or non-classical cooperativity, when consisting of heteromers. It has been shown that information processing within a RM depends not only on its receptor composition, but also on the topology and the order of receptor activation determined by the concentrations of the ligands and the receptor properties. The general function of RM has also been demonstrated to depend on allosteric regulators (e.g., homocysteine) of the receptor subtypes present. RM as integrative nodes for receptor-receptor interactions in conjunction with membrane associated proteins may form horizontal molecular networks in the plasma membrane coordinating the activity of multiple effector systems modulating the excitability and gene expression of the cells. The key role of electrostatic epitope-epitope interactions will be discussed for the formation of the RM. These interactions probably represent a general molecular mechanism for receptor-receptor interactions and, without a doubt, indicate a role for phosphorylation-dephosphorylation events in these interactions. The novel therapeutic aspects given by the RMs will be discussed in the frame of molecular neurology and psychiatry and combined drug therapy appears as the future way to go. The concept of intramembrane receptor-receptor interactions and evidence for their existence was introduced by Agnati and Fuxe in 1980/81 suggesting the existence of heteromerization of receptors. In 1982, they proposed the existence of aggregates of multiple receptors in the plasma membrane and coined the term receptor mosaics (RM). In this way, cell signaling becomes a branched process beginning at the level of receptor recognition at the plasma membrane where receptors can directly modify the ligand recognition and signaling capacity of the receptors within a RM. Receptorreceptor interactions in RM are classified as operating either with classical cooperativity, when consisting of homomers or heteromers of similar receptor subtypes having the same transmitter, or non-classical cooperativity, when consisting of heteromers. It has been shown that information processing within a RM depends not only on its receptor composition, but also on the topology and the order of receptor activation determined by the concentrations of the ligands and the receptor properties. The general function of RM has also been demonstrated to depend on allosteric regulators (e.g., homocysteine) of the receptor subtypes present. RM as integrative nodes for receptor-receptor interactions in conjunction with membrane associated proteins may form horizontal molecular networks in the plasma membrane coordinating the activity of multiple effector systems modulating the excitability and gene expression of the cells. The key role of electrostatic epitope-epitope interactions will be discussed for the formation of the RM. These interactions probably represent a general molecular mechanism for receptor-receptor interactions and, without a doubt, indicate a role for phosphorylation-dephosphorylation events in these interactions. The novel therapeutic aspects given by the RMs will be discussed in the frame of molecular neurology and psychiatry and combined drug therapy appears as the future way to go.

Selectivity of Oxomemazine for the $M_1$ Muscarinic Receptors

Lee, Shin-Woong,Woo, Chang-Woo,Kim, Jeung-Gu The Pharmaceutical Society of Korea 1994 Archives of Pharmacal Research Vol.17 No.6

The binding characteristics of pirenzepine and oxomemazine to muscarinic receptor were studied to evaluate the selectivity of oxomemazine for the muscarinic receptor subtypes in rat cerebral microsomes. Equililbrium dissociation constant $(K_D){\;}of{\;}(-)[^3H]$quinuclidinyl benzilate$([^3H)QNB)$ determined from saturation isotherms was 64-pM. Analysis of the pirenzepine inghibition curve of [$^3H$]QNB binding to cerebral microsome indicatd the presence of two receptor subtypes with high $(K_1 = 16 nM, M_1 receptor)$two receptor subypes with about 20-fold difference in the affinity for high $(k_1 = 84nM, {\;} O_H receptor){\;} and {\;}low{\;} (K_1{\;} ={\;} 1.65\muM, {\;} O_L receptor$) affinity sites. The percentage populations of $M_1{\;} and M_3$, receptors to the total receptors were 61 : 39, and those of $O_H{\;} and{\;} O_L$ receptors 39 : 61, resepectively. Both pirenzepine and oxomemazine increaed the $K_D$ value for $[^3H]QNB$ without affecting the binding site concentrations and Hii coefficient for the $[^3H]QNB$ without affecting the binding site concentractions and Hill coefficient for the [$^{3}$H]QNB binding. Oxomemazine had a 10-fold higher affinity at $M_1$ receptors than at $M_3$ receptors, and pirenzepine a 8-fold higher affinity at $O_H$ receptors were of $O_H$ receptors and 71% of $M_3$ receptors. However, $M_3$for oxomemazine and $O_H$for pirenzepine were composed of a uniform population. These results suggest that oxomemazine could be classified as a selective drug for $M_1$ receptors and also demonstrate that rat cerebral microsomes contain three different subtypes of $M_1{\;} M_3$ and the other site which is different from $M_1, {\;} M_2$, receptors.

Noonan syndrome-associated SHP2 mutation differentially modulates the expression of postsynaptic receptors according to developmental maturation

Oh, Jun-Young,Rhee, Sangmyung,Silva, Alcino J.,Lee, Yong-Seok,Kim, Hyong Kyu Elsevier 2017 Neuroscience Letters Vol.649 No.-

<P><B>Abstract</B></P> <P>Glutamate is the major excitatory neurotransmitter in the central nervous system, and related signaling involves both AMPA and NMDA subtype receptors. The expression of glutamate receptors is dynamically regulated during development. Recent studies showed that the dysregulation of glutamate receptor expression and function is associated with neurodevelopmental disorders including intellectual disability. Previously, a Noonan syndrome (NS)-associated SHP2 mutation (SHP2<SUP>D61G</SUP>) was shown to increase the synaptic delivery of AMPA receptor, subsequently impairing synaptic plasticity and learning in adult mice. However, how the mutant SHP2 affects glutamate receptor expression during development is not known. Here, we found that the SHP2<SUP>D61G</SUP> differentially regulates the expression of AMPA and NMDA receptors depending on the stage of neuronal maturation. In cultured neurons (immature stage; DIV 6), overexpression of SHP2<SUP>D61G</SUP> significantly increased the average size and the number of NMDA receptor-containing particles, but not those with AMPA receptors. In early matured neurons (DIV 12), SHP2<SUP>D61G</SUP> significantly increased only the average size of AMPA receptor particles, and subsequently increased their number in matured neurons (DIV 18). Importantly, all the changes described above for SHP2<SUP>D61G</SUP> neurons were reversed by inhibiting MAPK. These data demonstrate that the increased activation of MAPK signaling pathway by SHP2<SUP>D61G</SUP> could deregulate the surface expression of synaptic receptors during neuronal development, which likely contributes to cognitive impairments in NS patients.</P> <P><B>Highlights</B></P> <P> <UL> <LI> A Noonan syndrome-associated mutation in SHP2 increases NMDA receptor expression in premature neurons. </LI> <LI> This mutation increases the size of AMPA receptor clusters in early maturing neurons. </LI> <LI> The SHP2 mutation also increases both the size and the number of AMPA receptor clusters in mature neurons. </LI> <LI> The altered expressions of glutamate receptors by mutant SHP2 can be reversed by inhibiting the MAPK signaling pathway. </LI> </UL> </P>