Plasma Membrane as the Target Site of Cholic Acid Analogs

Tian Hong Zhang,Zhen Qing Zhang,Chun Guang Liu,Jin Xiu Ruan 대한약학회 2011 Archives of Pharmacal Research Vol.34 No.7

Although the mechanism is unknown, Calculus Bovis and its active components, cholic acid analogs (CAAs), have been used in China to treat a wide range of diseases. Based on the previous finding that the potency of CAA is strongly dependent on the intrinsic surface activity, this paper aimed to investigate the role of the plasma membrane in the pharmacological activity of CAAs. First, CAAs (0.1 mM) caused a surface activity-dependent depression on ATPase activity in the cell membrane extract, but it had no effects on other cellular extracts, suggesting an indispensable role of the membrane environment for pharmacological activity. Second, CAAs lowered the membrane fluidity of cultured Caco-2 cells with the same rank-order of potency sequence. Third, the hypothesis that any functional protein located on the membrane is influenced by changes in cellular membrane fluidity was supported by: ileal contraction that was induced by acetylcholine and mediated by the muscarinic receptor (M-receptor) or the relaxation induced by adrenaline and mediated by the β-adrenergic receptor (β-receptor) was inhibited by CAAs. They also had similar rank-order of potency and the effects on the plasma membrane. Collectively, the plasma membrane may be a target for the CAAs to exert the multiple pharmacological effects which are mediated by the alteration of the membrane mobility and the function of integral membrane proteins.

Heterogeneity in the Molecular Composition of Excitatory Postsynaptic Sites during Development of Hippocampal Neurons in Culture

Rao, Anuradha,Kim, Eunjoon,Sheng, Morgan,Craig, Ann Marie 부산대학교 유전공학연구소 1998 분자생물학 연구보 Vol.14 No.-

To dertermine their roles in the assemly of glutamatergic postsynaptic sites, we studied the distributions of NMDA- and AMPA-type glutamate receptors; the NMDA receptorinteracting proteins α-actinin-2, PSD-95, and chapsyn;and the PSD-95-associated protein GKAP during the development of hippocampal nerons in culture. NMDA receptors first formed nonsynaptic proximal dendrite shaft clusters within 2-5 d. AMPA receptors were diffuse at this stage anf began to cluster on spines at 9-10 d. NMDA receptor clusters remained partially nonsynaptic and mainly distinct from AMPA receptor clusters until after 3 weeks in culture, when the two began to colocalize at spiny synatic sites. thus, the localization of NMDA and AMPA receptors must be regulated by different mechanisms. α-Actinin-2 colocalize with the NMDA receptor only at spiny synaptic clusters, but not at shrft nonsynaptic or synaptic clusters, suggesting a modulatory role in the anchoring of NMDA receptor at spines. PSD-95, chapsyn, and GKAP were present at some, but not all, nonsynaptic NMDA receptor clusters during the first 2 weeks, indicating that none is essential for NMDA receptor cluster formation. When NMDA receptor clusters becames synaptic, PSD-95 and GKAP were always present, consistent with an essential function in synaptic localization of NMDA receptors. Furthermore, PSD-95 and GKAP clustered opposite presynaptic terminals serveral days before either NMDA or AMPA receptors clustered at these presumptive postsynaptic sites. These results suggest that synapse development proceeds by formation of a postsynaptic scaffold containing PSD-95 and GKAP in concert with presynaptic vesicle clustering, followed by regulated attachment of glutamate receptor subtypes to this scaffold.

P2X7 Receptor-mediated Membrane Blebbing in Salivary Epithelial Cells

황성민,구나연,최세영,Gae-Sig Chun,김중수,박경표 대한약리학회 2009 The Korean Journal of Physiology & Pharmacology Vol.13 No.3

High concentrations of ATP induce membrane blebbing. However, the underlying mechanism involved in epithelial cells remains unclear. In this study, we investigated the role of the P2X7 receptor (P2X7R) in membrane blebbing using Par C5 cells. We stimulated the cells with 5 mM of ATP for 1∼2 hrs and found the characteristics of membrane blebbing, a hallmark of apoptotic cell death. In addition, 500μM Bz-ATP, a specific P2X7R agonist, induced membrane blebbing. However, 300μM of Ox-ATP, a P2X7R antagonist, inhibited ATP-induced membrane blebbing, suggesting that ATP- induced membrane blebbing is mediated by P2X7R. We found that ATP-induced membrane blebbing was mediated by ROCK I activation and MLC phosphorylation, but not by caspase-3. Five mM of ATP evoked a biphasic [Ca2+]i response; a transient [Ca2+]i peak and sustained [Ca2+]i increase secondary to ATP-stimulated Ca2+ influx. These results suggest that P2X7R plays a role in membrane blebbing of the salivary gland epithelial cells.

P2X7 Receptor-mediated Membrane Blebbing in Salivary Epithelial Cells

Hwang, Sung-Min,Koo, Na-Youn,Choi, Se-Young,Chun, Gae-Sig,Kim, Joong-Soo,Park, Kyung-Pyo The Korean Society of Pharmacology 2009 The Korean Journal of Physiology & Pharmacology Vol.13 No.3

High concentrations of ATP induce membrane blebbing. However, the underlying mechanism involved in epithelial cells remains unclear. In this study, we investigated the role of the P2X7 receptor (P2X7R) in membrane blebbing using Par C5 cells. We stimulated the cells with 5 mM of ATP for 1${\sim}$2 hrs and found the characteristics of membrane blebbing, a hallmark of apoptotic cell death. In addition, 500 ${\mu}M$ Bz-ATP, a specific P2X7R agonist, induced membrane blebbing. However, 300 ${\mu}M$ of Ox-ATP, a P2X7R antagonist, inhibited ATP-induced membrane blebbing, suggesting that ATP-induced membrane blebbing is mediated by P2X7R. We found that ATP-induced membrane blebbing was mediated by ROCK I activation and MLC phosphorylation, but not by caspase-3. Five mM of ATP evoked a biphasic $[Ca^{2+}]_i$ response; a transient $[Ca^{2+}]_i$ peak and sustained $[Ca^{2+}]_i$ increase secondary to ATP-stimulated $Ca^{2+}$ influx. These results suggest that P2X7R plays a role in membrane blebbing of the salivary gland epithelial cells.

고혈압쥐에서 성장에 따른 신장 세포막 Na, K-ATPase와 Adrenergic Receptor의 변동에 관한 연구

박성규,이재흔 충남대학교 의과대학 지역사회의학연구소 1993 충남의대잡지 Vol.20 No.2

To investigate the developmental changes of Na, K-ATPase and adrenrgic receptors in the kidney basolateral membrane(BLM) of hypertensive rat, Na, K-ATPase activities, [^3H]ouabain, [^3H]prazosin and [^3H]dihydroalprenolol(DHA) binding sites were measured in the kideny basolateral membrane prepared from 1, 3, 5, 10 and 15 weeks old Sprague-Dawley rats(SD) and spontaneously hypertensive rats(SHR). Results are as follows ; 1. Systolic blood pressure in SHR was not elevated at 3 and 5 weeks old, but elevated significantly to 185 mmHg at 10 or 15 weeks old. 2. Na, K-ATPase activity of BLM in 3 weeks old SD was markedly increased compared to that in 1 week old and there was little change in 5, 10 or 15 weeks old rat. 3. Na, K-ATPase activity of BLM in SHR was also markedly increased at 3 weeks old, but continuously increased by 15 weeks old. Although the Na, K-ATPase activity in BLM of 1, 3 or 5 weeks old SHR was lesser, that of 10 or 15 weeks old SHR was higher than that observed in corresponding age of SD. 4. The numbers of ouabain binding sites were increased in both groups with increasing age. In the SHR, the numbers of ouabain binding; sites were lower in 1, 3 or 5 weeks old but higher in 10 or 15 weeks old, than those in corresponding age of SD. 5. In the dose-response curves of ouabain sensitive Na,K-ATPase activity and the ouabain binding experiments, Na, K-ATPase in the BLM of SD or SHR was consistent with the putative existence of low affinity site(IC_50 : 35 - 55 μM, Ed : 40 - 90 μM) 6. α1-Adrenergic receptor densities([^3H] prazosin binding sites) were markedly increased by 3 weeks after birth and further increased slightly by 5, 10 or 15 weeks old SD. 7. The numbers of prazosin binding sites in SHR were also increased with increase of age. However those were lower in 1, 3 or 5 weeks old, higher in 10 or 15 weeks old SHR than those in corresponding age of SD. 8. In the prazosin binding experiments, Ed value was 0.1 - 0.4 nM and there was no difference between in Kd values of SD and SHR. 9. β-Adrenergic receptor densities([^3H] DHA binding sites) in SD were markedly increased in 3 weeks old compared to those in 1 week old and there were no further changes of receptor densities in 5, 10 or 15 weeks old. But those in SHR were still increased until 15 weeks old. The numbers of DHA binding sites in 1, 3 or 5 weeks old SHR were slightly lower but almost same in 10 or 15 weeks old, compared to those in corresponding age of SD. 10. In the DHA binding experiments, Kd value was 1.0 - 1.8 nM and there was no difference between in Kd values of SD and SHR. From the above results, it is suggested that as increase of age, Na, K-ATPase, α1 and β-adrenergic receptors in the kidney basolateral membrane of SHR were increased with different pattern from normotensive SD ; lower in young age, but same or higher in hypertensive period of SHR than SD. These observed changes in Na,K-ATPase activity and adrenergic receptor densities of kindney basolateral membrane may partly contribute to the regulation of blood pressure in SHR.

Effects of ${\alpha}_1-Adrenergic$ Receptor Stimulation on Intracellular $Na^+$ Activity and Twitch Force in Guinea-Pig Ventricular Muscles

Chae, Soo-Wan,Gong, Q.Y.,Wang, D.Y.,Lee, Chin-O. The Korean Physiological Society 1995 대한생리학회지 Vol.29 No.2

The effects of ${\alpha}_1-adrenergic$ receptor stimulation on membrane potential, intracellular $Na^+$ activity, and twitch force were investigated in ventricular muscles from guinea-pig hearts. Action potentials, intracellular $Na^+$ activity, and twitch force of ventricular papillary muscles were measured simultaneously under various experimental conditions. Stimulation of the ${\alpha}_1-adrenergic$ receptor by phenylephrine produced variable changes in action potential duration, a slight hyperpolarization of the diastolic membrane potential, a decrease in intracellular $Na^+$ activity, and a biphasic inotropic response in which a transient negative inotropic response was followed by a sustained positive inotropic response. These changes were blocked by prazosin, an antagonist of the ${\alpha}_1-adrenergic$ receptor, but not by atenolol, an antagonist of the ${\beta}-adrenergic$ receptor. This indicates that the changes in membrane potential, intracellular $Na^+$ activity, and twitch force are mediated by stimulation of the ${\alpha}_1-adrenergic$ receptor, but not by stimulation of ${\beta}-adrenergic$ receptor. The decrease in intracellular $Na^+$ activity was not observed in quiescent muscles, depending on the rate of the action pontentials in beating muscles. The intracellular $Na^+$ activity decrease was substantially inhibited by tetrodotoxin. However, the decrease in intracellular $Na^+$ activity was not affected by an inhibition of the $Na^+-K^+$ pump. Therefore, the decrease in intracellular $Na^+$ activity mediated by the ${\alpha}_1-adrenergic$ receptor appears to be due to a reduction of $Na^+$ influx during the action potential, perhaps through tetrodotoxin sensitive $Na^+$ channels. Our study also revealed that the decrease in intracellular $Na^+$ activity might be related to the transient negative inotropic response. The intracellular $Na^+$ activity decrease could lower intracellular $Ca^{2+}$ through the $Na^+-Ca^{2+}$ exchanger and thereby produce a decline in twitch force.

Membranous Insulin-like Growth Factor-1 Receptor (IGF1R) Expression Is Predictive of Poor Prognosis in Patients with Epidermal Growth Factor Receptor (EGFR)-Mutant Lung Adenocarcinoma

박은향,정진행,박수영,김효진,Pingli Sun,Yan Jin,조석기,김관민,이춘택 대한병리학회 2015 Journal of Pathology and Translational Medicine Vol.49 No.5

Background: Insulin-like growth factor-1 receptor (IGF1R) is a membrane receptor-type tyrosine kinase that has attracted considerable attention as a potential therapeutic target, although its clinical significance in non-small cell lung cancer (NSCLC) is controversial. This study aimed to clarify the clinical significance of IGF1R expression in human NSCLC. Methods: IGF1R protein expression was evaluated using immunohistochemistry in 372 patients with NSCLC who underwent curative surgical resection (146 squamous cell carcinomas [SqCCs] and 226 adenocarcinomas [ADCs]). We then analyzed correlations between expression of IGF1R and clinicopathological and molecular features and prognostic significance. Results: Membranous and cytoplasmic IGF1R expression were significantly higher in SqCCs than in ADCs. In patients with SqCC, membranous IGF1R expression was associated with absence of vascular, lymphatic, and perineural invasion; lower stage; and better progression-free survival (PFS) (hazard ratio [HR], 0.586; p = .040). In patients with ADC, IGF1R expression did not have a significant prognostic value; however, in the subgroup of epidermal growth factor receptor (EGFR)-mutant ADC, membranous IGF1R expression was associated with lymphatic and perineural invasion, solid predominant histology, and higher cancer stage and was significantly associated with worse PFS (HR, 2.582; p = .009). Conclusions: Lung ADC and SqCC showed distinct IGF1R expression profiles that demonstrated prognostic significance. High membranous IGF1R expression was predictive of poor PFS in EGFR-mutant lung ADC, while it was predictive of better PFS in SqCC. These findings will help improve study design for subsequent investigations and select patients for future anti-IGF1R therapy.

Membranous Insulin-like Growth Factor-1 Receptor (IGF1R) Expression Is Predictive of Poor Prognosis in Patients with Epidermal Growth Factor Receptor ( <i>EGFR</i> )-Mutant Lung Adenocarcinoma

Park, Eunhyang,Park, Soo Young,Kim, Hyojin,Sun, Ping-Li,Jin, Yan,Cho, Suk Ki,Kim, Kwhanmien,Lee, Choon-Taek,Chung, Jin-Haeng The Korean Society of Pathologists and the Korean 2015 Journal of Pathology and Translational Medicine Vol.49 No.5

<P><B>Background:</B></P><P>Insulin-like growth factor-1 receptor (IGF1R) is a membrane receptor-type tyrosine kinase that has attracted considerable attention as a potential therapeutic target, although its clinical significance in non-small cell lung cancer (NSCLC) is controversial. This study aimed to clarify the clinical significance of IGF1R expression in human NSCLC.</P><P><B>Methods:</B></P><P>IGF1R protein expression was evaluated using immunohistochemistry in 372 patients with NSCLC who underwent curative surgical resection (146 squamous cell carcinomas [SqCCs] and 226 adenocarcinomas [ADCs]). We then analyzed correlations between expression of IGF1R and clinicopathological and molecular features and prognostic significance.</P><P><B>Results:</B></P><P>Membranous and cytoplasmic IGF1R expression were significantly higher in SqCCs than in ADCs. In patients with SqCC, membranous IGF1R expression was associated with absence of vascular, lymphatic, and perineural invasion; lower stage; and better progression-free survival (PFS) (hazard ratio [HR], 0.586; p = .040). In patients with ADC, IGF1R expression did not have a significant prognostic value; however, in the subgroup of epidermal growth factor receptor (<I>EGFR</I>)-mutant ADC, membranous IGF1R expression was associated with lymphatic and perineural invasion, solid predominant histology, and higher cancer stage and was significantly associated with worse PFS (HR, 2.582; p = .009).</P><P><B>Conclusions:</B></P><P>Lung ADC and SqCC showed distinct IGF1R expression profiles that demonstrated prognostic significance. High membranous IGF1R expression was predictive of poor PFS in <I>EGFR</I>-mutant lung ADC, while it was predictive of better PFS in SqCC. These findings will help improve study design for subsequent investigations and select patients for future anti-IGF1R therapy.</P>

Identification of a gene encoding a membrane-anchored toll-like receptor 5 (TLR5M) in <i>Oplegnathus fasciatus</i> that responds to flagellin challenge and activates NF-κB

Umasuthan, Navaneethaiyer,Bathige, S.D.N.K.,Thulasitha, William Shanthakumar,Jayasooriya, R.G.P.T.,Shin, Younhee,Lee, Jehee Elsevier 2017 Fish & shellfish immunology Vol.62 No.-

<P><B>Abstract</B></P> <P>Toll-like receptor 5 (TLR5) recognizes bacterial flagellin and induces the downstream signaling through the myeloid differentiation primary response gene 88 (MyD88) protein to produce proinflammatory cytokines. In this study, we describe a TLR5 membrane form (OfTLR5M) and its adaptor protein MyD88 (OfMyD88) in rock bream, <I>Oplegnathus fasciatus.</I> Both <I>Oftlr5m</I> (6.7 kb) and <I>Ofmyd88</I> (3.7 kb) genes displayed a quinquepartite structure with five exons and four introns. Protein structure of OfTLR5M revealed the conventional architecture of TLRs featured by an extracellular domain with 22 leucine rich repeats (LRR), a transmembrane domain and an endodomain with TIR motif. Primary OfTLR5M sequence shared a higher homology with teleost TLR5M. The evolutional analysis confirmed that TLR5 identified in the current study is a membrane receptor and the data further suggested the co-evolution of the membrane-anchored and soluble forms of TLR5 in teleosts. Inter-lineage comparison of gene structures in vertebrates indicated that the <I>tlr5m</I> gene has evolved with extensive rearrangement; whereas, the <I>myd88</I> gene has maintained a stable structure throughout the evolution. Inspection of 5′ flanking region of these genes disclosed the presence of several transcription factor binding sites including NF-κB. Quantitative real-time PCR (qPCR) detected <I>Oftlr5m</I> mRNA in eleven tissues with the highest abundance in liver. <I>In vivo</I> flagellin administration strongly induced the transcripts of both <I>Oftlr5m</I> and <I>Ofmyd88</I> in gills and head kidney tissues suggesting their ligand-mediated upregulation. In a luciferase assay, HEK293T cells transiently transfected with <I>Oftlr5m</I> and <I>Ofmyd88</I> demonstrated a higher NF-κB activity than the mock control, and the luciferase activity was intensified when cells were stimulated with flagellin. Collectively, our study represents the genomic, evolutional, expressional and functional insights into a receptor and adaptor molecules of teleost origin that are involved in flagellin sensing.</P> <P><B>Highlights</B></P> <P> <UL> <LI> <I>Oftlr5m</I> and <I>Ofmyd88</I> genes in rock bream display quinquepartite structure. </LI> <LI> While gene structure of <I>tlr5m</I> is evolved with rearrangements, <I>myd88</I> is preserved. </LI> <LI> <I>Oftlr5m</I> and <I>Ofmyd88</I> showed similar tissue mRNA profile with highest level in liver. </LI> <LI> <I>Oftlr5m</I> and <I>Ofmyd88</I> were induced by ultrapure flagellin in gill and head kidney. </LI> <LI> They individually and synergetically activated NF-κB upon flagellin-stimulation. </LI> </UL> </P>

Evolutionary Signature of Information Transfer Complexity in Cellular Membrane Proteomes

김종민,김병기,오석준 한국유전체학회 2009 Genomics & informatics Vol.7 No.2

Cell membrane proteins play crucial roles in the cell's molecular interaction with its environment and within itself. They consist of membrane-bound proteins and many types of transmembrane (TM) proteins such as receptors, transporters, channel proteins, and enzymes. Membrane proteomes of cellular organisms reveal some characteristics in their global topological distribution according to their evolutionary positions, and show their own information transfer complexity. Predicted transmembrane segments (TMSs) in membrane proteomes with HMMTOP showed near power-law distribution and frequency characteristics in 6-TMS and 7-TMS proteins in prokaryotes and eukaryotes, respectively. This reaffirms the important roles of membrane receptors in cellular communication and biological evolutionary history.