• Proceedings of the Korean Society of Applied Pharmacology
• /
• 1996.04a
• /
• pp.206-206
• /
• 1996

The C-terminus ends of the second putative transmembrane domains of both m1 and m2 muscarinic receptors contain a triplet of amino acid residues consisting of leucine (L), tyrosine (Y) and threonine (T), 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 drug-receptor interaction and cellular function at m1 muscarinic receptor. Mutation of the LYTTYL sequence of m1 receptors to the corresponding m2 receptor LYTLYT sequence, however, did not result in a significant change in the binding affinity of the agonist carbachol or in the affinity of the majority of a series of receptor antagonists which are able to discriminate between wild-type m1 and m2 receptors. Surprisingly, the LYTLYT ml 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+/. These changes were not due to alterations in the rate of receptor. desensitization or sequestration, On the other hand, the reverse LYTLYT-LYTTYL mutation in the m2 receptor did not alter its coupling to inhibition of adenylate cyclase, but slightly enhanced its coupling to stimulation of PI hydrolysis, Our data suggest that the LYTTYL/LYTLYT sequence difference between ml and n12 muscarinic receptors is not involved in determining receptor pharmacology. On the other hand, while these differences might play a role in the modulation of muscarinic receptor coupling to PI hydrolysis, they are not important for specifying coupling of various subtypes of muscarinic receptors to different cellular signaling pathways.

• The Korean Journal of Physiology and Pharmacology
• /
• v.1 no.1
• /
• pp.19-25
• /
• 1997

The C-terminus ends of the second putative transmembrane domains of both $M_1$ and $M_2$ muscarinic receptors contain a triplet of amino acid residues consisting of leucine (L), tyrosine (Y) and threonine (T). This triplet is repeated as LYT-TYL in $M_1$ receptors at the interface between the second transmembrane domain and the first extracellular loop. Interestingly, however, it is repeated in a transposedfashion (LYT-LYT) in the sequence of $M_2$ receptors. In our previous work, we investigated the possible significance of this unique sequence diversity for determining the distinct differential receptor function at the two receptor subtypes. However, we found mutation of the LYTTYL sequence of $M_1$ receptors to the corresponding $M_2$ receptor LYTLYT sequence demonstrated markedly enhanced the stimulation of phosphoinositide (PI) hydrolysis by carbachol without a change in its coupling to increased cyclic AMP formation. In this work, thus, the enhanced stimulation of PI hydrolysis in the LYTLYT $M_1$ receptor mutant was further investigated. The stimulation of PI hydrolysis by carbachol was enhanced in the mutant $M_1$ receptor, and this change was not due to alterations in the rate of receptor desensitization or sequestration. The observed larger response to carbachol at mutant $M_1$ receptors was also not due to an artifact resulting from selection of CHO cells which express higher levels of G-proteins or phospholipase C. Our data suggest that although the LYTTYL sequence in $M_1$ muscarinic receptors is not involved in determining receptor pharmacology, mutation of the sequence enhanced the coupling of $M_1$ receptors to the stimulation of phospholipase C.

• The Korean Journal of Physiology and Pharmacology
• /
• v.5 no.2
• /
• pp.139-146
• /
• 2001

L-type $Ca^{2+}$ channels play an important role in regulating cytosolic $Ca^{2+}$ and thereby regulating hormone secretions in neuroendocrine cells. Since hormone secretions are also regulated by various kinds of protein kinases, we investigated the role of some kinase activators and inhibitors in the regulation of the L-type $Ca^{2+}$ channel currents in rat pituitary $GH_3$ cells using the patch-clamp technique. Phorbol 12,13-dibutyrate (PDBu), a protein kinase C (PKC) activator, and vanadate, a protein tyrosine phosphatase (PTP) inhibitor, increased the $Ba^{2+}$ current through the L-type $Ca^{2+}$ channels. In contrast, bisindolylmaleimide I (BIM I), a PKC inhibitor, and genistein, a protein tyrosine kinase (PTK) inhibitor, suppressed the $Ba^{2+}$ currents. Forskolin, an adenylate cyclase activator, and isobutyl methylxanthine (IBMX), a non-specific phosphodiesterase inhibitor, reduced $Ba^{2+}$ currents. The above results show that the L-type $Ca^{2+}$ channels are activated by PKC and PTK, and inhibited by elevation of cyclic nucleotides such as cAMP. From these results, it is suggested that the regulation of hormone secretion by various kinase activity in $GH_3$ cells may be attributable, at least in part, to their effect on L-type $Ca^{2+}$ channels.

• The Korean Journal of Physiology and Pharmacology
• /
• v.1 no.2
• /
• pp.195-200
• /
• 1997

Water transport in highly-permeable membranes is facilitated by some specialized pathways, which are called aquaporins (AQP). AQP1 (AQP-CHIP) is the first recognized aquaporin identified from red cells and renal proximal tubules. Up until now 4 other aquaporin homologs have been reported. Each aquaporin has its unique tissue distribution and regulatory mechanims. To elucidate molecular mechanisms for their transcription regulation and tissue-specific expression isolation of aquaporin genes is required. To clone promoters of the AQP family mouse genomic library was screened by the 1st exon-specific probe of AQP4, and 5 different plaques were positively hybridized. Phage DNAs were purified and characterized by restriction mapping and sequencing. One of them is the mouse AQP-CD gene. The gene was consisted of 4 exons and the exon-intron boundaries of mouse AQP-CD gene were identified at identical positions in other related genes. The 5'-flanking region of AQP-CD gene contains one classic TATA box, a GATA consensus sequence, an E-box and a cyclic AMP-responsive element. The cloning of the mouse AQP-CD gene, of which product is expressed in the collecting duct and is responsible for antidiuresis by vasopressin, will contribute to understand the molecular mechanisms of tissue-specific expression and regulation of AQP-CD gene under various conditions.

• Journal of Microbiology and Biotechnology
• /
• v.26 no.3
• /
• pp.627-636
• /
• 2016

The causative agent of pandemic cholera, Vibrio cholerae, infects the anaerobic environment of the human intestine. Production of cholera toxin (CT), a major virulence factor of V. cholerae, is highly induced during anaerobic respiration with trimethylamine N-oxide (TMAO) as an alternative electron acceptor. However, the molecular mechanism of TMAO-stimulated CT production is not fully understood. Herein, we reveal that CT production during anaerobic TMAO respiration is affected by glucose fermentation. When the seventh pandemic V. cholerae O1 strain N16961 was grown with TMAO and additional glucose, CT production was markedly reduced. Furthermore, an N16961 Δcrp mutant, devoid of cyclic AMP receptor protein (CRP), was defective in CT production during growth by anaerobic TMAO respiration, further suggesting a role of glucose metabolism in regulating TMAO-mediated CT production. TMAO reductase activity was noticeably decreased when grown together with glucose or by mutation of the crp gene. A CRP binding region was identified in the promoter region of the torD gene, which encodes a structural subunit of the TMAO reductase. Gel shift assays further confirmed the binding of purified CRP to the torD promoter sequence. Together, our results suggest that the bacterial ability to respire using TMAO is controlled by CRP, whose activity is dependent on glucose availability. Our results reveal a novel mechanism for the regulation of major virulence factor production by V. cholerae under anaerobic growth conditions.

• BMB Reports
• /
• v.53 no.3
• /
• pp.142-147
• /
• 2020

Lipid accumulation in white adipose tissue is the key contributor to the obesity and orchestrates numerous metabolic health problems such as type 2 diabetes, hypertension, atherosclerosis, and cancer. Nonetheless, the prevention and treatment of obesity are still inadequate. Recently, scientists found that brown adipose tissue (BAT) in adult humans has functions that are diametrically opposite to those of white adipose tissue and that BAT holds promise for a new strategy to counteract obesity. In this study, we evaluated the potential of sinapic acid (SA) to promote the thermogenic program and lipolysis in BAT. SA treatment of brown adipocytes induced the expression of brown-adipocyte activation-related genes such as Ucp1, Pgc-1α, and Prdm16. Furthermore, structural analysis and western blot revealed that SA upregulates protein kinase A (PKA) phosphorylation with competitive inhibition by a pan-PKA inhibitor, H89. SA binds to the adenosine triphosphate (ATP) site on the PKA catalytic subunit where H89 binds specifically. PKA-cat-α1 gene-silencing experiments confirmed that SA activates the thermogenic program via a mechanism involving PKA and cyclic AMP response element-binding protein (CREB) signaling. Moreover, SA treatment promoted lipolysis via a PKA/p38-mediated pathway. Our findings may allow us to open a new avenue of strategies against obesity and need further investigation.

• The Korea Journal of Herbology
• /
• v.30 no.3
• /
• pp.13-17
• /
• 2015

Objectives : Taraxacum platycarpum H. Dahlstedt has been reported to have several biological properties such as skin hydration and antiinflammation. The purpose of this study was to examine the antidepressive effects of water extract of T. platycarpum (WTP) on an animal model of depression. Methods : In the present study, normal ICR mice (4 weeks) were used, and orally administered with WTP (25, 50 and 100 mg/kg). Depression-like behavior was monitored the forced swimming test (FST) and tail suspension test (TST) in mice. The locomotor activity was evaluated to eliminate the false-positive activity in the open field test (OFT). Fluoxetine, the selective serotonin reuptake inhibitor, as a positive control was intraperitoneally administered at a dose of 15 mg/kg at 30 min before starting the behavioral test. Moreover, we evaluated the effects of WTP on the expression of brain-derived neurotrophic factor (BDNF) and the extracellular signal-regulated kinase (ERK)/ cyclic AMP response-element binding protein (CREB) signaling pathway in the hippocampus using Western blot. Results : The administration of WTP (50 and 100 mg/kg) significantly (P < 0.05, respectively) reduced the immobility time during FST and TST without accompanying changes in locomotor activity by OFT. Furthermore, WTP at dose of 100 mg/kg increased the BDNF expression and the phosphorylation of ERK and CREB in the hippocampus region. Conclusions : These results suggest that WTP has a useful anti-depressant effect through the regulation of BDNF/ERK/CREB signaling pathway.

• Pediatric Gastroenterology, Hepatology & Nutrition
• /
• v.23 no.3
• /
• pp.189-230
• /
• 2020

The metabolic syndrome, by definition, is not a disease but is a clustering of individual metabolic risk factors including abdominal obesity, hyperglycemia, hypertriglyceridemia, hypertension, and low high-density lipoprotein cholesterol levels. These risk factors could dramatically increase the prevalence of type 2 diabetes and cardiovascular disease. The reported prevalence of the metabolic syndrome varies, greatly depending on the definition used, gender, age, socioeconomic status, and the ethnic background of study cohorts. Clinical and epidemiological studies have clearly demonstrated that the metabolic syndrome starts with central obesity. Because the prevalence of obesity has doubly increased worldwide over the past 30 years, the prevalence of the metabolic syndrome has markedly boosted in parallel. Therefore, obesity has been recognized as the leading cause for the metabolic syndrome since it is strongly associated with all metabolic risk factors. High prevalence of the metabolic syndrome is not unique to the USA and Europe and it is also increasing in most Asian countries. Insulin resistance has elucidated most, if not all, of the pathophysiology of the metabolic syndrome because it contributes to hyperglycemia. Furthermore, a major contributor to the development of insulin resistance is an overabundance of circulating fatty acids. Plasma fatty acids are derived mainly from the triglycerides stored in adipose tissues, which are released through the action of the cyclic AMP-dependent enzyme, hormone sensitive lipase. This review summarizes the latest concepts in the definition, pathogenesis, pathophysiology, and diagnosis of the metabolic syndrome, as well as its preventive measures and therapeutic strategies in children and adolescents.

• Molecules and Cells
• /
• v.37 no.8
• /
• pp.613-619
• /
• 2014

The optic nerve often suffers regenerative failure after injury, leading to serious visual impairment such as glaucoma. The main inhibitory factors, including Nogo-A, oligodendrocyte myelin glycoprotein, and myelin-associated glycoprotein, exert their inhibitory effects on axonal growth through the same receptor, the Nogo-66 receptor (NgR). Oncomodulin (OM), a calcium-binding protein with a molecular weight of an ~12 kDa, which is secreted from activated macrophages, has been demonstrated to have high and specific affinity for retinal ganglion cells (RGC) and promote greater axonal regeneration than other known polypeptide growth factors. Protamine has been reported to effectively deliver small interference RNA (siRNA) into cells. Accordingly, a fusion protein of OM and truncated protamine (tp) may be used as a vehicle for the delivery of NgR siRNA into RGC for gene therapy. To test this hypothesis, we constructed OM and tp fusion protein (OM/tp) expression vectors. Using the indirect immunofluorescence labeling method, OM/tp fusion proteins were found to have a high affinity for RGC. The gel shift assay showed that the OM/tp fusion proteins retained the capacity to bind to DNA. Using OM/tp fusion proteins as a delivery tool, the siRNA of NgR was effectively transfected into cells and significantly down-regulated NgR expression levels. More importantly, OM/tp-NgR siRNA dramatically promoted axonal growth of RGC compared with the application of OM/tp recombinant protein or NgR siRNA alone in vitro. In addition, OM/tp-NgR siRNA highly elevated intracellular cyclic adenosine monophosphate (cAMP) levels and inhibited activation of the Ras homolog gene family, member A (RhoA). Taken together, our data demonstrated that the recombinant OM/tp fusion proteins retained the functions of both OM and tp, and that OM/tp-NgR siRNA might potentially be used for the treatment of optic nerve injury.

• Journal of Physiology & Pathology in Korean Medicine
• /
• v.19 no.6
• /
• pp.1563-1567
• /
• 2005

Mori Cortex Radicis is distributed in Northwestern China, northern Asia, northern Europe, North America, and Korea. This extracts drops sugar in bloods and inhibits cyclic AMP phophodiesterase. In this study, we investigated whether Mori Cortex Radicis would cause apoptotic death of A549 lung cancer cells. To examine the apoptotic effect of Mori Cortex Radicis, cytotoxicity assay, DNA fragmentation analysis, caspase-3 activity assay, and Western blotting for caspase-3, caspase-9 and poly(ADP-ribose) polymerase (PARP) and cytochrome c were performed. Treatment of cells with Mori Cortex Radicis was shown to induce cell death in a dose-dependent manner. DNA fragmentation was made in response to Mori Cortex Radicis. The active fragments of caspase-3, caspase-9 and PARP were almost completely induced and cytochrome c was released following exposure to Mori Cortex Radicis. To elucidate the apoptotic mechanisms, RT-PCR and Western blot analyses for the expression of Bcl-2, Bu and Cox-2 were carried out. Treatment with Mori Cortex Radicis was expressed the reduction of Bcl-2 and Cox-2 and the induction of Bax. Especially p21 and p53 were increased prior to untreated control, while cyclin E and cyclin D1 decreased in the cytosol. These results suggest that the effect Mori Cortex Radicis is associated with the cell cycle arrest and pro-apoptotic cell death in A549 lung cancer cells.