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Molecular Mechanisms That Underlie The [Cl-]I-Sensitive Kinases-Mediated Regulation of Cftr Anion Selectivity
Poster Presentations (8.1%),diarrhea caused by clostridium difficile: 3 (1.4%) and four cases of medication error. Conclusions: The incidence of admission by adverse reactions is similar to other studies performed in other countries, around 5.7%, being the most frequent gastrointestinal bleeding associated with antiplatelet drugs or oral anticoagulants.
Molecular Mechanisms That Underlie The [Cl-]I-Sensitive Kinases-Mediated Regulation of Cftr Anion Selectivity I. Jun1; J. Jang2; J. Jung1; Y. Kim1; and M.G. Lee1 Department of Pharmacology and Brain Korea 21 PLUS Project for Medical Sciences, Yonsei University College of Medicine, Seoul 120-752, Korea; and 2Department of Hematology, Yonsei University College of Medicine, Seoul 120-752, Korea Introduction: CFTR is cAMP activated anion channel which secretes chloride and bicarbonate in airway, exocrine pancreas, intestine, and genitourinary systems. Human pancreas secretes pancreatic juice which contains as much as 140 mM bicarbonate (HCO3-) via CFTR. Recently, we have shown that [Cl-]i-sensitive activation of WNK1OSR1/SPAK pathway plays a critical role in pancreatic HCO3secretion by increasing the bicarbonate permeability (PHCO3/PCl) of CFTR. However, how [Cl-]i-sensitive kinases modulate PHCO3/ PCl of CFTR remains elusive. Material and Methods: Overexpression and knockdown of each kinase in HEK293T cells were performed. Using patch clamp, we measured permeability of bicarbonate and halide ions. We did pulldown assay between truncated WNK1 and CFTR at 150mM and 0mM Cl- concentrations. Results: WNK1 affects permeability of other anions as well as bicarbonate in patch clamp recordings. Especially, the interval of relative permeabilities (Px/PCl) between each anion was greatly narrowed by WNK1. Consequently, WNK1 increased the dielectric constant of the hypothetical selectivity filter of CFTR. And we measured the pore size of CFTR at each state. WNK1 makes CFTR pore size larger. We figured out that WNK1 kinase domain binds to CFTR and truncated forms of WNK1 respond to low Cl- concentrations. Conclusions: These findings suggest that WNK1 increases the bicarbonate permeability of CFTR by modulating the polarizability of anion selectivity filter and provide insight into the fundamental question of how ion selectivity of anion channels can be regulated by cytosolic signaling at the molecular level. Furthermore, WNK1 senses low [Cl-]i, then kinase domain is exposed and binds to CFTR. 1
Tmem16f (Ano6) Activation Is Accelerated By Selective Serotonin Reuptake Inhibitors I. Jun1; J. Jang2; J. Jung1; M.G. Lee1; and J.H. Nam3 1 Department of Pharmacology and Brain Korea 21 PLUS Project for Medical Sciences, Yonsei University College of Medicine, Seoul 120-752, Korea; 2Department of Hematology, Yonsei University College of Medicine, Seoul 120-752, Korea; and 3Department of Physiology, Dongguk University College of Medicine Introduction: ANO6 acts as Ca2+-activated Cl− channels and generates outward-rectifying ionic currents in response to intracellular Ca2+ increase. ANO6 is involved in platelet function by phospholipid scrambling required for blood coagulation. Selective serotonin reuptake inhibitors (SSRIs) are used for the treatment of major depressive disorders. Although chronic treatment of SSRI can increase the risk of upper gastrointestinal bleeding, at
August 2015
the earlier stage of intake, which is 1–7 days after the treatment, the possibility of blood coagulation might also increase, but transiently. Therefore, in this study, we investigated whether SSRI affected the Cl− current or phospholipid scrambling activity of ANO6. Material and Methods: HEK293T and PANC-1 cells were used. Plasmids expressing hANO6 were transfected to HEK293T cells. siRNA of hANO6 was transfected to PANC1 cells for knockdown of hANO6. We measured anion channel activities using whole cell and inside-out patch clamp techniques with and without treatment of SSRI. Flow cytometry was performed to evaluate phospholipid scrambling. Results: In the whole-cell patch mode, SSRIs facilitated Ca2+dependent activation of IANO6 in ANO6-transfected cells, as evidenced by a significant decrease in the delay of IANO6 generation. On the other hand, in the inside-out patch clamp configuration, SSRIs showed an inhibitory effect on ANO6 currents, suggesting that SSRIs activate ANO6 via an indirect mechanism in intact cells. SSRIs also facilitated Ca2+-dependent PS exposure and α -thrombin-induced platelet aggregation. Conclusions: SSRIs at clinically relevant concentrations promote Ca2+- dependent activation of ANO6, which may have potential clinical implications such as the underlying mechanism of SSRI induced adverse drug reactions.
A Review of New Drugs Approved By The Spanish Agency of Medicines and Medical Devices (Aemps): A Quick Method To Explore Therapeutic Innovation A. Pejenaute1; N. Ramos1; J.A. Arnaiz2,3; D. García-Cinca1; and G. Calvo2,3 1 Fundació Clínic Recerca Biomèdica, Barcelona, Spain; 2Hospital Clínic i Provincial, Barcelona, Spain; and 3Universitat de Barcelona, Barcelona, Spain Introduction: It has been questioned whether drug innovation meets societal needs. We undertook a critical review of new approved drugs by the AEMPS from September 2013 to December 2014. Methods: A revision of the monthly bulletin published at the AEMPS website was performed to gather information on new drugs approved from September 2013 to December 2014. Key features of new drugs approved were summarized. The following indicators of innovation were considered: Direct indicators: a) drugs with new mechanism of action and b) lack of therapeutic alternatives for the approved indication. Indirect indicators: a) advanced therapy; b) orphan designation; c) first line vs. second or further line therapies; d) pediatric indication. New indications and extension of indications were not considered. Results: A total of 82 drugs were approved. Indications fell into the following areas: (16, 19%) infectious diseases, (14, 17%) oncology, (10, 12%) endocrinology, (6, 7%) neurology and haematology each one, (5, 6%) pneumology, (4, 5%) cardiovascular diseases, (3, 4%) psychiatry, dermatology, digestive system, ophthalmology, gynecology and immunology each one, 1% (n = 1) nephrology, metabolism and urology each one. Most were single-active substance drugs (67, 82%). Fixe-dosed combinations were mainly antiretrovirals (n = 4) and hypoglycemic agents (n = 3). Only 2 drugs targeted specifically pediatric indications and 13 were addressed to both adult and pediatric population. Overall, 22 (27%) were orphan drugs. First-line agents accounted for 70% of the total (16% in restricted patient populations). Conclusion: A detailed analysis of newly approved drugs may be used to conduct an at first glance analysis of therapeutic innovation.
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Molecular Mechanisms That Underlie The [Cl-]I-Sensitive Kinases-Mediated Regulation of Cftr Anion Selectivity I. Jun1; J. Jang2; J. Jung1; Y. Kim1; and M.G. Lee1 Department of Pharmacology and Brain Korea 21 PLUS Project for Medical Sciences, Yonsei University College of Medicine, Seoul 120-752, Korea; and 2Department of Hematology, Yonsei University College of Medicine, Seoul 120-752, Korea Introduction: CFTR is cAMP activated anion channel which secretes chloride and bicarbonate in airway, exocrine pancreas, intestine, and genitourinary systems. Human pancreas secretes pancreatic juice which contains as much as 140 mM bicarbonate (HCO3-) via CFTR. Recently, we have shown that [Cl-]i-sensitive activation of WNK1OSR1/SPAK pathway plays a critical role in pancreatic HCO3secretion by increasing the bicarbonate permeability (PHCO3/PCl) of CFTR. However, how [Cl-]i-sensitive kinases modulate PHCO3/ PCl of CFTR remains elusive. Material and Methods: Overexpression and knockdown of each kinase in HEK293T cells were performed. Using patch clamp, we measured permeability of bicarbonate and halide ions. We did pulldown assay between truncated WNK1 and CFTR at 150mM and 0mM Cl- concentrations. Results: WNK1 affects permeability of other anions as well as bicarbonate in patch clamp recordings. Especially, the interval of relative permeabilities (Px/PCl) between each anion was greatly narrowed by WNK1. Consequently, WNK1 increased the dielectric constant of the hypothetical selectivity filter of CFTR. And we measured the pore size of CFTR at each state. WNK1 makes CFTR pore size larger. We figured out that WNK1 kinase domain binds to CFTR and truncated forms of WNK1 respond to low Cl- concentrations. Conclusions: These findings suggest that WNK1 increases the bicarbonate permeability of CFTR by modulating the polarizability of anion selectivity filter and provide insight into the fundamental question of how ion selectivity of anion channels can be regulated by cytosolic signaling at the molecular level. Furthermore, WNK1 senses low [Cl-]i, then kinase domain is exposed and binds to CFTR. 1
Tmem16f (Ano6) Activation Is Accelerated By Selective Serotonin Reuptake Inhibitors I. Jun1; J. Jang2; J. Jung1; M.G. Lee1; and J.H. Nam3 1 Department of Pharmacology and Brain Korea 21 PLUS Project for Medical Sciences, Yonsei University College of Medicine, Seoul 120-752, Korea; 2Department of Hematology, Yonsei University College of Medicine, Seoul 120-752, Korea; and 3Department of Physiology, Dongguk University College of Medicine Introduction: ANO6 acts as Ca2+-activated Cl− channels and generates outward-rectifying ionic currents in response to intracellular Ca2+ increase. ANO6 is involved in platelet function by phospholipid scrambling required for blood coagulation. Selective serotonin reuptake inhibitors (SSRIs) are used for the treatment of major depressive disorders. Although chronic treatment of SSRI can increase the risk of upper gastrointestinal bleeding, at
August 2015
the earlier stage of intake, which is 1–7 days after the treatment, the possibility of blood coagulation might also increase, but transiently. Therefore, in this study, we investigated whether SSRI affected the Cl− current or phospholipid scrambling activity of ANO6. Material and Methods: HEK293T and PANC-1 cells were used. Plasmids expressing hANO6 were transfected to HEK293T cells. siRNA of hANO6 was transfected to PANC1 cells for knockdown of hANO6. We measured anion channel activities using whole cell and inside-out patch clamp techniques with and without treatment of SSRI. Flow cytometry was performed to evaluate phospholipid scrambling. Results: In the whole-cell patch mode, SSRIs facilitated Ca2+dependent activation of IANO6 in ANO6-transfected cells, as evidenced by a significant decrease in the delay of IANO6 generation. On the other hand, in the inside-out patch clamp configuration, SSRIs showed an inhibitory effect on ANO6 currents, suggesting that SSRIs activate ANO6 via an indirect mechanism in intact cells. SSRIs also facilitated Ca2+-dependent PS exposure and α -thrombin-induced platelet aggregation. Conclusions: SSRIs at clinically relevant concentrations promote Ca2+- dependent activation of ANO6, which may have potential clinical implications such as the underlying mechanism of SSRI induced adverse drug reactions.
A Review of New Drugs Approved By The Spanish Agency of Medicines and Medical Devices (Aemps): A Quick Method To Explore Therapeutic Innovation A. Pejenaute1; N. Ramos1; J.A. Arnaiz2,3; D. García-Cinca1; and G. Calvo2,3 1 Fundació Clínic Recerca Biomèdica, Barcelona, Spain; 2Hospital Clínic i Provincial, Barcelona, Spain; and 3Universitat de Barcelona, Barcelona, Spain Introduction: It has been questioned whether drug innovation meets societal needs. We undertook a critical review of new approved drugs by the AEMPS from September 2013 to December 2014. Methods: A revision of the monthly bulletin published at the AEMPS website was performed to gather information on new drugs approved from September 2013 to December 2014. Key features of new drugs approved were summarized. The following indicators of innovation were considered: Direct indicators: a) drugs with new mechanism of action and b) lack of therapeutic alternatives for the approved indication. Indirect indicators: a) advanced therapy; b) orphan designation; c) first line vs. second or further line therapies; d) pediatric indication. New indications and extension of indications were not considered. Results: A total of 82 drugs were approved. Indications fell into the following areas: (16, 19%) infectious diseases, (14, 17%) oncology, (10, 12%) endocrinology, (6, 7%) neurology and haematology each one, (5, 6%) pneumology, (4, 5%) cardiovascular diseases, (3, 4%) psychiatry, dermatology, digestive system, ophthalmology, gynecology and immunology each one, 1% (n = 1) nephrology, metabolism and urology each one. Most were single-active substance drugs (67, 82%). Fixe-dosed combinations were mainly antiretrovirals (n = 4) and hypoglycemic agents (n = 3). Only 2 drugs targeted specifically pediatric indications and 13 were addressed to both adult and pediatric population. Overall, 22 (27%) were orphan drugs. First-line agents accounted for 70% of the total (16% in restricted patient populations). Conclusion: A detailed analysis of newly approved drugs may be used to conduct an at first glance analysis of therapeutic innovation.
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