Two-pore domain K_1hn+ channel TASK-1 in human pulmonary artery smooth muscle cells and its regulation by the vasoconstrictor endothelin-1 [Elektronische Ressource] / vorgelegt von Tang, Bi

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+Two-pore domain K channel TASK-1 in human pulmonary artery smooth muscle cells and its regulation by the vasoconstrictor endothelin-1 Inauguraldissertation Zur Erlangung des Grades eines Doktors der Medizin des Fachbereichs Medizin der Justus-Liebig Universität Giessen vorgelegt von Tang, Bi MD aus Ling Bi, Anhui province, China Gießen, 2010 Aus dem Zentrum für Innere Medizin, Medizinische Klinik und Poliklinik II, Pneumologie und Intensivmedizin Direktor: Prof. Dr. Werner Seeger Gutachter: Prof. DDr. T. Braun Gutachter: Prof. Dr. A. Olschewski Tag der Disputation: 06. 07. 2010 My wife, Pingping Sun, for her constant interest and her continuous helpfulness My family for the continuous support and encouragement during my doctoral dissertation Table of contents 1. Introduction and question……………………………………………………. ……1 2. Potassium channels in human pulmonary artery smooth muscle cells (PASMCs)………………………………………………………………………………6 3. Background two-pore domain potassium channels (K )……… ….………7 2P4. TASK-1 channels in PASMCs……………………………………………………..10 5. Role of endothelin-1 in pulmonary hypertension (PH) .……………………..12 6. Materials and Methods…………………………………………………………….14 6.

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• Published by: justus-liebig-universitat_giessen
• Published: 01 January 2010
• Reads: 9
• Language: English
• Document size: 1 MB

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+Two-pore domain K channel TASK-1 in human
pulmonary artery smooth muscle cells and its
regulation by the vasoconstrictor endothelin-1





Inauguraldissertation
Zur Erlangung des Grades eines Doktors der Medizin
des Fachbereichs Medizin
der Justus-Liebig Universität Giessen











vorgelegt von Tang, Bi MD
aus Ling Bi, Anhui province, China
Gießen, 2010




























Aus dem Zentrum für Innere Medizin, Medizinische Klinik und Poliklinik II,
Pneumologie und Intensivmedizin
Direktor: Prof. Dr. Werner Seeger














Gutachter: Prof. DDr. T. Braun
Gutachter: Prof. Dr. A. Olschewski


Tag der Disputation: 06. 07. 2010


































My wife, Pingping Sun,
for her constant interest and her continuous helpfulness



My family for the continuous support and
encouragement during my doctoral dissertation





















Table of contents

1. Introduction and question……………………………………………………. ……1
2. Potassium channels in human pulmonary artery smooth muscle cells
(PASMCs)………………………………………………………………………………6
3. Background two-pore domain potassium channels (K )……… ….………7 2P
4. TASK-1 channels in PASMCs……………………………………………………..10
5. Role of endothelin-1 in pulmonary hypertension (PH) .……………………..12
6. Materials and Methods…………………………………………………………….14
6.1 Patch-clamp technique………………………………………………. 14
6.2 Electrophysiology in the present study……………….……………...17
6.3 Preparation of human pulmonary artery smooth muscle cells……19
6.4 Solutions and chemicals………………………………………………21
6.5 Relative mRNA Quantification………………………………………..23
6.6 Design and transfection of siRNA for human TASK-1….………….25
6.7 Immunoprecipitation…………………………………………………...25
6.8 Immunofluorescence staining……………………………….……..…26
6.9 Isolated, perfused and ventilated mouse lungs…..……………..….27
6.10 Statistical analysis………………………………………………….….29
7. Results ……………………………………………………………………….….30
7.1 Characteristics of K channels in hPASMCs………………………30 2P
7.1.1 Expression of TASK-1 channels…………………………….30
7.1.2 The Pharmacological properties of TASK-1 channels…….32
7.1.3 The effects of TASK-1 knockdown in hPASMCs…………..37
7.1.4 Acute hypoxia inhibits TASK-1………………………………40
7.2 Modulation of TASK-1 by endothelin-1 in hPASMCs.……………...42
7.2.1 Endothelin-1 inhibits TASK-1………………………………...42
7.2.2 Signaling pathway of ET-1……………………………………47
8. Discussion …………………………………………………………………………...55
8.1 Two-pore domain potassium channels in hPASMC………………..55 8.2 TASK-1 regulation by endothelin-1…………………………………..58
9. Conclusion and outlook……………………………………………………………62
10. Appendix……………………………………………………………………………...64
10.1. Reference……………………………………………………………….64
10.2. Figure Index…………………………………………………………….79
10.3. Table Index……………………………………………………………..80

Summary (English)..…………………………………………………………………….81
Summary (German)..……………………………………………………………………83
Acknowledgments………………………………………………………………………85
Curriculum vitae…………………………………………………………………………86
Abbreviation:
DAG: diacylglycerol
EGTA: ethyleneglycol bis(ß-aminoethyl ether)-N,N,N',N'-tetraacetic acid
E : resting membrane potential m
ET-1: endothelin-1
FITC: fluorescein isothiocyanate-conjugated
HEPES: 4-(2-Hydroxyethyl) piperazine-1-ethanesulfonic acid
HPV: hypoxic pulmonary vasoconstriction
+ I : noninactivating K current KN
K : ATP-sensitive potassium channels AT P
+ K : two-pore domain potassium (K ) channels2P
KCa: calcium-activated potassium channels
K : voltage-dependent potassium channels V
PH: pulmonary hypertension
PAH: pulmonary arterial hypertension
PAP: pulmal pressure
PASMC: pulmonary arterial smooth muscle cell
PCR: polymerase chain reaction
pHo: extracellular pH
PIP : phosphatidylinositol 4,5-biphosphate 2
PKA: protein kinase A
PKC: protein kinase C
PLC: phospholipase C
siRNA: small interfering RNA
+TASK-1: TWIK-related acid-sensitive K ; TWIK, for tandem P domains in a weak
+ inwardly rectifying K
TEA: tetraethylammonium