The cystic fibrosis transmembrane conductance regulator (CFTR) was identified to be involved in acidosis-induced ATP release from skeletal myocytes in vitro and from contracting muscle in vivo. My PhD studies aimed to investigate the underlying mechanism and identify the pathway for ATP release in acidosis-induced CFTR-regulated ATP release. Lactic acid (10 mM) decreased the intracellular pH of L6 skeletal myocytes to 6.87 ± 0.12 after 3 hours, and the lowered pH resulted in the elevation of ATP release from skeletal myocytes. The acidosis-induced ATP release was totally abolished by GlyH-101 (40 μM), an open-channel CFTR blocker, suggesting that CFTR was involved. The cAMP/PKA signaling pathway was involved in the CFTR-regulated ATP release from skeletal myocytes: 1). Forskolin increased the extracellular ATP and the phosphorylation of CFTR; IBMX, a phosphodiesterase inhibitor, further enhanced the forskolin-induced extracellular ATP and phosphorylation of CFTR; 2). Inhibition of PKA by its selective inhibitor KT-5720 abolished the acidosis-induced ATP release and the forskolin-induced phosphorylation of CFTR. In addition, the inhibition of Na+/H+ exchanger (NHE) by amiloride, or inhibition of Na+/Ca2+ exchanger (NCX) by its specific inhibitors SN-6 and KB-R7943 abolished the lactic-acid-induced ATP release from skeletal myocytes, indicating that NHE and NCX might be involved. Previous studies demonstrated that Connexin hemichannels and Pannexin channels were able to conduct ATP in response to stimuli. This study found that connexin 43 (Cx43) was strongly expressed on skeletal myocytes, while Pannexin 1 (Panx1) showed a strong expression in gastrocnemius muscle. Investigation of the role that Cx43 may play in acidosis-induced cAMP/PKA-activated CFTR-regulated ATP release from myocytes showed that: 1). Cx43 was immunoprecipitated with CFTR suggesting a physical interaction; 2). The opening of Cx hemichannels was increased by lactic acid and this lactic-acid-induced opening was inhibited by CFTRinh-172, suggesting the mediation of CFTR; 3). Inhibition of Cxs and Panxs with carbenoxolone abolished the acidosis-induced ATP release; moreover, specific silencing of the Cx43 gene using siRNA decreased both basal and acidosis-induced ATP release, suggesting that Cx43 was involved; 4). Overexpression of CFTR alone did not elevate the acidosis-induced ATP release, while overexpression of Cx43 alone doubled the acidosis-induced ATP, and co-overexpression of CFTR and Cx43 further elevated the acidosis-induced ATP release, supporting the concept that Cx43 functionally interacted with CFTR to induce the acidosis-induced ATP release. Panx1 was studied in native skeletal muscle, and found to be coimmunoprecipitated with CFTR. Inhibition of Panxs with gadolinium or probenecid abolished the muscle-contraction-induced ATP release, while inhibition with carbenoxolone or quinine reduced it to less than 10% of control, suggesting that Panx1 may be involved in the acidosis-induced ATP release during muscle contraction. All the in vitro and in vivo studies suggested that Cxs and Panx were involved in the acidosis-induced CFTR-regulated ATP release from skeletal myocytes and skeletal muscle. / published_or_final_version / Physiology / Doctoral / Doctor of Philosophy

Kattaya-Annappa-Seema. / Thesis submitted in: December 2006. / "2+" and "+" in the title are superscripts. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 162-188). / Abstracts in English and Chinese. / Abstract --- p.i / Acknowledgements --- p.viii / Abstracts published based on work in this thesis --- p.ix / Table of contents --- p.x / Chapter Chapter 1: --- Introduction / Chapter 1.1 --- Pulmonary hypertension / Chapter 1.1.1 --- Pulmonary circulation and its functions --- p.1 / Chapter 1.1.2 --- Pulmonary vascular diseases and symptoms --- p.3 / Chapter 1.2 --- Muscarinic Receptor functions --- p.5 / Chapter 1.3 --- Acetylcholine (ACh) and its function --- p.7 / Chapter 1.4 --- ACh receptors in pulmonary vascular bed --- p.11 / Chapter 1.5 --- Potassium channel classification and functions --- p.12 / Chapter 1.5.1 --- "Importance of High-conductance, Ca2+ activated potassium channel (BKca) in vascular smooth muscle functions" --- p.15 / Chapter 1.5.2 --- Modulation of BKca channel by various cations --- p.18 / Chapter 1.6 --- Calcium signaling and homeostasis --- p.20 / Chapter 1.7 --- Role of sodium in hypertension --- p.22 / Chapter 1.8 --- Na+-H+ exchanger (NHE) functions --- p.25 / Chapter 1.9 --- Na+-Ca2+ exchanger (NCX) in vascular smooth muscle cells --- p.29 / Chapter 1.10 --- Spontaneously hypertensive rat (SHR) / Chapter 1.10.1 --- Hypertension in SHR --- p.32 / Chapter 1.10.2 --- BKca in smooth muscle vasculature of SHR --- p.33 / OBJECTIVES OF THE STUDY --- p.34 / Chapter Chapter 2: --- Material and methods / Chapter 2.1 --- Material / Chapter 2.1.1 --- Solutions and Drugs --- p.35 / Chapter 2.1.2 --- Chemicals and Enzymes --- p.39 / Chapter 2.2 --- Methods / Chapter 2.2.1 --- Isolation of single pulmonary arterial smooth muscle cells --- p.40 / Chapter 2.2.2 --- Electrophysiological measurement --- p.42 / Chapter 2.2.3 --- Data analysis --- p.44 / Chapter Chapter 3: --- Receptor-mediated activation of BKca Channel / Chapter 3.1 --- BKCa activation by ACh/ Carbachol (CCh) --- p.45 / Chapter 3.2 --- Role of extracellular sodium ([Na+]o）on BKca activation --- p.49 / Chapter 3.3 --- Receptor-mediated activation of BKca in a [Na+]o-containing solution --- p.51 / Chapter 3.4 --- Receptor-mediated activation of BKca in a [Na+]o-free solution --- p.55 / Chapter Chapter 4: --- Non-receptor mediated activation of BKCa Channel / Chapter 4.1 --- Effect of different concentrations of sodium nitroprusside (SNP) on BKCa activation --- p.60 / Chapter 4.2 --- Effect of SNP on BKca activation in a [Na+]o-containing and [Na+]o-free solutions --- p.62 / Chapter Chapter 5: --- Role of NHE in modulating activation of BKCa Channel / Chapter 5.1 --- Effect of Monensin on BKca activation / Chapter 5.1.1 --- Effect of monensin on CCh-mediated activation of BKca in a [Na+]o-containing solution --- p.70 / Chapter 5.1.2 --- Effect of monensin on CCh-mediated activation of BKca in a [Na+]o-free solution --- p.74 / Chapter 5.1.3 --- Effect of monensin on SNP- mediated activation of BKca in [Na+]o-containing and [Na+]o-free solutions --- p.78 / Chapter 5.2 --- Effect of 5-(N-ethyl-N-isopropyI) amiloride (EIPA) on BKCa activation / Chapter 5.2.1 --- Effect of EIPA on CCh-mediated activation of BKca in a [Na+]o-containing solution --- p.85 / Chapter 5.2.2 --- Effect of EIPA on CCh-mediated activation of BKca in a [Na+]。-free solution --- p.89 / Chapter 5.2.3 --- Effect of EIPA on SNP-mediated activation of BKCa in [Na+]o-containing and [Na+]o-free solutions --- p.93 / Chapter Chapter 6: --- Role of NCX in modulating activation of BKCa Channel / Chapter 6.1 --- Effect of KB-R7943 on CCh-mediated activation of BKCa in a [Na+]o-containing solution --- p.100 / Chapter 6.2 --- Effect of KB-R7943 on CCh-mediated activation of BKCa in a [Na+]o-free solution --- p.104 / Chapter 6.3 --- Effect of KB-R7943 on SNP-mediated activation of BKca in [Na+]o-containing and [Na+]o-free solutions --- p.109 / Chapter Chapter 7: --- Effect of intracellular sodium ([Na+]i) on BKCa channel activation / Chapter 7.1 --- Effect of CCh on BKCa channel activation with elevated [Na+]i pipette solution --- p.117 / Chapter 7.2 --- Effect of SNP on BKca channel activation with elevated [Na+]j pipette solution --- p.130 / Chapter Chapter 8: --- Discussion / Chapter 8.1 --- Modulatory effect of ACh and SNP --- p.138 / Chapter 8.2 --- Role of ion exchangers: NHE and NCX in modulating BKca channel function --- p.144 / Chapter 8.3 --- Modulatory effect of elevated [Na+]i on BKca activation --- p.153 / CONCLUSION --- p.161 / References --- p.162