Contracting skeletal muscle releases ATP into the interstitial space where it is subsequently broken down to adenosine by the action of ecto-5’-nucleotidase. Both ATP and adenosine are vasodilators that contribute to the exercise hyperaemia. However, the mechanism for the release of ATP from muscle during exercise remains unknown. Cystic fibrosis transmembrane conductance regulator (CFTR) is involved in ATP release from muscle at low intracellular pH: this study was performed to investigate whether CFTR was involved in the ATP release from skeletal muscle during contractions. Experiments were performed in rats anaesthetised with sodium pentobarbitone and breathing spontaneously. A microdialysis probe was placed in one gastrocnemius muscle: ATP was determined in interstitial microdialysate samples using a bioluminescence assay. The sciatic nerve was stimulated to induce two bouts of muscle contractions, separated by a recovery period of 40 mins; one of the inhibitors was administered prior to the second bout of contractions. Muscle contractions elevated the interstitial ATP by 1500 to 3000%. In the control experiments, no drug was given: both the contractile force and the increase in interstitial ATP were reproducible in repeated contraction bouts. Infusion of a specific inhibitor of CFTR, CFTRinh-172, did not alter the contractile force, but significantly lowered the interstitial ATP during muscle contractions, suggesting that CFTR was involved in the contraction-induced ATP release. Similarly, infusion of the Protein Kinase A inhibitor, KT5720, significantly reduced interstitial ATP during muscle contractions without altering contractile force, suggesting that CFTR in skeletal muscle is activated through the cAMP/PKA pathway. The increase in interstitial ATP during muscle contraction was also inhibited by the Na/H exchanger inhibitor, amiloride, or the Na/Ca exchanger inhibitor, SN6. It has been also shown that two gap junction hemichannel inhibitors, gadolinium and carbenoxolone, could attenuate the increase of ATP during muscle contraction. These data suggest that CFTR, activated through the cAMP/protein kinase A pathway, is involved in the ATP release during muscle contraction, and that activation of the Na/H exchanger and Na/Ca exchanger was also required, indicating that the signal transduction mechanism for CFTR activation during muscle contractions may be similar to that which is reported to occur at low pH. The preliminary data showed that the gap junction hemichannels might mediate the ATP release from skeletal muscle cells during muscle contraction. / published_or_final_version / Physiology / Master / Master of Philosophy

Proton nuclear magnetic resonance (NMR) spectroscopy has been used to investigate the interaction of yeast phosphoglycerate kinase (PGK) with its phosphate containing substrates, ATP and 3-phosphoglycerate (3-PG). The application of one-dimensional and, for the first time, two-dimensional proton NMR techniques to this large protein has enabled specific resonance assignments to be made. Assignment has been aided by the investigation of specifically deuterated protein and site-specific mutant forms of the protein, including the isolated N- and C-domains. The effects of ATP and 3-PG binding on the proton NMR spectrum of yeast PGK have been characterised and the assigned resonances used as local probes of structural and dynamic changes. Two binding sites have been determined for the nucleotide substrate, ATP, the occupancies of which are dependent on Mg²⁺ concentration. One site corresponds to the catalytic site determined crystallographically. A single binding site was found for 3-PG. This binding was shown to cause highly specific conformational changes throughout the N-domain and the interdomain region, which involve the relative movement of at least three α-helices. Investigation of 3-PG binding to several site-specific mutant forms of yeast PGK revealed a critical role for arginine 168 in the propagation of these changes. The general binding of anions to yeast PGK was investigated using the paramagnetic probes [Cr(CN)₆]^3- and [Fe(CN)₆]^3-, and the diamagnetic anion [Co(CN)₆]^3-. The primary anion binding site was determined from [Cr(CN)₆]^3- broadening data and found to share some side-chains involved in 3-PG binding, namely histidine 62 and arginine 168. Evidence for a secondary anion site was found. The anion binding data is discussed in view of the complex activation/inhibition effects of anions on the catalytic activity. Investigation of the isolated N- and C-domains showed that both can fold independently and confirmed that the C-domain is a nucleotide binding domain. It appears that the presence of the interdomain residues and/or the C-terminal peptide are necessary for 3-PG binding to the N-domain. This work shows that the specificity of the substrates is in binding, as expected, but also in the motions induced in the protein as a whole.

Thesis (Ph. D.)--Georgia State University, 2007. / Title from file title page. Chun Jiang, committee chair; Teryl Frey, Deborah Baro, Delon Barfuss, committee members. Electronic text (168 p. : ill. (some col.)) : digital, PDF file. Description based on contents viewed Jan. 30, 2008. Includes bibliographical references (p. 146-151).

Dissertation (Ph.D.) -- University of Texas Southwestern Medical Center at Dallas, 2006. / Vita. Bibliography: p. 92-107.

Thesis (Ph. D.)--Cornell University, January, 2009. / Vita. Includes bibliographical references (leaves 264-277).

Thesis (M.S.)--University of Missouri-Columbia, 2006. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file (viewed on April 21, 2009) Includes bibliographical references.

Thesis (Ph. D.)--Duke University, 2008. / Includes bibliographical references.

Thesis (Ph. D.)--University of Missouri--Columbia, 1998. / "July 1998." Typescript. Vita. Includes bibliographical references (leaves 145-165). Also available on the Internet.

Thèses (M.Sc.)--Université de Sherbrooke (Canada), 2003. / Titre de l'écran-titre (visionné le 20 juin 2006). Publié aussi en version papier.

Thesis (Ph.D. in Neuroscience) -- University of Colorado at Denver and Health Sciences Center, 2005. / Typescript. Includes bibliographical references (leaves 114-131).