published_or_final_version / Physiology / Master / Master of Medical Sciences

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

In living systems, ion channels are membrane transport proteins that provide pathways for the passive diffusion of ions through lipid membranes. The flow of ions across membranes is the basis of many important physiological processes, including but not limited to the regulation of membrane potential, transepithelial transport and cell volume. While many efforts have been made to understand the biological roles of natural ion channels, the biological activities of artificial ion channels remain largely unknown. Recently, it was reported that a small molecule 1, which forms synthetic chloride (Cl–) channels in membranes via self-assembly, is capable of modulating vascular functions. In this thesis, novel small molecules that are structurally similar to 1 are shown to form artificial ion channels in membranes. Together with 1, the effects of these small molecules on the contractile activities of smooth muscles and epithelial ion transport are explored. The therapeutic implications of the findings are also discussed. A collection of small molecules was screened using liposome-based fluorescence assays. In these assays, the ability of the synthetic compounds to modulate membrane potential was monitored. The screening yielded compound 3 that formed synthetic potassium (K+) channels in liposomal membranes, although the liposome-based fluorescence experiments suggested that 3 also transported Cl–. Two derivatives of 3, namely, compounds 2 and 4 were also examined. Single-channel recording experiments suggested that 2 forms synthetic Cl– channels in liposomal membranes. The effects of compounds 2 and 3 on the functions of the vascular smooth muscle are explored. Using confocal imaging, it was shown that both 2 and 3 counteracted the effects of high-K+ depolarizing solution on membrane potential and intracellular Ca2+ concentration ([Ca2+]i) in cultured vascular smooth muscle cells. 2 and 3 also relaxed mice aortic rings pre-contracted with high-K+ solution. These observations can be explained in terms of the Cl– transporting functions of 2 and 3. To determine the potential for developing the compounds into bronchodilators, the effects of compounds 1 and 3 on the contractile activities of the airway smooth muscle (ASM) were explored using organ bath technique. The contractile activities of the trachea isolated from Sprague-Dawley (SD) rats were first characterized. Among the contractile agents used, only potassium chloride (KCl), cholinergic agonists, serotonin and endothelin-1 were contractile to the SD rat trachea. 1 and 3 relaxed the ASM pre-contracted with KCl, whereas the contractions induced by other agonists were not affected. The ability of compounds 2, 3 and 4 to modulate ion transport across cultured epithelia was tested by the short-circuit current measurement technique. It was shown that the compounds were capable of inducing Cl– secretion when applied to the apical side of airway and colonic epithelia. Importantly, the synthetic compounds induced apical Cl– secretion in immortalized cystic fibrosis (CF) bronchial epithelia. This suggests that the synthetic compounds may be used to correct the anion transport defect in CF epithelia. In summary, the small-molecule based synthetic ion channels demonstrated two important general functions of natural ion channels, namely, the regulation of membrane potential and epithelial ion transport. / published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

In addition to leukocytes, secondary lymphoid organs are populated by non-hematopoietic stromal cells. This diverse group of cells supports lymphocyte migration and homing, facilitates antigen delivery, and promotes T cell survival. However, there is relatively little known about the specific molecules governing the roles that these cells play in regulating dendritic cell (DC) motility and lymph node architecture. Here, we examine the interaction between two molecules, CLEC-2 and podoplanin (PDPN), that are critical for DC migration and maintaining structural integrity of lymph nodes. Together, these studies identify novel functions of lymph node stromal cells and a unique function for PDPN in the immune system. In response detecting an potentially harmful antigen, DCs in peripheral tissues mature and travel to downstream lymph nodes by following chemokine gradients secreted by lymphatic endothelial cells (LECs) and fibroblastic reticular cells (FRCs) present in the lymph node paracortex. We discovered that, in addition to chemokines, DC migration requires CLEC-2 on DCs, as engagement of CLEC-2 with PDPN, which is expressed by LECs and FRCs, incites DC motility and is required for DC entry into the lymphatics, efficient arrival in the lymph node, and migration along the FRC network within the lymph node. Next, we examined the effect of this interaction with respect to the stromal cell. Through a combination approaches, we discovered that PDPN is a master regulator of contractility in FRCs. The fact that FRCs are contractile cells was previously reported, but our study is the first to identify a function for this contractility: upon blockade of PDPN-mediated contractility, lymph nodes became enlarged, the FRC network became more sparse, and there were increased numbers of lymphocytes in the lymph node. Importantly, during an immune response, these changes resulted in more proliferation of antigen-specific T cells and impaired contraction of the lymph node upon resolution of inflammation. Finally, we found that CLEC-2 binding PDPN recapitulated the effect of PDPN deletion. Thus, during an immune response, CLEC-2+ DCs would use PDPN to efficiently migrate to the lymph node and simultaneously cause FRCs to relax and prepare the lymph node for expansion.

No description available.

No description available.

To examine the effect of acute beetroot juice supplementation on the rate of fatigue as measured by changes in peak torque. Placebo-controlled, double-blind, cross-over study, 35 recreationally active subjects consumed beetroot (BR) juice or black currant juice (PL) 12 and 2.5 hours before the exercise procedure. Peak torque was measured on the BIODEX dynamometer by performing 50, maximal effort, concentric knee extensions at 90°/s. Blood pressure (BP) was recorded before and after exercise. No significant difference between BR and PL in the rate of fatigue measured by change in peak torque. By stage 3, subjects retained 87.6±6.9% of strength with BR and 86.7±6.3% with PL (p= 0.363). Stages 10 was as follows: BR 47.9±12.6 vs. PL 46.9±12.9% (p= 0.419). The rate of work fatigue showed no significant differences. By stage 4, mean percent work fatigue showed 20.6±9% with BR and 21.8±10.1% with PL (p= 0.224). Stage 10 was as follows: BR 52.5±12.6% vs. PL 53.2±13% (p= 0.571). Post-exercise diastolic BP (BR: 67.2±9.8 vs. PL: 64.5±7.9mmHg, p= 0.039) and MAP (BR: 91.6±9.3 vs. PL: 88.8±8.2mmHg, p= 0.011) were higher with BR supplementation. Acute bouts of beetroot juice supplementation had no significant effect on knee extensor muscle fatigue measured during isokinetic contractions

La contraction du muscle cardiaque et squelettique est activée par la libération du calcium (Ca²[indice supérieur +]) du réticulum sarcoplasmique (RS), en réponse à la dépolarisation du sarcolemme pendant la propagation du potentiel d'action (PA) le long des tubules transverses (tubules T). Ce processus s'appelle le couplage excitation-contraction (couplage EC). Le couplage EC dans le muscle cardiaque est différent de celui squelettique en ce sens qu'il nécessite du Ca²[indice supérieur +] extracellulaire ce qui n'est pas le cas dans le couplage EC dans le muscle squelettique. Le but de mon projet de Maîtrise a été principalement de développer et de perfectionner une nouvelle méthode de mesure de la concentration totale de Ca²[indice supérieur +] dans le muscle cardiaque et le muscle squelettique ([Ca[indice inférieur T]]MUSCLE) de différentes espèces (rats, souris et grenouilles); dont la plus grande fraction est emmagasinée à l'intérieur du RS. Cette mesure quantitative a pour objectif à long terme, dans le cas du muscle cardiaque de comprendre les résultats apparemment contradictoires concernant le mécanisme principal de couplage EC et dans le cas du muscle squelettique, de confirmer que la concentration totale de Ca²[indice supérieur +] dans la préparation des cellules isolées correspond au niveau physiologique. De surcroît, dans ce dernier cas, la [Ca[indice inférieur T]]MUSCLE dans les fibres musculaires squelettiques de grenouille obtenu avec la technique de EGTA-Rouge de phénol effectué par Pape et al. (1995) est similaire à celle obtenue à partir de cette nouvelle méthode dans le muscle squelettique entier. Les résultats obtenus en relation avec le poids du muscle sur les souris C57BL6 montrent qu'il y a une grande dépendance du contenu total de Ca²[indice supérieur +] sur le poids du muscle. En effet, le poids du muscle varie de 12.7 mg à 5.2 mg ce qui correspond à 1.34 mM et 4.14 mM respectivement. Ces résultats suggèrent la possibilité d'un mécanisme pour la régulation du [Ca[indice inférieur T]]MUSCLE où le plus petit muscle augmente [Ca[indice inférieur T]]MUSCLE afin d'augmenter sa force spécifique (force normalisée pour la grandeur) pour produire une force similaire aux muscles plus grands. La calséquestrine est une protéine qui tamponne le Ca²[indice supérieur +] à l'intérieur du RS est la source principale de Ca²[indice supérieur +] impliquée dans le couplage EC. En effet, Fenelon et al. (2012) ont estimé que 95% du Ca²[indice supérieur +] dans le RS est lié, avec 5% dans le forme libre (i.e. Ca²[indice supérieur +]), et que plus de 80% du Ca²[indice supérieur +] lié paraît être associé avec la calséquestrine. La raison principale pour développer cette nouvelle méthode a été d'évaluer si le contenu total de Ca intracellulaire est largement réduit dans les muscles KO en CSQ afin de mieux résoudre la controverse sur ce sujet. Contrairement à nos attentes [Ca[indice inférieur T]]MUSCLE a été proche de 2mM dans les muscles contrôles, ce qui est proche de la moyenne mesurée pour les muscles KO en CSQ. Notre hypothèse est qu'il y a une "uprégulation" d'une ou plusieurs protéines de liaison de Ca²[indice supérieur +] dans le RS.

No description available.

The purpose of this investigation was to examine the contractile properties of individual slow and fast myofibers in response to various training periods throughout a collegiate cross-country season in male runners. Muscle biopsies were obtained from the gastrocnemius of five runners at three time points during the season; after a summer aerobic training phase (T1); after an 8 week period of intense training (T2) and after a 4 week taper period (T3). Absolute (4.6±0.3 L/min) and relative (71±0.7 mI/kg/min) maximal oxygen consumption were unchanged during the study duration. Run performance (8 km) improved 3% from T1 to T2 (27:26 to 26:38 min:sec) and 1% from T2 to T3 (26:21 min:sec). A total of 328 individual myofibers were isolated and studied at 15°C. MHC I diameter was 3% lower (P < 0.05) at T2 compared to T1 and 4% smaller at T3 compared to T2. MHC I and Ila fibers were 18 and 11 % stronger (P < 0.05) respectively, from T1 to T2. MHC I fibers produced 9% less force (P < 0.05) after the taper (T2-T3) while MHC Ila fibers were 9% stronger (P < 0.05). Specific tension (Po/CSA) increased (P < 0.05) 38 and 26% for MHC I and Ila fibers, respectively from T1 to T2 and was unchanged with the taper. Maximal shortening velocity (Vo) of the MHC I fibers decreased (P < 0.05) 23% from T1 to T2 and 17% from T2 to T3. MHC I peak power decreased (P < 0.05) 20% from T1 to T2 and 25% from T2 to T3. Power corrected for cell size decreased (P < 0.05) 15% from T2 to T3 and was 24% lower at T3 compared to T1 for the MHC I fibers only. MHC Ila diameter, Vo, peak and normalized power were unaltered during the study duration. These data suggest that myocellular function is sensitive to changes in run training and it appears that MHC I fibers are moreaffected than MHC IIa fibers. Furthermore, the increase in interval training with the taper may have offset the modest reduction in training volume and prevented any positive adaptations at the cellular level. / Human Performance Laboratory