Few studies have quantified resting mouse muscle metabolism and even fewer studies have separated the contribution of sarcoplasmic reticulum (SR) Ca2+ pumping to resting metabolic rate. Furthermore, the studies that have attempted to quantify the contribution of Ca2+ pumping have used indirect methods to inhibit SR Ca2+ ATPase activity. The purpose of this study is to directly quantify resting muscle oxygen consumption and the contribution of SR Ca2+ pumping to resting oxygen consumption in mouse hindlimb muscles by using CPA to specifically inhibit Ca2+ pump activity in intact muscles at rest. The TIOX system was used to measure resting muscle VO2 of extensor digitorum longus (EDL) and soleus (SOL) muscles at 30oC and 20oC. C57BL mice aged 8-12 weeks were used with an average whole body mass of 23.8 g and EDL and SOL dry weights averaging 1.88 mg and 1.8 mg, respectively. All muscle VO2 measurements are expressed per gram dry weight. There were no differences (P>0.1) in resting muscle VO2 between EDL and SOL muscles at either 30oC (EDL, 2.05 µL/g/s; SOL, 2.27 µL/g/s) or 20oC (EDL, 0.62 µL/g/s; SOL, 0.71 µL/g/s). The average Q10 (3.1) was determined from EDL and SOL VO2 measures at 20oC and 30oC. The contribution of Ca2+ pumping by the sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) was measured at 30oC using a range of CPA concentrations (1-15 µM) . There was a concentration-dependent effect of CPA on oxygen consumption with increasing CPA concentrations up to 10 µM resulting in progressively greater reductions in muscle oxygen consumption. Specifically, 1, 5, 10, and 15 µM CPA caused an 11, 35.4, 49.5, and 50.3% reduction in VO2. There were no differences (P>0.1) between 10 and 15 µM CPA indicating that 10 µM CPA induces maximal inhibition of SERCA in isolated muscle preparations. The results indicate that the Ca2+ pumping by SERCA is responsible for ~50% of oxygen consumption in resting mouse EDL and SOL muscle. This is the first study to use a direct inhibitor of SERCA to quantify the contribution of Ca2+ cycling to resting oxygen consumption and therefore is a more accurate reflection of the actual contribution of SERCA to resting muscle oxygen consumption compared to previous findings. These results suggest that SERCA energy consumption accounts for a large portion of resting muscle metabolism and may represent a potential therapeutic target for metabolic alterations to oppose obesity.

Reproduction de : Thèse de doctorat : Sciences de la Vie et de la Santé : Lille 1 : 2005. / N° d'ordre (Lille 1) : 3677. Titre provenant de la page de titre du document numérisé. Bibliogr. p. 185-215.

Thesis (M.S.)--University of Toledo, 2009. / "In partial fulfillment of the requirements for the degree of Master of Science in Biomedical Science." Title from title page of PDF document. Bibliography: p. 39-48.

Thesis (Ph.D.)--Ohio University, November, 2009. / Release of full electronic text on OhioLINK has been delayed until December 1, 2014. Title from PDF t.p. Includes bibliographical references.

All cells maintain essential metal nutrients at optimal levels by metal homeostasis. P-type ATPases, a crucial superfamily of integral membrane proteins, are involved in the active transport of metal ions across biological membranes driven by the motive force of ATP- hydrolysis. The PIB-type ATPase subfamily, also called CPx-ATPases, fulfills a key role in heavy metal homoeostasis among the most widespread species from bacteria to human. In humans, the defect in copper transporters is the direct cause of severe neurological and hepatic disorders such as Wilson and Menkes diseases, therefore, understanding the molecular function of these pumps is of paramount importance in human health. Cu+-ATPases have two transmembrane metal binding sites (TM-MBS) and three cytosolic domains, namely the actuator (A-domain) and phosphorylation and nucleotide-binding domain (PN), and regulatory N-terminal heavy metal binding domain (HMBD). Here, we have studied the Legionella pneumophila CopA (LpCopA) and its isolated cytosolic domains to improve our understanding of the functional interaction of the protein domains during metal transport relate this to the known structure of this ATPase. To elucidate how cytosolic ligands (Cu+ and nucleotide) stimulate the interactions among the cytosolic domains and may transmit conformational changes to the TM-MBS, the interactions among recombinant isolated cytosolic domains were first examined biochemically by co-purification and spectroscopically by circular dichroism, time-resolved fluorescence and site-directed fluorescent labeling assays. The Cu+-dependent interaction between the A-domain and HMBD has been postulated as a mechanism for activating the ATPase cycle. This question was addressed here by studying copper-dependent interactions between the isolated expressed domains. Spectroscopic evidence is provided that an HMBD-A complex is formed in the presence of Cu+ which binds with 100-200 nM affinity to the recombinant HMBD. In contrast, the A-domain interacts with the PN domain in a nucleotide-dependent fashion. This molecular recognition is required for the dephosphorylation step in the catalytic cycle. The interaction was investigated in more detail by the use of a decameric peptide derived from the PN-binding interface of the A-domain and carrying the conserved TGE-motif involved in dephosphorylation. Its binding to the isolated PN domain in a weakly nucleotide-dependent manner, is demonstrated here by stopped-flow fluorescence spectroscopy. Several ATPase assays were modified to assess the functionality of the PN-domain and full length LpCopA. The peptide was found to reduce the catalytic turnover of full length LpCopA. This agrees with the expected slowing down of the reformation of the PN-A-domain interaction since the peptide occupies their binding interface. Thus, the synthetic peptide provides a means to study specifically the influence of PN-A-domain interactions on the structure and function of LpCopA. This was done by time-correlated single photon counting (TCSPC) method. The time-dependent Stokes shift of the environmentally sensitive fluorophore BADAN which was covalently attached to the conserved CPC-motif in the TM-MBS was measured. The data indicate that the interior of the ATPase is hydrated and the mobility of the intra-protein water varies from high to low at C382 at the “luminal side” and C384 at the “cytosolic side” of the TM-MBS, respectively. This finding is consistent with the recent MD simulation of LpCopA, bringing the first experimental evidence on a luminal-open conformation of E2~P state. The A-domain-derived decapeptide, although binding to the cytosolic head piece, induces structural changes also at the TM-MBS. The peptide-stabilized state (with a disrupted PN-A interface) renders the C384 environment more hydrophobic as evidenced by TCSPC. Taken together, the data from cytosolic domain interactions, ATPase assays and of time-dependent Stoke shift analyses of BADAN-labeled LpCopA reveal the presence of hydrated intramembraneous sites whose degree of hydration is regulated by the rearrangement of cytosolic domains, particularly during the association and dissociation of the PN-A domains. Copper affects this arrangement by inducing the linkage of the A-domain to the HMBD. The latter appears to play not only an autoinhibitory but also a chaperone-like role in transferring Cu+ to the TM-MBS during catalytic turnover.

We hypothesized that alterations in cardiac myofilaments are associated with hereditary canine DCM. DCM myofilaments demonstrated a reduction in EC50 and a modest decrease in maximum activity compared to non-failing dog samples. Treatment of myofilaments with the calcium sensitizer, bepridil, showed a reduction in EC50. Desmin and tropomyosin phosphorylation was increased in DCM. Desmin protein levels were increased in DCM. Total troponin I phosphorylation was unchanged, but S23/S24 phosphorylation was reduced in DCM. Myofilament-associated PKC-δ and -ζ were elevated in DCM, PKC- ε was modestly reduced, and PKC-α showed no change. These data are the first investigation of cardiac myofilaments in naturally occurring canine DCM, and support the hypothesis that alterations in cardiac myofilaments are associated with DCM. / OVC Pet Trust (operating funds)

Most vertebrates suffer permanent damage after minutes of anoxia. Many insects however, have part of their life cycle in anoxia or constant hypoxia, such as during their egg-hatching phase, by living as deep burrowers, or at high altitudes. Insects are able to survive in anoxia from hours to days, or even months by developing various strategies through evolution. For example, the locusts (Locusta migratoria) enter a reversible coma during anoxia that is associated with an arrest of ventilation, and a reinitiation of ventilation when returned to normoxia. This coma is correlated with a surge in the concentration of extracellular potassium ions ([K+]o), and recovery from this reversible coma is dependent on re-establishing the functional [K+]o. Prior exposure to a sublethal heatshock (HS)-preconditioning grants locusts a temporary resilience to anoxia; however, the molecular mechanisms of this protection are still unclear. This project investigated the effects of HS-preconditioning on locusts’ ventilation, the total enzymatic activity of the Na+/K+-ATPase, as well as its distribution within the metathoracic ganglion and tested the hypothesis that HS-preconditioning alters locusts’ ventilation and increases the totally Na+/K+-ATPase activity and its concentration within neuronal membranes. I recorded electromyograms of locusts’ ventilatory motor patterns in the presence and absence of anoxic coma by placing a copper wire electrode on ventilatory muscles 161 or 173 in control and HS-preconditioned animals. In addition, I studied the enzymatic activity of the Na+/K+-ATPase using a pyruvate kinase/lactate dehydrogenase assay and the localization of the Na+/K+-ATPase using immunohistochemistry in control and HS-preconditioned locusts at different stages of coma. I found that the ventilatory cycle period was decreased and the ventilatory muscle burst duration was increased after recovery from anoxic coma in HS-preconditioned locusts. I also found that anoxia did not affect the activity or the localization of the Na+/K+-ATPase. However, HS-preconditioning increased the total activity of the Na+/K+-ATPase and the localization of the Na+/K+-ATPase within the neuronal membranes. From this project, I concluded that HSpreconditioning affected locusts’ ventilatory motor pattern after recover from anoxia and increased the total activity and the neuronal membrane localization of the Na+/K+-ATPase. / Thesis (Master, Biology) -- Queen's University, 2011-09-26 13:14:48.472

Neurons perform complex computations, communications and precise transmissions of information in the form of action potentials (APs). The high level of heterogeneity and complexity at all levels of organization within a neuron and the functional requirement of highly permeable cell membranes leave neurons exposed to damage when energy levels are insufficient for the active maintenance of ionic gradients. When energy is limiting the ionic gradient across a neuron’s cell membrane risks being dissipated which can have dire consequences. Other researchers have advocated “generalized channel arrest” and/or “spike arrest” as a means of reducing the neuronal permeability allowing neurons to adjust the demands placed on their electrogenic pumps to lower levels of energy supply. I investigated the consequences of hypoxia on the propagation of a train of APs down the length of a fast conducting axon capable of transmitting APs at very high frequencies. Under normoxic conditions I found that APs show conduction velocities and instantaneous frequencies nearly double that of neurons experiencing energy limiting hypoxic conditions. I show that hypoxia affects AP conduction differently for different lengths of axon and for APs of different instantaneous frequencies. Action potentials of high instantaneous frequency in branching lengths of axon within ganglia were delayed more significantly than those in non-branching lengths contained within the connective and fail preferentially in branching axon. I found that octopamine attenuates the effects of hypoxia on AP propagation for the branching length of axon but has no effect on the non-branching length of axon. Additionally, for energetically stable cells, application of the anti-diabetic medication metformin or the hyperpolarization-activated cyclic nucleotide-gated (HCN) channel blocker ZD7288 resulted in a reduced performance similar to that seen in neurons experiencing energetic stress. Furthermore both metformin and ZD7288 affect the shape of individual APs within an AP train as well as the original temporal sequence of the AP train, which encodes behaviourally relevant information. I propose that the reduced performance observed in an energetically compromised cell represents an adaptive mechanism employed by neurons in order to maintain the integrity of their highly heterogeneous and complex organization during periods of reduced energy supply. / Thesis (Master, Biology) -- Queen's University, 2011-10-07 14:41:46.972

Previous studies have shown that ischemia-reperfusion (I/R) injury is associated with cardiac dysfunction and depression in sarcolemmal Na+-K+-ATPase activity. This study was undertaken to evaluate the role of proteases in these alterations by subjecting rat hearts to different times of global ischemia, and reperfusion after 45 min of ischemia. Decreases in Na+-K+-ATPase activity at 60 min of global ischemia were associated with augmented activities of both calpain and MMPs and depressed protein content of β1- and β2-subunits, without changes in α1- and α2-subunits of the enzyme. However, reperfusion of ischemic heart produced depression in Na+-K+-ATPase activity, no change in the augmented calpain activity, but decreases in augmented MMP-2 activity and Na+-K+-ATPase content. MDL28170, a calpain inhibitor, was more effective in attenuating I/R-induced alterations than doxycycline, an MMP inhibitor. Incubation of control SL preparation with calpain, unlike MMP-2, depressed Na+-K+-ATPase activity and decreased α1, α2 and β2 without changes in β1. These results support the view that activation of calpain is involved in depressing Na+-K+-ATPase activity and degradation of its subunits in hearts subjected to I/R injury.

The developmental profiles of mRNA and protein expression for ornithine decarboxylase (ODC), the Na$ sp+$-dependent glucose co-transporter (SGLT1), sucrase isomaltase (SI), and the Na$ rm sp+K sp+$ ATPase $ alpha sb1$ and $ beta sb1$ subunit isoforms in the postnatal rat small intestine, as well as the effects of exogenous spermidine on their precocious development, were examined. Postnatal age had a significant effect with all enzymes and the nutrient transporter maturing around weaning. Consecutive exposure to exogenous spermidine during suckling precociously induced ODC mRNA, SI protein, and SGLT1 gene expression in the proximal and distal small intestine. Levels of Na$ rm sp+K sp+$ ATPase $ alpha sb1$ and $ beta sb1$ subunit isoform mRNA were precociously induced in the proximal small intestine only. These findings show that exposure to exogenous spermidine can promote precocious alterations in intestinal enzyme and nutrient transporter expression; however, it appears that spermidine must be continuously supplied for these alterations to persist in suckling rats.