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Coupled and Uncoupled Operational Mode of Nitric Oxide Synthase and the Regulation of the Sarcolemmal Na+- K+ ATPase: Receptor and Non-receptor Pathways

ARAN - Access to Research at NUI Galway

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dc.contributor.advisor Daly, Kieran
dc.contributor.author White, Caroline
dc.date.accessioned 2012-09-14T08:26:17Z
dc.date.available 2012-09-14T08:26:17Z
dc.date.issued 2012-05-09
dc.identifier.uri http://hdl.handle.net/10379/2969
dc.description.abstract The membrane Na+-K+ pump transports 3 Na+ ions out and 2 K+ ions into cells against their electrochemical gradient, using energy derived from hydrolysis of ATP. The Na+ and K+ ion gradients generated by the pump serve in secondary co- and counter transport processes critical for cell function. This thesis examined the hypothesis that synthesis of radical oxygen species/radical nitrogen species (ROS/RNS), coupled to hormone receptors and their intracellular messenger pathways, can regulate Na+-K+ pump activity in cardiac myocytes. A whole cell patch clamp technique was used measure the membrane current generated by the Na+-K+ pump (Ip) of isolated rabbit cardiac myocytes. Supplementing the patch pipette solution with L-arginine (L-Arg), a known substrate of nitric oxide synthase(NOS) stimulated Na+-K+ pump activity in a manner sensitive to the soluble guanylase cyclise inhibitor 1H-[1,2,4]Oxadiazole[4,3-a]quinoxalin-1-one (ODQ). In contrast, uncoupling NOS by exposing myocytes to the chemical paraquat, caused significant Na+-K+ pump inhibition which was abolished by ROS/RNS scavengers, ebselen, and superoxide dismutase(SOD). Since Angiotensin II (AngII) activates nicotinamide adenine dinucleotide phosphate hydrogen (NAD(P)H) oxidase, we tested the hypothesis that NAD(P)H oxidase mediates Ang II-induced pump inhibition. Ang II significantly inhibited the Na+-K+ pump. This was abolished by the addition of apocynin and gp91ds potent NAD(P)H oxidase inhibitors. The effect was also abolished by the addition of ¿ protein kinase C (¿PKC) inhibitor. Forskolin, an activator of adenyl cyclase was used to mimic ¿1/¿2 adrenergic receptor activation. It 4 decreased electrogenic Na+-K+ pump current (Ip). Interestingly, in addition to the expected role of protein kinase A (PKA) in this inhibition, inclusion of specific inhibitors implicated PKC and NADPH oxidase, i.e. cross talk between PKA and the same oxidative signalling pathway we had observed for Ang II. In summary, we have shown that PKC and PKA activate NADPH oxidase dependent inhibition of the Na+-K+ pump. The data provides a link between ROS/RNS and signalling mediated by neurohormonal activation in the control of cardiac myocyte Na+, and by inference, Ca2+ and may have important therapeutic implications in heart failure. This work has led to the very recent identification by our laboratory of the specific molecular mechanism by which oxidant signalling inhibits the Na+-K+ pump en_US
dc.subject Na+- K+ ATPase en_US
dc.subject Nitric oxide synthase en_US
dc.subject Whole cell patch clamp technique en_US
dc.subject Reactive nitrogen species en_US
dc.subject Reactive oxygen species en_US
dc.title Coupled and Uncoupled Operational Mode of Nitric Oxide Synthase and the Regulation of the Sarcolemmal Na+- K+ ATPase: Receptor and Non-receptor Pathways en_US
dc.type Thesis en_US
dc.local.note Cardiac cells generate an electrical current that controls contraction by the exchange of Na+, K+ and Ca2+ ions. This thesis examines the relationship between Nitric Oxide (NO) generation and its effects on the Na+-K+ pump molecule. It also examines the effects of oxygen radicals and reactive nitrogen species. en_US
dc.local.final Yes en_US

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