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

White, Caroline
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
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Attribution-NonCommercial-NoDerivs 3.0 Ireland