Abstract 162: Angiotensin II Regulates the Na+-K+ Pump in Rabbit Ventricular Myocytes via the ϵ-Isoform of Protein Kinase C, NAD(P)H Oxidase and Reactive Oxygen/Nitrogen Species
Phosphorylation of the Na+-K+ pump provides an unsatisfactory explanation for its regulation because putative phosphorylation sites are thought to be poorly accessible to regulatory kinases. However, the Na+-K+ pump molecule contains sites easily oxidised by reactive oxygen/nitrogen species (ROS/RNS) and the RNS peroxynitrate (ONOO-) inhibits the enzymatic equivalent of the pump, Na+-K+ ATPase, in isolated membrane fragments. We therefore examined if ROS/RNS regulate the pump in intact cells. This hypothesis is important for our understanding of pathophysiology and treatment of heart failure, a condition characterised by raised levels of myocardial ROS/RNS and intracellular Na+. We internally perfused myocytes with patch pipette solution that included 10 mM Na+ and, to support endogenous nitric oxide (NO) synthesis, 10 μ M L-arginine. Electrogenic pump current (Ip, arising from the 3:2 Na+: K+ exchange ratio and normalized for membrane capacitance) was identified as the shift in membrane current induced by 100 μ M ouabain at a holding potential of −40 mV. Ip (mean ± SEM) of controls was 0.48 ± 0.02 pA/pF, N = 7. Since Angiotensin II (Ang II) receptors are coupled to activation of NAD(P)H oxidase, the main sarcolemmal source of superoxide (O2· −), we exposed myocytes to 100 nM Ang II. Ip (0.36 ± 0.03 pA/pF, N = 6) was lower than Ip of controls (P<0.01). Superoxide dismutase (200 U/ml) included in pipette solutions abolished the Ang II-induced decrease in Ip (0.48 ± 0.05 pA/pF, N = 5). The decrease was also abolished by the inclusion of the gp91ds peptide (10 μ M) in pipette solutions to block assembly and hence activation of NAD(P)H oxidase (Ip=0.58 ± 0.06 pA/pF, N = 6). Since ONOO−., formed in the O2· − + NO → ONOO− reaction, should permeate the pump molecules’ hydrophobic milieu much more readily than O2· − we included the ONOO− scavenger ebselen (10 μ M) in pipette solutions. It abolished the Ang II-induced decrease in Ip. Since Ang II activates NAD(P)H oxidase via PKC we included in pipette solutions a specific peptide inhibitor (100 nM) of the most abundant protein kinase C (PKC) isoform in rabbit myocytes, ϵPKC. It abolished the Ang II-induced decrease in Ip. In conclusion, Ang II can inhibit the Na+-K+ pump via ϵPKC, NAD(P)H oxidase and O2· −/ONOO− in cardiac myocytes.