Abstract 196: Involvement of Peroxynitrite Formation in Angiotensin II Induced Changes in Na+K+ATPase Activity in HK2 Cells

Angiotensin II (AngII) induces both superoxide (O 2 - ) and nitric oxide (NO) generation forming peroxynitrite (ONOO - ) in biological systems. To determine the role of ONOO - in AngII induced sodium excretory responses, we examined Na + K + ATPase (NKA) activity in cultured HK2 cells (human kidney proximal tubule cell line) incubated for 30 min with a wide range (10 pM to 200 μM) of AngII concentrations (conc) in the presence or absence of a ONOO - scavenger, mercapto-ethyl-guanadine (MEG; 200μM). Post incubation HK2 cellular membrane fractions were used for measurement of NKA activity via colorimetric assay capable of detecting inorganic phosphate (Pi). Baseline value of NKA activity in these HK2 cells was measured as 10.3 ±0.7 μmoles of Pi liberated/mg protein/hr (n=12). AngII exerts dose-dependent differential effects on NKA activity. Compared to the baseline value, NKA activity was increased at lower conc (13.7±1.3% at 10 pM to 19.6±1.5% at 100nM) and decreased at higher conc (-6.0±0.8% at 1 μM to -38±2.1% at 200 μM ) without any significant effect at 500 nM conc (-1.2±0.6%) of AngII. Interestingly, MEG treatment markedly attenuated these AngII induced changes in NKA activity, both at lower conc (activity increased only to 7.8±0.67% at 10 pM and to 8.9±0.57% at 100nM; an average reduction of 33.2±2.4% in stimulatory effects) and at higher conc (activity decreased to -3.0±0.7% at 1 μM and to -20±0.57% at 200 μM; an average reduction of 54.4±4.1% in inhibitory effects). Co-incubation with O 2 - scavenger, tempol (1 mM) or NO synthase inhibitor, nitro-L-arginine methyl ester (100 μM) did not alter these AngII induced responses in HK2 cells indicating that neither O 2 - , nor NO, was directly involved in mediating these responses. AT 1 receptor (AT 1 R) blocker, losartan (10μM) treatment prevented these AngII induced changes on NKA activity confirming the involvement of AT 1 R signaling in these responses. These findings demonstrate a direct contributory role for concomitant ONOO - generation in mediating AngII induced changes in NKA activity in the proximal tubular cells. These data also suggest a reno-protective role for ONOO - in minimizing sodium retaining action of AngII by the renal tubules, particularly in the conditions associated with enhanced renin-angiotensin system.