Abstract P616: Evidence That The Extracellular Domain Of Na + /K + Atpase Is The Receptor For Cyclic Gmp-Induced Natriuresis

Abstract

Previous studies from our laboratory have shown that extracellular renal interstitial (RI) cyclic guanosine 3’5’-monophosphate (cGMP) increases urine sodium (Na + ) excretion (U Na V) at the renal proximal tubule (RPT) in rats via activation of Src family kinase. Extracellular cGMP engenders this response through an unknown receptor. We hypothesized that cGMP binds to the extracellular domain of Na + /K + -ATPase (NKA) on basolateral membranes of RPT cells inhibiting Na + transport. In the present study, we evaluated the effect of RI infusion of rostafuroxin (RF), a digitoxigenin derivative that specifically displaces oubain (OUA) binding from NKA, on U Na V in the presence of RI cGMP infusion. Volume expanded, uninephrectomized, 12-week-old female Sprague-Dawley rats received RI infusions of vehicle (D 5 W) (N=8), RI cGMP (18, 36, and 72 μg/kg/min; each dose for 30 min; N=10), or RI cGMP + RF (0.012 μg/kg/min; N=5) for 90 min following a 30 min control period with RI infusion of vehicle D 5 W. RI cGMP infusion induced a significant natriuresis from 0.39 ± 0.06 μmol/min to 1.03 ± 0.21 (P<0.05), 1.17 ± 0.19 (P<0.01), and 1.94 ± 0.16 (P<0.001) μmol/min at 18, 36, and 72 μg/kg/min cGMP, respectively. RI co-infusion of cGMP + RF abolished the cGMP-induced natriuresis at all doses (F=16.05, P<0.001). There was no change in mean arterial pressure during any infusion. To further demonstrate that cGMP binds to NKA, we performed a series of competitive binding studies in isolated RPTs from normal rat kidneys (N=4 for each) with bodipy-OUA (2 μM) + cGMP (10 μM) and 8-[Biotin]-AET-cGMP (2 μM) + OUA (10 μM). In the presence of cGMP, bodipy-OUA fluorescence intensity was reduced from 1422.1 ± 63 to 1072.5 ± 64 relative fluorescent units (RFU, P<0.01). In the presence of OUA, 8-[Biotin]-AET-cGMP staining was reduced from 1916.3 ± 144 to 1492.2 ± 84 RFU (P<0.05). Serving as control, biotinylated cAMP (N=2) did not demonstrate any fluorescence above background. Together, these data suggest that cGMP may compete with RF for binding on NKA and that the extracellular domain of NKA may serve as the receptor for cGMP-induced natriuresis.