Abstract P437: The Extracellular Domain of Na + /K + ATPase Serves as a Receptor for Cyclic GMP in Mediating Natriuresis

Abstract

Previous studies from our laboratory have shown that extracellular renal interstitial (RI) cyclic guanosine 3’5’-monophosphate (cGMP) inhibits renal proximal tubule (RPT) sodium reabsorption and induces natriuesis via activation of Src family kinase in vivo . The cellular mechanisms by which extracellular cGMP regulates this process are unknown. Previous immunofluorescence competitive binding studies in isolated RPTs from normal rat kidneys demonstrated that cGMP and ouabain (OUA) compete for binding to Na + /K + -ATPase (NKA). We hypothesized that cGMP binds to the extracellular domain of the α1-subunit of NKA on basolateral membranes of RPT cells thereby inhibiting Na + transport. In the present study, we cross-linked cGMP to isolated RPTs from normal rat kidneys (N=6). We incubated RPT cells with 4-N 3 ;-PET-8-Biotin-11-cGMP (azido cGMP) or 8-N 3 -6-Biotin-10-cAMP (azido cAMP; both at 2 μM) in the presence or absence of UV light to induce cross linking; azido cAMP served as a negative control. Immunoprecipitation was then performed using strepavidin beads followed by Western blot analysis probing for NKA. RPTs with cross linked azido cGMP exhibited a strong signal for NKA that was significantly weaker for non-cross linked azido cGMP (33.4 ± 6%; P<0.0001) and cross linked (45.8 ± 8%; P<0.001) or non-cross linked (35.2 ± 6%; P<0.001) azido cAMP samples. We further demonstrated that in the presence of OUA (10 μM) NKA was reduced in cross linked azido cGMP RPTs, providing confirmation for previous immunofluorescence competition studies. Azido cGMP cross linked samples (N=3) were subsequently separated in a Tris-HCl gel and stained with Coomassie Blue. The protein band corresponding to NKA was excised and processed for mass spectrometry. NKA was identified as the second most ubiquitous protein in that band with 50 unique peptides for NKA represented covering 47% of all the amino acids in NKA. Together, these data demonstrate that cGMP competes with OUA for binding on NKA and that NKA serves as a receptor for cGMP thereby mediating natriuresis.