PP2B upregulation mediates increased NO production independent of NOS3 phosphorylation in the renal medullary thick ascending limb during diabetes mellitus

Abstract

The Ser/Thr phosphatase PP2B is known to regulate NO synthase 3 (NOS3) activity. Diabetes increases renal medullary NOS3 activity and reduces its phosphorylation (Thr495) in the medullary thick ascending limb (mTAL). We hypothesized that diabetes upregulates PP2B in the mTAL, which stimulates NO production by dephosphorylating NOS3. Three weeks after induction of diabetes (streptozotocin; STZ), immunoblotting revealed increased PP2B‐Aβ (catalytic subunit) in mTAL suspensions from STZ vs Sham rats (3.2±0.6 vs 1.7±0.1 RDU, P<0.05) and a trend for increased PP2B‐B (regulatory subunit; 0.47±0.12 vs 0.24±0.02 RDU, P=0.07). NO production by STZ mTALs was greater than Sham (977±161 vs 397±116 pmol/30min/mg prot., P<0.05) and reduced by CsA (551±67 pmol/30min/mg prot.; P<0.05 vs untreated). Although CsA decreased PP2B activity in STZ mTALs (64±14 vs 21±6 nmol PO4/mg prot., P<0.05), it did not alter NOS3 phosphorylation at Thr495, Ser1177, or Ser633 in Sham or STZ mTALs. NOS3 phosphorylation at Ser114 and Ser617 was not apparent in mTALs by immunostaining, although evident in other renal cells. We conclude that PP2B upregulation accelerates NO production by the mTAL during diabetes, but this does not involve PP2B‐mediated changes in NOS3 phosphorylation. We speculate that the PP2B‐dependent NO production may involve phosphorylation of another protein that, in turn, regulates NOS activity. (Supported by the AHA)