The β‐adrenergic stimulation of the renal NaCl cotransporter is mediated via a cAMP‐dependent activation of protein phosphatase 1 inhibitor 1

Abstract

Sympathetic β‐adrenergic activation of the thiazide‐sensitive NaCl cotransporter (NCC) in the renal distal convoluted tubule (DCT) was suggested to contribute to the development of salt‐sensitive hypertension. Previous studies demonstrated that β‐adrenergic agonists (e.g. norepinephrine and isoproterenol) increase cytoplasmic cAMP and the phosphorylation and hence the activity of NCC in the mouse DCT. Recently, we identified the endogenous inhibitor 1 (I1) of protein phosphatase 1 (PP1) as a DCT enriched gene product that controls NCC phosphorylation and blood pressure. As I1 is activated by cAMP‐dependent protein kinase A (PKA), we now tested the hypothesis that β‐adrenergic agonists inhibit PP1 via a cAMP/PKA‐dependent activation of I1 ultimately stimulating NCC phosphorylation. To test this hypothesis we performed experiments on MDCK cells stably transfected with NCC and on ex vivo preparations of kidneys from wildtype (WT) and I1‐knockout (I1‐KO) mice. The exposure of isolated DCTs to the cAMP‐elevating drugs forskolin and IBMX rapidly increased the phosphorylation of NCC via a protein kinase A (PKA)‐dependent pathway. The forskolin/IBMX‐induced phosphorylation of NCC was paralleled by I1 phosphorylation at its PKA‐consensus phosphorylation site (T35). Moreover, the forskolin/IBMX‐induced phosphorylation of NCC was diminished in kidney slices of from I1‐KO mice. The transgenic overexpression of a phosphomimetic I1 mutant (T35D) in kidneys of I1‐KO mice restored NCC phosphorylation, but made NCC resistant to forskolin/IBMX stimulation. Using an unbiased yeast‐two‐hybrid screen and co‐immunoprecipitation assays, we demonstrate that NCC and PP1 physically interact in transfected MDCK cells. Finally, the isoproterenol stimulated increase of NCC‐phosphorylation was impaired in I1‐KO mice as assessed in kidney slices and isolated‐perfused mouse kidneys. Remarkably, this occurred independent from any change in the phosphorylation of the kinases SPAK/OSR1. Thus, our data establish a complete signal transduction pathway by which cAMP increases NCC phosphorylation via a PKA‐dependent phosphorylation of I1 and subsequent inhibition of PP1. This pathway likely accounts for beta‐adrenergic NCC activation and hence may contribute to salt‐sensitive hypertension in patients with sympathetic hyperactivity.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.