Protein Phosphatase 1 Inhibitor–1 Mediates the cAMP-Dependent Stimulation of the Renal NaCl Cotransporter

Abstract

Background: A number of cAMP-elevating hormones stimulate phosphorylation (and hence activity) of the NaCl cotransporter (NCC) in the distal convoluted tubule (DCT). Evidence suggests that protein phosphatase 1 (PP1) and other protein phosphatases modulate NCC phosphorylation, but little is known about PP1's role and the mechanism regulating its function in the DCT. Methods: We used ex vivo mouse kidney preparations to test whether a DCT-enriched inhibitor of PP1, protein phosphatase 1 inhibitor-1 (I1), mediates cAMP's effects on NCC, and conducted yeast two-hybrid and coimmunoprecipitation experiments in NCC-expressing MDCK cells to explore protein interactions. Results: Treating isolated DCTs with forskolin and IBMX increased NCC phosphorylation via a protein kinase A (PKA)dependent pathway. Ex vivo incubation of mouse kidney slices with isoproterenol, norepinephrine, and parathyroid hormone similarly increased NCC phosphorylation. The cAMP-induced stimulation of NCC phosphorylation strongly correlated with the phosphorylation of I1 at its PKA consensus phosphorylation site (a threonine residue in position 35). We also found an interaction between NCC and the I1-target PP1. Moreover, PP1 dephosphorylated NCC in vitro, and the PP1 inhibitor calyculin A increased NCC phosphorylation. Studies in kidney slices and isolated perfused kidneys of control and I1-KO mice demonstrated that I1 participates in the cAMP-induced stimulation of NCC. Conclusions: Our data suggest a complete signal transduction pathway by which cAMP increases NCC phosphorylation via a PKAdependent phosphorylation of I1 and subsequent inhibition of PP1. This pathway might be relevant for the physiologic regulation of renal sodium handling by cAMP-elevating hormones, and may contribute to salt-sensitive hypertension in patients with endocrine disorders or sympathetic hyperactivity. I1 mediates cAMP stimulation of NCC Significance statement:The thiazide-sensitive NaCl cotransporter (NCC) in the distal convoluted tubule (DCT)is critical for the renal control of ion homeostasis and blood pressure. NCC phosphorylation, and hence activity, is increased by cAMP-elevating stimuli, including β-adrenergic agonists and PTH. We tested the hypothesis that the inhibitor 1 (I1) of protein phosphatase 1 (PP1) mediates the effects of cAMP-elevating hormones on NCC. Using several in vitro and ex vivo approaches, we propose a novel signaling pathway in which a PKA-dependent phosphorylation of I1 inhibits the PP1-dependent dephosphorylation of NCC. This novel pathway may contribute to the physiological regulation of NCC and the development of arterial hypertension in the context of abnormal hormonal stimulation. I1 mediates cAMP stimulation of NCC Abstract Background: The NaCl cotransporter (NCC) in the distal convoluted tubule (DCT) is critical for renal Na + reabsorption and blood pressure control. A number of cAMPelevating hormones stimulate NCC phosphorylation, and hence activity. Methods: Using a variety of experiments on mouse ex vivo kidney preparations, we t...

Regulation of the Renal NaCl Cotransporter and Its Role in Potassium Homeostasis

“…The role of PP1 was confirmed when the endogenous inhibitor of PP1, I-1, was deleted in mice, and pNCC levels decreased independent of total NCC abundance (242). Furthermore, PP1 interacts with NCC and can dephosphorylate it in vitro (239). The current view is that changes in cAMP, which can occur via, e.g., the ␤-adrenergic receptor or the vasopressin type 2 receptor (V2R), stimulates protein kinase A (PKA) to phosphorylate I-1, which inhibits PP1 and ultimately leads to an increase in pNCC.…”

Role of α2-Adrenoceptors in Hypertension: Focus on Renal Sympathetic Neurotransmitter Release, Inflammation, and Sodium Homeostasis

“…Activation of α1-adrenoceptors inhibits the suppression of NCC during high salt intake via a WNK/SPAK/ OxSR1-dependent signaling pathway in rat kidneys (Frame et al, 2019). Dephosphorylation of NCC by the protein phosphatase 1 can be inhibited through a protein kinase A-dependent activation of the protein phosphatase 1 inhibitor via β1-adrenergic receptor activation (Penton et al, 2019).…”

Modified HEK cells simulate DCT cells in their sensitivity and response to changes in extracellular K

“…In one of our earlier in vitro studies in Xenopus oocytes, NCC activity was found to be regulated by Protein phosphatase 4 (PP4) through its interaction with a conserved N‐terminal amino acid threonine 58 of NCC, and this regulation was independent of WNK4 regulated NCC trafficking to the membrane (Glover, Mercier Zuber, Figg, & O'Shaughnessy, ). More recently, signaling pathways independent of the WNK4/SPAK cascade have been shown to regulate NCC phosphorylation (and therefore its activity) in the DCT (Penton et al, ). In vitro studies showed that protein phosphatase 1 (PP1) dephosphorylates NCC whereas the PP1 inhibitor calyculin A had the opposite effect, and protein kinase A (PKA)‐dependent phosphorylation of the PP1 inhibitor 1 abolished the PP1‐mediated dephosphorylation of NCC in ex vivo mouse kidney preparations (Penton et al, ).…”

“…More recently, signaling pathways independent of the WNK4/SPAK cascade have been shown to regulate NCC phosphorylation (and therefore its activity) in the DCT (Penton et al, ). In vitro studies showed that protein phosphatase 1 (PP1) dephosphorylates NCC whereas the PP1 inhibitor calyculin A had the opposite effect, and protein kinase A (PKA)‐dependent phosphorylation of the PP1 inhibitor 1 abolished the PP1‐mediated dephosphorylation of NCC in ex vivo mouse kidney preparations (Penton et al, ). A comprehensive study of PP inhibitors in our cells has not been done to confirm if either PP1 or PP4 are important controllers of NCC dephosphorylation in our model HEK cells.…”