Stimulation of the β2 adrenergic receptor rapidly increases phosphorylation of the Na+‐Clcotransporter (NCC)

Abstract

Adrenergic signaling is proposed to modulate activity of the thiazide‐sensitive Na+‐Clcotransporter (NCC) by direct effects on the renal distal convoluted tubule (DCT). However, there are conflicting data on whether, α1, β1 or β2‐adrenoceptors mediate these effects by activation of the with‐no‐lysine kinases (WNKs) and STE20/SPS1‐related proline alanine‐rich kinase (SPAK) kinase pathway or by other signaling pathways. Here, we determined using RT‐PCR that the β2‐adrenergic receptor was abundantly expressed in mpkDCT cells (a model of the DCT) and in DCT cells isolated from parvalbumin EGFP mice by fluorescence‐activated cell sorting. In mice administered salbutamol (β2 adrenergic receptor agonist used to treat asthma) for 30 min, phosphorylated NCC levels increased, indicating increased activity. However, mice developed hypokalemia, a condition that also increases NCC phosphorylation. To investigate direct effects of β2 adrenergic signaling on DCT cells independent of K+, the effects of salbutamol on mpkDCT cells and in kidney slices/tubule suspensions were examined. Salbutamol gradually increased NCC phosphorylation in the pharmacologically relevant concentration range of 0.01–10 μM, an effect observed after 15 min and maintained at 60 min. Changes in NCC phosphorylation were associated with increased cAMP levels in tubule suspensions, kidney slices and mpkDCT cells. A role for the NCC modulatory PKA/I‐1/PP1 pathway was suggested by attenuated effects of salbutamol on phosphorylated NCC during PKA/PP1 pathway inhibition or in slices from I‐1 KO mice. Inhibition of the WNK‐SPAK pathway with a WNK inhibitor also attenuated, but did not prevent, the salbutamol‐induced phosphorylation of NCC. In conclusion, β2 adrenergic receptor signaling modulates NCC phosphorylation in vivo and ex vivo via PKA and WNK‐dependent pathways. We speculate that this response may be a compensatory mechanism to limit electrogenic renal K+ secretion during β2 adrenergic receptor stimulation of muscle. Furthermore, frequent use of salbutamol in e.g. asthmatic patients may lead to renal NaCl retention and influence blood pressure.Support or Funding InformationThe Novo Nordisk Foundation, The Leducq Foundation and the Danish Medical Research Council.