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Beta‐adrenergic receptor stimulation enhances phosphorylation of NKCC2 at threonine96/101 via cAMP but independently of PKA (892.21)
Author(s) -
Haque Mohammed,
Ares Gustavo,
Ortiz Pablo
Publication year - 2014
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.28.1_supplement.892.21
Subject(s) - stimulation , phosphorylation , rolipram , medicine , forskolin , cotransporter , endocrinology , ibmx , phosphodiesterase , chemistry , protein kinase a , receptor , kinase , sodium , biology , biochemistry , enzyme , organic chemistry
Beta‐adrenergic receptor (β‐AR) stimulation enhances phosphorylation of NKCC2 at threonine96/101 via cAMP but independently of PKA Mohammed Z. Haque, Gustavo R. Ares and Pablo A. Ortiz The thick ascending limb (TAL) reabsorbs 30% of the filtered sodium via the apical Na‐K‐2Cl cotransporter NKCC2. NKCC2 activity and trafficking to the apical surface are enhanced by β‐AR stimulation via cAMP. NKCC2 activity may also be stimulated by phosphorylation at threonine96/101 (Thr96/101) by SPAK/OSR1 kinases and serine126 (Ser126) by PKA. However, it is not known whether SPAK and NKCC2 phosphorylation (P‐NKCC2) at Thr96/101 are enhanced by cAMP and stimulation of β‐ARs in the TAL. We hypothesized that β‐AR stimulation increases Thr96/101 P‐NKCC2 via cAMP and SPAK. To maximally stimulate β‐ARs signaling we treated TALs (20 min) with 1µM isoproterenol and the phosphodiesterase4 inhibitor rolipram (ISO). ISO enhanced Thr96/101 and Ser126 P‐NKCC2 by 79±28% and 71±28% ( p <0.05, n=6). ISO also enhanced phosphorylated SPAK at Ser373 by 50±4 % ( p <0.05, n=6). Inhibition of PKA (H‐89,10 µM) did not inhibit β‐AR stimulated Thr96/101 P‐NKCC2. We then tested whether cAMP enhanced Thr96/101 P‐NKCC2 by stimulating endogenous cAMP with forskolin and the phosphodiesterase inhibitor IBMX (20 min). cAMP increased Thr96/101 by 30± 8% ( p <0.05, n=4) and this was not blocked by PKA inhibition; whereas Ser126 P‐NKCC2 was enhanced by 32±6 fold ( p <0.05) and PKA inhibition blocked this effect by 84±4%. We conclude that β‐adrenergic receptor stimulation enhances NKCC2 phosphorylation at Thr96/101 via cAMP and SPAK/OSR1 whereas PKA mediates the effect of cAMP on Ser126. Grant Funding Source : AHA/NIH