Protein kinase A induces recruitment of active Na⁺,K⁺‐ATPase units to the plasma membrane of rat proximal convoluted tubule cells

Abstract

The aim of this study was to investigate the mechanism of control of Na+,K+‐ATPase activity by the cAMP‐protein kinase A (PKA) pathway in rat proximal convoluted tubules. For this purpose, we studied the in vitro action of exogenous cAMP (10−3 M dibutyryl‐cAMP (db‐cAMP) or 8‐bromo‐cAMP) and endogenous cAMP (direct activation of adenylyl cyclases by 10−5 M forskolin) on Na+,K+‐ATPase activity and membrane trafficking. PKA activation stimulated both the cation transport and hydrolytic activity of Na+,K+‐ATPase by about 40 %. Transport activity stimulation was specific to the PKA signalling pathway since (1) db‐cAMP stimulated the ouabain‐sensitive 86Rb+ uptake in a time‐ and dose‐dependent fashion; (2) this effect was abolished by addition of H‐89 or Rp‐cAMPS, two structurally different PKA inhibitors; and (3) this stimulation was not affected by inhibition of protein kinase C (PKC) by GF109203X. The stimulatory effect of db‐cAMP on the hydrolytic activity of Na+,K+‐ATPase was accounted for by an increased maximal ATPase rate (Vmax) without alteration of the efficiency of the pump, suggesting that cAMP‐PKA pathway was implicated in membrane redistribution control. To test this hypothesis, we used two different approaches: (1) cell surface protein biotinylation and (2) subcellular fractionation. Both approaches confirmed that the cAMP‐PKA pathway was implicated in membrane trafficking regulation. The stimulation of Na+,K+‐ATPase activity by db‐cAMP was associated with an increase (+40 %) in Na+,K+‐ATPase units expressed at the cell surface which was assessed by Western blotting after streptavidin precipitation of biotinylated cell surface proteins. Subcellular fractionation confirmed the increased expression in pump units at the cell surface which was accompanied by a decrease (‐30 %) in pump units located in the subcellular fraction corresponding to early endosomes. In conclusion, PKA stimulates Na+,K+‐ATPase activity, at least in part, by increasing the number of Na+‐K+ pumps in the plasma membrane in proximal convoluted tubule cells.

C-Peptide stimulates Na+,K+-ATPase activity via PKC alpha in rat medullary thick ascending limb

“…On the other hand, the cell surface expression of the Na + -pump was not altered by C-peptide, therefore increased Na + ,K + -ATPase V max values relies on an increased turnover rate. In agreement with this observation, cAMP synthesis, which results in stimulation of Na + ,K + -ATPase activity through an increase of Na + ,K + -ATPase cell surface expression, was not altered by C-peptide [21,33].…”

“…Phosphorylation of the catalytic α-subunit was assessed by Na + ,K-ATPase immunoprecipitation after 32 P labelling of the α-subunit, as described elsewhere [21]. Outer medullary tubule suspensions were preincubated at 30°C for 2 h in oxygenated solution containing 37.10 Bq/µl [ 32 P]-orthophosphoric acid and then incubated with or without drugs and/or hormones.…”

“…The Na + ,K + -ATPase cell surface expression was calcualted as previously described [21]. After preincubation in the absence of or with C-peptide, pools of 60 microdissected MTALs were incubated with 1.5 mgml −1 sulfo-NHS-S-S-Biotin for 1 hour at 4°C.…”

Analysis of Na+,K+-ATPase Motion and Incorporation into the Plasma Membrane in Response to G Protein–coupled Receptor Signals in Living Cells

“…The action of these networks ultimately results in activation of protein kinases and phosphorylation of the Na ϩ ,K ϩ -ATPase catalytic ␣-subunit (Therien and Blostein, 2000). Depending on the type and isoform of the kinase their action could lead to a decrease or increase in cell Na ϩ ,K ϩ -ATPase activity (Carranza et al, 1998;Efendiev et al, 1999Efendiev et al, , 2000Ridge et al, 2002). However, phosphorylation may not affect the intrinsic properties of the Na ϩ ,K ϩ -ATPase (i.e., catalytic activity) but their subcellular distribution.…”

Regulation of Renal Potassium Channels by Protein Kinases and Phosphatases

“…This report demonstrated that the functional coupling of CaN and PKA modulated Ca 2+ release in ventricular myocytes (Santana et al, 2002). It has also been demonstrated that Na + -K + www.intechopen.com pump at the basolateral membrane of kidney tubular epithelia was inhibited by CyA (Tumlin & Sands, 1993) and stimulated by PKA (Carranza et al, 1998). On the other hand, a cardiac Na + /Ca 2+ exchanger was reported to be regulated by CaN and PKC (Shigekawa et al, 2007).…”