Signaling to and from the sodium pump : effects of insulin and cardiotonic steroids

Insulin and proinsulin-connecting peptide (C-peptide) stimulate Na+,K+-ATPase activity. The molecular mechanism by which insulin and C-peptide regulate the Na-pump in differentiated primary human skeletal muscle cells (HSMCs) and primary human renal tubular cells (HRTCs) was determined. Insulin- and C-peptide stimulated Na+,K+-ATPase activation, as assessed by ouabain-sensitive 86Rb+ uptake and 3H-ouabain binding, phosphorylation of α- and translocation of α- and β-subunits to plasma membrane from intracellular stores. These effects were abolished by the MEK1/2 inhibitor (PD98059).

Furthermore, inhibitors of PI3 kinase (wortmannin) and PKC (GF109203X) had similar effects. Notably, insulin-stimulated ERK1/2 phosphorylation was abolished by wortmannin and GF109203X. Insulin led to an increase in phosphorylation of the α1- and α2-subunits. C-peptide caused phosphorylation of the human α1-subunit on a Thr-Pro amino acid motif that confers a specific ERK phosphorylation site. ERK1 and 2 kinases were able to phosphorylate the α-subunit of purified human Na-pump in vitro. Thus, insulin and C-peptide activate Na+,K+-ATPase via a MAP kinase signaling pathway.

Exercise and in vitro skeletal muscle contraction leads to a significant increase in the plasma membrane abundance of the Na-pump α1- and α2-subunits via an ERK1/2 MAPK-dependent mechanism.

Cardiotonic steroids (CTS) initiate signaling cascade through the Na+,K+-ATPase, and increase growth and proliferation of different cell types. The effects of cardiotonic steroids, ouabain and marinobufagenin, on glucose metabolism in HSMC and rat skeletal muscle were explored in an effort to clarify the mechanisms of CTS signal transduction. Ouabain at a low dose increased glycogen synthesis additively to the effect of insulin. This effect was independent of PI3-kinase inhibitor LY294002, and abolished in the presence of either the MEK1/2 inhibitor PD98059 or the Src inhibitor PP2. Insulin-stimulated Akt phosphorylation was unaffected by ouabain.

Ouabain increased Src-dependent tyrosine phosphorylation of the α1- and α2-subunits and promoted a direct interaction of α-subunits with Src and formation of signaling complex. Phosphorylation of ERK1/2, GSK3α/β and p90rsk activity, were increased in response to ouabain independently of insulin action. These effects were prevented in the presence of PD98059 and PP2.

Incubation of cells with ouabain increased phosphorylation of the α-subunits at a Thr-Pro motif. Ouabain treatment decreased cell surface abundance of the α2-subunit, whereas the abundance of the α1-subunit was unchanged. Marinobufagenin (10 nM), an endogenous vertebrate bufadienolide cardiotonic steroid, increased glycogen synthesis in HSMC and this effect was similar to that observed in cells exposed to 100 nM ouabain. Incubation of skeletal muscle with ouabain did not induce metabolic stress.

In conclusion, activation of a MAPK signaling cascade stimulates Na+,K+-ATPase activity via phosphorylation of the α-subunits and translocation to plasma membrane. Cardiotonic steroids ouabain and marinobufagenin stimulate glycogen synthesis additively to insulin in skeletal muscle. The effect of cardiotonic steroids is mediated by activation of Src-, ERK1/2-, p90rsk-, and GSK3-dependent signaling pathway.

List of scientific papers

I. Al-Khalili L, Kotova O, Tsuchida H, Ehren I, Feraille E, Krook A, Chibalin AV. (2004). ERK1/2 mediates insulin stimulation of Na(+),K(+)-ATPase by phosphorylation of the alpha-subunit in human skeletal muscle cells. J Biol Chem. 279(24): 25211-8.
https://pubmed.ncbi.nlm.nih.gov/15069082

II. Zhong Z, Kotova O, Davidescu A, Ehren I, Ekberg K, Jornvall H, Wahren J, Chibalin AV. (2004). C-peptide stimulates Na+, K+-ATPase via activation of ERK1/2 MAP kinases in human renal tubular cells. Cell Mol Life Sci. 61(21): 2782-90.
https://pubmed.ncbi.nlm.nih.gov/15549182

III. Kotova O, Galuska D, Essen-Gustavsson B, Chibalin AV, (2006). Metabolic and signaling events mediated by cardiotonic steroid ouabain in rat skeletal muscle. Cell Mol Biol. [Accepted]
https://pubmed.ncbi.nlm.nih.gov/17535736

IV. Kotova O, Al-Khalili L, Talia S, Hooke C, Fedorova OV, Bagrov AY, Chibalin AV. (2006). Cardiotonic steroids stimulate glycogen synthesis in human skeletal muscle cells via a Src- and ERK1/2-dependent mechanism. J Biol Chem. 281(29): 20085-94.
https://pubmed.ncbi.nlm.nih.gov/16714287