Insulin-induced Stimulation of Na⁺,K⁺-ATPase Activity in Kidney Proximal Tubule Cells Depends on Phosphorylation of the α-Subunit at Tyr-10

Abstract

Phosphorylation of the ␣-subunit of Na ϩ ,K ϩ -ATPase plays an important role in the regulation of this pump. Recent studies suggest that insulin, known to increase solute and fluid reabsorption in mammalian proximal convoluted tubule (PCT), is stimulating Na ϩ ,K ϩ -ATPase activity through the tyrosine phosphorylation process. This study was therefore undertaken to evaluate the role of tyrosine phosphorylation of the Na ϩ ,K ϩ -ATPase ␣-subunit in the action of insulin. In rat PCT, insulin and orthovanadate (a tyrosine phosphatase inhibitor) increased tyrosine phosphorylation level of the ␣-subunit more than twofold. Their effects were not additive, suggesting a common mechanism of action. Insulin-induced tyrosine phosphorylation was prevented by genistein, a tyrosine kinase inhibitor. The site of tyrosine phosphorylation was identified on Tyr-10 by controlled trypsinolysis in rat PCTs and by site-directed mutagenesis in opossum kidney cells transfected with rat ␣-subunit. The functional relevance of Tyr-10 phosphorylation was assessed by 1) the abolition of insulin-induced stimulation of the ouabain-sensitive 86 Rb uptake in opossum kidney cells expressing mutant rat ␣1-subunits wherein tyrosine was replaced by alanine or glutamine; and 2) the similarity of the time course and dose dependency of the insulin-induced increase in ouabain-sensitive 86 Rb uptake and tyrosine phosphorylation. These findings indicate that phosphorylation of the Na ϩ ,K ϩ -ATPase ␣-subunit at Tyr-10 likely participates in the physiological control of sodium reabsorption in PCT.

Activities of Three Erythrocyte Enzymes of Hyperglycemic Rats (Rattus norvegicus) Treated with Allium sativa Extract.

“…[11] The relatively reduced levels of erythrocyte Na + /K + -ATPase activity in hyperglycemic rats ( Figure 3) was consistent with the findings of previous authors. Soulis-Liparota et al, [64] reported reduced Na + / K + -ATPase activity streptozotocin-induced diabetic rats with nephropathy, whereas others [65,66] reported impairment in the enzyme activity in diabetic rats and mice with retinopathy.…”

“…[11,13,72] Hyperglycemia with associated depressed glucose utilization in diabetic states results in low intracellular ATP concentration, insufficient for the required obligatory phosphorylation of the enzyme. The dose dependent increase in erythrocyte Na + /K + -ATPase activity in hyperglycemic rat treated with extract of A. sativa as an instrument of glycemic control was an indication of improved glucose utilization exemplified in hyperglycemic rats treated with the standard anti-diabetic drug.…”

“…[11,12] The regulation of this pump activity is dependent on the phosphorylation of the α-subunit of Na + /K + -ATPase. [11,13] Allium sativa has been widely reported to exhibit the rapeutic benefits to numerous pathologic states whose etiology is linked to oxidative stressors and electrophiles [14] such as diabetes mellitus, [15][16][17][18] atherosclerosis, [19,20] hyperlipidemia [20,21] thrombosis, [22] hypertension. [23] Phytochemical and biochemical profiles of A. sativa have been reported elsewhere.…”

Structural Basis for α1 Versus α2 Isoform-distinct Behavior of the Na,K-ATPase

“…The significance of this difference is underscored by the observation that in transfected opossum kidney cells (24) protein kinase C␤-mediated phosphorylation of ␣1 at these residues promotes its translocation to the plasma membrane. Similarly, in ␣1 but not ␣2, the presence of Tyr-5 within a consensus sequence for phosphorylation by tyrosine kinases of the Src family has been implicated in the insulin stimulation of pump activity in the proximal convoluted tubules of the rat kidney (25). Furthermore, although insulin acts via tyrosine kinases in rat proximal convoluted tubules to stimulate pump activity by increasing the apparent Na ϩ affinity (26) in skeletal muscle, it promotes translocation of ␣2 to the plasma membrane (27) via the action of protein kinase C (28).…”

Blood Na+/K+ and Cl- Levels of Hyperglycemic Rats Administered with Traditional Herbal Formulations

“…5,6 Additionally, Na + /K + -ATPase activity is regulated by insulin action. 7,8 Therefore, low circulating insulin level compromises Na + /K + -ATPase activity with concomitant poor Na + and K + metabolism and transport across biomembranes as well as hindered facilitative uptake of monosaccharides by intestinal epithelia. 9,10 In diabetes mellitus (DM), hyperglycemia imposes glucose induced osmotic diuresis with resultant loss of body fluids and electrolytes.…”