Abstract
The balance and cross-talk between natruretic and antinatruretic hormone receptors plays a critical role in the regulation of renal Na+ homeostasis, which is a major determinant of blood pressure. Dopamine and angiotensin II have antagonistic effects on renal Na+ and water excretion, which involves regulation of the Na+,K+-ATPase activity. Herein we demonstrate that angiotensin II (Ang II) stimulation of AT1 receptors in proximal tubule cells induces the recruitment of Na+,K+-ATPase molecules to the plasmalemma, in a process mediated by protein kinase Cbeta and interaction of the Na+,K+-ATPase with adaptor protein 1. Ang II stimulation led to phosphorylation of the alpha subunit Ser-11 and Ser-18 residues, and substitution of these amino acids with alanine residues completely abolished the Ang II-induced stimulation of Na+,K+-ATPase-mediated Rb+ transport. Thus, for Ang II-dependent stimulation of Na+,K+-ATPase activity, phosphorylation of these serine residues is essential and may constitute a triggering signal for recruitment of Na+,K+-ATPase molecules to the plasma membrane. When cells were treated simultaneously with saturating concentrations of dopamine and Ang II, either activation or inhibition of the Na+,K+-ATPase activity was produced dependent on the intracellular Na+ concentration, which was varied in a very narrow physiological range (9-19 mm). A small increase in intracellular Na+ concentrations induces the recruitment of D1 receptors to the plasma membrane and a reduction in plasma membrane AT1 receptors. Thus, one or more proteins may act as an intracellular Na+ concentration sensor and play a major regulatory role on the effect of hormones that regulate proximal tubule Na+ reabsorption.
Highlights
The balance and cross-talk between natruretic and antinatruretic hormone receptors plays a critical role in the regulation of renal Na؉ homeostasis, which is a major determinant of blood pressure
We observed that treatment of opossum kidney (OK) cells with angiotensin II (Ang II) induced a concentrationdependent increase in Rbϩ transport, and a maximal activation was achieved with 1 pM Ang II (Fig. 1A)
The present study demonstrates that changes in [Naϩ]i modulate the number of DA and Ang II receptors present at the plasma membrane
Summary
Materials—Cells culture supplies were purchased from Invitrogen and Hyclone Laboratories (Logan, UT). Determination of Rbϩ Transport—Measurements of NKA-mediated transport by Rbϩ uptake were performed with attached cells as described previously [22,23,24]. Anti-␣1 antibody was added, and the suspension was incubated for 1 h at 4 °C with end-over-end shaking and overnight with protein A/G-agarose, which had been pre-washed three times with phosphate-buffered saline and once with immunoprecipitation buffer containing 1% Triton X-100. The precipitated material was separated by SDS-PAGE, and the proteins were transferred to a piece of polyvinylidene difluoride membrane This was assayed by Western blot analysis with anti-AP1 antibody. Equal amounts of solvents were added to control and test samples These amounts were minimal, and they did not appreciably change the ouabain-sensitive NKA-mediated Rbϩ transport. Determinations of protein concentration and immunoprecipitation of NKA were performed as described previously [24]. Comparisons between groups were performed by either Student’s t test for unpaired data or analysis of variance, as indicated in the figure legends
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