Brefeldin A influences the cell surface abundance and intracellular pools of low and high ouabain affinity Na+, K+-ATPase cx subunit isoforms in articular chondrocytes

Abstract

The catalytic a isoforms of the Na+, K+- ATPase and stimuli controlling the plasma membrane abundance and intracellular distribution of the enzyme were studied in isolated bovine articular chondrocytes which have previously been shown to express low and high ouabain affinity a isoforms (a1 and a 3 respectively; al>>a3). The Naf, K+-ATPase density of isolated chondrocyte preparations was quantified by specific 3 ~ - ouabain binding. Long-term elevation of extracellular medium [Na+] resulted in a significant (31%; p<0.05) upregulation of Na+, K+-ATPase density and treatment with various pharmacological inhibitors (Rrefeldin A, monensin and cycloheximide) significantly @<0.001) blocked the upregulation. The subcellular distribution of the NaC, K+-ATPase a isoforms was examined by immunofluorescence confocal laser scanning microscopy which revealed predominantly plasma membrane immunostaining of a subunits in control chondrocytes. In Brefeldin A treated chondrocytes exposed to high [Na+], Na+, K+-ATPase a isoforms accumulated in juxta-nuclear pools and plasma membrane Na+, K+- ATPase density monitored by 3~-ouaba inb inding was significantly down-regulated due to Brefeldin A mediated disruption of vesicular transport. There was a marked increase in intracellular a 1 and a3 staining suggesting that these isoforms are preferentially upregulated following long-term exposure to high extracellular [Na+]. The results demonstrate that Na+, K+-ATPase density in chondrocytes is elevated in response to increased extracellular [Na+] through de novo protein synthesis of new pumps containing a 1 and a 3 isoforms, delivery via the endoplasrnic reticulum- Golgi complex constitutive secretory pathway and insertion into the plasma membrane.