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

The catalytic alpha 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 previousl y been shown to express low and high ouabain affinity alpha isoforms (alpha 1 and alpha 3 respectively; alpha 1>>alpha 3). The Na+, K+-ATPase density of isolated chondrocyte preparations was quantified by specific H-3-ouabain binding. Long-term elevation of extracellular medium [Na+] resulted in a s ignificant (31%; p<0.05) upregulation of Na+, K+-ATPase density and treatme nt with various pharmacological inhibitors (Brefeldin A, monensin and cyclo heximide) significantly (p<0.001) blocked the upregulation. The subcellular distribution of the Na+, K+-ATPase alpha isoforms was examined by immunofl uorescence confocal laser scanning microscopy which revealed predominantly plasma membrane immunostaining of alpha subunits in control chondrocytes. I n Brefeldin A treated chondrocytes exposed to high [Na+], Na+, K+-ATPase al pha isoforms accumulated in juxta-nuclear pools and plasma membrane Na+, K-ATPase density monitored by 3H-ouabain binding was significantly down-regu lated due to Brefeldin A mediated disruption of vesicular transport. There was a marked increase in intracellular alpha 1 and alpha 3 staining suggest ing that these isoforms are preferentially upregulated following long-term exposure to high extracellular [Na+]. The results demonstrate that Nat, K+- ATPase density in chondrocytes is elevated in response to increased extrace llular [Na+] through de novo protein synthesis of new pumps containing alph a 1 and alpha 3 isoforms, delivery via the endoplasmic reticulum-Golgi comp lex constitutive secretory pathway and insertion into the plasma membrane.