Ammonium chloride affects receptor number and lateral mobility of the vasopressin V₂-type receptor in the plasma membrane of LLC-PK₁ renal epithelial cells: Role of the cytoskeleton

The acidotropic agent ammonium chloride (NH₄Cl) not only affects receptor metabolism by inhibiting lysosomal acidification, but can also affect the targeting of proteins to specific membranes in polarized cells, possibly through effects mediated by the cytoskeleton. The present study examines the effects of NH₄Cl and perturbers of cytoskeleton structure on vasopressin V₂ receptor expression in LLC-PK₁ renal epithelial cells. Surprisingly, long-term pretreatment of cells with NH₄Cl or short-term treatment with the actin perturber cytochalasin B resulted in an up to 70% increase in specific Arg-8-vasopressin binding compared to control cells, which was independent of the presence of NH₄Cl in the binding test, and apparently the result of increased V₂ receptor expression. Perturbers of microtubules such as colchicine and vinblastine had no such effect. A rhodamine-labeled analog of vasopressin was used to fluorescently label the V₂ receptor of LLC-PK₁ cells, and microscopic measurements of membrane-localized fluorescence confirmed the increased V₂ receptor expression in the basal plasma membrane subsequent to NH₄Cl pretreatment. Lateral mobility of the V₂ receptor was measured in living cells using the technique of microphotolysis (photobleaching). The fraction of mobile receptors was 0.2 in cells pretreated with NH₄Cl, markedly reduced compared to that of 0.9 in untreated cells. The apparent lateral diffusion coefficient D was about 3 × 10^-10 cm²/s in both pretreated and untreated cells. Results for fluorescence labeling of the actin cytoskeleton indicate that NH₄Cl pretreatment of LLC-PK₁ cells results in pertubation of microfilament structure. All results imply that the cytoskeleton plays a central role in V₂ receptor expression and lateral mobility.