Ammonium chloride affects receptor number and lateral mobility of the vasopressin V 2-type receptor in the plasma membrane of LLC-PK 1 renal epithelial cells: Role of the cytoskeleton

Abstract

The acidotropic agent ammonium chloride (NH 4Cl) 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 NH 4Cl and perturbers of cytoskeleton structure on vasopressin V 2 receptor expression in LLC-PK 1 renal epithelial cells. Surprisingly, long-term pretreatment of cells with NH 4Cl 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 NH 4Cl in the binding test, and apparently the result of increased V 2 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 V 2 receptor of LLC-PK 1 cells, and microscopic measurements of membrane-localized fluorescence confirmed the increased V 2 receptor expression in the basal plasma membrane subsequent to NH 4Cl pretreatment. Lateral mobility of the V 2 receptor was measured in living cells using the technique of microphotolysis (photobleaching). The fraction of mobile receptors was 0.2 in cells pretreated with NH 4Cl, markedly reduced compared to that of 0.9 in untreated cells. The apparent lateral diffusion coefficient D was about 3 × 10 −10 cm 2/s in both pretreated and untreated cells. Results for fluorescence labeling of the actin cytoskeleton indicate that NH 4Cl pretreatment of LLC-PK 1 cells results in pertubation of microfilament structure. All results imply that the cytoskeleton plays a central role in V 2 receptor expression and lateral mobility.