Target Cell Polarity and Membrane Phosphorylation in Relation to the Mechanism of Action of Antidiuretic Hormone

Abstract

The plasma membrane of the bovine renal collecting duct epithelial cell has been resolved into its apical (luminal) and basal-lateral (contraluminal) components by free flow electr4iphoresis. The contraluminal, but not the luminal, membrane was found to contain antidiuretic hormone-sensitive adenylate cyclase. The luminal membrane was found to contain a cyclic 3': 5'-adenosine monophosphate-sensitive self-phosphorylating system consisting of a membrane-bound protein kinase and its membrane-bound substrate(s); this intrinsic protein kinase was not present in the contraluminal membrane.These Recently we have shown that parathyroid hormone (PT hormone)-sensitive adenylate cyclase is localized in the contraluminal (basal-lateral) but not in the luminal (apical) plasma membranes of rat renal cortical epithelial cells, whereas cAMP-dependent phosphorylation occurs in the luminal but not the contraluminal membranes (9, 10). To determine whether a similar enzymatic distribution occurs in other segments of the nephron, we have investigated the localization of antidiuretic hormone (AD hormone)-sensitive adenylate cyclase and of cAMP-dependent protein kinase in the papillary collecting duct epithelium. The results of these studies indicate that AD hormone-stimulated cAMP generation is localized in the contraluminal plasma membrane, whereas cAMP-mediated membrane phosphorylation is localized in the luminal plasma membrane. The latter localization-in conjunction with the well-established luminal site 10 mM triethanolamine-HCl, pH 7.6); all subsequent procedures were carried out at 0-4'. The kidneys were freed from adherent connective tissues and the papillae were removed by careful dissection of the renal pelvis. Six grams of tissue were minced in a small volume of ST-buffer and then diluted to a final volume of 30 ml. Aliquots (10 ml) of the suspension were homogenized in a Dounce homogenizer by five strokes with a loose-fitting pestle. The homogenates were combined and filtered through two layers of cheese cloth and further homogenized by 15 strokes with a tight-fitting pestle. This homogenate was centrifuged at 700 X g for 10 min; the supernatant was centrifuged at 10,000 X g for 10 mmi. The second supernatant was saved and the pellet resuspended in 5 ml of STbuffer. Then 10 ml of ST-buffer was added and the suspension centrifuged for 10 min at 10,000 X g. The resulting supernatant was combined with the "saved" supernatant and the solution was centrifuged at 100,000 X g for 1 hr. The pellet was suspended in 4 ml of electrophoresis buffer (8.5 mM acetic acid, 8.5 mM triethanolamine, and 280 mM sucrose adjusted to pH 7.4 with 2 M NaOH), homogenized with 10 strokes in the tight-fitting Dounce honiogenizer, and diluted to a final volume of 10 ml with the electrophoresis buffer. The resulting preparation, observed by electron microscopy to consist of membrane fragments and vesicles, was used to study the properties of papillary plasma membranes prior to electrophoretic separation.To prepare for electrophoresis, we cent...

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