IMMUNOLOCALIZATION OF THE SECRETORY ISOFORM OF NA-K-CL COTRANSPORTER IN RAT RENAL INTERCALATED CELLS

Two bumetanide-sensitive ion cotransporters that carry Na+, K+, and Cl - in a coupled fashion have been identified. One type, the ''absorptiv e'' isoform, carries these ions across the apical plasma membrane of;t he thick ascending limb of Henle's loop. Another isoform, the ''secret ory'' cotransporter, has been identified in a number of epithelial tis sues by physiological means, but its sites of expression in the kidney have not been fully characterized, Complementary DNA believed to code for the secretary isoform (called ''BSC2'' or ''NKCC1'') have recentl y been cloned, This study used a specific affinity-purified antipeptid e antibody to this protein for immunolocalization in the rat kidney. I mmunoblot studies using this antibody show abundant immunoreactivity a gainst bands of 140-190 and 120 kd in the parotid gland, colon, and st omach, sites where the secretory form of the cotransporter has been id entified by physiological techniques. This distribution supports the h ypothesis that this isoform represents the secretory form of the cotra nsporter. Studies in the kidney revealed that the same bands are assoc iated with membrane fractions chiefly in the outer medulla, Immunoloca lizations show that immunoreactivity is selectively and intensely loca lized to the basolateral plasma membrane of a subfraction of outer med ullary collecting duct cells. An independently produced monoclonal ant ibody (T4) specific for Na-K-Cl cotransporter displays the same locali zation. Dual localizations of cotransporter antibody with respect to a ntibody specific for principal cells (aquaporin-2) and intercalated ce lls (band 3 and H+-ATPase) show that cotransporter immunoreactivity is localized to alpha-intercalated cells of the outer medullary collecti ng duct in the rat. This distinctive localization suggests that the se cretory form of the cotransporter may play a role in renal NH4+ and/or acid secretion by this cell type.