Borke, James L.

The syncytiotrophoblast represents the primary cellular barrier between maternal and fetal circulations in the placenta. Large amounts of Ca2+ are transported across this barrier by mechanisms that are not clearly understood. To further understand this phenomenon, we examined rat and human placenta by immunohistochemical and protein blotting techniques with a monoclonal antibody raised against the human erythrocyte plasma membrane Ca2+ pump. Immunohistochemistry with this antibody showed specific staining in the human placenta of the basal (fetal facing) surface of the syncytiotrophoblast. In the rat placenta, immunohistochemistry also showed specific staining of the innermost (fetal facing) layer of the trophoblast and the basal surface of the endoderm of the intraplacental yolk sac. In Western blots of placental homogenates and membranes, the monoclonal antibody bound to a 140,000-mol wt band, characteristic of Ca2+ pumps in other tissues. Western blots of isolated basal membranes showed more intense staining than isolated microvillous membranes, confirming the results of the immunohistochemistry. In addition, Ca2+ transport in basal membrane vesicles from human placenta was inhibited by polyclonal antibodies prepared against the erythrocyte Ca2+ pump. We conclude that basal (fetal facing) layers of human and rat placentas contain a high-affinity Ca2+ pump situated to transport Ca2+ from the maternal to the fetal circulation. calcium-magnesium-adenosinetriphosphatase; calcium transport; immunohistochemistry; rat and human placenta Submitted on November 14, 1988 Accepted on March 27, 1989

In an effort to extend our studies on Ca2+ pumps to animal models, we developed a new monoclonal antibody (5F10) prepared against the human erythrocyte Ca2+-Mg2+-adenosinetriphosphatase (ATPase) that recognizes a protein of approximately 140 kDa in rat kidney homogenates. Enzyme-linked immunosorbent assays show that monoclonal antibody 5F10 binds purified Ca2+-Mg2+-ATPase and rat kidney membrane extracts in a concentration-dependent manner. In paraffin-embedded tissue sections, antibody 5F10 binds to an epitope in the basolateral membranes of rat kidney distal convoluted tubule principal cells. The antibody does not bind to intercalated cells. The latter cells were characterized by the presence of large amounts of carbonic anhydrase C. Polyclonal antibodies directed against chick intestinal 28-kDa vitamin D-dependent calcium binding protein (28-kDa CaBP) also bind epitopes in distal convoluted tubule cells, connecting tubules, and portions of collecting duct but not intercalated cells. Western blot and 45Ca blot analysis of renal cytosolic proteins showed that the polyclonal 28-kDa CaBP-directed antibody detects a protein which also binds calcium. Western blot analysis with monoclonal antibody 5F10 shows binding to both the authentic purified erythrocyte Ca2+ pump (approximately 138 kDa) and to tryptic fragments of this pump. Antibody JA3, previously used for staining of human kidney tubules, reacts with a different set of tryptic fragments, showing that the two antibodies are directed against different regions or conformational determinants on the pump molecule. We show that Ca2+-Mg2+-ATPase and 28-kDa CaBP are present in the principal cells of the distal convoluted tubule of the rat and are absent in intercalated cells.(ABSTRACT TRUNCATED AT 250 WORDS)