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Background: Epithelial cells of the rat coagulating gland secrete a minor fraction of proteins by means of an alternative export mode named apocrine secretion. Thereby, proteins are released by means of membrane bounded blebs or "aposomes" arising from the apical plasma membrane. Little is known about the composition of the aposomal membrane and whether or not proteins located in the apical plasma membrane are integrated into the aposomes., Methods: To show expression and localization of Ca(2+)-ATPase in rat coagulating gland, reverse transcriptase-polymerase chain reaction, Western blotting experiments, and Ca(2+)-ATPase activity assays, as well as immunofluorescence studies were performed., Results: Ca(2+)-ATPase is located in the apical plasma membrane of the epithelial cells of the rat coagulating gland and is also included in the aposomes. Mg(2+)-dependent and Mg(2+)-independent Ca(2+)-ATPase activity was observed in coagulating gland primary epithelial cells and tissue. Gene expression of plasma membrane Ca(2+)-ATPase isoforms 1 and 4 was detected in cultured primary epithelial cells of the rat coagulating gland and coagulating gland tissue., Conclusions: We show for the first time that Ca(2+)-ATPase as an important, functionally active membrane protein of the apical plasma membrane is incorporated into the aposomal membranes and is released from the cells during apocrine secretion process., (Copyright 2002 Wiley-Liss, Inc.)

Two different pathways for protein secretion are described for epithelial cells of rat coagulating gland and dorsal prostate: the classical merocrine and the alternative apocrine release mode. Apocrine-secreted proteins are synthesized on cytoplasmic polyribosomes and are subsequently exported in protrusions on the apical cell surface (aposomes). In this article we report the identification and purification to homogeneity of a 29-kD protein from the secretion of rat coagulating gland. N-terminal amino acid sequence analyses revealed 100% identity to rat brain carbonic anhydrase II (CAH II). In addition, the 29-kD protein showed CAH enzyme activity. On Western blot analysis, a polyclonal anti-CAH II antibody raised in rabbit reacted specifically with the rat and human but not bovine CAH II isoforms. Immunohistochemical studies on rat coagulating gland showed strong labeling for CAH II protein in aposomes. Immunoelectron microscopy confined CAH II protein to the cytoplasm and aposomes, whereas no staining was visible in the compartments of the classical merocrine route, the endoplasmic reticulum and Golgi apparatus. The resident cytoplasmic protein lactate dehydrogenase, however, was not found in the secretion. Taken together, the morphological and biochemical data clearly indicate that cytoplasmic CAH II from rat coagulating gland is specifically selected and then secreted via the apocrine pathway.