Salisbury, J. L.

A number of peptide hormones have been shown to undergo receptor-mediated endocytosis (RME). RME involves the internalization of receptor-ligand complexes followed by delivery to an intracellular compartment, the endosome, from which ligands or receptors can be delivered to lysosomes or other cellular destinations. Vasopressin, a peptide hormone that plays a role in kidney and vascular physiology, has recently been demonstrated to undergo RME in LLC-PK1 and A10 cells, which express V2- and V1-type vasopressin receptors, respectively. Fluorescent vasopressin analogues are internalized by RME from the basolateral surface of polarized LLC-PK1 cells. The precise role of RME in vasopressin action is uncertain, but it is likely that it is involved in the desensitization of target cells by altering the number of cell surface vasopressin receptors. Alterations in the rate of RME may alter the response of the cell to vasopressin. Fluorescent and biotinylated vasopressins are useful tools for the study of this process.

Abstract Centrin is an evolutionarily conserved 20 kDa, Ca+2-binding, calmodulin-related protein associated with centrioles and basal bodies of phylogenetically diverse eukaryotic cells. Earlier studies have shown that residual centrosomes of non-rodent mammalian spermatozoa retain centrin and, in theory, could contribute this protein for the reconstruction of the zygotic centrosome after fertilization. The present work shows that CEN2 and CEN3 mRNA were detected in germinal vesicle-stage (GV) oocytes, MII oocytes, and pre-implantation embryos from the two-cell through the blastocyst stage, but not in spermatozoa. Boar ejaculated spermatozoa possess centrin as revealed by immunofluorescence microscopy and western blotting. Immature, GV oocytes possess speckles of centrin particles in the perinuclear area, visualized by immunofluorescence microscopy and exhibit a 19 kDa band revealed by western blotting. Mature MII stage oocytes lacked centrin that could be detected by immunofluorescence or western blotting. The sperm centrin was lost in zygotes after in vitro fertilization. It was not detectable in embryos by immunofluorescence microscopy until the late blastocyst stage. Embryonic centrin first appeared as fine speckles in the perinuclear area of some interphase blastocyst cells and as putative centrosomes of the spindle poles of dividing cells. The cells of the hatched blastocysts developed centrin spots comparable with those of the cultured cells. Some blastomeres displayed undefined curved plate-like centrin-labeled structures. Anti-centrin antibody labeled interphase centrosomes of cultured pig embryonic fibroblast cells as distinct spots in the juxtanuclear area. Enucleated pig oocytes reconstructed by electrofusion with pig fibroblasts displayed centrin of the donor cell during the early stages of nuclear decondensation but became undetectable in the late pronuclear or cleavage stages. These observations suggest that porcine zygotes and pre-blastocyst embryonic cells lack centrin and do not retain exogenously incorporated centrin. The early embryonic centrosomes function without centrin. Centrin in the blastocyst stage embryos is likely a result of de novo synthesis at the onset of differentiation of the pluripotent blastomeres.

To determine whether receptor-mediated endocytosis occurs in vasopressin-responsive cells, we developed a model system using synthetic fluorescent-labeled vasopressin analogs and A10 (smooth muscle) and LLC-PK1 (kidney epithelial) cells in culture; these cell lines express V1 and V2 vasopressin cell surface receptor types, respectively. We used epifluorescence microscopy to examine the binding, internalization, and intracellular destination of [1-(2-mercapto)propionic acid,8-lysine-N6-carboxytetramethylrhodamine] vasopressin (R-MLVP) and [1-(2-mercapto)propionic acid,8-lysine-N6-carboxyfluorescein]vasopressin (F-MLVP) in these cells. The rhodamine-labeled fluorescent vasopressin analog, R-MLVP, initially bound in a diffuse manner at the cell surface of both A10 and LLC-PK1 cells and could be displaced by excess unlabeled [8-arginine]vasopressin. After incubation at 37 degrees C, bound ligand rapidly aggregated into small clusters or patches, which were internalized in a manner consistent with receptor-mediated endocytosis. Subsequent processing of internalized ligand-receptor complexes appeared to differ between A10 and LLC-PK1 cells. In the case of LLC-PK1 cells, ligand was delivered to a tightly focused lysosome compartment in the perinuclear region of the cell, and receptor molecules were replenished at the cell surface. The lysosomal location of ligand was supported by the quenching of fluorescence in the internalized vesicles when F-MLVP was used as a fluorescent tracer. In the case of A10 cells, ligand became localized to a vesicular compartment and reappearance of receptor at the cell surface was limited. Our data are consistent with the occurrence of receptor-mediated endocytosis of vasopressin in cells with V1 and V2 receptors.

Abstract PHEX, a phosphate-regulating gene with homologies to endopeptidases on the X chromosome, is mutated in X-linked hypophosphatemia (XLH) in humans and mice (Hyp). Although recent observations indicate that Phex protein is expressed primarily in bone and may play an important role in osteoblast function and bone mineralization, the pattern of the Phex protein expression in the developing skeleton and its subcellular localization in osteoblasts remain unknown. We examined the ontogeny of the Phex protein in the developing mouse embryo and its subcellular localization in osteoblasts using a specific antibody to the protein. Immunohistochemical staining of mouse embryos revealed expression of Phex in osteogenic precursors in developing vertebral bodies and developing long bones on day 16 postcoitum (pc) and thereafter. Calvaria from day 18 pc mice showed Phex epitopes in osteoblasts. No Phex immunoreactivity was detected in lung, heart, hepatocytes, kidney, intestine, skeletal muscle, or adipose tissue of mouse embryos. Interestingly, embryonic mouse skin showed moderate amounts of Phex immunostaining. In postnatal mice, Phex expression was observed in osteoblasts and osteocytes. Moderate expression of Phex was seen in odontoblasts and slight immunoreactivity was observed in ameloblasts. Confocal microscopy revealed the presence of immunoreactive PHEX protein in the Golgi apparatus and endoplasmic reticulum of osteoblasts from normal mice and in osteoblasts from Hyp mice transduced with a human PHEX viral expression vector. PHEX protein was not detected in untransduced Hyp osteoblasts. These data indicate that Phex protein is expressed in osteoblasts and osteocytes during the embryonic and postnatal periods and that within bone, Phex may be a unique marker for cells of the osteoblast/osteocyte lineage.