We previously demonstrated that inhibition of Na-dependent phosphate (P(i)) transport in osteoclasts led to reduced ATP levels and diminished bone resorption. These findings suggested that Na/P(i) cotransporters in the osteoclast plasma membrane provide P(i) for ATP synthesis and that the osteoclast may utilize part of the P(i) released from bone resorption for this purpose. The present study was undertaken to define the cellular localization of Na/P(i) cotransporters in the mouse osteoclast and to identify the proteins with which they interact. Using glutathione S-transferase (GST) fusion constructs, we demonstrate that the type IIa Na/P(i) cotransporter (Npt2a) in osteoclast lysates interacts with the Na/H exchanger regulatory factor, NHERF-1, a PDZ protein that is essential for the regulation of various membrane transporters. In addition, NHERF-1 in osteoclast lysates interacts with Npt2a in spite of deletion of a putative PDZ-binding domain within the carboxy terminus of Npt2a. In contrast, deletion of the carboxy-terminal TRL amino acid motif of Npt2a significantly reduced its interaction with NHERF-1 in kidney lysates. Studies in osteoclasts transfected with green fluorescent protein-Npt2a constructs indicated that Npt2a colocalizes with NHERF-1 and actin at or near the plasma membrane of the osteoclast and associates with ezrin, a linker protein associated with the actin cytoskeleton, likely via NHERF-1. Furthermore, we demonstrate by RT/PCR of osteoclast RNA and in situ hybridization that the type III Na/P(i) cotransporter, PiT-1, is also expressed in mouse osteoclasts. To examine the cellular distribution of PiT-1, we infected mouse osteoclasts with a retroviral vector encoding PiT-1 fused to an epitope tag. PiT-1 colocalizes with actin and is present on the basolateral membrane of the polarized osteoclast, similar to that previously reported for Npt2a. Taken together, our data suggest that association of Npt2a with NHERF-1, ezrin, and actin, and of PiT-1 with actin, may be responsible for membrane sorting and regulation of these Na/P(i) cotransporters in the osteoclast.

We have identified a second isoform of the catalytic A subunit of the vacuolar HI pump in chicken osteoclasts. In this isoform (A2) a 72-bp cassette replaces a 90-bp cassette present in the classical Al isoform. The Alspecific cassette encodes a region of the protein that contains one of the three ATP-binding consensus sequences (the Ploop) identified in this polypeptide, as well as the pharmacologically relevant Cys254. In contrast, the A2-specific cassette does not contain any of these features. These two isoforms, which appear to be ubiquitously expressed, are encoded by a single gene and are generated by alternative splicing of two mutually exclusive exons. The alternative RNA processing involves the recognition of a single site, the boundary between the A2-and Al-specific exons, as either acceptor (in Al) or donor (in A2) splice site.Vacuolar H+-ATPases (V-ATPases) are a family of enzymes involved in generating HI gradients (1, 2). V-ATPases are found in all eukaryotic cells, where they are responsible for acidification of intracellular organelles including lysosomes, endosomes, the Golgi apparatus, secretory vesicles, and clathrin-coated vesicles, as well as plant and fungal vacuoles. They are also located in the apical membrane of cells specialized in HI secretion, such as osteoclasts (OCs), kidney intercalated cells, and insect midgut (3-5). It has been suggested that V-ATPases are also present in the plasma membrane of macrophages and neutrophils, where they could play a role in cytoplasmic pH regulation (6). In bone, the OC V-ATPases are responsible for acidification of the resorbing compartment, a process required for bone resorption and involved in the growth, remodeling, and repair of the skeleton and in Ca2+ homeostasis. These V-ATPases are polarized to the apical ruffled-border membrane of the OC (5, 7, 8).V-ATPases are multisubunit complexes with two distinct domains: a peripherally associated catalytic sector (V1) and a membrane-spanning HI channel (VO). The peripheral Vl domain is composed of three copies of each of the A and B catalytic subunits, plus one copy of each of four accessory peptides (C-F). The A subunit contains the catalytic ATP binding site, while the B subunit, which binds but does not hydrolyze ATP, is thought to play a regulatory role. The integral VO domain contains the HI conducting channel, formed by six copies of a 17-kDa proteolipid, plus one copy of a set of three to four accessory subunits (9). The genes encoding most of these subunits have been cloned from a wide variety of mammalian, insect, plant, and fungal cells, and sequence analysis shows that they are highly conserved in evolution (10, 11). In yeast, disruption of any of the known genes that encode the V-ATPase subunits leads to conditional lethality (12, 13).The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.Although the V-ATPases which are located...

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Portal hypertension was produced experimentally in rats by partial constriction of the portal vein. Twelve rats were injected daily with indomethacin, 4 mg/kg body weight, and 12 with the vehicle (80% ethanol, 0.5 ml/day). There were no differences in portal-systemic shunts nor systemic or splanchnic haemodynamics between indomethacin-treated and untreated rats. These results suggest that cyclo-oxygenase products do not play a significative role in haemodynamic alterations shown by portal-ligated rats.

Osteoclasts are multinucleated cells derived from the mononuclear phagocyte system in the hematopoietic bone marrow. Their function is to resorb bone during skeletal growth and remodeling. They perform this function by acidifying an enclosed extracellular space, the bone resorbing compartment. Analysis of proton transport by inside-out vesicles derived from highly purified chicken osteoclast membranes has revealed the presence of a novel type of multisubunit vacuolar-like H(+)-ATPase. Unlike H(+)-ATPases derived from any other cell type or organelle, proton transport and ATPase activity in osteoclast vesicles are sensitive to two classes of inhibitors, namely V-ATPase inhibitors [N-ethyl-maleimide (NEM) and bafilomycin A1] and vanadate (IC50 100 mumol l-1), an inhibitor previously found to affect only P-ATPases. The osteoclast V-ATPase morphologically resembles vacuolar proton pumps and contains several vacuolar-like subunits (115 x 10(3), 39 x 10(3) and 16 x 10(3)M(r)), demonstrated by Western blot analysis. Subunits A and B of the catalytic domain of the enzyme, however, differ from that of other V-ATPases. In osteoclasts, subunit A has an M(r) of 63 x 10(3) instead of 67 x 10(3)-70 x 10(3); in contrast, monocytes, macrophages and kidney microsomes, which contain a vanadate-insensitive H(+)-ATPase, express the classical subunit A (70 x 10(3)M(r)). Moreover, two types of 57 x 10(3)-60 x 10(3)M(r) B subunits are also found: they are differentially recognized by antibodies and one is expressed predominantly in osteoclasts and the other in bone marrow cells and in kidney microsomes. Preliminary cloning data have indicated that the B subunit expressed in osteoclasts may be similar to the brain isoform. The osteoclast proton pump may, therefore, constitute a novel class of V-ATPase, with a unique pharmacology and specific isoforms of two subunits in the catalytic portion of the enzyme.