HLA-DR associates with specific stress proteins and is retained in the endoplasmic reticulum in invariant chain negative cells.

The major histocompatibility complex class II molecules are composed of two polymorphic chains which, in cells normally expressing them, transiently associate with a third, nonpolymorphic molecule, the invariant chain (Ii). To determine differences in the biology of class II molecules synthesized in the presence or absence of Ii, a comparative study was performed of BALB/c 3T3 cells that had been transfected with human class II HLA-DR molecules with or without cotransfection with human Ii. It was observed that in the absence of Ii, at least three high molecular weight proteins coimmunoprecipitate with HLA-DR molecules. These proteins did not coimmunoprecipitate with HLA-DR from cells cotransfected with Ii, nor did they coimmunoprecipitate with class I molecules from any of the transfectants. NH2-terminal sequence and/or Western blot analysis revealed the identity of two of the proteins as the endoplasmic reticulum (ER) resident stress proteins GRP94 and ERp72. Neither of these proteins was found to have an increased level of synthesis in the Ii- versus the Ii+ transfectants, indicating that their synthesis was not induced over constitutive levels. Fluorescence microscopy revealed that in the Ii- transfectants, the majority of the HLA-DR molecules were present in the ER, whereas in the Ii+ transfectants, the HLA-DR molecules were found in vesicular structures. We hypothesize that in the absence of Ii, ER resident stress proteins bind to class II molecules and retain them in the ER. This process, in turn, could prevent class II molecules from exiting the ER with endogenous peptides bound in their peptide binding cleft, and therefore could minimize autoimmune responses to endogenously processed self-peptides.

Protein transduction: unrestricted delivery into all cells?

Several proteins can traverse biological membranes through protein transduction. Small sections of these proteins (10-16 residues long) are responsible for this. Linking these domains covalently to compounds, peptides, antisense peptide nucleic acids or 40-nm iron beads, or as in-frame fusions with full-length proteins, lets them enter any cell type in a receptor- and transporter-independent fashion. Moreover, several of these fusions, introduced into mice, were delivered to all tissues, even crossing the blood-brain barrier. These domains thus might let us address new questions and even help in the treatment of human disease.

Gelsolin deficiency blocks podosome assembly and produces increased bone mass and strength.

Osteoclasts are unique cells that utilize podosomes instead of focal adhesions for matrix attachment and cytoskeletal remodeling during motility. We have shown that osteopontin (OP) binding to the alpha(v)beta(3) integrin of osteoclast podosomes stimulated cytoskeletal reorganization and bone resorption by activating a heteromultimeric signaling complex that includes gelsolin, pp(60c-src), and phosphatidylinositol 3'-kinase. Here we demonstrate that gelsolin deficiency blocks podosome assembly and alpha(v)beta(3)-stimulated signaling related to motility in gelsolin-null mice. Gelsolin-deficient osteoclasts were hypomotile due to retarded remodeling of the actin cytoskeleton. They failed to respond to the autocrine factor, OP, with stimulation of motility and bone resorption. Gelsolin deficiency was associated with normal skeletal development and endochondral bone growth. However, gelsolin-null mice had mildly abnormal epiphyseal structure, retained cartilage proteoglycans in metaphyseal trabeculae, and increased trabecular thickness. With age, the gelsolin-deficient mice expressed increased trabecular and cortical bone thickness producing mechanically stronger bones. These observations demonstrate the critical role of gelsolin in podosome assembly, rapid cell movements, and signal transduction through the alpha(v)beta(3) integrin.

Osteoclast cytosolic calcium, regulated by voltage-gated calcium channels and extracellular calcium, controls podosome assembly and bone resorption.

The mechanisms of Ca2+ entry and their effects on cell function were investigated in cultured chicken osteoclasts and putative osteoclasts produced by fusion of mononuclear cell precursors. Voltage-gated Ca2+ channels (VGCC) were detected by the effects of membrane depolarization with K+, BAY K 8644, and dihydropyridine antagonists. K+ produced dose-dependent increases of cytosolic calcium ([Ca2+]i) in osteoclasts on glass coverslips. Half-maximal effects were achieved at 70 mM K+. The effects of K+ were completely inhibited by dihydropyridine derivative Ca2+ channel blocking agents. BAY K 8644 (5 X 10(-6) M), a VGCC agonist, stimulated Ca2+ entry which was inhibited by nicardipine. VGCCs were inactivated by the attachment of osteoclasts to bone, indicating a rapid phenotypic change in Ca2+ entry mechanisms associated with adhesion of osteoclasts to their resorption substrate. Increasing extracellular Ca2+ ([Ca2+]e) induced Ca2+ release from intracellular stores and Ca2+ influx. The Ca2+ release was blocked by dantrolene (10(-5) M), and the influx by La3+. The effects of [Ca2+]e on [Ca2+]i suggests the presence of a Ca2+ receptor on the osteoclast cell membrane that could be coupled to mechanisms regulating cell function. Expression of the [Ca2+]e effect on [Ca2+]i was similar in the presence or absence of bone matrix substrate. Each of the mechanisms producing increases in [Ca2+]i, (membrane depolarization, BAY K 8644, and [Ca2+]e) reduced expression of the osteoclast-specific adhesion structure, the podosome. The decrease in podosome expression was mirrored by a 50% decrease in bone resorptive activity. Thus, stimulated increases of osteoclast [Ca2+]i lead to cytoskeletal changes affecting cell adhesion and decreasing bone resorptive activity.

Peripheral metabolism of intact parathyroid hormone. Role of liver and kidney and the effect of chronic renal failure.

The plasma disappearance rate (metabolic clearance rate) of administered intact parathyroid hormone (intact PTH) was analyzed in awake dogs with indwelling hepatic and renal vein catheters. The metabolic clearance rate (MCR) of intact PTH was found to be very rapid, 21.6 +/- 3.1 ml/min per kg in 11 normal dogs. The liver accounted for the greatest fraction of the MCR of intact PTH (61 +/- 4%) by virtue of an arterial minus venous (a - v) difference across the liver of 45 +/- 3%. The renal uptake of intact PTH accounted for 31 +/- 3% of the MCR of intact PTH. The renal a - v difference for intact PTH of 29 +/- 2% was significantly greater than the filtration fraction indicating renal uptake of intact PTH at sites independent of glomerular filtration. Together, the hepatic and renal clearances of intact PTH accounted for all but a small fraction of the MCR of intact PTH. The MCR of intact PTH, rendered biologically inactive by oxidation, was markedly decreased to 8.8 +/- 1 ml/min per kg. The a - v difference of oxidized intact PTH was reduced both in the liver and kidney. These data suggested that the high uptake rates of intact PTH are dependent, at least in part, upon sites recognizing only biologically active PTH. Chronic renal failure (CRF) decreased the MCR of intact PTH to 11.3 +/- 1.3 ml/min per kg (n = 10). Both the hepatic and renal a - v differences of intact PTH were reduced in dogs with CRF. This resulted in reductions in the hepatic and renal clearances of intact PTH. These studies identify the liver as a major extrarenal site of PTH metabolism affected by CRF. They suggest that CRF impairs the function of the major uptake sites involved in intact PTH metabolism.

Calcium transport in canine renal basolateral membrane vesicles. Effects of parathyroid hormone.

The effects of parathyroid hormone were studied on Ca2+ fluxes in canine renal proximal tubular basolateral membrane vesicles (BLMV). Efflux of Ca2+ from preloaded BLMV was found to be stimulated by an external Na+ gradient, and this was inhibited by the Na+ ionophore, monensin, and enhanced by intravesicular negative electrical potentials, which indicated electrogenic Na+/Ca2+ exchange activity. There was a Na+ gradient independent Ca2+ flux, but membrane binding of Ca2+ was excluded from contributing to the Na+ gradient-dependent efflux. The Na+ gradient-dependent flux of Ca2+ was very rapid, and even 2- and 5-s points may not fully represent absolute initial rates. It was saturable with respect to the interaction of Ca2+ and Na+ with an apparent (5 s) Km for Na+-dependent Ca2+ uptake of 10 microM, and an apparent (5 s) Vmax of 0.33 nmol/mg protein per 5 s. The Na+ concentration that yielded half maximal Ca2+ efflux (2 s) was 11 mM, and the Hill coefficient was two or greater. Both Na+ gradient dependent and independent Ca2+ efflux were decreased in BLMV prepared from kidneys of thyroparathyroidectomized (TPTX) dogs, and both were stimulated by parathyroid hormone (PTH) infusion to TPTX dogs. BLMV from TPTX dogs exhibited significantly reduced maximal stimulation of Na+ gradient-dependent Ca2+ uptake with an apparent (5 s) Vmax of 0.23 nmol/mg protein per 5 s, but the apparent Km was 8 microM, which was unchanged from normal. The Na+ gradient independent Ca2+ uptake was also reduced in BLMV from TPTX dogs compared with normal. Thus, PTH stimulated both Na+/Ca2+ exchange activity and Na+ independent Ca2+ flux. In vivo, the latter could result in an elevation of cytosolic Ca2+ by PTH, and this might contribute to the observed decrease in solute transport in the proximal tubule.

Stretch-induced atriopeptin secretion in the isolated rat myocyte and its negative modulation by calcium.

Cellular mechanism(s) regulating atriopeptin secretion and processing by the atrial myocyte are currently unknown. Osmotic stretch of isolated atrial myocytes as well as potassium chloride depolarization were potent stimuli of atriopeptin secretion. Release was potentiated by buffering either extracellular calcium with EGTA or intracellular calcium with the intracellular chelator, BAPTA AM. Atrial release of atriopeptin was inhibited after administration of ionomycin which elevates intracellular calcium. Fetal or early neonatal ventricular myocytes actively synthesize atriopeptin. Atriopeptin secretion by ventricular myocytes was also markedly potentiated by osmotic stretch as well as KCl depolarization. Only the 126 amino acid prohormone was secreted by the stretch-stimulated atrial and ventricular myocyte. These data suggest that stretch of the myocyte plasma membrane is a major stimulus for atriopeptin secretion and that atriopeptin secretion is not stimulated by raising intracellular calcium and appears to be negatively modulated by this cation. Like the atrial myocyte, the ventricular myocyte possesses the cellular mechanism(s) necessary to secrete atriopeptin by a regulated mechanism.

Stimulation of inositol trisphosphate and diacylglycerol production in renal tubular cells by parathyroid hormone.

Parathyroid hormone (PTH) produced a dose-dependent immediate stimulation of inositol triphosphate and diacylglycerol production in the opossum kidney cell line, primary culture proximal tubular cells, and basolateral membranes from canine proximal tubular segments. The increase in inositol triphosphate production was accompanied by a minor increase in inositol phosphate and no significant increase in inositol bisphosphate production. Associated with the changes in inositol triphosphate and diacylglycerol, there was an immediate hydrolysis of phosphatidylinositol 4'5-bisphosphate. The effect on phospholipid hydrolysis was followed by stimulation of phosphorylation of phosphatidylinositol 4' monophosphate and phosphatidylinositol. PTH produced a sudden increase in cytoplasmic Ca2+ in opossum kidney cells that persisted for approximately 1 min. Inositol triphosphate transiently increased cytoplasmic Ca2+ in saponin-treated opossum kidney and primary culture proximal tubule cells. The effects of PTH were not mimicked by cyclic nucleotides. In fact, cyclic AMP appeared to diminish inositol triphosphate production. These results demonstrate that PTH may activate renal tubular epithelial cells by the production of inositol triphosphate and diacylglycerol.

Regulation of sodium-dependent phosphate transport in osteoclasts.

Osteoclasts are the primary cells responsible for bone resorption. They are exposed to high ambient concentrations of inorganic phosphate (Pi) during the process of bone resorption and they possess specific Pi-transport system(s) capable of taking up Pi released by bone resorption. By immunochemical studies and PCR, we confirmed previous studies suggesting the presence of an Na-dependent Pi transporter related to the renal tubular "NaPi" proteins in the osteoclast. Using polyclonal antibodies to NaPi-2 (the rat variant), an approximately 95-kD protein was detected, localized in discrete vesicles in unpolarized osteoclasts cultured on glass coverslips. However, in polarized osteoclasts cultured on bone, immunofluorescence studies demonstrated the protein to be localized exclusively on the basolateral membrane, where it colocalizes with an Na-H exchanger but opposite to localization of the vacuolar H-ATPase. An inhibitor of phosphatidylinositol 3-kinase, wortmannin, and an inhibitor of actin cytoskeletal organization, cytochalasin D, blocked the bone-stimulated increase in Pi uptake. Phosphonoformic acid (PFA), an inhibitor of the renal NaPi-cotransporter, reduced NaPi uptake in the osteoclast. PFA also elicited a dose-dependent inhibition of bone resorption. PFA limited ATP production in osteoclasts attached to bone particles. Our results suggest that Pi transport in the osteoclast is a process critical to the resorption of bone through provision of necessary energy substrates.

Effects of glucose and parathyroid hormone on the renal handling of myoinositol by isolated perfused dog kidneys.

The effects of glucose and parathyroid hormone (PTH) on the transport and metabolism of myoinositol (MI) and [2-(3)H]MI were studied in isolated perfused dog kidneys. Studies during perfusion of kidneys with normal and elevated glucose concentrations demonstrated that under normal conditions the isolated kidney reabsorbed 94.7+/-0.2% of the filtered MI, and the renal production of (3)H-metabolities of MI was 117.9+/-6% of the filtered MI load. This indicated that entry of MI into tubular cells by reabsorption was not the sole pathway for entry into the pool of MI within the kidney undergoing catabolism. High glucose perfusate decreased MI reabsorption to 68.6+/-4.7% and thus decreased delivery of [2-(3)H]MI into the catabolic pool from the reabsorptive pathway. In the high glucose experiments, the rate of [2-(3)H]MI catabolism exceeded [2-(3)H]MI reabsorption by the same fraction as in normal glucose experiments, which indicates that high glucose did not affect nonreabsorptive access of MI to the catabolic site. In contrast to the effects of glucose, PTH administration resulted in an increase in perfusate MI concentration and a decrease in the perfusate [2-(3)H]MI specific activity. Concomitantly, urinary MI and [2-(3)H]MI concentrations were increased, again with a decrease in [2-(3)H]MI specific activity. These results indicate that PTH caused a release of MI into the urine (not the same as decreased MI reabsorption, which would not affect urinary [(3)H]MI specific activity) and into the perfusate of the isolated kidneys. These effects on MI release were about coincidental with the increase in urinary cyclic 3',5'-AMP after PTH and preceded the peak phosphaturic effect of PTH. There was no detectable effect of PTH on MI synthesis from glucose as a source of the MI released into the urine and perfusate. However, PTH temporarily halted accumulation of tritiated MI catabolites. There was no effect of inactivated PTH on urinary cyclic 3',5'-AMP or on MI transport, which indicates that the PTH effect on MI handling was a specific hormonal effect. These studies clarify the renal metabolism of MI, and they demonstrate heretofore unknown effects of PTH on the renal handling and metabolism of MI. The effects of PTH on renal MI metabolism have important implications in renal carbohydrate metabolism and phospholipid turnover.

The renal handling of parathyroid hormone. Role of peritubular uptake and glomerular filtration.

The mechanisms of uptake of parathyroid hormone (PTH) by the kidney was studied in anesthetized dogs before and after ureteral ligation. During constant infusion of bovine PTH (b-PTH 1-84), the renal arteriovenous (A-V) difference for immunoreactive PTH (i-PTH) was 22+/-2%. After ureteral ligation and no change in renal plasma flow, A-V i-PTH fell to 15+/-1% (P < 0.01), indicating continued and significant uptake of i-PTH at peritubular sites and a lesser role of glomerular filtration (GF) in the renal uptake of i-PTH. Since, under normal conditions, minimal i-PTH appears in the final urine, the contribution of GF and subsequent tubular reabsorption was further examined in isolated perfused dog kidneys before and after inhibition of tubular reabsorption by potassium cyanide. Urinary i-PTH per 100 ml GF rose from 8+/-4 ng/min (control) to 170+/-45 ng/min after potassium cyanide. Thus, i-PTH is normally filtered and reabsorbed by the tubular cells. The physiological role of these two mechanisms of renal PTH uptake was examined by giving single injections of b-PTH 1-84 or synthetic b-PTH 1-34 in the presence of established ureteral ligation. After injection of b-PTH 1-84, renal A-V i-PTH was 20% only while biologically active intact PTH was present (15-20 min). No peritubular uptake of carboxyl terminal PTH fragments was demonstrable. In contrast, after injection of synthetic b-PTH 1-34, renal extraction of N-terminal i-PTH after ureteral ligation (which was 13.4+/-0.6% vs. 19.6+/-0.9% in controls) continued for as long as i-PTH persisted in the circulation. These studies indicate that both GF and peritubular uptake are important mechanisms for renal PTH uptake. Renal uptake of carboxyl terminal fragments of PTH is dependent exclusively upon GF and tubular reabsorption, whereas peritubular uptake can only be demonstrated for biologically active b-PTH 1-84 and synthetic b-PTH 1-34.

Selective uptake of the synthetic amino terminal fragment of bovine parathyroid hormone by isolated perfused bone.

Studies from our laboratory have shown that the metabolic clearance rate of carboxy terminal immunoreactive parathyroid hormone (i-PTH) can be accounted for by extraction of i-PTH by liver and kidney. In contrast, there was no demonstrable hepatic uptake of the synthetic amino terminal bovine PTH fragment (syn b-PTH 1-34) and the kidney accounted for only 45% of the metabolic clearance rate of amino terminal i-PTH. This suggested that another major site, presumably bone, played a role in the metabolism of syn b-PTH 1-34. Extraction of i-PTH by isolated perfused bone was studied during infusion of purified bovine PTH (b-PTH) 1-84 or syn b-PTH 1-34. In five studies during infusion of syn b-PTH 1-34 the arteriovenous difference for i-PTH across bone was 36%. In contrast, no significant uptake of carboxy terminal i-PTH was observed in nine studies during infusion of b-PTH 1-84. In addition, when H(2)O(2)-oxidized (biologically inactive) syn b-PTH 1-34 was used no arteriovenous difference was observed. These findings correlated with the ability of these PTH preparations to stimulate cyclic AMP production by the perfused bone. Syn b-PTH 1-34 increased cyclic AMP production at perfusate PTH concentrations of 1-5 ng/ml, whereas b-PTH 1-84 evoked only a minimal response at concentrations of 10-20 ng/ml. We conclude that bone is a major site of metabolism of the amino terminal PTH fragment, syn b-PTH 1-34. In addition, these data suggest that cleavage of the intact hormone, with the production of amino terminal PTH fragments by peripheral organs (liver and kidney), may play a major role in the regulation of PTH effects on bone.

Degradation of parathyroid hormone and fragment production by the isolated perfused dog kidney. The effect of glomerular filtration rate and perfusate CA++ concentrations.

The renal degradation of intact bovine parathyroid hormone (b-PTH 1-84) was studied with the isolated perfused dog kidney. Disappearance of b-PTH 1-84 from the perfusate occurred concomitantly with the appearance of smaller molecular weight forms of immunoreactive parathyroid hormone (PTH). These smaller molecular weight PTH fragments included both carboxyl and amino terminal regions of the PTH peptide. Perfusate from kidneys with lower glomerular filtration rates (GFR) contained b-PTH 1-84 for longer periods of time than kidneys with higher GFRs, and perfusate from kidneys with lower GFRs demonstrated greater accumulation of carboxyl terminal PTH fragments. Perfusate containing high Ca++ concentrations retarded, and perfusate with low Ca++ concentrations accelerated the rate of degradation of b-PTH 1-84 by the kidney. These studies, therefore document the production of PTH fragments during the course of intact hormone degradation by the kidney. They also demonstrate renal clearance of the PTH fragments produced and define the effects of glomerular filtration rate and calcium concentrations on degradation of intact hormone and the clearance of PTH fragments.

Effect of intact parathyroid hormone on hepatic glucose release in the dog.

The liver has been shown to remove parathyroid hormone (PTH) from its arterial circulation by a mechanism that is selective for the intact form of the peptide (PTH 1-84). The present studies demonstrate that PTH has biologic effects on the liver in vivo. Bovine PTH 1-84 stimulated hepatic glucose release in dogs with indwelling hepatic vein catheters from basal values of 31+/-8 to 68+/-9 mg/min per kg after bolus injections of PTH. The effect on hepatic glucose release was apparent by 5 min and persisted for the 80 min of observation. The NH(2)-terminal PTH fragment (syn b-PTH 1-34) had no effect. Bovine PTH 1-84 administered in doses designed to produce circulating levels of immunoreactive PTH similar to the endogenous levels observed in uremic dogs also increased the incorporation of (14)C from infused [(14)C]alanine into glucose, and increased estimated hepatic uptake of both chemical and [(14)C]alanine, while increasing hepatic glucose release. Thus, administration of "physiologic levels" of b-PTH 1-84 stimulated hepatic glucose release in part through increased gluconeogenesis in vivo, whereas syn b-PTH 1-34 had no demonstrable effect. Circulating levels of insulin rose after PTH administration, an increase which presumably represents a secondary response to the rise in glucose release. These results suggest that the liver is a target organ of PTH, and that PTH might potentially alter carbohydrate metabolism during hypersecretion. They also suggest that hepatic uptake of PTH may be related in part to production of a specific biologic effect rather than just simple peptide degradation.

Metabolism in immunoreactive parathyroid hormone in the dog. The role of the kidney and the effects of chronic renal disease.

The role of the kidney in the metabolism of parathyroid hormone (PTH) was examined in the dog. Studies were performed in awake normal and uremic dogs after administration of bovine parathyroid hormone (b-PTH) or synthetic amino terminal tetratricontapeptide of b-PTH (syn b-PTH 1-34). The renal clearance of immunoreactive PTH was determined from the product of renal plasma flow and the percent extraction of PTH immunoreactivity by the kidney. Blood levels of circulating immunoreactive PTH were determined by radioimmunoassay. The normal dog kidney extracted 20 plus or minus 1% of the immunoreactive b-PTH delivered to it, and renal clearance (RC) of immunoreactivity was 60 ml/min. When RC was compared to an estimate of total metabolic clearance (MCR) of immunoreactivity, it accounted for 61% of the total. Both MCR and RC were markedly decreased in dogs with chronic renal disease. However, the percent extraction of immunoreactive PTH was unchanged in chronic renal disease, and the observed decrease in RC was due to changes in renal plasma flow. The largest portion of the reduction in total MCR was accounted for by the decrease in RC, and there was no compensation for the decrease in RC by extrarenal sites of PTH metabolism.

Extracellular protons acidify osteoclasts, reduce cytosolic calcium, and promote expression of cell-matrix attachment structures.

Because metabolic acids stimulate bone resorption in vitro and in vivo, we focused on the cellular events produced by acidosis that might be associated with stimulation of bone remodeling. To this end, we exposed isolated chicken osteoclasts to a metabolic (butyric) acid and observed a fall in both intracellular pH and cytosolic calcium [( Ca2+]i). These phenomena were recapitulated when bone resorptive cells, alkalinized by HCO3 loading, were transferred to a bicarbonate-free environment. The acid-induced decline in osteoclast [Ca2+]i was blocked by either NaCN or Na3VO4, in a Na+-independent fashion, despite the failure of each inhibitor to alter stimulated intracellular acidification. Moreover, K+-induced membrane depolarization also reduced cytosolic calcium in a manner additive to the effect of protons. These findings suggest that osteoclasts adherent to bone lack functional voltage-operated Ca2+ channels, and they reduced [Ca2+]i in response to protons via a membrane residing Ca-ATPase. Most importantly, acidosis enhances formation of podosomes, the contact areas of the osteoclast clear zone, indicating increased adhesion to substrate, an early step in bone resorption. Thus, extracellular acidification of osteoclasts leads to decrements in intracellular pH and calcium, and appears to promote cell-matrix attachment.

Osteopontin deficiency produces osteoclast dysfunction due to reduced CD44 surface expression.

Osteopontin (OPN) was expressed in murine wild-type osteoclasts, localized to the basolateral, clear zone, and ruffled border membranes, and deposited in the resorption pits during bone resorption. The lack of OPN secretion into the resorption bay of avian osteoclasts may be a component of their functional resorption deficiency in vitro. Osteoclasts deficient in OPN were hypomotile and exhibited decreased capacity for bone resorption in vitro. OPN stimulated CD44 expression on the osteoclast surface, and CD44 was shown to be required for osteoclast motility and bone resorption. Exogenous addition of OPN to OPN-/- osteoclasts increased the surface expression of CD44, and it rescued osteoclast motility due to activation of the alpha(v)beta(3) integrin. Exogenous OPN only partially restored bone resorption because addition of OPN failed to produce OPN secretion into resorption bays as seen in wild-type osteoclasts. As expected with these in vitro findings of osteoclast dysfunction, a bone phenotype, heretofore unappreciated, was characterized in OPN-deficient mice. Delayed bone resorption in metaphyseal trabeculae and diminished eroded perimeters despite an increase in osteoclast number were observed in histomorphometric measurements of tibiae isolated from OPN-deficient mice. The histomorphometric findings correlated with an increase in bone rigidity and moment of inertia revealed by load-to-failure testing of femurs. These findings demonstrate the role of OPN in osteoclast function and the requirement for OPN as an osteoclast autocrine factor during bone remodeling.

Parathyroid hormone inhibition of phosphate transport in renal brush border vesicles from phosphate-depleted dogs.

Dietary phosphate (Pi) restriction increases renal Pi reabsorption and induces resistance to the phosphaturic action of parathyroid hormone. Na+-gradient-stimulated Pi transport in membrane vesicles isolated from the renal brush border of experimental animals has been shown to parallel changes in renal Pi reabsorption induced by dietary Pi restriction and in vivo administration of parathyroid hormone. Dietary Pi restriction has been shown to markedly inhibit the phosphaturic response to parathyroid hormone in rats and dogs. Parathyroid hormone has been reported not to decrease the Na+-gradient-stimulated transport of Pi in brush border membrane vesicles isolated from dietary Pi restricted rats unless the rats were administered an acute Pi load prior to killing, however, thyroparathyroidectomy of rats fed a low Pi diet has been reported to increase Na+-gradient-stimulated Pi transport. Using the dietary Pi restricted dog, we demonstrated no significant decrease in renal reabsorption of Pi in response to parathyroid hormone administration. However, significant decreases in Pi transport in brush border membrane vesicles isolated from the kidneys of dietary Pi restricted dogs were observed in response to in vivo parathyroid hormone administration. These data demonstrate that the resistance to the phosphaturic action of parathyroid hormone observed in vivo does not include resistance to the inhibitory effect of parathyroid hormone on Pi transport in brush border membrane vesicles. Thus, the data suggest that parathyroid hormone continues to alter Pi transport characteristics of the brush border membrane in states of Pi depletion despite the resistance to parathyroid hormone seen in vivo.

Mechanism of stimulation of renal phosphate transport by 1,25-dihydroxycholecalciferol.

Vitamin D has been shown to stimulate renal phosphate transport and to alter membrane phospholipid composition. The present studies examine the possibility that the effects of 1,25(OH)2D3 on phosphate transport are related to its effects on membrane lipids. Arrhenius plots, which relate maximum rates of sodium dependent phosphate uptake into brush-border membrane vesicles to temperature were constructed. Phosphate transport was studied using brush-border membrane vesicles from normal, vitamin D-deficient, and physiologically replete (15 pmol/100 g body weight per 24 h) rats. These plots were triphasic with characteristic, lipid-dependent, slopes (M1,M2,M3) representing activation energies and transition temperatures (T1,T2). Physiologic 1,25(OH)2D3 repletion normalized these plots by stimulating phosphate transport at all temperatures, increasing T2 from 18 +/- 0.7 to 23.5 +/- 0.9 degrees C and decreasing M2 and M3 from -5.8 +/- 0.2 and -10.2 +/- 0.4 to -4.5 +/- 0.4 and -7.7 +/- 0.3, respectively. Pharmacologic (1.2 nmol/100 g per 3 h) 1,25(OH)2D3 treatment resulted in a change in the Arrhenius plot of phosphate transport to a biphasic one with a transition temperature of 30 degrees C. This effect was not blocked by cycloheximide. The Arrhenius plots of glucose transport were triphasic and unchanged with vitamin D repletion. These data support a liponomic mechanism of action for 1,25(OH)2D3 on phosphate transport.

1,25-Dihydroxycholecalciferol stimulates renal phosphate transport by directly altering membrane phosphatidylcholine composition.

Controversy exists regarding the mechanisms by which 1,25-dihydroxycholecalciferol (1,25(OH2)D3) alters membrane lipid composition and ion transport. Recent studies have demonstrated stimulation of the transfer of 1-acyl-2-(N-4-nitrobenz-2-oxa-1,3-diazole)aminocaproylphosphatidylcholine (NBD-PC) by 1,25(OH)2D3. In the present studies, brush border membrane vesicle phosphatidylcholine content was increased after incubation with liposomes composed of dioleoylphosphatidylcholine or beta-linoleyl, gamma-palmitoyl phosphatidylcholine and 1,25(OH)2D3 (10(-7) M). Vesicular phosphatidylcholine content was increased from control levels of 49.5 micrograms/mg protein to 56.9 and 58.5, respectively, after treatment with the liposomes containing 1,25(OH)2D3, P less than 0.05. When the vesicles were incubated with liposomes composed of beta-linoleyl, gamma-palmitoyl phosphatidylcholine, phosphate transport was stimulated from 231 +/- 20 to 431 +/- 41 pmol/mg protein per 15 s in the presence of 1,25(OH)2D3 if the vesicles were derived from vitamin D deficient rats and from 443 +/- 33 to 601 +/- 42 pmol/mg protein per 15 s if the vesicles were prepared from normal rats. Despite phosphatidylcholine transfer, incubation with liposomes composed of dioleoylphosphatidylcholine and 1,25(OH)2D3 did not stimulate phosphate transport. Furthermore, incubation of vesicles with liposomes and 1,25(OH)2D3 did not alter glucose transport.