Biomedical subjects

R Baron

Publications and source records attributed to R Baron.

At least 145 records · Page 8Linked to original sources

PubMed1997

Colony-stimulating factor-1 induces cytoskeletal reorganization and c-src-dependent tyrosine phosphorylation of selected cellular proteins in rodent osteoclasts.

Colony-stimulating factor-1 (CSF-1) stimulates motility and cytoplasmic spreading in mature osteoclasts. Therefore, we examined the cellular events and intracellular signaling pathways that accompany CSF-1-induced spreading in normal osteoclasts. To explore the role c-src plays in these processes, we also studied osteoclasts prepared from animals with targeted disruption of the src gene. In normal osteoclasts, CSF-1 treatment induces rapid cytoplasmic spreading, with redistribution of F-actin from a well-delineated central attachment ring to the periphery of the cell. CSF-1 increases membrane phosphotyrosine staining in osteoclasts and induces the phosphorylation of several cellular proteins in cultured, osteoclast-like cells, including c-fms, c-src, and an 85-kD Grb2-binding protein. Src kinase activity is increased threefold after CSF-1 treatment. In src- cells, no attachment ring is present, and CSF-1 fails to induce spreading or a change in the pattern of F-actin distribution. Although c-fms becomes phosphorylated after CSF-1 treatment, the 85-kD protein is significantly less phosphorylated in src- osteoclast-like cells. These results indicate that c-src is critical for the normal cytoskeletal architecture of the osteoclast, and, in its absence, the spreading response induced by CSF-1 is abrogated, and downstream signaling from c-fms is altered.

Amino Acid Sequence↗

PubMed1997

Identification of an osteoclast transcription factor that binds to the human T cell leukemia virus type I-long terminal repeat enhancer element.

Transgenic mice expressing human T cell leukemia virus type I (HTLV-I)-tax under the control of HTLV-I-long terminal repeat (LTR) promoter develop skeletal abnormalities with high bone turnover and myelofibrosis. In these animals, Tax is highly expressed in bone with a pattern of expression restricted to osteoclasts and spindle-shaped cells within the endosteal myelofibrosis. To test the hypothesis that lineage-specific transcription factors promote transgene expression from the HTLV-I-LTR in osteoclasts, we first examined tax expression in transgenic bone marrow cultures. Expression was dependent on 1alpha,25-dihydroxycholecalciferol and coincided with tartrate-resistant acid phosphatase (TRAP) expression, a marker of osteoclast differentiation. Furthermore, Tax was expressed in vitronectin receptor-positive mononuclear precursors as well as in mature osteoclast-like cells (OCLs). Consistent with our hypothesis, electrophoretic mobility shift assays revealed the presence of an OCL nuclear factor (NFOC-1) that binds to the LTR 21-base pair direct repeat, a region critical for the promoter activity. This binding is further enhanced by Tax. Since NFOC-1 is absent in macrophages and conserved in osteoclasts among species including human, such a factor may play a role in lineage determination and/or in expression of the differentiated osteoclast phenotype.

Acid Phosphatase↗

PubMed1997

Targeted ablation of the vitamin D receptor: an animal model of vitamin D-dependent rickets type II with alopecia.

Vitamin D, the major steroid hormone that controls mineral ion homeostasis, exerts its actions through the vitamin D receptor (VDR). The VDR is expressed in many tissues, including several tissues not thought to play a role in mineral metabolism. Studies in kindreds with VDR mutations (vitamin D-dependent rickets type II, VDDR II) have demonstrated hypocalcemia, hyperparathyroidism, rickets, and osteomalacia. Alopecia, which is not a feature of vitamin D deficiency, is seen in some kindreds. We have generated a mouse model of VDDR II by targeted ablation of the second zinc finger of the VDR DNA-binding domain. Despite known expression of the VDR in fetal life, homozygous mice are phenotypically normal at birth and demonstrate normal survival at least until 6 months. They become hypocalcemic at 21 days of age, at which time their parathyroid hormone (PTH) levels begin to rise. Hyperparathyroidism is accompanied by an increase in the size of the parathyroid gland as well as an increase in PTH mRNA levels. Rickets and osteomalacia are seen by day 35; however, as early as day 15, there is an expansion in the zone of hypertrophic chondrocytes in the growth plate. In contrast to animals made vitamin D deficient by dietary means, and like some patients with VDDR II, these mice develop progressive alopecia from the age of 4 weeks.

Alopecia↗

PubMed1997

Bcl-2 lies downstream of parathyroid hormone-related peptide in a signaling pathway that regulates chondrocyte maturation during skeletal development.

Parathyroid hormone-related peptide (PTHrP) appears to play a major role in skeletal development. Targeted disruption of the PTHrP gene in mice causes skeletal dysplasia with accelerated chondrocyte maturation (Amizuka, N., H. Warshawsky, J.E. Henderson, D. Goltzman, and A.C. Karaplis. 1994. J. Cell Biol. 126:1611-1623; Karaplis, A.C., A. Luz, J. Glowacki, R.T. Bronson, V.L.J. Tybulewicz, H.M. Kronenberg, and R.C. Mulligan. 1994. Genes Dev. 8: 277-289). A constitutively active mutant PTH/PTHrP receptor has been found in Jansen-type human metaphyseal chondrodysplasia, a disease characterized by delayed skeletal maturation (Schipani, E., K. Kruse, and H. Jüppner. 1995. Science (Wash. DC). 268:98-100). The molecular mechanisms by which PTHrP affects this developmental program remain, however, poorly understood. We report here that PTHrP increases the expression of Bcl-2, a protein that controls programmed cell death in several cell types, in growth plate chondrocytes both in vitro and in vivo, leading to delays in their maturation towards hypertrophy and apoptotic cell death. Consequently, overexpression of PTHrP under the control of the collagen II promoter in transgenic mice resulted in marked delays in skeletal development. As anticipated from these results, deletion of the gene encoding Bcl-2 leads to accelerated maturation of chondrocytes and shortening of long bones. Thus, Bcl-2 lies downstream of PTHrP in a pathway that controls chondrocyte maturation and skeletal development.

Animals↗

PubMed1997

1,25-Dihydroxyvitamin D3 selectively induces increased expression of the Na,K-ATPase beta 1 subunit in avian myelomonocytic cells without a concomitant change in Na,K-ATPase activity.

Treatment of avian myelomonocytic cells with 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) results in an approximately two fold increase in levels of Na,K-ATPase beta 1 subunit mRNA and protein (both total and plasma membrane-associated). The changes in beta 1 subunit expression occur in the absence of a detectable increase in expression of any of the three alpha subunit isoforms or in Na,K-ATPase activity. The selective induction of the expression of the beta subunit in avian myelomonocytic cells by 1,25(OH)2D3 reveals a previously unobserved feature of the regulation of Na,K-ATPase expression, while the targeting of beta subunit polypeptides to the plasma membrane in the absence of a corresponding increase in active Na,K-ATPase suggests that, in these cells, transport of the beta subunit to the plasma membrane may be independent of its binding to the alpha subunit.

Animals↗

PubMed1997

Role of c-Src in cellular events associated with colony-stimulating factor-1-induced spreading in osteoclasts.

We and others have observed that in response to treatment with Colony Stimulating Factor-1 (CSF-1) neonatal rat osteoclasts demonstrate rapid cytoplasmic spreading. The receptor for CSF-1, c-Fms, is expressed in osteoclasts, possesses intrinsic tyrosine-kinase activity, and signals via rapid phosphorylation of selected proteins. It has been reported previously that c-Src becomes tyrosine phosphorylated following CSF-1 treatment of fibroblasts overexpressing c-Fms. We therefore examined the cellular events associated with CSF-1-induced spreading in osteoclasts and what role, if any, c-Src played in these processes. Confocal microscopic studies using phosphotyrosine (P-tyr) monoclonal antibodies demonstrated that CSF-1 induced a significant dose- and time-dependent increase in P-tyr labeling of neonatal rat osteoclasts. Phalloidin staining was consistent with partial to complete disassembly of the actin attachment ring with redistribution of actin to the spreading cytoplasmic edge of the cell. Quantitation of cellular F-actin using NBD-phallicidin confirmed a decrease in polymerized actin following exposure to CSF-1. In contrast, CSF-1 failed to induce any cytoplasmic spreading in osteoclasts isolated from mice with targeted disruption of the src gene. Further, in src- osteoclasts no well defined attachment ring could be identified. To investigate cell-signaling events associated with osteoclast spreading, detergent lysates were made from purified multinucleated osteoclast-like cells (OCLs) obtained by coculturing murine bone marrow and osteoblasts with calcitriol. Western blot analyses of lysates from control and CSF-1-treated normal cells indicated that several proteins were specifically phosphorylated in response to CSF-1, most notably proteins of 165, 60, and 85-90 kDa. Immunoprecipitation studies revealed that the 165 and 60 kDa proteins were, respectively, c-Fms and c-Src. The c-Src kinase activity was increased 2.9-fold following CSF-1 treatment. The 85-90 kDa protein is as yet unidentified. Since activated receptor tyrosine kinases may induce spreading in part by reducing phosphoinositol 4,5-bisphosphate (PIP2) binding to actin-associated proteins, a monoclonal antibody to PIP2 was used to assess the nature of PIP2 binding proteins in OCLs. Proteins of 85-90 kDa, 43 kDa, and 30 kDa were consistently demonstrated to bind PIP2. Further, the PIP2 content of the 85-90 kDa protein appeared to decrease with CSF-1 treatment. Whether this protein represents the phosphoprotein of the same M.W. is unclear. We also examined the effect of CSF-1 on the PIP2 content of alpha-actinin. Alpha-actinin showed low-level PIP2 binding, which was demonstrable only after immuno-precipitation and did not change with CSF-1 treatment. However, CSF-1 did cause a significant decline in the phosphotyrosine content of alpha-actinin. In contrast, in src- OCLs, CSF-1 induced more prolonged phosphorylation of c-Fms, and the 85-90 kDa protein was markedly hypophosphorylated. Further, alpha-actinin did not dephosphorylate in src- cells. We conclude that CSF-1-induced osteoclast spreading is accompanied by rapid reorganization of the actin cytoskeleton and phosphorylation of several cellular substrates, including c-Fms and c-Src. PIP2 binding to at least one protein appears to decrease with CSF-1 treatment, which may favor actin depolymerization. The reduced tyrosine phosphorylation of alpha-actinin could effect its ability to bind to actin. Thus c-Src may play an important role in these cellular events since in its absence, osteoclasts do not spread and signaling events downstream are altered. Whether these changes relate in part to the basal abnormalities in the cytoskeletal organization of src- osteoclasts remains to be determined.

Actinin↗

PubMed1997

[Vasculitis course of neuroborreliosis with thalamic infarct].

A-20-year-old man without vascular risk factors presented with paraesthesia of the left side of the body with acute onset. Cerebral magnetic resonance imaging showed an infarction in the right thalamus. Intra-arterial digital subtraction angiography revealed stenosis of the right thalamic vessels. Recent infection by Borrelia burgdorferi was demonstrated by typical findings in the cerebrospinal fluid: lymphocytic pleocytosis and intrathecal synthesis of borrelial-specific antibodies. The diagnosis of a borrelial-induced vasculitis with secondary thalamic infarction was made from these findings. After antibiotic treatment with cefrtriaxone, the patient was discharged without residual complaints.

Adult↗

PubMed1997

Afferent large fiber polyneuropathy predicts the development of postherpetic neuralgia.

Acute zoster infection may be followed by a chronic pain syndrome, i.e., postherpetic neuralgia (PHN). Besides older age, the intensity of pain and neuronal damage within the acutely affected body region are regarded as predictors or risk factors for PHN. As an alternative approach an underlying peripheral polyneuropathy may be considered as potential co-factor. Is a preexisting generalized impairment of certain fiber classes important in initiating chronic pain states after subsequent localized nerve lesion due to zoster infection? Neurophysiological tests of different efferent and afferent small and large fiber systems were performed prospectively at unaffected body regions in patients with acute herpes zoster. Patients that were still in pain 6 months later (PHN, n = 17) and pain free patients (non-PHN, n = 17) were compared regarding the results obtained during the acute phase. Both groups were age matched. Nociceptive C-fiber function was assessed at the forearm by quantitative measurement of the axon reflex vasodilatation and flare induced by histamine iontophoresis. Mechanosensitive A beta-fibers were tested at all extremities by quantitative vibrametry. Parasympathetic small fiber function was studied by heart rate variability tests. No clinically manifest polyneuropathy was present. However, in PHN risk patients considerably higher vibration detection thresholds in hands and feet were detected compared with non-PHN patients. Pathologic test results of vibration sense at the lower extremity predicted PHN with a sensitivity of 70%. Nociceptive C-fiber and parasympathetic fiber function demonstrated no significant differences in both groups. Acute zoster pain was slightly more intense in the PHN group. We concluded that (i) a mild generalized impairment of afferent A beta-fiber function (A beta-polyneuropathy) seems to be an important co-factor in the development of PHN and (ii) impairment of vibration sense, i.e., impairment of afferent A beta-fiber function, may be used as a predictor of PHN.

Acute Disease↗

PubMed1997

Assessment of metabolic response and functional changes after anterior cruciate ligament surgery.

The main purpose of this study was to investigate metabolic changes after anterior cruciate ligament reconstruction and to find out whether the statements on training intensity in the literature can also be applied to patients in rehabilitation. Twenty-four male patients, 16 wk (range: 11-21 wk) postoperative, volunteered to participate in the study. A new four-stage test with constant time and load increments on the legpress in the closed kinetic chain (CKC) was performed. Evaluative variables were the aerobic and anaerobic threshold, force at exhaustion, and the maximum strength. Besides the four-stage test, the other parameters evaluated were functional tests (one-legged single hop, one-legged triple hop), rating scales (Lysholm, subjective feeling scale), thigh circumference, peak torque (PT), work and explosive force in the open kinetic chain (OKC), and muscle activity via electromyographic examination. Positive correlations were established for the maximum force in the CKC and PT (P < 0.01) and maximum force in the CKC and the thigh circumference (P < 0.05). The Lysholm score correlated with the anaerobic threshold (P < 0.05) and with the functional tests (P < 0.01). Further correlations were established between the PT and the work (P < 0.05) and between the single hop and the triple hop (P < 0.001). While the maximum force of the uninvolved leg was significantly higher than that of the involved leg (P < 0.01), the forces measured at termination and at the aerobic and anaerobic threshold (given as percent of maximum force) were significantly higher in the involved leg (P < 0.01). It seems that the oxidative potential increases or remains constant in this phase of rehabilitation and therefore the intensity of strength training must be adapted to the needs of the patient. Our easy-to-perform method makes it possible to establish an optimal intensity of training.

Anaerobic Threshold↗

PubMed1997

Phase II study of Spherex (degradable starch microspheres) injected into the hepatic artery in conjunction with doxorubicin and cisplatin in the treatment of advanced-stage hepatocellular carcinoma: interim analysis.

Patients with advanced-stage unresectable hepatocellular carcinoma (HCC) were treated with intrahepatic arterial doxorubicin 30 mg/m2 plus escalating doses of cisplatin up to 100 mg/m2 in conjunction with rapid bolus injection of Spherex (degradable starch microspheres; Kabi Pharmacia, Lund, Sweden) into the hepatic artery, until slowing or reversal of blood flow. Treatments were repeated every 4 to 6 weeks until progression, or were continued indefinitely if there was disease stability or response. Thirty-five evaluable patients have so far been accrued to the study. Objective tumor responses have occurred in 22 patients (63%), of whom 20 had partial responses and two had complete responses. Four of the patients had reversal of tumor-induced portal vein thrombus. Toxicities included death, one patient (and a death of uncertain cause in an additional patient); hepatitis, two patients; pancreatitis, one patient; dyspnea/hypotension, two patients; and hepatic artery nontransient thrombosis in four patients. Six patients have survived 2 years and an additional 10 patients have survived 1 year. The addition of Spherex to intrahepatic arterial chemotherapy for advanced-stage HCC appears to be relatively safe and is well tolerated even in patients with portal vein thrombosis, which represent the majority of patients with advanced-stage HCC.

Antineoplastic Combined Chemotherapy Protocols↗

PubMed1997

Stress fracture of the ulna in a female table tennis tournament player.

A stress fracture of the diaphyseal ulna occurred in a 19-year-old female competitive table tennis player with a history of secondary amenorrhea. Stress fractures are one of more common problems occurring in vigorous sports. Stress fractures of the ulna can result from any repeated forearm flexor muscle activity. In our case stress was caused by increased athletic activity by additional training twice a day six times a week starting eighth weeks before first signs of pain and by rhythmic repeated submaximal work load in a new job additional to training. Physical training is often associated with menstrual irregularity. Hormonal factors were known for six months. Spontaneous onset of pain in women and previous history of amenorrhea should include stress fractures as differential diagnosis.

Adult↗

PubMed1996

Identification of a novel 135-kDa Grb2-binding protein in osteoclasts.

The tyrosine kinase receptor for macrophage colony-stimulating factor and the non-receptor tyrosine kinase c-Src play critical roles in osteoclast differentiation and function. Since the ubiquitously expressed adaptor protein Grb2 plays an important role in several tyrosine kinase signal transduction pathways, we used a filter binding assay to identify osteoclast proteins that bind to Grb2. In osteoclasts, there were three major Grb2-binding proteins, two of which, mSos and c-Cbl (p120), have been previously identified as Grb2-binding proteins in many cell types. The third protein, p135, had a restricted pattern of expression and was present at high levels in authentic osteoclasts and osteoclast-like cells formed in an in vitro co-culture system. In addition to binding Grb2 in the filter binding assay, p135 was isolated in complexes with endogenous Grb2 from osteoclast cell extracts. The association of p135 and Grb2 was dependent on an intact Src homology 3 domain and furthermore, was shown to preferentially interact with the N-terminal Src homology 3 domain of Grb2, which is similar to the interaction of mSos and Grb2 in other cell types. p135 was not recognized by antibodies against several known Grb2-binding proteins and thus may be a novel Grb2-binding protein.

Adaptor Proteins, Signal Transducing↗

PubMed1996

The deletion of 14 amino acids in the seventh transmembrane domain of a naturally occurring calcitonin receptor isoform alters ligand binding and selectively abolishes coupling to phospholipase C.

The cDNA that encodes the rabbit calcitonin receptor was cloned by screening a rabbit osteoclast library. Reverse transcription-polymerase chain reaction amplification of calcitonin receptor sequences from rabbit osteoclast RNA yielded cDNAs that encode two isoforms of the calcitonin receptor. One isoform is homologous to the C1a isoform previously identified in multiple cell types and species, while the second, designated CTRDeltae13, is a previously unidentified isoform that is apparently generated by alternative splicing during mRNA processing that deletes exon 13, resulting in the absence of 14 amino acids in the predicted seventh transmembrane domain. Expression of mRNA transcripts encoding the two isoforms varies in a tissue-specific manner, with CTRDeltae13 accounting for less than 15% of the total calcitonin receptor mRNA in osteoclasts, kidney, and brain, but comprising at least 50% of the transcripts in skeletal muscle and lung. The two isoforms were expressed, and the ligand binding and signal transduction properties were characterized. Deletion of the residues in the seventh transmembrane domain in CTRDeltae13 reduced the binding affinity for salmon and human calcitonin by more than 10-fold and approximately 2-fold, respectively, resulting in a receptor that failed to discriminate between the two forms of calcitonin. Both isoforms activated adenylyl cyclase, with EC50 values consistent with the difference in ligand affinities. In contrast, only the C1a isoform, but not the CTRDeltae13 isoform, activated phospholipase C. Thus, while the CTRDeltae13 remains active despite the deletion of a significant portion of its seventh transmembrane domain, it has significantly altered ligand recognition and signal transduction properties.

Amino Acid Sequence↗

PubMed1996

c-Cbl is downstream of c-Src in a signalling pathway necessary for bone resorption.

The primary defect in mice lacking the c-src gene is osteopetrosis, a deficiency in bone resorption by osteoclasts. Osteoclasts express high levels of the c-Src protein and the defect responsible for the osteopetrotic phenotype of the c-src-deficient (src-) mouse is cell-autonomous and occurs in mature osteoclasts. However, the specific signalling pathways that require c-Src expression for normal osteoclast activity have not been elucidated. We report here that the proto-oncogene product c-Cbl is tyrosine-phosphorylated in a Src-dependent manner in osteoclasts, where the two proteins colocalize on some vesicular structures. In vitro bone resorption by osteoclast-like cells (OCLs) is inhibited by both c-src and c-cbl antisense oligonucleotides. Furthermore, tyrosine phosphorylation of c-Cbl and the localization of c-Cbl-containing structures to the peripheral cytoskeleton are impaired in resorption-deficient c-src- OCLs, as well as in wild-type OCLs that have been treated with c-src antisense oligonucleotides. These results indicate that c-Cbl may act downstream of c-Src in a signalling pathway that is required for bone resorption.

3T3 Cells↗

PubMed1996

Targeted overexpression of parathyroid hormone-related peptide in chondrocytes causes chondrodysplasia and delayed endochondral bone formation.

Parathyroid hormone-related peptide (PTHrP) was initially identified as a product of malignant tumors that mediates paraneoplastic hypercalcemia. It is now known that the parathyroid hormone (PTH) and PTHrP genes are evolutionarily related and that the products of these two genes share a common receptor, the PTH/PTHrP receptor. PTHrP and the PTH/PTHrP receptor are widely expressed in both adult and fetal tissues, and recent gene-targeting and disruption experiments have implicated PTHrP as a developmental regulatory molecule. Apparent PTHrP functions include the regulation of endochondral bone development, of hair follicle formation, and of branching morphogenesis in the breast. Herein, we report that overexpression of PTHrP in chondrocytes using the mouse type II collagen promoter induces a novel form of chondrodysplasia characterized by short-limbed dwarfism and a delay in endochondral ossification. This features a delay in chondrocyte differentiation and in bone collar formation and is sufficiently marked that the mice are born with a cartilaginous endochondral skeleton. In addition to the delay, chondrocytes in the transgenic mice initially become hypertrophic at the periphery of the developing long bones rather than in the middle, leading to a seeming reversal in the pattern of chondrocyte differentiation and ossification. By 7 weeks, the delays in chondrocyte differentiation and ossification have largely corrected, leaving foreshortened and misshapen but histologically near-normal bones. These findings confirm a role for PTHrP as an inhibitor of the program of chondrocyte differentiation. PTHrP may function in this regard to maintain the stepwise differentiation of chondrocytes that initiates endochondral ossification in the midsection of endochondral bones early in development and that also permits linear growth at the growth plate later in development.

Animals↗

PubMed1996

Respiratory modulation of blood flow in normal and sympathectomized skin in humans.

Sympathetic vasoconstrictor neurons innervating hairless skin of the cat show a respiratory rhythm of activity discharging in inspiration. The following questions arise: (1) Is it possible to detect respiratory variations in cutaneous blood flow in humans? (2) Are these variations actively mediated by rhythmic activity in vasoconstrictor neurons (active rhythms), or do they depend on blood flow changes induced passively due to respiratory blood pressure waves (passive rhythms)? Three patients who had been sympathectomized unilaterally and four healthy controls were studied. Cutaneous blood flow was measured bilaterally using a laser-Doppler flowmeter during physiological breathing (14/min, tidal volume 500-600 ml. minute volume 81/min) and during slower respiratory rate with a higher tidal and smaller minute volume (5/min, 11, 51/min). The temporal pattern of skin blood flow was analyzed with respect to respiration by constructing peri-event-time histograms after summation and averaging of 10-15 respiratory cycles. During physiological breathing no or minimal variation of cutaneous blood flow could be detected. During slower respiratory rate with higher tidal and smaller minute volume a potentiation of variations appeared. In controls the inspiratory phase was followed by a considerable decrease in cutaneous blood flow with a latency of 4.6 s. Identical rhythms were also present on the unoperated side of the patients. In contrast, on the sympathectomized side a respiratory rhythm appeared that was lower in amplitude and phase shifted by about half a cycle. We conclude: (1) Respiration related cutaneous blood flow variations can be detected, in particular if slower respiratory rates, higher tidal and smaller minute volumes are present. (2) Passive oscillations can be differentiated from active rhythms due to sympathetic vasoconstrictor activity by their temporal pattern. (3) The observations suggest that the neurons responsible for the active rhythm discharge during inspiration.

Adult↗

PubMed1996

Vanadate inhibits vacuolar H(+)-ATPase-mediated proton transport in chicken kidney microsomes by an ADP-dependent mechanism.

Recent reports indicate that vacuolar-type proton ATPases from chicken osteoclasts (Chatterjee et al. (1992) Proc. Natl. Acad. Sci. USA 89, 6257-6261), yeast vacuoles and chromaffin granules (Beltran and Nelson (1992) Acta Physiol. Scand. Suppl. 607, 41-47) can be inhibited by vanadate, albeit at a concentration much higher than that required to inhibit P-type ATPases. We have characterized the mechanism by which vanadate inhibits vacuolar-type ATPase-mediated proton transport by chicken kidney microsomes. The initial rate of proton transport is somewhat less sensitive to vanadate than the total acidification, with IC50 values of 1.58 mM and 0.78 mM vanadate, respectively. The inhibition of both the initial rate and total acidification is noncompetitive with respect to ATP. The inhibition is abolished when ADP is removed by an ATP-regenerating system, and the addition of exogenous ADP increases the vanadate inhibition of proton transport in a synergistic manner, thus demonstrating that inhibition by vanadate is dependent on the presence of ADP and explaining the lower effect of vanadate on the initial rate of acidification. Phosphate protects proton transport activity from inhibition by vanadate. These effects of ADP and phosphate suggest that inhibition by vanadate may involve the formation of a complex with ADP at a nucleotide binding site, possibly at the catalytic site of the enzyme.

Adenosine Diphosphate↗

PubMed1996

The bisphosphonate tiludronate is a potent inhibitor of the osteoclast vacuolar H(+)-ATPase.

Although bisphosphonates have been shown to be potent inhibitors of osteoclast-mediated bone resorption in vivo and in vitro and are used as therapeutic agents in hyper-resorptive bone diseases such as Paget disease or hypercalcemia of malignancy, their exact biochemical target(s) and mode(s) of action are for the most part still unknown. The resorption of bone requires solubilization of the mineral component of the matrix, achieved by acidification of the resorbing compartment by a vacuolar-type proton ATPase (V-ATPase) present in the ruffled border membrane of osteoclasts. Since we have shown that the V-ATPase is inhibited by both ADP and phosphate, which share structural characteristics with bisphosphonates, we hypothesized that inhibition of the osteoclast V-ATPase could be one of the mechanism(s) by which bisphosphonates inhibit bone resorption. Pyrophosphate and the bisphosphonates etidronate, alendronate, and YM-175 inhibited proton transport in membrane vesicles derived from chicken kidney and osteoclasts but with very low potency (IC50 > or = 5 mM). In contrast, the ability of tiludronate to inhibit proton transport was 5-fold higher in kidney-derived vesicles (IC50 = 1.1 mM) and 10,000-fold higher in vesicles derived from osteoclasts (IC50 = 466 nM). Tiludronate also potently inhibited proton transport in yeast microsomal preparations (IC50 = 3.5 microM) and inhibited the activity of purified yeast V-ATPase. The inhibition of the osteoclast V-ATPase-mediated proton transport by tiludronate was rapid, pH-dependent, and reversible. No change in membrane vesicle permeability to protons was detected. The inhibition was noncompetitive with respect to ATP, and tiludronate did not protect the pump from inactivation by N-ethylmaleimide, strongly suggesting that tiludronate does not bind to the catalytic site of the enzyme. It is concluded that tiludronate is a significantly more potent inhibitor of V-ATPase than other bisphosphonates and that it has a significant degree of selectivity for the avian osteoclast V-ATPase relative to the avian kidney V-ATPase.

Alendronate↗