Beyond Reproduction: Pituitary Hormone Actions on Bone.

The long-held belief that pituitary hormones act solely on master targets was first questioned when we documented G protein-coupled receptors for thyroid-stimulating hormone, follicle-stimulating hormone, adrenocorticotrophic hormone, oxytocin, and vasopressin on bone cells. These evolutionarily conserved hormones and their receptors are known to have primitive roles, and exist in invertebrate species as far down as coelenterates. It is not surprising therefore that each such hormone has multiple hitherto unrecognized functions in mammalian integrative physiology, and hence, becomes a potential target for therapeutic intervention. Here we discuss the skeletal actions of pituitary hormones.

Thyroid and bone: macrophage-derived TSH-ß splice variant increases murine osteoblastogenesis.

It is now firmly established that TSH may influence the physiology and patho-physiology of bone by activating osteoblasts and inhibiting osteoclast activity resulting in relative osteoprotection. Whether this influence is directly exerted by pituitary-derived TSH in vivo is less certain, because we have previously reported that the suppression of pituitary TSH does not remove such protection. Here, we have characterized the functional relevance of a novel form of the TSH-ß subunit, designated TSH-ßv, known to be produced by murine bone marrow cells. We found that fresh bone marrow-derived macrophages (MØs) preferentially produced TSH-ßv and, when cocultured with CHO cells engineered to overexpress the full-length TSH receptor, were able to generate the production of intracellular cAMP; a phenomenon not seen in control CHO cells, such results confirmed the bioactivity of the TSH variant. Furthermore, cocultures of MØs and osteoblasts were shown to enhance osteoblastogenesis, and this phenomenon was markedly reduced by antibody to TSH-ß, suggesting direct interaction between MØs and osteoblasts as observed under the electron microscope. These data suggest a new paradigm of local modulation of bone biology by a MØ-derived TSH-like molecule and raise the question of the relative contribution of local vs pituitary-derived TSH in osteoprotection.

Calcium signalling and calcium transport in bone disease.

Calcium transport and calcium signalling mechanisms in bone cells have, in many cases, been discovered by study of diseases with disordered bone metabolism. Calcium matrix deposition is driven primarily by phosphate production, and disorders in bone deposition include abnormalities in membrane phosphate transport such as in chondrocalcinosis, and defects in phosphate-producing enzymes such as in hypophosphatasia. Matrix removal is driven by acidification, which dissolves the mineral. Disorders in calcium removal from bone matrix by osteoclasts cause osteopetrosis. On the other hand, although bone is central to management of extracellular calcium, bone is not a major calcium sensing organ, although calcium sensing proteins are expressed in both osteoblasts and osteoclasts. Intracellular calcium signals are involved in secondary control including cellular motility and survival, but the relationship of these findings to specific diseases is not clear. Intracellular calcium signals may regulate the balance of cell survival versus proliferation or anabolic functional response as part of signalling cascades that integrate the response to primary signals via cell stretch, estrogen, tyrosine kinase, and tumor necrosis factor receptors.

Epidermal growth factor activates m-calpain (calpain II), at least in part, by extracellular signal-regulated kinase-mediated phosphorylation.

How m-calpain is activated in cells has challenged investigators because in vitro activation requires near-millimolar calcium. Previously, we demonstrated that m-calpain activation by growth factors requires extracellular signal-regulated kinase (ERK); this enables tail deadhesion and allows productive motility. We now show that ERK directly phosphorylates and activates m-calpain both in vitro and in vivo. We identified serine 50 as required for epidermal growth factor (EGF)-induced calpain activation in vitro and in vivo. Replacing the serine with alanine limits activation by EGF and subsequent cell deadhesion and motility. A construct with the serine converted to glutamic acid displays constitutive activity in vivo; expression of an estrogen receptor fusion construct produces a tamoxifen-sensitive enzyme. Interestingly, EGF-induced m-calpain activation occurs in the absence of increased intracellular calcium levels; EGF triggers calpain even in the presence of intracellular calcium chelators and in calcium-free media. These data provide evidence that m-calpain can be activated through the ERK cascade via direct phosphorylation and that this activation may occur in the absence of cytosolic calcium fluxes.

Recent advances in osteoclast biology and pathological bone resorption.

The osteoclast is a bone-degrading polykaryon. Recent studies have clarified the differentiation of this cell and the biochemical mechanisms it uses to resorb bone. The osteoclast derives from a monocyte/macrophage precursor. Osteoclast formation requires permissive concentrations of M-CSF and is driven by contact with mesenchymal cells in bone that bear the TNF-family ligand RANKL. Osteoclast precursors express RANK, and the interaction between RANKL and RANK (which is inhibited by OPG) is the major determinant of osteoclast formation. Hormones, such as PTH/PTHrP, glucocorticoids and 1,25(OH)2D3, and humoral factors, including TNFalpha, interleukin-1, TGFss and prostaglandins, influence osteoclast formation by altering expression of these molecular factors. TNFalpha, IL-6 and IL-11 have also been shown to promote osteoclast formation by RANKL-independent processes. RANKL-dependent/independent osteoclast formation is likely to play an important role in conditions where there is pathological bone resorption such as inflammatory arthritis and malignant bone resorption. Osteoclast functional defects cause sclerotic bone disorders, many of which have recently been identified as specific genetic defects. Osteoclasts express specialized proteins including a vacuolar-type H+-ATPase that drives HCl secretion for dissolution of bone mineral. One v-ATPase component, the 116 kD V0 subunit, has several isoforms. Only one isoform, TCIRG1, is up-regulated in osteoclasts. Defects in TCIRG1 are common causes of osteopetrosis. HCl secretion is dependent on chloride channels; a chloride channel homologue, CLCN7, is another common defect in osteopetrosis. Humans who are deficient in carbonic anhydrase II or who have defects in phagocytosis also have variable defects in bone remodelling. Organic bone matrix is degraded by thiol proteinases, principally cathepsin K, and abnormalities in cathepsin K cause another sclerotic bone disorder, pycnodysostosis. Thus, bone turnover in normal subjects depends on relative expression of key cytokines, and defects in osteoclastic turnover usually reflect defects in specific ion transporters or enzymes that play essential roles in bone degradation.

Defining thyrotropin-dependent and -independent steps of thyroid hormone synthesis by using thyrotropin receptor-null mice.

The thyrotropin (TSH) receptor (TSHR) is a member of the heterotrimeric G protein-coupled family of receptors whose main function is to regulate thyroid cell proliferation as well as thyroid hormone synthesis and release. In this study, we generated a TSHR knockout (TSHR-KO) mouse by homologous recombination for use as a model to study TSHR function. TSHR-KO mice presented with developmental and growth delays and were profoundly hypothyroid, with no detectable thyroid hormone and elevated TSH. Heterozygotes were apparently unaffected. Knockout mice died within 1 week of weaning unless fed a diet supplemented with thyroid powder. Mature mice were fertile on the thyroid-supplemented diet. Thyroid glands of TSHR-KO mice produced uniodinated thyroglobulin, but the ability to concentrate and organify iodide could be restored to TSHR-KO thyroids when cultured in the presence of the adenylate cyclase agonist forskolin. Consistent with this observation was the lack of detectable sodium-iodide symporter expression in TSHR-KO thyroid glands. Hence, by using the TSHR-KO mouse, we provided in vivo evidence, demonstrating that TSHR expression was required for expression of sodium-iodide symporter but was not required for thyroglobulin expression, suggesting that the thyroid hormone synthetic pathway of the mouse could be dissociated into TSHR-dependent and -independent steps.

Osteoclasts secrete the chemotactic cytokine mim-1.

Osteoclasts are terminally differentiated, multinucleated cells of monocytic origin. In this study, we report that osteoclasts secrete a 35 kD protein and that phorbol myristate acetate treatment stimulates secretion dramatically. Peptide digests of the protein were analyzed by mass spectroscopy. The protein was identified as myb induced myeloid protein-1 precursor (mim-1 protein). Mim-1 is expressed specifically by hematopoietic cells and has no known function. It is homologous with the neutrophil chemokine, chondromodulin II, which stimulates proliferation of osteoblasts and chondrocytes. Western analysis showed that osteoclasts secrete mim-1 into culture media. Immunofluorescence studies demonstrated a cytoplasmic and perinuclear distribution of mim-1 in both avian osteoclasts and human osteoclast-like cells. Expression and secretion of a chemokine-like protein by osteoclasts suggests a novel signaling pathway in the bone microenvironment that may be involved in coordinating bone remodeling.

Regional trabecular bone matrix degeneration and osteocyte death in femora of glucocorticoid- treated rabbits.

Glucocorticoids at pharmacological concentrations cause osteoporosis and aseptic necrosis, particularly in the proximal femur. Several mechanisms have been proposed, but the primary events are not clear. We studied changes in the bone structure and cellular activity in femora of glucocorticoid-treated rabbits before the occurrence of fracture or collapse. In rabbits treated 28 days with 4 micromol/kg.day of methylprednisolone acetate, changes in the cortical bone were minor. However, metabolic labeling showed that bone formation was virtually absent in the subarticular trabecular bone, and scanning electron microscopy showed resorption of 50-80% of the trabecular surface. Thus, reduction in bone synthesis and increased resorption were involved in bone loss. Vascular changes, which have been hypothesized to mediate glucocorticoid damage, were not seen, but histological changes suggested that trabecular bone was damaged. Matrix integrity was examined using laser scanning confocal microscopy to detect passive tetracycline adsorption. In treated animals, but not controls, tetracycline was adsorbed, in a novel lamellar pattern, in 50--200 microm regions extending deep into trabeculae. This showed that the matrix, which is normally impervious, was exposed at these sites. TUNEL assays showed that matrix damage correlated with cell death in the subarticular trabecular bone of treated animals. The pattern of cell death involving cohorts of osteoblasts and osteocytes comprised up to half of the bone volume in affected regions and is consistent with an apoptotic mechanism. Small numbers of TUNEL-labeled osteoblasts, but no osteocytes, were detected in control bone. We conclude that exposure of bone matrix permeability and that regional cell death consistent with apoptosis is an early event in glucocorticoid-induced bone damage.

Proteinase expression during differentiation of human osteoclasts in vitro.

Osteoclasts are macrophage-derived polykaryons that degrade bone in an acidic extracellular space. This differentiation includes expression of proteinases and acid transport proteins, cell fusion, and bone attachment, but the sequence of events is unclear. We studied two proteins expressed at high levels only in the osteoclast, cathepsin K, a thiol proteinase, and tartrate-resistant acid phosphatase (TRAP), and compared this expression with acid transport and bone degradation. Osteoclastic differentiation was studied using human apheresis macrophages cocultured with MG63 osteosarcoma cells, which produce cytokines including RANKL and CSF-1 that mediate efficient osteoclast formation. Immunoreactive cathepsin K appeared at 3-5 days. Cathepsin K activity was seen on bone substrate but not within cells, and cathepsin K increased severalfold during further differentiation and multinucleation from 7 to 14 days. TRAP also appeared at 3-5 d, independently of cell fusion or bone attachment, and TRAP activity reached much higher levels in osteoclasts attached to bone fragments. Two proteinases that occur in the precursor macrophages, cathepsin B, a thiol proteinase related to cathepsin K, and an unrelated lysosomal aspartate proteinase, cathepsin D, were also studied to determine the specificity of the differentiation events. Cathepsin B occurred at all times, but increased two- to threefold in parallel with cathepsin K. Cathepsin D activity did not change with differentiation, and secreted activity was not significant. In situ acid transport measurements showed increased acid accumulation after 7 days either in cells on osteosarcoma matrix or attached to bone, but bone pit activity and maximal acid uptake required 10-14 days. We conclude that TRAP and thiol proteinase expression begin at essentially the same time, and precede cell fusion and bone attachment. However, major increases in acid secretion and proteinases expression continue during cell fusion and bone attachment from 7 to 14 days.

Tandem repeat of C/EBP binding sites mediates PPARgamma2 gene transcription in glucocorticoid-induced adipocyte differentiation.

Bone marrow stromal stem cells differentiate into many different types of cells including osteoblasts and adipocytes. Long-term glucocorticoid treatment decreases osteoblastic activity but increases adipocytes. We investigated the mechanism of glucocorticoid-induced PPARgamma2 transcription. Treatment of human bone marrow stromal cells with dexamethasone induced the differentiation of these cells into adipocytes as measured by oil-red O staining, and Northern blot analysis showed that dexamethasone strongly induced PPARgamma2 mRNA expression in cells cultured in adipocyte induction medium. Moreover, the mRNA of C/EBPdelta, an adipocyte-promoting transcription factor, was also induced by dexamethasone in the presence of induction medium. Gel mobility shift assays using purified GST-C/EBPdelta fusion protein showed that C/EBPdelta specifically binds to a 40-base pair DNA element from PPARgamma2 promoter, which was found to contain a tandem repeat of C/EBP binding sites. Transfection studies in mouse mesenchymal C3H10T1/2 cells showed that it is the tandem repeat of the C/EBP binding site in PPARgamma2 promoter region that regulates dexamethasone-mediated PPARgamma2 gene activation. We conclude that glucocorticoid-induced adipogenesis from bone marrow stromal cells is mediated through a reaction cascade in which dexamethasone transcriptionally activates C/EBPdelta; C/EBPdelta then binds to PPARgamma2 promoter and transactivates PPARgamma2 gene expression. This activated master regulator, in turn, initiates the adipocyte differentiation.

Intracellular calcium puffs in osteoclasts.

We studied intracellular calcium ([Ca(2+)](i)) in acid-secreting bone-attached osteoclasts, which produce a high-calcium acidic extracellular compartment. Acid secretion and [Ca(2+)](i) were followed using H(+)-restricted dyes and fura-2 or fluo-3. Whole cell calcium of acid-secreting osteoclasts was approximately 100 nM, similar to cells on inert substrate that do not secrete acid. However, measurements in restricted areas of the cell showed [Ca(2+)](i) transients to 500-1000 nM consistent with calcium puffs, transient (millisecond) localized calcium elevations reported in other cells. Spot measurements at 50-ms intervals indicated that puffs were typically less than 400 ms. Transients did not propagate in waves across the cell in scanning confocal measurements. Calcium puffs occurred mainly over regions of acid secretion as determined using lysotracker red DND99 and occurred at irregular periods averaging 5-15 s in acid secreting cells, but were rare in lysotracker-negative nonsecretory cells. The calmodulin antagonist trifluoperazine, cell-surface calcium transport inhibitors lanthanum or barium, and the endoplasmic reticulum ATPase inhibitor thapsigargin had variable acute effects on the mean [Ca(2+)](i) and puff frequency. However, none of these agents prevented calcium puff activity, suggesting that the mechanism producing the puffs is independent of these processes. We conclude that [Ca(2+)](i) transients in osteoclasts are increased in acid-secreting osteoclasts, and that the puffs occur mainly near the acid-transporting membrane. Cell membrane acid transport requires calcium, suggesting that calcium puffs function to maintain acid secretion. However, membrane H(+)-ATPase activity was insensitive to calcium in the 100 nM-1 microM range. Thus, any effects of calcium puffs on osteoclastic acid transport must be indirect.

Expression of stem cell factor by osteoblasts in normal and hyperparathyroid bone: relation to ectopic mast cell differentiation.

Mast cells accumulate in hyperparathyroid bone, but the reason is not clear. We compared the distribution of mast cells and related growth factors in normal and hyperparathyroid bone. Mast cell formation was strongly affected by proximity to bone-forming surfaces of hyperparathyroid bone. Hyperparathyroidism greatly increased the production by active, bone-synthesizing osteoblasts of stem cell factor (SCF) but not of IL-3. Osteoblast SCF was distributed to the basolateral cell membranes, and its cDNA sequence (GenBank AF119835) is homologous to the murine membrane-bound SCF. Quiescent osteoblasts did not produce detectable SCF. Synthetic osteoblasts in normal bone were SCF positive, but comprised a much smaller population of cells, in keeping with the slow turnover of normal bone. Major SCF isoforms on immunoblot analysis of osteoblast-fraction proteins from high-turnover bone had M(r)s of about 48 and 40 kDa. Similar SCF isoforms were produced by MG63 osteoblast-derived cells and were identified by several anti-SCF antibodies. SCF is expressed in several mesenchymal cell types in a complementary fashion with cells bearing its receptor. SCF potently facilitates differentiation of mast cells, so the increase in paratrabecular mast cells in hyperparathyroid bone is probably driven by osteoblastic SCF. However, since mast cells are not normal components of bone, osteoblastic SCF probably regulates other cells, with mast cell differentiation occurring as a side effect greatly increased osteoblastic activity.

Parathyroid hormone-regulated production of stem cell factor in human osteoblasts and osteoblast-like cells.

We investigated stem cell factor (SCF) expression in osteoblasts because mast cells, which occur ectopically in hyperparathyroid bone, are induced by SCF. Nontransformed osteoblasts and Saos2 or MG63 cells expressed SCF in response to PTH. Western analysis showed only large, cell-associated isoforms, Mrs approximately 40-48 kD. Transfection of MG63 cells with plasmids expressing antisense SCF mRNA eliminated immunoreactive SCF. Sequencing osteoblast SCF cDNAs showed that exon 6 was omitted. mRNAs without exon 6 produce membrane-associated SCF isoforms in rodents, suggesting that human SCFs are processed similarly. The major osteoblastic SCF mRNA, approximately 5 kB, was augmented by PTH. Neither protein or mRNA was increased by vitamin D, however, 6-7 kB transcripts were predominant in other tissues but not detectable in osteoblasts. We conclude that osteoblasts express SCF in response to PTH, with mRNA and protein processing differences relative to other cells. SCF stimulates osteoclasts, suggesting that PTH-induced osteoblastic SCF functions to accelerate bone turnover. Mast cells may occur due to SCF overexpression at extreme PTH levels.

Nitric oxide regulation of cGMP production in osteoclasts.

Bone resorption by osteoclasts is modified by agents that affect cyclic guanosine monophosphate (cGMP), but their relative physiological roles, and what components of the process are present in osteoclasts or require accessory cells such as osteoblasts, are unclear. We studied cGMP regulation in avian osteoclasts, and in particular the roles of nitric oxide and natriuretic peptides, to clarify the mechanisms involved. C-type natriuretic peptide drives a membrane guanylate cyclase, and increased cGMP production in mixed bone cells. However, C-type natriuretic peptide did not increase cGMP in purified osteoclasts. By contrast, osteoclasts did produce cGMP in response to nitric oxide (NO) generators, sodium nitroprusside or 1-hydroxy-2-oxo-3,3-bis(3-aminoethyl)-1-triazene. These findings indicate that C-type natriuretic peptide and NO modulate cGMP in different types of bone cells. The activity of the osteoclast centers on HCI secretion that dissolves bone mineral, and both NO generators and hydrolysis-resistant cGMP analogues reduced bone degradation, while cGMP antagonists increased activity. NO synthase agonists did not affect activity, arguing against autocrine NO production. Osteoclasts express NO-activated guanylate cyclase and cGMP-dependent protein kinase (G-kinase). G-kinase reduced membrane HCI transport activity in a concentration-dependent manner, and phosphorylated a 60-kD osteoclast membrane protein, which immunoprecipitation showed is not an H+-ATPase subunit. We conclude that cGMP is a negative regulator of osteoclast activity. cGMP is produced in response to NO made by other cells, but not in response to C-type natriuretic peptide. G-kinase modulates osteoclast membrane HCI transport via intermediate protein(s) and may mediate cGMP effects in osteoclasts.

Tyrosine kinase inhibitor effects on avian osteoclastic acid transport.

We found that tyrosine kinase pp60(c-src) coisolates with acid-transporting osteoclast membranes and hypothesized that this kinase regulates hydrochloric acid transport. We assayed the membrane acid transport and bone degradation effects of tyrosine kinase inhibitors in avian osteoclasts. Isoflavone, tyrphostin, and benzoquinonoid inhibitors were compared with inactive analogues to determine nonspecific effects. Acid-secreting membranes, isolated by nitrogen cavitation, were assayed as reconstituted vesicles by using acridine orange to indicate ATP-dependent hydrogen ion transport. The soy isoflavone genistein and the benzoquinonoid antibiotic herbimycin inhibited hydrochloric acid transport with 50% inhibition at approximately 10 and approximately 2 micromol/L, respectively; effects appeared in <2 min and were reversible. In membrane incubated with inhibitors, the herbimycin effect also inhibited Cl- transport by variable amounts, suggesting that this compound affects Cl- channel activity. However, genistein and tyrphostins did not produce chloride dependent effects. After 30 min with ATP, tyrphostin A47 irreversibly inhibited hydrochloric acid transport with 50% inhibition at approximately 10 micromol/L. Tyrphostin A25 and controls, tyrphostin A1 and daidzein (a genistein congener), were inactive despite preincubation. Osteoclastic bone resorption was more sensitive to the inhibitors over 3-5-d assays than was membrane acid transport, except for tyrphostins. Herbimycin and genistein inhibited bone resorption with half maximal effects at 0.5 and 10 micromol/L and complete inhibition at 3 d in 1 and 20 micromol/L, respectively. None of the tyrphostins, including A47, nor daidzein inhibited resorption to >20 micromol/L. We conclude that tyrosine kinase inhibition directly inhibits osteoclast membrane hydrochloric acid transport; differences among inhibitors may reflect chemical reactivity and permeability.

How the osteoclast degrades bone.

Osteoclasts are multinucleated monocyte-macrophage derivatives that degrade bone. Their specialized role is central to a process that continuously removes and replaces segments of the skeleton in the higher vertebrates. Osteoclasts allow skeletal mineral to be used to manage extracellular calcium activity, which is an important adaptation for life on land, and solid skeletal structure to be replaced by hollow architecture that has a superior strength-to-weight ratio. Degrading bone also allows periodic repair and remodeling for ordered growth and efficient response to mechanical loads. A fairly comprehensive view of osteoclastic ontogeny and function is emerging from recent studies. Osteoclasts dissolve bone mineral by massive acid secretion and secrete specialized proteinases that degrade the organic matrix, mainly type I collagen, in this acidic milieu. The site of bone dissolution is a high-calcium environment; removal of degradation products by transcytosis of membrane vesicles allows the osteoclast to maintain a normal intracellular calcium. Osteoclastic differentiation is normally balanced with bone formation, although bone formation is the function of unrelated stromal cell-derived osteoblasts. Interactions between osteoclast precursors and bone-forming cells are believed to control osteoclast differentiation under most circumstances, preserving bone architecture over many cycles of bone replacement.

Differential effects of tamoxifen-like compounds on osteoclastic bone degradation, H(+)-ATPase activity, calmodulin-dependent cyclic nucleotide phosphodiesterase activity, and calmodulin binding.

We studied effects of calmodulin antagonists on osteoclastic activity and calmodulin-dependent HCl transport. The results were compared to effects on the calmodulin-dependent phosphodiesterase and antagonist-calmodulin binding affinity. Avian osteoclast degradation of labeled bone was inhibited approximately 40% by trifluoperazine or tamoxifen with half-maximal effects at 1-3 microM. Four benzopyrans structurally resembling tamoxifen were compared: d-centchroman inhibited resorption 30%, with half-maximal effect at approximately 100 nM, cischroman and CDRI 85/287 gave 15-20% inhibition, and l-centchroman was ineffective. No benzopyran inhibited cell attachment or protein synthesis below 10 microM. However, ATP-dependent membrane vesicle acridine transport showed that H(+)-ATPase activity was abolished by all compounds with 50% effects at 0.25-1 microM. All compounds also inhibited calmodulin-dependent cyclic nucleotide phosphodiesterase at micromolar calcium. Relative potency varied with assay type, but d- and l-centchroman, surprisingly, inhibited both H(+)-ATPase and phosphodiesterase activity at similar concentrations. However, d- and l-centchroman effects in either assay diverged at nanomolar calcium. Of benzopyrans tested, only the d-centchroman effects were calcium-dependent. Interaction of compounds with calmodulin at similar concentrations were confirmed by displacement of labeled calmodulin from immobilized trifluoperazine. Thus, the compounds tested all interact with calmodulin directly to varying degrees, and the observed osteoclast inhibition is consistent with calmodulin-mediated effects. However, calmodulin antagonist activity varies between specific reactions, and free calcium regulates specificity of some interactions. Effects on whole cells probably also reflect other properties, including transport into cells.

Characterization of the osteoclast ruffled border chloride channel and its role in bone resorption.

Bone resorption by osteoclasts requires massive transcellular acid transport, which is accomplished by the parallel action of a V-type proton pump and a chloride channel in the osteoclast ruffled border. We have studied the molecular basis for the appearance of acid transport as avian bone marrow mononuclear cells acquire a bone resorptive phenotype in vitro. We demonstrate a critical role for regulated expression of a ruffled border chloride channel as the cells become competent to resorb bone. Molecular characterization of the chloride channel shows that it is related to the renal microsomal chloride channel, p64. In planar bilayers, the ruffled border channel is a stilbene sulfonate-inhibitable, outwardly rectifying chloride channel. A mechanism by which outward rectification of the single channel chloride current could allow efficient regulation of acidification by the channel is discussed.

Regulation of osteoclastic bone resorption by glucose.

Osteoclasts degrade bone by pumping molar quantities of HCl to dissolve the calcium salts of bone, an energy intensive process evidently supported by abundant mitochondria. This is the first study to directly examine the ability of various metabolites to serve as potential energy sources for osteoclastic bone resorption. Glucose, and to a lesser extent lactate, supported osteoclastic bone degradation. However, fatty acids (palmitate, myristate and stearate), essential amino acids plus 20 mM alanine, or ketone bodies (acetoacetate, beta-hydroxybutyrate and alpha-ketoglutarate) did not support bone degradation. Resorption declined to 10-30% of glucose controls when fatty acids or ketoacids were substituted for glucose. Resorption was glucose concentration dependent, with maximal activity at approximately 7 mM (K(M) approximately 3 mM). Glucose transport was linear for approximately 15 minutes, specific for D-glucose, and inhibited by cytochalasin B. Osteoclasts cultured on bone transported glucose at almost twice the rate of those off bone (Vmax 23 versus 13 nmols/mg/min, respectively) and medium acid accumulation paralleled glucose uptake, while the K(M) was unchanged. We conclude that glucose is the principal energy source required for bone degradation. Further, characteristics of glucose transport are consistent with the hypothesis that fluctuations in serum glucose concentration are an important component in regulation of osteoclastic bone degradation.