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We provide the first demonstration for a Na+/Ca2+ exchanger, NCX-1, in the osteoclast. We speculate that by using Na+ exchange, NCX-1 couples H+ extrusion with Ca2+ fluxes during bone resorption. Microspectrofluorimetry of fura-2-loaded osteoclasts revealed a rapid and sustained, but reversible, cytosolic Ca2+ elevation upon Na+ withdrawal. This elevation was abolished by the cytosolic introduction (by gentle permeabilization) of a highly specific Na+/Ca2+ exchange inhibitor peptide, XIP, but not its inactive analogue, sXIP. Confocal microscopy revealed intense plasma membrane immunofluorescence with an isoform-specific monoclonal anti-NCX-1 antibody applied to gently permeabilized osteoclasts. Electrophysiological studies using excised outside-in membrane patches showed a low-conductance, Na+-selective, dichlorobenzamil-sensitive, amiloride-insensitive channel that we tentatively assigned as being an NCX. Finally, to examine for physiological relevance, an osteoclast resorption (pit) assay was performed. There was a dramatic reduction of bone resorption following NCX-1 inhibition by dichlorobenzamil and XIP (but not with S-XIP). Together, the results suggest that a functional NCX, likely NCX-1, is involved in the regulation of osteoclast cytosolic Ca2+ and bone resorption., (Copyright 2001 Academic Press.)

We report the molecular cloning and functional characterization of a novel member of the CD38 family of cyclic ADP-ribose (cADPr)-generating cyclases. We cloned a cDNA insert that encoded a 298-amino-acid-long protein (M(w) approximately 39 kDa). The predicted protein displayed 69, 61, and 58% similarity, respectively, to mouse, rat, and human CD38. Rabbit CD38 was also 28% homologous to Aplysia ADP-ribosyl cyclase and leukocyte CD157 (another ADP-ribosyl cyclase); the three cyclases shared 10 cysteine and 2 adjacent proline residues. We then transfected CD38-negative NIH3T3 cells with cDNA encoding a CD38-EGFP fusion protein. Epifluorescence microscopy showed intense EGFP fluorescence confirming CD38 expression. We finally confirmed the ADP-ribosyl cyclase activity of the expressed CD38 by measuring its ability to catalyze the cyclization of the nicotinamide adenine dinucleotide (NAD(+)) surrogate, NGD(+), to its fluorescent nonhydrolyzable derivative, cGDPr., (Copyright 2000 Academic Press.)

Nitric oxide (NO) triggers marked osteoclast retraction which closely resembles that due to Ca2+. The effect of Ca2+ has been attributed to a stimulated release of NO. Here, we show for the first time, by direct measurement with a microsensor, that osteoclasts do indeed produce NO and that this production is enhanced by a high Ca2+. We also show that the Ca2+ ionophore, A23187, mimics the latter. Furthermore, osteoclasts on dentine produce more NO than osteoclasts on glass and NO release from dentine-plated osteoclasts is much less sensitive to stimulation by Ca2+. Finally, the microsomal Ca2+ store-depleting agent, thapsigargin, attenuates NO release only from osteoclasts on glass, suggesting that stored Ca2+ has the dominant effect in modulating NO release from non-resorbing cells. NO is a powerful inhibitor of bone resorption: a direct demonstration of its production is therefore strong evidence for a role in modulating osteoclast function., (Copyright 1999 Academic Press.)

We provide the first molecular evidence for the presence of a functional serine/threonine phosphatase, calcineurin-A (CN-A), in the osteoclast. Polymerase chain reaction (PCR) of an osteoclast cDNA library, together with restriction mapping, revealed two isoform sequences, alpha and beta. We then examined the functionality of the detected CN-A by assessing the effect of a classical antagonist, cyclosporin A (CsA), in the osteoclast resorption (pit) assay. CsA (0.1 and 1 microg ml-1) potently inhibited bone resorption. The presence of lymphocytes, with or without prior exposure to CsA in vivo, failed to reverse the CsA-induced resorption-inhibition. Expectedly, CsA had no direct effect on cytosolic Ca2+ levels in fura-2-loaded osteoclasts. These studies are a prelude to further investigations into the possible role of CN-A in osteoclast regulation. Finally, mechanistic studies on the bone effects of CsA, a widely used immunosupressant, should proceed from these observations., (Copyright 1999 Academic Press.)

Upon removal of its sialic acid or galactose residue, vitamin D-binding protein (DBP) becomes a potent macrophage-activating factor, DBP-MAF. Here we document a new function of DBP-MAF and its parent molecule, DBP, in osteoclast control. We show that all DBPs potently inhibit extracellular Ca2+ (cation) sensing at low nanomolar concentrations with the following rank order of potency: native DBP = sialidase-treated DBP > beta-galactosidase-treated DBP. This attenuation remains unaffected despite co-incubation either with the native DBP ligand, 1,25-dihydroxyvitamin D3, or with an asialoglycoprotein receptor modulator, asialoorosomucoid. Taken together, the results suggest that circulating DBP may play a role in the systemic control of osteoclastic bone resorption, a hitherto unrecognized action of the protein.

We demonstrate for the first time that cellular aging in vitro is accompanied by a dramatic elevation in the levels of ryanodine receptor-bearing Ca2+ channels. These channels normally reside within microsomal membranes and gate Ca2+ release from intracellular stores. We therefore measured cytosolic Ca2+ levels in 'young' (30 mean population doublings, MPDs) and 'senescent' (53 to 58 MPDs) human diploid fibroblasts (HDFs). Application of the known ryanodine receptor modulators, caffeine or cyclic adenosine diphosphate-ribose (cADPr), triggered cytosolic Ca2+ signals in both young and senescent cells. The signal magnitude however was significantly greater in senescent compared with young HDFs. In parallel, incubation with a highly specific anti-ryanodine receptor antiserum resulted in specific immunofluorescence only in senescent HDFs. We envisage that elevated levels of functional ryanodine receptors may underlie the defective Ca2+ handling and cellular degeneration that occurs with aging., (Copyright 1998 Academic Press.)

We report that the divalent cation Ni2+ elicits elevations in the cytosolic free Ca2+ concentration ([Ca2+]) in cultured enterocytes. These elevations were monophasic, each response consisting of a rapid initial transient rise of cytosolic [Ca2+] to a peak value followed by an exponential decline. The magnitude of the cytosolic [Ca2+] elevation varied with the concentration of applied Ni2+. In some cells, a single application of Ni2+ induced oscillatory changes in cytosolic [Ca2+]. There was also evidence for use-dependent inactivation: a conditioning application of Ni2+ substantially attenuated the response resulting from its subsequent application. Our findings thus demonstrate the existence of a divalent cation-sensing "receptor" on the enterocyte. This putative receptor may play a role in regulating mineral absorption across the enterocyte membrane.

We report a sensitivity of the osteoclast cell surface Ca2+ receptor to extracellular protons. Freshly isolated rat osteoclasts were exposed to the known agonists of the Ca2+ receptor, Ca2+ and Ni2+, in extracellular solutions set at different pH values. Decreasing the extracellular pH from 7.8 to 4.0 units markedly potentiated the cytosolic Ca2+ signals elicited in response to Ca2+ receptor activation by either Ni2+ (50 microM, 500 microM or 5 mM) or Ca2+ (5 mM). Each response consisted of a rapid and usually transient elevation of cytosolic [Ca2+]. Maximal cytosolic [Ca2+] responses were obtained at pH values of 6.6 (for 5 mM-[Ni2+]) and 4.0 units (for 5 mM-[Ca2+]). Finally, the effects of extracellular pH persisted in Ca(2+)-free, EGTA-containing solutions, suggesting a modulation of intracellular Ca2+ release.