Parallel solid-phase synthesis of vitronectin receptor (alphavbeta3) inhibitors.

A combinatorial approach for rapid optimization of a vitronectin receptor (alphavbeta3) inhibitor lead was accomplished by solid-phase synthesis. Orthogonally bis protected 2,3-diaminopropionic acid was used to immobilize the C-terminus of the molecule. Selective deprotection and functionalization of the alpha-amino group followed by acyl resorcinol scaffold attachment and N-terminus diversification was used to explore structure activity relationship (SAR).

Expression of meltrin-alpha mRNA is not restricted to fusagenic cells.

Meltrin-alpha is a myoblast gene product reported to be required for cell fusion [Yagami-Hiromasa et al. (1995): Nature 377:652-656]. Because Northern blots revealed expression only in muscle and bone, the suggestion was made that meltrin-alpha is expressed exclusively by fusagenic cells in these tissues (myoblast and osteoclast). We studied expression of meltrin-alpha mRNA in a panel of tissues and cell lines using the polymerase chain reaction and found it widely expressed. Meltrin-alpha mRNA was readily detected in the osteoblast, the most abundant cell type in bone. In situ hybridization analysis on sections of neonatal mice revealed high levels of expression in the trabecular meshwork of long bones, the basal regions of the dermis and its underlying mesenchyme. We conclude that expression of meltrin-alpha mRNA is not restricted to fusagenic cells and that, in bone, the osteoblast is the major source.

Lack of significant effect of carboxyl-terminal parathyroid hormone-related peptide fragments on isolated rat and chick osteoclasts.

Parathyroid hormone-related protein (PTHrP) fragments 107-111, 107-138, and 107-139 were all found to be without consistent significant inhibitory effect on the resorptive activity of isolated rat and chick osteoclasts over the dose range 10(-13) M-10(-9) M. In the rat, these results contrasted with a strong and significant inhibition (100%) by calcitonin. Our results differ from that previously reported for C-terminal fragments of PTHrP by Fenton et al. [1-3], who noted up to a 70% inhibition of resorptive activity of isolated rat or chick osteoclasts at femtomolar doses and greater. It is possible that the lack of response observed in our osteoclast assays is due to unknown variables in the bone slice assay that influence the responsiveness of isolated osteoclasts to these fragments.

Stimulation of bone resorption and osteoclast clear zone formation by low pH: a time-course study.

The significance of low pH-induced stimulation of osteoclastic bone resorption has recently been questioned following the finding that embryonic chick osteoclasts were only weakly stimulated by extremely low pH (6.5) and that the effect was transient, apparently due to cytotoxicity. Although low pH in the range 6.8-7.2 is known to stimulate rat osteoclasts over 24 h, the long-term effects of low pH on mammalian osteoclasts are not known. We have therefore conducted time-course studies over 72 h on the effect of pH in the range 6.3-7.3 on bone resorption and cytotoxicity in both rat and chick osteoclasts. In neonatal rat osteoclasts, lowering extracellular pH produced a powerful and significant stimulation of resorption over 24 h. Detailed analysis of the resorption focus revealed that this was due mainly to a higher proportion of active osteoclasts at lower pH. In addition, osteoclasts excavated slightly larger pits at low pH. Stimulation was no longer significant at 72 h, however, due to a pH-dependent slowing of resorption at acid pH associated 1) with cytotoxicity primarily of nonosteoclastic cells and 2) with an acceleration of bone resorption after 24 h at more alkaline pH. Resorption stimulated by low pH was associated with the formation of actin-rich "clear zones" within the osteoclast. Chick osteoclasts were less sensitive to low pH than rat osteoclasts but nonetheless showed a consistently higher level of resorption at low pH over 24-72 h. These results suggest that protons play an important regulatory role in neonatal rat osteoclasts, and stimulate the formation of clear zones. The lower sensitivity of the chick osteoclast to acid pH may be due to a species difference or the chick osteoclast's higher basal level of resorption.

Effects of phorbol myristate acetate on rat and chick osteoclasts.

The role of protein kinase C in the regulation of osteoclast function is not known. We therefore compared the effect of phorbol myristate acetate (PMA), which activates protein kinase C, on the resorptive function, motility, and morphology of osteoclasts from rat and chick. PMA caused a significant reduction in resorption pit number in both species; rat osteoclasts were more sensitive, being significantly inhibited at doses of 10(-9)-10(-6) M compared with 10(-7)-10(-6) M for chick osteoclasts. The inactive analog PMA-alpha was without significant effect, and inhibition was not blocked by 10(-6) M indomethacin. In time course experiments, inhibition at 24 h was similar to or greater than inhibition at 6 h, indicating a persistent or progressive effect on bone resorption. Removal of PMA after 6 h prompted partial recovery of bone-resorptive ability in chick osteoclasts but not rat, at least over a 48 h incubation. In time-lapse video studies of rat osteoclasts, 10(-6) M PMA produced an immediate but transient cessation of motility and retraction of the cell margin into prominent filopodia. Motility resumed within 2.5 h after addition, but the osteoclasts remained partially contracted. Chick osteoclasts behaved similarly but showed no formation of filopodia at the cell periphery and a more rapid recovery of motility than rat osteoclasts; chick osteoclasts also underwent a transient vacuolation following PMA exposure, whereas rat osteoclasts did not. Despite differences in the sensitivity of rat and chick osteoclasts to PMA, these results suggest a fundamental role for protein kinase C in the inhibition of osteoclasts from both species.

The effects of parathyroid hormone (PTH) and PTH-related peptide on osteoclast resorption of bone slices in vitro: an analysis of pit size and the resorption focus.

The mechanism whereby PTH, a potent stimulator of bone resorption, may under certain circumstances exert anabolic effects on bone is not known, but it is possible that it involves reduction of the size of osteoclast resorption lacunae. We have therefore made a detailed in vitro study of the effects of PTH and PTH-related peptide (PTHrP) on resorption by neonatal rat osteoclasts paying particular attention to the plan area of resorption pits. In order to distinguish between increased resorption at a particular site and increased numbers of sites, we have used an eyepiece graticule to define a focus of resorption, namely an area occupying 1/116th of the bone slice, which may contain either one or several pits. In addition we have studied the relationship between the number of pits in a resorption focus and the total area of bone resorbed at the focus. We found that PTH and PTHrP, at doses between 2 x 10(-10) M and 2 x 10(-8) M, while exerting significant stimulatory effects on bone resorption, caused a reduction in the median plan area of pits. An increase in the number of resorption foci was the primary stimulatory effect of PTH and PTHrP, occurring within 6 h in the case of PTH. However, the plan area of bone resorbed at a focus showed no significant increase, despite an increase in the number of pits per focus, because as more pits were formed at a focus, the pits were smaller, thus partially dissipating the stimulatory effect of PTH on resorption. These results are consistent with the activation of new remodeling sites by PTH in vivo. Furthermore, the formation of smaller pits under the resorptive influence of PTH may, together with the maintenance of coupling between formation and resorption, play a role in the preservation of cancellous bone recorded in cases of primary hyperparathyroidism and the anabolic effect of exogenous PTH.

The effects of stimulators of intracellular cyclic AMP on rat and chick osteoclasts in vitro: validation of a simplified light microscope assay of bone resorption.

The aim of this study was to investigate whether a cyclic AMP-mediated inhibitory mechanism is present in embryonic chick osteoclasts and to extend data implicating cyclic AMP in the inhibition of neonatal rat osteoclasts. Dibutyryl cyclic AMP ((Bu)2cAMP) (5 x 10(-4) M and above) and isobutylmethylxanthine (IBMX) (10(-4) M and above) reduced the number of pits made in slices of devitalized bovine cortical bone by chick osteoclasts over 24 h. The effect of forskolin (FSK) on chick osteoclasts was biphasic, 10(-5) M producing a weak and variable reduction in pit number while 10(-6) M and 10(-7) M stimulated resorption. Doses of FSK (10(-5) M) and (Bu)2cAMP (3 x 10(-4) M), which individually produced no consistent significant effect, produced a synergistic and highly significant reduction in pit number when used in combination, implying that these agents were acting through a common mechanism, presumably cyclic AMP. Stimulatory doses of FSK were associated with increased osteoclast numbers, implicating cyclic AMP in the formation of osteoclasts. In comparative experiments using neonatal rat osteoclasts, (Bu)2cAMP (10(-4) M and above), IBMX (10(-3) M) and FSK (10(-7) M and above) all reduced the number of pits excavated. Strongly inhibitory doses of these agents caused contraction of chick osteoclasts into a hemispherical shape; contraction of rat osteoclasts into a stellate shape occurred with (Bu)2cAMP and FSK, but not with IBMX. Our results implicate cyclic AMP in the inhibition of both rat and chick osteoclasts, and show that pit counting in the light microscope is a valid method of analyzing the disaggregated osteoclast resorption assay.

Bone resorption by isolated human osteoclasts in vitro: effects of calcitonin.

Human osteoclasts were isolated from 12- to 17-week-old fetal tissue and from transiliac crest bone biopsies for an in vitro study of their biology. A hypodermic needle was used to flush either the fetal long bones or the trabeculae of the iliac crest bone biopsy with tissue culture medium and the resulting cell suspension sedimented briefly either onto the surface of plastic tissue culture dishes, for time-lapse microcinematography, or onto slices of devitalized bovine cortical bone for quantitative assay of bone resorption. The osteoclasts were motile, tartrate-resistant acid phosphatase positive and capable of excavating pits in slices of devitalized bovine cortical bone. Human calcitonin, at doses of 1 ng/ml and 1 microgram/ml, caused a 70% inhibition of bone resorption by human fetal osteoclasts over a 24 h period but had no apparent effect on the morphology or motility of either fetal or adult osteoclasts.

Microelectrode studies on the acid microenvironment beneath adherent macrophages and osteoclasts.

Osteoclasts and activated macrophages in culture were shown to generate an acidic microenvironment specifically in the attachment zone between the cell and the base of the culture dish. Measurements using pH microelectrodes revealed that osteoclasts, when firmly attached, could achieve a pH fall of about 1 unit min-1 to a limit value of pH 3.0 or less. Activated macrophages produced a slower fall of 0.5-2 pH units h-1 and a limit value of pH 3.6-3.7 was generally detected. The method of activation was relatively unimportant, but where macrophages formed clumps the pH effect was reinforced. Osteoclasts were also examined in situ in osteoporotic bone fragments in rabbit ear chambers, using a combination H+ and Ca2+ double-barrelled electrode. The pH readings reached a lower limit of 4.7 and the calcium concentration rose to a maximum of 40 mM in the erosion sites. In vivo such acid conditions would favor the direct extracellular action of secreted lysosomal proteinases in the degradation of collagen by both cell types.

Biological activity of chicken calcitonin: effects on neonatal rat and embryonic chick osteoclasts.

Chicken calcitonin (cCT) has recently been synthesized according to nucleotide sequence data. We have investigated the in vitro effects of this hormone on the activity of disaggregated, neonatal rat and embryonic chick osteoclasts. While synthetic cCT inhibited bone resorption by neonatal rat osteoclasts at concentrations as low as 0.1 pg/ml, it failed to produce a significant reduction in bone resorption by embryonic chick osteoclasts, even at concentrations as high as 1 microgram/ml. Similarly, cCT at 1 pg/ml reproducibly produced the rapid and dramatic inhibition of rat osteoclast motility characteristic of other calcitonins, as judged by time-lapse video recording, but did not impair the motile behavior of chick osteoclasts at concentrations up to a million-fold higher. Previous studies showing that embryonic chick osteoclasts were unresponsive to synthetic salmon calcitonin left open the possibility that the native hormone was required to produce an inhibitory effect. We conclude that the osteoclast is not a target cell for calcitonin in the embryonic chick; further studies will be required to define a role, if any, for this potent but enigmatic hormone in birds.