Age-dependent stimulation or inhibition of calcium release from bone cultures by interleukin-1 beta.

Previous studies have shown that interleukin-1 beta (IL-1 beta) is a potent bone resorption stimulating agent in cultures of fetal or neonatal bones. In the present study, evidence has been provided showing that this cytokine failed to stimulate bone resorption in cultured 75-day-old mouse calvaria maintained in a chemically defined medium for 14 days, as determined by measuring calcium release into the medium and histological examination of cultured bones. Moreover, the cytokine significantly inhibited basal bone resorption in cultured 75-day-old mouse calvaria, a finding reminiscent of the paradoxical effect observed with 1 alpha,25-dihydroxycholecalciferol. Since IL-1 beta did not alter the number of osteoclasts present in the cultured older calvaria as compared to the untreated control, we hypothesized that in such cultured older bones the cytokine affects primarily the function rather than proliferation/differentiation of osteoclasts, either directly or indirectly through its action on other cells in bone tissue, such as osteoblasts or stromal cells. Also, it is possible that the cytokine affects the formation and/or function of macrophages that have been shown to participate in the bone resorption process. These findings support the concept that at different stages of host maturation, bone tissue may exhibit a different response to the same osteotropic agent.

Calcium 'homeostatic' effect of 1 alpha,25-dihydroxycholecalciferol on young adult mouse calvaria in organ culture.

The effect of 1 alpha,25-dihydroxycholecalciferol on calcium release from cultured young adult (75-day-old) mouse calvaria in the presence of indomethacin or dexamethasone was studied. Indomethacin (2.8 x 10(-7) M) or dexamethasone (10(-8) M) abolished or considerably inhibited basal calcium release. At 2.6 x 10(-7) M, 1 alpha,25-dihydroxycholecalciferol prevented the effect of either indomethacin or dexamethasone, resulting in higher calcium release levels approximating those observed in cultures containing only 1 alpha,25-dihydroxycholecalciferol. We have shown previously that in contrast to its effect on neonatal mouse calvaria in culture, 1 alpha,dihydroxycholecalciferol either failed to enhance or even inhibited basal calcium release from cultured 75-day-old mouse calvaria. Furthermore, 1 alpha,25-dihydroxycholecalciferol inhibited PTH- or PGE2-enhanced calcium release from such cultures. These studies demonstrated that, at the same concentration, 1 alpha,25-dihydroxycholecalciferol decreases high calcium release due to various bone resorption-stimulating factors and increases low calcium release values due to various bone resorption-inhibiting factors. This suggested that this vitamin D3 metabolite directly regulates the degree of bone resorption at the bone tissue level resulting in a local calcium 'homeostasis'.

H(+)-stimulated release of prostaglandin E2 and cyclic adenosine 3',5'-monophosphoric acid and their relationship to bone resorption in neonatal mouse calvaria cultures.

The addition of protons to the medium of neonatal mouse calvaria cultures stimulated bone resorption and release of calcium into the medium. In addition, added protons significantly increased the release of prostaglandin E2 (PGE2) and cyclic adenosine 3',5'-monophosphoric acid (cAMP) from the bones. Indomethacin significantly inhibited the release of calcium, PGE2 and cAMP from proton-treated cultures. The positive control, parathyroid hormone (PTH)-treated cultures, also gave rise to bone resorption and calcium release into the medium. However, unlike the addition of protons, the addition of PTH did not stimulate PGE2 release nor did indomethacin inhibit calcium release from PTH-treated cultures. In addition, indomethacin only slightly inhibited cAMP release from PTH-treated cultures, as compared to the marked inhibition by indomethacin of cAMP release from proton-treated cultures. These findings indicate that bone resorption due to added protons is dependent on both PGE2 and cAMP production, whereas bone resorption due to PTH only involves cAMP production.

Age-dependent stimulation or inhibition of calcium release from bone cultures by 1 alpha, 25-dihydroxyvitamin D3.

The effect of 1 alpha, 25-dihydroxyvitamin D3 (1 alpha, 25-(OH)2D3) on calcium release from neonatal and young adult mouse calvaria in tissue culture was studied. At concentrations from 2.6 X 10(-6) M to 2.6 X 10(-9) M, 1 alpha 25-(OH)2D3 enhanced calcium release from 5-day-old calvaria. At the same concentrations, 1 alpha, 25-(OH)2D3 either inhibited or failed to enhance calcium release from 75-day-old calvaria. Parathyroid hormone-enhanced calcium release from 75-day-old calvaria was inhibited considerably by 1 alpha, 25-(OH)2D3 at 2.6 X 10(-6) M and 2.6 X 10(-7) M. These findings suggest that cells involved in the bone resorption process may respond differently to the same stimulus at different stages in the development of the organism. This direct inhibitory effect of 1 alpha, 25-(OH)2D3 on bone resorption in cultured calvaria of older animals may explain, in part, its beneficial effect in the treatment of osteoporosis.

Inhibition of A23187-stimulated bone resorption in tissue culture by serum and indomethacin.

The present study addresses the controversy that currently exists concerning the effect of the divalent cation ionophore A23187 on bone resorption in tissue culture. We show here that this compound is ineffective if serum is present in the culture medium. However, in a serum-free medium it effectively stimulates bone resorption in neonatal mouse calvaria cultures. This finding does account for the various conflicting reports in the literature. Furthermore, the stimulation of bone resorption by the ionophore was accompanied by increased prostaglandin E2 production. Indomethacin inhibited both phenomena, indicating that prostaglandin E2 biosynthesis is involved in mediating the bone resorption-stimulating effect of A23187.

H+ stimulation of cell-mediated bone resorption in tissue culture.

The addition of protons in the form of hydrochloric acid (10.5, 17.2, or 26.6 meq/l resulting in an initial media pH of 7.28, 7.15, and 6.94, respectively) to neonatal mouse calvaria maintained in a chemically defined medium in tissue culture for 1 wk increased calcium release in a dose-response fashion. The same amounts of protons added to the media of devitalized calvaria caused no increase in calcium release into the medium. The net cell-mediated calcium release resulting from the addition of 26.6 meq/l of protons amounted to approximately 50% of the initial calvarial calcium content. Hydroxyproline determinations revealed that active resorption was taking place, wherein both mineral and organic matrix are removed simultaneously. Histological examination of the extensively resorbed calvaria demonstrated the presence of numerous osteoclasts in different stages of bone destruction. The addition of indomethacin (100 ng/ml) strongly inhibited the increase in calcium release by added protons, suggesting that prostaglandin synthesis is involved in the phenomenon. The addition of thyrocalcitonin also inhibited proton-induced calcium release, providing additional evidence that the calcium release from cultures exposed to added protons involved osteoclastic activity.

Effect of age on calcium release from mouse calvaria in tissue culture.

The effect of age on calcium release in organ culture was studied using calvaria from 5-, 14-, 30-, and 75-day-old mice. The results demonstrated a different and characteristic calcium release pattern over the 14-day culture period for untreated calvaria of different ages. Since calcium release in all cultures was abolished by procedures such as boiling, multiple freezing and thawing, maintaining cultures in an oxygen-free gas phase, or maintaining cultures in a non-nutritive medium, it was concluded that the calcium release from older calvaria was due to a cell-mediated process. Histological observations demonstrating the presence of Howship's lacunae and active osteoclastic resorption confirmed that calcium release from older calvaria was due to an active bone resorption process. Parathyroid hormone did not substantially alter the general pattern of calcium release exhibited by different aged calvaria. However, it tended to exaggerate the magnitude of the response. Indomethacin and dexamethasone inhibited calcium release from untreated 5- and 75-day-old calvaria suggesting that prostaglandin biosynthesis was involved in the calcium release process. Direct measurements of PGE2 and PGI2 released into the culture medium gave results consistent with this hypothesis, although it is conceivable that indomethacin and dexamethasone might have influenced calcium release by other mechanisms.

Inhibition of bone resorption in tissue culture by H1-receptor antagonists.

Mouse bone culture studies show that several representative H1-receptor antagonists, promethazine hydrochloride, pyrilamine maleate, tripelennamine hydrochloride, and diphenhydramine hydrochloride, inhibit parathyroid extract-stimulated bone resorption. The H2-receptor antagonists, metiamide and cimetidine, are ineffective. In view of the finding that histamine and histamine agonists did not stimulate bone resorption, it is unlikely that histamine receptors are involved in mediating parathyroid extract-stimulated bone resorption. Because H1-receptor antagonists bind to phospholipids and have been shown to influence membrane structure and function, it is suggested that they inhibit bone resorption by a mechanism that depends on their membrane-stabilizing effect.