Biomedical subjects

U H Lerner

Publications and source records attributed to U H Lerner.

At least 73 records · Page 4Linked to original sources

PubMed1992

Haptoglobin-stimulated bone resorption in neonatal mouse calvarial bones in vitro.

OBJECTIVE: To study the in vitro effects of human haptoglobin (Hp) on bone resorption and prostanoid formation. METHODS: Parietal bones were dissected out from neonatal mice that had been injected with 45Ca, and were cultured in chemically defined medium with or without test substances. Bone resorption was assessed by analysis of 45Ca release. Prostanoid formation was quantified by analysis of the amount of prostaglandin E2 (PGE2) in culture medium. RESULTS: Hp phenotype 2-1, in quantities greater than or equal to 0.17 mg/ml, stimulated the release of 45Ca and the biosynthesis of PGE2, in a time- and dose-dependent manner. Hp-induced PGE2 formation was abolished by indomethacin and flurbiprofen, whereas the stimulation of 45Ca release was only partially reduced. CONCLUSION: These observations suggest that the acute-phase reactant Hp may contribute, by a humoral mechanism, to the bone resorption seen in chronic inflammatory processes.

Animals↗

PubMed1992

Human cystatin C, a cysteine proteinase inhibitor, inhibits bone resorption in vitro stimulated by parathyroid hormone and parathyroid hormone-related peptide of malignancy.

The effect of human recombinant cystatin C, a cysteine proteinase inhibitor, on bone resorption in vitro was evaluated. Bone resorption was assessed by analyzing the release of 45Ca and 3H from mouse calvarial bones prelabeled in vivo by injections with 45Ca or [3H]proline, respectively. In 24 h cultures, cystatin C (50 micrograms/ml) significantly inhibited the release of 45Ca and 3H stimulated by parathyroid hormone (PTH, 15 nmol/liter) or parathyroid hormone-related peptide of malignancy (PTHrP, 15 nmol/liter). The degree of inhibition caused by cystatin C in these 24 h cultures was similar to that caused by calcitonin (30 ng/ml). The inhibitory effect of cystatin C on 45Ca release induced by PTH was sustained in 96 h cultures, whereas the initial inhibition caused by calcitonin was transient. Cystatin C, 10-100 micrograms/ml, caused a dose-dependent inhibition of PTH (15 nmol/liter), and PTHrP (15 nmol/liter) stimulated 45Ca release. Addition of 50 micrograms/ml of cystatin C to mouse bone cultures inhibited the release of 45Ca induced by PTH and PTHrP at a wide range of submaximal and maximal concentrations of hormones (0.01-10 nmol/liter). No effect of cystatin C on 45Ca release in dead bones could be observed, nor did the inhibitor decrease the release of calcium in control bones. The inhibition by cystatin C on PTH-induced mineral mobilization was reversible. Cystatin C (1-100 micrograms/ml) did not affect protein synthesis or mitotic activities in mouse calvarial bones as assessed by the incorporation of [3H]proline and [3H]thymidine, respectively. These data show that cystatin C is a potent inhibitor of mineral mobilization and matrix degradation in cultured bones stimulated to resorb by PTH and PTHrP and that this effect is not due to general cytotoxicity.

Bone Resorption↗

PubMed1992

Neuroendocrine regulation of cyclic AMP formation in osteoblastic cell lines (UMR-106-01, ROS 17/2.8, MC3T3-E1, and Saos-2) and primary bone cells.

The effect of four different neuropeptides and norepinephrine (NE) on cyclic AMP formation in four different osteoblastic cell lines and in isolated neonatal mouse calvarial bone cells has been examined. In the rat osteosarcoma cell line UMR-106-01, vasoactive intestinal polypeptide (VIP, 0.001-1 microM), calcitonin gene-related peptide (CGRP, 0.3-30 nM), and NE (0.1-300 microM), but not neuropeptide Y (NPY, 0.001-1 microM) or substance P (SP, 0.1-10 microM), caused a dose-dependent stimulation of cyclic AMP formation. The stimulatory effects were synergistically potentiated by forskolin (0.1-3 microM). The effects of NE and VIP were time dependent, with an optimal effect seen at 5 minutes. The amount of cyclic AMP accumulated in cells stimulated with NE and VIP was in the same range. The amplitude of the cyclic AMP response induced by CGRP was smaller than that caused by VIP and NE. In the human osteosarcoma cell line Saos-2, NE (0.1 microM) and VIP (0.3 microM) stimulated cyclic AMP formation, and the effect was synergistically potentiated by forskolin. In the absence of forskolin, no effect of CGRP (30 nM) could be seen in the Saos-2 cells, but in the presence of forskolin (3 microM) a stimulatory effect was observed. SP and NPY did not change basal cyclic AMP levels in Saos-2 cells. In the osteoblastic osteosarcoma cell line of rat, ROS 17/2.8, NE (0.1 microM) caused a significant stimulatory action on cyclic AMP formation that was synergistically potentiated by forskolin (3 microM), VIP, CGRP, and SP did not affect the cellular content of cyclic AMP in ROS 17/2.8.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Caffeine has the capacity to stimulate calcium release in organ culture of neonatal mouse calvaria.

In view of the possible association between ingestion of caffeine (a constituent of coffee, tea, and several beverages) and osteoporosis, we have studied the effect of caffeine on bone resorption in vitro. Caffeine caused a dose-dependent increase of the spontaneous release of 45Ca from neonatal mouse calvarial bones. The effect of caffeine was less pronounced than that of parathyroid hormone (PTH), but of the same magnitude as that of theophylline, a structurally related methylxanthine. The enhancement of 45Ca release induced by caffeine and PTH was observed in 5 days culture. In 2 days culture, however, only PTH stimulated mineral mobilization. The delayed stimulatory effect of caffeine in long-term cultures was abolished by indomethacin and flurbiprofen. In indomethacin-treated bones, however, caffeine potentiated the stimulatory effect on 45Ca release induced by choleratoxin and forskolin. In contrast, caffeine did not potentiate 45Ca release stimulated by PTH. These data show that caffeine can stimulate calcium release from bone in vitro and that this effect is due to potentiation of a stimulatory action of a bone resorptive agonist acting via the adenylate cyclase-cyclic AMP system.

Animals↗

PubMed1992

Effects of parathyroid hormone on cyclic AMP-formation and cytoplasmic free Ca2+ in the osteosarcoma cell line UMR 106-01.

The effects of parathyroid hormone (PTH) on cytoplasmic free Ca2+ (Cai2+) and cAMP-formation were investigated in the rat osteosarcoma cell line UMR 106-01. In fura-2 loaded adherent single cells bPTH 1-34 (10 nM - 1 microM) induced a rapid transient increase in Cai2+ in 11% of the studied cells. In fura-2 tracings from UMR 106-01 cells in suspension, bPTH 1-34 (0.1 microM) induced a transient increase in Cai2+ in 20% of the experiments. The transient increase in Cai2+ seen in suspensions of cells was not abolished by addition of EGTA (2.5 mM) prior to challenge with PTH, suggesting that the increase in Cai2+ was derived from intracellular stores. A marked rapid increase in cAMP-formation was observed in all experiments with cells in suspension, also in the experiments where PTH did not affect Cai2+. These data show that PTH causes a release of Ca2+ from intracellular stores in a small percentage of osteosarcoma UMR 106-01 cells, and that PTH is capable of inducing an increase in cAMP-formation without affecting Cai2+ in osteoblasts.

Animals↗

PubMed1992

On the signal transducing mechanisms involved in the synergistic interaction between interleukin-1 and bradykinin on prostaglandin biosynthesis in human gingival fibroblasts.

Recombinant human interleukin-1 beta (IL-1 beta) and bradykinin (BK) synergistically stimulate prostaglandin E2 (PGE2) formation in human gingival fibroblasts cultured for 24 h. Neither BK or IL-1 beta per se, nor their combinations, caused any acute stimulation of cellular cyclic AMP accumulation. BK, but not IL-1 beta, caused a rapid, transient rise of intracellular Ca2+ concentration ([Ca2+]i), as assessed by recordings of fura-2 fluorescence in monolayers of prelabelled gingival fibroblasts. IL-1 beta did not change the effect of BK on [Ca2+]i. Ionomycin and A23187, two calcium ionophores, synergistically potentiated the stimulatory effect of IL-1 beta on PGE2 formation. Three different phorbol esters known to activate protein kinase C also synergistically potentiated the action of IL-1 beta on PGE2 formation. Exogenously added arachidonic acid significantly enhanced the basal formation of PGE2. In IL-1 beta treated cells, the enhancement of PGE2 formation seen after addition of arachidonic acid, was synergistically upregulated by IL-1 beta. These data show that i) the synergistic interaction between IL-1 beta and BK on PGE2 formation is not due to an effect linked to an upregulation of cyclic AMP or [Ca2+]i; ii) the signal transducing mechanism by which BK interacts with IL-1 beta, however, may be linked to a BK induced stimulation of [Ca2+]i and/or protein kinase C; iii) the mechanism involved in the action of IL-1 beta may, at least partly, be due to enhancement of the biosynthesis of prostanoids mediated by an upregulation of cyclooxygenase activity.

Arachidonic Acid↗

PubMed1992

Bradykinin induces a B2 receptor-mediated calcium signal linked to prostanoid formation in human gingival fibroblasts in vitro.

The aim of the study was to determine the effect of bradykinin (BK) on the level of cytoplasmic-free Ca2+, [Ca2+]i, in human gingival fibroblasts and its relation to BK-induced prostanoid formation. BK, but not des-Arg9-BK, induced a significant rapid (within seconds) and transient increase in [Ca2+]i, that was not dependent on extracellular Ca2+. The stimulatory effect of BK was seen in concentrations at or above 10(-8) M, with the most pronounced effect at 10(-6) M. D-Arg0-Hyp3-Thi5,8-DPhe7-BK, a BK B2 receptor antagonist, but not des-Arg9-Leu8-BK, a BK B1 receptor antagonist, blocked BK-induced rise in [Ca2+]i. The BK B2 receptor antagonist also significantly reduced BK-induced PGE2 formation. When extracellular Ca2+ in the incubation medium was depleted, either by addition of EGTA or by omission of Ca2+ addition, BK still caused a significant stimulation of PGE2 formation. The calcium ionophores A23187 and ionomycin, similar to BK, caused a burst of PGE2 formation. The two phorbol esters phorbol 12,13-dibutyrate and 4-beta-phorbol-didecanoate positively amplified calcium ionophore A23187-induced PGE2 formation. The results indicate that BK-induced PGE2 formation in gingival fibroblasts is coupled to an increase in [Ca2+]i mediated by the BK B2 receptor, and which is independent of extracellular Ca2+.

Bradykinin↗

PubMed1992

Phenytoin potentiates interleukin-1-induced prostaglandin biosynthesis in human gingival fibroblasts.

1. The effect of phenytoin (PHT) on prostaglandin E2 (PGE2) biosynthesis in human gingival fibroblasts stimulated by interleukin-1 (IL-1 alpha, IL-1 beta) or by tumour necrosis factor alpha (TNF alpha) was studied. 2. IL-1 alpha (1.5-6.0 ng ml-1) and IL-1 beta (30-300 pg ml-1), dose-dependently, stimulated PGE2 formation, in 24 h cultures, with IL-beta being the most potent agonist. 3. PHT (2.5-20 micrograms ml-1) did not induce PGE2 formation itself but potentiated IL-1 alpha- and IL-1 beta-induced PGE2 formation in the gingival fibroblasts in a manner dependent on the concentrations of both IL-1 and PHT. 4. IL-1 beta (0.1-1.0 ng ml-1) induced release of [3H]-arachidonic acid ([3H]-AA) from prelabelled fibroblasts that was potentiated by PHT (20 micrograms ml-1). 5. TNF-alpha (greater than or equal to 0.01 micrograms ml-1) significantly stimulated the biosynthesis of PGE2 by a process that was potentiated by PHT. 6. Addition of exogenous arachidonic acid (AA) (greater than or equal to 1 microM) caused an increase of PGE2 formation in the fibroblasts that was not potentiated by PHT (20 micrograms ml-1). 7. The results indicate that treatment with PHT results in upregulation of prostaglandin biosynthesis in gingival fibroblasts challenged with IL-1 or TNF alpha, at least partly due to enhanced level of phospholipase A2 activity.

Arachidonic Acid↗

PubMed1992

Enhanced prostaglandin biosynthesis in human gingival fibroblasts isolated from patients treated with phenytoin.

Prostaglandin E2 (PGE2) formation was studied in human gingival fibroblasts derived from three epileptic patients before and after 9 months of phenytoin (PHT) therapy. Interleukin 1 (IL-1 alpha; 0.3-6.0 ng/ml), (IL-1 beta; 10-1000 pg/ml) and tumour necrosis factor (TNF alpha; 0.01-0.1 microgram/ml) dose-dependently stimulated the formation of PGE2 in 24 h cultures. In fibroblasts, derived after 9 months of PHT therapy, IL-1 alpha, IL-1 beta and TNF alpha induced a significantly higher formation of PGE2 compared to that in fibroblasts derived before PHT therapy. IL-1 beta induced a significantly higher release also of 3H-arachidonic acid (3H-AA) from prelabelled PHT fibroblasts compared to that in prelabelled gingival fibroblasts isolated before the drug therapy. Addition of exogenous AA caused a spontaneous increase of PGE2 formation in PHT fibroblasts compared to that in fibroblasts isolated before the PHT treatment. The results indicate that PHT medication results in an upregulation of prostanoid formation in gingival fibroblasts partly due to an increased phospholipase A2 activity and partly due to an increased cyclooxygenase activity.

6-Ketoprostaglandin F1 alpha↗

PubMed1991

Haptoglobin synergistically potentiates bradykinin and thrombin induced prostaglandin biosynthesis in isolated osteoblasts.

Haptoglobin of two different phenotypes (Hp 1-1 and Hp 2-1) dose-dependently (1-4 mg/ml) stimulated the formation of prostaglandin E2 (PGE2) in osteoblast-like cells isolated from neonatal mouse calvarial bones. The degree of stimulation obtained by haptoglobins (4 mg/ml) on PGE2 biosynthesis was in the same range as that caused by bradykinin (1 mumol/l). Pretreatment of osteoblasts with Hp 1-1 or Hp 2-1 (1-4 mg/ml) resulted in a dose-dependent, synergistic potentiation of the stimulatory effect of bradykinin (1 mumol/l) on PGE2 formation. Thrombin (7 U/ml) stimulated PGE2 formation in the osteoblast-like cells by a mechanism that was also synergistically potentiated by haptoglobin (2 mg/ml). These data show that haptoglobin per se stimulates PGE2 biosynthesis in isolated osteoblasts and, in addition, synergistically potentiates the effect of bradykinin and thrombin. Consequently, the enhanced production of haptoglobin seen in different inflammatory processes may contribute to the destruction of bone by inducing the formation of prostanoids capable of stimulating bone resorption.

Animals↗

PubMed1991

Bradykinin synergistically potentiates interleukin-1 induced bone resorption and prostanoid biosynthesis in neonatal mouse calvarial bones.

Interleukin-1 (IL-1) alpha and beta dose-dependently stimulated the release of 45Ca and the formation of prostaglandin E2 (PGE2) and PGI2 in cultured mouse calvarial bones, with IL-1 beta being the most potent agonist. Bradykinin (BK; 10 nmol/l) synergistically potentiated the effect of IL-1 alpha (10 pg/ml) and IL-1 beta (5 pg/ml) both on 45Ca release and on biosynthesis of PGE2 and PGI2. The capacity of BK to potentiate IL-1 beta induced 45Ca release and PGE2 formation was seen at concentrations of BK from 1-1000 nmol/l. These data indicate that BK and IL-1, which are formed in inflammatory processes, may act in concert to stimulate bone resorption in the vicinity of inflammatory lesions.

Animals↗

PubMed1991

Bradykinin induces formation of inositol phosphates and causes an increase in cytoplasmic Ca2+ in the osteoblastic cell line MC3T3-E1.

Recordings of fura-2 fluorescence from single osteoblastic MC3T3-E1 cells showed that bradykinin (BK, 1 microM) induced a rapid increase in cytoplasmic free Ca2+ (Cai2+, from 114 +/- 13 to 239 +/- 17 nM, mean +/- SEM). Following this initial transient (less than 1 minute) increase there was a second slow increase in Cai2+ (from 117 +/- 11 to 151 +/- 12 nM). Incubation in buffer with no Ca2+ did not affect the first rapid BK-induced increase in Cai2+ but eliminated the second slow increase. Addition of indomethacin or hydrocortisone to the incubation buffer did not inhibit the effect of BK on Cai2+. BK caused a dose-dependent initial rapid increase in Cai2+ with threshold at 1 nM and a maximal effect (241 +/- 30% of basal Cai2+ concentration) at 0.1 microM. The B1 BK receptor agonist des-Arg9-BK (1 microM) caused only a small increase in Cai2+ in MC3T3-E1 cells (from 101 +/- 20 to 140 +/- 4 nM). BK dose and time dependently stimulated the formation of inositol phosphates in MC3T3-E1 cells with EC50 at 2.4 nM and a significant increase in inositol trisphosphate already seen after 15 s. The Ca2+ ionophore ionomycin induced a rapid increase in Cai2+ and prostaglandin E2 (PGE2) formation in MC3T3-E1 cells. Forskolin (10-30 microM) increased cyclic AMP accumulation but did not affect Cai2+ or PGE2 formation. Depletion of extracellular Ca2+ significantly reduced (but did not abolish) BK-induced PGE2 formation. The initial action of BK on Cai2+ is probably due to an inositol-(1,4,5)-trisphosphate-mediated rapid release of Ca2+ from intracellular stores in osteoblasts and is followed by an influx of extracellular Ca2+. The effect is due to B2 BK receptor occupancy and is not secondary to the prostaglandin synthesis. The BK-induced breakdown of phosphatidylinositol-(4,5)-bisphosphate with a subsequent increase in Cai2+ may be involved in BK-induced prostaglandin formation in osteoblasts.

Animals↗

PubMed1991

In vitro studies on bone resorption in neonatal mouse calvariae using a modified dissection technique giving four samples of bone from each calvaria.

Bone resorption in a modified bone culture system, based on incubation of small fragments from neonatal mouse calvarial bones, has been studied. Four bone fragments were dissected out from each mouse calvaria and were thereafter cultured in CMRL 1066 medium in plastic multiwell dishes. Bone resorption was assessed by 45Ca release from prelabeled bones. The rate of bone resorption in response to parathyroid hormone (PTH) was less in the anterior part of the calvaria compared to the posterior part. After removing the anterior region, four parietal bone fragments that showed identical basal and PTH-stimulated release of 45Ca could be dissected out from each mouse. Excretion of lactate dehydrogenase and beta-glucuronidase was the same in bones cultured submerged or on grids. Uptake of [3H]thymidine in bones cultured submerged was 54% of [3H]thymidine uptake in bones cultured on grids. Dose-response curves, established by using parietal bone fragments, showed that the sensitivity and the magnitude of the increase in 45Ca release seen after stimulation with PTH, prostaglandin E2, and 1 alpha-hydroxyvitamin D3 were the same for bones cultured submerged or on grids. The 45Ca release in response to stimulation with PTH, prostaglandin E2, and 1 alpha-OHD3 was the same in calvarial fragments cultured submerged and those previously obtained with calvarial halves cultured on grids. Thus, even though the rate of DNA synthesis was slower in bones cultured submerged, the rate and the magnitude of resorption were the same in bones cultured on grids or submerged. These data show that it is possible to perform studies on bone resorption with small fragments of neonatal mouse parietal bones.

Animals↗

PubMed1991

On the role of cyclic AMP as a mediator of bone resorption: gamma-interferon completely inhibits cholera toxin- and forskolin-induced but only partially inhibits parathyroid hormone-stimulated 45Ca release from mouse calvarial bones.

The effects of gamma-interferon (gamma-IFN) on bone resorption and cyclic AMP formation stimulated by parathyroid hormone (PTH), forskolin, and cholera toxin have been studied in cultured neonatal mouse calvarial bones. Bone resorption was assessed by the release of 45Ca from prelabeled mouse calvarial bone fragments. Cyclic AMP formation was quantified by analyzing the amount of the nucleotide in calvarial bone tissue. gamma-IFN completely blocked the 45Ca release response to forskolin and cholera toxin in 96 h cultures. In contrast, the 45Ca release response to PTH was only partially inhibited, an effect that was seen over a wide range of PTH concentrations. The inhibitory effect of gamma-IFN was dose dependent, with a threshold for action at 10 U/ml. Forskolin-stimulated 45Ca release could only be inhibited when gamma-IFN was added simultaneously with forskolin; gamma-IFN added to bones prestimulated with forskolin had no effect. The inhibitory effect of gamma-IFN on PTH-stimulated 45Ca release was seen first after a time lag of 48 h. In contrast calcitonin caused an inhibition after only 3 h. PTH and cholera toxin stimulation of radioactive calcium release was also inhibited by gamma-IFN in bones treated with indomethacin. gamma-IFN inhibited forskolin-induced 45Ca release in bones treated with the mitotic inhibitor hydroxyurea. No effect of gamma-IFN on cyclic AMP formation induced by PTH, cholera toxin, or forskolin could be seen. These data show that gamma-IFN inhibits forskolin- and cholera toxin-induced bone resorption by a mechanism unrelated to prostaglandin production or mitotic activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1991

Bradykinin-induced burst of prostaglandin formation in osteoblasts is mediated via B2 bradykinin receptors.

The receptor subtype mediating the bradykinin (Bk)-induced burst of prostaglandin formation in osteoblasts has been studied. Bk, but not des-Arg9-Bk, induced the formation of prostaglandin E2 and prostacyclin in neonatal mouse calvarial bones incubated for 30 minutes. Bk-induced prostaglandin synthesis is neonatal mouse calvarial bones was significantly inhibited by the B2 Bk receptor antagonist D-Arg0[Hyp3,Thi5,8,D-Phe7]-Bk. The B2 Bk receptor agonists Bk and Lys-Bk, but not the B1 Bk receptor agonist des-Arg9-Bk, caused a rapid burst (5 minutes) of prostaglandin E2 and prostacyclin formation in isolated osteoblast-like cells from neonatal mouse calvarial bones and in the murine osteoblastic cell lineage MC3T3-E1. When comparing the relative potency of different kinin analogs on their stimulatory effect on prostaglandin formation in isolated osteoblast-like cells and in MC3T3-E1 cells, we found that Bk = Lys-Bk greater than Met-lys-Bk much much greater than Des-Arg9-Bk. Bk-induced prostaglandin synthesis in isolated osteoblast-like cells and in MC3T3-E1 cells was inhibited by D-Arg0[Hyp3,Thi5,8,D-Phe7]-Bk, whereas the B1 Bk receptor antagonist des-Arg9-Leu8-Bk had no effect. Total binding of 3H-Bk (3-20 nM; 4 degrees C) to whole MC3T3-E1 cells reached a maximum after 4-5 h. An excess of nonradioactive Bk (1 microM) reduced cell-associated radioactivity by 20-30%. The B2 Bk receptor agonist Bk and the B2 Bk receptor antagonist D-Arg0[Hyp3,Thi5,8,D-Phe7]-Bk were able to reduce specific binding, but the B1 Bk receptor agonist des-Arg9-Bk was unable to reduce the specific binding of 3H-Bk to whole MC3T3-E1 cells. These findings indicate the presence of B2 Bk receptors on osteoblasts coupled to enhanced prostaglandin synthesis.

Amino Acid Sequence↗

PubMed1991

Bradykinin B1 and B2 receptor agonists synergistically potentiate interleukin-1-induced prostaglandin biosynthesis in human gingival fibroblasts.

The interactions between bradykinin (BK) and interleukin-1 (IL-1) on prostaglandin formation in human gingival fibroblasts have been studied. IL-1 alpha and IL-1 beta stimulated prostaglandin E2 (PGE2) formation in the gingival fibroblasts with IL-1 beta being the most potent agonist. The effects of both IL-1 alpha and IL-1 beta on PGE2 biosynthesis was synergistically potentiated by BK, in a dose-related manner. The synergistic interaction between IL-1 beta and BK on PGE2 production was seen both with B1 (des-Arg9-BK) and B2 (BK, Lys-BK) BK receptor agonists. No synergistic interaction between BK and IL-1 beta was seen on arachidonic acid release. These data suggest that BK and IL-1 act in concert to enhance prostanoid formation in inflammatory lesions and that the level of interaction is distal to phospholipase activity.

Arachidonic Acid↗

PubMed1991

Inhibitory effects of gamma-interferon on bradykinin-induced bone resorption and prostaglandin formation in cultured mouse calvarial bones.

The effects of mouse recombinant gamma-interferon (gamma-IFN) and indomethacin on bone resorption stimulated by bradykinin, Lys-bradykinin, Met-Lys-bradykinin, des-Arg9-bradykinin and prostaglandin E2 (PGE2) have been studied using cultures of neonatal calvarial bones and analyzing the release of 45Ca from prelabelled bones as a parameter of bone resorption. In addition, the effects of gamma-IFN and indomethacin on formation of PGE2 in bone cultures stimulated by bradykinin was analyzed. Indomethacin (1 mumol/l) totally abolished bradykinin (1 mumol/l) induced 45Ca release. The inhibitory effect of indomethacin could be fully reversed by addition of PGE2 (1 mumol/l). gamma-IFN (1000 U/ml) almost totally inhibited 45Ca release stimulated by bradykinin (1 mumol/l), but the inhibitory effect could only be partially overcome by PGE2. gamma-IFN and indomethacin also inhibited the stimulatory effects of Lys-bradykinin, Met-Lys-bradykinin and des-Arg9-bradykinin (1 mumol/l) on 45Ca release. The stimulatory effects of PGE2 (1 mumol/l) on radioactive calcium mobilization was partially inhibited by gamma-IFN (1000 U/ml), whereas indomethacin (1 mumol/l) was without effect. The inhibitory effect of gamma-IFN on 45Ca release stimulated by bradykinin and PGE2 was dose-dependent with threshold for action at 3-30 U/ml. Comparative dose-response curves showed that gamma-IFN was most potent as inhibitor of bradykinin induced 45Ca release. Bradykinin (1 mumol/l) significantly stimulated PGE2 formation by a mechanism that was completely inhibited by indomethacin (1 mumol/l). gamma-IFN (1000 U/ml) partially inhibited the stimulatory effect of bradykinin on PGE2 formation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1991

Effects of phorbol esters and pertussis toxin on calcitonin-stimulated accumulation of cyclic AMP in neonatal mouse calvarial bones.

Calcitonin (CT) is a well-known inhibitor of osteoclastic bone resorption both in vivo and in vitro. The effect is mediated by activation of adenylate cyclase and subsequent increased levels of cyclic AMP (cAMP). We report here that CT-induced (30 nmol/liter) accumulation of cAMP in cultured neonatal mouse calvaria is enhanced two-fold by 12-O-tetradecanoylphorbol-13-acetate (TPA; 100 nmol/liter) and phorbol 12,13-dibutyrate (PDBU; 100 nmol/liter), two protein kinase C (PKC)-activating phorbol esters, whereas phorbol 13-monoacetate (phorb-13; 100 nmol/liter), a related compound that does not activate PKC, has no effect. The ability of TPA and PDBU to enhance CT-stimulated cAMP accumulation was obtained also in the presence of indomethacin (1 mumol/liter). Kinetic studies revealed that TPA enhanced the cAMP response to CT at all the time points at which CT had a significant effect per se and that TPA did not alter the time-course of the cAMP response to CT. Treatment with pertussis toxin (100 ng/ml) enhanced cAMP response to parathyroid hormone (10 nmol/liter) and prostaglandin E2, but not to CT. From these data it is concluded that PKC, but not pertussis toxin-sensitive guanyl nucleotide-binding proteins (G-proteins), can interact with and modify the signal transducing system for CT in osteoclasts.

Adenylate Cyclase Toxin↗