Prostaglandin E2 binding and cyclic AMP production in isolated bone cells

Summary The binding of prostaglandin E₂ (PGE₂) to bone cells was studied to provide direct evidence for the existence of specific receptors in bone. Bone cells were isolated by collagenase digestion of fetal and newborn rat calvaria. Isolated cells were incubated with³H-PGE₂ and collected on Millipore filters. Specific binding was determined by subtracting the binding that occurred with 10⁻⁶ M non-radioactive PGE₂ and³H-PGE₂ from that with³H-PGE₂ alone. With heterogeneous cell preparations and at PGE₂ concentrations from 10⁻⁹ − 1.7 × 10⁻⁸ M at 37°C, specific binding reached steady state within 10 min. Bound³H-PGE₂ was displaced by the addition of increasing amounts of unlabeled PGE₂. Inhibition of PGE₂ binding was observed with PGE₁ and the endoperoxide analog, U44069, but not with PGE_2α, a lipopolysaccharide, or 13,14-dihydro 15-keto PGE₂. Studies with bone cell populations, obtained by sequential digestions, indicated that an osteoclastic population binds 30-fold more PGE₂ than osteoblastic cells. Scatchard analyses revealed that the osteoclastic cells have an affinity constant for PGE₂ binding similar to that obtained with heterogeneous populations. However, the PGE₂ binding capacity in this osteoclastic population was fivefold greater than in the heterogeneous population. The osteoclastic population responded with an increase in cyclic AMP to lower concentrations of PGE₂ than the osteoblastic populations. These studies suggest that differences in the binding capacity of PGE₂ receptors exist among bone celltypes and that these differences are reflected in the cellular cyclic AMP response.     

Development of the cyclic AMP response to parathyroid hormone and prostaglandin E2 in the embryonic chick limb

Summary The developing chick limb was studied to determine the ability of parathyroid hormone (PTH) and prostaglandin E₂ (PGE₂) to increase intracellular cyclic AMP (cAMP) during various stages of development. All developmental stages examined (stages 20–21, 24–25, and 26–28) responded to PGE₂ when the cells were assayed immediately following the removal of the limbs from the embryos. In contrast, only stage 26–28 limb cells responded to PTH when assayed in a similar manner. The response to PTH was temporally correlated with the appearance of cartilage matrix in vivo. Undifferentiated limb cells were also cultured and assayed at various times for hormone responsiveness. Stage 24–25 high-density cell cultures responded initially to PGE₂ but not to PTH. However, by 36 h and in all subsequent itme intervals tested, the response to PTH was significantly greater than that to PGE₂. The PTH receptor, in contrast to that of PGE₂, was shown to be sensitive to trypsin treatment, but could be regenerated during subsequent cell culture. The majority of the hormoneresponsive cells were found in cartilaginous regions of the limb, and were shown to respond to both hormones in a dose-dependent manner. The PTH-induced cAMP response was affected by low cell density and mouse serum, both of which significantly inhibit the chondrogenic potential of cultured limb cells. These findings are consistent with a temporal correlation between the development of the PTH response and chondrogenesis in vivo.     

Effect of low-dose infusion of prostaglandin E1 on vecuronium-induced neuromuscular blockade

The effect of low-dose (20 ng·kg⁻¹·min⁻¹) infusion of prostaglandin E₁ (PGE₁) on vecuronium-induced neuromuscular blockade was studied. The study population consisted of 24 elderly patients (65–75 years old) and 24 younger adult patients (25–56 years old). They were randomly assigned to the control and PGE₁ groups. The steady-state dose requirement (SSDR) of vecuronium was derived from ondemand infusion of the drug which produced a stable twitch height of 20% of its baseline reading, and recovery time after steady-state infusion was defined as the time for recovery from twitch height from 25% to 75%. The patients in the PGE₁ group received an infusion of PGE₁ 20 ng·kg⁻¹·min⁻¹, while those in the control group received an infusion of normal saline. The SSDR (23.2±9.1 and 34.2±5.9 μg·kg⁻¹. hr⁻¹, respectively;P=0.02) was significantly less and the recovery time (35.0±9.5 and 19.9±4.2 min, respectively;P=0.01) was significantly longer in the elderly than in the younger patients. However, low-dose infusion of PGE₁ significantly increased the SSDR (23.2±9.1 to 37.4±3.7 μg· kg⁻¹·hr⁻¹;P=0.01) and shortened the recovery time (35.0±9.5 to 23.5±4.0 min;P=0.02) in elderly patients. We concluded that low-dose infusion of PGE₁ is effective in preventing the prolonged action of vecuronium in elderly patients.     

Inhibitory effect of prostaglandin E1 on gastric secretion during general anesthesia in humans

The present study was undertaken to clarify the effects of prostaglandin E₁ (PGE₁) on gastric secretion during general anesthesia. Thirty-three patients, 16 with (PGE₁ group) and 17 without (control group) PGE₁ administration, scheduled for selective surgery were studied during general anesthesia with nitrous oxide (67%) and enflurane (1%–2% inspired). PGE₁ was administered at a rate of 50–200 ng·kg⁻¹·min⁻¹ when hypotensive medication was required. In the PGE₁ group, gastric juice was collected serially three times before and during administration and 60 min after discontinuation of PGE₁. In the control group, it was collected three times corresponding to those in the PGE₁ group. The pH of gastric juice increased significantly, and the acidity and pepsin activity decreased after the beginning of the administration of PGE₁, and these changes were observed even 1h after discontinuation. There was significant differences in the pH, acidity, and pepsin activity between the two groups after administration of PGE₁. The results indicate that PGE₁ inhibits gastric secretion at doses that produce a sufficient hypotensive effect under general anesthesia.     

The effect of prostaglandin E1 on the increase of serum lactate and plasma granulocyte elastase activity during radical surgery for esophageal cancer

Serum lactate concentrations and the lactate/pyruvate (L/P) ratio were measured in two groups of patients undergoing radical esophagectomy, as an indicator of tissue hypoxia, and β-glucuronidase and granulocyte elastase as indicators of tissue damage. One group received prostaglandin E₁ (PGE₁) and the other group received nothing. Serum lactate concentrations and the L/P ratio increased significantly 30 min after starting thoracotomy in the patients who were not treated with PGE₁. On the contrary, intravenous drip infusion of PGE₁ (0.04 μg·kg⁻¹·min⁻¹) suppressed the increases in serum lactate concentratons and L/P ratios. Plasma granulocyte elastase activity increased significantly at the end of surgery in both groups. There was no change in serum β-glucuronidase activity in both groups. This study suggests that low doses of PGE₁ maintain organ blood flow without affecting blood pressure. However, these low doses of PGE₁ could not suppress granulocyte elastase release.     

Effects of prostaglandin E2 and indomethacin on 25-hydroxyvitamin D3-1α-hydroxylase activity in isolated kidney cells of normal and streptozotocin-induced diabetic rats

Summary The effects of prostaglandin E₂ (PGE₂) (1 μM) and indomethacin (IN) (20 μM) on 1,25 dihydroxyvitamin D₃ production were studied in renal cell suspensions isolated from control, stroptozotocin-induced diabetic, and insulin-treated diabetic rats. Renal cortex cells were isolated by enzymatic digestion, and preincubated for 30 min at 37°C with the appropriate additive(s) followed by a 1 h incubation with 8 nM 25-hydroxyvitamin D₃ in serumfree medium. Radioactivity incorporated into that fraction of the cell suspension extract co-eluting with synthetic 1,25-dihydroxyvitamin D₃ on high pressure liquid chromatography was determined All animals were raised for 5 weeks on a vitamin D-deficient diet. Isolated kidney cells from vitamin D-deficient rats showed dose-dependent response of 1,25 dihydroxyvitamin D₃ production to PGE₂. Cells from control animals demonstrated a stimulatory effect of PGE₂ (P<0.05) and a suppressive effect of IN (P<0.01) on 1,24 dihydroxyvitamin D₃ production. In contrast, cells from diabetic rat kidneys failed to respond to these agents, alterations which were reversed by insulin treatment. The accumulated data suggest that depressed synthesis of 1,24 dihydroxyvitamin D₃ previously observed in the experimental diabetic rat is due, at least in part, to an impaired production and response to PGE₂-like prostaglandins.     

Regulation of hormone-induced cyclic AMP response to parathyroid hormone and prostaglandin E2 in cells cultured from human giant cell tumors of bone

Summary Cells dispersed from human giant cell tumors of bone and grown in monolayer culture increase intracellular cyclic AMP (cAMP) when incubated with parathyroid hormone (PTH) or prostaglandin E₂ (PGE₂). When cells are continuously exposed to PTH, cAMP levels increase acutely but then decrease rapidly to pretreatment values despite continued presence of hormone or addition of new hormone. Preincubation of cells with PTH for periods as short as 10 min results in a decrease in the capacity of cells to increase cAMP content when re-exposed to maximal stimulatory concentrations of PTH. The decrease in the magnitude of the PTH-induced cAMP response observed in cells pretreated with this hormone is dependent on the concentration of PTH present during the pre-incubation. The loss of cAMP response in cells pre-treated with either PGE₂ or PTH is hormone specific in that cells made refractory by pretreatment with one hormone still increase cAMP content when exposed to the other. Although the cells are not releasing measurable amounts of prostaglandins into the medium, pretreatment with indomethacin results in an increase in the magnitude of the cAMP response to PGE₂. The PTH-induced cAMP response is not affected by indomethacin pre-treatment. The loss of PTH responsiveness produced by hormone preincubation is consistent with the phenomenon of “down-regulation” observed with ligand-receptor interactions in a variety of tissues.     

Increased bone growth by local prostaglandin E2 in rats

Summary The effects of prostaglandin E₂ (PGE₂) on bone growth were investigated in rats. Daily injection of PGE₂ (1, 10, and 100 pmol) was given via local intraosseous route into the metaphysis of the left tibia for 14 days. The contralateral right tibia injected with vehicle and saline was for the control. The rats receiving no injection provided as normal control. The results obtained indicated that PGE₂ slightly but significantly decreased the body weight increment without effect on tibial length. The most prominent effect of PGE₂ was the increase of metaphyseal bone trabeculae by 45–81% in a dose-dependent manner. The microscopic examination revealed that PGE₂ unequivocally increased the new woven bone formation. The bone cell population study showed no difference between the number of osteoblasts and osteoclasts in primary spongiosa of the PGE₂-injected limbs and those of contralateral limbs. However, the numbers of osteoblasts and osteoclasts were markedly increased in secondary spongiosa in the PGE₂-injected limbs. This finding confirmed a stimulatory role of PGE₂ in the bone formation. The local intraosseous injection of PGE₂ was proven to be a good model for the study of local growth factors on bone metabolism with a lower effective dose which eliminates the systemic side effects.     

Effect of prostaglandin E2 on mineralization of bone nodules formed by fetal rat calvarial cells

The effects of PGE₂ on mineralized bone nodule formation were studied in fetal rat calvarial (RC) cells in vitro. Continuous exposure of RC cells to 3x10^-8M PGE₂ induced a twofold increase in mineralized bone nodule formation and a 1.5-fold increase in alkaline phosphatase (ALPase) activity without affecting RC cell growth. These stimulatory effects were evoked by concentrations of 3x 10^-9-3x10^-6 M PGE₂ and the maximal effect was observed with 3x10^-8 M PGE₂. The in vitro effects of PGE₂ were evident when RC cells were exposed to it on days 8–14 and 8–21, which correspond to the post-confluent culture stage, but no effects were observed when the cells were exposed on days 1–7, the growth stage. The ALPase activity was also higher (1.2–1.4-fold) when 3x10^-8 M PGE₂ was added during the post-confluent stage. In order to determine the effect of PGE₂ during the mineralization phase of bone nodules in the presence of a large population of osteoprogenitor cells, RC cells were exposed to dexamethasone for 7 days before PGE₂ was added during the post-confluent stage. A significantly higher percentage of nodules mineralized were observed with 3x10^-8-3x10^-9 M PGE₂ (1.6-and 1.4-fold, respectively), than in control cultures. Analysis of the mineral-related proteins by EDTA extraction of bone nodules followed by electrophoresis and Stains-All staining revealed an increased total amount of osteopontin extracted from the mineralized matrix after PGE₂ treatment. The osteopontin content was highest after 3x10^-8 M PGE₂, with a 73% increase of the densitometric intensity of the bands, although this increase, reflected the increased number of mineralized bone nodules due to PGE₂. These findings suggest that PGE₂ may increase the proportion of functional osteoblasts able to produce mineralized bone nodules in the population by stimulating differentiation during the postconfluent stage of RC cell culture.     

Gadolinium chloride inhibits lipopolysaccharide-induced mortality and in vivo prostaglandin E2 release by splenic macrophages

The monocytic phagocytic system, consisting primarily of tissue macrophages of the liver and spleen, produces prostaglandin E₂ (PGE₂), a modulator of the septic response. Macrophages are known to internalize gadolinium chloride (GD), a lanthanide metal, which inhibits phagocytic function. Thus we studied the effect of in vivo GD on lipopolysacchride (LPS)-induced mortality and on LPS-stimuIated PGE₂ release by cultured splenic macrophages. GD (7 mg/kg intravenously) given on the two days prior to LPS challenge (30 mg/kg intravenously) completely prevented the uniform mortality in rats. This protective effect was transient since rechallenge with LPS 10 days later was uniformly lethal. Previous work in this laboratory has established a critical role of arginine concentration on macrophage behavior in vitro. Therefore, to establish culture conditions reflective of the milieu within the portal venous system, alanine and arginine levels were measured in the portal and hepatic veins of normal and endotoxemic (LPS, 10 mg/kg intraperitoneally) rats. In contrast to alanine levels, which were not altered by endotoxemia, there was a reduction of arginine concentrations from a range of 50 to 250 μmol/L in normal rats to a range of 10 to 50 μmol/L after LPS challenge. Consequently subsequent in vitro assays of splenic macrophage secretory behavior were performed in concentrations of 1200 μmol/L arginine (in standard RPMI-1640), as well as in concentrations reflective of physiologic arginine levels (10 and 100 μmol/L in modified RPMI-1640). Rat splenic macrophages harvested after two consecutive days of either in vivo saline or GD injection (7 mg/kg intravenously) were stimulated with LPS (0.025 to 2.5 μg/ml). At 72 hours of culture, the release of PGE₂ by splenic macrophages from GD-treated rats was significantly (P<0.0001) reduced at all LPS concentrations. Increased PGE₂ production was not present when the splenic macrophages were cultured in the supraphysiologic arginine (1200 μmol/L) concentration. The results demonstrate the relevance of physiologic arginine concentrations in cell culture studies and suggest that the protection conferred by GD against septic mortality may be related to downregulation of the release of immunosuppressive PGE₂ by the monocytic phagocytic system. Supported by the following grants: NIH RO1 28480, the Association for Academic Surgery, and the American Liver Foundation. Presented at the Thirty-Ninth Annual Meeting of The Society for Surgery of the Alimentary Tract, New Orleans, La., May 17–20, 1998.     

Cyclic nucleotides and the rapid inhibitions of bone45Ca uptake in response to bovine parathyroid hormone and 16,16-dimethyl prostaglandin E2 in chicks

Summary Intravenous injection of chicks with bovine parathyroid hormone (1–34) (3.3 μg/100 g body wt.) or 16,16-dimethyl PGE₂ (5 μg/100 g body wt.) caused rapid (3 minute) net inhibition of⁴⁵Ca uptake into femur and calvarium. These agents also elevated bone adenosine 3′,5′-cyclic monophosphate (cAMP) but not guanosine 3′,5′-cyclic monophosphate (cGMP) levels at this time. Methylxanthine phosphodiesterase inhibitors (MXPI), caffeine, theophylline, and 3-isobutyl-1-methylxanthine (IBMX) (0.3–5 mg/100 g body wt.) similarly inhibited net⁴⁵Ca uptake into femur and to a lesser extent calvarium. Plasma⁴⁵Ca and total Ca levels were unaltered or showed a slight tendency to be increased over control values 3 minutes after injection. However, the effects of the non-MXPI, dibutyryl-cAMP (0.5–5 mg/100 g body wt.) on bone⁴⁵Ca uptake were negligible. Of the MXPI, only IBMX elevated total cAMP levels in chick bone at 3 minutes. These data implicate but do not confirm a mediatory role for cAMP in the rapid inhibitory actions of PTH and PGEs on bone net⁴⁵Ca uptake in chicks.     

The transduction of mechanical force into biochemical events in bone cells may involve activation of phospholipase A2

Summary Mechanical forces applied to cultured bone cells induce the production of cAMP via stimulation of the formation of prostaglandin E₂ (PGE₂) and its release into the medium, resulting in stimulation of adenylate cyclase. In this paper we show that either the antibiotic gentamycin (100 μg/ml) or antiphospholipid antibodies (0.1%) which bind to membrane phospholipids abolish cAMP formation induced by mechanical forces; exogenously added arachidonic acid or PGE₂ stimulates cAMP formation, even in the presence of these agents. Addition of exogenous phospholipase A₂ (but not phospholipase C) causes an increase in the formation of cAMP in bone cells, a response that is also inhibited by gentamycin or antiphospholipase antibodies. These observations suggest that mechanical forces exert their effect on bone cells via the following chain of events: (1) activation of phospholipase A₂, (2) release of arachidonic acid, (3) increased PGE synthesis, (4) augmented cAMP production.     

Effect of prostaglandin E1 on arterial ketone body ratio in hepatectomy

We evaluated the effect of prostaglandin E₁ (PGE₁) administration during hepatectomy on arterial ketone body ratio (AKBR), which is an indicator of liver function, and on other liver functions in the postoperative period. Eighteen patients were divided into two groups: Continuous intravenous administration of PGE₁ (0.02 μg·kg⁻¹·h⁻¹) was started immediately before hepatic resection and ceased at the end of operation in nine patients (PGE₁ group); the other nine did not receive PGE₁ (control group). After hepatic resection, a significant increase in AKBR was observed in the PGE₁ group. However, no change was observed in the control group. In the PGE₁ group, total bilirubin and SGOT recovered more rapidly to the preoperative level than in the control group. These findings suggested that PGE₁ might have a protective effect on the liver.     

The effects of prostaglandin E2 on DNA and collagen synthesis in osteoblastsIn Vitro

Summary The effects of prostaglandin E₂(PGE₂) on DNA and collagen synthesis in two separate cell populations were investigated. In view of their morphology, ALPase activity, DNA and collagen synthesis, and response to PGE₂, one population was in an undifferentiated state consisting of preosteoblast-like (PL) cells and the other was in a differentiated state consisting predominantly of osteoblastlike (OB) cells. As parameters of bone-forming activity, the incorporation of³H-thymidine into DNA and the incorporation of³H-proline into collagenase digestible protein were measured to assess DNA and collagen synthesis. The cells were treated with PGE₂ in the presence of indomethacin (IM) to avoid the influence of endogenous prostaglandins. At 24 hours, IM stimulated the DNA synthesis in both cell populations. Furthermore, a greater stimulation was found in the PL cells than in the OB cells. On the other hand, exogenously supplemented PGE₂ reversed the IM-induced stimulation of DNA synthesis. In contrast, high concentrations of PGE₂ alone increased the DNA synthesis. With respect to collagen synthesis, IM showed an inhibitory effect, especially in the PL cells. This inhibitory effect was also reversed by the addition of PGE₂. In addition to the stimulation of collagen synthesis, PGE₂ enhanced the proportion of protein synthesized as collagen. In the PL cells, the percentage of collagen synthesis was markedly decreased when cultured with IM for 48 hours. These results suggested that the effects of IM were mediated in part via its ability to reduce biosynthesis of prostaglandins, and that PGE₂ is a multifunctional autocrine regulator of bone formation.     

Variation of increases in free cytosolic calcium ion induced by prostaglandin E2 in mouse osteoblast clone,MC3T3-E1—Single cell assay using microspectro-fluorometry

Osteoblasts exhibit multiple phenotypic expression in response to prostaglandin E₂ (PGE₂). Intracellular calcium concentration ([Ca²⁺] _i ) was elevated by PGE₂ treatment in the mouse osteoblast clone, MC3T3-E1, but the degree of elevation was varied by the day after subculturing. To study the different response to PGE₂, we have used microspectrofluorometry to measure [Ca²⁺] _i in a single MC3T3-E1 cell loaded with fura-2. In the presence of extracellular Ca²⁺, the increase in [Ca²⁺] _i in the osteoblast exhibited multiple patterns. The patterns were roughly classified into four groups by the time reached maximum level; “transient”, “gradual”, “transient and gradual” and “no response”. Within 2 days after subculturing, the cells showing “gradual” and “no response” were predominant, whereas after day 3 the cells showing “transient” and “transient and gradual” were predominant. We also investigated the daily change in the maximum level of [Ca²⁺] _i in the cells showed “transient” in response to PGE₂. The magnitude of [Ca²⁺] _i increase was also varied in cultivating period. These data suggest that there are phenotypic variations in a single cell even in a cloned cell line and this phenotype may change in the stage of cell proliferation.     

Regulation of creatine kinase activity in rat osteogenic sarcoma cell clones by parathyroid hormone,prostaglandin E2, and vitamin D metabolites

Summary We have previously shown that both parathyroid hormone (PTH) and prostaglandin E₂ (PGE₂) stimulate the activity of creatine kinase BB (CKBB) in rat bone cells in culture. Therefore, morphologically distinct rat osteogenic sarcoma cells in culture were tested for stimulation of CKBB activity by hormones that regulate skeletal tissues. PTH stimulated CKBB in the osteoblast-like clone ROS 17/2; 1α,25(OH)₂D₃ inhibited this activity while PGE₂, CT and 24R,25(OH)₂D₃ had no significant effect. PGE₂ stimulated CKBB activity in the fibroblast-like clone ROS 24/1, which was unresponsive to PTH, CT and Vitamin D metabolites. 24R,25(OH)₂D₃ as well as PGE₂ (but not PTH, CT or 1α25(OH)₂D₃) stimulated CKBB in clone ROS 25/1, suggesting that this fibroblast-like clone has some chondroblast-like character. Both PTH and PGE₂ stimulated the brain type isoenzyme of CK (CKBB), although the osteogenic sarcoma cell clones contain a significant proportion of the muscle type of CK (CKMM). Thus, increased CKBB activity can serve as an additional characteristic marker for the action of steroid and polypeptide hormones and for prostaglandins.     

Systemic effects of prostaglandin E2 on vertebral trabecular remodeling in beagles used in a healing study

Summary Prostaglandin E₂ (PGE₂) at a dose of 10 mg/kg was administered orally to beagles used in a study of rib fracture and drill hole defect healing. Double fluochrome labels were given prior to surgical manipulation and before necropsy at 30 days. Bone remodeling was evaluated in trabecular bone of the fourth lumbar vertebra. There was a decrease in the number and extent of posttreatment labels (P<0.05) in the controls, with decreased mineral apposition rate (P<0.05) and decreased active bone formation rate (P<0.01). In dogs given PGE₂ for 30 days following surgery, the extent of posttreatment labels (P<0.05) and bone formation rate (P<0.01) were increased. There was no difference found, however, in static morphometric parameters, including osteoid and osteoblast-covered surface, indicating that the stimulation of bone formation may have been transitory and matrix synthesis had declined. In another group of dogs given PGE₂ for 5 days prior to surgical manipulation and between the first and second pretreatment labels, the extent of the double-labeled surface was increased (P<0.05) indicating an acute PG effect to sustain formation at remodeling sites. These studies show that PGE₂ given orally has a systemic effect on bone remodeling in vertebral trabeculae that involves the stimulation of formation activity.     

Hemodynamic effects of prostaglandin E1 during cardiopulmonary bypass in infants and children

Effects of prostaglandin E₁ (PGE₁) and phenoxybenzamine (POB) on the hemodynamics during cardiopulmonary bypass (CPB) were studied in 30 infants and children. Patients were grouped into three; PGE₁ was given to ten patients, POB to another ten, and the other ten patients served as the control. Vasodilative drugs were witheld. PGE₁ was infused at 0.01 to 0.02 μg/kg/min during CPB, and POB at 1.0 mg/kg within the initial 10 minutes of bypass. There was a significant drop in arterial and venous pressure at the time of initiation of bypass in both the PGE₁ and POB groups. In the PGE₁ group in particular, such a stable hemodynamic condition of over 60 mm Hg in mean arterial pressure, 7.5 to 12.5 cmH₂O in central venous pressure, 1300 to 1700 dynes·sec·cm⁻⁵ in systemic vascular resistance was maintained throughout CPB, as compared with the other two groups. PGE₁ contributed to an adequate diuresis and the preservation of platelets. Our findings indicate that PGE₁ has potential clinical advantages for application during CPB.     

Interleukin-6 does not mediate the stimulation by prostaglandin E2, parathyroid hormone,or 1,25 dihydroxyvitamin D3 of osteoclast differentiation and bone resorption in neonatal mouse parietal bones

The cytokine interleukin-6 (IL-6) was produced by neonatal mouse parietal bones during a 6- or 48-hour culture period in response to prostaglandin E₂ (PGE₂) and bovine parathyroid hormone (PTH) 1-34 fragment but not 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃]. At the same time there was an increase in tartrate-resistant, acid phosphatasepositive osteoclasts (TRAP+OC) with all three osteotropic effectors over 6 hours, and an increase in ⁴⁵Ca release over 48 hours. TRAP+OC numbers on PGE₂-stimulated bones were positively correlated with IL-6 concentration. Our aim was to determine if IL-6 mediated this response. Recombinant human IL-6 (rhIL-6) was added to parietal bones in culture at concentrations within the range that PGE₂ or PTH would produce during incubation. However, over 6 or 48 hours, rhIL-6 did not stimulate TRAP+OC to increase in number nor did it cause an increase in calcium release over 48 hours. Adding an antibody against mouse IL-6 to bone cultures stimulated with PTH or PGE₂ neutralized the resulting IL-6 bioactivity by up to 92% but did not inhibit TRAP+OC formation. We conclude that although IL-6 is produced in response to two important stimulators of bone resorption, it does not mediate osteoclast differentiation or bone resorption in this model.Part of this work has been presented as an abstract to the Bone and Tooth Society Winter Meeting on 6/12/93 at The Royal College of Obstetricians and Gynaecologists, London.     

6-keto-prostaglandin E1-stimulated bone resorption in organ culture

Summary Previous investigations have shown that prostaglandin E₂ (PGE₂), 13,14-dihydro-PGE₂, and prostacyclin (PGI₂) are among the most potent prostaglandin stimulators of bone resorption. 6-Ketoprostaglandin F_1α (6-keto-F_1α; also called 6-oxo-prostaglandin F_1α), a metabolite of PGI₂ formed by spontaneous hydrolysis, has little bone resorptive or other biological activity. The present study demonstrated that another metabolite of PGI₂, 6-keto-prostaglandin E₁ (6-keto-PGE₁; also called 6-oxo-prostaglandin E₁), was active in stimulating bone resorption in fetal rat long bone organ culture. 6-Keto-PGE₁ stimulated significant release of previously incorporated⁴⁵Ca over the concentration range of 10⁻⁹ to 10⁻⁵ M. The potency of 6-keto-PGE₁ was one-twelfth that of PGE₂. If 6-keto-PGE₁ is formed by bone or adjacent tissues, or reaches bone through the circulation, it could significantly affect bone mineral metabolism.