The effect of prostaglandins E1, E2 and F2 alpha and indomethacin on the sensitivity of glycolysis and glycogen synthesis to insulin in stripped soleus muscles of the rat.

Prostaglandins E1 and E2 increased the sensitivity of glycolysis to insulin in the isolated stripped soleus muscle of the rat, but prostaglandin F2 alpha had no effect. Indomethacin, which inhibits prostaglandin formation, markedly decreased the sensitivity of glycolysis to insulin. These findings suggest that prostaglandins of the E series increase the sensitivity of muscle glycolysis to insulin in vivo.     

Retroviral-based gene therapy with cyclooxygenase-2 promotes the union of bony callus tissues and accelerates fracture healing in the rat

BACKGROUND: An in vivo gene therapy strategy was developed to accelerate bone fracture repair. METHODS: Direct injection of a murine leukemia virus-based vector targeted transgene expression to the proliferating periosteal cells arising shortly after fracture. Cyclooxygenase-2 (Cox-2) was selected because the transgene for its prostaglandin products that promote angiogenesis, bone formation and bone resorption, are all required for fracture healing. The human (h) Cox-2 transgene was modified to remove AU-rich elements in the 3'-untranslated region and to improve protein translation. RESULTS: In vitro studies revealed robust and sustained Cox-2 protein expression, prostaglandin E(2) and alkaline phosphatase production in rat bone marrow stromal cells and osteoblasts transgenic for the hCox-2 gene. In vivo studies in the rat femur fracture revealed that Cox-2 transgene expression produced bony union of the fracture by 21 days post-fracture, a time when cartilage persisted within the fracture tissues of control animals and approximately 1 week earlier than the healing normally observed in this model. None of the ectopic bone formation associated with bone morphogenetic protein gene therapy was observed. CONCLUSIONS: This study represents the first demonstration that a single local application of a retroviral vector expressing a single osteoinductive transgene consistently accelerated fracture repair.     

Mechanisms involved in prostaglandin-induced increase in bone resorption in neonatal mouse calvaria

Prostaglandins (PG) E1, E2 and F2alpha induce bone resorption in isolated neonatal parietal bone cultures, and an associated increase in interleukin-6 (IL-6) production. Indomethacin had little effect on the response to PGE2, or the relatively non-selective EP receptor agonists 11-deoxy PGE1 and misoprostol, but blocked the effects of PGF2alpha and the F receptor agonist fluprostenol, indicating an indirect action via release of other prostaglandins. It is more likely that there is positive autoregulation of prostaglandins production in this preparation mediated via stimulation of F receptors. The effects of selective EP receptor agonists sulprostone (EP1,3) and 17-phenyl trinor PGE2(EP1), indicated the involvement of EP2 and/or EP4 receptors, which signal via cAMP. The relatively weak increase in IL-6 production by misoprostol (with respect to resorption) suggests that these responses are controlled by different combination of EP2 and EP4 receptors. The PKA activator, forskolin, induced small increases in bone resorption at lower concentrations (50-500 ng/ml) but a reversal of this effect, and inhibition of resorption induced by other stimuli (PTH, PGE2), at higher concentrations (0.5-5 microg/ml). IL-6 production was markedly increased only at the higher concentrations. The inhibitory effect of forskolin may be a calcitonin-mimetic effect. PMA induced both resorption and IL-6 production which were both blocked by indomethacin, indicating a role for PKC in the control of prostaglandin production.     

E and F alpha series prostaglandins in developing muscles

Prostaglandins are known to affect myoblast proliferation and fusion in vitro and are putative regulators of in vivo myogenesis. The levels of E and F alpha series prostaglandins in the thigh muscles of chicken embryos were measured by radioimmunoassays and correlated with indicators of muscle development. Just prior to the onset of secondary myogenesis, the amounts of PGE1, PGE2 and PGF1 alpha plus PGF2 alpha per mg of protein were high. In temporal association with myotube formation, the amount of PGE1 and PGE2 per mg of protein decreased. PGF alpha levels also fell, but at a slower rate than observed with the E series prostaglandins. The decreases in the amounts of prostaglandins per mg protein appeared to be due to a decline in the total amount of prostaglandin within each muscle. These observations are consistent with prostaglandins being one of the factors that controls in vivo muscle formation.     

Stimulation of bone resorption by various prostaglandins in organ culture.

The ability of E, F, A and B prostaglandins to stimulate bone resorption was demonstrated in organ culture. All of the compounds tested were able to increase the release of previously incorporated 45Ca from fetal rat bone by 60 to 135 per cent at maximally effective doses, but prostaglandins of the E series were 10- to 100- fold more potent than F, A or B prostaglandins. Compounds with two double bonds in the side chain were usually more potent than those with one double bond. PGE2 stimulation of bone resorption increased linearly with the logarithm of the medium concentration over the range of 10(-9)M to 10(-5)M, then decreased at higher concentrations. PGE2 stimulated bone resorption more slowly than did parathyroid hormone but caused complete resorption after six days in the culture system. Equilibrium dialysis studies showed no significant binding of F, and 16-34% binding of E and A prostaglandins to bovine serum albumin, which was present in the medium at 1 mg/ml. These differences in albumin binding could not account for differences in potency.     

Stimulation of bone resorption by various prostaglandins in organ culture

The ability of E,F,A and B prostaglandins to stimulate bone resorption was demonstrated in organ culture. All of the compounds tested were able to increase the release of previously incorporated ⁴⁵Ca from fetal rat bone by 60 to 135 per cent at maximally effective doses, but prostaglandins of the E series were 10- to 100- fold more potent than F,A or B prostaglandins. Compounds with two double bonds in the side chain were usually more potent than those with one double bond. PGE₂ stimulation of bone resorption increased linearly with the logarithm of the medium concentration over the range of 10⁻⁹M to 10⁻⁵M, then decreased at higher concentrations. PGE₂ stimulated bone resorption more slowly than did parathyroid hormone but caused complete resorption after six days in the culture system. Equilibrium dialysis studies showed no significant binding of F, and 16–34% binding of E and A prostaglandins to bovine serum albumin, which was present in the medium at 1 mg/ml. These differences in albumin binding could not account for differences in potency.     

Stimulation of bone resorption by various prostaglandins in organ culture

The ability of E,F,A and B prostaglandins to stimulate bone resorption was demonstrated in organ culture. All of the compounds tested were able to increase the release of previously incorporated ⁴⁵Ca from fetal rat bone by 60 to 135 per cent at maximally effective doses, but prostaglandins of the E series were 10- to 100- fold more potent than F,A or B prostaglandins. Compounds with two double bonds in the side chain were usually more potent than those with one double bond. PGE₂ stimulation of bone resorption increased linearly with the logarithm of the medium concentration over the range of 10⁻⁹M to 10⁻⁵M, then decreased at higher concentrations. PGE₂ stimulated bone resorption more slowly than did parathyroid hormone but caused complete resorption after six days in the culture system. Equilibrium dialysis studies showed no significant binding of F, and 16–34% binding of E and A prostaglandins to bovine serum albumin, which was present in the medium at 1 mg/ml. These differences in albumin binding could not account for differences in potency.     

A novel method to 'exercise' rats: making rats rise to erect bipedal stance for feeding - raised cage model

We employed a novel method to exercise rats: making them rise to bipedal stance for feeding using raised cages. We studied its effects on the skeletons of 6 and 10-month-old intact or orchidectomized (ORX) rats. Body and hindlimb muscle weights, tibial BMC and periosteal cortical bone formation increased after housing in raised cages, but more so in 6-month-old animals than in 10-month-old ones. In 6-month-old orchidectomized rats, raised cages partially prevented ORX-induced bone loss by stimulating periosteal cortical bone (TX) formation and decreased bone resorption next to marrow. In 10-month-old male orchidectomized rats, raised cages also decreased the endosteal and trabecular bone resorption, but not enough to prevent completely ORX-induced net bone losses. Because the osteogenic effects of raised cages alone were only partial, we also studied the interaction between raised cage and prostaglandin E(2) (PGE(2)) in 10-month-old retired female breeders. When treated with combined raised cage and PGE(2), both cortical (TX) and trabecular bone mass of the proximal tibial metaphysis and lumbar vertebral body increased over either raised cages or PGE(2) treatment alone, that was accompanied by dramatic increased bone formation at periosteal and endosteal surfaces. Thus making rats rise to erect bipedal stance for feeding helps to prevent bone loss after orchidectomy; it amplifies the anabolic effects of PGE(2), and it provides an inexpensive, non-invasive and reliable way to increase mechanical loading of certain bones of the rat skeleton.     

Continuous infusion of PGE2 is catabolic with a negative bone balance on both cancellous and cortical bone in rats

It is well documented that intermittent PGE(2) treatment increases both trabecular and cortical bone mass. However, the effects of continuous PGE(2) administration remain undocumented. The aim of the study was to investigate the effects of continuous prostaglandin E(2) (PGE(2)) on different bone sites in skeletally mature rats. Six-month-old Sprague Dawley rats were treated with PGE(2) at 1 or 3 mg/kg/d continuously via infusion pump for 21 days. Two other groups of rats received PGE(2) at the same doses by intermittent (daily) subcutaneous injections and served as positive controls. Histomorphometry was performed on cancellous bone of the proximal tibial metaphysis and cortical bone of the tibial shaft. As expected, intermittent PGE(2) treatment increased both cancellous and cortical bone mass by stimulating bone formation at the cancellous, periosteal and endocortical bone surfaces. In contrast, continuous PGE(2) treatment decreased cancellous bone mass with bone resorption exceeding bone formation. In addition, continuous PGE(2) treatment increased endocortical and intracortical bone remodeling, inducing bone loss which was partially offset by stimulating periosteal expansion. We conclude that continuous PGE(2) treatment induces overall catabolic effects on both cancellous and cortical bone envelopes, which differs from intermittent PGE(2) treatment that is anabolic. Lastly, we speculate that superior bone mass may be achieved by co-treatment of continuous PGE(2) in combination with an anti-catabolic agent.     

Cooxidation of steroidal and non-steroidal estrogens by purified prostaglandin synthase results in a stimulation of prostaglandin formation

Estrone (E1), estradiol (E2), the catechol estrogens 2-OHE1 and 2-OHE2, and diethylstilbestrol (DES) were incubated with purified prostaglandin synthase (PHS) in vitro in the presence of arachidonic acid and their PHS-catalyzed cooxidation was determined. 2-OHE1, 2-OHE2, and DES were extensively metabolized by PHS peroxidase activity, E1 and E2 to a lesser extent. The cooxidation of the estrogens is accompanied by an increased prostaglandin formation and an increase in cyclooxygenase activity in vitro; progesterone and nylestriol are without effect. Prostaglandins have been proposed to play a role in events related to early estrogen action in tissues such as the uterus. The cooxidation of estrogens and their metabolites by prostaglandin hydroperoxidase might represent one type of interaction between the hormones and the arachidonic acid cascade that could lead to changes in prostaglandins.     

Differential activity of granulocyte-macrophage and macrophage colony stimulating factors on bone resorption in fetal rat long bone organ cultures.

In this study, the ability of recombinant human macrophage (M) and murine granulocyte-macrophage (GM) colony stimulating factor (CSF) to affect both basal and stimulated bone resorption in fetal rat long-bone organ cultures was assessed. It was found that M-CSF does not affect basal bone resorption or bone resorption stimulated by parathyroid hormone, recombinant human interleukin 1 beta, prostaglandin E2 (PGE2), and 1,25 dihydroxy vitamin D3. Specifically, M-CSF at concentrations as high as 30 nM (1 microgram/mL) did not modulate 45Ca release from fetal rat long bones stimulated by these agents. The addition of recombinant murine GM-CSF (at equal molar concentration to M-CSF) also did not affect bone resorption stimulated by parathyroid hormone and interleukin 1 beta. On the other hand, GM-CSF stimulated basal bone resorption over a 120-h period and augmented the resorption mediated by exogenous PGE2 over a 48-h incubation. In addition, GM-CSF was shown to stimulate production of endogenous PGE2 in cultures of bone rudiments. These effects on bone resorption were blocked by the addition of prostaglandin synthesis inhibitors and specific antibodies to murine GM-CSF. These data indicate that M-CSF does not act as a regulator of bone turnover, but GM-CSF may cause bone resorption by stimulating the synthesis of PGE2 in bone.     

Effect of prostaglandins on the sensitivity of cerebral cortical neurons in rabbits to cholinoreceptor agonists

The effect of prostaglandins F2a and E2 on the reaction of rabbit's brain cortex neurons provoked by arecoline and nicotine (stimulators of M- and N-cholinereceptors) has been investigated by using a microionophoretic technique. As a rule, prostaglandin F2a decreased and prostaglandin E2 increased the effects of arecoline. Prostaglandins rarely changed the effect of nicotine. The data obtained confirms the supposition that prostaglandin F2a has inhibitory effects on the synthesis in neurons of the cyclical guanosinemonophosphate. Some hypothesis of the prostaglandins participation in integrative activity mechanisms of neurons are supposed.     

Prostaglandins in human semen during fish oil ingestion: evidence for in vivo cyclooxygenase inhibition and appearance of novel trienoic compounds

Marine oils may offer cardiovascular benefits, but inhibition of prostaglandin E and prostaglandin F synthesis by fish oil has been found in animal studies, and such effects could alter physiological responses in man to a clinically significant degree. Since greater amounts of E and F-type prostaglandins are made in human seminal vesicles than in the rest of the body combined, the influence of n-3 supplements upon semen prostaglandins was assessed in 10 subjects before and after one month of taking 50 ml menhaden oil daily. Prostaglandins E1, E2 and their 19-hydroxy derivatives were measured by HPLC-UV as PGB's, and prostaglandin E3, 19-OH PGE3, and analogous PGF's by gas chromatography/mass spectrometry. Fish oil ingestion reduced concentrations of one- and two series prostaglandins (mean reduction in PGE's = 37%, in PGF's = 20%, p less than 0.05), while more than doubling the low amounts of PGE3 and PGF3 alpha, and their previously undescribed 19-hydroxy derivatives. Semen phospholipids were enriched in eicosapentaenoic acid after dietary fish oil, but sperm counts and motility were not altered during the study. Since dietary fish oil reduces prostaglandin concentration in semen, clinical trials of n-3 fatty acids should also evaluate other possible results of in vivo cyclooxygenase inhibition.     

Deletion of the P2X7 nucleotide receptor reveals its regulatory roles in bone formation and resorption

The P2X7 nucleotide receptor is an ATP-gated ion channel expressed widely in cells of hematopoietic origin. Our purpose was to explore the involvement of the P2X7 receptor in bone development and remodeling by characterizing the phenotype of mice genetically modified to disrupt the P2X7 receptor [knockout (KO)]. Femoral length did not differ between KO and wild-type (WT) littermates at 2 or 9 months of age, indicating that the P2X7 receptor does not regulate longitudinal bone growth. However, KO mice displayed significant reduction in total and cortical bone content and periosteal circumference in femurs, and reduced periosteal bone formation and increased trabecular bone resorption in tibias. Patch clamp recording confirmed expression of functional P2X7 receptors in osteoclasts from WT but not KO mice. Osteoclasts were present in vivo and formed in cultures of bone marrow from KO mice, indicating that this receptor is not essential for fusion of osteoclast precursors. Functional P2X7 receptors were also found in osteoblasts from WT but not KO mice, suggesting a direct role in bone formation. P2X7 receptor KO mice demonstrate a unique skeletal phenotype that involves deficient periosteal bone formation together with excessive trabecular bone resorption. Thus, the P2X7 receptor represents a novel therapeutic target for the management of skeletal disorders such as osteoporosis.     

Bone resorption and prostaglandin production by mouse calvaria in vitro: response to exogenous prostaglandins and their precursor fatty acids

Mouse calvaria were maintained in organ culture for 96 h and endogenous prostaglandin production and active bone resorption (45Ca release) measured. After a lag phase of 12 h, active resorption increased over the 96 h period. The amounts of prostaglandins released into the culture medium (measured by radioimmunoassay) were highest in the first 24 h of culture. Unless these were removed by preculturing for 24 h, or suppressed by indomethacin, no response to exogenous PGE2, or prostaglandin precursors could be demonstrated. Bone resorption was stimulated after preculture by both PGE2 and PGF2 alpha in a dose-dependent manner (10-8M-10-5M), with PGE2 being the more potent. Collagen synthesis was unaffected by PGF2 alpha, whereas PGE2 (10-5M) had an inhibitory effect. Eicosatrienoic acid did not stimulate bone resorption at lower concentrations (10-7M-1-5M), but was inhibitory at 10-4M. Arachidonic acid also inhibited resorption at 10-4m, but at lower concentrations (10-7M-10-5M) increased active resorption. This was concomitant with a rise in PGE2 and PGF2 alpha levels, PGE2 production being significantly higher than PGF2 alpha. The effects of PGE2 (10-8M) and PGF2 alpha (10-8M) appeared additive; there was no evidence of synergistic or antagonistic effects when varying ratios of PGE2: PGF2 alpha were employed.     

Changes in tissue concentration of prostaglandins during endochondral bone differentiation

Prostaglandins are known to be involved in bone metabolism as evidenced by the ability of PGE2 to induce bone resorption. It was, therefore, of interest to determine if there was an association of specific prostaglandin metabolites with the various stages of developing bone by utilizing the matrix-induced endochondral bone formation system. During mesenchymal cell proliferation a peak of endogenous thromboxane B2 was detected. In the subsequent stages of chondrogenesis and chondrolysis PGF2 alpha was in high concentration, whereas during bone formation PGE2, 6-Keto-PGF1 alpha and thromboxane B2 were elevated. These changes in the peak levels of the various prostaglandin metabolites may reflect differences in the cell populations and function associated with various stages of endochondral bone formation.     

Human osteoclast ontogeny and pathological bone resorption

Monocytes and macrophages are capable of degrading both the mineral and organic components of bone and are known to secrete local factors which stimulate host osteoclastic bone resorption. Recent studies have shown that monocytes and macrophages, including those isolated from neoplastic and inflammatory lesions, can also be induced to differentiate into cells that show all the cytochemical and functional characteristics of mature osteoclasts, including lacunar bone resorption. Monocyte/macrophage-osteoclast differentiation occurs in the presence of osteoblasts/bone stromal cells (which express osteoclast differentiation factor) and macrophage-colony stimulating factor and is inhibited by osteoprotegerin. Various systemic hormones and local factors (e.g. cytokines, growth factors, prostaglandins) modulate osteoclast formation by controlling these cellular and humoral elements. Various pathological lesions of bone and joint (e.g. carcinomatous metastases, arthritis, aseptic loosening) are associated with osteolysis. These lesions generally contain a chronic inflammatory infiltrate in which macrophages form a significant fraction. One cellular mechanism whereby pathological bone resorption may be effected is through generation of increased numbers of bone-resorbing osteoclasts from macrophages. Production of humoral factors which stimulate mononuclear phagocyte-osteoclast differentiation and osteoclast activity is also likely to influence the extent of pathological bone resorption.     

Computational simulation of spontaneous bone straightening in growing children

Periosteal surface pressures have been shown to inhibit bone formation and induce bone resorption, while tensile strains perpendicular to the periosteal surface have been shown to inhibit bone resorption and induce new bone deposition. A new computational model was developed to incorporate these experimental findings into simulations of spontaneous bone straightening in children with congenital posteromedial bowing of the tibia. Three-dimensional finite element models of the periosteum were used to determine the relationships between the defect angle and the distribution of bone surface pressures and strains due to growth-generated tensile strains in the periosteum. These relationships were incorporated into an iterative simulation to model development of a growing, bowed tibia with an initial defect angle of 27°. When periosteal loads were included in the simulation, the defect angle decreased to 10° after 2 years, and the bone straightened by an age of 25 years. When periosteal loads were not included in the simulation, the defect angle decreased to 23° after 2 years, and a defect angle of 9° remained at an age of 25 years. A “modeling drift” bone apposition/resorption pattern appeared only when periosteal loads were included. The results suggest that periosteal pressures and tensile strains induced by bone bowing can accelerate the process of bone straightening and lead to more complete correction of congenital bowing defects. Including the mechanobiological effects of periosteal surface loads in the simulations produced results similar to those seen clinically, with rapid straightening during the first few years of growth.     

Hypercalcemia and cancer: an update.

Hypercalcemia is a not infrequent complication of cancer that every physician should be aware of. It is a significant factor in the morbidity and mortality of cancer patients. Almost invariably hypercalcemia is associated with accelerated resorption of bone, which is thought to be mediated by humoral or metabolic factors. Three such factors, parathyroid hormone, E2 prostaglandins and osteoclast activating factor, have been strongly implicated in the pathogenesis of hypercalcemia in cancer patients. Other mechanisms for the hypercalcemia may exist. Accurate diagnosis of the disorder is important in therapy, and current research into the various mechanisms for hypercalcemia in cancer patients may well lead to new modes of therapy that are more specific and perhaps less toxic.     

Prostaglandins suppress VLDL secretion in primary rat hepatocyte cultures: relationships to hepatic calcium metabolism.

In primary cultures of rat hepatocytes, prostaglandin E2 and prostaglandin D2 (PGE2 and PGD2) inhibited the secretion of very low density lipoprotein (VLDL)-associated apoB, triacylglycerol, and cholesterol. These effects were concentration-dependent and remained apparent for at least 3 days of culture without an effect on the apoB/triacylglycerol ratio of the secreted VLDL. Prostaglandins had no effect on the overall synthesis of triacylglycerol but triacylglycerol accumulated within the cells, without intracellular accumulation of apoB. PGE2, when added to the medium together with glucagon, increased the inhibition of VLDL secretion, compared to that observed with glucagon alone. However, PGE2 did not increase the stimulatory effect of glucagon on ketogenesis. Unlike glucagon, the prostaglandins did not inhibit fatty acid synthesis nor did they stimulate ketogenesis or production of cAMP. Thus, of all the parameters of hepatic lipid metabolism studied, PGE2 and PGD2 selectively affected VLDL. Selective inhibition of VLDL secretion was also observed with the calcium antagonist verapamil. The divalent cation ionophore A23187 also inhibited VLDL release but, in contrast, also inhibited fatty acid and cholesterol synthesis. The results suggest that VLDL secretion is modulated at some optimal cell calcium concentration that may be mediated selectively by agents such as prostaglandins.