Starvation of a clonal osteoblast-like cell line, MOB 3-4-F2, down-regulates prostaglandin E2 receptors but increases cAMP response to prostaglandin E2.

Prostaglandin E2 (PGE2) stimulated cAMP production in the MOB 3-4-F2 cell line, a subclone of the osteoblast-like MOB 3-4 cell line. After being cultured in alpha-minimum essential medium supplemented with 10% heat-inactivated foetal calf serum (HIFCS), cells responded to PGE2 (greater than or equal to 50 ng/ml) with a small, but significant, increase in cAMP production. This response did not vary with duration of culture. In 2% HIFCS-containing medium, despite their lower basal cAMP level, cells responded to PGE2 (greater than or equal to 5 ng/ml) with strikingly increased cAMP production. In addition, prolonged culture in this serum-deficient medium enhanced this response. On the other hand, culture of cells in 2% HIFCS-containing medium decreased the apparent number of PGE2 receptors, which was also enhanced by prolonged culture, without effect on their apparent affinity. Their number in 10% HIFCS-containing medium, more than that in 2% HIFCS-containing medium, was almost constant, independent of the culture period. Starvation of MOB 3-4-F2 cells in serum-deficient medium, therefore, appeared to down-regulate PGE2 receptors but increase the cAMP response to PGE2. Moreover, prolonged starvation of cells appeared to facilitate these phenomena. Our findings suggest that cAMP response to PGE2 does not always reflect the number of available PGE2 receptors in the cells.     

Aluminum enhances the stimulatory effect of NaF on prostaglandin E2 synthesis in a clonal osteoblast-like cell line, MOB 3-4, in vitro

The effect of NaF on prostaglandin E2 (PGE2) synthesis in a clonal osteoblast-like cell line, MOB 3-4, was examined in the presence of Al3+. The MOB 3-4 cell line, which was derived from neonatal mouse calvaria, displays many osteoblastic characteristics, including the biosynthesis of PGE2. In the absence of Al3+, 1 mM NaF increased PGE2 synthesis (per well) to about 340% of the control level, whereas NaF at lower concentrations (below 0.1 mM) did not show such a significant effect. In the presence of 10 microM Al3+, NaF concentrations ranging from 0.01 to 1 mM increased PGE2 synthesis in a dose-dependent manner, though 10 microM Al3+ had no effect by itself. Similar effects were observed on alkaline phosphatase (ALP) activity per well, but a stimulatory effect of NaF on protein synthesis was observed only in the presence of 10 microM Al3+. These data demonstrated that PGE2 synthesis per protein was increased by NaF alone, and this effect was markedly enhanced by the addition of AlCl3. ALP activity per protein was, however, significantly increased by NaF in the absence of AlCl3. Taken together with our previous finding that Al3+ enhances the NaF-induced Ca2+ mobilization in MOB 3-4 cells, these results suggest that F- combined with Al3+ (i.e., AlF4-) is a more potent stimulator of PGE2 synthesis in cells than F- alone, and that the AlF4- -enhanced PGE2 synthesis may be caused by an increase in cytosolic free Ca2+ concentration during activation of the G protein by AlF4-.     

Effect of prostaglandin E2 on PZ-peptidase and several other peptidase activities in a clonal osteoblast-like cell line derived from newborn mouse calvaria

The effects of prostaglandin E2(PGE2) on the degradation of collagen and non-collagenous peptides in clonal osteoblastic MC3T3-E1 cells were investigated by using highly sensitive assay methods for PZ-peptidase, collagenase-like peptidase (CL-peptidase), dipeptidyl-aminopeptidase (DAP), leucine aminopeptidase (LAP), and post-proline cleaving enzyme (PPCE). PGE2, at concentrations of 0.1 to 4.0 micrograms/ml, doubled the PZ-peptidase and CL-peptidase activities in the cells on 24 h culturing in a dose-dependent manner. PGE2, at a concentration of 2.0 micrograms/ml, enhanced the specific activities of PZ-peptidase, CL-peptidase, DAP, LAP, and PPCE for 75 h after the start of PGE2 stimulation. The time dependent changes in PZ-peptidase and CL-peptidase activities showed similar patterns, and 3- and 2-fold increases were seen after 48 h, respectively. The protein and DNA contents gradually increased after addition of PGE2. Since the PZ-peptidase and CL-peptidase, involved in degradation of collagen peptides, were significantly induced by PGE2 in comparison with LAP and PPCE, involved in the degradation of non-collagenous peptides, these results show that PGE2 specifically stimulates induction of collagen catabolizing enzymes in clonal osteoblasts.     

Effects of indomethacin, prostaglandin E2, prostaglandin F2 alpha and 6-keto-prostaglandin F1 alpha on hatching of mouse blastocysts

The present study has been performed to investigate how PGs would participate the hatching process. Effects of indomethacin, an antagonist to PGs biosynthesis, on the hatching of mouse blastocysts were examined in vitro. Furthermore, it was studied that prostaglandin E2 (PGE2), prostaglandin F2 alpha (PGF2 alpha) or 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha) were added to the culture media with indomethacin. The hatching was inhibited by indomethacin yet the inhibition was reversible. In the groups with indomethacin and PGE2, no improvement was seen in the inhibition of hatching and the inhibition was irreversible. In the groups with indomethacin and PGF2 alpha, inhibition of hatching was improved in comparison with the group with indomethacin. In the groups with indomethacin and 6-keto-PGF1 alpha, no improvement was seen. The above results indicated that PGF2 alpha possibly had an accelerating effect on hatching and a high concentration of PGE2 would exert cytotoxic effect on blastocysts.     

Effect of phorbol myristate acetate on release of arachidonic acid and its metabolites in the osteoblastic MOB 3-4 cell line and its subclone, MOB 3-4-F2.

We examined the effect of phorbol 12-myristate 13-acetate (PMA) on release of arachidonic acid (AA) and its metabolites in osteoblastic cells in an attempt to study mechanism of the regulation of phospholipase A2 (PLA2) activity. In the MOB 3-4-F2 cell line, a subclone of the clonal osteoblastic MOB 3-4 cell line, PMA (0.1-100 nM) changed its appearance and increased AA release in a dose- and time-dependent manner, whereas 4 alpha-phorbol 12,13-didecanoate (4 alpha-PDD) did not show a significant affect on the release. The addition of 1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA, greater than or equal to 1.5 mM), a Ca2+ chelator, almost completely inhibited the PMA-induced AA release without affecting the intrinsic AA release. Preincubation with staurosporine (5-20 nM), an inhibitor of protein kinase C (PKC), partially (approximately 60%) blocked the AA release. However, 30-min preincubation with H-7 (50-200 microM), an inhibitor of PKC, failed to block the AA release. PMA, thus, appeared to stimulate AA release partially by a staurosporine-sensitive mechanism, probably an activation of PKC, in an external Ca(2+)-dependent manner. On the other hand, MOB 3-4 cells responded to PMA with an increased AA release but not with a drastic change of its shape. Both staurosporine and BAPTA exerted similar inhibitory effects. Prolonged exposure (48 h) to PMA (0.1-10 nM) enhanced DNA synthesis of MOB 3-4-F2 cells, but not MOB 3-4 cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Platelet-activating factor stimulates production of prostaglandin E2 in murine osteoblast-like cell line MC3T3-E1.

We found that platelet-activating factor (PAF) stimulated the production of prostaglandin (PG) E2 in MC3T3-E1 cells in a time- and dose-dependent manner. 1.0 microM PAF gave a maximal stimulation of PGE2 production by MC3T3-E1 cells after a 4 hr PAF-treatment. Furthermore, the PAF-induced PGE2 production was abolished by the pre-treatment of the cells with a PAF receptor antagonist, 1-O-hexadecyl-2-acetyl-sn-glycero-3-phospho(N,N,N-trimethyl)hexanolamine, which occupied the same receptor site as PAF. These results suggest that PAF stimulates the PGE2 synthesis through a PAF receptor mediated pathway. Possibly PAF modulates bone metabolism by stimulating PGE2 synthesis.     

Protein kinase C activation amplifies prostaglandin F2 alpha-induced prostaglandin E2 synthesis in osteoblast-like cells.

In cloned osteoblast-like cells, MC3T3-E1, prostaglandin F2 alpha (PGF2 alpha) stimulated arachidonic acid (AA) release in a dose-dependent manner in the range between 1 nM and 10 microM. 12-O-tetradecanoylphorbol-13-acetate (TPA), a protein kinase C (PKC) activator, which by itself had little effect on AA release, markedly amplified the release of AA stimulated by PGF2 alpha in a dose-dependent manner. 4 alpha-phorbol 12,13-didecanoate, a phorbol ester which is inactive for PKC, showed little effect on the PGF2 alpha-induced AA release. 1-oleoyl-2-acetylglycerol (OAG), a specific activator for PKC, mimicked TPA by enhancement of the AA release induced by PGF2 alpha. H-7, a PKC inhibitor, markedly suppressed the effect of OAG on PGF2 alpha-induced AA release. Quinacrine, a phospholipase A2 inhibitor, showed partial inhibitory effect on PGF2 alpha-induced AA release, while it suppressed the amplification by OAG of PGF2 alpha-induced AA release almost to the control level. Furthermore, TPA enhanced the AA release induced by melittin, known as a phospholipase A2 activator. On the other hand, TPA inhibited the formation of inositol trisphosphate stimulated by PGF2 alpha. Under the same condition, PGF2 alpha indeed stimulated prostaglandin E2 (PGE2) synthesis and TPA markedly amplified the PGF2 alpha-induced PGE2 synthesis as well as AA release. These results indicate that the activation of PKC amplifies PGF2 alpha-induced both AA release and PGE2 synthesis through the potentiation of phospholipase A2 activity in osteoblast-like cells.     

Effects of vitamin D3 on signaling by prostaglandin E2 in osteoblast-like cells

We investigated the effects of vitamin D₃ on the signaling pathways by prostaglandin E₂ (PGE₂) in osteoblast-like MC3T3-E1 cells. The pretreatment with 1,25-dihydroxyvitamin D₃ (1,25-(OH)₂D₃), an active form of vitamin D₃, significantly inhibited cAMP accumulation induced by 10 μM PGE₂ in a dose-dependent manner in the range between 1 pM and 1 nM. This effect of 1,25-(OH)₂D₃ was dependent on the time of pretreatment up to 8 h. 1,25-(OH)₂D₃ also inhibited the cAMP accumulation induced by NaF, a GTP-binding protein activator, or forskolin which directly activates adenylate cyclase. On the other hand, 1,25-(OH)₂D₃ significantly inhibited PGE₂-induced IP₃ formation in a dose-dependent manner between 10 pM and 1 nM. However, 1,25-(OH)₂D₃ had little effect on NaF-induced IP₃ formation. The pretreatment with 24,25-dihydroxyvitamin D₃, an inactive form of vitamin D₃, affected neither cAMP accumulation nor IP₃ formation induced by PGE₂. These results strongly suggest that 1,25-(OH)₂D₃ modulates the signaling by PGE₂ in osteoblast-like cells as follows: the inhibitory effect on the cAMP production is exerted at a point downstream from adenylate cyclase and the inhibitory effect on the phosphoinositide hydrolysis is exerted at the point between the PGE₂ receptor and GTP-binding protein, probably G_i2.     

Effects of analogues of prostaglandin E2 and F2 alpha on the decidual cell reaction in the rat

The identity of the prostaglandins (PGs) involved in the decidual cell reaction is uncertain. In the present study we investigated the ability of analogues of PGE2 and PGF2 alpha, 16,16-dimethyl-prostaglandin E2, methyl ester (16,16Me2PGE2) and 15(S)-15-methyl-prostaglandin F2 alpha (15MePGF2 alpha) respectively, to bring about decidualization when infused into the uterine lumen of rats sensitized for the decidual cell reaction. As indicated by uterine weights 5 days after the commencement of the infusions into rats in which endogenous PG production had been inhibited by treatment with indomethacin, 16,16Me2PGE2 produced decidualization which was equivalent to that produced by PGE2. By contrast, the infusion of 15MePGF2 alpha inhibited decidualization, even when PGE2 was infused concomitantly. As indicated by uterine radioactivity concentrations after i.v. administration of 125I-labeled bovine serum albumin, the PGF2 alpha analogue also inhibited the endometrial vascular permeability increase which precedes decidualization. Compared to PGE2, 16,16Me2PGE2 was slightly less effective at displacing 3H-PGE2 from an endometrial membrane preparation; by contrast 15MePGF2 alpha was considerably less effective. These data suggest that PGE2 mediates the decidual cell reaction, and that the decidualization obtained in response to PGF2 alpha may involve its conversion within the uterus to PGE2.     

Effects of indomethacin, prostaglandin E2 and prostaglandin F2 alpha on mouse blastocyst attachment and trophoblastic outgrowth in vitro

A study was performed in order to investigate the participation of prostaglandins (PGs) during implantation. The effects of indomethacin on mouse blastocyst attachment and trophoblastic outgrowth were examined in vitro. Studies were also carried out on cultures supplemented with PGE2 and/or PGF2 alpha along with indomethacin. (1) Blastocyst attachment and trophoblastic outgrowth were inhibited by indomethacin dose-dependency. (2) In the cultures supplemented with indomethacin and PGE2 or PGF2 alpha, respectively, the inhibitory effects of indomethacin were reduced. (3) In the cultures supplemented with all three substances with treatment (1) and (2), inhibition of indomethacin was partially reversed, but still lower than control group without indomethacin. The above results indicate that both PGE2 and PGF2 alpha have a promoting effect on implantation, and PGF2 alpha was more effective than PGE2.     

Effects of prostaglandin E2 and F2 alpha on peri-implantation development of mouse embryos in vitro.

The effects of exogenous prostaglandin (PG) E2 and F2 alpha on the morphology and lactate dehydrogenase (LDH) activities of pre-implantation mouse embryos in vitro were studied. A 24-hour exposure from 0.01 to 10 micrograms/ml of PGE2 at the 4-cell or morula stages had no effect on the morphology of embryos during the 144 hours in culture. Exposure to 10 micrograms/ml PGE2 at the blastocyst stage accelerated and enhanced spreading of the trophoblast in vitro. Embryos treated at 0.01 to 10 micrograms/ml PGE2 at various stages all showed a more rapid decline in LDH activity from morula to blastocysts. Treatment with 50 or 100 micrograms/ml PGE2 led to abnormal morphology of embryos in vitro. In contrast, continuous treatment with 0.01 to 100 micrograms/ml PGF2 alpha from 4-cell to early post-implantation (day 8) had no effect on the morphology of embryos, although breakdown of LDH was again accelerated. These results suggest that the peak of PGE2 secretion on day 4 of pregnancy in mice may enhance trophoblastic outgrowth, and the lower levels of PGE2 and PGF2 alpha secreted earlier in pregnancy modulate the development of pre-implantation mouse embryos.     

Urinary excretion of prostaglandin E2 and prostaglandin F2alpha in potassium-deficient rats

Potassium-deficiency was induced in rats by dietary deprivation of potassium. The animals became polyuric and urine osmolality decreased more then three-fold compared to controls. Urinary excretion of prostaglandin E2 (PGE2) and prostaglandin F2alpha (PGF2alpha) did not increase during 2 weeks of potassium depletion. Partial inhibition of renal prostaglandin synthesis by meclofenamate did not increase the urine osmolality after water deprivation. These results make unlikely the hypothesis that the polyuria of potassium-deficiency, is the result of enhanced renal synthesis of prostaglandins with subsequent antagonism of the hydro-osmotic effect of vasopressin. Male animals consistently excreted less PGE2 than female animals.     

Influence of coronary vasodilators on prostaglandin E, F2alpha, I2, and adenosine metabolism.

We investigated the influence of oxyfedrine, verapamil, prenylamine, sodium nitrite, nitroglycerin, papaverine and carbochromen on the release of prostaglandins (PG) and adenosine from Langendorff heart preparations of guinea pigs and rabbits and on the PG biosynthesis in the heart and renal medulla of rabbits, in the bovine seminal vesicle homogenate, and in the rat stomach fundus. Oxyfedrine stimulated the release of PGE and PGF2alpha from the Langendorff hearts and increased the PG biosynthesis in all organs investigated. Compared with the other drugs, oxyfedrine showed the strongest stimulation of PGI2-synthesis in the rat stomach fundus. Prenylamine depressed the PG biosynthesis in all organs investigated but increased the PGE release from Langendorff hearts. Nitroglycerin, verapamil and, to some degree, also carbochromen increased the PGI2 synthesis in rat stomach fundus. Sodium nitrite was without influence on the PG release from Langendorff hearts and on the PG biosynthesis. Papaverine did not influence the PGI2 synthesis. Independently of the PG release, oxyfedrine, prenylamine and sodium nitrite increased the adenosine efflux from the rabbit hearts.     

Thrombin increases cytoplasmic Ca2+ and stimulates formation of prostaglandin E2 in the osteoblastic cell line MC3T3-El

Using microfluorometric analysis in individual, fura-2 loaded cells, we found that thrombin (0.1–10 U/ml) caused a dose-dependent (EC₅₀ ≈ 0.5 U/ml), rapid (within seconds), transient increase in cytoplasmic Ca²⁺ in the osteoblastic cell line MC3T3-El. The thrombin induced rise in cytoplasmic Ca²⁺ was not dependent on extracellular Ca²⁺ and was unaffected by indomethacin. In MC3T3-El cells, thrombin (0.3–10 U/ml) caused a rapid and dose-dependent (EC₅₀ ≈ 0.5 U/ml) stimulation of PGE₂ formation. The calcium ionophore A23187 (2 μmol/l) also rapidly stimulated an increase in cytoplasmic Ca²⁺ and the formation of PGE₂ in MC3T3-El cells. These data indicate that thrombin mobilizes Ca²⁺ from intracellular stores and that Ca²⁺ may serve as a second messenger in thrombin induced stimulation of PGE₂ biosynthesis in osteoblasts.     

Stereospecificity of enzymatic reduction of prostaglandin E2 to F2alpha.

Antibodies directed toward PGF2beta were prepared in rabbits. The serologic specificity of the immune reaction was determined by inhibition of sodium borohydride-reduced (3H) PGE2 anti-PGF2beta binding by several prostaglandins. The antibodies to PGF2beta recognize the beta-hydroxyl configuration in the cyclopentane ring of PGF2beta. With the use of both anti-PFG2alpha and anti-PFG2beta, the product of PGE2 reduction by 9-ketoreductase purified from chicken heart was identified as PGF2alpha. Guinea pig liver and kidney homogenates were examined for PGE 9-ketoreductase activity. Although enzyme activity was present, no evidence of PGF2beta production was found.     

Stimulatory effect of prostaglandin F(2alpha) on Na-dependent phosphate transport in osteoblast-like cells

Prostaglandin F(2alpha) (PGF(2alpha)) has been reported to activate protein kinase C (PKC) through both phospholipase (PL) C and D, resulting in the proliferation of osteoblast-like cells. In addition, it has also been reported that Erk mitogen-activated protein kinase is also involved in the mechanism of PGF(2alpha)-induced proliferation of these cells. Recently, we have reported that several growth factors stimulate Na-dependent phosphate transport (Pi transport) activity of osteoblast-like cells, which has been recognized to play an important role in their mineralization. In the present study, we investigated the effect of PGF(2alpha) on Pi transport in MC3T3-E1 osteoblast-like cells. PGF(2alpha) stimulated Na-dependent Pi transport dose dependently in the range between 1nM and 10 micro M in MC3T3-E1 cells. The effect was time dependent up to 24h. Kinetic analysis revealed that PGF(2alpha) induces newly synthesized Pi transporter. Pretreatment with actinomycin D and cycloheximide suppressed PGF(2alpha)-induced enhancement of Pi transport. Combined effect of PMA and PGF(2alpha) was not additive in Pi transport. Calphostin C, a PKC inhibitor, dose-dependently suppressed Pi transport induced by PGF(2alpha). On the contrary, U0126, which inhibits an upstream kinase of Erk (MEK), did not affect PGF(2alpha)-induced enhancement of Pi transport. In conclusion, PGF(2alpha) stimulates Pi transport through activation of PKC in osteoblast-like cells.     

Determination of prostaglandin F2 alpha, E2, D2 and 6-keto-F1 alpha in human cerebrospinal fluid.

Prostaglandin (PG)F2 alpha, E2, D2 and 6-keto-F1 ALha were determined in human cerebrospinal fluid by a mass spectrometric technique. The samples were obtained from 12 patients with suspected intracranial disease. A 64 fold variation in PG levels was observed. The major PG was 6-keto-F1 alpha (0.12--15 ng/ml). PGF2 alpha and PGE2 were present in lower concentrations PGD2 was below the level of detection (0.05 ng/ml) except in one patient with extremely high total levels of PGs.     

Effects of prostaglandin F2 alpha prostaglandin E2 and arachidonic acid on the induction of oviposition in vivo and in vitro in oviparous lizards.

Gravid females of four different species of oviparous lizard were treated in vivo with varying doses of prostaglandin F2 alpha (PGF2 alpha), prostaglandin E2 or arachidonic acid (AA). In contrast to previous studies examining birds and viviparous lizards, no dosage induced oviposition in any of the treated females. All females, however, did exhibit behaviors associated with oviposition. Intact oviducts removed from gravid females and placed in organ culture did oviposit when treated with 30 or 100 ng PGF2 alpha/ml of culture media. Arachidonic acid at similar concentrations also was effective in stimulating birth. These data suggest that prostaglandins can stimulate oviposition in oviparous lizards but further suggest that their action may be inhibited by oviducal innervation until just prior to natural birth.