Cell Cycle activation and ouabain-sensitive ion movements of 3T3 and C3H-10T½ fibroblasts

The introduction of either PGF_2α (10^?7 M) or TPA (10^?7 M) stimulated, ouabain-sensitive ⁸⁶Rb⁺ influx at 30 min in postconfluent 3T3-4 mouse fibroblast cultures by 117% and 124%, respectively. Both TPA and PGF_2α at these concentrations stimulated the incorporation of ³H-TdR into DNA. TPA had the greatest stimulatory effect, which was similar to that obtained with 10% fetal calf serum. In accord with the idea that modulation of membrane processes such as Na⁺/K⁺ pump activity in fibroblasts may reflect important events related to the initiation of DNA synthesis, it was observed that in both 3T3-4 and C3H-1 0T½ cells there were parallel increases in ³H-TdR incorporation and ouabain-sensitive ⁸⁶Rb⁺ influxes with 10^?7 M TPA, whereas PGF_2α stimulated a significant increase in ³H-TdR incorporation in 3T3-4 but not C3H-10T½ cells and only marginal increases in ouabain-sensitive ⁸⁶Rb⁺ influx in both. Therefore, although there appears to be a close correlation between Na⁺/K⁺ pump activation and subsequent S-phase entry following TPA stimulation, a similar correlation for PGF_2α cannot be confirmed.     

Retinoic acid enhances the initiation of DNA synthesis stimulated by prostaglandin F2α in Swiss 3T3 cells

Retinoic acid at concentrations of 10⁻⁸–10⁻⁶M increases the initiation of DNA synthesis stimulated by prostaglandin F_2α (PGF_2α), alone or with insulin, in confluent resting Swiss mouse 3T3 cells. Analogues of retinoic acid had a similar effect. Adding retinoic acid, alone or with insulin, did not stimulate DNA synthesis. The synergistic effect of PGE₁ or PGE₂ with PGF_2α and insulin was not further enhanced by retinoic acid. Neither the synergistic effect of retinoic acid nor that of PGE₁ or PGE₂ with PGF_2α was affected by indomethacin, an inhibitor of prostaglandin synthesis. The results suggest that the synergy of retinoic acid with PGF_2α is not mediated through an increase of prostaglandin synthesis and that retinoic acid stimulates event(s) in common with those of PGE₁ and PGE₂ leading to an increase in the initiation of DNA synthesis.     

The stimulation of the initiation of DNA synthesis by fibroblast growth factor in swiss 3T3 cells: Interactions with hormones during the pre-replicative phase

Fibroblast Growth Factor (FGF) stimulates quiescent Swiss 3T3 cells to initiate DNA synthesis and divide. Cells begin to enter the S-phase after a lag of 13–15 hr, and the rate of initiation of DNA synthesis in the population can be quantified by a first order rate constant, k. A subsaturating concentration of FGF may establish the lag phase, while the value of k is dependent on the FGF concentration present during the second half of the lag phase. Insulin and hydrocortisone enhance the effect of FGF by increasing k without changing the lag phase, and they can act when added at any time after FGF. Prostaglandin E₁ (PGE₁) causes a decrease in k and a lengthening of the lag phase, and acts only when added during the first 8 hr. None of these agents stimulate DNA synthesis in the absence of FGF. These results show that the stimulation of growth by FGF follows the same basic pattern as was previously shown with Prostaglandin F_2α (PGF_2α). However, since hydrocortisone inhibits stimulation by PGF_2α when added during the first 4 hr of the lag phase, there are clearly differences in some events stimulated by the two growth factors.     

Epidermal growth factor initiates DNA synthesis after a time-dependent sequence of regulatory events in Swiss 3T3 cells—interactions with hormones and growth factors

Epidermal growth factor (EGF) stimulates the initiation of DNA synthesis in Swiss 3T3 cells after a constant prereplicative period of 14–15 hours. The final rate of initiation follows apparent first-order kinetics and can thus be quantified by a rate constant k. The value of k can be changed by later additions during the prereplicative period: When cells stimulated by a very low concentration of EGF, alone or with insulin, which results in a relatively low value of k, receive a saturating amount of EGF at 15 hours, then k is markedly increased after 4–6 hours. Insulin alone (up to 200 ng/ml) is unable to set the lag phase, but does have a synergistic effect on the value of k given by EGF. When added at 15 hours, insulin also increases k, but after a delay of 4–6 hours. In contrast, both hydrocortisone and prostaglandin E₁ (PGE₁) inhibit the stimulation of DNA synthesis by EGF only during the first 8 hours of the prereplicative period of decreasing the value of k. Prostaglandin F_2α (PGF_2α), which stimulates DNA synthesis in a similar mode as EGF, when added with EGF has a synergistic effect on DNA synthesis. This suggests that EGF and PGF_2α, nevertheless, act through different regulatory events.     

Stimulation of hepatocyte DNA synthesis by prostaglandin E2 and prostaglandin F2α additivity with the effect of norepinephrine,and synergism with epidermal growth factor

Previous data obtained in vivo and in vitro suggest that both prostaglandins (PGs) and catecholamines may have a role in promoting hepatocyte proliferation, and PGE₂ and PGFF_2α have also been implicated as mediators of the mitogenic actions of epidermal growth factor (EGF) (and transforming growth factor alpha [TGFα]). We have studied the effects of PGs and norepinephrine on DNA synthesis in serum-free primary cultures of rat hepatocytes, and compared the PG effects with those of norepinephrine. PGE₂, PGF_2α, PGD₂, and the synthetic analog dimethyl-PGE₂ markedly enhanced the DNA synthesis. A more quantitative analysis of the effects of PGE₂ and PGF_2α on the DNA synthesis, in the presence and absence of EGF, indicated that these PGs interacted in an essentially multiplicative manner with the effect of EGF. The effects of PGE₂ and PGF_2α showed almost complete additivity with the stimulation of DNA synthesis produced by maximally effective concentrations of norepinephrine. The data suggest (a) that PGE₂ and PGF_2α facilitate and synergize with, rather than mediate, the actions of EGF in hepatocytes, and (b) that this effect of the PGs occurs by mechanisms that are at least partly distinct from those of norepinephrine. © 1994 wiley-Liss, Inc.     

Prostaglandin F2alpha and insulin stimulate phosphate uptake and (Na+, K+) ATPase activity in resting mouse fibroblast cultures.

The (Na⁺, K⁺)ATPase transport system in resting 3T3 Swiss mouse fibroblasts is rapidly activated by prostaglandin F₂α and insulin, which initiate DNA synthesis in these cells. Prostaglandin F₂α, but not insulin, promotes a rapid increase in P_i uptake which is partially coupled to the Na⁺ pump. This rapid activation of both transport systems occurs by a mechanism which does not require fluctuation of cyclic AMP levels or new protein synthesis. A subsequent protein synthesis-dependent increase in P_i uptake is stimulated by insulin and prostaglandin F₂α. These results suggest that different types of control of membrane transport occur during growth stimulation.     

Regulation of inositol phosphate levels by prostaglandins in cultured endometrial cells

Stimulation of cultured rabbit endometrial cells by one of the rabbit endometrial cell culture proliferation factors, prostaglandin F_2α (PGF_2α), resulted in a very rapid increase in the intracellular levels of [³H]-inositol triphosphate (IP₃), [³H]-inositol biphosphate (IP₂), and [³H]-inositol monophosphate (IP₁) in cells prelabeled with [³H]-inositol. These increases in inositol phosphate levels were detected in periods of stimulation as short as 30 seconds, reached a maximum by 1 1/2?2 min and declined to control levels by 6–10 min. The stimulation was dose-dependent with maximal increases observed near 10^?6 M PGF_2α. The cholinergic agent, carbachol, also led to time and dose-independent increases in IP₃. Lithium, cadmium, silver, copper, and zinc ions had no effect either on the breakdown of IP₃ or on the accumulation of IP₁. In contrast, vanadate at 10^?6 or 10^?5 M did lead to a decrease in the breakdown of IP₁ and a concomitant increase in IP₁, IP₂, and IP₃. PGF_2α was found previously to induce an increase in rabbit endometrial cell DNA synthesis which was inhibited by concomitant or prior addition of prostaglandin E₁ (PGE₁). PGE₁, in a dose-dependent manner, was found to inhibit the observed IP₃ increase by PGF_2α at 1 1/2 min of stimulation. PGF_2α treated and control cultures did not differ in cAMP or cGMP levels, cellular ⁴⁵Ca uptake, nor cellular ²²Na uptake. We propose that IP₃ may be one of the intracellular messenger(s) synthesized following the treatment of rabbit endometrial cell cultures with the proliferation agent PGF_2α and that it may play a crucial role with cAMP in growth regulation.     

Mevalonate dependency of the early cell cycle mitogenic response to epidermal growth factor and prostaglandin F2α in Swiss mouse 3T3 cells

Lovastatin (LOV), a hydroxy-methylglutaryl-coenzyme A (HMGCoA) reductase competitive inhibitor, blocks epidermal growth factor (EGF)— or prostaglandin F_2α (PGF_2α)—induced mitogenesis in confluent resting Swiss 3T3 cells. This inhibition occurs even in the presence of insulin, which potentiates the action of these mitogens in such cells. LOV exerts its effect in a 2–80 μM concentration range, with both mitogens attaining 50% inhibition at 7.5 μM. LOV exerted its effect within 0–8 h following mitogenic induction. Mevanolactone (10–80 μM) in the presence of LOV could reverse LOV inhibition within a similar time period. LOV-induced blockage of PGF_2α response is reflected in a decrease in the rate of cell entry into S phase. Neither cholesterol, ubiquinone, nor dolichols of various lengths could revert LOV blockage. In EGF- or PGF_2α-stimulated cells, LOV did not inhibit [³H]leucine or [³H]mannose incorporation into proteins, while tunicamycin, an inhibitor of N′ glycosylation, prevented this last phenomenon. Thus, it appears that LOV exerts its action neither by inhibiting unspecific protein synthesis nor by impairing the N′ glycosylation process. These findings strongly suggest that either EGF or PGF_2α stimulations generate early cell cycle signals which induce mevalonate formation, N′ glycoprotein synthesis, and proliferation. The causal relationship of these events to various mechanisms controlling the onset of DNA synthesis is also discussed. © 1995 Wiley-Liss, Inc.     

Comitogenicity of eicosanoids and the peroxisome proliferator ciprofibrate in cultured rat hepatocytes

Several hypolipidemic drugs and environmental contaminants induce hepatic peroxisome proliferation and hepatic tumors when administered to rodents. These chemicals increase the expression of the peroxisomal β-oxidation pathway and the cytochrome P-450 4A family, which metabolize lipids, including eicosanoids and their precursor fatty acids. We previously found that the peroxisome proliferator ciprofibrate decreases the level of eicosanoids in the liver and in cultured hepatocytes. In this study, we examined the effect of prostaglandins E₂ and F_2α (PGE₂ and PGF_2α), leukotriene C₄ (LTC₄) and the peroxisome proliferator ciprofibrate on DNA synthesis in cultured hepatocytes. Primary rat hepatocytes were cultured on collagen gels in serum-free L-15 medium with varying concentrations of eicosanoids and ciprofibrate, and the absence or presence of growth factors. Ciprofibrate lowered hepatocyte eicosanoid concentrations; the addition of eicosanoids restored their levels. After a 48-h exposure with [³H]-thymidine, DNA synthesis was determined by measuring [³H]-thymidine incorporation into DNA. The addition of PGE₂, PGF_2α, and LTC₄ to cultures along with ciprofibrate increased DNA synthesis, whereas treatment with ciprofibrate or eicosanoids alone resulted in a much smaller increase. The addition of epidermal growth factor (EGF) to the eicosanoid-ciprofibrate combination increased DNA synthesis more than EGF or the eicosanoid-ciprofibrate combination alone. The PGF_2α-ciprofibrate combination also was comitogenic with transforming growth factor-α and hepatocyte growth factor. The addition of both ciprofibrate and prostaglandins also blocked the growth inhibitory effect of transforming growth factor-β on DNA synthesis induced by EGF. These results show that the eicosanoids PGE₂, PGF_2α, and LTC₄ are comitogenic with the peroxisome proliferator ciprofibrate in cultured rat hepatocytes. © 1996 Wiley-Liss, Inc.     

Colchicine enhances the effect of growth factors on the initiation of DNA synthesis in sparse and confluent Swiss 3T3 cells

We have studied the effect of colchicine on the initiation of DNA synthesis stimulated by serum or by growth factors such as EGF, FGF or PGF_2α in sparse and confluent resting Swiss 3T3 cells. Colchicine in combination with FGF or with low concentrations of serum is more effective in stimulating DNA synthesis than with EGF or PGF_2α. Only in the presence of insulin do these latter two growth factors give with colchicine a response comparable to that of FGF with colchicine. This phenomenon was observed both in sparse and in confluent resting cells. We conclude that the synergistic effect of colchicine with growth factors or with serum does not depend on the cellular saturation density at which Swiss 3T3 cells become quiescent.     

On the mechanisms of the growth-promoting effect of prostaglandins in hepatocytes: The relationship between stimulation of DNA synthesis and signaling mediated by adenylyl cyclase and phosphoinositide-specific phospholipase C

While many observations indicate that prostaglandins may act as positive regulators of hepatocyte proliferation, the underlying mechanisms are not known. We have examined some of the signal pathways in the growth response induced by prostaglandins in hepatocytes, with particular focus on adenylyl cyclase and phosphoinositide-specific phospholipase C. Adult rat hepatocytes were cultured as primary monolayers in serum-free medium in the presence of EGF and insulin. PGE₂ or PGF_2α (added 0-3 h after plating) enhanced the incorporation of [³H]-thymidine into DNA (measured at 50 h); at 100 γM the stimulation was about threefold. PGI₂ and PGD₂ also showed significant but smaller stimulatory effects. No significant increase in the level of cyclic AMP (cAMP) was detected in response to any of the prostaglandins. Low concentrations of glucagon (0.1-10 nM), a potent activator of hepatic adenylyl cyclase, or 8-bromo-cAMP (0.1-10 γM) enhanced the DNA synthesis. When 8-bromo-cAMP was used in maximally effective concentrations, no further stimulation was obtained by combining it with glucagon, whereas the effects of PGE₂ and 8-bromo-cAMP were completely additive. All the prostaglandins also showed additivity with the effect of glucagon on the DNA synthesis. PGE₂, PGF_2α, PGI₂, and PGD₂ increased intracellular inositol-1,4,5-trisphosphate (InsP₃), with a relative order of efficacy roughly corresponding to their activity as stimulators of DNA synthesis. Increases in cytosolic free Ca²⁺, as measured in single cells, were elicited in a majority of the hepatocytes by all these prostaglandins at 1 γM. Supramaximal concentrations of vasopressin, a strong activator of phospholipase C in hepatocytes, acted additively with PGE₂ on the DNA synthesis. Pretreatment of the hepatocytes with a concentration of pertussis toxin that prevented the inhibitory effect of PGE₂ on glucagon-induced cAMP accumulation did not abolish the ability of PGE₂ to stimulate the DNA synthesis. The results do not support a role for adenylyl cyclase activation in the stimulatory effect of prostaglandins on hepatocyte growth. While the data are compatible with an involvement of phosphoinositide-specific phospholipase C in the growth-promoting effect of prostaglandins in cultured rat hepatocytes, they suggest this may not be the sole mechanism. © 1995 Wiley-Liss, Inc.     

The effect of serum,EGF, PGF2α and insulin on S6 phosphorylation and the initiation of protein and DNA synthesis

To test the connection between S6 phosphorylation and the activation of protein and DNA synthesis, we compared the effects of serum, epidermal growth factor (EGF), prostaglandin F_2α (PGF_2α) and insulin (which is not mitogenic in these cells). Increasing concentrations of serum or EGF produced roughly parallel effects on all three processes, though the maximum response elicited by EGF (10^?9 M) was only a portion of that caused by saturating levels of serum (7.5% to 10%). PGF_2α (8.5 × 10^?7 M) alone acted similarly to EGF (10^?9 M) and with EGF produced a synergistic effect on all three processes. Insulin (10^?9 M) alone stimulated both S6 phosphorylation and protein synthesis to approximately the same level as EGF or PGF_2α, but had no effect on initiation of DNA synthesis. Thus neither stimulation of S6 phosphorylation nor activation of protein synthesis is sufficient for initiation of DNA synthesis. The requirement for S6 phosphorylation could not be dissociated from the activation of protein synthesis. Ribosomes containing the most highly phosphorylated forms of S6 appear to have a selective advantage in entering polysomes.     

Cell growth and ouabain-sensitive Rb uptake and (Na + K)-ATPase activity in 3T3 and SV40 transformed 3T3 fibroblasts

The uptake of ouabain-sensitive ⁸⁶Rb⁺ uptake measured at 5 min and the uptake measured at 60 min was 4.5- and 2.7-fold greater respectively for SV40 transformed 3T3 cells compared to 3T3 cells during the late log phase of growth. This uptake, however, varied markedly with cell growth. Ouabain-sensitive ⁸⁶Rb⁺ uptake was found to be a sensitive indicator of protein synthesis as measured by total protein content. Cessation of cell growth as measured by total protein content was associated with a decline in ouabain-sensitive ⁸⁶Rb⁺ uptake in both cell types. This increased ouabain-sensitive cation transport was reflected in increased levels of (Na⁺ + K⁺)-ATPase activity for SV40 3T3 cells, which showed a 2.5-fold increase V but the same K_rmm as 3T3 cells.These results are compared with the results of related work. Possible mechanisms for these effects are discussed and how changes in cation transport might be related to alterations in cell growth.     

Prostaglandin-stimulated second messenger signaling in bone-derived endothelial cells is dependent on confluency in culture

New bone formation is associated with an increase in blood flow by the invasion of capillaries. Endothelial cells that line the capillaries can produce paracrine factors that affect bone growth and development, and in turn, could be affected by products produced by bone cells, in particular the osteoblasts. Since osteoblasts produce prostaglandins E₂ and F_2α (PGE₂, PGF_2α), it was investigated if these PGs were agonists to bone-derived endothelial cells (BBE) by assessing changes in cAMP and free cytosolic calcium concentration ([Ca²⁺]i) second messenger generation. We found that confluent cultures of BBE cells, a clonal endothelial cell line derived from bovine sternal bone, responded to 1 μM PGE₂ by an increase in cAMP. PGF_2α at the same concentration was less potent in stimulating an increase in cAMP production in confluent BBE cells. Subconfluent cells with a morphology similar to that of fibroblastic cells were not as sensitive to PGE₂-stimulated cAMP generation. PGF_2α failed to elicit any cAMP production in subconfluent cultures. PGE₂ and PGF_2α both stimulated an increase in [Ca²⁺]i concentration in a dose-dependent manner. The potency of PGE₂ was similar to that of PGF_2α in stimulating an increase in [Ca²⁺]i. The Ca²⁺ response was mostly independent of extracellular Ca⁺, was unchanged even with prior indomethacin treatment, was unaffected by caffeine pretreatment, but was abolished subsequent to thapsigargin pretreatment. The PG-induced increase in [Ca²⁺]i was also dependent on the confluency of the cells. In a subconfluent state, the responses to PGE₂ or PGF_2α were either negligible, or only small increases in [Ca²⁺]i were noted with high concentrations of these two PGs. Consistent, dose-dependent increases in [Ca²⁺]i were stimulated by these PGs only when the cells were confluent and had a cobblestoned appearance. Since it was previously demonstrated that BBE cells respond to parathyroid hormone (PTH) by the production of cAMP, we tested if bovine PTH(1-34) amide bPTH(1—34) also increased [Ca²⁺]i in these cells. No change in [Ca²⁺]i was found in response to bPTH (1—34), although bPTH (1—34) stimulated a nine to tenfold increase in cAMP. We conclude that BBE cells respond to PGE₂ and PGF_2α but not to bPTH(1—34) by an increase in [Ca²⁺]i probably secondary to stimulation of phospholipase C and that the cAMP and [Ca²⁺]i second messenger responses in BBE cells are dependent on the state of confluency of the cells. © 1994 Wiley-Liss, Inc.     

Stimulative effect of serum on the growth of HeLa cells suppressed in a K+-deficient chemically defined medium

Growth of HeLa cells cultured with a chemically defined medium was slightly stimulated in the presence of 5% dialyzed calf serum. The growth-promoting action of serum was more conspicuous when cell growth was suppressed in the same medium, in which K⁺ was replaced by Rb⁺ to various ratios. The growth-promoting factors(s) of serum was heat-labile. Upon addition of dialyzed serum, passive K⁺ or Rb⁺ influx was increased, whereas the active cation uptake was unaffected and cell K⁺ was rather decreased. The serum did not alter uptake of [³H] amino acids. Also, protein synthesis inhibited in the Rb⁺-substituted medium was not stimulated significantly, except that observed only when the external K⁺/Rb⁺ ratio was $\text{1}\text{5}$. From the distinct effects of serum on cell growth and protein synthesis, we conclude that (i) the serum-induced stimulation of cell growth, which is suppressed in the Rb⁺-substituted medium, is not a result of the direct effect of serum on synthesis of bulk protein, but a reflection of the effect on another mechanism(s) required for cell growth; and that (ii) this action is basically identical with the growth-promoting action on cells cultured in the normal medium.     

Prostaglandins of the F series are extremely powerful growth factors for primary neonatal rat hepatocytes

At low concentrations (i.e., 10^?12–10^?9 mol/l), PGF_1α and PGF_2α very intensely stimulated both the DNA-synthetic and mitotic activities of hepatocytes in 4-day-old primary cultures of neonatral rat liver. DNA replication was more intensely enhanced by PGF_2α than by PGF_1α, whereas mitotic activity was nearly equally affected by the two prostaglandins. On the whole, the growth-promoting activity of PGF_1α used by itself or in equimolar mixtures with other prostaglandins ($\text{e}$. $\text{g}$., A₁, E₁, $\text{etc}$.) mimicked that of arachidonic acid we previously reported (1). On a molar basis, PGF_2α by itself stimulated hepatocytes′ DNA synthesis is more powerfully than arachidonate did, and when used in equimolar mixtures with other prostaglandins was at least as potent as arachidonic acid. These observations establish prostaglandins of the F series as quite powerful commitment factors and, though by a lesser degree, also intracycle regulators for neonatal rat hepatocytes in primary culture. However, the understanding of the role(s) of prostaglandins of F and other series in the physiological control of hepatocytes′ proliferative activation must wait the clarification of their interaction(s) with other arachidonate derivative(s) and polypeptide growth factor(s) which also may be involved in the process.     

Stretch-induced growth of skeletal myotubes correlates with activation of the sodium pump

Skeletal myotubes responded to passive stretch by increased amino acid uptake (as measured with [³H]α-aminoisobutyric acid), increased incorporation of amino acids into total cellular protein and myosin heavy chains, and increased accumulation of total cellular protein and myosin heavy chains. These alterations were preceded by an increase in the uptake of ouabain-sensitive rubidium-86 (⁸⁶Rb⁺), a potassium tracer used to measure membrane sodium pump activity (Na⁺K⁺ATPase). This stretch-induced stimulation of ⁸⁶Rb⁺ uptake resulted from a 60-70% increase in the V_max of the Na pump with little change in the K_m. [³H] ouabain binding studies showed no stretch-induced change in the number of membrane Na pumps, indicating that stretch activates the Na pumps that are already present on the cell surface. Since the stretch-induced increases in amino acid transport and amino acid incorporation into proteins were inhibited by ouabain, Na pump activation may be involved in stretch-induced cell growth of skeletal muscle cells by hypertrophy.     

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 (⁴⁵ Ca 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, PGF_2α or prostaglandin precursors could be demonstrated. Bone resorption was stimulated after preculture by both PGE₂ and PGF_2α in a dose-dependent manner (10^?18M – 10^?5M), with PGE₂ being the more potent. Collagen synthesis was unaffected by PGF_2α, whereas PGE₂ (10^?5M) had an inhibitory effect. Eicosatrienoic acid did not stimulate bone resorption at lower concentrations (10^?7M – 10^?5M_, but was inhibitory at 10^?4M. Arachidonic acid also inhibited resorption at 10^?4M, but at lower concentrations (10^?7M – 10^?5M0 increased active resorption. This was concomitant with a rise in PGE₂ and PGF_2α levels, PGE₂ production being significantly higher than PGF_2α. The effects of PGE₂ (10^?8M) and PGF_2α (10^∞M appeared additive: there was no evidence of synergistic or antagonistic effects when varying ratios of PGE₂ : PGF_2α were employed.