Endogenous prostaglandin production by established cultures of neoplastic rat mammary epithelial cells

The production and release of prostaglandins (PGs) into the growth medium by established cultures of neoplastic, mammary epithelial cells derived from (a) N-nitrosomethylurea (NMU)-induced and (b) 7,12-dimethylbenz(a)anthracene (DMBA)-induced mammary tumors, was assessed using radioimmunoassay techniques. Prostaglandin production was determined, to a considerable extent, by in vitro conditions and the tumor line analyzed. In medium supplemented with bovine calf serum (10%), NMU cells synthesized and released nanogram quantities of PGE2, PGE1, and PGF2 alpha (6.7, 4.7, and 1.7 ng/10(6) cells per 48 h, respectively). Concentrations of the two stable protanoid metabolites, 6-keto-PGF1 alpha and TXB2, were indistinguishable from controls. In cells derived from the DMBA-induced tumor (RBA cells), no net production of immunoreactive PGs was detected. In contrast, in media supplemented with fetal bovine serum (10%), both RBA and NMU cells synthesized and released nanogram quantities of PGE2 (1 and 4 ng/10(6) cells per 48 h, respectively). PGE2 production by both NMU and RBA cells was inhibited by ibuprofen, an inhibitor of cyclooxygenase (EC 1.14.99.1). The pattern of PG inhibition by ibuprofen differed in the two cell lines. In NMU cells, a linear dose-response inhibitory pattern was discernable, whereas in RBA cells a biphasic pattern was observed; PGE2 levels increased at low concentrations of ibuprofen and then decreased at higher concentrations. At 100 micrograms/ml ibuprofen, PG synthesis and release was inhibited by 90 and 100% and cell growth by 64 and 66% in NMU and RBA cells, respectively. There was no obvious dose-response relationship between ibuprofen concentration and cell growth inhibition in either cell line. These results underline the importance of the serum component of growth medium when analyzing PG production in vitro and suggest that the epithelial cell components of experimental mammary tumors are capable of producing physiologically relevant amounts of PGS.     

Effect of prolonged prostaglandin exposure on prostaglandin synthesis by human lung fibroblasts

Pretreatment of human lung fibroblasts with PGE₂ but not PGF_2α enhanced synthesis of prostaglandins (PGs). The effect of the pretreatment on PG synthesis was related to the concentration of PGE₂ that was added to the culture medium. Pretreatment with PGE₂ at 5 × 10⁻¹²M did not enhance PG synthesis whereas pretreatment with PGE₂ at 5 × 10⁻⁶M induced a maximal effect. Production of PGs was increased following 1 day of pretreatment with PGE₂ and was increased further following 3 days of pretreatment. The PGE₂ treated cells showed only a slight increase in the bradykinin-induced release of radioactivity from cells prelabeled with [³H]arachidonic acid but showed a dramatic increase in the bradykinin-induced synthesis of radio-labeled PGs. The conversion of free arachidonate to PGs in both intact cells and in a cell-free preparation was increased by PGE₂ pretreatment. The presence of cyclohexamide during the pretreatment did not inhibit the PGE₂-induced activation of PG synthesis. Taken together, the results indicate that pretreatment of cells with PGE₂ increased PG synthesis by augmenting the conversion of arachidonate to PGs.     

3H] prostaglandins binding to dispersed bovine luteal cells: evidence for discrete prostaglandin receptors.

Suspensions of dispersed bovine luteal cells prepared by collagenase digestion of luteal tissue specifically bound [³H]Prostaglandin (PG) E₁ and [³H]PGF_2α. While the number of sites per cell (～ 1.8 × 10⁵) were about the same for both [³H]PGs, the apparent Kds were different: [³H]PGE₁ ? 2.4 nM; [³H]PGF_2α ? 11 nM. The [³H]PGs binding was inhibited in a dose-dependent manner in the presence of increasing concentrations of unlabeled PGs. Potency order for inhibition of [³H]PGE₁ binding was: PGE₂ > PGE₁ > PGF_2α > PGF_1α. The corresponding data for [³H]PGF_2α was: PGF_2α > PGF_1α > PGE₂ > PGE₁. While [³H]PGE₁ and [³H]PGF_2α bind to their own receptors with high affinity, their affinities for each other's binding were extremely low. Thus, these results demonstrate that luteal cells, like plasma membranes isolated from luteal tissue, contain receptors for PGEs and PGF_2α which are discrete with respect to specificity and affinity.     

Radioimmunoassay measurement of ACTH-facilitated PGE2 and PGF2α release from isolated cat adrenocortical cells

Prostaglandin (PG) biosynthesis by trypsin-dispersed cat adrenocortical cells was studied by radioimmunoassay (RIA). Parallel assays of incubation media using PGF_2α and PGF_1α antisera established that PGF_2α is the primary PGF released by feline cortical cells. Following the reduction of PGE to PGF with sodium borohydride (NaBH₄) these same two antisera were also used to identify PGE₂ as the primary PGE released. RIA using a PGE antiserum confirmed the presence of PGE in the incubation medium. Steroidogenic concentrations of ACTH (50–250μU) enhanced PGE and PGF release, and indomethacin suppressed the ACTH-facilitated release. These studies provide additional evidence for ACTH-induced PG synthesis by feline cortical cells, and support the hypothesis that PGs play some role in the steroidogenic action of ACTH.     

Patterns of prostaglandin synthesis and degradation in isolated superficial and proliferative colonic epithelial cells compared to residual colon

Prostaglandin (PG) synthesis and degradation were examined in different regions (epithelial versus non-epithelial structures) of the rat distal colon by both HPLC analysis of [¹⁴C] arachidonate (AA) metabolites and by specific radioimmunoassays. Intact isolated colonic epithelial cells synthesized mainly PGF₂α and TXA₂, as monitored from the formation of its stable degradation product TXB₂ (PGF_2α > TXB₂ > 6-keto-PGF_1α, the stable degradation product of PGI₂=PGD₂=PGE₂=13,14-dihydro-15-keto-PGF_2α). The profile of PG products of isolated surface epithelial cells was identical to that of proliferative epithelial cells. However, generation of PGs by surface epithelium was 2 to 3-fold higher than by proliferative cells both basally and in the presence of a maximal stimulating concentration (0.1 mM) of AA. The latter implied a greater synthetic capacity of surface epithelium, rather than differences due to endogenous AA availability. The major sites of PG synthesis in colon clearly resided in submucosal structures; the residual colon devoid of epithelial cells accounted for at least 99% of the total PGs produced by intact distal colon. The profile of AA metabolites formed by submucosal structures also differed markedly from that of the epithelium. The dominant submucosal product was PGE₂. PGE₂ and its degradation product 13,14-dihydro-15-keto-PGE₂ accounted for 63% of the PG products formed by submucosal structures (PGE₂ PGD₂ > 13,14-dihydro-15-keto-PGE₂ > PGF_2α=TXB₂=6-keto-PGF_1α > 13,14-dihydro- 15-keto-PGF_2α). By contrast, epithelial cells, and particularly surface epithelium, contributed disproportionately to the PG degradative capacity of colon, as assessed from the metabolism of either PGE₂ or PGF_2α. When expressed as a percentage, epithelial cells accounted for 71% of total colonic PGE₂ degradative capacity but only 23% of total colonic protein. Approximately 15% of [³H] PGE₂ added to the serosal side of everted colonic loops crossed to the mucosal side intact. Thus, at least a portion of the PGE₂ formed in the submucosa reaches, and thereby can potentially influence functions of the epithelium.     

Cytokine regulation of adult human osteoblast-like cell prostaglandin biosynthesis

Prostaglandin (PG) biosynthesis by cytokine stimulated normal adult human osteoblast-like (hOB) cells was evaluated by thin layer chromatography, high performance liquid chromatography, and specific immunoassays. PGE₂ was the predominant PG formed under all incubation conditions tested. Control samples produced measurable amounts of PGE₂, and the measured level of this metabolite increased by 22-fold (from 7 to 152 ng/ml) following a 20 h treatment with the combination of TGFβ and tumor necrosis factor-α(TNF). The production of 6-keto-PGF_1α (the stable metabolite of prostacyclin) and of PGF_2α were each increased by about five-fold (from about 0.5 to 2.5 ng/ml) in samples treated with the cytokines. Thus, TGFβ and TNF exerted a regulation of hOB cell PG biosynthesis that was principally directed towards an increased PGE₂ biosynthesis, with lesser effects on the production of other PG metabolites. COX-2 mRNA levels were increased within 2 h of cytokine stimulation, reached a maximum at 6–12 h, and levels had appreciably diminished by 24 h after treatment. Both TGFβ and TNF could independently increase COX-2 mRNA levels and PG biosynthesis. However, the increased production of PGE₂ resulting from TNF stimulation was blocked by the addition of an interleukin-1β (IL-1β) neutralizing antibody, suggesting that TNF regulation of hOB cell PG synthesis was secondary to its capacity to increase hOB cell IL-1β production. TGFβ regulation of PG production was not affected by the addition of the neutralizing antibody. These studies support the proposition that PGs can be important autocrine/paracrine mediators of bone biology, whose production by hOB cells is responsively regulated by osteotropic cytokines. J. Cell. Biochem. 64:618–631. © 1997 Wiley-Liss, Inc.     

Prostaglandin-induced enhancement of the anamnestic response

The ability of various prostaglandins (PGs) to affect the anamnestic immune response of keyhole limpet hemocyanin (KLH)-primed rabbit popliteal lymph node cells was investigated. Of the four PGs studied (PGA₁, PGE₂ and PGF_2α), PGE₁ was found to have a stimulatory effect, whereas PGA₁, PGE₂ and PGF_2α were ineffective in stimulating or inhibiting the anamnestic response. Under the conditions studied, a 3.5-fold increase in antibody production was obtained in PGE₁-treated, KLH-stimulated cultures. Maximum enhancement was obtained when 0.2 μg of PGE₁ were added at the time of culture initiation and were allowed to remain in contact with the lymph node cells for 24 hours.     

Interrelationships among prostaglandins, vasopressin and cAMP in renal papillary collecting tubile cells in culture

To determine the influence of prostaglandins on cAMP metabolism in renal papillary collecting tubule (RPCT) cells, intracellular cAMP levels were measured after incubating cells with prostaglandins (PGs) alone or in combination with arginine vasopressin (AVP). PGE₁, PGE₂ and PGI₂, but not PGD₂ or PGF_2α, increased intracellular cAMP concentrations. At maximal concentrations (10⁻⁵ tthe effects of PGE₂ plus PGI₂ (or PGE₁), but not of PGI₂ plus PGE₁, were additive suggesting that at least two different PG receptors may be present in RPCT cell populations. Bradykinin treatment of RPCT cells caused an accumulation of intracellular cAMP which was blocked by aspirin and was quantitatively similar to that observed with 10⁻⁵ PGE₂. PGs, when tested at concentrations (e.g. 10⁻⁹ ) which had no independent effect on intracellular cAMP levels, did not inhibit the AVP-induced accumulation of intracellular cAMP in RPCT cells. These results indicate that PGs do not block AVP-induced accumulation of intracellular cAMP in RPCT cells at concentrations of PGs which have been shown to inhibit the hydroosmatic effect of AVP on perfused collecting tubule segments. However, at higher concentrations of PGs (e.g. 10⁻⁵ ), the effects of AVP plus PGE₁, PGE₂, PGI₂ or bradykinin on intracellular cAMP levels were not additive. Thus, under certain conditions, there is an interaction between PGs and AVP at the level of cAMP metabolism in RPCT cells.     

Catechol oestrogens stimulate and direct prostaglandin synthesis

We have tested the action of a catechol oestrogen -2,3,17β- trihydroxy oestra-1,3,5 (10)-triene (2-OH oestradiol) in stimulating prostaglandin (PG) production by an homogenate of rat uterus. Marked and dose dependent stimulation was observed in PGF_2α and PGE₂ production using 20–250 μM concentrations of catechol oestrogen; a concentration of 250 μM 2-OH oestradiol resulted in a 23 fold increase in PGF_2α production with a 50% reduction in the synthesis of 6-keto PGF_1α. Tryptophan, catechol and glutathione were without effect on PGF_2α and PGE₂ production whereas adrenalin stimulated the production of all PGs, although the increase was less than that seen with 2-OH oestradiol. Oestradiol had a slight stimulatory action on PGF_2α production which reached a maximum at around 40 μM but had a more marked stimulation of 6-keto PGF_1α formation. Stimulation of prostaglandin production by oestradiol and 2-OH oestradiol showed no variation at different stages of the rat oestrous cycle. The use of 5 to 100 mg of tissue/ml gave similar product distribution although the effect of catechol oestrogen both in terms of stimulation of E and F formation (expressed per mg of tissue) and in its action on product distribution was more marked at lower concentrations of tissue.     

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.     

Pharmacological modulation of prostaglandin production by phagocytic cells of the thymic reticulum in relation to immunoregulation

We cultured phagocytic cells derived from the thymic reticulum in order to study the regulation of prostaglandin (PG) production by antiinflammatory or immunostimulating agents. The kinetics of PGE₂, 6-keto-PGF_1α and PGF_2α production were measured by specific radioimmunoassays of the supernatants harvested from cells treated with dexamethasone, a steroidal antiinflammatory drug and by two non steroidal inhibitors (indomethacin and sulindac) or by various immunostimulating agents, one of them, RU 41740 is currently being used in humans. Our results revealed that ech of these drugs exerts a differential effect on the PG production, with a striking action on PGE₂ synthesis, a lesser effect on 6-keto-PGF_1α production and almost no effect on PGF_2α synthesis. The possible mechanisms responsible for this complex regulation of PG production are discussed.     

Prostaglandin-induced enhancement of the in vitro anamnestic response

The ability of various prostaglandins (PGs) to affect the $\text{in vitro}$ anamnestic immune response of keyhole limpet hemocyanin (KLH)-primed rabbit popliteal lymph node cells was investigated. Of the four PGs studied (PGA₁, PGE₂ and PGF_2α), PGE₁ was found to have a stimulatory effect, whereas PGA₁, PGE₂ and PGF_2α were ineffective in stimulating or inhibiting the $\text{in vitro}$ anamnestic response. Under the conditions studied, a 3.5-fold increase in antibody production was obtained in PGE₁-treated, KLH-stimulated cultures. Maximum enhancement was obtained when 0.2 μg of PGE₁ were added at the time of culture initiation and were allowed to remain in contact with the lymph node cells for 24 hours.     

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.     

Prostaglandin-induced enhancement of the in vitro anamenstic response

The ability of various prostaglandins (PGs) to affect the in vitro anamnestic immune response of keyhole limpet hemocyanin (KLH)-primed rabbit popliteal lymph node cells was investigated. Of the four PGs studied (PGA₁, PGE₂ and PGF_2α), PGE₁ was found to have a stimulatory effect, whereas PGA₁, PGE₂ and PGF_2α were ineffective in stimulating or inhibiting the in vitro anamnestic response. Under the conditions studied, a 3.5-fold increase in antibody production was obtained in PGE₁-treated, KLH-stimulated cultures. Maximum enhancement was obtained when 0.2 μg of PGE₁ were added at the time of culture initiation and were allowed to remain in contact with the lymph node cells for 24 hours.     

Prostaglandin actions in established insect cell lines

Prostaglandins (PGs) are oxygenated metabolites of arachidonic acid (AA) and two other C20 polyunsaturated fatty acids that serve as biochemical signals mediating physiological functions. We reported that PGs influence protein expression in insect cell lines, which prompted the question: do PGs influence cell proliferation or viability in insect cell lines? Here, we report on the outcomes of experiments designed to address the question in cell lines from three insect orders: Hemiptera (squash bug, Anasa tristis, BCIRL-AtE-CLG15A), Coleoptera (red flour beetle, Tribolium castaneum, BCIRL-TcA-CLG1), and Lepidoptera (tobacco budworm, Heliothis virescens, BCIRL-HvAM1). Treating the insect cell lines with PGA₁, PGA₂, or PGD₂ led to dose-dependent reductions in cell numbers. All three cell lines were sensitive to PGA₁ and PGA₂ (IC₅₀s = 9.9 to 26.9 μM) and were less sensitive to PGD₂ (IC₅₀s = 31.6 to 104.7 μM). PG treatments also led to cell death at higher concentrations, as seen in mammalian cell lines. PGE₁, PGE₂, and PGF_2α treatments did not influence AtE-CLG15A or HvAM1 cell numbers at lower concentrations, but led to dose-related reductions in TcA-CLG1 cells at higher concentrations. Similar treatments with pharmaceutical inhibitors of PG biosynthesis also led to reduced cell numbers: MAFP (inhibits phospholipase A₂), indomethacin (inhibits PG biosynthesis), and esculetin (inhibits lipoxygenase). Because these pharmaceuticals are used to relieve inflammation and other medical issues in human medicine, they are not toxic to animal cells. We infer PGs are necessary in optimal quantities for ongoing homeostatic functions in established cell lines; in quantities outside the optimal concentrations, PGs are deleterious.     

Elevation of rhesus monkey plasma luteinizing hormone levels in response to E series prostaglandins

Experiments were carried out to assess the influence of prostaglandins (viz. PGE₁, PGE₂ and PGF_2α) on plasma concentrations of FSH and LH in the female rhesus monkey. Monkeys were ovariectomized and treated with estradiol benzoate to suppress endogenous gonadotropin levels prior to these experiments. Femoral venous blood was taken at intervals following a single carotid arterial injection of the PG in anesthetized monkeys. FSH and LH concentrations, determined by radioimmunoassay, were not significantly altered in 4 control animals receiving saline (2) or ethanol-saline (2), the vehicles for PGF_2α and for the E series PGs, respectively. PGE₁ (5mg) effected dramatic elevations of LH within 5 min in 3 animals and the high plasma concentrations were maintained at least for 60 min. Similarly, 5.0 mg of PGE₂ effected rapid elevation of LH concentrations, from 2- to 7-fold pre-injection levels in 3 animals. In contrast, FSH levels were not so markedly altered by PGE₁ and PGE₂, but in general, appeared to be somewhat decreased by these treatments. PGF_2α had no effect on plasma FSH and LH concentrations. These data demonstrate the ability of PGs of the E series to elevate plasma LH concentrations in the rhesus monkey and support studies in other species suggesting a modulating role for PGs on gonadotropin secretion or release.     

Synthesis of prostaglandins by pig blastocysts cultured in medium containing estradiol or catechol estrogen

Two experiments were conducted to determine the effects of 2-hydroxy-estradiol-17β (2---OH---E₂; 0, 50 and 100 μM) and estradiol-17β (E₂; 0, 25 and 50 μM) on prostaglandin (PG) E and PGF₂α synthesis by day-10 pig blastocysts (day 0 is first day of estrus). Blastocysts were incubated in a modified Krebs-Ringer bicarbonate medium, supplemented with bovine serum albumin (4 mg/ml) and the vitamins and amino acids (essential and nonessential) in Minimum Essential Medium (without phenol red or antibiotics). The incubations were conducted at 39°C for three 2-h periods; the second and third periods included an E₂ or catechol estrogen treatment. Release of PGF₂α into the culture medium decreased (p<0.001) linearly with increasing concentrations of 2---H---E₂ in both periods. Release of PGE was not affected by 2---OH---E₂, therefore 2---OH---E₂ increased (p<0.06) the PGE:PGF₂α. When E₂ was added to the medium, release of PGE was decreased (p<0.01) during the second and third periods. Release of PGF₂α also was decreased (p<0.05) by E₂ during period 2, but E₂ did not alter the PGE:PGF₂α. Content of PGs in blastocysts at recovery was less than 10% of the PGs released in vitro. Therefore, these studies demonstrate effects of both the primary and catechol forms of E₂ on the synthesis of PGE and PGF₂α. Catechol estrogens and E₂ may inhibit PG synthesis and modify the PGE:PGF₂α during the establishment of pregnancy in pigs.     

Effect of prostaglandins F2α and E2 on milk ejection, blood pressure and blood flow through the mammary artery in the cow

The influence of prostaglandins (PG) F₂α and E₂ on milk ejection, mammary artery blood flow and arterial blood pressure was studied in lactating cows. Injections of both PG in the jugular vein or the carotid artery induced milk ejection after a relatively long latency period. The minimal effective dose amounted to 1 to 5 μg and to 100 to 300 μg for PGF₂α and PGE₂ respectively. In several cases with PGF₂α and once with PGE₂ milk ejection was accompanied with a simultaneous increase in blood flow through the mammary artery whereas arterial blood pressure remained unchanged. Both routes of administration showed the same response. It was suggested that the effect of the PG on the bovine myoepithelium is indirect, possibly secondary to a release of oxytocin from the neurohypophysis.     

Primary colonic epithelial cell culture of the rabbit producing prostaglandins

We have established primary colonic epithelial cell culture from adult rabbits and examined effects of anti-inflammatory drugs on prostaglandin (PG) E₂ production. Colonic epithelium of adult rabbits was scraped and minced into small pieces. They were incubated for isolation in Hanks' balanced salt solution with 0.35 % collagenase and Earle's solution with 1 mM EDTA. Isolated cells were cultured in Coon's modified Ham's F-12 medium with 10 % fetal bovine serum and antibiotics on collagen coated cell wells. The medium was refed twice a week. The production of PGs was assessed by high pressure liquid chromatography (HPLC). PGE₂ and PGF_2α were measured by radioimmunoassay. Within 24 hours after inoculation, the cell clumps attached to the surface of the wells and cells began to spread out and grow. Monolayer cultures became confluent in 4 days. Phase contrast microscopy showed that these cells consisted of a homogeneous population of epithelial cells with large oval nuclei, polyhedral shape, and organized sheet-like growth pattern. HPLC profile showed synthesis of 6-keto-PGF_1α, thromboxane B₂, PGF_2α, PGE₂, and PGD₂ by cultured cells. Quantitatively, 117±7 ng/mg-protein/hour PGE₂ by 7.4±0.7 ng/mg-protein/hour PGF_2α were produced. While hydrocortisone (10⁻⁴-10⁻² M) did not show a significant effect on PGE₂ production, indomethacin (10⁻⁸-10⁻⁶ M), and 5-aminosalicylic acid (2×10⁻⁴-5×10^{−3 M}) inhibited PGE₂ production. We have established relatively convenient procedure for primary culture of colonic epithelial cells from adult rabbits. Different actions of anti-inflammatory drugs on PGE₂ synthesis suggest that these cultured cells might be a good tool for the various cellular functional studies of normal colonic epithelial cells.     

Restoration by Prostaglandins E2 and F2 α of Resveratrol-Induced Suppression of Hepatoma Cell Invasion in Culture

In our previous study, resveratrol, a polyphenolic compound in grapes with antioxidative property, and resveratol-loaded rat serum (RS) were found to suppress the invasion of AH109A cells, an ascite hepatoma cell line. The aim of the present study was to investigate whether and which prostaglandins (PGs) would be involved in the invasion of AH109A cells and its suppression by resveratrol and resveratrol-loaded RS, using an in vitro invasion assay system. Not only PGE₂ but also PGF_{2 α} stimulated the spontaneous invasion of AH109A cells.They also canceled the resveratrol-induced suppression of hepatoma cell invasion. Results obtained suggest an involvement of PGs, especially PGE₂, in the invasion of hepatoma cells.