Prostaglandin metabolism during circulatory shock

The rates of metabolic degradation and the patterns of metabolite formation of tritium-labeled prostaglandins E₂ and F_2α were assessed in vitro in tissues obtained from normal rabbits and from rabbits subjected to hemorrhagic or endotoxic shock. Normal rabbit tissues metabolized prostaglandin E₂ at the following rates: renal cortex 479 ± 34, liver 389 ± 95, and lung 881 ± 93 pmol of PGE₂ metabolized/mg soluble protein per min at 37°C (mean ± S.E.). Prostaglandin F_2α metabolism proceeded in normal animal tissues at rates of 477 ± 39, 324 ± 95, and 633 ± 69 pmol of PGF_2α metabolized/mg soluble protein per min for renal cortex, liver and lung, respectively. There were no significant differences between these rates of PGE₂ and PGF_2α metabolism when compared to rates in tissues obtained from animals subjected to either hemorrhagic or endotoxic shock. In addition, no significant differences were observed between the rate of PGE₂ metabolism and that of PGF_2α metabolism for any tissue. However, the lung was able to metabolize PGE₂ and PGF_2α significantly more rapidly than the liver, and to degrade PGE₂ at a significantly greater rate than the renal cortex. Although slightly different patterns of metabolite production were observed between lung and kidney homogenates, only the liver metabolized prostaglandins almost exclusively to more polar metabolites. While hemorrhagic or endotoxic shock induced slight changes in the patterns of PGE₂ metabolite formation in all three tissues studied, PGF_2α metabolite formation patterns were not significantly altered by circulatory shock. Thus, prostaglandin metabolism is not significantly impaired during the first 2 h of hemorrhagic or endotoxic shock in rabbit tissues. Therefore, impairment of prostaglandin metabolism is not the major factor responsible for the early increase in circulating prostaglandin concentrations in these forms of shock.     

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.     

Temporal effect of prostaglandins E and F on human adrenal cAMP and cortisol output.

The $\text{in vitro}$ modulating effects of the E and F series prostaglandins upon the cAMP and cortisol output of normal human adrenal dice were evaluated with time and compared to the effects of ACTH. PGE₁ and PGE₂ significantly increased human adrenal cAMP levels; cortisol output increased in a dose related manner. Although the cortisol levels produced by E prostaglandins and ACTH were quantitatively similar, on a molar basis ACTH was greater than 10⁶ fold more effective. PGE_1α, PGF_2α, PGF_1β and PGF_2β depressed adrenal cAMP, except PGF_2α and PGF_2β at 100 μg/ml. PGF_1α and PGF_2α depressed cortisol levels at all doses. Similarly, PGF_1β and PGF_2β also depressed cortisol output, except PGF_2β at 100 μg/ml which was stimulatory. In both series of prostaglandins the temporal responses were dose related in regard to the cyclic nucleotide and steroid alterations. The findings demonstrate the E and F series prostaglandins act antagonistically in respect to cAMP and cortisol output. In addition, as the cAMP level and cortisol output are not always correlated, it appears that these prostaglandin mediated events are separable. The relationship between adrenal prostaglandins and cyclic nucleotides, therefore, invites a more sophisticated second messenger concept in terms of adrenocortical function, than currently proposed.     

Relation between prostaglandin E2, F2α, and cyclic nucleotides levels in rat brain and induction of convilsions

Fully convulsant doses of pentamethylenetetrazole cause marked increase in rat brain cortical PGF_2α, PGE₂, cGMP and cAMP during seizures, whereas subconvulsant doses cause an increase of rat brain cortical PGF_2α without affecting the other biochemical parameters considered. Rat cerebellar prostaglandins were not modified by the convulsant agent at either dosage.     

Effect of prostaglandins on cyclic nucleotide levels in cultured keratinocytes

Guinea pig ear epidermal cells (keratinocytes) were established in primary cultures using trypsin, and treated in their proliferative phase of growth with prostaglandins E₁, D₁, F_1α, E₂, D₂, or F_2α. This phase is induced by the addition of retinoic acid during cell plating. Intracellular content of cAMP and cGMP was measured by radioimmunoassay at various times after treatment.Maximum stimulation of cAMP levels was observed with PGD₂, smaller increases with PGE₂ and relatively transient rises with PGF_2α which were of low significance, but confirm earlier data. Similar results were observed with PGD₁, PGE₁, and PGF_1α with smaller increases. The effects of D and E PGs were biphasic. Significant increases in cGMP were immediately observed with PGD₂ and PGE₂. With PGF_2α, maximum cGMP levels were noted after some delay.All PGs tested showed some effect in elevating cyclic nucleotides in keratinocytes. The most striking result was the increase in cAMP on PGD₂ treatment.     

A radioimmunoassay for 6-keto- prostaglandin F1α

A radioimmunoassay for 6-keto-prostaglandin F_1α has been developed. The assay is accurate and sensitive but since the antiserum cross-reacts 5–10% with prostaglandins (PGs) of the E and F series, solvent extraction and thin layer chromatography are required fo absolute specifity. The assay has been validated by comparison with a radiochemical assay and by the use of an inhibitor of 6-keto PGF_1α formation, 15-hydroperoxy arachidonic acid. 6-Keto PGF_1α was found to have a low cross reaction with antisera directed against PGE₂, PGF_2α and thromboxane B₂.     

Cerebral endothelial cell culture II. Adenylate cyclase response to prostaglandins and their interaction with the adrenergic system

The response of endothelial adenylate cyclase (AC) to prostaglandins (PGE₁, PGE₂, PGF_1α, PGF_2α, PGD₂ and PGI₂) and the relationship of PGE₂ to adrenergic systems were investigated in cerebrovascular endothelial cultures. E-type prostaglandins and PGI₂ were more effective in stimulating endothelial AC (EC₅₀ = 3 × 10^?7M, and 3 × 10^?7M, respectively) than prostaglandins of the F-series and PGD₂ which activated AC at high doses only. A modulation of endothelial AC response to either PGE₂ or norepinephrine (NE) was observed in the presence of both agents in the system. It was manifested by a dose-dependent NE inhibition of the PGE₂-stimulated formation of cAMP, which was partially restored by phentolamine. Alpha and β-adrenergic agonists (α, clonidine and 6-fluoronorepinephrine; β, isoproterenol) also partly blocked while forskolin and PGE₂ synergistically stimulated the production of cAMP in the endothelial cultures. These findings strongly suggest that the interaction of prostaglandins and α- and β-adrenergic agonists with the AC system in cerebrovascular endothelium may play a role in the regulation of the cerebral microcirculation and/or blood pressure.     

Elevations of intracellular cAMP result in a change in cell shape that resembles dome formation in cultured rat glomerular epithelial cells

Summary Cultured glomerular epithelial cells form a continuous monolayer of polyhedral-shaped cells. PGE₂ (1 μg/ml) in the presence of the phosphodiesterase inhibitor isobutylmethylxanthine (MIX) markedly raises intracellular and medium cyclic AMP (cAMP) levels at 20 min (intracellular: MIX alone, 112 ± 6.6 pmol cAMP/mg protein, MIX plus PGE₂, 2252±63 pmol cAMP/mg protein; medium: MIX, 20.6±2.1 pmol cAMP/mg protein; MIX plus PGE₂, 117±3.8 pmol cAMP/mg protein). By 2 h, when cellular and medium cAMP levels were still elevated, the cells underwent a change in shape that was similar to dome formation (15 to 20% of the monolayer changing shape). Derivatives of cAMP [i. e. dibutyryl and 8-(4-chlorophenylthio)-cAMP], when added to the incubation medium also caused shape change in glomerular epithelial cells at 2 h; cAMP itself did not. The formation of domes has been used as a morphological indicator of the vertorial transport of salt and water in other cultured epithelial cells. This work was supported by grant AM 29787 from the National Institutes of Health, Bethesda, MD.     

Analysis of A,B, E,and F prostaglandins as pentafluorobenzyl esters by electron-capture gas-liquid chromatography

A new and sensitive method is described for the simultaneous analysis of a mixture containing PGE₁, PGE₂, PGF_1α, and PGF_2α by electron-capture gas-liquid chromatography. During derivatization of the mixture, PGE₁ and PGE₂ were converted to PGB₁ and PGB₂, respectively, yielding a mixture of PGB₁, PGB₂, PGF_1α, and PGF_2α trimethylsilyl ether pentafluorobenzyl esters. Gas chromatographic resolution of all four derivatives is sufficient for quantitation of each prostaglandin. The A prostaglandins were analyzed by similar conversion to the respective B prostaglandin derivatives. Minimum detection limits for the B and F prostaglandin derivatives were 10 pg and 1 pg, respectively. Samples of rabbit kidney medulla were incubated and analyzed for A, B, E, and F prostaglandins. The results indicate that the method is capable of high recovery and reproducibility.     

Influence of angiotensins (I,II & III) on prostaglandin production by minced rat renal medulla

Minced rat renal medulla was incubated for 30 min at 37 °C in the presence of angiotensin, I, II or III (100 ng/ml) to determine the existence of a direct stimulating effect on prostaglandin (PG) production. PGE₂, PGF_2α, 6-keto PGF_1α and Thromboxane B₂ (TXB₂)_were determined by radioimmunoassay.For analysis of data variance, the results were separated according to whether the net output of PGE₂ was above or below 1.5 ng PGE₂ equivalent/mg tissue/30 min. Under low-output conditions, angiotensin I, II or III stimulated PGE₂ production significantly (p<0.02) and tended to augment PGF_2α production, while under high-output conditions no effect on PGE₂ or PGF_2α production was observed.Under either output condition, angiotensin I, II or III had no effect on 6-keto PGF_1α and TXB₂.     

Determination of prostaglandin F2α and E2 contents in human cerebrospinal fluid by the radioisotope dilution method

Prostaglandin F_2α and E₂ contents in human cerebrospinal fluid were determined by the radioisotope dilution method. The mean values of PGF_2α and PGE₂ of men were 9.8±0.87 ng/ml and 6.5±1.39 ng/ml, respectively. Those of women were 8.3±1.4 ng/ml and 6.9±1.72 ng/ml, respectively. The correlation between age and PG was significantly with PGE₂ of men and with PGF_2α of women.     

Cyclic AMP formation of isolated human corpora lutea in response to HCG — Interference by PGF2α

Human corpora lutea of defined ages were excise at operation cut into pieces and incubated in the presence of HCG, PGF_2α and PGE₂ alone or in combination. Following incubation cAMP formation in tissue and medium was determined. HCG-stimulated tissue cAMP content was most pronounced at a corpus luteum age of 7–10 days after ovulation. This stimulation was antagonized by PGF_2α in corpora lutea older than 6 days. PGE₂ stimulated cAMP formation $\text{per se}$ and this effect was more pronounced when HCG and PGE₂ were combined. A possible role for PGF_2α as a luteolytic substance in the human is suggested.     

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.     

A radioimmunoassay for 6-keto-prostaglandin F1α utilizing an antiserum against 6-methoxime-prostaglandin F1α: Application to study renal in vitro synthesis of prostacyclin

PGI₂ and 6-keto-PGF_1α were converted to 6-methoxime-PGF_1α (6-MeON-PGF_1α) by treatment with methoxyamine HCl in acetate buffer. The formed 6-MeON-PGF_1α was measured by radioimmunoassay. Antisera were raised in rabbits after immunization against 6-MeON-PGF_1α-BSA conjugate. Diluted 1:20.000 to bind 50% of the tracer (³H-6-MeON-PGF_1α, 100 Ci/mmol), the antiserum cross reacted 0.8% with PGE₂, 1% with PGF_2α and less than 0.2% with PGD₂, PGF_1α, PGF_2β and TXB₂. The radioimmunoassay was used to estimate release of PGI₂ and 6-keto-PGF_1α from chopped rabbit renal medulla and cortex incubated in Krebs-Ringer bicarbonate buffer (37°C, 30 min). The 6-keto-PGf_1α radioimmunoassay was validated in biological samples by mass fragmentography. The chopped medulla (n=5) released 38±9 ng/g/min and the cortex (n=5) 4.7±2.0 ng/g/min, while the release of immunoreactive PGE₂ (iPGE₂) and iPGF_2α was 171±26 and 74±13 ng/g/min from the medulla and 4.3±1.3 and 2.7±0.3 ng/g/min from the cortex, respectively. The results confirm previous findings, which indicate that in the renal medulla prostaglandin endoperoxides are mainly transformed to prostaglandins, while in the cortex transformation to PGI₂ seems to be of greater $\text{relative}$ importance.     

Prostaglandin F2α antagonizes thromboxane A2-induced human platelet aggregation

The effects of prostaglandin F_2α on human blood platelet function were investigated. PGF_2α at 15 μM completely blocked platelet aggregation induced by 500 μM arachidonic acid or 3 μM U46619 but had no effect on aggregatin induced by 7.5 μM ADP. A similar specificity of action was not obtained with either PGI₂ or PGE₂. Thus concentrations of PGI₂ (3 nM) or PGE₂ (20 μ M) which inhibited U46619-induced aggregation by 100% also blocked ADP-stimulated aggregation.The inhibitory properties of PGF_2α were not related to increases in platelet cAMP, since direct measurement of intracellular cAMP revealed that 15 μ M PGF_2α produced no substantial change in cAMP levels. This finding was in direct contrast to results obtained using either PGI₂ or PGE₂. Both PGI₂ (3 nM) and PGE₂ (20 μ M) induced significant increases in platelet cAMP levels.The possibility that PGF_2α directly interacts at the platelet TXA₂/PGH₂ receptor was investigated by measuring [³H]PGF_2α binding to isolated platelet membranes. It was found that [³H] PGF_2α binding reached equilibrium within 30 min at room temperature and could be 90% displaced by addition of 1000 fold excess of unlabelled PGF_2α. Furthermore, when 1000 fold excess of either the TXA₂/PGH₂ “mimetic” U46619 or the TXA₂/PGH₂ antagonist 13-azaprostanoic acid was added, specific [³H] PGF_2α binding was displaced by 95% and 85% respectively. In contrast, the same molar excess of 6-keto-PGF_1α, azo analog 1, or TXB₂, caused displacement of only 15%, 20% or 25% of the [³H] PGF_2α binding. Scatchard analysis indicated that [³H] PGF_2α has two binding sites; i.e., a high affinity binding site with an apparent Kd of 50 nM and a low affinity binding site with apparent Kd of 320 nM. These results suggest that the selective inhibition by PGF_2α of AA or U46619-induced aggregation may be mediated through interaction at the platelet TXA₂/PGH₂ receptor.     

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.     

High performance liquid chromatography of prostaglandins: Biological applications

Two procedures are described for separation and purification of prostaglandins by high performance liquid chromatography. Both systems show excellent resolution of PGA₂, PGE₂ and PGF_2α. Peak definition on the micro-particle silicic acid system is particularly good with the PGs appearing in 2–3 ml of organic effluent. Studies on reproducibility showed that PGE₂ and PGF_2α could be recovered with a retention volume of 54.2±0.76 ml and 64±0.6 ml, respectively (n=7, mean ±SD) with good recovery. The column can be run in about one hour and can be regenerated indefinitely (>200 times). The degree of purification is compatible with analysis by gas chromatography-mass spectrometry. Examples showing the application of this chromatographic method to human seminal fluid, human renal tissue, platelet rich plasma and human urine samples indicate that it makes possible analysis of these samples even at low levels.     

Measurement of Prostaglandins in the Cerebrospinal Fluid in Cat, Dog, and Man

Prostaglandins are involved in the modulation of various central functions (neurotransmitters and hypothalamic hormone release, thermoregulation, cerebro-vascular tone) and their levels increase in pathological situations [subarachnoid hemorrhage (SAH), stroke, convulsive disorders, etc.]. This study, using sensitive and specific antibodies, examined levels of four eicosanoids, Prostaglandins E₂ and F_2α(PGE₂, PGF_2α); and the metabolites of PGI₂, 6-keto-prostaglandin F_1α (6-keto-PGF1_α) and of thromboxane A₂, thromboxane B₂ (TxB₂), in the cerebrospinal fluid (CSF) obtained atraumatically from three species (human, canine, and feline). An assessment of the methodologic procedures (extraction and radioimmunoassay) was carried out. Human lumbar cerebrospinal fluid was shown to contain PGF_2α (15–44 pg/ml), 6-keto-PGF_1α (undetectable to 39 pg/ml), and TxB₂ (un-detectable to 28 pg/ml), whereas PGE₂ was undetectable (>18 pg) in all cases. In both animals species the eico-sanoid concentrations were 3-to 30-fold higher than humans for every prostaglandin examined. Although the prostaglandin profile for a given species remained constant (cat, PGE₂:6-keto-PGF_1α:TxB₂:PGF_2α; dog, TxB₂:PGE₂:6-keto-PGF_1α:PGF_2α), the absolute levels were found to be lower in the pentobarbital-anesthetized animals than in conscious cats. The correspondence of the prostaglandin profiles found in cerebrospinal fluid with the profiles reported in the literature in brain homogenates for the same species supports the hypothesis that cerebrospinal fluid levels of prostaglandins reflect the relative rates of synthesis in neural tissue.     

Tiludronate inhibits interleukin-6 synthesis in osteoblasts: Inhibition of phospholipase D activation in MC3T3-E1 cells

In previous studies, we have reported that PGF_2α stimulates phosphoinositide hydrolysis by phospholipase C and phosphatidylcholine hydrolysis by phospholipase D through heterotrimeric GTP-binding protein in osteoblast-like MC3T3-E1 cells, and that PGF_2α and PGE₁ induce interleukin-6 (IL-6) synthesis via activation of protein kinase C and protein kinase A, respectively. In the present study, we investigated the effect of tiludronate, a bisphosphonate known to inhibit bone resorption, on the PGF_2α- and PGE₁-induced IL-6 synthesis in these cells. Tiludronate significantly suppressed the PGF_2α-induced IL-6 secretion in a dose-dependent manner in the range between 0.1 and 30 μM. However, the IL-6 secretion induced by PGE₁ or (Bu)₂cAMP was hardly affected by tiludronate. The choline formation induced by PGF_2α was reduced by tiludronate dose-dependently in the range between 0.1 and 30 μM. On the contrary, tiludronate had no effect on PGF_2α-induced formation of inositol phosphates. Tiludronate suppressed the choline formation induced by NaF, known as an activator of heterotrimeric GTP-binding protein. However, tiludronate had little effect on the formation of choline induced by TPA, a protein kinase C activator. Tiludronate significantly inhibited the NaF-induced IL-6 secretion in human osteoblastic osteosarcoma Saos-2 cells. These results strongly suggest that tiludronate inhibits PGF_2α-induced IL-6 synthesis via suppression of phosphatidylcholine-hydrolyzing phospholipase D activation in osteoblasts, and that the inhibitory effect is exerted at the point between heterotrimeric GTP-binding protein and phospholipase D. J. Cell. Biochem. 69:252–259, 1998. © 1998 Wiley-Liss, Inc.