Metabolism and action of the prostaglandin endoperoxide PGH2 in rat kidney

Kidney membrane fractions metabolized [1-¹⁴C]PGH₂ to TXB₂, PGE₂, PGF_2α, PGD₂, 6-keto PGF_1α, and HHT. TXA₂, as measured by TXB₂, was enzymatically formed in cortex microsomes and was identified by thin layer chromatography and gas chromatography - mass spectrometry. PGH₂ caused a labile inhibition of cortical PGE₂-stimulated adenylate cyclase. PGE₂, PGF_2α, and PGD₂ are stimulators of cortical adenylate cyclase. The inability of two thromboxane synthetase inhibitors, imidazole and 9,11-azoprosta-5,13 dienoic acid, to block PGH₂ inhibition suggested that TXA₂ was not an obligatory intermediate in this process. Therefore, a potential function of cortical PGH₂ is inhibition of adenylate cyclase.     

Prostaglandins and myogenic control of tension in lower esophageal sphincter in vitro

Active tension is produced by the lower esophageal sphincter (LES) of North American opossum $\text{in vitro}$ by a myogenic mechanism. Strips of LES, but not those from the esophageal body, contracted to prostaglandin (PG)F_2α, stable expoxymethano derivatives of PGH₂ and to thromboxane B₂. Stable endoperoxides were more than 500 times more potent than PGF_2α. PGI₂ and 6-keto PGF_1α were weak relaxants of LES strips. LES strips transformed arachidonic acid into contractile substances. This transformation was prevented by agents which interfere with PG synthesis by inhibiting cyclo-oxygenase [indomethacin (IDM), 5,8,11,14-eicosatetraynoic acid (ETA) or thromboxane synthetase [imidazole]. Tranylcypromine 500 μg/ml also inhibited contractions to arachidonic acid. These agents also reduced muscle tone, so that endogenous PG formation may contribute to active tension in the LES. ETA and IDM increased tone before inhibiting it, and this effect was prevented by prior treatment with ETA or imidazole. There may also be an endogenous PG which inhibits LES tone. The possibility that this may be PGI₂ is discussed.     

PGE2 and PGF2α biosynthesis in stimulated and nonstimulated peritoneal preparations containing macrophages

The biosynthesis of PGE₂ and PGF_2α was measured in intact peritoneal exudate preparations obtained fom $\text{C. parvum}$-treated and control C₃H mice. Although both the control and stimulated preparations biosynthesized PGF_2α and PGE₂ from [1–14C] arachidonic acid, the stimulated preparations generated more of both prostaglandins than did nonstimulated preparations, probably as a result of increased synthesis within macrophages. Increased transformation of PGE₂ into PGF_2α 9-ketoreductase was noted in stimulated preparations when compared to that in control cells. The data suggest that stimulated macrophages are capable of generating increased quantities of PGF_2α and therefore might function as one source of this substance in resolving inflammatory reactions.     

The in vitro production of prostanoids by cultured bovine articular chondrocytes

Bovine articular chondrocytes, cultured as cell suspensions and monolayers, produced prostaglandin (PG) E₂ and PGI₂ (assayed as 6 keto PGF₁α), rather less PGF₂α and irregular quantities of thromboxane (Tx) B₂. Addition of foetal calf serum to the medium greatly stimulated PG production (a sixfold increase in PGE₂ and a twofold increase in 6 keto PGF₁α).Prostanoid production by cell suspension grown in serum-free medium generally plateaued after 24 hours. In the presence of 20% foetal calf serum, prostanoid production in long-term monolayer cultures increased during the first 6 days of culture. Levels of PGE₂α levels remained high. Indomethacin (10-⁶M) inhibited chondrocyte PG production both in the presence and absence of added arachidonic acid (10-⁴M). Prostanoids produced by chondrocytes may play a role in the modulation of cartilage metabolism $\text{in vivo}$.     

Prostaglandin endoperoxides IV. Effects on smooth muscle

The effect on smooth muscle of the endoperoxides PGG₂ and PGH₂, which are intermediates in prostaglandin biosynthesis, was studied in different systems $\text{in vitro}$ and $\text{in vivo}$. On gastrointestinal smooth muscle (gerbil colon, rat stomach) PGG₂ and PGH₂ produced contractions comparable to those of PGE₂ and PGF_2a whereas contractions elicited on vascular (rabbit aorta) and airway (guinea-pig trachea) smooth muscle were considerably greater than those of PGE₂ and PGF_2a respectively. On intravenous injection into guinea-pigs PGG₂ and PGH₂ caused a triphasic change in blood pressure and were 8–10 times more effective than PGF_2a in producing an increase in tracheal insufflation pressure. When given as aerosols the unstable endoperoxides were less effective than PGF_2a. It is concluded that the endoperoxides are potent smooth muscle stimulants and that they are more effective than their degradation products (PGD₂, PGE₂, PGF_2a) in some systems.     

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.     

Mechanism of the inhibition of platelet aggregation produced by prostaglandin F2α

The inhibition of human platelet aggregation produced by PGF_2α is not specific for thromboxane A₂ mimetics. Aggregation waves induced by PAF and thrombin are also inhibited by PGF_2α (8 μM); ADP is unaffected. These effects are still seen in platelets from aspirin-treated donors and platelets desensitized to thromboxane-like agonists (e.g. 11,9-epoxymethano PGH₂). In contrast the thromboxane receptor antagonist EP 045 (up to 20 μM) had no effect on primary aggregation induced by PAF, thrombin and ADP. We have previously shown that EP 045 (IC₅₀ = 0.5 μM), displaces the specific binding of [³H] 9,11-epoxymethano PGH₂ to washed human platelets.PGF_2α produces small increases in cAMP levels, and both this effect and the anti-aggregation are diminished by the adenyl cyclase inhibitor SQ 22536. The rise in cAMP induced by PGF_2α is inhibited to a greater extent by the presence of ADP than by thrombin, PAF or a thromboxane mimetic. The ability of aggregating agents to inhibit this increase correlates inversely with their sensitivity to inhibition by PGF_2α.We suggest that the very weak effect of PGF_2α on cyclic AMP_ production is sufficient to account for its inhibitory activity, and it is unlikely to be a competitive antagonist at the platelet thromboxane receptor as suggested by others.     

Effect of oestradiol 17 β on prostaglandin biosynthesis by the uterus of ovariectomized rat and guinea pig

$\text{In vitro}$ prostaglandin biosynthesis by uteri of ovariectomized rats and guinea pigs treated or untreated with oestradiol 17 β, administered subsutaneously, was measured by R.I.A. of PGF_2α and PGE₂. Incubations with [1-¹⁴C] arachidonic acid were also performed and labelled metabolites were analyzed by TLC. The main metabolite in rats was 6 keto PGF_1α and in decreasing order of magniture, PGF_2α and PGE₂. In guinea pig PGF_2ga was the main product. Ovariectomy in rats completely changed the pattern of synthesized prostanoids: PGI₂ production was doubled when compared to cycling rats and PGE₂ increased 10 fold. PGF_2α walues were similar to the mean value measured during the cycle. OE₂ treatment almost completely inhibited PGI₂ synthesis and reduced PGE₂ by half. Total PG synthesis in OE₂ treated animals was decreased by 5 fold when compared to spayed rats. Endogenous PGF_sα synthesis was slightly stimulated. In the guinea pig OE₂ treatment of ovariectomized animals increased the total synthesis from 50 per cent. PGF_2α was always the main metabolite. In conclusion OE₂ regulation of uterine PG synthesis is depending on the animal species and cannot be explained by a unique effect on the cyclooxyhenase, but rather by an interplay on the various enzymes of the arachidonic acid cascade.     

Prostaglandins and in vitro ovarian progestin biosynthesis

Prostaglandin F_2α (PGF_2α) did not alter the $\text{in vitro}$ biosynthesis of progesterone by slices of luteinized rat ovaries when used in concentrations from 1 to 10,000 ng/ml of incubation medium; likewise, PGF_2α did not affect the incorporation of acetate-1-¹⁴C into progestins. PGF_1α, 15-keto PGF_2α, and PGE₁ did not alter the biosynthesis of progesterone by luteinized rat ovaries; PGE₂ inhibited the production of progesterone when used at a concentration of 10 μg/ml, but not at lower doses. PGF_2α in combination with luteinizing hormone (LH) enhanced the metabolism of progesterone to 20α-hydroxypregn-4-en-3-one in luteinized rat ovaries. Interestingly, PGF_2α, at a high concentration of 10 μg/ml, did stimulate progesterone biosynthesis by slices of ovarian tissue from immature rats hormonally primed to simulate “pseudopregnancy,” suggesting a steroidogenic action of prostaglandins on the ovarian follicular or interstitial cell. PGF_2α (10 μg/ml) did not stimulate the $\text{in vitro}$ biosynthesis of progesterone or 20α-hydroxypregn-4-en-3-one by slices of rabbit corpora lutea or rabbit ovarian interstitial tissue. It is concluded that prostaglandins do not stimulate progestin biosynthesis in rat luteal tissue.     

Modulation of human platelet adenylate cyclase by prostacyclin (PGX)

Prostacyclin (PGX) ($\text{5Z}\text{)-9-}\text{deoxy}\text{-6,9α-}\text{epoxy}\text{-Δ}{}^5\text{-}\text{PGF}{}_{\mathrm{1\alpha }}$ has been found to be a potent stimulator of cAMP accumulation in human platelet rich plasma (PRP), and a direct stimulator of platelet microsome adenylate cyclase. Prostacyclin is, on a molar basis, at least 10 times more potent a stimulator of cAMP accumulation in platelets than PGE₁. The prostacyclin stimulation of platelet cAMP accumulation can be antagonized by the prostaglandin endoperoxide PGH₂, and a PGH₂-induced platelet aggregation is antagonized by prostacyclin. A model of platelet homeostasis is proposed that suggests platelet aggregation is controlled by a balance between the adenylate cyclase stimulating activity of prostacyclin, and the cAMP lowering activity of PGH₂.     

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.     

Effects of 19-hydroxy-prostaglandins on oviductal and uterine motility

The effects of 19-hydroxy-prostaglandins (19-OH-PGs) were tested $\text{in}$$\text{vivo}$ on the rabbit oviduct and uterus and on the rhesus monkey ($\text{Macaca}$$\text{mulatta}$) uterus. The 19-OH-PGEs suppressed spontaneous oviductal and uterine activity in the rabbit. The qualitative effect on the rabbit oviduct of 19-OH-PGEs was similar to that of PGE₂. However, the typical response of the rabbit uterus to PGE₂ was an increase in muscle activity. With regard to the rabbit oviduct, 19(R)-OH-PGE₂ was as potent as PGE₂, but 19(S)-OH-PGE₂ was approximately $\text{1}\text{2}$ as potent as PGE₂. Based on the dose of 19-OH-PGEs usually required to cause a minimal suppression and the dose of PGE₂ required to cause a minimal stimulation of rabbit uterine activity, 19(R)-OH-PGE₂ was twice as potent as PGE₂ while 19(S)-OH-PGE₂ was $\text{1}\text{2}$ as potent as PGE₂. Stimulatory effects on the rabbit oviduct and uterus were observed following administration of 19-OH-PGEs and PGF_2α. The potency on the rabbit oviduct of 19(S)-OH-PGF_2α was about $\text{1}\text{5}$ to $\text{1}\text{10}$ that of PGF_2α; the potency of 19(R)-OH-PGF_2α was about $\text{1}\text{10}$ to $\text{1}\text{20}$ that of PGF_2α. Both 19-OH-PGFs were approximately $\text{1}\text{5}$ to $\text{1}\text{10}$ as potent as PGF_2α on the rabbit uterus. At the doses tested 19-OH-PGFs were inactive on the monkey uterus. Thus, these compounds are at least $\text{1}\text{5}$ as active as PGF_2α. In contrast, 19(R)-OH-PGE₂ had approximately the same potency as PGE₂ in stimulating monkey uterine activity; but 19(S)-OH-PGE₂ was approximately $\text{1}\text{3}$ as potent as PGE₂.     

Concentrative accumulation of 3H-prostaglandins by some rabbit tissues in vitro: The chemical nature of the accumulated 3H-labelled substances

Following incubation with [³H]-PGF_2α, 73–91% of the ³H activity accumulated by rabbit uterus, choroid plexus or anterior uvea was shown to remain associated with PGF_2α on two different chromatographic systems. The tissue to medium ratios, calculated on the basis of chromatographically identified [³H]-PGF_2α, were greater than unity (2.3–10.4) for all three tissues and the extracted ³H activity could be effectively accumulated by these tissues for a second time. Under conditions when 85% of authentic [³H]-PGF_2α and only 8% of [³H]-15-keto-PGF_2α was adsorbed on rabbit anti-PGF_2α serum, 60–75% of the extracted ³H was adsorbed onto the antiserum. Following incubation with a mixture of 5,6-[³H]-PGE₁ and 2-[¹⁴C]-PGE₁, the anterior uvea and the uterus showed similar $\text{T}\text{M}$ ratios for ³H and ¹⁴C and the ${}^3\text{H}{}^{14}\text{C}$ ratios were essentially constant in their respective homogenates, extracts and chromatographic fractions, indicating insignificant β-oxidation of the accumulated PGE₁. In the case of the kidney cortex, a substantial fraction of the accumulated ¹⁴C did not extract as a PG presumably as a result of β-oxidation. It is concluded that metabolic alteration of the accumulated PG molecule does occur in some tissues, but such chemical alterations are not an integral part of the PG accumulative process. These results are consistent with the concept that some vertebrate tissues can accumulate PGs against a concentration gradient by an active transport mechanism.     

Transport-active renal tubular epithelial cells (MDCK and LLC-PK1) in culutre. Prostaglandin biosynthesis and its regulation by peptide hormones and ionophore

Renal tubular epithelial cells isolated from dog and pig kidney (MDCK and LLC-PK₁, respectively) transport water and electrolytes in culture. MDCK cells resemble collecting tubule cells by additional, but not all, morphologic and biochemical criteria. It has previously been reported that PGE₂ appears to regulate transport activity by MDCK cells as well as their proliferation. We investigated prostaglandin biosynthesis by MDCK and LLC-PK₁ cells and assessed the effects of peptide hormones, bradykinin and vasopressin, on the cells' prostaglandin biosynthesis. Thin-layer chromatography of radioactive products released by MDCK cells labelled with octatritiated of [¹⁴C] arachidonic acid indicated the presence of materials comigrating with PGE₂, PGI₂ (detected as 60oxo0PGF₁α) and PGF₂α, in decreasing order of abundance. Maclofenamate inhibited the biosynthesis of all radioactive peaks comigrating with PGs, thus confirming their identities as product of fatty acid cyclo-oxygenase activity. The chemical identities of [³H] PGE₂ and [³H] 6-oxo-PGF₁α made by the cells were further confirmed by treatment with KOH. Radioimmunoassay of culture fluids incubated with MDCK cells verified that PGE₂ was the most abundant prostaglandin. Tranylcypromine, thought to be a specific inhibitor of prostacyclic synthetase, inhibited PGE₂ as well as PGI₂ biosynthesis indicating a lack of specificity of the inhibitor. The observation of PGE₂ and PGF₂α as respectively the most and least abundant prostaglandinds made by MDCK was in disagreement with results from another laboratory in which the reverse order of abundance was found. This suggests the presence of more than one cell line identified as MDCK but having different biochemical properties.Bradykinin stimulated acylhydrolase activity as well as PGE₂ and PGI₂ biosynthesis in MDCK cells while vasopressin had little or no effect. These results support the hypothesis that bradykinin's natriuretic effects may be mediated by prostaglandinds and that vasopressin is unlikely to acutely stimulate prostaglandin biosynthesis in collecting tubule cells $\text{in}$$\text{vivo}$. Endogenous PGE₂ may also regulate the proliferation of MDCK cells in culture.In contrast to MDCK cells, LLC-PK₁ cells lacked significant prostaglandin biosynthetic capability as documented by radiometric thin-layer chromatography and radioimmunoassay. This suggests that prostaglandins may have a modulatory rather than an obligatory role in regulating transport activity by tubular epithelial cells.     

Stimulatory effect of prostaglandin E1 on thyroliberin-induced α-melanotropin release from perifused neuro-intermediate lobes of frog pituitary gland

A possible direct effect of prostaglandins on α-melanotropin (α-MSH) release at the level of the intermediate lobe of the frog pituitary was investigated $\text{in vitro}$ using a perifusion system technique. The effect of prostaglandins was studied on both spontaneous and TRH-stimulated α-MSH secretion. No significant effect of PGE₁, PGE₂, PGF_1α or PGF_2α on basal release of α-MSH could be detected. Indomethacin did not alter the α-MSH release induced by TRH. Conversely a significant increase in TRH-induced α-MSH secretion was observed in the presence of 1 x 10^?6M PGE₁. This magnifying effect was directly related to the concentration of TRH for doses ranging from 1 x 10^?8M to 1 x 10^?6M.     

Imidazole: A selective inhibitor of thromboxane synthetase

Imidazole inhibits the enzymatic conversion of the endoperoxides (PGG₂ and PGH₂) to thromboxane A₂ by platelet microsomes (IC₅₀: 22 μg/ml; determined by bioassay). The inhibitor is selective, for prostaglandin cyclo-oxygenase is only affected at high doses. Radiochemical data confirms that imidazole blocks the formation of ¹⁴C-thromboxane B₂ from ¹⁴C-PHG₂. Several imidazole analogues and other substances were tested but only 1-methyl-imidadole was more potent that imidazole iteself. The use of imidazole of inhibit thromboxane formation could help to elucidate the role of thromboxanes in physiology or pathophysiology.     

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.     

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.     

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.     

Effects of prostaglandin PGE2 on the synthesis of placental proteins and human placental lactogen (HPL)

The biosynthesis of placental proteins and placental lactogen (HPL) was studied $\text{in vitro}$ in 10–12 week, 16–18 week and term human placenta in the presence and absence of PGE_2α. The highest ¹⁴C-leucine incorporation was detected in 10 to 12 weeks old placentas. Addition of PGE_2α to the induction medium depressed the rate of incorporation of ¹⁴C-leucine into placental proteins on a dose dependent manner. Placentas most sensitive to this action of PGE_2α were those obtained at 18 weeks gestation followed by placentas at term. $\text{In vivo}$ application of PGE_2α for tharapeutic induction of abortions resulted in the marked inhibition of placental protein synthesis $\text{in vitro}$.