Macromolecular binding of (5,6-3H) prostaglandin E1 in liver cytosol in vitro

[³H] Prostaglandin E₁ (PGE₁) is bound extensively to macromolecules in liver cytosol $\text{in vitro}$. A principal binding protein accounts for 80% of the binding. This macromolecule is saturated at about 10^?10 M PGE₁. The partially purified protein has a molecular weight of 50,000 by gel filtration and a pI of about 3.5 by isoelectrofocusing. Binding is primarily noncovalent and the dissociated ligand behaves similarly to the parental [³H] PGE₁ on thin layer chromatography. Possible significance of this interaction is discussed.     

Prostaglandin E2 receptor in the myometrium: distribution in subcellular fractions

[³H]Prostaglandin (PG) E₂ bound specifically to several subcellular fractions from bovine myometrium. The binding was temperature dependent, rapid, and reversible. PGE₂ and PGE₁ competed for the [³H]PGE₂ binding site. The PGs inhibited in the following decreasing order: PGE₂ = PGE₁ ? PGF_2α > PGA₂ > PGF_1β > PGB₂. No competitive effect could be found for oxytocin. Scatchard analysis of the binding data were interpreted as showing a single high-affinity binding constant. There was no difference in the binding constant between the various fractions. The average molar dissociation constant was 2.74 ± 0.14 × 10^?9. Quantitative differences in the maximum number of binding sites were observed between fractions. One plasma membrane fraction contained 21.4 ± 2.3 × 10^?11 and the sarcoplasmic reticulum contained 11.2 ± 0.8 × 10^?11 mol binding sites/g. The results suggest that there is a high-affinity PGE₂ receptor present in both plasma membrane and sarcoplasmic reticulum.     

The effect of prostaglandin E2-induced echinocytic transformation on the optassium loss,viscosity and osmotic fragility of normal and sickle cell erythrocytes

Prostaglandin E₂ (PGE₂)-induced discocyte → echinocytic transformation has no effect om the viscosity or osmotic fragility of normal or stickle cell erythrocytes. Membrane permeability, reflected, reflected as potassium efflux, is significantly affected in normal erythrocytes when >90% of the cells are morphologically transformed to the enchinocytic III stage (PGE₂ concentration of 1–2×10⁶ ng/ml blood). This potassium loos is significant in sickle erythrocytes when 50–70% of the cell population has been transformed (PGE₂ concentration, 5×10⁵ ng/ml blood). This change in membrane permeability reprensents one-half to one-third the flux that occurs with sickling (i.e., >80% of the erythrocytes sickled).     

The influence of calcium on the cyclic AMP-mediated stimulation of DNA synthesis and cell proliferation by prostaglandin E 1

Prostaglandin type E₁ (PGE₁) rapidly stimulates cyclic AMP formation and the initiation of deoxyribonucleic acid (DNA) synthesis in rat thymic lymphocytes suspended in vitro by reactions which are not affected by wide variations in the extracellular calcium concentration. On the other hand, the operation of the associated reaction(s) responsible for the subsequent progression of the stimulated cells into mitosis is profoundly affected by the extracellular calcium level. If the maximum intracellular cyclic AMP concentration is in the lower range of stimulatory values (e.g., 150 × 10^?8 picomoles per cell as produced by an exposure to 0.5 μg of PGE₁ per milliliter of medium), an extracellular calcium concentration of 0.5 to 1.0 mM is needed to obtain maximum cell proliferation, but not the maximum stimulation of DNA synthesis. Contrariwise, if the cellular cyclic AMP content is raised to a much higher level (260 × 10^?8 picomoles per cell) by exposure to a greater PGE₁ concentration (5.0 μg per millilter), cell proliferation is maximally stimulated in calcium-free medium and increasing the extracellular calcium concntration above 0.2 mM actually prevents the stimulation of cell proliferation (but does not affect the stimulation of DNA synthesis). Thus, the ultimate translation of PGE₁'s early cyclic AMP-mediated reactions into increased cell proliferation is determined by both the intracellular cyclic AMP level and the extracellular calcium concentration.     

Antagonism of prostaglandin-promoted pituitary cyclic amp accumulation and growth hormone secretion in vitro by 7-oxa-13-prostynoic acid

The studies reported here confirm the previously observed potent stimulus to growth hormone (GH) secretion by prostaglandin E₁ (PGE₁). Proportional increments in GH secretion were observed following $\text{in vitro}$ addition of PGE₁ over a concentration range of 10^?7 to 10^?5 M. Growth hormone secretion could not be further stimulated by higher concentrations of prostaglandin. Prostaglandin E₁ also increased cyclic AMP concentration in the pituitary explants in a proportional fashion, which correlated closely with its potency as a growth hormone secretogogue. In order to define more precisely the mechanism by which prostaglandin acts, the effects of prostaglandin antagonist, 7-oxa-13-prostynoic acid, on GH secretion and cyclic AMP accumulation were investigated. Addition of the antagonist alone had no consistent effects on GH secretion or cyclic AMP levels in the pituitary. However, the antagonist significantly reduced the stimulation of hormone release and cyclic AMP accumulation found following addition of PGE₁. Increasing the concentration of antagonist further diminished prostaglandin stimulated hormone release and nucleotide accumulation. The antagonist failed to block the stimulatory effects of theophylline and dibutyryl cyclic AMP on GH release, indicating that the inhibition observed occurred prior to intracellular accumulation of the cyclic nucleotide. These results are consistent with the hypothesis that a prostaglandin receptor on the pituitary somatotrope is linked to the adenyl cyclase-cyclic AMP system.     

Receptors for prostaglandins and gonadotropins in the cell membranes of bovine corpus luteum

The cell membranes exhibited specific binding to ³H-prostaglandin E₁ (³H-PGE₁) and ¹²⁵I-human chorionic gonadotropin (¹²⁵I-HCG). Unlabeled PGE₁,PGE₂ (1.4 × 10^?7M), PGF_1α and PGF_2α (1.4 × 10^?5M) decreased ³H-PGE₁ binding by more than 80%. The binding of ¹²⁵I-HCG was completely inhibited by 5 × 10^?8M unlabeled HCG. However, the unlabeled PGE₁ (1.15 × 10^?6M) and HCG (8.4 × 10^?7M) had no effect on ¹²⁵I-HCG and ³H-PGE₁ binding respectively. A PG antagonist, 7-oxa-13-prostynoic acid, inhibited only ³H-PGE₁ binding but not ¹²⁵I-HCG binding. These results suggest the presence of specific receptors for PGE₁ and HCG in the cell membranes and that the binding occurs either at two different sites on the same receptor or that each binds to a “different” receptor molecule.     

Effect of Noradrenaline and Prostaglandin E<Subscript>1</Subscript> on Adenosine 3′,5′;-Monophosphate Formation in Isolated Pericardial Fat Cells of Man

NORADRENALINE increases the intracellular concentration of adenosine 3′,5′-monophosphate (cyclic AMP)^1,2 which, in turn, enhances glycogenosis³ and lipolysis^4,5 in adipose tissue by increasing Phosphorylase and lipase activities. Prostaglandin E₁ (PGE₁) antagonizes the induced increases in Phosphorylase activity^6,7 and glycerol release in human adipose tissues^8,9 and isolated adipocytes⁷. The finding that the stimulatory effects of the cyclic AMP analogue N⁶—O² dibutyryl cyclic AMP, which mimics the hormonal effect of noradrenaline in human fat cells, are not blocked by PGE₁⁷ suggests that noradrenaline and PGE₁ alter fat cell metabolism by acting on the adenyl cyclase system¹⁰. Whether noradrenaline and PGE₁ alter concentrations of cyclic AMP in human fat cells, however, has not been reported.     

Comparison of the effects of prostaglandin I2 and prostaglandin E2 stimulation of the rat kidney adenylate cyclase-cyclic AMP systems

Prostacyclin (Prostaglandin I₂) effects on the rat kidney adenylate cyclase-cyclic AMP system were examined. Prostaglandin I₂ and prostaglandin E₂, from 8 · 10^?4 to 8 · ^?7 M stimulated adenylate cyclase to a similar extent in cortex and outer medulla. In inner medulla, prostaglandin I₂ was more effective than prostaglandin E₂ at all concentrations tested. Both prostaglandin I₂ and prostaglandin E₂ were additive with antidiuretic hormone in outer and inner medulla. Prostaglandin I₂ and prostaglandin E₂ were not additive in any area of the kidney, indicating both were working by similar mechanisms. Prostaglandin I₂ stimulation of adenylate cyclase correlated with its ability to increase renal slice cyclic AMP content. Prostaglandin I₂ and prostaglandin E₂ (1.5 · 10^?4 M) elevated cyclic AMP content in cortex and outer medulla slices. In inner medulla, with Santoquin® (0.1 mM) present to suppress endogenous prostaglandin synthesis, prostaglandin I₂ and prostaglandin E₂ increased cyclic AMP content. 6-Ketoprostaglandin F_1α, the stable metabolite of prostaglandin I₂, did not increase adenylate cyclase activity or tissue cyclic AMP content. Thus, prostaglandin I₂ activates renal adenylate cyclase. This suggests that the physiological actions of prostaglandin I₂ may be mediated through the adenylate cyclase-cyclic AMP system.     

Presence of prostaglandins E2 and A2 in canine gastric secretions

The presence of prostaglandins (PGs) was determined in gastric juice obtained from 3 conscious dogs, provided with a chronic gastric fistula. Outputs of acid (mequiv min^?1) and PGs (pg min^?1) were measured in gastric secretions stimulated by pentagastrin (100 or 200 ng kg^?1min^?1). Prostaglandin activity was estimated, after extraction and thin layer chromatography, by radioimmuno-assay of the PGB formed by treatment with alkali. Tritiated PGs were added to gastric juice for the purpose of correcting for PGs recovery. Using this method, the minimum mass of PGB which could be satisfactorily distinguished from zero was 25 pg. Prostaglandins A₂ and E₂ were present in pentagastrin-activated gastric secretions and averaged (mean ± SE, n = 8) 200.7 ± 18.1 and 260.1 ± 18.0 pg min^?1 respectively. The identity of PGA₂ and PGE₂ was confirmed by gas liquid chromatography combined with mass spectrometry. The amount of PGE₂ converted to PGA₂ during extraction, separation and conversion procedures was estimated from the amount of [³H] PGA₂ found when only [³H] PGE₂ had been added to a sample of gastric juice and averaged 14.5% ± 2.0. Our preliminary results support the possibility that PGE₂ and PGA₂ may be of physiological importance in the regulation of canine gastric secretions.     

Studies of effects of cyclic adenosine 3′,5′-monophosphate in regulation of human hemopoiesis in vitro

Summary Prostaglandin E₁ (PGE₁), high concentrations of dibutyryl cyclic AMP (dbcAMP), and theophylline were strikingly inhibitory both to tritiated thymidine ([³H]TdR) incorporation into bone marrow deoxyribonucleic acid (DNA) in vitro and to granulocytic colony growth. Autoradiography revealed that lower concentrations of dbcAMP were stimulatory to red blood cell precursors. This study was supported in part by United States Public Health Service Grant AM15163, by Health Research Council of the City of New York Career Scientist Award I-683, and by a Veterans Administration Medical Investigatorship to V. H.     

Prostaglandins and renin release: III. Effects of PGE1, E2 F2α and D2 on renin release from rabbit renal cortical slices

We have investigated the direct effects of prostaglandins E₁, E₂, F_2α and D₂ on renin release from rabbit renal cortical slices. Prostaglandin E₁ (PGE₁) was the most potent stimulant of renin release, while PGE₂ was 20–30 fold less active. PGF_2α was found not to be an inhibitor of renin release as reported by others, but rather a weak agonist. PGD₂ up to a concentration of 10 μg/ml had no activity in this system. That the stimulation of renin release by PGE₁ is a direct effect is supported by the finding that PGE₁-induced release is not blocked by L-propranolol or by Δ^5,8,11,14-eicosatetraynoic acid (ETYA), a prostaglandin synthesis is inhibitor. The fatty acid precursor of PGE₁, Δ^8,11,14-eicosatrienoic acid, also stimulated renin release, an effect which was blocked by ETYA. In addition to the above findings, ethanol, a compound frequently used to dissolve prostaglandins, was shown to inhibit renin release.     

Ductus arteriosus responses to prostaglandin E1 at high and low oxygen concentrations

It previously has been suggested that prostaglandin E₁ (PGE₁) relaxes the ductus arteriosus in a low but not in an elevated oxygen environment. However, in the experiments reported here PGE₁ relaxed rings on fetal lamb ductus arteriosus at both low (14 to 20 torr) and high (680 to 720 torr) oxygen tensions. The threshold concentration for PGE₁ was 10⁻¹⁰ M in either PO₂ and the ED50's of PGE₁ relaxation in high and low oxygen were 8.5 ± 3.4 × 10⁻¹⁰ M and 5.5 ± 0.7 × 10⁻¹⁰ M respectively. The magnitude of the relaxation was greater for the oxygen contracted ductus arteriosus than for that exposed to low oxygen. It is suggested that earlier reports of the lack of response of the ductus arteriosus to PGE₁ in a high oxygen environment following relaxation in a low oxygen environment may be related to loss of response of the ductus arteriosus to repeated doses of PGE₁ rather than to differences in PO₂. Prostaglandin E₁ therefore may play a significant role in the regulation of ductus arteriosus tone in the elevated oxygen environment of the newborn as well as the low oxygen environment of the fetus.     

Effects of estradiol and progesterone on uterine prostaglandin levels in the pregnant rat

Prostaglandin D₂ was found to be a potent inhibitor of B-16 melanoma cell replication $\text{in vitro}$. The inhibition was dose-dependent between 3×10^?9M and 3×10^?6M (IC_50～ 0.3 μM after 6 days). On a molar basis, PGD₂ was a better inhibitor than PGA₂ or 16,16-dimethyl-PGE₂-methyl ester (di-M-PGE₂) and in higher concentrations (10^?6?10^?7M), comparable to retinoic acid. In higher concentrations, PGD₂ inhibited DNA, RNA and protein synthesis. The B-16 melanoma cell line which we used synthesized arachidonic acid metabolites which comigrated with PGA₂, PGD₂, PGE₂ and PGF_2α on a thin layer chromatography system.     

mTORC2 regulates PGE2-mediated endothelial cell survival and migration

Prostaglandin E₂ (PGE₂) promotes angiogenesis by in part inducing endothelial cell survival and migration. The present study examined the role of mTOR and its two complexes, mTORC1 and mTORC2, in PGE₂-mediated endothelial cell responses. We used small interfering RNA (siRNA) to raptor or rictor to block mTORC1 or mTORC2, respectively. We observed that down-regulation of mTORC2 but not mTORC1 reduced baseline and PGE₂-induced endothelial cell survival and migration. At the molecular level, we found that knockdown of mTORC2 inhibited PGE₂-mediated Rac and Akt activation two important signaling intermediaries in endothelial cell migration and survival, respectively. In addition, inhibition of mTORC2 by prolonged exposure of endothelial cells to rapamycin also prevented PGE₂-mediated endothelial cell survival and migration confirming the results obtained with the siRNA approach. Taken together these results show that mTORC2 but not mTORC1 is an important signaling intermediary in PGE₂-mediated endothelial cell responses.     

The in vitro effect of indomethacin on basal bone resorption, on prostaglandin production and on the response to added prostaglandins

Mouse calvaria were maintained in organ culture without serum additives. Basal active resorption, as measured by ⁴⁵Ca and hydroxyproline release, was significantly inhibited to 74% control levels by indomethacin (1.4 × 10⁻⁷ M). Prostaglandin F and prostaglandin E₂ production, determined by radioimmunoassay, were both significantly lowered by this concentration of indomethacin. DNA, protein and hydroxyproline synthesis, as indices of cell toxicity, were unaffected by low concentrations of indomethacin, while concentrations of 1.4 × 10⁻⁶M inhibited protein synthesis (p<0.005). In the presence of indomethacin (1.4 × 10⁻⁷M) both PGE₂ and PGF_2α stimulated resorption in a dose-dependent manner, with PGE₂ being the more potent. Neither prostaglandin affected hydroxyproline synthesis at low concentrations, but PGE₂ had a marked inhibitory action at a higher concentration (10⁻⁶M). In combination, the effects of PGE₂ and PGF_2α showed no evidence of synergism or any antagonistic action. The study shows that in vitro calcium and hydroxyproline resorption in the unstimulated mouse calvaria are inhibited by indomethacin at concentrations measured in serum during human therapy. The decreased PGF and PGE₂ production associated with this decreased bone resorption in the presence of non-toxic concentrations of indomethacin would suggest a role for these prostaglandins in maintaining the basal resorption of cultured bone.     

Endogenous prostaglandin E2 enhances polyclonal immunoglobulin production by tonically inhibiting T suppressor cell activity

The immunoregulatory effect of endogenous prostaglandin E₂ (PGE₂) on immunoglobulin production was studied in an in vitro culture system of human peripheral blood mononuclear cells, stimulated with pokeweed mitogen (PWM). Three different cyclooxygenase inhibitors (indomethacin, carprofen, and piroxicam) suppressed Ig synthesis by ~50%. This inhibitory effect could be reversed by adding low doses of exogenous PGE₂ (3 × 10^?9 to 3 × 10^?8M). These doses are endogenously produced in PWM-stimulated cultures, and a concentration of 2 × 10^?8M is reached after 48 hr of culture. When B cells were directly stimulated with helper factor, PGE₂ did not enhance Ig production and doses of 3 × 10^?7M to 3 × 10^?6M were inhibitory. The effects of indomethacin and PGE₂ were eliminated when T cells were irradiated or treated with mitomycin prior to culture. The enhancing effects of PGE₂ were substantially reduced after OKT8(+) T cells were removed from the system. PWM-stimulated cultures of lymphocytes from healthy subjects over age 70 were more sensitive to inhibition by indomethacin and to stimulation by PGE₂ than were cultures of lymphocytes from young controls. Thus the major role of endogenous PGE in polyclonal Ig production in vitro is to tonically inhibit a radiosensitive, OKT8(+) suppressor T cell. This tonic inhibition is increased in subjects over 70, which provides one explanation for decreased suppressor cell function in elderly subjects.     

Effect of guanyl nucleotides on the stimulation of adenyl cyclase activity in human thyroid plasma membranes by thyroid-stimulating hormone and prostaglandin E2

Thyroid homogenates and thyroid plasma membranes were prepared from human thyroid and the effects of thyroid-stimulating hormone (thyrotropin), NaF, and prostaglandins E₁ and E₂ on adenyl cyclase activity in these preparations were studied. The basal level of adenyl cyclase activity in plasma membranes was 5–8 times greater than that of the original homogenates. Adenyl cyclase activity in plasma membranes was stimulated 4.7-fold by 100 munits/ml of thyrotropin and 5-fold by 10 mM of NaF, but the activity in the homogenates was only stimulated 2-fold by either thyrotropin or NaF. Prostaglandin E₁ (10^?6?10^?3 M) and prostaglandin E₂ (10^?7?10^?4 M) failed to stimulate adenyl cyclase activity in plasma membranes, but they did stimulate adenyl cyclase activity in the homogenates. A marked stimulatory effect of prostaglandin E₂ (10^?5 M) on adenyl cyclase activity in plasma membranes resumed in the presence of GTP (10^?7?10^?4 M), although GTP itself only slightly stimulated enzyme activity. GDP and GMP were also effective in this respect, although their potencies varied from compound to compound. GTP potentiated slightly the action of thyrotropin on adenyl cyclase in plasma membranes, but it significantly depressed an increase of enzyme activity produced by NaF. Since GTP did not affect the ATP-regenerating system, it seems that GTP, GDP or GMP was required for the manifestation of prostaglandin E₂ action on adenyl cyclases of human thyroid plasma membranes.     

Specific prostaglandine E1 and A1 binding sites in rat a dipocyte plasma membranes

Rat adipocyte plasma membranes sacs have been shown to be a sensitive and specific system for studying prostaglandin binding. The binding of prostaglandin E₁ and prostaglandin A₁ increases linearly with increasing protein concentration, and is a temperature-sensitive process. Prostaglandin E₁ binding is not ion dependent, but is enhanced by GTP. Prostaglandin A₁ binding is stimulated by ions, but is not affected by GTP.Discrete binding sites for prostaglandin E₁ and A₁ were found. Scatchard plot analysis showed that the binding of both prostaglandins was biphasic, indicating two types of binding sites. Prostaglandin E₁ had association constants of 4.9 · 10⁹ 1/mole and 4 · 10⁸ 1/mole, while the prostaglandin A₁ association constants and binding capacities varied according to the ionic composition of the buffer. In Tris-HCl buffer, the prostaglandin A₁ association constants were 8.3 · 10⁸ 1/mole and 5.7 · 10⁷ 1/mole, while in the Krebs—Ringer Tris buffer, the results were 1.2 · 10⁹ 1/mole and 8.6 · 10⁶ 1/mole.Some cross-reactivity between prostaglandin E₁ and A₁ was found for their respective binding sites. Using Scatchard plot analysis, it was found that a 10-fold excess of prostaglandin E₁ inhibited prostaglandin A₁ binding by 1–20% depending upon the concentration of prostaglandin A₁ used. Prostaglandin E₁ competes primarily for the A prostaglandin high-affinity binding site. Similar Scatchard analysis using a 20-fold excess of prostaglandin A₁ inhibited prostaglandin E₁ binding by 10–40%. Prostaglandin A₁ was found to compete primarily for the E prostaglandin low-affinity receptor.All of the bound [³H]prostaglandin E₁, but only 64% of the bound [³H]-prostaglandin A₁ can be recovered unmetabolized from the fat cell membrane. There is no non-specific binding of prostaglandin E₁, but 10–15% of prostaglandin A₁ binding to adipocyte membranes is non-specific. Using a parallel line assay to measure relative affinities for the E binding site, prostaglandin E₁ > prostaglandin A₂ > prostaglandin F_2α. Prostaglandin E₂ and 16,16-dimethyl prostaglandin E₂ were equipotent with prostaglandin E₁, while other prostaglandins had lower relative affinities. 7-Oxa-13-prostynoic acid does not appear to antagonize prostaglandin activity in adipocytes at the level of the receptor.     

Effects of prostaglandin E1 on contractility and 45Ca release in rabbit aortic smooth muscle

Concentrations of prostaglandin E₁ (PGE₁; 10^?7 M) that do not elicit tension responses in aortic strips potentiate contractions induced by submaximal concentrations (10^?8 ? 10^?7 M) of norepinephrine (NE) or angiotensin III (Ang III) but not those of high K⁺ depolarization or maximal NE or Ang III concentrations. Higher concentrations of PGE₁ (10^?6 M and above) initiate contractions which are additive with submaximal responses to NE and Ang III but not to K⁺. These same concentrations of PGE₁ also decrease ⁴⁵Ca retention at high affinity La⁺⁺⁺-resistant sites in a manner similar to but not additive with NE and Ang III. Uptake of ⁴⁵Ca at low affinity La⁺⁺⁺-resistant sites (which is increased by high K⁺-depolarization) is not altered by 10^?6 M PGE₁. The effects of PGE₁ are not altered by decreased extracellular Ca⁺⁺ (0.1 mM), decreased temperature, phentolamine or meclofenamate. Thus, PGE₁ does not appear to increase uptake of extracellular Ca⁺⁺ in this smooth muscle tissue. Instead, PGE₁ increases mobilization of Ca⁺⁺ from the same high affinity La⁺⁺⁺-resistant sites affected by Ang III and NE and, in this manner, may increase responses to these two stimulatory agents.     

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.