Prostaglandin F2 alpha inhibition of epinephrine stimulated cyclic AMP and progesterone production by rat corpora lutea of various ages

Epinephrine can mimic the stimulatory effects of LH in vitro on cyclic AMP (cAMP) and progesterone production by isolated rat corpora lutea. The aim of the present study was to test whether the effects of epinephrine in vitro on the rat corpus luteum, as with LH, can be inhibited by prostaglandin F2 alpha (PGF2 alpha). The stimulatory effect of epinephrine on tissue levels of cAMP in 1-day-old corpora lutea was not inhibited by PGF2 alpha. A dose-dependent inhibition by PGF2 alpha (0.5-50 microM) was seen for 3-day-old corpora lutea and this inhibition could not be overcome by higher concentrations of epinephrine (0.165-165 microM). The stimulation by epinephrine on progesterone production was inhibited by PGF2 alpha (5 microM) in 3- and 5-day-old, but not in 1-day-old corpora lutea. Thus, PGF2 alpha can inhibit the stimulatory effect of epinephrine in 3- and 5-day-old corpora lutea, but not in the newly formed corpora lutea (1-day-old) and PGF2 alpha shows in this respect the same age dependent inhibitory pattern as in relation to LH stimulation.     

Prostaglandin F2α specific binding in equine corpora lutea

Preliminary studies indicate the presence of PGF_2α specific binding sites in membrane fractions prepared from equine corpora lutea. The equilibrium binding data indicate an apparent dissociation constant of 3.2 × 10^?9M and the concentration of binding sites of ～0.1 pmoles/mg membrane protein. Competition of several natural prostaglandins for equine luteal PGF_2α specific binding sites indicates specificity for the 9α-hydroxyl moiety and the 5,6-cis doublebond. Significant increases in relative binding affinities were demonstrated for PGF_2α analogs with a phenyl ring introduced at carbons 16 or 17. Specific PGF_2α binding was demonstrated in corpora lutea collected at known stages of the estrous cycle. There was no pattern in these values based on the stage of the cycle. While specific ³H-PGE₁ binding could be demonstrated, no high affinity sites could be quantitated. ³H-PGE₁ binding appeared unaffected by changes in temperature or time of incubation, whereas PGF_2α specific binding was significantly modified by both these factors.     

Inhibition by vanadate of cyclic AMP production in rat corpora lutea incubated in vitro

Vanadate, a normal constituent of cells, has been reported to affect a variety of enzymes involved in phosphate transfer; the findings regarding adenylate cycle vary with the tissue and experimental system. In the corpus luteum, cyclic AMP (cAMP) stimulates steroidogenesis; and prostaglandin F2 alpha, which induces luteal regression, inhibits luteinizing hormone (LH)-induced cAMP accumulation. We examined the influence of orthovanadate on cAMP concentration in isolated corpora lutea from pseudopregnant rats. With 2 mM vanadate, basal cAMP level was unaffected, but LH-induced cAMP accumulation was inhibited by 45-68%. Lower doses of vanadate (0.2-1 mM) were almost as effective. When added simultaneously with LH, vanadate was inhibitory within 25 min, but no inhibition occurred when vanadate was added for 30 min to tissue pretreated with LH for 60 min. The decrease in cAMP accumulation was observed also when corpora lutea were exposed to vanadate in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (0.5 mM), indicating that vanadate inhibits cAMP synthesis. Vanadate may increase cytosolic calcium by inhibiting ion pumps in cell membranes. Thus, we examined the effect of vanadate in corpora lutea incubated in calcium-depleted medium and found that vanadate still inhibited cAMP formation. Vanadyl sulfate (0.4 and 2 mM) reduced the LH-induced cAMP accumulation as effectively as vanadate. Thus, the use of vanadate as a tool for exploring physiological regulators of luteal adenylate cyclase should be considered.     

Effect of prostaglandin F2α on the hCG-stimulated progesterone production by human corpora lutea

The effect of prostaglandin PGF_2α on the hCG stimulated and basal progesterone production by human corpora lutea was examined . hCG (40 i.u./ml) stimulated progesterone formation in corpora lutea of early (days 16–19 of a normal 28 day cycle), mid (days 20–22) and late (days 23–27) luteal phases. This stimulation was inhibited by PGF_2α (10 μg/ml) in corpora lutea of mid and late luteal phases. PGF_2α alone did not show a consistent effect on basal progesterone production. The inhibition of hCG stimulated progesterone production by PGF_2α at times corresponding to luteolysis indicates a role for that prostaglandin in the process of luteolysis in the human corpus luteum.     

Prostaglandin I2 stimulation of granulosa cell cyclic AMP production

The ability of prostaglandin I₂ (PGI₂) to stimulate cyclic AMP production by granulosa cells, isolated from intact immature rats, has been demonstrated in vitro. The minimal effective dose was 15 ng/ml, which was comparable to the minimal effective dose for PGE₂. However, a concentration of 15 μg/ml PGI₂ was required to stimulate cyclic AMP production maximally, compared to a concentration of 1 μg/ml PGE₂, which produced the maximum response. It therefore appears that PGI₂ is not more effective than PGE₂ in stimulating cyclic AMP production in granulosa cells, and is possibly less effective. Submaximal concentrations of PGI₂ appeared to be able to modify the stimulation of cyclic AMP production by follicle- stimulating hormone (FSH), but whether or not PGI₂ plays any role in follicular function remains to be established.     

Abrogation by prostaglandin F2alpha of LH-stimulated cyclic AMP accumulation in isolated rat corpora lutea of pregnancy.

Corpora lutea explanted from rats on the sixth day of pregnancy responded to luteinizing hormone (LH; 5 μg/ml) in vitro with a two- to five-fold increase in cellular cyclic AMP (cAMP) concentration. The maximal cAMP level was reached within 60 min and maintained to the end of the 2 hr-incubation. On incubation with prostaglandin F_2α (PGF_2α) in addition to LH, this rise in cAMP accumulation was prevented. For significant suppression, 1.4 × 10^?5M PGF_2α was required. In the absence of LH, PGF_2α (4.2 × 10^?5M) caused no change in cellular cAMP. Addition of PGF_2α (4.2 × 10^?5M) to the incubation medium after the maximal response to LH was attained, caused the cAMP concentration to return to its basal level within 15 min. This abrogation of LH-stimulated cAMP accumulation represents the earliest and hence possibly the triggering event in PGF_2α-induced luteolysis.     

Prostaglandin E1 and F2α specific binding in bovine corpora lutea: Comparison with luteolytic effects

Preliminary characterization indicated the presence of separate prostaglandin (PG)E₁ and (PG)F_2α binding sites in membrane fractions prepared from bovine corpora lutea. These differ in the rate and temperature dependence of the specific binding. Equilibrium binding data indicate the apparent dissociation constants as 1.32 × 10⁻⁹M and 2.1 × 10⁻⁸M for PGE₁ and PGF_2α, respectively. Competition of several natural prostaglandins for the PGE₁ and PGF_2α bovine luteal specific binding sites indicates specificity for the 9-keto or 9α-hydroxyl moiety, respectively. Differences in relative ability to inhibit ³H-PG binding were found due to sensitivity to the absence or presence of the 5,6-cis-double bond as well.Bovine luteal function was affected following treatment of heifers with 25 mg PGF_2α as measured by reduced estrous cycle length, decreased corpus luteum size and significantly decreased plasma progesterone levels. In contrast, treatment with 25 mg PGE₁ resulted in cycle lengths comparable to those of non-treated herdmates with no apparent modification in corpus luteum size. However, plasma progesterone levels were increased significantly following PGE₁ treatment compared to pretreatment values. In so far as data obtained on PGF_2α relative binding affinity to the bovine CL can be compared to data obtained independently on PGF_2α induced luteolysis in the bovine, PGF_2α relative binding to the CL and luteolysis appeared to be associated. By similar reasoning, there was no apparent relationship between PGE₁ relative binding affinity in the luteal fractions and luteolysis in estrous cyclic cattle.     

Prostaglandin E1 and F2α specific binding in bovine corpora lutea: Comparison with luteolytic effects

Preliminary characterization indicated the presence of separate prostaglandin (PG)E₁ and (PG)F_2α binding sites in membrane fractions prepared from bovine corpora lutea. These differ in the rate and temperature dependence of the specific binding. Equilibrium binding data indicate the apparent dissociation constants as 1.32 × 10⁻⁹M and 2.1 × 10⁻⁸M for PGE₁ and PGF_2α, respectively. Competition of several natural prostaglandins for the PGE₁ and PGF_2α bovine luteal specific binding sites indicates specificity for the 9-keto or 9α-hydroxyl moiety, respectively. Differences in relative ability to inhibit ³H-PG binding were found due to sensitivity to the absence or presence of the 5,6-cis-double bond as well.Bovine luteal function was affected following treatment of heifers with 25 mg PGF_2α as measured by reduced estrous cycle length, decreased corpus luteum size and significantly decreased plasma progesterone levels. In contrast, treatment with 25 mg PGE₁ resulted in cycle lengths comparable to those of non-treated herdmates with no apparent modification in corpus luteum size. However, plasma progesterone levels were increased significantly following PGE₁ treatment compared to pretreatment values. In so far as data obtained in vitro on PGF_2α relative binding affinity to the bovine CL can be compared to data obtained independently in vitro on PGF_2α induced luteolysis in the bovine, PGF_2α relative binding to the CL and luteolysis appeared to be associated. By similar reasoning, there was no apparent relationship between PGE₁ relative binding affinity in the luteal fractions and luteolysis in estrous cyclic cattle.     

Stimulation with LH of progesterone production by rabbit corpora lutea in vitro

Quartered CL from 7-day pseudopregnant rabbits were incubated at 37 degrees C for 0-180 min in the presence of BSA, LH or adrenaline in Krebs-Ringer-bicarbonate buffer. Total progesterone at each time point was quantified in homogenates of tissue plus incubation media and expressed relative to CL protein. Progesterone increased linearly with time during the first 30 min of incubation in the presence of BSA. LH and adrenaline markedly accelerated progesterone accumulations relative to the BSA control. At 10 min, progesterone accumulation in the presence of LH and adrenaline were 2.4 and 5.9 times that in the absence of stimulators, respectively. Both hormones caused concentration-dependent increases in progesterone and the apparent ED50 was 0.75 microgram/ml for LH and adrenaline. The CL obtained from ovaries of 7-day pseudopregnant rabbits are therefore capable of an acute steroidogenic synthetic response to LH as well as adrenaline.     

Effect of prostaglandin E2 alpha on the hCG-stimulated progesterone production by human corpora lutea

The effect of prostaglandin PGF2 alpha on the hCG stimulated and basal progesterone production by human corpora lutea was examined in vitro. hCG (40 i.u./ml) stimulated progesterone formation in corpora lutea of early (days 16-19 of a normal 28 day cycle), mid (days 20-22) and late (days 23-27) luteal phases. This stimulation was inhibited by PGF2 alpha (10 micrograms/ml) in corpora lutea of mid and late luteal phases. PGF2 alpha alone did not show a consistent effect on basal progesterone production. The inhibition of hCG stimulated progesterone production by PGF2 alpha at times corresponding to luteolysis indicates a role for that prostaglandin in the process of luteolysis in the human corpus luteum.     

Prostaglandin E2: Effects on aggregation,shape change and cyclic AMP of rat platelets

PGE₂ inhibits intracellular cyclic AMP accumulation induced by PGE₁ in rat platelets. PGE₂ also counteracts PGE₁-inhibition of both platelet aggregation and shape change. However, in the presence of theophylline, PGE₂ acts like PGE₁ in inhibiting aggregation. The mechanism of interaction of the two closely related prostaglandins is discussed.     

Prostaglandin I2 stimulation of granulosa cell cyclic AMP production.

The ability of prostaglandin I2 (PGI2) to stimulate cyclic AMP production by granulosa cells, isolated from intact immature rats, has been demonstrated in vitro. The minimal effective dose was 15 ng/ml, which was comparable to the minimal effective dose for PGE2. However, a concentration of 15 microgram/ml PGI2 was required to stimulate cyclic AMP production maximally, compared to a concentration of 1 microgram/ml PGE2, which produced the maximum response. It therefore appears that PGI2 is not more effective than PGE2 in stimulating cyclic AMP production in granulosa cells, and is possibly less effective. Submaximal concentrations of PGI2 appeared to be able to modify the stimulation of cyclic AMP production by follicle-stimulating hormone (FSH), but whether or not PGI2 plays any role in follicular function remains to be established.     

Secretion of oxytocin and progesterone by ovine corpora lutea in vitro

The mechanisms involved in the control of oxytocin and progesterone secretion by the ovine corpus luteum have been investigated in vitro using luteal slice incubations. Oxytocin and progesterone were secreted at constant rates from luteal slices for 2 h of incubation (366 +/- 60 pg X mg X h and 18.9 +/- 0.18 ng X mg X h, respectively). Secretion of progesterone, but not of oxytocin, was significantly (p less than 0.02) stimulated in the presence of ovine luteinizing hormone. Incubation of luteal slices in medium containing 100 mM potassium, however, resulted in increased secretion of oxytocin and, to a lesser extent, of progesterone (294 +/- 59% and 142 +/- 15%, respectively, p less than 0.05). Basal oxytocin secretion was reduced during incubation in calcium-free medium, compared to secretion in the presence of calcium (70 +/- 15 and 175 +/- 25 pg X mg X 20 min, respectively, p less than 0.01), whereas progesterone secretion was not altered in the absence of calcium. Secretion of both hormones by luteal slices was stimulated by the addition of the calcium ionophore A23187 (p less than 0.05). Addition of prostaglandin F2 alpha (2.8 microM) had no effect on secretion of either oxytocin or progesterone. We have demonstrated that oxytocin and progesterone can be stimulated, independently, from corpus luteum slices incubated in vitro. The pattern of release is consistent with the proposal that oxytocin, but not progesterone, is associated with and actively released from luteal secretory granules. Our results also indicated that prostaglandin F2 alpha does not directly stimulate release of oxytocin or progesterone from luteal cells in vitro.     

Evidence for the preferential utilization of esterified cholesterol in progesterone production by rabbit corpora lutea

Our previous studies show that lipoproteins stimulate progesterone secretion by rabbit luteal cells in vitro and that estradiol modifies this effect. This study examines the relationship between estradiol and serum lipoproteins for progesterone production by rabbit corpora lutea in vivo. Using morphometric analysis, we determined that estrogen treatment of hysterectomized pseudopregnant (E-hyst) rabbits increased luteal lipid volume by mid-pseudopregnancy without altering serum progesterone levels. Treatment of E-hyst rabbits with 4-amino-3,4,pyrazolo pyrimidine (APP) during early to mid-pseudopregnancy reduced serum cholesterol levels without decreasing serum progesterone concentrations. However, 3-hydroxy-3 methyl glutaryl-CoA reductase activity was increased. Thus, in the presence of exogenous estrogen, serum cholesterol is esterified and stored rather than converted directly into progesterone. APP-treatment of E-hyst rabbits during late-pseudopregnancy, when estrogen receptor levels are low, increased serum progesterone levels and reduced intracellular lipid content. Thus, stored lipid is the primary source of cholesterol for progesterone synthesis. In addition, estrogen, via estrogen receptor, is important in maintaining steady progesterone output despite fluctuations in serum lipoprotein levels. A working model for cholesterol utilization by rabbit luteal cells is presented, which suggests that stored cholesterol esters, derived from both endogenous and exogenous sources, is the key source or cholesterol for progesterone production. Furthermore, we propose that estradiol regulates the uptake and storage of cholesterol and its rate of metabolism into progesterone.     

Prostaglandin A1 metabolism and inhibition of cyclic AMP extrusion by avian erythrocytes

Prostaglandins (PG) inhibit active cyclic AMP export from pigeon red cells, PGA1 and PGA2 most potently (Brunton, L.L., and Mayer, S.E. (1979) J. Biol. Chem. 254, 9714-9720). To probe the mechanism of this action of PGA1, we have studied the interaction of [3H]PGA1 with suspensions of pigeon red cells. The interaction of PGA1 with pigeon red cells is a multistep process of uptake, metabolism, and secretion. [3H] PGA1 rapidly enters red cells and is promptly metabolized (Vmax greater than or equal to 1 nmol/min/10(7) cells) to a compound(s) that remains in the aqueous layer after ethylacetate extraction. The glutathione-depleting agent, diamide, inhibits formation of the PGA1 metabolite. In agreement with the order of potency of other prostaglandins to inhibit cAMP efflux (A much greater than E congruent to B greater than F), PGA2 forms a polar adduct whereas prostaglandins E2, B1, and F2 alpha do not. The red cells secrete the polar metabolite of PGA1 by a saturable mechanism (at 37 degrees C, Km congruent to 0.6 microM, Vmax congruent to 0.5 pmol/min/10(7) cells) that lowered temperatures inhibit (Eact congruent to 21 kcal/mol). Because uptake and metabolism progress with much greater rates than metabolite secretion, red cells transiently concentrate the polar compound intracellularly. Onset and reversal of inhibition of cyclic AMP export by PGA1 coincide with accumulation and secretion of PGA1 metabolite, suggesting that the polar metabolite acts at an intracellular site to inhibit cyclic AMP efflux. In the accompanying Appendix, we present chromatographic and amino acid analyses demonstrating that the polar metabolite is a glutathione adduct of PGA1.     

Prostaglandin production by corpora lutea of rhesus monkeys: characterization of incubation conditions and examination of putative regulators

Prostaglandins (PGs) are produced by the corpus luteum (CL) of many domestic and laboratory species and may play a role in CL regulation. The production of PGs by luteal tissue of the rhesus monkey has yet to be clearly elucidated. The production of PGE2, PGF2 alpha, and 6-keto-PGF1 alpha by CL from rhesus monkeys and the incubation conditions (time and cell number) that permit assessment of their synthesis were examined. CL (n = 3 per characterization) were surgically removed from nonpregnant monkeys during the mid-luteal phase of the menstrual cycle (approximately 8-10 days after ovulation). Luteal tissue was dissociated and the cells were incubated at varying concentrations for increasing periods of time at 37 degrees C. Subsequent to defining incubation conditions, various exogenous factors were examined for their potential to modify PG production. Indomethacin, calcium ionophore, human chorionic gonadotropin (hCG), estradiol-17 beta (E2), progesterone (P), testosterone (T), dihydrotestosterone (DHT), and 1-4-6 androstatriene-3, 17-dione (ATD) were incubated with luteal cells in increasing doses. PG and P concentrations in the medium were determined by radioimmunoassay. PGs in the medium after 6 h incubation were detectable at all cell concentrations tested (50,000, 100,000, 200,000 cells/tube). Concentrations of PGs and P increased with cell number (p less than 0.05). Luteal cells (50,000 cells/tube) were incubated for times of 0-24 h. Concentrations of P, PGE2, and PGF2 alpha in the medium were relatively low prior to incubation (0 h), increased (p less than 0.05) linearly within the first 6-12 h, and plateaued through the remaining 24 h.(ABSTRACT TRUNCATED AT 250 WORDS)     

Secretion of prostaglandins and progesterone by cells from corpora lutea of mares

Corpora lutea (CL) were collected from mares during early (Day 4-5), mid- (Day 8-9), and late (Day 12-13) dioestrus. Dispersed cell suspensions were obtained by enzymic digestion of tissue. Two distinct luteal cell populations (large and small) were observed. The proportion of small luteal cells significantly increased as age of CL advanced. Cells (2 x 10(6)) from CL which were incubated for 24 h secreted prostaglandin (PG) F, PGE-2 and 6-keto-PGF-1 alpha (the stable metabolite of prostacyclin). Higher concentrations of all PGs were produced by cells from CL at early dioestrus than from those at mid- or late dioestrus. The ratio of PGF:PGE-2 increased from 0.33 in CL of early dioestrus to 1.34 in CL of mid-dioestrus, whereas ratios of PGF:6-keto-PGF-1 alpha remained relatively constant (approximately 0.6). The ratio of PGE-2:6-keto-PGF-1 alpha from CL decreased between early (3.27) and mid-dioestrus (0.43). Addition of LH, dbcAMP, or ionophore to cell cultures did not consistently affect secretion of progesterone or PGs by luteal cells. It is suggested that prostaglandins produced by luteal cells of mares may contribute to control of luteal function and that the changing ratios of prostaglandins may be more important in controlling the lifespan of the CL than absolute concentrations of each.     

Cardiovascular actions of prostaglandins F2α; 15-me-F2α; F1α; F2β and F1β in the cat

Prostaglandin F_2α (5μg/kg, i.v.) causes an increase in pulmonary arterial pressure, decrease in systemic arterial pressure, and reflex bradycardia in the anesthetized cat. The same dose of the 15-methyl analogue of PGF_2α produces the same triad of effects but of greater magnitude and duration. Although prostaglandins F_1α, F_2β and F_1β also cause the same cardiovascular effects as F_2α, there is a decrease in potency for all parameters measured, with PGF_2α>PGF_1α>PGF_2β>PGF_1β. When compared to the actions of PGF_2α in producing an increase in pulmonary arterial pressure, PGs F_1α, F_2β and F_1β were less potent by approximately 10, 100, and 1000 fold respectively.