Prostaglandin F2a 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 F_2a (PGF_2a. The stimulatory effect of epinephrine on tissue levels of cAMP in 1-day-old corpora lutea was not inhibited by PGF₂. A dose-dependent inhibition by PGF_2a (0.5–50 μM) was seen for 3-day-old corpora lutea and this inhibition could not be overcome by higher concentrations of epinephrine (0.165–165 μM). The stimulation by epinephrine on progesterone production was inhibited by PGF_2a (5 μM) in 3- and 5-day-old, but not in 1-day-old corpora lutea. Thus, PGF_2a 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 PGF_2a shows in this respect the same agedependent inhibitory pattern as in relation to LH stimulation.     

Prostaglandin F2alpha specific binding in equine corpora lutea

Preliminary studies indicate the presence of PGF2alpha specific binding sites in membrane fractions prepared from equine corpora lutea. The equilibrium binding data indicate an apparent dissociation constant of 3.2 X 10(-9)M and the concentration of binding sites of -0.1 pmoles/mg membrane protein. Competition of several natural prostaglandins for equine luteal PGF2alpha specific binding sites indicates specificity for the 9alpha-hydroxyl moiety and the 5,6-cis doublebond. Significant increases in relative binding affinities were demonstrated for PGF2alpha analogs with a phenyl ring introduced at carbons 16 or 17. Specific PGF2alpha 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 PGF2alpha specific binding was significantly modified by both these factors.     

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.     

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 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.     

Luteinizing hormone receptors and progesterone content in porcine corpora lutea after prostaglandin F2 alpha

The effect of prostaglandin F2 alpha (PGF2 alpha) on luteinizing hormone (LH) receptors, weight and progesterone content of corpora lutea (CL), and serum progesterone concentrations was studied in gilts. Fifteen gilts were hysterectomized between Days 9 to 11 of the estrous cycle. Twelve gilts were injected i.m. with 10 mg of PGF2 alpha and 3 with saline on Day 20. Ovaries were surgically removed from each of 3 gilts at 4, 8, 12 and 24 h following PGF2 alpha treatment and from the 3 control gilts 12 h following saline injection. Jugular blood samples for progesterone analysis were collected from all gilts at 0, 2 and 4 h following treatment and at 8, 12 and 24 h for gilts from which ovaries were removed at 8, 12 and 24 h, respectively. Mean serum progesterone and CL progesterone concentrations decreased within 4 h after PGF2 alpha treatment (P less than 0.05) and remained low through 24 h after treatment. The number of unoccupied LH receptors decreased by 4 h (P less than 0.05) and this trend continued through 24 h. There were no differences in luteal weight or affinity of unoccupied LH receptors of luteal tissue at 4, 8 12 and 24 h after PGF2 alpha when compared to luteal tissue from controls. These data indicate that during PGF2 alpha-induced luteolysis in the pig, luteal progesterone, serum progesterone concentrations and the number of LH receptors decrease simultaneously.     

Prostaglandin F2 alpha-induced release of oxytocin from bovine corpora lutea in vitro

Two experiments were conducted to study the in vitro effects of prostaglandins F2 alpha (PGF2 alpha), E2 (PGE2), and luteinizing hormone (LH) on oxytocin (OT) release from bovine luteal tissue. Luteal concentration of OT at different stages of the estrous cycle was also determined. In Experiment 1, sixteen beef heifers were assigned randomly in equal numbers (N = 4) to be killed on Days 4, 8, 12, and 16 of the estrous cycle (Day 0 = day of estrus). Corpora lutea were collected, an aliquot of each was removed for determination of initial OT concentration, and the remainder was sliced and incubated with vehicle (control) or with PGF2 alpha (10 ng/ml), PGE2 (10 ng/ml), or LH (5 ng/ml). Luteal tissue from heifers on Day 4 was sufficient only for determination of initial OT levels. Luteal OT concentrations (ng/g) increased from 414 +/- 84 on Day 4 to 2019 +/- 330 on Day 8 and then declined to 589 +/- 101 on Day 12 and 81 +/- 5 on Day 16. Prostaglandin F2 alpha induced a significant in vitro release of luteal OT (ng.g-1.2h-1) on Day 8 (2257 +/- 167 vs. control 1702 +/- 126) but not on Days 12 or 16 of the cycle. Prostaglandin E2 and LH did not affect OT release at any stage of the cycle studied. In Experiment 2, six heifers were used to investigate the in vitro dose-response relationship of 10, 20, and 40 ng PGF2 alpha/ml of medium on OT release from Day 8 luteal tissue.(ABSTRACT TRUNCATED AT 250 WORDS)     

Prostaglandin E1 and F2alpha specific binding in bovine corpora lutea: comparison with luteolytic effects.

Preliminary characterization indicated the presence of separate prostaglandin (PG)E1 and (PG)F2alpha 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 x 10(-9)M and 1.1 x 10(-8)M for PGE1 and PGF2alpha, respectively. Competition of several natural prostaglandins for the PGE1 and PGF2alpha bovine luteal specific binding sites indicates specificity for the 9-keto or 9alpha-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 PGF2alpha as measured by reduced estrous cycle length, decreased corpus luteum size and significantly decreased plasma progesterone levels. In contract, 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 PGF2alpha relative binding affinity to the bovine CL can be compared to data obtained independently in vitro on PGF2alpha induced luteolysis in the bovine, PGF2alpha 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.     

Lipid metabolism and production of progesterone and prostaglandin F during induced regression of porcine corpora lutea

Patterns of luteal lipid and arachidonic acid accumulation were examined in relation to luteal progesterone and prostaglandin F synthesis in 30 sows and gilts between days 8 and 18 of the estrous cycle. Net release of progesterone from luteal tissue declined from 722 ng/100 mg tissue at day 8 to 81 ng/100 mg tissue at day 18. Although statistical significance was not present, net prostaglandin F release increased slightly from 8.6 to 13.9 ng/100 mg tissue. Luteal free cholesterol, esterified cholesterol, and free fatty acid contents did not change between days 8 and 18 whereas triglycerides accumulated rapidly between days 14 and 18 of the estrous cycle. Phospholipids increased between days 8 and 12, plateaued at 20.2 mg/g between days 14 and 16, and decreased to 15.4 mg/g on day 18. Between days 12 and 18, arachidonic acid increased from 19.4 to 34.8% in cholesterol esters, from 10.1 to 22.5% in triglycerides, and from 12.3 to 27.2% in luteal free fatty acids. Arachidonic acid in luteal phospholipids increased from 21.3 to 25.1% between days 14 and 16 of the estrous cycle. Luteal regression was associated with conservation of arachidonic acid. Based on blood plasma lipid fatty acid compositions, the corpus luteum elongated and desaturated essential fatty acids. Within porcine corpora lutea, calculated free arachidonic acid content was adequate for maintenance of prostaglandin synthesis.     

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 F2 alpha on release of progesterone and leukotriene B-4 by cells from corpora lutea of mares.

Corpora lutea were recovered from mares either 4 to 5 days or 12 to 13 days after ovulation. Mixed populations of luteal cells were prepared by collagenase digestion and were incubated for 24 h in the presence or absence of prostaglandin (PG) F-2 alpha (250 ng/ml). PGF-2 alpha significantly (P = 0.03) reduced progesterone secretion by cells from late diestrous corpora lutea and tended (P = 0.06) to reduce secretion by early diestrous cells. PGF-2 alpha had no significant effect on leukotriene B-4 (LTB-4) production by cells from early diestrous corpora lutea, but significantly (P = 0.03) increased LTB-4 production by late diestrous luteal cells. It seems possible that LTB-4 could play a role as an intermediary in the action of PGF-2 alpha in luteolysis in the mare.     

Sensitivity of bovine corpora lutea to prostaglandin F2alpha is dependent on progesterone, oxytocin, and prostaglandins.

Prostaglandin (PG) F2alpha that is released from the uterus is essential for spontaneous luteolysis in cattle. Although PGF2alpha and its analogues are extensively used to synchronize the estrous cycle by inducing luteolysis, corpora lutea (CL) at the early stage of the estrous cycle are resistant to the luteolytic effect of PGF2alpha. We examined the sensitivity of bovine CL to PGF2alpha treatment in vitro and determined whether the changes in the response of CL to PGF2alpha are dependent on progesterone (P4), oxytocin (OT), and PGs produced locally. Bovine luteal cells from early (Days 4-5 of the estrous cycle) and mid-cycle CL (Days 8-12 of the estrous cycle) were preexposed for 12 h to a P4 antagonist (onapristone: OP; 10(-4) M), an OT antagonist (atosiban: AT; 10(-6) M), or indomethacin (INDO; 10(-4) M) before stimulation with PGF2alpha. Although OP reduced P4 secretion (p < 0.001) only in early CL, it reduced OT secretion in the cells of both phases examined (p < 0.001). OP also reduced PGF2alpha and PGE2 secretion (p < 0.01) from early CL. However, it stimulated PGF2alpha secretion in mid-cycle luteal cells (p < 0.001). AT reduced P4 secretion in early and mid-cycle CL (p < 0.05). Moreover, PGF2alpha secretion was inhibited (p < 0.05) by AT in early CL. The OT secretion and the intracellular level of free Ca2+ ([Ca2+]i) were measured as indicators of CL sensitivity to PGF2alpha. PGF2alpha had no influence on OT secretion, although [Ca2+]i increased (p < 0.05) in the early CL. However, the effect of PGF2alpha was augmented (p < 0.01) in cells after pretreatment with OP, AT, and INDO in comparison with the controls. In mid-cycle luteal cells, PGF2alpha induced 2-fold increases in OT secretion and [Ca2+]i. However, in contrast to results in early CL, these increases were magnified only by preexposure of the cells to AT (p < 0.05). These results indicate that luteal P4, OT, and PGs are components of an autocrine/paracrine positive feedback cascade in bovine early to mid-cycle CL and may be responsible for the resistance of the early bovine CL to the exogenous PGF2alpha action.     

Functional alpha 2-adrenoceptors in rat adipocytes: inhibition of cyclic AMP accumulation

Alpha 2-adrenoceptor activation inhibits cyclic AMP accumulation in fat cells from many species. However, the presence of alpha 2-adrenoceptors in rat adipocytes has been difficult to demonstrate. We observed that alpha 2-adrenergic activation inhibits forskolin-stimulated cyclic AMP accumulation both in rat and hamster adipocytes; UK 14304, p-amino clonidine and clonidine were the agents with higher efficacy. The effect of UK 14304 was blocked by yohimbine but not by prazosin demonstrating the involvement of alpha 2-adrenoceptors. Pertussis toxin blocked the alpha 2-adrenergic effect. Our results demonstrate the presence in rat fat cells of alpha 2-adrenoceptors coupled to adenylate cyclase via "Gi".     

Prostaglandin E2 and F2 alpha inhibit growth of human gastric carcinoma cell line KATO III with simultaneous stimulation of cyclic AMP production

The effects of prostaglandins (PGs) on the growth of human gastric carcinoma cell line KATO III were investigated. PGE2 as well as PGF2 alpha significantly and dose-dependently inhibited the growth of this gastric carcinoma cell line (PGE2 greater than PGF2 alpha). This inhibition of cell growth by the PGs was associated with the increase in cyclic AMP production (PGE2 greater than PGF2 alpha), whereas inositol-phospholipid turnover was not affected by either PGE2 or PGF2 alpha as assessed by the formation of 3H-inositol phosphates. Furthermore, the proliferation of these gastric carcinoma cells was also suppressed by the administration of forskolin as well as of dibutyryl cyclic AMP. These results suggest that PGE2 and PGF2 alpha inhibit the growth of cultured human gastric carcinoma cells KATO III via stimulation of cyclic AMP production.