Reproduction in the freshwater gastropod,Helisoma: involvement of prostaglandin in egg production

Summary

The introduction of nanogram quantities of prostaglandin E₂ (PGE₂) into the female genital opening of mated animals was found to increase both the number of egg masses produced and the number of eggs per mass. The intrahaemocoelic administration of a thousand-fold higher concentration of PGE₂ was without effect, suggesting an indirect role of prostaglandin(s) in the regulation of egg production.

Prostaglandin (PG) synthetase activity in both the bursa copulatrix and ovotestis varied with the reproductive status. High PG synthetase activity was present in virgin animals, and lower activity was found in mated animals.

Prostaglandins produced by the bursa copulatrix are proposed here to be involved in the production of a matedness factor, which acts upon the brain to initiate or modulate egg production. PG produced by the ovotestis may be involved in ovulation. A model is proposed for the involvement of PG in the regulation of reproduction in Helisoma.     

Prostaglandins in the isolated testicular capsule of immature and young adult rats

The presence of prostaglandin (PGs) E₁, E₂, F_1α, F_2α and the metabolite 13, 14-Dihydro-15-keto PG Fa (1+2) has been studied in the isolated testicular capsule of Wistar rats (22 to 90 days of age). Handling and homogenization of tissues were controlled and the in vitro synthesis and degradation were prevented by immediated freezing of samples and homogenization in a solution containing of PG synthetase inhibitor. The PGs were measured by specific and sensitive radioimmuoassay. The isolated rat testicular capsule was found to contain mainly PG E₂ and PG_2α at concentrations about 100 times higher than those in decapsuled testes (ng/g of tissue).     

Prostaglandin in teh uterus: Modulation by steroid hormones

Phospholipase A₂ (PLA₂), one of the enzymes considered to be rate-limiting in generating free arachidonic acid for prostaglandin (PG) synthesis, endogenous concentrations and $\text{in vitro}$ production of PGs in the rat uterus were studied under various experimental conditions. Uterine PLA₂ activity showed a 167-fold increase in ovariectomized rats bearing estradiol-17 β (E₂)-implants as compared to those treated with vehicle only. On the other hand, dexamenthasone treatment reduced the E₂-stimulable PLA₂ activity by about 24-fold. The uterine PLA₂ activity in the ovariectomized rat uterus was low and not altered by instillation of progesterone (P₄) implants or by administration of dexamethasiome. On the contrary, simultaneously placement of E₂- and P₄-implants prevented significantly the rise in PLA₂ activity as observed under upposed E₂ exposure. Dexamethasome treatment further reduced the activity. The endogenous concentration of uterine PGF was several fold higher in the E₂-implanted ovariectomized rats as compared to those without the E₂-implants or carrying only P₄-implants. The simulataneous treatment of the E₂-implanted rats with P₄ and/or dexamethasone reduced the uterine PGF concentrations considerably. The uterine PGF concentration was always lower in the ovariectomized rats under any condition if they were not treated with E₂. Uterine PGE-A concentration did not change significantly between the ovariectomized rats and the ovariectimized rats carrying E₂-implants. The treatment with P₄ and/or dexamethasome, however, tended to decrease the PGE-A concentration. The production of PGF by the uterine homogenate increased by several fold in ovariectomized rats implanted with E₂-silastic capsules as compared to those without the e₂ implants. The treatments of the E₂-implanted rats with P₄ or dexamethasome did not alter this production. However, simultaneous exposure of E₂-implanted rats to P₄ and dexamethasone lowered the production rate of PGF in the uterus. The treatment of the ovariectomized rats with dexanethasome or P₄ tended to elevate the uterine PGF production. The uterine PGE-A production followed more or less the same pattern. The analysis of our present data suggest that although a relationship exists between uterine PLA₂ activity and PGD concentration, the role of PG synthetase could also be important in regulating PGF synthesis. Our study with dexamethasome, which showed inhibition of uterine PLA₂ activity and decline in endogenous but not $\text{in vitro}$ production of PGs, indicate that cellular integrity is essential for PLA₂ of function as a rate-limiting step in PG synthesis. The present findings also imply that alteration in PLA₂ or PG synthesis has little influence on PGE formation and the latter is not sensitive, to any great extent, to steroid hormonal changes. We considere that fluctuation in PGF/PGE ratio is mainly due to fluctuation in PGF level under various conditions.     

Studies of prostaglandins and prostaglandin antagonists on bovine pulmonary vein in vitro

Acetylsalicylic acid (ASA), indomethacin, sodium meclofenamate (FEN), phenylbutazone (PB), phloretin phosphates (PP), SC-19220, and diethylcarbamazine citrate (DECC) were screened against histamine, 5-hydroxytryptamine (5-HT), bradykinin, acetylcholine, and prostaglandins (PG) E₁, E₂, and F_2α to determine their specificity in antagonizing PG's on the bovine pulmonary vein. PG E₂ relaxed the smooth muscle preparation at low concentrations and induced contraction at higher concentrations. PG E₁ consistently evoked dose-related relaxations, whereas PG F_2α contracted the bovine pulmonary vein. Studies with inhibitors suggest that the different actions of prostaglandins could be mediated through different receptors. Sodium meclofenamate and PP dimer blocked PG E₂-induced contractions, whereas relaxations were not blocked. DECC inhibited the relaxant effect of PG E₂. DECC also antagonized histamine, 5-HT, and PG F_2α, suggesting the drug is rather non-specific. Phenylbutazone antagonized the actions of both PG E₂ and PG F_2α on the bovine pulmonary vein. By classifying receptors by antagonism the bovine pulmonary vein appears to contain PG E₂ (PP-type), PG E₂ (FEN-type), PG E₂ (PB-type), and PG F_2α (PB-type) receptors. An absence of SC-type PG-receptors is noted.     

Paradoxical endogenous synthesis of a coronary dilating substance from arachidonate

Isolated bovine, canine, and human coronary arteries exhibited dose dependent contractions to prostaglandin (PG) E₂ and F_2α (50 ng/ml to 10 μg/ml). The ED₅₀ value for both PGE₂ and PGF_2α was 500 ng/ml in the bovine and human coronary arteries. Paradoxically, although PGE₂ and PGF_2α are vasoconstrictors, administration of their precursor, arachidonate (100 ng/ml to 10 μg/ml) caused relaxation of the bovine, canine and human coronary arteries. This observation suggests that arachidonate is not being converted by the coronary PG synthetase to PGE₂ or PGF_2α. However, the arachidonate induced coronary relaxation was inhibited by pretreatment with PG synthetase inhibitors, indomethacin, meclofenemate and aspirin. Indomethacin addition to the strips previously relaxed by arachidonate caused contraction. In contrast to other PGs (E₂ and F_2α), PGE₁ (10 ng/ml to 10 μg/ml) caused dose dependent relaxation of the bovine coronary arteries (ED₅₀ = 100 ng/ml). Indomethacin induced further relaxation of the blood vessels previously relaxed by PGE₁. Since PGE₁ cannot arise from arachidonate, the arachidonate coronary dilation and reversal by indomethacin must be independent of PGE₁ formation. Linolenate (100 ng/ml to 10 μg/ml) and oleate (100 ng/ml to 10 μg/ml) also caused relaxation of the bovine coronary blood vessels both before and after indomethacin, thereby eliminating a direct non-specific fatty acid effect as the cause of the arachidonate relaxation. These results suggest that in isolated coronaries, arachidonate undergoes a novel conversion, possibly by PG synthetase, to a dilating substance which exerts different contractile effects than exogenously administered PGE₂, PGF_2α and PGE₁.This work was supported by (USPHS) training grants NS 05221, RCDA (P.N.) HL-19586, HL-11771A, HL-14397 and SCOR grant HL-17646, HL-17646-0.     

UP-regulaton of prostaglandin E2 binding and of prostanoid release in rabbits producing antibodies

German Giant rabbits successfully immunized agianst prostaglandin (PG) E₂ as shown by a rise in antibody titers developed gastric mucosal lesions. Enzymatically dispersed gatric mucosal cells of these animals had a significantly enhanced production of PG₂ and PG I₂ as measured by specific radioimmunoassays. This may be explained by an increased supply with endogenous arachidonic acid (as indicated by an enhanced phospholipase A₂/LAT ratio) and by a higher activity of the subsequent PG forming enzymes (as indicated by a more effectvie stimulation of PG production by exogenous arachidonic acid). Gastric mucosal plasma membranes of immunized rabbits had significantly high PG E₂ binding capacity (108 ± 9 fmol/mg protein) than those of nomimmunized rabbits (72 ± 5 fmol/mg protein). The ligand affinity was not afected by immunization. Neither histamine-stimulated ¹⁴C-aminopyrine uptake of isolated parietal cells as a marker for acid production nor its inhibition by PG E₂ were influenced by receptor up-regulation. The increased eicosanoid release can be regarded as an endogenous defense emchanism against increased mucosal vulnerability caused by PG E₂ scavenging. The potential role of PG E₂ receptor up-regullation in support of this process remains to be established.     

The Role of Progestogens in Regulating Matrix Metalloproteinase Activity in Macrophages and Microglial Cells

Although the systemic effects of progestogens have been extensively studied, little is known in regards to the cellular effects of these compounds. Using a cellular model for vascular (macrophages) and brain (microglial) cells, we studied the effects of various progestogens, either alone or in combination with 17β-estradiol (E₂) on the activity of matrix metalloproteinase-9 (MMP-9), a proteolytic enzyme involved in vascular remodeling and plaque destabilization in cardiovascular events, blood–brain barrier breakdown in stroke and brain regeneration and neurovascular remodeling during repair phases of brain injury. In the absence of E₂, medroxyprogesterone acetate (MPA), a synthetic progestogen and progesterone (PG) metabolites tended to increase MMP-9 enzyme activity in macrophages and microglial cells, whereas PG decreased such activity in macrophages; exceptions being that MPA and the PG metabolite, pregnanediol (Pdiol) had no effect on macrophage MMP-9 enzyme activity and PG had no effect on microglial cell MMP-9 enzyme activity. In the presence of E₂, an opposite affect was observed whereby MPA and the PG metabolites tended to decrease MMP-9 enzyme activity from macrophages and microglial cells, whereas PG had no effect; exceptions being that MPA and Pdiol had no effect on macrophage MMP-9 enzyme activity. In conclusion, these results demonstrate that the effects of PG, PG metabolites and MPA on MMP-9 enzyme activity differ across vascular and brain cells when administered alone or in combination with E₂ which could have important mechanistic implications for hormone therapy.     

Characterization of prostaglandin synthesis in the housefly,Musca domestica (L.)

The in vitro formation of prostaglandins (PG) was examined in the housefly Musca domestica. PG synthetase activity was detected in homogenates of whole insects and in head and thorax, abdomen, ovary and male reproductive tissues. Studies to determine the sub-cellular localization of PG synthetase indicated that the microsomal fraction contained the highest activity. Products obtained from radiolabeled arachidonic acid (20:4) and 8,11,14-eicosatrienoic acid (20:3, n-6) were PGE₂ and PGE₁, respectively, with lower amounts of the PGF series also present. In microsomal preparations from whole insects and reproductive tissues from both males and females, 20:3(n-6) was 2–2.5 times more efficiently converted to PG than was 20:4. Non-steroidal anti-inflammatory drugs (NSAID) fed to houseflies did not inhibit PG production from 20:4, whereas when they were included in microsomal preparations at high levels, they inhibited PG synthesis.     

Prostaglandin and prostaglandin synthetase in the cricket, Acheta domesticus

Prostaglandin (PG) synthetase was present in the testes, seminal vesicles, and spermatophores of the male house cricket, Acheta domesticus. The enzyme was not detected in bursa copulatrix, spermatheca, spermathecal canal, and oviducts from virgin females, while substantial activity was measured in the same tissue from mated females. The female appears to receive the enzyme from the spermatophore. A PGE₂-like material was detected by radioimmunoassay in A. domesticus testes and to a lesser extent in the remainder of the male reproductive tract. PG went undetected in virgin female reproductive tissues, while the same tissues from mated females contained an average of 589 pg of PGE₂-like material per female. In in vivo studies, injected PGE₁, PGE₂, and to a smaller degree PGF_2α stimulated oviposition by virgin females. Moreover, N-acetyl-p-aminophenol, a PG synthetase inhibitor, suppressed oviposition in mated females. Post-copulatory PG biosynthesis in the female reproductive tract might be partially responsible for triggering oviposition in A. domesticus. Since PG synthetase appears to be acquired from the male, it could be considered a primer pheromone.     

Action of luteinizing hormone-releasing hormone and synthesis of prostaglandin in the pituitary gland

The possibility that prostaglandin E₂ (PGE₂) may play a role in luteinizing hormone (LH) release was examined using an model. Addition of luteinizing hormone-releasing hormone (LH-RH) to the culture medium stimulated cyclic AMP accumulation and LH-release by incubated hemipituitaries, but did not affect the level of PGE₂ or prostaglandin synthetase activity in the gland. Aspirin and indomethacin reduced both prostaglandin synthetase activity and PGE₂ content in the pituitary, but did not impair the stimulatory action of LH-RH on either cyclic AMP accumulation or LH-release. Flufenamic acid on its own caused LH-release, but the drug abolished the effect of LH-RH on cyclic AMP accumulation. The mechanism of this action of flufenamic acid is not understood.It is concluded that the stimulatory action of LH-RH on pituitary cyclic AMP production and LH release is not mediated by prostaglandins.     

Prostaglandis show marked differences when tested against early ischemic dysrhythmias in rat

The effects of prostaglandins (PGs) F_1α, I₂, F_2β, and E₂, together with acetylsalicylic acid (ASA) and quinidine were compared on arrhythmias following ligation of the left anterior descending coronary artery in anesthetized rats. An objective method of assessing arrhythmias based on the severity and duration of a sudden fall in blood pressure (BP) was used together with changes in the electrocardiogram (ECG) to quantify the data. When infused at 2 mcg/kg/min pre-ligation, PGs F_1α and I₂ increased the severity of the arrhythmias while F_2β and E₂ decreased the severity. Only the results with PGE₂ were statistically significant. The most effective PG, E₂, produced a 69 per cent decrease in arrhythmic score compared to a 93 per cent decrease with quinidine. ASA produced a non statistically significant 43 per cent decrease in arrhythmic score. PGE₂ had no significant effect when infused post-ligation. None of the PGs had any marked effect on maximum following frequency in $\text{in vivo}$ rat heart when infused 1–10 mcg/kg/min. These data indicate that PGs show marked specificity depending on the prostaglandin in their action on early ischemic arrhythmias. The site of PG antiarrhythmic activity may be in the occluded zone, and the antiarrhythmic action of PGs cannot be explained by their effects on electrical refractoriness in the heart.     

Prostaglandin synthetase and prostaglandin e2 levels in human breast carcinoma

Prostaglandin synthetase activity and tissue prostaglandin E levels were measured in 53 human breast carcinoma specimens. Each set of data is lognormally distributed. There is a weak but statistically significant correlation between prostaglandin synthetase activity and tissue prostaglandin E₂ levels of individual specimens. In addition, in a series of four rat mammary tumors, a rough correlation between prostaglandin synthetase activity and tissue prostaglandin E₂ levels was observed. The data suggest a multifactorial explanation for abnormalities of prostaglandin metabolism in human breast cancer.     

Prostaglandin biosynthesis and catabolism in the developing fetal sheep kidney

The enzymatic capacity to form and degrade prostaglandins was studied in kidneys from fetal sheep (gestational ages 40,44,49,77,116 and 140 days). The prostaglandin system was detectable at all ages. Only prostaglandin F_2α was formed by renal homogenates at 40 and 44 days gestation; prostaglandin E₂ was first formed by the 77 day kidney and became the major prostaglandin by 116 days (3 fold relative to prostaglandin F_2α). Prostaglandin catabolism took place via the PG 15-hydroxy dehydrogenase and PG 13-reductase pathways. Catabolism was first detected at 40 days gestation and rose with age to an activity (15-PGDH) approximately 80 ng/min/mg protein in the term kidney. Only PG 15-hydroxydehydrogenase activity was detected at 40 days gestation, but PG 13-reductase activity became evident by 116 days and persisted until term. As with fetal sheep lungs (see preceding publication) PG 13-reductase activity was saturated quickly. These results confirm our observations with other tissues that prostaglandin catabolism is variable during ontogeny.     

Properties of solubilized prostaglandin synthetase from sheep vesicular glands.

Prostaglandin synthetase activity associated with the microsomal fraction from sheep vesicular glands has been solubilized by treatment with the non-ionic detergents Tween 20, Lubrol Px and Lubrol Wx. Approx.8 fold purification from microsomes is obtained and over 90% of the activity is recovered in the detergent solubilized fraction. The solubilized synthetase activity is stable at pH 5.0 but is gradually lost at pH 8.0; it is also heat and acid labile. The relative amounts of prostaglandins E₂, D₂ and F_2α formed by the microsomal-bound synthetase and by the solubilized synthetase are similar. Also similar are the pH optima (7.9–8.5) of the two synthetase preparations. The solubilization process appears to yield a fully active enzymatic preparation which could be employed for further purification and characterization of the prostaglandin synthetase complex.     

Influence of prostaglandins and of cyclic nucleotides on the metabolism of 25-hydroxyvitamin D3 in primary chick kidney cell culture

Prostaglandins (PG) are likely to be involved in a number of regulatory mechanisms in the kidney, which may be mediated by cyclic nucleotides. The present study describes the effects of prostaglandins and cyclic nucleotides on the hydroxylases of 25-hydroxycholecalciferol (25(OH)D₃) in a primary chick kidney cell culture. 3–30 nM PG E₂ produced significant increases in the 25(OH)D₃-1-hydroxylase associated with decreases in the 25(OH)D₃-24-hydroxylase at 6 hours but not at 1 hour. PG F_2α, in concentrations between 0.3 nM and 3 μM affected the hydroxylases in a similar manner. A significant increase of 1-hydroxylase activity was observed with 0.1 mM cyclic AMP (cAMP) or dibutyryl cyclic AMP (dbcAMP) in 6 hours, but again no effect on either hydroxylase was observed when the incubation time was reduced to 1 hour. These results suggest that PG E₂ and PG F_2α might be involved in the regulation of renal 25(OH)D₃ metabolism, and that the effects on the 25(OH)D₃-hydroxylases might be mediated by cAMP.     

Evidence for a cytosolic-dependent light induction of chloroplastic glutamine synthetase during greening of etiolated rice leaves

During the greening of etiolated rice leaves, total glutamine synthetase activity increases about twofold, and after 48 h the level of activity usually observed in green leaves is obtained. A density-labeling experiment with deuterium demonstrates that the increase in enzyme activity is due to a synthesis of the enzyme. The enhanced activity obtained upon greening is the result of two different phenomena: there is a fivefold increase of chloroplastic glutamine synthetase content accompanied by a concommitant decrease (twofold) of the cytosolic glutamine synthetase. The increase of chloroplastic glutamine synthetase (GS₂) is only inhibited by cycloheximide and not by lincomycin. This result indicates a cytosolic synthesis of GS₂. The synthesis of GS₂ was confirmed by a quantification of the protein by an immunochemical method. It was demonstrated that GS₂ protein content in green leaves is fivefold higher than in etiolated leaves.Abbreviations AbH heavy chain of antibodies - AbL light chain of antibodies - AP acid phosphatase - CH cycloheximide - G6PDH glucose-6-phosphate dehydrogenase - GS glutamine synthetase - GS₁ cytosolic glutamine synthetase - GS₂ chloroplastic glutamine synthetase - LC lincomycin - NAD-MDH NAD malate dehydrogenase - NADP-G3PDH NADP glyceraldehyde-3-phosphate dehydrogenase     

Characterization of imidazo[1,5-a]pyridine-5-hexanoic acid (CGS 13080) as a selective thromboxane synthetase inhibitor using in vitro and in vivo biochemical models

CGS 13080 inhibited cell-free thromboxane synthetase with an IC₅₀ of 3 nM. It was at least five orders of magnitude less potent toward other key enzymes involved in arachidonic acid metabolism. Submicromolar concentrations inhibited calcium ionophore-induced formation of thromboxane B₂ by intact human platelets with concomitant accumulation of prostaglandin E₂. Oral doses lower than 1 mg/kg in rats suppressed the elevations of plasma thromboxane B₂ induced by calcium ionophore. This was attended by shunting of endoperoxide substrate to 6-keto-prostaglandin F₁α and prostaglandin E₂. CGS 13080 is one of the most potent and selective thromboxane synthetase inhibitors yet identified.     

Prostaglandin synthetase activity of goat vesicular microsomes: Co-factor requirement and effect of calcium ions

The effects of several co-factors and bivalent cations on the activity of prostaglandin synthetase isolated from goat seminal vesicles were studied. Ca²⁺ appears to play a regulatory role in the biosynthesis of prostaglandin E₂ by goat vesicular microsomes as the normal parabolic time course of synthesis changed to a sigmoid curve in the presence of 4 mM Ca²⁺ and to nearly a hyperbolic pattern when the microsomes were preincubated with the metal ions. The Ca²⁺ modulated reaction showed increased rate of prostaglandin E₂ synthesis only when the period of incubation was extended beyond 30 min. The co-factor requirement of the goat enzyme was similar to that of the bovine and ovine prostaglandin synthetase systems.     

Neurological and electroencephalographic changes in newborns treated with prostaglandins E2 and E2

Six newborns with obstructive right heart lesions were examined neurologically and electroencephalographically during treatment with prostaglandin (PG) E₁ or E₂ given to maintain patency of the ductus arteriosus and to increase pulmonary blood flow. PG was administered intravenously or intraarterially in the aortic isthmus proximal to the ductus arteriosus. Besides a rise in arterial oxygen saturation, all patients had some sign of central nervous system involvement. The electroencephalogram showed minor changes suggestive of sedation. In addition, three patients in whom PG given intravenously presented various combinations of neurological abnormalities (“myoclonic jerks”, apnoeic spells, hiccup) of subcortical origin. Side-effects subsided after stopping the treatment anf posed no problem in the management of the patients. These findings confirm the usefulness and safety of the PG therapy and indicate that the intraaortic route of administration is preferable.     

Prostaglandin (PG) analysis in urine of humans and rats by different radioimmunoassays: Effect on PG-excretion by PG-synthetase inhibitors, laparotomy and furosemide

Thw radioimmunological (RIA) determination of prostaglandin (PG) E₂ and of PGF_2α in urine humans and rats is described in detail. After extraction and chromatography PGE₂ was determined by using a PGE specific antibody or by using either PGB or PGF_2α specific antibodies after the respective conversion procedures. The three different RIA procedures were compared to each other. PGF_2α was determined by a specific antibody to PGF_2α. Basal excretion of PGE₂ and of PGF_2α in healthy women on free diet was 9.3 ng/hour ± 0.96 and 18.3 ng/hour ± 2.5 respectively. Furosemide increased the excretion of PGE₂ and of PGF_2α in humans significantly, while PG-excretion rates decreased on indomethacin. In rat urine PGE₂ and PGE_2α increased markedly from 46.2 pg/min ± 9.3 and 27 ± 3.4 to 253.8 ± 43.3 and 108 ± 12.6 pg/min (per one kidney) in the anesthetized-laparotomized animal. This increase was abolished after giving two different PG synthetase inhibitors.