Effect of prostaglandin F2 alpha (PGF2 alpha) on oviductal nitric oxide synthase (NOS) activity: possible role of endogenous NO on PGF2 alpha-induced contractions in rat oviduct

The objective was to analyse differences in the epidemiological pattern of sudden death in infancy during two time periods--the Sudden Infant Death Syndrome (SIDS) 'epidemic': 1984-1989, and the period of rapid decline in the SIDS rate 1990-1996. Sex distribution, age, sleeping position, signs of infection, day of the week and place of death were registered and compared for the two time periods studied in all SIDS cases autopsied at the Institute of Forensic Medicine, Oslo. There were significantly more deaths in the age group under four months in the period 1984-89 than in the second period. Prone sleeping position, signs of infection, death outdoors and during the winter were more frequent during the first period than in the second. These features also were more frequent in the age group under four months than in the older babies during the first period. The shift in the epidemiological pattern after 1990, when the risk factor campaign was launched, indicates that prone sleeping position, cold climate, sleeping outdoors and infections seem to be risk factors that are particularly harmful to the youngest infants.     

Functional characterization of the ocular prostaglandin f2alpha (PGF2alpha) receptor. Activation by the isoprostane, 12-iso-PGF2alpha

Prostaglandin F2alpha (PGF2alpha) is a product of cyclooxygenase-catalyzed metabolism of arachidonic acid. Recently, PGF2alpha analogs have been hypothesized to reduce intraocular pressure via relaxation of the ciliary muscle. To investigate the molecular basis of PGF2alpha receptor (FP) activation in the eye, we cloned the FP from a human ciliary body (hcb) cDNA library. The open reading frame of the hcb-FP cDNA was identical to the uterine FP cDNA. The hcb-FP appeared to be predominantly membrane-localized, as visualized by an FP-specific peptide antibody, and coupled to inositol phosphate formation when stably expressed in HEK 293 cells. Interestingly, the hcb-FP could also be activated by the F2 isoprostane, 12-iso-PGF2alpha, in addition to its cognate ligand, PGF2alpha. 12-iso-PGF2alpha was less potent (EC50 = 5 microM) than PGF2alpha (EC50 = 10 nM) in generating inositol phosphates via the hcb-FP in HEK 293 cells. Both ligands also stimulated mitogenesis in NIH 3T3 cells. Although 12-iso-PGF2alpha caused a dose-dependent activation of the FP, it failed to activate the recombinant human prostacyclin receptor and caused only minimal activation of the thromboxane receptor isoforms stably expressed in HEK 293 cells. Four additional F2 isoprostanes, 8-iso-PGF2alpha, IPF2alpha-I, IPF2alpha-III, and 9beta,11beta-PGF2, caused trivial, or no, activation of the FP. Consistent with these observations, only PGF2alpha and 12-iso-PGF2alpha caused rapid homologous desensitization of FP and also exhibited cross-desensitization, with PGF2alpha resulting in a maximum of approximately 60% desensitization. The human FP may thus be activated specifically, by the free radical-catalyzed F2 isoprostane, 12-iso-PGF2alpha, in addition to the cyclooxygenase product, PGF2alpha. Incidental receptor activation by isoprostanes may complement the actions of PGF2alpha in clinical syndromes where oxidant stress and augmented prostaglandin biosynthesis coincide.     

Effect of mifepristone and antiestrogens on uterine PGF2 alpha and PGE2 concentrations in ovariectomized and pregnant rats

To clarify the role of histamine in uterine contractility, the effect of this biogenic amine on the myometrium of cyclic mature gilts was investigated by an isometric tension recording study in vitro. In addition, using crude membrane preparations isolated from the longitudinal (LM) and circular muscle (CM), the distribution of H1 histamine receptors was characterized by 3H-pyrilamine binding assay. Histamine caused a tetrodotoxin-resistant contractile response of LM and CM in Krebs solution, but LM (-logEC50 = 6.34) was more sensitive than CM (-logEC50 = 5.4). Pyrilamine decreased the excitatory response of histamine in both muscle layers. In pyrilamine-treated LM, a high concentration of histamine (1-30 microM) caused a slight inhibition of spontaneous contraction, and this inhibition was abolished by ranitidine. On the other hand, histamine did not cause any inhibition in the pyrilamine-treated CM preparations. Dimaprit (10-300 microM) concentration-dependently inhibited the spontaneous contraction of LM but not of CM. In the presence of pyrilamine and ranitidine, N alpha-methylhistamine, even at 10 microM, did not affect the spontaneous and electrical field stimulation (5Hz)-induced contraction of LM and CM layers. Specific 3H-pyrilamine binding sites were distributed heterogeneously in the swine myometrium. The maximum number of binding sites in LM (132.5 +/- 9.9 fmol/mg protein, n = 10) was 2.5 times higher than that in CM (52.2 +/- 3.2 fmol/mg protein, n = 6). These results indicate that there is a muscle layer-dependent difference of histamine-induced response in the swine myometrium. In the LM layer, histamine acts on both H1 and H2 histamine receptors, and causes contraction (via H1 receptors at a low concentration) or relaxation (via H2 receptors at a high concentration in the presence of pyrilamine). However, histamine causes only a contraction in the CM layer, likely the result of the absence of H2 histamine receptors. Histamine-induced contraction is conspicuous in the LM layer, because of the heterogeneous distribution of H1-receptors between LM and CM.     

Effect of prostaglandin F2 alpha-and gonadotropin releasing hormone-induced luteinizing hormone releases on ovulation and corpus luteum function of beef cows

Luteinizing hormone (LH) concentrations were measured in suckled beef cows treated during the postpartum period with prostaglandin F2 alpha (5 mg Alfaprostol; PGF2 alpha) and then gonadotropin releasing hormone (100 micrograms Cystorelin 30 h after PGF2 alpha; GnRH). The objective was to determine if PGF2 alpha would cause a release of LH in the absence of progesterone and affect the GnRH-induced LH release and ovulation (Experiment 1). LH concentrations increased (P < 0.05) after PGF2 alpha treatment in both anestrous and cyclic cows but to a greater extent (P < 0.05) in anestrous cows. The GnRH-induced LH release and ovulation response in previously anestrous cows were greater (P < 0.05) when PGF2 alpha was administered 30 h earlier. In Experiment 2, 49 beef cows received PGF2 alpha (5 mg Alfaprostol) and GnRH (100 micrograms Cystorelin) 30 h later to determine if the profile of the preovulatory LH surge was associated with the occurrence of subnormal luteal phases in postpartum beef cows suckling calves. Cows that had normal luteal phases had a greater (P < 0.05) mean area under the GnRH-induced LH response curve and a greater (P < 0.05) mean GnRH-induced LH peak amplitude than cows that had subnormal luteal phases. In summary, results suggest that PGF2 alpha may exert a fertility effect by causing a LH release independent of progesterone withdrawal; administration of PGF2 alpha 30 h before GnRH elevated the GnRH-induced LH release and ovulation response. In addition, cows with subnormal luteal phases had GnRH-induced LH surges of less area and peak amplitude than cows with normal luteal phases.     

Reduction by arachidonic acid of prostaglandin I2-induced cyclic AMP formation. Involvement of prostaglandins E2 and F2 alpha

Arachidonic acid reverses the increase in cyclic AMP levels of washed human platelets exposed to prostaglandin (PG)I2, under conditions where the PGH2 analogue U46619 is ineffective. This effect of arachidonic acid was inhibited by aspirin, a cyclooxygenase inhibitor, but not by the thromboxane (Tx) synthase inhibitor Ridogrel, which induces, by inhibiting the conversion of PGH2 into TxA2, an overproduction of PGE2, PGD2 and PGF2 alpha. Addition of PGE2 or PGF2 alpha, which share a receptor with PGI2, to washed human platelets also induced a decrease in cyclic AMP levels, but PGD2, which interacts with a different receptor, had no effect. Thus neither PGD2, PGG2, PGH2, TxA2 nor TxB2 formed from arachidonic acid via the cyclooxygenase pathway is involved in the decrease in cyclic AMP levels. These findings were confirmed using forskolin, a diterpene from the labdane family, which enhanced the formation of cyclic AMP synergistically with the PGs. Also, arachidonic acid, unlike U46619, is able to reverse the inhibition of platelet aggregation by PGI2 after a lag phase of about 4 min. Our data indicate that arachidonic acid decreased cyclic AMP levels through its cyclooxygenase metabolites PGE2 and PGF2 alpha probably interacting competitively with the receptor of PGI2. In addition, intracellular cyclic AMP levels and the degree of aggregation of platelets by arachidonic acid seem to be inversely correlated.     

The effect of PGE2 and PGF2alpha on the response of guinea pig myometrium to adenine nucleotides in vitro

Prostaglandins E2 and F2alpha potentiate contractile effect induced by adenine nucleotides ATP, ADP and AMP in guinea pig myometrium in vitro. Prostaglandins and nucleotides were added to the organ bath in minute concentrations which have been proved ineffective or slightly contractile when both groups of substances were administered separately. The data of the present work, together with our previously published studies (9, 10, 13), where the action of exogenous adenine nucleotides, NAD and adenosine on rabbit's jejunum in vitro has been proved antagonistic to the contractile effect of various prostaglandins, suggest that prostaglandins and adenine nucleotides appear to block selectively or augment each other's action on various organs. The initial hypothesis that there is a regulatory correlation between endogenous prostaglandins and the function of purinergic nerves also is reinforced.     

Inducing labor with intracervical prostaglandin (PG) gel administration. A comparative analysis of 171 patients with 2 different preparations of PGE2 with regard to course of labor and fetal outcome

In the present study we examined whether the difference in viscosity of two prostaglandins applied intracervically for induction of labour influences course of labour and fetal outcome. A comparative analysis was carried out in 171 patients, which received either Cerviprost (n = 85) or Prepidil (n = 86) at 6 hour intervals. The results show that the viscosity of the PG E2 has no influence on the course of induction of labour, the mode of delivery or on fetal outcome. The low viscosity does also not present an increased risk for uterine overstimulation, even in case of intracervical application.     

Gene expression in a subpopulation of luteinizing hormone-releasing hormone (LHRH) neurons prior to the preovulatory gonadotropin surge

Gene expression in luteinizing hormone-releasing hormone (LHRH) neurons was analyzed during the periovulatory period to (1) characterize temporal patterns of LHRH gene expression and their relationship(s) to gonadotropin surges, and (2) determine if any such changes are uniform or dissimilar at different rostrocaudal levels of the basal forebrain. The number of neurons expressing mRNA for the decapeptide, and the relative degree of expression per cell were analyzed using in situ hybridization and quantitative image analysis. Rats were killed at 1800 hr on metestrus (Met), 0800 hr, 1200 hr, 1800 hr, and 2200 hr on proestrus (Pro), or 0200 hr, 0800 hr, and 1800 hr on estrus (E; n = 5-6 rats/group). All sections were processed for LHRH mRNA in a single in situ hybridization assay. Sections were atlas matched and divided into four rostrocaudal groups for analysis: vertical limb of the diagonal band of Broca (DBB), rostral preoptic area/organum vasculosum of the lamina terminalis (rPOA/OVLT), medial preoptic area (mPOA), and suprachiasmatic/anterior hypothalamic area (SCN/AHA). Plasma LH and FSH levels from all animals were analyzed by RIA. The labeling intensity per cell was similar among all time points at all four rostrocaudal levels. The number of cells expressing LHRH mRNA, however, varied as a function of time of death during the estrous cycle, and this temporal pattern varied among the four anatomical regions. At the level of the mPOA, the number of cells was highest at 1200 hr on Pro, and then declined and remained low throughout the morning of E. At the level of the rPOA/OVLT, the greatest number of LHRH neurons was noted later in Pro, at 1800 hr, dropping rapidly to lowest numbers at 2200 hr. No significant changes in LHRH cell number occurred at the DBB or SCN/AHA levels. At all anatomical levels, the secondary surge of FSH was unaccompanied by any change in the number of neurons expressing LHRH mRNA. These data demonstrate that (1) the number of detectable LHRH mRNA-expressing cells fluctuates during the periovulatory period and (2) peak numbers of LHRH-expressing cells are attained in the mPOA before the onset of the LH surge and before peak LHRH cell numbers are seen at more rostral levels. A model is proposed in which gene expression in this subpopulation of LHRH neurons may be activated by preovulatory estrogen secretion and acutely reduced following the proestrous surge of progesterone.     

The effects of prostaglandin F2alpha on sperm output in boars

The effect of prostaglandin F2alpha (PGF2alpha) on the sperm output of six boars was investigated in two studies. Although PGF2alpha did not significantly affect sperm numbers in the ejaculate, a significantly longer (P less than 0.05) ejaculation of the sperm rich fraction occurred following injection of PGF2alpha. In the second study it was found that PGF2alpha produced a 49% increase (P less than 0.05) in the number of sperm in the sperm rich fraction of the ejaculate. The implications of these results on artificial breeding are discussed.     

Acute effects of prostaglandin F2 alpha, luteinizing hormone, and estradiol on second messenger systems and on the secretion of oxytocin and progesterone from granulosa and early luteal cells of the ewe

Previous reports have suggested that gonadotropins, estradiol, and prostaglandin F2 alpha (PGF2 alpha) have varying effects on progesterone and oxytocin synthesis or secretion in cultured granulosa and luteal cells collected at different stages of the estrous cycle. The experiments reported here were designed to investigate whether effects of these agonists on secretion of hormones and their coupling to second messenger systems changed around the time of ovulation. Granulosa cells and Day 2 luteal cells of the ewe were cultured for three days and then treated for 30 min with varying doses of PGF2 alpha, LH, or estradiol. LH increased intracellular cAMP at both stages, but granulosa cells were more responsive in terms of both minimum effective dose (10 compared with 100 ng/ml) and degree of stimulation. LH caused no change in intracellular inositol phosphate levels. Both granulosa and early luteal cells responded to LH treatment by an increase in progesterone output in a dose-responsive fashion. PGF2 alpha increased inositol phosphate accumulation in cells collected at both stages of the cycle. All doses tested (10(-6)-10(-8) M) stimulated the release of oxytocin into the culture medium from both granulosa and luteal cells. Progesterone secretion was also increased, but only at the highest dose (10(-6) M). Estradiol treatment (10(-6) M) did not affect either the inositol phosphate or cAMP second messenger systems, but it did inhibit the secretion of oxytocin from granulosa cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibitory effect of prostaglandin F2 alpha on oxytocin release and on milk ejection in lactating rats

Eleven Southdown male lambs averaging 19.8 kg were randomly allotted to two groups and fed diets containing 7.7% (low-N) or 15.8% (high-N) crude protein. All of the supplemental nitrogen in the high-N diet was supplied as urea. Intake of the low-N and high-N diets averaged 372.3 g and 340.5 g/day, respectively. Findings at the end of the thirty-day trial were: (1) mean body weights unchanged for the two groups; (2) plasma urea nitrogen three-fold higher in the high-N (19.07 mg/100 ml) than the low-N (6.57) animals; (3) similar hepatic activity levels of three urea cycle enzymes (ornithine transcarbamylase, argininosuccinase, arginase) in the two groups, and (4) similar liver weights and liver protein concentration. The absence of adaptive change in enzyme levels suggests the hypothesis that addition of non-protein nitrogen to maintenance diets may cause ammonia intoxication by exceeding the liver's reserve capacity for urea synthesis.     

Site-directed mutagenesis of recombinant equine chorionic gonadotropin/luteinizing hormone: differential role of oligosaccharides in luteinizing hormone- and follicle-stimulating hormone-like activities

Equine chorionic gonadotropin (eCG) consists of highly glycosylated alpha- and beta-subunits and belongs to the glycoprotein hormone family that includes LH and FSH. eCG is a unique member of the gonadotropin family because it elicits response characteristics of both FSH and LH in other species than the horse. To determine the biological role of the N-linked oligosaccharide at Asn 56 of the alpha-subunit and O-linked oligosaccharides at the carboxyl-terminal peptide (CTP) of the beta-subunit, two mutant eCGs, in which Asn 56 of the alpha-subunit was replaced with Gln (eCG alpha 56/beta) or CTP was deleted (eCG alpha/ beta-CTP), were produced by site-directed mutagenesis and transfecting chinese hamster ovary (CHO-K1) cells. LH- and FSH-like activities were assayed in terms of testosterone production and aromatase activity in primary cultured rat Leydig cells and granulosa cells, respectively. The wild type eCG showed similar LH- and FSH-like activities to native eCG in the in vitro bioassays. The LH-like activity of eCG alpha 56/beta was greatly reduced, whereas that of eCG alpha/beta-CTP was unaffected, demonstrating that the oligosaccharide at Asn 56 of the alpha-subunit of eCG plays an indispensable role in LH-like activity. Interestingly, the FSH-like activity of eCG alpha 56/beta was increased markedly in comparison with the wild type, and that of eCG alpha/beta-CTP was also considerably increased. These data indicate that the dual activities of eCG, LH- and FSH-like activities, could be separated by removal of the N-linked oligosaccharide on the alpha-subunit Asn 56 or CTP-associated O-linked oligosaccharides.     

The effects of levonorgestrel, desogestrel and gestodene on the pulsatile release of luteinizing hormone in oral contraceptive users

The dynamics of luteinizing hormone (LH) and follicle stimulating hormone (FSH) release were investigated in 60 long-term oral contraceptive (OC) users. Five different types of OC, all containing the same amount of estrogens were studied: three monophasic preparations containing levonorgestrel, desogestrel and gestodene, respectively, and two triphasic formulations, containing levonorgestrel or gestodene. Thirteen healthy, normally cycling volunteers served as controls. Blood sampling was performed at 10-min intervals during a 6-h period to determine the pulsatile release of LH. LH and FSH were measured using a sensitive immunoradiometric assay. Pulse patterns were classified on the basis of the overall LH level, as well as on the character of the LH pulses, according to both frequency and amplitude characteristics. Pulsatile LH release was maintained during OC use. After the 7-day pill-free interval, FSH levels as well as the LH pulse patterns were comparable to those of early follicular-phase controls. FSH levels and FSH release in response to a gonadotropin releasing hormone (GnRH) challenge were profoundly suppressed in all OC users, as early as day 8 of the pill cycle. LH release during the pill cycle was characterized by either a low frequency (median 1 pulse/6 h), high amplitude (median 2.5 IU/l) pulse pattern or by a pattern of low-amplitude pulses (median 0.2 IU/l) and low basal LH levels (median 0.2 IU/l). The distribution of these pulse patterns showed marked differences between different OC preparations and depended on both the type and dose regimen of the gestagenic component of the OC.     

Early onset of parturition induced by acute alcohol exposure in C57BL/6J mice: role of uterine PGE and PGF2alpha

These studies were designed to determine the effect of acute alcohol treatment on gestational length and to probe for a mechanism underlying alcohol-induced early onset of parturition (EOP) in mice. Experiment 1: alcohol increases the incidence of EOP. Pregnant C57BL/6J mice were given alcohol (0, 4, 5 or 6 g kg(-1), i.g.) on Gestational Day (GD) 10, 15, 16, 17 or 18. Deliveries were monitored every 6 h from GD 18. Results indicated that 6 g kg(-1) alcohol treatment on GD 17 or 18 increased the incidence of EOP. Experiment 2: prostaglandins (PGs) play roles in parturition. The purpose of Experiment 2 was to determine whether PGs mediate alcohol-induced EOP in mice. The results indicated that pretreatment on GD 17 with aspirin, a prostaglandin synthesis inhibitor, prevented alcohol-induced EOP. These data suggest that alcohol-induced EOP in mice may be mediated by PGs. Experiment 3: PGs are influenced by alcohol and are triggers of labour. Experiment 3 measured uterine PGs associated with the onset of alcohol-induced EOP in mice. Alcohol increased uterine PGE and PGF2alpha, with PGE levels higher than control before labour, and elevated PGF2alpha levels correlating with labour. Changes in gestational length have important implications for pregnancy outcome, as well as for normal fetal growth and development.     

Metabolism of PG in man: effect of furosemide on the excretion of the main metabolite of PG F2alpha

Forty-five out-patients with clinically manifested anxiety were tested in order to study the effects of 2 weeks' treatment with placebo, diazepam (5-10 mg three times daily) or thioridazine (25-50 mg three times daily) on their psychomotor skills related to driving. When compared with placebo, diazepam increased the number of mistakes in reaction and co-ordination tests and also decreased ability to discriminate the fusion of flickering light. When compared to other groups, reactive and co-ordinative skills were more impaired in patients treated with thioridazine which also impaired divided attention. Aubjectively thioridazine was not experienced as effective an anxiolytic as diazepam.     

A receptor binding site identified in the region 81-95 of the beta-subunit of human luteinizing hormone (LH) and chorionic gonadotropin (hCG)

Two series of overlapping peptides comprising the entire sequences of the beta-subunits of human lutropin (LH) and choriogonadotropin (hCG) were prepared by a comprehensive synthetic strategy in order to identify all linear regions of the subunit that may participate in binding of the hormone to its receptor. Each series of peptides (15 residues in length) spanned the entire amino acid sequences of the two beta-subunits. The peptides were tested for their ability to inhibit the binding of 125I-labeled hCG or LH to rat ovarian membranes and for their ability to inhibit hCG-stimulated progesterone production in a Leydig cell bioassay. The most potent inhibitor of LH/hCG binding was a peptide containing the sequence beta 81-95, a receptor binding site of the LH/hCG beta subunit not previously described. The concentration at which LH/hCG binding was inhibited at 50% (IC50) was 20 microM and 30 microM for hCGbeta 81-95 and LH beta 81-95, respectively. These peptides also inhibited the stimulation of progesterone production by hCG in Leydig cell bioassays. In order to determine important residues that inhibit binding within this region, a third set of peptides was synthesized in which each residue of hCG beta 81-95 was sequentially replaced with the residue L-alanine. Five residues (Leu-86, Cys-88, Cys-90, Arg-94, and Arg-95) were critical for maximal inhibition of hCG binding by CG beta 81-95. In addition to site beta 81-95, other sites that inhibited hCG/LH binding but with significantly lower potencies included hCG beta 1-15, LH beta 41-55, and LH beta 91-105.(ABSTRACT TRUNCATED AT 250 WORDS)