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Prostaglandin synthesis in rat adrenocortical cells.

The biosynthesis of prostaglandins by isolated rat adrenocortical cells has been studied by determinations of products formed during incubations with labeled arachidonic acid and by radioimmunoassays. Analysis by thin-layer chromatographic separation of silicic acid column fractions indicated that PGE2, PGA2, (B2) and PGF2 alpha were the predominant prostaglandins formed by rat adrenocortical cells. Approximately 75% of the incorporated isotope was associated with the prostaglandins of the PGE pathway [PGE2 + PGA2 (B2)]. This was a consistent finding whether cells were incubated directly with arachidonic acid or with cells prelabeled with the substrate prior to study. ACTH did not affect the uptake or oxidation of [1-14C]-arachidonate, but did significantly increase incorporation of labeled substrate into [14C]prostaglandins. Of the ACTH-induced increase, 92% was accounted for by an increase in prostaglandins of the E pathway. Studies with prelabeled cells indicated that 77% of the prostaglandins synthesized in both control and ACTH-stimulated adrenocortical cells was released into the incubation medium during the 2-hr study. These had the same composition [88% PGE2 + PGA2 (B2)] as did the intracellular prostaglandins. Analysis by radioimmunoassays gave comparable data on the distribution of E- and F-type prostaglandins in control cells and cells incubated with ACTH or dibutyryl cyclic AMP. Thus, with these techniques, 88-92% of the increased prostaglandin synthesis due to ACTH or cyclic AMP was produced by the PGE2 rather than the PGF2 alpha pathway.

Adrenal Cortex

Gas chromatography of the prostaglandins.

Gas chromatography is the most widely used chromatographic technique. Its strength lies in the fact that three distinct operations--separation, detection, and quantitation--can be performed on all the components of mixtures within a reasonable time, and that qualitative information about the compound identity is associated with its retention time. Despite a prominent position in other areas of biochemistry, its success in the prostaglandin field has been rather limited. The technique itself is not at fault since samples with reasonable levels of prostaglandins have been analyzed without overwhelming difficulty. However, many samples demand a sensitivity exceeding the limits of either electron capture or flame ionization detectors. Shifts in research problems have contributed to its decline, but it is fair to add that these shifts were associated with the appearance of more sensitive and accurate analytical methods. Let us not despair for gas chromatography because its competitors, radioimmunoassay and gas chromatography-mass spectroscopy, also have deficiencies. In this chapter we have described several traditional gas chromatographic methods for prostaglandin analysis and outlined their strengths and weaknesses. Hopefully, this will prevent the misapplication of a useful analytical tool and also serve as an incentive to provoke contributions to this somewhat neglected technology. In conclusion, it cannot be stressed enough that all instrumental methods are at the mercy of chemistry. Chemistry can be exploited to enhance the value of a technique, or it can be disregarded to discredit the technique. The rise of sophisticated instruments has falsely diminished the value of chemistry in analytical methods development. It is imperative that we remind ourselves of its place and use it properly.

Animals

Spontaneous motility and distribution of prostaglandins in different segments of human Fallopian tubes.

The "in vitro" spontaneous magnitude and stability with time of the isometric developed tension (IDT) and frequency of contractions of ampullar longitudinal and ampullar circular as well as isthmic longitudinal and isthmic circular muscle, from human Fallopian tubes, were explored. The effects of polyphloretin-phosphate (PPP), an inhibitor of prostaglandins (PG), upon the IDT and frequency of different tubal segments, were also explored. We looked for PGE, and F2alpha in extracts of ampullar and isthmic tubal segments. The initial IDT and frequency (recorded at 10 minutes following equilibration) as well as the stability with time (explored afterward during 40 minutes) were comparable among all the preparations, with the exception of the isthmic longitudinal muscle, which suffered a progressive decrement of IDT. PPP inhibited significantly the IDT of the isthmic circular muscle but had no effect upon the ampullar circular muscle. Whereas PGF2alpha was frequently detected in extracts from the isthmic region, those from the tubal ampullar region showed the consistent presence of PGE1. The results suggested a possible relationship between isthmic PGF2alpha and its spontaneous motility. Also, the peculiar distribution of PGE1 and PGF2alpha could have some bearing on the regulation of the different functions of isthmic and ampullar regions of human Fallopian tubes.

Fallopian Tubes

New concepts in dysmenorrhea.

The etiology of primary dysmenorrhea, which is the most common gynecologic complaint and cause of lost working hours, remains obscure but merits careful scientific investigation. Recent studies suggest that increased endometrial prostaglandin production and release may be responsible for dysmenorrhea. Prostaglandins cause myometrial contractility that, if excessive, leads to uterine ischemia and pain. This hypothesis has led to clinical trials of antiprostaglandin agents such as indomethacin and fenamates, which inhibit the synthesis of prostaglandin through the prostaglandin synthetase system as well as antagonize their action at the cell receptor level. The good response of dysmenorrhea to other conventional forms of therapy such as oral contraceptives and dilatation of the cervix can be partly explained on the basis of a reduced level of prostaglandins in the menstrual fluid with such therapy. There is a definite need for further evaluation of the antiprostaglandin compounds in the treatment of dysmenorrhea so that sound formulations can be evolved for the elimination of this incapacitating disorder.

Adrenergic beta-Agonists

The measurement of E and 19-hydroxy E prostaglandins in human seminal plasma.

A method is described which measures the four main prostaglandins of human semen (PGE1, E2, 19-hydroxy PGE1, and 19-hydroxy PGE2). For routine measurements E1 and E2 are measured together as are 19-OH E1 and 19-OH E2. These are measured by forming oximes in aqueous solution extraction, methylation and trimethyl silylation followed by gas chromatography. The method has sufficient sensitivity to measure the levels found in the majority of semen samples. The normal range in men with proven fertility was 90 to 260 mug/ml of 19-hydroxy Es and 30-200 mug/ml of Es.

Adult

The kinetics of extraamniotically injected prostaglandins to induce midtrimester abortion.

In an attempt to improve the extraamniotic administration of prostaglandins in effecting abortion, the movement of injected prostaglandins in a high or low viscosity medium has been observed by radiological studies and radioimmunoassy of PGE and PGFalpha in amniotic fluid and maternal plasma in association with intrauterine tocographic responses. Results obtained suggest that the immediate response by the uterus is a reflection of the speed of absorption of prostaglandins from the chorio-decidual space and that this influences the outcome in inducing abortion within 24 hours. By giving the prostaglandins in a viscous medium a slower release of prostaglandins was produced in most cases, which probably accounts for the greater efficacy obtained using single injections of prostaglandins in this vehicle compared with normal saline.

Abortion, Induced

Plasma levels of 15(S)15-methyl PGF2alpha following administration via various routes for induction of abortion.

Plasma levels of 15(S)15-methyl PGF2alpha were measured by gas chromatography - mass spectrometry following intravenous, intramuscular and subcutaneous administration. During intravenous infusion of 1.0 and 2.5 microgram/min of the drug for six hours the plasma levels were relatively constant around 600 and 1200 picog/ml, respectively. At an infusion rate of 5 microgram/min the plasma level continuously increased during the administration. Intramuscular injections of 100-400 microgram of 15(S)15-methyl PGF2alpha gave maximum levels in plasma (700-1700 picog/ml) after 15-20 minutes followed by a gradual decrease during more than three hours. This rapid resorption of drug into plasma could be delayed by addition of 5 microgram epinephrine to the injected solution. Analyses during a series of intramuscular injections demonstrated that a therapeutical plasma level could be maintained by injections at two to three hour intervals.

Abortion, Induced

Cyclic AMP formation of isolated human corpora lutea in response to HCG - interference by PGF2 alpha.

Human corpora lutea of defined ages were excised at operation, cut into pieces and incubated in the presence of HCG, PGF2 alpha and PGE2 alone or in combination. Following incubation cAMP formation in tissue and medium was determined. HCG-stimulated tissue cAMP content was most pronounced at a corpus luteum age of 7-10 days after ovulation. This stimulation was antagonized by PGF2 alpha in corpora lutea older than 6 days. PGE2 stimulated cAMP formation per se and this effect was more pronounced when HCG and PGE2 were combined. A possible role for PGF2 alpha as a luteolytic substance in the human is suggested.

Adult

The effect of intrauterine progesterone treatment on the endometrial prostaglandin F content in the rabbit.

The effect on rabbit endometrial prostaglandin F caused by progesterone delivered directly to the uterus was investigated. Four groups of animals were used in the experiment: (1) no treatment (control); (2) an empty Silastic capsule (as an intrauterine device [IUD]) was inserted in one horn and the other horn was sham-operated; (3) a Silastic capsule releasing 150 microng of progesterone/day was placed in one horn and the other horn was sham-operated; (4) a Silastic capsule releasing progesterone was placed in one horn and the opposite horn received an empty Silastic capsule. In group 1, which received no treatment, no difference was noted. In group 2, the prostaglandin content of the horn containing an empty IUD was significantly higher than that of the sham-operated horn. In group 3, the same significant difference was noted between the prostaglandin content of the IUD-containing, progesterone-treated horn and the sham-operated horn. In group 4, no significant difference was observed between the horn containing an inert IUD and that containing a progesterone-releasing device. The addition of progesterone to an IUD does not significantly affect the elevated prostaglandin content of the endometrium caused by an inert IUD.

Animals

Prostaglandin F2alpha and human prostatic affinity for testosterone.

Earlier work had shown that the lactogen, LTH and HPL, foster testosterone binding by the prostate. This study was undertaken to see if prostaglandin F2alpha would oppose the effect of the lactogen on the prostate as it does the luteotrophic action of the hormone on the corpus luteum. When it was found instead that the PGF increases steroid binding and that its interaction with lactogen was neither antagonistic nor additive, attention was directed to further characterization of prostaglandin's effect. A dosage/response study of F2alpha alone showed that concentrations of 4 ng/ml and 40 ng/ml increased binding but that 400 ng/ml did not. Glands with stromal hyperplasia and/or inflammation were not responsive than those with epithelial hyperplasia. Assays of water extracts of the tissue revealed concentrations of about 340 ng of F2alpha per gram fresh weight and that the concentration varied inversely as the beta-glucuronidase activity. If the enzyme level is considered an index of the epithelial cell density within the specimen, the inverse relationship suggest a non-epithelial (stromal) site of prostaglandin concentration.

Dose-Response Relationship, Drug

Steroid and prostaglandin concentrations in the plasma of pregnant ewes during infusion of adrenocorticotrophin or dexamethasone to intact or hypophysectomized foetuses.

Catheters were implanted into 16 ewes and their foetuses between days 110 and 124 of gestation. Hypophysectomy was attempted in eight of these foetuses. Continuous infusion of synthetic ACTH (10 microgram/h) or dexamethasone (1mg/24 h) into the foetus, starting between days 124 and 129, induced premature parturition. The concentration of progesterone in the maternal peripheral plasma decreased before parturition in all animals while the level of oestradiol increased in ewes with intact foetuses or in those in which hypophysectomy was incomplete. When hypophysectomy was complete, no increase in the maternal level of oestradiol occurred before delivery. The concentration of 13,14-dihydro-15-oxo-prostaglandin F2alpha increased in the peripheral plasma of ewes with intact or hypophysectomized foetuses infused with ACTH. It is suggested that an intact foetal pituitary gland is required for the rise in the level of oestrogen prepartum, but that this rise is not essential for increased prostaglandin production of parturition.

Adrenocorticotropic Hormone