Effect of progesterone on the contractile response of isolated pulmonary artery in rabbits.

The purpose of this study was to assess the direct effect of progesterone on rabbit pulmonary arteries and to examine the mechanism of its action. Rings of pulmonary artery from male rabbits were suspended in organ baths containing Krebs solution, and isometric tension was measured. The response to progesterone was investigated in arterial rings contracted with noradrenaline (NA), KCl, and CaCl2. The effects of endothelium, nitric oxide (NO), prostaglandins, cyclic GMP (cGMP), and the adrenergic beta-receptor on progesterone-induced relaxation were also assessed. Progesterone inhibited the vasocontractivity to NA, KCl, and CaCl2, and relaxed rabbit pulmonary artery. The relaxing response of progesterone in pulmonary artery was significantly reduced by removal of endothelium, inhibitors of nitric oxide synthase and guanylate cyclase, but not by prostaglandin synthase inhibitor and blockage of the adrenergic beta-receptor. In Ca2+-free (0.1 mM EGTA) Krebs solution, progesterone inhibited NA-induced contraction that was intracellular Ca2+-dependent, but didn't affect the contraction of extracellular Ca2+-dependent component. Our results suggest that progesterone induces relaxation of isolated rabbit pulmonary arteries partially via NO and cGMP. Progesterone may also inhibit Ca2+ influx through potential-dependent calcium channels (PDCs) and Ca2+ release from intracellular stores.     

The response of large and small luteal cells from the pregnant rat to substrates and secretagogues

Large (greater than 22 microns) and small (12-21 microns) luteal cells from Day 8 pregnant rats were separated by elutriation after enzyme dissociation. Aliquots of cells were incubated for 4 h at 37 degrees C in Medium 199 alone (control) or with medium containing dibutyryl cyclic adenosine 3', 5'-monophosphate (cAMP) at 0.5 mM or 5 mM; rat luteinizing hormone (LH) at doses of 1, 10, 100, or 1000 ng/ml; 10 micrograms/ml 25-OH-cholesterol; or 10 ng/ml testosterone. Production of progesterone, testosterone, and estradiol was measured by radioimmunoassay. Both cell types showed a similar increase in estradiol synthesis when stimulated with LH (1 microgram/ml) or dibutyryl cAMP (5 mM); however, large luteal cells aromatized exogenous testosterone, whereas small luteal cells did not. Large luteal cells produced increased amounts of progesterone at lower doses of dibutyryl cAMP (0.5 mM) and LH (10 ng/ml), compared to small cells, which required 5 mM dibutyryl cAMP or 1 microgram/ml LH for minimal stimulation. Dibutyryl cAMP (5 mM) also resulted in an increase of testosterone release from small luteal cells. Progesterone synthesis in both cell types was enhanced by 25-OH-cholesterol. These results suggest that the two cell types differ functionally with respect to steroidogenesis during pregnancy, and that the large luteal cells appear to be the primary site of progesterone and estradiol production at this stage of pregnancy.     

The reversible inhibition of steroid-induced sexual behavior by intracranial cycloheximide

Cycloheximide(Cyclo), an inhibitor of protein synthesis by a direct action on protein synthesis at the ribosomal level, was used to reversibly inhibit estrogen-induced sexual receptivity. Cyclo (100 μg per rat) was infused into the preoptic area(POA) of ovariectomized rats at varying times before, simultaneously with, and after 3 μg of subcutaneous estradiol benzoate (EB). All animals received 0.5 mg progesterone (P) 36 hr after EB, and were tested for sexual receptivity 4–6 hr after P. The females were placed with stud males and a lordosis quotient was computed for each female (lordosis quotient = number of lordosis responses/20 mounts by the male × 100). Females receiving Cyclo 6 hr before, simultaneously with, or 12 hr after EB showed significantly lower levels of sexual receptivity when compared to females receiving Cyclo 36 hr before and 18 and 24 hr after EB. When those animals that showed low levels of sexual behavior after Cyclo infusion were reprimed with EB and P 7 days later and presented with a male they showed high levels of sexual receptivity. Thus, the effect of Cyclo was reversible. Only Cyclo infusions into the POA (bilateral) and third ventricle were effective in suppressing sexual behavior. Caudate nucleus, lateral ventricle, and unilateral POA infusions were without effect.The data presented are in agreement with earlier work that utilized actinomycin D to inhibit steroid-induced sexual behavior. Cyclo was found to be less toxic than actinomycin D. All of the available evidence is consistent with the hypothesis that estrogen stimulates RNA and/or protein synthesis in its facilitation of sexual behavior in the female rat.     

Mechanism of mifepristone-induced spasmolytic effect on isolated rat uterus

Mifepristone, a synthetic 19-norsteroid, relaxed the KCl-induced tonic contraction in isolated rat uterus in a concentration-dependent way and CaCl2 (0.1 to 10 mM) counteracted it. This effect was similar to other steroids although the mechanisms involved are unclear. Before adding the contracturant, tissue was incubated with actinomycin D (10 microM), cycloheximide (300 microM), TPCK (3 and 10 microM), Rp-cAMPS (30 microM), DDA (100 microM) and H-7 (1 microM). None of these modified the relaxing effect of mifepristone. Incubation with drugs that interfere with cGMP such as a nucleotide analogue DDG (100 microM), a soluble guanylyl cyclase inhibitor ODQ (1 microM) and an inhibitor of protein kinase G 8pCPTcGMPS (1 microM) significantly modified the effect of mifepristone, increasing its IC50.     

Cell cycle analysis in asynchronous cultures using the BUdR-Hoechst technique.

During synchronized germination of spores of Dictyostelium discoideum, protein synthesis begins almost concomitantly with syntheses of messenger-like RNA (mlRNA) and 4–5S RNA (presumably tRNA) in the swollen spore stage and the initiation of ribosomal RNA (rRNA) synthesis is somewhat delayed. DNA synthesis occurs in the early stages of the amoeba emergence phase. Cycloheximide (200 μg/ml) blocked spore germination as well as total protein synthesis, whereas actinomycin D (60 μg/ml) did not affect either. This concentration of actinomycin D selectively inhibited formation of rRNA but did not influence the synthesis of mlRNA. Examinations of RNA labeled with [¹⁴C]uracil during germination indicated that polysomes initially detectable in the course of the germination process contain ¹⁴C-labeled mlRNA. It was concluded that at least some of mRNA synthesized during germination of D. discoideum spores is involved in protein synthesis required for the germination.     

Steroidogenesis by isolated porcine oocyte-cumulus complexes: Lack of an inhibitory effect of low molecular weight fraction of porcine follicular fluid on conversion of androgen to estrogen

The ability of isolated porcine oocyte-cumulus complexes to secrete progesterone and convert androgens to estrogen during two days of culture was examined. We studied the effects of steroids, as well as a partially purified fraction of follicular fluid oocyte maturation inhibitor (Sephadex Peak A OMI), on the ability of oocyte-cumulus complexes to mature and convert androgen to estrogen. The addition of 0.014, 0.14 or 1.4 μg/ml androstenedione to the culture medium resulted in a substrate dose-dependent accumulation of estrogen in the culture medium after two days. Oocyte-cumulus cell complexes secreted more estrogen in the presence of androstenedione than in the presence of testosterone (P < 0.05). The addition of 1.4 μg/ml testosterone, androstenedione, or estradiol, but not dihydrotestosterone, inhibited cumulus cell progesterone secretion (P < 0.001 versus untreated control culture). Oocyte maturation was not altered by the addition of steroids in doses up to and including 1.4 μg/ml. The Sephadex Peak A OMI fraction of pFFL inhibited oocyte maturation 51% (P < 0.01) and progesterone secretion 91% (P < 0.01) but had no effect on the conversion of androgens to estrogens. Cumulus cell monolayer formation was inhibited 71.5% (P < 0.01) by the Sephadex Peak A OMI fraction and 35.4% (P < 0.05) by the Sephadex Peak A OMI fraction plus androstenedione. These studies indicate that porcine oocyte-cumulus complexes can convert androgens to estrogens and that partially purified OMI does not inhibit conversion of androgens to estrogen.     

Effect of verapamil on the contractions produced by high potassium and by noradrenaline in human isolated saphenous vein

The effect of a calcium channel blocker, e.g. verapamil, on the contractions produced by high potassium (K+) and noradrenalne (NA), was studied in the isolated saphenous vein in man. The aim of the present experiments was to see which of the two types of contractions was more sensitive to blockade by a calcium channel blocker, e.g. verapamil, and if verapamil had a differential effect on KCl and NA, whether this could be interpreted in terms of the presence of two calcium activation mechanisms in human saphenous vein. The results of the present investigation showed that KCl and NA contracted whereas verapamil relaxed the human saphenous vein. NA produced larger contraction (3.4 g tension) than did KCl (1.3 g tension). Lowering the calcium concentration in the external medium, from 2.5 mM to 1 mM, resulted in a reduced contraction in both NA and KCl responses, indicating dependence on influx of calcium. However, verapamil (1 microM) produced greater reduction in the KCl than NA-induced contraction, indicating that the NA contraction may involve additional mechanism, i.e. dependence on the release of calcium from intracellular Ca2+ stores. These results are in favour of the suggestion that the KCl-induced contraction was due to depolarization and voltage-dependent activation of calcium channels, whereas the NA-induced contraction was due to both depolarization and receptor-activation of the calcium channels, the latter being less sensitive to calcium channel blockers, e.g. verapamil. Thus, the KCl and NA-induced contractions in human saphenous vein may be due to two different calcium activation mechanisms; one is more sensitive (KCl) than the other (NA) to the presence of the calcium antagonist, verapamil.     

Effects of steroid hormones on total brain Na(+)-k+ ATPase activity in Oreochromis mossambicus

The effects of administration of cortisol, corticosterone, testosterone, progesterone and a synthetic estrogen. diethylstilbestrol (DES) on total brain Na(+)-K+- ATPase were investigated in tilapia, O. mossambicus. Exogenous administration of 0.125 and 0.25 microg/g body weight of glucocorticoids and 0.125, 0.25 and 0.5 microg/g body weight of DES for 5 days significantly stimulated Na+(-) K+ ATPase activity by 14-41% in the brain, while 0.5 microg/g body weight of glucocorticoids did not evoke any response on the activity of the enzyme. Progesterone (0.125 and 0.25 microg/g body weight) administration significantly decreased the enzyme activity by 21-36% and high dose (0.5 microg/g body weight) was ineffective. Testosterone exhibited a biphasic effect on Na(+)-K+ ATPase activity--a low dose stimulated by 14% while middle and high doses inhibited it by 19-24%. The results seem to be the first report on the effect of steroids on brain ATPase activity in a teleost. When 0.25microg/g body weight of actinomycin D or puromycin was administered prior to the treatment of similar doses of hormones, the inhibitors significantly inhibited the effect of the hormones by 24-52%. This clearly shows that the effect of the hormones was sensitive to the action of inhibitors suggesting a possible genomic mode of action under long-term treatment. The results suggest that cortisol, corticosterone and DES may possibly stimulate the co-transport of glucose and excitation of membrane potential while progesterone and testosterone inhibit them in the brain of O. mossambicus by regulating the activity of Na(+)-K+ ATPase.     

Ability of cortisol and progesterone to mediate the stimulatory effect of adrenocorticotropic hormone upon testosterone production by the porcine testis

The influence of corticosteroids and progesterone upon porcine testicular testosterone production was investigated by administration of exogenous adrenocorticotropic hormone (ACTH), cortisol and progesterone, and by applying a specific stressor. Synthetic ACTH (10 micrograms/kg BW) increased (P less than 0.01) peripheral concentrations of testosterone to peak levels of 5.58 +/- 0.74 ng/ml by 90 min but had no effect upon levels of luteinizing hormone (LH). Concentrations of corticosteroids and progesterone also increased (P less than 0.01) to peak levels of 162.26 +/- 25.61 and 8.49 +/- 1.00 ng/ml by 135 and 90 min, respectively. Exogenous cortisol (1.5 mg X three doses every 5 min) had no effect upon circulating levels of either testosterone or LH, although peripheral concentrations of corticosteroids were elevated (P less than 0.01) to peak levels of 263.57 +/- 35.03 ng/ml by 10 min after first injection. Exogenous progesterone (50 micrograms X three doses every 5 min) had no effect upon circulating levels of either testosterone or LH, although concentrations of progesterone were elevated (P less than 0.01) to peak levels of 17.17 +/- 1.5 ng/ml by 15 min after first injection. Application of an acute stressor for 5 min increased (P less than 0.05) concentrations of corticosteroids and progesterone to peak levels of 121.32 +/- 12.63 and 1.87 +/- 0.29 ng/ml by 10 and 15 min, respectively. However, concentrations of testosterone were not significantly affected (P greater than 0.10). These results indicate that the increase in testicular testosterone production which occurs in boars following ACTH administration is not mediated by either cortisol or progesterone.     

Chloroform Extract of Underground Parts of Ferula heuffelii: Secondary Metabolites and Spasmolytic Activity

Plants from the genus Ferula L. (Apiaceae) were used for various purposes in traditional medicine of different nations throughout the history. Ferula heuffelii Griseb . ex Heuffel is a perennial species endemic for Balkan peninsula. Ten compounds which belong to classes of prenyl‐furocoumarin‐, prenyl‐dihydrofurochromone‐, prenyl‐benzoyl‐ and prenyl‐benzoylfuranone‐type sesquiterpenoids, as well as sesquiterpene coumarins and phenylpropanoids, were, for the first time, isolated from the CHCl₃ extract of the underground parts of this plant and identified. Furthermore, extract and three isolated compounds, i.e., latifolone ( 1 ), dshamirone ( 4 ), and (2S*,3R*)‐2‐[(3E)‐4,8‐dimethylnona‐3,7‐dien‐1‐yl]‐2,3‐dihydro‐7‐hydroxy‐2,3‐dimethylfuro[3,2‐c]coumarin ( 6 ) were, for the first time, evaluated for their in vitro antispasmodic activities in three experimental models: spontaneous contraction, and ACh‐ and KCl‐induced contraction of an isolated rat ileum. The extract (0.1–1.3 mg/ml) and compound 6 (1–10 μg/ml) exhibited dose‐dependent effect in all three models. Compound 1 (1–6 μg/ml) affected spontaneous contractions and those induced by KCl, while compound 4 (8 μg/ml) displayed only moderate activity with ACh‐induced contractions. It can be concluded that tested compounds contribute to exhibited antispasmodic activity of crude extract. Additionally, extract (0.1–1.3 mg/ml) was tested for in vitro relaxant activity on an isolated rat trachea, and relaxed the KCl‐induced contractions in a dose‐dependent manner.     

Regulation of sugar transport in cultured diploid human skin fibroblasts

The regulation of hexose transport under glucose-starvation conditions was studied in cultured human skin fibroblasts. Glucose starvation enhanced the transport of 2-DG and 3-O-methyl-D-glucose (3-OMG) but not of L-glucose. Glucose-starvation enhanced transport was inhibited by cytochalasin B (10 μM). The starvation-induced change in 2-DG transport was due to an increase in the V_max of both the high and low affinity transport sites (2.8- and 2.4-fold, respectively) with no effect on their K_ms. The presence of 5.55 mM galactose, fructose, or L-glucose in the medium resulted in transport increases similar to those seen in glucose-starved cells, while the presence of 5.55 mM glucose, mannose, or 3-OMG repressed 2-DG transport. Glucose-starvation enhancement of 2-DG transport was blocked by cycloheximide (20 μg/ml) but not by actinomycin D (0.03 μg/ml) or α-amanitin (3.5 μM). Readdition of glucose (5.55 mM) for six hours to glucose-starved cells led to a rapid decrease in hexose transport that could be blocked by cycloheximide but not actinomycin D. Although readdition of 3-OMG to glucose-starved cells had little effect on reversing the transport increases, glucose plus 3-OMG were more effective than glucose alone. Serum containing cultures (10% v/v) of glucose-fed or glucose-starved cells exhibited rapid decreases in 2-DG transport when exposed to glucose-containing serum-free medium. These decreases were prevented by employing glucose-free, serum-free medium. The data indicate that hexose transport regulation in cultured human fibrob asts involves protein synthesis of hexose carriers balanced by interactions of glucose with a regulatory protein(s) and glucose metabolism as they affect the regulation and/or turnover of the carrier molecules.     

Progesterone is a mediator in the ovulatory process of the in vitro-perfused rat ovary

The role of steroids in the ovulatory process of the rat was explored in an in vitro perfusion system. Immature rat ovaries were primed with pregnant mare's serum gonadotropin (20 IU) and perfused in a recirculating perfusion system for up to 20 h. Unstimulated ovaries did not ovulate whereas the addition of luteinizing hormone (LH; 0.1 micrograms/ml) plus 3-isobutyl-1-methylxanthine (IBMX; 0.2 mM) resulted in 13.6 +/- 1.0 ovulations per treated ovary. Addition of an inhibitor of 3 beta-hydroxysteroid dehydrogenase (Compound A; 10 micrograms/ml) significantly (p less than 0.01) decreased the number of ovulations after LH plus IBMX stimulation (1.6 +/- 0.8 ovulations per treated ovary). This inhibition was reversed by the addition of progesterone, with 6.6 +/- 2.1 ovulations at approximately 100 ng/ml progesterone in the perfusion medium and 15.2 +/- 3.4 ovulations at approximately 3000 ng/ml progesterone. The addition of testosterone (10 micrograms/ml) did not reverse the inhibition of ovulations by Compound A. High levels of progesterone in the perfusion medium (greater than 3000 ng/ml) did not significantly (p greater than 0.05) increase the number of ovulations after stimulation with LH plus IBMX (20.2 +/- 4.8 ovulations), and progesterone (greater than 3000 ng/ml) was not by itself able to induce ovulations. Addition of LH plus IBMX resulted in a marked increase in the levels of progesterone, testosterone, and estradiol in the perfusion medium. The production of these steroids was almost completely inhibited by the addition of Compound A, and the levels of testosterone and estradiol were restored by the addition of high concentrations of progesterone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Progestins stimulate the differentiation of 3T3-L1 preadipocytes

The effect of progesterone on the differentiation of the 3T3-L1 preadipocytes was investigated and compared with other sex steroids (estradiol and testosterone), with cortisol, with the synthetic progestin R5020 and with the progestin/glucocorticoid antagonist RU38486. At 10⁻⁸ M, progesterone stimulated the activity of glycerol-3-phosphate dehydrogenase and triglyceride deposition. Progesterone, R5020, cortisol, and RU38486 increased triglycerides about 2-fold at 10⁻⁷ M. Only minimal effects were observed with testosterone and estradiol even at 10⁻⁶ M. When the cells were cultured in presence of 10⁻⁵ M metyrapone the effect of progesterone was unchanged, suggesting that the progesterone was not metabolized to a glucocorticoid. Progesterone, R5020 and RU38486 competed efficiently with [³H]dexamethasone for the glucocorticoid receptor in 3T3-L1 cytosol. These results indicate a significant, reproducible dose-dependent effect of progestins on differentiation of the preadipocytes, which appears to be mediated via the glucocorticoid receptor.     

Circadian rhythms of the sex steroids of female Papio hamadryas in long-term hypokinesia

Radioimmunoassay was used to study the effect of restricted motor activity on circadian rhythms of blood plasma sexual steroids and cortisol in female Papio hamadryas during different phases of the menstrual cycle. Instact animals manifested clear-cut circadian rhythms of the testosterone and cortisol content during both the phases and those of estradiol and progesterone during the follicular phase of the cycle. Two-week immobilization did not produce any changes in the general pattern of circadian rhythms of cortisol and testosterone but led to a decrease in their mean daily concentrations, with that decrease being more pronounced for testosterone of females immobilized during the luteal phase. The authors established unbalance of sexual steroids with a dramatic fall of the mean daily concentrations of estradiol and progesterone and desynchronism of their circadian rhythms regardless of the cycle phase at the moment of immobilization. The mechanisms responsible for alterations in circadian rhythms of sexual steroids in hypokinetic females are discussed.