Lower E2 Concentrations Research Articles

Beta-adrenergic modulation of the polymorphonuclear leukocyte respiratory burst is dependent upon the mechanism of cell activation.

Although inhibition of polymorphonuclear leukocyte activation by beta-adrenoceptor agonists has been recognized for over a decade, effects have only been observed at high drug concentrations and in the presence of theophylline. In this study, catecholamine and prostaglandin modulation of the respiratory burst was evaluated with respect to the mechanism of polymorphonuclear leukocyte activation. Very low concentrations of isoproterenol and prostaglandin E2 inhibited the respiratory burst when induced by chemotactic peptide (N-formyl-methionyl-leucyl-phenylalanine) or calcium ionophore (A23187, ionomycin), but not when initiated by synthetic diacylglycerol. Because formyl-methionyl-leucyl-phenylalanine and ionophore mobilize calcium and arachidonic acid generation follows an increase in intracellular calcium, the arachidonic acid metabolite leukotriene B4 was studied. Isoproterenol at a very low (0.1 nM) concentration also rapidly inhibited leukotriene B4 generation. Since cyclic AMP was increased by isoproterenol regardless of the means of cell activation, modulation of intracellular calcium was evaluated with the fluorescent probe indo-1. A transient increase in calcium after formyl-methionyl-leucyl-phenylalanine or ionophore (but not oleoyl acetylglycerol) cell activation was inhibited by isoproterenol or prostaglandin E2. These results suggest that adrenergic agonists specifically modulate calcium-dependent polymorphonuclear leukocyte function. Because marked inhibition was observed at very low drug concentrations, cyclic AMP-dependent effects may be important in both homeostatic and therapeutic modulation of inflammatory response.

PgE 2 modulating effects on the cell-mediated and humoral immune response to sperm antigens

1.1. Rat epidydimal fat pads and isolated fat cells release arachidonic acid as well as prostaglandin E2 during lipolysis.2.2. The enzyme system converting suitable essential fatty acids into prostaglandins E is present in adipose tissue of rat and ram and in isolated fat cells.3.3. Low concentrations of prostaglandin E1, prostaglandin E2 and ω-homoprostaglandin E1 efficiently inhibit stimulated lipolysis; biologically inactive prostaglandins E have no such effect in comparable concentrations.4.4. In essential fatty acid-deficient adipose tissue of the rat there is a decreased release of prostaglandin E2, an increase in lipolysis and a pronounced inhibition of the stimulated lipolysis by added prostaglandin E.5.5. These observations are compatible with a physiological role of prostaglandins E in the regulation of lipolysis, possibly by a feed-back mechanism.

Effects of prostaglandins and thromboxane analogues on bullock and dog iris sphincter preparations.

1 The bullock iris sphincter was contracted by low concentrations of prostaglandin E2 (PGE2), 16, 16-dimethyl PGE2 and 17,18,19,20-tetranor-16-p-chlorophenoxy PGE2. Other compounds with thromboxane-like actions, for example 11,9-epoxymethano PGH2, were also potent spasmogens, ZK 36374, a stable carbacyclin, was a partial agonist on the PGE-sensitive system of this tissue. 2 The thromboxane antagonist, EP 045, had little effect on the action of PGE2 and 16,16-dimethyl PGE2 on the bullock iris. 3 The dog iris sphincter was sensitive to PGF2 alpha but not to PGE2 and 11,9-epoxymethano PGH2. 4 16,16-dimethyl PGE2 had very low activity on the dog iris in contrast to its high activity on the bullock iris. The reverse was found with the 17,18,19,20-tetranor-16-m-trifluoromethylphenoxy analogue of PGF2 alpha (ICI 81008). This indicates a considerable selectivity of action of the two analogues. 5 The results are discussed in relation to the existing knowledge of prostanoid receptors.

Effects of inflammatory agents on endothelial lysosomal fragility and their inhibition by anti-inflammatory drugs.

1. Endothelial cells from human umbilical veins were maintained in tissue culture. The fragility of lysosomal membranes were studied by microdensitometry. 2. Histamine (50 microM to 10 mM), 4-methylhistamine (100 nM to 10 mM) and dimaprit (100 nM to 10 mM) increased lysosomal fragility. 2-Thiazolylethylamine and 2-pyridylethylamine (100 nM to 10 mM) had no effect. 3. Prostaglandins E1 and E2 (3 nM to 30 microM) and prostaglandin F2 alpha (2 nM to 20 microM) had no direct effect. Low concentrations of prostaglandins E1 and E2 inhibited the fragility induced by histamine 100 microM. 4. Bradykinin (100 nM to 100 microM) decreased fragility. 5. The increase in fragility induced by histamine 100 microM or dimaprit 100 microM was inhibited by cimetidine (100 microM to 1 mM) but not by mepyramine (1 microM to 1 mM). 6. Pretreatment with indomethacin, hydrocortisone, ibuprofen and sodium salicylate caused a dose-dependent inhibition of histamine-induced fragility. Threshold concentrations were 1 pM, 100 pM, 10 nM and 10 microM, respectively. 7. Lignocaine (1 microM to 1 mM) had no direct effect and did not decrease histamine-induced fragility.

Differential effects of low serum levels of estradiol-17 beta on hypothalamic LHRH levels and LH secretion in castrated male rats.

In castrated male rats, gonadal steroid treatment raises the medial basal hypothalamic (MBH) LHRH levels and suppresses LH release. We have now examined whether the steroid-induced MBH LHRH response could occur concomitantly with normal pituitary LH secretion. Of the various concentrations tested, serum estradiol-17 beta (E2) levels in the range of 9-16 pg/ml consistently and selectively stimulated the accumulation of LHRH in the MBH after a long latency period of 60-72 h. These low E2 levels did not suppress LH release at any time interval. Serum LH levels and the episodic pattern of LH secretion were similar to those of castrated control rats. It is unlikely that enhanced pituitary sensitivity in E2-treated rats may have compensated for a decrease in LHRH release since the LH response to exogenous LHRH was similar in E2-treated and control rats. These studies show that dissociation of the feedback effects on the MBH LHRH and pituitary gland LH release may be obtained with low serum concentrations of E2 in male rats; low E2 levels stimulate LHRH accumulation in the MBH by mechanisms which may not involve inhibition of LHRH release.

Effect of agents that alter cyclic AMP on arachidonate-induced platelet aggregation in the dog

Recent studies in our laboratory demonstrated that 30% of normal mongrel dogs have platelets that aggregate when stirred with sodium arachidonate (ASP), while the majority have platelets that do not aggregate under similar circumstances (AIP). In the current study, we confirmed previous observations that indicated that AIP formed aggregating products of prostaglandin metabolism (endoperoxides and/or thromboxane A2) when stirred with sodium arachidonate. We also determined that the difference between ASP and AIP was attributable to the platelets rather than the plasma. To further define the mechanism responsible for the difference between ASP and AIP, we studied the response of dog platelets to agents known to alter platelet cyclic AMP, and we measured platelet cyclic AMP. Radioimmunoassay of cyclic AMP in platelet-rich plasma (PRP) and in washed platelets demonstrated no differences between unstimulated ASP and AIP. However, agents that lower elevated platelet cyclic AMP (epinephrine, norepinephrine, adenosine diphosphate, serotonin and low concentrations of prostaglandin E2) uniformly converted AIP to ASP. Agents that elevate platelet cyclic AMP (prostaglandins E1' D2' l2' theophylline and dibutyryl cyclic AMP) uniformly converted ASP to AIP. The addition of epinephrine, following prostaglandin l2 (PGI2) restored the ability of ASP to aggregate when stirred with sodium arachidonate. In vitro PGI2 (0.14-2.1 nM) converted ASP to AIP without inhibiting adenosine diphosphate (ADP) or collagen-induced aggregation, thereby duplicating the behavior of naturally occurring AIP. PGI2 infusions produced the same result in vivo. The results of these studies suggest that cyclic AMP plays an important role in regulating the response of dog platelets to the aggregating products of prostaglandin metabolism. AIP have a cyclic AMP-dependent mechanism that modulates their response to the aggregating products of prostaglandin metabolism that is different from ASP and human platelets. We postulate that AIP retain the effects of in vivo elevation of platelet cyclic AMP stimulated by exposure to PGI2. The mechanism by which this occurs is uncertain.

Effect of agents that alter cyclic AMP on arachidonate-induced platelet aggregation in the dog

Abstract Recent studies in our laboratory demonstrated that 30% of normal mongrel dogs have platelets that aggregate when stirred with sodium arachidonate (ASP), while the majority have platelets that do not aggregate under similar circumstances (AIP). In the current study, we confirmed previous observations that indicated that AIP formed aggregating products of prostaglandin metabolism (endoperoxides and/or thromboxane A2) when stirred with sodium arachidonate. We also determined that the difference between ASP and AIP was attributable to the platelets rather than the plasma. To further define the mechanism responsible for the difference between ASP and AIP, we studied the response of dog platelets to agents known to alter platelet cyclic AMP, and we measured platelet cyclic AMP. Radioimmunoassay of cyclic AMP in platelet-rich plasma (PRP) and in washed platelets demonstrated no differences between unstimulated ASP and AIP. However, agents that lower elevated platelet cyclic AMP (epinephrine, norepinephrine, adenosine diphosphate, serotonin and low concentrations of prostaglandin E2) uniformly converted AIP to ASP. Agents that elevate platelet cyclic AMP (prostaglandins E1' D2' l2' theophylline and dibutyryl cyclic AMP) uniformly converted ASP to AIP. The addition of epinephrine, following prostaglandin l2 (PGI2) restored the ability of ASP to aggregate when stirred with sodium arachidonate. In vitro PGI2 (0.14-2.1 nM) converted ASP to AIP without inhibiting adenosine diphosphate (ADP) or collagen-induced aggregation, thereby duplicating the behavior of naturally occurring AIP. PGI2 infusions produced the same result in vivo. The results of these studies suggest that cyclic AMP plays an important role in regulating the response of dog platelets to the aggregating products of prostaglandin metabolism. AIP have a cyclic AMP-dependent mechanism that modulates their response to the aggregating products of prostaglandin metabolism that is different from ASP and human platelets. We postulate that AIP retain the effects of in vivo elevation of platelet cyclic AMP stimulated by exposure to PGI2. The mechanism by which this occurs is uncertain.

Threatened abortion studied by estradiol-17 beta in serum and ultrasound.

One hundred women were admitted to the hospital between 7 and 18 weeks' gestation because of vaginal bleeding and threatened abortion. Serum concentration of estradiol-17 beta was determined by radioimmunoassay. Ultrasound examination using a gray-scale scanner was performed as well. The values for prediction of abortion or successful outcome of pregnancy were calculated for estradiol (E2) and ultrasound separately and in combination. Prediction of abortion from a low E2 concentration combined with an ultrasound examination showing no fetal life was found to be totally reliable. Prediction of successful outcome of pregnancy from either a normal E2 or an ultrasound examination showing fetal life, or both, was 79.5% reliable. If no signs of fetal life were found by ultrasound examination from 10 weeks' gestation on, the pregnancy was doomed. Before this gestational age, measurements of E2 in serum gave supplementary information concerning the outcome of the threatened pregnancy.

Saturation kinetics applied to in vitro effects of low prostaglandin E2 and F2α concentrations on ion transport across human jejunal mucosa

Inhibition of ion transport by exogenous prostaglandins (PGs) has previously been accomplished only by using “pharmacologic” doses (10−7–10−4 M). Therefore, experiments were performed in which the in vitro effects of low (physiologic) doses (10−11–10−7 M) were assessed in the Ussing chamber preparation of human jejunal mucosa, both with and without blockade of endogenous PG-production by indomethacin (2.9 × 10−5 M). Serosal application of PGE2 caused a prompt, dose-dependent rise in short-circuit current (Isc), decreased net Na flux (JnetNa), elicited Cl secretion, and slightly depressed conductance (G). Dose-response curves for the early effects on Isc and JnetNa were performed. The percentage maximal response (Rmax) and the concentration for half-maximal response (km) were determined by nonlinear regression analysis using the normal Michaelis-Menten function (iterative evaluation by “least squares”). In the presence of indomethacin, the threshold concentration for effect of PGE2 was 10−10 M; Rmax: stimulation by 26% and inhibition by 58% of control values; and km: 10−9 M and 0.7 × 10−9 M for Isc and JnetNa, respectively. In the absence of indomethacin, the threshold concentration for exogenous PGE2 was 10−9 M; Rmax: stimulation by 15% and inhibition by 46% of control values; and km 4.7 × 10−8 M and 1.2 × 10−8 M for Isc and JnetNa, respectively. The mean concentration of endogenous PGE2 released into the serosal bath was approximately 2 × 10−11 M for indo-methacin-blocked tissues, and 1.8 × 10−9 M for unblocked tissues. The clearance of exogenous PGE2 from the serosal bath was about 20% during the experimental period, while less than 1% diffused across the membrane into the mucosal bath. The initial rise in Isc rapidly declined so that PGE2 had a transient effect that cannot be explained by clearance of PGE2, but is rather probably due to the activation of self-controlling feedback mechanisms in the tissue. Since Jm−sNa and G during control periods were higher with indomethacin blockade than without, and since Isc during experimental periods with high PGE2 concentrations usually reached a stable level above baseline before the effect dissipated, PGE2 appeared to have a “steady-state” effect as well, although less sensitive. Similar responses in Isc and JnetNa, were observed following mucosal PGE2 application, but required concentrations larger than 10−8 M. PGF2α had no effect on ion transport up to a concentration of 10−7 M. In conclusion, suppression of PG-biosynthesis by indomethacin increased the initial sensitivity of the tissue to exogenous PGE2. The extremely low dose-response suggests a “true” physiologic role for PGs in the modulation of intestinal ion transport. Since km of the response to PGE2 appeared to be below the threshold for activation of the intestinal adenylatecyclase-cAMP system, the role of cAMP in physiologic responses to PGs remains to be established.

Phospholipase A activity in the skin. Modulators of arachidonic acid release from phosphatidylcholine.

The distribution of the hydrolysis of 1-acyl-2-[1-14C]arachidonoyl-sn-glycero-3-phosphocholine and the simultaneous biosynthesis of prostaglandins by subcellular fractions from human and rat skin membrane preparations were determined. The phospholipase A2 activity was distributed among the subcellular particulate preparations with the highest specific activity in the 105000g particulate fraction. The activity was optimal at pH 7.5 in the presence of 1.0 mM-CaCl2 and was inhibited by EDTA. The hydrolysis of phosphatidylcholine by the skin 105000g particulate fraction was inhibited by cortisol and triamcinolone acetonide and it was stimulated by histamine, bradykinin, retinoic acid and cholera enterotoxin (freeze-dried Vibrio cholerae). Furthermore hydrolysis of phosphatidylcholine by the skin phospholipase A was also enhanced by low concentrations of prostaglandin E2 and prostaglandin F2 alpha. These last results suggest that the amplication of the hydrolysis of phosphatidylcholine by prostaglandin E2 and prostaglandin F2 alpha, with the consequent release of arachidonic acid (the substrate of prostaglandin synthesis) is likely a positive-feedback regulation of the arachidonic acid-prostaglandin cascade.

Human Endometrial Cells in Primary Tissue Culture: Estrogen Interactions and Modulation of Cell Proliferation*

Human endometrium, obtained from premenopausal women after hysterectomy, was dissociated into cells and placed in culture in order to examine estrogen responsiveness in a model in vitro system. Experiments which utilized cell proliferation as an index of responsiveness spanned at least 7 days and were performed in Dulbecco's modified Eagle's medium supplemented with 10% steroid-depleted serum from castrate-adrenalectomized calves. The addition of 2 × 1(10−7 M 17β-estradiol (E2) or diethylstilbestrol (DES) resulted in a 2- to 5-fold optimal acceleration in the rate of proliferation, with stimulation first being detected at 2-4 days. Ten-fold higher or lower concentrations of E2 or DES resulted in little stimulation, and high (2 × 10−5 M) concentrations of either estrogen stopped cell proliferation. The addition of E2 or DES (2 × 10−8−2 × 10−6 M) to cultures of a putative nontarget cell line (Detroit- 98) derived from human sternum failed to elicit any increase in the rate of proliferation, although high E2...

5 - Steroidogenesis in the Postmenopausal Ovary

Sex differences exist for stress reactivity as well as for the prevalence of depression, which is more frequent in women of reproductive age and often precipitated by stressful events. In animals, the differential effect of stress on male's and female's emotional behavior has been well documented. Crosstalk between the gonadal and stress hormones, in particular between estrogens and glucocorticoids, underlie these sex differences on stress vulnerability.We have previously shown that corticosteroid binding globulin (CBG) deficiency in a mouse model (Cbg k.o.) leads, in males, to an increased despair-like behavior caused by suboptimal corticosterone stress response. Because CBG displays a sexual dimorphism and is regulated by estrogens, we have now investigated whether it plays a role in the sex differences observed for emotional reactivity in mice.By analyzing Cbg k.o. and wild-type (WT) animals of both sexes, we detected sex differences in despair-like behavior in WT mice but not in Cbg k.o. animals. We showed through ovariectomy and estradiol (E2) replacement that E2 levels explain the sex differences found in WT animals. However, the manipulation of E2 levels did not affect the emotional behavior of Cbg k.o. females. As Cbg k.o. males, Cbg k.o. females have markedly reduced corticosterone levels across the circadian cycle and also after stress. Plasma free corticosterone levels in Cbg k.o. mice measured immediately after stress were blunted in both sexes compared to WT mice. A trend for higher mean levels of ACTH in Cbg k.o. mice was found for both sexes. The turnover of a corticosterone bolus was increased in Cbg k.o. Finally, the glucocorticoid-regulated immediate early gene early growth response 1 (Egr1) showed a blunted mRNA expression in the hippocampus of Cbg k.o. mutants while mineralocorticoid and glucocorticoid receptors presented sex differences but equivalent mRNA expression between genotypes.Thus, in our experimental conditions, sex differences for despair-like behavior in WT mice are explained by estrogens levels. Also, in both sexes, the presence of CBG is required to attain optimal glucocorticoid concentrations and normal emotional reactivity, although in females this is apparent only under low E2 concentrations. These findings suggest a complex interaction of CBG and E2 on emotional reactivity in females.

Androgen-Uterine Interaction: Nuclear Translocation of the Estrogen Receptor and Induction of the Synthesis of the Uterine-Induced Protein (IP) by High Concentrations of Androgens in Vitro but not in Vivo

High concentrations of androgens in vitro [10(-6) and 10(-7)M 5alpha-dihydrotestosterone (DHT) and testosterone (T)] translocate the estrogen receptor from cytoplasmic to the nuclear fraction of the immature rat uterus, and the androgen translocated sites are capable of eliciting the synthesis of the specific uterine "induced protein" (IP), formerly attributed to estrogenic compounds only. The magnitude of receptor translocation and IP synthesis induction is related to the concentration of androgen, and, at equal concentrations, DHT is more effective than T. Competitive protein-binding asssays with cell-free uterine cytosol indicate that DHT and T bind with barely detectable affinity to the cytosol estrogen receptor detectable affinity to the cytosol estrogen receptor [relative binding ability ca. 0.001% that of estradiol (E2)], and radioactive E2 uptake into whole uterus after pretreatment with androgens indicates that the androgen-translocated nuclear sites are readily filled by E2. DHT and T translocate the estrogen receptor to the nucleus in vitro, and the salt-extracted nuclear receptor is present as a 5 S ("transformed") receptor; however, concentrations of DHT and T that effect translocation are unable to elicit the heat-activated transformation of the estrogen receptor in cell free cytosol under conditions in which low concentrations of E2 fully transform the receptor. Under in vivo conditions, high levels of androgens (5--2,000 mug DHT or T) do not evoke any detectable translocation of the estrogen receptor and do not elicit any IP synthesis induction although uterine weight is increased (greater than 200 mug androgen) in long-term (2--3 day) assays. The analysis of androgen uptake and metabolism indicates that 500 mug or higher doses of DHT or T in vivo result in uterine concentrations of unmetabolized DHT or T equal to those seen after exposure to 5 X 10(-7) - 1 X 10(-6)M DHT or T in vitro. Hence, under conditions where in vivo and in vitro tissue uptake of androgen is equivalent, in vivo androgens are unable to affect the estrogen receptor system as is seen in vitro. These studies indicate that the in vitro and in vivo effects of androgens on the uterus are clearly different and suggest that the actions of androgens on the uterus in vivo are probably not directly mediated through the estrogen receptor system.

Inhibition of prostaglandin biosynthesis as the mechanism of analgesia of aspirin-like drugs in the dog knee joint.

A method has been developed to measure the analgesic action of aspirin-like drugs in knee joints of anaesthetized dogs. Bradykinin, injected into the joint cavity, induced a reflex rise in blood pressure which was dose-dependent; this was used as a measure of nociceptive activity. The joint cavity became more sensitive to bradykinin as the experiment proceeded, or when a low concentration of prostaglandin E1 or E2 was infused locally. The increase in sensitivity with time was prevented by local injection of aspirin or indomethacin, but that induced by exogenous prostaglandin infusion was not. Injections of carrageenin into dog knee joints increased the prostaglandin E2 content of synovial fluid by up to 160 ng per joint; indomethacin prevented this increase. These experiments support our previous conclusion that local biosynthesis of a prostaglandin (induced by mild trauma) sensitizes pain receptors to mechanical or chemical stimuli. Aspirin-like drugs are analgesic because they prevent prostaglandin biosynthesis, thereby preventing this sensitization.

Endogenous prostaglandins and osmotic water flow in the toad bladder.

Endogenous prostaglandins and osmotic water flow in the toad bladder.