S1R agonist modulates rat platelet eicosanoid synthesis and aggregation.

Sigma-1 receptor (S1R) is detected in different cell types and can regulate intracellular signaling pathways. S1R plays a role in the pathomechanism of diseases and the regulation of neurotransmitters. Fluvoxamine can bind to S1R and reduce the serotonin uptake of neurons and platelets. We therefore hypothesized that platelets express S1R, which can modify platelet function. The expression of the SIGMAR1 gene in rat platelets was examined with a reverse transcription polymerase chain reaction and a quantitative polymerase chain reaction. The receptor was also visualized by immunostaining and confocal laser scanning microscopy. The effect of S1R agonist PRE-084 on the eicosanoid synthesis of isolated rat platelets and ADP- and AA-induced platelet aggregation was examined. S1R was detected in rat platelets both at gene and protein levels. Pretreatment with PRE-084 of resting platelets induced elevation of eicosanoid synthesis. The rate of elevation in thromboxane B2 and prostaglandin D2 synthesis was similar, but the production of prostaglandin E2 was higher. The concentration-response curve showed a sigmoidal form. The most effective concentration of the agonist was 2 µM. PRE-084 increased the quantity of cyclooxygenase-1 as detected by ELISA. PRE-084 also elevated the ADP- and AA-induced platelet aggregation. S1R of platelets might regulate physiological or pathological functions.

Excessive alcohol consumption induces methane production in humans and rats.

Various studies have established the possibility of non-bacterial methane (CH4) generation in oxido-reductive stress conditions in plants and animals. Increased ethanol input is leading to oxido-reductive imbalance in eukaryotes, thus our aim was to provide evidence for the possibility of ethanol-induced methanogenesis in non-CH4 producer humans, and to corroborate the in vivo relevance of this pathway in rodents. Healthy volunteers consumed 1.15 g/kg/day alcohol for 4 days and the amount of exhaled CH4 was recorded by high sensitivity photoacoustic spectroscopy. Additionally, Sprague-Dawley rats were allocated into control, 1.15 g/kg/day and 2.7 g/kg/day ethanol-consuming groups to detect the whole-body CH4 emissions and mitochondrial functions in liver and hippocampus samples with high-resolution respirometry. Mitochondria-targeted L-alpha-glycerylphosphorylcholine (GPC) can increase tolerance to liver injury, thus the effects of GPC supplementations were tested in further ethanol-fed groups. Alcohol consumption was accompanied by significant CH4 emissions in both human and rat series of experiments. 2.7 g/kg/day ethanol feeding reduced the oxidative phosphorylation capacity of rat liver mitochondria, while GPC significantly decreased the alcohol-induced CH4 formation and hepatic mitochondrial dysfunction as well. These data demonstrate a potential for ethanol to influence human methanogenesis, and suggest a biomarker role for exhaled CH4 in association with mitochondrial dysfunction.

Rosuvastatin enhances anti-inflammatory and inhibits pro-inflammatory functions in cultured microglial cells.

Microglial activation results in profound morphological, functional and gene expression changes that affect the pro- and anti-inflammatory mechanisms of these cells. Although statins have beneficial effects on inflammation, they have not been thoroughly investigated for their ability to affect microglial functions. Therefore the effects of rosuvastatin, one of the most commonly prescribed drugs in cardiovascular therapy, either alone or in combination with bacterial lipopolysaccharide (LPS), were profiled in pure microglial cultures derived from the forebrains of 18-day-old rat embryos. To reveal the effects of rosuvastatin on a number of pro- and anti-inflammatory mechanisms, we performed morphometric, functional and gene expression studies relating to cell adhesion and proliferation, phagocytosis, pro- and anti-inflammatory cytokine (IL-1ß, tumor necrosis factor α (TNF-α) and IL-10, respectively) production, and the expression of various inflammation-related genes, including those related to the above morphological parameters and cellular functions. We found that microglia could be an important therapeutic target of rosuvastatin. In unchallenged (control) microglia, rosuvastatin inhibited proliferation and cell adhesion, but promoted microspike formation and elevated the expression of certain anti-inflammatory genes (Cxcl1, Ccl5, Mbl2), while phagocytosis or pro- and anti-inflammatory cytokine production were unaffected. Moreover, rosuvastatin markedly inhibited microglial activation in LPS-challenged cells by affecting both their morphology and functions as it inhibited LPS-elicited phagocytosis and inhibited pro-inflammatory cytokine (IL-1ß, TNF-α) production, concomitantly increasing the level of IL-10, an anti-inflammatory cytokine. Finally, rosuvastatin beneficially and differentially affected the expression of a number of inflammation-related genes in LPS-challenged cells by inhibiting numerous pro-inflammatory and stimulating several anti-inflammatory genes. Since the microglia could elicit pro-inflammatory responses leading to neurodegeneration, it is important to attenuate such mechanisms and promote anti-inflammatory properties, and develop prophylactic therapies. By beneficially regulating both pro- and anti-inflammatory microglial functions, rosuvastatin may be considered as a prophylactic agent in the prevention of inflammation-related neurological disorders.

Tocopherol inhibits the relaxing effect of terbutaline in the respiratory and reproductive tracts of the rat: the role of the oxidative stress index.

AIMS: Reactive oxygen species play a role in the signal transduction of beta-adrenergic receptors. We investigated whether an antioxidant (tocopherol) can reduce the effect of terbutaline in beta-2-adrenergic receptor (ß2-AR)-regulated smooth muscles. MAIN METHODS: Contractility of the tissues from nonpregnant (trachea) and 22-day-pregnant (myometrium and cervix) rats was investigated in an isolated organ bath. The tracheal and uterine ß2-AR expressions were increased by 17-beta-estradiol valerate (E2) and progesterone (P4), respectively. The accumulation of cyclic-AMP (cAMP), and the total oxidant (TOS) and total antioxidant status (TAS) were also measured. The oxidative stress index (OSI) was defined as the ratio of TOS and TAS. KEY FINDINGS: Terbutaline (10(-10)-10(-5)M) decreased the contractions in the nontreated and the P4-pretreated myometria, but tocopherol (10(-7)M) did not alter these actions. Terbutaline (10(-6)M) increased the cervical resistance both in the nontreated and in the P4-treated samples, while tocopherol reduced this action only in the P4-treated cervices. Terbutaline (10(-9)-10(-4)M) reduced the tracheal tones both in the nontreated and in the E2-treated tissues, while tocopherol reduced these effects. The changes in the intracellular cAMP levels of the tissues were in harmony with the isolated organ results. The OSI was highest in the trachea and lowest in the pregnant myometrium. SIGNIFICANCE: A higher OSI is linked to a higher tocopherol sensitivity of beta-mimetic-induced relaxation. Our results suggest that the antiasthmatic effect of beta-mimetics may worsen, while their tocolytic effect may remain unchanged during parallel tocopherol administration.

Inflammatory activation after experimental cardiac tamponade.

BACKGROUND/PURPOSE: Cardiac tamponade is a medical emergency situation associated with a high rate of life-threatening complications, even after immediate interventions. Our aim was to characterize the acute inflammatory consequences of this event in a clinically relevant large animal model. METHODS: Cardiac tamponade was induced for 60 min in anesthetized, ventilated and thoracotomized minipigs by intrapericardial fluid administration, the mean arterial pressure (MAP) being maintained in the interval of 40-45 mm Hg (n = 8). A further group (n = 7) served as sham-operated control. The global macrohemodynamics, including the right- and left-heart end-diastolic volumes (RHEDV and LHEDV), the pulmonary vascular resistance index (PVRI) and the superior mesenteric artery (SMA) flow, were monitored for 240 min, and the intestinal microcirculatory changes (pCO2 gap) were evaluated by indirect tonometry. Blood samples were taken for the determination of cardiac troponin T and vasoactive inflammatory mediators, including histamine, nitrite/nitrate, big-endothelin, superoxide and high-mobility group box protein-1 levels in association with intestinal leukocyte and complement activation. RESULTS: The cardiac tamponade induced significant decreases in MAP, cardiac output, LHEDV and SMA flow, while the PVRI and the pCO2 gap increased significantly. After the removal of fluid from the pericardial sac, the MAP and the LHEDV were decreased, while the PVRI and the pCO2 gap remained elevated when compared with those in the sham-operated group. In the posttamponade period, the abrupt release of inflammatory mediators was accompanied by a significant splanchnic leukocyte accumulation and complement activation. CONCLUSIONS: The macrocirculatory and splanchnic microcirculatory disturbances were accompanied by a significant proinflammatory reaction; endothelin and the complement system may be significant components of the inflammatory cascade that is activated in this porcine model of pericardial tamponade.

Prediction of gestational diabetes mellitus in a high-risk group by insulin measurement in early pregnancy.

AIMS: We hypothesized that an increased serum insulin level in early pregancy reflects an increased demand on the compensatory capacity of the pregnant woman, and can serve as a predictor of gestational diabetes mellitus (GDM). METHODS: A 2-h, 75-g oral glucose tolerance test (OGTT), with fasting and 2-h postprandial serum insulin determination, was performed in 71 pregnant women with one or more risk factors for GDM before gestation week 16. In 64 patients, subsequent OGTTs were performed at gestation weeks 24-28, and in the event of a negative result, at gestation weeks 32-34. RESULTS: Insulin determination at fasting and at 120 min had sensitivities of 69.2% and 92.3%, and specificities of 96.4% and 85.7%, respectively, for the prediction of GDM at gestation weeks 24-28. The sensitivities decreased to 33.3% and 75.0%, respectively, for the prediction of GDM at gestation weeks 32-34. Insulin determination at fasting and at 120 min had positive predictive values of 0.90 and 0.75, respectively, for the prediction of GDM at gestation weeks 32-34. The negative predictive values of fasting and 120-min serum insulin determination at gestation week < or = 16 were 0.87 and 0.96, respectively, for the prediction of GDM at gestation weeks 24-28. Increased serum insulin levels both at fasting and 120 min before gestation week 16 were very strong predictive factors for GDM by gestation weeks 32-34 with an odds ratio of 16.6 and 13.3, respectively. CONCLUSIONS: Serum insulin determination at gestation week < or = 16 is an easy and reliable method with which to predict GDM in a high-risk group. Despite a negative OGTT, patients with an elevated fasting and/or 120-min serum insulin level at gestation week < or = 16 should be managed in the same way as those with GDM. Considering the very high negative predictive value of the method, patients with a normal fasting and/or 120-min serum insulin level at gestation week < or = 16 should undergo an OGTT only at gestation weeks 32-34.

The involvement of dopamine and nitric oxide in the endocrine and behavioural action of endomorphin-1.

Previous publications have demonstrated a prominent central and corticotropin releasing hormone-mediated action of the endomorphins (EMs) on both open-field behaviour and the hypothalamo-pituitary-adrenal (HPA) axis. In the present experiments, the direct action of endomorphin-1 (EM1) on pituitary adrenocorticotropic hormone (ACTH) release, adrenal corticosterone secretion and the roles of nitric oxide (NO) and dopamine (DA) in the HPA and behavioural responses elicited by EM1 were investigated in mice. In vitro perifusion studies indicated that the action of EM1 on the HPA system appears to be confined to the hypothalamus, as EM1 did not influence the corticosterone secretion from adrenal slices and moderately attenuated the ACTH release from anterior pituitary slices. In in vivo experiments, NG-nitro-L-arginine (L-NNArg) pretreatment brought about a profound inhibition of both the endocrine and the behavioural responses. On the other hand, haloperidol completely abolished the increases in square crossing and rearing, without affecting corticosterone release. The direct action of EM1 on striatal DA release was therefore also investigated in an in vitro superfusion system. Although EM1 did not influence the basal release of tritiated DA, it significantly enhanced the transmitter release evoked by electric impulses and pretreatment with L-NNArg resulted in a considerable inhibition of the release elicited by EM1. In conclusion, our endocrine studies suggest an important role of NO in the mediation of the EM1-evoked corticosterone secretion. They also indicate that EM1 activates the HPA axis at a hypothalamic level and dopamine is not involved in this process. In contrast, the behavioural experiments reflect that the locomotor activation induced by EM1 is mediated by NO and dopamine, and the superfusion studies demonstrate that NO transmits the dopamine release enhancing effect of EM1.

Characterization of late-pregnant rat uterine contraction via the contractility ratio in vitro significance of alpha1-adrenoceptors.

The aim of this study was to characterize the ability of late-pregnant (days 15-22) rat uterine tissue rings to contract in response to electric field stimulation in vitro. For this purpose, maximum rhythmic contractions were elicited by optimum choice of the period time and the pulse width, the two main parameters of electric field stimulation. In parallel, the plasma 17beta-estradiol and progesterone levels were determined. It was found that the contractility ratio, i.e. the quotient of the optimum pulse width and the period time, is a good parameter with which to express the contractility. The larger the contractility ratio, the better the ability to contract. Evaluation of the area under the curve did not furnish information relating to the contractility in this method. A very close correlation was observed between the contractility ratio and the quotient of the 17beta-estradiol and progesterone levels on different days, demonstrating that the in vitro ability characterized by the contractility ratio is in keeping with the physiological regularity. There was also a very close correlation between the contractility ratio and the quotient of the alpha1- and beta-adrenergic receptors, suggesting the main role of the numbers of alpha1-receptor in pregnant uterine contractility. It is believed that this is the first in vitro model to give a numerical measure concerning the ontogeny of uterine contractility in late pregnancy.

[Gonadotropin stimulation of progesterone production of granulosa cells derived from in vitro fertilization procedures: comparison of the in vitro biopotency of recombinant follicle stimulating hormone, recombinant luteinizing hormone nad recombinant human chorionic gonadotropin]. / A rekombináns gonadotropinok biológiai hatásainak összehasonlító vizsgálata az in vitro fertilizáció során nyert humán granulóza sejtek progeszteron bioszintézisére sejtkultúrában.

The aim of this study was to investigate the effects of recombinant human gonadotrophins (follicle stimulating hormone [rFSH], luteinizing hormone [rLH] and human chorionic gonadotrophin [rhCG]) on granulosa-lutein cell progesterone production in long-term culture. Cultures were maintained for 9 days including a preincubation period for 3 days. Cells obtained from gonadotrophin-stimulated cycles produced increasing amounts of progesterone during the preincubation period reaching a maximum concentration on day 3. Granulosa cells in absence of serum secreted significantly lower levels of progesterone than in its presence and showed moderate responses to rhCG. Addition of serum (foetal calf serum, FCS) to the culture medium enhanced progesterone output in both control and rhCG-stimulated cultures in a dose-dependent manner. However, in absence of rhCG, granulosa cell progesterone production declined towards the end of culture even in the presence of constantly high FCS levels. All three recombinant gonadotrophins stimulated progesterone accumulation. Recombinant FSH and rLH, applied in the dose interval of 0.001-0.01-0.1 IU/ml, caused clear dose-related increases in progesterone production. Progesterone accumulation was also significantly augmented by the presence of rhCG (range of doses 0.01-0.1-1-10 IU/ml), but this effect was dose-dependent only between 0.1-10 IU/ml dose intervals with the maximum stimulation occurred at the dose of 0.1 IU/ml rhCG. From our results it can be concluded that granulosa cells require both serum supplementation and gonadotrophin stimulation for optimal progesterone synthesis. Recombinant FSH, completely devoid of LH activity, was equivalent to rLH and rhCG in terms of stimulation of progesterone production of luteinized human granulosa cells.

Oestradiol production by luteinized human granulosa cells: evidence of the stimulatory action of recombinant human follicle stimulating hormone.

In this study the effect of recombinant human follicle stimulating hormone (rFSH) on oestradiol production by human granulosa-lutein cells was examined in long-term culture, in the presence or absence of androgens. Cells were harvested at the time of follicular aspiration after ovarian hyperstimulation for in-vitro fertilization and cultured for 9 days. Granulosa cells were capable of secreting oestradiol spontaneously even without androgen and gonadotrophin support. Basal oestradiol secretion was relatively high and variable (421.3 +/- 159.3 pg/ml, mean +/- SEM, n = 13) on the first day and decreased gradually to 16.7 +/- 3.1 pg/ml on day 9. Addition of androgens (testosterone or androstenedione) to the incubation medium enhanced dose-dependently basal oestrogen production on days 5, 7 and 9. The androgen/oestrogen conversion rate remained constantly high during the culture, even without rFSH. After pre-incubation for 3 days, addition of rFSH resulted in a dose- and time-dependent increase in granulosa-lutein cell oestrogen production in the absence of exogenous androgens. Testosterone supplementation caused considerably higher basal oestradiol concentrations, however rFSH failed to further stimulate the oestrogen release from granulosa-lutein cells, suggesting that these cells cultured in vitro possess high aromatase activity even without rFSH support.

Determination of RU486 (mifepristone) in blood by radioreceptorassay; a pharmacokinetic study.

A human progesterone receptor assay has been developed for the measurement of the biologically active molecular fraction of RU486 (RU486 binding equivalent) for studying its pharmacokinetic properties. Thirty-nine healthy pregnant volunteers with amenorrhoea of 49 days or less receiving a single oral dose of 200 mg, 400 mg or 600 mg RU486 orally in a single dose were involved in this study. Blood samples were collected within 48 hours for the analysis. It was found that the pharmacokinetics of the RU486 binding equivalent followed an open two-compartment model. The dose was rapidly absorbed and peak serum concentrations were measured within 1-2 hours after ingestion of the drug. The distribution was also rapid, but the elimination was slow, the elimination half-life ranging between 83 and 90 hours. Significant differences were found between the peak plasma values for the 200 mg and 600 mg doses (p < 0.05) and between the AUCs for the 200 mg and 600 mg doses (p < 0.01) and the 400 mg and 600 mg doses (p < 0.05). It can be concluded that this newly developed radioreceptor assay satisfies the requirements of radioligand binding techniques and can be used to determine the serum levels of RU486 and its metabolites, which are able to bind to human myometrial progesterone receptors. The pharmacokinetics for the RU486 binding equivalent is similar to that for RU486, with the exception of very slow elimination, which may originate from the measurement of the biologically active metabolites together with the parent compound.

[Pharmacokinetics of RU 486 and its active metabolites in humans]. / Az RU 486 és aktív metabolitjainak farmakokinetikai vizsgálata radioreceptor analitikai mérések alapján.

The pharmacokinetics of RU 486 and its active metabolites were studied in 31 women who received a single oral dose of 200 mg (n = 9), 400 mg (n = 10) or 600 mg (n = 12) of RU 486 for termination of an early unwanted pregnancy. The serum levels were measured within 48 hours after the intake by radioligand binding assay using human myometrial cytosolic progesterone receptor as binding protein. The assay is based on the competitive replacement of 3H-ORG-2058 by the active molecular fraction of RU 486 present in the serum. The results were expressed as RU 486 equivalent. It was found that pharmacokinetics of the RU 486 equivalent followed two-compartment open model. The pharmacokinetic parameters were calculated by MEDUSA computer programme. Rapid absorption and distribution was followed by relatively slow elimination. The elimination half-life ranged between 80-90 hours. No significant difference was found between the parameters of the absorption distribution and elimination processes of three different doses. Thus, the RU 486 equivalent followed first-order kinetic. Peak serum concentrations were reached within 1-2 hours after the ingestion of the drug. Significant differences were found between 200 and 600 mg doses in the peak plasma values (p < 0.05) and in the areas under the curve (p < 0.01). However, these differences were not directly proportional to the increase of the dose.

[Radioreceptor assay for determination of the anti-progestogen, RU-486 and its active metabolites in the blood]. / Radioreceptor analitikai módszer az antiprogesztogén RU-486 és aktív metabolitjainak vérböl történö meghatározására.

RU-486 is an antiprogesterone 19-norsteroid with high binding affinity for the progesterone receptor. It can be used with success for termination of early human pregnancy. Since the metabolites of RU-486 are often biologically active, their concentrations in blood can also have clinical significance. We have developed a rapid sensitive radio receptor assay for determination of RU-486 and its active metabolites. Human myometrium progesterone receptor was used as binding protein. The assay is based on the competitive replacement of 3H-ORG-2058 by the active molecular fraction of RU-486 present in the serum. The analytical parameters of this assay are the followings: intraassay percent of coefficient of variation (CV%) is ranged between 7.6 and 10.4%. The interassay CV% was from 7.1 to 16.3. The sensitivity of the receptor assay was 8.7 pmol/tube and the acceptable range was between 10 and 120 pmol/tube. The recovery ranged between 95 and 122% (r = 0.983). These parameters are suitable for the requirement of radioligand binding techniques based on the immunoassays.

[Changes in serum levels of lipid and lipoprotein fractions during breast feeding under the effect of hormonal contraceptives]. / A lipid és a lipoprotein frakciók szérum szintjének változása a szoptatás alatti hormonális fogamzásgátlás hatására.

During the first 3 months of the full-time lactation the level of the lipid and lipoprotein fractions does not change in the mother's blood. The levonorgestrel contraceptive pills taken during the lactation do not influence considerably the lipid-metabolism either. There is only one minute deviation in the levonorgestrel group, which is the decreasing though not significant change of the HDL level. The contraceptive pills containing levonorgestrel can be given during the lactation without any danger from the point of view of mother's lipid-metabolism, too.