Biomedical subjects

A Gravanis

Publications and source records attributed to A Gravanis.

At least 55 records · Page 3Linked to original sources

PubMed1996

The PC12 rat pheochromocytoma cell line expresses the prodynorphin gene and secretes the 8 kDa dynorphin product.

Most adrenal chromaffin cells synthesize opioids derived from proenkephalin but not from prodynorphin. However, human pheochromocytomas and the PC12 rat pheochromocytoma cell line synthesize dynorphins. The aim of this study was to confirm the presence of the authentic prodynorphin transcript and its dynorphin product in PC12 cells. We have found that the sequence of a 458 bp cDNA fragment derived from RT-PCR amplification of total PC12 RNA was in complete accordance with the published sequence of the equivalent region of the prodynorphin gene. It encodes the potent endogenous kappa opioid agonists alpha-neo-endorphin, dynorphin A and dynorphin B. Furthermore, immunoaffinity-purified PC12 cell extracts were subjected to RP-HPLC. Most of its IR-dynorphin eluted on a peak exhibiting the retention time of similarly treated rat anterior pituitary. The expression of the prodynorphin gene in pheochromocytomas can be explained as either the result of (a) the process of dedifferentiation of chromaffin cells to pheochromocytoma which may thus cause the expression of a previously unexpressed prodynorphin or that (b) those pheochromocytomas expressing the prodynorphin gene derive from the few, centrally located chromaffin cells, which express this gene even under normal conditions.

Adrenal Medulla↗

PubMed1996

Corticotropin releasing hormone (CRH) in normal and pregnant uterus: physiological implications.

Corticotropin-releasing hormone, is a hypothalamic neuropeptide, responsible not only for the endocrine but also the autonomic, immunological and behavioural responses of mammalian organisms to stress. CRH is also expressed in female reproductive tissues, such as placenta and uterus. Multiple sites within the pregnant uterine cavity express the CRH gene, including the trophoblasts, fetal membranes (chorion, amnion) and decidua. The trophoblastic syncytium appears to be the major source of placental CRH. It is postulated that placental CRH influences the HPA axis of either mother or fetus and participates at the initiation of labour. Recent findings show that human and rat uterus express the CRH gene. Epithelial cells of both species are the main source of endometrial CRH, while stroma does not seem to express it, unless it differentiates to decidua. Estrogens and glucocorticoids inhibit and prostaglandin E2 stimulates the promoter of human CRH gene in transfected human endometrial cells, suggesting that endometrial CRH gene expression is under the control of these agents. Moreover, in rats, endometrial CRH expression is significantly higher at the implantation sites, compared to that at the inter-implantation uterine regions. Given the proinflammatory/vasoregulatory properties of CRH, we hypothesize that endometrial CRH may participate in the regulation of intrauterine phenomena, such as blastocyst implantation, endometrial vascularization and myometrial contractility.

Animals↗

PubMed1996

Dexamethasone alters rapidly actin polymerization dynamics in human endometrial cells: evidence for nongenomic actions involving cAMP turnover.

Glucocorticoids, in addition to their well characterized effects on the genome, may affect cell function in a manner not involving genomic pathways. The mechanisms by which the latter is achieved are not yet clear. A possible means for this action may involve the actin cytoskeleton, since the dynamic equilibrium of actin polymerization changes rapidly following exposure to several stimuli, including hormones. The aim of the present work was to find out if glucocorticoids exert rapid, nongenomic effects on actin polymerization in Ishikawa human endometrial cells, which represent a well characterized in vitro cell model expressing functional glucocorticoid receptors. Short term exposure of the cells to the synthetic glucocorticoid dexamethasone resulted in an overall decrease of the G/total-actin ratio in a time- and dose-dependent manner. Specifically, in untreated Ishikawa cells the G/total-actin ratio was 0.48 +/- 0.01 (n = 26). It became 0.35 +/- 0.01 (n = 13, P < 0.01) following exposure to 10(-7) M dexamethasone for 15 min. This was induced by a significant decrease of the cellular G-actin level, without affecting the total actin content, indicating a rapid actin polymerization. This conclusion was fully confirmed by direct fluorimetry measurements, that showed a significant increase of the F-actin content by 44% (n = 6, P < 0.001) in cells treated with dexamethasone (10(-7)M, 15 min). The rapid dexamethasone-induced alterations of the state of actin polymerization were further supported by fluorescence microscopy. The latter studies showed that the microfilaments of cells pretreated with 10(-7)M dexamethasone for 15 min were more resistant to various concentrations of the antimicrofilament drug cytochalasin B, compared to untreated cells, implying microfilament stabilization. The action of dexamethasone on actin polymerization seems to be mediated via specific glucocorticoid binding sites, since the addition of the glucocorticoid antagonist RU486 completely abolished its effect. Moreover, it appears to act via non-transcriptional pathways, since actinomycin D did not block the dexamethasone-induced actin polymerization. In addition, cell treatment with 10(-7)M dexamethasone for 15 min fully reversed the forskolin-, but not the 8-bromo-cAMP-induced actin depolymerization. In line with these findings, the cAMP content of Ishikawa cells was decreased by 29.2% after a 15 min treatment with 10(-7)M dexamethasone (n = 4, P < 0.01). In conclusion, our results showed that dexamethasone induces rapid, time-, and dose-dependent changes in actin polymerization dynamics in Ishikawa cells. This action seems to be mediated via cAMP, involving probably nongenomic pathways. The above findings offer new perspectives for the understanding of the early cellular responses to glucocorticoids.

Actin Cytoskeleton↗

PubMed1996

Opioids inhibit dopamine secretion from PC12 rat pheochromocytoma cells in a naloxone-reversible manner.

Opioids inhibit the release of catecholamines in the nervous system. Normal adrenal chromaffin cells produce delta opioids and they respond to them by suppressing the release of their catecholamines. Chromaffin cell tumors, the pheochromocytomas, produce mainly kappa opioids. The aim of this work was: (a) to test if pheochromocytomas retain the response of normal chromaffin cell catecholamines to delta opioids and to naloxone (a general opioid antagonist), and (b) to test if kappa opioids exert any specific effect on catecholamine release from these tumors. Since we have previously shown that, in common with human pheochromocytomas, the PC12 rat pheochromocytoma cells express the prodynorphin gene and secret its kappa opioid products, we used these cells to examine the effect of several opioid agonists and of naloxone on basal, nicotine-, and KCl-induced dopamine release. Dopamine is the main PC12 catecholamine. We have found that the specific kappa opioid agonist U-69593 inhibited the release of dopamine in a dose-dependent manner (IC50=0.5 x 10(-8)M). Under basal conditions the mean concentration of dopamine in the culture media was 11.25 +/- 0.57 ng/mg of total cellular protein (n=13). A 30 min exposure to U-69593 at 10(-6) M suppressed basal dopamine release to 58 +/- 2% (n=7) of controls. A 12 hr pre-incubation with U-69593 caused the same degree of suppression. The effect of the synthetic kappa opioid agonist dynorphin A was indistinguishable from that of U-69593. DADLE (a mu and delta synthetic opioid agonist) was significantly less effective in suppressing dopamine release (IC50=10(-7)M). The concentration of dopamine following exposure to 10-6 M of DADLE for 30 min was 74 +/- 5% of the controls (n=4). The mu opioid agonist DAGO was ineffective. The suppressive effect of all opioid agonists was blocked by naloxone suggesting that conventional opioid receptors were involved.

Analgesics↗

PubMed1996

Kappa opioids exert a strong antiproliferative effect on PC12 rat pheochromocytoma cells.

Pheochromocytomas synthesize several types of opioids and their receptors. Opioids affect the proliferation rate of normal and tumoral cells. We have previously shown that the PC12 rat pheochromocytoma cells synthesize multiple opioids. The aim of the present work was to study the effect of opioids on the proliferation of these pheochromocytoma cells. Thus, the effect of several opioid agonists and antagonists was examined on basal and EGF-induced PC12 cell proliferation. The kappa opioid agonists dynorphin A, U-69593, and U-50488 suppressed basal proliferation in a dose-dependent manner. The effect of kappa opioids was blocked by the general opioid antagonist naloxone and the selective kappa antagonist nor-binaltorphimine. Furthermore, both opioid antagonists given alone had a strong stimulatory effect, a findings suggesting that the proliferation of PC12 cells is under tonic inhibition by locally produced kappa opioids. Finally, the mu-opioid agonist DAGO and the delta and mu agonists DADLE and DSLET were ineffective.

Animals↗

PubMed1996

Identification, characterization and localization of corticotropin-releasing hormone receptors in human placenta.

The purpose of this work was to identify, characterise, and localise specific CRH binding sites in whole vaginally delivered term human placental membranes as well as in dispersed and purified trophoblastic membranes. We have found that whole placenta membranes contained specific and saturable CRH-binding sites. Maximum specific binding was obtained at pH 7.2-7.4, 22 C, for 2 h. The order of potency of CRH analogs was h/rCRH > alpha-helCRH > oCRH. Scatchard analysis revealed a single population of high affinity binding sites with a dissociation constant of 1.25 nM and maximum binding capacity of 119 fmol/mg of protein. Purified syncytiotrophoblast membranes contained high affinity CRH binding sites exhibiting the same dissociation constant and maximum binding capacity as whole placental membranes, suggesting that the CRH binding sites are expressed almost exclusively by the syncytiotrophoblast. Our findings indicate that CRH binding sites in the human placenta are exhibit similar characteristics to that described for anterior pituitary, brain and adrenals.

Corticotropin-Releasing Hormone↗

PubMed1996

Regulation of the promoter of the human corticotropin-releasing hormone gene in transfected human endometrial cells.

Corticotropin-releasing hormone (CRH) is expressed in several peripheral tissues, including normal epithelial cells of the human and rodent uterus. However, the biological role of endometrial CRH is known in neither species. As a first step to clarify this role, we studied the regulation of CRH promoter in endometrial cells. We performed homologous transfection experiments in Ishikawa cells, a human endometrial cell line, using a 0.9-kb fragment of the 5'-flanking region of the human CRH gene coupled to luciferase. Transfected cells were exposed for 18 h to 8-bromo cyclic adenosine monophosphate, forskolin, epidermal growth factor, steroids (estradiol, progesterone, and the synthetic glucocorticoid dexamethasone and their antagonists), and prostaglandin E2; then the activity of the luciferase reporter was determined in the cell lysates. We found that the activity of the 5'-flanking region of the CRH gene was stimulated by cyclic adenosine monophosphate and epidermal growth factor and inhibited in a receptor-mediated, dose-dependent fashion by estradiol and dexamethasone. The antiglucocorticoid RU 486 acted as a glucocorticoid agonist, suppressing the CRH gene activation, while progesterone was devoid of any activity. Prostaglandin E2 stimulated the CRH activation, and the prostanoid inhibitor indomethacin suppressed it, most probably by inhibiting endogenous prostaglandins. These findings suggest that endometrial CRH gene expression may be under the negative control of estrogens and glucocorticoids and under the positive control of prostaglandin E2.

Adenocarcinoma↗

PubMed1995

PC12 cells as a model to study the effects of opioids on normal and tumoral adrenal chromaffin cells.

Normal adrenal chromaffin cells produce delta opioid peptides while at the same time they have mainly kappa opioid receptors. This paper describes our date regarding the expression of the prodynorphin gene, the precursor of a family of endogenous kappa opioid ligands, in the PC12 rat pheochromocytoma cell line, and the effects of synthetic kappa opioid agonists and of Naloxone on various aspects of PC12 cell function including their secretion of catecholamines, proliferation and differentiation. This is the first part of a series of projects aimed at studying, (a) the conditions under which the prodynorphin gene is expressed in normal adrenomedullary cells, and (b) the physiological role of the endogenous kappa opioids in the physiology of the adrenal medulla.

Adrenal Medulla↗

PubMed1995

Corticotropin-releasing hormone (CRH) is expressed at the implantation sites of early pregnant rat uterus.

We have shown previously that the epithelial cells of human endometrium produce CRH. The biological role of endometrial CRH is not yet known. Among other things, CRH appears to be involved in the inflammatory process, acting as an autocrine/paracrine proinflammatory regulator. Since the reaction of endometrium to the invading blastocyst has characteristics of an aseptic inflammatory reaction, we have hypothesized that endometrial CRH may participate in the inflammatory phenomena taking place at the implantation site of blastocyst. In the present study we demonstrate a higher content of immunoreactive (IR)-CRH and CRH mRNA in the implantation sites of early pregnant rat uterus compared to the inter-implantation regions. Specifically we have found that: a) rat uterus contained a 1.3 kb CRH transcript, similar or identical in size to that present in human placenta, b) the size of the IR-CRH present in uterine extracts was similar to authentic hypothalamic CRH, c) Northern blot analysis showed that the content of CRH mRNA in uterus at the implantation sites was 3.5 fold higher compared to that in the inter-implantation regions and finally, d) immunohistochemical localization of IR-CRH in early pregnant rat uterus revealed positive staining of the luminal epithelial cells in both implantation and inter-implantation uterine regions, while decidualized stromal cells were positive only at the implantation sites. Our data suggest that endometrial CRH may play a role in the implantation of blastocyst.

Animals↗

PubMed1995

Hormonal anti-implantation agents: antiprogestins.

Oestrogens and progestogens separately or in combination are able to prevent implantation with high efficiency, thus acting as interceptive agents. Current interceptive medical regimens include high-dose oestrogens, the association of oestrogens with progestogens or progestogens alone. Compounds with antiprogesterone properties, such as RU 486 (mifepristone) or ZK 98734 (lilopristone), also exhibit a strong interceptive action which, as shown in animal models, is proportional to the dose and the day(s) of administration. Recent clinical studies show that RU 486 can be used successfully for postcoital interception. The regimen applied for this purpose consists of the intake of a single dose of 600 mg RU 486 within 72 h of a single act of unprotected intercourse. This treatment was found to be highly effective and to have a more favourably side-effect profile in comparison with the oestrogen-progestogen interceptive regimen. However, because of the induced irregularities of the cycle, the mifepristone regimen, as with the other hormonal methods, should not be used on a regular basis. Currently, all interceptive hormonal regimens are emergency methods. Their occasional use to prevent unwanted pregnancies may reduce the number of therapeutic abortions. However, the frequency and extent of their side-effects do not allow for a repeated postcoital use after every act of unprotected intercourse. Obviously, the development of an effective and safe 'morning after pill' requires further basic and clinical investigations.

Animals↗

PubMed1995

The role of prostaglandins in blastocyst implantation.

This review summarizes the information available on the involvement of prostaglandins in blastocyst implantation, and examines their interactions with three other inflammatory mediators, platelet-activating factor (PAF), interleukin (IL-1) and corticotrophin-releasing factor (CRF). Essential elements of this information, consistent with the assumption that prostaglandins play an important role in implantation, appear to be: (i) the burst of endometrial prostaglandin production, following the blastocyst signal(s) or an artificial stimulus; (ii) the main localization of this production at the luminal epithelium and release towards the stroma; and (iii) the presence at the stromal level of specific progesterone-dependent binding sites for prostaglandin E2. In addition, accumulated data indicate a paracrine interaction at the endometrial level between PAF and prostaglandin E2, which could serve, among others, to amplify the embryonic signal(s). Pro-inflammatory cytokines IL-1 alpha and IL-1 beta may also play a significant role in endometrial response via the modulation of prostaglandin E2 production. Prostaglandins and IL-1 induce the expression of CRF, which acts as an autocrine/paracrine inflammatory regulator. CRF exhibits a strong vasoactivity in skin tests, inducing a local increase of capillary permeability at a concentrations of 10(-10) M. Levels of CRF and its mRNA were found to be higher in rat implantation sites compared with those in the interimplantation regions. Stromal cells were found to be positive for immunoreactive CRF at the implantation sites only. It is suggested that CRF may be involved in the local increase of capillary permeability seen in implantation sites, and that its production by stromal cells may be the consequence of a paracrine action of epithelial prostaglandin, released under the effect of PAF and IL-1, derived from or produced by blastocysts in endometrial cells.

Blastocyst↗

PubMed1995

The corticotropin-releasing hormone (CRH) in normal and tumoral epithelial cells of human endometrium.

CRH is produced by several intrauterine sites, including placenta and desidua, during pregnancy. However, no data are available regarding the presence of CRH in the nonpregnant uterus. We now report that CRH is produced in the epithelial cells of normally cycling human uterus and in an endometrial epithelial cell-derived tumor. Specifically, we have found that: 1) Northern blot hybridization analysis of normal glandular endometrium as well as of Ishikawa human endometrial adenocarcinoma cells showed the presence of the CRH messenger RNA; the size of the transcript seemed to be identical to that present in human placenta and rat hypothalamus; 2) immunoreactive CRH (ir-CRH) was detectable in normal dispersed glandular endometrial cells as well as in the Ishikawa adenocarcinoma cells; 3) gel filtration chromatography of normal glandular endometrial and Ishikawa cell extracts and their culture media showed that most ir-CRH present had the mol wt of the authentic CRH peptide; in addition, a larger form of ir-CRH was also present in both normal and tumoral endometrial epithelial cell extracts; the latter most probably correspondents to CRH precursor molecules; and 4) immunofluorescence staining of CRH in normal glandular endometrial and Ishikawa cells revealed a cytoplasm rich in granules positive for ir-CRH. Our findings suggest that CRH may play an important role in the physiological events taking place within the uterine cavity, since CRH seems to be present in nonpregnant as well as pregnant uteri. Since CRH is expressed in normal endometrial epithelial cells and in an epithelial tumoral cell line, we propose the use of the Ishikawa cell line as a convenient model for the in vitro study of endometrial CRH.

Adenocarcinoma↗

PubMed1995

Identification and characterization of opioid-binding sites present in the Ishikawa human endometrial adenocarcinoma cell line.

Normal epithelial cells of human endometrium, and Ishikawa human endometrial adenocarcinoma cells (an in vitro model for the study of steroid hormone effects on human endometrium) have been found to express and secrete opioid peptides deriving from proenkephalin, prodynorphin, and proopiomelanocortin. These opioids may act locally, affecting the uterine tissues. In the present study, we identified and characterized opioid-binding sites on the Ishikawa cell line, producing evidence for the mechanism of local opioid action. We used an acid shock before the receptor assay to dissociate any endogenously bound peptide. The acidification improved specific binding by 2- to 4.5-fold. Characterization of opioid binding using different radiolabeled opioids and effectors has shown the existence of a low concentration of delta-sites (Kd, 6.20 nmol/L; 4,890 sites/cell), no mu-sites, low affinity kappa 1-sites (Kd, 10.8 nmol/L; 276,000 sites/cell), kappa 2-sites with high affinity for ethylketocyclazocine (Kd, approximately 1 nmol/L) and low affinity for diprenorphine (Kd, approximately 8 nmol/L) at a concentration of 93,000 sites/cell, and high affinity kappa 3-sites (Kd, 3.6 nmol/L; 77,000 sites/cell). In conclusion, our report characterizes opioid sites in a particular and homogeneous cell type of human endometrium, i.e. in epithelial cells. The coexistence of opioid sites and their endogenous ligands in the Ishikawa cell line makes these cells a good model for the study of autocrine/paracrine interactions of opioids in nonneural tissues.

Acids↗

PubMed1994

Interaction between steroid hormones and endometrial opioids.

The opioids beta-endorphin and the dynorphins belong to two separate families of endogenous opioid peptides (EOP). They are produced not only in the central nervous system but also in nonneural tissues where, as it appears, they act locally via paracrine mechanisms. These opioids have been shown to be produced at multiple sites along the mammalian reproductive tract including the intrauterine cavity. The aim of the present work was to find out if the well differentiated human endometrial cell line of Ishikawa, which has been shown to be a good in vitro model for the study of the effects of steroid hormones on human epithelial endometrium, expresses these two EOP. Northern blot hybridization of RNA from these cells showed the presence of a 1.2-kb POMC and a 2.4-kb PDYN transcript. Radioimmunoassay and gel filtration chromatography characterization of the immunoreactive (IR) opioid peptides present in the culture media showed the presence of IR-beta-endorphin and IR-dynorphins. The apparent molecular weight of IR-beta-endorphin was that of authentic beta-endorphin while the bulk of the IR-dynorphin had an apparent molecular weight of 8 kd. The secretion of both opioids could be increased by KCl-induced depolarization. Estrogen and glucocorticoids decreased, in a dose- and time-dependent manner, the secretion of beta-endorphin from the Ishikawa cells while progesterone and dihydrotestosterone did not have a statistically significant effect. The antiprogestin-antiglucocorticoid RU486 acted as an agonist, i.e., it diminished beta-endorphin secretion possibly via glucocorticoid receptors. On the other hand, the secretion of dynorphins was not affected by any of the steroids tested while LHRH, the inducer of gonadotropins and anterior pituitary dynorphins secretion, provoked a time- and dose-dependent increase of their secretion without affecting that of beta-endorphin. These data suggest that the regulation of endometrial opioids production is type-specific. Thus, it is possible that each type of endometrial opioid participates in different local homeostatic loops and exerts distinct paracrine effects.

Adenocarcinoma↗

PubMed1994

Cytochalasin B may shorten actin filaments by a mechanism independent of barbed end capping.

It is generally accepted that cytochalasin B (CB), as well as other cytochalasins, shorten actin filaments by blocking monomer addition at the fast-growing ("barbed") end of these polymers. Despite the predominance of this mechanism, recent evidence suggests that other interactions may also occur between CB and F-actin. To investigate this possibility further we have employed an actin derivative, prepared by substitution at Cys374 by a glutathionyl residue. We demonstrate here that CB did not significantly bind to glutathionyl F-actin under several ionic conditions. We further show that in the presence of CB the glutathionyl-F-actin exhibits a significantly higher ATPase activity than the non-modified F-actin. These data argue that the incorporation of glutathionyl groups prevents the high-affinity binding of CB to the barbed end of actin filaments, probably due to a decreased hydrophobicity of the CB binding site by the introduction of the hydrophilic glutathionyl residue. Despite the lack of substantial binding at equilibrium, we have found that the addition of CB to glutathionyl-F-actin results in extensive fragmentation of the filaments, as demonstrated by electron microscopy and by a significant reduction of the relative viscosity of actin solutions. These results are consistent with the idea that CB shortens glutathionyl-actin filaments by a mechanism distinct from barbed end capping. Glutathionyl F-actin offers an interesting model to study the complex mechanism of interaction of actin filaments with cytochalasins and with the physiologically important actin capping/severing proteins.

Actins↗

PubMed1994

Differences in the G/total actin ratio and microfilament stability between normal and malignant human keratinocytes.

The state of polymerization of actin and the organization of actin filaments is widely believed to be related to cellular transformation. Since the intracellular monomer (G) and filamentous (F) actin content reflects the state of microfilament polymerization, we measured the G/total actin ratio in primary cultures of normal and malignant human keratinocytes. In normal keratinocytes the mean value of this ratio was 0.30 +/- 0.03 (mean +/- SE, n = 15), while in basal cell carcinoma (BCC) keratinocytes it was 0.49 +/- 0.03 (n = 8) and in squamous cell carcinoma keratinocytes (SCC) 0.5 +/- 0.07 (n = 4), indicating a 1.7-fold increase of the G/total actin ratio in malignant cells. These results imply that the proportion of polymerized actin is decreased markedly in malignant keratinocytes, suggesting alterations of microfilament structures which probably occur during the transformation process. This was supported by the morphological changes of microfilament structures as assessed by fluorescence microscopy. A different distribution of actin filaments in normal and malignant cells became evident; stress-fibres were converging in patches at several points in SCC cells, when compared to normal keratinocytes. Furthermore, incubation of normal and malignant keratinocytes with cytochalasin B indicated differences in the resistance of their microfilament networks. After 1 h exposure to 10(-6) and 10(-5) M cytochalasin B, microfilaments in normal cells appeared to be less affected than their counterparts in neoplastic cells. Even in a high excess of cytochalasin B (10(-4) M), normal keratinocytes preserved their shape, while both basal cell and SCC were totally disrupted.(ABSTRACT TRUNCATED AT 250 WORDS)

Actin Cytoskeleton↗

PubMed1994

In vitro differential effects of the antiglucocorticoid RU486 on the release of lymphokines from mitogen-activated normal human lymphocytes.

The synthetic antiglucocorticoid RU486 has multiple effects on the immune system. We have recently reported that RU486 suppresses normal lymphocyte proliferation and downregulates interleukin-2 receptors (IL-2R) by decreasing the accumulation of the beta-chain IL-2R mRNA in normal human lymphocytes in culture. To further explore the mechanism of the immunoregulatory actions of RU486, in the present study, we investigated the effects of this molecule on the release of lymphokines from phytohemagglutinin (PHA)-activated normal human peripheral blood lymphocytes (NPBL) in culture. We have found that RU486 differentially regulates the release of lymphokines from PHA-activated NPB lymphocytes. Specifically, RU486 (at concentrations of 1-100 nM) exerts pure antagonist actions by almost completely reversing the inhibitory effects of the glucocorticoid dexamethasone (Dex) on the release of monocyte/macrophages-derived lymphokines, such as IL-1, IL-6, IL-8 and tumor necrosis factor-alpha (TNF-alpha). Dex decreased in a dose-dependent manner the release of the above four lymphokines, with an ID50 of 0.9 +/- 0.1, 4.76 +/- 0.4, 9.8 +/- 1.8, and 1.16 +/- 0.2 nM for IL-1, IL-6, IL-8 and TNF-alpha, respectively. Conversely, RU486 exhibits both agonist and antagonist effects on the release of T-lymphocyte-derived lymphokines. RU486 given alone, exerts agonist/glucocorticoid effects, by decreasing in a dose-dependent manner the release of IL-2 and -3. The maximal inhibitory effect of RU486 was observed at 10 nM and was 64.5 +/- 4.3% of the control value, (n = 6, P < 0.02) for IL-2 and 59.2 +/- 6.3% (n = 6, P < 0.02) for IL-3. The ID50 of RU486 for the release of IL-2 and -3 were 14.6 +/- 2.0 and 11.6 +/- 1.9 nM, respectively, i.e. almost similar with those of Dex. Interestingly, when high doses of RU486 (1 microM) were combined with Dex RU486 exhibited antagonist actions by significantly counteracting the inhibitory effects of Dex on IL-2 and -3 release. In conclusion, the antiglucocorticoid RU486 exhibits complex regulatory actions on lymphokine secretion, dependent upon the type of the lymphokine-producing cell. A pure antagonist effect was observed on the release of monocyte-derived IL-1, IL-6, IL-8 and TNF-alpha. However, when RU486 was given alone it acted as a glucocorticoid agonist on the secretion of T-lymphocyte-derived IL-2 and -3, while combined with the agonist (Dex) it exhibits antagonist effects on the release of the above lymphokines.(ABSTRACT TRUNCATED AT 400 WORDS)

Cells, Cultured↗

PubMed1994

Effects of cholic acid and 'Protectaid' formulations on human sperm motility and ultrastructure.

Cholic acid (sodium cholate) and the other active ingredients of F-5 gel preparations in use for the impregnation of a new vaginal sponge (Protectaid) with contraceptive and anti-sexually transmitted disease properties, were assessed for their effects on human sperm motility and ultrastructure. Cholic acid (CA) produced an inhibition of motility which was both dose- and time-dependent. A complete suppression of motility was obtained at 30 s by a CA concentration of 1.25%. Nonoxynol-9 (NX9) compared with benzalkonium chloride (BZC) showed no significant difference at the concentration required (0.025%) to give a total inhibition of sperm motility after exposure for 30 s. The addition of F-5A gel containing 0.5% of each one of the spermicide ingredients (CA, NX9 and BZC) produced the total suppression of sperm motility within 30 s at a dilution of 1/50. Another preparation, F-5B gel, containing the spermicide ingredients at different concentrations (1.25% CA, 0.125% NX9 and 0.05% BZC) produced this same effect with a 1/10 dilution. Exposure of semen to a CA concentration of 1.25% or to 1/10 dilutions of F-5A gel for 30 s led to profound changes of sperm ultrastructure studied by scanning (SEM) and transmission (TEM) electron microscopy. SEM and TEM findings indicate that CA acts as a spermicide through its 'natural detergent' properties, damaging the outer plasma membrane of sperm cells. Protectaid formulations affect sperm motility and viability in a similar way.

Acrosome↗