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P C MacDonald

Publications and source records attributed to P C MacDonald.

At least 37 records · Page 2Linked to original sources
PubMed1996

Modulation of the levels of interleukin-8 messenger ribonucleic acid and interleukin-8 protein synthesis in human endometrial stromal cells by transforming growth factor-beta 1.

Selected functions of uterine endometrium of ovulatory women before and during pregnancy appear to be modulated by cytokines and other paracrine-acting factors. Some of these functions are regulated, in turn, by cyclic changes in ovarian steroid secretion or by pregnancy-induced endocrine and paracrine factors. The recruitment of specific types and numbers of bone marrow-derived cells into the endometrium occurs in a predictable manner with hormonal changes of the ovarian cycle, during the process of endometrial decidualization, at the time of blastocyst implantation, and during pregnancy, parturition, and the puerperium. As part of an investigation of the regulation of the leukocyte population of endometrium/decidua, this study was conducted to evaluate further the regulation of interleukin-8 (IL-8) gene expression by transforming growth factor-beta (TGF beta). IL-8 is a neutrophil chemoattractant/activating and T cell chemotactic factor as well as a chemotactic factor for fibroblasts. IL-8 is produced by mesenchymal cells of many tissues, including human endometrial stromal cells in culture. The level of IL-8 messenger ribonucleic acid (mRNA) in endometrial stromal cells and the accumulation of immunoreactive IL-8 in medium are increased by TGF beta 1 treatment of these cells. This response to TGF beta 1 is attributable primarily to an increase in the stability of IL-8 mRNA through a process that is dependent on protein synthesis. Transcription of the IL-8 gene in endometrial stromal cells is not increased, but, rather, is slightly decreased, by treatment with TGF beta 1. The findings of this study indicate that TGF beta may act in endometrial stroma to modulate the stability of IL-8 mRNA.

Base Sequence↗
PubMed1995

Regulation of monocyte chemotactic protein-1 gene expression in human endometrial cells in cultures.

Bone marrow-derived leukocytes are present in human endometrium/decidua and are believed to serve a variety of functions in this tissue. The number and type of leukocytes in endometrium/decidua vary with the hormonal milieu of the ovarian cycle, with blastocyst implantation, and during pregnancy. The factors that regulate the recruitment of specific leukocytes to the endometrium and those that modulate the function or replication of leukocytes in this tissue are not well defined. In this study, we evaluated the potential for synthesis of monocyte chemotactic protein-1 (MCP-1), a polypeptide with monocyte/macrophage chemotactic and activating properties, in human endometrium and in separated endometrial stromal and epithelial cells. MCP-1 mRNA was readily detected by northern analysis of total RNA isolated from human endometrial tissue (n = 39 tissues from ovulatory women; n = 3 atrophic endometria from anovulatory women; n = 6 from women ingesting oral contraceptives or medroxyprogesterone acetate) and decidua parietalis at midtrimester (n = 6 pregnancies) and at term (n = 6 pregnancies). The levels of MCP-1 mRNA varied considerably among tissues; but in this relatively small number of samples, there was no apparent relationship between day of cycle, endocrine status, or duration of pregnancy and the level of MCP-1 mRNA. MCP-1 mRNA was detected in separated endometrial stromal cells and epithelial cells in culture. In confluent human endometrial stromal cells in the absence or presence of fetal bovine serum (10%, v/v), MCP-1 mRNA was detected by northern analysis of total RNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1994

Parathyroid hormone-related protein expression in gynecic squamous carcinoma cells.

BACKGROUND: The regulation of parathyroid hormone-related protein (PTH-rP) mRNA levels and immunoreactive (ir)PTH-rP formation by peptide growth factors, particularly transforming growth factor-beta (TGF-beta) and epidermal growth factor (EGF), in squamous cell carcinomas of gynecologic origin is largely unknown. METHODS: PTH-rP mRNA levels were evaluated by Northern analysis in A431 cells (derived from a human vulvar epidermoid carcinoma) and ME-180 cells (derived from a human papillomavirus-infected squamous cell carcinoma of the cervix). PTH-rP protein levels in cell culture media were evaluated using both radioimmunoassay and immunoradiometric assay techniques. These results were compared with those from a lung carcinoid cell line known to produce PTH-rP, namely, NCI-H727 cells. RESULTS: TGF-beta 1 or EGF treatment caused an increase in the levels of PTH-rP mRNA in A431 cells; these increases in PTH-rP mRNA were detectable after 60 minutes of treatment, were maximal at approximately 4-8 hours, and were approximately additive. Immunoreactive PTH-rP was not detectable (using two different PTH-rP immunoassays) in the culture medium or cell sonicates of A431 cells before or after treatment with TGF-beta 1, EGF, or TGF-beta 1 plus EGF. ME-180 cells responded to EGF (but not to TGF-beta 1) with an increase in the level of PTH-rP mRNA as early as 2 hours; irPTH-rP was present (by use of either immunoassay) in the medium of these cells at 8 and 24 hours. In NCI-H727 (human lung carcinoid) cells, TGF-beta 1 and EGF acted alone and synergistically to effect increases in PTH-rP mRNA and the accumulation of irPTH-rP. CONCLUSIONS: TGF-beta 1 and EGF regulation of PTH-rP gene expression in squamous cell carcinomas of gynecologic origin is unique for each cell line studied and different from that in human lung carcinoid cells.

Base Sequence↗
PubMed1994

17 beta-Hydroxysteroid dehydrogenase type 2: chromosomal assignment and progestin regulation of gene expression in human endometrium.

The cDNAs for two separate human 17 beta-hydroxysteroid dehydrogenases (17 beta-HSD) have been isolated and sequenced. The well-studied human placental cytosolic 17 beta-HSD (also referred to as estradiol dehydrogenase) preferentially catalyzes the reduction of estrone to estradiol-17 beta and the reduction of the C-20-ketone of progesterone to 20 alpha-dihydroprogesterone. This isoform of the enzyme has been referred to as 17 beta-HSD type 1 and localized to chromosome 17. A second 17 beta-HSD isoform (referred to as type 2) is localized in the endoplasmic reticulum of human trophoblast and is characterized by the preferential oxidation of the C-17 beta-hydroxyl group of C18- and C19-steroids and the C-20 alpha-hydroxyl group of 20 alpha-dihydroprogesterone. In this study, we determined the chromosomal localization of human 17 beta-HSD type 2, the expression of this gene in human endometrium, and the tissue distribution of the mRNA. We found that the human 17 beta-HSD type 2 gene is localized on chromosome 16, 16q24. 17 beta-HSD type 2 mRNA (approximately 1.5 kb) was identified in human endometrial tissues by Northern analysis of total RNA (10 micrograms). The highest levels of 17 beta-HSD type 2 mRNA were found in endometrial tissues obtained during the mid- to late secretory phase of the ovarian cycle (i.e., during the time of high plasma levels of progesterone). 17 beta-HSD type 2 mRNA levels were much greater in glandular epithelium than in the stromal cells isolated from secretory phase endometrium. The levels of 17 beta-HSD type 2 mRNA in secretory phase endometrium were approximately one-tenth that in villous trophoblast tissue from human placenta. We did not detect 17 beta-HSD type 1 mRNA in endometrial tissue by Northern analysis of total (10 micrograms) RNA. These findings are consistent with the view that the progestin-regulated 17 beta-HSD of the glandular epithelium of the human endometrium is primarily, if not exclusively, the product of the 17 beta-HSD type 2 gene. 17 beta-HSD type 2 mRNA was present in human placenta, liver, and small intestine; much smaller amounts, barely detectable by Northern analysis of poly(A)+ RNA, were present in prostate, kidney, pancreas, and colon, but not in heart, brain, skeletal muscle, spleen, thymus, ovary, or testis.

17-Hydroxysteroid Dehydrogenases↗
PubMed1994

Human fetal membrane contribution to the prevention of parturition: uterotonin degradation.

This investigation was conducted to evaluate the potential capacity of the human fetal membranes-decidua parietalis, and in particular the chorion laeve, to degrade uterotonins that are produced in amnion, are present in amniotic fluid, or both. The four uterotonins that have been evaluated most frequently as myometrial contractants potentially involved in the initiation of human parturition are prostaglandins, oxytocin, endothelin-1, and platelet-activating factor. We assessed the levels of mRNA and the specific activities (SAs) of enkephalinase (the plasma membrane endopeptidase that degrades endothelins) and prostaglandin dehydrogenase (PGDH) in human fetal membranes, i.e. amnion and chorion leave, and in decidua parietalis. The SA of oxytocinase (which inactivates oxytocin) in these tissues also was determined. The SA of enkephalinase in chorion laeve from all anatomical sites (singleton and diamnionic-dichorionic twin placentae) in all pregnancies studied (mean +/- SEM, 95 +/- 7.9 ng/min.mg protein; n = 28) is similar to that in human fetal kidney (89.5 +/- 2.8; n = 6). Kidney tissue is believed to be one of the richest sources of enkephalinase. The SAs of enkephalinase in amnion (18.3 +/- 2.3 nmol/min.mg protein; n = 29) and in decidua parietalis (31.8 +/- 6.7; n = 20) also were high, but significantly less than that in chorion leave. The level of enkephalinase mRNA in chorion laeve in singleton pregnancies is high, as is the SA of enkephalinase (111.9 +/- 10.6 nmol/min.mg protein; n = 17). In paired chorion laeve tissues from five diamnionic-dichorionic twin placentae, the SAs of enkephalinase in reflected chorion laeve (74 +/- 12.8; P < 0.06 compared with singletons) and fused chorion laeve (64.8 +/- 6.5; P < 0.001 compared with singletons) were similar. The SA of PGDH in reflected chorion leave (46.3 +/- 6.9 nmol/min.mg protein; n = 19) was significantly greater than that in decidua (16 +/- 5.5; n = 15). There was a significant correlation between the levels of PGDH mRNA and PGDH enzyme SA. In fused chorion laeve of diamnionic-dichorionic twin placentae, the SA of PGDH (14.9 +/- 7.3; n = 4) was much less than that in reflected chorion laeve of the same twin pregnancy (70.5 +/- 14.7; n = 4). PGDH mRNA was not detectable in amnion tissue (n = 5) by northern analysis, and the SA of PGDH (< 1.2 +/- 1.0; n = 6) in amnion was undetectable or near the lower limit of assay detection.(ABSTRACT TRUNCATED AT 400 WORDS)

1-Alkyl-2-acetylglycerophosphocholine Esterase↗
PubMed1994

Steroid secretory characteristics of a virilizing adrenal adenoma in a woman.

A tumour of the left adrenal gland was identified in a woman who presented with virilization and secondary amenorrhea. Preoperatively, the plasma levels of dehydroepiandrosterone sulphate, dehydroepiandrosterone, androstenedione, testosterone, 5 alpha-dihydrotestosterone and 5-androstene-3 beta,17 beta-diol were elevated two- to fourfold whereas those of urinary 17-ketosteroids were elevated more than tenfold. The production rate of dehydroepiandrosterone sulphate was more than 16 times that in normal women whereas those of dehydroepiandrosterone, testosterone and androstenedione were approximately twofold greater; plasma testosterone was derived almost entirely from the peripheral conversion of androstenedione. Blood was obtained by catheterization of the ovarian veins, left adrenal gland vein and inferior vena cava (at two different sites) and plasma steroid levels were determined: testosterone and cortisol levels were elevated in all blood samples whereas those of androstenedione, dehydroepiandrosterone sulphate and 11-desoxycortisol were approximately six- to eightfold, 1.5-fold and nine- to 22-fold higher in the effluent on the left adrenal gland/tumour compared with the levels in the other compartments. Blood was collected hourly for 24 h to determine steroid levels under basal conditions and, also, after ACTH treatment. Plasma cortisol levels increased markedly upon ACTH administration and fell to very low levels 11 h later, but those of androstenedione, testosterone, dehydroepiandrosterone, 5-androstene-3 beta,17 beta-diol and dehydroepiandrosterone sulphate were not affected by ACTH treatment. A histological diagnosis of cortical adenoma of the extirpated tumour was made. Tissue explants and adenoma cells were maintained in culture to characterize the steroid-metabolizing properties of the tumour. The secretion of dehydroepiandrosterone sulphate by tissue explants was highly initially, but declined to almost undetectable levels after 5 days in culture. In the presence of ACTH, dehydroepiandrosterone sulphate secretion remained elevated throughout the entire study up to 5 days. Basal secretion of dehydroepiandrosterone sulphate, androstenedione, 11-desoxycortisol, cortisol, testosterone and 11 beta-hydroxyandrostenedione by adenoma cells was either very low or undetectable. In the presence of ACTH, dibutyryl cyclic AMP or cholera toxin the secretion of dehydroepiandrosterone sulphate, androstenedione and 11-desoxycortisol increased markedly with time in culture up to 3 days, whereas the other steroids were undetected in the medium. A homogenate of adenoma tissue metabolized testosterone to androstenedione, but the conversion of androstenedione to testosterone was minimal. The findings of this study served to establish that virilization in this woman was due at least in part, to excess testosterone--and testosterone-derived 5 alpha-dihydrotestosterone--produced at extra-adrenal tissue sites almost exclusively through metabolism of tumour-secreted androstenedione.(ABSTRACT TRUNCATED AT 400 WORDS)

17-Ketosteroids↗
PubMed1993

Regulation of interleukin-8 gene expression in human endometrial cells in culture.

In this study, we investigated the regulation of interleukin-8 (IL-8) gene expression in separated endometrial stromal and epithelial cells of human endometrium. This research was conducted as part of an analysis of the role of these cells in regulating the recruitment of leukocytes to the endometrium. Well-characterized model systems were used to study the regulation of endometrial IL-8 gene expression, namely, stromal cells in monolayer culture after first passage and glandular epithelium in primary culture. The levels of IL-8 mRNA and the accumulation of immunoreactive IL-8 in the medium of endometrial stromal cells is culture increased in a time- and concentration-dependent manner upon treatment with IL-1 alpha, tumor necrosis factor-alpha, or serum. The effects of IL-1 alpha plus serum on IL-8 mRNA levels were at least additive. Serum treatment caused a modest stimulation of IL-8 gene transcription (evaluated after 6 h of treatment) in endometrial stromal cells, but serum also acted in these stromal cells to prolong the half-life of IL-8 mRNA by more than 2.5-fold. The regulation of the levels of IL-8 mRNA in endometrial epithelial cells is distinctly different from that in stroma. First, the levels of IL-8 mRNA in non-treated epithelial cells in serum-free medium were much greater than those in stromal cells under similar conditions. Second, whereas the levels of IL-8 mRNA in endometrial epithelial cells also increased in response to serum and to IL-1 in the absence of serum, in the presence of serum, IL-1 treatment caused no appreciable change in the levels of IL-8 mRNA as was the case in endometrial stromal cells.

Base Sequence↗
PubMed1993

Cellular localization of membrane metalloendopeptidase (enkephalinase) in human endometrium during the ovarian cycle.

Previously, we found that membrane metalloendopeptidase (MMEP; enkephalinase) is present in human endometrium where it may inactivate certain bioactive peptides such as endothelin. The specific activity of MMEP in endometrium is correlated positively with the level of plasma progesterone. In the present study, we determined the cellular distribution of MMEP immunoreactivity (IR) in samples of endometrium from 31 ovulatory women at different phases of the menstrual cycle and from 5 postmenopausal women. At all times, MMEP-IR was localized in the endometrial stromal cells and was absent from myometrium, epithelia, and vessels. The presence of the enzyme on the stromal cell plasma membrane was demonstrated by immunostaining of freshly isolated endometrial stromal cells. During the endometrial cycle, there were dramatic changes in MMEP-IR in the zona functionalis: In the proliferative phase when plasma progesterone levels were low, MMEP-IR was weak; but after ovulation, during the early and midsecretory phases, when progesterone levels were increasing or high, MMEP-IR was strong. During the late secretory phase, in the stromal areas that were decidualized, contiguous with spiral arterioles and beneath the surface epithelium, most of the MMEP-IR was lost, whereas MMEP in the other stromal areas remained strong. In the premenstrual phase, there was a more generalized decline in MMEP-IR in the stromal cells. MMEP-IR in the zona basalis was weak to moderate and was relatively unchanged during the ovarian cycle; the findings in postmenopausal tissues were similar to those of proliferative phase tissues. These results are consistent with the previous findings of increased MMEP mRNA, protein, and activity in endometrial tissue of midsecretory phase and in cultured endometrial stromal cells in response to progestin. Furthermore, in this study, we demonstrate that in the late secretory phase in decidualizing stroma, MMEP-IR is decreased considerably compared with midsecretory stromal cells. These changes in MMEP-IR are consistent with the possibility that endothelin, also produced in endometrium, is spared inactivation in the premenstrual phase and may act on the spiral arterioles. These findings are supportive of a role for MMEP in paracrine interactions affecting vascular homeostasis in the endometrium.

Endometrium↗
PubMed1993

The accumulation of prostaglandins (PG) in amniotic fluid is an aftereffect of labor and not indicative of a role for PGE2 or PGF2 alpha in the initiation of human parturition.

The accumulation of prostaglandins (PGs) in amniotic fluid (AF) during labor is cited frequently as one line of evidence in support of a role for these eicosanoids in the initiation of human parturition. In this study, we evaluated an alternate possibility, viz. that PGs entering AF at parturition are produced as a sequela of labor-associated processes. During labor, the AF normally becomes separated into two compartments, viz. the forebag and the upper compartment, by the obstruction produced as the descending fetal presenting part is engaged in the maternal pelvis. We theorized that the PGs that enter AF are produced in traumatized tissues lining the forebag, which is formed as the result of labor-driven cervical dilatation. In addition, these traumatized tissues are exposed to and bathed by the vaginal fluid, which contains many potent stimuli of PG formation, viz. large numbers of microorganisms and bacterial toxins. AF was collected at term before labor (n = 50) and from the upper compartment during labor (n = 47) by transuterine amniocentesis, and AF was collected by direct needle aspiration of the forebag during labor (n = 143). PGF2 alpha, 13,14-dihydro-15-keto-PGF2 alpha (PGFM), and PGE2 were quantified by RIA. The concentrations (nanomoles per L mean +/- SEM) of PGs in AF of the forebag (PGF2 alpha, 85.6 +/- 10.6; PGFM, 20.8 +/- 2.58; PGE2, 26.9 +/- 2.73) were much greater than those in the AF before labor (PGF2 alpha, 0.56 +/- 0.05; PGFM, 0.9 +/- 0.08; PGE2, 5.89 +/- 1.13) or in AF of the upper compartment during labor (PGF2 alpha, 7.14 +/- 1.64; PGFM, 5.11 +/- 0.82; PGE2, 8.74 +/- 1.71). The concentrations of PGs in AF of the upper compartment during early labor (< or = 2.5-cm cervical dilatation) were no greater than those in AF before labor began. The concentration and total content of PGs in AF of the forebag increased as a function of cervical dilatation until delivery. At 3- to 5-cm cervical dilatation, the levels of PGs in AF of the upper compartment were greater than those before labor, but significantly less than those in AF of the forebag at the same stage of labor progress. After 3-5 cm, the levels of PGs in the upper compartment did not increase further. These findings indicate that PGF2 alpha, PGFM, and PGE2, which enter AF in increased amounts during parturition, are produced during, not before, labor in tissues (principally decidua) lining for forebag.(ABSTRACT TRUNCATED AT 250 WORDS)

Amniotic Fluid↗
PubMed1993

Endothelin levels in human amniotic fluid at mid-trimester and at term before and during spontaneous labor.

Endothelin (ET)-1 is synthesized in human amnion and immunoreactive (ir) ET is present in amniotic fluid in concentrations 10- to 100-times those found in plasma. ET-1 is a potent uterotonin; therefore, the possibility must be considered that ET-1, derived from amnion/amniotic fluid, serves to promote the uterine contractions of human labor. In term pregnancies, after labor begins, the amniotic fluid normally becomes divided into the upper and forebag compartments as the fetal presenting part is engaged in the maternal pelvis. The forebag tissues are exposed in the vagina because of cervical dilatation. Vaginal fluid contains microorganisms, bacterial toxins, and cytokines, e.g., interleukin-1 beta, that oblige an inflammatory reaction. Increased ET-1 formation in these tissues of the forebag would be indicative that the greater rate of ET-1 formation and entry into amniotic fluid was an aftereffect of labor, not a cause of parturition. The levels of irET in amniotic fluid during the midtrimester of human pregnancy, 93.3 +/- 7.4 pmol/L (mean +/- SEM, n = 38), were significantly greater than those in amniotic fluid at term before the onset of labor, 39.8 +/- 4.1 (n = 33, p < 0.01). The levels of irET in the upper compartment during labor, 45.5 +/- 3.5 pmol/L (n = 40), were not significantly different from those in amniotic fluid before labor, but were significantly less (p < 0.01) than those in amniotic fluid of the forebag, 82.1 +/- 5.2 pmol/L (n = 125). These findings are suggestive that increases in the concentration of ET in amniotic fluid at parturition are confined to the forebag and are the result of ET formation after labor begins. Inflammation of the tissues lining the forebag compartment of the amniotic fluid is a normal consequence of labor. Therefore, the entry of inflammatory response mediators, some of which are uterotonins, viz., ET and prostaglandins, into forebag amniotic fluid is an aftereffect of labor and not indicative of a role for these agents (in amniotic fluid) in the initiation of parturition. In a subset of the amniotic fluids from normal pregnancies at term, prostaglandin (PG) levels also were determined. There was a highly significant correlation between the levels of irET and PGE2 in the forebag compartment (p < 0.0001); there was no correlation between irET and PGE2 levels in the upper compartment or in amniotic fluid collected at term prior to labor onset.(ABSTRACT TRUNCATED AT 400 WORDS)

Amniotic Fluid↗
PubMed1993

Transforming growth factor-beta 1 inhibits enkephalinase (EC 3.4.24.11) gene expression in human endometrial stromal cells and sex skin fibroblasts in culture.

This study was conducted to evaluate the negative regulation of enkephalinase by an endogenously produced peptide, namely transforming growth factor-beta 1 (TGF beta 1). We found that TGF beta 1 acts in human endometrial stromal cells and sex skin fibroblasts in culture to cause a striking decrease (60% to > 90% in 3-7 days) in the specific activity of enkephalinase (membrane metalloendopeptidase; EC 3.4.24.11) by reducing the levels of enkephalinase mRNA and protein. Platelet-derived growth factor caused a slight reduction in enkephalinase specific activity in endometrial stromal cells; epidermal growth factor caused a slight decrease in enkephalinase specific activity in sex skin fibroblasts. In studies in which TGF beta 1 treatment and [35S]methionine labeling were conducted simultaneously, radiolabeling of enkephalinase was decreased. When proteins were radiolabeled with [35S] methionine before treatment with TGF beta 1, the extent of enkephalinase radiolabeling was similar to that in nontreated cells. These findings are indicative that TGF beta 1 acts to decrease enkephalinase activity by a reduction in gene transcription or mRNA stability and not by accelerated degradation of enkephalinase protein.

Blotting, Northern↗
PubMed1993

Regulation of parathyroid hormone-related protein gene expression in human endometrial stromal cells in culture.

The regulation of PTH-related protein (PTH-rP) gene expression by human endometrial stromal cells in monolayer culture was evaluated. 17 beta-Estradiol (E2), interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha (TNF alpha), endothelin-1 (ET-1), transforming growth factor-beta 1 (TGF beta 1), and serum stimulated PTH-rP production (evaluated by immunoassay of PTH-rP in the culture medium) by these cells. Treatment of endometrial stromal cells with E2 plus a progestin, medroxy-progesterone acetate (MPA), caused a decrease in PTH-rP production. PRL, epidermal growth factor, and dexamethasone, which are known to affect PTH-rP production (increase or decrease) in other cells, were ineffective in altering PTH-rP production in endometrial stromal cells, as were a number of other growth factors, viz. TGF alpha, platelet-derived growth factor, and basic fibroblast growth factor, and metabolites of progesterone. The effects of E2 and MPA on PTH-rP gene expression in these cells were investigated in detail. E2 (50 nM) increased PTH-rP mRNA levels (at 2, 4, 8, 12, and 24 h). When serum (10%) was present in the medium, E2 (50 nM) did not increase PTH-rP production or PTH-rP mRNA levels, and in the presence of serum, E2 plus MPA did not decrease PTH-rP production. In cells that were pretreated with E2 (50 nM) for 48 h before treatment (18 h) with ET-1, TNF alpha, tetradecanoylphorbol acetate, and TGF beta 1, the production of PTH-rP was greater than that in cells not pretreated with estrogen. The effect of treatment of endometrial stromal cells with MPA on PTH-rP mRNA levels (at 2, 4, 8, and 12 h) was variable, but in all studies conducted, PTH-rP production after 18 or 24 h of progestin treatment was not increased compared with that in nontreated cells. We conclude that estrogen acts directly in endometrial stroma cells to increase PTH-rP mRNA levels and PTH-rP protein production. In addition IL-1 beta, TNF alpha, ET-1, TGF beta 1, and serum stimulate PTH-rP production by these cells. With the exception of serum, the effects of most of the other agents identified in this study to stimulate PTH-rP production were at least additive with that of E2.

Blood↗
PubMed1993

Interleukin-1 beta, -1 alpha, and -6 and prostaglandins in vaginal/cervical fluids of pregnant women before and during labor.

Interleukin-1 beta (IL-1 beta) is not detected in the amniotic fluid of normal human pregnancies before the initiation of parturition, but during labor, both at term and preterm, this cytokine is present in the amniotic fluid of 25-40% of pregnancies. A critical question, however, is whether this finding is indicative of a role for IL-1 beta (directly or indirectly) in the initiation of parturition or is the result of IL-1 beta formation and entry into amniotic fluid as a natural sequela of normal labor. The forebag of the amniotic sac is formed during labor in response to cervical dilatation, and on the decidual surface, the tissues of this structure become exposed and bathed by vaginal fluids as the cervix opens. Microorganisms and bacterial toxins are present in vaginal fluid before labor begins; these agents should act upon the exposed tissues of the forebag to cause inflammation and evoke an inflammatory response. This study was conducted to examine the likelihood that the inflammatory mediators found in amniotic fluid in increased amounts at parturition are produced in forebag tissues after the onset of labor because of obliged inflammation in these tissues. Vaginal/cervical fluids were collected by lavage from nonpregnant women and from pregnant women at term before and during labor. The amount of immunoreactive IL-1 beta in vaginal/cervical fluids of pregnant women during labor (mean +/- SEM, 91.5 +/- 16.9 ng; n = 17) was significantly greater (P < 0.001) than that in fluids collected before labor (7.8 +/- 3 ng; n = 14). The in vivo rate of IL-1 beta secretion directly from the decidua lining the forebag during labor was brisk (1.71 +/- 0.88 ng/cm2.min; n = 4), consistent with previous observations of higher levels of pro-IL-1 beta mRNA in decidual tissues adherent to the forebag compared with those in decidua adherent to chorion laeve of the upper compartment of the amnionic sac. The vaginal fluid content of prostaglandins (PGs) during labor [PGE2, 82.1 +/- 16.4 ng; PGF2 alpha, 141.5 +/- 30.5 ng; PGFM, 35.2 +/- 5.8 ng (mean +/- SEM; n = 13)] was significantly greater for PGE2 and PGF2 alpha (P < 0.05 and 0.004, respectively) than that before labor (PGE2, 42.6 +/- 9.4 ng; PGF2 alpha, 35.3 +/- 8.5 ng; PGFM, 21.7 +/- 4.6 ng; n = 12). In addition, there was a significant increase in the ratio of PGF2 alpha to PGE2 (P < 0.03) in vaginal fluids during labor.(ABSTRACT TRUNCATED AT 250 WORDS)

Amniotic Fluid↗
PubMed1993

The metabolic disposition of plasma 5 alpha-dihydroprogesterone (5 alpha-pregnane-3,20-dione) in women and men.

This study was conducted 1) to ascertain whether the high levels of plasma 5 alpha-dihydroprogesterone (5 alpha DHP) during the luteal phase of the human ovarian cycle and pregnancy are attributable to high rates of production or, alternatively, low rates of clearance, and 2) to estimate the relative distribution of the irreversible metabolism of 5 alpha DHP, i.e. hepatic compared with extrahepatic clearance of plasma 5 alpha DHP. The concentration of 5 alpha DHP in plasma of women during the luteal phase of the ovarian cycle and pregnancy is very high, viz. 12-40% that of progesterone. Thus, a potentially large source of steroid precursor is available for the formation of bioactive 5 alpha-pregnanolone metabolites. We found that the MCR of 5 alpha DHP in women and men is 4187 +/- 312 L plasma/24 h (range, 3181-5506; n = 6). The MCR of 5 alpha DHP as a function of body surface area was 2406 +/- 240 L/24 h.m2. The MCR of 5 alpha DHP, therefore, is the greatest of any steroid reported, except for that of the catechol estrogens, which are metabolized intravascularly by erythrocyte catechol-O-methyltransferase. Based on estimated rates of liver plasma flow (1500 L/24 h) and hepatic extraction (75-85%) of lipophilic steroids that are not specifically bound in plasma with high affinity to binding proteins as determined by other investigators (e.g. 5 alpha DHP), we estimate that approximately 1200 L plasma/24 h are cleared of 5 alpha DHP by liver and approximately 2800 L plasma/24 h are cleared of 5 alpha DHP (70%) by metabolism in extrahepatic tissues. Thus, 5 alpha DHP can serve as a precursor for bioactive 5 alpha-pregnanolone(s).

5-alpha-Dihydroprogesterone↗
PubMed1993

Role of brain cytochrome P450 in regulation of the level of anesthetic steroids in the brain.

The role of brain cytochrome P450 (P450) in regulating the levels of the potent anesthetic steroid 3 alpha-hydroxy-5 alpha-pregnan-20-one (3 alpha-OH-DHP) has been investigated. By analogy with the elimination of androgen from its target tissues, we present evidence that it is 3 beta-hydroxy-5 alpha-pregnan-20-one (3 beta-OH-DHP) and not 3 alpha-OH-DHP that represents the major pathway for the formation of more polar metabolites and thus the elimination of the 5 alpha-reduced metabolites of progesterone from target tissues. No polar metabolites were formed when 3 alpha-OH-DHP was incubated with microsomal fractions prepared from rat brain, but 3 beta-OH-DHP was hydroxylated at the 6 alpha- and 7 alpha-positions. These 3 beta-diols were not formed to any detectable extent in the liver or kidney but were formed in prostate, pituitary, brain, and breast. The highest catalytic activity, 512 nmol of products formed/g of tissue/hr, was found in the prostate. The corresponding rates in the pituitary, brain, and breast were 71.9, 28.1, and 6.7 nmol/g/hr, respectively. These hydroxylations were confirmed to be P450-catalyzed reactions by solubilization of the P450 from prostate, brain, and breast microsomes and reconstitution of the catalytic activity with NADPH-P450 reductase (EC 1.6.2.4) and lipid. Because 5 alpha-androstane-3 beta,17 beta-diol (3 beta-Adiol) has been shown to be a good substrate for prostate and brain P450, competition experiments were performed to determine whether the same form of P450 is involved in the elimination of 3 beta-Adiol and 3 beta-OH-DHP in the brain. These two substrates competed with each other for metabolism in microsomal fractions and in reconstitution experiments with P450 extracted from the brain or prostate. To test the hypothesis that the hydroxylation of 3 beta-OH-DHP represents a pathway for regulation of the level of 3 alpha-OH-DHP in the brain, the effect of inhibition of the hydroxylation of 3 beta-OH-DHP on the duration of 3 alpha-OH-DHP-induced anesthesia was examined. The nonanesthetic steroid 3 beta-Adiol was used as a competitive inhibitor of the metabolism of 3 beta-OH-DHP. The duration of anesthesia upon intravenous administration of 3 alpha-OH-DHP was increased by 33% when 3 beta-Adiol was coadministered.(ABSTRACT TRUNCATED AT 400 WORDS)

Androstane-3,17-diol↗
PubMed1992

Endothelin-1 gene expression and biosynthesis in human endometrial HEC-1A cancer cells.

In this study, evidence was obtained that endothelin-1 (ET-1) is produced by an established endometrial cancer (HEC-1A) cell line. PreproET-1 mRNA is present in HEC-1A cells, and immunoreactive endothelin is secreted into the medium of these cells maintained in culture. Cycloheximide treatment of these cells caused superinduction of preproET-1 mRNA. Transforming growth factor-beta acts in these cells to increase the levels of preproET-1 mRNA. This effect of transforming growth factor-beta on preproET-1 mRNA accumulation was accompanied by an increase in the amount of immunoreactive endothelin secreted into the culture medium. ET-1, added to the culture medium, did not act as a mitogen in HEC-1A cells. We speculate that ET-1 (which is known to stimulate fibroblast proliferation) produced by endometrial adenocarcinoma cells may participate in the angiogenic process that occurs during the establishment of this carcinoma in vivo.

Adenocarcinoma↗
PubMed1992

Culture characteristics of human endometrial glandular epithelium throughout the menstrual cycle: modulation of deoxyribonucleic acid synthesis by 17 beta-estradiol and medroxyprogesterone acetate.

OBJECTIVE: The purpose of this study was to evaluate the culture characteristics and cell proliferation of human endometrial glandular epithelial cells in primary culture on extracellular matrix and to evaluate sex steroid modulation of this process. STUDY DESIGN: We examined the culture characteristics in 53 endometrial gland preparations obtained throughout the menstrual cycle and determined the incorporation of tritiated thymidine in endometrial epithelial cells as a measure of deoxyribonucleic acid synthesis (cell proliferation) in the presence and absence of 17 beta-estradiol and medroxyprogesterone acetate. RESULTS: Good culture maintenance of endometrial epithelial monolayers on extracellular matrix was observed from glands derived from proliferate phase endometrium (21 of 23), whereas poor adherence and culture maintenance was observed in all 30 of specimens from the secretory phase. With 17 beta-estradiol treatment for 22 hours, tritiated thymidine incorporation was not different from control, but medroxyprogesterone acetate treatment for 22 hours was associated with diminished tritiated thymidine incorporation by 36% (p < 0.004). When 17 beta-estradiol (10(-8) mol/L) was included in the incubation medium from the time of initial culture, tritiated thymidine incorporation on day 4 was 40% of control (p < 0.006), and tritiated thymidine was not suppressed further by 22 hours of treatment with medroxyprogesterone acetate (10(-7) mol/L). In spite of cellular spread of endometrial epithelial cells to subconfluence, deoxyribonucleic acid content per well did not increase over time in culture. CONCLUSIONS: We conclude that exposure of endometrial glands to progesterone in vivo inhibits adherence and the establishment of cell monolayers cultured on a thin layer of extracellular matrix. Because 17 beta-estradiol treatment in culture for 22 hours does not increase tritiated thymidine incorporation, it is possible that 17 beta-estradiol exerts its proliferative effect on the endometrial epithelium by an indirect action mediated by stromal cells. The effects of 17 beta-estradiol and medroxyprogesterone acetate on deoxyribonucleic acid synthesis in endometrial epithelial cells may represent an action on a stem cell population of dividing cells or a terminally differentiated cell population that undergoes programmed cell death.

Cells, Cultured↗