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Biomedical subjects

M L Casey

Publications and source records attributed to M L Casey.

At least 55 records · Page 3Linked to original sources
PubMed1993

Vanadate, epidermal growth factor and prostaglandin E2 production in human amnion cells.

We have investigated the effects of vanadate (VAN) and mouse epidermal growth factor (mEGF) on prostaglandin (PG) E2 production in human amnion cells in monolayer culture that served as a model system. The secretion of PGE2 into the culture medium was quantified by radioimmunoassay. The rate of conversion of [14C] arachidonic acid to [14C] PGE2 (PGE2 synthase) was determined in cell sonicates under optimal in vitro conditions. After a treatment period of 4 h we observed an increase in PGE2 production in the presence of mEGF (4.7-fold), VAN (1.8-fold) or both agents (6.8-fold) over control samples. Similarly, the specific activity of PGE2 synthase was stimulated maximally after 4 h with mEGF (2.1-fold), VAN (1.7-fold) or with both (4.8-fold) compared with untreated tissue samples. In the presence of cycloheximide those effects were abolished. We suggest, that VAN and mEGF act by a mechanism(s) that involves de novo protein synthesis or that alters the phosphorylation state of enzymes that are requisite for the conversion of arachidonic acid to PGE2.

Amnion↗
PubMed1993

The effects of vanadate and epidermal growth factor on the specific activities of phospholipase A2 and phosphatidylinositol-specific phospholipase C in human amnion cells.

We have evaluated the specific activity of phospholipase A2 (PLA2) and phosphatidylinositol-specific phospholipase C (PLC) in human amnion cells in monolayer culture that had not (CTL) or that had been treated with vanadate (VAN) and/or mouse epidermal growth factor (mEGF) for 4 h. It is known that both agents stimulate prostaglandin (PG) E2 synthesis in these cells. Phospholipase enzyme activities were determined in the 750 x g supernatant fraction of amnion cell homogenates under optimal in vitro conditions. The specific activity of PLA2 ranged from 1.1 to 1.25 nmol/mg protein/0.5 h and that of phosphatidylinositol-specific PLC from 1.04 to 1.2 mumol/mg protein/h. Treatment of amnion cell cultures with VAN and/or mEGF had no statistically significant effect on the specific activities of either phospholipases. Thus, we conclude that the stimulation of PGE2 production by VAN and mEGF is not due to an increase in the release of arachidonic acid from glycerophospholipid storage forms in human amnion cells.

Amnion↗
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↗
PubMed1992

Endothelin-1 gene expression and protein biosynthesis in human endometrium: potential modulator of endometrial blood flow.

We present evidence that endothelin-1 (ET-1) is produced by two distinct cell types (other than vascular endothelial cells) in human endometrial tissue. The supportive findings of this investigation are summarized as follows: 1) prepro-ET-1 mRNA is present in endometrial tissue and in separated endometrial stromal and glandular epithelial cells in culture; 2) immunoreactive ET is secreted into the medium of isolated endometrial stromal cells and glandular epithelium maintained in culture; and 3) the level of prepro-ET-1 mRNA in endometrial tissues obtained at the premenstrual-menstrual phase of the endometrial cycle is greater than that in tissues from the proliferative or early and midsecretory phases. We also found that transforming growth factor-beta and interleukin-1 alpha act to increase the levels of prepro-ET-1 mRNA in endometrial stroma cells in monolayer culture. We speculate that ET-1 derived from endometrial stromal cells may act on the adventitial surface of contiguous spiral arterioles of the endometrium to modulate endometrial blood flow.

Amino Acid Sequence↗
PubMed1992

Massive amounts of immunoreactive endothelin in human seminal fluid.

We found that immunoreactive endothelin (ET) is present in seminal fluid in very large amounts (500-5000 ng/L; quantification based on ET-1 standard). This immunoreactive ET was detected by use of a radioimmunoassay system in which the N-terminal portion of ET-1 and ET-2 (and big ET-1 and big ET-2) are recognized. Thus, the immunoreactive ET in seminal fluid may include the precursors of ET-1 or ET-2 (i.e., big ET) as well as metabolites of ET-1 or ET-2 in which the N-terminal region is intact. The levels of immunoreactive ET in seminal fluid from men with normal semen analyses and that in seminal fluid of vasectomized men were within the same range. Using a different radioimmunoassay system in which the C-terminal portion of ET-1, ET-2, and ET-3 is recognized, we found that the levels of immunoreactive ET were much lower (or undetectable). We speculate that bioactive ET may be produced and act to promote sperm transport in the male reproductive tract; thereafter, bioactive ET may be metabolized by membrane metalloendopeptidase (which is present in male reproductive tissues and semen) to immunoreactive, inactive products. Alternatively, big ET in seminal fluid may be processed in tissues of the female internal genitalia to bioactive ET, which could act to promote sperm transport through the uterine cavity by stimulating myometrial contractions.

Adult↗
PubMed1992

Contractile effects of prostaglandins, oxytocin, and endothelin-1 in human myometrium in vitro: refractoriness of myometrial tissue of pregnant women to prostaglandins E2 and F2 alpha.

Whereas there is much evidence in support of a role for prostaglandins (PG) in the parturitional process, it has not been demonstrated unequivocally that PGs are the physiological uterotonins involved in the induction of the myometrial contractions of spontaneous labor in women. This study was conducted to evaluate the contractile responsiveness of human myometrial tissue in vitro to PGs and to compare this response with that of other uterotonins, viz. oxytocin and endothelin-1. We found that treatment of uterine smooth muscle strips obtained from nonpregnant and pregnant women with PGE2 (10(-8)-10(-6) M) caused a biphasic response characterized by an initial single contraction of increased amplitude and duration, followed by relaxation and a long period (10-15 min) of quiescence. Conversely, PGE2 acted in rat myometrium to cause a monophasic response of increased contractile frequency and force. Whereas uterine smooth muscle from nonpregnant women was responsive to PGF2 alpha, the contractile responsiveness of myometrium from pregnant women was weak. This weak response to PGF2 alpha was found in myometrium of women in labor and in myometrium of women not in labor. 15-Methyl-PGF2 alpha evoked a small response in myometrium from pregnant women. Under identical in vitro conditions, PGF2 alpha (10(-8)-10(-6) M) and 15-methyl-PGF2 alpha (10(-6) M) caused sustained contractions in human vascular smooth muscle tissues (fetal aorta and arterial smooth muscle from chorionic vessels). Similarly, oxytocin and endothelin-1 (in myometrium from pregnant women) were effective in stimulating the force and frequency of myometrial contraction in vitro. We conclude that the myometrium of pregnant women, as evaluated in vitro, is refractory to the contractile effects of PGE2 and PGF2 alpha.

Analysis of Variance↗
PubMed1992

Parathyroid hormone-related protein mRNA in avascular human amnion.

Previously, we found that preproendothelin-1 mRNA is present in human avascular amnion tissue. In this investigation, we evaluated the possibility that another vasoactive protein, namely, parathyroid hormone-related protein (PTH-rP), also is produced in amnion. Using a specific cDNA probe, we identified PTH-rP mRNA in amnion tissue and found that the level of PTH-rP mRNA in placental amnion was greater than that in reflected amnion tissue obtained from the same pregnancy. PTH-rP mRNA also was demonstrable in chorion laeve and in decidua, but the levels of this mRNA in these tissues were much lower than that in amnion. By radioimmunoassay, we found that human amnion cells in primary monolayer culture secrete immunoreactive PTH-rP into the medium. Importantly, the placental amnion covers the chorionic vessels that traverse over the chorionic plate prior to branching into the cotelydons; specifically, there is no intervening tissue between placental amnion and the adventitial tissue of the chorionic vessel wall. Thus, the potential exists for the production of endothelin-1, a potent vasocontractant, and PTH-rP, a vasorelaxant, in placental amnion for export to the adventitial surface of the chorionic vessels. Moreover, amnion cells respond to a number of agents (e.g., transforming growth factor-beta) that effect changes in the expression of these two peptides. Therefore, the possibility should be considered that amnion-derived vasoactive proteins may modulate fetal chorionic, umbilical, and villous vessel tone and thereby fetal-placental blood flow.

Amnion↗
PubMed1991

Progesterone-regulated cyclic modulation of membrane metalloendopeptidase (enkephalinase) in human endometrium.

Membrane metalloendopeptidase (MMEP; EC 3.4.24.11; enkephalinase) catalyzes the degradation of endothelins, enkephalins, atrial natriuretic factor, substance P, and other small bioactive peptides. We found that MMEP is present in human endometrium, localized primarily in stromal cells of this tissue, and that the specific activity of MMEP (and immunoreactive MMEP protein) in endometrial tissue is correlated in a highly significant positive manner with the concentration of progesterone in plasma. In estrogen-treated, human endometrial stromal cells in monolayer culture, the specific activity of MMEP increases in response to treatment with progestin; and, this increase is accompanied by increases in immunoreactive MMEP protein, newly synthesized MMEP, and MMEP mRNA.

Adult↗
PubMed1991

Endothelin-1 gene expression and regulation of endothelin mRNA and protein biosynthesis in avascular human amnion. Potential source of amniotic fluid endothelin.

We demonstrated previously that preproendothelin mRNA is present in avascular human amnion tissue and in human amnion cells maintained in primary monolayer culture. In this investigation we sought to identify the specific endothelin (ET) gene that is expressed in amnion and to determine whether endothelin is produced by amnion. Using oligonucleotides specific for ET-1, ET-2, and ET-3 mRNA, we identified preproET-1 mRNA in human amnion tissue. By radioimmunoassay of ET we found that human amnion tissue explants and amnion cells in culture secrete immunoreactive ET into the medium. PreproET mRNA levels and immunoreactive ET production by human amnion cells in monolayer culture are increased in response to treatment with agents that are known to be present in human amniotic fluid, i.e. epidermal growth factor, interleukin-1, and tumor necrosis factor-alpha. We found that the level of preproET mRNA in amnion cells was low compared with that in human umbilical endothelial cells; treatment with cycloheximide together with a stimulus of ET-1 gene transcription led to a striking increase in the level of preproET mRNA in amnion cells compared with a much weaker response in endothelial cells. These findings suggest that protein synthesis-dependent mechanisms may be of great importance in maintaining low levels of preproET mRNA in amnion tissue and in regulating the amount of preproET mRNA in amnion exposed to stimuli of ET-1 transcription. In addition, we demonstrated that immunoreactive ET is present in human amniotic fluid at the midtrimester of pregnancy and at term. Thus, it is likely that the avascular fetal amnion is one tissue site of origin of ET in amniotic fluid during human pregnancy.

Amnion↗
PubMed1991

Responsiveness of human endometrial stromal cells to cytokines.

Cytokines are known to act in a variety of tissues, most commonly in a paracrine manner, to effect a number of biochemical processes. Previously, we found that human endometrial stromal cells respond to the action of interleukin-1 (IL-1) with an increase in the production of prostaglandins. In these investigations, we also found that IL-1 acts in endometrial stromal cells to stimulate the synthesis of IL-1 and IL-6 mRNA and protein. Specifically, in human endometrial stromal cells maintained in monolayer culture, treatment with IL-1 alpha leads to a striking increase in the synthesis of IL-1 beta mRNA and protein; this increase is IL-1 alpha-dose- and time-dependent. The pro-IL-1 beta produced, however, is not secreted into the culture medium but is retained within the stromal cell. The failure of secretion of IL-1 beta is characteristic of non-monocyte/macrophage cell types; this obtains because the enzyme that effects processing of pro-IL-1 beta (31 kDa) to the mature, secreted form of IL-1 beta (17 kDa) is believed to be present only in monocytes/macrophages. We also find that IL-1 and tumor necrosis factor-alpha (TNF-alpha) act in endometrial stromal cells to stimulate the synthesis of interleukin-6 (IL-6) mRNA and protein; the IL-6 produced by these cells is secreted into the culture medium. In addition, we find that IL-1 acts in endometrial stromal cells to inhibit the expression of mRNA for connexin43, a gap junction protein that is believed to be the principal component of gap junctions in cardiac and smooth muscle. Thus, it is likely that IL-1 action leads to a decrease in gap junction-dependent intercellular communication among endometrial stromal cells. Based on these findings, we conclude that endometrial stromal cells are responsive to the actions of IL-1 and TNF-alpha. These cells synthesize both IL-1 and IL-6; and, IL-6 is released into the extracellular medium. Thus, the possibility exists that the synthesis and action of cytokines may be involved in the mechanisms that serve to regulate the mesenchymal-epithelial interactions between endometrial stromal and glandular components; and, the formation and action of cytokines in decidua may serve to modulate immunological and infectious challenges encountered by this tissue in pregnancy.

Cells, Cultured↗
PubMed1991

Decidual activation in parturition: examination of amniotic fluid for mediators of the inflammatory response.

The accumulation of bioactive agents (characteristic of an inflammatory-type response) in amniotic fluid is common during term and preterm labor, viz., interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-alpha). In addition, prostaglandins, including PGE2, PGF2 alpha, and PGFM, also accumulate in amniotic fluid in some cases of term and preterm labor. From these observations, a number of critical questions arise. Namely, 1) what is the tissue source of origin of these agents?; 2) what are the stimuli that evoke this inflammatory response?; and, 3) are these bioactive agents of inflammation involved in the commencement of labor or else a natural accompaniment of the parturition process? It is reasonable to suspect that the decidua is activated during parturition as the membranes-decidua are exposed after cervical dilation to the vaginal/cervical secretions. Amnion and chorion laeve, in the human, are avascular tissues that produce PGE2 but not PGF2 alpha. Therefore, the accumulation of PGF2 alpha and PGFM in amniotic fluid during labor cannot be attributed to a fetal membrane origin. Moreover, the fetal membranes and decidua do not convert PGE2 to PGF2 alpha. In addition, the fetal membranes do not produce mature, i.e., secreted 17kD IL-1 beta. On the other hand, the decidua does produce PGF2 alpha and PGFM and is stimulated to do so by agents in the vaginal secretions, namely, bacterial endotoxin and IL-1 beta. After the fetal membranes and contiguous decidua are exposed during the time of cervical dilatation, these tissues are acted upon to cause 1) an influx of mononuclear phagocytes into the forebag compartment of the amniotic fluid; 2) to produce PGF2 alpha and PGFM; and 3) to produce cytokines, including IL-1 beta, IL-6, and TNF-alpha. Exposure of the fetal membranes-decidua to bioactive agents in vaginal/cervical secretions will effect an inflammatory response both in vivo and in vitro. We conclude that the accumulation of bioactive agents characteristic of the inflammatory response in amniotic fluid during term and preterm labor is usually an accompaniment of parturition and not its cause.

Amniotic Fluid↗
PubMed1991

Effects of cGMP on [Ca2+]i, myosin light chain phosphorylation, and contraction in human myometrium.

Adenosine 3',5'-cyclic monophosphate (cAMP) is believed to be an important mediator of myometrial relaxation, and there is evidence to suggest that guanosine 3',5'-cyclic monophosphate (cGMP) is a mediator of smooth muscle relaxation in vascular and probably nonvascular tissues. To investigate the biochemical mechanisms involved in regulation of human myometrial contractility, we studied the effects of analogues of cAMP and cGMP, as well as activators of adenylate and guanylate cyclases, on uterine smooth muscle contractile activity. We found that myometrial smooth muscle cells in culture respond to analogues of cGMP and cAMP, as well as activators of guanylate cyclase, with a significant decrease in the resting and endothelin-induced increase in [Ca2+]i. Treatment of human uterine smooth muscle strips with sodium nitroprusside or isoproterenol results in diminished force and frequency of contraction as well as a decrease in the rate and extent of myosin light chain phosphorylation in spontaneous contractions of human myometrium. cGMP did not effect relaxation of endothelin-stimulated contractions of human myometrium, but the relaxation effects of cGMP were dramatic in precontracted bovine tracheal and human fetal aortic smooth muscles. Whereas cGMP and cAMP act to promote a decrease in the force and frequency of spontaneous contractions in human myometrium, this tissue is not as responsive to the actions of cyclic nucleotides as are other types of smooth muscle.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1990

Expression and regulation of endothelin precursor mRNA in avascular human amnion.

Posttranslational processing of preproendothelin in endothelial cells gives rise to endothelin, a 21 amino acid polypeptide that is a potent vasoconstrictor. Endothelin production is believed to be mediated principally by transcriptional mechanisms. Previously, preproendothelin mRNA expression has been detected only in vascular endothelial tissue and cells. In this study, we found that preproendothelin mRNA is expressed in an avascular human tissue, namely, amnion, an extraembryonic fetal membrane. Preproendothelin mRNA was not detected in avascular chorion laeve tissue (also an extraembryonic fetal membrane), in the highly vascularized fetal trophoblast, or in maternal uterine tissues. Furthermore, we found that preproendothelin gene expression is retained in human amnion cells maintained in primary monolayer culture. Using the amnion cells in primary monolayer culture to investigate the regulation of preproendothelin mRNA expression, we found that epidermal growth factor (EGF) and interleukin-1 (IL-1) act to stimulate preproendothelin mRNA levels; in addition, the induction of preproendothelin mRNA by either of these agents is enhanced upon simultaneous treatment with cycloheximide. These findings are indicative that preproendothelin gene expression in amnion is regulated positively by EGF and IL-1 and that inhibition of protein synthesis leads to superinduction of preproendothelin mRNA. In human umbilical cord endothelial cells, neither IL-1 nor EGF stimulate preproendothelin mRNA expression but inhibition of protein synthesis does lead to increased levels of preproendothelin mRNA. The amnion, therefore, provides a useful system for expansion of our understanding of the tissue specific expression and regulation of preproendothelin mRNA.

Amnion↗
PubMed1990

Endothelin increases cytoplasmic calcium and myosin phosphorylation in human myometrium.

Endothelin, a recently discovered sarafotoxin-like peptide secreted by endothelial cells, is a potent stimulator of vascular smooth muscle contraction. We found that the action of endothelin is not restricted to the vasculature; we demonstrated that endothelin causes an increase in the concentration of intracellular Ca++ and phosphorylation of the 20 kd light chain of myosin in human uterine smooth muscle cells in culture. In the absence of Ca++ in the buffer medium of myometrial cells, the effects of endothelin on intracellular Ca++ and myosin light chain phosphorylation are attenuated but not abolished. Endothelin also increases the frequency of contraction of human uterine smooth muscle (longitudinal and circular). The contractile effects of endothelin on myometrial strips are diminished in the presence of nifedipine. We conclude that (1) human myometrium is responsive to endothelin, (2) endothelin promotes contraction in myometrium by effecting an increase in intracellular Ca++ and thus an increase in myosin light chain phosphorylation, and (3) endothelin acts in myometrium by stimulating Ca++ influx as well as Ca++ release from intracellular stores.

Calcium↗