Ovine fetal renal development impacted by multiple fetuses and uterine space restriction.

Intrauterine growth restriction (IUGR) from uteroplacental dysfunction causes impaired nephrogenesis and ultimately hypertension, but it is unknown whether IUGR caused by insufficient space for placental development seen in uterine anomalies and/or multifetal gestation exerts the same effects. Fetal renal development and metabolism were studied in an ovine space-restriction model by combining unilateral horn surgical ligation and/or multifetal gestation. Reduced placental attachment sites and placental weight per fetus defined space-restricted (USR) v. control nonrestricted (NSR) fetuses. Space-restricted fetuses exhibited evidence for decreased plasma volume, with higher hematocrit and plasma albumin at gestational day (GD) 120, followed by lower blood pO2, and higher osmolarity and creatinine at GD130, P < 0.05 for all. By combining treatments, fetal kidney weight relative to fetal weight was inversely related to both fetal weight and plasma creatinine levels, P < 0.05 for both. At GD130, space-restricted fetal kidney weights, cortical depths and glomerular generations were decreased, P < 0.05 for all. Space-restricted kidneys underwent an adaptive response by prolonging active nephrogenesis and increasing maculae densa number, P < 0.05 for both. The major renal adaptations in space-restricted IUGR fetuses included immaturity in both development and endocrine function, with evidence for impaired renal excretory function.

Pregnancy, programming and preeclampsia: gap junctions at the nexus of pregnancy-induced adaptation of endothelial function and endothelial adaptive failure in PE.

The challenge of pregnancy to the mother requires that her own metabolic and endocrine needs be met while also taking on the literally growing demands of the unborn child. While all of the mother's organs require continued support, the uterus and now added placenta must also develop substantially. One critical area of adaptation is thus the ability to provide added blood flow over and above that already serving the preexisting maternal organs. Previous reviews have covered in detail how this is achieved from an endocrine or indeed vascular physiology standpoint and we will not repeat that here. Suffice it to say in addition to new vessel growth, there is also the need to achieve reduced vascular resistance through maintenance of endothelial vasodilation, particularly through NO and PGI2 production in response to multiple agonists and their associated cell signaling systems. In this review, we continue our focus on pregnancy adaptive changes at the level of cell signaling, with a particular emphasis now on the developing story of the critical role of gap junctions. Remapping of cell signaling itself beyond changes in individual hormones and respective receptors brings about global changes in cell function, and recent studies have revealed that such post-receptor changes in cell signaling are equally if not more important in the process of pregnancy adaptation of endothelial function than the upregulated expression of vasodilator synthetic pathways themselves. The principle significance, however, of reviewing this aspect of pregnancy adaptation of endothelial cell function is that these same gap junction proteins that mediate pregnancy-adapted changes in vasodilatory signaling function may also be the focal point of failure in diseased pregnancy, and clues as to how and why are given by comparing studies of Cx43 functional suppression at wound sites with studies of preeclamptic pregnancy. If preeclamptic pregnancy is indeed a pregnancy misconstrued by the body in endocrine terms to be a wound, then the kinases so activated that correspondingly suppress Cx43 function in the vascular endothelium may also be valid pharmacologic targets for novel therapies in the near future.

Suppression of protein phosphatase 2 differentially modulates VEGF- and FGF2-induced signaling in ovine fetoplacental artery endothelial cells.

Vascular endothelial growth factor (VEGF) and fibroblast growth factor 2 (FGF2) elicit cellular responses via activation of protein kinases and phosphatases. We have reported that the MEK1/2/ERK1/2 and PI3K/AKT1 pathways are critical for VEGF- and FGF2-stimulated ovine fetoplacental artery endothelial (OFPAE) cell proliferation. We have also shown that protein phosphatase 3 (PPP3) differentially modulates VEGF- and FGF2-stimulated cell proliferation and activation of ERK1/2 and AKT1 in OFPAE cells. Herein, we investigated if protein phosphatase 2 (PPP2) modulated VEGF- and FGF2-induced ERK1/2, AKT1, and p38 MAPK activation and VEGF- and FGF2-stimulated cell proliferation in OFPAE cells. Small interfering RNA (siRNA) specifically targeting human PPP2CA catalytic subunit alpha (PPP2CA) was used to suppress PPP2CA expression in OFPAE cells. When compared with scrambled siRNA, PPP2CA siRNA decreased (p<0.05) PPP2CA protein levels (approximately 70%) and activity (approximately 50%) without altering protein levels of PPP3 catalytic subunit alpha (PPP3CA), nitric oxide synthase 3 (NOS3), ERK1/2, AKT1, and p38 MAPK. FGF2, but not VEGF rapidly (< or =5 min) induced p38 MAPK phosphorylation. Suppression of PPP2CA enhanced (p<0.05) VEGF-induced AKT1, but not ERK1/2 phosphorylation, whereas inhibited (p<0.05) FGF2-induced ERK1/2 and p38 MAPK and slightly attenuated FGF2-induced AKT1 phosphorylation. Suppression of PPP2CA did not significantly affect VEGF- and FGF2-stimulated OFPAE cell proliferation. Thus, suppression of PPP2CA alone differentially modulated VEGF- and FGF2-induced ERK1/2, AKT1, and p38 MAPK activation, without altering VEGF- and FGF2-stimulated cell proliferation in OFPAE cells. These data also suggest that signaling molecules other than ERK1/2, AKT1, and p38 MAPK are important mediators for VEGF- and FGF2-stimulated OFPAE cell proliferation after PPP2CA suppression.

Microarray analysis of BeWo and JEG3 trophoblast cell lines: identification of differentially expressed transcripts.

Trophoblast cell lines are important research tools used as a surrogate for primary trophoblast cells in the study of placental function. Because the cellular origins of transformed trophoblasts are likely to be diverse, it would be of value to understand the unique and shared phenotypes of the cells on a global scale. We have compared two widely used cell lines, BeWo and JEG3, by microarray analysis in order to identify differentially expressed genes. Results indicated that approximately 2700 genes were differentially expressed between the cell lines, with principal differences observed in the biological processes of response to stress, cell adhesion, signal transduction, and protein and nucleobase metabolisms. These data suggest that BeWo and JEG3 cell lines, and perhaps other trophoblast cell lines, are sufficiently dissimilar from each other such that they will be differentially suited for specific experimental paradigms.

Activity and expression of soluble and particulate guanylate cyclases in myometrium from nonpregnant and pregnant women: down-regulation of soluble guanylate cyclase at term.

The role of cGMP in the regulation of human myometrial smooth muscle contractility is at present unclear. cGMP can be synthesized by a cytoplasmic, soluble guanylate cyclase (sGC), which is stimulated by nitric oxide and carbon monoxide, and by particulate membrane-bound GC, which are activated by natriuretic peptides. The aim of this study was to determine whether sGC or pGC are present in nonpregnant and pregnant human myometrium, and whether the activity and expression of these enzymes and the cGMP content change during pregnancy and with labor. Myometrium was obtained from nonpregnant women (n = 12) and pregnant women who were preterm (25-34 wk gestation; n = 12), term (>38 wk) not in labor (n = 14), or term in active labor (n = 12). The cGMP content in myometrium obtained from preterm deliveries was significantly higher than that in tissue obtained from nonpregnant women and decreased at term, especially in laboring groups. Protein and mRNA for sGC, particulate GC-A, GC-B, and the clearance receptor were detected in human myometrium. cGMP in pregnant human myometrium, however, appears to be produced predominantly by sGC and possibly by GC-B, as GC-A was only weakly expressed. sGC activity was greater in myometrium from preterm (nonlabor) deliveries compared those taken at term (in labor), but was down-regulated compared with activity in nonpregnant myometrium. Neither atrial natriuretic peptide nor C-type natriuretic peptide (agonists for GC-A and GC-B, respectively) altered contractility in vitro of myometrium from women at term (not in labor). We conclude that the cGMP/guanylate cyclase system in human myometrium is gestationally regulated and potentially plays an important role in mediating quiescence during early pregnancy. A reduction in cGMP availability may contribute to the switch to contractile activity at term.

Pregnancy-specific enhancement of agonist-stimulated ERK-1/2 signaling in uterine artery endothelial cells increases Ca(2+) sensitivity of endothelial nitric oxide synthase as well as cytosolic phospholipase A(2).

Uterine artery endothelial cells (UAEC) from pregnant ewes (P-UAEC) demonstrate generally enhanced ability to couple growth factor and G protein-coupled receptors to the ERK-1/2 signaling pathway and stimulate NO production independently of elevated [Ca(2+)]. Herein we investigate the signaling and vasodilator responses to ATP, an agonist that also elevates [Ca(2+)](i) in both NP and P-UAEC, to determine the relative importance of Ca(2+) vs. ERK-1/2 in the activation of eNOS. We observed in both NP-UAEC and P-UAEC that ATP acts through G protein-coupled P(2Y) receptors to activate phospholipase C and dose-dependently elevate [Ca(2+)](i) independently of extracellular Ca(2+). The small reduction in the [Ca(2+)](i) response in NP vs. P-UAEC did not, however, account for the difference in NO production by P-UAEC>>NP-UAEC. ATP had no stimulatory effect on Akt phosphorylation but rapidly stimulated ERK-1/2 phosphorylation in P-UAEC>>NP-UAEC in a manner that correlated with NO production. In both NP- and P-UAEC, both ERK-1/2 and Ca(2+) were absolutely required for eNOS as well as cPLA(2) activation and the Ca(2+) sensitivity of eNOS was enhanced through the cytosolic [Ca(2+)](i) range in P-UAEC>>NP-UAEC. Thus ERK-1/2 may regulate the Ca(2+) sensitivity of eNOS to an even greater extent than is known to occur for cPLA(2).

Endothelium-derived nitric oxide synthase protein expression in ovine placental arteries.

During the third trimester, fetoplacental and uterine blood flows increase dramatically to meet the high metabolic demands of the growing fetus. We hypothesized that the expression of endothelial nitric oxide synthase (eNOS) in fetoplacental artery endothelium and the concentrations of nitric oxide (NO) and cyclic GMP (cGMP) in amniotic fluid (AF) are increased during the third trimester of ovine gestation. Placental arteries and AF were collected from ewes at 110, 120, 130, and 142 days of gestation (n = 24; mean +/- SEM term = 145 +/- 3 days). Expression of eNOS protein was measured in intact and denuded placental arteries and in endothelium-derived protein by Western analysis and confirmed by immunohistochemistry. Concentrations of NO (nitrates plus nitrites) and cGMP were determined in AF. Placental artery eNOS protein expression was localized to the endothelium, where it was markedly greater than in vascular smooth muscle. Placental artery endothelium-derived eNOS expression and AF cGMP concentrations were similar at 110 and 120 days of gestation; however, both peaked at 130 days at levels two- to threefold above baseline (P < 0.05) before returning to baseline at 142 days of pregnancy. The AF NO (nitrates plus nitrites) levels, however, increased progressively between 120 days of gestation and term (P < 0.05). We concluded that endothelium-derived placental artery eNOS levels, AF NO (nitrates plus nitrites), and AF cGMP were markedly increased during the third trimester, thus supporting a role for NO-mediated elevations in cGMP in the control of fetoplacental blood flow.

Ovine caveolin-1: cDNA cloning, E. coli expression, and association with endothelial nitric oxide synthase.

Caveolin-1 (Cav-1), the principal coat protein of caveolae, plays an obligatory role in regulating the activity of endothelial nitric oxide (NO) synthase (eNOS). We propose that Cav-1 may be critical to eNOS-NO mediated uterine vasodilatation during pregnancy and estrogen replacement therapy. To test this hypothesis in the sheep model, we isolated the full-length cDNA of ovine Cav-1 (oCav-1) from a Lambda ZAP cDNA library of ovine placental artery endothelial cells. Thirty-two positive oCav-1 clones were recognized by a partial oCav-1 cDNA from this library, of which eight were sequenced. Restriction digestion of these clones revealed that the cDNAs of oCav-1 ranged from approximately 2.1 to 2.7 kb. Northern analysis of Cav-1 mRNAs in ovine uterine artery endothelial cells (UAEC) showed two transcripts of approximately 2.1 and 2.7 kb, respectively. Immunoreactive Cav-1 protein, but not caveolin-2 or caveolin-3, was detected in UAEC. Sequence analysis revealed that in addition to a 537-bp open reading frame encoding a 178 amino acid oCav-1 protein, full-length oCav-1 cDNAs apparently possess a approximately 1.6-2.1 kb 3'-untranslated region. Database searches with oCav-1 cDNA revealed that the coding region of mammalian Cav-1 genes is highly conserved. We prepared a recombinant full-length oCav-1 protein in which six consecutive histidine residues were tagged at the end of its COOH-terminus and developed a [His]6-tagged oCav-1 'pull-down assay' for studying the association of eNOS with Cav-1. Incubation of exogenous [His]6-tagged oCav-1 with resting UAEC extracts led to the formation of a [His]6-tagged oCav-1-eNOS complex. In the presence of a synthetic caveolin-scaffolding domain (CSD, aa 82-101) peptide, but not a mutated CSD peptide, [His]6-tagged oCav-1 associated eNOS was dose (0-10 microM)-dependently inhibited. eNOS association with Cav-1 in UAEC was further confirmed by the facts that eNOS co-immunoprecipitated with Cav-1 and vice versa, and that eNOS co-existed with Cav-1 during the isolation of caveolae membranes. Because dissociation of eNOS from Cav-1 is required for the activation of eNOS, eNOS association with Cav-1 in UAEC suggests an important role of Cav-1 in regulating UAEC production of NO and possibly NO-mediated uterine vasodilatation.

Endothelial vasodilator production by uterine and systemic arteries. VI. Ovarian and pregnancy effects on eNOS and NO(x).

Normal pregnancy and the follicular phase of the ovarian cycle are both estrogen-dominated physiological states that are characterized by elevations in uterine blood flow and endothelial nitric oxide synthase (eNOS) protein expression in the uterine artery (UA) endothelium. It is unknown if elevations in mRNA level account for the changes in protein or eNOS activity. We tested the hypothesis that pregnancy and the follicular phase are associated with increases in eNOS mRNA and the consequent elevated expression of eNOS protein results in increased circulating nitric oxide (NO) levels. UA were obtained from pregnant (PREG; n = 8; 110-130 days gestation; term = 145 +/- 3 days), nonpregnant luteal (LUT; n = 6), nonpregnant follicular (FOL; n = 6), and nonpregnant ovariectomized (OVEX; n = 6) sheep. Circulating NO levels were analyzed as total NO(2)-NO(3) (NO(x)). Western analysis performed on UA endothelial-isolated proteins demonstrated that eNOS protein levels were OVEX = LUT < or = FOL < PREG (P < 0.05), whereas eNOS mRNA expression (RT-PCR) in UA endothelial cells obtained by limited collagenase digestion was OVEX < LUT < FOL < PREG (P < 0.05). Pregnancy dramatically elevated eNOS protein (4.1- to 6.9-fold) and mRNA (2.4- to 6.9-fold) over LUT controls (P < 0.01). Circulating NO(x) levels were not altered by ovariectomy or the ovarian cycle but were elevated from 4.4 +/- 1.1 microM in LUT to 12 +/- 4, 22 +/- 3, and 41 +/- 3 microM at 110, 120, and 130 days gestation (P < 0.01). Systemic NO(x) levels in singleton (12.5 +/- 1.6 microM) were less (P < 0.01) than in multiple (twin 27.6 +/- 6.5 microM; triplet = 46 +/- 10 microM) pregnancies. Therefore, the follicular phase and, to a much greater extent, pregnancy are associated with elevations in UA endothelium-derived eNOS expression, although significant increases in systemic NO(x) levels were only observed in the PREG group (multiple > singleton). Thus, although UA endothelial increases in eNOS protein and mRNA levels are associated with high estrogen states, increases in local UA NO production may require additional eNOS protein activation to play its important role in the maintenance of uterine blood flow in pregnancy.

Endothelial vasodilator production by uterine and systemic arteries. VII. Estrogen and progesterone effects on eNOS.

Uterine blood flow (UBF) and uterine artery endothelial nitric oxide synthase (eNOS) expression are greatest during the follicular vs. luteal phase. 17 beta-Estradiol (E(2)beta) increases UBF and elevates eNOS in ovine uterine but not systemic arteries; progesterone (P(4)) effects on E(2)beta changes of eNOS remain unclear. Nonpregnant ovariectomized sheep received either vehicle (n = 10), P(4) (0.9 g Controlled Internal Drug Release vaginal implants; n = 13), E(2)beta (5 microg/kg bolus + 6 microg x kg(-1) x day(-1); n = 10), or P(4) + E(2)beta (n = 12). Reproductive (uterine/mammary) and nonreproductive (omental/renal) artery endothelial proteins were procured on day 10, and eNOS was measured by Western analysis. P(4) and E(2)beta alone and in combination increased (P < 0.05) eNOS expression in uterine artery endothelium (vehicle = 100 +/- 16%, P(4) = 251 +/- 59%, E(2)beta = 566 +/- 147%, P(4) + E(2)beta = 772 +/- 211% of vehicle). Neither omental, renal, nor mammary artery eNOS was altered, demonstrating the local nature of steroid-induced maintenance of uterine arterial eNOS. In the myometrial microvasculature, eNOS was increased slightly (P = 0.06) with E(2)beta and significantly with P(4) + E(2)beta. Systemic NO(x) was increased with P(4) and P(4) + E(2)beta, but not E(2)beta, suggesting differential regulation of eNOS expression and activity, since P(4) increased eNOS in uterine artery endothelium while E(2)beta and the combination further increased eNOS protein.

Upregulation of eNOS in pregnant ovine uterine arteries by chronic hypoxia.

We tested the hypothesis that chronic high-altitude (3,820 m) hypoxia during pregnancy was associated with the upregulation of endothelial nitric oxide (NO) synthase (eNOS) protein and mRNA in ovine uterine artery endothelium and enhanced endothelium-dependent relaxation. In pregnant sheep, norepinephrine-induced dose-dependent contractions were increased by removal of the endothelium in both control and hypoxic uterine arteries. The increment was significantly higher in hypoxic tissues. The calcium ionophore A23187-induced relaxation of the uterine artery was significantly enhanced in hypoxic compared with control tissues. However, sodium nitroprusside- and 8-bromoguanosine 3',5'-cyclic monophosphate-induced relaxations were not changed. Accordingly, chronic hypoxia significantly increased basal and A23187-induced NO release. Chronic hypoxia increased eNOS protein and mRNA levels in the endothelium from uterine but not femoral or renal arteries. In nonpregnant animals, chronic hypoxia increased eNOS mRNA in uterine artery endothelium but had no effects on eNOS protein, NO release, or endothelium-dependent relaxation. Chronic hypoxia selectively augments pregnancy-associated upregulation of eNOS gene expression and endothelium-dependent relaxation of the uterine artery.

Expression of endothelial and inducible nitric oxide synthases and nitric oxide production in ovine placental and uterine tissues during late pregnancy.

We evaluated the expression of endothelial (eNOS) and inducible (iNOS) nitric oxide (NO) synthases, NO production, and the role of angiotensin II (ANG II) in regulating NO production during late ovine pregnancy (day 110-142). Samples of the following tissues were obtained: fetal [cotyledonary (COT)] and maternal [caruncular (CAR)] portions of the placentoma, intercotyledonary fetal chorioallantoic membrane (ICOT) and intercaruncular maternal endometrium (ICAR). Using immunohistochemistry, eNOS positive staining was detected in all four tissues, primarily in the endothelium, chorioallantoic membrane, and luminal and glandular epithelium. For iNOS, the positive staining was observed primarily in stromal cells in ICOT and ICAR. Expression of eNOS and iNOS proteins was confirmed in COT using Western immunoblot. eNOS protein levels increased (P< 0.05) approximately 3.5-fold from day 110 to 130 and then declined at term, whereas no change in iNOS protein levels was observed throughout the days studied. The tissue explants of COT, CAR, ICOT and ICAR were cultured in media in the absence or presence of ANG II (10(-9)or 10(-7) m) for 24 h. Total NO (nitrate and nitrite) levels in the explant-conditioned media were determined by chemiluminesence. In fetal COT, total NO levels increased (P< 0.05) 3.5-fold from day 110 to 130 and then declined (P< 0.05) at term. In ICOT, total NO levels exhibited a gradually increasing trend (r(2)=0.96, P< 0.01) from day 110 to days 130 and 142. In maternal CAR, total NO levels were higher (P< 0.05) on day 130 than those on days 120 and 142, whereas no change in total NO levels was observed in ICAR. ANG II at 10(-7) m treatment decreased (P< 0.05) total NO levels in COT on day 130. Thus, during late ovine pregnancy: (1) eNOS is expressed in COT, CAR, ICOT and ICAR while iNOS is primarily seen in stromal cells of ICOT and ICAR; (2) NO production by COT exhibits a biphasic pattern and parallels the changes in eNOS, but not iNOS protein levels, suggesting that eNOS is a predominant NOS isoform for the NO production; and (3) ANG II may contribute partially to decreases in NO production by COT at term.

Ethanol exposure induces oxidative stress and impairs nitric oxide availability in the human placental villi: a possible mechanism of toxicity.

OBJECTIVE: We undertook this investigation to explore the effects of ethanol exposure on nitric oxide synthase levels and nitric oxide release. Our hypothesis was that ethanol exposure modifies nitric oxide activity within the placenta as a result of oxidative stress. STUDY DESIGN: Four 10-g samples of term normal human placental villous tissue were perifused with nonrecirculating Dulbecco's modified Eagle's medium and 25-mmol/L N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid] with 0-, 50-, 100-, or 200-mmol/L ethanol. After 2 hours of exposure, tissue was removed, fixed, and frozen for analysis. Immunohistochemical analysis was performed for subtype I or neuronal nitric oxide synthase (nNOS), subtype II or inducible nitric oxide synthase (iNOS), and subtype III or endothelial nitric oxide synthase (eNOS) localization. Western blot analysis was performed for eNOS quantitation. Cyclic guanosine monophosphate and copper-zinc superoxide dismutase levels were measured by electroimmunoassay and kinetic assay, respectively. Nitric oxide release was analyzed by a Sievers nitric oxide analyzer. RESULTS: Immunohistochemical examination confirmed that only eNOS was localized to the syncytiotrophoblasts. After ethanol exposure, eNOS protein expression increased 2.5- to 3.0-fold over that of the control. Tissue cyclic guanosine monophosphate content and nitric oxide release into the effluent were decreased, whereas superoxide dismutase levels were increased at higher ethanol levels (P <.05). CONCLUSION: Ethanol exposure appears to induce oxidative stress, which may account for the decreased nitric oxide release, because nitric oxide may be shunted toward scavenging free radicals. Increased eNOS protein expression may be a response to the increased demand for nitric oxide. Decreased nitric oxide availability could adversely affect placental blood flow regulation, which could, in turn, account for the growth restriction seen in ethanol-exposed fetuses.

Pregnancy-dependent changes in cell signaling underlie changes in differential control of vasodilator production in uterine artery endothelial cells.

During pregnancy, the uterine vasculature shows a marked increase in vasodilator production [prostacyclin (PGI2) and nitric oxide (NO)] in response to a number of agonists including angiotensin II (AII) and ATP. As a consequence vascular resistance is kept low, and uterine blood flow is maximized to meet the needs of the growing fetus. Studies of the molecular basis underlying this change in control of endothelial NO and PGI2 production have been hampered by the lack of availability of a suitable cell model. To that end we have developed and characterized a new ovine uterine artery endothelial cell (UAEC) culture model derived from nonpregnant (NP) or pregnant (P) ewes. Endothelial cells were isolated from pregnant (120-130 days; n = 6) and nonpregnant (n = 4) ewes and maintained in primary culture. Endothelial cells at passage 4 showed uniform expression of endothelial nitric oxide synthase (eNOS; an endothelial marker) as well as AII type 1 receptor and growth factor receptors and uniform uptake of acetylated low density lipoprotein (a property of endothelial cells not shared by fibroblasts or vascular smooth muscle cells), thus demonstrating cell purity. Expressions of eNOS, cyclooxygenase-1, PGI2 synthase, cytosolic phospholipase A2, AII type 1 receptor, and growth factor receptors are also maintained at passage 4. Mitogenesis is maintained in response to basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), and vascular endothelial growth factor (VEGF) in both NP-UAEC and P-UAEC. The differential production of vasodilators by NP-UAEC and P-UAEC is maintained in a manner similar to that previously reported in vivo. Thus, P-UAEC make NO in response to AII, ATP, bFGF, EGF, and VEGF, whereas NP-UAEC make NO in response to bFGF, EGF, and VEGF only. Similarly, P-UAEC make PGI2 in response to AII, ATP, bFGF, and VEGF, whereas NP-UAEC make PGI2 only in response to ATP and VEGF. As both cytosolic phospholipase A2 and eNOS may be regulated by both Ca2+ and protein kinases, we investigated the effects of these agonists on Ca2+ mobilization and ERK-1/2 phosphorylation. ATP consistently elevates Ca2+ levels in both P-UAEC and NP-UAEC. All other agonists were without acute (0-4 min) effect on Ca2+ in P-UAEC or NP-UAEC. In contrast, all agonists stimulated an acute (10 min) phosphorylation of ERK-1/2 in P-UAEC, whereas only EGF stimulated activation in NP-UAEC. P-UAEC production of PGI2 by agonists of both heptahelical receptors and growth factor receptors correlates closely with ERK-2 phosphorylation alone. For NO, this correlation holds for heptahelical receptor agonists, but additional signaling pathways are also implicated for bFGF and VEGF. In contrast, in NP-UAEC the lack of ERK-2 phosphorylation in response to all agonists other than EGF, and the dissociation between NO or PGI2 production and ERK-2 phosphorylation suggest that alternate pathways play a predominant role.

Endothelial vasodilator production by uterine and systemic arteries. IV. Cyclooxygenase isoform expression during the ovarian cycle and pregnancy in sheep.

Uterine artery endothelial production of the potent vasodilator, prostacyclin, is greater in pregnant versus nonpregnant sheep and in whole uterine artery from intact versus ovariectomized ewes. We hypothesized that uterine artery cyclooxygenase (COX)-1 and/or COX-2 expression would be elevated during pregnancy (high estrogen and progesterone) and the follicular phase of the ovarian cycle (high estrogen/low progesterone) as compared to that in luteal phase (low estrogen/high progesterone) or in ovariectomized (low estrogen and progesterone) ewes. Uterine and systemic (omental) arteries were obtained from nonpregnant luteal-phase (LUT; n = 10), follicular-phase (FOL; n = 11), and ovariectomized (OVEX; n = 10) sheep, as well as from pregnant sheep (110-130 days gestation; term = 145 +/- 3 days; n = 12). Endothelial and vascular smooth muscle (VSM) COX-1 protein levels and uterine artery endothelial cell COX-1 mRNA levels were compared. Using immunohistochemistry and Western analysis, the primary location of COX-1 protein was the endothelium; that is, we observed 2.2-fold higher COX-1 protein levels in intact versus endothelium-denuded uterine artery and a 6.1-fold higher expression in the endothelium versus VSM (P < 0.05). COX-2 protein expression was not detectable in either uterine artery endothelium or VSM. COX-1 protein levels were observed to be higher (1.5-fold those of LUT) in uterine artery endothelium from FOL versus either OVEX or LUT nonpregnant ewes (P < 0.05), with substantially higher COX-1 levels seen in pregnancy (4.8-fold those of LUT). Increases in uterine artery endothelial COX-1 protein were highly correlated to increases in the level of COX-1 mRNA (r(2) = 0.66; P < 0.01) for all treatment groups (n = 6-8 per group), suggesting that increased COX-1 protein levels are regulated at the level of increased COX-1 mRNA. No change in COX-1 expression was observed between groups in a systemic (omental) artery. In conclusion, COX-1 expression is specifically up-regulated in the uterine artery endothelium during high uterine blood flow states such as the follicular phase and, in particular, pregnancy.

Endothelial vasodilator production by uterine and systemic arteries. V. Effects of ovariectomy, the ovarian cycle, and pregnancy on prostacyclin synthase expression.

Prostacyclin (PGI(2)) is a potent vasodilator, the level of which is increased during pregnancy, and is the main eicosanoid of which production is elevated in the endothelium and vascular smooth muscle (VSM) of both uterine and omental (systemic) arteries. We tested the hypothesis that during physiologic states that have high uterine blood flow, such as pregnancy and the follicular phase of the ovarian cycle (versus luteal phase and ovariectomized ewes), there is an increased level of prostacyclin synthase (PGIS) expression in ovine uterine and omental artery endothelium and VSM. To investigate this, the cellular localization and PGIS protein expression level in uterine and systemic arteries was examined by immunohistochemistry as well as by Western immunoblot analysis of endothelial-isolated protein and denuded vessels (VSM). Whole uterine, but not omental (systemic), arteries from the pregnant ewes showed an increase (P < 0.001) in PGIS expression. Further localization of PGIS protein by immunohistochemistry and quantification by Western analysis showed PGIS to be somewhat higher in the uterine artery VSM (69 +/- 7%) than endothelium (31 +/- 7%). PGIS protein levels in uterine and omental artery endothelial isolated protein were not altered by ovariectomy or the ovarian cycle, although they were both significantly elevated by pregnancy. Uterine and omental artery VSM PGIS expression levels also were not altered by ovariectomy or the ovarian cycle, whereas PGIS expression, in uterine but not omental artery VSM showed a significant elevation during pregnancy. Thus, the rise in PGI(2) production by uterine arteries observed in ovine pregnancy is paralleled by an elevation in PGIS expression in both endothelium and VSM, whereas those seen in omental arteries is associated with increases in endothelial PGIS.

Basic FGF decreases clearance receptor of natriuretic peptides in fetoplacental artery endothelium.

Atrial natriuretic peptide (ANP) is present in the fetoplacental circulation of humans and sheep. The ANP-A receptor is the specific membrane receptor for ANP, which produces cGMP. The clearance receptor of natriuretic peptide (CR) is postulated to modulate local concentrations of ANP, thereby modulating cGMP production through the ANP-A receptor. Recently we reported that fetoplacental basic fibroblast growth factor (bFGF) and cGMP levels are increased dramatically during the third trimester of ovine gestation. Therefore we hypothesized that bFGF will downregulate CR expression in cultured ovine fetoplacental artery endothelial (OFPAE) cells via the mitogen-activated protein kinase (MAPK) signal cascade mechanism, thereby causing augmentation of ANP-mediated cGMP production. Western analysis and/or RT-PCR of CR expression were performed after treatment of OFPAE cells with bFGF (10 pg/ml-1 microgram/ml) with or without 50 microM PD-98059, a selective inhibitor of MAPK kinase. To investigate the possible effects of CR downregulation on the functional modulation of ANP-A receptor activation, cGMP production (20 min) by OFPAE cells was measured in response to ANP (10 pM-1 microM) with or without pretreatment (24 h) of 10 ng/ml bFGF. CR expression in OFPAE cells was dose dependently downregulated by 1-10 ng/ml bFGF treatment (maximum -69%), which was completely reversed by pretreatment with PD-98059. Treatment of OFPAE cells with 10 ng/ml bFGF (24 h) did not alter maximum ANP-A activity (cGMP production/20 min), but decreased the apparent ED(50) of ANP to stimulate cGMP production from 2.5 to 0.83 nM, suggesting the possibility that bFGF-mediated downregulation of CR may elevate ANP-mediated cGMP production responses. Thus bFGF downregulates CR mRNA and protein expressions via the MAPK cascade in OFPAE cells.

Effects of angiogenic growth factors on endothelium-derived prostacyclin production by ovine uterine and placental arteries.

Uteroplacental and fetoplacental arteries produce substantial amounts of prostacyclin (PGI2). Because angiogenic growth factors such as basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), and epidermal growth factor (EGF) are increased in pregnancy, we hypothesized that treatment of uterine and fetoplacental arteries with bFGF, VEGF, and EGF would further enhance the pregnancy-induced increase in PGI2 production. Duplicate uterine (UA) and fetoplacental (PA) artery (primary branch off of the umbilical cord = pPA; cotyledonary or tertiary = tPA) explants from seven late gestation sheep were placed in tissue culture (RPMI; 37 degrees C) for 24 h alone or with (1-100 ng/mL) bFGF, VEGF, or EGF. To evaluate the endothelial contribution to basal and stimulated PGI2 production and to determine whether it is de novo, arteries with and without endothelium from three additional late gestation ewes, tissues were incubated in the absence or presence of growth factors with or without meclofenamate (1 microM). The stable metabolite of PGI2, 6-keto-PGF1 alpha, was measured in culture media and expressed as ng/mg wet wt 24 h. PGI2 production by UA increased (p < 0.05) from 5.43 +/- 0.26 at control to 8.93 +/- 0.99 with 100 ng/mL bFGF. Although VEGF produced a similar response, EGF did not increase PGI2 production in UA. In pPA, 100 ng/mL bFGF induced a 2.2-fold increase (p < 0.01) in PGI2 production from 1.94 +/- 0.14 to 4.20 +/- 0.31; VEGF and EGF were without effect. In tPA, 50 and 100 ng/mL bFGF increased PGI2 production from 1.98 +/- 0.14 to 3.5 +/- 1.05 and 3.96 +/- 0.46 (p < 0.02). In tPA, VEGF did not increase PGI2 production; however, 10, 50, and 100 ng/mL EGF, enhanced (p < 0.03) PGI2 production from 1.98 +/- 0.14 to 3.39 +/- 0.62, 3.62 +/- 0.26, and 2.93 +/- 0.20. Endothelium removal and meclofenamate treatment caused a 90% and 100% decrease, respectively, in basal PGI2 production, with no recovery after treatment with growth factors. We conclude that PGI2 production is augmented by bFGF in UA, pPA and tPA, by VEGF in UA, and by EGF in tPA during ovine pregnancy. Basal and stimulated PGI2 secretion is endothelium-derived via de novo synthesis. bFGF, VEGF, and EGF, in addition to angiogenesis, may modulate PGI2 production, further enhancing blood flow to the growing uteroplacental bed.

Endometrial and myometrial expression of nitric oxide synthase isoforms in pre- and postmenopausal women.

Expression of nitric oxide synthase (NOS) protein was examined by Western immunoblot analysis and immunohistochemistry in the endometrium and myometrium of 19 premenopausal and 18 postmenopausal women undergoing hysterectomy for benign gynecological reasons. The predominant isoform of NOS in the human uterus was endothelial NOS (eNOS). Using immunohistochemistry, eNOS was localized predominantly to the glandular epithelium and endometrial microvascular endothelium. eNOS was scant and inconsistently detected in endometrial stromal cells. In the myometrium, eNOS was predominantly found in smooth muscle cells (myocytes) and was also detected in the microvascular endothelium. Neuronal NOS was not detectable by immunohistochemical techniques, and inducible NOS (iNOS) was only detectable in occasional specimens, although more often in secretory specimens. iNOS, when present, was predominantly found in glandular epithelium and occasional stromal cells. Myometrial iNOS was scant and not consistently detected. By Western immunoblot analysis, neuronal NOS or iNOS was not detected. We observed a unique menstrual cycle-dependent expression of eNOS that was different in the endometrium compared to the myometrium and was independent of uterine pathology. In the endometrium, there was 62% higher expression of eNOS during the secretory phase (P = 0.00085) compared to the proliferative phase, whereas in the myometrium, there was 74% greater expression of eNOS in the proliferative phase (P = 0.03) compared to the secretory phase. Within the secretory phase, maximal endometrial eNOS expression was found in the midportion, whereas in the myometrium, highest eNOS expression occurred during the late secretory phase. In postmenopausal women not treated with hormones, a significant reduction in endometrial and myometrial expression of eNOS occurred, which was reversed by continuous hormone replacement therapy. In summary, both endogenous ovarian steroids and exogenous sex hormones influence uterine eNOS expression. Our results suggest that estrogen may regulate myometrial eNOS, whereas progesterone or a combination of estrogen and progesterone may be more important in regulating endometrial eNOS, and NO may be a critical mediator of sex steroid actions in the human uterus.

Repeated use of betamethasone in rabbits: effects of treatment variation on adrenal suppression, pulmonary maturation, and pregnancy outcome.

OBJECTIVE: The aim of the study was to determine whether reduced birth weight, adrenal suppression, and lung maturation occur in parallel and are cumulative with increasing courses of betamethasone. STUDY DESIGN: Time-bred rabbits were assigned to a control group or to receive saline solution or 1, 2, or 3 courses of betamethasone (early treatment, beginning day 19). Two additional groups (n = 5 per group) were given 1 or 2 late courses (late treatment). Birth weight, serum cortisol, adrenal 17alpha-hydroxylase (P450c17) messenger ribonucleic acid and fetal lung surfactant proteins A and B were quantified on day 27. RESULTS: Fetal weight was inversely proportional to the number of courses, with late treatment having a greater effect. Maternal cortisol and P450c17 levels were progressively suppressed with each early course, but fetal cortisol and P450c17 levels were only suppressed after 3 courses. A single late treatment profoundly suppressed both maternal and fetal cortisol and P450c17 messenger ribonucleic acid levels. In contrast, fetal lung surfactant proteins A and B increased progressively with betamethasone courses, regardless of timing. CONCLUSIONS: Time from last injection to delivery determined adrenal suppression, whereas total betamethasone courses determined surfactant protein production. Lower birth weight was dependent on the number of courses and was greater with late treatment.