Expression and cellular localisation of chloride intracellular channel 3 in human placenta and fetal membranes.

Chloride channels regulate the movement of a major cellular anion and are involved in fundamental processes that are critical for cell viability. Regulation of intracellular chloride is achieved by multiple classes of channel proteins. One class of putative channels are the chloride intracellular channel (CLIC) family. Evidence suggests that several CLICs are expressed in human placenta, although their roles in this tissue are not certain. Northern blot analysis has shown that CLIC3 is highly expressed in placenta relative to other human tissues; however, its cellular distribution is not known. This study used microarray expression profiling to clarify which CLICs are expressed in human placenta and RT-PCR, Western blot and immunohistochemistry to determine the expression pattern of CLIC3 in human placenta and fetal membranes. Placentas and fetal membranes were obtained from term pregnancies after delivery and placental tissue was obtained from first trimester following either chorionic villous sampling or elective pregnancy termination. Trophoblast cells were isolated from first trimester and term placentas and placental endothelial cells were isolated from term placentas. Microarray expression profiling identified high expression of mRNA for CLICs 1, 3 and 4 in the isolated first trimester and term trophoblast cells. High mRNA expression in the isolated endothelial cells was also found for CLICs 1 and 4, but not CLIC3. Low expression was found for CLIC5 in all three types of isolated cells. RT-PCR confirmed that CLIC3 mRNA was expressed in trophoblast cells at both gestational ages, but was not present in endothelial cells. CLIC3 mRNA was also identified in whole placental extracts at both gestational ages and in term amnion and choriodecidua. Immunohistochemistry using a chicken anti-human CLIC3 antibody localised strong CLIC3-specific staining to the syncytiotrophoblast and villous cytotrophoblast cells in both first trimester and term placentas, and weaker staining in extravillous trophoblast cells in first trimester. In fetal membranes at term strong CLIC3-specific staining was localised to chorionic trophoblast cells, with weaker staining in amniotic epithelial and decidual cells. It was previously shown that chloride uptake was increased into cells that had been transfected with CLIC3. CLIC3 may facilitate chloride ion movement and the regulation of cellular processes associated with the movement of chloride in the placental and fetal membrane cells in which it is expressed.

Utility of Computed Tomography versus Abdominal Ultrasound Examination to Identify Iliosacral Lymphadenomegaly in Dogs with Apocrine Gland Adenocarcinoma of the Anal Sac.

BACKGROUND: Apocrine gland adenocarcinoma of the anal sac (AGAAS) is associated with high rates of iliosacral lymph node metastasis, which may influence treatment and prognosis. Magnetic resonance imaging (MRI) recently has been shown to be more sensitive than abdominal ultrasound examination (AUS) in affected patients. OBJECTIVE: To compare the rate of detection of iliosacral lymphadenomegaly between AUS and computed tomography (CT) in dogs with AGAAS. ANIMALS: Cohort A: A total of 30 presumed normal dogs. Cohort B: A total of 20 dogs with AGAAS that underwent AUS and CT. METHODS: Using cohort A, mean normalized lymph node : aorta (LN : AO) ratios were established for medial iliac, internal iliac, and sacral lymph nodes. The CT images in cohort B then were reviewed retrospectively and considered enlarged if their LN : AO ratio measured 2 standard deviations above the mean normalized ratio for that particular node in cohort A. Classification and visibility of lymph nodes identified on AUS were compared to corresponding measurements obtained on CT. RESULTS: Computed tomography identified lymphadenomegaly in 13 of 20 AGAAS dogs. Of these 13 dogs, AUS correctly identified and detected all enlarged nodes in only 30.8%, and either misidentified or failed to detect additional enlarged nodes in the remaining dogs. Despite limitations in identifying enlargement in all affected lymph nodes, AUS identified at least 1 enlarged node in 100% of affected dogs. CONCLUSION AND CLINICAL IMPORTANCE: Abdominal ultrasound examination is an effective screening test for lymphadenomegaly in dogs with AGAAS, but CT should be considered in any patient in which an additional metastatic site would impact therapeutic planning.

Choline transport across a carbon tetrachloride phase containing a chloroform-methanol extract of brain.

The presence of a chloroform-methanol extract of cat brain in a carbon tetrachloride phase separating two aqueous phases resulted in an increased passage of [3H]choline across the organic phase which was inhibited by the choline transport inhibitor hemicholinium-3 and by high concentrations of non-radioactive choline. In the absence of cat brain extract, [3H]choline passage across carbon tetrachloride was neither inhibited by hemicholinium-3, nor by non-radioactive choline.

Expression of GLUT12 in the fetal membranes of the human placenta.

The aim of this study was to characterize the expression of the novel glucose transporter GLUT12 in the fetal membranes of the human placenta. RT-PCR and Western blotting of extracts of amnion and choriodecidua from four normal term placentas identified GLUT12 mRNA and protein expression. In all four samples the signals for GLUT12 were markedly stronger in the choriodecidua than in the amnion, whereas the signals for GLUT1, a glucose transporter know to be expressed in fetal membranes, were similar for the two tissues. In further studies, paraffin sections of fetal membranes were analyzed by immunohistochemistry with GLUT12 and GLUT1-specific polyclonal antibodies. GLUT12 immunoreactivity was localized predominantly to the trophoblast cells in the chorion and to a lesser extent to decidual cells and to epithelial and fibroblast cells of the amnion. GLUT1 was localized to chorionic trophoblast cells and amniotic epithelial and fibroblast cells. GLUT12 expression was predominantly cytoplasmic, whereas GLUT1 was associated with the membrane of the cells. These results show that GLUT12 is expressed in cells of human fetal membranes and suggest that GLUT12 may play a role in the facilitation of glucose transport into these cells.

In vivo detection of small subsurface melanomas in athymic mice using noninvasive fiber optic confocal imaging.

Fiber optic confocal imaging, following intravenous administration of fluorescently labeled antibodies and Texas Red-dextran, enabled in vivo detection of melanoma and surrounding blood vessels in athymic mice. Human melanoma cells (three cell lines) and cultured normal human skin cells were implanted intradermally into the haunch skin of anesthetized athymic BALB/C mice and allowed to grow to a maximum size of 2 mm diameter. Using three different fluorescein-isothiocyanate-labeled antimelanoma antibodies, single channel confocal images of melanoma cells were obtained in vivo. Using noninvasive techniques, the overall in vivo melanoma detection rate for tumors within 0.2 mm of the skin surface was 84% (27 of 32 tumors). Normal cultured human skin cells were found to have little or no fluorescence after administration of the fluorescein-isothiocyanate-labeled antibodies and tumors were not labeled by an isotype control antibody. Dual channel imaging of the implanted melanoma tumor and surrounding dermal vasculature in vivo showed increased blood vessel density at the melanoma site. Conventional immunoperoxidase histology confirmed that fiber optic confocal imaging was able to detect melanoma tumors up to 0.2 mm below the skin surface, in vivo.

Parathyroid hormone-related protein (PTHrP) mRNA splicing and parathyroid hormone/PTHrP receptor mRNA expression in human placenta and fetal membranes.

During human pregnancy, parathyroid hormone-related protein (PTHrP) and parathyroid hormone (PTH)/PTHrP receptor are produced by the uterus, placenta, fetal membranes (amnion and chorion) and developing fetus. PTHrP alternative 3' mRNA splicing results in transcripts which encode three PTHrP isoforms and have been identified in amnion. Uteroplacental PTHrP expression is greatest in amnion and increases dramatically during late pregnancy. The aims of this study were to determine PTH/PTHrP receptor mRNA expression at preterm and term gestations and to determine 3' alternative splicing patterns in placenta, amnion and choriodecidua at preterm and term gestations. Using semiquantitative reverse transcription-polymerase chain reaction, PTHrP and PTH/PTHrP receptor transcripts were identified in preterm (n=5) and term (n=7) gestational tissues. PTH/PTHrP receptor mRNA expression did not differ between tissue types or change with advancing gestation. In contrast, PTHrP expression in the same tissues increased with advancing gestation and was significantly greater in amnion than in placenta and choriodecidua. Thus PTHrP, although produced predominantly in amnion, may act in amnion and other tissues including placenta, choriodecidua and myometrium. In amnion over placenta, transcripts encoding PTHrP 1-139 and 1-173 were detected in some preterm and all term samples and those encoding PTHrP 1-141 were detected in all samples. Similar results were obtained for reflected amnion. In placenta and choriodecidua, PTHrP 1-139 and 1-173 transcripts were undetectable or of low abundance. PTHrP 1-141 transcripts were detected in some placenta and choriodecidua samples. In summary, transcripts encoding PTHrP 1-141 appeared to be more abundantly expressed than those encoding PTHrP 1-139 or 1-173. However, the up-regulation of PTHrP expression in amnion at term may involve each of the alternative 3' mRNA splicing pathways since transcripts for each isoform appeared to be more consistently expressed at term.

Effects of glucose, insulin or aldose reductase inhibition on responses to endothelin-1 of aortic rings from streptozotocin-induced diabetic rats.

1. This study investigated the constrictor responsiveness to endothelin-1 (ET-1, 0.1 nM-0.1 microM) of aortic rings (under 10 g resting tension in Krebs solution) from 2- and 6-week streptozotocin (STZ, 60 mg kg-1, i.v.)-induced diabetic rats and vehicle-treated control rats. 2. In aortae from 2- and 6-week STZ-treated rats, and their corresponding controls, removal of endothelium caused leftward shifts of ET-1 concentration-response curves without affecting maximum responses. 3. Maximum responses to ET-1 were reduced in aortae from both 2- and 6-week STZ-treated rats compared to those from control rats. Such reductions were still evident after removal of the endothelium. 4. Decreased responsiveness to ET-1 of aortae from 2-week STZ-treated rats was still evident after chronic treatment with the aldose reductase inhibitor epalrestat, but not after chronic insulin treatment or in aortae bathed in high glucose (30 mM) Krebs solution. 5. Decreased responsiveness to ET-1 of aortae from 6-week STZ-treated rats (compared with those from controls) was still evident after chronic epalrestat treatment and in high glucose Krebs solution. 6. These data suggest that the decreased responsiveness to ET-1 observed in aortae from 2- and 6-week STZ-induced diabetic rats is not due to abnormal activity of the polyol pathway. The altered responsiveness in aortae from 2-week diabetic rats (compared with those from control rats) may possibly be a manifestation of changes (adaptive or otherwise) which occur as a result of high glucose concentrations in vivo.However, in aortae from rats with diabetes of longer duration, other mechanisms may also play a role in the altered responsiveness, since it was no longer reversible by bathing in high glucose Krebs solution.

Changes in cardiovascular sensitivity of alloxan-treated diabetic rats to arachidonic acid.

Arachidonic acid (AA, 0.125-2.0 mg kg-1) administered intravenously to male Wistar rats produced a dose-dependent fall in diastolic blood pressure. However AA (0.125-1.0 mg kg-1) injected into the autoperfused hindquarters via the aorta produced a dose-dependent increase in perfusion pressure. Both these responses to AA were inhibited by indomethacin (5 mg kg-1). The thromboxane A2 receptor antagonist AH23848 (5 mg kg-1, i.v.) inhibited pressor responses to AA in the autoperfused hindquarters, but potentiated depressor responses to AA (0.125-0.5 mg kg-1) in the whole animal. Alloxan-treated diabetic rats (14 days after a single s.c. injection of alloxan, 175 mg kg-1) displayed reduced sensitivity to the depressor effects of AA (1-2 mg kg-1) in the whole animal, increased sensitivity to the pressor effects of AA (0.5-1.0 mg kg-1) in the perfused hindquarters, and reduced sensitivity to the pressor effects of the thromboxane A2 mimetic U46619 (0.5-8.0 micrograms kg-1, i.a.) in the perfused hindquarters. These results suggest that AA can be predominantly converted to either pressor or depressor metabolites depending on the vasculature. In the diabetic state the ratio of the metabolites formed appears to change favouring a major pressor metabolite, which is probably thromboxane A2.

Methoxyphenamine inhibits basal and histamine-induced nasal congestion in anaesthetized rats.

1. Nasal resistance in anaesthetized rats was assessed by measuring air overflow during ventilation of the nasal passages at constant pressure. Nasal basal resistance was reduced in a dose-dependent manner by methoxyphenamine hydrochloride (0.01-30 mg kg-1, i.v.), pseudoephedrine hydrochloride (0.03-3 mg kg-1, i.v.) and adrenaline bitartrate (0.01-3 micrograms kg-1, i.v.). Both methoxyphenamine and pseudoephedrine were less potent and less efficacious than adrenaline but caused longer-lasting responses. 2. Nasal congestion induced by histamine (0.2% nebulised solution passed into the nasal passages for 15 s) was inhibited by i.v. administration of methoxyphenamine, pseudoephedrine, adrenaline, methoxamine or tyramine: the ID50s against 0.2% histamine-induced nasal congestion were 1.16 (95% confidence limits; 0.5, 1.8) mg kg-1, 0.25 (0.19, 0.33) mg kg-1, 0.037 (0.018, 0.06) micrograms kg-1, 8.12 (6.74, 9.65) micrograms kg-1 and 30.6 (26.1, 35.8) micrograms kg-1 respectively. 3. The inhibitory effects of both methoxyphenamine and tyramine on histamine-induced nasal congestion were reduced after administration of desmethylimipramine (0.1 and 1 mg kg-1, i.v.) or prazosin (0.1 and 0.3 mg kg-1, i.v.). Similarly, the inhibitory effects of methoxamine were reduced after prazosin (0.1 and 0.3 mg kg-1). 4. These results indicate that methoxyphenamine (1 mg kg-1, i.v.) inhibits histamine-induced nasal congestion in the rat. This action, at least in part, is probably indirect being mediated by release of neuronal noradrenaline which then acts on alpha 1-adrenoceptors.

Attenuated responses to endothelin-1, KCl and CaCl2, but not noradrenaline, of aortae from rats with streptozotocin-induced diabetes mellitus.

1. This study investigated the responsiveness to vasoconstrictor agents (including endothelin-1, ET-1) of aortic rings from rats with two-week streptozotocin (STZ, 60 mg kg-1, i.v.)-induced diabetes and vehicle-treated control rats. The basal tension was 10 g, which was estimated to be more physiological than the tension of 1-2 g that has been previously used for most studies of aortic rings from diabetic rats. 2. Maximum responses to ET-1 (0.13-18 nM), KCl (2-20 mM) or CaCl2 (10 microM-10 mM) were reduced in aortae from STZ-treated rats compared to those from control rats. Such reductions were still evident after removal of the endothelium. 3. Responses to noradrenaline (NA, 0.1 nM-26 microM) of aortae from STZ-treated rats were not significantly different from responses of aortae of control rats. 4. Removal of endothelium resulted in a significant reduction in the EC50 values for NA of rings from both STZ-treated rats (6.90 +/- 0.13 and 8.17 +/- 0.35 (-log M) with and without endothelium, respectively, n = 5) and control rats (6.90 +/- 0.15 and 8.37 +/- 0.44 (-log M) with and without endothelium, respectively, n = 5). 5. In calcium-free medium (with 1 mM EGTA), responses to NA and ET-1 were reduced compared with those in normal Krebs solution and maximum responses were less in rings from STZ-treated compared with control rats. 6. Indomethacin (5 microM) did not prevent the reduced maximum responsiveness to ET-1 in rings from STZ-treated rats compared with those from controls.7. This study indicates that changes in vascular responsiveness to ET-1, KCI and CaCl2 (but not NA) occur in aortae of two-week STZ-treated rats. The endothelium does not appear to play a major role in mediating changes in responsiveness to ET-1.

Acetylcholine output and foetal vascular resistance of human perfused placental cotyleda.

The foetal villous vessels of single cotyleda of human placentae have been perfused with a constant flow of Krebs solution, recording inflow pressure and passing the venous perfusate in cascade over guinea-pig ileum and rat stomach strip preparations in vitro. Each cotyledon released for at least 4 h a substance that was probably acetylcholine. The perfusate caused contractions of both preparations which were inhibited by atropine or hyoscine and potentiated by physostigmine. Contractile activity was destroyed after incubation at 37 degrees C of perfusate with acetylcholinesterase but not with acetylcholinesterase plus physostigmine. When the perfusion temperature was lowered to 34 degrees C or below, acetylcholine output was reduced, the extent depending on the fall in temperature. No change in resistance of the villous vessels occurred during the changes in temperature or in the presence at 37 degrees C of atropine, hyoscine, hexamethonium, (+)-tubocurarine, hemicholinium-3 or bretylium. Submaximal vasoconstrictor responses of the villous vessels to the thromboxane A2-mimetic U46619 were not affected by reduction of the perfusion temperature to 30 degrees C, which lowered acetylcholine-like output by approximately 70%. Responses to U46619, at 37 degrees C, were unchanged during the presence of atropine or hyoscine. Acetylcholine is released into the foetal circulation of the human placenta but no evidence could be obtained that it affects villous vascular smooth muscle tone or vasoconstrictor responses.

Effects of morphine, H-Tyr-D-Arg-Phe-Lys-NH2 (DALDA) and B-HT920 on non-cholinergic nerve-mediated bronchoconstriction in pithed guinea-pigs.

1. Electrical stimulation (1 ms, 5 Hz, 80 V) for 5 or 15 s at the level of C4-T1 in the spinal canal of artificially respired pithed guinea-pigs (which had received intravenously (i.v.) (+)-tubocurarine chloride 2 mg kg-1, atropine sulphate 2 mg kg-1 and pentolinium tartrate 5 mg kg-1) caused constriction of airways, indicated by increased insufflation pressure. 2. This non-cholinergic constriction was inhibited by morphine (1-3 mg kg-1, i.v.), the peripherally acting mu-receptor agonist, H-Tyr-D-Arg-Phe-Lys-NH2 (DALDA, 0.1-1 mg kg-1, i.v.) or the alpha 2-adrenoceptor agonist B-HT920 (1-3 mg kg-1, i.v.). 3. The effects of either morphine (3 mg kg-1, i.v.) or DALDA (1 mg kg-1, i.v.) were inhibited by naloxone (3 mg kg-1, i.v.). Idazoxan (3 mg kg-1, i.v.) inhibited the anti-constrictor effect of B-HT920 (3 mg kg-1, i.v.), but not that of DALDA (0.1 mg kg-1, i.v.). 4. Thus activation of peripheral mu-opioid receptors or alpha 2-adrenoceptors inhibits airways constriction induced by non-cholinergic nerve stimulation in the pithed guinea-pig. This preparation therefore provides a further method for the in vivo examination of the effects of drugs on non-cholinergic tracheobronchial constrictor nerve function.

Plasma levels of atrial natriuretic peptides during pregnancy and post partum in the rat.

Plasma concentrations of atrial natriuretic peptides (ANP) in female Wistar rats were measured by radioimmunoassay at oestrus, during pregnancy, during parturition and between 3 h and 4 days post partum. Concentrations of ANP in rats on days 10, 15 and 17 of pregnancy were not significantly different from those in non-pregnant animals in oestrus (32.5 +/- 2.2 pmol/l; mean +/- S.E.M., n = 9), but levels near term (days 20 and 21 of pregnancy) were reduced by approximately 50%. However, plasma concentrations of ANP at 6, 12 and 24 h post partum were approximately twice those of non-pregnant animals in oestrus, but returned to normal levels within 4 days after parturition. Maternal plasma volume increased significantly during pregnancy, and fell 15-20% 6-24 h post partum. These results suggest that the relationship between plasma volume and the plasma concentration of ANP is reset during pregnancy and changes rapidly post partum. The results do not necessarily, however, imply any changes in the relationship between atrial pressure and the concentration of ANP.

The expression of parathyroid hormone-related protein mRNA and immunoreactive protein in human amnion and choriodecidua is increased at term compared with preterm gestation.

Parathyroid hormone-related protein (PTHrP) gene expression and/or immunoreactive protein have previously been identified in the uterus and intrauterine gestational tissues. The putative roles of PTHrP during pregnancy include vasodilatation, regulation of placental calcium transfer, uterine smooth muscle relaxation and normal fetal development. The aims of this study were 1) to determine the tissue-specific and temporal expression of PTHrP mRNA and immunoreactive protein in human gestational tissues collected at preterm and term; and 2) to determine the effect of labour on PTHrP expression by collecting these tissues from women undergoing elective caesarean section (before labour), intra-partum caesarean section during spontaneous-onset labour (during labour), and women with spontaneous labour and normal vaginal delivery (after labour). Total RNA and protein were extracted from placenta, amnion (over placenta and reflected) and choriodecidua for analysis by Northern blot (using a specific human PTHrP cDNA probe), and by N-terminal PTHrP RIA respectively. In amnion over placenta, reflected amnion and choriodecidua both PTHrP mRNA relative abundance and immunoreactive protein were significantly elevated at term compared with preterm (P < 0.01). At term, both PTHrP and its mRNA were significantly greater in amnion than in placenta and choriodecidua (P < 0.05). Also, both PTHrP and its mRNA were significantly elevated in amnion over placenta compared with reflected amnion (P < 0.05). The expression of PTHrP and its mRNA did not change in association with term labour or rupture of the fetal membranes, therefore this study provides no evidence for a specific PTHrP role in the onset and/or maintenance of term labour. However, the significant up-regulation of PTHrP mRNA and protein in the fetal membranes at term compared with preterm suggests an important role in late human pregnancy.

The effects of Al3+, Cd2+ and Mn2+ on human erythrocyte choline transport.

The effects of Al3+, Cd2+ and Mn2+ on human erythrocyte choline transport, Na-K-ATPase, Ca-Mg-ATPase and intracellular K+ levels were examined. The concentrations used were below the levels which caused significant haemolysis (less than or equal to 300 microM). All three cations inhibited concentrative choline accumulation over 3 hr [IC50 values at 1 microM choline were 35 microM (AlCl3), 250 microM (CdCl2) and 300 microM (MnCl2)] but at the concentrations tested, none decreased initial rates of choline uptake. The effects of Cd2+ and Mn2+ (but not Al3+) on choline accumulation were reversed by removing the cations from the extracellular medium by washing. All three cations also inhibited efflux of choline, at 1 microM choline, 30% inhibition being produced by 33 microM AlCl3, 81 microM CdCl2 and 111 microM MnCl2. At subhaemolytic concentrations, only CdCl2 inhibited Na-K-ATPase, (IC50 = 147 microM) and none of the cations significantly inhibited Ca-Mg-ATPase. Intracellular K+ levels were only reduced by the highest concentration of AlCl3 used (100 microM). These results suggest that inhibition of choline accumulation and efflux in erythrocytes by Al3+, Cd2+ and Mn2+ is not explicable solely in terms of either inhibition of Ca-Mg-ATPase, or inhibition of Na-K-ATPase causing reduced intracellular K+. Our conclusions are similar to those previously obtained using synaptosomes and provide support for the hypothesis that inhibition of choline transport by Al3+ may contribute to a number of disease states.

Secretory type II PLA2 immunoreactivity and PLA2 enzymatic activity in human gestational tissues before, during and after spontaneous-onset labour at term.

Arachidonic acid mobilization by phospholipase A2 (PLA2) and subsequent prostaglandin synthesis is thought to be a pivotal event in the onset and/or maintenance of human labour. The purpose of this study was to quantify secretory PLA2 (sPLA2) and non-secretory PLA2 enzymatic activity and type II sPLA2 immunoreactivity in human gestational tissues before, during and after labour of spontaneous onset. Placental tissue and fetal membranes were collected from women before, during and after spontaneous-onset labour at term and stored at - 80 degrees C. PLA2 activity in supernatants was quantified by radiolabelled substrate assay (14C-phosphatidylethanolamine) with and without 12.5 mm dithiothreitol (DTT) to separate enzymatic activity contributed by secretory and non-secretory PLA2 components. Immunoreactive type II PLA2 in supernatants was determined by a monoclonal, non-competitive sandwich ELISA. Total PLA2 enzymatic activity in amnion, choriodecidua and placenta was not significantly different before, during and after labour (n=18-20). Likewise, non-secretory enzymatic activity was not significantly different before (n=9), during (n=10) and after labour (n=9) in any of the three types of gestational tissue examined. Although immunoreactive type II PLA2 was significantly higher in the placenta (P<0.01) compared to amnion and choriodecidua, there was no significant difference in immunoreactive type II PLA2 within each tissue group according to labour status (n=18-20). Overall, no change in PLA2 secretory or non-secretory enzymatic activity or immunoreactive type II PLA2 could be detected throughout the peripartum period.

Human placental choline acetyltransferase activity at parturition.

Choline acetyltransferase (ChAT) activity measured in human placentae obtained during labour, or after labour of spontaneous onset, was significantly lower than that measured in placentae obtained before the onset of labour. The reduction in ChAT activity may partly account for the reduction in placental acetylcholine (ACh) content and release into maternal vessels at this time. When cycloheximide (10 micrograms/ml) was infused for 3 h into both fetal and maternal vessels of the perfused placental lobule, it did not significantly alter placental ChAT activity or ACh output from the fetal vessels, though it significantly reduced placental beta HCG release from the maternal side of the perfused lobule. These results suggest that regulation of placental ChAT activity at the time of human parturition may be due to endogenous modulators of its activity, rather than to acute changes in the synthesis of this enzyme.

Antisense oligonucleotide inhibition of type II phospholipase A2 expression, release and activity in vitro.

Phospholipase A2 (PLA2) enzymes that regulate the release of arachidonic acid from cell membrane phospholipids represent a crucial rate-limiting step for the prostaglandin biosynthetic pathway. The aim of this study was to determine the mechanism of action and effects of type II PLA2 antisense oligonucleotides on type II PLA2 mRNA relative abundance, and the release of PLA2 enzymatic activity and prostaglandin F2alpha (PGF2alpha) in vitro. A human placental explant system was used to evaluate the effects of the type II PLA2 specific antisense oligonucleotides A (5'-GGGTGGGTATAGAAGGGCTCC-3', complementary to the base sequence 697-717 of the type II PLA2 gene) and B (5'-TTTTTGATTTGCTAATTGCTT-3', complementary to the base sequence 821-841 of the type II PLA2 gene). PLA2 activity released from explants was quantified by radiolabelled substrate assay using 14C-phosphatidylethanolamine (PE), and PGF2alpha content was analyzed by radioimmunoassay. Compared with control, the release of PLA2 activity and PGF2alpha was significantly reduced over the 24-h period by treatment with both antisense oligonucleotides (P< 0.05). At this concentration, type II PLA2 mRNA abundance was also significantly reduced by both antisense oligonucleotides A and B (P< 0.05). This data demonstrates the efficacy of antisense oligonucleotide inhibition of secretory PLA2 (sPLA2) expression and activity, and the contribution of sPLA2 to placental prostaglandin production.

Effect of the choline acetyltransferase inhibitor (2-benzoylethyl)-trimethylammonium iodide (BETA) on human placental prostaglandin release and phospholipase A2 activity.

We investigated the effect of an inhibitor of acetylcholine (ACh) synthesis, (2-benzoylethyl)trimethylammonium iodide (BETA), on prostaglandin (PG)E2 and PGF2 alpha release from incubated placental explants in the presence or absence of ACh or arachidonic acid (AA). BETA alone (100 microM) significantly reduced both PGE2 and PGF2 alpha release. However, this inhibitory effect of BETA was not reversed in the presence of ACh (10 microM to 1 mM). The addition of AA (10 to 100 microM) increased both PGE2 and PGF2 alpha release, and simultaneously overcame the inhibition of PGE2 but not PGF2 alpha release by BETA. These results are compatible with the hypothesis that BETA may be inhibiting the phospholipase A2 (PLA2) step rather than prostaglandin H synthase (PGHS) step in the enzymatic pathway of PG generation. This hypothesis was supported by evidence showing a lack of effect of BETA (10 and 100 microM) on ovine placental microsome PGHS activity. Moreover, human placental homogenate PLA2 activity was reversibly inhibited by BETA (100 microM). In the presence of BETA (100 microM), addition of exogenous ACh (100 microM) had no significant effect on placental PLA2 activity. These results indicate that the inhibitory effect of BETA on placental PG release was unlikely to be via an action on ACh synthesis, but rather via a reversible effect on PLA2 activity.

Effects of opioids on acetylcholine output of perfused human placental cotyleda.

(1) Output of acetylcholine (ACh) from fetal vessels of Krebs-perfused human placental cotyleda was estimated by bioassay using the rat stomach strip. (2) Infusion of high concentrations of the opioids pethidine, morphine or ethylketocyclazocine, but not [D-Ala2]-methionine enkephalinamide (0.1-300 mumol/l), reduced ACh output. Fifty per cent inhibition of output occurred in the presence of concentrations of greater than or equal to 300 mumol/l (morphine and ethylketocyclazocine) and 338 (317, 353; 95 per cent c.l., n = 6) mumol/l (pethidine). (3) ACh output was inhibited by infusion of 100 mumol/l naloxone or 10 mumol/l naltrexone, but was not affected by lower concentrations of either antagonist. (4) These results suggest that the therapeutic concentrations of morphine and pethidine likely to occur in vivo would not affect placental ACh output into fetal vessels. The finding that high concentrations of synthetic opioids or opioid antagonists were required to inhibit output suggests that they may not be acting specifically, and provides no evidence for the hypothesis that endogenous opioids play a role in control of ACh release into fetal vessels of human placentae.