Insulin-mediated endothelin signaling is antiviral during West Nile virus infection.

IMPORTANCE: Arboviruses, particularly those transmitted by mosquitoes, pose a significant threat to humans and are an increasing concern because of climate change, human activity, and expanding vector-competent populations. West Nile virus is of significant concern as the most frequent mosquito-borne disease transmitted annually within the continental United States. Here, we identify a previously uncharacterized signaling pathway that impacts West Nile virus infection, namely endothelin signaling. Additionally, we demonstrate that we can successfully translate results obtained from D. melanogaster into the more relevant human system. Our results add to the growing field of insulin-mediated antiviral immunity and identify potential biomarkers or intervention targets to better address West Nile virus infection and severe disease.

Interactions between the complement and endothelin systems in normal pregnancy and following placental ischemia.

Preeclampsia is characterized by new onset hypertension and fetal growth restriction and is associated with aberrant activation of the innate immune complement system and stressed or ischemic placenta. Previous studies have suggested a role for both endothelin and complement system activation products in new onset hypertension in pregnancy, but inter-relationships of the pathways are unclear. We hypothesized that complement activation following placental ischemia stimulates the endothelin pathway to cause hypertension and impair fetal growth. The Reduced Uterine Perfusion Pressure (RUPP) model results in hypertension and fetal growth restriction in a pregnant rat due to placental ischemia caused by mechanical obstruction of blood flow to uterus and placenta. The effect of inhibitor of complement activation soluble Complement Receptor 1 (sCR1) and endothelin A receptor (ETA) antagonist atrasentan on hypertension, fetal weight, complement activation (systemic circulating C3a and local C3 placental deposition) and endothelin [circulating endothelin and message for preproendothelin (PPE), ETA and endothelin B receptor (ETB) in placenta] in the RUPP rat model were determined. Following placental ischemia, sCR1 attenuated hypertension but increased message for PPE and ETA in placenta, suggesting complement activation causes hypertension via an endothelin independent pathway. With ETA antagonism the placental ischemia-induced increase in circulating C3a was unaffected despite inhibition of hypertension, indicating systemic C3a alone is not sufficient. In normal pregnancy, inhibiting complement activation increased plasma endothelin but not placental PPE message. Atrasentan treatment increased fetal weight, circulating endothelin and placental ETA message, and unexpectedly increased local complement activation in placenta (C3 deposition) but not C3a in circulation, suggesting endothelin controls local placental complement activation in normal pregnancy. Atrasentan also significantly decreased message for endogenous complement regulators Crry and CD55 in placenta and kidney in normal pregnancy. Results of our study indicate that complement/endothelin interactions differ in pregnancies complicated with placental ischemia vs normal pregnancy, as well as locally vs systemically. These data clearly illustrate the complex interplay between complement and endothelin indicating that perturbations of either pathway may affect pregnancy outcomes.

Expression of inflammatory and apoptosis factors following coronary stent implantation in coronary heart disease patients.

We investigated the changes in characteristics of neutrophil CD11b, monocyte CD11b, platelet CD62P, endothelin (ET), and neutrophil CD178 in patients with coronary heart disease (CHD) before and after primary coronary stenting. A total of 41 patients with CHD who underwent coronary stenting and 40 control subjects were enrolled in the study. In CHD patients, peripheral blood samples were taken 24 h before and 30 min, 24 h, and 72 h after successful coronary stenting. All markers were significantly elevated in patients with CHD compared with controls (P<0.05). Time-course studies revealed that the expressions of neutrophil CD11b, monocyte CD11b, platelet CD62P, and ET were lower at 30 min post-operation (PO) compared with that at 24 h before operation (BO) (P<0.05). All levels significantly increased from 30 min PO to 24 h PO (P<0.05) and decreased thereafter until 72 h PO (P>0.05). Time course changes in neutrophil CD11b levels after coronary stenting were significantly higher in patients with unstable angina pectoris than in patients with stable angina pectoris (P<0.05). CD11b levels were related to CD62P in patients with CHD (P<0.05). Neutrophil CD11b and monocyte CD11b levels were significantly increased in patients with CHD who underwent coronary stenting compared with controls (P<0.05). Results show that CD11b levels increased, meanwhile, the levels of CD62P and ET increased in CHD patients after coronary stenting. In addition, neutrophil CD178 levels of apoptosis factor in patients, which is important for regression of inflammation, remained high for a period of time after coronary stenting.

[A comparison study of post-cerebral hemorrhage stress ulcer with two interventional treatments aimed at endogenous endothelin in rat].

OBJECTIVE: To observe the respective effects of intervention either with endothelin (ET) antibody or with ET receptor antagonist on acute stress ulcer (ASU) subsequent to cerebral hemorrhage in rats. METHODS: Forty Sprague-Dawley (SD) rats were randomly divided into control group (group A, n=10), model group (group B, n=10), ET antibody (group C, n=10), and ET receptor antagonist group (group D, n=10). Right intracerebral hemorrhage was reproduced by injection of 200 microl autologous venous blood. Normal saline, ET antibody, or ET receptor antagonist was respectively administered intravenously per day for designated group. The rats were sacrificed at 3 days of the experiment. The incidence of ASU and ulcer index were assessed, serum ET-1 level, malondialdehyde (MDA) content and superoxide dismutase (SOD) activity in serum, Rsvmit (Rsv) and Vvmit (Vv) of cerebral and gastric mucosa were measured, and pathological examination of the cerebral tissue and gastric mucosa was performed with light microscopy and electron microscopy. RESULTS: In group B, serum ET-1 level did not changed. MDA content were markedly increased in serum, cerebral and gastric mucosa, SOD activity were markedly decreased, cerebral water content were markedly increased; Rsv in neuron and gastric parietal cell, Vv in gastric parietal cell both were markedly decreased (P<0.05 or P<0.01). ASU was only observed in group B (the incidence was 30%, ulcer index was 15). It was not observed in other groups. Compared with group B, MDA content were significantly decreased, and SOD activity were significantly increased in serum, cerebral and gastric mucosa in groups C and D, cerebral water content in group C were dramatically decreased (all P<0.01), but these were not statistically different between groups C and D. Rsv and Vv in neuron and gastric parietal cell in groups B, C and D were not statistically different, and serum ET-1 level were not statistically different among the groups (all P>0.05). CONCLUSION: Intervention of ET antibody and ET receptor antagonist can both reduce occurrence and development of ASU subsequent to cerebral hemorrhage in rats.

Endothelin: 20 years from discovery to therapy.

Since its identification as an endothelial cell-derived vasoconstrictor peptide in 1988, endothelin-1, the predominant member of the endothelin peptide family, has received considerable interest in basic medical science and in clinical medicine, which is reflected by more than 20 000 scientific publications on endothelin research in the past 20 years. The story of endothelin is unique as the gene sequences of endothelin receptors and the first receptor antagonists became available within only 4 years of the identification of the peptide sequence. The first clinical study in patients with congestive heart failure was published only 3 years thereafter. Yet, despite convincing experimental evidence of a pathogenetic role for endothelin in development, cell function, and disease, many initial clinical studies on endothelin antagonism were negative. In many of these studies, study designs or patient selection were inadequate. Today, for diseases such as pulmonary hypertension, endothelin antagonist treatment has become reality in clinical medicine, and ongoing clinical studies are evaluating additional indications, such as renal disease and cancer. Twenty years after the discovery of endothelin, its inhibitors have finally arrived in the clinical arena and are now providing us with new options to treat disease and prolong the lives of patients. Possible future indications include resistant arterial hypertension, proteinuric renal disease, cancer, and connective tissue diseases.

EndothelinA receptor antagonist BSF-208075 causes immune modulation and neuroprotection after stroke in gerbils.

UNLABELLED: Leukocytes contribute to the ischemia-reperfusion injury. Recent studies suggested endothelins could be important mediators for leukocyte activation in stroke. We tested if the endothelinA receptor antagonist BSF-208075 (ambrisentan) could reduce an ischemic lesion by modulation of leukocyte-endothelium interactions. Twenty-four gerbils underwent either a sham operation (n=6) or 15 min of bilateral carotid artery occlusion resulting in global cerebral ischemia. Ischemic animals received normal saline (n=6), 5 mg/kg BSF-208075 (n=6) or 30 mg/kg (n=6) administered intravenously at 10 min of reperfusion. Leukocytes rolling or adhering to endothelium were counted by intravital microscopy in parietal subsurface venules through a closed cranial window. BSF-208075 dose-dependently reduced postischemic leukocytes rolling (7.3+/-2.3 vs. 3.3+/-1.4 vs. 0.7+/-0.7 [n/100 microm/min]; p<0.05) and adhering (5.3+/-1.4 vs. 2.7+/-1.6 vs. 1.3+/-0.5 [n/100 microm/min]; p<0.05). Cerebral blood flow was not significantly changed by BSF-208075. Cortical neurons [n/mm2] in an area corresponding to the in vivo microscopy were dose-dependently preserved 7 days after ischemia (2456+/-687 vs. 3254+/-245 vs. 3780+/-168; p<0.05). CONCLUSION: Endothelins mediate leukocyte activation in ischemic stroke. The endothelinA receptor antagonist BSF-208075 administered during reperfusion reduces the postischemic leukocyte activation and causes neuroprotection.

Autoantibodies against EPCR are found in antiphospholipid syndrome and are a risk factor for fetal death.

The antiphospholipid syndrome (APS) is associated with thrombosis and fetal death but the pathologic mechanisms are poorly understood. Since endothelial protein C receptor (EPCR) plays a role in the anticoagulant system and in placental development, we hypothesized that anti-EPCR autoantibodies may be involved in clinical manifestations of APS and in fetal loss. The levels of immunoglobulin M (IgM) and IgG anti-EPCR autoantibodies were analyzed by enzyme-linked immunosorbent assay (ELISA) in 43 patients with APS and 43 controls. Anti-EPCR levels were higher in APS patients than in controls. Interestingly, one of the IgM anti-EPCR autoantibodies inhibited the generation of activated protein C on endothelium. Since markedly high anti-EPCR levels were found in women with fetal death, 87 patients with a first episode of unexplained fetal death were subsequently analyzed and their anti-EPCR levels were compared with 87 matched controls. We found that anti-EPCR autoantibodies constitute an independent risk factor for a first fetal death episode: the adjusted odds ratios (ORs) for anti-EPCR autoantibodies above the 95th percentile were 23.0 (95% confidence interval [CI], 2.0-266.3) for IgM and 6.8 (95% CI, 1.2-38.4) for IgG. Anti-EPCR autoantibodies can be detected in APS patients and are independent risk factors for fetal death.

Activated protein C inhibits bronchial hyperresponsiveness and Th2 cytokine expression in mice.

Asthma is one of the most common diseases and is characterized by airway obstruction, airway inflammation, and increased airway responsiveness. Glucocorticoids are very effective in treatment, but their long-term use is associated with several side effects, so that new anti-inflammatory drugs are in development. Activated protein C (APC) is a serine protease with potent anti-inflammatory effects. This study evaluated the effect of inhaled APC on airway inflammation and hyperresponsiveness in a murine asthma model. Asthma was induced in BALB/c mice by exposure to chicken egg ovalbumin (OVA), and the effect of inhaled APC was assessed by administering prior to OVA exposure. Inhalation of APC significantly inhibited the expression of T helper 2 (Th2) cytokines, immunoglobulin E (IgE), eosinophilic inflammation, and hyperresponsiveness. APC also significantly suppressed the expression of Th2 cytokines and IgE from lymphocytes isolated from OVA-sensitized/challenged animals. In addition, binding of signal transducer and activator of transcription 6 (STAT6) and nuclear factor kappa B (NF-kappa B) oligonucleotides to lung nuclear proteins was significantly reduced in mice treated with inhaled APC. In brief, the exogenous supplementation of APC inhibits the immunologic and inflammatory responses induced by Th2 cytokines in a mouse model of asthma and may represent a novel anti-inflammatory treatment.

Hypotensive activity of ultralow doses of antibodies to factors involved in the regulation of vascular tone.

Hypotensive activity of ultralow doses of antibodies to some endogenous substances involved in the regulation of vascular tone was studied on NISAG rats with hereditary stress-induced arterial hypertension. It was found that antibodies to angiotensin II and its receptor in ultralow doses markedly reduced systolic blood pressure, which was reproducible after repeated treatment. The course of peroral treatment with antibodies to endothelin and endothelial NO synthase in ultralow doses did not decrease systolic blood pressure.

Activated protein C alters cytosolic calcium flux in human brain endothelium via binding to endothelial protein C receptor and activation of protease activated receptor-1.

Activated protein C (APC) exerts endothelial protein C receptor (EPCR)-dependent neuroprotective effects in a brain focal ischemia model and direct cellular effects on human umbilical vein endothelial cells (HUVECs) via protease-activated receptor-1 (PAR-1). Because PAR receptors are expressed in brain endothelium and mediate intracellular calcium concentration ([Ca2+]i) signaling, we hypothesized that APC may regulate intracellular [Ca2+] flux in human brain endothelial cells (BECs) via EPCR and PAR-1. Primary cortical BECs derived from human autopsies (early passage) and HUVECs were used for [Ca2+]i imaging fluorometry. Cells were exposed for 1 minute to APC, protein C zymogen, or mutant Ser360Ala-APC, and [Ca2+]i was monitored in the presence or absence of antibodies against PAR-1, PAR-2, PAR-3, or EPCR. APC, but not protein C zymogen or the active site mutant Ser360Ala-APC, induced dose-dependent [Ca2+]i release in human BECs (Delta[Ca2+]i max = 278.3 +/- 19.5 nM; EC50 for APC = 0.23 +/- 0.02 nM, n = 70 measurements). APC-induced [Ca2+]i signaling was abolished by a cleavage site blocking anti-PAR-1 antibody, whereas anti-PAR-2 and -PAR-3 antibodies were without effect. Antibody RCR252 that ablates APC binding to EPCR blocked APC-mediated [Ca2+]i signaling, whereas anti-EPCR antibody RCR92 that does not block APC binding did not abolish the APC-induced [Ca2+]i response. Experiments using HUVECs confirmed the findings for BECs. Thapsigargin inhibited the APC-induced [Ca2+]i signal, implicating the endoplasmic reticulum as a major source for the APC-induced [Ca2+]i release. These data suggest that APC regulates [Ca2+]i in human brain endothelium and in HUVECs by binding to EPCR and signaling via PAR-1.

Localization of immunoreactive endothelin and characterization of its receptors in aortic cusps.

BACKGROUND AND AIM OF THE STUDY: Aortic valve cusp tissue has been shown to have contractile properties in response to a range of common vasoactive agents. Of these, endothelin (ET) is both the most potent and efficacious. METHODS: In an attempt to define the mechanism of action and localization of ET, the response of porcine aortic valve cusps to ET and the selective ET(B) receptor agonist sarafotoxin 6c (S6c) was examined, in the presence and absence of ET(A) and ET(B) receptor antagonists. An attempt was made, using immunocytochemical techniques, to localize ET in cusp tissue. RESULTS: Addition of 90 mM KCl produced a mean contractile response of 1.02+/-0.09 mN (n = 27). ET (10- to 10(-7)M) produced a concentration-dependent contraction of aortic valve cusps, with a maximum response of 116.7+/-12.7% (n = 6) of that obtained with 90 mM KCl. In a similar manner, 10(-5)M of the selective ET(A) receptor antagonist BQ123 (n = 4) and 10(-5)M of the selective ET(B) receptor antagonist BQ788 (n = 4) each partially inhibited the effect of ET. The ET(B)-selective agonist S6c (10(-9) to 10(-7)M) also induced a concentration-dependent contraction of valve cusps (n = 4), with a maximum response of 99.1+/-11.1%. This response was completely inhibited by 10(-5)M BQ788 (n = 4). Immunoreactive ET was localized to the endothelial cells that lined both the ventricular and aortic side of the cusps. CONCLUSION: These results show that aortic valve cusps contract to ET via an action at both ET(A) and ET(B) receptors. The presence of immunoreactive ET in the endothelial cells of the cusps also suggests that it might play a role in valve function. Further studies are required to elucidate the role of these receptors in the physiology and pathophysiology of the aortic valve.

[Berger's disease in childhood]. / La maladie de Berger chez l'enfant.

Berger's disease or IgA nephropathy (NIgA) is the most common form of glomerulonephritis in the world. In children macroscopic haematuria is the first sign in about 80% of the patients. Renal failure appears in 20% of cases after twenty years of follow-up. The most important prognosis indicators are a nephrotic syndrome at the onset, a proteinuria > 1 g/24 hours, diffuse tubulo-interstitial lesions and extracapillary proliferation with crescents in more than 50% of the glomeruli. The pathogenic mechanisms are just emerging and involve a disrupted process of the systemic tolerance to mucosal antigen with abnormal mucosal gamma delta T cell repertoire, abnormally glycosylated IgA1 molecules and a down-regulation of Fc alpha receptors on blood cells. After IgA deposition, the mechanisms of mesangial cell damage and activation involve vascular factors as endothelin/nitric oxide system, cytokines and growth factors such as interleukine-6, platelet derived growth factor and transforming growth factor beta. There is no curative treatment but steroids are useful in diffuse proliferative extracapillary forms, when histological activity score is high with a short delay between diagnosis and treatment, or for moderately severe NIgA with normal renal function.

Endothelin immunocytochemistry: indications of false-positive labeling patterns and non-detectable antigen concentrations.

Endothelin is an endothelium-derived peptide with potent vasoconstrictor and mitogenic properties. Since studies concerning the immunocytochemical localization of endothelin are often inconsistent we tried to clear up some of these discrepancies by comparing specificity and labeling patterns of different endothelin antibodies. Monoclonal and polyclonal endothelin antibodies ( n=7) were examined concerning their reactivity with endothelins and heart tissues by immunoblotting. Using immunofluorescence microscopy reactivities with human non-failing hearts, failing hearts, and cultured endothelial cells were examined. Specificity of labelings was assessed by absorption controls and functional controls using endothelial cells conditioned to produce different amounts of endothelin. As shown by immunoblotting five out of seven endothelin antibodies revealed both specific endothelin reactivity and negligible non-specific reactivities with heart proteins. Immunocytochemistry showed vascular reactivity of N-terminal endothelin antibodies to be associated with alpha-smooth muscle actin expression. One N-terminal antibody showed additional nuclear reactivity, and one C-terminal antibody vimentin-like labeling patterns. Although these reactivities were abolished in absorption controls these labelings had to be graded non-specific because functional specificity of endothelin antibodies could not be proven. The remaining antibodies revealed no endothelial reactivity even after tyramide signal amplification. Cellular endothelin concentrations ranged around 2,000-fold below the detection limit of immunocytochemical methods. Discrepancies in endothelin immunocytochemistry may originate from false-positive results and from expression levels of endothelin below the detection limit of immunocytochemical methods.

Amelioration of post-ischaemic renal injury by contralateral uninephrectomy: a role of endothelin-1.

BACKGROUND: Previous studies showed that unilateral renal damage is attenuated by prior contralateral uninephrectomy (Nx) in ischaemia-induced acute renal failure (ARF). Since renal ischaemia increases endothelin-1 (ET-1) production in the kidney, we examined whether the alteration of renal ET-1 content may contribute to the nephrectomy-induced attenuation of renal injury. METHODS: Ischaemic renal injury was provoked by 60-min left renal artery occlusion (RAO). Removal of the right kidney was performed just before RAO in the Nx group. Forty-eight hours after release of the clamp, renal ET-1 content was measured in both non-nephrectomized and unilaterally nephrectomized rats. We also examined the effects of a selective ET(A) receptor (FR139317) and monoclonal ET antibody (AwETN40) on the RAO-induced changes in renal haemodynamics at 2 and 48 h after RAO respectively. RESULTS: The plasma concentration of ET-1 did not change in the two groups of ARF rats, but the cortical content of ET-1 increased to a lesser extent in Nx animals after ischaemia. Prior removal of the right kidney significantly facilitated the percentage recovery of left renal blood flow (RBF) during the first 2 h after release of the clamp. The percentage recovery of inulin clearance (Cin) by the kidney was also significantly better in Nx than sham-Nx rats at 48 h after RAO. Continuous administration of FR139317 (50 mg/kg/day) using osmotic minipumps for 3 days significantly attenuated exogenous ET-1-induced decrease in Cin and RBF. Infusion of FR139317 restored the decrease in RBF to control values during first 2 h in sham-Nx rats. However, FR139317 and AwETN40 did not ameliorate the RAO-induced decline of Cin in sham-Nx or Nx rats at 2 and 48 h after ischaemia respectively. CONCLUSIONS: Contralateral uninephrectomy prior to ischaemia-induced ARF attenuated the increase in cortical ET-1 content and subsequent renal response to ischaemic injury. This beneficial effect of unilateral nephrectomy, however, was not mediated through well-preserved RBF due to reduced intrarenal ET-1 action.

Endothelin is a potent inhibitor of matrix metalloproteinase-2 secretion and activation in rat mesangial cells.

We examined the effects of endothelin (ET) on the activity of matrix metalloproteinase-2 (MMP-2) in cultured MCs. Addition of the ET(A) receptor antagonists or neutralizing anti-endothelin antibody into MC cultures markedly augmented the secretion and activation of MMP-2. On the contrary, addition of the exogenous ET-1 into MC culture significantly inhibited the synthesis of MMP-2 in both basal and cytokines (tumor necrosis factor-alpha and interferon-gamma) plus lipopolysaccharide-stimulated conditions. Furthermore, pretreatment of cells with exogenous ET-1 obviously prevented cytochalasin D-elicited activation of MMP-2, an effect that was completely abolished by ET(A) receptor antagonist, FR139317. In addition, ET-1 was found to be able to suppress the expression of membrane type-1 MMP (MT1-MMP) and promote the conversion of tissue inhibitor of matrix metalloproteinase-2 (TIMP-2) from cell associated form to secreted form. The addition of recombinant TIMP-2 into the culture abrogated dose-dependently the cytochalasin D-elicited activation of MMP-2. These results suggest that ET is a potent inhibitor of MMP-2 secretion and activation in MCs. These novel findings may help us understand the subtle regulation of the synthesis and activation of MMP-2 in MCs. It also provides us with further insight into the pathophysiological mechanisms involving ET in the regulation of matrix turnover in glomerulus.

Biphasic release of immunoreactive endothelins following acute pulmonary thromboembolism in pigs.

The purpose of this investigation was to study the role of endothelins (ETs) in the pathogenesis of acute pulmonary thromboembolism (PTE). Eighteen piglets (20 +/- 3 kg) were anesthetized and ventilated with 100% oxygen, five of them then served as controls. Acute thromboembolic injury in the lung was induced by injecting 15-25 ml of preformed clots into the left lower lobar pulmonary artery during thoracotomy. Pulmonary arterial pressure (Ppa) increased by at least 2.5-fold from baseline. During the subsequent 8 h, seven blood samples were collected from the left atrium and assayed for immunoreactive ETs. The results showed that following PTE: (1) Ppa remained elevated but cardiac output remained constant throughout the experiments; (2) plasma level of immunoreactive ETs increased in the embolized group compared to controls and the profile of immunoreactive ET release suggested a biphasic response. We conclude that the release of these vasocontractile and bronchoconstrictive mediators after PTE may contribute to ventilation perfusion mismatching and account for the pulmonary hypertension and deterioration of gas exchange that are often seen clinically.

Placental endothelin gene expression and endothelin concentration in fetal fluids of the first trimester gestational sac.

We have investigated the distribution of immunoreactive endothelins (irET) in fetal fluids and expression of ET precursor genes in villous tissue during the first trimester. Samples of maternal plasma (n = 6), coelomic fluid (n = 28), amniotic fluid (n = 23) and villous tissue (n = 3) were obtained from 30 pregnancies immediately before surgical termination at 7-12 weeks gestation. irET concentration was measured in plasma and fluids using two different radioimmunoassay kits, i.e. RPA 545 and RPA 555 and high performance liquid chromatography (HPLC). Total RNA was extracted and purified from villous tissue, reverse transcription and polymerase chain reaction (RT-PCR) were performed to evaluate the expression of ET-related genes. The irET concentration as evaluated by both kits was significantly higher (P<0.005) in maternal plasma than in coelomic or amniotic fluid and significantly higher (P<0.005) in coelomic fluid than in amniotic fluid using the RPA 555 kit. The profile of ET obtained by the HPLC- radioimmunoassay (RPA 555 kit) method confirmed significantly (P<0.005) higher ET concentration in coelomic than in amniotic fluid, although a similar distribution pattern for the three ET was observed in both embryonic fliud cavities. ET-3 was the predominant isoform in both fluids, reaching 19.4+/-2.0 pg/ml and 6.3+/-1.6 pg/ml in coelomic and amniotic fluid, respectively. Coelomic or amniotic fluid irET concentration did not change with gestational age irrespective of the kit used. RT-PCR demonstrated that first trimester placenta expresses the genes encoding for prepro-ET-1, -ET-2 and -ET-3. The similar ET distribution pattern in both fluid cavities could reflect their origin from the villous tissue and suggests that ET may play a role in the development of placenta and other fetal organs during organogenesis.

Specific sandwich-type enzyme immunoassays for smooth muscle constricting novel 31-amino acid endothelins.

We established highly sensitive and specific sandwich-enzyme immunoassays (EIAs) for three newly discovered bioactive 31-amino acid endothelins [ETs(1-31)], which can detect as little as 0.16 pg/well of ET-1(1-31), 0.39 pg/well of ET-2(1-31), and 0.16 pg/well of ET-3(1-31). The EIAs showed no crossreactivity with 21-amino acid endothelins [ETs(1-21)] or big ETs at the usual assay concentrations below 1-5 ng/ml. In reversed-phase HPLC, immunoreactive ETs(1-31) in the granulocytes of normal human subjects eluted at the exact positions of authentic ETs(1-31), except for the presence of one additional unknown immunoreactive ET-1(1-31). The results also indicate that ETs(1-31) exist in the granulocytes at levels higher than or similar to those of ETs(1-21). This study is the first to establish EIAs for novel bioactive ETs(1-31). These assays can be utilized to assess the pathophysiological roles of ETs(1-31).