The interaction of endothelin receptor responses in the isolated perfused rat lung

To gain more insight into the complex pulmonary interactions of endothelins (ET), we studied airway and vascular responses to endothelins in isolated perfused rat lungs in the presence of the novel ET(B)-receptor antagonist BQ788. In particular we focused on airway responses and on prostacyclin release. The effectiveness of BQ788 in our system was shown by its ability to concentration-dependently prevent vasoconstriction (IC50 0.1 microM), bronchoconstriction (IC50 0.1 microM) and prostacyclin production (IC50 < 0.1 microM) induced by the ET(B)-receptor agonist IRL1620 (1 nmol). Airway responses to ET-1: ET-1-induced bronchoconstriction was aggravated by BQ123 (1 or 8 microM), while BQ788 pretreatment (1 or 8 microM) showed no significant effect. Simultaneous treatment with 8 microM BQ123 and BQ788 attenuated the ET-1-induced bronchoconstriction. Vascular responses to ET-1: ET-1 (1 nmol)-induced vasoconstriction was potentiated by BQ788 (1 or 8 microM), but attenuated by the ET(A)-receptor antagonist BQ123 (1 microM). In the presence of BQ788 diminished amounts of the stable prostacyclin metabolite 6-keto-PGF1alpha were detected in the perfusate. Simultaneous treatment with 8 microM BQ123 and BQ788 completely prevented the ET-1-induced vasoconstriction. Conclusions: Both ET(A)- and ET(B)-receptors contribute to ET-1-induced vasoconstriction and bronchoconstriction. The ET-1-induced vasoconstriction is attenuated by stimulation of ET(B)-receptors, a response that is partly mediated by prostacyclin. Due to the mutual interactions between ET(A)- and ET(B)-receptors, simultaneous inhibition of both receptors is required to prevent the deleterious effects of ET-1 on lung functions.     

Effects of neutral endopeptidase inhibition and combined angiotensin converting enzyme and neutral endopeptidase inhibition on angiotensin and bradykinin peptides in rats

The respective roles of apoptosis and accidental cell death after thermal injury were evaluated in normal human epidermal keratinocytes. By coupling the LIVE/DEAD fluorescence viability assay with the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) method and ultrastructural morphology, these two processes could be distinguished. Cells were grown on glass coverslips with a microgrid pattern so that the results of several staining procedures performed sequentially could be visualized in the same cells after heating at temperatures of up to 72 degrees C for 1 second. After exposure to temperatures of 58 to 59 degrees C, cells died predominantly by apoptosis; viable cells became TUNEL positive, indicating degradation of DNA. After exposure to temperatures of 60 to 66 degrees C, both TUNEL-positive viable cells and TUNEL-positive nonviable cells were observed, indicating that apoptosis and accidental cell death were occurring simultaneously. Cells died almost immediately after exposure to temperatures above 72 degrees C, presumably from heat fixation. The fluorescent mitochondrial probe MitoTracker Orange indicated that cells undergoing apoptosis became TUNEL positive before loss of mitochondrial function. Nucleosomal fragmentation of DNA analyzed by enzyme-linked immunosorbent assay and gel electrophoresis occurred after exposure to temperatures of 58 to 59 degrees C. The characteristic morphological findings of cells undergoing apoptosis, by transmission electron microscopy, included cellular shrinkage, cytoplasmic budding, and relatively intact mitochondria. Depending on temperature and time of exposure, normal human epidermal keratinocytes may die by apoptosis, accidental cell death, or heat fixation.     

Endothelin-1 induces vasoconstriction and nitric oxide release via endothelin ET(B) receptors in isolated perfused rat liver

Endothelin-1 (0.1, 1 and 10 nM) induced a significant increase in portal pressure and nitric oxide (NO) release in the isolated rat liver. The endothelin ET(B) receptor agonist, IRL 1620 (Suc-[Glu9,Ala(11,15)]endothelin-1-(8-21)) (0.1, 1 and 10 nM) also elicited a marked increase in portal pressure and NO release. The potency of endothelin-1 was higher than that of IRL 1620. The endothelin ET(A) receptor antagonist, BQ-123 (cyclo(-D-Trp-D-Asp-Pro-D-Val-Leu)) (1 and 10 microM), had no effect on the endothelin-1-induced change in portal pressure and NO current. In contrast, the endothelin ET(B) receptor antagonist, BQ-788 (N-cis-2,6-dimethylpiperidinocarbonyl-L-gamma-methyl-leucyl-D-1-++ +methoxycarbonyltryptophanyl-D-norleucine) (1 and 10 nM), attenuated the endothelin-1-induced change in portal pressure and NO current. Administration of N(G)-monomethyl-L-arginine (L-NMMA), a NO synthase inhibitor, completely abolished the endothelin-1- or IRL 1620-induced NO release. L-NMMA enhanced the increase in portal pressure and decrease in O2 consumption caused by endothelin-1. These results indicated that endothelin ET(B) receptors mediate both vasoconstriction and NO release and that NO plays a significant role in stabilizing microcirculation in isolated perfused rat liver.     

Antihypertensive and natriuretic effects of CGS 30440, a dual inhibitor of angiotensin-converting enzyme and neutral endopeptidase 24.11

Dual angiotensin-converting enzyme (ACE)/neutral endopeptidase (NEP) inhibitors, by decreasing angiotensin-II production and by preventing the degradation of atrial natriuretic peptide (ANP), may be useful for the treatment of hypertension and congestive heart failure. The thiol dipeptide CGS 30440 (prodrug of CGS 30008, IC50: ACE/NEP = 19/2 nM) administered to rats (10 mg/kg p.o.) inhibited lung tissue ACE activity by 98% and 61% at 1 and 24 hr (P < .001) and inhibited the angiotensin-I pressor response by 75 to 90% for more than 6 hr. Renal tissue NEP activity was reduced by 80% at 1 hr and 73% at 24 hr (P < .001). In rats supplemented with exogenous ANP, CGS 30440 (1 mg/kg p.o.) elevated the concentration of circulating ANP (133%, P < .025) for 4 hr and increased the excretion of urine (300%, P < .001), sodium (194%, P < .025) and cyclic GMP (238%, P < .005). CGS 30440 (10 mg/kg p.o.) administered to hypertensive rats with aortic ligation between the renal arteries (mean arterial blood pressure, 209 +/- 4 mm Hg) produced a 48 mm Hg blood pressure reduction (P < .001) within 4 hr. CGS 30440 given to cynomolgus monkeys at 2 mg/kg inhibited plasma ACE activity by 96% within 1 hr (P < .001), and this inhibition was maintained for 7 and 21 days in monkeys receiving the compound orally at 2.5 mg/kg b.i.d. These studies demonstrate that CGS 30440 is an orally active agent which produces tissue ACE and NEP inhibition in rats and plasma ACE inhibition in primates and suggest that the compound may be useful in the treatment of hypertension and congestive heart failure.     

Characterization of the enzyme involved in the processing of big endothelin-1 in human lung epithelial cells

Biosynthesis of active endothelin-1 (ET-1) implies an enzymatic processing of the inactive precursor Big ET-1 (1-39) into the mature, 21 amino acid peptide. The aim of this study was to characterize in airway and alveolar epithelial cells the enzymes responsible for this activation. BEAS-2B and A549 cells, which both produce ET-1, were studied in vitro as models for bronchiolar and alveolar cells, respectively. Both cell lines were able to convert exogenously added Big ET-1 (0.1 microM) into ET-1, suggesting a cell surface or an extracellular processing. The conversion was inhibited by phosphoramidon in both cell lines with an IC50 approximately 1 microM, but not by thiorphan, a specific inhibitor of neutral endopeptidase 24.11 (NEP). The endogenous production of serum-stimulated BEAS-2B and A549 cells was not inhibited by thiorphan, and phosphoramidon showed inhibition only at high concentration (>100 microM). Western blotting following electrophoresis in reducing conditions demonstrated a protein of MR 110 corresponding to the ECE-1 monomer in both BEAS-2B and A549 cells, as well as in whole lung extracts. By RT-PCR we revealed the mRNA encoding for the ECE-1b and/or -1c subtype, but not ECE-1a, in both cell lines. We conclude that BEAS-2B and A549 cells are able to process either endogenous or exogenous Big ET-1 by ECE-1 and that isoforms 1b and 1c could be involved in this processing with no significant role of NEP.     

PGF2 alpha causes bronchoconstriction and pulmonary vasoconstriction via thromboxane receptors in rat lung

OBJECTIVE: The present study set out to determine general levels of distress, anxiety and depression in an obstetric-gynaecology inpatient population and to ascertain levels of patient satisfaction with services. METHOD: 200 English-speaking consecutive admissions to the hospital filled out a package of questionnaires consisting of a demographic data form, the 30-item General Health Questionnaire, the Speilberger State-Trait Anxiety Inventory, the Inventory to Diagnose Depression and a Patient Satisfaction with Services Questionnaire. 134 packages were fully completed. RESULTS: The most significant findings were: first, that high levels of anxiety prevailed, with approximately 1/3 of patients at or above the 75th percentile for anxiety levels; and second, that those patients born in non-English speaking countries were significantly more dissatisfied with their care. CONCLUSIONS: The results of this study suggest that there is a need for increased awareness of and further research into the psychological needs of obstetric-gynaecology patients.     

Renal and depressor activities of inhibitors of neutral endopeptidase and angiotensin converting enzyme in monkeys infused with angiotensin II

In a previous study, the depressor activity of combined selective inhibitors of neutral endopeptidase EC 3.4.24.11 (NEP) and angiotensin-converting enzyme (ACE) depended on the level of ACE inhibition, whereas the renal responses were determined by NEP inhibition. Our study confirmed that a mixed NEP/ACE inhibitor BMS-182657 ([S-(R*,R*)]-2,3,4,5-tetrahydro-3-[(2-mercapto-1-oxo-3- phenylpropyl)amino]-2-oxo-1H-benzazepine-1-acetic acid) reduced mean arterial pressure (MAP) when renin release was reduced by a sodium load, suggesting that the depressor response did not require suppression of endogenous angiotensin II generation. Furthermore, a pressor dose of 30 ng/min of angiotensin II was required to block the depressor response to BMS-182657 in the presence or absence of exogenous human atrial natriuretic peptide (hANP 99-126). Thirty ng/min of angiotensin II also significantly enhanced the natriuresis induced by hANP 99-126 after BMS-182657 administration. In contrast, a nonpressor dose of angiotensin II (3 ng/min) reduced basal sodium excretion and the natriuretic responses to exogenous hANP 99-126 in the presence or absence of BMS-182657. The potentiation of the urinary ANP and cyclic guanosine monophosphate (cGMP) responses to hANP 99-126 by BMS-182657 was similar for all doses of angiotensin II; therefore angiotensin did not alter the effects of BMS-182657 on ANP metabolism or cGMP accumulation in the kidney. In summary, the renal responses to mixed metalloprotease inhibitors were apparently mediated by ANP potentiation and were modulated by angiotensin II. The depressor activity depended on ACE inhibition but was not mediated solely by reductions in endogenous angiotensin II levels.     

Neutral endopeptidase inhibition increases the potency of ANP in isolated rat pulmonary resistance vessels and isolated blood perfused lungs

We have investigated the effects of the neutral endopeptidase inhibitor, SCH 42354, on the vasoreactivity of atrial natriuretic peptide (ANP) in rat isolated pulmonary resistance vessels (PRV) and isolated perfused lungs (IPL). PRV (n = 37) were mounted onto the jaws of a myograph and precontracted with PGF2alpha (100 mu M). Concentration-responses to ANP (0.17 to 340 nM) were determined before and after the addition of SCH 42354 (10, 30 and 100 nM). Each concentration of SCH 42354 caused a significant increase in potency (- log EC50) of ANP in isolated PRV. Lungs from normoxic rats (n = 13) were isolated and perfused with whole blood. An increase in pulmonary artery pressure was achieved by ventilating with an hypoxic gas mixture and concentration-responses obtained by incremental additions of ANP (40 nM to 12 mu M), before and after the addition of SCH 42354 (100 nM). SCH 42354 significantly increased the potency (- log EC50) of ANP in the rat IPL. ANP is partly metabolized by NEP. That an inhibitor of NEP increased the potency of ANP in isolated pulmonary vessels, and in isolated perfused whole lungs, suggested that SCH 42354 may be having a local action within the pulmonary vasculature.     

Comparison of the effects of nicorandil, pinacidil and nitroglycerin on hypoxic and hypercapnic pulmonary vasoconstriction in the isolated perfused lung of rat

Viral genome DNA from four different multiple-enveloped nuclear polyhedrosis virus isolates, obtained from naturally infected larvae of satin moth (Stilpnotia salicis), a pest of poplar tree (Populus) was analysed. Larvae were collected over a period of 11 years, from 1978 to 1989. The genomic DNA restriction patterns pointed to heterogeneity of these wild-type viruses. The differences observed in isolates of several years revealed limited restriction fragment length polymorphism and showed that these viruses contained distinct, but closely related genotypes. The genome size of SsMNPV was established as 128-134 kb, based on HindIII and SacI restriction analysis.     

Platelets enhance contractility in perfused rat mesenteric arteries: involvement of endothelin-1

We investigated the effects of platelet supernatant on pressor responses to norepinephrine in isolated perfused rat mesenteric arteries. Perfusion of the arteries with platelet supernatant for 2 h markedly enhanced the pressor responses to norepinephrine (10(-6) and 3 x 10(-6) M). This enhancement was significantly inhibited by phosphoramidon (10(-4) M), an endothelin converting enzyme inhibitor. Both BQ788 [N-cis-2,6-dimethylpiperidinocarbonyl-L-gamma-methylleucyl-D -1-methoxycarbonyltryptophanyl-D-norleucine] (10(-6) M), an endothelin ET(B) receptor antagonist, and bosentan (Ro47-0203, 4-tert-butyl-N-[6-(2-hydroxy-ethoxy)-5-(2-methoxy-phenoxy)-2,2-bipyri midin-4-yl]-benzenesulfonamide) (10(-5) M), a nonselective endothelin receptor antagonist, also prevented the potentiation of responses to norepinephrine evoked by platelet supernatant, but FR 139317 ((R)2-[(R)-2-[(S)-2-[[1-(hexahydro-1H-azepinyl)]carbonyl]amino-4-+ ++methylpentanoyl] amino-3-[3-(1-methyl-1H-indoyl)]propionyl]amino-3-(2-pyridyl) propionic acid) (10-6 M), an endothelin ET(A) receptor antagonist, had little effect. Suppressor doses of endothelin-1 (3 x 10(-10) M) or sarafotoxin S6c (S6c) (3 x 10(-10) M) potentiated significantly the norepinephrine-induced vasoconstriction, in the same preparation. Moreover, supernatant-induced enhancement of pressor responses to norepinephrine was markedly suppressed by TGF-beta1 neutralizing antibody. Transforming growth factor-beta1 (TGF-beta1) (40 pM) also significantly enhanced the pressor responses to norepinephrine (10(-6) M) and this enhancement was significantly inhibited by phosphoramidon. These results suggest that platelet-derived TGF-beta1 stimulates the vascular production of endothelin-1 and thereby enhances vasoconstrictor responses to norepinephrine. Platelet-induced enhancement of vasoconstrictor responses to norepinephrine seems to be mainly mediated by endothelin ET(B) receptor, in rat mesenteric arteries.     

Characterisation of the processing by human neutral endopeptidase 24.11 of GLP-1(7-36) amide and comparison of the substrate specificity of the enzyme for other glucagon-like peptides

The post-secretory processing of the potent insulinotropic peptide hormone, GLP-1(7-36)amide, probably involves one or more of a small group of membrane-bound ectopeptidases. Reported here, is the characterisation of the endoproteolysis of human GLP-1(7-36)amide by the recombinant human form of neutral endopeptidase (NEP) 24.11, which is one of the best characterised and widely-distributed of ectopeptidases and is involved in the processing of other peptide hormones. The products of the limited endoproteolysis were characterised by mass and primary structure following fractionation using high performance liquid chromatography. The rate of this endoproteolysis by NEP 24.11 was estimated and compared to that of GLP-1(7-36)amide-related peptides. GLP-1(7-36)amide appears to be good substrate for NEP 24.11 with most, but not all potential target bonds being cleaved. Also, the structurally-related peptides, secretin and glucagon appear to be good substrates whereas GIP and exendin-4 are very poor substrates. That the GLP-1(7-36)amide super-agonist, exendin-4 is a poor substrate for NEP 24.11 is significant for the possible use of this peptide as a prototype for the development of clinically-useful peptide agonists. Further studies should reveal whether NEP 24.11 is important for the metabolic clearance of GLP-1(7-36)amide and will be highly relevant for the attempts to realise the suggested therapeutic value of GLP-1(7-36)amide.     

Characterization of methylumbilliferone (mendiaxon r)-induced choleresis in the isolated perfused rat liver

The influence of the choleretic drug methylumbilliferone on bile formation in the isolated perfused rat liver is characterized. The compound induces rapidly an elevation of bile flow, bile acid secretion and soium excretion. The increased production of bile is of canalicular origin. The choleretic effect was defined as "bile acid like" choleresis due to excretion of the drug into the bile. It is discussed that the excretion of methylumbilliferone can influence the transport of bile in form of a positive cooperation on transport mechanism.     

Comparable potent coronary constrictor effects of endothelin-1 and big endothelin-1 in humans

BACKGROUND: Endothelin-1 (ET-1) is a potent vasoconstrictor produced from the precursor big ET-1 in endothelial cells. The coronary effects of these peptides in humans in vivo are unknown. Therefore, the effects of ET-1 and big ET-1 on coronary blood flow in relation to plasma ET-1 and big ET-1 levels were compared in healthy subjects. METHODS AND RESULTS: The peptides were infused intravenously at the rates of 0.2, 1, and 8 pmol/kg per minute. Each dose administered for 20 minutes except the highest dose of ET-1, which was administered for 10 minutes. ET-1 and big ET-1 evoked dose-related increases in mean arterial blood pressure from 93 +/- 4 to 107 +/- 4 mm Hg and from 89 +/- 2 to 122 +/- 5 mm Hg, respectively, at the highest dose. ET-1 and big ET-1 reduced coronary sinus blood flow, measured with thermodilution by a maximum of 25 +/- 4% and 28 +/- 8% and increased coronary vascular resistance by 50 +/- 9% and 107 +/- 26%, respectively. Coronary sinus, but not arterial, oxygen saturation was reduced in parallel with the coronary sinus blood flow. The effects of ET-1 and big ET-1 were similar at corresponding time points. During infusion of ET-1, a 19 +/- 5% extraction of ET-1 was observed over the coronary vascular bed (P < .05). Administration of big ET-1 elevated arterial plasma ET-1 levels by 2.4-fold, and after correction for the local extraction of ET-1, a myocardial production of ET-1 was observed. CONCLUSIONS: ET-1 and big ET-1 induce comparable increases in blood pressure and coronary constriction in humans in vivo. The results also suggest a net local removal of circulating ET-1 and big ET-1 and a local conversion of big ET-1 into ET-1 within the coronary vascular bed.     

Effects of nimodipine and isradipine on endothelin-1-induced contraction of pregnant rat myometrium

Bladder malignancy in the renal transplant recipient is an infrequent occurrence. The 11 previously reported cases reflect an aggressive tumor growth with invasion, requiring partial or complete cystectomy with or without conduit diversion. We report an additional case in a 40-yr-old woman with a living related renal transplant, who experienced rapid progression of her tumor over 3 wk from initial hematuria to a pelvic mass involving the anterior bladder. Her allograft ureter and native ureters, as well as her left iliac vein, became obstructed with tumor in another 2 wk. Biopsy showed poorly differentiated, invasive transitional carcinoma. Attempted resection was abandoned because of finding tumor involvement in most of the pelvis. Chemotherapy was not attempted. She died 2 wk after her attempted resection from tumor burden. Our report presents a collective review of these previously reported 11 cases plus our case. These bladder tumors demonstrate a rapid progression of invasive disease and respond poorly to chemotherapy. There is a possible association of bladder tumors with cyclophosphamide immunosuppression. An aggressive surgical approach should be followed, especially since these tumors present in a younger age group.