Biomedical subjects
Publications and source records attributed to R Corder.
A molecular model has been developed for human Big Endothelin-1, which is the immediate precursor to the potent vasoconstrictor polypeptide endothelin-1 and the target of the highly specific endothelin converting enzyme. This model is produced by a threading algorithm protocol and is consistent with all the currently available structural and biochemical data for this molecule.
The importance of big endothelin-1 (big ET-1) retaining a specific conformation for its conversion to ET-1 has yet to be determined. As a prelude to developing affinity labels for studying the interaction between big ET-1 and endothelin converting enzyme (ECE), the effect on biological activity of modifying human big ET-1 with the N-hydroxysuccinimide esters of 3-(p-hydroxyphenyl)propionic acid (HPP) or S-acetylthioglycolic acid (ATG) was investigated. Mono-derivatized HPP-big-ET-1 and ATG-big-ET-1, and the corresponding ET-1 molecules, were purified by HPLC. The identity of the modified big ET-1 and ET-1 molecules were confirmed by mass spectrometry. Comparison of the pressor activities with big ET-1 (1 nmol/kg) in anaesthetized rats showed the responses to equivalent doses of HPP-big-ET-1 and ATG-big-ET-1 to be reduced by 67% and 73%, respectively. In contrast, the same modifications to ET-1 had no significant effect on blood pressure responses or vasoconstrictor activity on the isolated rat thoracic aorta. To evaluate the effect of these modifications on the conversion of big ET-1 to ET-1, cultured bovine aortic smooth muscle (BASMC) and endothelial (BAEC) cells were used as sources of endothelin converting enzyme activity. After a 4-hr incubation of the modified molecules with intact cells, the quantity of ET-1 immunoreactivity generated was compared to that from unmodified big ET-1. The amount of conversion, relative to big ET-1 (1 microM), for HPP-big-ET-1 was reduced by 21% for BAEC and by 50% for BASMC. The corresponding decreases for ATG-big-ET-1 were 79% and 82%. Because of the large decreases in the level of conversion, the linear big ET-1 molecule S-carboxy-amidomethylated big ET-1 (CM-big-ET-1) was prepared for comparison. Incubations of CM-big-ET-1 with BAEC and BASMC yielded only 53% and 23%, respectively, of the ET-1 immunoreactivity obtained with unmodified big ET-1. Thus, incorporation of the HPP or ATG groups, or removal of disulphide bridges decreases the ability of plasma membrane ectoenzyme ECE activities to hydrolyze the Trp21-Val22 bond of big ET-1. This indicates that the conformation of big ET-1 is important for obtaining an optimal rate of hydrolysis by ECE activities in vivo and in vitro. Further evidence of secondary structure was obtained from studies of the crossreactivity of big ET-1 in two RIAs recognising the ET-1 sequence.
Explore the source record for details and available documents.
Vesicles containing endothelin 1 (ET-1) were isolated from bovine aortic endothelial cells (BAECs) by fractionation of homogenates on sucrose density gradients by ultracentrifugation. The vesicles were localized at the 1.0/1.2 M sucrose interface using a specific anti-ET-1-(16-21) RIA. Identification of ET-1 and big ET-1 in this fraction was confirmed by HPLC analysis combined with RIA. Morphological examination of the ET-1-enriched fraction by electron microscopy identified clusters of vesicles approximately 100 nm in diameter. Immunostaining of ultrathin cryosections prepared from the vesicle fraction for ET-1 or big ET-1 showed clusters of 15-nm gold particles attached to or within vesicles. Immunofluorescence staining of whole BAECs using a specific ET-1-(16-21) IgG purified by affinity chromatography revealed punctate granulation of the cell cytoplasm viewed under light microscopy. This distinct pattern of staining was shown by confocal light microscopy to be intracellular. Immunofluorescence staining of whole cells with a polyclonal antiserum for big ET-1-(22-39) showed a defined perinuclear localization of precursor molecule. Hence, several different approaches have demonstrated that ET-1 and big ET-1 are localized within intracellular vesicles in BAECs, suggesting that these subcellular compartments are an important site for processing of big ET-1 by endothelin-converting enzyme.
Treatment of rats with cytokines has been associated with an increase in the circulating levels of endothelin 1 (ET-1). Here we show that administration of tumor necrosis factor alpha (TNF-alpha; 4 micrograms.kg-1) to anesthetized rats caused within 15 min a strong elevation in the circulating levels of ET-1. This was associated with a striking coronary vasoconstriction in hearts from these animals when they were removed and perfused in vitro by the Langendorff technique. This vasoconstriction was largely overcome by treatment with either the endothelin type A (ETA) receptor antagonist FR 139317 or antibody against ET-1. Furthermore, it was mimicked by in vivo exposure to exogenous ET-1. Endogenously produced TNF-alpha may also cause such a coronary vasoconstriction, for treatment with interleukin 2 (600 micrograms.kg-1) produced an increase in coronary perfusion pressure that correlated with the increases in circulating TNF-alpha. This coronary vasoconstriction was substantially reversed by treatment either with antibody against TNF-alpha or with FR 139317. We suggest, therefore, that cytokine-driven changes in the production of ET-1 are key events in the development of vascular pathologies.
Subcellular fractionation of the phosphoramidon sensitive membrane-bound endothelin converting enzyme (ECE-1) activity from homogenates of bovine aortic endothelial cells and the human endothelial cell line EA.hy 926, combined with studies of intact cells, shows ECE-1 to be localised primarily to the plasma membrane with the topology of an ectoenzyme. To overcome the problem of neutral endopeptidase 24.11 contaminating the human ECE-1 activity solubilised from the plasma membrane fractions of EA.hy 926, we have used isoelectric focusing to simultaneously solubilise and separate these activities. The metallopeptidase ECE-1 obtained displayed a neutral pH optimum, a molecular weight of 250 kDa on gel filtration chromatography and was inhibited by phosphoramidon with an IC50 of 0.8 microM.
Compared with endothelin-1 (ET-1), big endothelin-1 (big ET-1) is only weakly active on isolated vascular smooth muscle preparations. However, on systemic administration high doses of big ET-1 (1 nmol.kg-1) are approximately equipotent to ET-1, indicating the existence of an endothelin converting enzyme in the circulation that rapidly converts big ET-1 to ET-1. In this study arterial blood levels of big ET-1 and ET-1 immunoreactivity were measured after bolus i.v. administration of big ET-1 (1 or 3 nmol.kg-1) or ET-1 (1 nmol.kg-1) in anaesthetised male Wistar rats. In addition, the effect of phosphoramidon (10 mg.kg-1) on the pressor response to big ET-1 and its disappearance rate from the circulation were examined. After big ET-1 injection, circulating ET-1 concentrations did not exceed 2% of the big ET-1 level. Phosphoramidon reduced the pressor response to big ET-1 by 93%, but did not alter its rate of clearance from the circulation. Thus exogenous big ET-1 is converted locally in the vasculature and its disappearance from the circulation is not dependent on conversion to ET-1.
1. In vivo the effects of endothelin-1 (ET-1) are limited by its rapid removal from the circulation and possibly by its metabolism by enzymes such as neutral endopeptidase 24.11, deamidase or carboxypeptidase A. Here, using as a model the isolated perfused mesenteric arterial bed of the rat, we have examined the involvements of these enzymatic activities in the vascular responses to ET-1. 2. Samples of Krebs buffer which had been recirculated through the mesenteric arterial bed for 30 min rapidly destroyed the activity of ET-1 as assessed either by bioassay on rings of rat thoracic aorta or by high performance liquid chromatography (h.p.l.c.). For instance, after 15 min incubation with the recirculated-Krebs solution (recirc-K) the contraction induced by 3 x 10(-9) M ET-1 was reduced by more than 90%. Contractions induced by sarafotoxin 6b (3 x 10(-9) M) were similarly suppressed by preincubation with recirc-K whereas those to Arg-vasopressin (3 x 10(-9) M) were unaffected. 3. The degradation of ET-1 by recirc-K was prevented by 1,10-phenanthroline (10(-3) M), abolished by heating the recirc-K solution to 90 degrees C for 15 min, and reduced by EGTA (5 x 10(-3) M) or ET-1(16-21) (10(-5) M). For instance, in the presence of ET-1(16-21) (n = 6) the contraction induced by ET-1 was reduced by only 40% after 15 min incubation with recirc-K buffer. Leupeptin (3 x 10-4 M), dichloroisocoumarin(5 x 10-5 M), phenylmethyl-sulphonyl fluoride (10-3 M), a combination of bacitracin (300 mg ml-1),bestatin (10-5 M), captopril (10-5 M), phosphoramidon (10-4 M) and thiorphan (10-4 M) or Polypep (aproprietary protein digest) did not inhibit the degradation of ET-1 by recirc-K.4. In experiments examining directly the vascular responses of the isolated perfused mesentery of the rat, the addition of cumulative concentrations of ET-1 to the recirculating Krebs solution caused small concentration-dependent increases in perfusion pressure. The inclusion of ET-1(16-2l), ET-1(17-21), or ET-1(18-21) (10-5M) greatly potentiated these responses, but not those to Arg-vasopressin or methoxamine.The effects of 1,10-phenanthroline or EGTA could not be examined in this system because these agents both depressed non-specifically the vasoconstrictor responses of the mesenteric vascular bed.5. Thus, the rat mesentery releases an enzyme that very rapidly destroys ET-1 or the very closely related peptide, sarafotoxin 6b but not Arg-vasopressin. This enzyme is most probably a metallopeptidase because of its sensitivity to inhibition by 1,10-phenanthroline or EGTA. It is particularly interesting that a simple vascular bed such as the mesentery produces such a powerful endothelin metabolising enzyme. It is tempting, therefore, to speculate that the endothelin degrading enzyme active at neutral pH that- we have found is important in the metabolism of ET-1 throughout the vasculature.
A single addition of 3 x I0-7 M ET-1, ET-2 or ET-3 produced contractions that reached a steady state in 28.2 +/- 4.2, 21.1 +/- 1.3 and 24.0 +/- 3.8 min, respectively and took 2.7 +/- 0.4, 2.1 + 0.1 and 1.6 +/- 0.1 min to reach half of this steady-state response.4. Contractions induced by 3 x I0-7 M big ET-11-38 or big ET-11- 39 reached a plateau in 38.5 +/- 3.6 and 35.6 +/- 3.3 min, respectively, and half of these responses were attained in 12.0 +/- 2.5 and 7.1 +/- 1.1 min.Thus, these contractions developed more slowly than those induced by ET-1. Contractions induced by 3 x 10-7 M big ET-21-38 were also much slower to develop than those to ET-2, for these took 49 +/- 2 min to reach plateau and 19.4 +/- 2.1 min to attain half that response. Contractions induced by 3 x 10-7 M big ET-31-41 amide took 50.2 +/- 3.7 min to reach a plateau and 27.3 +/- 3.0 min to reach half of this response.5. Phosphoramidon (0.1, 1 and 3 x 10-4 M) inhibited contractions induced by big ET-11.39. For instance,the contractions induced by 3 x 10-7 M big ET-11-39 were inhibited by 10-4 M or 3 x 10-4 M of phosphoramidon by 62.8 +/- 6.7% or 74.5 +/- 4.6%, respectively. Similarly, contractions induced by ET-21-38 were inhibited by 91.3 +/- 5.4% and the small response induced by big ET-3l-4l amide was abolished by 3 x 10-4M phosphoramidon. Conversely, the neutral endopeptidase (EC 24.11) inhibitor DL-thiorphan(3 x 10-4 M) had no effect. Captopril (10-5 M), pepstatin A (10-5 M), phenylmethylsulphonylfluoride(PMSF, 10-3 M), aprotinin (10-5 M), E-64 (10-5 M), cystatin (10-6 M), leupeptin (10-4 M),chymostatin (10-4 M), or bestatin (10-5 M) did not inhibit but rather increased to a similar, but small degree the contractions induced by 3 to 30 x 10-9 M big ET-11-39. Only captopril (10-5 M) or leupeptin(10-4 M) increased the contraction induced by 3 x 10-7 M big ET-11-39. Phosphoramidon (10-4 M),pepstatin (10-5 M) or PMSF (10-3 M) did not affect contractions induced by ET-1.6. Removal of the epithelium increased by 70% the size of the contraction induced by 5 microM histamine(1.08 +/- 0.05 g; n = 160 to 1.84 +/- 0.14 g; n = 12) but did not affect, in absolute terms, the contraction induced by ET-1 (as a % of the response to histamine, these responses were, of course, apparently depressed). Epithelium removal did, however, increase the size of the contractions induced by 3 to 30 x 10-9 M big ET-1 -39 which was very similar to the effect of the protease inhibitors.7. In competition binding studies on membranes prepared from the guinea-pig gallbladder, 10-11 MET-1 inhibited by 76.9 +/- 3.1% the binding of [125]-ET-I while porcine big ET-11-39 caused no inhibition(0.7 +/- 3.0; n = 3). ET-1 (10-6 M) inhibited binding by 95.7 =/- 1.1% (n = 3) while at this much higher concentration, big ET-11-39 inhibited binding by only 16.8 +/- 4.2% (n = 3). This clearly suggests that big ET-11-39 does not bind directly to ET receptors.8. Thus, a phosphoramidon-sensitive endothelin-converting enzyme (ECE), different from neutral endopeptidase (NEP; EC 24.11) and not located on the epithelium, converts big ET-1 into ET-1 in the gallbladder of the guinea-pig. This ECE appears to act preferentially on big ET-1 or big ET-2 over bigET-3.
Direct immunoassay of plasma corticotrophin-releasing hormone (CRH) is potentially subject to interference from high levels of CRH-binding protein (CRH-BP) that exist in the human circulation. In this study, we tested the effect of CRH-free, native CRH-BP (6.4 nmol/l) purified from human plasma, CRH-BP diluent alone, normal human plasma (containing 5.8 nmol endogenous CRH-BP/l) and normal sheep plasma (containing no CRH-BP) on the binding of 125I-labelled CRH tracer to five N-terminal and four C-terminal CRH antibodies. All anti-(1-20)CRH N-terminal antibody dilution curves displayed marked inhibition of binding in the presence of purified CRH-BP and human plasma in comparison with the curves with the control diluent or sheep plasma. Almost no inhibition of binding was obtained with any of the C-terminal antibodies (all directed against epitopes within the last six amino acids of CRH) and the four dilution curves were nearly superimposable. Liquid-phase CRH IRMAs were then developed with different combinations of two of each of the N- and C-terminal antibodies, using radiolabelled IgG prepared from purified C-terminal antisera as tracer and raw N-terminal antisera as the link antibodies to the separating system. The addition of dilutions of purified CRH-BP over the range 1.25-20 nmol/l to the IRMA standard curve in assay buffer resulted in a dose-dependent reduction in the signal; with 5 nmol CRH-BP/l, a level commonly found in human plasma, the reduction in binding was 67% and 81% in two different IRMAs at a CRH concentration of 631 pmol/l.(ABSTRACT TRUNCATED AT 250 WORDS)
In many pathologic conditions, elevations in the circulating level of endothelin-1 (ET-1) are linked to increased production of cytokines. Therefore, we have investigated the relationship between cytokines and ET-1. Administration of tumor necrosis factor-alpha (TNF-alpha, 4 micrograms/kg) to anesthetized rats caused, within 15 min, a marked elevation in the circulating plasma level of ET-1. This was associated with pronounced coronary vasoconstriction in hearts removed from these animals and perfused in vitro by the Langendorff technique. This coronary vasoconstriction was attenuated by in vivo pretreatment with either ETA receptor antagonists or with an antibody against ET-1 (ET-Ab). Treatment of the anesthetized rats with interleukin-2 (IL-2) to increase the endogenous release of TNF-alpha also elevated the in vitro coronary vascular resistance, an effect that was greatly reduced by administration of an antibody against TNF-alpha (hr-TNF-alpha-Ab) or FR 139317. Finally, in rats with adjuvant-induced arthritis, in which the levels of circulating cytokines are greatly elevated, we also found large increases in the ex vivo coronary perfusion pressure. Significantly, these were abolished by in vivo treatment for 2 days with the nonpeptide ET receptor antagonist SB209670. Cytokines cause marked and acute changes in the production of ET-1, leading to pronounced coronary vasoconstriction. Cytokine-driven changes in the production of ET-1 may therefore be key events in the development of vascular pathologies.
Elevated plasma levels of endothelin-1 (ET-1) in patients with septic shock have implicated ET-1 in the vascular hypoperfusion and organ dysfunction, including renal failure, that occur in this condition. Administration of endotoxin to rats induces renal dysfunction characterized by increased blood urea nitrogen (BUN) and plasma creatinine (Cr) levels. Here, we have found that neither SB 209670 (an ETA and ETB receptor antagonist) nor an affinity-purified ET antibody blocked the endotoxin-induced changes in renal function, suggesting that ET-1 is not involved in the pathophysiology of endotoxin-induced renal dysfunction.
The action of cytokines on the endothelium is a pivotal event in a number of vascular pathologies and inflammatory conditions. Here we have tested the effect of tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-1 beta, and IL-2 on endothelial-1 (ET-1) release from cultured bovine aortic endothelial cells. TNF-alpha and IL-1 beta caused concentration-dependent increases in ET-1 release. IL-2 up to concentrations of 100 ng/ml did not stimulate ET-1 release. The induction of ET-1 synthesis by cytokines may be of underlying importance in the endothelial cell dysfunction observed in a number of vascular diseases.
Explore the source record for details and available documents.
The antagonism by PD 145065 (Ac-D-Bhg-L-Leu-L-Asp-L-Ile-L-Ile-L-Trp; D-Bhg = 5H-dibenzyl[a,d]cycloheptene-10,11-dihydroglycine), a non-selective endothelin ETA and ETB receptor antagonist, of the pressor and renal haemodynamic responses to an infusion of endothelin-1 was investigated in the anaesthetised rat. Infusion of endothelin-1 at 2 or 4 pmol/min/rat for 2 h produced a significant increase in mean arterial blood pressure and renal vascular resistance. Glomerular filtration rate was reduced by both infusion rates of endothelin-1 (-34% for the lower rate and -72% for the higher rate), but only the high rate significantly reduced renal blood flow (-64%). These effects of endothelin-1 were completely blocked by infusion of PD 145065 (0.05 mg/min/rat), demonstrating the efficacy of this antagonist in preventing the actions of endothelin-1 in vivo. These results lend support to an involvement of endothelin ETB receptors in the renal effects of endothelin-1.
The effects were examined of mechanical stretch on the release of endothelin-1 (ET-1) and prostacyclin (measured as 6-keto-prostaglandin (PG) F1 alpha) from cultured endothelial cells. Stretching (0.2 Hz) for 20, 60 or 360 min caused immediate (< or = 20 min) and secondary (up to 360 min) increases in ET-1 release. The secondary but not immediate release of ET-1 was prevented by actinomycin D (8 x 10(-7) M) or cycloheximide (3.6 x 10(-6) M). Neither compound affected the release of ET-1 from unstretched cells over 360 min. Stretching of the endothelial cells increased the accumulation of 6-keto-PGF1 alpha at 360 min but not at 20 min, suggesting that stretch does not produce a rapid, non-selective increase in autacoid production from endothelial cells. The intracellular amounts of ET-1 were approximately 20 times greater than those of big ET-1. Thus, endothelial cells contain stores of ET-1 that are released rapidly by stretch.
1. To characterize the receptor subtype(s) mediating the renal vasoconstrictor effects of the endothelin (ET) and sarafotoxin (SX) peptides in the isolated perfused kidney of the rat, we have examined the effects of endothelin-1 (ET-1), sarafotoxin 6b (SX6b) and sarafotoxin 6c (SX6c) as agonists, BQ-123 and FR 139317 as selective ETA receptor antagonists, and PD 145065 as a non-selective (ETA and ETB) receptor antagonist. We have also compared in the anaesthetized rat the systemic pressor and renal vasoconstrictor effects of ET-1 and SX6c alone or after pretreatment with PD 145065. 2. In the isolated perfused kidney, ET-1, SX6b and SX6c all gave similar concentration-dependent increases in perfusion pressure. The ETA receptor selective antagonists, BQ-123 and FR 139317, both partially blocked the increase in perfusion pressure induced by ET-1. In contrast, PD 145065 completely blocked the increase in perfusion pressure caused by ET-1. 3. Indomethacin (10 microM) had no effect on the ET-1-induced increases in perfusion pressure but significantly reduced the vasoconstriction induced by low concentrations of SX6c, without affecting responses to high concentrations. In the anaesthetized rat, indomethacin (5 mg kg-1) did not modify the systemic pressor or renal vasoconstrictor effects of ET-1 or SX6c. 4. In anaesthetized rats, bolus intravenous injections of ET-1 or SX6c (0.1, 0.25, 0.5 or 1.0 nmol kg-1) produced initial transient depressor responses followed by sustained and dose-dependent increases in mean arterial pressure (MAP). Both peptides caused an equipotent fall in renal blood flow (RBF).PD 145065 (5 mg kg-1) partially antagonized the systemic pressor effects of ET-1 and SX6c but completely blocked the fall in RBF and rise in renal vascular resistance (RVR) induced by ET-1 and SX6c. PD 145065 also antagonized the transient depressor effect following the bolus administration of either ET-1 or SX6c.5. These results indicate that ET/SX induced renal vasoconstriction is mediated via ETA and ETB-like receptors with ETB receptors having a predominant role in vivo. This may be of therapeutic relevance for an ETA receptor-selective antagonist may offer only limited protection against the deleterious renal effects of endogenous ETs.
1. Phosphoramidon, a potent inhibitor of endopeptidase-24.11 (E-24.11) and thermolysin, has been shown to reduce the hypertensive effect of exogenous big endothelin-1 (big ET-1) in rats. To examine whether E-24.11 or thermolysin convert big ET-1 to endothelin-1 (ET-1) and C-terminal fragment (CTF), the effects on porcine and human big ET-1 of each of the purified enzymes were compared in vitro. 2. For E-24.11, the relative rates of hydrolysis were ET-1 > CTF >> big ET-1. The relative half-lives for hydrolysis of 3 nmol of each peptide by 200 ng enzyme were: big ET-1 > 24 h; ET-1, 37 min; CTF, 57 min. For comparison, the half-life for hydrolysis of substance P under similar conditions was 2.1 min. 3. For thermolysin the relative rates of hydrolysis were found to be big ET-1 > CTF > ET-1. The relative half-lives for hydrolysis of 3 nmol peptide by 50 ng enzyme were: big ET-1, 25 min; ET-1, 56 min; CTF, 47 min. 4. Because the low rate of conversion of big ET-1 to ET-1 by E-24.11 did not yield sufficient ET-1 for h.p.l.c. quantification a RIA specific for ET-1(16-21) was used to study further the hydrolysis of big ET-1 by E-24.11. Incubation of big ET-1 (0.2-2 nmol) with E-24.11 (4-400 ng) generated ET-1 levels of between 1.7 and 33 pmol measured by RIA. Incubation of big ET-1 (2 nmol) with E-24.11 (40 ng) for 8 h showed that steady state levels of ET-1 were achieved after 4 h indicating that the rate of ET-1 degradation was then equal to the formation of new ET-1. Characterization of the immunoreactivity by h.p.l.c. and RIA confirmed that authentic ET-1 had been produced, but the yield was insufficient for verification by mass spectrometry.5. Both ET-l-like and CTF-like peaks were detected at 214 nm when the products of big ET-1 hydrolysis by thermolysin were resolved by h.p.l.c. RIA and mass spectrometry confirmed the production of ET-1 with amounts in the range 120-160 pmol.6. The hydrolysis profile of ET-1 by E-24.11 and thermolysin shows that both enzymes have some common cleavage sites consistent with their similar specificities hydrolysing on the amino side of a hydrophobic residue.7. Thermolysin, for which 3D structural information is available, may represent a better model for endothelin converting enzyme (ECE) action than E-24.11 and could be useful for the design of ECE inhibitors. Since E-24.11 can both synthesize and hydrolyse ET-1, the presence of E-24.11 in membrane fractions or in partially purified ECE preparations may produce misleading estimates of ECE activity.