Search

Endothelin A and B receptors, together with sodium-glucose cotransporter-2 (SGLT-2) channels are important targets in improving endothelial function and intervention with inhibitors has been the subject of multiple mechanistic and clinical outcome trials over recent years. Notable successes include the treatment of pulmonary hypertension with endothelin receptor antagonists, and the treatment of heart failure and chronic kidney disease with SGLT-2 inhibitors. With distinct and complementary mechanisms, in this review, we explore the logic of combination therapy for a number of diseases which have endothelial dysfunction at their heart., (© 2024 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Hypertension is a major risk factor in the development of cardiovascular disease. Adenovirus gene transfer of endothelin-1 (Ad.CMV.ET-1) in rats produced significant (5-fold) increases in plasma ET-1 and systemic blood pressure (46%) 4 days after viral administration, compared with beta-galactosidase (Ad.CMV.beta-gal) injected as control. The density (B(max)) of the ET receptor ET(A) measured in aortas was reduced significantly by more than 50% to 17+/-2 fmol.mg(-1) of protein for the Ad.CMV.ET-1 group compared with 39+/-6 fmol x mg(-1) of protein for the control. There was no change in the density of the smaller population of the ET(B) sub-type. In agreement, the ratio of ET(A) mRNA to cyclophilin mRNA (a housekeeping gene) measured by Northern analysis was reduced in Ad.CMV.ET-1 rats compared with controls. The ratio of mRNA encoding the ET(B) sub-type did not change. ET-1 vasoconstriction was significantly reduced (P<0.05) in aortas from Ad.CMV.ET-1-treated rats [pD(2)=8.67+/-0.14 (where pD(2) is -log(10)EC(50)); n=11] versus the control (pD(2)=9.11+/-0.06; n=14) but there was no significant difference in the potency of two other vasoconstrictors tested (noradrenaline and Arg-vasopressin), indicating this was a specific effect on ET receptors. There was no change in the affinity of ET-1 binding to either receptor sub-type in the experimental group compared with the control, demonstrating that the attenuation in the constrictor response is the result of the reduced density of receptors rather than a change in affinity. The results show that ET(A) (but not ET(B)) receptors are modulated in this experimental model of hypertension and provide further evidence for selective blockade of the ET(A) receptor as a therapeutic strategy.

Plasma endothelin (ET) is elevated in patients with vasospasm following subarachnoid haemorrhage (SAH). However, systemic levels provide no indication regarding local production in the brain, and late elevation may be a consequence rather than a cause of vasospasm. We measured arteriojugular (AJ) gradients of ET-1 in 17 patients over the first week after SAH, and related these to the subsequent development of vasospasm. Daily, paired arterial and jugular bulb blood samples were obtained up to seven days post SAH, and assayed for ET-1 using radioimmunoassay. Systemic levels and AJ gradients were compared in patients with and without vasospasm. Significant AJ gradients were observed for ET-1 (P<0.01). These differences remained significant in the subgroup of patients who developed vasospasm (0.12+/-0.05 pmol/l; P<0.05), in whom AJ gradients represented 25+/-7% of systemic levels (0.84+/-0.05 pmol/l). AJ gradients did not reach significance in patients who did not develop vasospasm (0.09+/-0.07 pmol/l; P=0.2). Systemic ET-1 levels and AJ gradients were unrelated to SAH grade, surgical or endovascular interventions, or extracranial complications. AJ gradients in the first week following SAH suggest early production of ET-1 in the cerebrovascular bed. However, early systemic ET-1 levels did not discriminate between patients with and without vasospasm. Larger AJ differences may predict vasospasm, but further work is needed to confirm this observation. AJ gradient measurement may provide a useful technique for investigating the role of other peptides in acute brain injury.

Positron emission tomography (PET) is a powerful technique with the sensitivity to image and quantify receptor-bound radioligands in vivo. Recent progress in PET scanner technology has resulted in the development of dedicated tomographs designed for small animals, with resolution that allows the delineation of discrete organs and their larger substructures in rats and mice. Our aim was to determine whether endothelin-1 (ET-1) could be labelled with (18)F, and whether the resulting (18)F-ET-1 would have the required pharmacokinetic properties to permit binding and imaging of ET receptors in vivo. (18)F-ET-1 could be produced in a total radiochemical yield of 5.9+/-0.7% in 207+/-3 min (n=20). Specific radioactivities were in the range 220-370 GBq/micromol, and the radiochemical purity of the isolated (18)F-ET-1 was >95%. In vivo distribution in the rat was studied using microPET. High levels of (18)F-ET-1 uptake were found in lung and kidney, whereas liver showed moderate levels of uptake. The resolution of the microPET scanner was sufficient to differentiate heterogeneous uptake in subrenal structures in the rat.

The effect of previous nitrate therapy on vascular responses to endothelin-1 (ET-1) and NO was investigated in human internal mammary artery (IMA) in vitro. Cumulative concentration-response curves to ET-1 were constructed in rings of IMA and the data grouped into IMA from patients given nitrates prior to the bypass graft operation (nitrate group) and IMA from patients who were not prescribed nitrates (control group). No significant differences were observed between the two groups, either in EC(50) value [P>0.05; 3.5 nM (2.4-5.3 nM; 95% confidence interval) and 4.8 (2.2-10 nM), nitrate and control groups respectively] or E(max) (P>0.05; 78+/-7.5% and 85+/-9.5%, nitrate and control group respectively). No significant differences in concentration-response curves to the NO-donor diethylamine NONOate (DEA/NO) in rings of IMA pre-constricted with 10 nM ET-1 were observed between control and nitrate groups [P>0.05; EC(50) values 0.59 (0.21-1.7) microM and 0.17 (0.03-0.87) microM; E(max) 110+/-5.7% and 112+/-4.5%, nitrate and control groups respectively]. Concentration-response curves to DEA/NO constructed in normal coronary artery were not significantly different from those in coronary artery obtained from patients with ischaemic heart disease (IHD) [P>0.05; E(max) 124+/-11% and 138+/-20%; EC(50) 0.08 (0.02-0.30) microM and 0.23 (0.02-24) microM, normal and IHD respectively]. These data indicate that nitrate therapy does not induce long-term changes in the ET signalling pathway. Furthermore, the tolerance to nitrate therapy is likely to be because of impaired bio-transformation of the drug rather than reduced sensitivity of the media to NO. The similar responses to DEA/NO in normal and atherosclerotic coronary artery suggests that the reduced vasodilator responses in IHD is because of a dysfunctional endothelium and is not mediated by changes in the NO signalling pathway of the smooth muscle.

We have determined the ability of the endothelin (ET)(A) receptor antagonist, PD156707 (CI 1020), to inhibit intimal proliferation in human saphenous veins maintained in organ culture. After 28 days in culture, veins exposed to 1 microM PD156707 exhibited a significant reduction in intima to intima-plus-media ratio (I:I+M ratio) (0.14+/-0.02, n=15) and an increase in lumen area (3.1+/-0.8 mm(2)) compared with veins cultured without the antagonist (I:I+M, 0.29+/-0.02; lumen area, 2.5+/-0.7 mm(2); n=23) but were not significantly different from pre-cultured controls (I:I+M, 0.15+/-0.02; lumen area, 4.4+/-1.2 mm(2); n=17) (Dunn's test for non-parametric multiple comparisons: alpha<0.05). In organ bath experiments, ET-1 and 5-hydroxytryptamine constricted pre-cultured control vessels with pD(2) values (where pD(2) is defined as the negative logarithm of the molar EC(50) value of an agonist) of 8.9+/-0.4 and 7.0+/-0.4 (n=3) and E(max) (efficacy) values of 86+/-3% and 71+/-13% (compared with constriction induced by KCl) respectively. There was no difference in the responsiveness of veins cultured for 14 days to either agonist, indicating that the vessels maintained in organ culture remain viable. Crucially, vein segments cultured with 1 microM PD156707 (a concentration that antagonized ET-1 responses in pre-cultured control vessels) contracted to ET-1 with a potency comparable to that obtained in vessels cultured in the absence of the antagonist (pD(2)=8.9+/-0.4 and 8.0+/-0.6 respectively, n=3) confirming that PD156707 was not toxic to the tissue at the concentration used. In conclusion we have shown that the ET(A)-selective antagonist, PD156707, completely blocked intimal hyperplasia in human saphenous veins in organ culture, suggesting that ET(A) antagonists may be beneficial in preventing or delaying saphenous vein graft disease in patients receiving bypass grafts for coronary artery disease.

Endothelin (ET)-1 and thromboxane (Tx) levels are increased in human atherosclerosis. One of the aims of this study was to understand how receptors for a peptide mediator (ET-1) with a long physiological half life, would differ from a lipid mediator (TxA(2)), with a short physiological half life, in human coronary artery disease (CAD). Secondly, to determine if receptor protein is present in human coronary artery vascular smooth muscle for the recently adopted peptide orphan receptors for urotensin-II, apelin and ghrelin. The ET(A) receptor subtype predominated in the medial smooth muscle layer of both non-diseased coronary artery (NCA) and CAD. However, this subtype was present at relatively low density in the proliferated intimal layer of CAD. The ET(B) receptor protein was not altered with CAD, compared with NCA. Tx receptor density was significantly (P<0.05) increased in both the media and intima of CAD, compared with NCA. There was no alteration in receptor density, on the medial smooth muscle for urotensin-II and apelin with CAD. Interestingly, receptor density for the novel vasodilator peptide ghrelin was significantly (P<0.05) increased (approx. 4 fold) with CAD, compared with NCA. The alteration of receptor density with disease for Tx and ghrelin provides novel therapeutic targets for the treatment of atherosclerosis. In conclusion, while some GPCR are altered, others remain unchanged with human atherosclerosis. The increase in vasoconstrictor Tx receptor density with disease suggests the importance of Tx receptor antagonism. Intriguingly, the increase in receptor density for the novel vasodilator ghrelin, identified from post-genomic research, may potentially be beneficial with human atherosclerosis.

We investigated the binding characteristics of angiotensin receptors and used this assay to determine the predominant enzyme capable of converting angiotensin I in the human left ventricle. In homogenates of human left ventricle, (125)I-[Sar(1),Ile(8)]angiotensin II bound with sub-nanomolar affinity, with a corresponding K(D) of 0.42+/-0.09 nM, a B(max) of 11.2+/-2.3 fmol.mg(-1) protein and a Hill slope of 1.04+/-0.04. The rank order of inhibitory potency of competing ligands for the (125)I-[Sar(1),Ile(8)]angiotensin II binding site was CGP42112>angiotensin II> or =angiotensin III=angiotensin I>losartan. The angiotensin type II (AT(2)) receptor predominated in the human left ventricle over the angiotensin type I (AT(1)) receptor, with an approximate AT(1)/AT(2) receptor ratio of 35:65. No specific (125)I-angiotensin IV binding sites could be detected in the human left ventricle. Using competitive radioligand binding assays, we were able to demonstrate that the chymase/cathepsin G enzyme inhibitor chymostatin was more potent than the angiotensin-converting enzyme (ACE) inhibitor captopril at inhibiting the conversion of angiotensin I in the human left ventricle. Aprotonin (an inhibitor of cathepsin G but of not chymase) had no effect on angiotensin I conversion, suggesting that the majority of the conversion was mediated by chymase. Thus, although the current therapies used for the renin-angiotensin system have focused on ACE inhibitors and AT(1) receptor antagonists, the left ventricle of the human heart expresses mainly AT(2) receptors and the tissue-specific conversion of angiotensin I occurs predominantly via chymase rather than ACE.

1. We have examined the expression of endothelin isoforms and their precursors in the human heart using RIA, HPLC, immunocytochemistry and reverse transcriptase-polymerase chain reaction assays. 2. Highly specific RIAs were used to measure the levels of mature endothelin and big endothelin-1 immunoreactivity in extracts of human right ventricle. There was no significant difference between samples from patients with ischaemic heart disease and idiopathic dilated cardiomyopathy. 3. HPLC coupled with RIAs allowed the separation and identification of the three mature isoforms of endothelin, big endothelin-1 and the C-terminal fragment of big endothelin-1. In extracts of human endocardial endothelial cells, peaks of immunoreactivity that co-eluted with authentic endothelin-1, big endothelin-1 and C-terminal fragment were found. 4. Intense immunocytochemical staining of mature endothelin immunoreactivity was detected in the cytoplasm of endothelial cells of all regions of the heart tested. Big endothelin-1 immunoreactivity mirrored that of the mature peptide and, in two of three individuals tested, big endothelin-2 immunoreactivity was also detected. No big endothelin-3 immunoreactivity was detected in any of the tissues examined. 5. Reverse transcriptase-polymerase chain reaction assays demonstrated endothelin-1 and endothelin-2 mRNA in all three samples of human left ventricle tested. In two of the individuals, additional bands were also detected with the endothelin-2 primers which corresponded to splice variants. There was no evidence for the expression of endothelin-3 mRNA. 6. These data suggest that endothelin-1 is the predominant isoform of endothelin in the human heart and is probably largely synthesized by the endothelial cells within the heart. If released from the endothelial cells in vivo, this potent cardiotonic peptide may play an important paracrine role in human cardiovascular function.

1. Two-site enzyme-linked immunosorbent assays have been developed for the rapid, sensitive and non-isotopic measurement of endothelin-1 and big endothelin-1. The sensitivities of detection were 0.5 and 0.3 fmol/well, with ED50 values of 13 and 12 fmol/well for the endothelin-1 and big endothelin-1 assays, respectively. Each assay is highly selective for its corresponding antigen. The ET-1 assay showed no detectable cross-reactivity with ET-1-(1-20), indicating that the assay only recognizes the 21-amino acid biologically active peptide. 2. The two assays were used to measure the effects of two classes of protease inhibitor on the basal release of enothelin-1 and big endothelin-1 from cultured first-passage human umbilical vein endothelial cells. 3. The secretion of both peptides was time-dependent over 12 h. The metalloprotease inhibitor phosphoramidon (1 x 10(-4) mol/l) significantly reduced the amount of endothelin-1 secreted into the medium (P < 0.05), with a concomitant increase in the secreted levels of big endothelin-1 (P < 0.01). The aspartyl protease inhibitor, pepstatin A, also caused a significant decrease in the secretion of endothelin (P < 0.05). However, unlike phosphoramidon, there was no increase in the levels of big ET-1 compared with the controls. At these concentrations, neither inhibitor affected the viability of the cells as indicated by Trypan Blue exclusion. 4. The two assays permit the direct measurement of endothelin-1 and its precursor, and will be of use in the elucidation of the putative human endothelin-converting enzyme(s).(ABSTRACT TRUNCATED AT 250 WORDS)

1. A peptide-protein mobility shift assay has been developed, using native polyacrylamide-gel electrophoresis, that enables the isolation of de-natured receptor proteins from small amounts of human cardiac tissue. 2. Radiolabelled endothelin-1 and related peptides were used to identify and isolate endothelin receptors from partially purified membrane extracts of human atrial tissue. 3. Binding analysis using radiolabelled endothelin-1 gave an equilibrium dissociation constant (Kd) of 2 nmol/l, similar to results from binding experiments conducted directly on tissue. 4. Peptide-receptor complexes were electroeluted from native gels and dissociated. Receptor material was characterized by dot-immunobinding analysis of eluates using an antibody raised against a predicted human endothelin receptor sequence.

1. Binding sites for calcitonin-gene-related peptide were localized and characterized in porcine coronary arteries using quantitative autoradiography, and the density of binding sites was compared between large epicardial and small intramyocardial coronary arteries. 2. A single class of binding sites for calcitonin-gene-related peptide with a dissociation constant of 2.1 +/- 0.2 nmol/l was detected in both the large and small coronary arteries. The density of specific binding sites was higher (maximum binding site density 231 +/- 14 fmol/mg of protein) in the small coronary arteries than in the large epicardial coronary arteries (maximum binding site density 108 +/- 5 fmol/mg of protein). beta-Human calcitonin-gene-related peptide showed higher affinity than alpha-human calcitonin-gene-related peptide for the binding sites. Most of the specific binding sites for both peptides in the large coronary artery were localized in the intima and media. 3. In coronary artery from patients with coronary heart disease, there were more binding sites for calcitonin-gene-related peptide in the smooth muscle layer of atheromatous segments (7.2 +/- 0.7 amol/mm2) than in that of normal segments (3.0 +/- 0.3 amol/mm2, P < 0.002). 4. The present findings lend further support to the theory of regional variation in the vasodilator response to calcitonin-gene-related peptide in porcine coronary arteries, which seems to be due to different densities of a single type of receptor for calcitonin-gene-related peptide.

1. A radioimmunoassay has been developed for measuring endothelin-like immunoreactivity in human plasma using an antibody raised against endothelin-1 which also cross-reacts with big endothelin-1 and endothelin-2 but not endothelin-3. 2. The sensitivity of the assay was 1 fmol/tube with inter- and intra-assay coefficients of variation of 13% and 9%, respectively. Cross-reactivity with endothelin-3 and non-endothelin peptides was less than 1%. 3. Endothelin-like immunoreactivity was present in the plasma of hypertensive patients (n = 25) at a concentration of 5.7 +/- 0.5 pmol/l (mean +/- SEM), which was not significantly different from that of age-matched control subjects (5.1 +/- 0.5 pmol/l). At these levels, endothelin-1 is unlikely to function as a circulating hormone. 4. Within the normotensive group, the concentration of endothelin-like immunoreactivity in plasma was positively correlated with mean arterial blood pressure, but in hypertensive patients it showed a significant negative correlation.

1. Quantitative receptor autoradiography in vitro has been used to determine the distribution and density of specific binding sites for 125I-labelled endothelin-1 in human, porcine and rat kidneys. Immunocytochemistry was used to visualize von Willebrand factor-positive endothelial cells in adjacent sections to those used for autoradiography. 2. High levels of specific binding were detected in the vasa recta and papilla of all three species with lower levels in the medulla and cortex. 3. A major difference between the species was observed within the glomeruli, where high levels of binding were found in the rat but no detectable binding sites in pig or man.