Search

1 The angiogenic activity of four vasoactive peptides with a range of vasodilator and vasoconstrictor properties, i.e. vasoactive intestinal peptide (VIP), endothelin-1, endothelin-3 and angiotensin II, were investigated in a rat sponge model. Neovascularization was assessed by the 133Xe clearance technique and confirmed by histological studies. 2 Daily doses of the vasodilator peptide, VIP (1000 pmol), caused intense neovascularization, but a lower dose (10 pmol) produced no apparent effect. However, the lower dose of VIP, when given with a subthreshold dose of interleukin-1 alpha (0.3 pmol), produced an angiogenic response similar to that seen with the higher dose of VIP. The neovascular response induced by co-administration of VIP and interleukin-1 alpha was inhibited by simultaneous administration of 100 pmol VIP (10-28), a specific VIP receptor antagonist. 3 In contrast, daily doses of 10, 100 or 1000 pmol endothelin-3 (a mixed vasoconstrictor and vasodilator with more marked vasodilator activity) or of 100 or 1000 pmol endothelin-1 (also with mixed activity but with much more pronounced vasoconstrictor response) produced no apparent effect on sponge-induced angiogenesis. 4 The vasoconstrictor peptide, angiotensin II, in daily doses of 1000 pmol, caused an intense neovascularization like VIP but lower doses of angiotensin II (10 or 100 pmol) produced no apparent effect. The lowest dose of angiotensin II (10 pmol) when administered with the subthreshold dose of interleukin-1 alpha (0.3 pmol) had no effect on the basal neovascular response in the sponges. The angiotensin II-induced neovascular response was inhibited by co-administration of 100 nmol of the specific AT1 receptor antagonist, losartan, but not by the AT2 receptor antagonist, PD 123319. 5 These data show that VIP and angiotensin II possess angiogenic activity. However, endothelin-1 and endothelin-3 had no activity at the doses used. Thus the angiogenic response is not related to local vasoconstriction or vasodilatation in the sponges. The blockade of VIP- and angiotensin II-induced angiogenesis at the receptor level suggests that receptor modulation could provide a strategy for the management of angiogenic diseases.

An in vitro model of endothelial cell injury was used to investigate the role of endothelins and related peptides in endothelial repair. Endothelin-3 (10-100 nM) enhanced wound repair over an 18 h period by promoting proliferation, an effect not inhibited by the specific ETA receptor antagonist BQ-123 (100 nM) or the mixed ETA/ETB antagonist PD142893 (10 microM). Like endothelin-3, the ETB selective agonists [Ala1,3,11,15]endothelin-1 and sarafotoxin S6c were able to enhance wound repair over the same dose range. Neither endothelin-1 nor endothelin-2, however, had any effect on endothelial cell wound healing. Inhibition of cyclo-oxygenase or neutralisation of basic fibroblast growth factor did not inhibit this endothelin-3-mediated event. These results suggest that endothelin-3 might have a direct role in endothelial cell proliferation as a response to injury which is not mediated by either of the currently defined ETA and ETB receptors.

SK-N-MC cells, derived from a human neuroblastoma, respond to endothelin (ET) peptides with an increase in the free intracellular calcium concentration. The response is biphasic, with the secondary plateau phase being abolished or reduced by removal of extracellular Ca2+ or by the presence of 100nM nitrendipine. Restoration of Ca2+ to the bathing solution in cells stimulated by ET-1 in the absence of Ca2+ caused the plateau to reappear. The order of potency of ET family peptides was ET-2 greater than or equal to sarafotoxin S6b greater than or equal to ET-1 much greater than ET-3, suggesting that ETA receptors mediate the response. Sarafotoxin S6c and the C-terminal hexapeptide endothelin (16-21) were inactive in these cells. [Ala1,3,11,15]ET-1, a linear analogue of ET-1 which has been suggested to be a selective ETB receptor agonist, was a weak competitive antagonist of the actions of ET-1 in these cells. However, BQ-123, recently introduced as a selective and competitive antagonist at ETA receptors, was a potent non-competitive antagonist of ET-1 giving a 50% reduction in the maximum response at 6nM.

1. The rat isolated superior mesenteric bed, perfused with Krebs-Henseleit solution containing 10 microM indomethacin and precontracted with 100 microM methoxamine, was used to study the vasorelaxation produced by some fragments of endothelin-1, by two alanyl-substituted analogues, and by human and porcine proendothelins. The systemic cardiovascular effects of some of these peptides were also studied in anaesthetized rats. 2. Endothelin (16-21) was an endothelium-dependent vasodilator about 10 times less potent than acetylcholine and its amide was about 500 times less potent than the free acid. Human proendothelin-1 and porcine proendothelin-1 also caused endothelium-dependent relaxations but neither [Ala3,11]endothelin-1 nor [Ala1,3,11,15]endothelin-1 produced significant reductions in the methoxamine-induced tone. Sodium nitroprusside was approximately 200 times less potent than acetylcholine in the presence of the endothelium and was the only vasorelaxant to be active after destruction of the endothelium by perfusion with 0.3% CHAPS; in the absence of the endothelium it was 3.7 times more potent as a vasodilator than in its presence. 3. Porcine proendothelin-1 had weak contractile activity in the isolated mesenteric bed but porcine endothelin (22-39), endothelin (16-21) and endothelin (16-21) amide did not have vasoconstrictor actions. 4. Endothelin-3, [Ala3,11]endothelin-1, [Ala1,3,11,15]endothelin-1 and endothelin (16-21) all had systemic blood pressure effects in the anaesthetized rat. Although endothelin (16-21) did not cause vasoconstriction in vitro, it increased mean arterial pressure in vivo but was at least 100 times less potent than endothelin-3. Despite causing no vasorelaxation in vitro, [Ala1,3,11,15]endothelin-1 and [Ala3,11]endothelin-1 induced short-lived systemic depressor responses which were followed by pressor responses. Endothelin-3 was more potent as a systemic depressor agent than as a pressor agonist and, as a vasodepressor agent, it was 3-4 times more potent than either of the alanyl-substituted analogues. Endothelin-3, [Ala3,11]endothelin-1 and [Ala1,3,11,15]endothelin-1 were equipotent vasopressor agents. Porcine endothelin (22-39) had no systemic blood pressure effects. 5. This study shows directly that the presence of both disulphide bonds is required in the endothelins for them to be able to cause endothelium-dependent relaxation in the mesenteric vascular bed and that this response is mediated by different receptors from those causing contraction of the vascular smooth muscle in the mesentery. Vasorelaxation caused by endothelin (16-21) and its amide suggests that there is a receptor on the endothelium similar to one reported in the guinea-pig trachea. Finally, the ability of endothelin peptides to cause systemic vasodepressor responses is not related to their ability to cause endothelium-dependent relaxation, at least in the mesenteric circulation, and, for endothelin (16-21), its systemic pressor response is not likely to be the result of vasoconstriction in the mesenteric bed.

1. The isolated superior mesenteric arterial bed of the rat, perfused with Krebs-Henseleit solution containing 10 microM indomethacin, was used to study the effects of reducing dissolved O2 tension on the pressor responses to endothelin-1, endothelin-3 and sarafotoxin S6b. The modulation of these responses by the endothelium was investigated by removing the intima with the detergent CHAPS and, for endothelin-1, by inhibiting nitric oxide production with N omega-nitro-L-arginine methyl ester (L-NAME). Comparison was made with the effects of lowering O2 tension on the pressor responses to noradrenaline and 5-hydroxytryptamine. 2. Lowering the perfusate O2 tension from 551 +/- 2 mmHg to 14.0 +/- 0.5 mmHg did not change the ED50 for endothelin-1 but its maximal responses (Rmax) were increased by 2.1 and 2.7 fold, respectively, in the presence and absence of endothelium. The Rmax values for endothelin-3 were also greater in hypoxia either in the presence (by 2.3 fold) or absence of the endothelium (by 1.6 times) but those for sarafotoxin S6b were only enhanced significantly by hypoxia in the absence of the intima. hypoxia reduced the potencies of endothelin-3 and sarafotoxin S6b whether or not endothelium was present. 3. Endothelial destruction, whether in hypoxic or oxygenated conditions, increased the Rmax values for endothelin-1 and endothelin-3; at both O2 tensions those for endothelin-3 increased more than those for endothelin-1. The ED50 for endothelin-1 was unchanged by destroying the endothelium but endothelin-3 was less potent in the absence of an endothelium than in its presence. Removal of the endothelium did not change the R.ax for sarafotoxin S6b but increased its potency in both hypoxic and oxygenated tissues. 4. In hypoxia, and in the presence of both the endothelium and 100 microM L-NAME, the Rmax for endothelin-1 was 1.6 times greater than that in hypoxia in the absence of L-NAME. Co-infusion of 100 microM L-arginine, but not of 100 mircoM D-arginine, with 100 microM L-NAME reversed this effect. The presence of L-NAME decreased the potency of endothelin-1. 5. Destroying the endothelium did not affect the Rmax for noradrenaline in either oxygenated conditions or hypoxia. Changing 02 tension when the endothelium was intact had no effect on the Rmax but it was 11% greater in oxygenated, than in hypoxic, endothelium denuded preparations. Endothelial destruction decreased the potency of noradrenaline in hypoxia but increased it in oxygenated tissues. In hypoxia, L-NAME had no effect on the ED50 relative to control preparations with endothelium but the Rmax was 30% greater. 6. 5-Hydroxytryptamine gave very small pressor responses in the presence of endothelium in both oxygenated and hypoxic tissues but the Rmax was 1.7 times greater in hypoxia. L-NAME increased the R,,x by 9.8 times in oxygenated preparations and 6.3 fold in hypoxia. The ED5o values were the same in all conditions. 7. It is concluded that, although hypoxia generally increased the R.. for the endothelin/sarafotoxin peptides, the changes could not be explained by a simple increase in receptor number since hypoxia decreased the potency of endothelin-3 and sarafotoxin S6b. Thus alterations in receptor binding or activation properties, or both, also occurred. The changes associated with hypoxia were not common to all vasoconstrictor agonists since, in the absence of endothelial function, hypoxia did not affect the Rmax values for either noradrenaline or 5-hydroxytryptamine. Also, the pressor responses to the peptides and both the amines can be modulated by the endothelium in hypoxia as well as in oxygenated conditions.

Radioligand binding studies were used to define the binding constants for inhibition of [125I]endothelin-1 binding by endothelin-1 (ET-1), endothelin-3 (ET-3) and a synthetic analogue of ET-1, [Ala1,3,11,15]ET-1 in rat aorta and cerebellum. The inhibition curves for the three substances were similar in the cerebellum (Ki, ET-1 = ET-3 = [Ala1,3,11,15]ET-1) but differed in the aorta (Ki, ET-1 much less than [Ala1,3, 11,15]ET-1) with the ET-3 inhibition curves being biphasic, indicating binding to two sites. In functional studies in the aorta, in contrast to findings in the cerebellum, [Ala1,3,11,15]ET-1 was inactive and ET-3 was a partial agonist. These findings support the idea of heterogeneity of endothelin receptors. Autoradiographic studies were performed to identify the cellular localisation of endothelin-1 receptors in the two tissues. In the aorta binding was to all elements of the vasculature including the endothelium, smooth muscle, periadventitial connective tissue and nerves. In the cerebellum binding sites were located predominantly over the neuronal elements of the granular cell layer. The cellular localisation of the high and low affinity sites for ET-3, and the functional significance of the high affinity binding site for ET-3, remain to be determined.

1. [125I]-endothelin-1, over the concentration range 6 pM-10 nM, bound to a single site in homogenates of rat cerebellum with high affinity (Kd = 2.8 +/- 0.6 x 10(-10) M). The site was present in a concentration of 321 +/- 58 fmol mg-1 protein. 2. The rates of association and dissociation of [125I]-endothelin-1 with the binding site were slow (at 25 degrees C, k+1 = 8.0 +/- 1.3 x 10(5) M-1s-1; k-1 = 2.6 x 10(-4)s-1) and, on addition of a maximally displacing concentration of endothelin-1 (100 nM), 94.0 +/- 8.4% of the [125I]-endothelin-1 was still bound after 14 h. 3. [125I]-endothelin-1 binding was inhibited by a number of naturally occurring or genetically encoded members of the endothelin/sarafotoxin family of peptides. The order of potency was endothelin-3 = sarafotoxin S6b greater than endothelin-2 = endothelin-1 much greater than porcine proendothelin1-39. 4. Binding was also inhibited by analogues in which either one or both of the cystine disulphide bridges had been replaced by substitution with 2 or 4 alanine residues. The tetra-alanyl substituted analogue, [Ala1,3,11,15]endothelin-1, was equipotent with endothelin-1 at inhibiting the binding of [125I]-endothelin-1. [Ala3,11]endothelin-1 and [Ala1,15]endothelin-1, analogues which each contained one of the disulphide bridges from the parent peptide, were respectively 3 and 14 times less potent than the parent peptide. An analogue in which the Glu10 residue had been anisylated was 25 fold less potent than endothelin-1. 5. It is concluded that the structural requirements for binding to the cerebellar sites for [1251]_ endothelin-1 do not require the presence of the disulphide bridges characteristic of the endothelin/ sarafotoxin family. Rather, the binding may be more sensitive to the presence of bulky side chain substituents, at least in the smaller intramolecular loop.

1. The vasoconstrictor activities of endothelin-2, endothelin-3, sarafotoxin S6b, human proendothelin1-38 and mouse vasoactive intestinal contractor (VIC) were studied in the isolated Krebs-Henseleit perfused mesenteric arterial bed of the rat in the presence and absence of the endothelium. The vasoconstrictor properties of endothelin-1 were studied in control preparations and in preparations treated with methylene blue or N omega-nitro-L-arginine methyl ester (NAME). Finally, the direct vasodilator properties of endothelin-2, endothelin-3 and sarafotoxin S6b were studied in preparations preconstricted with methoxamine. 2. In the presence of an intact endothelium, all of the peptides caused dose-dependent increases in perfusion pressure and sarafotoxin S6b was a full agonist relative to the other peptides studied (maximum increase in perfusion pressure, Rmax = 106 +/- 11 mmHg). Endothelin-1, endothelin-2 and VIC were more potent vasoconstrictors (ED50 93.0 +/- 40.0, 90.8 +/- 20.5 and 106 +/- 63 pmol, respectively) than endothelin-3 and sarafotoxin S6b, which were found to be equipotent (ED50 values 411 +/- 195 and 345 +/- 86 pmol, respectively). A full dose-response relationship could not be constructed for proendothelin, but the highest dose used (4 nmol) increased the perfusion pressure by 15.4 +/- 1.6 mmHg. 3. Destruction of the endothelium with the zwitterionic detergent 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulphonate (CHAPS) significantly enhanced the pressor activity of all 5 peptides. The Rmax for sarafotoxin S6b was not significantly altered by removal of the endothelium but its potency was significantly increased (ED50 = 115 +/- 15 pmol). Although their R,,, values were significantly increased, endothelin-2 and VIC were still partial agonists relative to sarafotoxin S6b in CHAPSpretreated preparations; their potencies were unchanged (ED5o values 118 + 53 and 416 + 196pmol, respectively). Removal of the endothelium significantly reduced the potency of endothelin-3 (ED5o, 6.3 + 2.2 nmol) but this peptide then exhibited full agonist activity (R..x = 106 + 14 mmHg). After endothelial cell destruction, the pressor responses to proendothelin were increased; 4 nmol gave a response of 38.8 + 5.5 mmHg. 4. Exposure of preparations to either 100 microM NAME (R,,,X = 42.6 + 2.4mmHg and EDSo = 57.5 + 13.7 pmol) or 10 microM methylene blue (R,,,. = 36.0 + 5.1 mmHg and ED50 = 81.5 + 26.1 pmol) significantly enhanced the maximum pressor responses to endothelin-l (control: R.,=X = 22.5 + 2.6 mmHg; ED5o = 93.0 + 40.Opmol). The values in the presence of NAME or methylene blue were not significantly different from those found previously for endothelin-1 after removal of the endothelium with CHAPS. 5. Endothelin-2, endothelin-3 and sarafotoxin S6b all caused vasorelaxation in preparations which had been precontracted with 100 microM methoxamine. This action was endothelium-dependent as it was abolished by perfusing the mesentery with CHAPS. Endothelin-3 and sarafotoxin S6b caused relaxation at much lower doses than were needed with endothelin-1 and endothelin-2. 6. The endothelium significantly modulates the vasoconstrictor activity of all the endothelin-like peptides studied, including the precursor peptide proendothelin (which was the least potent of the peptides). This modulation is likely to be due to the release of endothelium-derived relaxing factor, since similar results to destruction of the endothelium were obtained when endothelin-l was investigated in the presence of either methylene blue or NAME (an inhibitor of nitric oxide formation) in the perfusion fluid. The vasodilator effects of the peptides were also endothelium-dependent. There was a different order of potency for vasoconstriction and vasodilatation supporting the suggestion that there are sub-types of receptor for the endothelin-like peptides in the vasculature; one type on the vascular smooth muscle and a second type on the endothelium.