Regional distribution of endothelin receptor in porcine cardiovascular tissues

To clarify effecting sites of endothelin (ET) in a circulation system, we have identified specific receptors for porcine ET(ET-1) and investigated the distribution in the porcine cardiovascular tissues. Scatchard analysis of 125I-porcine ET binding indicated the presence of a single class of high-affinity binding sites. The binding was highly specific for ET-1, because (1) none of the other various peptides or Ca2+-channel antagonists affected the binding, (2) the scission of disulfide bonds, the digestion of the C-terminal 6-amino acid residues, or nitrophenylsulfenylization of the C-terminal Trp21 of ET-1 markedly reduced the binding ability and, (3) ET-1 showed the highest affinity for the vascular receptor among three ET isopeptides. Cardiac atria possessed the highest density (2.7 pmol/mg protein) of ET receptors of all the tissues examined, including thoracic aorta, cardiac atria and cardiac ventriculi, basilar, renal, coronary and pulmonary branch arteries, coronary, renal and jugular veins, and small vessels of pia mater encephali. Small vessels, renal and coronary arteries also showed relatively high density (0.8-1.4 pmol/mg protein). Various veins examined also showed considerable density (0.45-0.74 pmol/mg protein). The apparent Kd of cardiac ET receptors (0.76 nM) was significantly greater than that of the receptors of the other tissue (0.06-0.14 nM). The extensive distribution and the local enrichment of ET receptor in a cardiovascular system strongly suggests that ET is one of the essential endogenous substances to control the tone of the vasculature.     

Functional endothelin/sarafotoxin receptors in rat heart myocytes: structure-activity relationships and receptor subtypes

Functional receptors for the peptides of the endothelin (ET) and sarafotoxin (SRTX) family were characterized in newborn rat heart myocytes using human and rat endothelins (ET-1 and ET-3, respectively), SRTX-b and SRTX-c. Binding studies in intact cells and homogenates revealed significantly higher affinities of ET-1 and SRTX-b than of ET-3 and SRTX-c towards these receptors. This binding profile of ET/SRTX peptides points to their interaction with the receptor subtype designated E-S alpha. All four peptides induced time- and dose-dependent phosphoinositide hydrolysis with the following rank order of potency: ET-1 greater than SRTX-b greater than SRTX-c greater than ET-3. Thus, ET-3 which possesses an intermediate affinity toward the receptor was the least effective with regard to this response. These results confirm and extend our earlier report that the ET/SRTX peptides interact with a newly characterized receptor(s) associated with phosphoinositide metabolism and Ca2+ mobilization. The initiation of inositol phosphate formation is largely independent of extracellular Ca2+, verapamil and nifedipine, indicating that the ET/SRTX peptides are not agonists for the voltage-dependent Ca2+-channels.     

Functional endothelin/sarafotoxin receptors in the rat uterus

Functional receptors for the peptides of the endothelin (ET) and sarafotoxin (SRTX) families were detected in the rat uterus. These receptors specifically bind 125I-SRTX-b (Bmax = 220 fmol/mg protein), as well as ET-1, ET-3 and SRTX-c (IC50's 10, 5, 300 and 780 nM, respectively). Activation of the uterine ET/SRTX receptors induced dose-dependent phosphoinositide (PI) hydrolysis and three typical contractile responses: 1) increase in the muscle tonic tension; 2) increase in frequency of the spontaneous rhythmic contractions; 3) decrease of relaxation in each spontaneous rhythmic cycle. All three effects appeared at doses as low as 0.5-1 nM. Dose responses yield ED50 values of 5.5, 30 and 680 nM for ET-1, SRTX-b and ET-3, respectively. SRTX-c was the least effective peptide in achieving decrease in relaxation. In view of these results, and since the uterine responses to the peptides were almost immediate and reversible, we suggest that the functional ET/SRTX receptor of the rat uterus that is coupled to PI hydrolysis may be of physiological significance.     

Interaction of synthetic sarafotoxin with rat vascular endothelin receptors

The effects of synthetic analogs of sarafotoxin (STX) S6b, a snake venom peptide with a high sequence homology to the endothelium-derived vasoconstrictor endothelin (ET), on ET receptor binding activity and cytosolic free Ca2+ concentration [( Ca2+]i) were studied in cultured rat vascular smooth muscle cells. Binding studies revealed that [Cys1-15, Cys3-11] STX competed with 125I-ET for the binding to its vascular receptors with lower affinity than that of ET, but was far more effective than [Cys1-11, Cys3-15]STX in inhibiting the binding. [Cys1-15, Cys3-11]STX had a less potent effect on increasing [Ca2+]i than ET, whereas [Cys1-11, Cys3-15]STX was inactive. These data suggest that there may exist heterogenous subpopulations of the vascular ET/STX receptors, and that the proper double cyclic structure of STX is essential for interacting with its putative receptors to induce the [Ca2+]i response.     

Multiple subtypes of endothelin receptors in porcine tissues: characterization by ligand binding, affinity labeling and regional distribution.

To clarify the existence and the distribution of endothelin (ET) receptor subtypes, we have examined the pharmacological properties and the molecular weight (Mr) of 125I-ET-1 and 125I-ET-3 binding sites in various tissues of pigs. ET-1 and ET-2 showed almost identical potencies in displacing the bound 125I-ET-1 in all the tissues examined. ET-3, sarafotoxin S6b (SRT-b) and sarafotoxin S6c (SRT-c) displaced the 125I-ET-1 with the same sensitivity as ET-1 (IC50 = 0.1-1.4 nM) in brain, kidney, liver and adrenal, whereas the three peptides showed very weak competition (IC50 = 40-500 nM) against 125I-ET-1 binding in cardiac atria, aorta, lung, stomach and uterus. The computer analyses of the binding data suggested the presence of high (Kd1 = 0.04-0.29 nM) and low (Kd2 = 60-190 nM) affinity binding sites for ET-3 and SRT-b in lung and stomach. 125I-ET-3 bound to the high affinity sites in lung and stomach was displaced by ET/SRT isopeptides almost equipotently. Two proteins with Mr of 47,000 and 35,000 were affinity-labeled with 125I-ET-1 in cerebellum, while a protein with Mr of 123,000, in addition to the two proteins, was predominantly labeled in lung. The above findings indicated that two distinct subclasses of ET receptors, namely, ET-1-specific and ET/SRT family-common receptors were distributed in various proportions in mammalian tissues, and suggested that their molecular forms are also different.     

Immunological and structural characterization of sarafotoxin/endothelin family of peptides

A highly specific and sensitive radioimmunoassay (RIA) was developed for the potent vasoconstrictor peptides, sarafotoxin-b and human endothelin. The antigenic determinants of the antibodies employed in studies with these assays were found to be localized within the amino acid sequence at positions 4-7. This was confirmed by CNBr cleavage of the methionyl residue at position 6 in the sarafotoxin and at position 7 in the endothelin. The chemically characterized modified peptides showed very low cross reactivity in the RIAs. On the other hand, the binding properties as well as the ability to induce phosphoinositide hydrolysis were very similar in the modified and native peptides, indicating that despite cleavage of the peptide bond the biologically active conformation responsible for either binding or phosphoinositide hydrolysis is retained, probably because of the disulfide bonds. Thus, structural alteration might be a valuable means of curtailing some of the various activities induced by the sarafotoxin/endothelin family of peptides.     

A comparative study of endothelin and sarafotoxin action in vascular and non-vascular smooth muscle

The effects of endothelin and sarafotoxin on smooth muscle tone have been examined in the rat aorta and anococcygeus muscle and their actions compared to those of norepinephrine. The contractions elicited by endothelin and sarafotoxin (10 nM), or norepinephrine (1 μM) were approximately equieffective in terms of tension development and correspond to EC₅₀ values and these concentrations were thus used throughout the study. In calcium-free Krebs the three agonists generated approximately similar levels of tone in the aorta and the anococcygeus corresponding to 20 and 5% of the maximum response, respectively. Nifedipine, 10 μM, significantly inhibited responses to endothelin and norepinephrine in the aorta but only norepinephrine in anococcygeus; the responses to sarafotoxin were however not significantly affected in either tissue. A combination of 10 μM ryanodine and nifedipine caused near complete inhibition of the response to endothelin in the aorta and also significantly reduced the response to both endothelin and norepinephrine in the anococcygeus. The lipoxgenase inhibitor, nordihydroguaiaretic acid, inhibited the response to endothelin in the aorta and endothelin and norepinephrine in the anococcygeus muscle. The cyclooxygenase inhibitor, indomethacin, however, had no effect on the responses to any of the three agonists in either the aorta or anococcygeus. At concentrations greater than 30 nM both endothelin and sarafotoxin induced myogenic activity in normally quiescent anococcygeus muscle. As determined by the loss of myogenic activity the tissues recovered more rapidly from sarafotoxin than endothelin with complete recovery apparent after 2.62 ± 0.85 and 5.22 ± 0.06 h respectively. Omitting Ca²⁺ from the Krebs solution reduced recovery times to 1.62 ± 0.2 and 2.4 ± 0.51 h respectively.

Overall the results suggest that endothelin and sarafotoxin activate different cell signaling systems to differing extents in rat aorta versus anococcygeus suggesting that the membrane receptors mediating the responses to endothelin and sarafotoxin are not necessarily identical.     

SRTX-d, a new native peptide of the endothelin/sarafotoxin family

The primary structure of a new sarafotoxin, SRTX-d, from the venom of Atractaspis engaddensis is described. SRTX-d differs from SRTX-b in two substitutions: Ile19 instead of Val and Thr2 instead of Ser. The toxicity of SRTX-d and its vasoconstriction potency are very low in comparison to SRTX-a and SRTX-b, whereas its IC50 for 125I-SRTX-b binding is similar to that of SRTX-b. It is suggested that the Thr to Ser substitution, which is shared by two additional weak members of the endothelin/sarafotoxin family, SRTX-c and ET-3, affects the biological activity of SRTX-d as well.     

Competitive interaction between endothelin and sarafotoxin: Binding and phosphoinositides hydrolysis in rat atria and brain

Binding studies with the structurally similar vasoconstrictor peptides 125I-endothelin and 125I-sarafotoxin b, the former of mammalian origin and the latter derived from snake venom, reveal their mutually exclusive binding to rat atrium and various regions of the rat brain. In these tissues endothelin, like sarafotoxin, induces phosphoinositide hydrolysis which is in part Ca2+-independent. It is suggested that endothelins and sarafotoxins share common binding sites and mechanisms of action.     

Conversion of porcine big endothelin to endothelin by an extract from the porcine aortic endothelial cells

Conversion of porcine big endothelin (big ET) to endothelin (ET) by an extract from cultured porcine aortic endothelial cells was investigated using a radioimmunoassay (RIA) specific for ET and reverse-phase high performance liquid chromatography (RP-HPLC). When big ET was incubated with the extract at an acid pH in the presence of E-64, a cysteine protease inhibitor, the amount of immunoreactive-ET (IR-ET) in the incubation mixture was greatly increased and the optimum pH for this increased reaction was 4.0. The extract-induced increase in IR-ET was completely inhibited by pepstatin-A. These immunoreactive alterations correlated with those in the vasoconstrictor activity. When the incubation mixture of big ET with the cell extract was applied to the RP-HPLC, the IR-ET eluted at the same retention time as seen with synthetic porcine ET. We suggest that a pepstatin-sensitive aspartic protease is involved in the conversion of big ET to ET in vascular endothelial cells.     

A comparison of the binding of endothelin to various tissues from spontaneously hypertensive and Wistar-Kyoto rats

The binding affinities for endothelin-1 and endothelin-3 to membrane preparations of various tissues of spontaneously hypertensive rats and normotensive Wistar-Kyoto rats were compared by competition binding of the peptides with [¹²⁵I]endothelin-1. Endothelin-1 binding data obtained using membrane preparations from brain, heart, kidney, liver, lung and spleen of both strains were better fit with a one-site model. The brain tissue demonstrated the highest affinity for endothelin-1 in both strains with the same IC₅₀ of 0.11 nM, while the kidney and lung tissues showed the lowest affinities in both strains with IC₅₀ values ranged between 1.4 and 4.1 nM. Only the kidney tissues of these two strains showed a statistically significant difference in binding affinities for endothelin-1; the IC₅₀ values were 1.4 ± 0.1 nM (mean ± SE, N = 3) and 3.2 ± 0.4 nM (n = 4) for the spontaneously hypertensive and normotensive rats, respectively. Endothelin-3 binding data obtained using membrane preparations from brain, kidney and lung of both strains were also better fit with a one-site model. In contrast, a two-site model was more suitable for analyzing endothelin-3 binding results obtained using membrane preparations from heart, liver and spleen of both strains. Again, only the kidney tissues of the two strains showed a statistically significant difference in binding affinities for endothelin-3. The ratio of IC₅₀ value of the major endothelin-3 binding site to that of endothelin-1 in each tissue varied from approx. 1.5 in brain, kidney and liver to greater than 500 in heart and spleen of both strains. Scatchard analysis of saturation binding data showed that [¹²⁵I]endothelin-1 bound to a single class of binding sites in brain, heart, liver and spleen of both rat strains and in kidney of the spontaneously hypertensive rats. Specific binding to the kidney membrane preparation of the normotensive rats was not saturable at radioligand concentrations up to about 2 nM. These results suggest that the tissues of both strains investigated have different affinities as well as different selectivities for endothelin-1 and endothelin-3. Furthermore, kidney is the only tissue examined which showed higher binding affinity in the spontaneously hypertensive rats than that of the normotensive ones.     

Obesity-associated activation of angiotensin and endothelin in the cardiovascular system

The renin-angiotensin system (RAS) and the endothelin system have been implicated in the pathogenesis of human cardiovascular and renal diseases, and inhibition of the RAS markedly improves morbidity and survival. Obesity in humans is associated with an increased risk for the development of hypertension, atherosclerosis and focal-segmental glomerulosclerosis, however the exact mechanisms underlying these pathologies in obese individuals are not known. This article discusses the clinical importance of obesity and the current evidence for local activation of the renin-angiotensin system and its interactions with the endothelin system in obesity and the cardiovascular pathologies associated with it.     

Cadmium directly acts on endothelin receptor and inhibits endothelin binding activity.

The binding of endothelin (ET) to human placenta ET receptor was strongly inhibited by cadmium ions (Cd2+) (IC50 = 2 x 10(-5) M). Experiments with affinity cross-linking showed that the major 40 kDa receptor was inhibited to form a [125I]ET-1/receptor complex. The mode of inhibition was noncompetitive with respect to ET-1. The inhibitory effect of Cd2+ on solubilized ET receptor was partially reversed by the chelating agent, ethylenediaminetetraacetic acid (EDTA), whereas the effect was irreversible for the membrane-associated receptor. The rat aorta contractions by ET were prevented by pretreatment or addition of Cd2+.     

Endothelin-1 and endothelin receptor antagonists in cardiovascular remodeling.

Endothelins build a peptide family composed of three isoforms, each of them containing 21 amino acids. Endothelin-1 is the isoform mainly responsible for any cardiovascular action and therefore the sole scope of this review. Endothelin-1 is the most potent endogenous vasoconstrictor known; in addition it acts as a potent (co)mitogen. There is a substantial body of experimental evidence that endothelin-1 may contribute not only to sustained vasoconstriction, but also to remodeling within the cardiovascular system. Thus, with the help of endothelin receptor antagonists (available for a few years) the involvement of mainly ETA receptors in structural diseases such as heart failure, pulmonary hypertension, atherosclerosis, restenosis, systemic hypertension, and chronic renal failure has been shown. These data make endothelin receptor antagonists, and especially those selective for the ETA receptor, promising agents for the treatment of chronic cardiovascular diseases associated with remodeling. Currently several chemically distinct, orally available members of this novel class of therapeutic agents are under clinical investigation.     

Endothelin/sarafotoxin receptor heterogeneity: evidence for different glycosylation in receptors from different tissues.

Neuraminidase was used in an attempt to determine whether the endothelin (ET)/sarafotoxin (SRTX) receptor subtypes are glycoproteins and, if so, to determine the role of the carbohydrate moiety in the binding of ligands to the receptor. Incubation of rat cerebellar membranes with neuraminidase was accompanied by a decrease in the capacity of the receptors to bind ET-1 and SRTX-b. In contrast, treatment of the rat caudate putamen and strium or of guinea pig ileum with the enzyme did not affect the binding properties of these receptors. Following exposure of [125I]-ET-1 affinity-labeled receptor to neuraminidase, gel electrophoresis and autoradiography revealed a decrease in molecular mass in the cerebellar and atrial preparations of about 2.5-2.8 kDa. These data indicate that some of the ET/SRTX receptors are glycoproteins and that the sugar moiety is important for ligand binding. Thus, glycosylation might be responsible for the observed heterogeneity among ET/SRTX receptors.