Endothelin Isopeptides Research Articles

In rats, atrial natriuretic peptide secretion is regulated differently in the right and left atria.

Recent studies have suggested the secretion of Atrial Natriuretic Peptide (ANP) is regulated by receptor mediated activation of protein kinase C, which causes the autocrine release of prostaglandins. Prostaglandins stimulate ANP secretion via the adenylate cyclase second messenger system. This report examined the response of right and left atrial ANP secreting cells to the three endothelin isopeptides and to cyclooxygenase inhibition. Our results show that right atrial ANP secretion is stimulated by endothelin 1 and 2 but not 3. In addition, right atrial ANP secretion is reduced by inhibition of cyclooxygenase. In contrast, left atrial ANP secretion is stimulated by endothelin 2 and 3 but not 1. Inhibition of cyclooxygenase did not affect left atrial ANP secretion. These results show the regulation of ANP secretion is different between the two atrial chambers. Right atrial cells appear to contain the prostaglandin-mediated response to protein-kinase C activation, whereas left atrial cells regulate ANP secretion differently.

Endothelin peptides.

Application of gene knockout techniques to endothelin research has led to the revelation of unexpected and interesting roles for endothelin isopeptides and their receptors. Homozygous mice with null endothelin-1 and ETA receptors presented with craniofacial and cardiovascular malformations, indicating that these receptors are essential for the development of the first branchial arch derived from the neural crest. Homozygous mice with null endothelin-3 and ETB receptors presented with megacolon (Hirschsprung's disease) associated with spotted colour coats, indicating that these latter receptors are essential for the development of neural crest-derived cell lineage, enteric neuron and epidermal melanocytes. Isolation and characterization of endothelin-converting enzyme which cleaves big endothelin-1, an intermediate form, into mature endothelin-1 has revealed a type II integral membrane-bound metalloprotease that is structurally homologous to neutral endopeptidase. Development of selective inhibitors for endothelin-converting enzyme isoenzymes will facilitate a greater understanding of the role of endogenous endothelin isopeptides.

2421 Intimate relationship between endothelins and catecholamines through ET-A receptor

Ca2+ signaling by peptides of the endothelin (ET) gene family was studied in cultured glomerular mesangial cells. In addition to the increase in cytosolic free [Ca2+] ([Ca2+]i) previously described for ET-1, we also observed that ET-2, ET-3, and sarafotoxin S6b generate similar [Ca2+]i waveforms but with dissimilar potencies and kinetics. The prepro form of ET-1 was inactive, suggesting that mature ET peptides are constrained in an inactive conformation within the preproET species. ET isopeptides caused both release of Ca2+ from intracellular stores and Ca2+ influx via a voltage- and dihydropyridine-insensitive pathway. ET-mediated Ca2+ influx was independent of the increase in [Ca2+]i. Activation of protein kinase C inhibited ET-induced Ca2+ signaling, whereas addition of ET to protein kinase C-depleted cells resulted in enhanced [Ca2+]i waveforms. Mesangial cells also demonstrated a marked adaptive desensitization response to ET. These data demonstrate that Ca2+ signaling is a common response to different ET peptides in glomerular mesangial cells and that activation of protein kinase C down-regulates these Ca2+ signals.

Methoxamine-induced inhibition of the positive inotropic effect of endothelin via α1-adrenoceptors in the rabbit heart

The influence of methoxamine on the positive inotropic effect of endothelin was assessed in the isolated rabbit ventricular myocardium. Methoxamine by itself elicited a positive inotropic effect and it simultaneously inhibited the positive inotropic effects of endothelin-1 and endothelin-3 without affecting the acceleration of the hydrolysis of phosphoinositide that was induced by the endothelin isopetptides. By contrast, the positive inotropic effects induced by elevation of concentration of external Ca 2+ ions, by Bay k 8644 (methyl 1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl)-pyridine-5-carboxylate), by dihydroouabain and by forskolin were unaffected by methoxamine. The inhibitory action of methoxamine was abolished by α 1-adrenoceptor antagonists, such as prazosin, WB 4101 (2-(2,6-dimethoxyphenoxyethyl)aminomethyl-1,4-benzodioxane hydrochloride) and (±)-tamsulosin; and it was inhibited to a lesser extent by chlorethylclonidine. In addition, methoxamine did not modify the specific binding of [ 125I]endothelin-3 to ventricular membrane fraction. These results indicate that methoxamine antagonizes the positive inotropic effect of endothelin isopeptides at the level of the signal-transduction process, subsequent to acceleration of the hydrolysis of phosphoinositide, via activation of α 1-adrenoceptors in the rabbit ventricular myocardium.

Nature of endothelin binding in the porcine ovary.

We investigated the nature of endothelin (ET) binding in the porcine ovary. We demonstrated the presence of high affinity (Kd = 0.72; 95% confidence interval = 0.43-1.1 nM) binding sites for ET-1 in the porcine ovary. The binding capacity for this ET-1-specific binding site was 97 pmol/micrograms DNA (95% confidence interval = 90-107). Autoradiographic studies showed that putative ET receptors reside in the granulosa cell layer of the maturing Graafian follicle and in the vascular components of the corpora lutea. The relative abundance of ET receptors was greatest in granulosa cells of large antral follicles, whereas ET binding was absent in granulosa cells of preantral follicles and in luteal cells. ET binding by cultured granulosa cells was further characterized by RRA and shown to exhibit a rank order of binding affinities for different ET isopeptides. The observed rank order indicates that the ET receptors present on granulosa cells are of the ET(A) receptor subtype. The radioreceptor studies also indicated that granulosa cells collected from large antral follicles (9-10 mm in diameter) have a greater binding capacity for [125I]ET-1 in culture than granulosa cells collected from smaller follicles. When cultured granulosa cells were exposed to 100 nM ET-1 or 14 nM 12-O-tetradecanoylphorbol 13-acetate overnight, the percentage of specific [125I]ET-1 binding was reduced (12% and 50%, respectively), indicating a down-regulation of the ET receptor by these treatments. In summary, we have characterized the distribution, isopeptide specificity, relative abundance, and down-regulation of putative ovarian endothelin receptors of subtype ET(A) on swine granulosa cells. Such results in conjunction with other available literature strongly suggest that granulosa cells of maturing Graafian follicles are targeted by ET-1. An additional physiological role for ET-1 in the ovary is suggested by the presence of putative ET receptors in the vasculature of the corpus luteum.

Dissecting the factors involved in the pathogenesis of sodium retention in cirrhosis with ascites using TIPS: [formula omitted]. Depts of Medicine∗, Radiology† & Nuclear Cardiology‡, Toronto Hospital, Toronto, Canada

Endothelin (ET), a potent endothelium-derived vasoconstrictor peptide [1] contributes to the regulation of glomerular function both through changes in hemodynamics and through changes in glomerular configuration. Three structurally and pharmacologically distinct isoforms of the peptide have been identified with a molecular weight of 2.5 kDa, each consisting of 21 amino acids. These isopeptides share a common structure with a hydrophobic C-terminal end and two disulfide bonds, thus forming a hairpin loop at the amino terminus. ET-1 differs from ET-2 by only two amino acids, and from ET-3 by six amino acids. Each ET isopeptide is encoded by separate genes at distinct chromosomal loci [2].All ETs are synthesized via proteolytical cleavage from specific prohormones (Fig. 1). Prepro ETs are long polypeptide chains consisting of ∼203 amino acids [1] which exhibit specific differences among the isopeptides. ET isopeptide precursors are proteolytically cleaved by processing endopeptidases that recognize paired basic amino acids (Arg-Arg or Lys-Arg) and thus form pro or big ET. Pro ET-1 is then converted to mature ET-1 by cleavage of the Trp21-Val22 bond via a highly specific endothelin-1-converting enzyme, which is a Phosphoramidon-sensitive, membrane bound metalloprotease (Fig. 1) [3–5]. Pro ET-3 is less susceptible to this protease, so that the presence of other types of endothelin-converting enzyme are suspected [5].ETs bind to two G-protein coupled receptors of ∼43 to ∼73 kD molecular weight [6–8], consisting of ∼415 to ∼427 amino acids and having seven transmembrane domains [6, 7]. The ETA receptor binds ET-1 with 2- to 10-fold higher affinity than ET-2 and > 100-fold higher affinity than ET-3 [6–9], whereas the ETB receptor has similar affinities to all three peptides [7, 8].Little is known about the fate of ET. ETs are either inactivated by receptor internalization or by enzymatic cleavage of the peptide prior to receptor coupling [10–13].

Therapeutic effects of endothelin receptor antagonists in stroke.

The presence of the endothelin isopeptides and endothelin receptors on neurons, glial cells and brain capillary endothelium suggests that endothelins may play a significant role in neuromodulation, astrocytic function and in regulation of cerebral blood flow. Furthermore, endothelins may play a significant role in the central regulation of neuroendocrine and autonomic nervous system functions (i.e., plasma volume, cardiovascular and respiratory control). Endothelin has potent cerebrovascular and proliferative effects suggesting a pathogenic role in cerebrovascular diseases. Endothelin receptors may represent important therapeutic targets for the treatment of both hemorrhagic and ischemic stroke. A review of the available data on endothelin levels and the effects of endothelin antagonists in cerebrovascular diseases is provided in the present report. Most notably is evidence in support of increased brain endothelin levels in hemorrhagic and ischemic stroke both in animal models and in humans. Also, endothelin receptor antagonists exert significant efficacy in animal models of cerebrovascular disease. For example, SB 209670, a rationally designed, potent, nonpeptide endothelin receptor antagonist, exerts therapeutic efficacy in reducing vasospasm following subarachnoid hemorrhage and neuroprotection following ischemic stroke. Certainly the available data warrants further evaluation of novel, selective endothelin receptor antagonists or endothelin converting enzyme inhibitors in cerebrovascular diseases.

Role of G proteins in the vasodilator response to endothelin isopeptides in vivo

The purpose of the present study was to determine the influence of pertussis toxin (PTX) on the pulmonary and systemic vasodilator responses to endothelin (ET) isopeptides in the intact cat under conditions of constant pulmonary blood flow and left atrial pressure. When pulmonary vasomotor tone was actively increased by an intralobar arterial infusion of U-46619, intralobar arterial bolus injections of ET-1, ET-2, and ET-3 decreased lobar arterial pressure and systemic vascular resistance in a dose-related manner. The vasodilator responses to ET-1 and ET-2 in the cat lung were abolished by PTX pretreatment, whereas PTX pretreatment did not alter the pulmonary vasodilator response to ET-3 and cromakalim, a specific ATP-sensitive potassium (KATP) channel activator, and the systemic vasodilator responses to all ET isopeptides studied. Glipizide, an inhibitor of KATP channels, inhibited the pulmonary vasodilator responses to ET-1, ET-2, and ET-3, whereas the systemic vasodilator responses to these isopeptides were not changed. The present data are the first to provide a functional correlate in vivo suggesting the existence of different signal transduction mechanisms for two pulmonary vascular ET receptor subtypes, ETA-like that is PTX sensitive and has greater sensitivity to ET-1 and ET-2 (than to ET-3) and ETc-like that is PTX insensitive and has sensitivity to ET-3 (than to ET-1 and ET-2). However, both ET-receptor subtypes promote vasodilation in the adult pulmonary vascular bed by activating KATP channels.(ABSTRACT TRUNCATED AT 250 WORDS)

Endothelin-induced interleukin-6 production by rat aortic endothelial cells.

Endothelin (ET) produced by endothelial cells has recently been found to be a potent vasoconstricting hormone. In this paper, ET isopeptides, both ET-1 and ET-3, were shown to be potent stimulators of interleukin (IL)-6 production by a rat aortic endothelial cell clone, WAE-1. Semi-quantitative polymerase chain reaction analysis indicated that augmentation of IL-6 production is due to an increase in IL-6 messenger RNA level. Ligand binding assay indicated that most of the [125I]ET-1 binding sites correspond to ET receptor type A (ETAR). However, ET receptor type B (ETBR) was shown to be also present on this cell line by reverse transcription polymerase chain reaction using ETBR-specific primers and by ligand binding assay using [125I]ET-3, although the protein receptor level is much lower than that of ETAR. ET-1, but not ET-3, induced inositol 1,4,5-triphosphate production and an increase in intracellular Ca2+ concentration with similar dose response. These data suggest that ET-1 stimulates IL-6 production through ETAR-phospholipase C activation axis, whereas ET-3 stimulates IL-6 production through different signaling pathway. The results shown in this paper raise the possibility that ET plays a role in inducing inflammation in endothelium.

Effects of endothelin isopeptides in cultured human hepatic stellate cells (HSC)

Effects of endothelin isopeptides in cultured human hepatic stellate cells (HSC)

Endothelins stimulate mitogen-activated protein kinase cascade through either ETA or ETB

Endothelin (ET) has been shown to activate mitogen-activated protein kinase (MAPK). However, it has been unclear which of the ET receptors is coupled to MAPK activation. In the present study, we conducted experiments to determine which ET receptor is linked to MAPK activation. We found that both human ETA and ETB were coupled to the MAPK cascade in ETA or ETB cDNA-transfected Chinese hamster ovary cells. ET-1 was more potent than ET-3 in the activation of p42 MAPK, induction of MAPK kinase (MAPKK) gel retardation and uptake of [3H]thymidine in ETA-transfected cells, whereas sarafotoxin (S6c) showed no stimulatory effect on the kinases and [3H]thymidine uptake. ET-1, ET-3, and S6c had approximately the same potency to activate p42 MAPK, MAPKK gel retardation, and [3H]thymidine uptake in ETB-transfected cells. These data suggest that 1) ET isopeptides, through either ETA or ETB receptors, induce the MAPK cascade as well as cell proliferation; and 2) the different potencies of ET isopeptides for activation of the MAPK cascade and induction of cell growth are mainly due to their different affinities toward ETA and ETB.

Endothelin stimulates mitogen-activated protein kinase activity in mesangial cells through ETA.

Accumulating evidence suggests that endothelin (ET) contributes to the pathophysiology of such disorders as acute renal failure, cyclosporine-mediated renal and vascular toxicity, and perhaps even glomerular inflammation. The postreceptor signaling pathways that mediate the actions of ET in these pathophysiologic conditions may include activation of kinase cascades. Thus, the effects of ET isopeptides on p42 and p44 mitogen-activated protein (MAP) kinase activity in rat glomerular mesangial cells were examined. ET-1 activated both p42 and p44 MAP kinases with similar dose responses and different kinetics. The threshold for kinase activation was 10(-9) M ET-1. ET-1 stimulated p42 and p44 MAP kinases with similar rapid (5 min) but different sustained activation of p42 (3 to 6 h) and p44 (1 to 2 h). Endothelin-3 (ET-3) also activated both isoforms of MAP kinase but with a threshold at 10(-7) M. Compared with ET-1, ET-3 stimulated only a rapid increase of p42 MAP kinase activity. We further investigated which ET receptors are coupled to MAP kinase activation. BQ-123, an ETA blocker, completely blocked the responsiveness of the MAP kinase to either ET-1 or ET-3. In Chinese hamster lung fibroblasts transfected with ETA or ETB cDNA, both receptors showed a rapid stimulation of MAP kinase in response to ET-1. These results suggest that ET can activate MAP kinases through both ET receptors but act exclusively through ETA in glomerular mesangial cells.

A rapid and versatile method for screening endothelin converting enzyme activity

A flexible two-step protocol has been developed for high-throughput assays of endothelin converting enzyme (ECE) activity. The method is illustrated using endothelial cell mono-layers and crude cell extracts as enzyme sources for conversion of exogenous substrates (big endothelin isopeptides) to their respective vasoactive products (endothelin isopeptides). Cleavage conditions have been optimized to minimize substrate utilization (reducing cost) while maintaining sensitivity and selectivity for mature endothelin detection in receptor- and antibody-based assays. Endothelins are quantified in a separate step and comparable estimates of mature endothelin formation are obtained using both EIA and radioreceptor assays in 96-well formats. Human umbilical vein (HUVEC) and human aorta (HAEC) endothelial cells characterized by this method are found to preferentially convert the big endothelin-1 (big ET-1) isopeptide through a membrane-bound, thiorphan-insensitive, and phosphoramidon-sensitive zinc metalloendopeptidase. Both intact cells and membrane preparations used as the enzyme source predict similar IC 50 values for phosphoramidon inhibition of ECE (ca 1 μM). The procedure described is simple, rapid, and suitable for high-volume screening.

Characterization of endothelin (ET) receptors in the isolated gall bladder of the guinea-pig: evidence for an additional ET receptor subtype.

1. We have characterized the receptors mediating contractions induced by endothelin-1 (ET-1), ET-2, ET-3 and the ETB-selective receptor agonists, sarafotoxin 6c (SX6c), IRL 1620, BQ-3020, [Ala1,3,11,15]ET-1 and ET (16-21) in strips of the isolated gall bladder of the guinea-pig (GPGB). We used as antagonists BQ-123 (ETA receptor selective) and PD 145065 (ETA/ETB receptor non-selective). 2. ET-1, ET-2 and ET-3 (10(-10) M to 3 x 10(-7) M) caused similar slowly-developing concentration-dependent contractions of the GPGB. Contractile effects induced by ET-1, ET-2 or ET-3 (at 3 x 10(-7) M) were also similar (230 +/- 25, 241 +/- 7 and 287 +/- 37% of that to histamine at 5 x 10(-6) M, n = 7, 6, 12, respectively). However, the threshold concentration for ET-1 or ET-2 was 10(-10) M whereas it was 3 x 10(-9) M for ET-3. 3. SX6c (10(-10) M to 3 x 10(-7) M) also caused slowly-developing concentration-dependent contractions at a threshold concentration of 10(-10) M (n = 16). However, the contraction caused by SX6c at 3 x 10(-7) M was 116 +/- 9% of that to histamine at 5 x 10(-6) M, which was half of that induced by the same concentration of the ET isopeptides. The contraction induced by IRL 1620 at 3 x 10-7 M (n = 9) was 43 +/- 9% of that to histamine at 5 x 10-6 M, which was one fifth of that produced by the same concentration of ET-1. Contractions induced by BQ-3020 or [Ala1,3,11,15]ET-I at 3 x 10-7 M were even less than those produced by IRL 1620. ET (16-21) was inactive up to 10-5 M. Addition of a concentration of 3 x 10-7 M of ET-1 to tissues with developed contractions induced by the bolus addition of 3 x 10-7 M SX6c caused a further contraction of the GPGB to the level observed with ET-1 alone at 3 x 10-7M (n = 8).4. BQ-123 (10-5 M) did not affect the concentration-response curve to ET-1 and the contraction induced by 3 x 10- M was also not affected (n = 5; 239 +/- 19% of histamine at 5 x 10-6 M). PD 145065(10-5 M) shifted the ET-1 concentration-response curve to the right and the contraction induced by ET-1at 3 x 10-7 M was inhibited by 15% (n = 6; NS). A higher concentration of BQ-123 (10-4 M) caused a significant shift to the right of the ET-1 concentration-response curve similar to that caused by PD 145065 (10-s M) and caused a 24% (n = 6; NS) inhibition of the contractions induced by ET-1 at 3 x 10-7 M. PD 145065 (10-4 M) abolished contractions induced by ET-1 (up to 10- M) and inhibited the response to ET-1 at 3 x 10-7 M by 52% (n = 4; P< 0.05).5. Contractions induced by ET-3 were more sensitive to inhibition by the antagonists. BQ-123 (10-6,10-5 or 10-4 M) inhibited responses to 3 x 10-7 M ET-3 by 66, 71 and 83%, respectively (n = 5, 5, 3;P< 0.05). PD 145065 (10-6, 10-5 or 10-4 M) attenuated more strongly than did BQ-123 the contractions induced by ET-3. For instance, the contractions caused by ET-3 at 3 x 10-7 M were decreased by 73 and 80% (n = 5, 5; P<0.05) in the presence of PD 145065 (10-6 or 10-5 M, respectively). PD 145065(10-4 M) completely abolished contractions to ET-3 (n = 4; up to 3 x 10-7M).6. Contractions induced by SX6c, especially those observed at concentrations lower than 10-8 M, were attenuated by BQ-123 (up to 10-4 M). PD 145065 (10-5M) shifted to the right the concentration response curve to SX6c and inhibited by 38% (P<0.05) the contractions induced by 3 x 10-7M.However, the contractions induced by a bolus addition of a high concentration of SX6c (3 x 10-7 M)and the subsequent addition of an identical concentration of ET-1 on top of SX6c were not affected byBQ-123 (10-6 or 10-5 M).7. These results suggest that ETB receptors are involved in the contractions induced by endothelins in the GPGB. However, SX6c and other selective ETB agonists produced only half or less than half of the contractile response induced by non-selective agonists. In addition, the responses to ET-1 but not to ET-3, were insensitive to the antagonist action of BQ-123 at 10-5 M whereas BQ-123 or PD 145065 at 10-5 M strongly antagonized contractions induced by ET-3. Finally, BQ-123 at 10-4 M inhibited contractions to ET-1 and SX6c. Thus, within the GPGB there may well be additional ET receptor(s) not conforming to the established ETA/ETB receptor subtype classification, as well as ETB receptors.

Mutational analysis of human endothelin receptors ETA and ETB identification of regions involved in the selectivity for endothelin 3 or cyclo-(D-Trp-D-Asp-Pro-D-Val-Leu).

Two endothelin(ET)-receptor subtypes have been identified in mammals. They differ in their affinity towards the ET isopeptides with ETA displaying an ET-1-selective profile and ETB a non-selective one. To identify the regions responsible for the differential selectivity, chimeric forms were engineered by sequentially exchanging extracellular regions together with their flanking transmembrane domains. Two sets of reciprocal receptor mutants were thereby generated and analysed by expression in COS-7 cells. The recombinant receptor chimeras were characterised by direct and competitive radioligand-binding analysis. COS-7 cells transfected with vectors for the mutant receptors exhibited specific saturable [3-125I]iodotyrosyl ET-1 (125I-ET-1) binding, with affinities comparable to those of the wild-type receptors (apparent Ki approximately 1-6 x 10(-9) M). An average of 10(5)-10(6) binding sites/cell was calculated for the wild-type and mutant forms. In competition experiments using 125I-ET-1 and unlabeled ET-3, an ETB-selective agonist, we detected a clear switch from an ET-1-selective profile to a non-isopeptide-selective profile in ETA chimeras where the second extracellular loop and the flanking transmembrane domains IV and V, or the third extracellular loop and the flanking transmembrane domains VI and VII, had been exchanged for the corresponding parts of ETB. The opposite effect, namely a switch from a non-isopeptide-selective to an ET-1-selective binding, was observed for the mirror ETB chimeras where the symmetrical exchange had been operated. Using 125I-ET-1 and the ETA-specific antagonist cyclo-(D-Trp-D-Asp-Pro-D-Val-Leu) (BQ123), we were able to map the main determinants responsible for this selectivity to the N-terminal moiety of this receptor. Therefore, the ability for the interaction with ET-3 or BQ123 is governed by two different regions of the ET receptors.

Differential regulation of c-fos gene expression by two types of human endothelin receptor in Chinese hamster ovary cells

To investigate the nuclear signalling pathway induced by endothelin (ET) isopeptides, we have established permanent Chinese hamster ovary (CHO) cell lines, CHO-ETA/fos-lacZ and CHO-ETB/fos-lacZ, that produce both a c-fos-beta-galactosidase fusion protein and either the type A or the type B human ET receptor. These cell lines permitted a colorimetric measurement of c-fos expression, which was induced by the signal transduction system with ET receptors and ET isopeptides. We found that the ET-1-dependent c-fos expression was so efficient that it could respond to low concentrations (even a physiological concentration) of ET-1. For example, CHO-ETA/fos-lacZ and CHO-ETB/fos-lacZ responded to ET concentrations of 5 x 10(-9) M and 5 x 10(-13) M respectively. Using this highly sensitive system, the H-7 sensitive protein kinase was found to be involved in signal transduction mediated by ETA, and also partly in the ETB-mediated pathway. These lines of evidence suggest that c-fos expression occurs through at least two different pathways, depending on the concentration of ET in plasma.

Mutation of peptide binding site in transmembrane region of a G protein-coupled receptor accounts for endothelin receptor subtype selectivity.

The molecular basis for endothelin (ET) isopeptide selectivity between ETA and ETB receptors was studied by examining ligand binding to several site-specific mutants of the human ETA receptor. Based on a computer-built three-dimensional model of the ETA receptor, five non-conserved amino acids, clustered around the putative ligand binding site, were targeted for mutation to alanine. Expression of the wild-type and mutant ETA receptors in COS-7 cells revealed that the binding profile of one of the ETA mutants, Tyr129-->Ala, was characteristic of the ETB receptor. In the Tyr129-->Ala ETA receptor mutant the affinity of two ETB-selective agonists, endothelin-3 and sarafotoxin S6c, was increased 10-200-fold, whereas that for two ETA-selective antagonists, BQ-123 and BMS-182874, was reduced 350-2,000-fold. Thus, mutation of a single amino acid in the second transmembrane region of the wild-type ETA receptor results in subtype conversion. In addition, these data represent the first example of peptide interactions with a transmembrane region of a G protein-coupled receptor and indicate that Tyr129, located in the second transmembrane region of the ETA receptor, is a critical component for determination of endothelin receptor subtype-selective ligand binding.

Endothelin-stimulated secretion of natriuretic peptides by rat atrial myocytes is mediated by endothelin A receptors.

Endothelin (ET), a potent vasoconstrictor peptide, is known to enhance the secretion of atrial natriuretic factor (ANF) by the heart. In the present study, we investigated the potency of ET isopeptides to stimulate ANF and brain natriuretic peptide (BNP) secretion in primary cultures of neonatal atrial myocytes, and we characterized the receptor mediating these effects. All ET isopeptides caused a twofold increase of ANF and BNP secretion with the following order of potency: ET-1 approximately ET-2 > sarafotoxin 6b >> ET-3. Secretion of the natriuretic peptides was blocked by BQ-123, an ETA-receptor antagonist, but was not affected by either IRL-1620 or [Ala1,3,11,15]ET-1, two ETB-receptor agonists. ET receptors were localized by autoradiography on the surface of atrial myocytes, indicating that contaminating cells were not responsible for 125I-ET-1 binding. Competition binding analyses were then used to assess the ET-receptor subtype on atrial myocyte membrane preparations. A high-affinity (100 pmol/L) binding site with high density (approximately 1500 fmol/mg) was found to preferentially bind the ET isopeptides in the following order: ET-1 > or = ET-2 > or = sarafotoxin 6b > ET-3. Binding was totally displaced by BQ-123 but not by IRL-1620. The ET binding site therefore had the characteristics of an ETA-like receptor. Analysis by cross-linking and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that it possessed a molecular mass of approximately 50 kD. Northern blot analysis of both ETA- and ETB-receptor mRNAs allowed only the detection of the former, indicating that the ETB receptor may be expressed in very small amounts. These results demonstrate that ANF and BNP secretion by atrial myocytes is enhanced by ET via binding to an ETA-like receptor.

Renal actions of endothelin: Linking cellular signaling pathways to kidney disease

Endothelin (ET), a potent endothelium-derived vasoconstrictor peptide [1] contributes to the regulation of glomerular function both through changes in hemodynamics and through changes in glomerular configuration. Three structurally and pharmacologically distinct isoforms of the peptide have been identified with a molecular weight of 2.5 kDa, each consisting of 21 amino acids. These isopeptides share a common structure with a hydrophobic C-terminal end and two disulfide bonds, thus forming a hairpin loop at the amino terminus. ET-1 differs from ET-2 by only two amino acids, and from ET-3 by six amino acids. Each ET isopeptide is encoded by separate genes at distinct chromosomal loci [2]. All ETs are synthesized via proteolytical cleavage from specific prohormones (Fig. 1). Prepro ETs are long polypeptide chains consisting of ∼203 amino acids [1] which exhibit specific differences among the isopeptides. ET isopeptide precursors are proteolytically cleaved by processing endopeptidases that recognize paired basic amino acids (Arg-Arg or Lys-Arg) and thus form pro or big ET. Pro ET-1 is then converted to mature ET-1 by cleavage of the Trp 21 -Val 22 bond via a highly specific endothelin-1-converting enzyme, which is a Phosphoramidon-sensitive, membrane bound metalloprotease (Fig. 1) [3–5]. Pro ET-3 is less susceptible to this protease, so that the presence of other types of endothelin-converting enzyme are suspected [5]. ETs bind to two G-protein coupled receptors of ∼43 to ∼73 kD molecular weight [6–8], consisting of ∼415 to ∼427 amino acids and having seven transmembrane domains [6, 7]. The ET A receptor binds ET-1 with 2- to 10-fold higher affinity than ET-2 and > 100-fold higher affinity than ET-3 [6–9], whereas the ET B receptor has similar affinities to all three peptides [7, 8]. Little is known about the fate of ET. ETs are either inactivated by receptor internalization or by enzymatic cleavage of the peptide prior to receptor coupling [10–13].

Effects of vasoactive intestinal contractor on voltage-activated Ca2+ currents in feline parasympathetic neurons.

Intracellular current-clamp and single-electrode voltage-clamp techniques were used to study in vitro action potentials and the action of vasoactive intestinal contractor (VIC; 0.03-1 microM) on the high-voltage-activated Ca2+ currents (ICa) of neurons in feline colonic parasympathetic ganglia. In the current-clamp recording mode, action potential amplitude was depressed by cobalt (1 mM) and omega-conotoxin (300 nM) or in nominally Ca(2+)-free Krebs solutions. In the single-electrode voltage-clamp recording mode, the ICa was isolated by blocking the voltage-gated Na+ current with tetrodotoxin (1-3 microM) and by Krebs solutions containing a low Na+ concentration. The voltage-activated K+ currents were blocked by intracellular injection of cesium through a recording electrode filled with 2 M CsCl and external application of tetraethylammonium (30-50 mM) and barium (2 mM). The Ca(2+)-dependent Cl- current was blocked by replacement of Ca2+ (2 mM) with equimolar barium. Anomalous rectification was blocked by external application of 2 mM cesium. The ICa was evoked by depolarizing step commands more positive than -40 mV from holding potentials ranging between -80 and -60 mV. ICa was depressed by cobalt (1 mM), cadmium (100 microM), and omega-conotoxin (500 nM) but not by nifedipine (10 microM), nicardipine (10 microM), and verapamil (10 microM). BAY K 8644 (3-10 microM) also did not affect the ICa. VIC (0.1-1 microM), one of the endothelin (ET) isopeptides, caused an inward current followed by an outward current. The VIC-induced inward and outward currents were associated with an increase and decrease in membrane conductance, respectively. VIC also caused an initial depression followed by a long-lasting augmentation of the ICa. ET-1, ET-2, and ET-3 equally mimicked the action of VIC on both holding current and ICa. These data suggest that VIC activates a receptor-operated channel and modulates the omega-conotoxin-sensitive voltage-activated Ca2+ channels through ETB receptor subtypes of neurons in feline colonic parasympathetic ganglia.