Soon after the discovery of endothelin (ET), analysis of the ET gene revealed the existence of two other ETs, named ET-2 and ET-3. Endothelin-1 is the original ET which endothelial cells produce exclusively (2). Amino acid sequences of these three isoforms are very similar. In addition, all of these three endogenous ETs are distributed in various tissues and cells in different proportions. This result as well as the pharmacological data suggest multiple functions of ETs.
Interestingly, soon after the first publication on ET, the structure of a rare snake venom sarafotoxin (STX) was reported. Surprisingly, it was very similar to that of the ETs. At least four STXs, i.e. STXa, STXb, STXc and STXd which are similar to ETs in their structure, are known so far. They also have 21 amino acid residues. Interestingly, several amino acid residues of the carboxyl terminus of ETs and STXs are very similar. Those peptides bind to the receptor by this carboxyl terminus.
Further important progress in ET research came with the successful cloning of cDNA for the ET receptor. Two types of ET receptor were cloned, named Endothelin receptor-A (ETA) and endothelin receptor-B (ETB) (3,4). Both receptors belong to a family of the heptahelical G-protein coupled receptor. Two receptor subtypes are different in their affinities to endogenous ligands. ETA has a high affinity to ET-1 and ET-2 and a low affinity to ET-3. ETB has an equally potent affinity to all of the three ligands. In addition, distribution of the two subtypes in various types of vascular beds is different. Basically, ETA exists on smooth muscle cells and mediates vasoconstriction. Endothelin receptor-B is distributed on endothelial cells and mediates release of relaxing factors, such as prostacyclin and nitric oxide. However, recent research showed that ETB also exists on smooth muscle cells of several kinds of vein and mediates vasoconstriction. The ETB on the smooth muscle cells is pharmacologically different from the ETB on endothelium. Several nonselective ET receptor antagonists such as PD142893 can discriminate between those two ETB subtypes, but those two subtypes cannot be discriminated on the basis of their molecular biology. They named them ETB1 (endothelial ETB) and ETB2 (smooth muscle ETB). These two ETB subtypes are distributed throughout various blood vessels and species in different patterns. Additionally, recent reports demonstrated that activated ETB1 resulted in release of adrenomedullin and C-type natriuretic peptide as well as nitric oxide and prostacyclin from endothelium.
BIOSYNTHESIS OF ENDOTHELIN
In the initial publication of ET, we described a novel process of ET biosynthesis according to the sequence analysis of ET cDNA. The precursor of human ET has 212 amino acid residues. After cleavage of the signal peptide at the amino terminus, the resulting peptide, proendothelin was further cleaved by an endopeptidase specific for a pair of dibasic amino acids, resulting in an intermediate form, big ET-1, which has 38 amino acid residues. The big ET-1 is further cleaved to the mature ET-1 by an enzyme which was named ET-converting enzyme (ECE). Recent studies demonstrated that the former endopeptidase is a furin-like endopeptidase and that the cleavage of the proendothelin by this enzyme is an essential step for the cleavage by ECE.
Endothelin-converting enzyme was cloned in 1994. The first ECE was named ECE-1 (5,6). Endothelin-converting enzyme-1 is a highly glycosylated neutral metalloprotease which is 130 kDa in size. It has a single transmembrane domain near the amino-terminus. There are two types of ECE-1 which differ by their aminoterminal amino acid sequences, named ECE-1α/a and ECE-1β/b (7). Another enzyme termed ECE-2 was also identified. It is activated at acidic pH. Both ECE-1 and ECE-2 cleaved big ET-1 more efficiently than either big ET-2 or big ET-3. The ECE-1α is expressed ubiquitously, with highest levels of expression in the endothelium, lung, ovary, testis and adrenal medulla, while the ECE-1β/b is expressed in neural tissues. Very recently, another type of ECE was purified from bovine iris and named ECE-3, which selectively cleaved big ET-3.
G-PROTEIN AND SIGNAL TRANSDUCTION
As mentioned above, both ET-receptors are heptahelical G-protein coupled receptors. Endogenous ligand ET binds to the receptor, resulting in activation of the G-protein. At least three kinds of Gα-proteins can be coupled to the ET-receptor. Interestingly, a different domain of the ET-receptor structure is required for the coupling of different G-proteins. Gαs is coupled to the second intracellular loop of the receptor. The third intracellular loop is also involved to some extent in this coupling. Three cysteine residues in the cytoplasmic tail of the ETB receptor can be palmitoylated, resulting in formation of the fourth intracellular loop. Similarly, carboxyl-terminal cysteine residues of ETA can be palmitoylated and form the fourth loop. For the activation of Gαi protein, the third intracellular loop, the fourth intracellular loop and the carboxyl-terminal tail sequence of ETB are critical. For the Gαq coupling of ETA and ETB, the fourth intracellular loop is essential.
After the coupling of Gα-protein, the second messenger system is activated. When Chinese hamster ovary (CHO) cells transfected with human ETA were stimulated by ET-1, Gαs was activated, resulting in activation of adenylate cyclase. Gαq activated phospholipase C and probably nonselective cation channel. Endothelin-induced contraction via ETA is mediated by activation of the latter nonselective cation channel. Activation of ETB led to Gαi and Gαq activation. The Gαi activation led to the inhibition of adenylate cyclase and probably activation of voltage-opereted calcium channel (VOC).
MECHANISM OF ENDOTHELIN-INDUCED VASOCONSTRICTION
Endothelin induces characteristic sustained vasoconstriction. It is generally accepted that the major part of the sustained contraction by ET-1 requires the persistent entry of extracellular calcium ion.
After activation of the ETA receptor, the intracellular free calcium ion level increased transiently followed by a sustained phase at the raised level. The transient phase results from the calcium ion release from the intracellular calcium pool and the sustained phase is caused by influx of the exterior calcium ion via the calcium channel of the plasma membrane. At low concentration of ET-1, only the sustained phase can be observed. Then a question arises as to what kind of ion channel is involved in the calcium influx stimulated by ET.
We demonstrated that at least two types of nonselective cation channel as well as voltage-operated calcium channel were activated by ET-1. One was activated by low and the other by high concentrations of ET-1. Neither channel was sensitive to nifedipine. Those two channels can be discriminated by an inhibitor of nonselective cation channel, SK&F96365. The nonselective cation channel activated by a high concentration of ET-1 was inhibited by SK&F96365 but that activated by a low concentration of ET-1 was not affected. Interestingly, this nonselective cation channel activated by ET-1 was inhibited by nitric oxide and therefore ET and nitric oxide affect the same channel.
Another important function of the ET receptor is to mediate growth activity of cells such as smooth muscle or endothelial cells. This activity is mediated in two ways, i.e. classical protein kinase C-dependent and independent ways. It is accepted that the ET-induced cell growth activity is mediated by ETA. However, there are only a few reports that provided evidence that ETB also mediated cell growth activity. Furthermore in some cases ETB was reported to suppress cell growth. We demonstrated that in human melanoma cell A375, ETB mediated differentiation and apoptosis and suppressed cell growth in a cell cycle-dependent manner.
PHYSIOLOGICAL AND PATHOPHYSIOLOGICAL SIGNIFICANCE
What is the physiological function of ET in the vascular bed? Since the concentration of circulating ET-1 in plasma is very low, it may be impossible that the circulating ET-1 regulates the vascular tone. If endogenous ET-1 regulates the vascular function, it may act as a local hormone. It was demonstrated that most ET-1 was secreted from endothelium abluminally. The released ET-1 acts on neighboring endothelial cells themselves and underlying smooth muscle cells in a paracrine and autocrine manner. The ET-1, in turn, stimulates endothelium to release relaxing factors such as nitric oxide and prostacyclin, while it acts on smooth muscle to elicit contraction. Then a question arises as to whether ET-1 is a relaxing or a constricting factor.
Recently several papers have demonstrated that ET plays some role in maintenance of vascular tone in peripheral vascular beds. Webb's group demonstrated that when BQ-123, an ETA antagonist, was infused into the forearm of normal subjects, the forearm blood flow increased (8), suggesting that endogenous ET-1 plays a role in maintenance of vascular tone. Similarly, several groups demonstrated that infusion of ETB antagonist induces sustained hypertension, suggesting that ET plays some role in reduction of blood pressure via ETB. Vascular ET-1 has two faces, depending on the vascular beds examined. In the human forearm vascular bed, the constricting action of endogenous ET-1 probably predominates over the relaxing action.
Accumulating evidence has demonstrated that ET plays a harmful role in several disorders. At the Fifth International Conference on Endothelin held in Kyoto in 1997, a large part of the discussion was devoted to the pathophysiology in the cardiovascular system (9,10). A consensus emerged in the conference that ET antagonists showed a significant beneficial effect in several pathological conditions including: chronic heart failure, pulmonary hypertension, salt-sensitive hypertension, cerebral vasospasm after subarachnoid hemorrhage, renal insufficiency, etc. In these diseases the plasma level of ET-1 increases and the ET antagonist appears to show a beneficial effect.
In chronic heart failure, there are now many reports which demonstrate a beneficial effect of the ET antagonist. The symptoms of chronic heart failure are greatly improved by infusion with ETA or nonselective antagonists. Survival rate of chronic heart failure with infarction in the rat model was greatly improved by treatment with ET antagonist. Furthermore, in rats with infarction, cardiac myocytes hypertrophied and the surviving myocardium produced ET-1 markedly. The released ET-1 from the myocardium probably acts on neighboring cells in a paracrine and autocrine manner, resulting in cell growth. The ET antagonist improved this phenomenon.
Similarly, in atherosclerotic vascular tissues, production of ET-1 in smooth muscle cells was reportedly enhanced, and it might act on the neighboring cells to induce cell growth activity. The ET antagonist reportedly improved this process.
The hypertrophy is regarded as an adaptive phenomenon. However, excessive hypertrophy, is an aggravating effect of ET-1, which was totally protected against by ET antagonists.
Although ET antagonists show a beneficial effect in those diseases such as chronic heart failure, pulmonary hypertension and vasospasm after subarachnoid hemorrhage, the question whether the ETA antagonist or the ETA/ETB antagonist is more useful in humans remains to be solved.
Several papers have demonstrated that ET antagonists were effective in salt-sensitive hypertension. The kidney is also a target organ of ET. Acute renal failure induced by ischemia or cyclosporin administration elicited an up-regulation of ET-1 production. The ET-1 produced acts on the renal blood vessels via ETA and reduced glomerular filtration rate and renal blood flow. On the other hand, ET is known to affect sodium absorption. It was shown that adult ETB-deficient mice and rats exhibited significantly elevated blood pressure. In those mice, hypertension was shown to be salt-sensitive and resistant to ETA blockade. Those results suggest that ETB plays some role in the hypertension.
Ten years have passed since the discovery of ET. A large amount of information has been accumulated. However, the precise mechanism of action of ET is still unclear.
In the cardiovascular system, endogenous ET-1 might play some role in regulatory mechanism of peripheral blood flow, and an important role in progression of several disorders. It is generally recognized that ET induces both beneficial and harmful effects in the living body. Further experimental results are needed to elucidate further the physiological and pathophysiological roles of ET.
1. Yanagisawa M, Kurihara H, Kimura S. A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature
2. Inoue A, Yanagisawa M, Kimura S, Kasuya Y, Miyauchi T, Goto K, et al. The human endothelin family: three structurally and pharmacologically distinct isopeptides predicted by three separate genes. Proc Natl Acad Sci USA
3. Arai H, Hori S, Aramori I, Ohkubo H, Nakanishi S. Cloning and expression of a cDNA encoding an endothelin receptor. Nature
4. Sakurai T, Yanagisawa M, Takuwa Y, Miyazaki H, Kimura S, Goto K, et al. Cloning of a cDNA encoding a non-isopeptide-selective subtype of the endothelin receptor. Nature
5. Shimada K, Takahashi M, Tanzawa K. Cloning and functional expression of endothelin-converting enzyme from rat endothelial cells. J Biol Chem
6. Xu D, Emoto N, Giaid A, Slaughter C, Kaw S, deWit D, et al. ECE-1: A membrane-bound metalloprotease that catalyzes the proteolytic activation of big endothelin-1. Cell
7. Shimada K, Takahashi M, Ikeda M, Tanzawa K. Identification and characterization of two isoforms of an endothelin-converting enzyme-1. FEBS Letts
8. Haynes WG, Webb DJ. Contribution of endogenous generation of endothelin-1 to basal vascular tone. The Lancet
9. Webb DJ, Monge JC, Rabelink TJ, Yanagaisawa M. Endothelin: new discoveries and rapid progress in the clinic. Trends Pharmacol Sci
10. Endothelin V. J Cardiovasc Pharmacol
1998;31 suppl. 1.
Proceedings of a satellite symposium to the 17th Scientific Meeting of the International Society of Hypertension Padua, Italy; June 12-13, 1998
Publication of this supplement was made possible thanks to the generous support of the University of Padua and Bayer Ag, Germany.