Determination of the levels of novel 31-amino acid endothelins and endothelins in human lungs

An effective method for determination of the levels of newly discovered 31-amino acid endothelins [ETs(1-31)] as well as big ETs and 21-amino acid ETs [ETs(1-21)], in human lungs has been developed. About 85% of ETs in human lung homogenates were recovered on acid extraction 8 times. Most of the published protocols for the determination of tissue ETs involve a reverse-phase minicolumn to separate proteins from peptides, after which the levels of ETs are directly determined by enzyme immunoassay. The levels determined, however, include fairly high amounts of non-bioactive ET metabolites in tissues and the data reported are diverse. We established an effective methods for the extraction and the separation of nine different muscle constricting ETs from their metabolites on a reverse-phase C18 column. Using this protocol, the levels of ETs in human lungs were determined by means of a sandwich-enzyme immunoassay specific for each ET derivative. The levels of ET-2(1-21) were the highest among those of ETs, and the levels of ETs(1-31) were in a similar range to those of big ETs but were lower than those of ETs(1-21). This method can be utilized to assess the pathophysiological roles of ETs(1-31) in various human organs.     

Effect of endothelin-1(1-31) on p38 mitogen-activated protein kinase in cultured human mesangial cells

It was reported that human chymase cleaves big endothelins (ETs) at the Tyr31-Gly32 bond and produces 31-amino acid ETs(1-31). In this study, we investigated the effect of ET-1(1-31) on p38 mitogen-activated protein kinase (p38-MAPK) activity in human mesangial cells (HMCs). By measuring the kinase activity, we demonstrated that ET-1 (1-31) activated the p38-MAPK dose-dependently (10(-9) M to 10(-7) M), which was inhibited by SB203580. The p38-MAPK activation induced by ET-1(1-31) peaked at 10 minutes. BQ123 almost abolished ET-1(1-31)-induced p38-MAPK activation, whereas BQ788 failed to inhibit it. These findings suggest that the stimulatory effect of ET-1(1-31) on p38-MAPK activation is mediated through ET(A) or ET(A)-like receptor. In conclusion, ET-1(1-31) induced increase in p38-MAPK activation in cultured HMCs.     

Effect of endothelin-1(1-31) on intracellular free calcium in cultured human mesangial cells

We found that human chymase selectively produces 31-amino-acid length endothelins (1-31) (ETs(1-31)). We investigated the effect of synthetic ET-1(1-31) on intracellular free Ca2+ concentration ([Ca2+]i) in cultured human mesangial cells. ET-1(1-31) increased [Ca2+]i in a concentration-dependent manner to a similar extent as ET-1. The ET-1 (1-31)-induced [Ca2+]i increase was not influenced by removal of extracellular Ca2+ but was inhibited by thapsigargin. ET-1(1-31)-induced [Ca2+]i increase was not affected by phosphoramidon. It was inhibited by BQ123, but not by BQ788. These results suggest that ET-1(1-31) by itself exhibits bioactive properties probably through endothelin ET(A) or ET(A)-like receptors. Since human chymase has been reported to exist in the kidney, ET-1(1-31) may be a candidate substance for mesangium-relevant diseases.     

Endothelin receptors in human parathyroid gland.

We studied whether specific receptors for endothelins (ETs) exist in human parathyroid tissues and whether ETs may have any effect on secretion of PTH from parathyroid cells. Binding studies using [125I]ET-1 to the parathyroid membranes obtained from patients with hyperparathyroidism (2 adenomas, 2 hyperplasias) revealed that ET-1 competitively inhibited the binding of [125I]ET-1 to the membranes (the apparent Kd: 62 +/- 18 pM), whereas ET-3 showed biphasic and less steep inhibition curve than ET-1 in all tissue membranes examined. Northern blot analysis of poly(A)+ RNA from the parathyroid adenoma clearly demonstrated gene expression of both ETA and ETB receptors as well as preproET-1. ET-1 inhibited basal PTH secretion from dispersed adenoma cells more potently than ET-3. The present study clearly demonstrates the presence of both ETA and ETB receptor subtypes in human parathyroid tissues through which ETs may modulate PTH secretion in an autocrine and/or paracrine manner.     

Abundance of endothelin-3 in rat intestine, pituitary gland and brain

We established a highly sensitive and specific sandwich-enzyme immunoassay (EIA) for endothelin-3 (ET-3), which showed no crossreactivity with endothelin-1 (ET-1), endothelin-2 (ET-2) and big-endothelin-1 (big-ET-1). We had previously established a sensitive sandwich-EIA for ET-1, which fully crossreacted with ET-2, but not with ET-3 or big-ET-1. These EIAs were used to examine the tissue distribution of immunoreactive (ir-) ET-3 and compare them with those of ir-ET-1 (including ir-ET-2) in Sprague-Dawley rats. High concentrations of ir-ET-3 were found in the intestine, lung, pituitary gland and brain (greater than 100 pg/g wet tissue), ir-ET-1(ET-2) showed widespread distribution, with large amounts in the lung and colon (greater than 1000 pg/g wet tissue). The pituitary gland was the only organ containing higher amounts of ir-ET-3 than ir-ET-1 (ET-2). In reverse phase-high performance liquid chromatography coupled with EIAs, the ir-ET-3 was exclusively eluted at the position of synthetic ET-3, indicating that the ir-ET-3 was identical to ET-3. The abundance of ET-3 in the intestine, pituitary gland and brain indicates that ET-3 is a new brain-gut peptide which may have a physiological function in nervous and endocrine systems.     

Role of endothelin type B receptor in NO/cGMP signaling pathway in rat median eminence

We studied the effect of endothelins (ETs) on receptor-mediated NO/cGMP signaling in rat arcuate nucleus–median eminence (AN-ME) fragments, an hypothalamic structure known to contain a rich plexus of nitric oxide synthase (NOS)-containing neurons and fibers together with densely arranged ET_B-receptor-like immunoreactive fibers. NOS activity was determined measuring the conversion of [³H] arginine to [³H] citrulline, as an index of NO produced. cGMP production was determined by radio immunoassay. ET-1, ET-3, and the selective ET_B receptor agonist, IRL1620, significantly increased cGMP formation and NOS activity. Preincubation of AN-ME fragment with L-arginine analog, N-nitro-L-arginine (L-NAME), inhibited ET-1 or IRL1620-stimulated cGMP formation. The addition of the selective ET_B receptor antagonist, BQ788, blocked ET-1-, ET-3-, or IRL1620-induced increase in NOS activity and cGMP generation, while BQ123, a selective ET_A receptor antagonist, was ineffective. Our results demonstrate that in whole rat AN-ME fragments, ETs stimulate NO/cGMP signaling pathway through the interaction with the ET_B receptor subtype, supporting the concept that ETs may represent an important regulator of reproductive and neuroendocrine function.     

Short-term regulation of tyrosine hydroxylase activity and expression by endothelin-1 and endothelin-3 in the rat posterior hypothalamus

Brain catecholamines are involved in several biological functions regulated by the hypothalamus. We have previously reported that endothelin-1 and -3 (ET-1 and ET-3) modulate norepinephrine release in the anterior and posterior hypothalamus. As tyrosine hydroxylase (TH) is the rate-limiting enzyme in catecholamine biosynthesis, the aim of the present work was to investigate the effects of ET-1 and ET-3 on TH activity, total enzyme level and the phosphorylated forms of TH in the rat posterior hypothalamus. Results showed that ET-1 and ET-3 diminished TH activity but the response was abolished by both selective ET(A) and ET(B) antagonists (BQ-610 and BQ-788, respectively). In addition ET(A) and ET(B) selective agonists (sarafotoxin S6b and IRL-1620, respectively) failed to affect TH activity. In order to investigate the intracellular signaling coupled to endothelins (ETs) response, nitric oxide (NO), phosphoinositide, cAMP/PKA and CaMK-II pathways were studied. Results showed that N(omega)-nitro-l-arginine methyl ester and 7-nitroindazole (NO synthase and neuronal NO synthase inhibitors, respectively), 1H-[1,2,4]-oxadiazolo[4,3-alpha]quinozalin-1-one and KT-5823 (soluble guanylyl cyclase, and PKG inhibitors, respectively) inhibited ETs effect on TH activity. Further, sodium nitroprusside and 8-bromoguanosine-3',5'-cyclic monophosphate (NO donor and cGMP analog, respectively) mimicked ETs response. ETs-induced reduction of TH activity was not affected by a PKA inhibitor but it was abolished by PLC, PKC and CaMK-II inhibitors as well as by an IP(3) receptor antagonist. On the other hand, both ETs did not modify TH total level but reduced the phosphorylation of serine residues of the enzyme at positions 19, 31 and 40. Present results suggest that ET-1 and ET-3 diminished TH activity through an atypical ET or ET(C) receptor coupled to the NO/cGMP/PKG, phosphoinositide and CaMK-II pathways. Furthermore, TH diminished activity may result from the reduction of the phosphorylated sites of the enzyme without changes in its total level. Taken jointly present and previous results support that ET-1 and ET-3 may play a relevant role in the modulation of catecholaminergic neurotransmission in the posterior hypothalamus of the rat.     

Endothelin-1[1-31] acts as a selective ETA-receptor agonist in the rat adrenal cortex

Endothelin-1 (ET-1) is a 21-amino acid residue (ET-1[1-21]) hypertensive peptide, which together with its receptor subtypes A and B (ETA and ETB) is expressed in the rat adrenal cortex, where it stimulates steroid-hormone (aldosterone and corticosterone) secretion through the ETB receptor and the growth (proliferative activity) of the zona glomerulosa (ZG) through the ETA receptor. ET-1[1-21] is generated from bigET-1 by the endothelin-converting enzyme (ECE-1). However, recent evidence indicates the existence of an alternative chymase-mediated biosynthetic pathway leading to the production of an ET-1[1-31] peptide, which was found to reproduce the ETA receptor-mediated vascular effects of ET-1[1-21]. We found that ET-1[1-21], but not ET-1[1-31], concentration-dependently raised steroid secretion from dispersed rat adrenocortical cells, its effect being blocked by the ETB-receptor selective antagonist BQ-788. Both ET-1s concentration-dependently increased the number of "S-phase" cells (as detected by the 5-bromo-2'-deoxyuridine immunocytochemical method) in capsule-ZG strips within a 240 min incubation. The ZG proliferogenic action of both ET-1s was blocked by the ETA-receptor antagonist BQ-123, and ET-1[1-31] was found to be significantly more potent than ET-1[1-21]. Autoradiography showed that in the rat adrenal ET-1[1-21] displaced the binding of selective ligands to both ETA ([125I]PD-151242) and ETB receptors ([125I]BQ-3020), while ET-1[1-31] eliminates only the binding to ETA receptors. Collectively, our findings provide strong evidence that ET-1[1-31] acts in the rat adrenal glands as a selective ETA-receptor agonist, mainly involved in the stimulation of ZG proliferative activity.     

Cultured bovine endothelial cells convert big endothelin isopeptides to mature endothelin isopeptides

The incubation of big endothelin-1 (big ET-1), big ET-2 or big ET-3 with cultured bovine endothelial cells (ECs) resulted in their conversions to mature endothelins (ETs). These conversions apparently exhibited Michaelis-Menten kinetics as a function of each big ET isopeptide. The conversions of big ETs were abolished by phosphoramidon. These results indicate that vascular endothelium can convert exogenous big ET-1 to mature ET-1 through a phosphoramidon-sensitive metalloprotease, and that this enzyme has also high affinities for big ET-2 and big ET-3.     

Hyperglycemia triggers abnormal signaling and proliferative responses in Schwann cells

Peripheral neuropathy is a serious diabetic complication. Delayed nerve regeneration in diabetic animal models suggests abnormalities in proliferation/differentiation of Schwann cells (SC). We recently reported that endothelins (ETs) regulate proliferation and phenotype in primary and immortalized SC (iSC). We now investigated changes in the effects of ETs on SC proliferation and signaling in nerve segments from streptozotocin-induced diabetic rats and in iSC exposed to high glucose. Cultured explants from diabetic rats displayed a delay in the time-course of [³H]-thymidine incorporation as well as enhanced sensitivity to endothelin-1 (ET-1) or insulin. iSC cultured in high (25 mM) glucose-containing media also exhibited higher [³H]-thymidine incorporation, along with an enhanced activation of p38 mitogen-activated protein kinase and phospholipase C in response to ET-1 or platelet-derived growth factor as compared to controls (5.5 mM glucose). These studies support an extra-vascular role of ETs in peripheral nerves and SC. The increased sensitivity to ET-1 in nerves and iSC exposed to high glucose may contribute to abnormal SC proliferation characterizing diabetic neuropathy.     

An endothelin B receptor-selective antagonist: IRL 1038, [Cys11-Cys15]-endothelin-1(11-21)

In the inhibition of specific binding of [125I]endothelins (ETs) to membrane from various tissues of rats, guinea pigs, pigs and humans, [Cys11-Cys15]-ET-1(11-21), IRL 1038, has a much higher affinity for ETB receptors (Ki = 6-11 nM) than for ETA receptors (Ki = 0.4-0.7 microM). In contraction assays, with ET-3 as a stimulant, 3 microM IRL 1038 antagonized the ETB receptor-mediated contraction of guinea pig ileal and tracheal smooth muscle without any significant agonistic activity, but did not effect the ETA receptor-mediated contraction of rat aortic smooth muscle. IRL 1038 is therefore, considered to be the first antagonist selective to the ETB receptor.     

Endothelin-1 and -3 diminish neuronal NE release through an NO mechanism in rat anterior hypothalamus

The existence of endothelin binding sites on the catecholaminergic neurons of the hypothalamus suggests that endothelins (ETs) participate in the regulation of noradrenergic transmission modulating various hypothalamic-controlled processes such as blood pressure, cardiovascular activity, etc. The effects of ET-1 and ET-3 on the neuronal release of norepinephrine (NE) as well as the receptors and intracellular pathway involved were studied in the rat anterior hypothalamus. ET-1 (10 nM) and ET-3 (10 nM) diminished neuronal NE release and the effect blocked by the selective ET type B receptor antagonist BQ-788 (100 nM). N(omega)-nitro-L-arginine methyl ester (10 microM), methylene blue (10 microM), and KT5823 (2 microM), inhibitors of nitric oxide synthase activity, guanylate cyclase, and protein kinase G, respectively, prevented the inhibitory effects of both ETs on neuronal NE release. In addition, both ETs increased nitric oxide synthase activity. Furthermore, 100 microM picrotoxin, a GABA(A)-receptor antagonist, inhibited ET-1 and ET-3 response. Our results show that ET-1 as well as ET-3 has an inhibitory neuromodulatory effect on NE release in the anterior hypothalamus mediated by the ET type B receptor and the involvement of a nitric oxide-dependent pathway and GABA(A) receptors. ET-1 and ET-3 may thus diminish available NE in the synaptic gap leading to decreased noradrenergic activity.     

Endothelin expression in the uterus

The endothelins (ETs) comprise a family of 21 amino acid peptides, ET-1, ET-2 and ET-3, first demonstrated as products of vascular endothelium. Subsequent work showed that they are also found in non-endothelial cells from a variety of tissues such as breast, parathyroid and adrenal gland. At first, the ETs were recognized for their pressor effects. However, ET administration in vivo initially caused hypotension at low concentrations by triggering the paracrine release of endothelial-derived vasodilators. The ETs exert powerful contractile actions on myometrium and other types of smooth muscle and are mitogenic, or co-mitogenic for fibroblasts, vascular smooth muscle and other cells. Demonstration of extravascular ET in endometrium has revealed a powerful vasoconstrictor which might act on the spiral arterioles to effect a powerful and sustained contraction of vascular smooth muscle. ETs might also contribute to the process of endometrial repair. In addition, the ETs appear to play a fundamental role in the control of uterine function in pregnancy. Effects on myometrial contractility have been implicated in the mechanisms governing the onset of normal and pre-term labour, and the peptides are likely to be key determinants of placental blood flow by binding to vascular smooth muscle receptors in the placenta.     

Effect of a novel vasoconstrictor endothelin-1 (1-31) on human umbilical artery

To clarify the action of a novel endothelin-1 with 31 amino acids, ET-1 (1-31), on fetal circulation, its vasoconstrictive activity on human umbilical and uterine arteries was investigated in comparison with that of a conventional ET-1 (1-21). UFER micro-easy magnus was used for determination of vasoconstriction. The contraction of umbilical artery by KCl was significantly weaker than that of the uterine artery. In ETs, constriction by KCl was set as control, and the rate of constriction of uterine and umbilical arteries was used for comparison. The constriction of human uterine artery induced by ET-1 (1-31) was also significantly weaker than that by ET-1 (1-21). On the contrary, ET-1 (1-31) was a potent constrictor on the umbilical artery equally to ET-1 (1-21). The present study is the first to demonstrate that ET-1 (1-31) has a contractile activity on human vessels. Furthermore, the regulatory mechanism on constriction of umbilical artery is different from that observed in a systemic vessel, indicating a particularly important role of ET-1 (1-31) in fetal circulation.     

Proliferative and morphological effects of endothelins in Schwann cells: roles of p38 mitogen-activated protein kinase and Ca(2+)-independent phospholipase A2.

The regulation of Schwann cell (SC) proliferation and morphology is critical to nerve homeostasis. We have previously reported that endothelins (ETs) regulate the activity of different effectors in SC including adenylyl cyclase, phospholipases C and A2 and mitogen-activated protein kinases (MAPKs). These effects imply a possible participation of ETs in the regulation of SC phenotype. We have now investigated the effects of endothelins on the proliferation and morphology of SC, and compared them with the responses to platelet-derived growth factor (PDGF), a known mitogen in these cells. Both endothelin-1 (ET-1) and PDGF increased the incorporation of [3H]thymidine and the proportion of SC in S and G2/M, with a concomitant decrease in the G0/G1 stage cells. Treatment with ET-1 produced rapid changes in the morphology of the SC, characterized by the appearance of cell spreading with shorter processes. The response to ET-1 was considered to represent a proliferative phenotype, in contrast to the effects of forskolin, which decreased [3H]thymidine incorporation in immortalized SC (iSC) and lead to a differentiated morphology with longer extensions. While both ET-1 and PDGF displayed a proliferative effect on SC, treatment with PDGF did not affect the morphology of these cells to a significant extent. A role for p38 MAPK and Ca(2+)-independent phospholipase A2 in the changes in morphology and proliferation of iSC driven by ET-1 was suggested by the effects of selective inhibitors of these pathways [SB202190 and HELSS, respectively]. The unique pattern of signaling pathways recruited by ET-1 and its combined effects on regulation of phenotype and proliferation of SC suggest an important role for this peptide during nerve degeneration/regeneration.     

The endothelin-A receptor subtype transduces the effects of the endothelins in the anterior pituitary gland.

All members of the mammalian endothelin family of peptides exert significant effects on prolactin and luteinizing hormone release from dispersed anterior pituitary cells in vitro. The rank order of potency for the prolactin inhibiting effects of the endothelins is ET-1 = ET-2 much less than ET-3. This suggests an involvement of the ET-A receptor subtype. The selective ET-A receptor antagonist BQ-123 antagonized the effects of the ETs in a competitive fashion with pA2 values of 6.1 (ET-1), 5.7 (ET-2) and 6.4 (ET-3), when added simultaneously with the ETs. This suggests the involvement of the ET-A receptor subtype in the actions of the ETs within the anterior pituitary gland.     

Involvement of nitric oxide pathways in short term modulation of tyrosine hydroxylase activity by endothelins 1 and 3 in the rat anterior hypothalamus

The ability of endothelins 1 and 3 (ET-1 and ET-3) to reduce neuronal norepinephrine release through ETB receptor activation involving nitric oxide (NO) pathways in the rat anterior hypothalamus region (AHR) was previously reported. In the present work, we studied the effects of ET-1 and -3 on tyrosine hydroxylase (TH) activity and the possible involvement of NO pathways. Results showed that ET-1 and -3 (10 nM) diminished TH activity in AHR and this effect was blocked by a selective ETB receptor antagonist (100 nM BQ-788), but not by a ET(A) receptor antagonist (BQ-610). To confirm these results, 1 microM IRL-1620 (ET(B) agonist) reduced TH activity whereas 300 nM sarafotoxin S6b falled to modify it. N(omega)-Nitro-L-arginine methyl ester (10 microM), 7-nitroindazole (10 microM), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-ona (10 microM), KT5823 (2 microM), inhibitors of nitric oxide synthase, neuronal nitric oxide synthase, NO-sensitive-guanylyl cyclase, and protein kinase G, respectively, did not modify the reduction of TH activity produced by ETs. In addition, both 100 microM sodium nitroprusside and 50 microM 8-bromoguanosine-3',5'-cyclic monophosphate (NO donor and guanosine-3',5'-cyclic monophosphate analog, respectively) diminished TH activity. Present results showed that ET-1 and ET-3 diminished TH activity through the activation of ET(B) receptors involving the NO/guanosine-3',5'-cyclic monophosphate/protein kinase G pathway. Taken jointly present and previous results it can be concluded that both ETs play an important role as modulators of norepinephrine neurotransmission in the rat AHR.     

Aerobic exercise training reduces plasma endothelin-1 concentration in older women.

Endothelial function deteriorates with aging. On the other hand, exercise training improves the function of vascular endothelial cells. Endothelin-1 (ET-1), which is produced by vascular endothelial cells, has potent constrictor and proliferative activity in vascular smooth muscle cells and, therefore, has been implicated in regulation of vascular tonus and progression of atherosclerosis. We previously reported significantly higher plasma ET-1 concentration in middle-aged than in young humans, and recently we showed that plasma ET-1 concentration was significantly decreased by aerobic exercise training in healthy young humans. We hypothesized that plasma ET-1 concentration increases with age, even in healthy adults, and that lifestyle modification (i.e., exercise) can reduce plasma ET-1 concentration in previously sedentary older adults. We measured plasma ET-1 concentration in healthy young women (21-28 yr old), healthy middle-aged women (31-47 yr old), and healthy older women (61-69 yr old). The plasma level of ET-1 significantly increased with aging (1.02 +/- 0.08, 1.33 +/- 0.11, and 2.90 +/- 0.20 pg/ml in young, middle-aged, and older women, respectively). Thus plasma ET-1 concentration was markedly higher in healthy older women than in healthy young or middle-aged women (by approximately 3- and 2-fold, respectively). In healthy older women, we also measured plasma ET-1 concentration after 3 mo of aerobic exercise (cycling on a leg ergometer at 80% of ventilatory threshold for 30 min, 5 days/wk). Regular exercise significantly decreased plasma ET-1 concentration in the healthy older women (2.22 +/- 0.16 pg/ml, P < 0.01) and also significantly reduced their blood pressure. The present study suggests that regular aerobic-endurance exercise reduces plasma ET-1 concentration in older humans, and this reduction in plasma ET-1 concentration may have beneficial effects on the cardiovascular system (i.e., prevention of progression of hypertension and/or atherosclerosis by endogenous ET-1).     

Endothelin Signaling Pathways in Rat Adrenal Medulla

1. We further characterized the effect of endothelins (ETs) on receptor-mediated phosphoinositide (PI) turnover, nitric oxide synthase (NOS) activation, and cGMP formation in whole rat adrenal medulla.2. The PI hydrolysis was assessed as accumulation of inositol monophosphates (InsP₁) in the presence of 10 mM LiCl in whole tissue and the analysis of inositol-1-phosphate by Dowex anion exchange chromatography. NOS activity was assayed by monitoring the conversion of radiolabeled L-arginine to L-citrulline. Cyclic GMP formation was assessed as accumulation of cGMP in whole tissue in the presence of phosphodiesterase inhibition, and the amount of cGMP formed was determined by radioimmuno-antibody procedure.3. ET-1 and ET-3 increased PI turnover by 30% in whole adrenal medulla prelabeled with [³H] myoinositol. Both ETs isoforms, at equimolar doses, increased NOS activity and cGMP levels in similar degree. The selective ET_B receptor agonist, IRL-1620, also increased cGMP formation, mimicking the effects of ETs, while IRL-1620 did not alter the PI metabolism. ETs-induced InsP₁ accumulation and cGMP was dependent on extracellular calcium. The effect of ETs on PI turnover was inhibited by neomycin. The L-arginine analogue, N-nitro-L-arginine (L-NAME), and two inhibitors of soluble guanylyl cyclase, methylene blue and ODQ, significantly inhibited the increase in cGMP production induced by ETs or IRL-1620. The selective ET_A receptor antagonist, BQ 123, inhibited the ETs-induced increase in PI turnover, while the selective ET_B receptor antagonist, BQ 788, was ineffective. Likewise, BQ 788, significantly inhibited ET-1- or ET-3-induced NOS activation and cGMP generation but not ETs-induced InsP₁ accumulation.4. Our data indicate that stimulation of PI turnover and NO-induced cGMP generation constitutes ETs signaling pathways in rat adrenal medulla. The former action is mediated through activation of ET_A receptor, while the latter through the activation of ET_B receptor. These results support the role of endothelins in the regulation of adrenal medulla function.