Purification and molecular cloning of carp ovarian cystatin

Endothelin converting enzyme (ECE) from intact cells of a permanent human endothelial cell line, EA.hy926, was studied by examining the effects of phosphoramidon, an endothelin converting enzyme inhibitor, on the levels of secreted endothelin-1 and big endothelin-1. The specific ECE activity was demonstrated by a phosphoramidon dose-dependent decrease in ET-1 level with a concomitant increase in big ET-1 level. By using a specific neutral endopeptidase 24.11 (NEP 24.11) inhibitor, thiorphan, it was also shown that the phosphoramidon-sensitive ET-1 degrading activity in this cell line is due to the NEP 24.11 activity. Other serine, acid, and cysteine protease inhibitors had no effect on the endogenous synthesis of ET-1 and big ET-1 supporting the evidence that ECE is insensitive to these protease inhibitors as has been demonstrated with the isolated enzyme.     

Phosphoramidon, an inhibitor of endothelin-converting enzyme, prevents indomethacin-induced gastric mucosal damage in rats

Endothelin-1 (ET-1) is produced from inactive precursor big ET-1 by endothelin-converting enzyme-1 (ECE-1), a membrane-bound metalloprotease, structurally similar to another metalloprotease, neutral endopeptidase 24.11 (NEP). Although both phosphoramidon and thiorphan are metalloprotease inhibitors, the ECE activity is inhibited by phosphoramidon but not by thiorphan, a specific inhibitor of NEP. Therefore, to investigate whether an ECE inhibitor can prevent indomethacin-induced gastric mucosal damage in rats, we compared the effects between the two metalloprotease inhibitors on both gastric mucosal integrity and the levels of ET-1 and big ET-1 in gastric tissue. Phosphoramidon significantly decreased ET-1 levels, causing a concomitant big ET-1 increase and dose-dependently attenuated indomethacin-induced gastric mucosal damage. By contrast, thiorphan neither changed the ratio of ET-1/big ET-1 nor attenuated the damage. In conclusion, the prevention of gastric mucosal damage by an ECE inhibitor indicates that endogenous ET-1 may play an important role in the pathogenesis of indomethacin-induced gastric mucosal damage.     

Characterization of the enzyme involved in the processing of big endothelin-1 in human lung epithelial cells

Biosynthesis of active endothelin-1 (ET-1) implies an enzymatic processing of the inactive precursor Big ET-1 (1-39) into the mature, 21 amino acid peptide. The aim of this study was to characterize in airway and alveolar epithelial cells the enzymes responsible for this activation. BEAS-2B and A549 cells, which both produce ET-1, were studied in vitro as models for bronchiolar and alveolar cells, respectively. Both cell lines were able to convert exogenously added Big ET-1 (0.1 microM) into ET-1, suggesting a cell surface or an extracellular processing. The conversion was inhibited by phosphoramidon in both cell lines with an IC50 approximately 1 microM, but not by thiorphan, a specific inhibitor of neutral endopeptidase 24.11 (NEP). The endogenous production of serum-stimulated BEAS-2B and A549 cells was not inhibited by thiorphan, and phosphoramidon showed inhibition only at high concentration (>100 microM). Western blotting following electrophoresis in reducing conditions demonstrated a protein of MR 110 corresponding to the ECE-1 monomer in both BEAS-2B and A549 cells, as well as in whole lung extracts. By RT-PCR we revealed the mRNA encoding for the ECE-1b and/or -1c subtype, but not ECE-1a, in both cell lines. We conclude that BEAS-2B and A549 cells are able to process either endogenous or exogenous Big ET-1 by ECE-1 and that isoforms 1b and 1c could be involved in this processing with no significant role of NEP.     

Lyme disease in outdoor workers on Assateague Island: high tick-exposure but low disease risk

Endothelin-1 (ET-1)-converting enzyme (ECE) activity in the human serum lipoprotein fraction was studied using a sensitive enzyme immunoassay and reverse-phase high performance liquid chromatography. The ECE activity of cleaving synthetic human big ET-1 into ET-1 by the serum lipoprotein fraction was about 14-times greater than that by whole serum, and the activity was closely associated with lipoprotein itself. The lipoprotein ECE activity, which was optimal at pH 7.0, was inhibited by EDTA, o-phenanthroline, phosphoramidon, thiorphan, phenylmethanesulfonyl fluoride and chymostatin, but not by cysteine or aspartic proteinase inhibitors, suggesting metalloproteinase- and chymotrypsin-like properties. These results suggest that the serum lipoprotein ECE may be involved in the processing of big ET-1 to ET-1 in the circulatory system.     

Simulating the Langevin force by simple noise in nuclear one-body dynamics

1. The profile of haemoconcentration induced by big endothelin-1(big ET-1), a precursor of endothelin-1 (ET-1), was compared with that induced by endothelin-1 in mice. 2. ET-1(1.5 nmol kg-1, i.v.) increased haematocrit in mice, which reached a maximum at 5 min and then returned to the control value within 30 min after the administration, this occurred at the same time as changes in the plasma immunoreactive endothelin-1 and rat atrial natriuretic peptide (rANP)-like activities (IR-ET-1 and IR-rANP, respectively). 3. Big ET-1(2.5-15 nmol kg-1, i.v.) also caused a significant and dose-dependent increase in haematocrit, that lasted over 3 h although elevated plasma IR-ET-1 and IR-rANP had almost been restored to the initial levels within 10 min after big ET-1 injection. 4. A metalloproteinase inhibitor, phosphoramidon (10 mg kg-1, i.v.), which inhibits the activity of endothelin converting enzyme (ECE), delayed the onset of big ET-1-induced haemoconcentration, but failed to alter the maximal value and the duration of the haemoconcentration. 5. Pretreatment with phosphoramidon (10 mg kg-1, i.v.) did not affect the big ET-1-induced change in plasma IR-ET-1, while significant delay of the disappearance of plasma IR-rANP and significant suppression of a sustained increase in tissue IR-ET-1 were observed. 6. These results suggest that ET-1, not in plasma but in tissue, plays an important role in the pathogenesis of big ET-1-induced long-lasting haemoconcentration, in which unknown factors besides rANP are involved.     

Central pathology review in clinical trials for patients with malignant glioma. A Report of Radiation Therapy Oncology Group 83-02

We previously described delayed pressor response (DPR) 3 h after endothelin (ET)-1 injection in normotensive rats. In the current study, we examined effects of the ETA receptor antagonist BQ123 (0.01 mumol/kg/min intravenously, i.v.), phosphoramidon (100 mumol/kg i.v.), the neutral endopeptidase inhibitor SQ28603 (112 mumol/kg + 0.04 mumol/kg/min i.v.), the angiotensin-converting enzyme inhibitor enalaprilat (10 mumol/kg i.v.), and the thromboxane receptor antagonist, SQ29548 (0.5 mumol/kg + 0.5 mumol/kg/h i.v.) on DPR. Vehicle and ET-1 (1.0 nmol/kg i.v.) were administered on day 1; vehicle or drug and ET-1 were administered on day 2. BQ123 inhibited DPR 36% (vehicle 44 +/- 5, BQ123 28 +/- 3 mm Hg); phosphoramidon inhibited DPR 56% (vehicle 45 +/- 4, and phosphoramidon 20 +/- 5 mm Hg). DPR was unchanged after SQ28603 (vehicle 39 +/- 2 and SQ28603 44 +/- 2 mm Hg), enalaprilat (vehicle 39 +/- 2 and enalaprilat 38 +/- 7 mm Hg), or SQ29548 (vehicle 46 +/- 6 and SQ29548 43 +/- 3 mm Hg). The results suggest that DPR 3 h after ET-1 injection in rats is mediated in part through ETA receptors. DPR does not appear to involve thromboxane or synthesis of angiotensin II (AII), but may be related to synthesis of ET-1.     

Pharmacological profile of T-0201, a highly potent and orally active endothelin receptor antagonist

The authors studied the pharmacological properties of N-(6-(2-(5-bromopyrimidin-4-yl)-4-(2-hydroxy-1, 1-dimethylethyl)benzensulfonamide sodium salt sesquihydrate (T-0201), a new nonpeptide endothelin (ET) receptor antagonist, in vitro and in vivo. In binding studies, T-0201 competitively antagonized the specific binding of [125I]-ET-1 to human cloned ETA receptors (the Ki value was 0.015 +/- 0.004 nM). T-0201 weakly inhibited [125I]-ET-1-binding to human cloned ETB receptors; the Ki value was 41 +/- 21 nM. T-0201 shifted the concentration-response curve of ET-1-induced contraction of the isolated rat aorta (ETA receptors) to the right (pA2 = 9.0 +/- 0.2). In the isolated rat trachea, a selective ETB agonist sarafotoxin S6c-induced contraction was inhibited by T-0201 (pA2 = 6.8 +/- 0.3). T-0201 also caused the inhibition of ET-1-induced contraction of the isolated rabbit pulmonary artery (pA2 = 5.7 +/- 0.3). In anesthetized rats, T-0201 (0.01-1 mg/kg) inhibited the pressor response to exogenous big ET-1 (1 nmol/kg i.v.), after both i.v. and p.o. administration, in a dose-dependent manner. The significant inhibitory effect of orally administered T-0201 on big ET-1-induced pressor response lasted for 4 hr at 0.1 mg/kg and for 8 hr at 1 mg/kg. Thus the present study demonstrates that T-0201 is a highly potent, long-lasting, orally active and selective ETA receptor antagonist.     

Use of the endothelin antagonists BQ-123 and PD 142893 to reveal three endothelin receptors mediating smooth muscle contraction and the release of EDRF

1. We have compared the receptors mediating the contractions of rings of rat thoracic aorta or rabbit pulmonary artery and rat stomach strips in response to the endothelin/sarafotoxin (ET/SX) family of peptides and to those mediating endothelium-dependent vasodilations within the isolated perfused mesentery of the rat. To discriminate ETA receptors from ETB receptors we have used the criteria that ET-1 is more active than SX6c on ETA receptors, and that the ET/SX peptides are equiactive on ETB receptors. We have also assessed the effects of the ETA receptor-selective antagonist BQ-123, and the non-selective ET receptor antagonist PD 142893 on the responses of each preparation to the ET/SX peptides. 2. ET-1-induced constrictions of the rat thoracic aorta (EC50 3 x 10(-10) M), a prototypic ETA receptor-mediated response, or isolated perfused mesentery of the rat were antagonized by BQ-123 (10(-5) M) or PD 142893 (10(-5) M). SX6c did not constrict either the rat isolated perfused mesentery or the rat thoracic aorta. Thus, ETA receptors mediate these constrictions. 3. ET-1 and SX6c were approximately equipotent in constricting rabbit pulmonary artery rings (EC50S 3-6 x 10(-10) M). Neither BQ-123 (10(-5) M) nor PD 142893 antagonized the contractions induced by ET-1. These effects suggest mediation by ETB receptors but PD 142893 (10(-5) M) did give a 3 fold antagonism of constrictions induced by SX6c. 4. SX6c was more potent than ET-1 in contracting the rat stomach strip (threshold concentrations 10(-10) and 3 x 10(-10) M). Contractions to ET-1 or SX6c were unaffected by BQ-123 (10-5 M), again indicative of ETB receptor-mediated events. PD 142893 (10-5 M) was ineffective against ET-1 but produced a 3 fold antagonism of SX6c.5. In the rat isolated perfused mesentery ET-1 or SX6c (0.3-300pmol) were equipotent in producing dose-related vasodilatations that were unaffected by BQ-123 (10-6 M), indicative of an ETB receptor mediated response. In contrast to the other ETB-mediated responses, PD 142893 (10-6 M) strongly antagonized these vasodilatations.6. Thus, ETA receptors mediate constrictions of the rat thoracic aorta and rat isolated perfused mesentery whereas ETB receptors mediate constrictions of the rabbit pulmonary artery and rat stomach strip and endothelium-dependent dilatations within the mesentery. However, within the group of ETB receptor-mediated responses, endothelium-dependent vasodilatations are sensitive to PD 142893, whereas contractions of the isolated smooth muscle preparations are not. Thus, the receptor present on the endothelium responsible for the release of nitric oxide in response to the ET/SX peptides is most probably different from that present on smooth muscle that mediates BQ-123-insensitive contractions.     

Inhibition of neutral endopeptidase causes vasoconstriction of human resistance vessels in vivo

BACKGROUND: Neutral endopeptidase (NEP) degrades vasoactive peptides, including the natriuretic peptides, angiotensin II, and endothelin-1. Systemic inhibition of NEP does not consistently lower blood pressure, even though it increases natriuretic peptide concentrations and causes natriuresis and diuresis. We therefore investigated the direct effects of local inhibition of NEP on forearm resistance vessel tone. METHODS AND RESULTS: Four separate studies were performed, each with 90-minute drug infusions. In the first study, 10 healthy subjects received a brachial artery infusion of the NEP inhibitor candoxatrilat (125 nmol/min), which caused a slowly progressive forearm vasoconstriction (12+/-2%; P=0.001). In a second two-phase study, 6 healthy subjects received, 4 hours after enalapril (20 mg) or placebo, an intra-arterial infusion of the NEP inhibitor thiorphan (30 nmol/min). Thiorphan caused similar degrees of local forearm vasoconstriction (P=0.6) after pretreatment with both placebo (13+/-1%, P=0.006) and enalapril (17+/-6%, P=0.05). In a third three-phase study, 8 healthy subjects received intra-arterial thiorphan (30 nmol/min), the endothelin ETA antagonist BQ-123 (100 nmol/min), and both combined. Thiorphan caused local forearm vasoconstriction (13+/-1%, P=0.0001); BQ-123 caused local vasodilatation (33+/-3%, P=0.0001). Combined thiorphan and BQ-123 caused vasodilatation (32+/-1%, P=0.0001) similar to BQ-123 alone (P=0.98). In a fourth study, 6 hypertensive patients (blood pressure >160/100 mm Hg) received intra-arterial thiorphan (30 nmol/min). Thiorphan caused a slowly progressive forearm vasoconstriction (10+/-2%, P=0.0001). CONCLUSIONS: Inhibition of local NEP causes vasoconstriction in forearm resistance vessels of both healthy volunteers and patients with hypertension. The lack of effect of ACE inhibition on the vasoconstriction produced by thiorphan and its absence during concomitant ETA receptor blockade suggest that it is mediated by endothelin-1 and not angiotensin II. These findings may help to explain the failure of systemic NEP inhibition to lower blood pressure.     

Hybrid peptides constructed from RES-701-1, an endothelin B receptor antagonist, and endothelin; binding selectivity for endothelin receptors and their pharmacological activity

Hybrid peptides were constructed from endothelin B receptor (ET(B)) selective antagonist RES-701-1 (1) and endothelin (ET-1). They have N-terminal 10 amino acids derived from 1 and C-terminal 10 amino acids derived from ET-1. RES-701-1(1-10)-[Ala15]ET-1(12-21) and its analogues substituted or truncated at the residues derived from RES-701-1 had proved to possess high receptor binding activity selective for ETB as well as 1. Substitutions at the residues derived from ET-1 had produced some analogues that possessed high affinity not only for ETB but for ETA. Although all analogues had antagonistic effects on ETA, some analogues had proved to function as agonist on ETB confirmed by the changes in intracellular calcium concentrations of ET receptor-transfected COS-7 cells. We have found four types of ET receptor-binding peptides: (1) ETB-selective agonist with weak ETA antagonism (3, KT7421); (2) ETB-selective antagonist with weak ETA antagonism (29, KT7539); (3) ETB agonist with potent ETA antagonism (27, KT7538); and (4) non-selective ETA/ETB antagonist (26, KT7540).     

Endopeptidase 24.11 inhibition does not modify uterotonic effects of endothelins in rat uterus

We investigated effects of the endopeptidase 24.11 inhibitor, SCH 39370, on uterotonic effects of endothelins (ETs) and sarafotoxin S6b. Responses of uteri from non-pregnant rats were inhibited by the ETA receptor antagonist, BQ123 (1 microM) but not the ETB receptor antagonist, BQ 788 (1 microM). ET-1, sarafotoxin S6b and ET-2 were more potent than ET-3 in tissues from non-pregnant and pregnant rats. SCH 39370 (10 microM) did not affect uterotonic responses to these peptides in either group, but inhibited those of big ET-1 in non-pregnant rat tissues, indicating inhibition of conversion of big ET-1 to ET-1. These data indicate that endopeptidase 24.11 does not inactivate the endothelin peptides in the rat uterus.     

Increased prevalence of hypertension and long-term arsenic exposure

A possible role of endothelin (ET)-1 in mediating hypoxic pulmonary vasoconstriction (HPV) was examined by comparing haemodynamic differences between ET-1-induced vasoconstriction and HPV in isolated perfused rat lungs. An ETA receptor antagonist (BQ123) was also employed to assess the effects of ET-1. The pulmonary arterial pressure (Ppa) was significantly increased by alveolar hypoxia (3% O2) and by ET-1 (5 nM). The pulmonary microvascular pressure was not changed by hypoxia, but increased more than two-fold by ET-1 (P < 0.01). Hypoxia significantly increased pulmonary arterial resistance (P < 0.01) while ET-1 significantly increased pulmonary venous resistance (P < 0.01), and slightly increased arterial resistance. Lung weight was increased by ET-1 and decreased by hypoxia, accompanied by similar Ppa responses in both cases. BQ123 (10(-6) M and 10(-5) M) did not influence the changes in Ppa and lung weight induced by hypoxia or angiotensin II (0.3 micrograms). BQ123 did, however, suppress (P < 0.05) the increase in Ppa and lung weight induced by 5 nM ET-1. Thus, it appears unlikely that ET-1 is involved in changes in pulmonary vascular tone during acute HPV.     

Characterization of endothelin receptors in the human umbilical artery and vein

The aim of the present study was to characterize pharmacologically endothelin receptors that are present in human umbilical vessels. 2. Endothelin-1 (ET-1) and endothelin-2 (ET-2) are potent stimulants of both the human umbilical artery (pEC50 7.9 and 7.5) and vein (pEC50 8.1 and 8.0). Endothelin-3 (ET-3) is inactive on the artery but contracts the vein (pEC50 7.6). IRL1620 is inactive in both vessels. The order of potency of agonists is suggestive of a typical ET(A) receptor in the artery (ET-1 = ET-2 > > ET-3) and a mixture of ET(A) and ET(B) receptors in the vein (ET-1 = ET-2 > or = ET-3). 3. The selective ET(A) receptor antagonist, BQ123, competitively inhibits the effect of ET-1 in the human umbilical artery (pA2 6.9), while in the vein, only a mixture of BQ123 and BQ788 (a selective ET(B) antagonist) weakly displaces to the right of the cumulative concentration-response curve to ET-1. Contractions induced by ET-3 in the vein are inhibited by BQ788 (pA2 7.6), but not by BQ123. 4. Inhibition of Ca2+ channels by nifedipine (0.1 microM) is accompanied by a significant decrease of the maximal response to ET-1 by 40% in the artery and by 30% in the vein. The response of the vein to ET-3 is almost abolished by nifedipine. 5. The results indicate that: (i) endothelins contract the human isolated umbilical artery via stimulation of an ET(A) receptor type; (ii) the contraction induced by ET-1 in the vein is mediated by both ET(A) and ET(B) receptors, while ET-3 stimulates the ET(B) receptor; (iii) the contribution of Ca2+ channels to the contraction mediated by the ET(B) receptor appears to be more important than to that mediated by the ET(A) receptor.     

Binding of the nonpeptide antagonist, SB 209670, to endothelin receptors on cultured neurons

We studied the binding characteristics of a novel, nonpeptide endothelin antagonist, SB 209670, to two subtypes of endothelin (ET) receptor in cultured rat cerebellar granule cell neurons. Displacement binding studies of [125I]ET-1 performed in the presence of the ETB receptor-selective agonist, sarafotoxin 6c (S6c), allowed us to measure a Ki of 4.0 +/- 1.5 nM for (+/-)SB 209670 at the ETA receptor (n = 4). Similarly, binding studies in the presence of the ETA receptor-selective antagonist, BQ123, allowed us to measure a Ki of 46 +/- 14 nM for (+/-)SB 209670 at the ETB receptor (n = 4). These studies indicate that the novel endothelin antagonist, SB 209670, has high affinity for both types of neuronal endothelin receptor.     

Effects of endothelin on phospholipases and generation of second messengers in cat iris sphincter and SV-CISM-2 cells

In both immortalized cat iris sphincter smooth muscle cells (SV-CISM-2 cells) and cat iris sphincter, endothelin-1 (ET-1) markedly increased the activities of phospholipase A2 (PLA2), as measured by the release of arachidonic acid (AA), phospholipase C (PLC), as measured by the production of inositol trisphosphate (IP3), and phospholipase D (PLD), as measured by the formation of phosphatidylethanol (PEt). In SV-CISM-2 cells, ET-1 induced AA release, IP3 production and PEt formation in a dose- and time-dependent manner. The dose-response studies showed that the peptide is more potent in activating PLD (EC50 = 1.2 nM) than in activating PLC (EC50 = 1.5 nM) or PLA2 (EC50 = 1.7 nM). The time course studies revealed that ET-1 activated the phospholipases in a temporal sequence in which PLA2 was stimulated first (t1/2 = 12 s), followed by PLC (t1/2 = 48 s) and lastly PLD (t1/2 = 106 s). In SV-CISM-2 cells, in contrast to the intact iris sphincter, sarafotoxin-c, an ETB receptor agonist, had no effect on the phospholipases, and indomethacin, a cyclooxygenase inhibitor, had no effect on the stimulatory effect of ET-1 on the phospholipases. These results suggest that in this smooth muscle cell line, ET-1 interacts with the ETA receptor subtype to activate, via G proteins, phospholipases A2, C and D in a temporal sequence.     

Discovery of TBC11251, a potent, long acting, orally active endothelin receptor-A selective antagonist

Previously we reported the discovery of amidothiophenesulfonamides as endothelin receptor-A antagonists with high potency and selectivity. Replacement of an amide group in this class of compounds with an acetyl group maintained the in vitro binding affinity and in vivo activity while providing a compound with oral bioavailability and longer duration of action. The optimal compound discovered during these studies, 15q (TBC11251), binds competitively to human ETA receptors with a Ki of 0.43 +/- 0.03 nM and an IC50 of 1.4 nM (IC50 for ETB = 9800 nM). This compound inhibits ET-1-induced stimulation of phosphoinositide turnover with a Ki of 0.686 nM and a pA2 of 8.0. The compound has a serum half-life in the rat and the dog of 6-7 h and 60-100% oral bioavailability. This compound is one of the most selective ETA antagonists reported and therefore is suitable for additional pharmacological and clinical investigation of the role of ETA receptors in diseases.     

The endothelin system and endothelin-converting enzyme in the brain: molecular and cellular studies

The biologically active vasoactive peptides, the endothelins (ETs), are generated from inactive intermediates, the big endothelins, by a unique processing event catalysed by the zinc metalloprotease, endothelin converting enzyme (ECE). In this overview we examine the actions of endothelins in the brain, and focus on the structure and cellular locations of ECE. The heterogeneous distribution in the brain of ET-1, ET-2, and ET-3 is discussed in relation to their hemodynamic, mitogenic and proliferative properties as well as their possible roles as neurotransmitters. The cellular and subcellular localization of ECE in neuronal and in glial cells is compared with that of other brain membrane metalloproteases, neutral endopeptidase-24.11 (neprilysin), angiotensin converting enzyme and aminopeptidase N, which all function in neuropeptide processing and metabolism Unlike these ectoenzymes, ECE exhibits a dual localisation in the cell, being present on the plasma membrane and also, in some instances, being concentrated in a perinuclear region. This differential localization may reflect distinct targeting of different ECE isoforms, ECE-1 alpha, ECE-1 beta, and ECE-2.     

Effects of endothelin-1 on Ca2+ signaling and secretion in parathyroid cells

It has been previously reported that parathyroid cells express endothelin (ET) receptors and secrete ET-1 in an extracellular Ca2+ concentration ([Ca2+]e)-dependent manner. Here, we examined the effects of ET-1 on intracellular signaling and parathyroid hormone (PTH) secretion in dispersed bovine parathyroid (bPT) cells, which comprise several cell types including epithelial and endothelial cells, in two cell lines, the rat parathyroid epithelial (PT-r) and the bovine parathyroid endothelial (BPE-1) cells. An RNA-polymerase chain reaction analysis revealed that both ETA and ETB receptors are expressed in bovine parathyroid tissue and BPE-1 cells, and only the ETA receptor is expressed in PT-r cells. PT-r cells also expressed an inositol 1,4,5-trisphosphate (Ins[1,4,5]P3) receptor, and ionomycin induced an increase in the intracellular Ca2+ concentrations ([Ca2+]i) in a Ca(2+)-deficient medium, indicating the presence of an operative intracellular Ca2+ pool in these cells. In cells bathed in 1 mM [Ca2+]e, ET-1 induced a rapid and transient increase in the Ins(1,4,5)P3 production, which was associated with a similar profile of increase in [Ca2+]i and with a peak response of about 800 nM. No changes in the profile of [Ca2+]i responses were observed in ET-1-stimulated cells in the presence of Ca2+ channel blockers, or in Ca(2+)-deficient medium, indicating that Ca2+ mobilization was not associated with Ca2+ entry. Furthermore, a sustained stimulation with ET-1 induced a decrease in [Ca2+]i below the prestimulatory level in a large population of cells, and the percentage of the cell population that shows the sustained decrease of [Ca2+]i increased in higher ET-1 concentrations. [Ca2+]i in PT-r cells was also controlled by a [Ca2+]e-dependent mechanism that changed [Ca2+]i from 28 to 506 nM in a 0.1-3 mM concentration range with an EC50 of 1.2 mM, which is comparable to that reported for bPT cells. In the same range of [Ca2+]e, PTH secretion from bPT cells was inhibited with an IC50 of 1 mM, and ET-1 increased PTH release in a dose-dependent manner but without affecting the IC50 for the [Ca2+]e-dependent inhibition. Thus, the parathyroid epithelial cells appear to respond to ET-1 in a unique way, and the ET autocrine system can be regarded as a possible mechanism to modulate the sensitivity of [Ca2+]e-dependent PTH release.