Alteration in endothelin receptor sub-type responsiveness and in the endothelin-TXA(2) mimetic U46619 interaction, in type-2 hypertensive diabetic Zucker rats

BACKGROUND: Type-2 diabetes is characterized by endotheliopathy, which increases target organ damage and mortality. There is excessive endothelin-1 and TXA(2) production, and abnormal vascular reactivity to endothelin-1, manifested as a paradoxical hypotensive action in Zucker diabetic, but not lean rats. We examined the hypothesis that there is an alteration in the ET-A/ET-B receptor subtype sensitivity, and/or the interaction or cross-talk between ET-1 and TXA(2) in type-2 diabetes, using Zucker diabetic rats and their lean littermates. MATERIALS AND METHODS: Hemodynamic studies were performed in lean and Zucker fatty diabetic rats of both sexes. Laser doppler flowmetry was used to measure renal cortical (RCF) and medullary blood flow (MBF) responses. Dose response curves for mean arterial blood pressure (MAP), MBF and RCF in response to ET-1, U46619, acetylcholine, and L-NAME (25mg/kg) were constructed after pre-treatment of the rats with either BQ610 1mg/kg i.v. or BQ788 0.5mg/kg i.v. The effects of BQ610 and BQ788 on whole blood impedance aggregation were also assessed. RESULTS: BQ788, but not BQ610 abolished both the paradoxical hypotensive action of ET-1 in Zucker diabetic rats (n=7 each, P<0.001 ANOVA) as well as the dose-dependent rise in MBF (P<0.001 ANOVA). BQ788, but not BQ610 abolished the difference in response to ET-1 between lean and diabetic Zucker rats. U46619 caused a hypotensive action in male Zucker rats which was abolished by L-NAME 25mg/kg or indomethacin 10mg/kg i.v. The U46619 interaction with BQ788 on both MAP and MBF was significantly (P<0.03 ANOVA) different between lean and diabetic Zucker rats. BQ788, but not BQ610 attenuated both the MAP and MBF responses to acetylcholine or L-NAME P<0.02 ANOVA). However, BQ610 dose-dependently attenuated the slope of platelet aggregation in both lean and Zucker diabetic rats (P<0.02 ANOVA). CONCLUSION: ET-B receptor antagonism abolished the abnormal vascular reactivity and MBF responses to ET-1, and also normalized the vasoactive responses to the level seen in healthy lean Zucker rats. ET-1 receptor blockade influences the responses to TXA(2) receptor activation. In the systemic and renal circulation, this interaction appears to be mostly ET-B receptor mediated, whilst in platelets, ET-A receptor role may be predominant. The interaction or cross-talk between ET-1 and TXA(2) is altered in the type-2 diabetic state. Collectively, these pathophysiological changes may contribute to the vicious circle of diabetic endotheliopathy.     

Stimulation of colorectal cancer cell line growth by ET-1 and its inhibition by ET(A) antagonists

BACKGROUND: The vasoactive peptide endothelin 1 (ET-1) acts via two receptors, endothelin receptors A (ET(A)) and B (ET(B)). ET-1 is overexpressed by human cancers in vivo and in vitro and may be mitogenic for cancer cells. METHOD: To elucidate if ET-1 is a growth regulator the following were investigated in human colorectal cancer cell lines (LIM1215 and HT29): ET-1 production by ELISA; ET receptor expression using radioligand autoradiographic techniques; and responsiveness to ET-1, and to ET(A) and ET(B) antagonism by growth measurements. RESULTS: ET-1 was produced by LIM1215 and HT29 cells (21.3 and 41.7 fmol/ml/10(6) cells (24 hours); 22.6 and 71.7 fmol/ml/10(6) cells (48 hours), respectively). ET(A) and ET(B) receptors were expressed by both cell lines. Addition of ET-1 resulted in a dose dependent increase in cell numbers which was significant at 10(-8)-10(-9) M for LIM1215, with the greatest increase at 10(-8) M (32.7% and 28.4% increase above controls at 48 hours and 72 hours; p<0.05) and at 10(-8)-10(-9) M for HT29, with the greatest increase at 10(-9) M (13.4% and 15.7% increase above controls at 48 hours and 72 hours; p<0.05). ET(A) antagonists BQ123 and BQ610, but not the ET(B) antagonist BQ788, inhibited ET-1 induced proliferation of both LIM1215 and HT29 (p<0.05). CONCLUSION: ET-1 can stimulate the proliferation of colorectal cancer cell lines via the ET(A), but not the ET(B), receptor.     

Importance of local production of endothelin-1 and of the ET(B)Receptor in the regulation of pulmonary vascular tone

Interaction between locally released endothelin-1 (ET-1) and the endothelial ET(B)receptor could modulate pulmonary vascular tone. We evaluated pulmonary ET-1 clearance and ET-1-ET(B)receptor interaction in the modulation of pulmonary vascular tone. Controls and rats with Monocrotaline (MCT)-induced pulmonary hypertension (PH) were studied. Lungs were isolated and perfused under constant pressure. The effect of the selective ET(B)antagonist BQ-788 (10(-12)-10(-8)mole) on perfusion flow rate and(125)I-ET-1 extraction was determined. Baseline(125)I-ET-1 extraction was reduced from 62+/-5% in controls to 49+/-10% in PH (P=0.012). BQ-788 inhibited extraction with a higher half-inhibitory dose in the MCT group (-Log ID(50)= 8.9+/-0.4 vs. 9.5+/-0.1, P=0.03). BQ-788 induced a mild reduction in perfusion flow rate of 0.7+/-0.3 ml/min in controls. In the MCT group, this occurred at a lower dose and was more pronounced with a maximal reduction of 3.3+/-0.7 ml/min (P<0.01 vs. control). ET-1 was undetectable in the effluent at baseline but was present in similar concentrations in both groups after ET(B)blockade. Addition of 2 pg/ml ET-1 to lung perfusate did not modify pulmonary ET-1 clearance or the effect of BQ788 on perfusion flow rate in control lungs. In normal rat lungs, the ET(B)receptor plays a minor regulatory role on vascular tone. In MCT hypertension however, despite a reduction in ET(B)mediated extraction, luminal production of ET-1 attenuates the increase in pulmonary vascular tone.     

Comparison of selective ET(A) and ET(B) receptor antagonists in patients with chronic heart failure

BACKGROUND: The vasoconstrictor action of endothelin-1 (ET-1) is mediated through ET(A) and ET(B) receptor subtypes on vascular smooth muscle. ET(B) receptors are also present on the vascular endothelium where they mediate vasodilation. Animal studies suggest that the ET(B) receptor also acts as a clearance receptor for endothelin. AIMS: To investigate the effects of a selective ET(A) and a selective ET(B) receptor antagonist alone and in combination on haemodynamics and circulating concentrations of ET-1 in patients with chronic heart failure. RESULTS: Infusion of BQ-123 (n=10), a selective ET(A) receptor antagonist, led to systemic vasodilation and did not change plasma ET-1 concentrations (1.38+/-0.82 to 1.38+/-0.91 fmol/ml, ns). Infusion of BQ-788 (n=8) led to systemic vasoconstriction with a rise in plasma ET-1 (1.84+/-1.06 to 2.73+/-0.99 fmol/ml, p<0.01). The addition of BQ-123 to BQ-788 led to systemic and pulmonary vasodilation with no further increase in plasma ET-1 concentrations (2.80+/-1.14 to 2.90+/-1.20 fmol/ml, ns). CONCLUSION: The rise in plasma ET-1 concentrations in response to selective blockade of ET(B) receptors and the associated adverse haemodynamic effects suggest that ET(B) receptors have a role in the clearance of ET-1 in man and that their blockade may not be advantageous for patients with heart failure.     

Endothelin-1 stimulates proliferation of human coronary smooth muscle cells via the ET(A) receptor and is co-mitogenic with growth factors.

We investigated the effects of endothelin-1 (ET-1) on growth of cultured human coronary artery smooth muscle cells (cSMC). ET-1 alone stimulated DNA synthesis in growth-arrested cSMC as measured by [3H]thymidine incorporation, with a maximum 63 +/- 23% increase above control by 10(-7) M (P < 0.05). ET-1 (10(-7) M) also stimulated increases in cyclin D1 protein levels after 24 h, and in absolute cell number after 4 days. Furthermore, ET-1 stimulated protein synthesis (maximum 73 +/- 32% increase in [3H]leucine incorporation by 10(-7) M (P < 0.05)), as well as triggering intracellular calcium transients in human cSMC, as visualised under fura-2 fluorescence microscopy. The selective ET(A) receptor antagonist BQ123 inhibited the increases in DNA synthesis, cell number, protein synthesis and intracellular calcium concentration in response to ET-1, whereas the ET(B) receptor antagonist BQ788 had no such effects. Furthermore, the ET(B) agonist sarafotoxin 6c had no effect on cSMC DNA synthesis. In addition, co-incubation of ET-1 with threshold concentrations of the growth factors, platelet-derived growth factor-BB (PDGF-BB), basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF), resulted in pronounced synergistic increases in DNA synthesis over that observed with the factors alone. In conclusion, we have shown that ET-1 stimulates proliferation of human cSMC via the ET(A) receptor and is also a co-mitogen with the growth factors tested. These findings indicate a role for ET-1 in the development of coronary intimal hyperplasia in man.     

Endothelins enhance prostaglandin (PGE(2) and PGF(2alpha)) biosynthesis and release by human luteal cells: evidence of a new paracrine/autocrine regulation of luteal function

We have previously shown that endothelin-1 (ET-1) is normally found in human luteal cells, where it is able to significantly inhibit both basal and hCG-induced progesterone production. To further expand our comprehension of the possible roles of endothelins (ETs) in luteal physiology, in this study we used primary cultures of luteal cells exposed to graded doses of ET-1 and ET-3; PGF(2alpha) and PGE(2) were assayed in the culture medium to investigate whether ETs also influence cyclooxygenase activity in these cells. We found that both ETs are able to significantly stimulate PGF(2alpha) and PGE(2) release in a dose- and time-dependent manner. ET-1 was always more effective than ET-3. Experiments with two endothelin receptor antagonists (the BQ485 and BQ788 compounds, which block the ET-A and ET-B receptors, respectively) showed that the two endothelins induce PG production through different receptors and signaling pathways. In conclusion, here we demonstrate the ability of ETs to influence PG synthesis and release from human luteal cells. As PGs are deeply involved in corpus luteum activity, and ETs were also able to influence progesterone production, the present new data suggest an interesting interplay among progesterone, PGs, and ETs in the control of corpus luteum physiology.     

The effect of chronic hypoxia on endothelin receptor subtype-mediated responses in rat isolated airways.

Contractile responses to endothelin-1 (ET-1) were investigated in isolated trachea from rats previously exposed to chronic hypoxia (10% O(2)) or room air for 14 days. Concentration-response curves were constructed to ET-1 (10(-11)-3x10(-7)m ) in the presence and absence of the ET(A)receptor antagonist FR 139317 (10(-8), 10(-7)and 10(-6)m ), the ET(B)receptor antagonist BQ 788 (10(-6)and 3x10(-6)m ), the non-selective ET receptor antagonist SB 209670 (10(-7)and 10(-6)m ) and a combination of FR 139317 (10(-6)m ) and BQ 788 (10(-6)m ). Concentration-response curves were also conducted to the ET(B)receptor agonist sarafotoxin S6c (10(-11)-3x10(-7)m ). In addition, responses to ET-1 (10(-11)-3x10(-7)m ) were examined in the presence and absence of the nitric oxide synthase inhibitor, L-NAME. In control rat trachea, both FR 139317 and BQ 788 failed to inhibit ET-1-induced contractions and, indeed, FR 139317 (10(-8)m ) and BQ 788 actually potentiated responses. In trachea from chronic hypoxic rats, FR 139317 did not alter ET-1 responses whereas BQ 788 again potentiated ET-1-induced contractions. The non-selective ET receptor antagonist SB 209670 attenuated ET-1-evoked contractions in trachea from control and chronically hypoxic rats. A combination of FR 139317 (10(-6)m ) and BQ 788 (10(-6)m ) also attenuated ET-1 responses in control rat trachea, but not trachea from chronically hypoxic rats. In trachea from both control and chronically hypoxic rats, L-NAME significantly potentiated responses to ET-1. To investigate ET receptor-mediated relaxation, tissues were preconstricted with methacholine and concentration-response curves were conducted to ET-1 (10(-13)-10(-8)m ) in the presence and absence of BQ 788 (10(-6)m ) and to the ET(B)receptor agonist sarafotoxin S6c (10(-13)-10(-8)m ). In trachea from control and chronic hypoxic rats, ET-1 and sarafotoxin S6c evoked only very small, non-reproducible relaxatory responses.     

Combined endothelin receptor blockade evokes enhanced vasodilatation in patients with atherosclerosis

Endothelin (ET)-1 causes vasoconstriction via ET(A) and ET(B) receptors located on vascular smooth muscle cells and vasodilatation via ET(B) receptors on endothelial cells. Studies in vitro indicate an upregulation of ET(B) receptors in atherosclerosis. The present study investigated the vascular effects evoked by endogenous ET-1 in atherosclerotic patients. Forearm blood flow (FBF) was measured with venous occlusion plethysmography in 10 patients with atherosclerosis and in 10 healthy control subjects during intra-arterial infusion of selective ET receptor antagonists. The ET(B) receptor antagonist BQ788 evoked a significant increase in FBF (31+/-13%) in the patients, whereas a 20+/-9% reduction was observed in the control subjects. The ET(A) receptor antagonist BQ123 combined with BQ788 evoked a marked increase in FBF (102+/-25%) in the patients compared with no effect in the control subjects (-3+/-9%, P<0.001 versus patients). The ET(A) receptor antagonist BQ123 increased FBF to a similar degree in patients (39+/-11%) as in control subjects (41+/-11%). The increase in FBF evoked by selective ET(A) receptor blockade was significantly (P<0.05) less than that evoked by combined ET(A)/ET(B) receptor blockade in the atherosclerotic patients. These observations suggest an enhanced ET-1-mediated vascular tone in atherosclerotic patients, which is at least partly due to increased ET(B)-mediated vasoconstriction.     

Mechanisms of big endothelin-1-induced diuresis and natriuresis : role of ET(B) receptors

Endothelin-1 (ET-1) at high concentrations has marked antidiuretic and antinatriuretic activities, whereas its precursor, big endothelin-1 (big ET-1), has surprisingly potent diuretic and natriuretic actions. The mechanisms underlying the excretory effects of big ET-1 have not been fully elucidated. To explore these mechanisms, we examined the effects of a highly selective ET(B) antagonist (A-192621.1), a calcium channel blocker (verapamil), a nitric oxide synthase inhibitor (N-nitro-L-arginine methyl ester [L-NAME]), and a cyclooxygenase inhibitor (indomethacin) on the systemic and renal actions of big ET-1 in anesthetized rats. An intravenous bolus injection of incremental doses of big ET-1 (0.3, 1. 0, and 3.0 nmol/kg) produced a significant hypertensive effect that was dose dependent and prolonged (from 113+/-7 mm Hg to a maximum of 148+/-6 mm Hg). The administration of big ET-1 induced marked diuretic and natriuretic responses (urinary flow rate increased from 8.5+/-1 to 110+/-14 microL/min, and fractional excretion of sodium increased from 0.38+/-0.13% to 7.51+/-1.24%). Glomerular filtration rate and renal plasma flow significantly decreased only at the highest dose of big ET-1. Pretreatment with A-192621.1 (3 mg/kg plus 3 mg. kg(-1). h(-1)) significantly abolished the diuretic (17+/-5 microL/min to a maximum of 19+/-3 microL/min) and natriuretic (0. 29+/-0.1% to a maximum of 1.93+/-0.37%) responses induced by big ET-1. Moreover, A-192621.1 potentiated the decline in glomerular filtration rate and renal plasma flow and the increase in mean arterial blood pressure produced by the low doses of big ET-1. Similar to A-192621.1, pretreatment with a nitric oxide synthase inhibitor (L-NAME, 10 mg/kg plus 5 mg. kg(-1). h(-1)) significantly and comparably reduced the diuretic and natriuretic actions of big ET-1 and augmented the hypoperfusion/hypofiltration and systemic vasoconstriction induced by high doses of the peptide. Pretreatment with verapamil (2 mg. kg(-1). h(-1)) slightly inhibited the diuretic/natriuretic effects of the high-dose big ET-1 and completely prevented the increase in mean arterial blood pressure provoked by the peptide. Unlike verapamil and L-NAME, only indomethacin administration was associated with significant natriuretic/diuretic responses and did not influence the pressor effect and renal actions of big ET-1. Taken together, these results suggest that big ET-1-induced diuretic and natriuretic responses are mediated mainly by stimulation of nitric oxide production coupled to ET(B) receptor subtype activation.     

Up-regulation in endothelin-1 by Helicobacter pylori lipopolysaccharide interferes with gastric mucin synthesis via epidermal growth factor receptor transactivation

OBJECTIVE: Endothelin-1 (ET-1), a key mediator of inflammatory processes associated with bacterial infection, is a 21-amino acid peptide produced from a biologically inactive big ET-1 by the action of endothelin-converting enzyme-1 (ECE-1) that acts through G protein-coupled ET(A) and ET(B) receptors. Here we report on the role of ET-1 in the mediation of the detrimental influence of Helicobacter pylori on the synthesis of gastric mucin. MATERIAL AND METHODS: Rat gastric mucosal cells were exposed to H. pylori key virulence factor, lipopolysaccharide (LPS). RESULTS: The LPS inhibitory effect on gastric mucin synthesis was accompanied by a marked increase in ET-1 generation and enhancement in ECE-1 activity. Inhibition of ECE-1 with phosphoramidon not only led to the impedance of LPS-induced ET-1 generation, but also countered the detrimental effect of LPS on mucin synthesis. Moreover, the LPS inhibitory effect on mucin synthesis was blocked by ET(A) receptor antagonist BQ610, but not by ET(B) receptor antagonist BQ788. Furthermore, the LPS-induced suppression in gastric mucin synthesis was countered in a concentration-dependent fashion by PD153035 (81.7%), a specific inhibitor of epidermal growth factor receptor (EGFR) kinase as well as PP2 (69.8%), a selective inhibitor of tyrosine kinase Src responsible for ligand-independent EGFR transactivation. CONCLUSIONS: Our findings are the first to show that the detrimental effect of H. pylori on gastric mucin synthesis is intimately linked to the events associated with ECE-1 up-regulation, enhancement in ET-1 production, and G protein-coupled ET(A) receptor activation that triggers the EGFR transactivation.     

Heterozygous knock-Out of ET(B) receptors induces BQ-123-sensitive hypertension in the mouse

Homozygous knock-out of ET(A) or ET(B) receptor genes results in lethal developmental phenotypes in the mouse. Such deleterious phenotypes do not occur in heterozygous littermates. However, it remains to be determined whether mice partially defective in ET(A) or ET(B) receptors display significant alterations in their responses to exogenous or endogenous endothelin-1 (ET-1). Furthermore, the anesthetized ET(B) (+/-) knock-out mice showed a significantly higher mean arterial blood pressure than the ET(A) (+/-) knock-out or their wild-type littermates. The pressor response to ET-1 but not to a selective ET(B) agonist, IRL-1620, was significantly reduced in the ET(A) (+/-) knock-out mice. In ET(B) (+/-) knock-out mice, the pressor effect of IRL-1620 was more markedly altered than those induced by ET-1. In wild-type mice, both ET(A) and ET(B) receptors were found to be involved in the pressor effect of ET-1, as confirmed by the significant and specific antagonism induced by either BQ-123 (ET(A) antagonist) or BQ-788 (ET(B) antagonist). Also, ET(A)-selective or mixed ET(A)/ET(B)- but not ET(B)-selective antagonists reversed the hypertensive state of the ET(B) (+/-) knock-out mice to the level of wild-type littermates. Finally, radiolabeled ET-1 plasmatic clearance was altered in ET(B) (+/-) but not ET(A) (+/-) knock-out mice when compared with wild-type animals. Thus, heterozygous knock-out of ET(B) receptors results in a hypertensive state, suggesting an important physiological role for that particular receptorial entity in opposing the endogenous ET-1-dependent pressor effects in the mouse.     

IL-15 mediates immune inflammatory hypernociception by triggering a sequential release of IFN-gamma, endothelin, and prostaglandin

IL-15 is closely associated with inflammatory diseases. IL-15 targeting is effective in treating experimental and clinical rheumatoid arthritis (RA). Because hyperalgesia accompanies RA, we investigated the ability of IL-15 to induced nociceptor sensitization (hypernociception). We report here that IL-15 induced time- and dose-dependent mechanical hypernociception in mice. IL-15-induced hypernociception was inhibited by treatment with a dual endothelin receptor type A (ET(A))/endothelin receptor type B (ET(B)) antagonist (bosentan), ET(A) receptor antagonist (BQ123), or cyclooxygenase inhibitor (indomethacin). Moreover, IL-15 failed to induce hypernociception in IFN-gamma(-/-) mice, suggesting that IL-15 mediated hypernociception via an IFN-gamma-, endothelin (ET)-, and prostaglandin-dependent pathway. Consistent with this finding, IFN-gamma and ET-1 induced dose- and time-dependent mechanical hypernociception that was inhibited by BQ123 or indomethacin but not BQ788 (an ET(B) receptor antagonist). IFN-gamma induced the production of ET-1 and the expression of its mRNA precursor (preproET-1, PPET-1). Moreover, IL-15 also induced ET-1 production and PPET-1 mRNA expression in an IFN-gamma-dependent manner. Prostaglandin E(2) (PGE(2)) production was induced by IL-15, IFN-gamma, or ET-1. We also found that hypernociception induced by ovalbumin (OVA) in OVA-immunized mice was significantly diminished by treatment with sIL-15Ralpha (soluble IL-15 receptor alpha-chain), bosentan, BQ123, or indomethacin. Furthermore, OVA challenge induced the expression of PPET-1 mRNA in WT mice but not in IFN-gamma(-/-) mice. The PPET-1 mRNA expression was also inhibited by sIL-15Ralpha pretreatment. Therefore, our results demonstrate the sequential mechanical hypernociceptive effect of IL-15 --> IFN-gamma --> ET-1 --> PGE(2) and suggest that these molecules may be targets of therapeutic intervention in antigen-induced hypernociception.     

ET(A) and ET(B) receptors modulate the proliferation of human pulmonary artery smooth muscle cells

We determined the distribution of ET(A) and ET(B) receptors in pulmonary arteries from pulmonary hypertensive patients and control subjects, using in vitro autoradiography, and investigated their role in mediating the proliferative effects of endothelin-1 (ET-1) on distal human pulmonary artery smooth muscle cells (PASMCs). Distal arteries possessed more medial [(125)I]-ET-1 binding sites (105 +/- 10 versus 45 +/- 6 amol/mm(2); p < 0.001) and a greater proportion of ET(B) receptors than proximal arteries (36 +/- 3% versus 3 +/- 1%; p < 0.001). Receptor density in distal arteries and lung parenchyma was twofold greater (p < 0.05) in pulmonary hypertensive patients than in control subjects. ET-1 (10(-9)-10(-7) mol/L) stimulated DNA synthesis (147 +/- 10% of control subjects; p < 0.05) and attenuated the antiproliferative action of cicaprost and forskolin on PASMCs, these effects being mediated via ET(A) and ET(B) receptors. Serum-stimulated proliferation was attenuated by inhibiting either endogenous ET-1 release with phosphoramidon (10(-5) mol/L) or its action with PD145065 (10(-5) mol/L). Cicaprost (10(-10)-10(-7) mol/L) inhibited ET-1 release from PASMCs (49 +/- 16% of control after 24 h; p < 0.001) and increased intracellular cAMP levels, whereas ET(B) receptor stimulation selectively reduced cAMP levels. In conclusion, ET(A) and ET(B) receptors are differentially distributed in human pulmonary arteries. Both receptors promote the proliferation of PASMCs in vitro and may contribute to vascular remodeling in pulmonary hypertension.     

Renal endothelin ET(A)/ET(B) receptor imbalance differentiates salt-sensitive from salt-resistant spontaneous hypertension

It is unclear why a subgroup of patients with essential hypertension develop salt-sensitive hypertension with progression of target organ damage over time. We evaluated the role of the renal endothelin (ET) system in the stroke-prone spontaneously hypertensive rat (SHRSP) model of salt-sensitive spontaneous hypertension (SS-SH) compared with the spontaneously hypertensive rat (SHR) model of salt-resistant spontaneous hypertension (SR-SH). Both strains were studied after either sham-operation on a normal diet (Sham) or after unilateral nephrectomy and high NaCl loading (NX-NaCl) with 4% NaCl in diet for 6 weeks (n=10, respectively). Systolic blood pressure (SBP) increased only in SHRSP-NX-NaCl compared with SHRSP-Sham (250+/-6 versus 172+/-5 mm Hg, P:<0.0001). SBP remained unchanged in SHR-NX-NaCl compared with SHR-Sham. In SHRSP-NX-NaCl animals, urinary albumin and ET-1 excretion, renal ET-1 mRNA expression, glomerulosclerosis index, and tubulointerstitial damage index were elevated compared with SHRSP-Sham (P:<0.05, respectively), whereas no significant changes were found in SHR after NX-NaCl. Urinary sodium excretion (U(Na(+))) was significantly reduced by 38% in SHRSP-NX-NaCl compared with SHR-NX-NaCl (P:<0.005, respectively). SHR animals showed a similar increase in both renal ET(A) and ET(B) receptor densities after NX-NaCl (2.2-fold, P:<0.05). In contrast, SHRSP-NX-NaCl developed a significantly more pronounced increase in ET(A) compared with ET(B) binding (4.7-fold versus 2.4-fold, P:<0.05, compared with SHRSP-Sham, respectively), resulting in a significant 2.1-fold increase in ET(A)/ET(B) receptor ratio only in the SHRSP-NX-NaCl (P:<0.05). Thus, activation of the renal ET system together with an increased ET(A)/ET(B) receptor ratio may contribute to the development and progression of SS-SH.     

BQ‐788, A Selective Endothelin ETB Receptor Antagonist

We describe characteristics of a selective endothelin (ET) ET_B receptor antagonist, BQ‐788 [N‐cis‐2,6‐dimethylpiperidinocarbonyl‐L‐γ‐methylleucyl‐D‐1‐methoxycarbonyltryptophanyl‐D‐norleucine], which is widely used to demonstrate the role of endogenous or exogenous ETs in vitro and in vivo. In vitro, BQ‐788 potently and competitively inhibited ¹²⁵I‐labeled ET‐1 binding to ET_B receptors in human Girrardi heart cells (hGH) with an IC₅₀ of 1.2 nM, but only poorly inhibited the binding to ET_A receptors in human neuroblastoma cell line SK‐N‐MC cells (IC₅₀, 1300 nM). In isolated rabbit pulmonary arteries, BQ‐788 showed no agonistic activity up to 10 μ and competitively inhibited the vasoconstriction induced by an ET_B‐selective agonist (pA₂, 8.4). BQ‐788 also inhibited several bioactivities of ET‐1, such as bronchoconstriction, cell proliferation, and clearance of perfused ET‐1. Thus, it is confirmed that BQ‐788 is a potent, selective ET_B receptor antagonist. In vivo, in conscious rats, BQ‐788, 3 mg/kg/h, i.v., completely inhibited a pharmacological dose of ET‐1‐ or sarafotoxin6c (S6c) (0.5 nmol/kg, i.v.)‐induced ET_B receptor‐mediated depressor, but not pressor responses. Furthermore, BQ‐788 markedly increased the plasma concentration of ET‐1, which is considered an index of potential ET_B receptor blockade in vivo. In Dahl salt‐sensitive hypertensive (DS) rats, BQ‐788, 3 mg/kg/h, i.v., increased blood pressure by about 20 mm Hg. It is reported that BQ‐788 also inhibited ET‐1‐induced bronchoconstriction, tumor growth and lipopolysaccharide‐induced organ failure. These data suggest that BQ‐788 is a good tool for demonstrating the role of ET‐1 and ET_B receptor subtypes in physiological and/or pathophysiological conditions.     

Effect of endothelin-1 on human platelet shape change: reversal of activation by naftidrofuryl

Naftidrofuryl (Praxilene; NAF) significantly improves claudication distance in patients with peripheral vascular disease (PVD). Endothelin-1 (ET-1) is a powerful endogenous vasoconstrictor and the circulating levels of ET-1 are elevated in patients with vascular disease. Platelet rich plasma (PRP) was prepared from healthy volunteers. NAF at concentrations similar to therapeutic levels (3.5-14 micromol/l), inhibited (P < 0.02) platelet activation (as indicated by a fall in median platelet volume, MPV) induced by ET-1 (0.4 micromol/l) alone. NAF also inhibited (P <0.0001) shape change (PSC; an early phase of platelet activation, characterised by an increase in MPV) induced by ET-1 (0.4 micromol/l) in combination with ADP (0.05-0.15 micromol/l) or serotonin (0.03-0.13 micromol/ l). We assessed the effect of ET(A) (BQ123, 50 nmol/l) or ET(B) (BQ788, 50 nmol/l) receptor antagonists on PSC induced by ET-1 alone. Both antagonists significantly inhibited PSC. We conclude that ET-1 activates human platelets. Both ET(A) and ET(B) receptors probably contribute to this response by a complex mechanism that requires further elucidation. NAF antagonises the action of ET-1 on human platelets. These actions may contribute to the beneficial effects of NAF in PVD.     

Endothelin-1 (ET-1)-potentiated insulin secretion: involvement of protein kinase C and the ET(A) receptor subtype

Endothelin-1 (ET-1), a potent vasoconstrictor peptide of endothelial origin, is capable of influencing hormone secretion from endocrine tissues, eg, pancreatic islet cells. We have shown a direct stimulatory effect of ET-1 on insulin secretion from isolated mouse islets of Langerhans. However, it is unknown as to whether the peptide acts through specific receptors on the islet cells and which mechanisms are involved in this insulinotropic action. We have therefore used the specific ET(A) receptor antagonist BQ123, the ET(B) receptor agonist BQ3020, and classic alpha- and beta-adrenergic and cholinergic antagonists. ET-1 (100 nmol/L) stimulated insulin secretion from islets incubated at 8.3, 11.1, 16.7, and 25 mmol/L glucose (P < .05). At 3.3 mmol/L glucose, no alteration in insulin secretion was found. The cholinergic receptor antagonist atropine (5 micromol/L) or the adrenergic receptor antagonists propranolol (5 micromol/L) or phentolamine (5 micromol/L) did not affect ET-1 (100 nmol/L)-stimulated insulin secretion. BQ123 (10 pmol/L to 10 nmol/L) and BQ3020 (1 nmol/L to 1 micromol/L) had no effect on glucose (16.7 mmol/L)-stimulated insulin secretion, but BQ123 counteracted the stimulatory effect of ET-1 (100 nmol/L) at concentrations of 1 nmol/L to 10 micromol/L (P < .01). We also studied the relative role of protein kinase C (PKC) and a Wortmannin-sensitive pathway for ET-1-induced insulin secretion using 12-O-tetradecanoyl phorbol-13-acetate (TPA), Calphostin C, and Wortmannin, respectively. At 5.6 mmol/L glucose, ET-1 (100 nmol/L) had no effect per se, whereas in the presence of 1 micromol/L TPA, which acutely stimulates PKC, the peptide did potentiate insulin secretion (P < .05). Furthermore, the insulinotropic effect of ET-1 at 16.7 mmol/L glucose was counteracted by the PKC inhibitor Calphostin C (P < .05) and by downregulation of PKC by 24 hours of exposure of islets to TPA (0.5 micromol/L, P < .05). Wortmannin (1 micromol/L) did not alter ET-1-potentiated insulin secretion. In conclusion, our results suggest that ET-1 acts through specific ET-1 receptors, most likely the ETA subtype. Furthermore, PKC plays an essential role in the insulinotropic action of ET-1 in mouse islets.     

Effect of endothelin-1 on human platelet shape change: reversal of activation by naftidrofuryl

Naftidrofuryl (Praxilene; NAF) significantly improves claudication distance in patients with peripheral vascular disease (PVD). Endothelin-1 (ET-1) is a powerful endogenous vasoconstrictor and the circulating levels of ET-1 are elevated in patients with vascular disease. Platelet rich plasma (PRP) was prepared from healthy volunteers. NAF at concentrations similar to therapeutic levels (3.5-14 w mol/l), inhibited (P < 0.02) platelet activation (as indicated by a fall in median platelet volume, MPV) induced by ET-1 (0.4 w mol/l) alone. NAF also inhibited (P <0.0001) shape change (PSC; an early phase of platelet activation, characterised by an increase in MPV) induced by ET-1 (0.4 w mol/l) in combination with ADP (0.05-0.15 w mol/l) or serotonin (0.03-0.13 w mol/ l). We assessed the effect of ET A (BQ123, 50 nmol/l) or ET B (BQ788, 50 nmol/l) receptor antagonists on PSC induced by ET-1 alone. Both antagonists significantly inhibited PSC. We conclude that ET-1 activates human platelets. Both ET and ET B receptors probably contribute to this response by a complex mechanism that A requires further elucidation. NAF antagonises the action of ET-1 on human platelets. These actions may contribute to the beneficial effects of NAF in PVD.     

ET(B) receptor blockade potentiates the pressor response to big endothelin-1 but not big endothelin-2 in the anesthetized rabbit

The precursor of endothelin-1, big endothelin-1, is considered to be a more reliable marker of systemic production of vasoactive peptide. However, it is largely unclear whether ET(B) receptor-dependent clearance and endothelium-derived relaxing factors affect the precursor in a similar manner to mature ET-1. These ET(B)-dependent modulations of big ET-1 and big ET-2 pressor properties were therefore studied in the anesthetized rabbit. When injected into the left cardiac ventricle, ET-1 and ET-2 (0.01 to 1 nmol/kg) each induced biphasic responses (a depressor followed by a pressor response), whereas big ET-1 and big ET-2 (0.1 to 3 nmol/kg) caused only protracted pressor responses. The highest dose of big ET-1 caused significantly greater responses than ET-1, ET-2, or big ET-2. A selective ET(A) receptor antagonist, BQ-123 (1 mg/kg), markedly reduced pressor responses to all 4 peptides, whereas blockade of ET(B) receptors with BQ-788 (0.25 mg/kg) sharply potentiated the responses to ET-1, ET-2, and big ET-1, but not to big ET-2. Indomethacin (10 mg/kg) sharply potentiated the pressor response to ET-1 (1 nmol/kg), but not big ET-1, at all time points. In control animals, ET-1, but not big ET-1, also triggered an indomethacin-sensitive increase in circulating prostacyclin. Finally, systemically administered big ET-1, but not big ET-2, induced a phosphoramidon-sensitive increase in plasma IR-ET. Our results suggest a significant limiting role of ET(B) receptors on pressor responses to big ET-1. In contrast, the same receptor entities do not modulate the hemodynamic properties of the ET-2 precursor, given that, unlike big ET-1, it is poorly converted in the pulmonary or systemic circulation in anesthetized rabbits.