ETB Receptor Agonist Research Articles

Characterization and comparison of the responses of equine digital arteries and veins to endothelin-1.

To compare the responses of equine digital arteries (EDAs) and equine digital veins (EDVs) to endothelin-1 (ET-1) and determine the role of the endothelium and type of receptors involved in the modulation and mediation of those responses, respectively. 5 to 9 palmar digital vessels/experiment from 28 healthy horses. Rings of dissected vessels were mounted under tension between force transducer wires in organ baths containing Krebs-Henseleit solution at 30 degrees C. Responses of EDAs and EDVs (with intact [+e] or denuded [-e] endothelium) to cumulative concentrations of ET-1 (10(-10) to 3 X 10(-7) M) were compared. For (+e)EDAs and (+e)EDVs precontracted with a thromboxane-mimetic (U44069; 10(-8) M) and (-e)EDAs and (-e)EDVs, responses to an ETB receptor agonist (S6c; 10(-10) to 3 X 10(-7) M) were evaluated. Responses to ET-1 (10(-7) M) in (-e)EDAs and (-e)EDVs were evaluated after incubation with an ETA receptor antagonist (BQ-123; 3 X 10(-7) M), an ETB receptor antagonist (BQ-788; 3 X 10(-7) M), or vehicle solution. Endothelin-1 induced a concentration-dependent contraction of endothelium-intact and -denuded EDAs and EDVs; EDVs were more sensitive. Neither vessel type relaxed in response to S6c, although 2 of the (-e)EDAs contracted mildly. Whereas BQ-123 inhibited the (-e)EDA and (-e)EDV responses to ET-1, BQ-788 had no effect. Endothelin-1 induced digital vasoconstriction (marked constriction in veins). This action was unaffected by endothelium and mediated predominantly by ETA receptors. These findings suggest ET-1 can induce selective digital venoconstriction.

Intracellular pathways involved in upregulation of vascular endothelin type B receptors in cerebral arteries of the rat.

Previous studies have shown that contractile endothelin type B (ETB) receptors are upregulated in cerebral arteries after experimental focal cerebral ischemia. The aim of this study was to examine the upregulation of contractile ETB receptors in cerebral arteries after organ culture and to elucidate the intracellular pathways involved. Rat middle cerebral arteries (MCAs) were incubated with or without inhibitors. The vessels were mounted in myographs, and the contractile responses to endothelin-1 (ET-1) (ETA and ETB receptor agonist) and sarafotoxin 6c (ETB receptor agonist) were measured. Levels of ETB receptor mRNA were measured with real-time polymerase chain reaction. In fresh MCA, sarafotoxin 6c had no contractile effect. However, after organ culture, a strong concentration-dependent contraction was induced. ET-1 produced a strong contraction, in which the Emax was unaffected by organ culture but the EC50 was decreased with time. The sarafotoxin 6c-induced contraction after 24 hours of organ culture was attenuated by the transcriptional inhibitor actinomycin D and the translational inhibitor cycloheximide as well as the protein kinase C inhibitor Ro-31-8220. Real-time polymerase chain reaction revealed that the mRNA levels of the ETB receptor were increased after organ culture compared with fresh vessels. Actinomycin D and Ro-31-8220 diminished the enhanced mRNA levels considerably. The results suggest that, in fresh MCA, the ETA receptor is the most prominent subtype, while after organ culture ETB receptors also contribute to the contraction. This upregulation is due to de novo transcription of receptors. Protein kinase C is involved in the upregulation as Ro-31-8220 attenuates the contraction and the mRNA increase.

Endothelin causes contraction of human esophageal muscularis mucosae through interaction with both ETA and ETB receptors

Endothelin (ET) causes contraction of the muscularis mucosae in the guinea pig esophagus, but its role in the human esophagus remains unknown. To investigate effects of ET in the human esophagus, we measured contraction of isolated human esophageal muscularis mucosae strips caused by ET related peptides and binding of 125I-ET-1 to cell membranes prepared from the human esophageal muscularis mucosae. Autoradiography demonstrated specific binding of 125I-ET-1 to the muscularis mucosae and muscularis propria (muscularis externa) of the human esophagus. ET-1 caused tetrodotoxin and atropine-insensitive contraction of muscularis mucosae strips. In terms of the maximal tension of contraction, ET-1 and ET-2 were equal in efficacy. The relative potencies for ET related peptides to cause contraction were ET-1=ET-2>ET-3>sarafotoxin S6c (SX6c), an ETB receptor agonist. ET-1 caused contraction was mildly inhibited by BQ-123, an ETA receptor antagonist, and not by BQ-788, an ETB receptor antagonist. It was moderately inhibited by the combination of both antagonists, indicating synergistic inhibition. Furthermore, desensitization to SX6c with SX6c pretreatment failed to abolish the contractile response to ET-1, which was completely inhibited by BQ-123. These indicate the involvement of both ETA and ETB receptors in the contraction. Binding of 125I-ET-1 to cell membranes of the muscularis mucosae was saturable and specific. Analysis of dose-inhibition curves demonstrated the presence of ETA and ETB receptors. This study demonstrates that, the muscularis mucosae of the human esophagus, similar to that of the guinea pig esophagus, possesses both ETA and ETB receptors mediating muscle contraction.

ETA receptor-mediated Ca2+ signaling in thin descending limbs of Henle's loop: Impairment in genetic hypertension

Endothelins (ET) have diuretic and natriuretic actions via ETB receptors that are found in most renal tubular segments, although the thin limbs have not been studied. Data also suggest that dysfunction of the renal ET system may be important in the pathogenesis of hypertension. The present study was aimed at determining the presence and nature of ET receptors in the thin limbs of Henle's loop and their ability to activate a Ca2+-dependent signaling pathway, as well as whether ET-induced Ca2+ signals are altered in hypertension. Reverse transcription-polymerase chain reaction (RT-PCR) and Fura 2 fluoreselected strains of Lyon rats with low-normal (LL), normal (LN), and high (LH) blood pressure. In SD rats, ET induced Ca2+ signals in DTL of long-looped nephrons, but not in DTL of short loops, or in ascending thin limbs. Ca2+ increases were abolished by BQ123, an antagonist of the ETA receptor, but not by BQ788, an antagonist of the ETB subtype. Endothelin-3 and sarafotoxin 6c, two ETB receptor agonists, were both inactive. RT-PCR showed the presence of both ETA and ETB receptor mRNA. Ca2+ signals measured scence measurements of [Ca2+]i were made to characterize ET receptors in descending thin limbs (DTL) of Sprague-Dawley rats, spontaneously hypertensive (SH) rats, and control Wistar-Kyoto (WKY) rats, and the three in DTL of WKY LL and LN rats were similar to those in Sprague-Dawley rats, but were significantly diminished (LH) or abolished (SH) in hypertensive rats. A functional ETA receptor activating a Ca2+-dependent pathway is expressed in DTL. This ETA-induced calcium signaling is impaired in two strains of genetically hypertensive rats.

Alterations in ET-1, not nitric oxide, in 1-week-old lambs with increased pulmonary blood flow.

Altered pulmonary vascular reactivity is a source of morbidity and mortality for children with congenital heart disease and increased pulmonary blood flow. Nitric oxide (NO) and endothelin (ET)-1 are important mediators of pulmonary vascular reactivity. We hypothesize that early alterations in endothelial function contribute to the altered vascular reactivity associated with congenital heart disease. The objective of this study was to characterize endothelial function in our lamb model of increased pulmonary blood flow at 1 wk of life. Eleven fetal lambs underwent in utero placement of an aortopulmonary vascular graft (shunt) and were studied 7 days after delivery. The pulmonary vasodilator response to both intravenous ACh (endothelium dependent) and inhaled NO (endothelium independent) was similar in shunted and control lambs. In addition, tissue NO(x), NO synthase (NOS) activity, and endothelial NOS protein levels were similar. Conversely, the vasodilator response to both ET-1 and 4Ala-ET-1 (an ET(B) receptor agonist) were attenuated in shunted lambs, and tissue ET-1 concentrations were increased (P < 0.05). Associated with these changes were an increase in ET-converting enzyme-1 protein and a decrease in ET(B) receptor protein levels (P < 0.05). These data demonstrate that increased pulmonary blood flow induces alterations in ET-1 signaling before NO signaling and suggest an early role for ET-1 in the altered vascular reactivity associated with increased pulmonary blood flow.

Simultaneous changes in intracellular calcium and tension induced by endothelin-1 and sarafotoxin S6c in guinea pig isolated gallbladder: influence of indomethacin

The relationships between changes in intracellular Ca2+ and smooth muscle tension triggered by endothelin-1 and the selective endothelin ETB receptor agonist sarafotoxin S6c, as well as their susceptibility to modification by the nonselective cyclooxygenase blocker indomethacin, were assessed in guinea pig isolated gallbladder strips. Cumulative additions of either agonist (1, 10, and 100 nM) induced simultaneous graded, strongly correlated, slowly developing, and sustained changes in tension and intracellular Ca+2 (Fura-2 technique). Sarafotoxin S6c was more effective than endothelin-1 in raising intracellular Ca2+ at 1 or 10 nM, but their abilities to cause contractions were similar at all concentrations. Indomethacin (5.6 microM) markedly inhibited the changes in both intracellular Ca2+ and tension caused by all concentrations of sarafotoxin S6c (in response to 100 nM, increases in Ca2+ fluorescence intensity and tension were inhib ited from 7.7 +/- 0.7 to 4.0 +/- 0.4% and from 460 +/- 100 to 160 +/- 40 mg, respectively) but only reduced the contraction triggered by 100 nM endothelin-1 (from 560 +/- 100 to 230 +/- 70 mg). Endothelin-1 caused greater prostacyclin release from gallbladder than sarafotoxin S6c (at 100 nM, 6-keto-PGF1alpha levels in the medium rose 4.8- and 2.8-fold, respectively; P < 0.05) and slightly increased thromboxane A2 release (1.6-fold; P < 0.05). Thus, gallbladder contractions triggered by combined ETA/ETB or selective ETB receptor stimulation (with endothelin-1 or sarafotoxin S6c, respectively) are strongly correlated with increases in intracellular Ca2+ but differentially affected by indomethacin. It remains to be assessed if this difference is because endothelin-1 triggers greater prostacyclin release than sarafotoxin S6c and (or) is due to the coupling of ETA and ETB receptors to distinct patterns of generation of cyclooxygenase-derived eicosanoids.

LPS-Induced Imbalanced Expression of Hepatic Vascular Stress Genes in Cirrhosis: Possible Mechanism of Increased Susceptibility to Endotoxemia

Cirrhosis predisposes the liver to secondary stresses such as endotoxemia possibly via dysregulation of the hepatic portal circulation secondary to imbalanced upregulation of vascular stress genes. In this study we determined the effect of cirrhosis on hepatic vasoregulatory gene expression in response to endotoxin (LPS, i.p., 1 mg/kg). Cirrhosis was induced by bile duct ligation (BDL) for 21 days in male Sprague-Dawley rats. Plasma and liver samples were taken 6 h following an injection of LPS for alanine aminotransferase (ALT) assays and RT-PCR analysis of mRNA levels for genes of interest: endothelin (ET-1), its receptors ET(A) and ET(B), endothelial nitric oxide synthase (eNOS), inducible NOS (iNOS), and heme oxygenase-1 (HO-1). ALT release increased by 5.5-fold in the BDL animals and 9.9-fold in BDL + LPS compared to sham. ET-1 mRNA was increased by either LPS or BDL treatment alone and increased significantly more in BDL + LPS compared to sham + LPS. mRNA levels for ET(B) receptors showed no change, whereas ETA transcripts decreased in BDL animals compared to sham, with no significant difference between the saline and LPS treatment groups. The resultant increased ratio of ET(B) over ET(A) in BDL animals was reflected functionally in the portal pressure responses to ET(A) and ET(B) agonists ET-1 and IRL-1620 (a specific ETB receptor agonist). The pressor response to ET-1 was attenuated, while the response to IRL-1620 was similar in BDL and sham. eNOS mRNA levels did not increase in response to either BDL or LPS or a combination of both compared to sham. The increase in iNOS mRNA was attenuated in BDL + LPS compared to sham + LPS. HO-1 expression increased significantly in sham + LPS, but failed to increase in BDL + LPS. Taken collectively, significantly greater induction of the constrictor ET-1 over the dilation forces (i.e., eNOS, iNOS, and HO-1) was observed in BDL + LPS. This suggests a compromised ability of the cirrhotic liver to upregulate sufficient dilatory forces to counterbalance the constrictive effect of ET-1 upon a secondary insult of endotoxemia. These results may partly explain the increased susceptibility of cirrhotic livers to injury as a result of endotoxemia.

Design of ET(B) receptor agonists: NMR spectroscopic and conformational studies of ET7-21[Leu7, Aib11, Cys(Acm)15

In a previous report we have shown that the endothelin-B receptor-selective linear endothelin peptide, ET-1[Cys (Acm)1,15, Ala3, Leu7, Aib11], folds into an alpha-helical conformation in a methanol-d3/water co-solvent [Hewage et al. (1998) FEBS Lett., 425, 234-238]. To study the requirements for the structure-activity relationships, truncated analogues of this peptide were subjected to further studies. Here we report the solution conformation of ET7-21[Leu7, Aib11, Cys(Acm)15], in a methanol-d3/water co-solvent at pH 3.6, by NMR spectroscopic and molecular modelling studies. Further truncation of this short peptide results in it displaying poor agonist activity. The modelled structure shows that the peptide folds into an alpha-helical conformation between residues Lys9-His16, whereas the C-terminus prefers no fixed conformation. This truncated linear endothelin analogue is pivotal for designing endothelin-B receptor agonists.

Mechanisms transducing the aldosterone secretagogue signal of endothelins in the human adrenal cortex

Evidence has been provided that the 21-amino acid hypertensive peptide endothelin (ET)-1 exerts a potent secretagogue effect on human adrenocortical zona glomerulosa (ZG), acting through two receptor subtypes, called ETA and ETB, the signaling mechanism(s) of which has (have) not yet been investigated. Collagenase dispersed human ZG cells were obtained from normal adrenals of patients undergoing nephrectomy/adrenalectomy for renal cancer. The selective ETA- and ETB-receptor activation was obtained by exposing dispersed cells to ET-1 plus the ETB-receptor antagonist BQ-788 and to the ETB-receptor agonist BQ-3020, respectively. The phospholipase (PL) C inhibitor U-73122 abolished ETA receptor-mediated secretory response, but only partially prevented the ETB receptor-mediated one. The phosphatidylinositol 3-kinase inhibitor wortmannin, the calmodulin inhibitor W-7 and the protein kinase (PK) C inhibitor calphostin-C significantly blunted the secretory responses ensuing from the activation of both receptor subtypes. When added together, calphostin-C and wortmannin or W-7 abolished ETA-mediated secretory response, but only decreased ETB-mediated one. The ETB receptor-, but not the ETA receptor-mediated aldosterone response was partially reversed by the cyclooxygenase (COX) inhibitor indomethacin, which when added together with U-73122 abolished it. ETA-receptor activation raised inositol triphosphate (IP3) production from dispersed ZG cells, while ETB-receptor stimulation enhanced both IP3 and prostaglandin-E2 production. Collectively, our findings indicate that ETs stimulate aldosterone secretion from human ZG cells, acting through ETA receptors exclusively coupled to PLC/PKC-dependent pathway and ETB receptors coupled to both PLC/PKC- and COX-dependent cascades.

Human endothelin subtype A receptor enhancement during tissue culture via de novo transcription.

Endothelin (ET) has, since its discovery, increasingly been considered a key player in the pathophysiological processes of cerebral vasospasm in the course of subarachnoid hemorrhage, although it remains unclear how ET is involved. We present data that indicate an inherent capacity of human cerebral arteries to change their sensitivity to ET. Human cerebral arteries were obtained from patients undergoing intracranial tumor surgery. The vessels were divided into segments and subjected to organ culture for 48 hours. The vessels were then examined by using in vitro pharmacological methods and molecular biological techniques. After organ culture of the cerebral arteries, both the sensitivity to and potency of ET were enhanced (maximal response, 152 +/- 9%; -log (50% effective concentration), 10.3 +/- 0.3), in comparison with data for fresh cerebral arteries. Contractions were inhibited by both FR139317 (a specific ET(A) receptor antagonist) and bosentan (a mixed ET(A) and ET(B) receptor antagonist), in a manner indicating the sole presence of contractile ET(A) receptors. An inconsistent dilative response to the selective ET(B) receptor agonist sarafotoxin 6c was observed; the response was preserved in some segments and abolished in others, and potentiation of the precontraction was observed in yet other segments. No isolated contractile response to sarafotoxin 6c was observed, however. In reverse transcription-polymerase chain reaction assays, both ET(A) and ET(B) receptor messenger ribonucleic acid was detected. These results demonstrate that human cerebral arteries are capable of enhancing the function of ET(A) receptors.

Hemorrhagic shock primes the hepatic portal circulation for the vasoconstrictive effects of endothelin-1.

To test whether hemorrhagic shock and resuscitation (HSR) alters the vascular responsiveness of the portohepatic circulation to endothelins (ETs), we studied the macro- and microcirculatory effects of the preferential ET(A) receptor agonist ET-1 and of the selective ET(B) receptor agonist sarafotoxin 6c (S6c) after 1 h of hemorrhagic hypotension and 5 h of volume resuscitation in the isolated perfused rat liver ex vivo using portal pressure-flow relationships and epifluorescence microscopy. Although HSR did not cause major disturbances of hepatic perfusion per se, the response to ET-1 (0.5 x 10(-9) M) was enhanced, leading to greater increases in portal driving pressure, total portal resistance, and zero-flow pressures and more pronounced decreases in portal flow, sinusoidal diameters, and hepatic oxygen delivery compared with time-matched sham shock controls. In sharp contrast, the constrictive response to S6c (0.25 x 10(-9) M) remained unchanged. Thus HSR primes the portohepatic circulation for the vasoconstrictive effects of ET-1 but does not alter the effects of the ET(B) receptor agonist S6c. The enhanced sinusoidal response may contribute to the subsequent development of hepatic microcirculatory failure after secondary insults that are associated with increased generation of ET-1.

ETB Receptor Activates Adenylyl Cyclase via a c-PLA2-Dependent Mechanism: A Novel Counterregulatory Mechanism of ET-Induced Contraction in Airway Smooth Muscle

Endothelin-1 (ET-1) contracted the rabbit tracheal smooth muscle (RTSM), yielding a bell-shaped tension–concentration curve. Moreover, ET-1 induced concentration- and time-dependent increases in cAMP concentrations in RTSM (EC50, 58 nM; t1/2, 2.4 min). Pretreatment with the AC inhibitors, SQ-22536, or 2′–5′-dideoxyadenosine, enhanced contraction to ET-1 and converted its bell-shaped tension curve into a sigmoidal one, but left contraction to carbachol and KCl unaltered. The potent ETB-receptor agonists, ET-3 or sarafotoxin-c, mimicked ET-1's effects on cAMP levels (EC50 values 55 and 50 nM). Further, cAMP formation by ETs was inhibited by BQ-788 (selective ETB receptor blocker; IC50, 8 nM), but not by BQ-610 (selective ETA receptor blocker). Removal of the epithelium did not prevent ET-induced increases in cAMP levels. Unlike isoproterenol, ETs failed to activate AC in membrane fractions from RTSM. In intact RTSM, the c-PLA2 inhibitor, AACOCF3, and the cyclooxygenase inhibitor, indomethacin, blocked ET-induced increases in cAMP levels. These findings reveal a novel, nonepithelial, c-PLA2-mediated, regulatory mechanism downstream from ETB receptors.

Signaling pathways involved in the A and B receptor-mediated cortisol secretagogue effect of endothelins in the human adrenal cortex.

Endothelins (ETs) are a family of 21-amino acid hypertensive peptides, which together with their receptors ETA and ETB are expressed in human adrenal cortex. Evidence has been provided that ETs exert a potent secretagogue effect on human adrenocortical cells, acting through both ETA and ETB receptors. Therefore, it seemed worthwhile to study the signaling cascades mediating the cortisol secretagogue effect of the two receptor subtypes. Normal adrenal glands were obtained from consenting patients undergoing unilateral nephrectomy with ipsilateral adrenalectomy for renal cancer. Dispersed zona fasciculata-reticularis (ZF/R) cells were obtained by collagenase digestion and mechanical disaggregation. The selective activation of ETA and ETB receptors was obtained by exposing dispersed cells to ET-1 plus the ETB receptor antagonist BQ-788 and to the selective ETB receptor agonist BQ-3020, respectively. ETA and ETB receptors about equally contributed to the cortisol response of dispersed ZF/R cells to ETs. The phospholipase (PL) C inhibitor U-73122 abolished ETA-mediated secretory response, but only partially prevented the ETB-mediated one. The phosphatidylinositol 3-kinase inhibitor wortmannin and the protein kinase (PK) C inhibitor calphostin-C significantly blunted the secretory responses ensuing from the activation of both receptor subtypes, while the Ca(2+)-channel blocker nifedipine was ineffective. The ETB receptor-, but not the ETA receptor-mediated cortisol response was partially reversed by the cyclooxygenase (COX) inhibitor indomethacin, which when added together with U-73122 abolished it. The inhibitors of adenylate cyclase, PKA, tyrosine kinase and lipoxygenase did not affect the secretory response to the activation of either receptor subtype. ETA-receptor activation raised inositol triphosphate (IP3) production from dispersed ZF/R cells, while ETB-receptor stimulation enhanced both IP3 and prostaglandin-E(2) production. Collectively, our findings indicate that ETs stimulate cortisol secretion from human ZF/R cells, acting through ETA receptors exclusively coupled with PLC/PKC-dependent pathway and ETB receptors coupled with both PLC/PKC- and COX-dependent cascades.

Endothelins stimulate aldosterone secretion from dispersed rat adrenal zona glomerulosa cells, acting through ETB receptors coupled with the phospholipase C-dependent signaling pathway

Compelling evidence indicates that endothelins (ETs) stimulates aldosterone secretion from rat zona glomerulosa (ZG) cells, acting through the ETB receptor subtype. We have investigated the mechanisms transducing the aldosterone secretagogue signal elicited by the pure activation of ETB receptors. Aldosterone response of dispersed rat ZG cells to the selective ETB-receptor agonist BQ-3020 was not affected by inhibitors of adenylate cyclase/protein kinase (PK)A, tyrosine kinase-, mitogen-activated PK-, cyclooxygenase- and lipoxygenase-dependent pathways. In contrast, the inhibitor of phospholipase C (PLC) U-73122 abrogated, and the inhibitors of PKC, phosphatidylinositol trisphosphate (IP 3)-kinase and calmodulin (calphostin-C, wortmannin and W-7, respectively) partially prevented aldosterone response to BQ-3020. When added together, calphostin-C and wortmannin or W-7 abolished the secretagogue effect of BQ-3020. BQ-3020 elicited a marked increase in the intracellular Ca 2+ concentration ([Ca 2+]i) in dispersed rat ZG cells, and the effect was abolished by the Ca 2+-release inhibitor dantrolene. The Ca 2+ channel blocker nifedipine affected neither aldosterone nor Ca 2+ response to BQ-3020. Collectively, our findings suggest that (1) ETs stimulate aldosterone secretion from rat ZG cells through the activation of PLC-coupled ETB receptors; (2) PLC stimulation leads to the activation of PKC and to the rise in [Ca 2+]i with the ensuing activation of calmodulin; and (3) the increase in [Ca 2+] is exclusively dependent on the stimulation of IP 3-dependent Ca 2+ release from intracellular stores.

The endothelin ET(B) receptor agonist [125I]BQ-3020 binds predominantly to nerves in the bovine retractor penis muscle and penile artery.

Preliminary pharmacological experiments have suggested that in the bovine retractor penis muscle there are relaxation-mediating endothelin ET(B) receptors, at least part of which are located on the inhibitory nitrergic nerves. The present work was undertaken to test this hypothesis by means of receptor autoradiography and additional pharmacological experiments. In the retractor penis muscle and the penile artery, specific binding of the ETB receptor-selective agonist [125I]BQ-3020 took place predominantly to nerve trunks and minor nerve branches. The situation was the same in the dorsal metatarsal artery, that was included as a reference because of its different innervation. Throughout the nerves the silver grains were evenly distributed over the nuclei of Schwann cells and the spaces between them. In the retractor penis there was also a small amount of specific binding to smooth muscle. No specific endothelial binding was observed in any of the tissues examined. The pharmacological studies confirmed that the relaxation of the retractor penis muscle induced by the ET(B) receptor-selective agonist, sarafotoxin S6c, is susceptible to tetrodotoxin as well as to inhibition of nitric oxide synthase. The relaxation was also characterized by inconsistency, weakness and tachyphylaxis. The electrical field stimulation-induced submaximal relaxation of the retractor penis was unaffected by stimulation or blockade of ET(B) receptors. The autoradiography suggests that in all the three bovine tissues studied there are ET(B) receptors located on nerves independently of the type of efferent nerve. The pharmacological experiments do not support the concept that in the bovine retractor penis muscle neuronal ET(B) receptors exert important immediate effects on the functioning of the penile erection-mediating nitrergic nerves.

The Endothelin ETB Receptor Agonist [125I]BQ‐3020 Binds Predominantly to Nerves in the Bovine Retractor Penis Muscle and Penile Artery

Abstract:Preliminary pharmacological experiments have suggested that in the bovine retractor penis muscle there are relaxation‐mediating endothelin ETB receptors, at least part of which are located on the inhibitory nitrergic nerves. The present work was undertaken to test this hypothesis by means of receptor autoradiography and additional pharmacological experiments. In the retractor penis muscle and the penile artery, specific binding of the ETB receptor‐selective agonist [125I]BQ‐3020 took place predominantly to nerve trunks and minor nerve branches. The situation was the same in the dorsal metatarsal artery, that was included as a reference because of its different innervation. Throughout the nerves the silver grains were evenly distributed over the nuclei of Schwann cells and the spaces between them. In the retractor penis there was also a small amount of specific binding to smooth muscle. No specific endothelial binding was observed in any of the tissues examined. The pharmacological studies confirmed that the relaxation of the retractor penis muscle induced by the ETB receptor‐selective agonist, sarafotoxin S6c, is susceptible to tetrodotoxin as well as to inhibition of nitric oxide synthase. The relaxation was also characterized by inconsistency, weakness and tachyphylaxis. The electrical field stimulation‐induced submaximal relaxation of the retractor penis was unaffected by stimulation or blockade of ETB receptors. The autoradiography suggests that in all the three bovine tissues studied there are ETB receptors located on nerves independently of the type of efferent nerve. The pharmacological experiments do not support the concept that in the bovine retractor penis muscle neuronal ETB receptors exert important immediate effects on the functioning of the penile erection‐mediating nitrergic nerves.

Prolonged endothelin B receptor blockade causes pulmonary hypertension in the ovine fetus.

Endothelin (ET)-1 contributes to regulation of pulmonary vascular tone and structure in the normal ovine fetus and in models of perinatal pulmonary hypertension. The hemodynamic effects of ET-1 are due to activation of its receptors. The ET(A) receptor mediates vasoconstriction and smooth muscle cell proliferation, whereas the ET(B) receptor mediates vasodilation. In a lamb model of chronic intrauterine pulmonary hypertension, ET(B) receptor activity and gene expression are decreased. To determine whether prolonged ET(B) receptor blockade causes pulmonary hypertension, we studied the hemodynamic effects of selective ET(B) receptor blockade with BQ-788. Animals were treated with an infusion of either BQ-788 or vehicle for 7 days. Prolonged BQ-788 treatment increased pulmonary arterial pressure and pulmonary vascular resistance (P < 0.05). The pulmonary vasodilator response to sarafotoxin 6c, a selective ET(B) receptor agonist, was attenuated after 7 days of BQ-788 treatment, demonstrating pharmacological blockade of the ET(B) receptor. Animals treated with BQ-788 had greater right ventricular hypertrophy and muscularization of small pulmonary arteries (P < 0. 05). Lung ET-1 levels were threefold higher in the animals treated with BQ-788 (P < 0.05). We conclude that prolonged selective ET(B) receptor blockade causes severe pulmonary hypertension and pulmonary vascular remodeling in the late-gestation ovine fetus.

Positive inotropic responses mediated by endothelin ETA and ETB receptors in human myocardial trabeculae

The aim of the present study was to determine possible inotropic effects mediated by endothelin ETA and ETB receptors in human myocardial trabeculae from the right atrium and the left ventricle. Isolated trabeculae from human hearts were paced at 1.0 Hz in tissue baths, and changes in isometric contractile force upon exposure to agonist were studied. Endothelin-1 (ET-1) and ET-3 had a strong positive inotropic effect in all trabeculae. ET-1 was significantly more potent than ET-3 in both atrial (P &amp;lt; 0.001) and ventricular (P &amp;lt; 0.05) trabeculae. Preincubation with the ETA receptor antagonist FR139317 (1 μM) decreased significantly (P &amp;lt; 0.005) the potency of ET-1 in both atrial and ventricular trabeculae, without any significant changes in Emax (maximum effect obtained with an agonist). The ETB receptor agonist IRL 1620 had a positive inotropic effect only in some trabeculae, and the ETB receptor antagonist BQ 788 (1 μM) almost completely blocked this effect. These results suggest that both ETA and ETB receptors mediate positive inotropic responses at both the atrial and ventricular level in the human heart.

Endothelin Inhibits Histamine-Induced Cyclic AMP Accumulation in Bovine Brain Vessels

We have studied whether endothelin isopeptides have any effects on histamine-induced cyclic AMP in [3H]adenine-prelabeled brain vessels isolated from bovine brain. Basal levels of [3H]cyclic AMP were enhanced by histamine in a concentration-dependent manner (EC50 = 1.1 ± 0.3 μM). Endothelin-1 inhibited histamine-elicited [3H]cyclic AMP generation with an IC50 value of 3 ± 2.5 nM. Sarafotoxin 6c, an ET-B receptor agonist, had no effect. ET-1 inhibition of histamine-induced [3H]cyclic AMP was reversed by the ET-A receptor antagonist BQ-123 while the ET-B receptor antagonist BQ-788 had no effect. The levels of [3H]cyclic AMP induced by isoprenaline were not altered by endothelin-1. Taken together, these results show that endothelins modulate the actions of histamine on the blood–brain barrier, probably by type A endothelin receptors.

Constriction to ETB receptor agonists, BQ-3020 and sarafotoxin s6c, in human resistance and capacitance vessels in vivo.

The aim of the study was to examine the effects of the ETB receptor selective agonists sarafotoxin S6c (SFTX6c) and BQ-3020 on the forearm resistance and capacitance vessels in healthy subjects in vivo. The local response to intra-arterial or intravenous infusion of SFTX6c (5 pmol min-1) or BQ-3020 (50 pmol min-1) was assessed, on separate occasions, in eight healthy men (aged 20-28 years). Data (mean +/- s.e.mean) were examined by ANOVA. Results are expressed as percentage change from baseline at 90 min. SFTX6c and BQ-3020 reduced forearm blood flow, following local intra-arterial infusion (-25 +/- 7% and -27 +/- 7%, respectively; P < 0.001) and reduced hand vein diameter, following local intravenous infusion (-30 +/- 8% and -16 +/- 7%, respectively; P < 0.001). We have shown that locally active infusions of the selective ETB receptor agonists SFTX6c and BQ-3020 cause arterial constriction and venoconstriction in healthy human blood vessels in vivo. These results indicate that ETB receptor stimulation may mediate vasoconstriction in humans.