Obligatory role of the endocardial endothelium in the increase of myocardial distensibility induced by endothelin-1

This study investigated how the endocardial endothelium (EE) and particularly endothelial type B (ET(B)) receptors influence the effects of endothelin-1 (ET-1) on diastolic distensibility. ET-1 (0.1, 1, and 10 nM) was tested in rabbit papillary muscles (Krebs-Ringer; 1.8 mM CaCl2, 35 degrees C) (i) with intact EE (n = 10), (ii) with damaged EE (0.5% Triton X-100, n = 11), and (iii) in the presence of RES-701-1 (selective endothelial ET(B1) receptor antagonist, 1 microM, n = 6). Additionally, increasing doses (0.1 nM to 1 microM) of Sarafotoxin S6c (SRTXc, a selective ET(B) receptor agonist) and IRL-1620 (a selective endothelial ET(B1) agonist) were studied (i) in muscles with intact EE (n = 7 and n = 6, respectively) and (ii) after damaging the EE (n = 8 and n = 7, respectively). In papillary muscles with intact EE, ET-1 induced dose-dependent positive inotropic and lusitropic effects. At 10 nM, active tension (AT) increased 78% +/- 17%, maximum velocity of tension rise (dT/dt(max)) increased 82% +/- 10%, and maximum velocity of tension decline (dT/dt(min)) increased 77% +/- 17%. These effects were maintained when ET-1 was given after damaging the EE (AT increased 70% +/- 12%, dT/dt(max) increased 93% +/- 14%, and dT/dt(min) increased 56% +/- 14%), but were significantly reduced in the presence of RES-701-1 (AT increased 30% +/- 6%, dT/dt(max) increased 37% +/- 7%, and dT/ dt(min) increased 29% +/- 9%). ET-1 reduced resting tension (RT) and increased diastolic distensibility by 3% +/- 1%, 5% +/- 1%, and 9% +/- 2% (at 0.1, 1, and 10 nM, respectively) in muscles with intact EE. This effect was abolished after damaging the EE or in the presence of RES-701-1. In muscles with intact EE, SRTXc had no significant effects, whereas, when given after damaging the EE, SRTXc (1 microM) increased inotropy and lusitropy (AT increased 116% +/- 24%, dT/dt(max) 110% +/- 28%, and dT/dt(min) 88% +/- 19%) without affecting RT. IRL-1620 dose-dependently decreased AT, dT/dt(max), and dT/dt(min) in muscles with intact EE-effects that were abolished after EE damage. No significant effects were elicited by IRL-1620 in RT. ET-1-induced increase in myocardial distensibility, previously shown to be mediated by ET(A) receptor stimulation, requires an intact EE and active endothelial ET(B1) receptors.     

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.     

Impaired response to ET(B) receptor stimulation in heart failure: functional evidence of endocardial endothelial dysfunction?

Inotropic effects of selective ET(B) receptor stimulation depend on the functional integrity of the endocardial endothelium (EE), which is negative when it is intact and positive when it is damaged. These results have been attributed to the existence of two subtypes of ET(B) receptors in the heart: (i) ET(B1), located on the EE, decreases inotropy; (ii) ET(B2), located on myocardial cells, increases inotropy. In the present study we investigated the functional integrity of the EE in a heart failure (HF) model (doxorubicin-induced cardiomyopathy) by evaluating the contractile response to ET(B1) receptor stimulation. New Zealand White rabbits were treated with doxorubicin (DOX-HF, 1 mg/kg, iv, twice weekly for 8 weeks) or with saline. Contractile effects of increasing doses of a selective agonist of endothelial ET(B) receptors, IRL-1620 (10(-9) to 10(-6) M), were studied in papillary muscles (Krebs-Ringer: 1.8 mM CaCl2, 35 degrees C) from control (n = 10) and DOX-HF rabbits (n = 7). Isotonic and isometric twitches were recorded and analyzed. Reported parameters included active tension (AT) and maximum velocities of tension rise (dT/dt(max)) and decline (dT/dt(min)). On echocardiography, DOX-HF rabbits had increased left ventricular (LV) end-diastolic and end-systolic diameters and reduced ejection fraction (52% +/- 2% vs. 61% +/- 1%). Contrary to control papillary muscles, DOX-HF muscles showed a steady decrease in contractility between 1 and 4 Hz. In the control group, IRL-1620 induced dose-dependent negative inotropic and lusitropic effects that decreased at 10(-6) M: 26% +/- 3%, AT; 17% +/- 3%, dT/dt(max); and 16% +/- 5%, dT/dt(min). In the DOX-HF group, these effects were significantly reduced. At the same concentration, IRL-1620 decreased AT (8% +/- 3%) and dT/dt(max) (8% +/- 3%), without significantly affecting dT/dt(min). This study showed an impaired response to endothelial ET(B) receptor stimulation, providing for the first time strong evidence of the occurrence of EE dysfunction in the failing heart and further highlighting the potential use of ET(B) receptor stimulation as a marker of EE function.     

Endothelin 1 and 3 enhance neuronal nitric oxide synthase activity through ETB receptors involving multiple signaling pathways in the rat anterior hypothalamus

We have previously reported that endothelin 1 and 3 (ET-1, ET-3) through the ETB receptor decrease norepinephrine release in the anterior hypothalamus and activate the nitric oxide (NO) pathway. In the present work we sought to establish the receptors and intracellular mechanisms underlying the increase in nitric oxide synthase (NOS) activity stimulated by ET-1 and ET-3 in the rat anterior hypothalamus. Results showed that ETs-stimulated NOS activity was inhibited by a selective ETB antagonist (BQ-788), but not by a selective ETA antagonist (BQ-610). In addition, NOS activity was not altered in the presence of an ETA agonist (sarafotoxin 6b), but it was enhanced in the presence of a ETB agonist (IRL-1620). Both Nomega-nitro-L-arginine methyl ester (NOS inhibitor), and 7-nitroindazole (neuronal NOS inhibitor) diminished ETs-stimulated NOS activity. The stimulatory effect of ETs on NOS activity was inhibited in the presence of PLC, PKC, PKA and CaMK-II inhibitors (U-73122, GF-109203X, H-89 and KN-62, respectively), and the IP3 receptor selective antagonist, 2-APB. Our results showed that both ET-1 and ET-3 modulate neuronal NOS activity through the ETB receptor in the rat anterior hypothalamus involving the participation of the PLC-PKC/IP3 pathway as well as PKA and CaMK-II.     

Nitric oxide and prostaglandins - important players in endothelin-1 induced myocardial distensibility

This study investigated whether endothelin (ET)-1-induced increase in myocardial distensibility is preserved in heart failure (HF) and whether it is modulated by nitric oxide (NO) and prostaglandins. New Zealand white rabbits were treated with doxorubicin (1 mg/kg, intravenously twice a week for 8 weeks, DOX-HF group) or saline (control group). Effects of ET-1 (0.1, 1, 10 nM) were tested in papillary muscles from the DOX-HF group and a control group in the presence of: i) intact endocardial endothelium (EE); ii) damaged EE; iii) N(G)-nitro-L-arginine (L-NNA; NO synthase inhibitor), and iv) indomethacin (INDO; cyclooxygenase inhibitor). In the presence of an intact EE, ET-1 promoted concentration-dependent positive inotropic and lusitropic effects that were maintained after damaging the EE, in the presence of L-NNA or INDO and in the DOX-HF Group. ET-1 reduced resting tension at the end of the isometric twitch (increased diastolic distensibility) by 3.2+/-1.3 %, 6.0+/-1.6 % and 8.8+/-2.7 % (at 0.1, 1 and 10 nM, respectively), in muscles with intact EE, effect that was completely abolished after damaging EE, in the presence of L-NNA or INDO or in the DOX-HF Group. This study demonstrated that the increase in myocardial distensibility induced by ET-1 is absent in HF and is dependent of NO and prostaglandin release.     

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.     

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

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

Nitric oxide and endothelin in oxygen-dependent regulation of vascular tone of human umbilical vein

We investigated the possible contribution of nitric oxide (NO) and endothelin (ET) to oxygen-dependent regulation of human umbilical vein vascular tone by simultaneous registration of intracellular membrane potential and isometric tension of vessel strips with and without NO synthase inhibition [10-4 M N omega-nitro-L-arginine methyl ester (L-NAME)], ETA receptor blockade (10(-5) M BQ-123), or ETB receptor blockade (10(-7) M BQ-788) at Po2 values in the bath solution between 5 and 104 mmHg. Increasing PO2 above the physiological intrauterine range resulted in depolarization and an increase of isometric tension, whereas lowering PO2 resulted in hyperpolarization and a decrease in isometric tension. Removal of the endothelium reversed these effects. At PO2 values below 39 mmHg, intact preparations treated with either L-NAME, BQ-788, or BQ-123 were more depolarized than controls. In the case of treatment with L-NAME or BQ-123, this was accompanied by an increase in isometric tension. We conclude that it is NO that mediates the hypoxic hyperpolarization and vasodilatation of the human umbilical vein and that ET, via activation of ETB1 receptors on endothelial cells, contributes to this effect.     

Upregulation of endothelin-1 binding in tissues of salt-loaded stroke-prone spontaneously hypertensive rats

Upon maintained on a 1% NaCl drinking solution beginning at 7 weeks of age, the stroke-prone spontaneously hypertensive rat (SHRsp) developed severe hypertension and stroke; most died by 16 weeks. The mechanism by which these diseases evolve remains unclear. Endothelin-1 (ET-1) is a potent, peptidic vasoconstrictor and is implicated in the pathogenesis of various cardiovascular, renal, and central nervous system diseases. The purpose of the present study was to compare the binding of [125I]ET-1 to the brain, heart, kidney, liver, and spleen membrane preparations of 16-week-old SHRsp and age-matched normotensive Wistar-Kyoto rats (WKY). The KD values for [125I]ET-1 binding to the corresponding tissues of the two strains were not significantly different, except in the brain (SHRsp: 17 +/- 1 pM; WKY: 24 +/- 1 pM). In contrast, the Bmax values measured in the brain, heart, kidney, and liver of SHRsp were 1.5- to 2.1-fold greater than those of their WKY counterparts. Competition of [125I]ET-1 binding to the membrane preparations by the specific ETA receptor antagonist BQ-123 or the specific ETB receptor agonist sarafotoxin S6c revealed a similar proportion of ETA and ETB receptor subtypes in the corresponding tissues of the two rat strains. These results indicate that ET-1 binding is upregulated in SHRsp and suggest that ET-1 may play a pathophysiological role in this animal model of genetic hypertension.     

Venous smooth muscle contains vasoconstrictor ETB-like receptors.

Two endothelin (ET) receptor subtypes have been identified to date: the ETA receptor which preferentially binds ET-1 over ET-3, and the ETB receptor which is non-selective. This study characterized the ET receptor subtypes present in several vascular smooth muscle preparations using standard in vitro techniques. In all but one of the arteries tested, ET-3 was significantly less potent than ET-1. In contrast, the potency of ET-3 was very similar to that of ET-1 in all of the veins. The selective ETA receptor antagonist BQ-123 blunted the ET-1 contractions in rabbit carotid artery, but not in saphenous vein. The selective ETB receptor ligand sarafotoxin S6c contracted the rabbit saphenous vein, but not the carotid artery. These data suggest that vascular smooth muscle cells express ETA and ETB receptors. Stimulation of either receptor subtype can result in force development.     

Nonpeptidic antagonists of ETA and ETB receptors reverse the ET-1-induced sustained increase of cytosolic and nuclear calcium in human aortic vascular smooth muscle cells

Our previous work showed that ET-1 induced a concentration-dependent increase of cytosolic Ca2+ ([Ca]c) and nuclear Ca2+ ([Ca]n) in human aortic vascular smooth muscle cells (hVSMCs). In the present study, using hVSMCs and 3-dimensional confocal microscopy coupled to the Ca2+ fluorescent probe Fluo-3, we showed that peptidic antagonists of ETA and ETB receptors (BQ-123 (10(-6) mol/L) and BQ-788 (10(-7) mol/L), respectively) prevented, but did not reverse, ET-1-induced sustained increase of [Ca]c and [Ca]n. In contrast, nonpeptidic antagonists of ETA and ETB (respectively, BMS-182874 (10(-8)-10(-6) mol/L) and A-192621 (10(-7) mol/L)) both prevented and reversed ET-1-induced sustained increase of [Ca]c and [Ca]n. Furthermore, activation of the ETB receptor alone using the specific agonist IRL-1620 (10(-9) mol/L) induced sustained increases of [Ca]c and [Ca]n, and subsequent administration of ET-1 (10(-7) mol/L) further increased nuclear Ca2+. ET-1-induced increase of [Ca]c and [Ca]n was completely blocked by extracellular application of the Ca2+ chelator EGTA. Pretreatment with the G protein inhibitors pertussis toxin (PTX) and cholera toxin (CTX) also prevented the ET-1 response; however, strong membrane depolarization with KCl (30 mmol/L) subsequently induced sustained increase of [Ca]c and [Ca]n. Pretreatment of hVSMCs with either the PKC activator phorbol-12,13-dibutyrate or the PKC inhibitor bisindolylmaleimide did not affect ET-1-induced sustained increase of intracellular Ca2+. These results suggest that both ETA- and ETB-receptor activation contribute to ET-1-induced sustained increase of [Ca]c and [Ca]n in hVSMCs. Moreover, in contrast to the peptidic antagonists of ET-1 receptors, the nonpeptidic ETA-receptor antagonist BMS-182874 and the nonpeptidic ETB-receptor antagonist A-192621 were able to reverse the effect of ET-1. Nonpeptidic ETA- and ETB-receptor antagonists may therefore be better pharmacological tools for blocking ET-1-induced sustained increase of intracellular Ca2+ in hVSMCs. Our results also suggest that the ET-1-induced sustained increase of [Ca]c and [Ca]n is not mediated via activation of PKC, but via a PTX- and CTX-sensitive G protein calcium influx through the R-type Ca2+ channel.     

Sarafotoxin S6c: an agonist which distinguishes between endothelin receptor subtypes.

In contrast to endothelin-1 (ET-1) and several of its analogues, sarafotoxin S6c (S6c) was a much more potent inhibitor of [125I]-ET-1 binding in rat hippocampus and cerebellum (Ki approximately 20 pM) than in rat atria and aorta (Ki approximately 4500 nM), suggesting the existence of ET-1 receptor subtypes (aorta/atria, ETA; hippocampus/cerebellum, ETB). S6c was a potent activator of PI turnover in hippocampus (EC50 approximately 10 nM) but not atria (EC50 greater than 1 microM), unlike ET-1 which was active in both tissues. S6c, therefore, is a highly selective ETB agonist. Furthermore, S6c was a potent pressor agent in the pithed rat (ED25 mm Hg approximately 0.1 nmoles/kg, i.v.), suggesting that the ETB receptor subtype may be important in cardiovascular function.     

High affinity interaction of endothelin-3 with recombinant ETA receptors

Pharmacological evidence has suggested that endothelin-3 (ET-3) may act via a novel form of ET receptor that is shared by ETA receptor antagonists but not by ETB receptor selective agonists. This study analyses the properties of interaction of ET-3 with recombinant bovine ETA receptor. Apparent Kd(ET-3) values as low as 50 nM were defined from [125I]ET-1 binding experiments performed at low (5 microg/ml) protein concentrations in the assays. Larger (up to 1 microM) values were artefactually obtained in experiments performed at larger protein concentrations. The three monoiodo ET-3 derivatives were synthetized. ([125I]Y14)ET-3 did not recognize ETA receptors. ([125I]Y6)ET-3 labelled 18% of [125I]ET-1 binding sites with a Kd value of 320 pM. ([125I]Y13)ET-3 labelled 44% of [125I]ET-1 binding sites with a Kd value of 130 pM. High affinity ([125I]Y6)ET-3 and ([125I]Y13)ET-3 bindings were prevented by ET-1 (Kd = 5-7 pM), ET-3 (Kd = 70-250 pM), BQ-123 (Kd = 2 nM) and FR139317 (Kd = 2 nM) but not by low concentrations of 4-AlaET-1, sarafotoxin S6c or IRL1620. The three monoiodo ET-3 derivatives bound to recombinant rat ETB receptors with a pM affinity. The results suggest that ET-3, ([125I]Y6)ET-3 and ([125I]Y13)ET-3 should not be considered as ETB receptor specific ligands.     

Functional cross-talk of Ca2+-mobilizing endothelin receptors in C6 glioma cells

There are conflicting results concerning the receptor subtype(s) involved in calcium-mediated endothelin signaling in the glial cells. In order to elucidate the role of endothelin A and B receptors in these processes, we have studied the effect of a complex spectrum of endothelin receptor ligands on intracellular calcium concentration changes in proliferating and differentiated C6 rat glioma cells. Cell differentiation was induced by dibutyryl-cAMP and assessed by the glial fibrillar acidic protein content. Intracellular calcium changes were measured in cell suspensions using fluorescent probe Fura-2. The specific endothelin B receptor agonists sarafotoxin S6c and IRL-1620 did not influence the intracellular calcium concentration. However, calcium changes induced by endothelin-1 and especially by endothelin-3 after the pretreatment of cells with one of these endothelin B receptor specific agonists were significantly enhanced even above the values attained by the highest effective endothelin concentrations alone. Such endothelin B-receptor ligand-induced sensitization of calcium signaling was not observed in differentiated C6 cells. Moreover, endothelin-induced calcium oscillations in differentiated C6 cells were less inhibited by BQ-123 and BQ-788 than in their proliferating counterparts. In conclusion, the specific activation of endothelin B receptor in C6 rat glioma cells does not affect intracellular calcium per se, but probably does so through interaction with the endothelin A receptor. The pattern and/or functional parameters of endothelin receptors in C6 rat glioma cells are modified by cell differentiation.     

Identification of high affinity endothelin-1 receptor subtypes in human tissues.

Two subtypes of the high affinity endothelin-1 (ET-1) receptor were identified in human tissue, and were distinguished by their differential affinities for sarafotoxin S6c (S6c). Uterus contains mostly the ETA subtype, with low affinity for S6c (Ki greater than 7300 nM), while the predominant subtype in hippocampus is ETB, with high affinity for S6c (Ki = 0.25 nM). These subtypes also have different affinities for [Ala1,3,11,15]-ET-1, which was found to be ETB selective. The two subtypes distinguished by these ligands in human tissue correspond to the subtypes previously identified in rat. Differential stimulation of phosphatidyl inositol turnover in rat tissue slices by ET-1 and S6c indicates that both ETA and ETB subtypes represent functional receptors.     

Chromatic effects of endothelin family peptides in non-innervated fish, Synbranchus marmoratus, melanophores

The biological activity of endothelins (ETs) in non-innervated Synbranchus marmoratus melanophores was demonstrated. These peptides induced a dose-dependent pigment aggregation (lightening skin) in these cells. However, they presented EC50's (effective concentration required to produce 50% of response) 26, 106 and 35 times higher than, respectively, the melanin concentrating hormone (MCH) EC50, and exhibited a characteristic temporal and dose-dependent autodessensibilization of the aggregative effect on the melanophores of this fish.The receptor characterization suggested the presence of the ET(B) subtype, since BQ-788 (selective antagonist of ET(B)) but not BQ-485 (selective antagonist of ET(A)) blocked the aggregative effect of the hormones. Confirming these data, sarafotoxin (SRTX) S6c, a toxin selective for ET(B), induced maximal aggregation of pigment granules. S6c presented an EC50 6.8 times higher than the MCH EC50, and 3.9, 15.6 and 5.1 times lower than the EC50's ETs, respectively.The melanotropic effect of SRTX S6b and vasoactive intestinal contractor (VIC) were demonstrated for the first time in this work. SRTX S6b induced a dose-dependent pigment aggregation and presented an EC50 2.54 and 17.2 times higher than the S6c and MCH EC50's, respectively. Compared to the ETs it was 1.53, 6.19 and 2.03 times lower, respectively.     

Selective autoregulation of endothelins in primary astrocyte cultures: endothelin receptor-mediated potentiation of endothelin-1 secretion.

Observations that primary rat astrocytes express high-affinity binding sites for endothelins and, in addition, are capable of producing not only endothelin-3 but also endothelin-1 prompted the investigation of a possible relation between endothelin peptides and receptors in these cells. Sarafotoxin S6b, an endothelin receptor agonist, was used as a tool to study endothelin receptor-mediated changes in the secretion of endothelin-1 and -3. The effects of sarafotoxin S6b and endothelin-1 in stimulating inositolphospholipid turnover as well as in inducing AP1 in primary astrocyte cultures were found to be similar. A low cross-reactivity of sarafotoxin S6b with endothelin-1 and -3 in the endothelin radioimmunoassays used here, along with a distinctly different elution position in high-performance liquid chromatography, allowed a clear discrimination between sarafotoxin and endothelins in the culture media. Stimulation of primary rat astrocytes with 10(-7) M sarafotoxin S6b for 1 hour resulted in a substantial increase in endothelin-1 immunoreactivity in the medium. This immunoreactivity reached a peak at 3 hours and showed no further increase after 8 and 24 hours. Treatment of our cultures with phorbol myristate acetate, lipopolysaccharide, tumor necrosis factor alpha, and norepinephrine for 24 hours led to only a moderate elevation of endothelin-1 immunoreactivity. Immunoreactive endothelin-3 was not affected by any of the treatments tested. Thus, our data suggest that endothelins in primary rat astrocytes are subject to selective autoregulation, as demonstrated by the potentiation of endothelin-1 secretion after activation of glial endothelin receptors.     

Endothelin ET(A) and ET(B) receptors in postnatal intestine

We aimed to characterize endothelin (ET) receptors in the swine intestinal vasculature and to determine ischemia-reperfusion (I/R) effects on these receptors. Saturation and competitive binding assays were performed on mesenteric artery protein membranes from 1- and 40-day-old animals, both control and those subjected to 1 h of partial ischemia followed by 6 h of reperfusion in vivo. Scatchard analysis of saturation binding with (125)I-labeled ET-1 in membranes from endothelium-denuded (E(-)) vessels revealed that the maximum number of binding sites was greater in younger animals. Competitive (125)I-ET-1 binding was significant for a one-site model with ET-1, ET-3, and sarafotoxin S6c (S6c) in membranes from endothelium-intact (E(+)) and E(-) vessels in both age groups. The maximum number of ET-1 binding sites was significantly greater in younger animals. In the presence of the ET(A) receptor antagonist BQ-123, competitive (125)I-ET-1 binding was significant for a one-site model with ET-1 and S6c in membranes from E(+) vessels in both age groups. The maximum number of ET-1 binding sites was significantly greater in younger animals. After I/R, the maximum number of ET-1 binding sites was unchanged. In the presence of BQ-123, specific binding by ET-1 and S6c was eliminated in both age groups after I/R. These results suggest that both ET receptor populations are expressed to a greater degree in younger animals and I/R significantly affects the ET(B) receptor.     

Endothelin-1 causes pruritus in mice

Endothelin (ET)-1 evokes a burning pruritus sensation when injected intradermally in humans and nocifensive behavior when injected into the hind paw of rodents. Because pain and pruritus are clearly distinct nociceptive sensory modalities in humans, the current study evaluates the potential of ET-1 to elicit scratching behavior in mice. Mice received an intradermal injection of 1-30 pmol ET-1; 10 microg of the mast cell degranulator compound, 48/80; 100 nmol histamine; or vehicle into the scruff, and the number of scratching bouts displayed during the first 40 mins was recorded. ET-1 caused dose-dependent scratching bouts, which, like the responses to histamine and compound 48/80, occurred mainly during the first 5 to 10 mins of injection, but fewer episodes were also seen up to 35 mins. The effect of ET-1 was maximal at 10 pmol (total 40 +/- 7 bouts), a value similar to that caused by histamine (52 +/- 5 bouts) and compound 48/80 (53 +/- 6 bouts). The selective ET(B) receptor agonist, IRL-1620 (10 pmol), was not pruritic per se, and actually inhibited responses to histamine and ET-1. Pruritus induced by ET-1 was inhibited by the ET(A) receptor antagonists, 10 nmol BQ-123 (co-injected; net inhibition, 87%) and 10 mg/kg atrasentan (intraperitoneal administration; net inhibition, 83%), or the ET(B) receptor antagonist, 20 mg/kg A-192621 (intraperitoneal administration; net inhibition, 64%), but the response was augmented by co-injection of the ET(B) receptor antagonist, 3 nmol BQ-788 (net potentiation, 234%). Responses to compound 48/80 or responsiveness of vehicle-treated mice were unaffected by these antagonists. Thus, ET-1 displays potent pruritic actions in the mouse mediated to a substantial extent via local ET(A) receptors. The findings with IRL-1620 and BQ-788 suggest that local ET(B) receptors exert an antipruritic role, but, for reasons still unknown, the results obtained using systemic A-192621 injection are at variance with this view.     

ETA and ETB receptors are expressed in vascular adventitial fibroblasts

The adventitia has been recognized to play important roles in vascular oxidative stress, remodeling, and contraction. We recently demonstrated that adventitial fibroblasts are able to express endothelin (ET)-1 in response to ANG II. However, it is unclear whether ET-1 receptors are expressed in the adventitia. We therefore investigated the expression and roles of both ET(A) and ET(B) receptors in collagen synthesis and ET-1 clearance in adventitial fibroblasts. Adventitial fibroblasts were isolated and cultured from the mouse thoracic aorta by the explant method. Cultured cells were treated with ANG II (100 nmol/l) or ET-1 (10 pM) in the presence or absence of the ANG II type 1 receptor antagonist losartan (100 μM), the ET-1 receptor antagonists BQ-123 (ET(A) receptor, 1 μM) and BQ-788 (ET(B) receptor, 1 μM), and the ET(B) receptor agonist sarafotoxin 6C (100 nM). ET-1 peptide levels were determined by ELISA, whereas ET(A), ET(B), and collagen levels were determined by Western blot analysis. ANG II increased ET-1 peptide levels in a time-dependent manner. ANG II increased ET(A) and ET(B) receptor protein levels as well as collagen in a similar fashion. ANG II-induced collagen was reduced while in the presence of BQ-123, suggesting a role for the ET(A) receptor in the regulation of the extracellular matrix. ANG II treatment in the presence of BQ-788 significantly increased ET-1 peptide levels. Conversely, the ET(B) receptor agonist sarafotoxin 6C significantly decreased ET-1 peptide levels. These data implicate a role for the ET(B) receptor in the clearance of the ET-1 peptide. In conclusion, both ET(A) and ET(B) receptors are expressed in adventitial fibroblasts, which paves the ground for the biological significance of adventitial ET-1. The ET(A) receptor subtype mediates collagen I expression, whereas the ET(B) receptor subtype may play a protective role through increasing the clearance of the ET-1 peptide.