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.     

Inotropic effects of ETB receptor stimulation and their modulation by endocardial endothelium, NO, and prostaglandins

Endothelin (ET)-1 acts on ETA and ETB receptors. The latter include ETB1 (endothelial) and ETB2 (muscular) subtypes, which mediate opposite effects on vascular tone. This study investigated, in rabbit papillary muscles (n = 84), the myocardial effects of ETB stimulation. ET-1 (10(-9) M) was given in the absence or presence of BQ-123 (ETA antagonist). The effects of IRL-1620 (ETB1 agonist, 10(-10)-10(-6) M) or sarafotoxin S6c (ETB agonist, 10(-10)-10(-6) M) were evaluated in muscles with intact or damaged endocardial endothelium (EE); intact EE, in the presence of NG-nitro-L-arginine (L-NNA); and intact EE, in the presence of indomethacin (Indo). Sarafotoxin S6c effects were also studied in the presence of BQ-788 (ETB2 antagonist). ET-1 alone increased 64 +/- 18% active tension (AT) but decreased it by 4 +/- 2% in the presence of BQ-123. In muscles with intact EE, sarafotoxin S6c alone did not significantly alter myocardial performance. Sarafotoxin S6c (10(-6) M) increased, however, AT by 120 +/- 27% when EE was damaged and by 39 +/- 8% or 23 +/- 6% in the presence of l-NNA or Indo, respectively. In the presence of BQ-788, sarafotoxin S6c decreased AT (21 +/- 3% at 10(-6) M) in muscles with intact EE, an effect that was abolished when EE was damaged. IRL-1620 also decreased AT (22 +/- 3% at 10(-6) M) in muscles with intact EE, an effect that was abolished when EE was damaged or in the presence of L-NNA or Indo. In conclusion, the ETB-mediated negative inotropic effect is presumably due to ETB1 stimulation, requires an intact EE, and is mediated by NO and prostaglandins, whereas the ETB-mediated positive inotropic effect, observed when EE was damaged or NO and prostaglandins synthesis inhibited, is presumably due to ETB2 stimulation.     

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.     

Intrapericardial angiotensin II stimulates endothelin-1 and atrial natriuretic peptide formation of the in situ dog heart

Angiotensin (AT) II, endothelin (ET)-1, and atrial natriuretic peptide (ANP) play an important role in cardiovascular regulatory processes under physiologic and pathophysiologic conditions. All of these agents are present in the pericardial fluid, and alteration of their pericardial concentrations mirror changes in the myocardial interstitium. Moreover, the composition the pericardial fluid may also reflect the myocardial interaction of these agents. The local myocardial effects of AT II on cardiac ET-1 and ANP production, as well as on cardiovascular function, was studied by intrapericardial (ip) administration of AT II (0.125-1.0 microg/kg) to the in situ dog heart (n = 8). Big ET, ET-1, and ANP [1-28] fragment concentrations were measured by enzyme-linked immunosorbent assay in pericardial infusate samples and in peripheral blood before and after an AT II treatment of 15 mins. Systemic blood pressure (BP), heart rate (HR), and left ventricular contractility (dP/dt) were also recorded. In our studies, the pericardial big ET (but not ET-1) concentration was increased to a maximum value of 139 +/- 28 versus 74 +/- 12 pg/ml (control; P < 0.02) with ip AT II administration, with parallel elevations of the pericardial ANP levels (36.8 +/- 7.2 vs. 24.4 +/- 3.6 ng/ml; P < 0.05). The ip administration of AT II did not influence HR, and it elicited moderate changes in BP (BP(max), +14 +/- 2 mm Hg, P < 0.001; dP/dt(max), +10 +/- 3%, P < 0.02). The plasma levels of big ET, ET-1, and ANP did not change significantly. The results suggest that AT II promotes production of big ET and ANP in the heart. However, no detectable conversion of big ET-1 to ET-1 was observed within 15 mins. The myocardial formation of big ET-1 and ANP occurred, at least in part, independently of the changes in cardiovascular function.     

Effect of the ET(B) receptor agonist, IRL-1620, on paclitaxel plasma pharmacokinetics of breast tumor rats

Endothelin (ET)-B receptors are expressed in human breast carcinoma. We previously demonstrated that intravenous administration of the ET(B) receptor agonist, IRL-1620, to tumor-bearing rats, increased blood perfusion and enhanced delivery of paclitaxel to breast tumor tissue. The present study was conducted to determine whether IRL-1620 alters the pharmacokinetics of paclitaxel. Breast tumor-bearing rats were given 0.3 ml/kg saline or 3 nmol/kg IRL-1620 by intravenous (iv) administration. Fifteen minutes after saline or IRL-1620, 40 microCi/rat 3H-Paclitaxel was administered iv and serial plasma samples were collected until 24 hrs. 3H-Paclitaxel radioactivity in the plasma samples was measured by liquid scintillation counting. Data were fit to a three-compartment model and pharmacokinetic parameters were generated using WinNonlin software. IRL-1620 did not produce any change in the plasma paclitaxel pharmacokinetics of tumor-bearing rats. The AUC(0-infinity) (9.43 +/- 3.18 microg-hr/ml), clearance (0.69 +/- 0.17 l/hr/kg), volume of distribution (10.31 +/- 4.54 l/kg), and half-life (1.0 +/- 0.32 hrs) of paclitaxel were similar between rats treated with saline or IRL-1620. In conclusion, the ET(B) receptor agonist, IRL-1620, does not alter paclitaxel plasma pharmacokinetics and, therefore, could be used to augment the delivery of paclitaxel to the tumor tissue.     

Both endothelin-A and endothelin-B receptors are present on adult rat cardiac ventricular myocytes

Endothelin-A (ET(A)) and endothelin-B (ET(B)) receptors have been demonstrated in intact heart and cardiac membranes. ET(A) receptors have been demonstrated on adult ventricular myocytes. The aim of the present study was to determine the presence of ET(B) and the relative contribution of this receptor subtype to total endothelin-1 (ET-1) binding on adult ventricular myocytes. Saturation binding experiments indicated that ET-1 bound to a single population of receptors (Kd = 0.52 +/- 0.13 nM, n = 4) with an apparent maximum binding (Bmax) of 2.10 +/- 0.25 sites (x 10(5))/cell (n = 4). Competition experiments using 40 pM [125I]ET-1 and nonradioactive ET-1 revealed a Ki of 660 +/- 71 pM (n = 10) and a Hill coefficient (nH) of 0.99 +/- 0.10 (n = 10). A selective ET(A) antagonist, BQ610, displaced 80% of the bound [125I]ET-1. No displacement was observed by concentrations of an ET(B)-selective antagonist, BQ788, up to 1.0 microM. However, in the presence of 1.0 microM BQ610, BQ788 inhibited the remaining [125I]ET-1 binding. Similarly, in the presence of 1.0 microM BQ788, BQ610 inhibited the remaining specific [125I]ET-1 binding. Binding of an ET(B1)-selective agonist, [125I]IRL-1620, confirmed the presence of ET(B). ET(B) bound to ET-1 irreversibly, whereas binding to ET(A) demonstrated both reversible and irreversible components, and BQ610 and BQ788 bound reversibly. Reducing the incubation temperature to 0 degrees C did not alter the irreversible component of ET-1 binding. Hence, both ET(A) and ET(B) receptors are present on intact adult rat ventricular myocytes, and the ratio of ET(A):ET(B) binding sites is 4:1. Both receptor subtypes bind to ET-1 by a two-step association involving the formation of a tight receptor-ligand complex; however, the kinetics of ET-1 binding to ET(A) versus ET(B) differ.     

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.     

Regulation of vasopressin secretion by ETA and ETB receptors in compartmentalized rat hypothalamo-neurohypophysial explants

The endothelins (ET) have been implicated in vasopressin (AVP) release in vivo and in vitro. The effects of ET in this system are complex, and the net AVP secretory response likely depends on a unique combination of ET isoform, ET receptor subtype, and neural locus. The purpose of these studies was to examine the role of ET receptor subtypes at hypothalamic vs. neurohypophysial sites on somatodendritic and neurohypophysial AVP secretion. Experiments were done in cultured explants of the hypothalamo-neurohypophysial system of Long Evans rats. Either the whole explant (standard) or only the hypothalamus or posterior pituitary (compartmentalized) was exposed to log dose increases (0.01-10 nM) of the agonists ET-1 (ET(A) selective), ET-3 (nonselective), or IRL-1620 (ET(B) selective) with or without selective ET(A) (BQ-123, 2-200 nM) or ET(B) (IRL-1038, 6-600 nM) receptor antagonism. In standard explants, ET-1 and ET-3 dose-dependently increased, whereas IRL-1620 decreased net AVP release. Hypothalamic ET(B) receptor activation increased both somatodendritic and neurohypophysial AVP release. At least one intervening synapse was involved, as tetrodotoxin blocked the response. Activation of ET(A) receptors at the hypothalamic level inhibited, whereas ET(A) receptor activation at the posterior pituitary stimulated, neurohypophysial AVP secretion. Antagonism of hypothalamic ET(A) receptors potentiated the stimulatory effect of ET-1 and ET-3 on neurohypophysial secretion, an effect not observed with ET(B) receptor-induced somatodendritic release of AVP. Thus the response of whole explants reflects the net result of both stimulatory and inhibitory inputs. The integration of these excitatory and inhibitory inputs endows the vasopressinergic system with greater plasticity in its response to physiological and pathophysiological states.     

Positive inotropic and relaxant effects of papaverine on cat papillary muscle

The effects of papaverine and isoproterenol on the isometric twitch and high KCl-induced contractures were compared in papillary muscles from reserpinized cats. Papaverine (10(-5) M) significantly increased developed tension (T), maximal rate of rise of tension (+dT/dt max) and maximal velocity of relaxation (--dT/dt max) in 52.3 +/- 6.1, 74.1 +/- 6.7 and 82.1 +/- 12.1% respectively with respect to control values. Time to peak tension (TTP) and contracture tension decreased in 9.1 +/- 2.0% and 50.9 +/- 5.6% respectively with respect to controls (P less than 0.05). Isoproterenol in a dose (8 X 10(-10) M), that produced an increase in +dT/dt max non significantly different to the one elicited by papaverine (65.6 +/- 9.0%), increased (in % with respect to control values), T in 55.3 +/- 7.3, --dT/mx in 73.8 +/- 13.1 and decreased TTP in 6.6 +/- 1.1 and contracture tension in 40.7 +/- 6.3 (P less than 0.05). The effects of isoproterenol on all the parameters studied were not statistically different from the ones of papaverine. It is concluded that in cat papillary muscles, papaverine has a positive inotropic action and an isoproterenol-like relaxant effect.     

Identification of a binding domain of the endothelin-B receptor using a selective IRL-1620-derived photoprobe

On the basis of the structure of IRL-1620, a specific agonist of the endothelin-B receptor subtype (ET(B)), a few photosensitive analogues were developed to investigate the binding domain of the receptor. Among those, a derivative containing the photoreactive amino acid, p-benzoyl-l-phenylalanine in position 5 showed, as assessed with endothelin-A (ET(A)) and ET(B) receptor paradigms, pharmacological properties very similar to those of IRL-1620. The binding capacity of the probe was also evaluated on transfected Chinese hamster ovary (CHO) cells overexpressing the human ET(B) receptor. Data showed that binding of the radiolabeled peptide was inhibited by ET-1 and IRL-1620. Therefore, this photolabile probe was used to label the ET(B) receptor found in CHO cells. Photolabeling produced a ligand-protein complex appearing on SDS-PAGE at around 49 kDa. An excess of ET-1 or IRL-1620 completely abolished the formation of the complex, showing the selectivity of the photoprobe. Digestions of the [Bpa(5),Tyr((125)I)(6)]IRL-1620-ET(B) complex were carried out, and receptor fragments were analyzed to define the region of the receptor where the ligand interacts. Results showed that Endo Lys-C digestion gave a 3.8-kDa fragment corresponding to the Asp(274)-Lys(303) segment, whereas migration after V8 digestion revealed a fragment of 4.6 kDa. Because the fragments of these two digestions must overlap, the latter would be the Trp(275)-Asp(313) stretch. A cleavage with CNBr confirmed the identity of the binding domain by giving a fragment of 3.6 kDa, corresponding to Gln(267)-Met(296). Thus, the combined cleavage data strongly suggested that the agonist binding domain of ET(B) includes a portion of the fifth transmembrane domain, between residues Trp(275) and Met(296).     

Endothelin-3-dependent pulmonary vasoconstriction in monocrotaline-induced pulmonary arterial hypertension

Blockade of the endothelin (ET) system is beneficial in pulmonary arterial hypertension (PAH). The contribution of ET-3 and its interactions with ET receptors have never been evaluated in the monocrotaline (MCT)-induced model of PAH. Vasoreactivity of pulmonary arteries was investigated; ET-3 localization was determined by confocal imaging and gene expression of prepro-ET-3 quantified using RT-PCR. ET-3 plasma levels tended to increase in PAH. ET-3 localized in the media of pulmonary arteries, where gene expression of prepro-ET-3 was reduced in PAH. ET-3 induced similar pulmonary vasoconstrictions in sham and PAH rats. In sham rats, the ET(A) antagonist A-147627 (10nmol/l) significantly reduced the maximal response to ET-3 (E(max) 77+/-1 to 46+/-2%, mean+/-S.E.M., P<0.001), while the ET(B) antagonist A-192621 (1mumol/l) reduced the sensitivity (EC(50) 21+/-7 to 59+/-16nmol/l, P<0.05) without affecting E(max). The combination of both antagonists completely abolished ET-3-induced pulmonary vasoconstriction. In PAH, the ET(A) antagonist further reduced the maximal response to ET-3 and shifted the EC(50) (E(max) 23+/-2%, P<0.001, EC(50) 104+/-24nmol/l, P<0.05), while the ET(B) antagonist only shifted the EC(50) (123+/-36nmol/l, P<0.05) without affecting the E(max). In PAH, dual ET receptor inhibition did not further reduce constriction compared to selective ET(A) inhibition. ET-3 significantly contributes to pulmonary vasoconstriction by activating the ET(B) at low concentration, and the ET(A) at high concentration. The increased inhibitory effect of the ET(A) antagonist in PAH suggests that the contribution of ET(B) to ET-3-induced vasoconstriction is reduced. Although ET-3 is a potent pulmonary vasoconstrictor in PAH, its potential pathophysiologic contribution remains uncertain.     

Endothelin-1 increases intracellular Ca(2+) in rabbit pulmonary artery smooth muscle cells through phospholipase C

In freshly isolated rabbit pulmonary artery smooth muscle cells, endothelin (ET)-1 induced a transient increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) followed by a return to the initial [Ca(2+)](i). This response was not abolished by the voltage-dependent Ca(2+) channel blocker nicardipine or removal of Ca(2+) from the bath solution but was inhibited by ryanodine and thapsigargin. This finding suggested that the increase in [Ca(2+)](i) induced by ET-1 was attributable to release of Ca(2+) from ryanodine- and inositol 1,4,5-trisphosphate-sensitive intracellular Ca(2+) stores. The transient increase in [Ca(2+)](i) induced by ET-1 was also inhibited by pretreatment with antagonists of ET type A and B (ET(A) and ET(B)) receptors (BQ-123 and BQ-788, respectively). Furthermore, the ET(B) receptor agonist IRL-1620 induced an increase in [Ca(2+)](i) that was followed by a sustained increase in [Ca(2+)](i); the sustained increase in [Ca(2+)](i) was blocked by nicardipine. Using the nystatin-perforated patch-clamp technique, we found that IRL-1620 caused an increase in Ca(2+) current that was inhibited by addition of ET-1. ET-1 did not inhibit Ca(2+) current when cells were pretreated with BQ-123. These results suggested that when both receptor types are activated, the opposing responses lead to abolition of the sustained [Ca(2+)](i) increases induced by ET(B) receptor activation. Western blot analysis confirmed expression of ET(A) and ET(B) receptors. Finally, U-73122 inhibited the ET-1-induced [Ca(2+)](i) increase, indicating that phospholipase C was involved in modulation of the ET-1-induced [Ca(2+)](i) increase in rabbit pulmonary artery smooth muscle cells.     

Alpha 1-adrenergic stimulation increases the Vmax of isolated myocardial papillary muscles.

alpha-Adrenergic agonists have been shown to increase the tension developed by myocardial muscle. However, their effects on the maximum velocity of unloaded muscle shortening (Vmax) have not been rigorously examined. In this study, the contractile effects of the alpha-adrenergic agonist phenylephrine were examined in the presence of propranolol in papillary muscles of two species, the dog and the rabbit. In rabbit papillary muscles studied at physiological calcium concentrations (1.25 mM), phenylephrine increased all indices of contractility (Vmax, tension, and maximum rate of tension developed (dT/dt)) starting at 10(-8) M. The percent increase in Vmax (121 +/- 8%) was less than that of tension (188 +/- 20%, p less than 0.05) and dT/dt (262 +/- 35%, p less than 0.01). These findings occurred at both 29 and 35 degrees C and were inhibited by adding 10(-5) M prazosin. Increasing extracellular calcium concentration from 1.25 to 15 mM caused changes in twitch configuration that were significantly different from those of phenylephrine. Calcium increased all indices of contractility more than did phenylephrine. This was particularly true for dT/dt (502 +/- 82 vs. 262 +/- 35% for phenylephrine, p less than 0.01). Nonetheless, the ratio of increase in tension to increase in Vmax under both experimental conditions was similar (the increase in Vmax was 64% of that of tension with phenylephrine and 69% with increased calcium). At 1.25 mM calcium, the increase in contractility caused by phenylephrine was much smaller in dog myocardium as compared with rabbit myocardium. Rather, the effects of phenylephrine on dog myocardium studied at 1.25 mM calcium resembled that of rabbit myocardium studied at 15 mM calcium.(ABSTRACT TRUNCATED AT 250 WORDS)     

ET-1 increases distensibility of acutely loaded myocardium: a novel ETA and Na+/H+ exchanger-mediated effect

This study investigated, in rabbit papillary muscles (n = 61) and human auricular strips (n = 7), effects of endothelin-1 (ET-1; 0.1-10 nM) on diastolic myocardial properties. ET-1 (1 nM) was also given in the presence of selective ET(A) or ET(B) antagonism, nonselective ET(A)/ET(B) antagonism, and Na(+)/H(+) exchanger inhibition. Effects of 6.3 mM Ca(2+) were also studied. ET-1 dose dependently increased inotropism. In contrast to baseline, in the presence of ET-1, resting tension (RT) decreased, after an isometric twitch, 3.4 +/- 1.4, 6.9 +/- 1.5, and 12.5 +/- 3.1% with 0.1, 1, and 10 nM, respectively, reflecting an increase in myocardial distensibility. ET-1 effects were abolished with selective ET(A) as well as with nonselective ET(A)/ET(B) antagonism, whereas they were still present with ET(B) antagonism. Na(+)/H(+) exchanger inhibition abolished ET-1 effects on distensibility, whereas it only partially inhibited positive inotropic effect. Ca(2+) increased inotropism to a similar extent to ET-1 (1 nM) but did not affect distensibility. ET-1 therefore increased diastolic distensibility of acutely loaded human and nonhuman myocardium. This effect is mediated by ET(A) receptors, requires Na(+)/H(+) exchanger activation, and cannot be elicited by Ca(2+).     

Endothelin isoforms and the response to myocardial stretch

Myocardial stretch elicits a biphasic increase in developed force with a first rapid force response and a second slow force response (SFR). The rapid phase is due to an increase in myofilament Ca(2+) responsiveness; the SFR, analyzed here, is ascribed to a progressive increase in Ca(2+) transients. Experiments were performed in cat papillary muscles to further elucidate the signaling pathway underlying the SFR. Although the SFR was diminished by BQ-123, a similar endothelin (ET)-1-induced increase in force was not affected: 23 +/- 2 vs. 23 +/- 3% (not significant). Instead, BQ-123 suppressed the contractile effects of ET-2 or ET-3 (21 +/- 2 and 25 +/- 3% vs. -1 +/- 1 and -7 +/- 3% respectively, P < 0.05), suggesting that ET-2 or ET-3, but not ET-1, was involved in the SFR. Each isoform activated the Na(+)/H(+) exchanger (NHE-1), increasing intracellular Na(+) concentration by 2.0 +/- 0.1, 2.3 +/- 0.1, and 2.1 +/- 0.4 mmol/l for ET-1, ET-2, and ET-3, respectively (P < 0.05). The NHE-1 inhibitor HOE-642 prevented the increases in force and intracellular Na(+) concentration induced by all the ET isoforms, but only ET-2 and ET-3 effects were sensitive to BQ-123. Real-time RT-PCR measurements of prepro-ET-1, -ET-2, and -ET-3 were performed before and 5, 15, and 30 min after stretch. No changes in ET-1 or ET-2, but an increase of approximately 60% in ET-3, mRNA after 15 min of stretch were detected. Stretch-induced ET-3 mRNA upregulation and its mechanical counterpart were suppressed by AT(1) receptor blockade with losartan. These data suggest a role for AT(1)-mediated ET-3 released in the early activation of NHE-1 that follows myocardial stretch.     

Concomitant antagonism of endothelial and vascular smooth muscle cell ETB receptors for endothelin induces hypertension in the hamster

In the vascular system, endothelin (ET) type B (ET(B)) receptors for ET-1 are located on endothelial and on venous and arterial smooth muscle cells. In the present study, we investigated the hemodynamic effects of chronic ET(B) receptor blockade at low and high doses in the Syrian Golden hamster. After 16 days of gavage with A-192621 (0.5 or 30 mg.kg(-1).day(-1)), a selective ET(B) receptor antagonist, hamsters were anesthetized with a mixture of ketamine and xylazine (87 and 13 mg/kg im, respectively), and basal mean arterial blood pressure (MAP) and pressor responses to exogenous ET-1 were evaluated. The lower dose of A-192621 (0.5 mg.kg(-1).day(-1)) did not modify basal MAP, whereas the higher dose (30 mg.kg(-1).day(-1)) increased MAP and plasma ET levels. Radio-telemetry recordings confirmed the increase in MAP induced by the higher dose of A-192621 in conscious hamsters. On the other hand, although the lower dose of A-192621 was devoid of intrinsic pressor effects, it markedly reduced the transient hypotensive phase induced by intravenously injected IRL-1620, a selective ET(B) receptor agonist. Finally, A-192621 (0.5 mg.kg(-1).day(-1)) alone or A-192621 (30 mg.kg(-1).day(-1)) + atrasentan (6 mg.kg(-1).day(-1)), a selective ET(A) receptor antagonist, potentiated the pressor response to exogenous ET-1. Our results suggest that, in the hamster, ET(B) receptors on vascular smooth muscle cells are importantly involved in the clearance of endogenous ET-1, whereas the same receptor type on the endothelium is solely involved in the vasodilatory responses to the pressor peptide. Blockade of endothelial and vascular smooth muscle cell ET(B) receptors triggers a marked potentiation of ET(A)-dependent increases in systemic resistance.     

Comparison of binding characteristics of endothelin receptors on subpopulations of astrocytes.

Astrocytes in primary culture originating from different brain areas of the mouse embryo (striatum, cerebral cortex and mesencephalon) were compared for their [125I]-Endothelin-1 (ET-1) binding characteristics, in terms of affinity, binding capacity and specificity. Our results indicate that astrocytes from mesencephalon express about twice as many receptors as astrocytes from striatum or cortex (149,000 +/- 9700 vs 63,700 +/- 5600 and 81,900 +/- 5300, respectively), with similar affinities. Specificity patterns for the various peptides of the endothelins/sarafotoxins family (ET-1, -2, -3; SRTXa, b, c) are comparable in the three subpopulations of astrocytes: ET-1, -2 and SRTXb exhibit higher affinities than SRTXa and SRTXc. In addition, ET-3 and SRTXc seem to discriminate between different subsets of [125I]-ET-1 binding sites in the three subpopulations.     

PVN lesions prevent the endothelin 1-induced increase in arterial pressure and vasopressin

Endothelin (ET) acts within the central nervous system to increase arterial pressure and arginine vasopressin (AVP) secretion. This study assessed the role of the paraventricular nuclei (PVN) in these actions. Intracerebroventricular ET-1 (10 pmol) or the ET(A) antagonist BQ-123 (40 nmol) was administered in conscious intact or sinoaortic-denervated (SAD) Long-Evans rats with sham or bilateral electrolytic lesions of the magnocellular region of the PVN. Baseline values did not differ among groups, and artificial cerebrospinal fluid (CSF) induced no significant changes. In sham-lesioned rats, ET-1 increased mean arterial pressure (MAP) 15.9 +/- 1.3 mmHg in intact and 22.3 +/- 2.7 mmHg in SAD (P < 0.001 ET-1 vs. CSF) rats. PVN lesions abolished the rise in MAP: -0.1 +/- 2.8 mmHg in intact and 0.0 +/- 2.9 mmHg in SAD. AVP increased in only in the sham-lesioned SAD group 8.6 +/- 3.5 pg/ml (P < 0.001 ET-1 vs. CSF). BQ-123 blocked the responses. Thus the integrity of the PVN is required for intracerebroventricularly administered ET-1 to exert pressor and AVP secretory effects.     

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

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