Local formation and degradation of endothelin-1 in guinea pig airway tissues

Endothelin(ET)-1 and big ET-1 caused potent and sustained constriction of isolated guinea pig bronchus. The response to ET-1 was enhanced by phosphoramidon in a simple dose-related manner (0.01-1000 microM), while the response to big ET-1 was enhanced at lower doses (0.01-0.1 microM) but was suppressed at higher doses (100-1000 microM) of phosphoramidon. Big ET-1, when given intravenously (i.v.) to anesthetized guinea pigs, increased both bronchopulmonary inflation pressure and mean arterial blood pressure (2.5, 5, 10 nmol/kg i.v.). The pressor response to big ET-1 was attenuated by phosphoramidon dose-relatedly, while the pulmonary response was modified in a complex fashion composed of delayed onset and prolonged duration of action. These results suggest that ET converting as well as degrading enzymes coexist in the airway tissue and both enzymes are sensitive to phosphoramidon, so that phosphoramidon acts bifunctionally to reduce and stimulate the airway responses to big ET-1.     

Phosphoramidon inhibits the vasoconstrictor effects evoked by big endothelin-1 but not the elevation of plasma endothelin-1 in vivo

Intravenous injections of big endothelin (ET)-1 (700 pmol/kg) in the pig increased arterial plasma levels of ET-1-like immunoreactivity (ET-1-LI) from 11.1 +/- 0.7 pM to 46.3 +/- 6.7 pM in the control situation and from 11.5 +/- 0.4 pM to 58.2 +/- 17 pM in the presence of the neutral endopeptidase inhibitor phosphoramidon (3 mg/kg). Big ET-1 increased splenic vascular resistance by 29% in the control situation. The vasoconstriction evoked by big ET-1 in the spleen was reduced after phosphoramidon treatment whereas the elevation of arterial ET-1-LI was not influenced. Furthermore the splenic vasoconstriction evoked by ET-1 was reduced after phosphoramidon without influencing plasma ET-1-LI. Also in rats the pressor effect of big ET-1 (1 nmol/kg) was inhibited by phosphoramidon (5 mg/kg) whereas the elevation of plasma ET-1 was not influenced. It is concluded that the vasoconstrictor effects of both big ET-1 and ET-1 are inhibited, but the increase in plasma ET-1 is unaffected by phosphoramidon.     

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.     

Endogenous endothelin attenuates the pressor response to acute environmental stress via the ETA receptor

Clinical studies have documented an abrupt rise in plasma endothelin-1 (ET-1) coincident with an increase in mean arterial pressure (MAP) during the response to acute stress. We therefore examined the ET(A) and ET(B) receptor-dependent effects of ET-1 on the pressor response to acute environmental stress in ET-1-dependent hypertension. Stress was induced by administration of air jet pulses (3 min) in ET(B) receptor-deficient (ET(B) sl/sl) rats fed normal salt (NS; 0.8% NaCl), high salt (HS; 8% NaCl), and HS plus the ET(A) receptor antagonist ABT-627 (5 mg.kg(-1).day(-1)) on successive weeks. MAP was chronically monitored by telemetry. Total pressor response (area under the curve) was significantly reduced in ET(B) sl/sl rats maintained on a HS vs. NS diet [-6.8 mmHg (SD 18.7) vs. 29.3 mmHg (SD 8.1) x 3 min, P < 0.05]. Conversely, the total pressor response was augmented in both wild-type [34.2 mmHg (SD 29.2) x 3 min, P < 0.05 vs. NS] and ET(B) sl/sl rats [49.1 mmHg (SD 11.8) x 3 min, P < 0.05 vs. NS] by ABT-627. Blockade of ET(B) receptors in Sprague-Dawley rats caused an increase in basal MAP that was enhanced by HS and lowered by mixed ET(A)/ET(B) receptor antagonism; none of these treatments, however, had any effect on the pressor response. These data demonstrate that increasing endogenous ET-1 suppresses the pressor response to acute stress through ET(A) receptor activation in a genetic model of ET-1-dependent hypertension. These results are consistent with reports that ET-1 can attenuate sympathetically mediated responses.     

Endothelin-1 in hypertension in the baroreflex-intact SHR: a role independent from vasopressin release

This study sought to identify whether central endothelin (ET) receptor activation contributes to the elevated pressure in spontaneously hypertensive rats (SHR) and whether an ET-stimulated vasopressin (AVP) release mediates the increased pressure. In Wistar Kyoto (WKY) rats, intracerebroventricular ET-1 induced a dose-dependent pressor response that was shifted rightward in SHR. ET(A) antagonism decreased mean arterial pressure in baroreflex-intact SHR (P<0.01), consistent with inhibition of endogenous ET-1, and blocked the pressor response to exogenous ET-1 in both strains. ET-1 increased AVP only after sinoaortic denervation (P<0.05). Contrary to WKY, sinoaortic denervation was required to elicit a significant pressor response with 5 pmol ET-1 in SHR. Sinoaortic denervation permitted ET-1 to increase AVP in both strains, and peripheral V(1) blockade decreased pressure in denervated but not intact rats. After nitroprusside normalized pressure in SHR, the pressor and AVP secretory responses paralleled those in WKY. Thus endogenous ET(A) receptor mechanisms contribute to hypertension, independent of AVP, in baroreflex-intact SHR. Although blunted in the hypertensive state, the arterial baroreflex buffers the ET-1-induced pressor and AVP secretory responses in both strains.     

Pepsin, an aspartic protease, converts porcine big endothelin to 21-residue endothelin

Porcine big endothelin (big ET-39) at 1 nM, a concentration with no influence on contractile activity in isolated rat aorta, induced a slow-onset and sustained contraction by the pre-incubation with pepsin. When the incubation mixture of big ET-39 with pepsin was analyzed by high-performance liquid chromatography on an octadecyl silica column, two major products of pepsin hydrolysis were obtained; their amino acid sequences were identical with those of 21-residue endothelin (ET-21) and a C-terminal peptide of big ET-39, big ET (22-39), respectively. On the other hand, no degradation of ET-21 was observed by pepsin treatment. These results indicate that pepsin specifically cleaves a Trp21-Val22 bond in the big ET-39 molecule, producing ET-21 and big ET (22-39). Thus, the possibility that pepsin-like aspartic protease may participate in the conversion of big ET-39 to ET-21 in vivo warrants further attention.     

Endothelin-induced vascular and bronchial effects in pig airways: role in acute allergic responses.

The effects of endothelin (ET) agonists on airway mechanics and bronchial blood flow were studied as well as the effects of mixed ET-receptor antagonist bosentan on allergen-induced airway reactions in the pig. ET agonists [ET-1, ET-3, and the ET(B) receptor-selective agonist Sarafotoxin 6c (Sf6c)] were given as intravenous injections (0.4-200 pmol/kg) to eight anesthetized pigs. Bosentan (10 mg/kg iv) was then administered, and the injections were repeated. Only Sf6c caused a significant increase in airway resistance, and this response was blocked by bosentan. Sf6c and ET-1 (200 and 400 pmol/kg, respectively) were also given as aerosols to five pigs. Sf6c, but not ET-1, caused bronchoconstriction via this route. All agonists (intravenous) caused increases in bronchial vascular conductance, an effect that was blocked by an NO-synthase inhibitor (N(G)-nitro-L-arginine) but unaffected by a cyxlooxygenase inhibitor (diclofenac). Fourteen pigs were sensitized with ascaris suum antigen. Under anesthesia, eight pigs were pretreated with bosentan, and six pigs were controls. They were all challenged with allergen aerosol resulting in acute bronchoconstriction and elevation of ET-1 in bronchoalveolar lavage fluid. Bosentan did not affect the maximal acute airway obstruction but markedly increased baseline bronchial vascular conductance, suggesting a basal vascular tone regulated by ETs. In conclusion, ETs induce bronchoconstriction primarily via the ET(B) receptor in the pig. However, ETs are probably not involved in the allergen-induced acute bronchoconstriction in this model.     

ET-1 induces cortical spreading depression via activation of the ETA receptor/phospholipase C pathway in vivo

Recently, it has been shown that brain topical superfusion of endothelin (ET)-1 at concentrations around 100 nM induces repetitive cortical spreading depressions (CSDs) in vivo. It has remained unclear whether this effect of ET-1 is related to a primary neuronal/astroglial effect, such as an increase in neuronal excitability or induction of interastroglial calcium waves, or a penumbra-like condition after vasoconstriction. In vitro, ET-1 regulates interastroglial communication via combined activation of ET(A) and ET(B) receptors, whereas it induces vasoconstriction via single activation of ET(A) receptors. We have determined the ET receptor profile and intracellular signaling pathway of ET-1-induced CSDs in vivo. In contrast to the ET(B) receptor antagonist BQ-788 and concentration dependently, the ET(A) receptor antagonist BQ-123 completely blocked the occurrence of ET-1-induced CSDs. The ET(B) receptor antagonist did not increase the efficacy of the ET(A) receptor antagonist. Direct stimulation of ET(B) receptors with the selective ET(B) agonist BQ-3020 did not trigger CSDs. The phospholipase C (PLC) antagonist U-73122 inhibited CSD occurrence in contrast to the protein kinase C inhibitor G?-6983. Our findings indicate that ET-1 induces CSDs through ET(A) receptor and PLC activation. We conclude that the induction of interastroglial calcium waves is unlikely the primary cause of ET-1-induced CSDs. On the basis of the receptor profile, likely primary targets of ET-1 mediating CSD are either neurons or vascular smooth muscle cells.     

Insulin sensitivity and big ET-1 conversion to ET-1 after ETA- or ETB-receptor blockade in humans.

Cardiovascular diseases are characterized by insulin resistance and elevated endothelin (ET)-1 levels. Furthermore, ET-1 induces insulin resistance. To elucidate this mechanism, six healthy subjects were studied during a hyperinsulinemic euglycemic clamp during infusion of (the ET-1 precursor) big ET-1 alone or after ET(A)- or ET(B)-receptor blockade. Insulin levels rose after big ET-1 with or without the ET(B) antagonist BQ-788 (P < 0.05) but were unchanged after the ET(A) antagonist BQ-123 + big ET-1. Infused glucose divided by insulin fell after big ET-1 with or without BQ-788 (P < 0.05). Insulin and infused glucose divided by insulin values were normalized by ET(A) blockade. Mean arterial blood pressure rose during big ET-1 with or without BQ-788 (P < 0.001) but was unchanged after BQ-123. Skeletal muscle, splanchnic, and renal blood flow responses to big ET-1 were abolished by BQ-123. ET-1 levels rose after big ET-1 (P < 0.01) in a similar way after BQ-123 or BQ-788, despite higher elimination capacity after ET(A) blockade. In conclusion, ET-1-induced reduction in insulin sensitivity and clearance as well as splanchnic and renal vasoconstriction are ET(A) mediated. ET(A)-receptor stimulation seems to inhibit the conversion of big ET-1 to ET-1.     

Endothelin-1 decreases CD36 protein expression in vascular smooth muscle cells

Recent studies have shown that CD36 plays important roles as a major scavenger receptor for oxidized low-density lipoproteins and as a crucial transporter for long-chain fatty acids. CD36 deficiency might be associated with insulin resistance and abnormal dynamics of long-chain fatty acids. Endothelin-1 (ET-1), which is synthesized and secreted by vascular endothelial cells, is the most potent endogenous vasoconstrictor known and also stimulates the proliferation of vascular smooth muscle cells (VSMCs) and thus is believed to play an important role in the development of various circulatory disorders, including hypertension and atherosclerosis. The aim of the present study was to investigate the regulatory effect of ET-1 on CD36 expression in cultured VSMCs. VSMCs were treated for different times (0-24 h) with a fixed concentration (100 nM) of ET-1 or with different concentrations (0-100 nM) for a fixed time (24 h); then CD36 expression was determined using Western blots. CD36 expression was significantly decreased by ET in a time- and dose-dependent manner. This inhibitory effect was prevented by the ET(A) receptor antagonist BQ-610 (10 microM) but not the ET(B) receptor antagonist BQ-788 (10 microM). To further explore the underlying mechanisms of ET-1 action, we examined the involvement of the tyrosine kinase-mediated and MAPK-mediated pathways. The inhibitory effect of ET-1 on CD36 protein expression was blocked by inhibition of tyrosine kinase activation by use of genistein (100 microM) and by the ERK inhibitor PD-98059 (75 microM) but not by the p38 MAPK inhibitor SB-203580 (20 microM). In conclusion, we have demonstrated that ET-1, acting via the ET(A) receptor, suppresses CD36 protein expression in VSMCs by activation of the tyrosine kinase and ERK pathways.     

Aminopropionic acid receptors in paraventricular nucleus mediate pressor and vasopressin responses to endothelin-1 in subfornical organ

Endothelin-1 (ET-1) acts at selected brain loci to elicit a pressor response and secretion of vasopressin (AVP). Glutamatergic receptors of the N-methyl-D-aspartate (NMDA) subtype mediate ET-1-induced AVP secretion in vitro, but the role of glutamatergic receptors in the pressor response and the secretion of AVP in vivo has not been studied. We hypothesized that both the pressor response and AVP secretion in response to ET-1 microinjection into subfornical organ (SFO) would be suppressed by ionotropic glutamatergic receptor antagonists in the paraventricular nucleus (PVN). Sinoaortic denervated male Long Evans rats were equipped with intracerebral cannulae directed into the SFO and the magnocellular region of the PVN bilaterally. Experiments were performed 5 days later in conscious rats. Direct injection of 5 pmol ET-1 into the SFO resulted in a 20 +/- 3 mm Hg increase in mean arterial pressure (MAP) (+/- SE) and a 14.1 +/- 0.3 pg/ml increase in the mean plasma AVP level (+/- SE) (P < 0.001 vs. artificial CSF) that was blocked by selective ET(A) inhibition. Neither the pressor response nor the increase in plasma AVP in response to ET-1 was altered despite prior injection of the NMDA blocker diclozipine (5 microg, MK801) into PVN bilaterally. In contrast, bilateral PVN injection with 6-cyano-7-nitroquinoxaline-2,3-dione (40 nmol, CNQX) prevented the pressor response (MAP +/- SE, - 4 +/- 4 mm Hg) and also inhibited AVP secretion (mean AVP level +/- SE, 0.16 +/- 0.50 pg/ml) (P < 0.001 vs. vehicle in PVN after injection of ET-1 into SFO). These findings support the conclusion that both the pressor response and AVP secretion in response to ET-1 acting at the SFO are mediated by a non-NMDA, most likely an aminopropionic acid glutamatergic receptor within the PVN.     

Differential trafficking and desensitization of human ET(A) and ET(B) receptors expressed in HEK 293 cells

Endothelin-1 (ET-1) is a vasoconstrictor peptide that acts on ET(A) and ET(B) receptors on smooth muscle cells (SMCs). Because vascular SMCs can express both receptors, it is difficult to study the localization and properties of each subtype. Therefore, we investigated the localization and function of ET(A) and ET(B) receptors transfected into HEK 293 cells. Immunocytochemistry was used to examine colocalization of ET receptors with the plasma membrane marker, pan cadherin. In cells transfected with ET(A) receptors, 83 +/- 2% of these receptors colocalized with pan cadherin. In ET(B) receptor-transfected cells, 54 +/- 2% of the receptor colocalized with pan cadherin. When ET(A) and ET(B) receptors were cotransfected, 97 +/- 1% of ET(B) receptors colocalized with ET(A) receptors and 84 +/- 2% of ET(B) receptors colocalized with pan cadherin. ET-1 and sarafotoxin 6c (S6c, ET(B) receptor agonist) increased [Ca2+]i in cells transfected with ET(A) or ET(B) receptors; 100 nM of ET-1 and S6c caused maximal responses. When stimulated with ET-1, ET(B) receptors desensitized faster (t(1/2) = 21 +/- 1 sec) than ET(A) receptors (t(1/2) = 48 +/- 1 sec). S6c-induced increases in [Ca2+]i desensitized in cells expressing ET(B) receptors only (t(1/2) = 17 +/- 1 s). Desensitization was eliminated in cells cotransfected with ET receptors. We conclude that ET(A) receptors localize to the cell membrane, whereas ET(B) receptors are in the membrane and intracellular compartments. Coexpressed ET receptors are in the membrane. ET(B) receptors desensitize faster than ET(A) receptors, but receptor coexpression eliminates desensitization. Finally, ET(A) and ET(B) receptors interact to change receptor trafficking which may modify ET receptor function in vascular SMCs coexpressing these receptors.     

Endothelin type A receptor antagonist normalizes blood pressure in rats exposed to eucapnic intermittent hypoxia

We have reported that eucapnic intermittent hypoxia (E-IH) causes systemic hypertension, elevates plasma endothelin 1 (ET-1) levels, and augments vascular reactivity to ET-1 and that a nonspecific ET-1 receptor antagonist acutely lowers blood pressure in E-IH-exposed rats. However, the effect of chronic ET-1 receptor inhibition has not been evaluated, and the ET receptor subtype mediating the vascular effects has not been established. We hypothesized that E-IH causes systemic hypertension through the increased ET-1 activation of vascular ET type A (ET(A)) receptors. We found that mean arterial pressure (MAP) increased after 14 days of 7 h/day E-IH exposure (109 +/- 2 to 137 +/- 4 mmHg; P < 0.005) but did not change in sham-exposed rats. The ET(A) receptor antagonist BQ-123 (10 to 1,000 nmol/kg iv) acutely decreased MAP dose dependently in conscious E-IH but not sham rats, and continuous infusion of BQ-123 (100 nmol.kg(-1).day(-1) sc for 14 days) prevented E-IH-induced increases in MAP. ET-1-induced constriction was augmented in small mesenteric arteries from rats exposed 14 days to E-IH compared with those from sham rats. Constriction was blocked by the ET(A) receptor antagonist BQ-123 (10 microM) but not by the ET type B (ET(B)) receptor antagonist BQ-788 (100 microM). ET(A) receptor mRNA content was greater in renal medulla and coronary arteries from E-IH rats. ET(B) receptor mRNA was not different in any tissues examined, whereas ET-1 mRNA was increased in the heart and in the renal medulla. Thus augmented ET-1-dependent vasoconstriction via vascular ET(A) receptors appears to elevate blood pressure in E-IH-exposed rats.     

The effects of novel cathepsin E inhibitors on the big endothelin pressor response in conscious rats.

The aspartic protease, cathepsin E, has been shown to specifically cleave big endothelin (big ET-1) at the Trp21-Val22 bond to produce endothelin (ET-1) and the corresponding C-terminal fragment. To determine whether cathepsin E is a physiologically relevant endothelin converting enzyme (ECE), three novel and potent inhibitors of cathepsin E were administered to conscious rats prior to a pressor challenge with big ET-1. One of the inhibitors of cathepsin E, SQ 32,056 (3 mg/kg i.v.), blocked the big ET-1 response. However, this dose of SQ 32,056 also blocked the pressor response to ET-1. Phosphoramidon specifically inhibited the Big ET-1 pressor response. These results suggest that ECE is not cathepsin E.     

Hypoxia augments TNF-alpha-mediated endothelin-1 release and cell proliferation in human optic nerve head astrocytes

The effect of hypoxia (24 h) on TNF-alpha-mediated release of endothelin-1 (ET-1) from human optic nerve head astrocytes (hONAs) and TNF-alpha- and ET-1-induced hONA proliferation was determined. ET-1 synthesis and release was quantitated using ELISA while TNF-alpha (10 nM)- and ET-1 (100 nM)-mediated hONA proliferation was assessed by CellTiter 96 aqueous one-solution cell proliferation assay, respectively. hONAs appeared to be more rounded with fewer processes following 24 h hypoxia compared to thodr seen in normoxia. Hypoxia enhanced TNF-alpha-mediated ET-1 synthesis and release (by 5-fold) and also significantly increased TNF-alpha- and ET-1-mediated hONA proliferation. PD142893 (1 microM), an ET(A/B) receptor antagonist, blocked ET-1-mediated hONA proliferation both under normoxia and hypoxia, while doing so only under normoxia following TNF-alpha treatment. Also, U0126 (10 microM; an upstream ERK1/2 inhibitor) completely blocked agonist-induced hONA proliferation in normoxia and partially blocked the same in hypoxia. These results demonstrate for the first time that hONAs secrete ET-1 and that TNF-alpha and hypoxia can regulate its levels. Moreover, hypoxia augments the proliferative responses of hONAs to TNF-alpha and ET-1. These agonist-mediated effects following hypoxia could contribute to astroglial activation as seen in glaucomatous optic nerve heads.     

Effect of endothelin-1 on release of arginine-vasopressin from perifused rat hypothalamus

Endothelin-1 (ET-1) is an endothelium-derived vasoconstrictor peptide with potent pressor activity. We studied the effect of ET-1 on release of arginine-vasopressin (AVP) from perifused rat hypothalamus. ET-1 (10(-10) to 10(-8) M) significantly stimulated AVP release. The ET-1-induced AVP release was completely blocked in the presence of nicardipine. Our results suggest a possible involvement of ET in the regulation of AVP release.     

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.     

Calcium dependence of effects of endothelin on rat mesenteric microvessels

We investigated the calcium dependence of the effects of endothelin (ET) on resistance vessels (less than 300 microns lumen diameter) from the mesenteric vascular bed of the rat, mounted on a wire myograph. ET-1 induced a potent sustained contraction with an ED50 of 12 nmol/L. The response to ET-3 and big ET at the maximum concentrations used (100 nmol/L) was less than 40% of that to ET-1, with an estimated ED50 of 45 nmol/L. Relaxation of the ET-1-induced contraction was slow, and resulted in a reduction of the maximum response to a second challenge with ET-1 to 60% of the initial contraction after 3 h. Long-lasting tachyphylaxis to arginine vasopressin (AVP) induced contraction also occurred. The response to 100 nmol/L ET-1 produced an active tension 88% greater than that induced by 124 mmol/L KCl, and similar to that produced by norepinephrine and AVP. The response to 100 nmol/L ET-1 in the absence of calcium + 1 mmol/L EGTA in the medium for 30 min resulted in a maximum contraction of 43% of the response in the presence of calcium, followed by a faster relaxation rate. The addition of calcium produced a further contraction, and stimulation with 100 nmol/L ET-1 at this point did not result in further response. The calcium channel blocker nitrendipine in concentrations of 1-10 mumol produced increasing reductions of the responses to 100 nmol/L ET-1 to 35% at the higher concentration. Nitrendipine (3 mumol/L) partially blocked the response to calcium after ET-1 was added in the absence of calcium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Analysis of responses to endothelins in the rabbit pulmonary and systemic vascular beds

The effects of two isoforms of human endothelin (ET) on the pulmonary and systemic vascular beds were compared in the anesthetized intact-chest rabbit under conditions of constant pulmonary blood flow and left atrial pressure. Intralobar bolus injections of ET-1 (0.1-1 micrograms) and ET-3 (1-3 micrograms) produced modest vasoconstriction in the pulmonary vascular bed, whereas both peptides decreased systemic arterial pressure. The pulmonary vasoconstrictor response to ET-1 and ET-3 was inhibited by intralobar infusion of nitrendipine but was not altered by indomethacin. In contrast to the small effects of ET-1 and ET-3 on intact pulmonary resistance vessels, both peptides markedly contracted isolated pulmonary conductance vessels, with greater activity on venous than on arterial segments. Intravenous bolus injection of ET-1 (0.1-0.3 micrograms) or ET-3 (0.3-1 microgram) decreased systemic arterial pressure, increased cardiac output, and markedly decreased systemic vascular resistance. Higher doses of ET-1 produce a biphasic systemic vascular response with a prominent secondary pressor component. The present data suggest that the pulmonary vasoconstrictor activity of ET-1 is greater than that of ET-3 and their pressor activity depends on an extracellular source of calcium. The pulmonary and systemic hemodynamic effects of ET-1 and ET-3 in the rabbit do not depend on cyclooxygenase products. The systemic vasodilator response to ET-1 is not altered by first-pass lung transit. Furthermore the systemic vasodilator response to both peptides occurs independent of activation of muscarinic, beta 2-adrenergic, and platelet-activating factor receptors. Although ET-1 and ET-3 were initially reported as vasoconstrictor peptides, the present data suggest that, by having unique and potent systemic vasodilator activity, ET-1 and ET-3 act differently in the systemic and pulmonary vascular beds under resting conditions in the rabbit.     

Effects of endothelin-1 and homologous trout endothelin on cardiovascular function in rainbow trout

The cardiovascular effects of endothelin (ET)-1 and the recently sequenced homologous trout ET were examined in unanesthetized trout, and vascular capacitance curves were constructed to evaluate the responsiveness of the venous system to ET-1. A bolus dose of 667 pmol/kg ET-1 doubled ventral aortic pressure; produced a triphasic pressor-depressor-pressor response in dorsal aortic pressure (P(DA)); increased central venous pressure, gill resistance, and systemic resistance; and decreased cardiac output, heart rate, and stroke volume. These responses were dose dependent. Bolus injection of trout ET (333 or 1,000 pmol/kg) produced essentially identical, dose-dependent cardiovascular responses as ET-1. Dorsal aortic infusion of 1 and 3 pmol. kg(-1). min(-1) ET-1 and central venous infusion into the ductus Cuvier of 0.3 and 1 pmol. kg(-1). min(-1) produced similar dose-dependent cardiovascular responses, although the increase in P(DA) became monophasic. The heightened sensitivity to central venous infusion was presumably due to the more immediate exposure of the branchial vasculature to the peptide. Infusion of 1 pmol. kg(-1). min(-1) ET-1 decreased vascular compliance but had no effect on unstressed blood volume. These results show that ETs affect a variety of cardiovascular functions in trout and that branchial vascular resistance and venous compliance are especially sensitive. The multiplicity of effectors stimulated by ET suggests that this peptide was extensively integrated into cardiovascular function early on in vertebrate phylogeny.