LPS from Porphyromonas gingivalis increases the sensitivity of contractile response mediated by endothelin-B (ETB) receptors in cultured endothelium-intact rat coronary arteries

The purpose of our study was to examine if lipopolysaccharide (LPS) from Porphyromonas gingivalis (P.g.) modifies the vasomotor responses to Endothelin-1 (ET-1) and Sarafotoxin 6c (S6c) in rat coronary arteries. The arteries were studied directly or following organ culture for 24 h in absence and presence of 2.5 EU/ml LPS. The contractile responses of coronary arteries were investigated by using the selective ETB receptor agonist S6c (1 pM–0.3 μM) and ET-1 (1 pM–0.3 μM). The functional studies demonstrated an augmented contractile response only to S6c in isolated rat coronary arteries after organ culture (with or without LPS). These contractile responses by S6c were blocked by the selective ETB receptor antagonist BQ788 in both vessel groups. The augmented contractile response to S6c was supported by immunohistochemistry, where a significant increase in fluorescence intensity for ETB receptors in smooth muscle cells was observed after organ culture. The presence of LPS in the culture medium significantly increased the sensitivity of endothelium-intact coronary artery to S6c as compared to endothelium-denuded segments. Our results showed a significant increase in both ETB receptor protein levels and S6c-induced maximal contraction in coronary arteries upon 24 h of organ culture, which was further sensitized by LPS.     

Importance of ERK1/2 in upregulation of endothelin type B receptors in cerebral arteries

This study examines the importance of mitogen-activated protein kinases (MAPKs) in upregulation of endothelin type B (ETB) receptors. Rat middle cerebral arteries (MCAs) were incubated for 24 h with or without kinase inhibitors. Vessel segments were mounted in myographs and the contractile responses to endothelin-1 (ET-1; ETA and ETB receptor agonist) and sarafotoxin 6c (S6c; ETB receptor agonist) were studied. We used real-time PCR to measure the receptor mRNA levels. An ELISA assay showed the activation of ERK1/2 kinases after 3 h. Immunohistochemistry revealed the presence of ETB receptors on the vessels. After organ culture, S6c induced vasoconstriction. Incubation with the MEK/ERK inhibitors U0126 and SB386023 diminished the contractile response to S6c. The p38 MAPK inhibitor SB239063 did not affect the S6c-induced contraction. The ET-1-induced vasoconstriction was increased after incubation with SB386023 or SB239063, while unaffected by U0126. The ETB receptor mRNA levels were diminished by SB386023 and U0126. The ETA receptor mRNA levels were unaffected. The levels of activated ERK1/2 kinases were significantly higher after 3 h of organ culture as compared to fresh vessels. The level of ETB receptor protein on the smooth muscle cells of the MCA, visualised by immunohistochemistry, was somewhat diminished by SB386023. Our results show that the ERK1/2 MAPK is important in the upregulation of contractile ETB receptors in MCA after organ culture. Since there is a similar upregulation in models of focal ischaemia and subarachnoid haemorrhage, this may be an important pathophysiological event.     

ERK1/2通路参与大鼠肠系膜动脉平滑肌细胞ETB受体上调表达

目的探讨细胞外信号调节激酶1/2(ERK1/2)信号转导通路在血管平滑肌细胞内皮素-1 B型受体(ET_B)上调中的作用。方法用大鼠肠系膜上动脉离体培养模型，以敏感的离体小血管张力描记技术记录血管张力变化，实时PCR定量ET_B受体mRNA，PhosphoELISA法测定细胞内磷酸化的ERK1/2蛋白水平。结果大鼠肠系膜上动脉培养3 h，细胞内ERK1/2蛋白磷酸化水平明显增高，培养24 h ET_B受体mRNA表达水平显著上调，选择性ET_B受体激动剂蛇毒类似物(sarafotoxin 6c，S6c)引起的收缩增强；与特异性ERK1/2通路阻滞剂SB386023共同孵育24 h，S6c引起的最大收缩E_max明显下降，ET_B受体mRNA水平也显著降低。结论ERK1/2信号转导通路参与大鼠肠系膜上动脉离体平滑肌细胞ET_B受体上调过程。     

Comparison of endothelin B (ETB) receptors in rabbit isolated pulmonary artery and bronchus.

1. To explore potential differences between endothelin (ET) receptors in airway versus vascular smooth muscle from the same species, the ETB receptors mediating contractions produced by ET-1, ET-3 and the selective ETB ligands, sarafotoxin S6c (S6c) and BQ-3020, in rabbit bronchus and pulmonary artery were investigated by use of peptide and non-peptide ET receptor antagonists. 2. In rabbit pulmonary artery SB 209670 (10 microM), a mixed ETA/ETB receptor antagonist, was a more potent antagonist of contractions produced by S6c (pKB = 7.7; n = 9; P < 0.05), than those elicited by ET-1 (pKB = 6.7; n = 6) or ET-3 (pKB = 6.7; n = 5). BQ-788 (10 microM), an ETB receptor antagonist, inhibited responses produced by ET-3 (pKB = 5.1; n = 8), BQ-3020 (pKB = 5.2; n = 4) or S6c (pKB = 6.2; n = 9; P < 0.05 compared to potency versus ET-3- or BQ-3020-induced contractions), but was without inhibitory effect on ET-1-induced contractions (n = 5). RES-701 (10 microM), another selective ETB receptor antagonist, was without effect on contractions produced by S6c (n = 4) or ET-1 (n = 4), and potentiated ET-3- (n = 5) or BQ-3020-induced responses (n = 4). 3. The combination of BQ-788 (10 microM) and BQ-123 (10 microM), an ETA-selective receptor antagonist, antagonized contractions produced by lower concentrations of ET-1 (1 and 3 nM) in rabbit pulmonary artery, but was without effect on responses elicited by higher concentrations of ET-1 (n = 5). The combination of RES-701 (10 microM) and BQ-123 (10 microM) potentiated responses elicited by ET-1, producing a 3.7 fold shift to the left in the agonist concentration-response curve (n = 5). 4. In rabbit bronchus SB 209670 (3 microM) had similar potency for antagonism of contractions produced by ET-1 (pKB = 6.3; n = 6), ET-3 (pKB = 6.5; n = 6) or S6c (pKB = 6.1; n = 8). BQ-788 (3 microM) was without effect on responses elicited by ET-1, ET-3 or S6c (n = 6) but antagonized BQ-3020-induced contractions (pKB = 6.4; n = 4). RES-701 (3 microM) was without effect on contractions produced by S6c (n = 6) or BQ-3020 (n = 4), and potentiated rather than antagonized ET-1- or ET-3-induced responses (n = 6), reflected by a significant (about 6 fold) shift to the left in ET-1 or ET-3 concentration-response curves. The combination of BQ-788 (3 microM) and BQ-123 (3 microM) was without effect on contractions produced by ET-1 in rabbit bronchus (n = 6). The combination of RES-701 (3 microM) and BQ-123 (3 microM) potentiated responses elicited by ET-1, producing a 5.2 fold shift to the left in the agonist concentration-response curve (n = 5). 5. BQ-123 (3 or 10 microM), an ETA-selective receptor antagonist, was without effect on ET-1, ET-3 or S6c concentration-response curves (n = 3-6) in rabbit pulmonary artery or rabbit bronchus. 6. These data indicate that contractions induced by ET-1, ET-3, S6c and BQ-3020 in rabbit pulmonary artery or rabbit bronchus appear to be mediated predominantly via stimulation of ETB receptors. However, the qualitative and quantitative differences in the relative profiles of the various structurally diverse peptide and non-peptide antagonists examined suggests that responses produced by the ET ligands may not be mediated by a homogeneous ETB receptor population. In addition, the results suggest that differences exist in the ETB receptors mediating contraction in pulmonary vascular versus airway tissues in the same species. These receptors are not very sensitive to the standard ETB receptor antagonists, BQ-788 and RES-701. Furthermore, the results also provide further evidence that the potencies of ET receptor antagonists depend upon the ET agonist.     

The functional shift of endothelin receptor subtypes in dogs with heart failure produced by rapid ventricular pacing

To clarify the functional role of endothelin-A/endothelin- B (ETA/ETB) receptors in congestive heart failure (CHF), we examined the effects of a non-selective endothelin receptor agonist, endothelin-1 (ET-1), and a selective ETB receptor agonist, sarafotoxin S6c. CHF was induced in dogs by rapid ventricular pacing and resulted in decreased left ventricular dp/dtmax, decreased cardiac output and increased pulmonary vascular resistance. Sarafotoxin S6c (0.3 nmol/kg) resulted in decreased left ventricular dp/dtmax (-26 +/- 2%), decreased cardiac output (-47 +/- 3%) and increased pulmonary vascular resistance (+48 +/- 10%) in dogs without CHF. The effects of sarafotoxin S6c were attenuated in dogs with CHF (-12 +/- 5% in left ventricular dp/dtmax, -19 +/- 5% in cardiac output and +7 +/- 5% in pulmonary vascular resistance). In contrast, ET-1 (0.5 nmol/kg) had no effect on left ventricular dp/dtmax in dogs without CHF and increased left ventricular dp/dtmax by 16 +/- 3% in dogs with CHF. These data indicate that reduced cardiac contractile and pulmonary vasoconstrictor responses via the ETB receptor are attenuated and that responses mediated by the ETA receptor are more prominent in the context of CHF. This suggests a functional shift of endothelin receptor subtypes in CHF.     

EndothelinB receptor-mediated contraction in human pulmonary resistance arteries.

1. Using wire myography, we have examined the endothelin (ET) receptor subtypes mediating vasoconstriction to ET peptides in human pulmonary resistance arteries (150-200 microns, i.d.). 2. Cumulative concentration-response curves to ET-1, sarafotoxin 6c (SX6c) and ET-3 were constructed in the presence and absence of the selective antagonists FR 139317 (ETA-selective), BMS 182874 (ETA-selective) and BQ-788 (ETB-selective). 3. All agonists induced concentration-dependent contractions. However, the response curves to ET-1 were biphasic in nature. The first component demonstrated a shallow slope up to 1 nM ET-1. Above 1 nM ET-1 the response curve was markedly steeper. Maximum responses to ET-3 and SX6c were the same as those to 1 nM ET-1 and 30% of those to 0.1 microM ET-1. The order of potency, taking 0.3 microM as a maximum concentration was SX6c >> ET-3 > ET-1 (pEC50 values of: 10.75 +/- 0.27, 9.05 +/- 0.19, 8.32 +/- 0.08 respectively). Taking 1 nM ET-1 as a maximum, the EC50 for ET-1 was 10.08 +/- 0.13 and therefore ET-1 was equipotent to ET-3 and SX6c over the first component of the response curve. 4. Responses to ET-1 up to 1 nM were resistant to the effects of the ETA receptor antagonists, FR 139317 and BMS 182874 but were inhibited by the ETB receptor antagonist, BQ-788. Conversely, responses to ET-1 over 1 nM were inhibited by the ETA receptor antagonists, FR 139317 and BMS 182874 but unaffected by the ETB receptor antagonist, BQ-788. 5. The results suggest that at concentrations up to 1 nM, responses to ET-1 are mediated via the ETB receptor, whilst the responses to higher concentrations are mediated by ETA receptors.     

Vascular reactivity of mesenteric arteries and veins to endothelin-1 in a murine model of high blood pressure

We characterized vascular reactivity to endothelin-1 (ET-1) in mesenteric vessels from DOCA-salt hypertensive and SHAM control mice and assessed the effect that endothelial-derived vasodilators have on ET-1-induced vasoconstriction. Changes in the diameter of unpressurized small mesenteric arteries and veins (100- to 300-microm outside diameter) were measured in vitro using computer-assisted video microscopy. Veins were more sensitive than arteries to the contractile effects of ET-1. There was a decrease in arterial maximal responses (E(max)) compared to veins, this effect was larger in DOCA-salt arteries. The selective ET(B) receptor agonist, sarafotoxin 6c (S6c), contracted DOCA-salt and SHAM veins but did not contract arteries. The ET(B) receptor antagonist, BQ-788 (100 nM), but not the ET(A) receptor antagonist, BQ-610 (100 nM), blocked S6c responses. BQ-610 partially inhibited responses to ET-1 in mesenteric veins from DOCA-salt and SHAM mice while BQ-788 did not affect responses to ET-1. Co-administration of both antagonists inhibited responses to ET-1 to a greater extent than BQ-610 alone suggesting a possible functional interaction between ET(A) and ET(B) receptors. Responses to ET-1 in mesenteric arteries were completely inhibited by BQ-610 while BQ-788 did not affect arterial responses. Nitric oxide synthase inhibition potentiated ET-1 responses in veins from SHAM but not DOCA-salt mice. There was a prominent role for ET-mediated nitric oxide release in DOCA-salt but not SHAM arteries. In summary, these studies showed a differential regulation of ET-1 contractile mechanisms between murine mesenteric arteries and veins.     

ERK1/2通路参与ET_A和ET_B受体介导的离体肠系膜上动脉收缩研究

目的探讨细胞外信号调节激酶1/2(ERK1/2)信号转导通路在内皮素1(ET-1)的两个G蛋白偶联受体ET_A和ET_B介导的收缩机制。方法用大鼠肠系膜上动脉器官培养模型,以敏感的离体药理学实验方法记录培养前后血管平滑肌张力,实时定量的PCR测定培养前后受体mRNA表达水平的变化。结果S6c不引起新鲜的肠系膜上动脉收缩,培养后ETB受体mRNA表达水平上调,介导的收缩明显增强(P<005);而ETA受体介导的收缩功能和mRNA均变化不大。低浓度的SB386023(10-5mol·L-1)降低S6c引起的最大收缩(Emax从239%±26%降至89%±13%,P<001),而对ET1引起的最大收缩并无影响(Emax271%±19%vs251%±16%,P>005);高浓度的SB386023(10-4mol·L-1)明显抑制ETA受体介导的收缩。结论ET-1通过ET_A受体介导新鲜动脉的收缩;动脉培养后表达ET_B受体;ERK1/2信号转导通路对ET_B受体的作用强于ET_A受体。     

Different endothelin receptors involved in endothelin-1- and sarafotoxin S6B-induced contractions of the human isolated coronary artery.

1. Endothelin receptors, that mediate contraction of the human isolated coronary artery, were characterized by use of a number of agonists and antagonists. Contraction induced by the non-selective agonists, endothelin (ET)-1 and sarafotoxin S6b, was compared in endothelium-intact and endothelium-denuded ring segments. The effects of ET-1 and BQ-123 (an ETA receptor antagonist) were investigated both in ring segments and in spirally cut strips. Lastly, the effect of phosphoramidon was studied on contraction induced by big-ET-1. 2. The order of agonist potency (pD2) in endothelium-intact coronary artery ring segments was: ET-1 (8.27) approximately sarafotoxin S6b (8.16) > big-ET-1 (< 7.1) approximately ET-3 (< 6.9). [Ala1,3,11,15]ET-1 (ETB receptor agonist) caused significant contraction only at 1 microM, whereas 0.3 microM big-ET-3 had no effect. Removal of the endothelium in ring segments did not affect the contractile response to ET-1 or to sarafotoxin S6b. 3. After a full concentration-response curve had been obtained to ET-1 or sarafotoxin S6b, further contractions of the endothelium-intact coronary artery segments could only be achieved by applying ET-1 in segments exposed to sarafotoxin S6b, and not the reverse. 4. BQ-123 (0.1 microM) antagonized contractions of endothelium-intact ring segments induced by sarafotoxin S6b (pKB 7.86). Only 10 microM BQ-123 antagonized contractions induced by ET-1 (pKB 5.75). FR139317 was also more potent against sarafotoxin S6b (pKB 8.24-8.47) than against ET-1 (pKB 6.11). [Ala1,3,11,15]ET-1 (1 microM) had no effect on the contractile response to ET-1 or to sarafotoxin S6b.(ABSTRACT TRUNCATED AT 250 WORDS)     

Endothelin alters the reactivity of vasa vasorum: mechanisms and implications for conduit vessel physiology and pathophysiology

1 The walls of certain large blood vessels are nourished by the vasa vasorum, a network of microvessels that penetrate the adventitia and media of the vessel wall. The purpose of this study was to characterize endothelin-1 (ET-1)-mediated contraction of vasa and to investigate whether threshold concentrations of ET-1 alters the sensitivity to constrictors. Arterial vasa were dissected from the walls of porcine thoracic aorta and mounted in a tension myograph. 2 ET-1 and ETB-selective agonist, sarafotoxin 6c (S6c), produced concentration-dependent contraction. ETA receptor antagonist, BQ123 (10 microM), caused a biphasic rightward shift of ET-1 response curves. ETB receptor antagonist, BQ788 (1 microM), produced a rightward shift of response curves to ET-1 and S6c of 5- and 80 fold respectively. 3 ET-1 responses were abolished in Ca2+-free PSS but unaffected by selective depletion of intracellular Ca2+ stores. Nifedipine (10 microM), an L-type Ca2+ channel blocker, attenuated ET-1 responses by 44%. Inhibition of receptor-operated Ca2+ channels or non-selective cation entry using SKF 96365 (30 microM) and Ni2+ (1 mM) respectively, attenuated ET-1 contractions by 60%. 4 ET-1 (1-3 nM) enhanced responses to noradrenaline (NA) (4 fold) but not to thromboxane A2-mimetic, whilst K+ (10-20 mM) sensitized vasa to both types of constrictor. 5 Therefore, ET-1-induced contraction of isolated vasa is mediated by ETA and ETB receptors and involves Ca2+ influx through L-type and non-L-type Ca2+ channels. Furthermore elevation of basal tone of vasa vasorum alters the profile of contractile reactivity. These results suggest that ET-1 may be an important regulator of vasa vasorum reactivity.     

Characterization of endothelin receptor subtypes mediating Ca2+ mobilization and contractile response in rabbit iris dilator muscle.

1. We investigated the characteristics of endothelin (ET)-induced contraction and changes in intracellular Ca2+ concentration ([Ca2+]i) using the fura-2-loaded and non-loaded rabbit iris dilator. ET-1 and ET-2 (3-100 nM) and ET-3 (30-100 nM) caused contraction in a concentration-dependent fashion. 2. The selective ETB-receptor agonists, IRL1620 and sarafotoxin S6c produced only a small contraction or no contraction at a concentration of 1 microM. The rank order of potencies for the contraction (pD2 value) was ET-1 = ET-2 > ET-3 >> sarafotoxin S6c = IRL1620. 3. The contractile response to ET-3 was antagonized by pretreatment with BQ-123 (10 nM), a selective ETA receptor antagonist. The contractile responses to ET-1 and ET-2 were antagonized by pretreatment with BQ-123 (10 microM), but not at a concentration of 10 nM. 4. ETs increased [Ca2+]i and sustained muscle contraction. ET-1 (100 nM), ET-2 (100 nM), and ET-3 (1 microM) induced an elevation of [Ca2+]i consisting of two components: first a rapid and transient elevation to reach a peak, followed by a second, sustained elevation; a sustained contraction was produced without a transient contraction. The ETB receptor-selective agonist, IRL1620 (1 microM) and sarafotoxin S6c (1 microM) also induced a rapid and transient elevation of [Ca2+]i to reach a peak and a sustained elevation, together with only a small contraction or no contraction. 5. ET-1 (100 nM) induced a transient increase in [Ca2+]i in a Ca(2+)-free, 2 mM EGTA-containing physiological saline solution (Ca(2+)-free PSS), and a small sustained contraction which was significantly different from that induced by ET-1 (100 nM) in normal PSS. The ET-1-induced increase in [Ca2+]i and sustained contraction were not affected by the voltage-dependent Ca2+ channel blocker, nicardipine (10 microM). The ET-1-induced transient increase in [Ca2+]i was significantly reduced by the sarcoplasmic reticulum (SR) Ca(2+)-ATPase inhibitor, cyclopiazonic acid (30 microM); however, the ET-1-induced sustained contraction was not affected by this agent. 6. The selective ETA receptor antagonist, BQ-123 (100 nM) reduced the ET-3 (100 nM)-induced contraction, but did not affect the transient increase or elevation of the second phase of [Ca2+]i. However, this antagonist at 1 microM did not affect the ET-1 (100 nM)- and ET-2 (100 nM)-induced elevation of [Ca2+]i and contractile response, or the IRL1620-induced elevation of [Ca2+]i. 7. The selective ETB receptor antagonist, BQ-788 (1 microM) reduced the transient increase in [Ca2+]i induced by ET-1 (30 nM), ET-2 (30 nM), ET-3 (100 nM) and IRL1620 (1 microM), but did not affect the sustained elevation of [Ca2+]i and contractile responses produced by ET-1, ET-2 and ET-3. 8. Pretreatment with IRL1620 (1 microM) reduced the increase in [Ca2+]i induced by IRL1620 (1 microM) and sarafotoxin S6c (1 microM), as well as the ET-1 (100 nM)-, ET-2 (100 nM)- and ET-3 (1 microM)-induced elevation of [Ca2+]i, whereas in the presence of IRL1620, ET-1-, ET-2- and ET-3-induced contractions were unaltered. 9. These results suggest that ETA and ETB receptor subtypes exist in the rabbit iris dilator muscle, and that the ETA receptor is divided into: (1) BQ-123-sensitive ETA subtypes activated by ET-1, ET-2 and ET-3, and (2) BQ-123-insensitive ETA subtypes activated by ET-1 and ET-2, which cause the sustained increase of [Ca2+]i and contraction; in contrast, ETB receptor subtypes are activated by ET-1, ET-2, ET-3, IRL1620 and sarafotoxin S6c and cause the transient and sustained increase in [Ca2+]i which is not able to contract the smooth muscle.     

Arteries and veins desensitize differently to endothelin

Hypertension is accompanied by increased arterial endothelin-1 (ET-1) and decreased arterial contraction to ET-1. By contrast, veins remain responsive to ET-1 in hypertension. Isometric contraction was used to test the hypothesis that veins do not desensitize to ET-1 to the extent of arteries, possibly because of the presence of functional ETA and ETB receptors on veins and only functional ETA receptors on arteries. Contraction to ET-1 after exposure to ET-1 (100 nmol/L) was abolished in aortae, while in veins 36.3 +/- 0.2% of maximal contraction to ET-1 remained. Aortae were unresponsive to the ETA receptor agonist ET-1(1-31) (100 nmol/L) after ET-1 exposure, while 21.9 +/- 0.6% of maximum venous contraction to ET-1 (1-31) remained. In a similar manner, the venous ETB receptor did not lose responsiveness to the ETB receptor agonist sarafotoxin 6c (S6c, 100 nmol/L); aortae did not contract to S6c. In ET-1-desensitized veins, the ETB receptor antagonist BQ-788 (100 nmol/L) decreased maximum contraction to ET-1, but did not alter potency (-log EC50 control = 8.14 +/- 0.01 mol/L; BQ-788 = 8.13 +/- 0.04 mol/L). The ETA receptor antagonist atrasentan (100 nmol/L) blocked remaining venous contraction to ET-1 (control = 8.05 +/- 0.05 mol/L; atrasentan = unmeasurable). Maintained responsiveness to ET-1 in veins occurs primarily via the ETA receptor, while in arteries the ETA receptor is responsible for desensitization to ET-1.     

Endothelin-1 (ET-1)-induced contraction in rat isolated trachea: involvement of ETA and ETB receptors and multiple signal transduction systems.

1. Quantitative autoradiographic, biochemical and functional studies were performed to investigate the endothelin receptor subtypes and signal transduction systems that mediate endothelin-1 (ET-1)-induced contraction in rat isolated tracheal smooth muscle. 2. Specific binding of 0.5 nM [125I]-ET-1 to tracheal smooth muscle was inhibited by at least 40% in the presence of either the ETA receptor selective ligand BQ-123 (1 microM) or the ETB receptor-selective ligand sarafotoxin S6c (30 nM), indicating the presence of both ETA and ETB receptors in this tissue. 3. ET-1 and sarafotoxin S6c were both potent spasmogens of rat isolated tracheal smooth muscle preparations. Sarafotoxin S6c-induced contractions were unaffected in the presence of the ETA receptor antagonist BQ-123 (10 microM), but were markedly attenuated in tissue previously exposed to 100 nM sarafotoxin S6c to induce ETB receptor desensitization. ET-1-induced contractions were, at most, only partially attenuated either by blocking the ETA receptor-effector system (with 10 microM BQ-123) or by desensitizing the ETB receptor-effector system with sarafotoxin S6c. However, ET-1-induced contractions were markedly attenuated by blocking both receptor-effector systems simultaneously. These findings suggest that ET-1 could induce contraction by stimulating either ETA or ETB receptors. 4. ET-1 (10 microM) induced a 7 fold increase in intracellular [3H]-inositol phosphate accumulation over basal levels in rat isolated tracheal smooth muscle. In contrast, sarafotoxin S6c (2.5 microM) increased intracellular [3H]-inositol phosphate accumulation by only 2 fold. ET-1-induced accumulation of [3H]-inositol phosphates was abolished by 10 microM BQ-123.(ABSTRACT TRUNCATED AT 250 WORDS)     

Endothelin-1-induced potentiation of human airway smooth muscle proliferation: an ETA receptor-mediated phenomenon.

1. In this study the mitogenic effects in human cultured tracheal smooth muscle cells of endothelin-1 (ET-1), ET-3, and sarafotoxin S6c (S6c), the ETB receptor-selective agonist, were explored either alone or in combination with the potent mitogen, epidermal growth factor (EGF). 2. In confluent, growth-arrested human airway smooth, neither ET-1 (0.01 nM-1 microM) nor ET-3 (0.001 nM-1 microM) or S6c (0.01 nM-1 microM) induced cell proliferation, as assessed by [3H]-thymidine incorporation. In contrast, EGF (1.6 pM-16 nM) produced concentration-dependent stimulation of DNA synthesis (EC50 of about 0.06 nM). The maximum increase of about 60 fold above control, elicited by 16 nM EGF, was similar to that obtained with 10% foetal bovine serum (FBS). EGF (0.16-16 nM) also produced a concentration-dependent increase in cell counts, whereas ET-1 (1-100 nM) was without effect on this index of mitogenesis. 3. ET-1 (1-100 nM) potentiated EGF-induced proliferation of human tracheal smooth muscle cells. For example, ET-1 (100 nM), which alone was without significant effect, increased by 3.0 to 3.5 fold the mitogenic influence of EGF (0.16 nM). The potentiating effect of ET-1 on EGF-induced proliferation was antagonized by BQ-123 (3 microM), the ETA receptor antagonist, but was unaffected by the ETB receptor antagonist BQ-788 (10 microM). 4. Neither ET-3 (1-100 nM) nor S6c (1-100 nM) influenced the mitogenic effects of EGF (0.16-1.6 nM). 5. [125I]-ET-1 binding studies revealed that on average the ratio of ETA to ETB receptors in human cultured tracheal smooth muscle cells was 35:65 ( +/- 3; n = 4), confirming the predominance of the ETB receptor subtype in human airway smooth muscle. 6. These data indicate that ET-1 alone does not induce significant human airway smooth muscle cell proliferation. However, it potently potentiated mitogenesis induced by EGF, apparently via an ETA receptor-mediated mechanism. These findings suggest that ET-1, a mediator detected in increased amounts in patients with acute asthma, may potentiate the proliferative effects of mitogens and contribute to the airway smooth muscle hyperplasia associated with chronic severe asthma.     

ETA receptor-mediated constrictor responses to endothelin peptides in human blood vessels in vitro.

1. We have characterized the constrictor endothelin receptors present in human isolated blood vessels using ETA and ETB selective agonists and antagonists. 2. Monophasic dose-response curves were obtained for ET-1 with EC50 values of 6.8 nM in coronary artery, 3.9 nM in internal mammary artery, 17.4 nM in pulmonary artery, 14.5 nM in aorta and 3.2 nM in saphenous vein. In coronary artery, ET-2 was equipotent with ET-1 with an EC50 value of 5.7 nM. The non-selective peptide, sarafotoxin 6b, was 2-3 times less potent than ET-1 but the maximum responses to these two were comparable. 3. In each vessel ET-3 was much less active than ET-1. No response was obtained to ET-3 in aorta and pulmonary artery or in up to 50% of coronary artery, mammary artery and saphenous vein preparations. In those preparations that did respond, dose-response curves were incomplete at 300 nM. Variable contractions were also obtained with the ETB-selective agonist, sarafotoxin 6c (S6c). Where responses were detected, although S6c was more potent than ET-1 (EC50 values of 0.6-1.2 nM), the maximum response produced was always less than 20% of that to ET-1. 4. The synthetic ETB agonists, BQ3020 and [1,3,11,15Ala]-ET-1, were without effect in any of the five blood vessels at concentrations up to 3 microM. 5. ET-1-induced vasoconstriction was blocked by the ETA-selective antagonists, BQ123 and FR139317.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence that ET-1, but not ET-3 and S6b, ET(A)-receptor mediated contractions in isolated rat mesenteric arteries are modulated by co-activation of ET(B) receptors.

The effects of agonists with endothelin (ET) ET(A)-receptor activity have been analysed in relation to their interaction with ET(B) receptors in rat mesenteric arteries. ET-1, sarafotoxin 6b (S6b) and ET-3 induced large, slow-onset and sustained contractions whereas S6c induced weak transient contractions. However, following pre-contraction with U46619 and subsequent relaxation with forskolin, the effect of S6c was amplified, indicating a potential for powerful ET(B)-receptor mediated contraction. The selective ET(A)-receptor antagonist, FR139317, produced parallel rightward shifts of ET-1, S6b and ET-3 concentration-effect curves indicating that the contractions were mediated by ET(A) receptors. However, the corresponding FR139317 pK(B) values were significantly different between the agonists. As expected FR139317 had no effect on S6c responses. Pre-treatment with S6c to desensitize ET(B) receptors, increased ET-1 potency and the pK(B) value for FR139317. In contrast, neither the potency of S6b and ET-3 nor the pK(B) values for FR139317 estimated using these agonists were affected by ET(B)-receptor desensitization. Segments pre-contracted with submaximal concentrations of S6b and ET-3, but not ET-1, rapidly relaxed following wash-out or FR139317 administration. The results indicate that the small contractile response to selective ET(B) receptor activation, barely detectable under standard bioassay conditions, is greatly amplified when adenylate cyclase activity is elevated. Moreover, the response to ET(A) receptor activation by ET-1, but not ET-3 and S6b, is significantly modified by co-activation of ET(B) receptors. This interaction has a significant effect on the apparent affinity of ET(A)-receptor selective antagonists when ET-1 is used as agonist and decreases the potency of ET-1.     

Mechanism of rat uterine smooth muscle contraction induced by endothelin-1.

1. Endothelin (ET)-1 has been demonstrated to cause contraction of uterine smooth muscle. We investigated the role of ET receptor subtypes (ETA and ETB receptors) in ET-1-induced contraction of rat uterine smooth muscle by using the ETA receptor antagonist BQ-123 and the ETB receptor agonist BQ-3020. 2. ET-1 caused a contraction with superimposed oscillations of the rat isolated uterus suspended in Krebs-Ringer solution; both the amplitude of contraction as well as the oscillation frequency increased in a dose-dependent manner (10(-11)-10(-7)M). 3. BQ-123 (10(-6)M) markedly shifted the dose-response curve of ET-1 for both contractile effects and oscillation frequency to the right. 4. BQ-3020 (10(-11)-3 x 10(-7) M) did not cause uterine contraction; neither did it affect the dose-response curve of ET-1 for either the contractile effect or the increase in oscillation frequency. Thus, stimulation of ETB receptors is not involved in these responses. 5. The present findings suggest that ET-1-induced contractile responses and the increase in oscillation frequency in rat uterine smooth muscle is mediated through ETA receptors, and that ETB receptors play no role in these responses.     

Characterization of endothelin receptors in rat renal artery in vitro.

1. The aim of this study was to investigate the function and characteristics of endothelin receptors in rat main branch renal artery in vitro. 2. Endothelin(ET)-1 (mean EC50 = 9.8 nM) was approximately 12 fold more potent than ET-3 (mean EC50 = 120 nM) as a contractile agonist and produced a greater maximum response. In contrast, neither of the ETB receptor-selective agonists, alanine[1,3,11,15]ET-1 nor sarafotoxin S6c, (0.1 nM-1 microM), induced any contractile effect, or any relaxant effect in endothelium-intact preparations pre-contracted with the thromboxane A2 mimetic, U-46619. Sarafotoxin S6c (30 nM) also failed to induce any further contraction in tissues pre-contracted with an EC50 concentration of ET-1. 3. The ETA receptor-selective antagonist, BQ123, behaved as a weak and variable antagonist of the contractile effects of ET-1 (mean pA2 estimates in the range 5.8-6.3). In contrast, BQ123 antagonized ET-3 with a potency (mean pA2 = 7.6) consistent with its affinity for ETA receptors. Co-incubation of BQ123 (3 microM) with the putative ETB receptor-selective antagonist, IRL1038 (10 microM), produced no greater antagonism of ET-1 responses than was induced by BQ123 (3 microM) alone. 4. In conclusion, ETB receptors do not appear to be present in rat main branch renal artery. The contractile effects of ET-3 in this tissue seem to be mediated by ETA receptors. While ETA receptors partly mediate the contractile effects of ET-1, these data raise the possibility that a population of novel BQ123-insensitive endothelin receptors may also contribute to this response.