Cloning of cDNA encoding canine endothelin receptors and their expressions in normal tissues

The receptors for endothelin (ET) family, ETA and ETB, were molecularly cloned and the expression of ETA and ETB as well as preproendothelin-1 (PPET-1, precursor of ET-1) was examined in normal canine tissues by RT-PCR. The entire open reading frames of the canine ETA and ETB were shown to encode 427 and 442 amino acid residues, respectively, showing from 87.4 to 97.3% sequence similarity to human, mouse, and rat counterparts. ETA and ETB mRNAs were ubiquitously expressed in a variety of canine tissues in this study and PPET-1 mRNA was detected in the tissues except for heart and liver. It was speculated that ET could play an important role in physiological events in most of the organs.     

Prostaglandin F2alpha (PGF2alpha) independent and dependent regulation of the bovine luteal endothelin system

We have examined the genes of the endothelin system that are targets for regulation by prostaglandin F2alpha (PGF2alpha). The effects of a luteolytic dose of PGF2alpha ) on the mRNA encoding endothelin converting enzyme-1 (ECE-1), pre-pro endothelin-1 (pp ET-1) and the ET receptors ETA, ETB, in bovine corpus luteum (CL) during the early (days 1 and 4), mid (day 10) or late (day 17) luteal phases were examined. The effect of the PGF(2alpha) treatment on ECE-1 protein, Big ET-1 and the biologically active mature ET-1 peptide were also examined. Most importantly, the direct ECE-1 activity was determined. Before day 10 of the cycle, in a PGF2alpha-independent manner, the amounts of mRNA encoding ET-1, ECE-1, ETA, and ETB were increased steadily from day 1. After day 10 of the cycle, expression of mRNA encoding pp ET-1 and ETA acquired responsiveness to exogenous PGF2alpha and both genes were up-regulated by the PGF2alpha treatment. This effect of PGF2alpha was also detected for the proteins corresponding to the mature ET-1. The enzymatic activity of ECE-1 remained unchanged throughout the lifespan of the CL in spite of the detected changes in mRNA and protein. The results suggest that the luteal endothelin system is regulated in a PGF2alpha-independent and -dependent manner. Importantly, an alteration in luteal ET-1 availability is most likely achieved by modulating the expression of mRNA encoding pp ET-1 and not by the amount or activity of ECE-1. This interpretation is supported by the observation that the activity of ECE-1 remained unchanged throughout the ovarian cycle. The combined effects of greater ET-1 availability and gene expression encoding the ETA receptor in the late luteal phase could render the CL, at this developmental stage, more sensitive or responsive to ET-1. If the luteal tissue is responsive to the available ET-1 during the early phase of the ovarian cycle, an additional role for ET-1 should be considered beyond mediating the luteolytic actions of PGF2alpha. Agents blocking the actions of ET-1 might be the best approach to interfere with the luteal ET system and test its physiological role(s) in vivo.     

Oxidative Stress and Interaction of Endothelin Receptors in Airways of Clinically Healthy Horses

Endothelin-1 (ET-1), a physiological as well as an inflammatory mediator, causes contraction of airway smooth muscles by binding to endothelin-A (ETA) and endothelin-B (ETB) receptors. We investigated the interaction between endothelin receptors on contractions and oxidative stress of bronchial rings of clinically healthy horses. Rings for response studies were set in tissue chambers containing oxygenated Tyrode’s solution. Concentration-response relationships were determined for ET-1 on control rings, rings incubated with ETA antagonist (BQ123), rings incubated with ETB antagonists (IRL 1038 and BQ788), and rings incubated with all antagonists combined. For oxidative stress studies, rings were incubated for 30 minutes separately with ET-1, ETA antagonist + ET-1, ETB antagonists + ET-1, and all three antagonists + ET-1. The control rings were not treated with any agents. Electron paramagnetic resonance (EPR) method was used to measure total reactive oxygen species (ROS), superoxide, and peroxynitrite. Results showed that ET-1 caused dose-dependent contractions which were increased by ETA blockade and decreased by ETB blockade. Combined blockade significantly inhibited the response to ET-1. Regarding the oxidative stress, ET-1 and its antagonists significantly reduced the total ROS. Superoxide production was significantly decreased by ETA antagonist and completely abolished when ETA and ETB antagonists were combined. Peroxynitrite production was not changed significantly. The study suggested that an interaction between ET receptors exists in the expression of contractile responses and oxidative stress. ET-1 attenuated oxidative stress by decreasing total ROS. ETA receptors may be primarily responsible for superoxide production. It appears that ET-1 does not affect peroxynitrite production in normal equine tissues.     

Characterization of the in vitro responses of equine cecal longitudinal smooth muscle to endothelin-1

OBJECTIVE: To characterize the in vitro response of equine cecal longitudinal smooth muscle (CLSM) to endothelin (ET)-1 and assess the role of ETA and ETB receptors in those ET-1-induced responses. ANIMALS: 36 horses without gastrointestinal tract disease. PROCEDURE: To determine cumulative concentration-response relationships, CLSM strips were suspended in tissue baths containing graded concentrations of ET-1 (10(-9) to 10(-6)M) with or without BQ-123 (ETA receptor antagonist); with or without IRL-1038 (ETB receptor antagonist); or with both antagonists at concentrations of 10(-9), 10(-7), and 10(-5)M. To determine the percentage change in baseline tension of CLSM, the areas under the curve during the 3-minute periods before and after addition of each dose were compared. Also, the effects of ET-1 and a combination of selective ETA and ETB receptor antagonists on electrically evoked contractions were studied. RESULTS: ET-1 caused sustained increases in CLSM tension in a concentration-dependent manner. Contractile responses to ET-1 were not significantly inhibited by either BQ-123 or IRL-1038 alone at any concentration; however, responses were significantly inhibited by exposure to the antagonists together at a concentration of 10(-5)M. Electrical field stimulation did not change the spontaneous contractile activity of CLSM and did not significantly alter the tissue response to ET-1, BQ-123, or IRL-1038. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that ET-1 has a contractile effect on equine CLSM that is mediated via ETA and ETB receptors. In vitro spontaneous contractions of equine CLSM apparently originate in the smooth muscle and not the enteric nervous system.     

Endothelin-1 receptors and biosynthesis in the corpus luteum: molecular and physiological implications

Endothelin-1 (ET-1), a 21-amino acid peptide was initially identified as a potent vasoconstrictor, ET-1 plays an important role in the female reproductive cycle: its quick ascent during luteal regression, ability to inhibit steroidogenesis in vitro and in vivo, combined with the observation that the luteolytic effects of prostaglandin F2alpha (PGF2alpha) were delayed by pretreatment with ET-1 receptors type A (ETA) antagonists suggest that this peptide functions as an important element of the luteolytic cascade. The observation that ETA receptor expression was inversely correlated with steroidogenesis in luteal cells; namely factors which stimulated steroidogenesis inhibited ETA receptor levels is also in accord with the inhibitory role of ET-1 in corpus luteum (CL) function. Contrary to the mature mid cycle CL, the CL of early cycle is refractory to PGF2alpha-induced luteolysis. PGF2alpha administered at early luteal phase (day 4 of the cycle) failed to increase luteal ET-1 gene expression or its ETA receptors. In contrast, both genes were markedly induced in mid cycle CL exposed to PGF2alpha. ET-1 gene is transcribed as prepro ET-1 (ppET-1) and the active form of peptide is derived from the inactive intermediate big ET-1, by endothelin-converting enzyme-1 (ECE-1), therefore alterations in mature ET-1 levels can be achieved by modulating the expression of ppET-1 and/or ECE-1. Analysis using in situ hybridization and enriched luteal cell subpopulations showed that both steroidogenic and endothelial cells of the CL expressed high levels of ECE-1 mRNA. The ppET-1 mRNA, on the other hand, was only expressed by resident endothelial cells, suggesting that luteal parenchymal and endothelial cells cooperate in the biosynthesis of mature bioactive ET-1. A significant, four-fold elevation in ECE-1 expression (mRNA and protein levels) occurred during the transition of the CL from early to mid luteal phase. This increase was accompanied by a significant rise in ET-1 peptide. Surprisingly however, ppET-1 mRNA levels remained similar during early and mid luteal phase. Collectively, these studies demonstrate that: (a) the various components of ET-1 system (ET-1/ECE-1/ETA) are dynamically and independently regulated during bovine luteal life span. (b) The CL becomes PGF2alpha-responsive only when both ppET-1 and ECE-1 genes are expressed at a level which enable an uninterrupted ET-1 biosynthesis.     

Spasmogenic action of endothelin-1 on isolated equine pulmonary artery and bronchus

REASONS FOR PERFORMING STUDY: There is currently little published information about the effects of endothelin-1 (ET-1), a potent endogenous spasmogen of vascular and airway smooth muscle, on pulmonary vasculature and airways or which ET receptor subtypes mediate ET-1-induced vasoconstrictive and bronchoconstrictive action in the horse. OBJECTIVES: To investigate the effect of endothelin-1 (ET-1) on smooth muscle from isolated equine pulmonary artery and bronchus. In addition, the roles of ETA and ETB receptors in ET-1 mediated contraction in these tissues were assessed. METHODS: The force generation of ring segments from pulmonary arteries or third-generation airways (obtained from horses subjected to euthanasia for orthopaedic reasons) were studied in an organ bath at 37 degrees C in response to exogenous endothelin and selective endothelin A (BQ123) or B receptor (BQ788) antagonists. RESULTS: ET-1 produced concentration-dependent contractions of the equine pulmonary artery and bronchus. The threshold for contraction was 10(-10) and 10(-9) mol/l ET-1 for pulmonary artery and bronchus, respectively. The maximal contraction induced by the highest ET-1 concentration (10(-7) mol/l) was 173 and 194% of the contraction obtained with 100 mmol/l KCl in pulmonary artery and bronchus, respectively. ET-1 potency was 25 times greater in equine pulmonary artery than in equine bronchus (concentration of ET-1 producing 50% of maximal contraction [EC50] = 5.6 10(-9) mol/l and 2.2 10(-8) mol/l, respectively). In pulmonary artery, ET-1 induced contractions were significantly inhibited by the ETA receptor antagonist BQ123 (1 micromol/l; dose-response curve to ET-1 was shifted to the right by 5.4-fold), but not by the ETB antagonist BQ788. In bronchus, dose-responses curves to ET-1 were shifted to the right by BQ123 (1 micromol/l; 2.5-fold), but not by BQ788 (1 micromol/l). In the presence of both antagonists, the dose-response curve to ET-1 was shifted to the right by 4.5-fold. CONCLUSIONS: These functional studies demonstrate that ET-1 is a potent spasmogen of equine third generation pulmonary artery and bronchus, and that contractions are mediated via ETA receptors in the former and both ETA and ETB receptors in the latter. POTENTIAL CLINICAL RELEVANCE: Endothelin receptor antagonists may have potential for treating equine pulmonary hypertension or bronchoconstriction.     

In vitro evaluation of the contractile response to endothelin-1 of the circular and longitudinal myometrial layers of the uterine horn of nongravid mares

OBJECTIVE: To characterize the in vitro response of circular and longitudinal myometrial layers of the uterine horn (CMLH and LMLH, respectively) of horses to endothelin (ET)-1 by use of specific ETA (BQ-123) and ETB (IRL-1038) receptor antagonists. SAMPLE POPULATION: Uteruses from 10 nongravid mares in anestrus. PROCEDURE: Muscle strips from the CMLH and LMLH were suspended in tissue baths and connected to force-displacement transducers interfaced with a polygraph. Strips were incubated for 45-minute intervals with no antagonist (control specimens), and 3 concentrations (10(-9), 10(-7), and 10(-5)M) of BQ-123, IRL-1038, or BQ-123 and IRL-1038 before concentration-response curves to ET-1 were generated. Contractile response to cumulative concentrations of ET-1 (10(-9) to 10(-6)M) was quantified by measuring change in the area under the curve (AUC) for the 3-minute period after each ET-1 dose. RESULTS: ET-1 caused concentration-dependent contraction of the CMLH and LMLH specimens. Application of BQ-123 decreased AUC values for both layers. Application of IRL-1038 increased the AUC value for LMLH specimens but did not affect the CMLH value. The combination of BQ-123 and IRL-1038 decreased the AUC value for LMLH tissue and increased that for CMLH tissue. CONCLUSIONS AND CLINICAL RELEVANCE: ET-1 causes contraction of the CMLH and LMLH in nongravid horses. In both layers, ETA receptors mediate contraction but the role of ETB receptors remains unclear. In the LMLH, ETA receptors have a dominant role; the presence of another receptor or receptor subtype within this layer is suggested. These findings support a physiologic role for ET-1 in uterine contractility.     

Endothelin mediated contraction of equine laminar veins

REASONS FOR PERFORMING STUDY: Endothelin-1 (ET-1) may be a key mediator in the pathogenesis of laminitis, but endothelin-mediated responses in the venous microcirculation of the equine foot have yet to be fully characterised. OBJECTIVES: To characterise the response of equine laminar veins to ET-1 and evaluate the ET-1 receptor subtypes that mediate this response. METHODS: Small veins (150-500 microns) draining the equine digital laminae from healthy horses and ponies subjected to euthanasia at an abattoir were investigated using wire myography. Concentration response curves were constructed for ET-1 in the presence of ETA (BQ123) and ETB (BQ788) receptor antagonists, and L-NAME, a nitric oxide synthase blocker. The selective ETB receptor agonist BQ3020 was investigated alone and following incubation with L-NAME, with or without BQ788. RESULTS: Endothelin-1 contraction of laminar veins was significantly inhibited by BQ123 but not by BQ788. In the presence of L-NAME, sensitivity of laminar veins to ET-1 was enhanced 4-fold, and further addition of BQ788 did not alter this increased sensitivity. BQ3020 induced no venoconstriction; however, in the presence of L-NAME, it caused contraction of veins with approximately 30% of the efficacy of ET-1. The action of BQ3020 in the presence of L-NAME was abolished by BQ788. CONCLUSIONS: Both ETA and ETB receptors are involved in the net tonic response to ET-1 in normal laminar veins. A population of ETB receptors may be present on the vascular endothelium and on smooth muscle of laminar veins, and the action of ET-1 at these 2 sites is likely to be approximately equal and opposite. POTENTIAL RELEVANCE: Our results clarify the function of the ET-1 receptor subtypes in laminar veins from healthy horses. Further study of ET-1 receptors in laminitic horses is therefore warranted.     

Procalcitonin gene expression after LPS stimulation in the porcine animal model

Procalcitonin (PCT), recognised as a marker of sepsis, was investigated in a porcine model of endotoxic shock. The results showed that continuous IV infusion (1-4 h) of LPS (40 μg/kg) in pigs was able to induce a generalised increase of PCT expression in lung, heart, kidney and liver. The increase in PCT was significant only in kidney and was accompanied by an increase in IL-6 gene expression. In vitro results demonstrated that peripheral blood mononuclear cells (PBMCs), as well as endothelial cells, were potentially capable of contributing to in vivo extrathyroidal PCT production. These findings support previous data from pigs concerning the occurrence of widespread activation of PCT extrathyroidal gene expression during endotoxic shock in pigs. Nevertheless, the levels of PCT detected were very low, suggesting the need for additional studies to validate the pig as a reliable animal model for investigating the role of PCT in sepsis.     

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

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

Endothelin receptor mediating contraction of isolated bovine coronary artery

We studied endothelin (ET) receptors and their subtypes on isolated bovine coronary arteries. Endothelin receptors that mediated contraction of isolated bovine coronary artery were characterized by the use of antagonists and agonists. Contractions induced by the nonselective agonist ET-1 (10-10-10-7 M) were not affected by the removal of the endothelium (pEC50: 8.52, maximal contraction: 105% of that induced by 60 mM KCl). BQ-123 (3 x 10-7 M) antagonized contractions of endothelium-denuded coronary rings induced by low concentrations of ET-1 (10-10 or 10-9 M), but potentiated the contractions induced by higher concentrations of ET-1 (3 x 10-8 and 10-7 M). BQ-788 (10-6 M) potentiated contractions induced by ET-1 (3 x 10-10 and 10-7 M). In the presence of BQ-788 (10-6 M), BQ-123 (3 x 10-8-3 x 10-6 M) concentration - dependently inhibited contractions induced by ET-1 (3 x 10-10 and 10-7 M) (pA2: 6.61). Sarafotoxin S6b (10-9-3 x 10-7 M) evoked contractions in the denuded coronary artery (pEC50: 8.49, maximal contraction: 139% of 60 mM KCl). The BQ-123 caused a concentration-dependent rightward shift of contractions induced by sarafotoxin S6b (pA2: 7.89). The present study indicates that ET-1 and sarafotoxin S6b contract the isolated bovine coronary artery by stimulating ETA receptors on smooth muscle cells, and that ETB receptors might suppress the ET-1-induced contractions.     

Endothelin-1, endothelin converting enzyme-1 and endothelin receptors in the porcine corpus luteum

Porcine corpora lutea (CL) fail to show a luteolytic response to prostaglandin-F-2α (PGF-2α) (ie, luteolytic sensitivity [LS]) until about day 12-13 of the estrous cycle. Although little is known of the control of LS in any species, endothelin-1 (EDN1) is believed to play a role in LS control in ruminants. Therefore, we measured mRNA and protein expression and examined the cellular localization of EDN1 precursor (pre-pro EDN1, or ppEDN1), EDN-converting enzyme-1 (ECE1), and EDN receptors (A, EDNRA and B, EDNRB) in porcine CLs collected on days 4, 7, 10, 13, and 15 of the estrous cycle to look for differences between CLs displaying (days 13-15) versus those lacking (days 4-10) LS. Abundance of ppEDN1 mRNA was greatest (and significant vs all other days) on day 7 of the cycle, whereas EDN1 protein expression did not vary during the cycle and was localized exclusively to endothelial cells (EC). Abundance of ECE1 mRNA was also greatest on day 7 (vs all other days), but ECE1 protein was significantly elevated on day 10 (vs day 4) and was immunolocalized to ECs and large luteal cells (LLC). Abundance of EDNRA mRNA was also maximal on day 7 (vs all other days) of the cycle, whereas EDNRA protein expression was not significantly changed during the cycle and was observed in LLCs, ECs, and small luteal cells (SLC). On day 13, EDNRB mRNA was significantly decreased (versus day 7). Expression of EDNRB protein was decreased on day 10 (versus all other days), and on days 13-15 (vs day 4), and was primarily localized to ECs. In conclusion, the observed elevation in ECE1 protein concentrations on day 10 and the presence of EDNRA on LLC suggests a possible role for EDN1 (resulting from the actions of ECE1) acting via EDNRA in the control of LS in the pig.     

一氧化氮介导内毒素所致的外周血淋巴细胞凋亡及阳离子A的保护作用

采用内毒素（ET）所致家兔ET休克模型，分别在相应处理后3、4、8、12h检测血浆中NO含量的动态变化；用荧光显微镜观察外周血淋巴细胞（PBL）的凋亡情况并计算凋亡指数；用琼脂糖凝胶电泳检测PBL凋亡的特征性DNA降解，并观察阳离子A（CA）注射液对上述指标的影响。结果显示，模型组中各时间段的血浆中NO含量均显著高于对照组（P〈0．01），随着NO含量的增加，PBL凋亡指数明显上升（P〈0．01），其基因组DNA在处理后3、4、8h均发生180-200bp及其整数倍的“阶梯状”断裂，12h出现“弥散状”条带；CA保护组中NO水平和PBL凋亡指数均出现不同程度的降低（P〈0．01，P〈0．05），并且在电泳结果中无明显的“梯状”条带。提示ET可使血液中NO含量升高。NO参与了ET休克的发病机制，介导PBL凋亡，在晚期介导PBL坏死；而CA可有效地中和血液循环中的ET，明显降低机体内NO的含量，抑制PBL凋亡和坏死，对ET休克有一定的保护作用。     

Fasting influences steroidogenesis, vascular endothelial growth factor (VEGF) levels and mRNAs expression for VEGF, VEGF receptor type 2 (VEGFR-2), endothelin-1 (ET-1), endothelin receptor type A (ET-A) and endothelin converting enzyme-1 (ECE-1) in newly formed pig corpora lutea

This study was designed to verify whether fasting influences vascular endothelial growth factor (VEGF) production and VEGF, VEGF receptor-2 (VEGFR-2) as well as endothelin (ET) system members (endothelin converting enzyme-1, ECE-1; ET-1; endothelin receptor type A, ET-A) mRNA expression in pig corpora lutea; furthermore, we wanted to assess whether fasting affects steroidogenesis in luteal cells. Eight prepubertal gilts were induced to ovulate and were randomly assigned to two groups: (A) n = 4, normally fed; and (B) n = 4, fasted for 72 h starting 3 days after ovulation. At the end of fasting, ovaries were removed from all the animals and corpora lutea (CLs) were collected. VEGF and steroid levels in luteal tissue were determined by ELISA and RIA, respectively; VEGF, VEGFR-2, ET-1, ET-A and ECE-1 mRNAs expression was measured by real-time PCR. VEGF protein levels were similar in the two groups, while all steroid (progesterone, testosterone, estradiol 17beta) concentrations were significantly (P < 0.001) higher in CLs collected from fasted animals compared with those from normally fed gilts. VEGF, VEGFR-2, ET-1 and ECE-1 (but not ET-A) mRNA expression was significantly lower (P < 0.05) in fasted versus normally fed animals. The overall conclusion is that all the parameters studied are affected by feed restriction, but the mechanisms activated at luteal level are possibly not fully adequate to compensate for nutrient shortage.     

Gene expression of endothelin-1 and its receptors in the heart of broiler chickens with T(3)-induced pulmonary hypertension

To investigate the effect of T₃-induced pulmonary hypertension on endothelin (ET) production and genes expression of ET-1, ET_A and ET_B receptors (ET_AR and ET_BR) during rearing, semiquantitative RT-PCR and enzyme immunometric assay were performed in the heart ventricles and serum, respectively. The ET-1 and its receptor genes were expressed in the right and left ventricles of control and T₃-treated broilers at 12, 28 and 49 days of age. There were significant (P < 0.05) reductions of the relative amounts of ET-1 (in both ventricles) and ET_AR (in the right ventricle) mRNAs at 28 and 49 days of age, in T₃-treated broilers compared to controls. The relative amounts of ET_BR mRNA in the right and left ventricles did not significantly differ between control and T₃-treated broilers at any age. The serum level of ET was significantly (P < 0.05) increased in T₃-treated chickens at 28 and 49 days of age when compared with that of the control. It is concluded that ET-1, ET_AR and ET_BR genes are normally expressed in the heart ventricles of broilers. It is likely that increased serum level of ET and decreased ET-1/ET_AR genes expression in the ventricles are involved in the heart dysfunction of broiler chickens with developmental pulmonary hypertension.