Gq/G13 signaling by ET-1 in smooth muscle: MYPT1 phosphorylation via ETA and CPI-17 dephosphorylation via ETB

We analyzed the signaling pathways initiated by endothelin receptors ET_A and ET_B in intestinal circular and longitudinal smooth muscle cells. The response to endothelin-1 (ET-1) consisted of two phases in both cell types. The initial, transient phase of contraction and phosphorylation of 20-kDa myosin light chain (MLC₂₀) was mediated additively by ET_A and ET_B receptors and initiated by G_q-, Ca²⁺/calmodulin-dependent activation of MLC kinase. In contrast, the sustained phase was mediated selectively by ET_A receptors via a pathway involving sequential activation of G₁₃, RhoA, and Rho kinase, resulting in phosphorylation of MYPT1 at Thr⁶⁹⁶ and phosphorylation of MLC₂₀. Although PKC was activated, CPI-17 was not phosphorylated and hence did not contribute to inhibition of MLC phosphatase. The absence of CPI-17 phosphorylation by PKC reflected active dephosphorylation of CPI-17 by protein phosphatase 2A (PP2A). PP2A was activated via a pathway involving ET_B-dependent stimulation of p38 MAPK activity. CPI-17 phosphorylation was unmasked in the presence of the ET_B antagonist BQ-788, but not the ET_A antagonist BQ-123, and in the presence of a low concentration of okadaic acid, which selectively inactivates PP2A. The resultant phosphorylation of CPI-17 was blocked by bisindolylmaleimide, providing direct confirmation that it was PKC dependent. We conclude that the two phases of the intestinal smooth muscle response to ET-1 involve distinct receptors, G proteins, and signaling pathways. The sustained response is mediated via selective ET_A-dependent phosphorylation of MYPT1. In contrast, ET_B initiates an inhibitory pathway involving p38 MAPK-dependent activation of PP2A that causes dephosphorylation of CPI-17. endothelin receptor type A; endothelin receptor type B; myosin phosphatase targeting subunit     

Effect of progesterone on intracellular Ca2+ homeostasis in human myometrial smooth muscle cells

Although it iswell known that progesterone alters uterine contractility and plays animportant role in maintenance of pregnancy, the biochemical mechanismsby which progesterone alters uterine contractility in human gestationare less clear. In this investigation we sought to identifyprogesterone-induced adaptations in human myometrial smooth musclecells that may alter Ca²⁺signaling in response to contractile agents. Cells were treated withvehicle or the progesterone analog medroxyprogesterone acetate (MPA)for 5 days, and intracellular freeCa²⁺ concentration([Ca²⁺]_i)was quantified after treatment with oxytocin (OX) or endothelin (ET)-1.OX- and ET-1-induced increases in[Ca²⁺]_iwere significantly attenuated in cells pretreated with MPA in adose-dependent manner. Progesterone receptor antagonists prevented theattenuated Ca²⁺ transients inducedby MPA. ET_A andET_B receptor subtypes were expressed in myometrial cells, and treatment with MPA resulted insignificant downregulation of ET_Aand ET_B receptor binding. MPA didnot alter ionomycin-stimulated increases in[Ca²⁺]_iand had no effect on inositol trisphosphate-dependent or -independent release of Ca²⁺ from internalCa²⁺ stores. We conclude thatadaptations of Ca²⁺ homeostasis inmyometrial cells during pregnancy may include progesterone-inducedmodification of receptor-mediated increases in[Ca²⁺]_i.     

Ca(2+) channels activated by endothelin-1 in CHO cells expressing endothelin-A or endothelin-B receptors

We comparedthe Ca²⁺ channels activated by endothelin-1 (ET-1) inChinese hamster ovary (CHO) cells stably expressing endothelin type A(ET_A) or endothelin type B (ET_B) receptorsusing the Ca²⁺ channel blockers LOE-908 and SK&F-96365. Inboth CHO-ET_A and CHO-ET_B, ET-1 at 0.1 nMactivated the Ca²⁺-permeable nonselective cation channel-1(NSCC-1), which was sensitive to LOE-908 and resistant to SK&F-96365.ET-1 at 1 nM activated NSCC-2 in addition to NSCC-1; NSCC-2 wassensitive to both LOE-908 and SK&F-96365. ET-1 at 10 nM activated thesame channels as 1 nM ET-1 in both cell types, but inCHO-ET_A, it additionally activated the store-operatedCa²⁺ channel (SOCC), which was resistant to LOE-908 andsensitive to SK&F-96365. Up to 1 nM ET-1, the level of the formation of inositol phosphates (IPs) was low and similar in both cell types, but,at 10 nM ET-1, it was far greater in CHO-ET_A than inCHO-ET_B. These results show that, in CHO-ET_Aand CHO-ET_B, ET-1 up to 10 nM activated the sameCa²⁺ entry channels: 0.1 nM ET-1 activated NSCC-1, andET-1  1 nM activated NSCC-1 and NSCC-2. Notably, inCHO-ET_A, 10 nM ET-1 activated SOCCs because of the higherformation of IPs.

     

ET-1-induced bronchoconstriction is mediated via ETB receptor in mice

Nagase, Takahide, Tomoko Aoki, Teruaki Oka, YoshinosukeFukuchi, and Yasuyoshi Ouchi. ET-1-induced bronchoconstriction ismediated via ET_B receptor in mice.J. Appl. Physiol. 83(1): 46-51, 1997.Endothelin (ET)-1 is one of the most potent agonists of airwaysmooth muscle and can act via two different ET receptor subtypes, i.e.,ET_A andET_B. To determine the effects ofET-1 on in vivo pulmonary function and which ET receptors are involved in murine lungs, we investigated 1)the effects of ET and sarafotoxin S6c (S6c), a selectiveET_B agonist, on pulmonary functionand 2) the effects of BQ-123 andBQ-788, specific ET_A- andET_B-receptor antagonists, onET-1-induced bronchoconstriction. ICR mice were anesthetized and mechanically ventilated (frequency = 2.5 Hz, tidalvolume = 8 ml/kg, positive end-expiratory pressure = 3 cmH₂O). Intravenous ET-1, ET-2,and ET-3 increased lung resistance similarly and equipotently, whereasS6c elicited a greater degree of bronchoconstriction. Mice were thenpretreated with saline (Sal), BQ-123 (0.2, 1, and 5 mg/kg), or BQ-788(0.2, 1, and 5 mg/kg) before administration of ET-1(10⁷ mol/kg iv). No dose ofBQ-123 blocked ET-1-induced constriction, whereas pretreatment witheach dose of BQ-788 significantly inhibited ET-1-induced responses.There were significant differences in morphometrically assessed airwayconstriction between Sal and BQ-788 and between BQ-123 and BQ-788,whereas no significant difference was observed between Sal and BQ-123.There were no significant morphometric differences in the airway wallarea among the three groups. These observations suggest that theET_B- but notET_A-receptor subtype may mediatethe changes in murine pulmonary function in response to ET-1. Inaddition, the ET_B-receptorantagonist reduces ET-1-induced airway narrowing by affecting airwaysmooth muscle contraction in mice.

     

Role of endothelin type B receptor in NO/cGMP signaling pathway in rat median eminence

We studied the effect of endothelins (ETs) on receptor-mediated NO/cGMP signaling in rat arcuate nucleus–median eminence (AN-ME) fragments, an hypothalamic structure known to contain a rich plexus of nitric oxide synthase (NOS)-containing neurons and fibers together with densely arranged ET_B-receptor-like immunoreactive fibers. NOS activity was determined measuring the conversion of [³H] arginine to [³H] citrulline, as an index of NO produced. cGMP production was determined by radio immunoassay. ET-1, ET-3, and the selective ET_B receptor agonist, IRL1620, significantly increased cGMP formation and NOS activity. Preincubation of AN-ME fragment with L-arginine analog, N-nitro-L-arginine (L-NAME), inhibited ET-1 or IRL1620-stimulated cGMP formation. The addition of the selective ET_B receptor antagonist, BQ788, blocked ET-1-, ET-3-, or IRL1620-induced increase in NOS activity and cGMP generation, while BQ123, a selective ET_A receptor antagonist, was ineffective. Our results demonstrate that in whole rat AN-ME fragments, ETs stimulate NO/cGMP signaling pathway through the interaction with the ET_B receptor subtype, supporting the concept that ETs may represent an important regulator of reproductive and neuroendocrine function.     

Reduced sickle erythrocyte dehydration in vivo by endothelin-1 receptor antagonists

Elevated plasma levels of cytokines such as endothelin-1 (ET-1) have been shown to be associated with sickle cell disease (SCD). However, the role of ET-1 in the pathophysiology of SCD is not entirely clear. I now show that treatment of SAD mice, a transgenic mouse model of SCD, with BQ-788 (0.33 mg·kg^–1·day^–1 intraperitoneally for 14 days), an ET-1 receptor B (ET_B) antagonist, induced a significant decrease in Gardos channel activity (1.7 ± 0.1 to 1.0 ± 0.4 mmol·10¹³ cell^–1·h^–1, n = 3, P = 0.019) and reduced the erythrocyte density profile by decreasing the mean density (D₅₀; n = 4, P = 0.012). These effects were not observed in mice treated with BQ-123, an ET-1 receptor A (ET_A) antagonist. A mixture of both antagonists induced a similar change in density profile as with BQ-788 alone that was associated with an increase in mean cellular volume and a decrease in corpuscular hemoglobin concentration mean. I also observed in vitro effects of ET-1 on human sickle erythrocyte dehydration that was blocked by BQ-788 and a mixture of ET_B/ET_A antagonists but not by ET_A antagonist alone. These results show that erythrocyte hydration status in vivo is mediated via activation of the ET_B receptor, leading to Gardos channel modulation in SCD. cellular dehydration; Gardos channel; transgenic sickle mice     

L-type voltage-dependent Ca2+ channels in cerebral microvascular endothelial cells and ET-1 biosynthesis

We investigatedthe role of intracellular calcium concentration([Ca²⁺]_i) in endothelin-1 (ET-1) production,the effects of potential vasospastic agents on[Ca²⁺]_i, and the presence of L-typevoltage-dependent Ca²⁺ channels in cerebral microvascularendothelial cells. Primary cultures of endothelial cells isolated frompiglet cerebral microvessels were used. Confluent cells were exposed toeither the thromboxane receptor agonist U-46619 (1 µM),5-hydroxytryptamine (5-HT; 0.1 mM), or lysophosphatidic acid (LPA; 1 µM) alone or after pretreatment with the Ca²⁺-chelatingagent EDTA (100 mM), the L-type Ca²⁺ channel blockerverapamil (10 µM), or the antagonist of receptor-operated Ca²⁺ channel SKF-96365 HCl (10 µM) for 15 min. ET-1production increased from 1.2 (control) to 8.2 (U-46619), 4.9 (5-HT),or 3.9 (LPA) fmol/µg protein, respectively. Such elevated ET-1biosynthesis was attenuated by verapamil, EDTA, or SKF-96365 HCl. Toinvestigate the presence of L-type voltage-dependent Ca²⁺channels in endothelial cells, the [Ca²⁺]_isignal was determined fluorometrically by using fura 2-AM. Superfusionof confluent endothelial cells with U-46619, 5-HT, or LPA significantlyincreased [Ca²⁺]_i. Pretreatment ofendothelial cells with high K⁺ (60 mM) or nifedipine (4 µM) diminished increases in [Ca²⁺]_i inducedby the vasoactive agents. These results indicate that 1)elevated [Ca²⁺]_i signals are involved in ET-1biosynthesis induced by specific spasmogenic agents, 2) theincreases in [Ca²⁺]_i induced by thevasoactive agents tested involve receptor as well as L-typevoltage-dependent Ca²⁺ channels, and 3) primarycultures of cerebral microvascular endothelial cells express L-typevoltage-dependent Ca²⁺ channels.

     

Endothelin-1 decreases glutamate uptake in primary cultured rat astrocytes

Endothelin-1 (ET-1) is a potent vasoconstrictorpeptide that is also known to induce a wide spectrum of biologicalresponses in nonvascular tissue. In this study, we found that ET-1 (100 nM) inhibited the glutamate uptake in cultured astrocytes expressing the glutamate/aspartate transporter (GLAST); astrocytes did not expressthe glutamate transporter-1 (GLT-1). The V_maxand the K_m of the glutamate uptake were reducedby 57% and 47%, respectively. Application of the ET_A andET_B receptor antagonists BQ-123 and BQ-788 partly inhibitedthe ET-1-evoked decrease in the glutamate uptake, whereas thenonspecific ET receptor antagonist bosentan completely inhibited thisdecrease. Incubation of the cultures with pertussis toxin abolished theeffect of ET-1 on the uptake. The ET-1-induced decrease in theglutamate uptake was independent of extracellular free Ca²⁺concentration, whereas the intracellular Ca²⁺ antagoniststhapsigargin and 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester abolished the effect of ET-1 on the glutamate uptake. Incubation with the protein kinase C (PKC) antagonist staurosporine, but not withthe fatty acid-binding protein bovine serum albumin, prevented theET-1-induced decrease in the glutamate uptake. These results suggestthat ET-1 impairs the high-affinity glutamate uptake in culturedastrocytes through a G protein-coupled mechanism, involving PKC andchanges in intracellular Ca²⁺.

     

Endothelin receptor dimers evaluated by FRET, ligand binding, and calcium mobilization

Endothelin-1 (ET-1) mediates physiological responses via endothelin A (ET_A) and B (ET_B) receptors, which may form homo- and heterodimers with unknown function. Here, we investigated ET-receptor dimerization using fluorescence resonance energy transfer (FRET) between receptors tagged with CFP (donor) and receptors tagged with tetracysteine-FlAsH (fluorescein arsenical hairpin) (acceptor) expressed in HEK293 cells. FRET efficiencies were 15%, 22%, and 27% for ET_A/ET_A, ET_B/ET_B and ET_A/ET_B, respectively, and dimerization was further supported by coimmunoprecipitation. For all dimer pairs, the natural but nonselective ligand ET-1 rapidly (≤30 s) reduced FRET by >50%, but did not detectably reduce coimmunoprecipitation. ET-1 stimulated a transient increase in intracellular Ca²⁺ ([Ca²⁺]_i) lasting 1-2 min for both homodimer pairs, and these ET-1 actions on FRET and [Ca²⁺]_i elevation were blocked by the appropriate subtype-selective antagonist. In contrast, ET_A/ET_B heterodimers mediated a sustained [Ca²⁺]_i increase lasting >10 min, and required a combination of ET_A and ET_B antagonists to block the observed FRET and [Ca²⁺]_i responses. The sensitive CFP/FlAsH FRET assay used here provides new insights into endothelin-receptor dimer function, and represents a unique approach to characterize G-protein-coupled receptor oligomers, including their pharmacology.     

Endothelin contraction in pig coronary: Receptor types and Ca2+-mobilization

Endothelin is one of the most potent vasoconstrictors known. It plays an important role in the regulation of vascular tone and in the development of many cardiovascular diseases. This study focuses on the receptor types and the Ca²⁺ mobilization responsible for endothelin-1 (ET-1) contraction in de-endothelialized pig coronary artery rings. ET-1 contracted the artery rings with an EC₅₀ = 6.5 ± 1 nM and a maximum contraction which was 98.6 ± 9% of the contraction produced by 60 mM KCl. BQ123 (5 µM), an ET_A antagonist, reversed 78 ± 3% of the ET-1 contraction (50 nM). IRL1620, a selective ET_B agonist, produced 23 ± 3% of the total ET-1 contraction with an EC₅₀ = 12.7 ± 2 nM. More than 85% of the contraction due to 100 nM IRL 1620 was inhibited by 200 nMBQ788, an ET_B antagonist. Therefore, approximately 80% of the ET-1 contraction in this artery occurred via ET_A receptors, and the other 20% was mediated by ET_B receptors. To assess the Ca²⁺ pools utilized during the ET-1 response, ET-1 contraction was also examined in medium containing an L-type Ca²⁺ channel blocker nitrendipine, and in Ca²⁺ free medium containing 0.2 mM EGTA. In Ca²⁺ containing medium the contraction elicited by ET-1 was 98.6 ± 9% of the KCl contraction, however, in the presence 10 µM nitrendipine the ET-1 induced contraction was 54 ± 7% of the KCl contraction, and in Ca²⁺-free medium it was 13 ± 2%. Similarly, the IRL 1620 contractions in Ca²⁺ containing medium, in the presence of nitrendipine and in Ca²⁺-free medium were 22.4 ± 3%, 12 ± 3% and 11 ± 2% of the KCl response respectively. Thus, both ET_A and ET_B contractions utilize extracellular Ca²⁺ pools via L-type Ca²⁺ channels and other undefined route(s), as well as intracellular Ca²⁺ pools. In the pig coronary artery smooth muscle, ET-1 contractions occur predominantly via ET_A receptors, with ET_B receptors using similar Ca²⁺ mobilization pathways, but the ET_B receptors appear to use the intracellular Ca²⁺ stores to a greater extent.     

ETB receptor activation leads to activation and phosphorylation of NHE3

In OKP cellsexpressing ET_B endothelinreceptors, activation ofNa⁺/H⁺antiporter activity by endothelin-1 (ET-1) was resistant to low concentrations of ethylisopropyl amiloride, indicating regulation ofNa⁺/H⁺exchanger isoform 3 (NHE3). ET-1 increased NHE3 phosphorylation incells expressing ET_B receptors butnot in cells expressing ET_Areceptors. Receptor specificity was not due to demonstrable differencesin receptor-specific activation of tyrosine phosphorylation pathways orinhibition of adenylyl cyclase. Phosphorylation was associated with adecrease in mobility on SDS-PAGE, which was reversed by treatingimmunoprecipitated NHE3 with alkaline phosphatase. Phosphorylation wasfirst seen at 5 min and was maximal at 15-30 min. Phosphorylationwas maximal with 10⁹ MET-1. Phosphorylation occurred on threonine and serine residues atmultiple sites. In summary, ET-1 induces NHE3 phosphorylation in OKPcells on multiple threonine and serine residues.ET_B receptor specificity, timecourse, and concentration dependence are all similar betweenET-1-induced increases in NHE3 activity and phosphorylation, suggestingthat phosphorylation plays a key role in activation.     

Endothelin Signaling Pathways in Rat Adrenal Medulla

1. We further characterized the effect of endothelins (ETs) on receptor-mediated phosphoinositide (PI) turnover, nitric oxide synthase (NOS) activation, and cGMP formation in whole rat adrenal medulla.2. The PI hydrolysis was assessed as accumulation of inositol monophosphates (InsP₁) in the presence of 10 mM LiCl in whole tissue and the analysis of inositol-1-phosphate by Dowex anion exchange chromatography. NOS activity was assayed by monitoring the conversion of radiolabeled L-arginine to L-citrulline. Cyclic GMP formation was assessed as accumulation of cGMP in whole tissue in the presence of phosphodiesterase inhibition, and the amount of cGMP formed was determined by radioimmuno-antibody procedure.3. ET-1 and ET-3 increased PI turnover by 30% in whole adrenal medulla prelabeled with [³H] myoinositol. Both ETs isoforms, at equimolar doses, increased NOS activity and cGMP levels in similar degree. The selective ET_B receptor agonist, IRL-1620, also increased cGMP formation, mimicking the effects of ETs, while IRL-1620 did not alter the PI metabolism. ETs-induced InsP₁ accumulation and cGMP was dependent on extracellular calcium. The effect of ETs on PI turnover was inhibited by neomycin. The L-arginine analogue, N-nitro-L-arginine (L-NAME), and two inhibitors of soluble guanylyl cyclase, methylene blue and ODQ, significantly inhibited the increase in cGMP production induced by ETs or IRL-1620. The selective ET_A receptor antagonist, BQ 123, inhibited the ETs-induced increase in PI turnover, while the selective ET_B receptor antagonist, BQ 788, was ineffective. Likewise, BQ 788, significantly inhibited ET-1- or ET-3-induced NOS activation and cGMP generation but not ETs-induced InsP₁ accumulation.4. Our data indicate that stimulation of PI turnover and NO-induced cGMP generation constitutes ETs signaling pathways in rat adrenal medulla. The former action is mediated through activation of ET_A receptor, while the latter through the activation of ET_B receptor. These results support the role of endothelins in the regulation of adrenal medulla function.     

An endothelin type A receptor-expressing cell to characterize endothelin-1 binding and screen antagonist

Endothelin-1 (ET-1) induces contraction of vascular smooth muscle through binding to endothelin type A receptor (ET_AR). COS-7 cells stably expressing high levels of the ET_AR were established (designated COS-7(ET_AR)). The COS-7(ET_AR) cell bound [¹²⁵I]ET-1 with a K_d of 932 ± 161 pM and a B_max of 74 ± 13 fmol/2 × 10⁵ cells. [¹²⁵I]ET-1 binding was inhibited by ET-1 and the ET_AR antagonist BQ-610, but not by the endothelin type B receptor (ET_BR) antagonist BQ-788. In clones expressing two ET_AR mutants containing D46N or R53Q substitutions in the first extracellular domain of the receptor, [¹²⁵I]ET-1 binding activity was dramatically reduced. This suggests that these single amino acid substitutions alter the three-dimensional structure of the ligand-binding domain of the ET_AR. Using COS-7(ET_AR) cell, we showed that Ca²⁺ or Mg²⁺ was essential for ET-1 binding to the ET_AR and that ET-1 treatment induced postreceptor signaling, that is, intracellular accumulation of cyclic AMP (cAMP) and Ca²⁺ mobilization. The COS-7(ET_AR) established in this study will be a useful tool for screening ET-1 antagonists for treating hypertension.     

IP3 receptor blockade fails to prevent intracellular Ca2+ release by ET-1 and alpha -thrombin

The effect ofinositol 1,4,5-trisphosphate(IP₃) receptor blockade onplatelet-derived growth factor (PDGF), fibroblast growth factor (FGF),endothelin-1 (ET-1), or -thrombin receptor-mediated intracellularCa²⁺(Ca²⁺_i) release was examined using fura 2 microspectrofluorometry in single Chinese hamster ovary cells andmyoblasts. Blockade of the IP₃receptor was achieved by microinjection of heparin or monoclonalantibody (MAb) 18A10 into the IP₃type 1 receptor. Heparin completely inhibitedCa²⁺_i release after flash photolysis withcaged IP₃ and after exposure toPDGF and FGF. In contrast, heparin failed to blockCa²⁺_i release after -thrombin andET-1. After application of ligand, IP₃ levels were five- to sevenfoldhigher for -thrombin than for ET-1 or PDGF.IP₃ levels after PDGF and ET-1were comparable. Similar to heparin, MAb 18A10 blockedCa²⁺_i release after PDGF but failed toblock Ca²⁺_i release after ET-1 or-thrombin. These data suggest that the mechanisms of Ca²⁺_i release by tyrosine kinase andcertain 7-transmembrane receptors may differ. Although both receptortypes use the IP₃-signaling system, the ET-1 and -thrombin receptors may have a second,alternative mechanism for activatingCa²⁺_i release.

     

Calmodulin and protein kinase C regulate gap junctional coupling in lens epithelial cells

The mechanisms regulating the permeability of lens epithelial cell gap junctions in response to calcium ionophore or ATP agonist-mediated increases in cytosolic Ca²⁺ (Ca_i²⁺) have been investigated using inhibitors of calmodulin (CaM) and PKC. Cell-to-cell transfer of the fluorescent dye AlexaFluor594 decreased after the rapid and sustained increase in Ca_i²⁺ (to micromolar concentrations) observed after the addition of ionophore plus Ca²⁺ but was prevented by pretreatment with inhibitors of CaM but not PKC. In contrast, the delayed, transient decrease in cell-to-cell coupling observed after the addition of ATP that we have reported previously (Churchill G, Lurtz MM, and Louis CF. Am J Physiol Cell Physiol 281: C972-C981, 2001) could be prevented by either the direct or indirect inhibition of PKC but not by inhibition of CaM. Surprisingly, there was no change in the relative proportion of the different phosphorylated forms of lens connexin43 after this ATP-dependent transient decrease in cell-to-cell coupling. Although BAPTA-loaded cells did not display the ATP-dependent transient increase in Ca_i²⁺, the delayed, transient decrease in cell-to-cell dye transfer was still observed, indicating it was Ca_i²⁺ independent. Thus CaM-mediated inhibition of lens gap junctions is associated with sustained, micromolar Ca_i²⁺ concentrations, whereas PKC-mediated inhibition of lens gap junctions is associated with agonist activation of second messenger pathways that are independent of changes in Ca_i²⁺. calcium; connexin43; lens gap junctions     

Endothelins stimulate the production of stromelysin-1 in cultured rat astrocytes

The effects of endothelins (ETs) on the production of stromelysins, a sub-family of matrix metalloproteinases, were examined in cultured astrocytes. The treatment of cultured rat astrocytes with ET-1 increased stromelysin-1 mRNA levels, while stromelysin-2 and -3 mRNAs were not affected. Immunocytochemical observations showed that cultured astrocytes produced stromelysin-1 protein. ET-1 and Ala^1,3,11,15-ET-1, an ET_B receptor selective agonist, stimulated the release of stromelysin-1 from cultured astrocytes. Accompanying the increase in protein release, the peptidase activity of stromelysin-1 in the medium was also increased by ET-1. The effects of ET-1 on astrocytic stromelysin-1 expression were inhibited by PD98059, staurosporine, and Ca²⁺ chelation, but not by SB203580 or pyrrolidine dithiocarbamate. These results show that activation of astrocytic ET receptors stimulates the production of stromelysin-1, suggesting a role for ETs in stromelysin production in brain pathologies.     

Pulmonary clearance of circulating endothelin-1 in dogs in vivo: exclusive role of ETB receptors

Dupuis, Jocelyn, Carl A. Goresky, and Alain Fournier.Pulmonary clearance of circulating endothelin-1 in dogs in vivo: exclusive role of ET_B receptors.J. Appl. Physiol. 81(4):1510-1515, 1996.The pulmonary circulation plays an importantrole in the removal of circulating endothelin-1 (ET-1). Plasma ET-1levels are increased in pulmonary hypertensive states of variousetiologies (e.g., idiopathic, heart failure, and congenital anomalies)in proportion to the severity of pulmonary hypertension. It is possible that reduced pulmonary clearance of this peptide contributes to thehyperendothelinemia of those pathologies. TheET_A andET_B receptors are abundant in lungtissues: on the vascular endothelium, theET_B receptor is predominant andmay contribute to ET-1 extraction through receptor-mediatedendocytosis. We designed experiments to determine and quantify theimportance of the ET_A andET_B receptors in the pulmonaryextraction of circulating ET-1 in anesthetized dogs. The single-passcumulative tracer ET-1 extraction by the lung was measured with theindicator-dilution technique before and 5 min after intrapulmonaryinjection of the specific ET_Aantagonist BQ-123 (n = 5, 120-960nmol) and the specific ET_Bantagonist BQ-788 (n = 6, 1,000 nmol).The inhibitors had no significant effect on pulmonary and systemichemodynamics. Mean cumulative pulmonary ET-1 extraction was notmodified by BQ-123 [control (C): 36 ± 4%, antagonist (A): 34 ± 6%] but was completely abolished by BQ-788 (C: 34 ± 6%, A: 0 ± 2%, P < 0.001). Thepulmonary rate constant (K) for ET-1removal was also unaffected by BQ-123 (C: 0.050 ± 0.0085 s¹, A: 0.047 ± 0.012 s¹) but significantlydecreased and became close to zero after BQ-788 (C: 0.058 ± 0.014 s¹, A: 0.009 ± 0.007 s¹,P < 0.001). We conclude that theET_B receptor is completely andexclusively responsible for pulmonary ET-1 removal in vivo. Futurestudies are needed to show whether desensitization or downregulation ofthe ET_B receptor may contribute tothe increase in circulating ET-1 levels in conditions associated withpulmonary hypertension. This novel pulmonary endothelial cell functionmay play a protective role by modulating circulating ET-1 levels in thesystemic circulation.

     

Endothelin-1 induces intracellular [Ca2+] increase via Ca2+ influx through the L-type Ca2+ channel, Ca2+-induced Ca2+ release and a pathway involving ETA receptors, PKC, PKA and AT1 receptors in cardiomyocytes

Using fura-2-acetoxymethyl ester (AM) fluorescence imaging and patch clamp techniques, we found that endothelin-1 (ET-1) significantly elevated the intracellular calcium level ([Ca²⁺]_i) in a dose-dependent manner and activated the L-type Ca²⁺ channel in cardiomyocytes isolated from rats. The effect of ET-1 on [Ca²⁺]_i elevation was abolished in the presence of the ET_A receptor blocker BQ123, but was not affected by the ET_B receptor blocker BQ788. ET-1-induced an increase in [Ca²⁺]_i, which was inhibited 46.7% by pretreatment with a high concentration of ryanodine (10 μmol/L), a blocker of the ryanodine receptor. The ET-1-induced [Ca²⁺]_i increase was also inhibited by the inhibitors of protein kinase A (PKA), protein kinase C (PKC) and angiotensin type 1 receptor (AT1 receptor). We found that ET-1 induced an enhancement of the amplitude of the whole cell L-type Ca²⁺ channel current and an increase of open-state probability (NPo) of an L-type single Ca²⁺ channel. BQ123 completely blocked the ET-1-induced increase in calcium channel open-state probability. In this study we demonstrated that ET-1 regulates calcium overload through a series of mechanisms that include L-type Ca²⁺ channel activation and Ca²⁺-induced Ca²⁺ release (CICR). ETA receptors, PKC, PKA and AT1 receptors may also contribute to this pathway. Supported by the National Natural Science Foundation of China (Grant No. 200830870910).     

Endothelin- and Sarafotoxin-Induced Phosphoinositide Hydrolysis in Cultured Canine Tracheal Smooth Muscle Cells

Abstract: The endothelins (ETs) and sarafotoxin are two structurally related classes of potently contractile peptides. To understand the mechanism of action of ETs, we have examined the effect of ETs and sarafotoxin on phosphoinositide (PI) hydrolysis in cultured canine tracheal smooth muscle cells (TSMCs). ET-1, ET-2, ET-3, and sarafotoxin caused dose-dependent accumulation of inositol phosphates (IPs) and tracheal smooth muscle contraction. BQ-123, an ET_A receptor antagonist, had a high affinity to block the ET-1-induced IP accumulation and tracheal smooth muscle contraction with pK_B values of 7.3 and 7.4, respectively. Pretreatment of TSMCs with cholera toxin impaired the ability of ET-1 and ET-2 to stimulate IP formation, whereas there was no effect by treatment with pertussis toxin. Stimulation of PI turnover by these peptides required the presence of extracellular Ca²⁺ and was blocked by treatment with EGTA. The addition of Ca²⁺(3–620 nM) to digitonin-permeabilized TSMCs directly stimulated IP accumulation. A further Ca²⁺-dependent increase in IP formation was obtained by inclusion of either GTPrS or ET-1. The combined presence of GTPrS and ET-1 elicited an additive effect on IP formation. Short-term exposure to phorbol 12-myristate 13-acetate (PMA, 1 μM) abolished the stimulation of PI hydrolysis induced by these peptides. The inhibitory effect of PMA on ET-induced response was reversed by staurosporine, a protein kinase C (PKC) inhibitor, suggesting that the inhibitory effect of PMA is mediated through the activation of PKC. Prolonged incubation of TSMCs with PMA resulted in a recovery of receptor responsiveness that may be due to down regulation of PKC. Inactive phorbol ester, 4α-phorbol 12, 13-didecanoate at 1 μM, did not inhibit this response. The site of this response was further investigated by examining the effect of PMA on AIF₄^?-induced IP accumulation in canine TSMCs. AIF₄^?-induced IP accumulation was inhibited by PMA treatment, suggesting that G protein(s) can be directly activated by AIF₄^?, which was uncoupled to phospholipase C by PMA treatment. These data conclude that ET-stimulated PI hydrolysis and tracheal smooth muscle contraction are mediated by the activation of ET_Areceptors coupling to a G protein and dependent on the external Ca²⁺. The transduction mechanism of ETs is sensitive to feedback regulation by PKC.     

Sequential and opposite regulation of two outward K(+) currents by ET-1 in cultured striatal astrocytes

In the brain,astrocytes represent a major target for endothelins (ETs), a family ofpeptides that can be released by several cell types and that havepotent and multiple effects on astrocytic functions. Four types ofK⁺ currents (I_K) were detected invarious proportions by patch-clamp recordings of cultured striatalastrocytes, including the A-type I_K, theinwardly rectifying I_K IR, theCa²⁺-dependent I_K(I_K Ca), and the delayed-rectifiedI_K (I_K DR). Variationsin the shape of current-voltage relationships were related mainly todifferences in the proportion of these currents. ET-1 was found toregulate with opposite effects the two more frequently recorded outwardK⁺ currents in striatal astrocytes. Indeed, this peptideinduced an initial activation of I_K Ca(composed of SK and BK channels) and a delayed long-lasting inhibitionof I_K DR. In current-clamp recordings, theactivation of I_K Ca correlated with a transient hyperpolarization, whereas the inhibition ofI_K DR correlated with a sustaineddepolarization. These ET-1-induced sequential changes inmembrane potential in astrocytes may be important for the regulation ofvoltage gradients in astrocytic networks and the maintenance ofK⁺ homeostasis in the brain microenvironment.