Inhibitory Effect Of Endothelin-1 Research Articles

Endothelin ETA- and ETB-Receptor-Mediated Inhibition of Noradrenaline Release From Isolated Rat Stomach

We examined the effect of endothelin-1, endothelin-3 and sarafotoxin S6c on the release of noradrenaline from gastric sympathetic nerve terminals using an isolated, vascularly perfused rat stomach. The release of noradrenaline evoked by electrical stimulation of the gastric postganglionic sympathetic nerves (at 2.5 Hz for 1 min) was inhibited by endothelin-1 (10-10 – 10-8 M), endothelin-3 (10-9 – 10-8 M) and sarafotoxin S6c (a highly selective agonist of endothelin ETB receptors) (10-9 – 10-8 M) in a concentration-dependent manner; the inhibitory potencies were as follows: endothelin-1 > endothelin-3 > sarafotoxin S6c. The inhibitory effect of endothelin-1 (3 × 10-9 M) on noradrenaline release was abolished by BQ-123 (a selective antagonist of endothelin ETA receptors) in a dose-dependent manner (10-7 and 10-6 M), but not influenced by BQ-788 (a selective antagonist of endothelin ETB receptors) (10-7 and 10-6 M). The endothelin-1-induced inhibition of noradrenaline release was attenuated by pretreatment with pertussis toxin (10 μg/animal, i.v., 4 days before experiments), but not influenced by indomethacin (3 × 10-6 M). These results indicate that endothelin ETA and ETB receptors located on the sympathetic nerve terminals play a role in the inhibition of noradrenaline release from the rat stomach: endothelin ETA receptor-mediated inhibition is carried out by pertussis toxin-sensitive and indomethacin-insensitive mechanisms.

Blockade of sustained tumor necrosis factor in a transgenic model of progressive autoimmune encephalomyelitis limits oligodendrocyte apoptosis and promotes oligodendrocyte maturation

BackgroundTumor necrosis factor (TNF) is associated with several neurodegenerative disorders including multiple sclerosis (MS). Although TNF-targeted therapies have been largely unsuccessful in MS, recent preclinical data suggests selective soluble TNF inhibition can promote remyelination. This has renewed interest in regulation of TNF signaling in demyelinating disease, especially given the limited treatment options for progressive MS. Using a mouse model of progressive MS, this study evaluates the effects of sustained TNF on oligodendrocyte (OLG) apoptosis and OLG precursor cell (OPC) differentiation.MethodsInduction of experimental autoimmune encephalomyelitis (EAE) in transgenic mice expressing a dominant-negative interferon-γ receptor under the human glial fibrillary acidic protein promoter (GFAPγR1Δ) causes severe non-remitting disease associated with sustained TNF. Therapeutic effects in GFAPγR1Δ mice treated with anti-TNF compared to control antibody during acute EAE were evaluated by assessing demyelinating lesion size, remyelination, OLG apoptosis, and OPC differentiation.ResultsMore severe and enlarged demyelinating lesions in GFAPγR1Δ compared to wild-type (WT) mice were associated with increased OLG apoptosis and reduced differentiated CC1+Olig2+ OLG within lesions, as well as impaired upregulation of TNF receptor-2, suggesting impaired OPC differentiation. TNF blockade during acute EAE in GFAPγR1Δ both limited OLG apoptosis and enhanced OPC differentiation consistent with reduced lesion size and clinical recovery. TNF neutralization further limited increasing endothelin-1 (ET-1) expression in astrocytes and myeloid cells noted in lesions during disease progression in GFAPγR1Δ mice, supporting inhibitory effects of ET-1 on OPC maturation.ConclusionOur data implicate that IFNγ signaling to astrocytes is essential to limit a detrimental positive feedback loop of TNF and ET-1 production, which increases OLG apoptosis and impairs OPC differentiation. Interference of this cycle by TNF blockade promotes repair independent of TNFR2 and supports selective TNF targeting to mitigate progressive forms of MS.

Endothelins Inhibit Osmotic Swelling of Rat Retinal Glial and Bipolar Cells by Activation of Growth Factor Signaling.

Water accumulation in retinal glial (Müller) and neuronal cells resulting in cellular swelling contributes to the development of retinal edema and neurodegeneration. Here, we show that endothelin-1 (ET-1) dose-dependently inhibits the hypoosmotic swelling of Müller cells in freshly isolated retinal slices of control and diabetic rats, with a maximal inhibition at 100nM. Osmotic Müller cell swelling was also inhibited by ET-2. The effect of ET-1 was mediated by activation of ETA and ETB receptors resulting in transactivation of metabotropic glutamate receptors, purinergic P2Y1, and adenosine A1 receptors. ET-1 (but not ET-2) also inhibited the osmotic swelling of bipolar cells in retinal slices, but failed to inhibit the swelling of freshly isolated bipolar cells. The inhibitory effect of ET-1 on the bipolar cell swelling in retinal slices was abrogated by inhibitors of the FGF receptor kinase (PD173074) and of TGF-β1 superfamily activin receptor-like kinase receptors (SB431542), respectively. Both Müller and bipolar cells displayed immunoreactivities of ETA and ETB receptor proteins. The data may suggest that neuroprotective effects of ETs in the retina are in part mediated by prevention of the cytotoxic swelling of retinal glial and bipolar cells. ET-1 acts directly on Müller cells, while the inhibitory effect of ET-1 on bipolar cell swelling is indirectly mediated, via stimulation of the release of growth factors like bFGF and TGF-β1 from Müller cells.

Endothelin-1 suppresses insulin-stimulated Akt phosphorylation and glucose uptake via GPCR kinase 2 in skeletal muscle cells.

Endothelin-1 (ET-1) reduces insulin-stimulated glucose uptake in skeletal muscle, inducing insulin resistance. Here, we have determined the molecular mechanisms underlying negative regulation by ET-1 of insulin signalling. We used the rat L6 skeletal muscle cells fully differentiated into myotubes. Changes in the phosphorylation of Akt was assessed by Western blotting. Effects of ET-1 on insulin-stimulated glucose uptake was assessed with [(3) H]-2-deoxy-d-glucose ([(3) H]2-DG). The C-terminus region of GPCR kinase 2 (GRK2-ct), a dominant negative GRK2, was overexpressed in L6 cells using adenovirus-mediated gene transfer. GRK2 expression was suppressed by transfection of the corresponding short-interfering RNA (siRNA). In L6 myotubes, insulin elicited sustained Akt phosphorylation at Thr(308) and Ser(473) , which was suppressed by ET-1. The inhibitory effects of ET-1 were prevented by treatment with a selective ETA receptor antagonist and a Gq protein inhibitor, overexpression of GRK2-ct and knockdown of GRK2. Insulin increased [(3) H]2-DG uptake rate in a concentration-dependent manner. ET-1 noncompetitively antagonized insulin-stimulated [(3) H]2-DG uptake. Blockade of ETA receptors, overexpression of GRK2-ct and knockdown of GRK2 prevented the ET-1-induced suppression of insulin-stimulated [(3) H]2-DG uptake. In L6 myotubes overexpressing FLAG-tagged GRK2, ET-1 facilitated the interaction of endogenous Akt with FLAG-GRK2. Activation of ETA receptors with ET-1 suppressed insulin-induced Akt phosphorylation at Thr(308) and Ser(473) and [(3) H]2-DG uptake in a GRK2-dependent manner in skeletal muscle cells. These findings suggest that ETA receptors and GRK2 are potential targets for overcoming insulin resistance.

Increased interaction of connexin43 with zonula occludens-1 during inhibition of gap junctions by G protein-coupled receptor agonists

Astrocytes are extensively coupled through gap junctions (GJs) that are composed of channels mostly constituted by connexin43 (Cx43). This astroglial gap junctional intercellular communication (GJIC) allows propagation of ions and signaling molecules critical for neuronal activity and survival. It is drastically inhibited by a short-term exposure to endothelin-1 (ET-1) or to sphingosine-1-phosphate (S1P), both compounds being inflammatory mediators acting through activation of GTP-binding protein-coupled receptors (GPCRs). Previously, we have identified the GTPases G(i/o) and Rho as key actors in the process of S1P-induced inhibition. Here, we asked whether similar mechanisms underlied the effects of ET-1 and S1P by investigating changes in the phosphorylation status of Cx43 and in the molecular associations of Cx43 with zonula occludens (ZO) proteins and occludin. We showed that the inhibitory effect of ET-1 on GJIC was entirely dependent on the activation of G(i/o) but not on Rho and Rho-associated kinase. Both ET-1 and S1P induced dephosphorylation of Cx43 located at GJs through a process mediated by G(i/o) and calcineurin. Thanks to co-immunoprecipitation approaches, we found that a population of Cx43 (likely junctional Cx43) was associated to ZO-1–ZO-2–occludin multiprotein complexes and that acute treatments of astrocytes with ET-1 or S1P induced a G(i/o)-dependent increase in the amount of Cx43 linked to these complexes. As a whole, this study identifies a new mechanism of GJIC regulation in which two GPCR agonists dynamically alter interactions of Cx43 with its molecular partners.

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.

Peroxynitrites and impaired modulation of nitric oxide concentrations in embryos from diabetic rats during early organogenesis

Maternal diabetes significantly increases the risk of congenital malformation, a syndrome known as diabetic embryopathy. Nitric oxide (NO), implicated in embryogenesis, has been found elevated in embryos from diabetic rats during organogenesis. The developmental signaling molecules endothelin-1 (ET-1) and 15-deoxy delta(12,14)prostaglandin J2 (15dPGJ2) downregulate embryonic NO levels. In the presence of NO and superoxide, formation of the potent oxidant peroxynitrite may occur. Therefore, we investigated peroxynitrite-induced damage, ET-1 and 15dPGJ2 concentrations, and the capability of ET-1, 15dPGJ2 and prostaglandin E2 (PGE2) to regulate NO production in embryos from severely diabetic rats (streptozotocin-induced before pregnancy). We found intense nitrotyrosine immunostaining (an index of peroxynitrite-induced damage) in neural folds, neural tube and developing heart of embryos from diabetic rats (P < 0.001 vs controls). We also found reduced ET-1 (P < 0.001) and 15dPGJ2 (P < 0.001) concentrations in embryos from diabetic rats when compared with controls. In addition, the inhibitory effect of ET-1, 15dPGJ2 and PGE2 on NO production found in control embryos was not observed in embryos from severely diabetic rats. In conclusion, both the demonstrated peroxynitrite-induced damage and the altered levels and function of multiple signaling molecules involved in the regulation of NO production provide supportive evidence of nitrosative stress in diabetic embryopathy.

Endothelin-1 inhibits TNF alpha-induced iNOS expression in 3T3-F442A adipocytes.

1. Endothelin-1 (ET-1) and tumor necrosis factor alpha (TNFalpha) by their action on adipocytes have been independently linked to the pathogenesis of insulino-resistance. In isolated adipocytes, TNFalpha induces the expression of the inducible nitric oxide synthase (iNOS). The purpose of the present work was, in the 3T3-F442A adipocyte cell line, to characterise TNFalpha-induced iNOS expression and to determine whether or not ET-1 could influence TNFalpha-induced iNOS expression and NO production. 2. In differentiated 3T3-F442A, treatment with TNFalpha (20 ng ml(-1)) induced the expression of a functional iNOS as demonstrated by nitrite assay, Western blot, reverse transcription-polymerase chain reaction and Northern blot analysis. TNFalpha-induced iNOS expression requires nuclear factor kappaB activation, but does not necessitate the activation of the PI-3 kinase/Akt and P38-MAP kinase pathways. 3. ET-1, but not ET-3, inhibited the TNFalpha-induced expression of iNOS protein and mRNA as well as nitrite production. The effects of ET-1 were blocked by a specific ETA (BQ123, pA(2) 7.4) but not by a specific ETB receptor antagonist (BQ788). 3T3-F442A adipocytes express the mRNAs for prepro-ET-1 and the ET-A receptor subtype, but not for the ET-B subtype. 4. The inhibitory effect of ET-1 was not affected by bisindolylmaleimide, SB 203580 or indomethacin, inhibitors of protein kinase C, p38-MAP kinase and cyclooxygenase, respectively, and was not associated with cAMP production. However, the effect of ET-1 was partially reversed by wortmannin, suggesting the involvement of PI3 kinase in the transduction signal of ET-1. 5. Differentiated 3T3-F442A adipocytes did not release ET-1 with or without exposure to TNFalpha, although the mRNA for preproET-1 was detected in both pre- and differentiated adipocytes. 6. Thus, these results confirm that adipocytes are a target for circulating ET-1 and demonstrate that the activation of the ETA receptor subtype can prevent TNFalpha-induced iNOS expression.

Reduction of nitrite production by endothelin-1 in isolated porcine ciliary processes

It has been postulated that endothelin-1 and nitric oxide (NO) could participate in the modulation of aqueous humor dynamic in the eye. This study investigates whether endothelin-1 can reduce the production of nitrite (a stable metabolite of NO) in isolated porcine ciliary processes. Nitrite production was measured (Griess reaction) in the medium surrounding isolated ciliary processes before and after exposure to different drugs. Results are expressed in percent of basal nitrite production. In a concentration-dependent manner (0·1 n m to 1 μ m), endothelin-1 significantly decreased basal nitrite production (1 μ m: 81·7±3·5%; P<0·001). This effect was prevented by the endothelin type A (ET A)-receptor antagonist BQ123 (1 μ m: 95·6±4·3%; P<0·05), but not by the endothelin type B (ET B)-receptor antagonist BQ788 (1 μ m: 83·3±4·4%; P=0·86) or by the prostaglandin F 2α analog unoprostone (30 μ m: 86·7±3·7%; P=0·74). The adenylcyclase activator forskolin significantly increased basal nitrite production (1 μ m: 143·4±3·9%, P<0·001). This effect was prevented both by endothelin-1 (0·1 μ m; 113·9±6·7%: P<0·01) or the nitric oxide synthase inhibitor l-NAME (0·5 m m: 103·3±8·4%: P<0·001). The inhibitory effect of endothelin-1 on forskolin-induced nitrite production was significantly reversed by BQ123 (1 μ m: 132·4±5·1%; P<0·01), but not by BQ788 (1 μ m: 112·7±4·1%; P=0·64) or unoprostone (30 μ m: 109·3±4·8%; P=0·98). These results suggest that endothelin-1, through an ET A receptor activation, can reduce both basal and forskolin-induced nitrite production in isolated porcine ciliary processes.

Involvement of prostanoids in the inhibitory effect of endothelin-1 on progesterone production of ovarian granulosa cells.

The present study was undertaken to investigate the possible involvement of prostanoids in the inhibitory effect of endothelin-1 (ET-1) on progesterone production of ovarian granulosa cells. ET-1 (1 x 10(-7) M) decreased the basal and follicle-stimulating hormone- (FSH) stimulated progesterone production from both human and porcine granulosa cells. Indomethacin dose-dependently inhibited progesterone release, but did not alter the inhibitory effect of ET-1 (1 x 10(-7) M) on progesterone production of cultured ovarian granulosa cells. This study shows that ET-1 suppresses basal and FSH-stimulated progesterone production by ovarian granulosa cells, but this effect is not mediated by prostanoids.

Endothelin ET A- and ET B-receptor-mediated inhibition of noradrenaline release from isolated rat stomach.

We examined the effect of endothelin-1, endothelin-3 and sarafotoxin S6c on the release of noradrenaline from gastric sympathetic nerve terminals using an isolated, vascularly perfused rat stomach. The release of noradrenaline evoked by electrical stimulation of the gastric postganglionic sympathetic nerves (at 2.5 Hz for 1 min) was inhibited by endothelin-1 (10(-)(10) - 10(-)(8) M), endothelin-3 (10(-)(9) - 10(-)(8) M) and sarafotoxin S6c (a highly selective agonist of endothelin ET(B) receptors) (10(-)(9) - 10(-)(8) M) in a concentration-dependent manner; the inhibitory potencies were as follows: endothelin-1 > endothelin-3 > sarafotoxin S6c. The inhibitory effect of endothelin-1 (3 x 10(-)(9) M) on noradrenaline release was abolished by BQ-123 (a selective antagonist of endothelin ET(A) receptors) in a dose-dependent manner (10(-)(7) and 10(-)(6) M), but not influenced by BQ-788 (a selective antagonist of endothelin ET(B) receptors) (10(-)(7) and 10(-)(6) M). The endothelin-1-induced inhibition of noradrenaline release was attenuated by pretreatment with pertussis toxin (10 micro g/animal, i.v., 4 days before experiments), but not influenced by indomethacin (3 x 10(-)(6) M). These results indicate that endothelin ET(A) and ET(B) receptors located on the sympathetic nerve terminals play a role in the inhibition of noradrenaline release from the rat stomach: endothelin ET(A) receptor-mediated inhibition is carried out by pertussis toxin-sensitive and indomethacin-insensitive mechanisms.

Characterization of effects of endothelin-1 on the L-type Ca2+ current in human atrial myocytes.

The effects of endothelin-1 (ET-1) on the L-type Ca2+ current (I(Ca)) were examined in whole cell patch-clamped human atrial myocytes. Depending on the initial current density, ET-1 (10 nM) increased the amplitude of I(Ca) by 99 +/- 7% or decreased it by 33 +/- 2%. The stimulatory effect predominated on current of low density (2.3 +/- 0.2 pA/pF), whereas I(Ca) of higher density (5.8 +/- 0.3 pA/pF) was inhibited by ET-1. After I(Ca) stimulation by 1 microM isoproterenol, ET-1 always inhibited the current by 32 +/- 7% (P < 0.05), an effect that was suppressed by pretreating myocytes with pertussis toxin. Atrial natriuretic peptide (ANP) inhibited I(Ca) (41 +/- 3%) by reducing intracellular cAMP concentration. In ANP-treated myocytes, the stimulatory effect of ET-1 on I(Ca) predominated (52 +/- 7%). The inhibitory effect of ET-1 on I(Ca) was blocked by the ET(A) antagonist BQ-123, whereas the stimulatory effect was suppressed by the ET(B) agonist BQ-788. We conclude that ET-1 has opposite effects on I(Ca) depending on the baseline amplitude of current, and both subtype ET receptors are implicated in the signal transduction pathways.

Inhibitory effect of endothelin-1 on the isoproterenol-induced chloride current in human cardiac myocytes

It is still controversial whether the cAMP-activated Cl − current ( I Cl,cAMP) is expressed in human cardiomyocytes. The whole-cell configuration of the voltage-clamp technique was used to examine in detail the I Cl,cAMP in single human atrial and ventricular myocytes. Human cardiomyocytes were enzymatically isolated from atrial or ventricular specimens obtained from open-heart surgery or cardiac transplantation, respectively. Isoproterenol (1 μM) or forskolin (10 μM) was used to activate the cAMP second-messenger system. The isoproterenol- or forskolin-induced Cl − current was elicited in 12 of 54 atrial myocytes but was completely absent from ventricular myocytes. The isoproterenol-induced Cl − current in atrial myocytes was time-independent and had a reversal potential close to zero. Endothelin-1 (30 nM) inhibited the isoproterenol-induced Cl − current by 75±6% ( n=4). This inhibitory effect of endothelin-1 was attenuated by pretreating atrial myocytes with the endothelin ET A receptor antagonist, BQ485, but not with the ET B receptor antagonist, BQ-788. The results provide evidence that the I Cl,cAMP exists in human atria, but not ventricle, and is inhibited by endothelin-1.

Suppression of integrin alpha(v) expression by endothelin-1 in vascular smooth muscle cells.

Both integrins and endothelins (ETs) are known to play important roles in vascular remodeling via proliferation, apoptosis, and migration of vascular smooth muscle cells (VSMCs), whose dysfunctions have been implicated in the pathogenesis of end-organ damage associated with hypertension and arteriosclerosis. However, whether there is any interaction between endothelin-1 (ET-1) and integrins remains unknown. Therefore, the aim of the present study was to elucidate whether ET-1 regulates the expression of integrin alpha(v) in rat VSMCs. ET-1 dose- and time-dependently suppressed the integrin alpha(v) messenger RNA (mRNA) transcripts, as quantified by a real-time quantitative polymerase chain reaction (PCR) method, and decreased the transcriptional activity of integrin alpha(v) gene, as demonstrated by integrin alpha(v)-luciferase assay. The inhibitory effect of ET-1 on integrin alpha(v) gene expression was abrogated by an ETA receptor antagonist (BQ123) but not by an ET(B) receptor antagonist (BQ788). ET-1 also suppressed the cell surface expression of integrin alpha(v)beta5 and the adhesion to vitronectin, but not to fibronectin. These results demonstrate that the adhesion of vitronectin to rat VSMCs is inhibited by ET-1 via the ET(A) receptors by suppressing integrin alpha(v) gene transcription, suggesting that ET-1 is involved in regulation of vascular integrin alpha(v) gene expression.

Endothelin-1 inhibits secretin-stimulated ductal secretion by interacting with ETA receptors on large cholangiocytes.

We studied the expression of endothelin-1 (ET-1) receptors (ETA and ETB) and the effects of ET-1 on cholangiocyte secretion. The effects of ET-1 on cholangiocyte secretion were assessed in normal and bile duct-ligated (BDL) rats by measuring 1) basal and secretin-induced choleresis in vivo, 2) secretin receptor gene expression and cAMP levels in small and large cholangiocytes, and 3) luminal expansion in response to secretin in intrahepatic bile duct units (IBDU). ETA and ETB receptors were expressed by small and large cholangiocytes. ET-1 had no effect on basal bile flow or bicarbonate secretion in normal or BDL rats but decreased secretin-induced bicarbonate-rich choleresis in BDL rats. ET-1 decreased secretin receptor gene expression and secretin-stimulated cAMP synthesis in large cholangiocytes and secretin-induced luminal expansion in IBDU from normal or BDL rats. The inhibitory effects of ET-1 on secretin-induced cAMP synthesis and luminal duct expansion were blocked by specific inhibitors of the ETA (BQ-610) receptor. ET-1 inhibits secretin-induced ductal secretion by decreasing secretin receptor and cAMP synthesis, two important determinants of ductal secretion.

Endothelin activates large-conductance K+ channels in rat lactotrophs: reversal by long-term exposure to dopamine agonist.

Endothelin-1 (ET-1) inhibits PRL secretion from cultured rat lactotrophs. However, ET-1 stimulates PRL secretion after cultured lactotrophs have been exposed for 48 h to dopamine or D2 dopamine agonists. In the present study, we have used cell-attached and inside-out patch recordings to establish an ionic basis for these effects. Bath application of 20 nM ET-1 to untreated lactotrophs evoked a robust and persistent activation of large-conductance K+ channels in cell-attached patches. This effect of ET-1 had a long latency to onset, was maintained for as long as ET-1 was present, and required at least 10 min of washing in control saline before complete recovery was achieved. The stimulatory effect of 20 nM ET-1 on these channels was markedly attenuated in the presence of the selective ET(A) receptor antagonist BQ-610 (200 nM), or after pertussis toxin (200 ng/ml, 16 h) pretreatment. The unitary slope conductance of the ET-1 activated channels in cell attached patches was 165 and 95 pS when the recording electrodes contained 150 and 5.4 mM KCl, respectively. These channels were voltage-sensitive and their activity increased upon patch depolarization. Previously activated channels in cell-attached patches became quiescent immediately upon patch excision into Ca2+-free bath saline. Exposure of the intracellular surface to 0.1 microM Ca2+ restored the activity of these channels similar to the level seen before patch excision. In addition, preincubating the cells with the membrane-permeable Ca2+-chelator BAPTA-AM, or using Ca2+-free solution in the recording pipettes, prevented the activation of these channels by ET-1. The ET-1 activated large-conductance Ca2+-dependent K+ (BK(Ca)) channels were blocked by 20 mM tetraethylammonium but were insensitive to the K+ channel blockers apamin (1 microM), charybdotoxin (200 nM), or iberiotoxin (200 nM). Acute application of 10 microM dopamine and 20 nM ET-1 caused activation of BK(Ca) channels with indistinguishable kinetic properties, although the effect of dopamine occurred with shorter latency. After 48-h exposure to the specific D2 dopamine receptor agonist (+/-)-2-(N-phenyl-N-propyl) amino-5-hydroxytetralin hydrochloride (PPHT, 500 nM), bath application of 20 nM ET-1 resulted in inhibition of spontaneously active BK(Ca) channels. These data suggest that both the stimulatory and inhibitory effects of ET-1 on PRL secretion are mediated, at least in part, by actions on BK(Ca) channels, and that long term exposure to dopamine or D2 agonists alters the signaling pathways from the ET(A) receptor to BK(Ca) channels.

Endothelin Activates Large-Conductance K+ Channels in Rat Lactotrophs: Reversal by Long-Term Exposure to Dopamine Agonist

Endothelin-1 (ET-1) inhibits PRL secretion from cultured rat lactotrophs. However, ET-1 stimulates PRL secretion after cultured lactotrophs have been exposed for 48 h to dopamine or D2 dopamine agonists. In the present study, we have used cell-attached and inside-out patch recordings to establish an ionic basis for these effects. Bath application of 20 nm ET-1 to untreated lactotrophs evoked a robust and persistent activation of large-conductance K+ channels in cell-attached patches. This effect of ET-1 had a long latency to onset, was maintained for as long as ET-1 was present, and required at least 10 min of washing in control saline before complete recovery was achieved. The stimulatory effect of 20 nm ET-1 on these channels was markedly attenuated in the presence of the selective ETA receptor antagonist BQ-610 (200 nm), or after pertussis toxin (200 ng/ml, 16 h) pretreatment. The unitary slope conductance of the ET-1 activated channels in cell attached patches was 165 and 95 pS when the recording electrodes contained 150 and 5.4 mm KCl, respectively. These channels were voltage-sensitive and their activity increased upon patch depolarization. Previously activated channels in cell-attached patches became quiescent immediately upon patch excision into Ca2+-free bath saline. Exposure of the intracellular surface to 0.1 μm Ca2+ restored the activity of these channels similar to the level seen before patch excision. In addition, preincubating the cells with the membrane-permeable Ca2+-chelator BAPTA-AM, or using Ca2+-free solution in the recording pipettes, prevented the activation of these channels by ET-1. The ET-1 activated large-conductance Ca2+-dependent K+ (BKCa) channels were blocked by 20 mm tetraethylammonium but were insensitive to the K+ channel blockers apamin (1μ m), charybdotoxin (200 nm), or iberiotoxin (200 nm). Acute application of 10 μm dopamine and 20 nm ET-1 caused activation of BKCa channels with indistinguishable kinetic properties, although the effect of dopamine occurred with shorter latency. After 48-h exposure to the specific D2 dopamine receptor agonist (±)-2-(N-phenyl-N-propyl) amino-5-hydroxytetralin hydrochloride (PPHT, 500 nm), bath application of 20 nm ET-1 resulted in inhibition of spontaneously active BKCa channels. These data suggest that both the stimulatory and inhibitory effects of ET-1 on PRL secretion are mediated, at least in part, by actions on BKCa channels, and that long term exposure to dopamine or D2 agonists alters the signaling pathways from the ETA receptor to BKCa channels.

Inhibition of the acetylcholine-activated potassium current by endothelin-1 in atrial cardiomyocytes

Endothelin-1 (ET-1), is a 21 amino-acid peptide with potent inotropic and chronotropic actions in the heart. Relatively little is known about the underlying electrophysiological effects of the peptide. In this study the effects of ET-1 on the acetylcholine-activated potassium current (IK(Ach)) were investigated in the absence and presence of the receptor-selective antagonists, PD155080 (ETA receptor-selective) and RES-701 (ETB receptor-selective) in atrial cardiomyocytes. Cells were obtained from New Zealand White rabbits (2·5–3 kg) by enzymatic dissociation using collagenase. Potassium currents were recorded, in the presence of nifedipine (5 μM), using the whole cell ruptured patch-clamp technique. Low resistance electrodes (1–3 MΩ) were filled with potassium gluconate solution (pH 7·2). Cells were superfused with a modified Tyrode's solution. Following stabilization, control recordings were made using standard pulse protocols, and drugs were applied using a gravity fed microperfusion system. Data were expressed as mean±standard error, and comparisons were made using Friedman's ANOVA and Wilcoxon's Signed Ranks tests; p<0·05 was considered significant ET-1 (10 nM) alone did not affect the potassium current. Acetylcholine (Ach, 1 μM) increased (p<0·05) the potassium current to −1321±290 pA, from a control value of −955±191 pA, at a step potential of −100 mV. This change in current is attributed to IK(Ach) as it was blocked by atropine (10 μM). ET-1 (10 nM) inhibited (p<0.05) the IK(Ach) obtained in the presence of Ach (−882±88 compared to a control value of −870±98 pA) Ach also increased the holding current from +80±9 to ±242±38 pA, and this effect was blocked (p<0·05) in the presence of ET-1 (44±13 pA). The ETA receptor-selective antagonist, PD155080 (1 μM), prevented (p<0·05) the ET-1 induced inhibition of IK(Ach) (control vs Ach±ET-1+PD155080: −804±224 vs −1030±210 pA; at −100 mV; n=6). The ETB receptor-selective antagonist, RES-701 (1 μM), also prevented (p<0·05) the inhibitory effect of ET-1 on IK(Ach) (control vs Ach+ET-1+RES-701: −733±116 vs −913±137 pA; at −100 mV; n=6). However, unlike PD155080, RES-701 failed to sustain this inhibitory effect after a further 90 s exposure to Ach and ET-1 (control vs Ach+ET-1+RES-701; −733±116 vs 768±96 pA; at −100mV; n=6). In summary, ET-1 clearly inhibits the effects of Ach on IK(Ach) in rabbit atrial cardiomyocytes. This effect is mediated by an ETA receptor-subtype, and an RES-701 -sensitive ETB receptor subtype.

Endothelin-1-induced constriction inhibits nitric-oxide-mediated dilation in isolated rat resistance arteries.

Vascular endothelial cells release dilatory compounds like nitric oxide and prostacyclin, as well as contractile factors like endothelin-1 (ET-1). We investigated the interaction of ET-1 with nitric-oxide-mediated dilation in cannulated pressurized (75 mm Hg) arterioles from rat cremaster muscle (180 +/- 3 microm). Arterioles constricted spontaneously to 101 +/- 3 microm, while ET-1 (0.4 nM) increased constriction to 78 +/- 3 microm. Acetylcholine, an endothelium-dependent nitric-oxide-mediated vasodilator induced a dose-dependent dilation during spontaneous tone. After addition of ET-1, the response to acetylcholine was significantly impaired. Nitroprusside, an endothelium-independent nitric oxide donor, induced a dose-dependent dilation that was almost completely inhibited by ET-1. In contrast, 8-Br-cGMP-induced dilation was unaffected. Thus, ET-1 appears to inhibit nitric-oxide-mediated dilation at the level of cGMP formation or degradation. The effect of ET-1 appears to be specific for nitric oxide as responses to a prostacyclin analogue were impaired at low doses only. The inhibitory effect of ET-1 on nitric-oxide-mediated dilation could be mimicked with high potassium (65 +/- 6 microm), but not with phenylephrine (74 +/- 8 microm)-induced constriction. These data show a direct inhibitory effect of ET-1 on nitric-oxide-mediated dilation in isolated skeletal muscle arterioles.

Endothelin-1 mediated inhibition of the acetylcholine-activated potassium current from rabbit isolated atrial cardiomyocytes.

1. Endothelin-1 is a 21 amino acid peptide with potent inotropic and chronotropic actions in the heart. Relatively little is known about the underlying electrophysiological effects of the peptide. In this study, the effects of endothelin-1 (ET-1) on the acetylcholine-activated potassium current (IK(ACh) were investigated in the absence and presence of the receptor-selective antagonists, PD155080 (ETA receptor-selective) and RES-701 (ETB receptor-selective) in rabbit atrial cardiomyocytes. 2. Cells were obtained from New Zealand White rabbits (2.5-3 kg) by enzymatic dissociation with collagenase. Potassium currents were recorded, in the presence of nifedipine (5 microM), by use of the whole cell ruptured patch-clamp technique. Following stabilization, control recordings were made with standard pulse protocols, and drugs were applied by a gravity fed microperfusion system. 3. Endothelin-1 (10 nM) alone did not affect the "steady state' potassium current. Acetylcholine (1 microM) increased (P < 0.05) the potassium current to-1321 +/- 290 pA, from a control value of -955 +/- 191 pA, at a step potential of -100 mV. Acetylcholine also increased the holding current at -40 mV from +80 +/- 9 pA to +242 +/- 38 pA, and this effect was abolished (P < 0.05) in the presence of endothelin-1 (+44 +/- 13 pA). The responses to acetylcholine were attributed to activation of the atrial muscarinic-activated potassium current (IK(ACh)) as they were blocked by atropine (10 microM). Endothelin-1 (10 nM) in the presence of acetylcholine did not affect the "steady state' potassium current (-882 +/- 88 pA compared to a control value of -870 +/- 98 pA, at -100 mV). 4. The ETA receptor-selective antagonist, PD155080 (1 microM), prevented (P < 0.05) the ET-1 induced inhibition of IK(ACh) at all potentials. PD155080, in the presence of endothelin-1 and acetylcholine, increased the inward component of the "steady state' potassium current to -1030 +/- 210 pA from a control value of -804 +/- 224 pA at a step potential of -100 mV. Also the outward component was increased at a potential of -20 mV from +90 +/- 17 pA to +241 +/- 47 pA. 5. Unlike PD155080, the ETB receptor-selective antagonist, RES-701 (1 microM), only prevented (P < 0.05) the inhibitory effect of endothelin-1 on the inward component of the IK(ACh); at -100 mV, RES-701, in the presence of endothelin-1 and acetylcholine, increased the "steady state' potassium current to -913 +/- 137 pA from -733 +/- 116 pA. Furthermore, RES-701, in contrast to PD155080, failed to sustain this inhibitory effect as, in the presence of endothelin-1 and acetylcholine, the "steady state' potassium current returned to a value of -768 +/- 96 pA, at a step potential of -100 mV. 6. In conclusion, endothelin-1 clearly inhibits the effects of acetylcholine on IK(ACh) in rabbit atrial cardiomyocytes. This effect is primarily mediated by an ETA receptor-subtype, but is transiently and partially mediated by a RES-701-sensitive ETB receptor subtype. Inhibition of the IK(ACh) may account for the positive chronotropic properties of endothelin-1.