Receptor Agonist Sarafotoxin S6c Research Articles

High salt intake increases ETB receptor function in the renal medulla of rats

The present study was conducted to test the hypothesis that high salt intake would increase the diuretic and natriuretic effects of renal medullary ETB receptor stimulation in vivo. Two groups of male Sprague Dawley (SD) rats were compared; after being fed normal (0.3%) and high salt (4%) diet for 7 days. Rats were anesthetized and a catheter implanted in the renal medulla for interstitial infusion along with a ureteral catheter for urine collection. An adjustable occluder was placed around the aorta proximal to the left renal artery to maintain renal perfusion pressure throughout the protocol. After equilibration and two 20 min control periods, the urine flow (UV) and sodium excretion (UNaV) responses to medullary infusion of a low dose of the ETB receptor agonist sarafotoxin S6c (0.15 μg/kg/h) were determined over an 80 min infusion period. Intramedullary infusion of a relatively low dose of S6c (0.15 μg/kg/h) markedly increased the urine flow and sodium excretion in the high salt treated rats (UV: 5.6±0.7 to 30.7±3.1 μl/min; UNaV: 1.1±0.2 to 2.2±0.3 μmol/min; n=9), but not in the normal salt treated rats (UV: 8.6±2 to 11.6±1.9 μl/min; UNaV 0.5±0.1 to 0.5±0.1 μmol/min; n=8). We conclude that high salt intake enhances the diuretic and natriuretic effects of ETB receptor stimulation in vivo consistent with a role for the ETB receptor in maintaining fluid‐electrolyte balance.

Increased expression of vascular endothelin type B and angiotensin type 1 receptors in patients with ischemic heart disease

BackgroundEndothelin-1 and angiotensin II are strong vasoconstrictors. Patients with ischemic heart disease have elevated plasma levels of endothelin-1 and angiotensin II and show increased vascular tone. The aim of the present study was to examine the endothelin and angiotensin II receptor expression in subcutaneous arteries from patients with different degrees of ischemic heart disease.MethodsSubcutaneous arteries were obtained, by biopsy from the abdomen, from patients undergoing coronary artery bypass graft (CABG) surgery because of ischemic heart disease (n = 15), patients with angina pectoris without established myocardial infarction (n = 15) and matched cardiovascular healthy controls (n = 15). Endothelin type A (ETA) and type B (ETB), and angiotensin type 1 (AT1) and type 2 (AT2) receptors expression and function were examined using immunohistochemistry, Western blot and in vitro pharmacology.ResultsETA and, to a lesser extent, ETB receptor staining was observed in the healthy vascular smooth muscle cells. The level of ETB receptor expression was higher in patients undergoing CABG surgery (250% ± 23%; P < 0.05) and in the patients with angina pectoris (199% ± 6%; P < 0.05), than in the healthy controls (100% ± 28%). The data was confirmed by Western blotting. Arteries from CABG patients showed increased vasoconstriction upon administration of the selective ETB receptor agonist sarafotoxin S6c, compared to healthy controls (P < 0.05). No such difference was found for the ETA receptors. AT1 and, to a lesser extent, AT2 receptor immunostaining was seen in the vascular smooth muscle cells. The level of AT1 receptor expression was higher in both the angina pectoris (128% ± 25%; P < 0.05) and in the CABG patients (203% ± 41%; P < 0.05), as compared to the healthy controls (100% ± 25%). The increased AT1 receptor expression was confirmed by Western blotting. Myograph experiment did however not show any change in vasoconstriction to angiotensin II in CABG patients compared to healthy controls (P = n.s).ConclusionThe results demonstrate, for the first time, upregulation of ETB and AT1 receptors in vascular smooth muscle cells in ischemic heart disease. These receptors may play a role in the pathophysiology of ischemic heart disease and could provide important targets for pharmaceutical interventions.

Endothelin B Receptors on Vascular Smooth Muscle Cells of the Human Umbilical Vein Mediate Vasoconstriction

Objective: To test the hypothesis that smooth muscle cells of the human umbilical vein have vasoconstricting endothelin B (ETB) receptors. Methods: In strip preparations of human umbilical veins isometric tension after exposure to the selective ETB receptor agonist sarafotoxin S6c (S6c) was compared to the tension before S6c exposure (set as 100%). Results: In intact preparations S6c induced vasoconstriction only at the highest concentration applied (10^–8M; 149.5 ± 12.5 vs. 100%, p < 0.05). In contrast, in endothelium-denuded preparations S6c induced vasoconstriction already at the lowest S6c concentration investigated (10^–11M; 111.7 ± 4.3 vs. 100%, p < 0.05). Conclusions: The vasoconstricting effect of S6c in endothelium-denuded human umbilical vein preparations points to smooth muscle cell ETB receptor activation.

Renal medullary ETBreceptors produce diuresis and natriuresis via NOS1

Endothelin-1 (ET-1) plays an important role in the regulation of salt and water excretion in the kidney. Considerable in vitro evidence suggests that the renal medullary ET(B) receptor mediates ET-1-induced inhibition of electrolyte reabsorption by stimulating nitric oxide (NO) production. The present study was conducted to test the hypothesis that NO synthase 1 (NOS1) and protein kinase G (PKG) mediate the diuretic and natriuretic effects of ET(B) receptor stimulation in vivo. Infusion of the ET(B) receptor agonist sarafotoxin S6c (S6c: 0.45 microg x kg(-1) x h(-1)) in the renal medulla of anesthetized, male Sprague-Dawley rats markedly increased the urine flow (UV) and urinary sodium excretion (UNaV) by 67 and 120%, respectively. This was associated with an increase in medullary cGMP content but did not affect blood pressure. In addition, S6c-induced diuretic and natriuretic responses were absent in ET(B) receptor-deficient rats. Coinfusion of N(G)-propyl-l-arginine (10 microg x kg(-1) x h(-1)), a selective NOS1 inhibitor, suppressed S6c-induced increases in UV, UNaV, and medullary cGMP concentrations. Rp-8-Br-PET-cGMPS (10 microg x kg(-1) x h(-1)) or RQIKIWFQNRRMKWKK-LRK(5)H-amide (18 microg x kg(-1) x h(-1)), a PKG inhibitor, also inhibited S6c-induced increases in UV and UNaV. These results demonstrate that renal medullary ET(B) receptor activation induces diuretic and natriuretic responses through a NOS1, cGMP, and PKG pathway.

Adrenergic dependence of the endothelin‐mediated pressor response in Dahl salt‐resistant and salt‐sensitive rats

We have previously shown that selective endothelin A (ETA) receptor antagonism augments the stress-mediated rises in arterial pressure and norepinephrine in Dahl salt-resistant (DR), but not salt-sensitive (DS) rats. Since these effects were absent during mixed ETA/B receptor blockade, we tested the hypothesis that there is greater adrenergic dependence of the ETB-mediated pressor response in DR vs. DS rats. Pressor responses to the selective ETB receptor agonist sarafotoxin (S6c) were examined in anesthetized male DR and DS rats during ganglion blockade. S6c produced comparable dose-dependent increases in arterial pressure in both DR and DS rats. In DR rats, pretreatment with the selective α1 adrenergic antagonist prazosin (PZ; 1 mg/kg) blunted the peak pressor response to 0.1 (17±2 vs. 24±2 mmHg, PZ vs. untreated, p<0.05) and 1 nmol/kg S6c (23±2 vs. 35±4 mmHg, PZ vs. untreated, p<0.01). Peak pressor response to 1 nmol/kg S6c was also lower in DS rats treated with PZ (20±4 vs. 47±12 mmHg, PZ vs. untreated, p<0.01). There was a greater reduction in the steady-state response to 1 nmol/kg S6c in treated DS (-3+4 vs. 35±16 mmHg, PZ vs. untreated) than in DR rats (14±2 vs. 24±6 mmHg, PZ vs. untreated). Together, these data suggest that the pressor response to ETB receptor activation is partially dependent on α1 adrenergic stimulation; however, the adrenergic component is not enhanced in DR vs. DS rats.

The complex venous response to the ETB receptor (ETBR) agonist sarafotoxin S6C

Arteries and veins are both actively engaged in the control of cardiovascular function. However, they display many functional differences, such as the involvement in the endothelin system. In the endothelium, ETBR mediates nitric oxide (NO) release and relaxation, while on smooth muscle cells (SMCs) it mediates contraction. Although the ETBR is present on SMCs of both rat aorta (RA) and vena cava (RVC), stimulation with S6C, a selective ETBR agonist, leads to contraction of the RVC, but not RA. We hypothesized that the venous endothelial ETBR is less functionally active than the arterial one. Therefore we studied the ability of S6C to act as a relaxing agent when used on contracted, endothelium-intact RA and RVC. In PGF2α (20 μM)-contracted RA, S6C (100 nM) induced a relaxation (21.5±4.8% PGF2α-induced contraction). By contrast, in the RVC, S6C induced a four-phase response (short relaxation-short contraction-long relaxation-long contraction). BQ788, the ETBR antagonist (1 μM), blocked the S6C-induced relaxation in the aorta, while it only altered the short contractile phase of the VC response. The RVC response to S6C cannot be explained by a different ability of venous and arterial endothelium to produce NO, since ACh (10 μM) induced a similar relaxation of RA and RVC, which was completely blocked by LNNA (100 μM). These data highlight increasingly complex functions of the ETBR in the cardiovascular system.

ADP Ribosyl Cyclase and Ryanodine Receptors Mediate G‐Protein Coupled Receptor‐Induced Renal Vasoconstriction in vivo

Renal vasoconstriction is mediated by an increase in cytosolic calcium concentration ([Ca2+]i). ADP ribosyl cyclase (ADPR cyclase) activation leads to the formation of cyclic ADP ribose (cADPR) or nicotinic acid ADP (NAADP), both of which result in activation of ryanodine receptors (RYR) on the sarcoplasmic reticulum to cause Ca2+ release. We tested the hypothesis that interruption of the ADPR cyclase / RyR pathway inhibits angiotensin II (Ang II)-, norepinephrine (NE)-, and endothelin-1 (ET-1)- induced renal vasoconstriction. Using an ultrasonic flow transducer, we continuously measured renal blood flow in anesthetized Sprague-Dawley rats. Inhibitory agents were infused into the renal artery 2 min before a 10 μl injection of Ang II, NE, ET-1, or the ETB receptor agonist sarafotoxin (S6c). Infusion of ruthenium red, a RyR inhibitor (50 μg/kg/min), reduced Ang II,-, NE-, and S6c- induced renal vasoconstriction by 60 ± 8% and ET-1- induced constriction by 43 ± 12% (n ≥ 5/ group). Nicotinamide, an ADPR cyclase inhibitor (8 mg/kg/min), reduced Ang II-, NE-, and S6c- induced constriction by 75 ± 2% and ET-1- induced constriction by 43 ± 7%. These results show that Ang II, NE, and ET-1 activate ADPR cyclase and RyR signaling to produce renal vasoconstriction. ET-1 activation of this pathway in the renal microcirculation is likely mediated predominantly by ETB receptors. (This work was funded by NIH research grant HL-02334)

NADPH oxidase-derived superoxide augments endothelin-1-induced venoconstriction in mineralocorticoid hypertension.

Deoxycorticosterone acetate (DOCA)-salt hypertension is characterized by low renin/angiotensin but increased arterial superoxide levels. We have recently reported that the arterial endothelin-1 (ET-1) level is increased, resulting in NADPH oxidase activation and superoxide generation. However, the effect of ET-1 on venous superoxide production and its relation to venoconstriction are unknown. The present study tested the hypotheses that ET-1 stimulates venous NADPH oxidase and superoxide via its ET(A) receptors, resulting in enhanced venoconstriction in DOCA-salt hypertensive rats. Treatment with ET-1 (0.01 to 1 nmol/L), but not the selective ET(B) receptor agonist sarafotoxin s6c, of vena cavas of normal rats concentration-dependently increased superoxide levels, an effect that was abolished by the selective ET(A) receptor antagonist ABT-627. Although the ET-1 level was not increased in the vena cava and plasma, both venous NADPH oxidase activity and superoxide levels were significantly higher in DOCA-salt compared with sham rats. Moreover, ET-1 treatment (10(-9) mol/L, 10 minutes) of isolated vena cavas further elevated superoxide levels in DOCA-salt rats only but not sham rats, an effect that was abrogated by the superoxide scavenger tempol. Similarly, ET-1-induced contractions of isolated vena cavas of DOCA-salt but not sham rats were significantly inhibited by tempol. The NADPH oxidase inhibitor apocynin significantly reduced superoxide levels in vena cavas of DOCA-salt rats and in ET-1-treated vena cavas of normal rats. Finally, in vivo ET(A) receptor blockade by ABT-627 significantly lowered venous superoxide levels and blood pressure in DOCA-salt but not sham rats. These results suggest that superoxide contributes to ET-1-induced venoconstriction through an elevated venous NADPH oxidase activity in mineralocorticoid hypertension.

Simultaneous changes in intracellular calcium and tension induced by endothelin-1 and sarafotoxin S6c in guinea pig isolated gallbladder: influence of indomethacin

The relationships between changes in intracellular Ca2+ and smooth muscle tension triggered by endothelin-1 and the selective endothelin ETB receptor agonist sarafotoxin S6c, as well as their susceptibility to modification by the nonselective cyclooxygenase blocker indomethacin, were assessed in guinea pig isolated gallbladder strips. Cumulative additions of either agonist (1, 10, and 100 nM) induced simultaneous graded, strongly correlated, slowly developing, and sustained changes in tension and intracellular Ca+2 (Fura-2 technique). Sarafotoxin S6c was more effective than endothelin-1 in raising intracellular Ca2+ at 1 or 10 nM, but their abilities to cause contractions were similar at all concentrations. Indomethacin (5.6 microM) markedly inhibited the changes in both intracellular Ca2+ and tension caused by all concentrations of sarafotoxin S6c (in response to 100 nM, increases in Ca2+ fluorescence intensity and tension were inhib ited from 7.7 +/- 0.7 to 4.0 +/- 0.4% and from 460 +/- 100 to 160 +/- 40 mg, respectively) but only reduced the contraction triggered by 100 nM endothelin-1 (from 560 +/- 100 to 230 +/- 70 mg). Endothelin-1 caused greater prostacyclin release from gallbladder than sarafotoxin S6c (at 100 nM, 6-keto-PGF1alpha levels in the medium rose 4.8- and 2.8-fold, respectively; P < 0.05) and slightly increased thromboxane A2 release (1.6-fold; P < 0.05). Thus, gallbladder contractions triggered by combined ETA/ETB or selective ETB receptor stimulation (with endothelin-1 or sarafotoxin S6c, respectively) are strongly correlated with increases in intracellular Ca2+ but differentially affected by indomethacin. It remains to be assessed if this difference is because endothelin-1 triggers greater prostacyclin release than sarafotoxin S6c and (or) is due to the coupling of ETA and ETB receptors to distinct patterns of generation of cyclooxygenase-derived eicosanoids.

Functional Cross-talk of Ca2+-Mobilizing Endothelin Receptors in C6 Glioma Cells

There are conflicting results concerning the receptor subtype(s) involved in calcium-mediated endothelin signaling in the glial cells. In order to elucidate the role of endothelin A and B receptors in these processes, we have studied the effect of a complex spectrum of endothelin receptor ligands on intracellular calcium concentration changes in proliferating and differentiated C6 rat glioma cells. Cell differentiation was induced by dibutyryl-cAMP and assessed by the glial fibrillar acidic protein content. Intracellular calcium changes were measured in cell suspensions using fluorescent probe Fura-2. The specific endothelin B receptor agonists sarafotoxin S6c and IRL-1620 did not influence the intracellular calcium concentration. However, calcium changes induced by endothelin-1 and especially by endothelin-3 after the pretreatment of cells with one of these endothelin B receptor specific agonists were significantly enhanced even above the values attained by the highest effective endothelin concentrations alone. Such endothelin B-receptor ligand-induced sensitization of calcium signaling was not observed in differentiated C6 cells. Moreover, endothelin-induced calcium oscillations in differentiated C6 cells were less inhibited by BQ-123 and BQ-788 than in their proliferating counterparts. In conclusion, the specific activation of endothelin B receptor in C6 rat glioma cells does not affect intracellular calcium per se, but probably does so through interaction with the endothelin A receptor. The pattern and/or functional parameters of endothelin receptors in C6 rat glioma cells are modified by cell differentiation.

Cytokine enhancement of endothelin ET B receptor-mediated contraction in human temporal artery

Segments of human temporal artery were incubated in organ culture for 2 days in the absence or presence of cytokines. Thereafter, contractions were induced by the selective endothelin ET B receptor agonist sarafotoxin S6c, a peptide that does not induce contraction in fresh human temporal artery. Interleukin-1β was most potent in increasing the sarafotoxin-induced contraction in cultured segments. Tumour necrosis factor (TNF)-α increased the magnitude of contraction to a similar degree, but at a higher dose. A significant increase was also induced by interferon-γ, but not by interleukin-6 at the concentrations used. The results suggest that endothelin ET B receptor-mediated contraction can be enhanced by pro-inflammatory cytokines in a concentration-dependent manner, and this may have relevance for pathophysiological conditions where inflammation and vasoactivity are important.

Endothelin-1-induced ET(A) receptor-mediated nociception, hyperalgesia and oedema in the mouse hind-paw: modulation by simultaneous ET(B) receptor activation.

Endothelin-1 causes ET(A) receptor-mediated enhancement of capsaicin-induced nociception in mice. We have assessed if this hyperalgesic effect of endothelin-1 is also accompanied by other pro-inflammatory effects, namely nociception and oedema, and characterized the endothelin ET receptors involved. Intraplantar (i. pl.) hind-paw injection of endothelin-1 (0.3 - 30 pmol) induced graded nociceptive responses (accumulated licking time: vehicle, 20. 5+/-3.3 s; endothelin-1 at 30 pmol, 78.1+/-9.8 s), largely confined to the first 15 min. Endothelin-1 (1 - 10 pmol) potentiated ipsilateral capsaicin-induced (0.1 microgram, i.pl.; at 30 min) nociception (vehicle, 40.2+/-2.6 s; endothelin-1 at 10 pmol, 98.4+/-5.8 s, but 30 pmol was inactive), and caused oedema (increase in paw weight 5 min after capsaicin: vehicle, 46.3+/-2.3 mg; endothelin-1 at 30 pmol, 100.3+/-6.1 mg). Selective ET(B) receptor agonists sarafotoxin S6c (up to 30 pmol) and IRL 1620 (up to 100 pmol) were inactive, whereas endothelin-3 (up to 30 pmol) induced only modest oedema. ET(A) receptor antagonists BQ-123 (1 nmol, i.pl. ) or A-127722-5 (6 micromol kg(-1), i.v.) prevented all effects of endothelin-1 (10 pmol), but the ET(B) receptor antagonist BQ-788 (1 or 10 nmol, i.pl.) was ineffective. BQ-788 (10 nmol, i.pl.) unveiled hyperalgesic effects of 30 pmol endothelin-1 and endothelin-3. Sarafotoxin S6c (30 pmol, i.pl.) did not modify endothelin-1-induced (10 pmol) nociception or oedema, but abolished hyperalgesia. Thus, endothelin-1 triggers ET(A) receptor-mediated nociception, hyperalgesia and oedema in the mouse hind-paw. Simultaneous activation of ET(B) receptors by endothelin-1 or selective agonists can limit the hyperalgesic, but not the nociceptive or oedematogenic, effects of the peptide.

Characterization and distribution of endothelin receptors in the pulmonary circulation: investigation of isolated, perfused, and ventilated rabbit lungs.

The aim of the study was to investigate the distribution of 2 subtypes of endothelin-receptors, mediating the effects of endothelin-1 (ET-1) in the pulmonary circulation. Until now, it is still unclear, whether ET(A) receptors or ET(B) receptors or even both are localized in pulmonary vessels. The experiments were performed on 72 isolated and ventilated rabbit lungs that were perfused with a cell- and plasma-free buffer solution. The arterial pressure and the lung weight gain were continuously registered. Intermittently perfusate samples were taken for determination of thromboxane A2 (TXA2) and prostacyclin (PGI2). The injection of ET-1 (10(-8) M, n = 6) resulted in a biphasic increase in pulmonary arterial pressure (PAP) that was accompanied by the generation of TXA2 and PGI2. Pretreatment with the ET(A)-receptor antagonist LU135252 (10(-6) M, n = 6) suppressed the pressure response after ET-1 application (P < 0.01 at 120 min) and reduced the generation of TXA2 (P < 0.05 at 120 min) and PGI2 (P < 0.05 at 120 min). Pretreatment with the cyclooxygenase inhibitor diclofenac (10 microg/mL; n = 6) also reduced the PAP increase after ET-1 injection. In contrast to this, the pulmonary vascular pressure reaction after ET-1 application was elevated, when ET(B)-receptor antagonist BQ788 (10(-6) M; n = 6) was given. Furthermore, the PGI2 to TXA2 ratio was shifted from 2.3 to 0.9, reflecting a predominance of vasoconstrictive TXA2. The simultaneous application of LU135252 and BQ788 significantly reduced the PAP increase after ET-1 application, but no beneficial effects were observed compared with the application of LU135252 solely. The injection of the ET(B)-receptor agonist sarafotoxin S6c (S6c; 10(-8) M, n = 6) also induced an increase in PAP that was not attenuated by pretreatment with the ET(B)-receptor antagonist BQ788 (10(-6) M, n = 6). LU135252 (n = 6) as well as the application of LU135252 in combination with BQ788 (n = 6) failed to suppress the pressure response after S6c, whereas the cyclooxygenase inhibitor diclofenac (10 microg/mL, n = 6) alone and in combination with LU135252 and BQ788 (n = 6) was able to prevent the PAP increase after S6c injection (P < 0.001). The results demonstrate that the ET-1-induced increase in pulmonary vascular resistance is mainly mediated via ET(A) receptors, whereas ET(B) receptors seem to mediate vasodilation, which was shown by an imbalance of TXA2 and PGI2 generation. On the other hand, the ET(B)-receptor agonist S6c induced vasoconstriction, which was only attenuated by the cyclooxygenase inhibitor diclofenac. From the current results we conclude that, apart from vasoconstrictor ET(A) receptors, at least 2 ET(B)-receptor subtypes are expressed in the pulmonary circulation, one mediating vasoconstriction, which was not blocked by BQ788, and one mediating vasodilation, which was influenced by BQ788.

ET(A) receptors are the primary mediators of myofilament calcium sensitization induced by ET-1 in rat pulmonary artery smooth muscle: a tyrosine kinase independent pathway.

We have investigated the possibility that ET-1 can induce an increase in myofilament calcium sensitivity in pulmonary artery smooth muscle. Arterial rings were permeabilized using alpha-toxin (120 microg ml(-1)), in the presence of A23187 (10 microM) to 'knock out' Ca2+ stores, and pre-constricted with pCa 6.8 (buffered with 10 mM EGTA). In the presence of this fixed Ca2+ concentration, 1 microM ET-1 induced a sustained, reversible constriction of 0.15 mN. Pulmonary arterial rings were freeze-clamped at the peak of the induced constriction (time matched). Subsequent densitometric analysis revealed that ET-1 (1 microM) increased the level of phosphorylated myosin light chains by 34% compared to an 11% increase in the presence of pCa 6.8 alone. In contrast to ET-1, the selective ET(B) receptor agonist Sarafotoxin S6C (100 nM) failed to induce a significant constriction. The constriction induced by 1 microM ET-1 was reversibly inhibited when the preparation was preincubated (15 min) with the ETA receptor antagonist BQ 123 (100 microM). The constriction measured 0.13 mN in the absence and 0.07 mN in the presence of 100 microM BQ 123. In contrast, the constriction induced by 1 microM ET-1 measured 0.19 mN in the absence and 0.175 mN following a 15 min pre-incubation with the ET(B) antagonist BQ 788 (100 microM). The constriction induced by 1 microM ET-1 measured 0.14 mN in the presence and 0.13 mN following pre-incubation with the tyrosine kinase inhibitor Tyrphostin A23 (100 microM). We conclude that ET-1 induced an increase in myofilament calcium sensitivity in rat pulmonary arteries via the activation of ET(A) receptors and by a mechanism(s) independent of tyrosine kinase.

Activation of particulate guanylyl cyclase by endothelins in cultured SV-40 transformed cat iris sphincter smooth muscle cells

We investigated the effects of endothelins (ETs) on cGMP production in cultured SV-40 transformed cat iris sphincter smooth muscle (SV-CISM-2) cells. ET-3 increased cGMP formation in a concentration-dependent manner (EC 50 = 98nM), which was 2.5 times higher than that of ET-1. The ET B receptor agonists sarafotoxin-S6c and IRL 1620 also increased cGMP production, mimicking the effects of the ETs. The ET B receptor antagonist BQ 788, but not the ET A receptor antagonist BQ610, dose-dependently blocked ET-3-stimulated cGMP formation (IC 50 = 10nM). The phorbol ester, Phorbol 12, 13 — dibutyrate (PDBu), which inhibits particulate guanylyl cyclase in smooth muscle, dose-dependently inhibited ET-3-stimulated cGMP accumulation (IC 50 = 66nM). LY83583 and ODQ, inhibitors of soluble guanylyl cyclases, as well as inhibitors of the nitric oxide cascade and of intracellular Ca 2+ elevation had no appreciable effect on ET-3-induced cGMP production. ET-3 markedly inhibited carbachol-induced intracellular Ca 2+ mobilization. We conclude that ET-3 increases intracellular cGMP levels in SV-CISM-2 cells through activation of the ET B receptor subtype and subsequent stimulation of the membrane-bound guanylyl cyclase. Elevation of cGMP by ET and the subsequent inhibition of muscarinic stimulation of intracellular Ca 2+ mobilization by the cyclic nucleotide could serve to modulate the contractile effects of Ca 2+-mobilizing agonists in the iris sphincter smooth muscle.

Articular nociception induced by endothelin-1, carrageenan and LPS in naive and previously inflamed knee-joints in the rat: inhibition by endothelin receptor antagonists

Endothelin-1, unlike the selective endothelin ET B receptor agonist sarafotoxin S6c, causes nociception in the rat when injected intra-articularly into the naive knee-joint. By using selective antagonists, the present study further characterizes the receptors underlying the articular nociceptive actions of endothelin-1, as well as the possible contribution of endogenous endothelins towards nociception induced by carrageenan or E. coli lipopolysaccharide (LPS) in this tissue. Nociception was evaluated by placing the animal for 1 min each hour on a revolving (3 rpm) cylinder and measuring the increase in time the hindpaw of the limb affected by the intra-articular (i.a.) injection of the nociceptive agent, failed to touch its metallic surface (i.e. paw elevation time, PET). In naive joints, endothelin-1 (120 pmol) increased the area under the PET curve (AUC 0–6 h, in arbitrary units) from 61±3 (control) to 156±12. This nociceptive effect was reduced by prior intravenous (i.v.) injection of the mixed ET A/ET B receptor antagonist bosentan (by 54 and 73% with 10 and 30 mg/kg) or i.a. administration of the selective ET A receptor antagonist BQ-123 (cyclo [D-Asp-Pro-D-Val-Leu]; by ≅45% with 10 or 30 nmol), but was unaffected by the selective ET B receptor antagonist BQ-788 (N- cis-2,6-dimethyl-piperidinocarbonyl- l- γ-methyl-leucyl- d-1-methoxycarbonyl-tryptophanil-D-norleucine; 10 nmol). Prior joint challenge with carrageenan (300 μg) 72 h beforehand (i.e. priming) rendered the joint more sensitive to nociception induced by either endothelin-1 or sarafotoxin S6c (15, 30 and 60 pmol). Responses elicited by endothelin (30 pmol) in the primed joint were sensitive to inhibition by either BQ-123 or BQ-788 (each causing ≅80% inhibition at 10 nmol). Priming also enhanced PET responses to carrageenan itself and to LPS (1 μg) markedly and persistently, increasing the area under the curve (AUC 0–12 h, in arbitrary units) from 241±19 to 409±50 and from 312±40 to 466±25, respectively ( P<0.05), without changing that measured following vehicle injection (from 121±3 to 117±4). Bosentan (up to 30 mg/kg, i.v.) failed to modify nociception caused by carrageenan or LPS in naive joints, by carrageenan in the primed joint, or control PET responses. LPS-induced nociception in the primed joint, however, was inhibited by 52 to 56% by bosentan (3 or 10 mg/kg) or 59% by local injection of the selective endothelin ET B receptor antagonist BQ-788 (10 nmol, i.a.), but was unaffected by the selective endothelin ET A receptor antagonist BQ-123. Thus, nociception induced by endothelin-1 in the naive joint is mediated largely via endothelin ET A receptors, whereas both ET A and ET B receptors contribute to its action in the carrageenan-primed joint. Furthermore, LPS-induced nociception in the primed joint is mediated to a large extent via endothelin release and activation of ET B receptors within the joint itself. These findings may be relevant to the etiology of pain underlying chronic arthritic disease in humans.

Effects of endothelin-1 on capsaicin-induced nociception in mice

The influence of endothelin-1 on nociception induced by capsaicin was assessed in the mouse hindpaw. Local endothelin-1 injection (1 to 20 pmol/paw) 30 min prior to ipsilateral injection of capsaicin (0.1 μg/paw) increased, in a graded fashion, the time spent licking the injected paw. Maximal hyperalgesia was obtained with 10 pmol/paw of endothelin-1 (capsaicin-induced hindpaw licking time increased from 43±3 s to 114±7 s, n=6), but no hyperalgesia was evident following 30 pmol/paw of endothelin-1. The selective endothelin ET B receptor agonists sarafotoxin S6c (≤30 pmol/paw) and IRL 1620 (i.e., Suc[Glu 9,Ala 11,15]endothelin-1-(10–21); ≤100 pmol/paw) failed to induce hyperalgesia. Local treatment with BQ-123 (i.e., cyclo[ dTrp- dAsp-Pro- dVal-Leu] 1 nmol/paw selective endothelin ET A receptor antagonist), 10 min before endothelin-1 (10 pmol/paw), fully blocked the hyperalgesic response, whereas similar treatment with the selective endothelin ET B receptor antagonist BQ-788 (i.e., N- cis-2,6-dimethylpiperidinocarbonyl- l- γ-methylleucyl- d-1-methoxycarboyl- d-norleucine) was ineffective. Intravenous injection of bosentan (17 and 52 μmol/kg a non-peptidic mixed endothelin ET A/ET B receptor antagonist) or BMS 182874 (i.e., 5-[dimethylamino]- N-[3,4-dimethyl-5-isoxazolyl]-1-naphthalenesulphonamide; 10 and 30 μmol/kg; a non-peptidic selective endothelin ET A receptor antagonist), 1 h before endothelin-1, inhibited its hyperalgesic effect in a graded fashion and abolished the response at the higher doses. None of the antagonists modified nociception induced by capsaicin alone or the hyperalgesia induced by local injection of 5-hydroxytryptamine (5-HT; 2 nmol/paw, 30 min before capsaicin). Hyperalgesia induced by 5-HT was abolished by simultaneous injection of endothelin-1 or the endothelin ET B receptor agonist IRL 1620 (each at 30 pmol/paw). Therefore, local endothelin-1 exerts a dual influence in this model: at low doses it causes endothelin ET A receptor-mediated hyperalgesia (i.e., it potentiates capsaicin-induced nociception), whereas at higher doses it induces an anti-hyperalgesic effect against 5-HT which seems to be mediated via distinct endothelin ET (possibly ET B) receptors.

Endothelin receptor subtypes and their functional relevance in human small coronary arteries.

1. The potent constrictor peptide endothelin (ET) has been implicated in various cardiovascular disorders including myocardial infarction and atherosclerosis. We have investigated the nature of ET receptor subtypes present on human small coronary arteries. 2. Small coronary arteries were mounted in a wire-myograph for in vitro pharmacology. To investigate the ET receptor subtypes present in different segments of the coronary vascular tree, arteries were grouped according to internal diameter. Responses in arteries with small internal diameters (mean 316.7+/-7.9 microm; Group B) were compared to those in larger arteries (mean 586.2+/-23.1 microm; Group A). 3. ET-1 consistently and potently contracted arteries from Group A and B, with EC50 values of 1.7 (0.9-3.2) nM (n=15) and 2.3 (1.4-4.2) nM (n=14), respectively. No correlation was observed between ET-1 potency and internal diameter. The response to ET-1 was potently antagonized by the selective ET(A) receptor antagonist PD156707 in both Group A and Group B, yielding pA2 values of 8.60+/-0.12 (n=4-6) and 8.38+/-0.17 (n=4-6), respectively. Slopes from Schild regression were not significantly different from unity. 4. In contrast to ET-1, individual responses to ET-3 were variable. While all arteries from Group A responded to ET-3 (EC50 approximately 69 (23-210) nM) (n=12), no response was obtained in 5 of the 14 tested in Group B. Of those responding, many failed to reach a maximum at concentrations up to 1 microM. ET-1 was more potent than ET-3 in all arteries tested. A biphasic ET-3 response was observed in 8 arteries suggesting that a small ET(B) population was also present in some patients. The selective ET(B) receptor agonist sarafotoxin S6c had little or no effect up to 10 nM (n=4-6). 5. Responses to ET-1 and ET-3 were unaffected by removal of the endothelium in arteries from both groups suggesting a lack of functional, relaxant ET(B) receptors on endothelial cells (n=5). 6. Using autoradiography, specific high density binding of the non-selective, ET(A)/ET(B) ligand [125I]-ET-1 and selective ET(A) ligand [125I]-PD151242 was detected on the vascular smooth muscle layer of small intramyocardial coronary arteries (n=5). In contrast, little or no binding of the selective ET(B) receptor ligand [125I]-BQ3020 was observed (n=5). Similarly, [125I]-ET-1 binding to vascular smooth muscle was absent in the presence of the selective ET(A) receptor antagonist PD156707. 7. We conclude that human small epi- and intramyocardial coronary arteries express predominantly ET(A) receptors and it is these receptors which mediate ET-induced contractions. A constrictor ET(B) receptor population may exist in some patients. However, these receptors may have a limited role as contractions to ET-1 can be blocked fully by the selective ET(A) receptor antagonist PD156707.

Influence of applied tension and nitric oxide on responses to endothelins in rat pulmonary resistance arteries: effect of chronic hypoxia.

1. The effect of basal tension (transmural tensions 235 +/- 29 mg wt (low tension: equivalent to approximately 16 mmHg) and 305 +/- 34 mg wt (high tension: equivalent to 35 mmHg)) on rat pulmonary resistance artery responses to endothelin-1 (ET-1) and the selective ET(B)-receptor agonist sarafotoxin S6c (S6c) were studied. The effects of nitric oxide synthase inhibition with N(omega)-nitro-L-arginine methylester (L-NAME, 100 microM) on ET receptor-induced responses, as well as vasodilator responses to acetylcholine (ACh) and S6c, were also investigated. Changes with development of pulmonary hypertension, induced by two weeks of chronic hypoxia, were determined. 2. Control rat preparations showed greatest sensitivity for ET-1 when put under low tension (pEC50: 8.1 +/- 0.1) compared with at the higher tension (pEC50: 7.7 +/- 0.1) and there were significant increases in maximum contractile responses to S6c (approximately 80%) and noradrenaline (approximately 60%) when put under high tension. 3. In control pulmonary resistance arteries, both ET-1 and S6c produced potent vasoconstrictor responses. S6c was 12 fold more potent than ET-1 in vessels set at low tension (S6c pEC50: 9.2 +/- 0.1) and 200 fold more potent than ET-1 when the vessels were set at high tension (S6c pEC50: 9.0 +/- 0.1). Chronic hypoxia did not change the potencies of ET-1 and S6c but did significantly increase the maximum contractile response to ET-1 by 60% (at low tension) and 130% (at high tension). 4. In control rat vessels, L-NAME itself caused small increases in vascular tone (5-8 mg wt tension) in 33-56% of vessels. In the chronic hypoxic rats, in vessels set at high tension, L-NAME-induced tone was evident in 88% of vessels and had increased to 26.9 +/- 6.6 mg wt tension. Vasodilatation to sodium nitroprusside, in non-preconstricted vessels, was small in control rat vessels (2-6 mg wt tension) but increased significantly to 22.5 +/- 8.0 mg wt tension in chronic hypoxic vessels set at the higher tensions. Together, these results indicate an increase in endogenous tone in the vessels from the chronic hypoxic rats which is normally attenuated by nitric oxide production. 5. L-NAME increased the sensitivity to S6c 10 fold (low tension) and 6 fold (high tension) only in chronic hypoxic rat pulmonary resistance arteries. It had no effect on responses to ET-1 in any vessel studied. 6. Vasodilatation of pre-contracted vessels by ACh was markedly greater in the pulmonary resistance arteries from the chronic hypoxic rats (pIC50: 7.12 +/- 0.19, maximum: 72.1 +/- 0.2.0%) compared to their age-matched controls (pIC50: 5.77 +/- 0.15, maximum: 28.2 +/- 2.0%). There was also a 2.5 fold increase in maximum vasodilatation induced by ACh. 7. These results demonstrate that control rat preparations showed greatest sensitivity for ET-1 when set at the lower tension, equivalent to the pressure expected in vivo (approximately 16 mmHg). Pulmonary hypertension due to chronic hypoxia potentiated the maximum response to ET-1. Pulmonary resistance arteries from control animals exhibited little endogenous tone, but exposure to chronic hypoxia increased endogenous inherent tone which is normally attenuated by nitric oxide. Endogenous nitric oxide production may increase in pulmonary resistance arteries from chronic hypoxic rats and attenuate contractile responses to ET(B2) receptor stimulation. Relaxation to ACh was increased in pulmonary resistance arteries from chronic hypoxic rats.

Endothelins potentiate formalin-induced nociception and paw edema in mice

The present study investigates the influence of endothelin (ET) related peptides (0.3-30 pmol/paw) on both phases of nociception and on edema induced by intraplantar injection of formalin (0.5% in 20 microL) in the mouse hind paw. The first phase of nociception (0-5 min after injection) was significantly potentiated by simultaneous injection of either ET-1 (10 or 30 pmol/paw) or ET-3 (10 pmol/paw), but not of the selective ET3 receptor agonist sarafotoxin S6c (SRTX-c; up to 30 pmol/paw). All three peptides potentiated the second phase (10-30 min after injection) of formalin-induced nociception (at 3-30, 1-30, and 10-30 pmol/paw for ET-1, ET-3, and SRTX-c, respectively), whereas only ET-1 (10 or 30 pmol/paw) effectively enhanced edema caused by formalin (30 min after injection). Histamine also potentiated all three responses triggered by formalin, but was 30- to 100-fold less potent than ET-1. Treatment with the mixed ETA/ETB receptor antagonist bosentan (10 mg/kg i.p., 1 h beforehand) did not influence nociceptive and edematogenic responses to formalin or their potentiation by histamine (3 nmol/paw), but did inhibit the potentiations induced by ET-1 (10 pmol/paw). Thus, ET-1 potentiates formalin-induced nociception and edema in the mouse. These actions are possibly mediated via ETB and ETA receptors, respectively, but their true identity and the mechanisms involved still remain to be fully elucidated.