Pharmacological characterization of endothelin-induced rat pulmonary arterial dilatation

1. The aim of study was to characterize endothelin (ET)-induced vasodilatation in isolated extrapulmonary rat arteries (EPA) and in intrapulmonary arteries (IPA) preconstricted with 1 μM phenylephrine.
2. The ET-3 (1 nM–100 nM)- and ET-1 (10 nM–100 nM)-induced transient vasodilatations in EPA were more potent than those in IPA. The vasodilatation induced by ET-3 (100 nM) was larger than that induced by ET-1 (100 nM).
3. Both the ET_B antagonist, BQ788 (3 μM) and or endothelium denudation, but not the ET_A antagonist, BQ123 (3 μM), abolished the vasodilatation induced by ET-1 or ET-3 (100 nM each) in EPA and in IPA. The ATP-sensitive K+channel blocker, glibenclamide (20 μM) and the nitric oxide synthase inhibitor, N^G-monomethyl-L-arginine (L-NMMA, 1 mM) suppressed the ET-induced vasodilatation in EPA and in IPA.
4. We conclude that the vasodilatation induced by endothelins is markedly reduced in rat isolated IPA, and suggest that the endothelial ET_B-mediated vasodilatation varies depending on rat pulmonary arterial regions. Furthermore, ET_B-mediated vasodilatation involves activation of ATP-sensitive K⁺ channels and of nitric oxide synthase in rat isolated EPA and IPA.

     

Activation of endothelin ETA receptors masks the constrictor role of endothelin ETB receptors in rat isolated small mesenteric arteries

1. Endothelin-1 (ET-1) produces constriction of the rat mesenteric vascular bed in vivo via ET_A and ET_B receptor subtypes. The aim of this study was to investigate the relative roles of these receptor subtypes in rat isolated, endothelium-denuded, small mesenteric arteries, under pressure, by use of ET-1; the ET_A receptor antagonist, BQ-123; the ET_B receptor selective agonist, sarafotoxin S6c (SRTX S6c); the ET_B receptor selective antagonist, BQ-788; and the ET_A/ET_B antagonist, TAK-044.
2. In 3rd generation mesenteric arteries, ET-1 (10⁻¹³10⁻⁷ M) produced concentration-dependent contractions (pD₂ 9.86). SRTX S6c (10⁻¹²10⁻⁷ M) also induced concentration-dependent contractions in 53% of arteries studied, although the E_max was much less than that obtained with ET-1 (10.7±2.9% vs 101.9±2.6% of the 60 mM KCl-induced contraction).
3. Neither ET_B receptor desensitization, by a supra-maximal concentration of SRTX S6c (10⁻⁷ M), nor incubation with BQ-788 (3×10⁻⁸ M), had any significant effect on the ET-1 concentration-response curve, although both treatments tended to enhance rather than inhibit responses to ET-1.
4. In the presence of BQ-123 (10⁻⁶ M), responses to low concentrations of ET-1 (up to 10⁻¹⁰ M) were unaffected but responses to concentrations of ET-1 above 10⁻¹⁰ M were significantly inhibited.
5. SRTX S6c desensitization followed by incubation with BQ-123 (10⁻⁶ M) or co-incubation with BQ-788 (3×10⁻⁸ M) and BQ-123 caused inhibition of responses to all concentrations of ET-1, resulting in a rightward shift of the ET-1 concentration-response curve. The same effect was obtained by incubation with TAK-044 (10⁻⁸ M and 3×10⁻⁷ M).
6. Thus, responses of rat small mesenteric arteries to ET-1 are mediated by both ET_A and ET_B receptors. The relative role of ET_B receptors is greater than that predicted by the small responses to SRTX S6c or by resistance of ET-1-induced contraction to ET_B receptor desensitization or BQ-788. The effect of ET_B receptor desensitization or blockade is only revealed in the presence of ET_A receptor blockade, suggesting the presence of a ‘crosstalk'' mechanism between the receptors. These results support the concept that dual receptor antagonists, like TAK-044, may be required to inhibit completely constrictor responses to ET-1.

     

Involvement of ETA and ETB receptors in the activation of phospholipase D by endothelins in cultured rat cortical astrocytes

1. This study was performed to characterize the receptor subtypes involved in the endothelin stimulation of phospholipase D (PLD) in rat cortical astrocytes in primary culture. PLD activity was determined by measuring the formation of [³²P]phosphatidylbutanol in [³²P]orthophosphate prelabelled cells stimulated in the presence of 25 mM butanol.
2. The agonists endothelin-1 (ET-1), endothelin-3 (ET-3), sarafotoxin 6c (S6c) and IRL 1620 elicited PLD activation in a concentration-dependent manner. The potencies of ET-1, ET-3 and S6c were similar. The maximal effects evoked by the ET_B-preferring agonists, ET-3, S6c and IRL 1620, were significantly lower than the maximal response to the non-selective agonist ET-1.
3. The response to 1 nM ET-1 was inhibited by increasing concentrations of the ET_A receptor antagonist BQ-123 in a biphasic manner. A high potency component of the inhibition curve (24.2±3.5% of the ET-1 response) was defined at low (up to 1 μM) concentrations of BQ-123, yielding an estimated K_i value for BQ-123 of 21.3±2.5 nM. In addition, the presence of 1 μM BQ-123 significantly reduced the maximal response to ET-1 but did not change the pD₂ value.
4. Increasing concentrations of the ET_B selective antagonist BQ-788 inhibited the S6c response with a K_i of 17.8±0.8 nM. BQ-788 also inhibited the effect of ET-1, although, in this case, two components were defined, accounting for approximately 50% of the response, and showing K_i values of 20.9±5.1 nM and 439±110 nM, respectively. The ET-1 concentration-response curve was shifted to the right by 1 μM BQ-788, also revealing two components. Only one of them, corresponding to 69.8±4.4% of the response, was sensitive to BQ-788 which showed a K_i value of 28.8±8.9 nM.
5. Rapid desensitization was achieved by preincubation with ET-1 or S6c. In cells pretreated with S6c neither ET-3 nor S6c activated PLD, but ET-1 still induced approximately 40% of the response shown by non-desensitised cells. This remaining response was insensitive to BQ-788, but fully inhibited by BQ-123.
6. In conclusion, endothelins activate PLD in rat cortical astrocytes acting through both ET_A and ET_B receptors, and this response desensitizes rapidly in an apparently homologous fashion. The percentage contribution of ET_A and ET_B receptors to the ET-1 response was found to be approximately 20% and 80%, respectively, when ET_B receptors were not blocked, and 30–50% and 50–70%, respectively, when ET_B receptors were inhibited or desensitized. These results may be relevant to the study of a possible role of PLD in the proliferative effects shown by endothelins on cultured and reactive astrocytes.

     

Desensitization of ETA endothelin receptor-mediated negative chronotropic response in right atria–species difference and intracellular mechanisms

1. Desensitization of ET_A endothelin receptor (ET_AR) was compared between the rat and guinea-pig with regard to negative chronotropic response (NC) in the right atria (RA).
2. ET-1 (100 nM) produced distinct NC in the presence of BQ788 (300 nM), and positive chronotropic response (PC) in the presence of BQ123 (1 μM) in both species, showing that ET_AR and ET_B endothelin receptor (ET_BR) mediate NC and PC, respectively.
3. Repetitive applications of ET-1 (50 nM) desensitized PC, and the second application only induced a strong NC in both species. Later applications of ET-1 produced virtually no response in the rat RA, whereas they produced BQ123-sensitive NCs repetitively in guinea-pig RA, exhibiting marked species difference in desensitization of ET_AR-mediated NC.
4. Pretreatment with staurosporine (100 nM) prevented desensitization of ET_AR in the rat RA altogether. However, phorbol 12-myristate 13-acetate (PMA, 300 nM) failed to induce, but rather hampered, desensitization of ET_AR.
5. Partial amino acid sequencing of ET_ARs, spanning from the 2nd through the 4th intracellular loops, revealed that all the potential Ser/Thr phosphorylation sites, including a protein kinase C (PKC) site, are conserved among guinea-pigs, rats, rabbits, bovines and humans.
6. In guinea pig RA, pretreatment with okadaic acid (1 μg ml⁻¹) and PMA did not facilitate desensitization of ET_AR whereas these agents successfully desensitized ET_AR during combined stimulation of β-adrenoceptor and ET_AR by isoproterenol (300 nM) and ET-1 (100 nM).
7. These results suggest that species differences in desensitization of ET_AR are not caused by differences in the site(s) of, but caused by differences in the environment for phosphorylation of the receptor. Desensitization of ET_AR appears to require phosphorylation of the receptor by PKC as well as a kinase stimulated by β-adrenoceptor activation.

     

Influence of ETR-p1/f1 antisense peptide on endothelin-induced constriction in rat renal arcuate arteries

1. This study set out to examine the endothelin receptor subtypes mediating vasoconstriction in the rat renal arcuate artery. This was done in isolated vessels 120–200 μm in diameter, incubated with a selective agonist and the novel ‘antisense'' peptide to part of the human endothelin_A receptor.
2. Groups of vessels (n=6) were incubated with increasing concentrations of endothelin-1 (ET-1), from 1 to 100 nM, which caused a 65% maximal contraction at the highest dose with an pEC₅₀ of 8.16±0.11 M. By contrast, in six other vessels sarafotoxin 6c over the same dose range gave a minimal contraction (around 5% of maximum).
3. Preincubation of six vessels with the antisense peptide ETR p₁/f1 at 1 μM had no effect on the ET-1 induced vasoconstriction, in terms of displacement of the concentration-response curve or the maximal tension achieved by the agonist. In the six vessels exposed to 4 μM ETR p₁/f1, there was a significant shift of the concentration-response curve and a lower pEC₅₀ at 7.78±0.09 M (P<0.05). At the highest concentrations of ETR p₁/f1, there was a marked suppression of all responses to ET-1, which at the maximal concentrations tested, 0.1 μM, only reached some 10% of the maximal achievable contraction.
4. Increasing ET-1 concentrations up to 2 μM in vessels incubated with 40 μM ETR-p₁/f1 showed that the blockade could be overcome and that the relationship was shifted to the right (P<0.001) by approximately one log unit with a pEC₅₀ of 7.13±0.11 M. A Schild plot of the data indicated the antagonist to be acting competitively at a single population of receptors.
5. At the highest concentrations tested, 40 μM, ETR-p₁/f1 had no effect on noradrenaline-induced contractions, indicating a lack of non-specific actions.
6. Together, these data suggest that at the rat renal arcuate artery the endothelin_A receptor is the predominant functional receptor mediating contraction. Furthermore, this study has shown the potential usefulness of this novel type of ‘antisense'' peptide in blocking receptor activation.

     

Pharmacological characterization of endothelin-induced contraction in the guinea-pig oesophageal muscularis mucosae

1. In the oesophageal muscularis mucosae, we examined the effects of endothelin-1 (ET-1), endothelin-2 (ET-2), endothelin-3 (ET-3) and sarafotoxin S6c (SX6c) as agonists, and {"type":"entrez-nucleotide","attrs":{"text":"FR139317","term_id":"258103156","term_text":"FR139317"}}FR139317, BQ-123 and RES-701-1 as endothelin receptor antagonists.
2. All of the endothelins produced tonic contractions which were frequently superimposed on rhythmic motility in a concentration-dependent manner. The order of potency (−log EC₅₀) was ET-1 (8.61)=SX6c (8.65)>ET-2 (8.40)>ET-3 (8.18).
3. {"type":"entrez-nucleotide","attrs":{"text":"FR139317","term_id":"258103156","term_text":"FR139317"}}FR139317 (1–3 μM) and BQ-123 (1 μM) caused parallel rightward shifts of the concentration-response curve to ET-1, but at higher concentrations caused no further shift. RES-701-1 (3 μM) caused a rightward shift of the concentration-response curve to ET-1, while RES-701-1 (10 μM) had no additional effect. RES-701-1 (0.1–1 μM) concentration-dependently caused a rightward shift of the concentration-response curve to SX6c. The contraction to ET-1 (10 nM) in preparations desensitized to the actions of SX6c was greatly inhibited by pretreatment with {"type":"entrez-nucleotide","attrs":{"text":"FR139317","term_id":"258103156","term_text":"FR139317"}}FR139317 (10 μM).
4. Modulation of the Ca²⁺ concentration in the Krebs solution caused the concentration-response curve to ET-1 or SX6c to shift to the right and downward as external Ca²⁺ concentrations decreased. Verapamil (30 μM) abolished rhythmic motility induced by ET-1 or SX6c. Ni²⁺ (0.1 mM) weakly inhibited ET-1- or SX6c-induced tonic contraction. SK&F 96365 (60 μM) completely inhibited ET-1-induced contractions.
5. We conclude that there are two types of ET-receptors, excitatory ET_A- and ET_B-receptors in the oesophageal muscularis mucosae. These receptors mediate tonic contractions predominantly by opening receptor-operated Ca²⁺ channels (ROCs) and partly by opening T-type Ca²⁺ channels, and mediate rhythmic motility by opening L-type Ca²⁺ channels.

     

Development of endothelin receptors in perinatal rabbit pulmonary resistance arteries

1. Contractile responses to endothelin-1 (ET-1) and sarafotoxin S6c (S6c) were studied in pulmonary resistance arteries (∼320 μm i.d.) from fetal, 0–24 h, 4 day and 7 day rabbits. The effects of the ET_A-selective antagonist {"type":"entrez-nucleotide","attrs":{"text":"FR139317","term_id":"258103156","term_text":"FR139317"}}FR139317, the selective ET_B receptor antagonist BQ-788 and the non-selective ET_A/ET_B receptor antagonist SB 209670, on these responses, were determined. Acetylcholine-induced vasodilation and noradrenaline-evoked contractions were also examined.
2. ET-1 potency was in the following order (pEC₅₀ values): fetal (8.7) = 0–24 h (8.8) = 4 day (8.6) > 7 day (8.0). The order of potency for S6c was 7 days (11.1) = 4 days (10.8) >0–24 h (9.7) > fetal (8.6). Hence, S6c and ET-1 were equipotent in the fetus but S6c was increasingly more potent than ET-1 with increasing age, being some 1000 times more potent by 7 days. By 7 days, responses to ET-1 were also resistant to both {"type":"entrez-nucleotide","attrs":{"text":"FR139317","term_id":"258103156","term_text":"FR139317"}}FR139317 and BQ-788. {"type":"entrez-nucleotide","attrs":{"text":"FR139317","term_id":"258103156","term_text":"FR139317"}}FR139317 inhibited responses to ET-1 in vessels from 0–24 h and 4 day, but not fetal, rabbits (pK_b: 6.4 in 4 day rabbits). BQ-788 inhibited responses to ET-1 at all age points except for 7 days (pK_b: 6.7 at 0–24 h; 6.2 at 4 days). BQ-788 inhibited responses to S6c at all age points (pK_b: 8.5 at 4 days). SB 209670 inhibited responses to ET-1 and S6c at 0–24 h and 4 days (pK_b for ET-1: 8.3 and 8.0 respectively; pK_b for S6c: 9.2 and 10.2 respectively).
3. Acetylcholine (1 μM) induced vasodilation at all age points (inhibited by 100 μM L-N^ω-nitroarginine methylester) although the degree of vasodilation was significantly reduced (∼75%) at 0–24 h. Noradrenaline induced contraction at all age points except 7 days and its response was significantly enhanced at 0–24 h.
4. Over the first week of life, the potency of S6c increases whilst that to ET-1 decreases suggesting differential development of responses to ET-1 and S6c and heterogeneity of ET_A- or `ET_B-like'' receptor-mediated responses. There is no synergism between ET_A and ET_B receptors at birth but this is established by 7 days. Immediately after birth rabbit Pulmonary Resistance Arteries are hyperresponsive to ET-1 and noradrenaline but exhibit impaired nitric-oxide dependent vasodilation.

     

Modulation of relaxation to levcromakalim by S-nitroso-N-acetylpenicillamine (SNAP) and 8-bromo cyclic GMP in the rat isolated mesenteric artery

1. Levcromakalim caused concentration-dependent relaxations of methoxamine-induced tone in both endothelium-denuded and intact vessels. Its potency was reduced by the nitric oxide donor, S-nitroso-N-acetylpenicillamine (SNAP; 0.1 μM or 1 μM) in both denuded and intact vessels. The maximal relaxation (R_max) was reduced only in denuded vessels.
2. SNAP was more potent in endothelium-denuded than intact vessels but there were no differences in R_max. Glibenclamide (10 μM) did not affect relaxation to SNAP in endothelium-denuded or intact vessels.
3. The soluble guanylyl cyclase inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 10 μM) increased the potency and R_max of levcromakalim in endothelium-intact vessels. ODQ had no effect in denuded vessels.
4. ODQ (10 μM) reduced the vasorelaxant potency of SNAP in both intact and endothelium-denuded vessels by 190-fold and 620-fold, respectively.
5. 8-bromo cyclic GMP (10 or 30 μM) reduced both the potency and R_max of levcromakalim in de-endothelialized vessels, but had no effect in intact vessels although it reduced both the potency and R_max of levcromakalim in intact vessels incubated with ODQ (10 μM).
6. In the presence of ODQ (10 μM), SNAP (0.1 μM or 1 μM) reduced the potency of levcromakalim in intact vessels, without altering R_max, but had no effect in denuded vessels. SNAP (50 μM) reduced both the potency and R_max of levcromakalim in intact and endothelium-denuded vessels.
7. Therefore, although SNAP causes relaxation principally through generation of cyclic GMP, it can modulate the actions of levcromakalim through mechanisms both dependent on, and independent of, cyclic GMP; the former predominate in endothelium-denuded vessels and the latter in intact vessels.

     

Trophic effect of angiotensin II in neonatal rat cardiomyocytes: role of endothelin-1 and non-myocyte cells

1. Angiotensin II (AII) and the endothelins (ET) are known to be potent trophic stimuli in various cells including cardiomyocytes. In order to characterize further these effects we studied, in neonatal rat ventricular cardiomyocytes, the effects of several endothelin-receptor antagonists and the AT₁-receptor antagonist losartan on AII- and endothelin-induced inositol phosphate (IP)-formation (assessed as accumulation of total [³H]-IPs in myo-[³H]-inositol prelabelled cells) and increase in rate of protein synthesis (assessed as [³H]-phenylalanine incorporation).
2. Endothelin (10 pM–1 μM) concentration-dependently increased IP-formation (max. increase at 100 nM ET-1: 130±14% above basal, n=25) and [³H]-phenylalanine incorporation (max. increase at 1 μM: 52±4% above basal, n=16) with an order of potency: ET-1>>ET-3. Both effects were antagonized by the ET_A/ET_B-receptor antagonist bosentan and the ET_A-receptor antagonist BQ-123, but not affected by the ET_B-receptor antagonist IRL 1038 and the AT₁-receptor antagonist losartan.
3. Pretreatment of the cells with 500 ng ml⁻¹ pertussis toxin (PTX) overnight that completely inactivated PTX-sensitive G-proteins did not attenuate but rather enhance ET-1-induced IP-formation. On the other hand, in PTX-pretreated cardiomyocytes ET-1-induced [³H]-phenylalanine incorporation was decreased by 39±5% (n=5).
4. AII (1 nM–1 μM) concentration-dependently increased IP-formation (max. increase at 1 μM: 42±7% above basal, n=16) and [³H]-phenylalanine incorporation (max. increase at 1 μM: 29±2%, n=9). These effects were antagonized by losartan, but they were also antagonized by bosentan and BQ-123.
5. In well-defined cultures of cardiomyocytes (not contaminated with non-myocyte cells) AII failed to increase [³H]-phenylalanine incorporation; addition of non-myocyte cells to the cardiomyocytes restored AII-induced increase in [³H]-phenylalanine incorporation.
6. We conclude that, in rat neonatal ventricular cardiomyocytes, (a) the ET-1-induced increase in rate of protein synthesis (through ET_A-receptor stimulation) involves at least two signalling pathways: one via a PTX-insensitive G-protein coupled to IP-formation, and the other one via a PTX-sensitive G-protein, and (b) the trophic effects of AII are brought about via local ET-1 secretion upon AT₁-receptor stimulation in neonatal rat ventricular non-myocyte cells.

     

Effect of DMPPO,a phosphodiesterase type 5 inhibitor,on hypoxic pulmonary hypertension in rats

1. Cyclic guanosine 3′–5′-monophosphate (cyclic GMP) is the second messenger of important physiologically active mediators controlling the pulmonary vascular tone. To potentiate the effects of cyclic GMP on the pulmonary vasculature, we used DMPPO, a new selective PDE-5 inhibitor, and examined its action in a rat model of hypoxic pulmonary hypertension.
2. Levels of cyclic GMP measured during baseline conditions at 5 and 60 min of perfusion were similar in the perfusate of isolated lungs from normoxic and chronically hypoxic rats and did not differ with time. Pretreatment with DMPPO (1 μM) induced a larger increase in cyclic GMP concentration in the perfusate from chronically hypoxic rat lungs (319±36 at 5 min to 1821±83 pmol ml⁻¹ at 60 min) than in normoxic rat lungs (329±20 to 1281±127 pmol ml⁻¹, P<0.05).
3. In isolated lungs preconstricted with U-46619, pretreatment with DMPPO (1 μM) potentiated the vasodilator effects of atrial natriuretic peptide (100 pM–10 nM) and sodium nitroprusside (1 pM–10 nM), but did not alter vasodilation to isoproterenol.
4. In conscious rats previously exposed to 15 days hypoxia and studied under 10% O₂, DMPPO (0.01, 0.05 and 0.1 mg kg⁻¹, i.v. bolus) caused a dose-dependent decrease in pulmonary arterial pressure (Pap) with no change in systemic artery pressure (Sap) and cardiac output.
5. Continuous infusion of DMPPO (0.1 mg kg⁻¹ h⁻¹ i.v. by osmotic pumps) in rats exposed to 10% O₂ during 2-weeks reduced the Pap (P<0.05) and the degree of muscularization of pulmonary vessels at the alveolar wall (P<0.01) and alveolar duct levels (P<0.05) despite no significant change in right ventricular hypertrophy.
6. These results suggest that cyclic GMP phosphodiesterase inhibition may selectively dilate pulmonary circulation during chronic hypoxia.

     

Endothelin converting enzyme (ECE) activity in human vascular smooth muscle

1. We have characterized the human smooth muscle endothelin converting enzyme (ECE) present in the media of the endothelium-denuded human umbilical vein preparation.
2. Endothelin-1 (ET-1) and ET-2 were potent constrictors of umbilical vein with EC₅₀ values of 9.2 nM and 29.6 nM, respectively. ET-1 was at least 30 times more potent than ET-3 suggesting the presence of constrictor ET_A receptors. Little or no response was obtained to the ET_B-selective agonist sarafotoxin 6c. These data suggest that endothelin-mediated vasoconstriction is via ET_A receptors in this preparation.
3. Autoradiographical visualization of endothelin receptors with subtype selective ligands confirmed the predominance of the ET_A receptor in the media of umbilical vein. High density of binding was obtained with the ET_A selective [¹²⁵I]-PD151242, with much lower levels detected with the ET_B selective [¹²⁵I]-BQ3020.
4. Big ET-1 (EC₅₀=42.7 nM) and big ET-2_(1-38) (EC₅₀=99.0 nM) were less potent than ET-1 and ET-2, respectively. Big ET-2_(1-38) was more potent than its isoform big ET-2_(1-37) with concentration–response curves to big ET-2_(1-37) incomplete at 300 nM. No response was obtained to big ET-3 at concentrations up to 700 nM. The C-terminal fragments, big ET-1_(22-38) and big ET-2_(22-38) were inactive.
5. Responses to ET-1 were unaffected by either the neutral endopeptidase (NEP) inhibitor thiorphan (10⁻⁵ M) or by the dual NEP/ECE inhibitor phosphoramidon (10⁻⁵ M). Big ET-1 was also unaffected by thiorphan but antagonized in a concentration-dependent manner by phosphoramidon (10⁻⁵ M and 10⁻⁴ M).
6. Addition of all four big endothelin peptides to human umbilical vein preparations resulted in detectable amounts of ET-IR in the bathing medium. Therefore, although big ET-3 was functionally inactive this reflects the low potency of ET-3 at the ET_A receptor rather than the lack of ability of this smooth muscle ECE to convert big ET-3 to ET-3.
7. To conclude we have demonstrated the presence of a phosphoramidon-sensitive ECE on the smooth muscle layer of the human umbilical vein which can convert big ET-1, big ET-2_(1-37), big ET-2_(1-38) and big ET-3 to their mature biologically active forms. The precise subcellular localization of this enzyme and its physiological relevance remains to be determined.

     

Characterization of the endothelin receptor subtype mediating epithelium-derived relaxant nitric oxide release from guinea-pig trachea

1. The endothelin (ET) receptor subtype that mediates niric oxide (NO)-dependent airway relaxation in tracheal tube preparations precontracted with carbachol and pretreated with indomethacin was investigated. The release of NO induced by ET from guinea-pig trachea using a recently developed porphyrinic microsensor was also measured.
2. ET-1 (1 pM–100 nM) contracted tracheal tube preparations pretreated with the NO-synthase inhibitor, L-NMMA, and relaxed, in an epithelium-dependent manner, preparations pretreated with the inactive enantiomer D-NMMA. The effect of L-NMMA was reversed by L-Arg, but not by D-Arg.
3. The selective ET_B receptor agonists, IRL 1620 or sarafotoxin S6c, both (1 pM–100 nM) contracted tracheal tube preparations in a similar manner either after treatment with D-NMMA or with L-NMMA. In the presence of the ET_A receptor antagonist, {"type":"entrez-nucleotide","attrs":{"text":"FR139317","term_id":"258103156","term_text":"FR139317"}}FR139317 (10 μM), ET-1 administration resulted in a contraction that was similar after either L-NMMA or D-NMMA. In the presence of the ET_B receptor antagonist, BQ788 (1 μM), ET-1 relaxed and contracted tracheas pretreated with D-NMMA and L-NMMA, respectively.
4. Exposure of tracheal segments to ET-1 (1–1000 nM) caused a concentration-dependent increase in NO release that was reduced by L-NMMA. IRL1620 (1 μM) did not cause any significant NO release. {"type":"entrez-nucleotide","attrs":{"text":"FR139317","term_id":"258103156","term_text":"FR139317"}}FR139317 (10 μM), but not, BQ788 (1 μM), inhibited the NO release induced by ET-1.
5. These results demonstrate that in the isolated guinea-pig trachea activation of ET_B receptors results in a contractile response, whereas activation of ET_A receptors cause both a contraction, and an epithelium-dependent relaxation that is mediated by NO release.

     

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 μm; Group B) were compared to those in larger arteries (mean 586.2±23.1 μm; 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∼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 μM. 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.

     

Negative chronotropic actions of endothelin-1 on rabbit sinoatrial node pacemaker cells

1. The effects of endothelin-1 (ET-1) on sinoatrial (SA) node preparations of the rabbit heart were studied by means of whole-cell clamp techniques.
2. ET-1 at 1 nM slowed the spontaneous beating activity and rendered half of the cells quiescent. At a higher concentration of 10 nM, the slowing and cessation of spontaneous activity were accompanied by hyperpolarization.
3. In voltage-clamp experiments, ET-1 decreased the basal L-type Ca²⁺ current (I_Ca(L)) dose-dependently with a half-maximal inhibitory concentration (EC₅₀) of 0.42 nM and maximal inhibitory response (E_max) of 49.5%. The delayed rectifying K⁺ current (I_K) was also reduced by 33.2±11.1% at 1 nM. In addition, an inwardly rectifying K⁺ current was activated by ET-1 at higher concentrations (EC₅₀=4.8 nM). These ET-1-induced changes in membrane currents were abolished by BQ485 (0.3 μM), a highly selective ET_A receptor antagonist.
4. When I_Ca(L) was inhibited by ET-1 (1 nM), subsequent application of 10 μM ACh showed no additional decrease in I_Ca(L), suggesting the involvement of cyclic AMP in the effects of ET-1 on I_Ca(L). In contrast, 1 nM ET-1 further decreased I_Ca(L) in the presence of 10 μM ACh, suggesting that ET-1 activates some additional mechanism(s) which inhibit I_Ca(L). The ET-1-induced I_Ca(L) inhibition was abolished by protein kinase A inhibitory peptide (PKI, 20 μM) or H-89 (5 μM). However, the I_Ca(L) inhibition was not affected by methylene blue (10 μM), suggesting a minor role for cyclic GMP in the effect of ET-1 under basal conditions.
5. ET-1 failed to inhibit I_Ca(L) when the pipette contained GDPβS (200 μM). However, incubation of the cells with pertussis toxin (PTX, 5 μg ml⁻¹, >6 h) only reduced the ET-1-induced inhibition to 21.5±9.5%, whereas it abolished the inhibitory effect of ACh on I_Ca(L).
6. Intracellular perfusion of 8-bromo cyclicAMP (8-Br cyclicAMP, 500 μM) attenuated, but did not abolish the inhibitory effect of ET-1 on I_Ca(L). This 8-Br cyclicAMP-resistant component (17.5±14.4%, n=20) was not affected by combined application of 8-Br cyclicAMP with 8-bromo cyclicGMP (500 μM), ryanodine (1 μM) or phorbol-12-myristate-13-acetate (TPA; 50 nM).
7. In summary, ET-1 exerts negative chronotropic effects on the SA node via ET_A-receptors. ET-1 inhibits both I_Ca(L) and I_K, and increases background K⁺ current. The inhibition of I_Ca(L) by ET-1 is mainly due to reduction of the cyclicAMP levels via PTX-sensitive G protein, but some other mechanism(s) also seems to be operative.

     

Response of normoxic pulmonary arteries of the rat in the resting and contracted state to NO synthase blockade

1. The pulmonary vasculature is normally in a low resting state of tone. It has been hypothesized that this basal tone is actively maintained by the continuous release of a vasodilator in the resting state. However, evidence for basal release of nitric oxide (NO) is inconclusive.
2. We studied the release of NO in arteries from the pulmonary circulation of male Wistar-Kyoto rats by examining the effects of the L-arginine analogue N^G-nitro-L-arginine methyl ester (L-NAME) on resting pulmonary arteries and on vessels pre-contracted with prostaglandin F_2α (PGF_2α).
3. Rats (n=21) were killed by an overdose with pentobarbitone. Pulmonary arteries were dissected (mean internal diameter 459±11 μm) and mounted in a small vessel wire myograph. Resting tensions were set to simulate transmural pressures of 17.5 mmHg.
4. L-NAME (100 μM) was found to produce a contraction of 0.64±0.09 mN mm⁻¹ in resting pulmonary arteries when added alone to the myograph bath. This contraction was not produced following removal of the endothelium. Vessel contraction to PGF_2α (100 μM) was found to be significantly greater when carried out in the presence of L-NAME (100 μM)–1.37±0.15 mN mm⁻¹ compared with 1.96±0.17 mN mm⁻¹. Dilatation following acetylcholine (ACh) (1 μM) was abolished in the presence of L-NAME (100 μM).
5. Rat pulmonary artery contraction in response to the addition of L-NAME and the absence of contraction upon removal of the endothelium provides supportive evidence of the active release of nitric oxide for the maintenance of resting tone.

     

Effects of L-NG-nitro-arginine on noradrenaline induced contraction in the rat anococcygeus muscle

1. The influence of L-N^G-nitro-arginine (L-NOARG, 30 μM) on contractile responses to exogenous noradrenaline was studied in the rat anococcygeus muscle.
2. Noradrenaline (0.1–100 μM) contracted the muscle in a concentration-dependent manner. L-NOARG (30 μM) had no effect on noradrenaline responses.
3. Phenoxybenzamine (Pbz 0.1 μM) depressed by 46% (P<0.001) the maximum response and shifted to the right (P<0.001) the E/[A] curve to noradrenaline (pEC₅₀ control: 6.92±0.09; pEC₅₀ Pbz: 5.30±0.10; n=20).
4. The nested hyperbolic null method of analysing noradrenaline responses after phenoxybenzamine showed that only 0.61% of the receptors need to be occupied to elicit 50% of the maximum response, indicating a very high functional receptor reserve.
5. Contractile responses to noradrenaline after partial α₁-adrenoceptor alkylation with phenoxybenzamine (0.1 μM) were clearly enhanced by L-NOARG.
6. The potentiating effect of L-NOARG on noradrenaline responses after phenoxybenzamine was reversed by (100 μM) L-arginine but not by (100 μM) D-arginine.
7. These results indicate that spontaneous release of NO by nitrergic nerves can influence the α₁-adrenoceptor-mediated response to exogenous noradrenaline.

     

Potassium channel activation and relaxation by nicorandil in rat small mesenteric arteries

1. We used whole-cell patch clamp to investigate the currents activated by nicorandil in smooth muscle cells isolated from rat small mesenteric arteries, and studied the relaxant effect of nicorandil using myography.
2. Nicorandil (300 μM) activated currents with near-linear current-voltage relationships and reversal potentials near to the equilibrium potential for K⁺.
3. The nicorandil-activated current was blocked by glibenclamide (10 μM), but unaffected by iberiotoxin (100 nM) and the guanylyl cyclase inhibitor LY 83583 (1 μM). During current activation by nicorandil, openings of channels with a unitary conductance of 31 pS were detected.
4. One hundred μM nicorandil had no effect on currents through Ca²⁺ channels recorded in response to depolarizing voltage steps using 10 mM Ba²⁺ as a charge carrier. A small reduction in current amplitude was seen in 300 μM nicorandil, though this was not statistically significant.
5. In arterial rings contracted with 20 mM K⁺ Krebs solution containing 200 nM BAYK 8644, nicorandil produced a concentration-dependent relaxation with mean pD₂=4.77±0.06. Glibenclamide (10 μM) shifted the curve to the right (pD₂=4.32±0.05), as did 60 mM K⁺. LY 83583 caused a dose-dependent inhibition of the relaxant effect of nicorandil, while LY 83583 and glibenclamide together produced greater inhibition than either alone.
6. Metabolic inhibition with carbonyl cyanide m-chlorophenyl hydrazone (30 nM), or by reduction of extracellular glucose to 0.5 mM, increased the potency of nicorandil.
7. We conclude that nicorandil activates K_ATP channels in these vessels and also acts through guanylyl cyclase to cause vasorelaxation, and that the potency of nicorandil is increased during metabolic inhibition.

     

Modulation of AT1 receptor-mediated contraction of rat uterine artery by AT2 receptors

1. The aim of this study was to characterize the angiotensin II receptors in isolated uterine arteries from non pregnant and pregnant rats, since it has been reported from binding studies that ovine uterine arteries contain AT₂ receptors.
2. Uterine arterial segments were obtained from virgin, non-pregnant and late pregnant (18–21 days) Sprague-Dawley rats and mounted in small vessel myographs. Concentration-response curves were constructed to angiotensin II (1 nM–10 μM) in the absence and presence of various angiotensin II receptor subtype selective compounds. These included losartan (AT₁ antagonist; 1, 10 and 100 nM), PD 123319 (AT₂ antagonist; 1 μM) and CGP 42112 (AT₂ agonist; 1 μM). Responses to angiotensin II were measured as increases in force (mN) and expressed as a per cent of the response to a K⁺ depolarizing solution.
3. Losartan (1, 10 and 100 nM) caused significant concentration-dependent rightward shifts of the angiotensin II concentration-response curve in uterine arteries from non-pregnant and pregnant rats. The pA₂ values calculated from these data were 9.8 and 9.2, respectively, although the slope of the Schild plot in the non-pregnant group was less than unity.
4. PD 123319 (1 μM) caused significant 6- and 3 fold leftward shifts of the angiotensin II concentration-response curve in uterine arteries from non-pregnant and pregnant rats, respectively. In vessels from pregnant rats, PD 123319 also significantly increased the maximum response to angiotensin II.
5. CGP 42112 (1 μM) attenuated the response to angiotensin II of uterine arteries from non-pregnant rats. This was reflected by a 14 fold rightward shift of the angiotensin II concentration-response curve and a decrease in the maximum response. In uterine arteries from pregnant rats, CGP 42112 (1 μM) caused a 3 fold rightward shift of the angiotensin II concentration-response curve, but had no effect on the maximum response.
6. PD 123319 (1 μM) and CGP 42112 (1 μM) had no effect on the concentration-response curves to phenylephrine (PE) of uterine arteries from non-pregnant or pregnant rats. In addition, CGP 42112 (1 nM–1 mM) had no vasodilator effect on tissues precontracted with phenylephrine.
7. These results suggest that the contractile responses of the rat uterine artery are mediated by the AT₁ receptor. Furthermore, in this vascular preparation, the AT₂ receptor appears to inhibit the response mediated by the AT₁ receptor, although, this is not uniform between the non-pregnant and pregnant states.

     

Modulation by 5-HT1A receptors of the 5-HT2 receptor-mediated tachykinin-induced contraction of the rat trachea in vitro

1. In the Fisher 344 rat, tachykinins have been shown to cause the release of 5-hydroxytryptamine (5-HT) from airway mast cells, which then causes direct smooth muscle activation as well as the release of acetylcholine from cholinergic nerves. The aim of the present study was to examine the modulatory effects of 5-HT receptors on the neurokinin A (NKA)-induced release of endogenous 5-HT and airway smooth muscle contraction in the isolated Fisher 344 rat trachea.
2. The selective 5-HT₂ receptor antagonist ketanserin (0.1 μM) produced an almost complete inhibition of the contractions caused by NKA (n=4, P<0.0001, two-way ANOVA), and a significant rightward shift of the concentration-response curve to 5-HT (n=8, P<0.001, two-way ANOVA).
3. The partial agonist for 5-HT_1A receptors, 8-OH-DPAT (1 μM), and the full agonist for 5-HT₁ receptors, 5-CT (0.3 μM), potentiated the submaximal contractions induced by the 5-HT₂ receptor agonist α-methyl-5-HT (0.1 μM) (n=4; P<0.005 and P<0.05, respectively). 8-OH-DPAT (1 μM), as well as the 5-HT_1A receptor antagonists pMPPI, SDZ 216525 and NAN-190 (0.1 μM each), caused significant inhibition of the tracheal contractions induced both by NKA (10  nM–3  μM) and 5-HT (10 nM–10 μM) (n=4–10). This suggests that activation of 5-HT_1A receptors potentiates the 5-HT₂ receptor-mediated contractions.
4. SDZ 216525 (0.1 μM) significantly reduced the maximal contraction produced by 1 μM NKA (n=10, P<0.001), without affecting the release of endogenous 5-HT. These data rule out the involvement of a 5-HT_1A receptor-mediated positive feedback mechanism of the 5-HT release from mast cells.
5. Even in the presence of atropine (1 μM), 8-OH-DPAT (1 μM) further reduced the maximal NKA-induced contraction (n=4, P<0.0001), while the contractions of the rat isolated trachea induced by electrical field stimulation and the concentration-response curve to carbachol were unaffected by pMPPI (0.1 μM), SDZ 216525 (0.1 μM), NAN-190 (0.1 μM) and 8-OH-DPAT (1 μM) (n=4–6). These data demonstrate that the 5-HT_1A receptor-mediated potentiation of contractile responses is not due to non-specific inhibition of airway smooth muscle contraction or to modulation of postganglionic nerve activation.
6. The selective 5-HT_1B/1D receptor antagonist GR 127935, the selective 5-HT₃ receptor antagonist tropisetron and the selective 5-HT₄ receptor antagonists SB 204070 and GR 113808 (0.1 μM each) had no effect on the concentration-response curve for NKA (n=6–10), ruling out the involvement of 5-HT_1B/1D, 5-HT₃ and 5-HT₄ receptors.
7. The α-adrenoreceptor antagonist phentolamine (1 μM) had no effect on the 5-HT-induced contractions (n=4), ruling out the involvement of α-adrenoreceptors.
8. In conclusion, the tachykinin-induced contraction of the F334 rat isolated trachea is mediated by the stimulation of 5-HT₂ receptors. Activation of 5-HT_1A receptors located on airway smooth muscle potentiates the direct contractile effects of 5-HT₂ receptor activation. The 5-HT_1B/1D, 5-HT₃ and 5-HT₄ receptors are not involved in the NKA-induced contraction of rat airways.