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Neurokinins produce selective venoconstriction via NK-3 receptors in the rat mesenteric vascular bed

The vasoactive properties of the neurokinins (substance P (SP), neurokinin A (NKA), neurokinin B (NKB)) and some selective analogues were assessed in the arterial and venous mesenteric beds of the rat. Although both sides of the mesenteric vasculature displayed endothelium-dependent relaxation in response to acetylcholine (ACh) or bradykinin (BK) (1 and 10 nmol), SP and the selective NK-1 analogue, [Sar9,Met(O2)11]SP were inactive. Of the three selective neurokinin agonists used, [Sar9,Met(O2)11]SP (NK-1), [beta-Ala8]NKA-(4-10) (NK-2) and [MePhe7]NKB (NK-3), only the latter induced a dose-dependent pressor effect in the venous mesenteric vasculature. Injections of SP and the selective NK-1 and NK-2 analogues at high doses (10 nmol), did not change the perfusion pressure in the mesenteric bed even when the mesenteric vasculature was treated with methylene blue (50 microM) to inhibit the effects of endothelium-derived relaxing factor (EDRF) or with NG-nitro-L-arginine (L-NNA) (20 microM) to inhibit the formation of EDRF or with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate] (CHAPS 20 mM, 30 s) to remove the endothelial layer. In contrast, the vasoconstrictor effects of noradrenaline (NA), angiotensin II (ATII), NKB and [MePhe7]NKB on the venous side of the circulation were enhanced following treatment with L-NNA, methylene blue or CHAPS. The present results suggest that neurokinins act on the rat mesenteric bed by increasing the perfusion pressure of the venous vasculature via activation of NK-3 receptors. Neurokinins are inactive on the arterial mesenteric vasculature.(ABSTRACT TRUNCATED AT 250 WORDS)

Block of Endothelin‐1‐induced release of thromboxane A2 from the guinea pig lung and nitric oxide from the rabbit kidney by a selective ETB receptor antagonist, BQ‐788

1 The present study characterizes the receptors responsible for endothelin‐1‐induced release of thromboxane A2 from the guinea pig lung and of endothelium‐derived nitric oxide from the rabbit perfused kidney, by the use of the selective ETA receptor antagonist, BQ‐123, and a novel selective ETB receptor antagonist, BQ‐788. 2 In the guinea pig perfused lung, endothelin‐1 (ET‐1) (5 nm) induced a marked increase of thromboxane A2 which was reduced by 17 ± 5.0, 70 ± 1.0 and 93 ± 1.2% by BQ‐788 infused at concentrations of 1, 5 and 10 nm respectively. In contrast, BQ‐123 (0.1 and 1.0 μm) had little or no effect on the ET‐1‐induced release of thromboxane A2. 3 In the same perfused model, the selective ETB agonist, IRL 1620 (50 nm), stimulated the release of thromboxane A2, but not prostacyclin. The eicosanoid‐releasing properties of IRL 1620 were abolished by BQ‐788 at 10 nm, yet were unaffected by BQ‐123 (1 μm). 4 In the rabbit perfused kidney, BQ‐788 (10 nm) potentiated the increase of perfusion pressure induced by endothelin‐1 (1,5 and 10 nm) by approximately 90%, but not that induced by angiotensin II (1 μm). Furthermore, the selective ETB receptor antagonist did not reduce the release of prostacyclin triggered by either peptide. 5 In another series of experiments, pretreatment of the perfused kidney with a nitric oxide synthase inhibitor, L‐NAME (100 μm), potentiated the pressor responses to both endothelin‐1 and angiotensin II. Under L‐NAME treatment, BQ‐788 did not further potentiate the pressor response to endothelin‐1. 6 Our results illustrate the predominant role of ETB receptor activation in the release of thromboxane A2 and nitric oxide triggered by endothelin‐1 in the guinea pig perfused lung and rabbit kidney respectively.

Characterization of receptors for endothelins in the perfused arterial and venous mesenteric vasculatures of the rat.

1 Endothelin−1 and −3 induced marked arterial and venous constrictions in the perfused mesenteric vasculature of the rat with endothelin‐3 being at least 20 times less active than endothelin‐1, on both arterial and venous sides of the vasculature. 2 Two ETB selective agonists, BQ‐3020 and IRL 1620 (500 pmol), induced weak constrictions of the venous mesenteric vasculature and were inactive in the arterial side at doses up to 1000 pmol. 3 In mesenteric vasculatures precontracted with either methoxamine (arterial side) or the thromboxane A2‐mimetic, U46619 (venous side), acetylcholine or bradykinin produced vasodilatations of both arterial and venous vessels, whereas endothelin‐3 induced vasodilatations only on the arterial side. 4 A selective ETA receptor antagonist, BQ‐123, blocked, in a concentration‐dependent and reversible fashion, the vasoconstrictions induced by endothelin‐1 on both sides of the mesenteric circulation (IC50; arterial side: 0.013 μm; venous side: 0.032 μm). 5 In contrast, the vasodilator responses induced by endothelin‐3 on the arterial side of the precontracted mesenteric vasculature were not affected by BQ‐123. 6 The present study illustrates the presence of ETA receptors which are responsible for vasoconstriction by endothelins in the arterial and venous mesenteric vasculatures. Furthermore, we suggest that the vasodilatations induced by endothelin‐3 in the arterial vasculature uniquely, are ETB receptor‐mediated.

Endothelin-1 induces vasoconstriction and prostacyclin release via the activation of endothelin ETA receptors in the perfused rabbit kidney

Endothelin-1 (0.005 and 0.01 nmol) induced a dose-dependent increase in perfusion pressure in the perfused rabbit kidney. These pressor effects were markedly reduced by an endothelin ETA receptor antagonist, BQ-123 (0.1 microM). Similarly, the release of prostacyclin triggered by intra-arterial infusion of endothelin-1 (10 nM) was significantly reduced in a concentration-dependent manner when the kidney was pretreated with BQ-123 (0.5-1 microM). In contrast, two selective ETB receptor agonists, BQ-3020 and IRL 1620, were found to be inactive, both as pressor agents and releasers of prostacyclin at doses (for the pressor effects) and concentrations (for the prostacyclin generation) 50-100 times higher than those of endothelin-1. BQ-123 (1 microM) did not modify the pressor or prostanoid-releasing properties of angiotensin II. These results confirm our previous observations suggesting that pressor responses and prostanoid release induced by endothelin-1 are mediated via the selective activation of ETA receptors in the perfused rabbit kidney.

Human big-endothelin-1 and endothelin-1 release prostacyclin via the activation of ET1 receptors in the rat perfused lung.

Although ET1 and ET2 binding sites were found in rat lung membranes, a selective ET1 receptor antagonist, BQ‐123 (10 μm), did not displace [125I]‐endothelin‐1 ([125I]ET‐1) from ET2 sites, illustrating the selectivity of the angatonist for ET1 receptors. In rat perfused lungs, BQ‐123 (1 μm) markedly reduced the prostacyclin (PGI2) releasing properties of endothelin‐1 (ET‐1: 5 nm) and human big‐ET‐1 (100 nm) suggesting that both peptides induce the release of PGI2 via the selective activation of ET1 receptors.

Different pharmacological profiles of big-endothelin-3 and big-endothelin-1 in vivo and in vitro.

1 Human big‐endothelin‐1 (big‐ET‐1) and endothelin‐1 (ET‐1) are equipotent as pressor agents and produce a significant change in mean arterial blood pressure (MAP) in anaesthetized guinea‐pigs (2 nmol kg−1: peak Δ MAP: 23 ± 6 mmHg and 26 ± 5 mmHg, respectively). 2 Unlike big‐ET‐1, big‐endothelin‐3 (big‐ET‐3) (10 and 20 nmol kg−1) induces no pressor responses whereas endothelin‐3 (ET‐3) at 2 nmol kg−1induces a significant increase of blood pressure in anaesthetized guinea‐pigs (peak Δ MAP: 27 ± 5 mmHg) with a shorter duration than ET‐1 and big‐ET‐1. 3 Big‐ET‐1 at concentrations 40 times higher than those required for ET‐1 (2.5 nm) releases prostacyclin (PGI2) (maximal release: 2.7 ± 0.8 ng ml−1; 2.9 ± 0.9 ng ml−1, respectively) and thromboxane B2 (TxB2) (maximal release: 6.7 ± 1.3 ng ml−1; 6.8 ± 1.1 ng ml−1, respectively) from guinea‐pig perfused lungs. ET‐3 (2.5 nm) is also a potent releaser of PGI2 and TxB2 from the guinea‐pig lungs (maximal release: PGI2: 2.4 ± 1.0 ng ml−1; TxB2: 3.8 ± 0.6 ng ml−1). Conversely, big‐ET‐3 (100 nm) does not increase basal release of eicosanoids. 4 Phosphoramidon (50 μm), a metalloprotease inhibitor, markedly reduced the eicosanoid releasing properties of big‐ET‐1 (n = 4, P < 0.01) in guinea‐pig perfused lungs without affecting the release stimulated by ET‐1. 5 Our results suggest that big‐ET‐1 is converted to ET‐1 via a phosphoramidon‐sensitive endothelin converting enzyme (ECE) to release eicosanoids. The ECE is present in the guinea‐pig pulmonary vasculature. Furthermore, our results suggest that the ECE activity is specific for big‐ET‐1 and may not convert big‐ET‐3 to its active metabolite, ET‐3.

Presence of a phosphoramidon-sensitive endothelin-converting enzyme which converts big-endothelin-1, but not big-endothelin-3, in the rat vas deferens

SummaryEndothelin-1 (ET-1) enhanced field stimulation-evoked (0.1 Hz), nerve-mediated contractions of the prostatic portion of the rat vas deferens. The human precursor of ET-1, big-endothelin (1-38) (big-ET-1) was only two-fold less potent than ET-1 (pD2 values: 7.30 and 7.49, respectively). The threshold concentrations necessary to elicit an increase of the response to electrical stimulation was lower for ET-1 (5 nmol/l) than for big-ET-1 (25 nmol/l). Endothelin-3 (ET 3) also markedly enhanced the response of the tissue to field stimulation with a potency similar to ET-1 (pD2 value: 7.59). In contrast, the precursor of ET-3, big-endothelin (1–41) (big-ET-3), was inactive at concentrations up to 0.5 μmol/l. Treatment of the preparations with phosphoramidon (50 μmol/l) markedly reduced the twitch enhancement by big-ET-1 without affecting the response to ET-1. Our results suggest the presence of a specific phosphoramidon-sensitive endothelin-converting enzyme which converts big-ET-1 to ET-1 in the rat vas deferens.

Human big endothelin releases prostacyclin in vivo and in vitro through a phosphoramidon-sensitive conversion to endothelin-1.

Human big endothelin (big ET) and endothelin-1 (ET-1) induce similar increases in left ventricular systolic pressure in the anesthetized rabbit. Unlike ET-1, human big ET does not induce an initial transient hypotension. Human big ET (3 nmol/kg) inhibits ADP-induced platelet aggregation ex vivo by 60% whereas ET-1 at 1 nmol/kg inhibits platelet aggregation by more than 80%. The C-terminal fragment, big ET[22-38] (3 nmol/kg), has no antiaggregatory properties. Inhibition of ex vivo platelet aggregation by human big ET and ET-1 was not seen in rabbits pretreated with indomethacin (5 mg/kg). Human big ET (10(-7) M) or ET-1 (2.5 x 10(-9)-10(-8) M) induced the release of prostacyclin (PGI2) from rabbit, guinea pig, and rat lungs. Phosphoramidon (50 microM, infused 45 min prior to and during administration of peptides) inhibited the prostanoid-releasing properties of human big ET without affecting the release induced by ET-1. Intravascular administration of human big ET (1 nmol/kg) significantly increased the circulating levels of immunoreactive ET-1 (ir-ET-1) for 30 min whereas administration of ET-1 at the same concentration increased the plasma level of ir-ET-1 for 5 min only. Our results suggest that human big ET is converted to ET-1 in the rabbit in vivo. We further suggest that to induce the release of prostanoids in perfused lungs, human big ET needs to be converted to ET-1 by a phosphoramidon-sensitive endothelin-converting enzyme (ECE).

Phosphoramidon-Sensitive Effects of Big Endothelins in the Perfused Rabbit Kidney

The enzymatic conversion of human big endothelins (1, 2, and 3) to their respective active metabolites (endothelin-1, -2, and -3) was investigated in the perfused rabbit kidney through the pressor- and eicosanoid-releasing properties of these peptides. Intra-arterial bolus injections of endothelin-1 and -2 (5-50 pmol), endothelin-3 (100-250 pmol), and big endothelin-1 and -2 (100-250 pmol) into the kidney produced dose-dependent increases of perfusion pressure, whereas big endothelin-3 was inactive at doses up to 1,000 pmol. Endothelin-1 and -2 (10 nM), endothelin-3 (100 nM), and big endothelin-1 and -2 (100 nM) are potent enhancers of prostacyclin release without inducing any release of thromboxane B2 in the perfused kidney. In contrast, big endothelin-3 did not trigger the release of eicosanoids. A metalloprotease inhibitor, phosphoramidon (100 microM, 60 minutes), reduced the prostanoid release and pressor responses induced by big endothelin-1 and -2 without affecting the response induced by endothelin-1, -2, and -3. These results suggest the presence of a phosphoramidon-sensitive endothelin converting enzyme that converts the precursors of endothelin-1 and -2, but not of endothelin-3, in the renal vasculature of the rabbit.

Increased plasma levels of endothelin during anaphylactic shock in the guinea-pig

Intravenous injection of ovalbumin into actively and passively sensitized guinea-pig resulted in acute circulatory collapse. The plasma level of immunoreactive endothelin rose from 22 +/- 2 to 40 +/- 7 fmol/ml (n = 12, P < 0.01) and 29 +/- 5 fmol/ml (n = 12, P < 0.01) in actively and passively sensitized animals, respectively, within 5 min of antigen challenge, and it remained significantly higher in actively sensitized animals that survived for 15 min. The plasma immunoreactive endothelin level was inversely correlated with arterial blood pO2, but not with pH or pCO2, both in actively (rs = -0.585, n = 20, P < 0.05) and passively sensitized animals (rs = -0.558, n = 20, P < 0.05). When non-sensitized animals were bled (5 and 20 ml/kg body weight), the plasma immunoreactive endothelin level remained unchanged. These results suggest that the elevated plasma level of immunoreactive endothelin during anaphylactic shock is independent of hypotension, hypovolemia and respiratory insufficiency.