Audrey Claing

Processing of proendothelin-1 by human furin convertase.

Endothelin‐1 (ET‐1) is the most potent vasoactive peptide known to date. The peptide is initially synthesized as an inactive precursor (proET‐1) which undergoes proteolysis at specific pairs of basic amino acids to yield bigET‐1. Production of ET‐1 then proceeds by cleavage of bigET‐1 by the endothelin converting enzyme (ECE). Here, we demonstrate that the in vitro cleavage of proET‐1 by furin, a mammalian convertase involved in precursor processing, produced bigET‐1. Upon further processing, bigET‐1 was converted to biologically active ET‐1. Furthermore, we demonstrate that the furin inhibitor, decanoyl‐Arg‐ValLys‐Arg chloromethylketone, abolished production of ET‐1 in endothelial cells.

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.

Comparison of the contractile and calcium-increasing properties of platelet-activating factor and endothelin-1 in the rat mesenteric artery and vein.

In the present study, the properties of endothelin‐1 (ET‐1) and platelet‐activating factor (PAF) in inducing contraction and increased intracellular‐free calcium level in rat mesenteric arteries and veins were studied. Furthermore, measurements of cytosolic ([Ca]c) and nuclear ([Ca]n) Ca2+ were performed by confocal microscopy. PAF, at a concentration of 1 μM, and the selective ETB agonists, IRL‐1620 and sarafotoxin S6C (100 nM), induced a marked constriction and increase in [Ca]i in the mesenteric vein but not in the artery. On the other hand, endothelin‐1 (1–100 nM) induced a significant concentration‐dependent nifedipine‐insensitive increase in tension and [Ca]i in both arteries and veins. Those responses to endothelin‐1 were significantly reduced by the ETA receptor antagonist, BQ‐123 (10−6 M), on both types of vessels, whereas the selective ETB receptor antagonist, BQ‐788, inhibited only the venous responses. The mixed ETA/ETB receptor antagonist, SB 209670, reduced the ET‐1‐induced venous responses to the same level of that found in presence of BQ‐123 or BQ‐788. However, concomitant applications of BQ‐123 and BQ‐788 reduced the vasoconstriction below to that induced by ETA or ETB blockade without further affecting [Ca]i. PAF and the selective ETB agonists IRL‐1620, induced a sustained increase of [Ca]c and [Ca]n solely in venous cells and ET‐1 in both arterial and venous smooth muscle cells. Thus, PAF increases total intracellular calcium concentration and tension on the smooth muscle cells from venous origin only. Furthermore, ET‐1‐induced vasoactive as well as [Ca]i and [Ca]n increasing effects are mediated by distinct receptors on venous and arterial smooth muscles.

Characterization of receptors for kinins and neurokinins in the arterial and venous mesenteric vasculatures of the guinea-pig.

1 In the present work, we have studied the microvascular reactivity of the arterial and venous mesenteric beds of the guinea‐pig to bradykinin, neurokinins and other agents. 2 The vasoactive properties of three selective agonists for neurokinin receptors, namely [Sar9, Met (O2)11]SP (NK,1), [β‐Ala8]NKA(4–10) (NK2) and [MePhe7]NKB (NK3), were evaluated on precontracted arterial and venous mesenteric vasculatures of the guinea‐pig. The NK1selective agonist, [Sar9, Met(O2)11]SP (1 to 1000 pmol), induced an endothelium‐dependent and NΩ‐nitro‐L‐arginine methyl ester (L‐NAME)‐sensitive relaxation of the arterial vasculature precontracted with methoxamine, whereas the NK2 and NK3‐selective agonists were virtually inactive at high doses (1000 pmol). 3 The three selective neurokinin receptor agonists were inactive in the non‐precontracted arterial and venous mesenteric vasculatures as well as in the precontracted venous mesenteric vasculature. 4 Bradykinin (0.1 to 100 pmol) induced a marked dose‐ and endothelium‐dependent vasodilatation of the precontracted arterial and venous vasculatures. ED50 values were 5.5 pmol on the arterial side and 1.9 pmol on the venous side. In contrast, desArg9‐bradykinin was inactive at doses up to 1000 pmol. Furthermore, on the arterial and venous sides, a higher dose of bradykinin (1000 pmol), induced a biphasic effect, a transient constriction followed by a marked and sustained vasodilatation. The vasodilator effects of bradykinin were abolished by Hoe 140 (0.1 μm) and CHAPS, markedly reduced by L‐NAME and were unaffected by [Leu8]desArg9‐bradykinin (0.1 μm) on both sides of the mesenteric vasculature. Hoe 140 also abolished the arterial vasoconstrictions induced by high doses of bradykinin. 5 Noradrenaline, angiotensin II and endothelin‐1 produced contractions on both sides of the mesenteric circulation, while acetylcholine (arterial side) and sodium nitroprusside (arterial and venous sides) caused vasodilatation. 6 Our study supports the view that NK1 receptors responsible for vasodilatation are present solely in the endothelium of the arterial mesenteric vasculature of the guinea‐pig. On the other hand, bradykinin (0.1 to 100 pmol) exerts predominantly vasodilator effects on both sides of the mesenteric vasculature via selective activation of B2 receptors located on the endothelium. The same receptor type located on the smooth muscle appears to be responsible for the arterial and venous constriction with high doses of bradykinin.

Role of R‐type calcium channels in the response of the perfused arterial and venous mesenteric vasculature of the rat to platelet‐activating factor

1 The vasoactive properties of platelet‐activating factor (PAF) were studied in the arterial and venous vasculature of the rat double‐perfused mesenteric bed. Although PAF (0.01–0.3 pmol) induced a dose‐dependent vasodilatation of the arterial mesenteric vasculature, it triggered only vasoconstrictions on the venous side, with an intact endothelium as bradykinin induced a significant venodilatation. 2 NG‐nitro‐l‐arginine methyl ester (l‐NAME, 100 μm), a nitric oxide synthase inhibitor, markedly reduced the vasodilatation induced by PAF in the arterial mesenteric vasculature and potentiated the contractile responses of the venous side to the same agent. 3 The PAF antagonist, WEB‐2170, markedly reduced the response to PAF on both sides of the mesenteric vasculature. However, the IC50 of WEB‐2170 against PAF was reached at a much higher concentration (1 × 10−8 M) on the arterial side than on the venous side (5.3 × 10−11 M). Furthermore, a second antagonist of PAF receptors, SRI‐63441, although being less potent on the venous vasculature than WEB‐2170, was equipotent in antagonizing the venoconstriction and the arterial dilatation induced by PAF (IC50 of SRI‐63441, arterial side: 2.9 × 10−9 M; venous side: 3.1 × 10−9 M). 4 The dual L‐ and R‐calcium channel blocker, isradipine (PN 200–110), but not the L‐type calcium channel blocker, nifedipine, markedly reduced the PAF‐induced vasoactive properties on both sides of the mesenteric vasculature. 5 Our results illustrate the differential vasoactive properties of PAF in the mesenteric vasculature of the rat. These vasoactive responses occur following activation of specific receptors for PAF or, alternatively, through activation of R‐type calcium channels.