Nathalie Berthiaume

Heterozygous Knock-Out of ETB Receptors Induces BQ-123–Sensitive Hypertension in the Mouse

Homozygous knock-out of ETA or ETB receptor genes results in lethal developmental phenotypes in the mouse. Such deleterious phenotypes do not occur in heterozygous littermates. However, it remains to be determined whether mice partially defective in ETA or ETB receptors display significant alterations in their responses to exogenous or endogenous endothelin-1 (ET-1). Furthermore, the anesthetized ETB (+/−) knock-out mice showed a significantly higher mean arterial blood pressure than the ETA (+/−) knock-out or their wild-type littermates. The pressor response to ET-1 but not to a selective ETB agonist, IRL-1620, was significantly reduced in the ETA (+/−) knock-out mice. In ETB (+/−) knock-out mice, the pressor effect of IRL-1620 was more markedly altered than those induced by ET-1. In wild-type mice, both ETA and ETB receptors were found to be involved in the pressor effect of ET-1, as confirmed by the significant and specific antagonism induced by either BQ-123 (ETA antagonist) or BQ-788 (ETB antagonist). Also, ETA-selective or mixed ETA/ETB- but not ETB-selective antagonists reversed the hypertensive state of the ETB (+/−) knock-out mice to the level of wild-type littermates. Finally, radiolabeled ET-1 plasmatic clearance was altered in ETB (+/−) but not ETA (+/−) knock-out mice when compared with wild-type animals. Thus, heterozygous knock-out of ETB receptors results in a hypertensive state, suggesting an important physiological role for that particular receptorial entity in opposing the endogenous ET-1–dependent pressor effects in the mouse.

The kinin system mediates hyperalgesia through the inducible bradykinin B1 receptor subtype: evidence in various experimental animal models of type 1 and type 2 diabetic neuropathy.

Abstract Both insulin-dependent (type 1) and insulin-independent (type 2) diabetes are complex disorders characterized by symptomatic glucose intolerance due to either defective insulin secretion, insulin action or both. Unchecked hyperglycemia leads to a series of complications among which is painful diabetic neuropathy, for which the kinin system has been implicated. Here, we review and compare the profile of several experimental models of type 1 and 2 diabetes (chemically induced versus gene-prone) and the incidence of diabetic neuropathy upon aging. We discuss the efficacy of selective antagonists of the inducible bradykinin B1 receptor (BKB1-R) subtype against hyperalgesia assessed by various nociceptive tests. In either gene-prone models of type 1 and 2 diabetes, the incidence of hyperalgesia mostly precedes the development of hyperglycemia. The administration of insulin, achieving euglycemia, does not reverse hyperalgesia. Treatment with a selective BKB1-R antagonist does not affect basal nociception in most normal control rats, whereas it induces a significant time- and dose-dependent attenuation of hyperalgesia, or even restores nociceptive responses, in experimental diabetic neuropathy models. Diabetic hyperalgesia is absent in streptozotocin-induced type 1 diabetic BKB1-R knockout mice. Thus, selective antagonism of the inducible BKB1-R subtype may constitute a novel and potential therapeutic approach for the treatment of painful diabetic neuropathy.

Pharmacology of kinins in the arterial and venous mesenteric bed of normal and B2 knockout transgenic mice.

We have tested the vasoactive effects of kinins in addition to various other endothelium-dependent or independent agonists in the arterial and venous perfused mesenteric circuits of the mouse. Bradykinin (0.1 pmol-100 nmol), but not des-Arg9-bradykinin (10 nmol) induced a dose-dependent vasodilation of the precontracted arterial and venous mesenteric vasculature of the mouse. Furthermore, acetylcholine (2.5 nmol) also induced a marked arterial vasodilation but was without effect on the venous side. Other endothelium-dependent vasodilators, such as platelet-activating factor (PAF) (1 nmol), tachykinin NK1 selective agonist ([Sar9,Met(O2)(l1) ]substance P) (0.5 nmol) and adenosine diphosphate (5 nmol), were without effect on either side of the mesenteric bed of the mouse. The bradykinin B2 receptor selective antagonist (HOE 140) abolished the arterial and venous vasodilation induced by bradykinin without affecting that of acetylcholine or sodium nitroprusside. In addition, the bradykinin B1 receptor antagonist des-Arg9-[Leu8]bradykinin was without effect on the responses induced by bradykinin. A nitric oxide synthase inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME) markedly reduced, whereas removal of the endothelium with 3-[3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS) abolished dilatation to bradykinin and acetylcholine (arterial side only) without affecting that induced by sodium nitroprusside in the mouse arterial and venous mesenteric circuits. In the same two circuits of transgenic B2 knockout mice, the vasodilatory responses to bradykinin were absent, whereas the arterial circuit still responded to acetylcholine by a L-NAME-sensitive vasodilation. Our results suggest the exclusive contribution of B2 receptors located on the endothelium in the vasodilatory effects of bradykinin in the arterial and venous mesenteric circuits of the mouse.

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.

Pharmacology of endothelins in vascular circuits of normal or heterozygous endothelin-A or endothelin-B knockout transgenic mice

Endothelin-1 (ET-1; 0.001-1 nmol) and the ETB receptor agonist IRL-1620 (0.01-1 nmol) induced a dose-dependent vasoconstriction of the arterial and venous mesenteric circuits and of the kidney in normal mice. BQ-123 (10(-7) M) or BQ-788 (10(-7) M) abolished the vasoconstriction induced by ET-1 in the arterial mesenteric and renal vasculatures without affecting that of norepinephrine (NE). In the venous mesenteric vasculature, only BQ-123 reduced the response to ET-1 but not to NE. In other experiments we compared the mesenteric and renal vascular reactivities to ET-1 and IRL-1620 in ETA or ETB heterozygous knockout mice with those of the wild-type strain. We observed a significant reduction in vascular reactivity to ET-1 but not to IRL-1620 in the arterial mesenteric and renal but not the venous mesenteric circuits of ETA knockout mice. In contrast, there was a significant reduction in vascular reactivity to ET-1 and IRL-1620 in the arterial mesenteric and renal circuits of ETB knockout mice. In the venous mesenteric vasculature, only the vasoconstriction induced by IRL-1620 was significantly reduced in the same ETB knockout strain. Our results suggest that, in the mouse, arterial mesenteric and renal vasoconstriction to ET-1 is mediated by both subtypes of ET receptors, whereas venous mesenteric vasoconstriction appears to be mediated uniquely by the ETA receptor subtype. Knockout of only one allele of the ETA or ETB gene appears to be sufficient for reduction of the ET-1 or IRL-1620 vasoconstrictor effects in the mesenteric and renal vascular beds of the mouse.

Contribution of endogenous endothelin-1 and endothelin-A-receptors to the hypertensive state of endothelin-B heterozygous (+/-) knockout mice.

We observed that heterozygous knockout (+/-, KO) of either endothelin-A- (ET(A)) or -B- (ET(B)) receptors significantly reduced the pressor responses to systemically administered endothelin-1 (ET-1) in ET(A) or ET(B) (+/-) KO mice when compared to wild-type (WT) mice (data not shown). Also, we observed that basal mean arterial pressure (MAP) is significantly higher in ET(B) (+/-) (92.7 +/- 1.2 mmHg) (n = 53, p < 0.05) but not ET(A) (+/-) KO mice (70.6 +/- 1.8 mmHg) (n = 23) when compared to their anaesthetized WT littermates (70.1 +/- 0.7 mmHg) (n = 118). A 90 min treatment with either BQ-123 (10 mg/kg), an ET(A)-selective antagonist, or BQ-928 (10 mg/kg), a mixed ET(A)/ET(B) antagonist, administered intraperitoneally, significantly reduced basal MAP of ET(B) (+/-) KO mice almost to the level of their WT treated counterparts (94.9 +/- 4.9 mmHg) (n = 6) vs (+ BQ-123: 59.7 +/- 0.3 mmHg, n = 8); (+ BQ-928: 72.4 +/- 2.6 mmHg, n = 5). It is worthy of note that BQ-123 significantly reduced basal MAP in WT mice but to a lesser extent than in ET(B) (+/-) KO mice (69.6 +/- 2.3 mmHg, n = 8) vs (+ BQ-123: 57.3 +/- 1.4 mmHg, n = 8). In contrast, the ET(B)-selective antagonist, BQ-788 (10 mg/kg i.p.), had no significant effect on MAP even after 90 min of treatment (ET(B) (+/-) KO: (92.3 +/- 2.3 mmHg, n = 6) vs (+ BQ-788: 89.7 +/- 3.1 mmHg, n = 6); WT: (70.5 +/- 3.7 mmHg, n = 7) vs (+ BQ-788: 71.2 +/- 2.0 mmHg, n = 6). Therefore heterozygous KO of either ET(A)- or ET(B)-receptors significantly alters the phenotypic pressor properties of ET-1. We also suggest that there is less ET clearance in ET(B) (+/-) KO mice than in WT mice, which can explain the ET(A)-dependent hypertensive state of the former strain.We evaluated the role of endothelin-B- (ET(B)) receptor-mediated action in the development and maintenance of deoxycorticosterone acetate (DOCA)-salt-induced hypertension, cardiovascular hypertrophy and renal damage, using the spotting lethal (sl) rat which carries a naturally occurring deletion in the ET(B)-receptor gene. Homozygous (sl/sl) rats exhibit abnormal development of the neural crest-derived epidermal melanocytes and the enteric nervous system (ENS), and do not live beyond 1 month because of intestinal aganglionosis and resulting intestinal obstruction. Therefore, the dopamine-beta-hydroxylase (D betaH) promoter was used to direct ET(B) transgene expression in sl/sl rats to support normal ENS development. D betaH-ET(B) sl/sl rats live into adulthood and are healthy, expressing ET(B)-receptor in adrenals and other adrenergic neurons. When homozygous (sl/sl) and wild-type (WT) (+/+) rats, all of which were transgenic, were treated with DOCA and salt for 4 weeks, the homozygous rats exhibited significantly earlier and higher increases in systolic blood pressure than WT rats. The daily oral administration of ABT-627, a selective ET(A)-receptor antagonist, almost completely suppressed the DOCA-salt-induced hypertension in both groups. Renal dysfunction and histological damage induced by DOCA-salt treatment were more severe in homozygous than in WT rats. Increased and marked vascular hypertrophy of the aorta was also observed in homozygous rats, compared with WT rats. Renal and vascular injuries induced by DOCA and salt were significantly improved by ABT-627 administration. We propose that ET(B)-receptor-mediated actions are protective factors in the pathogenesis of DOCA-salt-induced hypertension. ET(A)-mediated actions are at least partly responsible for the increased susceptibility to DOCA-salt-induced hypertension and related tissue injuries in ET(B)-receptor-deficient rats.

Characterization of receptors for endothelins in the guinea pig mesenteric vasculature

Summary: In the present study we characterized the effects of and receptors for endothelins (ETs) in the guinea pig mesenteric arterial and venous vasculatures. Endo-thelin-1 (ET-1) (10–500 pmol) induced a dose-dependent increase of perfusion pressure of the arterial and venous beds. ET-2 (10–500 pmol) also induced a dose-dependent vasoconstriction on both sides of the mesenteric circulation but was less potent than ET-1. In contrast, ET-3 (10–1,000 pmol) and the selective ETB agonist IRL 1620 (1,000 pmol) were inactive. A nitric oxide (NO) synthase inhibitor, L-NAME (200 μM), markedly potentiated the vasoconstrictor response to ET-1 (100 pmol arterial side; 1,000 pmol venous side) on both sides of the mesenteric vasculature. In precontracted mesenteric vessels, ET-1 (0.1–5 pmol) and IRL-1620 (1,000 pmol) induced a small yet significant vasodilation only on the arterial side. Furthermore, BQ-123 (1 μM), an ETA receptor antagonist, significantly reduced the ET-1-induced venoconstriction and completely blocked the vasoconstriction on the arterial side. Hence, the arterial and venous mesenteric vessels of the guinea pig respond to ETs by activation of ETA receptors. Furthermore, the endothelium may act as a physiologic barrier to the constrictor effects of ETs by basally releasing NO.