Agatha Gambino

Vascular smooth muscle relaxation mediated by nitric oxide donors: a comparison with acetylcholine, nitric oxide andnitroxyl ion

Vasorelaxant properties of three nitric oxide (NO) donor drugs (glyceryl trinitrate, sodium nitroprusside and spermine NONOate) in mouse aorta (phenylephrine pre‐contracted) were compared with those of endothelium‐derived NO (generated with acetylcholine), NO free radical (NO·; NO gas solution) and nitroxyl ion (NO−; from Angelis salt). The soluble guanylate cyclase inhibitor, ODQ (1H‐(1,2,4‐)oxadiazolo(4,3‐a)‐quinoxalin‐1‐one; 0.3, 1 and 10 μM), concentration‐dependently inhibited responses to all agents. 10 μM ODQ abolished responses to acetylcholine and glyceryl trinitrate, almost abolished responses to sodium nitroprusside but produced parallel shifts (to a higher concentration range; no depression in maxima) in the concentration‐response curves for NO gas solution, Angelis salt and spermine NONOate. The NO· scavengers, carboxy‐PTIO, (2‐(4‐carboxyphenyl)‐4,4,5,5‐tetramethyl‐imidazoline‐1‐oxyl‐3‐oxide; 100 μM) and hydroxocobalamin (100 μM), both inhibited responses to NO gas solution and to the three NO donor drugs, but not Angelis salt. Hydroxocobalamin, but not carboxy‐PTIO, also inhibited responses to acetylcholine. The NO− inhibitor, L‐cysteine (3 mM), inhibited responses to Angelis salt, acetylcholine and the three NO donor drugs, but not NO gas solution. The data suggest that, in mouse aorta, responses to all three NO donors involve (i) activation of soluble guanylate cyclase, but to differing degrees and (ii) generation of both NO· and NO−. Glyceryl trinitrate and sodium nitroprusside, which generate NO following tissue bioactivation, have profiles resembling the profile of endothelium‐derived NO more than that of exogenous NO. Spermine NONOate, which generates NO spontaneously outside the tissue, was the drug that most closely resembled (but was not identical to) exogenous NO.

Vascular endothelial growth factor-B-deficient mice show impaired development of hypoxic pulmonary hypertension

OBJECTIVE To test the hypothesis that Vegf-B contributes to the pulmonary vascular remodelling, and the associated pulmonary hypertension, induced by exposure of mice to chronic hypoxia. METHODS Right ventricular systolic pressure, the ratio of right ventricle/[left ventricle+septum] (RV/[LV+S]) and the thickness of the media (relative to vessel diameter) of intralobar pulmonary arteries (o.d. 50-150 and 151-420 microm) were determined in Vegfb knockout mice (Vegfb(-/-); n=17) and corresponding wild-type mice (Vegfb(+/+); n=17) exposed to chronic hypoxia (10% oxygen) or housed in room air (normoxia) for 4 weeks. RESULTS In Vegfb(+/+) mice hypoxia caused (i) pulmonary hypertension (a 70% increase in right ventricular systolic pressure compared with normoxic Vegfb(+/+) mice; P<0.001), (ii) right ventricular hypertrophy (a 66% increase in RV/[LV+S]; P<0.001) and (iii) pulmonary vascular remodelling (a 27-36% increase in pulmonary arterial medial thickness; P<0.05). In contrast, in Vegfb(-/-) mice hypoxia did not cause any increase in either right ventricular systolic pressure or pulmonary arterial medial thickness; also right ventricular hypertrophy (41% increase in RV/[LV+S]; P<0.001) was less pronounced (P<0.05) than in Vegfb(+/+) mice. CONCLUSION Vegf-B may have a role in the development of chronic hypoxic pulmonary hypertension in mice by contributing to pulmonary vascular remodelling. If so, the effect of Vegf-B appears to be different from that of Vegf-A which is reported to protect against, rather than contribute to, hypoxia-induced pulmonary vascular remodelling.

The in vitro pulmonary vascular effects of FK409 (nitric oxide donor): a study in normotensive and pulmonary hypertensive rats.

Vasorelaxant responses to the nitric oxide (NO) donor, FK409 ((±)‐(E)‐4‐ethyl‐2‐[(E)‐hydroxyimino]‐5‐nitro‐3‐hexenamide), were evaluated on precontracted isolated ring preparations of main pulmonary artery and intralobar pulmonary artery from rats. On main pulmonary artery FK409 fully reversed the precontractions. Responses were inhibited by methylene blue but were independent of the endothelium. The potency (−log EC50) of FK409 was the same on preparations contracted with noradrenaline (7.62) or the thromboxane‐mimetic, U44619 (7.63). On intralobar pulmonary artery FK409 caused only 80% reversal of the precontraction and was 2 fold less potent than on main pulmonary artery. These differences in maximum response and potency between main and intralobar arteries are in keeping with previous findings with other NO donors. Pulmonary hypertension was induced in rats by chronic exposure to hypoxia (10% O2) for 1 or 4 weeks. Main pulmonary arteries from 1 week hypoxic rats had inherent tone and showed spontaneous contractile activity. In these arteries FK409 reversed not only the precontraction induced by noradrenaline but also the inherent tone. However, FK409 was 17 fold less potent than in control arteries, reflecting previous findings with other NO donors. Main pulmonary arteries from 4 week hypoxic rats had minimal inherent tone and were quiescent and FK409 was 4.5 fold less potent than in control arteries. In intralobar pulmonary arteries from 4 week hypoxic rats FK409 was 12 fold less potent than in controls. Treatment of arteries with either (a) in vitro hypoxic conditions (PO2 of solution in organ bath <10 mmHg) or (b) superoxide dismutase (SOD; 150 u ml−1) together with catalase (1200 u ml−1) significantly increased the potency of FK409 in preparations from hypoxic rats but had no effect on the potency in control preparations. Neither SOD nor catalase, alone, nor the nitric oxide synthase inhibitor, NG‐nitro‐L‐arginine methyl ester, had any effect on the potency of FK409 in preparations from control or hypoxic rats. It is concluded that the reduction in potency of FK409 seen in pulmonary arteries from rats with chronic hypoxic pulmonary hypertension may be due in part to the presence of one or more reactive oxygen species (either hydroxyl or superoxide plus hydrogen peroxide).

S‐Nitrosocaptopril: in vitro characterization of pulmonary vascular effects in rats

On rat isolated pulmonary arteries, vasorelaxation by S‐nitrosocaptopril (SNOcap) was compared with S‐nitrosoglutathione (GSNO) and nitroprusside, and inhibition by SNOcap of contractions to angiotensin I was compared with the angiotensin converting enzyme (ACE) inhibitor, captopril. SNOcap was equipotent as a vasorelaxant on main (i.d. 2–3 mm) and intralobar (i.d. 600 μm) pulmonary arteries (pIC50 values: 5.00 and 4.85, respectively). Vasorelaxant responses reached equilibrium rapidly (2–3 min). Pulmonary vasorelaxant responses to SNOcap, like GSNO, were (i) partially inhibited by the soluble guanylate cyclase inhibitor, ODQ (1H‐(1,2,4) oxadiazolo(4,3‐a)‐quinoxalin‐1‐one; 3 μM) whereas responses to nitroprusside were abolished and (ii) potentiated by hydroxocobalamin (HCOB; NO· free radical scavenger; 100 μM) whereas responses to nitroprusside were inhibited. The relative potencies for pulmonary vasorelaxation compared with inhibition of platelet aggregation were: SNOcap 7 : 1; GSNO 25 : 1; nitroprusside >2000 : 1. SNOcap, like captopril, concentration‐dependently and time‐dependently increased the EC50 for angiotensin I but not angiotensin II. The dependence on incubation time was independent of the presence of tissue but differed for SNOcap and captopril. This difference reflected the slow dissociation of SNOcap and instability of captopril, and precluded a valid comparison of the potency of the two drugs. After prolonged incubation (5.6 h) SNOcap was more effective than captopril. Thus, in pulmonary arteries SNOcap (i) possesses NO donor properties characteristic of S‐nitrosothiols but different from nitroprusside and (ii) inhibits ACE at least as effectively as captopril. These properties suggest that SNOcap could be valuable in the treatment of pulmonary hypertension.

The effect of the endothelin ETA receptor antagonist CI-1020 on hypoxic pulmonary vasoconstriction

The mechanism of Hypoxic Pulmonary Vasoconstriction is unknown. The role of endothelin-1 in hypoxic pulmonary vasoconstriction was studied in precontracted small and large pulmonary arteries using the endothelin ETA receptor antagonist sodium-2-benzol [1,3]dioxol-5-yl-4-(4-methoxyphenyl)-4-oxo-3-(3,4,5-trimethoxy-ben zyl)-but-2-enoate (CI-1020). Small rat pulmonary arteries exhibit a mixed endothelin ETA receptor and endothelin ETB2 receptor population whereas large rat pulmonary arteries contain only endothelin ETA receptors. CI-1020 inhibited endothelin-1 in small vessels via endothelin ETA receptor blockade (1 and 10 microM) and at high concentrations via endothelin ETA receptor and endothelin ETB2 receptor blockade (100 microM). CI-1020 (0.01, 0.1 and 1 microM) inhibited endothelin-1 in large vessels via endothelin ETA receptor blockade alone. CI-1020 (1, 10 and 100 microM) significantly reduced hypoxic pulmonary vasoconstriction in small vessels, by -9.8+/-1.4, -9.2+/-2.3 and -8.0+/-1.7% 80 mM K+, respectively, compared to +2.5+/-4.2% with vehicle (P < 0.05). CI-1020 (0.01, 0.1 and 1 microM) had no significant effect upon hypoxic pulmonary vasoconstriction in large vessels. In small, but not large, pulmonary arteries hypoxic pulmonary vasoconstriction is due in part to the action of endothelin-1 at the endothelin ETA receptor.

S-Nitrosocaptopril: acute in-vivo pulmonary vasodepressor effects in pulmonary hypertensive rats

The effects of S‐nitrosocaptopril (SNOcap), administered either intravenously or by oral gavage, on pulmonary artery pressure (PAP) were examined in anaesthetised normotensive rats and rats with hypoxic pulmonary hypertension (10% oxygen for 1 week). Mean PAP (MPAP) values in hypoxic and normoxic rats were (mmHg) 26± 1.7 and 15± 1.1, respectively. When given intravenously, 1 mg kg−1 SNOcap reduced MPAP by 28 and 32% in hypoxic and normoxic rats, respectively. The effects of 2 mg kg−1 were no greater than those of 1 mg kg−1. Pulmonary vasodepressor responses reached equilibrium in 1.7

Krn2391: Dual Action On Rat Pulmonary Artery And No Loss Of Potency In Pulmonary Hypertension

pL0.18 min following intravenous administration. When given orally 30 min before the measurement of PAP, 30 mg kg−1, but not 10 mg kg−1, significantly reduced MPAP in hypoxic rats to 17

Functional effects of 4-aminopyridine (4-AP) on pulmonary and systemic vessels from normoxic control and hypoxic pulmonary hypertensive rats

pL 1.5 mmHg. These in‐vivo data are consistent with previous in‐vitro data showing that SNOcap has direct pulmonary vasorelaxant properties in both large and small pulmonary arteries and also show that SNOcap causes pulmonary vasodepression in the setting of pulmonary hypertension. Since SNOcap also inhibits pulmonary vascular angiotensin converting enzyme (ACE) in pulmonary blood vessels (previous study), it would be an interesting drug with which to assess the benefits of direct pulmonary vasodilatation combined with ACE inhibition (which attentuates pulmonary vascular remodelling) in a long‐term study in pulmonary hypertension.

Potentiation of 5-hydroxytryptamine (5-HT) responses by a 5-HT uptake inhibitor in pulmonary and systemic vessels: effects of exposing rats to hypoxia

1. The pulmonary vasorelaxant properties of KRN2391 (N‐cyano‐N′‐(2‐nitroxyethyl)‐3‐pyridinecarboximidamide) were examined in isolated ring preparations of main (MPA) and intralobar (IPA) pulmonary artery from control and pulmonary hypertensive rats (exposure to hypoxia, 10% oxygen, for 1 week).