A role for nitroxyl (HNO) as an endothelium-derived relaxing and hyperpolarizing factor in resistance arteries

Abstract

Background and purpose: Nitroxyl (HNO) is emerging as an important regulator of vascular tone as it is potentially produced endogenously and dilates conduit and resistance arteries. This study investigates the contribution of endogenous HNO to endothelium-dependent relaxation and hyperpolarization in resistance arteries.

Experimental approach: Rat and mouse mesenteric arteries were mounted in small vessel myographs for isometric force and smooth muscle membrane potential recording.

Key results: Vasorelaxation to the HNO donor, Angeli's salt, was attenuated in both species by the soluble guanylate cyclase inhibitor (ODQ, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one), the voltage-dependent K(+) channel inhibitor, 4-aminopyridine (4-AP) and the HNO scavenger, L-cysteine. In mouse mesenteric arteries, nitric oxide (NO) synthase inhibition (with L-NAME, N(omega)-Nitro-L-arginine methyl ester) markedly attenuated acetylcholine (ACh)-mediated relaxation. Scavenging the uncharged form of NO (NO(*)) with hydroxocobalamin (HXC) or HNO with L-cysteine, or 4-AP decreased the sensitivity to ACh, and a combination of HXC and L-cysteine reduced ACh-mediated relaxation, as did L-NAME alone. ACh-induced hyperpolarizations were significantly attenuated by 4-AP alone and in combination with L-NAME. In rat mesenteric arteries, blocking the effects of endothelium-derived hyperpolarizing factor (EDHF) (charybdotoxin and apamin) decreased ACh-mediated relaxation 10-fold and unmasked a NO-dependent component, mediated equally by HNO and NO(*), as HXC and L-cysteine in combination now abolished vasorelaxation to ACh. Furthermore, ACh-evoked hyperpolarizations, resistant to EDHF inhibition, were virtually abolished by 4-AP.

Conclusions and implications: The factors contributing to vasorelaxation in mouse and rat mesenteric arteries are NO(*) = HNO > EDHF and EDHF > HNO = NO(*) respectively. This study identified HNO as an endothelium-derived relaxing and hyperpolarizing factor in resistance vessels.

Figure 1

Concentration–response curves to ACh in (A) MMA and (B) RMA in the absence (Control) or presence of ChTx (100 nmol·L⁻¹) & apamin (100 nmol·L⁻¹) in combination, l-NAME (100 µmol·L⁻¹) & ODQ (100 µmol·L⁻¹) in combination, l-NAME, ODQ, ChTx & apamin in combination and K⁺ (30 mmol·L⁻¹), l-NAME & ODQ in combination. Indomethacin (10 µmol·L⁻¹) was present throughout. Values are expressed as % reversal of pre-contraction and given as mean ± SEM, where n = number of animals. ***P < 0.001 for pEC₅₀ versus untreated control (one-way anova), ^†††P < 0.001 versus untreated control (two-way anova), ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 for response at 10 µmol·L⁻¹ or 30 µmol·L⁻¹ versus untreated control (one-way anova). ACh, acetylcholine; ChTx, charybdotoxin; EDHF, endothelium-derived hyperpolarizing factor; l-NAME, N^ω-Nitro-L-arginine methyl ester; MMA, mouse mesenteric arteries; NO, nitric oxide; ODQ, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one; RMA, rat mesenteric arteries.

Figure 2

Concentration–response curves to ACh in either (A) MMA in the absence (Control) or presence of HXC (100 µmol·L⁻¹), and l-NAME (100 µmol·L⁻¹) & HXC in combination or (B) RMA in the absence (Control) or presence of ChTx (100 nmol·L⁻¹) & apamin (100 nmol·L⁻¹) in combination (reproduced from Figure 1B for comparison), and ChTx, apamin & HXC in combination. Indomethacin (10 µmol·L⁻¹) was present throughout. Values are expressed as % reversal of pre-contraction and given as mean ± SEM, where n = number of animals. ^δδP < 0.01 for pEC₅₀ versus ChTx & apamin treatment (one-way anova), ^††P < 0.01, ^†††P < 0.001 versus untreated control (two-way anova), ^#P < 0.05, ^###P < 0.001 for response at 10 µmol·L⁻¹ or 30 µmol·L⁻¹ versus untreated control (one-way anova). ACh, acetylcholine; ChTx, charybdotoxin; HNO, nitroxyl; HXC, hydroxocobalamin; l-NAME, N^ω-Nitro-L-arginine methyl ester; MMA, mouse mesenteric arteries; NO, nitric oxide; RMA, rat mesenteric arteries.

Figure 3

Vasorelaxation responses to ACh in either (A) MMA in the absence (Control) or presence of 4-AP (1 mmol·L⁻¹), l-cysteine (3 mmol·L⁻¹) and l-cysteine & HXC (100 µmol·L⁻¹) in combination, or (B) RMA in the absence (Control; reproduced from Figure 1B for comparison) or presence of ChTx (100 nmol·L⁻¹) & apamin (100 nmol·L⁻¹) alone (reproduced from Figure 1B for comparison), or combined with 4-AP (1 mmol·L⁻¹), l-cysteine (3 mmol·L⁻¹) or l-cysteine & HXC (100 µmol·L⁻¹). Smooth muscle membrane potential recordings illustrating ACh-evoked hyperpolarization in MMA (C) and RMA (D) following depolarization with cirazoline. Hyperpolarizations were recorded in the absence and presence of 4-AP alone (C, E) or combined with l-NAME (C, E); or ChTx and apamin alone (D, F) or combined with 4-AP (D, F). In (C) and (D) // indicates a break in a continuous recording made in the same cell. Indomethacin (10 µmol·L⁻¹) was present throughout. Values are expressed as (A, B) % reversal of contraction or (E, F) % of maximal control hyperpolarization and given as mean ± SEM, where n = number of animals. ^†P < 0.05, ^††P < 0.01, ^†††P < 0.001 versus untreated control (two-way anova), ^‡‡P < 0.01, ^‡‡‡P < 0.001 versus ChTx & apamin treatment (two-way anova), ^##P < 0.01, ^###P < 0.001, for response at 10 µmol·L⁻¹ or 30 µmol·L⁻¹ versus untreated control (one-way anova). 4-AP, 4-aminopyridine; ACh, acetylcholine; ChTx, charybdotoxin; HNO, nitroxyl; HXC, hydroxocobalamin; l-NAME, N^ω-Nitro-L-arginine methyl ester; MMA, mouse mesenteric arteries; NO, nitric oxide; RMA, rat mesenteric arteries.

Figure 4

Concentration–response curves to ACh in either (A) MMA in the absence (Control; reproduced from Figure 3A for comparison) or presence of l-NAME (100 µmol·L⁻¹), l-NAME & HXC (100 µmol·L⁻¹), and l-NAME & l-cysteine (3 mmol·L⁻¹), or (B) RMA in the presence of l-NAME (100 µmol·L⁻¹), ChTx (100 nmol·L⁻¹) & apamin (100 nmol·L⁻¹) combined with either: 4-AP (1 mmol·L⁻¹), HXC (100 µmol·L⁻¹) or l-cysteine (3 mmol·L⁻¹). Indomethacin (10 µmol·L⁻¹) was present throughout. Values are expressed as % reversal of contraction and given as mean ± SEM, where n = number of animals. (A) ^‡‡‡P < 0.001 versus control (two-way anova), ^##P < 0.01 for response at 30 µmol·L⁻¹ versus Control (one-way anova). (B) ^ΨΨP < 0.01, ^ΨΨΨP < 0.001 versus l-NAME, ChTx & apamin treatment (two-way anova), ^###P < 0.001, for response at 10 µmol·L⁻¹ versus l-NAME, ChTx & apamin treatment (one-way anova). 4-AP, 4-aminopyridine; ACh, acetylcholine; ChTx, charybdotoxin; HNO, nitroxyl; HXC, hydroxocobalamin; l-NAME, N^ω-Nitro-L-arginine methyl ester; MMA, mouse mesenteric arteries; NO, nitric oxide; RMA, rat mesenteric arteries.