The nitroxyl anion (HNO) donor, Angeli's salt, does not develop tolerance in vivo

In addition to the free radical form of nitric oxide (NO^•), the reduced form of NO (NO^-/HNO) is also produced endogenously, causes vasorelaxation and has therapeutic potential in the treatment of heart failure. Given we have shown previously that, unlike the NO^• donor glyceryl trinitrate (GTN), the HNO donor Angeli's salt (AS) does not develop tolerance in vitro [1], this study aimed to determine whether AS was also resistant to the development of tolerance in vivo. Initial experiments using WKY rats confirmed that infusion with the HNO scavenger, N-Acetyl-L-cysteine (6.7 μmol/kg/min) significantly (P < 0.0001) attenuated the concentration dependent depressor responses to AS, whilst those to GTN or the additional NO^• donor, DEA/NO, were unchanged. Rats were instrumented with arterial and venous catheters and the depressor effects of GTN, AS and DEA/NO were examined in vehicle treated and GTN (10 μg/kg/min), AS (20 μg/kg/min) or DEA/NO (2 μg/kg/min) pre-treated animals (3 day infusion via osmotic mini pump). Following this, aortae were removed and ex vivo tolerance to all vasodilators assessed via examining vasorelaxation responses. GTN pre-treatment significantly blunted the depressor response to 50 μg/kg GTN (-9 ± 1 mmHg MAP) compared to vehicle treated rats (-40 ± 2 mmHg MAP; n = 8–9; P < 0.001) but did not alter the depressor responses to either AS or DEA/NO. Similarly, in vitro relaxation responses to GTN in GTN pre-treated rats showed a significant 6-fold decrease in sensitivity (pEC₅₀ = 6.72 ± 0.23; R_max = 79.4 ± 3.4%; n = 7) compared with vehicle treated rats (pEC₅₀ = 7.51 ± 0.14; R_max= 93.4 ± 2.1%; n = 6; P < 0.05) yet vasorelaxation to AS and DEA/NO was unchanged. In addition, vasorelaxation to acetylcholine (ACh) was attenuated in GTN pre-treated rats (R_max = 54.4 ± 5.4%; n = 6) compared to vehicle treated rats (R_max = 77.1 ± 2.3%; n = 5). In contrast, AS was resistant to in vivo tolerance development. Thus the depressor response to 200 μg/kg AS (-33 ± 4 mmHg MAP; n = 6–7) was unchanged following AS pre-treatment (-34 ± 3 mmHg MAP). Furthermore AS did not cause cross tolerance to either GTN or DEA/NO in vivo. Likewise, there was no change in the in vitro relaxation responses to AS in AS pre-treated rats (pEC₅₀ = 6.77 ± 0.15; R_max = 92.3 ± 1.7%; n = 7) compared with vehicle treated rats (pEC₅₀ = 6.77 ± 0.17; R_max = 93.0 ± 2.7%; n = 6), nor was there a reduced response to ACh. Like AS, DEA/NO did not develop tolerance either in vitro or in vivo. In conclusion, the HNO donor AS does not develop tolerance in vivo, thus suggesting that HNO donors may represent a novel class of nitrovasodilator for the treatment of cardiovascular disorders, such as heart failure and angina.