Effects of agents that inactivate free radical NO (NO*) on nitroxyl anion-mediated relaxations, and on the detection of NO* released from the nitroxyl anion donor Angeli's salt

Abstract

1. The effects of agents that inactivate free radical nitric oxide (carboxy-PTIO, hydroxocobalamin and pyrogallol) were tested on relaxations produced by the nitroxyl anion (NO(-)) donor Angeli's salt in rat aortic rings and anococcygeus muscles. The amount of NO(*) generated from Angeli's salt in the presence of these agents was measured using a NO(*)-selective electrode sensor. 2. Carboxy-PTIO (100, 300 microM), hydroxocobalamin (30, 100 microM) and pyrogallol (10, 30 microM) significantly reduced relaxations produced by Angeli's salt (0.3 microM) in aortic rings but not in anococcygeus muscles. 3. NO(*) generated from Angeli's salt (0.1 - 10 microM), as detected by the sensor electrode, was less than 0.5% of the amount of Angeli's salt added. Carboxy-PTIO (100 microM) and hydroxocobalamin (30 microM), but not pyrogallol significantly increased the amount of NO(*) detected. 4. In the presence of an oxidizing agent copper [II] (as CuSO(4) 100 microM), the amount of NO(*) detected from 0.3 microM of Angeli's salt increased from an undetectable level of 142.7+/-15.7 nM (equivalent to 47.6% of Angeli's salt added). Under these conditions, carboxy-PTIO, hydroxocobalamin and pyrogallol significantly reduced the amount of NO(*) detected from Angeli's salt as well as the signal generated by an equivalent amount of authentic NO (0.33 microM). 5. The difference in effects of these agents on relaxations to Angeli's salt in the aorta and the anococcygeus muscle may be explained by the ready conversion of NO(-) to NO(*) in the aorta through an unidentified mechanism, which makes NO(-) susceptible to inactivation by these agents. Furthermore, in addition to inactivating NO(*), carboxy-PTIO and hydroxocobalamin may themselves oxidize NO(-) to NO(*), albeit slightly.

Figure 1

Mean data on the effects of carboxy-PTIO (100, 300 μM), hydroxocobalamin (30, 100 μM) and pyrogallol (30, 100 μM) on relaxations produced by Angeli's salt (0.3 μM) in rat aortic rings (n=4 – 5) and anococcygeus muscles (n=4 – 7). Responses are expressed as a percentage of contractile tone produced by phenylephrine (1 μM) in aortic rings and by guanethidine (20 μM) plus clonidine (0.1 μM) in anococcygeus muscles. Columns represent means and T-bars indicate s.e.means. Asterisks indicate significant differences from the corresponding control values (P<0.05, paired t-test).

Figure 2

Traces illustrating the electrochemical detection of NO^• from Angeli's salt (0.1, 1, 10 μM) in PSS containing edetate (0.067 mM) in the absence (control) and presence of carboxy-PTIO (100 μM), hydroxocobalamin (30 μM) and pyrogallol (30 μM) using the amiNO-700 sensor electrode.

Figure 3

Mean data on the effects of carboxy-PTIO (100 μM), hydroxocobalamin (30 μM) and pyrogallol (30 μM) on NO^• generated from Angeli's salt (0.1, 1 and 10 μM) in PSS containing edetate (0.067 mM). NO^• is expressed in nanomoles. Symbols represent means and T-bars indicate s.e.means (n=4). Asterisks indicate significant differences from control (P<0.05, ANOVA).

Figure 4

Traces illustrating the detection of NO^• from Angeli's salt (0.3 μM) in PSS containing CuSO₄ (100 μM) in the absence (control) and presence of carboxy-PTIO (100 μM), hydroxocobalamin (30 μM) and pyrogallol (30 μM) using the amiNO-700 sensor electrode.

Figure 5

Mean data of the effects of carboxy-PTIO (100 μM), hydroxocobalamin (30 μM) and pyrogallol (30 μM) on NO^• generated from Angeli's salt (0.3 μM) in PSS containing CuSO₄ (100 μM). NO^• is expressed in nanomoles. Column heights represents mean and T-bars indicate s.e.means (n=4). Asterisks indicate significant differences from control values (P<0.05, paired t-test).