Comparison of the redox forms of nitrogen monoxide with the nitrergic transmitter in the rat anococcygeus muscle

Abstract

1. A sustained tone was produced in rat isolated anococcygeus muscles with guanethidine and clonidine and relaxant responses were elicited by electrical stimulation of its nitrergic nerves and by the three redox forms of nitrogen monoxide. 2. The nitroxyl anion (NO ) was donated by dissociation of Angeli's salt; the free radical (NO*) was from an aqueous solution of nitric oxide gas; the nitrosonium cation (NO+) was donated by dissociation of nitrosonium tetrafluoroborate. 3. The concentrations producing approximately 50% relaxations of the anococcygeus muscle were 0.3 microM for Angeli's salt (nitroxyl), 0.5 microM for NO* and 100 microM for nitrosonium tetrafluoroborate. Nitrergic nerve stimulation at 1 Hz for 10 s produced equivalent relaxant responses. 4. The superoxide generator pyrogallol (100 microM) had no effect on responses to nitrergic nerve stimulation or Angeli's salt but significantly reduced responses to NO* and nitrosonium tetrafluoroborate. 5. The NO* scavenger carboxy-PTIO (100 microM) had no effect on responses to nitrergic nerve stimulation or Angeli's salt but significantly reduced responses to NO* and nitrosonium tetrafluoroborate. 6. Hydroxocobalamin (30 microM) had no significant effect on responses to the nitrergic transmitter, enhanced the response to Angeli's salt, and significantly reduced responses to NO* and nitrosonium tetrafluoroborate. 7. The findings suggest that the nitroxyl anion donated by Angeli's salt is a better candidate than NO* to serve as the nitrergic transmitter in the rat anococcygeus muscle, although it still does not behave exactly like the transmitter.

Figure 1

Representative relaxations of anococcygeus muscles produced by electrical field stimulation of nitrergic nerves (EFS; 1 Hz for 10 s), nitroxyl (NO⁻) donated from Angeli's salt, NO^• from an aqueous solution of nitric oxide gas, and NO⁺ donated from nitrosonium tetrafluoroborate at the points indicated by the symbols in time-control experiments. The parameters of stimulation and concentrations used produced relaxations approximately equal to 50% of the pre-existing tone (see Figure 2).

Figure 2

Concentration-response curves for the relaxant actions of nitroxyl (NO⁻ donated from Angeli's salt), NO^• in aqueous solution and nitrosonium (NO⁺ donated from nitrosonium tetrafluoroborate) and frequency-response curve for electrical field stimulation (EFS; for 10 s periods). I-bars indicate standard errors of means (n=3–6 for each point).

Figure 3

Representative tracings of effects of ODQ on responses to electrical field stimulation of nitrergic nerves (EFS; 1 Hz for 10 s), NO⁻ donated from Angeli's salt, NO^• and NO⁺ donated from nitrosonium tetrafluoroborate.

Figure 4

Blockade of responses to nitrergic nerve stimulation (EFS; 1 Hz for 10 s), Angeli's salt (NO⁻, 0.3 μM), NO^• (0.5 μM) and nitrosonium tetrafluoroborate (NO⁺; 100 μM) by 1 μM ODQ. Column heights indicate means and I-bars are s.e.means with n indicated by the number at the foot of each column; ^* indicates significant difference from time control (unpaired t-test, P<0.05).

Figure 5

Representative tracings of effects of pyrogallol (100 μM) on responses to electrical field stimulation of nitrergic nerves (EFS; 1 Hz for 10 s). Angeli's salt (NO⁻; 0.3 μM), NO^• (0.5 μM) and nitrosonium tetrafluoroborate (NO⁺; 100 μM).

Figure 6

Effects of time controls, pyrogallol (100 μM), carboxy-PTIO (100 μM), and hydroxocobalamin (30 μM) on relaxations of rat anococcygeus muscles produced by nitrergic nerve stimulation (1 Hz for 10 s), Angeli's salt (NO⁻; 0.3 μM), NO^• (0.5 μM) and nitrosonium tetrafluoroborate (NO⁺; 100 μM) expressed as percentages of relaxations before addition of a modifying agent (or elapse of time for controls). Column heights indicate means and I-bars are s.e.means. The number of muscles contributing to each mean is indicated at the foot of each column.

Figure 7

Representative tracings of effects of carboxy-PTIO (100 μM) on responses to electrical field stimulation of nitrergic nerves (EFS; 1 Hz for 10 s), Angeli's salt (NO⁻; 0.3 μM), NO^• (0.5 μM) and nitrosonium tetrafluoroborate (NO⁺; 100 μM).

Figure 8

Representative tracings of effects of hydroxocobalamin (30 μM) on responses to electrical field stimulation of nitrergic nerves (EFS; 1 Hz for 10 s), Angeli's salt (NO⁻; 0.3 μM), NO^• (0.5 μM) and nitrosonium tetrafluoroborate (NO⁺; 100 μM).