Synchronous detection of vascular tension and nitric oxide release in pulmonary artery: A combined application of confocal wire myograph with confocal laser scanning microscopy

Abstract

Objectives

To detect the vascular tension and nitric oxide (NO) release synchronously in mice pulmonary artery, we perform two experiments and present a novel application of confocal wire myograph coupled with the confocal laser scanning microscopy.

Methods

In the first experiment, viable endothelium-intact mouse pulmonary artery (outer diameter 100–300 μM) rings underwent a one-hour preincubation with a NO-specific fluorescent dye, 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate Calbiochem (2.5 μM), and then precontracted with phenylephrine (Phen, 10⁻⁶ M), and subsequently dilated in acetylcholine (ACh, 10⁻⁶ M – 10⁻⁴ M). The endothelium-dependent vasorelaxation and NO generation in pulmonary artery rings were simultaneously recorded. In the second experiment, after 30-min incubation with the former NO fluorescent dye, the qualified pulmonary artery rings were co-incubated for another 30 min with a nitric oxide synthase inhibitor, 10⁻⁴ M Nω-nitro-L-arginine-methyl-ester (L-NAME), and then pretreated with Phen (10⁻⁶ M) followed by ACh (10⁻⁵ M). The Ach-induced vasodilation and NO release were recorded simultaneously.

Results

ACh (10⁻⁶ M – 10⁻⁴ M) promoted pulmonary artery relaxation and intracellular NO release in a dose-dependent manner. Additionally, L-NAME (10⁻⁴ M) significantly attenuated the vasodilatation and the intracellular NO release.

Conclusions

This combined application visually confirms that the synchronous changes in Ach induced vasodilation and NO release, which provides a new method for cardiovascular research.