NADPH diaphorase detects S-nitrosylated proteins in aldehyde-treated biological tissues

Abstract

NADPH diaphorase is used as a histochemical marker of nitric oxide synthase (NOS) in aldehyde-treated tissues. It is thought that the catalytic activity of NOS promotes NADPH-dependent reduction of nitro-blue tetrazolium (NBT) to diformazan. However, it has been argued that a proteinaceous factor other than NOS is responsible for producing diformazan in aldehyde-treated tissues. We propose this is a NO-containing factor such as an S-nitrosothiol and/or a dinitrosyl-iron (II) cysteine complex or nitrosated proteins including NOS. We now report that (1) S-nitrosothiols covalently modify both NBT and TNBT, but only change the reduction potential of NBT after modification, (2) addition of S-nitrosothiols or β- or α-NADPH to solutions of NBT did not elicit diformazan, (3) addition of S-nitrosothiols to solutions of NBT plus β- or α-NADPH elicited rapid formation of diformazan in the absence or presence of paraformaldehyde, (4) addition of S-nitrosothiols to solutions of NBT plus β- or α-NADP did not produce diformazan, (5) S-nitrosothiols did not promote NADPH-dependent reduction of tetra-nitro-blue tetrazolium (TNBT) in which all four phenolic rings are nitrated, (6) cytoplasmic vesicles in vascular endothelial cells known to stain for NADPH diaphorase were rich in S-nitrosothiols, and (7) procedures that accelerate decomposition of S-nitrosothiols, markedly reduced NADPH diaphorase staining in tissue sections subsequently subjected to paraformaldehyde fixation. Our results suggest that NADPH diaphorase in aldehyde-fixed tissues is not enzymatic but is due to the presence of NO-containing factors (free SNOs or nitrosated proteins such as NOS), which promote NADPH-dependent reduction of NBT to diformazan.

Pericyte control of pancreatic islet blood flow impacts glucose homeostasis

“…To assess local nitric oxide levels, we used NADPH-diaphorase histochemistry technique. NADPH diaphorase is commonly used as a histochemical marker of nitric oxide synthase and other nitric oxide-containing factors in aldehyde-fixed tissues 41 .…”

Photolytic Measurement of Tissue S-Nitrosothiols in Rats and Humans In Vivo

“…Thiol-NO adducts signal many processes in mammalian biology [1,2]. These include signaling in the central and peripheral nervous systems [3,10] as well as the cardiovascular [1,2], and respiratory systems [3,7]. S-Nitrosothiols can serve to stabilize and to transfer NO groups to target proteins in the form of post-translational protein modifications [20][21][22][23] and to bind as ligands to specific target proteins [24].…”

“…S-Nitrosothiols can serve to stabilize and to transfer NO groups to target proteins in the form of post-translational protein modifications [20][21][22][23] and to bind as ligands to specific target proteins [24]. Moreover, recent data demonstrate that they are stored in vascular and neuronal tissues in the form of vesicles [10]. However, in tissues and vesicles extracted for ex vivo analysis, they can be quite labile [15,25].…”

“…For example, hemoglobin R to T conformational switch caused by acidosis, hypercapnia, hypoxia, and other physiological phenomena causes formation of signaling S-nitrosothiols [9]. Recent evidence suggests that these molecules are also stored in endothelial and neuronal vesicles for subsequent release [10]. Abnormal S-nitrosothiol levels have been measured ex vivo in a variety of human conditions, ranging from diseases like asthma [11], pulmonary hypertension [12] and sepsis [7] to altitude acclimatization [13].…”

“…Here, we have developed a system for using chemiluminescence downstream from tissue photolysis to assay tissue S-nitrosothiols in living mammals (rats and humans) in situ (Figure 1). Nitric oxide released from S-nitrosothiols stored in endocytic vesicles [10] or blood [7] can diffuse through skin and other tissue to be recorded by the chemiluminescence device. Note that several authors have shown that low quantities of NO can be detected adjacent to blood in the pulmonary capillaries [19].…”