Fig 2: The effect of caveolae disruption by cholesterol depletion on the immunoreactivities of caveolin-1 and nNOS in smooth muscle cell membrane and the response to LNNA following tissue depolarization by KCl in BALB/c mouse small intestine. (A) immunostained cryosections of BALB/c mice small intestinal tissues showing the effects of Me-ß-CDX treatment (a–c) and Me-ß-CDX followed by WSC panels (d–f). caveolin-1 (a, red) and nNOS (b, green) immunoreactivities are very much reduced following Me-ß-CDX treatment. The immunoreactivities are restored by WSC (d for caveolin-1, red and e for nNOS, green). Panels c and f are merged images showing the colocalization of caveolin-1 and nNOS in smooth muscle cell membrane (yellow) that is restored after WSC treatment. Scale bars are 10 µm. (B) Representative tracings showing that cholesterol depletion reduced the increase in tone by LNNA and that this was restored following treatment with WSC to replenish membrane cholesterol. (C) increase in tone after LNNA is added following KCl normalized to the active tome before addition of KCl. n-values are 13 for the control and five for Me-ß-CDX and Me-ß-CDX/WSC treated tissues. Statistical analysis was done by anova followed by Bonferroni post hoc test and significance is denoted by *P < 0.05, **P< 0.01, and ***P< 0.001.

Fig 3: Immunostained cryosections (A) and whole mount preparations (B) of Cav1+/+ and Cav1-/- mice small intestine. In section (A), panels (a-c) show caveolin-1 (a, red) and nNOS (b, green) immunoreactivity and their colocalization (c, yellow) in the smooth muscle cell membrane of Cav1+/+ mice small intestinal tissue. Panels (d–f) show the absence of both caveolin-1 (d) and nNOS (e) immunoreactivities in Cav1-/- mice small intestine. Panel f is a merged image. The scale bar is 10 µm. icm, inner circular muscle layer; ocm, outer circular muscle layer and lm, longitudinal muscle layer. Arrows point to interstitial cells of Cajal in the myenteric plexus layer. Section (B) shows immunostained myenteric ganglia in Cav1+/+ (a–d) and Cav1-/- (e–h) mice. Myenteric neuron cell bodies were localized by staining with HuC/D antibodies that stains the cytoplasm of neuron cell bodies (a and e, red). Panels b and e show neuron cells in myenteric ganglia stained with anti-nNOS COOH terminal epitope (blue). Panels c and f are merged images. Scale bars are 10 µm.

Fig 4: Effects of AMN082 on TBI-induced neuronal nitrosative stress in the pericontusional cortex on the 3rd day after TBI. Expression of the markers Neu-N and n-NOS. #p < 0.05 compared with the TBI group; n = 6 in each group (a). Expression of the markers Neu-N and 3-NT. ***p < 0.001 compared with the sham group; ##p < 0.01 compared with the TBI group; n = 6 in each group (b)

Fig 5: Representative blot of nNOS precipitated from intestinal homogenate with anti-caveolin-1 antibodies in control conditions and in presence of calmodulin and a high calcium concentration in addition to IgG negative control antibody. The amount of nNOS pulled down in high calcium and calmodulin is very much reduced. Control mouse IgG was used to show the selectivity of the coimmunoprecipitation. The blot is a representative of four experiments.