Caveolin-1 gene knockout impairs nitrergic function in mouse small intestine

Abstract

Caveolin-1 is a plasma membrane-associated protein that is responsible for caveolae formation. It plays an important role in the regulation of the function of different signaling molecules, among which are the different isoforms of nitric oxide synthase (NOS). Nitric oxide (NO) is known to be an important inhibitory mediator in the mouse gut. Caveolin-1 knockout mice (Cav1(-/-)) were used to examine the effect of caveolin-1 absence on the NO function in the mouse small intestine (ileum and jejunum) compared to their genetic controls and BALB/c controls. Immunohistochemical staining showed loss of caveolin-1 and NOS in the jejunal smooth muscles and myenteric plexus interstitial cells of Cajal (ICC) of Cav1(-/-) mice; however, nNOS immunoreactive nerves were still present in myenteric ganglia. Under nonadrenergic noncholinergic (NANC) conditions, small intestinal tissues from Cav1(-/-) mice relaxed to electrical field stimulation (EFS), as did tissues from control mice. Relaxation of tissues from control mice was markedly reduced by N-omega-nitro-L-arginine (10(-4) M), but relaxation of Cav1(-/-) animals was affected much less. Also, Cav1(-/-) mice tissues showed reduced relaxation responses to sodium nitroprusside (100 microM) compared to controls; yet there were no significant differences in the relaxation responses to 8-bromoguanosine-3': 5'-cyclic monophosphate (100 microM). Apamin (10(-6) M) significantly reduced relaxations to EFS in NANC conditions in Cav1(-/-) mice, but not in controls. The data from this study suggest that caveolin-1 gene knockout causes alterations in the smooth muscles and the ICC, leading to an impaired NO function in the mouse small intestine that could possibly be compensated by apamin-sensitive inhibitory mediators.

Figure 1

An electron microscope image of the longitudinal muscle layer (LM) of mouse small intestine. Note the total absence of the flask-shaped caveolae on the plasma membrane of the longitudinal muscle cells of Cav1^−/− (b) as compared to the longitudinal muscle cells of the control BALB/c mouse (a). Caveolae are indicated by solid arrowheads. Small endoplasmic reticulum profiles (marked with thick arrows) and sarcoplasmic reticulum (marked with thin arrows) remain. Mitochondria are labeled with (M). Cav1^+/+ intestine showed similar caveolae profile to the BALB/c (data not shown). Length bars are 1 μm.

Figure 2

Immunohistochemical staining of cryosections from Cav1^−/− jejunum (a–c) and whole-mount preparations of myenteric plexus layer from Cav1^+/+ and Cav1^−/− jejunal tissues (d–k). Panels (a)–(c) show that there are no caveolin-1 and nNOS-C-immunoreactivities in all ICC and smooth muscles layers of Cav1^−/− jejunum. Panels (d) and (e) show myenteric ICC stained with anti-vimentin in Cav1^+/+ and Cav1^−/− myenteric plexus, respectively. ICCs are marked with asterisks. Note that the myenteric plexus ICC are equally present and similarly distributed in both strains. Panels (f)–(k) show double staining of whole-mount preparations for myenteric neurons HuC/D protein and nNOS-N. Panels (f) and (i) show myenteric neurons stained with HuC/D in Cav1^+/+ and Cav1^−/− myenteric plexus, respectively. Panels (g) and (j) show myenteric neurons and nerve fibers stained with nNOS-N in Cav1^+/+ and Cav1^−/− myenteric plexus, respectively. Panels (h) and (k) show myenteric neurons co-localized with HuC/D and nNOS-N in Cav1^+/+ and Cav1^−/− myenteric plexus, respectively. Note the persistence of nNOS-N in myenteric neurons in Cav1^−/− intestine. Length bars are 20 μm for panels (a)–(c), (d) and (e), and (f)–(k).

Figure 3

Effect of LNNA (100 μM) on the inhibitory responses obtained by EFS in NANC conditions at different stimulation frequencies (0.5 ms, 50 V cm⁻¹ for 10 s). The magnitudes of the EFS-evoked responses are expressed as the % change in the amplitude (the amplitude of the inhibitory phase normalized to the amplitude of the muscle activity directly precedent to the inhibitory stimulus) and the values shown are mean±s.e. (a, b) LNNA abolishes the EFS-induced inhibitions at all frequencies in BALB/c and Cav1^+/+, respectively. Significance is tested by ANOVA followed by Bonferroni's test (^***P<0.001, n-value=7 for BALB/c and 6 for Cav1^+/+). (c) LNNA significantly reduces the EFS-induced inhibitions only at 1 and 3 pps (^*P<0.05, n-value=11), and had no effect on the inhibitions at 10 and 30 pps in Cav^−/− mice.

Figure 4

The inhibitory effect produced by 100 μM SNP on longitudinal muscle preparations from the small intestine of Cav1^+/+, Cav1^−/−, and BALB/c mice. The inhibitory effects were more pronounced in wild-type mice compared to Cav1^−/−. The magnitudes of inhibition are expressed as the % inhibition of the amplitude (amplitude of the inhibitory phase normalized to the amplitude of the muscle activity directly precedent to the addition of SNP). The values are shown as mean±s.e. of six experiments. Significance was tested by ANOVA, followed by Bonferroni test (^**P<0.01).

Figure 5

The inhibitory effect produced by 100 μM BCGMP on longitudinal muscle preparations from the small intestine of Cav1^+/+, Cav1^−/−, and BALB/c mice. There were no significant differences among the magnitudes of inhibition, which are expressed as the % inhibition of the amplitude (amplitude of the contractions 5 min after the addition of BCGMP normalized to the amplitude of the muscle contraction directly precedent to its addition). The values shown are mean±s.e. of six experiments. Significance was tested by ANOVA, followed by Bonferroni test.

Figure 6

Effect of apamin (1 μM) on the inhibitory responses induced by EFS in the NANC conditions at different stimulation frequencies (0.5 ms, 50 V cm⁻¹ for 10 s). The magnitudes of the EFS-evoked responses are expressed as the % change in the amplitude (the amplitude of the inhibitory phase normalized to the amplitude of the muscle activity directly precedent to the inhibitory stimulus), and the values shown are mean±s.e. (a) Apamin significantly reduced the magnitude of EFS-induced inhibitions at all frequencies in Cav1^−/− mouse intestine. Significance was tested by ANOVA, followed by Bonferroni test (^*P<0.05; ***P<0.001, n-value=6). (b) Apamin did not produce a significant effect on the EFS-induced inhibitions in the Cav1^+/+ mouse intestine, except at 30 pps (^*P<0.05, n-value=6).