Fig. 1. A. Immunohistochemical staining of caveolin-1 and PMCA in cryosections from mouse small intestine and bovine tracheal smooth muscle. A. Cav1^+/+ tissue shows caveolin-1 (green) and PMCA (red) immunoreactivities that are co-localized (yellow) in smooth muscle membrane. Arrows indicate areas of co-localization in the longitudinal muscle membrane (large arrows). lm, longitudinal muscle layer; cm; circular muscle layer. Interstitial cells of Cajal in the deep muscular plexus and myenteric plexus also contain caveolin 1 and PMCA, labeled with arrowheads and small arrows respectively. A myenteric ganglion is present between the muscle layers. B. Cav1^−/− tissue shows only PMCA immunoreactivity (red). Scale bar is 10 µM for A and B. C. Control bovine tracheal smooth muscle tissue show caveolin-1 (green) and PMCA4b (red) immunoreactivities co-localized (yellow) in the cell membrane. D. Both caveolin-1 and PMCA immunoreactivities are almost abolished in the Me-β-CDX-treated bovine tracheal smooth muscle tissue (D). Scale bar is 5 µM for C and D.

Fig. 2. Electron micrographs showing the presence and distribution of caveolae in control bovine tracheal smooth muscle (top) and the disruption of caveolae after treatment with Me-β-CDX (bottom). Caveolae in control tissues are labeled with arrows and a cluster of caveolae is labeled with an asterisk. After with Me-β-CDX, only a few open caveolae (arrowheads). Scale bar is 0.5 µm.

Fig. 3. Distribution of PMCA4 in lipid raft-rich membrane fractions of mouse small intestinal and raft and non-raft membrane fractions from bovine tracheal smooth muscle. A. PMCA4 appears in the lipid raft-rich fraction in cav1^+/+ and cav1^−/−, however only PMCA4a is present in the cav1^−/− fraction. B. Upper two panels. PMCA4b distributes in lipid raft-rich fractions and heavy (non-raft) membrane fractions in control tissue. The numbers indicate the fraction in the sucrose density gradient. Fractions 1 and 2 were isolated from the 5% layer, fractions 3, 4, and 5 were isolated from the 35% layer, and fractions 6, 7, 8, 9 were isolated from the 45% layer. Lower two panels. PMCA4b was almost removed from the lipid raft-rich fractions from Me-β-CDX-treated tissues. The PMCA4b band intensity in the lipid raft-rich membrane fraction from three different control tissues and compared to bands from three different tissues treated with Me-β-CDX.

Fig. 4. Response of small intestinal segments from cav1^+/+ and cav1^−/− to Carbachol (1, 3, and 10 µM). A. The typical response to Carbachol consisted of a rapid phasic component and a sustained tonic component. The dot represents the time point at which Carbachol was added. B. The tonic response is significantly higher in cav1^−/− tissue segments only at 10 μM. n values were at least six for all experiments. Data are presented as means ± S.E.M.. Statistical significance was determined using ANOVA followed by Bonferroni post hoc test and denoted by P < 0.05.

Fig. 5. Effect of caloxin 1c2 (5 µM) on tissue response to 10 μM Carbachol in cav1^+/+ and cav1^−/− small intestinal tissue. A. Only cav1^+/+ segments treated with caloxin 1c2 maintained a high tonic response to Carbachol. The dot represents the time point when Carbachol was added. B. The response to Carbachol is higher in cav1^+/+ tissue segments treated with caloxin than in control tissues and cav1^−/− tissues. n values are 7 for cav1^+/+ segments and 6 for cav1^−/−. C. In calcium-free medium, caloxin 1c2 produces a higher tonic response to Carbachol only in cav1^+/+ tissue. n values are 7 for cav1^+/+ segments and 6 for cav1^−/− segments. Data are presented as means ± S.E.M.. Statistical significance was measured by ANOVA followed by the Bonferroni post hoc test and denoted by P < 0.05 and P < 0.01.

Fig. 6. Effect of caloxin 1c2 (5 µM) on the response to 0.1 µM Carbachol in bovine tracheal smooth muscle. A. Representative tracings showing the effect of caloxin 1c2 and the control peptide on Carbachol contraction. The dot represents the time point when Carbachol was added. B. The response to Carbachol only increases significantly after treatment with caloxin 1c2 and not the control peptide. The effect of caloxin is lost following cholesterol depletion by Me-β-CDX. n value is 7 for all experiments. Data are presented as means ± S.E.M..