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. 2007 Jun;151(3):332-40.
doi: 10.1038/sj.bjp.0707222. Epub 2007 Apr 23.

Effects of methyl beta-cyclodextrin on EDHF responses in pig and rat arteries; association between SK(Ca) channels and caveolin-rich domains

M Absi  1 , M P Burnham, A H Weston, E Harno, M Rogers, G Edwards
Affiliations

Effects of methyl beta-cyclodextrin on EDHF responses in pig and rat arteries; association between SK(Ca) channels and caveolin-rich domains

M Absi et al. Br J Pharmacol. 2007 Jun.

Abstract

Background and purpose: The small and intermediate conductance, Ca2+-sensitive K+ channels (SK(Ca) and IK(Ca), respectively) which are pivotal in the EDHF pathway may be differentially activated. The importance of caveolae in the functioning of IK(Ca) and SK(Ca) channels was investigated.

Experimental approach: The effect of the caveolae-disrupting agent methyl-beta-cyclodextrin (MbetaCD) on IK(Ca) and SK(Ca) localization and function was determined.

Key results: EDHF-mediated, SK(Ca)-dependent myocyte hyperpolarizations evoked by acetylcholine in rat mesenteric arteries (following blockade of IK(Ca) with TRAM-34) were inhibited by MbetaCD. Hyperpolarizations evoked by direct SK(Ca) channel activation (using NS309 in the presence of TRAM-34) were also inhibited by MbetaCD, an effect reversed by cholesterol. In contrast, IK(Ca)-dependent hyperpolarizations (in the presence of apamin) were unaffected by MbetaCD. Similarly, in porcine coronary arteries, EDHF-mediated, SK(Ca)-dependent (but not IK(Ca)-dependent) endothelial cell hyperpolarizations evoked by substance P were inhibited by MbetaCD. In mesenteric artery homogenates subjected to sucrose-density centrifugation, caveolin-1 and SK3 (SK(Ca)) proteins but not IK1 (IK(Ca)) protein migrated to the buoyant, caveolin-rich fraction. MbetaCD pretreatment redistributed caveolin-1 and SK3 proteins into more dense fractions. In immunofluorescence images of porcine coronary artery endothelium, SK3 (but not IK1) and caveolin-1 were co-localized. Furthermore, caveolin-1 immunoprecipitates prepared from native porcine coronary artery endothelium contained SK3 but not IK1 protein.

Conclusions and implications: These data provide strong evidence that endothelial cell SK(Ca) channels are located in caveolae while the IK(Ca) channels reside in a different membrane compartment. These studies reveal cellular organisation as a further complexity in the EDHF pathway signalling cascade.

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Figures

Figure 1
Figure 1
Typical traces showing the effect of MβCD on the SKCa and IKCa-induced myocyte hyperpolarizations in rat mesenteric artery segments. MβCD had no effect on (a, presence of apamin) the IKCa component of the response to ACh (b, presence of TRAM-34) but markedly reduced the SKCa component. (c, presence of TRAM-34) Similarly, the SKCa component of the hyperpolarization to NS 309 was also reduced by MβCD, an effect that was reversed by simultaneous exposure to cholesterol (chol).
Figure 2
Figure 2
Typical trace showing the effect of prolonged MβCD treatment on SKCa-dependent responses in rat mesenteric artery. Myocyte membrane potential was continuously recorded in the presence of TRAM-34. Responses to ACh were reduced after 30 min treatment with MβCD and these residual responses were essentially abolished by a further 30 min incubation with MβCD, as were responses to NS 309. Symbols used are identical to those in Figure 1. The broken lines in the membrane potential record indicate where a portion has been omitted for clarity; the cell remained impaled by the electrode.
Figure 3
Figure 3
Typical traces showing the effect of MβCD on IKCa- and SKCa-dependent vasorelaxation in rat mesenteric arteries. Arteries were mounted on a pressure myograph and pressurized to 70 mm Hg. Arteries were pre-incubated for 45 min with 5 mM MβCD and either (a) 100 nM apamin or (b) 10 μM TRAM-34. Arteries were constricted with 5–30 nM phenylephrine (PE) and 0.3–3 μM ACh applied. Full relaxation was induced with 100 μM papaverine. Mean data showing relaxations to ACh in the presence of (c) apamin and (d) TRAM-34 before and after incubation with MβCD (n=4).
Figure 4
Figure 4
(a) Sucrose density gradient fractions analysed by Western blot for the presence of caveolin-1 (Cav) and β-adaptin protein (a non-caveolae marker). Subsequently, fractions were pooled as fraction 1 (F1), fractions 2–5 (caveolin-rich, C), fractions 6–9 (non-caveolin 1, NC1) and fractions 10–13 (non-caveolin 2, NC2). (b) Effect of MβCD on SK3, IK1 and caveolin-1 protein distribution. Arteries were incubated with 5 mM MβCD for 30 min (lower panel) or left untreated (upper panel) before subjecting the resultant sucrose density gradient fractions to Western blotting. Note the migration of the SK3 and caveolin proteins into the NC fractions on MβCD treatment. (c) Western blot analysis showing the specificity of the anti-IK1 and anti-SK3 antibodies used in the sucrose density gradient experiments shown in (b) above. Note that each antibody recognizes only protein in HEK cells transfected with the appropriate subunit and does not react with any proteins in either untransfected HEK cells or those transfected with the alternative subunit. Molecular weight markers (kDa) are indicated. Images representative of three separate experiments are shown.
Figure 5
Figure 5
(a) Detection and localization of IK1 in sections of porcine coronary artery. Anti-IK1 immunoreactivity (red), nuclei (blue) and internal elastic lamina (green) are shown (representative of n=4 animals). All scale bars are 12.5 μm. Negative control experiment with anti-IK1 antibody pre-incubated with antigenic peptide (inset bottom right). Antibody specificity control data showing HEK cells expressing SK3 and IK1 immunolabelled with anti-SK3 and anti-IK1 antibodies (inset top left; representative of three separate experiments). (b) Deconvolution microscopy images of porcine coronary artery sections co-labelled with anti-caveolin-1 (Cav) and anti-eNOS, anti-IK1 or anti-SK3 antibodies. Low and high power magnification images shown are histogram-equalized z-projections of 25-image stacks. White lines enclose endothelial areas included in the co-localization analysis. (c) Graph of co-localization analysis mean data (eNOS, n=100 images, four animals; SK3, IK1, n=75 images, three animals) showing Pearson's correlation coefficient (r) versus red threshold for anti-eNOS, SK3 or IK1 with anti-caveolin-1 immunoreactivity (r=+1 indicates perfect co-localization, r=0 indicates no correlation and r=−1 indicates perfect negative correlation). (For colour figure see online version.)
Figure 6
Figure 6
Anti-SK3, IK1 and caveolin-1 Western blot analysis of anti-caveolin-1 immunoprecipitates prepared from porcine coronary artery endothelium. Immunoprecipitation (IP) was performed with and without anti-caveolin-1 antibody (+ve and −ve, respectively). Supernatants remaining after IP (SUP) were also subjected to Western blot analysis. Note that the SK3 protein co-precipitates with the caveolin-1 protein, whereas the IK1 protein does not. Images are representative of five (SK3) and three (IK1) experiments.
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References

    1. Alexander SPH, Mathie A, Peters JA.Guide to receptors and channels (GRAC) Br J Pharmacol 2007150Suppl 1S1–S168.2nd edition (2007 revision) - PMC - PubMed
    1. Burnham MP, Bychkov R, Félétou M, Richards GR, Vanhoutte PM, Weston AH, et al. Characterization of an apamin-sensitive small-conductance Ca2+-activated K+ channel in porcine coronary artery endothelium: relevance to EDHF. Br J Pharmacol. 2002;135:1133–1143. - PMC - PubMed
    1. Burnham MP, Johnson IT, Weston AH. Impaired small-conductance Ca2+-activated K+ channel-dependent EDHF responses in Type II diabetic ZDF rats. Br J Pharmacol. 2006a;148:434–441. - PMC - PubMed
    1. Burnham MP, Johnson IT, Weston AH. Reduced Ca2+-dependent activation of large-conductance Ca2+-activated K+ channels from arteries of Type 2 diabetic Zucker diabetic fatty rats. Am J Physiol. 2006b;290:H1520–H1527. - PubMed
    1. Busse R, Edwards G, Félétou M, Fleming I, Vanhoutte PM, Weston AH. EDHF: bringing the concepts together. Trends Pharmacol Sci. 2002;23:374–380. - PubMed

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