Introducción a las interfaces de usuario

The function of the BKCa channel is reported to be influenced by caveolar location and

interaction with caveolins. In this experiment, I tested whether the observed change in contraction by disrupting caveolae is mediated by the BKCa channel. As BKCa channels

were found in both endothelial and smooth muscle cells in rat femoral artery (see chapter 4), I designed two experiments to examine: (i) the effect of BKCa channel

inhibition by tetraethylammonium ions (TEA+) on the contraction of femoral arteries in response to 20 K/ Bay K before and after treatment with M-β-CD; (ii) the effect of BKCa

channel inhibition by TEA+ on the contraction of endothelium-denuded femoral arteries in response to 20 K/ Bay K before and after treatment with 5 mM M-β-CD.

5.3.9.1 Experimental protocol A

Endothelium-intact artery rings were contracted with 20 K/ Bay K. They were then pre- incubated in TEA+ (2 mM), a relatively selective BKCa channel inhibitor at this

concentration (Langton et al., 1991), for 10 min. Vessels were next contracted with 20 K/ Bay K in the presence of 2 mM TEA+. In order to disrupt caveolae, vessels were incubated with M-β-CD (5 mM) for 1 hour. Subsequently, the vessels were pre- incubated in TEA+ (2 mM) for 10 minutes and contracted with 20 K/ Bay K/ TEA.

5.3.9.2 Experimental protocol B

The endothelium was mechanically removed from the lumen of the artery and a similar protocol to that applied in Experimental protocol A was then used. Vessels were

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contracted with 20 K/ Bay K and ACh (10 μM) was added to confirm the absence of the endothelium. Vessels were pre-incubated in TEA+ (2 mM) for 10 min and contracted with 20 K/ Bay K/ TEA. Vessels were then incubated with M-β-CD (5 mM) for 1 hour, pre-incubated in TEA+ (2 mM) and contracted with 20 K/ Bay K/ TEA.

5.3.9.3 Analysis

Incubating artery rings with 2 mM TEA+ caused an increase in the resting force of the artery, and also the contraction force in response to 20 K/ Bay K (figure 5.10). This result suggests TEA+-sensitive K+ channels are contributing to the membrane potential at both resting state and when arteries are contacted with 20 K/ Bay K. The two data traces illustrated in figure 5.10 show different patterns of response. In the upper trace, spikes are superimposed on top of the increase in resting force, indicating TEA+ is inducing spontaneous oscillations in this vessel. As before, incubating arteries with 5 mM M-β-CD for 1 hour augmented contraction in response to 20 K/ Bay K (figure 5.10). After treatment with M-β-CD, TEA+ no longer caused significant additional contraction in response to 20K/ Bay K (figure 5.10). Analysis showed that treatment of intact femoral arteries with M-β-CD caused a significant increase in the force generated in response to 20 K/ Bay K, from 7.07 ± 2.05 mN to 15.29 ± 2.84 mN (n=8,**P< 0.01)

(figure 5.11). Increases in the resting tension caused by 2 mM TEA+ alone before and after M-β-CD treatment was 1.15 ± 0.68 mN and 2.15 ± 0.81 mN, respectively (ns,

figure 5.11). 2 mM TEA+ application significantly increased the contraction with 20 K/ Bay K from 7.07 ± 2.05 mN to 18.53 ± 3.25 mN (n=8,*P< 0.05). After the vessels had been incubated with M-β-CD, incubation with TEA+ (2 mM) caused a non-significant

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increase in the force generated in response to 20 K/ Bay K, from 18.53 ± 3.25 mN to 19.54 ± 2.22 mN (n=8, ns) (figure 5.11). The observation that TEA+ increased force before, but not after, M--CD treatment suggests caveolar disruption decreases the contribution of a TEA+-sensitive K+ channel to the membrane potential.

In endothelium-denuded femoral arteries, application of 2 mM TEA+ caused a small but non-significant increase in 20 K/ Bay K contraction (from 6.06 ± 1.44 mN to 8.01 ± 1.58 mN (n= 8, ns)) (figure 5.12 B). M-β-CD application had no significant effect on 20 K/ Bay K contraction (6.06 ± 1.44 mN to 6.19 ± 1.32 mN (n= 8, ns)). After arteries were incubated with M-β-CD, TEA+ caused a small, non-significant, additional contraction in response to 20 K/ Bay K /M-β-CD from 6.19 ± 1.32 mN to 7.23 ± 1.61 mN (n= 8,ns).

Overall, these results indicate that inhibition of the potassium channels, possibly BKCa

channels, by TEA+ effectively increases the contraction of rat femoral artery in the presence of endothelial cells. Caveolae disruption by M-β-CD and/or endothelium removal caused TEA+ to become much less effective at augmenting contraction to 20 K/ Bay K. M-β-CD treatment and removal of endothelium both seem to inhibit basal endothelial NO release. NO is a known activator of BKCa channels in SMCs, so reduced

basal NO release may lead to less active BKCa channels, so SMC depolarization, and

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Figure ‎5.10. Effect of TEA+ on femoral artery contractions to 20K/ Bay K. Traces show that incubation of arteries with 2 mM TEA+ by itself causes contraction, and also augments the contraction response to 20 K/ Bay K. In the upper trace (A), TEA+ triggered spontaneous oscillations in force. When arteries were subsequently incubated in M-β-CD, this caused an enhanced contraction in response to 20 K/ Bay K, but this effect of M-β-CD was absent when the arteries were treated with TEA+. Increases in the basal tension due to TEA+ application alone were similar before or after M-β-CD treatment.

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Figure ‎5.11. Mean data for effect of TEA+ on femoral artery contractions to 20 K/ Bay K before and after M-β-CD treatment. Data show a significant increase in the contraction in response to 20 K/ Bay K after incubation of the artery with 2 mM TEA+ before, but not after, treatment with M-β-CD. Statistical significance was examined using ANOVA: n=8, * P<0.05 and ** P< 0.01.

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Figure ‎5.12. Effect of TEA+ on contractions to 20 K/ Bay K in an endothelium-denuded artery. A. Application of2 mM TEA+ or M-β-CD had little effect on 20 K/ Bay K contraction.

B. Mean data showing a non-significant increase in the contraction response to 20 K/ Bay K by TEA+ or M-β-CD. Statistical significance was examined using ANOVA n=8.

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5.3.10 Effect of Iberiotoxin (IBTX) before and after disruption of caveolae by M-β-