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. 2005 Oct 15;568(Pt 2):483-95.
doi: 10.1113/jphysiol.2005.085019. Epub 2005 Aug 4.

Disruption of CFTR chloride channel alters mechanical properties and cAMP-dependent Cl- transport of mouse aortic smooth muscle cells

Affiliations

Disruption of CFTR chloride channel alters mechanical properties and cAMP-dependent Cl- transport of mouse aortic smooth muscle cells

Renaud Robert et al. J Physiol. .

Abstract

Chloride (Cl(-)) channels expressed in vascular smooth muscle cells (VSMC) are important to control membrane potential equilibrium, intracellular pH, cell volume maintenance, contraction, relaxation and proliferation. The present study was designed to compare the expression, regulation and function of CFTR Cl(-) channels in aortic VSMC from Cftr(+/+) and Cftr(-)(/)(-) mice. Using an iodide efflux assay we demonstrated stimulation of CFTR by VIP, isoproterenol, cAMP agonists and other pharmacological activators in cultured VSMC from Cftr(+/+). On the contrary, in cultured VSMC from Cftr(-)(/)(-) mice these agonists have no effect, showing that CFTR is the dominant Cl(-) channel involved in the response to cAMP mediators. Angiotensin II and the calcium ionophore A23187 stimulated Ca(2)(+)-dependent Cl(-) channels in VSMCs from both genotypes. CFTR was activated in myocytes maintained in medium containing either high potassium or 5-hydroxytryptamine (5-HT) and was inhibited by CFTR(inh)-172, glibenclamide and diphenylamine-2,2'-dicarboxylic acid (DPC). We also examined the mechanical properties of aortas. Arteries with or without endothelium from Cftr(-/-) mice became significantly more constricted (approximately 2-fold) than that of Cftr(+/+) mice in response to vasoactive agents. Moreover, in precontracted arteries of Cftr(+/+) mice, VIP and CFTR activators induced vasorelaxation that was altered in Cftr(-/-) mice. Our findings suggest a novel mechanism for regulation of the vascular tone by cAMP-dependent CFTR chloride channels in VSMC. To our knowledge this study is the first to report the phenotypic consequences of the loss of a Cl(-) channel on vascular reactivity.

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Figures

Figure 1
Figure 1. Expression of CFTR in mice vascular smooth muscle cells
Immunofluorescence study of CFTR proteins in VSMC from Cftr+/+ (A) and Cftr−/− mice (B) and from Cftr+/+ mice in absence of primary antibody (C). Scale bars are 20 μm. CFTR is stained green and nucleus (TOPRO-3 staining) red.
Figure 2
Figure 2. Functional analysis of CFTR and Ca2+-activated Cl channels in Cftr+/+ and Cftr−/− mice smooth muscle cells
The stimulation of iodide efflux as a function of time was evoked in solution containing 80 mm K+ by 100 nm angiotensin II (denoted AgII) and 1 μm isoproterenol (A), and cAMP agonists (10 μm forskolin, 500 μm IBMX and 500 μm cpt-cAMP) and 300 nm VIP (B) in Cftr+/+ (left) and Cftr−/− (right) mice as compared to basal. C, summary of the relative rates presented as means ±s.e.m.D, effect of 100 μm glibenclamide, 500 μm DPC and 100 nm calixarene on the efflux stimulated by 300 nm VIP or cAMP agonists in Cftr+/+ mouse smooth muscle cells as indicated. Basal was vehicle alone. Data are presented as means ±s.e.m. All experimental conditions have been repeated: n = 8. ***P < 0.001. ns: non-significant difference.
Figure 3
Figure 3. Pharmacological activation of CFTR Cl channels activity in mice cultured smooth muscle cells
Iodide efflux responses as a function of time evoked in solution containing 80 mm K+ by MPB-07 (100 μm) and MPB-91 (100 μm) (A), 30 μm genistein (denoted Gst) or 30 μm genistein plus 10 μm forskolin (denoted Gst + Fsk) (B) in Cftr+/+ (left) and Cftr−/− (right) mouse VSMC. All agents: n = 8. Basal was vehicle alone in high K+. C and D, summary of the data for each experimental condition with 80 mm K+ solution after stimulation by MPB-07 or MPB-91 (C) and by genistein or genistein + forskolin (D) in Cftr+/+ and Cftr−/− mice as indicated. Basal was vehicle alone. Data are presented as means ±s.e.m. and compared to the basal. ***P < 0.001. ns: non-significant difference.
Figure 4
Figure 4. Effect of CFTRinh-172 on stimulated iodide efflux in Cftr+/+ and Cftr−/− mouse smooth muscle cells
A, the stimulation of iodide efflux as a function of time was evoked in solution containing 80 mm K+ by 10 μm forskolin plus 30 μm genistein without or with 100 μm CFTRinh-172 as indicated. B, summary of the relative rates presented as means ±s.e.m. with statistical analysis. Basal was vehicle (DMSO). All experimental conditions have been repeated: n = 8. ***P < 0.001. ns: non-significant difference.
Figure 5
Figure 5. Chloride transport activity of CFTR in VSMC with 5-hydroxytryptamine (5-HT)
The stimulation of iodide efflux as a function of time was evoked in solution containing 10 μm 5-HT by 10 μm forskolin plus 30 μm genistein without or with 100 μm CFTRinh-172 as indicated for Cftr+/+ mice (A) and Cftr−/− mice (B). C, summary of the relative rates presented as means ±s.e.m. with statistical analysis. Basal was vehicle (DMSO). All experimental conditions have been repeated: n = 8. ***P < 0.001. ns: non significant difference.
Figure 6
Figure 6. Measurement of the wall tension of mice aorta
A, continuous traces from experiments performed with denuded aortic rings preconstricted with 80 mm K+ solution (denoted 80K beneath traces). B and C, bar graphs showing mean tensions in response to 80 mm K+ solution for Cftr+/+ and Cftr−/− mice with (B) or without (C) endothelium.
Figure 7
Figure 7. Effect of CFTR activators on the wall tension of mouse aorta
A, continuous traces from experiments performed with denuded aortic rings reversibly preconstricted with 80 mm K+ solution (denoted 80K beneath traces). The absence of endothelium was verified by addition of acetylcholine (ACh, 10−5m) as indicated. Then, 300 nm VIP was added to the bath for Cftr+/+ (top trace) and Cftr−/− (bottom trace) mice. B, bar graphs of mean percentage vasorelaxation determined 30 min after adding 300 nm VIP after constriction by 80 mm K+ solution for 7 Cftr+/+ and 5 Cftr−/− mice. C, concentration-dependent curves for MPB-07-dependent vasorelaxation of aortic rings constricted by 80 mm K+ for Cftr+/+(IC50= 37 ± 1.17 μm, n = 6) and Cftr−/− mice (n = 6). *P < 0.05, **P < 0.01, ***P < 0.001 between bars for B. In C statistical differences were calculated for a given concentration between Cftr+/+ and Cftr−/− mice.

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