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Investigation of the functional role of endothelial KCa channels in regulation of resistance artery diameter
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- Author / Creator
- Wei,Ran
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Background: The vascular endothelium regulates arterial diameter, and thus blood flow and pressure, through release of diffusible factors such as nitric oxide (NO), and spread of electrical charge to smooth muscle cells via myoendothelial gap junctions (MEGJs). Activation of these pathways is mediated by a rise in endothelial Ca2+ levels leading to recruitment of downstream effectors. Two of the most important effectors are NO synthase, and Ca2+ activated K+ (KCa) channels which mediate hyperpolarization of the endothelial cell membrane potential. This hyperpolarization spreads to surrounding smooth muscle cells to limit contraction by reducing the open probability of voltage-operated Ca2+ channels. The majority of research in this area has focused on activation of these mechanisms by stimuli acting directly on endothelial cells leading to the idea that KCa channels and NO mediate distinct pathways for vasodilation. However, our lab has shown that in resistance arteries stimulation of smooth muscle cells by ï¡1-adrenoceptor agonists activates the converse signaling pathway, termed myoendothelial feedback. Briefly, flux of inositol trisphosphate (InsP3) from smooth muscle to endothelial cells elicits localized increases in Ca2+, activation of intermediate conductance (IKCa) KCa channels located at MEGJs and production of NO. Elucidation of the myoendothelial feedback pathway indicates that the role of endothelial KCa channels and NO in modulating vasoconstriction may depend on the contractile stimulus, and rather than being independent, these vasorelaxant pathways may interact. Further support for this proposal comes from studies showing that blockers of endothelial small (SKCa) and IKCa channels inhibit vasorelaxation mediated by endothelium-derived NO, and activators of endothelial KCa channels can elicit vasodilation that is blocked by inhibitors of NO signaling. My over-arching goal has been to further explore the functional role of SKCa and IKCa channels in regulating resistance artery diameter, and investigate potential interactions between KCa channels and NO in mediating vasodilation. To this end, I have addressed three hypotheses: 1.The functional contribution of InsP3/IKCa channel-mediated myoendothelial feedback to limiting arterial diameter is determined by the ability of the vasoconstrictor stimulus to engage the endothelium. 2. Small molecule activators of endothelial KCa channels modulate myogenic reactivity at least in part through endothelium-derived NO. 3.NO facilitates KCa channel mediated, endothelium-dependent smooth muscle hyperpolarization. Methods: To test these hypotheses I have used a combination of functional (wire and pressure myography), electrophysiological (intracellular recording of endothelial and smooth muscle membrane potential using sharp electrodes), biochemical (immunohistochemical localization of K+ channel proteins and qPCR to examine expression of mRNA for these channels) techniques to address 3 aims: i.To investigate the contribution of myoendothelial feedback to endothelial modulation of resistance artery responses to contractile agonists, increases in pressure and stimulation of perivascular sympathetic nerves. ii.To investigate the mechanisms underlying vasodilation to endothelial KCa channel activators in myogenically active vessels. iii. To determine if endothelium-dependent hyperpolarization of vascular smooth is modulated by NO. Results: My data show firstly, a differential ability of vasoconstrictor stimuli to engage IKCa channel-mediated myoendothelial feedback, and that in the presence of constant flow, shear stress-induced activation of SKCa channels plays a dominant role in modulating resistance artery vasoconstriction. Second, that pharmacological activators of endothelial KCa channels can modulate myogenic reactivity, an effect dependent on inwardly rectifying K+ channels, and possibly, NO. Finally, interactions between NO- and KCa channel-dependent vasorelaxant pathways occur at the level of both the endothelium (dilation to an activator of SKCa channels is dependent on NO) and smooth muscle (NO facilitates endothelium-dependent smooth muscle hyperpolarization) Conclusion: To conclude, rather than separate pathways, endothelial modulation of resistance artery diameter results from the integrated activity of SKCa and IKCa channels, and NO to fine-tune the contractile state of smooth muscle cells in a stimulus- and context-dependent manner. Such integration of voltage-dependent and -independent pathways will contribute to coordinated spatial and temporal control of blood flow in resistance artery beds.
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- Graduation date
- Spring 2018
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- Type of Item
- Thesis
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- Degree
- Doctor of Philosophy
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- License
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