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Abstract
Despite increasing knowledge of the significance of calcium-activated potassium (KCa) and canonical transient receptor potential (TRPC) channels in endothelial physiology, no studies so far have investigated the link between these two distinct types of channels in the control of vascular tone in pathological conditions. We previously demonstrated that hypoxia-reoxygenation (H-R) inhibits endothelial KCa and TRPC3 channels in porcine coronary arteries (PCAs). The present study further investigated whether modulation of TRPC3 is involved in H-R-induced KCa channel inhibition and associated vasodilatory dysfunction using approaches of wire myography, whole-cell voltage-clamp, and coimmunoprecipitation. Pharmacological inhibition or siRNA silencing of TRPC3 significantly suppressed bradykinin-induced intermediate- and small-conductance KCa (IKCa and SKCa) currents in endothelial cells of PCAs (PCAECs). TRPC3 protein exists in physical association with neither IKCa nor SKCa. In H-R-exposed PCAECs, the response of IKCa and SKCa to bradykinin-stimulation and to TRPC3-inhibition was markedly weakened. Activation of TRPC3 channels restored H-R-suppressed KCa currents in association with an improved endothelium-derived hyperpolarizing factor (EDHF)-type vasorelaxation. We conclude that inhibition of TRPC3 channels contributes to H-R-induced suppression of KCa channel activity, which serves as a mechanism underlying coronary endothelial dysfunction in ischemia-reperfusion (I-R) injury and renders TRPC3 a potential target for endothelial protection in I-R conditions.
Highlights
Despite increasing knowledge of the significance of calcium-activated potassium (KCa) and canonical transient receptor potential (TRPC) channels in endothelial physiology, no studies so far have investigated the link between these two distinct types of channels in the control of vascular tone in pathological conditions
Pretreatment of porcine coronary arterial endothelial cells (PCAECs) for 30 min with the specific TRPC3 channel blocker Pyr[3] significantly suppressed bradykinin-induced whole-cell K+ currents (45.69 ± 2.77 vs. 65.45 ± 3.01 pA/pF, p < 0.01) with the IKCa channel current decreasing from 16.89 ± 0.93 pA/pF to 6.71 ± 0.85 pA/ pF (p < 0.001) and the SKCa channel current from 10.63 ± 1.69 pA/pF to 5.51 ± 1.17 pA/pF (p < 0.05) (Fig. 1a and b)
The present study demonstrated that in porcine coronary arteries (1) TRPC3 channels play a fundamental role in the regulation of KCa channel activity in endothelial cells; (2) the functional association between TRPC3 and KCa channels does not involve physical interaction; (3) reduction of membrane TRPC3 contributes to H-R-induced suppression of IKCa and SKCa channel activity; (4) activation of TRPC3 protects IKCa and SKCa channels from H-R-induced inhibition thereby preserving endothelium-derived hyperpolarizing factor (EDHF)-type vasorelaxation
Summary
Despite increasing knowledge of the significance of calcium-activated potassium (KCa) and canonical transient receptor potential (TRPC) channels in endothelial physiology, no studies so far have investigated the link between these two distinct types of channels in the control of vascular tone in pathological conditions. We previously demonstrated that hypoxia-reoxygenation (H-R) inhibits endothelial KCa and TRPC3 channels in porcine coronary arteries (PCAs). Pharmacological inhibition or siRNA silencing of TRPC3 significantly suppressed bradykinin-induced intermediate- and small-conductance KCa (IKCa and SKCa) currents in endothelial cells of PCAs (PCAECs). Activation of TRPC3 channels restored H-R-suppressed KCa currents in association with an improved endothelium-derived hyperpolarizing factor (EDHF)-type vasorelaxation. We conclude that inhibition of TRPC3 channels contributes to H-R-induced suppression of KCa channel activity, which serves as a mechanism underlying coronary endothelial dysfunction in ischemia-reperfusion (I-R) injury and renders TRPC3 a potential target for endothelial protection in I-R conditions. In our in vitro studies of I-R in porcine coronary arteries, we demonstrated that hypoxia-reoxygenation (H-R) suppresses IKCa and SKCa channel currents in endothelial cells and such inhibition is responsible for the compromised EDHF-mediated vasorelaxation and smooth muscle hyperpolarization[16]. Through inhibiting the expression of TRPC3 protein on endothelial cell membrane, H-R significantly reduces TRPC3 channel activity and Ca2+ influx via TRPC3, leading to decreased NO production and vasorelaxation[8]
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