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Ovariectomy-induced reductions in endothelial SK3 channel activity and endothelium-dependent vasorelaxation in murine mesenteric arteries.

Yap FC, Taylor MS, Lin MT - PLoS ONE (2014)

Bottom Line: The results from functional studies using isolated murine mesenteric arteries show that ovx reduces ACh-induced endothelium-dependent vasodilation due to decreased EDH and NO contributions, although the contribution of PGI2 is upregulated.The decreased EDH-mediated vasorelaxation in ovx vessels is due to reduced SK3 channel contribution to the pathway.Further, whole-cell recordings using dispersed endothelial cells also show reduced SK3 current density in ovx endothelial cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, University of South Alabama, Mobile, Alabama, United States of America.

ABSTRACT
Mesenteric artery endothelium expresses both small (SK3)- and intermediate (IK1)-conductance Ca(2+)-activated K(+) (KCa) channels whose activity modulates vascular tone via endothelium-dependent hyperpolarization (EDH). Two other major endothelium-dependent vasodilation pathways utilize nitric oxide (NO) and prostacyclin (PGI2). To examine how ovariectomy (ovx) affects the basal activity and acetylcholine (ACh)-induced activity of each of these three pathways to vasorelaxation, we used wire myograph and electrophysiological recordings. The results from functional studies using isolated murine mesenteric arteries show that ovx reduces ACh-induced endothelium-dependent vasodilation due to decreased EDH and NO contributions, although the contribution of PGI2 is upregulated. Both endothelial SK3 and IK1 channels are functionally coupled to TRPV4 (transient receptor potential, vanilloid type 4) channels: the activation of TRPV4 channels activates SK3 and IK1 channels, leading to EDH-mediated vascular relaxation. The decreased EDH-mediated vasorelaxation in ovx vessels is due to reduced SK3 channel contribution to the pathway. Further, whole-cell recordings using dispersed endothelial cells also show reduced SK3 current density in ovx endothelial cells. Consequently, activation of TRPV4 channels induces smaller changes in whole-cell current density. Thus, ovariectomy leads to a reduction in endothelial SK3 channel activity thereby reducing the SK3 contribution to EDH vasorelaxation.

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Ovariectomy reduces SK3 channel current density in endothelial cells.A: Representative traces recorded using conventional whole-cell recording on endothelial cells isolated from mesenteric arteries obtained from control mouse. Cells were voltage clamped at their resting membrane potential and a 200 ms voltage ramp from of −80 to +60 mV was delivered to elicit whole cell currents before (control) and after subsequent bath application of apamin (apamin) and apamin+tram34 (tram34). B: SK3 and IK1 current densities isolated from digital subtraction of the traces shown in (A) for control endothelial cells. C: Representative whole-cell current density obtained from ovx endothelial cells. D: SK3 and IK1 current densities isolated from digital subtraction of the traces in (C) for ovx endothelial cells. E: Summarized whole-cell SK3 and IK1 current densities from control (black) and ovx (grey) endothelial cells measured at +30 mV. F: Normalized SK3/IK1 ratios for control (black) and ovx (grey) recordings showing reduced SK3 channel activity in ovx endothelial cells. Asterisk (*) indicates statistical significance from control (P<0.05, t-test).
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pone-0104686-g005: Ovariectomy reduces SK3 channel current density in endothelial cells.A: Representative traces recorded using conventional whole-cell recording on endothelial cells isolated from mesenteric arteries obtained from control mouse. Cells were voltage clamped at their resting membrane potential and a 200 ms voltage ramp from of −80 to +60 mV was delivered to elicit whole cell currents before (control) and after subsequent bath application of apamin (apamin) and apamin+tram34 (tram34). B: SK3 and IK1 current densities isolated from digital subtraction of the traces shown in (A) for control endothelial cells. C: Representative whole-cell current density obtained from ovx endothelial cells. D: SK3 and IK1 current densities isolated from digital subtraction of the traces in (C) for ovx endothelial cells. E: Summarized whole-cell SK3 and IK1 current densities from control (black) and ovx (grey) endothelial cells measured at +30 mV. F: Normalized SK3/IK1 ratios for control (black) and ovx (grey) recordings showing reduced SK3 channel activity in ovx endothelial cells. Asterisk (*) indicates statistical significance from control (P<0.05, t-test).

Mentions: To directly test the hypothesis that ovariectomy reduces endothelial SK3 channel activity, we performed whole-cell patch clamp recordings. Vascular endothelial cells (ECs) were acutely dispersed from first- and second-order mouse mesenteric arteries. ECs were visually identified by their characteristic phase contrast under light microscopy as previously reported [7]. Whole-cell voltage clamp protocols were performed in the presence of 3 µM free internal Ca2+ to activate SK3 and IK1 channels and currents were elicited with a 200 ms voltage ramp from −80 to +60 mV, delivered every 30 seconds. We obtained current densities by normalizing recorded whole-cell currents to the membrane capacitance, calculated from a hyperpolarizing step (Fig. 5A and C). Whole cell current density averages measured at +30 mV for control and ovx ECs were 68±11 pA/pF (n = 10) and 55±13 pA/pF (n = 7; P>0.05), respectively. Bath applied apamin (300 nM) reduced current density and the SK3 current density was isolated by digital subtraction for both control and ovx ECs (Fig. 5B and D, respectively). Ovx ECs showed reduced SK3 current density (control: 15±2.9 pA/pF; ovx: 5.5±2.3 pA/pF; n = 6; P<0.05; Fig. 5E). Subsequent addition of tram34 (1 µM) further reduced whole-cell current density (Fig. 5A and C); however, digitally isolated IK1 current density was not different between control and ovx ECs (control: 32±2.1 pA/pF; ovx: 37±3.0 pA/pF; n = 6; P>0.05; Fig. 5B, D, and E). We further calculated the normalized contribution of SK3 and IK1 channels to EC current density and plotted the ratios of SK3/IK1 for both control and ovx ECs. Consistent with our functional studies, the significantly higher SK3/IK1 ratio in control ECs showed a 3-fold increase in SK3 channel activity as compared to that of ovx ECs (control: 0.54±0.04; oxv 0.17±0.05; P<0.05; Fig. 5F).


Ovariectomy-induced reductions in endothelial SK3 channel activity and endothelium-dependent vasorelaxation in murine mesenteric arteries.

Yap FC, Taylor MS, Lin MT - PLoS ONE (2014)

Ovariectomy reduces SK3 channel current density in endothelial cells.A: Representative traces recorded using conventional whole-cell recording on endothelial cells isolated from mesenteric arteries obtained from control mouse. Cells were voltage clamped at their resting membrane potential and a 200 ms voltage ramp from of −80 to +60 mV was delivered to elicit whole cell currents before (control) and after subsequent bath application of apamin (apamin) and apamin+tram34 (tram34). B: SK3 and IK1 current densities isolated from digital subtraction of the traces shown in (A) for control endothelial cells. C: Representative whole-cell current density obtained from ovx endothelial cells. D: SK3 and IK1 current densities isolated from digital subtraction of the traces in (C) for ovx endothelial cells. E: Summarized whole-cell SK3 and IK1 current densities from control (black) and ovx (grey) endothelial cells measured at +30 mV. F: Normalized SK3/IK1 ratios for control (black) and ovx (grey) recordings showing reduced SK3 channel activity in ovx endothelial cells. Asterisk (*) indicates statistical significance from control (P<0.05, t-test).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4126749&req=5

pone-0104686-g005: Ovariectomy reduces SK3 channel current density in endothelial cells.A: Representative traces recorded using conventional whole-cell recording on endothelial cells isolated from mesenteric arteries obtained from control mouse. Cells were voltage clamped at their resting membrane potential and a 200 ms voltage ramp from of −80 to +60 mV was delivered to elicit whole cell currents before (control) and after subsequent bath application of apamin (apamin) and apamin+tram34 (tram34). B: SK3 and IK1 current densities isolated from digital subtraction of the traces shown in (A) for control endothelial cells. C: Representative whole-cell current density obtained from ovx endothelial cells. D: SK3 and IK1 current densities isolated from digital subtraction of the traces in (C) for ovx endothelial cells. E: Summarized whole-cell SK3 and IK1 current densities from control (black) and ovx (grey) endothelial cells measured at +30 mV. F: Normalized SK3/IK1 ratios for control (black) and ovx (grey) recordings showing reduced SK3 channel activity in ovx endothelial cells. Asterisk (*) indicates statistical significance from control (P<0.05, t-test).
Mentions: To directly test the hypothesis that ovariectomy reduces endothelial SK3 channel activity, we performed whole-cell patch clamp recordings. Vascular endothelial cells (ECs) were acutely dispersed from first- and second-order mouse mesenteric arteries. ECs were visually identified by their characteristic phase contrast under light microscopy as previously reported [7]. Whole-cell voltage clamp protocols were performed in the presence of 3 µM free internal Ca2+ to activate SK3 and IK1 channels and currents were elicited with a 200 ms voltage ramp from −80 to +60 mV, delivered every 30 seconds. We obtained current densities by normalizing recorded whole-cell currents to the membrane capacitance, calculated from a hyperpolarizing step (Fig. 5A and C). Whole cell current density averages measured at +30 mV for control and ovx ECs were 68±11 pA/pF (n = 10) and 55±13 pA/pF (n = 7; P>0.05), respectively. Bath applied apamin (300 nM) reduced current density and the SK3 current density was isolated by digital subtraction for both control and ovx ECs (Fig. 5B and D, respectively). Ovx ECs showed reduced SK3 current density (control: 15±2.9 pA/pF; ovx: 5.5±2.3 pA/pF; n = 6; P<0.05; Fig. 5E). Subsequent addition of tram34 (1 µM) further reduced whole-cell current density (Fig. 5A and C); however, digitally isolated IK1 current density was not different between control and ovx ECs (control: 32±2.1 pA/pF; ovx: 37±3.0 pA/pF; n = 6; P>0.05; Fig. 5B, D, and E). We further calculated the normalized contribution of SK3 and IK1 channels to EC current density and plotted the ratios of SK3/IK1 for both control and ovx ECs. Consistent with our functional studies, the significantly higher SK3/IK1 ratio in control ECs showed a 3-fold increase in SK3 channel activity as compared to that of ovx ECs (control: 0.54±0.04; oxv 0.17±0.05; P<0.05; Fig. 5F).

Bottom Line: The results from functional studies using isolated murine mesenteric arteries show that ovx reduces ACh-induced endothelium-dependent vasodilation due to decreased EDH and NO contributions, although the contribution of PGI2 is upregulated.The decreased EDH-mediated vasorelaxation in ovx vessels is due to reduced SK3 channel contribution to the pathway.Further, whole-cell recordings using dispersed endothelial cells also show reduced SK3 current density in ovx endothelial cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, University of South Alabama, Mobile, Alabama, United States of America.

ABSTRACT
Mesenteric artery endothelium expresses both small (SK3)- and intermediate (IK1)-conductance Ca(2+)-activated K(+) (KCa) channels whose activity modulates vascular tone via endothelium-dependent hyperpolarization (EDH). Two other major endothelium-dependent vasodilation pathways utilize nitric oxide (NO) and prostacyclin (PGI2). To examine how ovariectomy (ovx) affects the basal activity and acetylcholine (ACh)-induced activity of each of these three pathways to vasorelaxation, we used wire myograph and electrophysiological recordings. The results from functional studies using isolated murine mesenteric arteries show that ovx reduces ACh-induced endothelium-dependent vasodilation due to decreased EDH and NO contributions, although the contribution of PGI2 is upregulated. Both endothelial SK3 and IK1 channels are functionally coupled to TRPV4 (transient receptor potential, vanilloid type 4) channels: the activation of TRPV4 channels activates SK3 and IK1 channels, leading to EDH-mediated vascular relaxation. The decreased EDH-mediated vasorelaxation in ovx vessels is due to reduced SK3 channel contribution to the pathway. Further, whole-cell recordings using dispersed endothelial cells also show reduced SK3 current density in ovx endothelial cells. Consequently, activation of TRPV4 channels induces smaller changes in whole-cell current density. Thus, ovariectomy leads to a reduction in endothelial SK3 channel activity thereby reducing the SK3 contribution to EDH vasorelaxation.

Show MeSH
Related in: MedlinePlus