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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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TRPV4 channel activation evokes a smaller response in ovx endothelial cells.A: Time course of the whole-cell current density evoked at +30 mV from control (solid) and ovx (open) endothelial cells using perforated patch clamp technique. Following a stable baseline, 30 nM GSK1016790 (GSK, 0 min) was added to the bath to activated TRPV4 channels, followed by bath application of 500 nM HC067047 (HC, 6 min) to block them. Time course was normalized to baseline. B: Averaged current density in the presence of GSK (3–5 min as shown in A) and GSK+HC (9–11 min), normalized to the control current density (−3 to −1 min). Asterisk (*) indicates statistical significance (P<0.05, t-test). C: Representative traces recorded from an endothelial cell obtained from control mesenteric artery at different conditions as shown in (A), before (control) and after subsequent bath addition of GSK (GSK) and GSK+HC (HC). Whole cell current density was elicited with −80 to +60 mV voltage-ramps. D: GSK-sensitive whole-cell current density isolated from digital subtraction of the traces (C) for control cells. E: Representative perforated whole-cell current density obtained from ovx endothelial cells. F: GSK-sensitive current density isolated from digital subtraction of the traces (E) for ovx cells.
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pone-0104686-g006: TRPV4 channel activation evokes a smaller response in ovx endothelial cells.A: Time course of the whole-cell current density evoked at +30 mV from control (solid) and ovx (open) endothelial cells using perforated patch clamp technique. Following a stable baseline, 30 nM GSK1016790 (GSK, 0 min) was added to the bath to activated TRPV4 channels, followed by bath application of 500 nM HC067047 (HC, 6 min) to block them. Time course was normalized to baseline. B: Averaged current density in the presence of GSK (3–5 min as shown in A) and GSK+HC (9–11 min), normalized to the control current density (−3 to −1 min). Asterisk (*) indicates statistical significance (P<0.05, t-test). C: Representative traces recorded from an endothelial cell obtained from control mesenteric artery at different conditions as shown in (A), before (control) and after subsequent bath addition of GSK (GSK) and GSK+HC (HC). Whole cell current density was elicited with −80 to +60 mV voltage-ramps. D: GSK-sensitive whole-cell current density isolated from digital subtraction of the traces (C) for control cells. E: Representative perforated whole-cell current density obtained from ovx endothelial cells. F: GSK-sensitive current density isolated from digital subtraction of the traces (E) for ovx cells.

Mentions: Results thus far suggest that the reduced SK3 channel activity underlies reduced EDH-mediated vasorelaxation in ovx vessels. This model predicts that activation of TRPV4 channels with GSK should induce a smaller SK3 channel-mediated current in ovx ECs. We tested this hypothesis using perforated whole-cell recordings, in which the intracellular [Ca2+] remained intact and the calcium activation of endothelial Ca2+-activation potassium (KCa) channels was examined. Perforated whole-cell current density was elicited using the same whole-cell voltage ramp protocol as described in Figure 5, and TRPV4 channels were activated with bath application of GSK. Changes in current density, due to TRPV4 channel activation, were quantified using the steady-state current density elicited at +30 mV and normalized to the baseline before GSK (Fig. 6A). Following a stable baseline recording, bath applied GSK (30 nM) increased the whole-cell current density of ECs isolated from control mice to 150±4% (n = 6; Fig. 6B). In contrast, the effect of GSK on whole-cell current density was reduced in ovx ECs (ovx: 116±2%; n = 6; P<0.05; Fig. 6A and B). Subsequently bath applied HC (500 nM) reduced the current density to 84±5% (control) and 96±3% (ovx) of baseline (n = 6; P>0.05). The whole-cell current density, induced by activation of TRPV4 channels and digitally isolated for control (Fig. 6C and D) and ovx ECs (Fig. 6E and F), reversing at very negative membrane potentials resembling K+ currents, suggested TRPV4-dependent activation of KCa channels. Notably, activation of TRPV4 channels caused a smaller increase in KCa current density in ovx ECs (Fig. 6B). Taken together, these results suggest TRPV4-induced SK3 channel activation is significantly reduced in ovx ECs due to reduced SK3 current density, resulting in a reduced EDH-mediated vasorelaxation in mesenteric vessels obtained from ovx mice.


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)

TRPV4 channel activation evokes a smaller response in ovx endothelial cells.A: Time course of the whole-cell current density evoked at +30 mV from control (solid) and ovx (open) endothelial cells using perforated patch clamp technique. Following a stable baseline, 30 nM GSK1016790 (GSK, 0 min) was added to the bath to activated TRPV4 channels, followed by bath application of 500 nM HC067047 (HC, 6 min) to block them. Time course was normalized to baseline. B: Averaged current density in the presence of GSK (3–5 min as shown in A) and GSK+HC (9–11 min), normalized to the control current density (−3 to −1 min). Asterisk (*) indicates statistical significance (P<0.05, t-test). C: Representative traces recorded from an endothelial cell obtained from control mesenteric artery at different conditions as shown in (A), before (control) and after subsequent bath addition of GSK (GSK) and GSK+HC (HC). Whole cell current density was elicited with −80 to +60 mV voltage-ramps. D: GSK-sensitive whole-cell current density isolated from digital subtraction of the traces (C) for control cells. E: Representative perforated whole-cell current density obtained from ovx endothelial cells. F: GSK-sensitive current density isolated from digital subtraction of the traces (E) for ovx cells.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0104686-g006: TRPV4 channel activation evokes a smaller response in ovx endothelial cells.A: Time course of the whole-cell current density evoked at +30 mV from control (solid) and ovx (open) endothelial cells using perforated patch clamp technique. Following a stable baseline, 30 nM GSK1016790 (GSK, 0 min) was added to the bath to activated TRPV4 channels, followed by bath application of 500 nM HC067047 (HC, 6 min) to block them. Time course was normalized to baseline. B: Averaged current density in the presence of GSK (3–5 min as shown in A) and GSK+HC (9–11 min), normalized to the control current density (−3 to −1 min). Asterisk (*) indicates statistical significance (P<0.05, t-test). C: Representative traces recorded from an endothelial cell obtained from control mesenteric artery at different conditions as shown in (A), before (control) and after subsequent bath addition of GSK (GSK) and GSK+HC (HC). Whole cell current density was elicited with −80 to +60 mV voltage-ramps. D: GSK-sensitive whole-cell current density isolated from digital subtraction of the traces (C) for control cells. E: Representative perforated whole-cell current density obtained from ovx endothelial cells. F: GSK-sensitive current density isolated from digital subtraction of the traces (E) for ovx cells.
Mentions: Results thus far suggest that the reduced SK3 channel activity underlies reduced EDH-mediated vasorelaxation in ovx vessels. This model predicts that activation of TRPV4 channels with GSK should induce a smaller SK3 channel-mediated current in ovx ECs. We tested this hypothesis using perforated whole-cell recordings, in which the intracellular [Ca2+] remained intact and the calcium activation of endothelial Ca2+-activation potassium (KCa) channels was examined. Perforated whole-cell current density was elicited using the same whole-cell voltage ramp protocol as described in Figure 5, and TRPV4 channels were activated with bath application of GSK. Changes in current density, due to TRPV4 channel activation, were quantified using the steady-state current density elicited at +30 mV and normalized to the baseline before GSK (Fig. 6A). Following a stable baseline recording, bath applied GSK (30 nM) increased the whole-cell current density of ECs isolated from control mice to 150±4% (n = 6; Fig. 6B). In contrast, the effect of GSK on whole-cell current density was reduced in ovx ECs (ovx: 116±2%; n = 6; P<0.05; Fig. 6A and B). Subsequently bath applied HC (500 nM) reduced the current density to 84±5% (control) and 96±3% (ovx) of baseline (n = 6; P>0.05). The whole-cell current density, induced by activation of TRPV4 channels and digitally isolated for control (Fig. 6C and D) and ovx ECs (Fig. 6E and F), reversing at very negative membrane potentials resembling K+ currents, suggested TRPV4-dependent activation of KCa channels. Notably, activation of TRPV4 channels caused a smaller increase in KCa current density in ovx ECs (Fig. 6B). Taken together, these results suggest TRPV4-induced SK3 channel activation is significantly reduced in ovx ECs due to reduced SK3 current density, resulting in a reduced EDH-mediated vasorelaxation in mesenteric vessels obtained from ovx mice.

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