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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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Reduced ACh-induced vasorelaxation due to decreased SK3 channel contribution in ovx vessels.A: (left panel) Representative force myograph recording showing tension (mN) plotted against time (s) using a mesenteric vessel obtained from control mouse. Addition of 3 µM PE increased tension and 1 µM ACh caused 64% vasorelaxation, normalized to the PE-induced tension. (right panel) Following bath washout, PE was added to pre-contract the vessel ∼50%, followed by the addition of 100 µM L-NAME and 1 µM ACh. L-NAME-induced 61% increase in PE-induced contraction and ACh reduced tension by 34%. B: Representative force myograph trace obtained from an ovx artery. C and D: Summarized results for (A and B) and for other selective inhibitors to block different vasorelaxation pathways to study their (C) change in tone and (D) contribution to ACh-induced relaxation for both control (black bars) and ovx (grey bars) vessels. C: Change in tone was obtained from tension increase in the presence of inhibitors normalized to the baseline tension (eg. 61% and 34% increase in the presence of L-NAME for control and ovx vessels, respectively, as shown in A and B). D: Contribution to ACh-induced relaxation was calculated from the difference in ACh relaxation before and after inhibitor treatment, normalized to the control (before) ACh relaxation. L-NAME blocks nitric oxide (NO) pathway; indomethacin blocks prostacyclin (PGI2) pathway; apamin (apa) and tram34 (tram) together block the EDH pathway.
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pone-0104686-g003: Reduced ACh-induced vasorelaxation due to decreased SK3 channel contribution in ovx vessels.A: (left panel) Representative force myograph recording showing tension (mN) plotted against time (s) using a mesenteric vessel obtained from control mouse. Addition of 3 µM PE increased tension and 1 µM ACh caused 64% vasorelaxation, normalized to the PE-induced tension. (right panel) Following bath washout, PE was added to pre-contract the vessel ∼50%, followed by the addition of 100 µM L-NAME and 1 µM ACh. L-NAME-induced 61% increase in PE-induced contraction and ACh reduced tension by 34%. B: Representative force myograph trace obtained from an ovx artery. C and D: Summarized results for (A and B) and for other selective inhibitors to block different vasorelaxation pathways to study their (C) change in tone and (D) contribution to ACh-induced relaxation for both control (black bars) and ovx (grey bars) vessels. C: Change in tone was obtained from tension increase in the presence of inhibitors normalized to the baseline tension (eg. 61% and 34% increase in the presence of L-NAME for control and ovx vessels, respectively, as shown in A and B). D: Contribution to ACh-induced relaxation was calculated from the difference in ACh relaxation before and after inhibitor treatment, normalized to the control (before) ACh relaxation. L-NAME blocks nitric oxide (NO) pathway; indomethacin blocks prostacyclin (PGI2) pathway; apamin (apa) and tram34 (tram) together block the EDH pathway.

Mentions: For these studies, both control and ovx arteries were pre-contracted with PE (3 µM; EC80) and subsequently exposed to 1 µM ACh. In control arteries, ACh induced 64% relaxation (Fig. 3A left). Following bath washout, ACh responses were assessed again in the presence of inhibitor. Notably, we reduced the second PE-induced precontraction (1 µM; EC50) in order to allow for any additional contractile effects of inhibitors (Fig. 3A right). The representative effect of L-NAME (NG-nitro-L-arginine methyl ester, an inhibitor for NO production) on tone and ACh-induced relaxation in vessels obtained from control mice is shown in Figure 3A (right). Addition of 100 µM L-NAME caused a 61% increase in force (normalized to the force difference between middle and bottom dashed lines as 100%; Fig. 3A right). This increase in force occurred over ∼15–20 min, revealing the presence of NO-dependent activity. Time controls for PE contractions showed less than 14% change in tone over the full course of the functional experiments (∼20 min). This experimental approach revealed the preexistence of NO activity in PE-contracted vessels. Contraction in response to L-NAME suggests regulation of mesenteric arterial tone by nitric oxide even in the absence of direct endothelial stimulation; thus, we attributed this increase to a preexisting activity of NO on vascular tone (summarized in Fig. 3C). In the presence of L-NAME, application of 1 µM ACh induced a 34% reduction in force (of top-to-bottom dashed lines; Fig. 3A right). Thus, ACh-induced vasorelaxation, in the presence of L-NAME, was reduced to 53% (34/64%), indicating that NO contributed 47% to ACh-induced vasorelaxation (summarized in Fig. 3D; see Methods).


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)

Reduced ACh-induced vasorelaxation due to decreased SK3 channel contribution in ovx vessels.A: (left panel) Representative force myograph recording showing tension (mN) plotted against time (s) using a mesenteric vessel obtained from control mouse. Addition of 3 µM PE increased tension and 1 µM ACh caused 64% vasorelaxation, normalized to the PE-induced tension. (right panel) Following bath washout, PE was added to pre-contract the vessel ∼50%, followed by the addition of 100 µM L-NAME and 1 µM ACh. L-NAME-induced 61% increase in PE-induced contraction and ACh reduced tension by 34%. B: Representative force myograph trace obtained from an ovx artery. C and D: Summarized results for (A and B) and for other selective inhibitors to block different vasorelaxation pathways to study their (C) change in tone and (D) contribution to ACh-induced relaxation for both control (black bars) and ovx (grey bars) vessels. C: Change in tone was obtained from tension increase in the presence of inhibitors normalized to the baseline tension (eg. 61% and 34% increase in the presence of L-NAME for control and ovx vessels, respectively, as shown in A and B). D: Contribution to ACh-induced relaxation was calculated from the difference in ACh relaxation before and after inhibitor treatment, normalized to the control (before) ACh relaxation. L-NAME blocks nitric oxide (NO) pathway; indomethacin blocks prostacyclin (PGI2) pathway; apamin (apa) and tram34 (tram) together block the EDH pathway.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0104686-g003: Reduced ACh-induced vasorelaxation due to decreased SK3 channel contribution in ovx vessels.A: (left panel) Representative force myograph recording showing tension (mN) plotted against time (s) using a mesenteric vessel obtained from control mouse. Addition of 3 µM PE increased tension and 1 µM ACh caused 64% vasorelaxation, normalized to the PE-induced tension. (right panel) Following bath washout, PE was added to pre-contract the vessel ∼50%, followed by the addition of 100 µM L-NAME and 1 µM ACh. L-NAME-induced 61% increase in PE-induced contraction and ACh reduced tension by 34%. B: Representative force myograph trace obtained from an ovx artery. C and D: Summarized results for (A and B) and for other selective inhibitors to block different vasorelaxation pathways to study their (C) change in tone and (D) contribution to ACh-induced relaxation for both control (black bars) and ovx (grey bars) vessels. C: Change in tone was obtained from tension increase in the presence of inhibitors normalized to the baseline tension (eg. 61% and 34% increase in the presence of L-NAME for control and ovx vessels, respectively, as shown in A and B). D: Contribution to ACh-induced relaxation was calculated from the difference in ACh relaxation before and after inhibitor treatment, normalized to the control (before) ACh relaxation. L-NAME blocks nitric oxide (NO) pathway; indomethacin blocks prostacyclin (PGI2) pathway; apamin (apa) and tram34 (tram) together block the EDH pathway.
Mentions: For these studies, both control and ovx arteries were pre-contracted with PE (3 µM; EC80) and subsequently exposed to 1 µM ACh. In control arteries, ACh induced 64% relaxation (Fig. 3A left). Following bath washout, ACh responses were assessed again in the presence of inhibitor. Notably, we reduced the second PE-induced precontraction (1 µM; EC50) in order to allow for any additional contractile effects of inhibitors (Fig. 3A right). The representative effect of L-NAME (NG-nitro-L-arginine methyl ester, an inhibitor for NO production) on tone and ACh-induced relaxation in vessels obtained from control mice is shown in Figure 3A (right). Addition of 100 µM L-NAME caused a 61% increase in force (normalized to the force difference between middle and bottom dashed lines as 100%; Fig. 3A right). This increase in force occurred over ∼15–20 min, revealing the presence of NO-dependent activity. Time controls for PE contractions showed less than 14% change in tone over the full course of the functional experiments (∼20 min). This experimental approach revealed the preexistence of NO activity in PE-contracted vessels. Contraction in response to L-NAME suggests regulation of mesenteric arterial tone by nitric oxide even in the absence of direct endothelial stimulation; thus, we attributed this increase to a preexisting activity of NO on vascular tone (summarized in Fig. 3C). In the presence of L-NAME, application of 1 µM ACh induced a 34% reduction in force (of top-to-bottom dashed lines; Fig. 3A right). Thus, ACh-induced vasorelaxation, in the presence of L-NAME, was reduced to 53% (34/64%), indicating that NO contributed 47% to ACh-induced vasorelaxation (summarized in Fig. 3D; see Methods).

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