Figure 1.
Ovariectomy reduces ACh-induced vasorelaxation.
Representative force myograph traces showing isometric tension (mN) plotted against time (s) for both control (A) and ovariectomized (ovx; B) mesenteric vessels, and their concentration responses to both phenylephrine (PE; left panel) and acetylcholine (ACh; right panel). A and B: (left panel) Arrowheads and arrow markers show addition of different [PE] (from left to right: 0.1, 0.3, 0.7, 1, 3, 7, 10, and 30 µM); the arrow indicates the addition of 3 µM PE. (right panel) Following PE washout the vessels were ∼50% pre-contracted with PE and different [ACh] were added (markers from left to right: 0.01, 0.03, 0.1, 0.3, 1, 3 µM), with the arrow indicating 1 µM ACh. Normalized concentration-response of PE (C) and ACh (D) for both control (solid circles) and ovx (empty squares) vessels. Data were fitted with sigmoidal curves for both control (solid lines) and ovx (dotted lines) vessels. Both plots were normalized to the maximum PE-induced tension.
Figure 2.
Compromised ACh-induced relaxation in ovx mesenteric vessels.
A: Representative force myograph traces showing isometric tension (mN) plotted against the time of recording (s) using control (top panel) and ovx (bottom panel) mesenteric vessels. Bath application of PE (3 and 5 µM) induced vasoconstriction, ACh (1 µM) induced vasorelaxation, was followed by bath solution replacement with physiological salt solution containing 60 mM KCl that induced vasoconstriction. B: Summary of isometric tension changes induced by PE (3 uM), KCl, and ACh for both control (black) and ovx (grey) vessels. Negative values show vasorelaxation. C: Tension changes normalized to the KCl-induced contraction for both PE and ACh. Ovx vessels show reduced ACh response. Asterisk (*) denotes statistical significance (P<0.05, t-test).
Figure 3.
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.
Figure 4.
IK1 channel activity mediates TRPV4-induced vasorelaxation in ovx vessels.
A: Summarized results from studies using 500 nM HC067047 (HC), a TRPV4 channel antagonist, on change in tone (left panel) and contribution to ACh-induced relaxation (right panel) using vessels obtained from both control (black) and ovx (grey) mice. B: Summarized results from studies using 300 nM GSK1016790, a TRPV4 channel agonist, on changes to vascular tension in the absence and presence of apamin (apa) and/or tram34 (tram). These studies were performed in the presence of L-NAME and indomethacin. Asterisk (*) denotes statistical significance (P<0.05, t-test).
Figure 5.
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).
Figure 6.
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.