ET-1-associated vasomotion and vasospasm in lymphatic vessels of the guinea-pig mesentery

Abstract

In vitro experiments were performed to investigate the actions of endothelin-1 (ET-1) on vasomotion and vasospasm in guinea-pig mesenteric lymphatics. ET-1 modulated lymphatic vasomotion independent of the endothelium, with lower concentrations (<or=10 nm) increasing lymphatic vasomotion and higher concentrations (>or=100 nm) causing vasospasm. ET-1-induced increases in vasomotion were accompanied by an increase in tonic [Ca2+]i. These actions were inhibited by the ETA receptor antagonist BQ-123 (1 microm), the phospholipase C (PLC) inhibitor U73122 (5 microm), removal of extracellular Ca2+, chelation of intracellular Ca2+ with BAPTA/AM (10 microm), the store Ca2+-ATPase inhibitor thapsigargin (1 microm), caffeine (10 mm) and the inositol 1,4,5-trisphosphate (IP3) receptor blocker heparin and 2-APB (30 microm). In contrast, the ETB receptor antagonist BQ-788 (1 microm), ryanodine (1 & 20 microm), pertussis toxin (PTx) or Cs+ had no significant actions on vasomotion or the magnitude of increase in tonic [Ca2+]i. ET-1-induced vasospasm was accompanied by a transient increase in smooth muscle [Ca2+]i followed by a sustained plateau, an action that was abolished by removal of extracellular Ca2+, but only marginally inhibited by nifedipine (1 microm). Caffeine (10 mm), SKF 96165 (30 microm) or U73122 (5 microm) together with nifedipine (1 microm) abolished ET-1-induced vasospasm and increase in [Ca2+]i. These results indicate that ET-1 increases lymphatic vasomotion by acting on smooth muscle ETA receptors and activation of G-protein-PLC-IP3 cascade, which is known to cause pacemaker Ca2+ release and resultant pacemaker potentials. High concentrations of ET-1 cause a failure in Ca2+ homeostasis causing vasospasm, triggered by excessive Ca2+ influx primarily through store-operated channels (SOCs) with l-Ca2+ voltage-operated channels (VOCs) also contributing, but to a much lesser extent.

Figure 1

Effects of ET-1 on lymphatic vasomotion and vessel tone as measured from vessel diameter. (a) Sample traces indicating ET-1-induced lymphatic vasomotion (upper trace) and lymphatic vasospasm (lower trace; upward deflections indicate constrictions). (b, c) ET-1 concentration–response curves for lymphatic constriction rate and vessel tone (measured as (D_i−D)/D_i) in vessels with or without endothelium. The double-sided arrow in (b) is to highlight the abrupt change from vasomotion tovasospasm. (d) Comparison of concentration–response curves for ET-1 on the lymphatic constriction rate and tone. Data were normalised with respect to the corresponding control lymphangion constriction rate and diameter (D_i), respectively, with n=9–12 lymphangions for all points.

Figure 2

Effects of ET-1 on vasomotion (measured using Ca²⁺ transients) and relative tonic [Ca²⁺]_i in lymphatic smooth muscle. (a) Sample traces showing fura-2 F₃₄₀ and F₃₈₀ fluorescence measurements with the F₃₄₀/F₃₈₀ ratio used to measure ET-1-induced increases in relative lymphatic smooth muscle [Ca²⁺]_i in response to low (1 nM) and higher ET-1 concentrations (100 nM). (b) ET-1 concentration–response curves for the % change in constriction rate and tonic [Ca²⁺]_i. (c) Comparison of ET-1 concentration–response curves for the % change in constriction rate measured either using fura-2-based Ca²⁺ transients or by video-recorded movement (i.e. the endothelium-denuded record of Figure 1b). (d) Comparison of ET-1 concentration–response curves for the % change in tonic [Ca²⁺]_i and normalised change in vessel tone (i.e. (D_i−D)/D_i; endothelium-denuded record of Figure 1c). All data presented here are from endothelium-denuded vessels. Data were normalised with respect to the corresponding control tissues with n=6–9. Vertical lines denote s.e.m. with n=9–12 lymphangions for all points.

Figure 3

Effects of ET-1 receptor antagonists on ET-1-induced vasomotion and relative [Ca²⁺]_i. BQ-123 (1 μM), an ET_A receptor blocker, inhibited vasomotion induced by 1 nM ET-1 (a) and prevented the associated increase in [Ca²⁺]_i (b). BQ-788 (1 μM), an ET_B receptor blocker, did not significantly alter vasomotion induced by 1 nM ET-1 (c) or associated increase in [Ca²⁺]_i (d). Direct application of the antagonists had no significant effects on either vasomotion or [Ca²⁺]_i. Data were normalised with respect to the corresponding control lymphangions with n=6–9. Vertical lines denote s.e.m. ^**P<0.01.

Figure 4

Application of the PLC inhibitor U73122 (5 μM) inhibited the increase in both vasomotion (a) and tonic [Ca²⁺]_i (b) induced by 1 nM ET-1. In contrast, the inactive analogue U73343 (5 μM) had no significant effect. Data were normalised with respect to the corresponding control lymphangions with n=9–12. Vertical lines denote s.e.m. ^**P<0.01.

Figure 5

Application of the store Ca²⁺ pump inhibitor thapsigargin (1 μM) inhibited the increase in both vasomotion (a) and tonic [Ca²⁺]_i (b) induced by 1 nM ET-1. Data were normalised with respect to the corresponding control lymphangions with n=9–12. Vertical lines denote s.e.m. ^**P<0.01 and ^*P<0.05.

Figure 6

Intracellular heparin inhibits the increase in vasomotion and tonic [Ca²⁺]_i induced by 1 nM ET-1. Lymphatic smooth muscle was preloaded with heparin (8 mg ml⁻¹) by a reversible permeabilisation procedure. The records show the fura-2 F₃₄₀/F₃₈₀ fluorescence ratio response to ET-1 in lymphatic smooth muscle without (a) and with heparin loading (b).

Figure 7

Effects of ryanodine and caffeine on the increase in vasomotion and tonic [Ca²⁺]_i induced by 1 nM ET-1. (A) The sample traces indicating the changes of the F₃₄₀/F₃₈₀ fluorescence ratio in the smooth muscle of the same lymphatic vessel exposed to 1 nM ET-1 (a), ryanodine (20 μM) (b), and ET-1 (1 nM), in the presence of ryanodine (20 μM) (c) are shown. Ryanodine did not prevent the responses to 1 nM ET-1. (B) Sample traces indicating the changes of the F₃₄₀/F₃₈₀ fluorescence ratio in the smooth muscle of a lymphatic vessel exposed to 1 nM ET-1 (a), 10 mM caffeine (b) and both 10 mM caffeine and 1 nM ET-1 (c) are shown. Caffeine inhibited the increase in vasomotion and tonic [Ca²⁺]_i induced by 1 nM ET-1.

Figure 8

Effect of Ca²⁺-free solution on ET-1-induced increase in [Ca²⁺]_i. Application of 100 nM ET-1 caused a large transient followed by sustained increase in [Ca²⁺]_i measured as the fura-2 F₃₄₀/F₃₈₀ fluorescence ratio in lymphatic smooth muscle. The sustained increase in [Ca²⁺]_i became transient when 100 nM ET-1 was applied in a Ca²⁺-free 1 mM EGTA-containing solution, with the tissue only briefly (i.e. ∼1 min) preincubated in this Ca²⁺-free solution.

Figure 9

Effects of nifedipine on ET-1- and K⁺-induced vasospasm. (a) Sample traces indicating the changes in lymphatic smooth muscle [Ca²⁺]_i measured as the F₃₄₀/F₃₈₀ fura-2 fluorescence ratio in response to 100 nM ET-1, 1 μM nifedipine and 100 nM ET-1 applied in the presence of 1 μM nifedipine. (b) Bar graph indicating the mean effect of 100 nM ET-1 on tonic [Ca²⁺]_i before and in the presence of 1 μM nifedipine. (c) Bar graph showing the mean effects of both 100 nM ET-1 and 100 mM KCl solution on relative lymphangion tone (i.e. measured as (D_i−D)/D_i)) in the absence and presence of 1 μM nifedipine. Data bars in (b) and (c) were normalised with respect to the corresponding controls with n=5 lymphangions for each value. Vertical lines denote s.e.m. ^**P<0.01.

Figure 10

Effects of SKF 96365 on ET-1-induced vasospasm. Sample traces indicating the changes in lymphatic smooth muscle [Ca²⁺]_i measured as the F₃₄₀/F₃₈₀ fura-2 fluorescence ratio in response to 100 nM ET-1 and both SKF 96365 (5 μM) and 100 nM ET-1.

Figure 11

Effects of caffeine and the PLC antagonist U73122 on ET-1-induced vasospasm. (a) The effects of 100 nM ET-1 on constriction of a lymphangion in control, caffeine (10 mM) and caffeine (10 mM) together with nifedipine (1 μM). (b) Bar graphs showing the effects of U73343 (5 μM) and U73122 (5 μM) on the relative tonic increase in lymphangion constriction in response to 100 nM ET-1 in the presence of nifedipine (1 μM). Data in (b) were normalised with respect to the corresponding control tissues with n=6 lymphangions for all points. Vertical lines denote s.e.m. ^**P<0.01.