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.