Low concentrations of sphingosylphosphorylcholine enhance pulmonary artery vasoreactivity: the role of protein kinase C delta and Ca2+ entry.

Sphingosylphosphorylcholine (SPC) is a powerful vasoconstrictor, but in vitro its EC(50) is approximately 100-fold more than plasma concentrations. We examined whether subcontractile concentrations of SPC (100 nmol/L of SPC, and independent of the endothelium, 2-aminoethoxydiphenylborane-sensitive Ca(2+) entry, and Rho kinase. It was abolished by the phospholipase C inhibitor U73122, the broad spectrum protein kinase C (PKC) inhibitor Ro31-8220, and the PKC delta inhibitor rottlerin, but not by Gö6976, which is ineffective against PKC delta. The potentiation could be attributed to enhancement of Ca(2+) entry. SPC also potentiated the responses to prostaglandin F(2 alpha) and U436619, which activate a 2-aminoethoxydiphenylborane sensitive nonselective cation channel in intrapulmonary arteries. In this case, potentiation was partially inhibited by diltiazem but abolished by 2-aminoethoxydiphenylborane, Ro31-8220, and rottlerin. SPC (1 micromol/L) caused translocation of PKC delta to the perinuclear region and cytoskeleton of cultured intrapulmonary artery smooth muscle cells. We present the novel finding that low, subcontractile concentrations of SPC potentiate Ca(2+) entry in intrapulmonary arteries through both voltage-dependent and independent pathways via a receptor-dependent mechanism involving PKC delta. This has implications for the physiological role of SPC, especially in cardiovascular disease, where SPC is reported to be elevated.

Sphingosylphosphorylcholine-induced vasoconstriction of pulmonary artery: activation of non-store-operated Ca2+ entry.

OBJECTIVE: Sphingosylphosphorylcholine (SPC) is an important lipid mediator that has been implicated in vascular disease. As it has not been studied in the pulmonary circulation, we examined its mechanisms of action in rat small intrapulmonary arteries (IPA). METHODS: IPA were mounted on a myograph for recording tension and intracellular Ca2+ concentration ([Ca2+]i). Ca2+ sensitisation was examined in alpha-toxin permeabilized IPA, and by Western blot analysis of MYPT1 phosphorylation. RESULTS: SPC induced a slow but powerful vasoconstriction in IPA associated with an elevation in [Ca2+]i, with an EC50 for vasoconstriction of 12+/-2 microM. Removal of extracellular Ca2+ increased the EC50 to 76+/-33 microM (p<0.01) and abolished the rise in [Ca2+]i. Endothelial denudation or inhibition of NO synthase with L-NAME enhanced vasoconstriction. Treatment with pertussis toxin or the PLC inhibitor U731223 had no effect on SPC-induced vasoconstriction. The Rho kinase inhibitor Y27632 reduced SPC-induced vasoconstriction by approximately 70% and abolished both SPC-induced Ca2+ sensitisation in permeabilized IPA and the associated increase in MYPT1 phosphorylation; Ca2+ sensitisation was substantially inhibited by GDPbetaS. La3+ and 2-APB, at concentrations previously shown to block capacitative Ca2+ entry in IPA, suppressed SPC-induced vasoconstriction to the same extent as removal of extracellular Ca2+; residual tension was abolished by Y27632. Diltiazem was relatively ineffective. 2-APB also abolished the SPC-induced rise in [Ca2+]i. However, treatment with thapsigargin to empty intracellular stores had no effect on the elevation of [Ca2+]i induced by SPC. CONCLUSION: We present evidence that SPC is a powerful vasoconstrictor of IPA and the novel finding that SPC-induced vasoconstriction in IPA is dependent on activation of a Ca2+ entry pathway with a similar sensitivity to La3+ and 2-APB as capacitative Ca2+ entry, although its activation is not dependent on emptying of PLC/IP3 or thapsigargin-sensitive intracellular stores.

Hypoxia, energy state and pulmonary vasomotor tone.

Vasomotor responses to hypoxia constitute a fundamental adaptation to a commonly encountered stress. It has long been suspected that changes in cellular energetics may modulate both hypoxic systemic artery vasodilatation (HSV) and hypoxic pulmonary artery vasoconstriction (HPV). Although limitation of energy has been shown to underlie hypoxic relaxation in some smooth muscles, the response to hypoxia in vascular smooth muscle does not appear to be a simple function of energy stores, but instead may involve perturbations of ATP or energy delivery to mechanisms controlling muscle force, and/or changes associated with anaerobic metabolism. Recent work in pulmonary vascular smooth muscle has demonstrated that energy stores are maintained during hypoxic pulmonary vasoconstriction, and that this is dependent on glucose availability and up-regulation of glycolysis. There is increasing evidence that glycolysis is preferentially coupled to a variety of membrane associated ATP dependent processes, including the Na(+) pump, Ca(2+)-ATPase, and possibly some protein kinases. These and other mechanisms may influence excitation-contraction coupling in both systemic and pulmonary arteries by effects on intracellular Ca(2+) and/or Ca(2+) sensitivity. Hypoxia has also been postulated to have major effects on other cytosolic second messenger systems including phosphatidylinositol pathways, cell redox state and mitochondrial reactive oxygen species production. This review examines the relationship between energy state, anaerobic respiration and hypoxic vasomotor tone, with a particular emphasis on hypoxic pulmonary vasoconstriction.