title: Sphingosylphosphorylcholine-induced vasoconstriction of pulmonary artery: activation of non-store-operated Ca2+ entry.
abstract: 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Asthma, Allergy and Respiratory Science, GKT School of Medicine, King's College London, Guy's Campus, London SE1 9RT, UK.
authors.forename: Gavin D
authors.initials: GD
authors.lastname: Thomas
authors.forename: Vladimir A
authors.initials: VA
authors.lastname: Snetkov
authors.forename: Rupal
authors.initials: R
authors.lastname: Patel
authors.forename: Richard M
authors.initials: RM
authors.lastname: Leach
authors.forename: Philip I
authors.initials: PI
authors.lastname: Aaronson
authors.forename: Jeremy P T
authors.initials: JP
authors.lastname: Ward
mesh_terms: D000577:Amides / Q000494:pharmacology; D000818:Animals; D015153:Blotting, Western / Q000379:methods; D002118:Calcium / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D020013:Calcium Signaling; D004305:Dose-Response Relationship, Drug; D051606:G-Protein-Coupled Receptor Kinase 1 / Q000037:antagonists & inhibitors; D066298:In Vitro Techniques; D007811:Lanthanum / Q000494:pharmacology; D008246:Lysophospholipids / Q000494:pharmacology; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000378:metabolism*; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors; D010767:Phosphorylcholine / Q000031:analogs & derivatives* / Q000494:pharmacology; D011651:Pulmonary Artery / Q000187:drug effects / Q000378:metabolism*; D011725:Pyridines / Q000494:pharmacology; D051381:Rats; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000494:pharmacology; D019284:Thapsigargin / Q000494:pharmacology; D010738:Type C Phospholipases / Q000494:pharmacology; D014661:Vasoconstriction
chemical_list: D000577:Amides; D002121:Calcium Channel Blockers; D008246:Lysophospholipids; D011725:Pyridines; C005356:sphingosine phosphorylcholine; D010767:Phosphorylcholine; C108830:Y 27632; D019284:Thapsigargin; D007811:Lanthanum; D019001:Nitric Oxide Synthase; D051606:G-Protein-Coupled Receptor Kinase 1; C496765:Grk1 protein, rat; D010738:Type C Phospholipases; D013110:Sphingosine; C032881:lysophosphatidic acid; D002118:Calcium; D019331:NG-Nitroarginine Methyl Ester