Sphingolipids differentially regulate mitogen-activated protein kinases and intracellular Ca2+ in vascular smooth muscle: effects on CREB activation

1 Related sphingolipids, sphingosine 1-phosphate (S1P) and sphingosylphosphorylcholine (SPC), have important effects on vascular smooth muscle. The aim of this study was to investigate the intracellular pathways regulated by S1P and SPC in rat cerebral artery.

2 In cerebral arteries, S1P increased extracellular signal-regulated kinase (ERK) 1/2 phosphorylation (5.2 +/- 1.4-fold increase) but did not activate p38 mitogen-activated protein kinase (p38MAPK) as assessed by immunoblotting. In contrast, SPC increased p38MAPK phosphorylation (3.0 +/- 0.3-fold increase) but did not stimulate ERK1/2. This differential activation was confirmed by measuring activation of heat shock protein (HSP) 27, a known downstream target of p38MAPK. Only SPC, but not S1P, activated HSP27.

3 In enzymatically dispersed cerebral artery myocytes, SPC increased [Ca2+](i) in a concentration-dependent manner ( peak response at 10 mu M: 0.4 +/- 0.02 ratio units) as determined using the Ca2+ indicator, Fura 2. In contrast to S1P, the SPC-induced [Ca2+](i) increase did not involve intracellular release but was due to Ca2+ influx via L-type Ca2+ channels.

4 Despite differences in signalling, both S1P and SPC phosphorylated the transcription factor cAMP response element-binding protein (CREB). S1P-induced CREB activation was dependent on ERK1/2 and Ca2+-calmodulin-dependent protein kinase ( CaMK) activation. CREB activation by SPC required both p38MAPK and CaMK activation, but not ERK1/2.

5 In conclusion, S1P and SPC activate distinct MAP kinase isoforms and increase [Ca2+](i) via different mechanisms in rat cerebral artery. This does not affect the ability of S1P or SPC to activate CREB, although this occurs via different pathways.