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

Abstract

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 microM: 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.

Figure 1

S1P- and SPC-induced phosphorylation of MAP kinases and HSP27 in rat cerebral arteries. (a) Representative immunoblots showing changes in phosphorylation of ERK1/2 by S1P and SPC in rat cerebral arteries. The bar graph represents fold increase in phosphorylation of ERK1 and ERK2 combined compared to basal levels. S1P (5 μM) significantly increased phosphorylation after 15 min stimulation (n=6). SPC (10 μM), however, did not significantly increase ERK1/2 phosphorylation after 15 min stimulation. (b) Representative immunoblots and bar graph demonstrating increased phosphorylation of p38MAPK by S1P and SPC in rat cerebral arteries. After 15 min of stimulation, SPC (10 μM) significantly increased phosphorylation (n=6) while S1P (5 μM) did not significantly increase p38MAPK phosphorylation (n=6). (c) Representative immunoblots and bar graph showing the fold increases in phosphorylation of HSP27 by S1P and SPC in the rat cerebral artery. Following 15 min stimulation, SPC (10 μM) significantly increased HSP27 phosphorylation; however, S1P (5 μM) did not increase HSP27 phosphorylation (n=5). Preincubation with the p38MAPK inhibitor, SB203580 (30 μM), for 30 min, the SPC (10 μM)-induced phosphorylation was significantly inhibited (n=5). Data shown are mean±s.e.m. Asterisk denotes P<0.05.

Figure 2

Sphingolipid-induced increases in [Ca²⁺]_i from cerebral artery myocytes. (a) Representative Fura-2 fluorescence ratio traces (FR_340/380) showing the changes in fluorescence produced upon application of S1P (5 μM, n=12) and SPC (10 μM, n=41) in freshly isolated myocytes from rat cerebral artery. (b) Bar graph showing the time taken for the fluorescence ratio to rise to peak after addition of S1P (5 μM, n=12) and SPC (10 μM, n=41). Data are mean±s.e.m. Asterisk denotes P<0.05.

Figure 3

Characteristics of the SPC-induced [Ca²⁺]_i increase in rat cerebral artery myocytes. (a) Mean data showing the increase in fluorescence ratio (peak) relative to resting levels induced by increasing concentrations of SPC. EC₅₀ could not be calculated as the curve was not saturable at soluble concentrations of SPC. (b) Mean data showing the increase in fluorescence ratio (relative to resting levels) induced by SPC (10 μM) following pretreatment with either 1 μM thapsigargin, 2 μM nifedipine or in the presence of no added extracellular Ca²⁺. Thapsigargin had no effect on SPC-induced [Ca²⁺]_i. However, both nifedipine and the removal of extracellular Ca²⁺ significantly decreased the Ca²⁺ increase induced by SPC. Resting Ca²⁺ levels at 10 μM SPC are 0.30±0.01 FR_340/380 (n=83). Data are mean±s.e.m. with numbers of cells in parentheses. Asterisk denotes P<0.05.

Figure 4

Phosphorylation of CREB by S1P and SPC in rat cerebral arteries. Representative immunoblot and bar graph showing changes in CREB phosphorylation by S1P and SPC. After 15 min of stimulation, both S1P (5 μM) and SPC (10 μM) significantly increased CREB phosphorylation (n=5). Data shown are mean±s.e.m. Asterisk denotes P<0.05.

Figure 5

Intracellular mechanism of CREB phosphorylation by S1P and SPC. (a) Representative immunoblot and bar graph showing the fold increases in CREB phosphorylation by S1P and SPC in the rat cerebral artery in the presence of the ERK1/2 inhibitor, PD98059 (30 μM). The S1P-induced phosphorylation was significantly inhibited in the presence of PD98059 while the SPC-induced phosphorylation was unaffected (n=4). (b) Representative immunoblot and bar graph showing the fold increases in CREB phosphorylation by S1P and SPC in the presence of the p38MAPK inhibitor, SB203580 (30μM). The SPC-induced phosphorylation was significantly inhibited in the presence of SB203580 while the S1P-induced phosphorylation was unaffected (n=6). (c) Representative immunoblot and bar graph showing the fold increases in CREB phosphorylation by S1P and SPC in the presence of the CaM kinase inhibitor, KN93 (30 μM). Both the S1P- and SPC-induced CREB phosphorylation was significantly inhibited by KN93 (n=4). (d) Typical immunoblot showing S1P-induced ERK1/2 activation following pretreatment with 2-APB and nifedipine. ERK1/2 phosphorylation was significantly decreased in the presence of inhibitors (S1P stimulation 5.6±0.5-fold increase of control; S1P stimulation following 2-APB/nifedipine pretreatment – 0.7±0.1-fold increase of control, n=3, P<0.05). Data shown are mean±s.e.m. Asterisk denotes P<0.05.