Sphingosine 1-Phosphate Receptors Mediate the Lipid-Induced cAMP Accumulation through Cyclooxygenase-2/Prostaglandin I₂Pathway in Human Coronary Artery Smooth Muscle Cells

Abstract

Sphingosine 1-phosphate (S1P) has been shown to exert a variety of biological responses through extracellular specific receptors or intracellular mechanisms. In the present study, we characterized a signaling pathway of S1P-induced cAMP accumulation in human coronary artery smooth muscle cells (CASMCs). S1P induced biphasic cAMP accumulation composed of a short-term and transient response (a peak at 2.5 min) and a late and sustained response (ϳ4 -6 h). The late phase of cAMP accumulation was parallel to the increment of cyclooxygenase-2 protein expression and was inhibited by N-[2-(cyclohexyloxyl)-4-nitrophenyl]-methane sulfonamide (NS398), a cyclooxygenase-2-specific inhibitor. We were surprised to find that the cyclooxygenase-2 inhibitor also inhibited short-term cAMP accumulation even when cyclooxygenase-2 protein expression was not yet increased. More interestingly, the short-term cAMP accumulation was also completely inhibited by pertussis toxin, an inhibitor of G i/o proteins. JTE-013, a specific antagonist for S1P 2 receptors, inhibited the S1P-induced cAMP accumulation. Furthermore, small interfering RNAs targeted for S1P 2 receptors significantly inhibited the S1P-induced cAMP accumulation. The cAMP response was also inhibited by specific inhibitors for phospholipase C, extracellular signal-regulated kinase pathways, and cytosolic phospholipase A 2 . S1P actually activated these enzyme activities and stimulated prostaglandin I 2 (PGI 2 ) synthesis. Finally, exogenously applied arachidonic acid and PGI 2 induced cAMP accumulation to a similar extent as S1P. In conclusion, S1P induced cAMP accumulation through S1P receptors, including S1P 2 receptor and G i/o protein-mediated stimulation of intracellular signaling pathways involving cyclooxygenase-2-dependent PGI 2 synthesis.

Ovarian cancer G protein-coupled receptor 1-dependent and -independent vascular actions to acidic pH in human aortic smooth muscle cells

“…The gene is also regulated by GPCRs in vascular SMCs (9,30,32). Activation of GPCRs has a potential to stimulate COX-2 expression through the family of heterotrimeric G proteins, including G s , G i , G q/11 , and G 12/13 proteins, and a variety of intracellular signaling pathways.…”

“…The anti-COX-2 antibody reacted with COX-2, which was detected as bands at ϳ72 kDa (30,37,53), but did not react with COX-1. The sources of all other reagents were the same as those previously described (9,52).…”

Extracellular acidification stimulates IL-6 production and Ca2+mobilization through proton-sensing OGR1 receptors in human airway smooth muscle cells

“…ASMCs were cultured on 24-well multiplates for IL-6 production, 6-cm dishes for antibody array, and mRNA measurement by TaqMan. In these experiments, the plates were coated with type I collagen (8). In some experiments, the cells were treated with siRNAs as shown below.…”

Stimulatory Role of Lysophosphatidic Acid in Cyclooxygenase-2 Induction by Synovial Fluid of Patients with Rheumatoid Arthritis in Fibroblast-Like Synovial Cells

“…To evaluate the expression level of mRNAs for LPA receptor subtypes (LPA 1 , LPA 2 , LPA 3, and LPA 4 /GPR23), quantitative RT-PCR was performed using real-time TaqMan technology with a sequence detection system (model 7700, Applied Biosystems) as described previously (25). The specific probes for LPA receptors were obtained from TaqMan gene expression assays (Applied Biosystems; ID numbers of the products are Hs00173500 for LPA 1 , Hs00173704 for LPA 2 , Hs00173857 for LPA 3 , and Hs99999905 for GAPDH).…”