Abstract
BackgroundSphingosine-1-phosphate (S1P) is a bioactive sphingolipid that acts through a family of five G-protein-coupled receptors (S1PR1–5) and plays a key role in regulating the inflammatory response. Our previous studies demonstrated that rat sensory neurons express the mRNAs for all five S1PRs and that S1P increases neuronal excitability primarily, but not exclusively, through S1PR1. This raises the question as to which other S1PRs mediate the enhanced excitability.MethodsIsolated sensory neurons were treated with either short-interfering RNAs (siRNAs) or a variety of pharmacological agents targeted to S1PR1/R2/R3 to determine the role(s) of these receptors in regulating neuronal excitability. The excitability of isolated sensory neurons was assessed by using whole-cell patch-clamp recording to measure the capacity of these cells to fire action potentials (APs).ResultsAfter siRNA treatment, exposure to S1P failed to augment the excitability. Pooled siRNA targeted to S1PR1 and R3 also blocked the enhanced excitability produced by S1P. Consistent with the siRNA results, pretreatment with W146 and CAY10444, selective antagonists for S1PR1 and S1PR3, respectively, prevented the S1P-induced increase in neuronal excitability. Similarly, S1P failed to augment excitability after pretreatment with either VPC 23019, which is a S1PR1 and R3 antagonist, or VPC 44116, the phosphonate analog of VPC 23019. Acute exposure (10 to 15 min) to either of the well-established functional antagonists, FTY720 or CYM-5442, produced a significant increase in the excitability. Moreover, after a 1-h pretreatment with FTY720 (an agonist for S1PR1/R3/R4/R5), neither SEW2871 (S1PR1 selective agonist) nor S1P augmented the excitability. However, after pretreatment with CYM-5442 (selective for S1PR1), SEW2871 was ineffective, but S1P increased the excitability of some, but not all, sensory neurons.ConclusionsThese results demonstrate that the enhanced excitability produced by S1P is mediated by activation of S1PR1 and/or S1PR3.
Highlights
Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid that acts through a family of five G-protein-coupled receptors (S1PR1–5) and plays a key role in regulating the inflammatory response
To explore the idea that S1PR1, R2, and R3 are key players in the S1P-mediated sensitization, short-interfering RNA (siRNA) targeted to these sphingosine-1-phosphate receptor (S1PR) were designed and their ability to reduce the expression of their respective receptor was measured by quantitative PCR (qPCR)
Our previous results showed that siRNA targeted to S1PR1 reduced its expression by about 75% [15]; this siRNA was used in experiments described below
Summary
Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid that acts through a family of five G-protein-coupled receptors (S1PR1–5) and plays a key role in regulating the inflammatory response. Our previous studies demonstrated that rat sensory neurons express the mRNAs for all five S1PRs and that S1P increases neuronal excitability primarily, but not exclusively, through S1PR1 This raises the question as to which other S1PRs mediate the enhanced excitability. Small-diameter sensory neurons treated with siRNA targeted to S1PR1 were unresponsive to the S1PR1 selective agonist SEW2871; treatment with the more global agonist, S1P, was still capable of increasing the excitability in approximately one third of the siRNA-treated neurons [15] These observations indicated that S1PR1 plays a prominent role in the S1P-induced neuronal sensitization, but there must be other S1P receptors capable of mediating the S1Pinduced enhancement of excitability. The studies described below show that, in addition to S1PR1, activation of S1PR3 can lead to the enhancement of excitability in sensory neurons
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