Lysophosphatidic acid activates peripheral glial cells

Background:

Chronic pruritus is a common symptom in hepatobiliary disorders which can dramatically reduce the quality of life of affected patients. Lysophosphatidic acid (LPA), which is synthesized extracellularly by autotaxin, could be identified as potential pruritogen. However, the molecular mechanisms resulting in neuronal activation remain elusive.

Methods:

Dorsal root ganglia (DRG) were isolated from C57BL/6, TRPV1-/-, TRPA1-/- mice as well as Wistar rats and served as culture of primary sensory neurons. A Schwann cell culture was established from sciatic nerve preparations. Transient receptor potential (TRP) A1 and V1 were cloned from murine and human mRNA. Fura-2 and Fluo-8 microfluometry was performed to quantify changes in cytosolic free calcium concentrations. Expression analyses were performed by qPCR. Immunohistochemistry was performed in whole DRG preparations.

Results:

Oleoyl-LPA elevated cytosolic free calcium concentrations in mouse and rat DRG cultures. While only a minor fraction (1.6%) of sensory neurons were activated by LPA, the majority of activated cells represented satellite glial cells (SGC) visualized by high-resolution images using real-time confocal microscope imaging. The magnitude of LPA-responses was inversely correlated with calcium responses to potassium and capsaicin. Besides satellite glial cells also Schwann cells responded to the application of LPA 18:1. Intriguingly, satellite glia cells responding to LPA were also activated by the TGR5 agonist INT-777. A striking cross-desensitization of LPA and established pruritogens could be observed. Heterologously expressed human and murine TRP channels in HEK293T cells showed at best a marginal involvement of TRP channels in cellular responses to LPA. The respective gial cells of TRPV1-/- and TRPA1-/- mice showed not relevant reduction in LPA-induced calcium responses. Various pre-treatments of wildtype DRG cultures using thapsigargin, the phospholipase C inhibitor (PLC) U73122, and the LPA receptor blockers indicated a G protein-coupled LPA receptor signaling via a PLC-IP3 pathway releasing calcium from intracellular stores. Real-time PCR experiments indicated a high expression of LPAR1 in mouse and rat DRGs and Schwann cells as well as a weak expression of LPAR4, LPAR5, and LPAR6. Immunohistochemistry in dorsal root ganglia revealed that LPAR1 expression co-localized with the glial marker glutamine synthetase.

Conclusion:

Oleoyl-LPA primarily activated satellite glia cells and peripheral Schwann cells mainly via LPAR1 rather than neuronal cells. Intriguingly, LPA responsive satellite glia cells were also activated by a TGR5 agonist, indicating that a cooperative effect of two or more pruritogens may be required for the onset of pruritus in hepatobiliary disorders.