Interleukin‐1 beta enhances endocytosis of glial glutamate transporters in the spinal dorsal horn through activating protein kinase C

Abstract

Excessive activation of glutamate receptors in spinal dorsal horn neurons is a key mechanism leading to abnormal neuronal activation in pathological pain conditions. Previous studies have shown that activation of glutamate receptors in the spinal dorsal horn is enhanced by impaired glial glutamate transporter functions and pro-inflammatory cytokines including interleukin-1 beta (IL-1β). In this study, we for the first time revealed that spinal glial glutamate transporter activities in the neuropathic animals are attenuated by endogenous IL-1β. Specifically, we demonstrated that nerve injury results in an increased expression of IL-1β and activation of PKC in the spinal dorsal horn as well as suppression of glial glutamate uptake activities. We provided evidence that the nerve-injury induced suppression of glial glutamate uptake is at least in part ascribed to endogenous IL-1β and activation of PKC in the spinal dorsal horn. IL-1β reduces glial glutamate transporter activities through enhancing the endocytosis of both GLT-1 and GLAST glial glutamate transporters. The IL-1β induced trafficking of glial glutamate transporters is through the calcium/PKC signaling pathway, and the dynamin-dependent endocytosis, which is dependent on the integrity of actin filaments. The signaling pathway regulating glial glutamate transporters revealed in this study provides novel targets to attenuate aberrant activation of glutamate receptors in the spinal dorsal horn, which could ultimately help the development of analgesics.

Adenosine Monophosphate–activated Protein Kinase Regulates Interleukin-1β Expression and Glial Glutamate Transporter Function in Rodents with Neuropathic Pain

“…The size of GTCs reflects the amount of transported glutamate, which has been widely used as an effective tool to study the function of glial GTs. 14,36,50 We recently reported that mice with pSNL had lower amplitudes of GTCs. 14 After recording baseline GTCs in spinal slices taken from neuropathic GFP-GFAP mice, we perfused the AMPK activator (AICAR, concentration in the bath: 10 μM) into the recording chamber and recorded GTCs again.…”

“…For measuring protein expression in the plasma membrane and cytosol in rat spinal slices, spinal slices of the spinal L4 to L5 segment were obtained as previously described. 14 Spinal slices were incubated with plain artificial cerebrospinal fluid (aCSF), aCSF plus IL-1β (10 ng/mL), or aCSF plus IL-1β (10 ng/mL) and AICAR (10 μM) bubbled with 95% O 2 and 5% CO 2 at 35°C for 15 min. The dorsal halves of the spinal slices were quickly frozen in liquid nitrogen and stored at −80°C.…”

“…14 Astrocytes in these mice were labeled by the green fluorescent protein which is under the control of the astrocyte-specific GFAP promoter. 23 Transverse spinal cord slices (400 μm thick) of the L4 to L5 segment were prepared 14,35 and pre-incubated in Krebs solution oxygenated with 95% O 2 and 5% CO 2 at 35°C. The Krebs solution contained (mM): 117.0 NaCl, 3.6 potassium chloride, 1.2 magnesium chloride, 2.5 calcium chloride, 1.2 monosodium phosphate, 11.0 glucose, and 25.0 sodium bicarbonate at 35°C.…”

“…GTCs were recorded at a holding potential of −80 mV in voltage clamp mode in the presence of blockers of γ-Aminobutyric acid A (GABA A ) receptor (10 μM bicuculline), glycine receptor (5 μM strychnine), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor/kainate receptors (10 μM DNQX), N-methyl-D-aspartate (NMDA) receptor (25 μM D-AP5), and tetrodotoxin (1 μM) at 35°C. 14,36 GTCs were evoked by puffing 50 μM L-glutamate onto the recorded astrocyte through a glass pipette connected to a Picospritzer controlled by a computer. Access resistance within the range of 10–20 MΩ was monitored continuously throughout the experiments.…”

“…Four sweeps of GTCs were averaged and the mean amplitude and charge transfer of GTCs were measured. 14,37 The commercial computer software Clampfit (Molecular Device, Sunnyvale, CA) was used for data analysis.…”