Painful nerve injury decreases sarco-endoplasmic reticulum Ca²⁺-ATPase activity in axotomized sensory neurons

Abstract

The sarco-endoplasmic reticulum Ca(2+)-ATPase (SERCA) is a critical pathway by which sensory neurons sequester cytosolic Ca(2+) and thereby maintain intracellular Ca(2+) homeostasis. We have previously demonstrated decreased intraluminal endoplasmic reticulum Ca(2+) concentration in traumatized sensory neurons. Here we examine SERCA function in dissociated sensory neurons using Fura-2 fluorometry. Blocking SERCA with thapsigargin (1 μM) increased resting [Ca(2+)](c) and prolonged recovery (τ) from transients induced by neuronal activation (elevated bath K(+)), demonstrating SERCA contributes to control of resting [Ca(2+)](c) and recovery from transient [Ca(2+)](c) elevation. To evaluate SERCA in isolation, plasma membrane Ca(2+) ATPase was blocked with pH 8.8 bath solution and mitochondrial buffering was avoided by keeping transients small (≤ 400 nM). Neurons axotomized by spinal nerve ligation (SNL) showed a slowed rate of transient recovery compared to control neurons, representing diminished SERCA function, whereas neighboring non-axotomized neurons from SNL animals were unaffected. Injury did not affect SERCA function in large neurons. Repeated depolarization prolonged transient recovery, showing that neuronal activation inhibits SERCA function. These findings suggest that injury-induced loss of SERCA function in small sensory neurons may contribute to the generation of pain following peripheral nerve injury.

Fig. 1

Sarco-endoplasmic reticulum Ca²⁺-ATPase (SERCA) regulates resting and activity-induced cytoplasmic Ca²⁺ ([Ca²⁺]_c) recovery in sensory neurons. Sample traces (A) showed that blocking SERCA by adding thapsigargin (1 μM; TG) to the bath solution (2 mM Ca²⁺) temporarily elevated resting [Ca²⁺]_c, which did not return completely to its baseline after 8 min, whereas no such changes occurred in time controls (No TG). Summary data (B) document a significant increase of resting [Ca²⁺]_c following TG administration. Also, K⁺-induced transients (50 mM for 0.3 s) recovered more slowly after TG administration (C). Mean±SEM; number in bars represents sample size; ***p<0.001.

Fig. 2

Blocking plasma membrane Ca²⁺-ATPase (PMCA) produced unstable cytoplasmic Ca²⁺ ([Ca²⁺]_c) in injured sensory neurons. Eliminating PMCA function by changing bath pH from 7.4 to 8.8 (A) increased [Ca²⁺]_c particularly in axotomized L5 neurons after spinal nerve ligation (SNL) and in Control neurons after thapsigargin treatment (1 μM; TG). [Ca²⁺]_c returned to baseline after returning bath pH to 7.4. Summary data of [Ca²⁺]_c (B) were obtained at baseline (pH 7.4) and after a fixed interval of 2 min following initiating pH 8.8 bath conditions, since a steady state level was not achieved. Significant increases were identified in all groups (paired t-test, ***p<0.001). Analysis of the incremental increases (ANOVA, p<0.001) showed that blocking PMCA increased [Ca²⁺]_c more in the SNL L5 and Control with TG groups compared to Control. Mean±SEM; number in bars represents the sample size.

Fig. 3

Increase of resting cytoplasmic Ca²⁺ ([Ca²⁺]_c) during plasma membrane Ca²⁺-ATPase (PMCA) blockade. (A) In bath with 2 mM Ca²⁺([Ca²⁺]_o=2 mM), the increase of [Ca²⁺]_c during bath change from pH 7.4 to 8.8 for 2 min was greater under baseline conditions (“−”) than when repeated in the same neurons with La³⁺ (5 μM) in the bath (“+”). Neurons were obtained from rats after spinal nerve ligation (SNL) from the axotomized L5 ganglion and the neighboring L4 ganglion, as well as TG-treated control neurons. Mean±SEM; ***p<0.001. (B) In SNL L5 neurons, the increase of [Ca²⁺]_c during bath change from pH 7.4 to 8.8 for 2 min was not significantly affected by blockers of VGCC subtypes L (5 μM nimodipine, “Nim”), N (200 nM ω-conotoxin GVIA “GVIA”), both N and P/Q (200 nM ω-conotoxin MVIIC, “MVIIC”), and T (1 μM 3,5-dichloro-N-[1-(2,2- dimethyltetrahudro-pyran-4-ylmethyl)-4-fluoro-piperidin-4-ylmethyl]-benzamide, TTA-P2, “TTA”). Bath change was performed at baseline (“BL”) and then repeated with the same neuron in the presence of the blocker. As a time control, bath change without blocker was performed at baseline (“1st”) and repeated (“2nd”). (C) Elevation of [Ca²⁺]_c during bath change from pH 7.4 to 8.8 was greatly reduced in bath with low Ca²⁺ ([Ca²⁺]_o=0.25 mM). Notice the different scale compared to panel A. Mean±SEM; number within or above the bars represents sample size.

Fig. 4

Injury reduced the amplitude of the Ca²⁺ transient induced by neuronal activation during plasma membrane Ca²⁺-ATPase (PMCA) blockade. Sample traces (A) showed smaller K⁺-induced transient amplitude in small (≤34 μm) spinal nerve ligation (SNL) L5 neurons during PMCA blockade (pH 8.8) in low Ca²⁺ (0.25 mM) external bath. Traces also demonstrate delayed transient recovery in SNL L5 neurons and thapsigargin-treated (TG, 1 μM) Control neurons, measured as the time constant for exponential fit (τ). Summary data (B) demonstrate a significantly diminished K⁺-induced transient amplitude in both large (>34 μm) and small SNL L5 neurons but not in TG-treated Control neurons during PMCA blockade. Mean±SEM; number in bars represents the sample size; **p<0.01, ***p<0.001.

Fig. 5

Injury prolonged recovery of activity-induced (K⁺, 35 mM, 1 s) transients recorded after plasma membrane Ca²⁺-ATPase (PMCA) blockade in low bath Ca²⁺ (0.25 mM) represented by the time constant for exponential fit (τ). (A) Summary data for small neurons (≤34 μm) from Control and spinal nerve ligation (SNL) animals, either with or without thapsigargin (TG, 1 μM) shows prolonged recovery of transients after axotomy (SNL L5 neurons), but no influence of injury in TG-treated neurons. Post hoc paired comparisons were evaluated between injury groups and their respective (with or without TG) control, and for each injury category between absence and presence of TG (e.g. SNL L4 with TG vs. SNL L4 without TG). (B) Transient recovery was not affected by transient amplitude in small Control, SNL L4 and SNL L5 neurons (regression p of 0.53, 0.59 and 0.38 for Control, SNL L4, and SNL L5, respectively; R² of 0.017, 0.014 and 0.021 for Control, SNL L4, SNL L5, respectively). Sample traces (C) showed that increased Ca²⁺ influx during longer K⁺-depolarizations induced larger transient amplitude but did not affect transient recovery (τ) in small Control neurons. Summary data demonstrate increasing Ca²⁺ influx with depolarization duration (D) but unaffected τ (E). In large neurons (F), recovery was not different in SNL L4 and SNL L5 neurons compared to Control. Rate of recovery in large neurons (G) was also unaffected by transient amplitude (regression p of 0.40, 0.55 and 0.66 for Control, SNL L4 and SNL L5, respectively; R² of 0.035, 0.033 and 0.017 for Control, L4, L5, respectively. Mean±SEM; number in bars represents the sample size; *p<0.05, ***p<0.001.

Fig. 6

Repeated neuronal activation leads to slowed transient recovery (τ) in neurons with blocked plasma membrane Ca²⁺-ATPase (PMCA). Sample trace (A) showed a higher transient amplitude and slower τ after repeated K⁺-depolarization in a control neuron. Summary data show that increased transient amplitude (B) and prolonged τ (C) in the 2nd K⁺-induced transient (35 mM, 1 s) in low Ca²⁺ (0.25 mM) external bath of small (≤34 μm) Control neurons. Mean±SEM; number in bars represents the sample size; ***p<0.001.