Axotomy Depletes Intracellular Calcium Stores in Primary Sensory Neurons

Abstract

Background The cellular mechanisms of neuropathic pain are inadequately understood. Previous investigations have revealed disrupted Ca2+ signaling in primary sensory neurons after injury. We therefore examined the effect of injury on intracellular Ca2+ stores of the endoplasmic reticulum, which critically regulate the Ca2+ signal and neuronal function. Methods Intracellular Ca2+ levels were measured with Fura-2 or mag-Fura-2 microfluorometry in axotomized fifth lumbar (L5) dorsal root ganglion neurons and adjacent L4 neurons isolated from hyperalgesic rats following L5 spinal nerve ligation, compared to neurons from control animals. Results Endoplasmic reticulum Ca2+ stores released by the ryanodine-receptor agonist caffeine decreased by 46% in axotomized small neurons. This effect persisted in Ca2+-free bath solution that removes the contribution of store-operated membrane Ca2+ channels, and after blockade of both the mitochondrial, sarco-endoplasmic Ca2+-ATPase, and the plasma membrane Ca2+ ATPase pathways. Ca2+ released by the sarco-endoplasmic Ca2+-ATPase blocker thapsigargin and by the Ca2+-ionophore ionomycin was also diminished by 25% and 41%, respectively. In contrast to control neurons, Ca2+ stores in axotomized neurons were not expanded by neuronal activation by K+ depolarization, and the proportionate rate of refilling by sarco-endoplasmic Ca2+-ATPase was normal. Luminal Ca2+ concentration was also reduced by 38% in axotomized neurons in permeabilized neurons. The adjacent neurons of the L4 dorsal root ganglia showed modest and inconsistent changes after L5 spinal nerve ligation. Conclusions Painful nerve injury leads to diminished releasable endoplasmic reticulum Ca2+ stores and a reduced luminal Ca2+ concentration. Depletion of Ca2+ stores may contribute to the pathogenesis of neuropathic pain.

Endoplasmic-Reticulum Calcium Depletion and Disease

“…Spinal-nerve ligation depletes ER Ca 2þ ) by a loss of ER and therefore of SERCA ). Rigaud et al (2009) suggested that this may trigger a UPR, but this was not directly shown. Depletion of ER Ca 2þ stores thus contributes to the pathogenesis of neuropathic pain.…”

Divergent effects of painful nerve injury on mitochondrial Ca2+ buffering in axotomized and adjacent sensory neurons

“…Although our prior investigations of Ca 2+ signaling in the SNL model have showed that injury effects are preferentially expressed in the axotomized SNL L5 population (e.g. depressed resting [Ca 2+ ] c (Fuchs et al, 2005), loss of ER Ca 2+ stores (Rigaud et al, 2009), elevated SOCE (Gemes et al, 2011), increased PMCA (Gemes et al, 2012b), and depressed SERCA function (Duncan et al, 2013)), our present study offers evidence that injury also alters Ca 2+ handling in the intact L4 population, in this case via amplified mitochondrial buffering. The underlying mechanism for this functional shift could involve greater Ca 2+ affinity of the Ca 2+ uniporter, elevated electrochemical drive for Ca 2+ uptake through the mitochondrial inner membrane, or a higher buffering capacity within the mitochondrial matrix.…”

“…Close mitochondrial association with ER provides for bidirectional exchange of Ca 2+ . On the one hand, since mitochondria provide an important source of Ca 2+ for refilling ER Ca 2+ stores (Arnaudeau et al, 2001; Malli et al, 2005), deficient mitochondrial Ca 2+ uptake in axotomized neurons may in part explain our prior finding of depleted ER Ca 2+ stores (Rigaud et al, 2009). On the other hand, mitochondria also receive Ca 2+ from ER (Montero et al, 2001).…”

“…The cytoplasmic Ca 2+ signal initiated by Ca 2+ influx is further shaped in sensory neurons by the simultaneous processes of Ca 2+ extrusion, sequestration, and release from stores. We have identified dysfunction of these processes in axotomized neurons following painful nerve injury, including elevated function of the plasma membrane Ca 2+ -ATPase (PMCA) (Gemes et al, 2012b), accompanied by decreased resting cytoplasmic Ca 2+ ([Ca 2+ ] c ) (Fuchs et al, 2005), reduced function of the sarcoplasmic/endoplasmic reticulum Ca 2+ -ATPase (SERCA) (Duncan et al, 2013) that reduces ER Ca 2+ stores (Gemes et al, 2009; Rigaud et al, 2009), with resulting elevation of store-operated Ca 2+ entry (SOCE) (Gemes et al, 2011) and diminished release of Ca 2+ from stores upon neuronal activity through the process of Ca 2+ -induced Ca 2+ release. Together, these disturbances of Ca 2+ signaling contribute to elevated generation and transmission of high-frequency trains of action potentials in the injured sensory neurons (Gemes et al, 2009; Gemes et al, 2012a; Hogan et al, 2008; Lirk et al, 2008; Sapunar et al, 2005; Tang et al, 2012).…”

Labat Lecture: The Primary Sensory Neuron

“…Injury of the peripheral axon depletes the Ca 2+ stores, both by decreasing the size of the ER and by decreasing the concentration of Ca 2+ within the ER. 19,20 This has the effect of limiting the stored Ca 2+ available for release during neuronal activation. Because the Ca 2+ released by Ca 2+ -induced Ca 2+ release is par-ticularly important in activating K + channels, the result is exacerbation of neuronal hyperexcitability.…”