Store-operated calcium entry in vagal sensory nerves is independent of Orai channels

Abstract

Vagal sensory nerves innervate the majority of visceral organs (e.g., heart, lungs, GI tract, etc) and their activation is critical for defensive and regulatory reflexes. Intracellular Ca(2+) is a key regulator of neuronal excitability and is largely controlled by the Ca(2+) stores of the endoplasmic reticulum. In other cell types store-operated channels (SOC) have been shown to contribute to the homeostatic control of intracellular Ca(2+). Here, using Ca(2+) imaging, we have shown that ER depletion in vagal sensory neurons (using thapsigargin or caffeine) in the absence of extracellular Ca(2+) evoked Ca(2+) influx upon re-introduction of Ca(2+) into the extracellular buffer. This store-operated Ca(2+) entry (SOCE) was observed in approximately 25-40% of vagal neurons, equally distributed among nociceptive and non-nociceptive sensory subtypes. SOCE was blocked by Gd(3+) but not by the Orai channel blocker SKF96365. We found Orai channel mRNA in extracts from whole vagal ganglia, but when using single cell RT-PCR analysis we found only 3 out of 34 neurons expressed Orai channel mRNA, indicating that Orai channel expression in the vagal ganglia was likely derived from non-neuronal cell types. Confocal microscopy of vagal neurons in 3 day cultures demonstrated rich ER tracker fluorescence throughout axonal and neurite structures and ER store depletion (thapsigargin) evoked Ca(2+) transients from these structures. However, no SOCE could be detected in the axonal/neurite structures of vagal neurons. We conclude that SOCE occurs in vagal sensory neuronal cell bodies through non-Orai mechanisms but is absent at nerve terminals.

Fig 1

Mean +/− SEM Ca²⁺ responses of WT (n=111, red) and TRPA1−/ − (n=74, black) vagal neurons to thapsigargin (10μM). Blocked line denotes the 60-s application of agonist. Data are presented as mean change in 340/380 ratio as a % of ionomycin peak. All neurons responded to KCl (75mM) applied immediately before ionomycin (not shown).

Fig 2

Left, mean +/− SEM Ca²⁺ responses of vagal neuron soma to thapsigargin (Thapsi, 10 μM, red, n=310), caffeine (Caff, 10mM, black, n=233) and vehicle (green, n=88) during changes in [Ca²⁺]_{extracellular}. Response to KCl (75mM) is included. Blocked line denotes the 60-s application of drugs. Right, proportion of vagal neurons responding to the Ca²⁺ addback.

Fig 3

(A) Left, mean +/− SEM Ca²⁺ responses of vagal neurons that responded to the Ca²⁺ addback following thapsigargin (1 μM, green, n=32 out of 108) or following thapsigargin (10 μM) in the absence (black, n=121 out of 310) or presence of SKF96365 (SKF, 10 μM, red, n=35 out of 102); right, proportion of vagal neurons responding to the Ca²⁺ addback. (B) Left, mean +/− SEM Ca²⁺ responses of vagal neurons that responded to the Ca²⁺ addback following caffeine (10 mM) under control conditions (black, n=55 out of 233) or in the presence of SKF96365 (10 μM, red, n=13 out of 33) or Gd³⁺ (10 μM, green, n=5 out of 117). Blocked line denotes the 60-s application of drugs. All neurons responded to KCl (75mM) applied immediately before ionomycin.

Fig 4

Representative data of single neuron RT-PCR detection of Orai channels. (A) Expression of housekeeping gene β-actin in individual neurons (1–4) and bath control (5B); top, with reverse transcription (RT+), and bottom, without reverse transcription (RT−). (B) Left, expression of TRPV1, Orai 1–3 in the same single neuron samples as (A). All samples were pooled for RT− for each gene. Right, expression of TRPV1, Orai 1–3 in whole nodose ganglia (NG).

Fig 5

Confocal images of an in vitro 3 day culture of dissociated vagal neuron soma (top) and terminus (bottom) labeled with ER-tracker (200 nM, green) and Mito-tracker (100 nM, red).

Fig 6

Mean +/− SEM Ca²⁺ responses of WT (n=7, red) and TRPA1−/ − (n=5, black) vagal neurites to thapsigargin (Thapsi, 10μM). Blocked line denotes the 60-s application of agonist. All neurite structures responded to KCl (75mM) applied immediately before ionomycin. (Insert) Mean +/− SEM Ca²⁺ responses of vagal neurites to the TRPA1 agonist AITC (100 μM).

Fig 7

Mean +/− SEM Ca²⁺ responses of vagal neurites to thapsigargin (10 μM, black, n=7) and vehicle (red, n=7) during changes in [Ca²⁺]_{extracellular}. Blocked line denotes the 60-s application of agonist. All neurites responded to KCl (75mM) applied immediately before ionomycin.