Physically disconnected non-diffusible cell-to-cell communication between neuroblastoma SH-SY5Y and DRG primary sensory neurons

Abstract

Background: Cell-cell communication occurs via a variety of mechanisms, including long distances (hormonal), short distances (paracrine and synaptic) or direct coupling via gap junctions, antigen presentation, or ligand-receptor interactions. We evaluated the possibility of neuro-hormonal independent, non-diffusible, physically disconnected pathways for cell-cell communication using dorsal root ganglion (DRG) neurons.

Methods: We assessed intracellular calcium ([Ca(2+)]) in primary culture DRG neurons that express ATP-sensitive P2X3, capsaicinsensitive TRPV1 receptors modulated by estradiol. Physically disconnected (dish-in-dish system; inner chamber enclosed) mouse DRG were cultured for 12 hours near: a) media alone (control 1), b) mouse DRG (control 2), c) human neuroblastoma SHSY-5Y cells (cancer intervention), or d) mouse DRG treated with KCl (apoptosis intervention).

Results: Chemosensitive receptors [Ca(2+)](i) signaling did not differ between control 1 and 2. ATP (10 μM) and capsaicin (100nM) increased [Ca(2+)](i) transients to 425.86 + 49.5 nM, and 399.21 ± 44.5 nM, respectively. 17β-estradiol (100 nM) exposure reduced ATP (171.17 ± 48.9 nM) and capsaicin (175.01±34.8 nM) [Ca(2+)](i) transients. The presence of cancer cells reduced ATP- and capsaicin-induced [Ca(2+)](i) by >50% (p<0.05) and abolished the 17β-estradiol effect. By contrast, apoptotic DRG cells increased initial ATP-induced [Ca(2+)](i), flux four fold and abolished subsequent [Ca(2+)](i), responses to ATP stimulation (p<0.001). Capsaicin (100nM) induced [Ca(2+)](i) responses were totally abolished.

Conclusion: The local presence of apoptotic DRG or human neuroblastoma cells induced differing abnormal ATP and capsaicin-mediated [Ca(2+)](i) fluxes in normal DRG. These findings support physically disconnected, non-diffusible cell-to-cell signaling. Further studies are needed to delineate the mechanism(s) of and model(s) of communication.

Keywords: Cell-cell communication; DRG; P2X3; SH-SY5Y; TRPV1.

Figure 1

Proposed mechanism of estradiol effect on ATP-induced [Ca²⁺]_i signaling in primary sensory neurons. ATP acts on P2X3 receptor resulting in activation of the L-type voltage-gated calcium channel (VGCC) stimulating an increase in [Ca²⁺]_i. Subsequent activation of voltage-gated sodium channels (VGSC) will lead to pain sensation. Capsaicin acting on nociceptive TRPV1 receptors directly induces [Ca²⁺]_i. Both pathways are attenuated by membraneassociated estrogen receptor-α (ERα). ERα agonist 17β-estradiol attenuated increases P2X3 and TRPV1-mediated [Ca²⁺]_i.

Figure 2

Experimental “dish in dish” system (patent pending). DRG sensory neurons were plated and cultured in the “center” dish. Other DRG neurons, SH-SY5Y neuroblastoma cells cultured under experimental condition or intervention media containing no cells were physically disconnected from the neurons in the center dish.

Figure 3

17β-estradiol inhibits ATP-induced calcium signaling in DRG neurons but not in the presence of physically isolated SH-SY5Y neuroblasoma cells. A: Typical ATP (10 μM)-induced [Ca²⁺]_i response (first ATP stimulation) and attenuated response after 5 min pretreatment with E2 (100 nM) (second ATP stimulation). ATP-induced [Ca²⁺]_i response rapidly attenuated in dorsal root ganglion cells. After wash-out with experimental medium, ATP response on [Ca²⁺]_i returned to initial (control) amplitude of stimulation (third ATP stimulation). B: Summary of ATP-induced [Ca²⁺]_i influxes in control state, in the presence of E2, and after wash-out of the E2 effect. E2 significantly decreased [Ca²⁺]_i response to ATP. Values are expressed as mean ± SEM (n=7). * indicates statistically significant difference from control, P<0.05. C: Response of DRG neurons after 12 hours incubation near but physically-disconnected from SH-SY5Y neuroblastoma cells. Initial response to ATP is markedly attenuated compared to panel A (first ATP stimulation). There was a slight non significant effect of 17β-estradiol (100 nM) on ATP (10 M)-induced [Ca²⁺]_i (second ATP stimulation) and a consistent reduced [Ca²⁺]_i response to ATP after washout (third ATP stimulation). D: Summary of ATP-induced [Ca²⁺]_i influxes in the presence of local physically-disconnected SH-SY5Y cells, in the presence of E2, and after wash-out of the E2 effect. E2 shows no effect on [Ca²⁺]_i response to ATP. Values are expressed as mean ± SEM (n=4).

Figure 4

Effect of 17β-estradiol on capsaicin-induced [Ca²⁺]_i increase in control and in the local presence of physically disconnected SH-SY5Y cells. A: Pre-incubation for 5 min with 17β-estradiol (100 nM) attenuates capsaicin (300 nM)- induced [Ca²⁺]_i response (first stimulation) compared with controlled capsaicin response (second stimulation). B: Summary of capsaicin- induced [Ca²⁺]_i increase in the presence of E2 and after wash-out with experimental medium. Values are expressed as mean ± SEM (n=8). * indicates statistically significant difference from control, P<0.05. C: Effect of 17β-estradiol (100 nM) on capsaicin (300 nM)-induced [Ca²⁺]_i increase was abolished after 12 hours incubation near but physically-disconnected from human neuroblastoma SH-SY5Y cells. D: Summary of capsaicin- induced [Ca²⁺]_i increase in the presence of E2 and after wash-out with experimental medium in sensory neurons cultured in the local presence of physically-disconnected SH-SY5Y cells. Values are expressed as mean ± SEM (n=4).

Figure 5

Pattern of multiple ATP-and capsaicin stimulations in isolated DRG neurons in the local presence of physically-disconnected DRGs treated with KCl to induce apoptosis and cell death. A: Typical indication of equal [Ca²⁺]_i responses to repeated ATP (10 μM) stimulation (indicated by arrow) with 10 min interval under control condition. B: ATP-induced [Ca²⁺]_i response in DRG neurons co-cultured near physically-disconnected DRG neurons (see Materials and Methods) treated with KCl (50 mM) for 12 hours. Under this experimental conditions the first ATP stimulation produces 3-4 times higher response in [Ca²⁺]_i increase but subsequent stimulations failed to induce any significant response compared to control. C: Typical indication of equal [Ca²⁺]_i response to repeat capsaicin (300 nM) stimulation (indicated by arrow) under control condition. D: Elimination of [Ca²⁺]_i to initial and repeat capsaicin stimulation in DRG neurons co-cultured near physically-disconnected DRG neurons treated with KCl (50 mM) for 12 hours.