Modulation of extracellular ATP content of mast cells and DRG neurons by irradiation: studies on underlying mechanism of low-level-laser therapy

Abstract

Low-level-laser therapy (LLLT) is an effective complementary treatment, especially for anti-inflammation and wound healing in which dermis or mucus mast cells (MCs) are involved. In periphery, MCs crosstalk with neurons via purinergic signals and participate in various physiological and pathophysiological processes. Whether extracellular ATP, an important purine in purinergic signaling, of MCs and neurons could be modulated by irradiation remains unknown. In this study, effects of red-laser irradiation on extracellular ATP content of MCs and dorsal root ganglia (DRG) neurons were investigated and underlying mechanisms were explored in vitro. Our results show that irradiation led to elevation of extracellular ATP level in the human mast cell line HMC-1 in a dose-dependent manner, which was accompanied by elevation of intracellular ATP content, an indicator for ATP synthesis, together with [Ca(2+)]i elevation, a trigger signal for exocytotic ATP release. In contrast to MCs, irradiation attenuated the extracellular ATP content of neurons, which could be abolished by ARL 67156, a nonspecific ecto-ATPases inhibitor. Our results suggest that irradiation potentiates extracellular ATP of MCs by promoting ATP synthesis and release and attenuates extracellular ATP of neurons by upregulating ecto-ATPase activity. The opposite responses of these two cell types indicate complex mechanisms underlying LLLT.

Figure 1

Extracellular ATP content of HMC-1 cells in response to 657 nm and 0.28 W/cm² laser irradiation. Relative changes of extracellular ATP level of HMC-1 cells induced by laser irradiation averaged for respective irradiation times of all samples. The data represent averages ± SEM from N = 3-4 independent experiments. ^** P < 0.01, compared to the preirradiation value.

Figure 2

Changes of intracellular ATP content in HMC-1 cells in response to 657 nm and 0.28 W/cm² laser irradiation. Relative changes of intracellular ATP content in HMC-1 cells induced by laser irradiation averaged for the respective irradiation times of all samples. The data represent averages ± SEM from N = 3–7 independent experiments. ^* P < 0.05, compared to the preirradiation value.

Figure 3

Response of [Ca²⁺]_i in HMC-1 cells induced by 657 nm and 0.28 W/cm² laser irradiation. (a) Representative MCs before and after irradiation. Irradiation times are given in the lower right corners. The pseudocolor calibration bar on the right illustrates [Ca²⁺]_i (in relative units). (b) Quantitative analysis from several sets of cells normalized to basal [Ca²⁺]_i-dependent fluorescence before irradiation (=100%). Prolongation of laser irradiation enhanced fluorescence intensity in HMC-1 cells. The data represent averages ± SEM from n = 15–17 cells. ^** P < 0.01, compared to control.

Figure 4

Response of MCs in rat tissue slices to 657 nm and 0.28 W/cm² laser irradiation. (a) MCs in connective tissue slices isolated from rat skin. The left graph shows their smooth and complete plasma membrane. The right graph presents the same MCs stained by 0.5% TB. (b) MCs in tissue slices showed smooth cell membranes before irradiation (left graph). Some MCs started to degranulate once they were exposed to irradiation. The cell membrane became rough, and some black granules were seen scattered around the cell (right graph). The arrow points to degranulating cell. Irradiation times are given in the lower right corners.

Figure 5

Extracellular ATP content of rat peritoneal MCs in response to 657 nm, 18 mW/cm², and 21 J/cm² laser irradiation. Changes of extracellular ATP content of rat peritoneal MCs induced by laser irradiation averaged for respective irradiation times of all samples. The data represent averages ± SEM from n = 12-13.

Figure 6

Extracellular ATP content of rat DRG neurons in response to 657 nm, 18 mW/cm², and 21 J/cm² laser irradiation. Relative changes of extracellular ATP level of rat DRG neurons induced by laser irradiation averaged for respective irradiation times of all samples. The data represent averages ± SEM from N = 3–7 independent experiments. ^** P < 0.01 compared to the preirradiation value; ^* P < 0.05 compared to the preirradiation in the presence of ARL 67156 value.