Effects of Low Power Laser Irradiation on Intracellular Calcium and Histamine Release
LLLT/Laser Therapy hass been shown to have positive bio-stimulation effects on living organisms both in vitro and in vivo. Enhanced histamine release in RBL-2H3 mast cells in vitro takes place after laser irradiation. Because of the multi-functions of histamine, such as vasodilation, increased vessel permeability, increased endothelial cell proliferation and nervous stimulation (13-17), and of the fact that mast cells exist in skin tissue in large extents and accumulate at the site of the wound, histamine release from mast cells is probably one of the reasons for the effect of laser irradiation on promoting wound healing.
LLLT/Laser Therapy hass been shown to have positive bio-stimulation effects on living organisms both in vitro and in vivo. Enhanced histamine release in RBL-2H3 mast cells in vitro takes place after laser irradiation. Because of the multi-functions of histamine, such as vasodilation, increased vessel permeability, increased endothelial cell proliferation and nervous stimulation (13-17), and of the fact that mast cells exist in skin tissue in large extents and accumulate at the site of the wound, histamine release from mast cells is probably one of the reasons for the effect of laser irradiation on promoting wound healing.
Long article, but here are the highlights:
From: Photochemistry and Photobiology Date: July 1, 2007 Author: Chen, Ji-Yao; Yu, Ji-Tong; Zhou, Lu-Wei; Yang, Wen-Zhong
Although laser irradiation has been reported to promote skin wound healing, the mechanism is still unclear. As mast cells are found to accumulate at the site of skin wounds we hypothesized that mast cells might be involved in the biological effects of laser irradiation. In this work the mast cells, RBL-2H3, were used in vitro to investigate the effects of laser irradiation on cellular responses. After laser irradiation, the amount of intracellular calcium ([Ca^sup 2+^]^sub i^) was increased, followed by histamine release, as measured by confocal fluorescence microscopy with Fluo-3/AM staining and a fluorescence spectrometer with o-phthalaldehyde staining, respectively. The histamine release was mediated by the increment of [Ca^sup 2+^]^sub i^ from the influx of the extracellular buffer solution through the cation channel protein, transient receptor potential vanilloid 4 (TRPV4). The TRPV4 inhibitor, Ruthenium Red (RR) can effectively block such histamine release, indicating that TRPV4 was the key factor responding to laser irradiation. These induced responses of mast cells may provide an explanation for Hie biological effects of laser irradiation on promoting wound healing, as histamine is known to have multi-functions on accelerating wound healing.
Low power laser irradiation has been shown to have positive bio-stimulation effects on living organisms both in vitro and in vivo, and various applications in the medical field (1-4). During the past two decades many reports have indicated that laser irradiation can promote wound healing, but the mechanism remains unclear (5-9). In recent years the key effects of mast cells on the process of wound healing have been revealed (10). Mast cells are known to be the main effector cells in allergic reaction and innate immunity (11,12). Under various stimuli, mast cells can be activated to degranulate and release mediators including histamine. The biological effects of histamine include vasodilatation, increased vessel permeability, endothelial cell proliferation and nervous stimulation (13-17). This may suggest that activation of mast cells may be required for cutaneous wound healing (18).
In the present study the effects of laser irradiation on the mast cell line (RBL-2H3) in vitro including intracellular calcium concentration ([Ca^sup 2+^ ]^sub i^) and histamine release were examined to explore the mechanism of laser irradiation in wound healing.
[provided by backspace... thanks!]
Cell culture and preparation. Rat basophilic leukemia (RBL-2H3) cells were obtained from the cell bank of Shanghai Science Academe. Cells were grown in minimal essential medium (MEM) with Earle’s salts containing 10% fetal bovine serum, 2% L-glutamine (all from Gibco), in an incubator with a humidified atmosphere (5% CO2) at 37°C. Cells in exponential phase of growth were used.
Fluorescence measurements of intracellular Ca^sup 2+^ . The cells were seeded on the cover-slips in 12-well culture plates and allowed to attach for 12 h. Before the measurements, cells were washed with Hank’s buffer solution (KH^sub 2^PO^sub 4^ 0.06 g, NaCl 8.0 g, NaHCO^sub 3^ 0.35 g, KCl 0.4 g, glucose 1.0 g. Na^sub 2^HPO^sub 4^*H^sub 2^O 0.06 g. CaCl^sub 2^ 0.14 g. MgSO^sub 4^*7-H^sub 2^O 0.20 g, add H2O to 1000 mL), and then incubated with 10 µM fluo-3/AM (Dojindo, Tokyo, Japan) at 37°C for 30 min. After three washes, these stained cells were sealed on the slides for fluorescence microscopical measurements.
Fluorescence images of intracellular [Ca^sub 2+^ ]^sub i^ were made by confocal fluorescence microscopy (DSU, IX-71; Olympus, Japan). The blue light (460-490 nm) filtered from an attached xenon lamp was selected for excitation. Fluorescence signals were acquired by an attached CCD detector (Evolution QEi, monochrome; Media Cybernetics, CA) with 510-550 nm band-pass filter. A pinhole disk was assembled in the microscope with a 60×/1.20 water-immersion objective lens. Confocal fluorescence images were obtained with the resolution of 0.5 µm in the z-axis and processed with the Image-Pro Plus software (version 5.1; Media Cybernetics). With such a system, the distribution and amount of [Ca^sup 2+^]^sub i^ can be studied with a good resolution in three dimensions. After 1-min irradiation, a series of fluorescence images of [Ca^sup 2+^]^sub i^ were captured with the exposure time of 300 ms in every 10 s for 2 min. To minimize the phut o bleaching effect, an ND filter (12%) was used to decrease the intensity of excitation.
Measurements of histamine release. Histamine release from RBL-2H3 cells was measured by the method of fluorescence staining with the highly specific reagent o-phthalaldehyde (OPA) (Sigma, USA), as initially described by Shore et al. (19) and later improved by others (20-22).
Cells were seeded in 96-well flat bottom plates (5 × 10^sup 5^ cells/well). Before laser irradiation the MEM medium was replaced by Hank’s buffer to give the lowest background in fluorescence measurements.
After laser irradiation, the supernatant in each well was collected and incubated with OPA (0.1 mM, 300 µL) for 20 min. The fluorescence spectra and relative intensities of the supernatants were measured, respectively, by an F-2500 fluorescence spectrophotometer (Hitachi, Japan) with the excitation wavelength of 352 nm. The cell viability after laser irradiation was measured by the MTT assay.
Laser irradiation. To study laser-induced changes in [Ca^sup 2+^]^sub i^, a 405 nm laser (Coherent, CA) was introduced into the microscope in order to irradiate a single cell. The total laser power of 0.96 mW on the cell was measured with a power meter (laser-check; Coherent). The irradiation area was about 28 µm^sup 2^ (irradiation radius was about 3 µm) on the focus of the objective (60×). The irradiation time was 60 s.
To examine the effect of laser on histamine release, the 405 or 532 nm laser (solid laser; CrystaLaser) was used. The diameter of the beam spot on the cell dish was about 3 mm, and the irradiation power was adjusted by a polarizer from 0 to 10 mW according to the designed experiments. Cells were divided into different groups (three wells for each group) and irradiated with different light doses. One group with nonirradiation as a control was also included.
Immunocytochemical detection of TRPV4 in RBL-2H3 cells. The transient receptor potential vanilloid 4 (TRPV4) is a cation channel protein localized in the membranes of some cell lines, which can be activated by diverse physical and chemical stimuli to mediate Ca^sup 2+^ influx (23). To verify the presence of TRPV4 in RBL-2H3 cells, an immunocytochemical method was used. RBL-2H3 cells were seeded onto glass coverslips in cell dishes. After 16 h cells were washed with phosphate-buffered saline (PBS) and fixed in 4% paraformaldehyde for 20 min at room temperature followed by permeation with 0.1% Triton-X-100 for 15 min. The permeation was blocked with 10% normal goat serum in PBS for 30 min. The primary antibody used was rabbit anti-rat TRPV4 (Sigma) and diluted 1:150 in 1% blocking solution and applied for 1 h at 37°C. Then, secondary antibody (goat anti-rabbit IgG-FITC conjugated, diluted 1:400 in PBS [Sigma]) was applied for another 1 h at 37°C. Cells were imaged by the above fluorescence microscopic system with excitation at 460-490 nm and fluorescence capture at 510-540 nm. The nuclei of cells were stained with Hoechst 33342 and imaged at the region of 400-460 nm with an excitation filter of 320-360 nm.
Influence of Ruthenium red (RR) on histamine release. Ruthenium red is known to inhibit the function of TRPV4 and thus was used to examine the effect of TRPV4 on histamine release induced by laser in RBL-2H3 cells. The cell preparation and histamine assay were the same as described above. The cells were divided into four groups including nonirradiation control, laser irradiation alone, irradiation plus RR (3 µM) and RR (12 µM). The cell culture dish was irradiated with 10 mW laser (532 nm) using the beam spot of 3 mm diameter. The irradiation time was 10 min.
In some experiments with measurements of [Ca^sup 2+^]^sub i^ and histamine, the Ca^sup 2+^-free saline solution (pH 7.0) was used as comparison, as the Hank’s buffer contains a large amount of Ca^sup 2+^.
Statistical analysis. Each set of experiments was repeated at least three times. Fluorescence intensities at the peak wavelength of the OPA-histamine complex for each group were calculated and expressed as mean ± SEM. Statistical comparisons between treated groups and an untreated one (control) were determined by using one-way ANOVA and significant difference was accepted when P < 0.05.
Effect of laser irradiation on intracellular [Ca^sup 2+^]^sub i^
After fluo-3/AM loaded RBL-2H3 cells in Hank's buffer had been irradiated with 405 nm laser for 60 s, the intracellular Ca^sup 2+^ fluorescence intensity was significantly increased in a total of 30 cells measured. The typical sequential confocal fluorescence images of the cells are shown in Fig. 1. Figure 2 shows a time-dependent profile of the fluorescence intensity in an irradiated cell. However, when Ca^sup 2+^-free saline solution was used instead of the Hank's buffer the effect of laser irradiation on [Ca^sup 2+^]^sub i^ increment was not seen (Fig. 3), indicating that the increased [Ca^sup 2+^]^sub i^ in Fig. 2 was from the extracellular Hank's buffer.
Effect of laser irradiation on histamine release
The typical histamine release in the RBL-2H3 cells in Hank's buffer after laser irradiation with 405 or 532 nm is shown in Fig. 4. Normally, the RBL-2H3 cells can spontaneously release histamine without laser irradiation, as found by others in rat peritoneal mast cells (24). However, the level of the spontaneously released histamines was low (Fig. 4), indicating that the irradiation by 405 or 532 nm laser can remarkably enhance histamine release.
The dependence of histamine release on irradiation dose was further studied by varying the irradiation power or irradiation time. The 532 nm laser was selected for the experiments, because the light distribution of the laser spot is much more uniform for the 532 nm laser than that of the 405 nm laser. When the irradiation time (10 min) and the size (d = 3 mm) of laser spot on cell sample remained unchanged but the laser power varied the histamine release was found to be irradiation power-dependent (Fig. 5). Compared with the control group, the histamine release after a lower than 2 mW laser power irradiation was not significant (P > 0.05). However, a significant release was found with the laser power larger than 2 mW (P < 0.05 or 0.01). Even with the highest irradiation power (10 mW) used, no cell damage was found by the MTT assay.
When the laser power was kept at 10 mW but the irradiation time varied, the histamine release was found to be irradiation dose-dependent (Fig. 6). The cells were remarkably releasing histamine in the first 10 min, and then the release was slowed down and finally stopped after 20 min irradiation. Such cell response to laser irradiation indicates that histamine release has a saturation level and over-dose irradiation does not enhance such a release.
Presence of the TRPV4 protein
Immunocytochemical staining was performed to verify the expression of TRPV4 protein in RBL-2H3 cells. Positive labeling of TRPV4 in the membrane of RBL-2H3 cells (Fig. 7a) in contrast to unlabeled negative control (Fig. 7d) is shown in Fig. 7, confirming the presence of TRPV4 in the membranes of the cells.
Influence of RR on histamine release
To elucidate the degranulation mechanism of RBL-2H3 cells invoked by laser irradiation, RR was used to block the TRPV4 channel. Table 1 shows that after 10 min laser irradiation on cells in Hank's buffer, histamine release was significantly increased (292.4 ± 15.13 vs 211.43 ± 10.17, P = 0.003). When the RR (low concentration of 3 µM) was added into cell dishes prior to irradiation, no significant inhibition of histamine release was found in irradiated cells (P = 0.174). However, the high concentration (12 µM) of RR suppressed significantly histamine release (152.43 ± 9.37 vs 292.4 ± 15.13,P = 0.0001). When Ca^sup 2+^-free saline solution was used as the extracellular buffer and cells were still irradiated by the laser with the same dose, the enhanced histamine release disappeared with the histamine level comparable to that of the un-irradiated control cells. These data demonstrate that histamine release was mediated by the Ca^sup 2+^ influx gated by TRPV4.
Cell viability
In each set of experiments, the cell viability was measured with the MTT assay after various treatments. The cell viability was found to remain at 90 ± 2%. a cell survival similar to that of the control group, indicating that the enhanced histamine release was a result of laser stimulation but not due to the leakage from cell damage.
Bio-stimulation effects of laser irradiation have been studied in many types of cells in vitro such as dermal fibroblasts and chondrocytes, but not in mast cells (25,26). The RBL-2H3 mast cell line was used in the present work to study laser-induced histamine release. We found that the significantly enhanced histamine release occurred when the laser power density was over 2 mW mm^sup -2^ and the irradiation dose was higher than 0.3 J mm^sup -2^ (Figs. 5 and 6). These values of laser irradiation may be considered as the threshold to induce histamine release in the RBL-2H3 cells.
The initial response of the cells (1 min) to laser irradiation was the elevation of [Ca^sup 2+^+]^sub i^. The increased cellular Ca^sup 2+^ could result either from the influx of extracellular Ca^sup 2+^ or from the release of sequestered Ca^sup 2+^ from the intracellular stores such as mitochondria and endoplasmic reticulum. In our case, as shown in Fig. 3, [Ca^sup 2+^], did not increase when the extracellular solution was Ca^sup 2+^-free, indicating that the increased intracellular Ca^sup 2+^ was the result of the Ca^sup 2+^ influx from the extracellular Hank's buffer.
It has been reported that the elevation of [Ca^sup 2+^]^sub i^ in RBL-2H3 cells can induce cell degranulation to release histamine (27). In our additional experiments, the reagent A23187, a divalent cation transport antibiotic and well-known Ca^sup 2+^ ionophore, was used to confirm this finding. After incubation with A23187 histamine was released from the cells, and such a release was proportional to the A23187 incubation time (data not shown). Furthermore, in experiments of laser irradiation, the results showing the initial increment of [Ca^sup 2+^]^sub i^ followed by histamine release suggest that histamine release may be mediated by the influx of Ca^sup 2+^. This is supported by the finding that this release can be abolished when Ca^sup 2+^-free saline solution was used (Table 1).
Our results showed that TRPV4 channel proteins gated the Ca^sup 2+^ influx in laser-stimulated cells. RR, an inhibitor of TRPV4, suppressed histamine release effectively, demonstrating that TRPV4 plays an important role in cellular responses to laser irradiation. Activated by laser irradiation. TRPV4 opened the Ca^sup 2+^ channel resulting in an elevation of intracellular[Ca^sup 2+^]^sub i^, which, in turn, mediated an enhanced histamine release.
As the TRP proteins were first found in the eye of a Drosophila mutant and established in photo-induction experiments(31), TRPV4 can probably absorb photons directly to be activated. However, so far the absorption spectra of isolated TRP proteins have not been found. On the other hand, several in vitro studies have demonstrated that some endogenous chromophores such as cytochromes and porphyrins can absorb the irradiation photons and produce reactive oxygen species (ROS) by photosensitization (32,33). These cellular ROS may react with channel proteins resulting in an increment of [Ca^sup 2+^]^sub i^ at least partially via the L-type calcium channel (34,35). In our case the activation of TRPV4 by ROS can not be excluded. The real mechanism of TRPV4 activation by laser irradiation needs to be investigated further.
It is evident that the enhanced histamine release in RBL-2H3 mast cells in vitro takes place after laser irradiation. Because of the multi-functions of histamine, such as vasodilatation, increased vessel permeability, increased endothelial cell proliferation and nervous stimulation (13-17), and of the fact that mast cells exist in skin tissue in large extents and accumulate at the site of the wound, histamine release from mast cells is probably one of the reasons for the effect of laser irradiation on promoting wound healing.
Low power laser irradiation has been shown to have positive bio-stimulation effects on living organisms both in vitro and in vivo, and various applications in the medical field (1-4). During the past two decades many reports have indicated that laser irradiation can promote wound healing, but the mechanism remains unclear (5-9). ...Under various stimuli, mast cells can be activated to degranulate and release mediators including histamine. The biological effects of histamine include vasodilatation, increased vessel permeability, endothelial cell proliferation and nervous stimulation (13-17).
...When the laser power was kept at 10 mW but the irradiation time varied, the histamine release was found to be irradiation dose-dependent (Fig. 6). The cells were remarkably releasing histamine in the first 10 min, and then the release was slowed down and finally stopped after 20 min irradiation. Such cell response to laser irradiation indicates that histamine release has a saturation level and over-dose irradiation does not enhance such a release.
...It is evident that the enhanced histamine release in RBL-2H3 mast cells in vitro takes place after laser irradiation. Because of the multi-functions of histamine, such as vasodilatation, increased vessel permeability, increased endothelial cell proliferation and nervous stimulation (13-17), and of the fact that mast cells exist in skin tissue in large extents and accumulate at the site of the wound, histamine release from mast cells is probably one of the reasons for the effect of laser irradiation on promoting wound healing.
Effects of Low Power Laser Irradiation on Intracellular Calcium and Histamine Release in RBL-2H3 Mast Cells
From: Photochemistry and Photobiology Date: July 1, 2007 Author: Chen, Ji-Yao; Yu, Ji-Tong; Zhou, Lu-Wei; Yang, Wen-Zhong
ABSTRACT
Although laser irradiation has been reported to promote skin wound healing, the mechanism is still unclear. As mast cells are found to accumulate at the site of skin wounds we hypothesized that mast cells might be involved in the biological effects of laser irradiation. In this work the mast cells, RBL-2H3, were used in vitro to investigate the effects of laser irradiation on cellular responses. After laser irradiation, the amount of intracellular calcium ([Ca^sup 2+^]^sub i^) was increased, followed by histamine release, as measured by confocal fluorescence microscopy with Fluo-3/AM staining and a fluorescence spectrometer with o-phthalaldehyde staining, respectively. The histamine release was mediated by the increment of [Ca^sup 2+^]^sub i^ from the influx of the extracellular buffer solution through the cation channel protein, transient receptor potential vanilloid 4 (TRPV4). The TRPV4 inhibitor, Ruthenium Red (RR) can effectively block such histamine release, indicating that TRPV4 was the key factor responding to laser irradiation. These induced responses of mast cells may provide an explanation for Hie biological effects of laser irradiation on promoting wound healing, as histamine is known to have multi-functions on accelerating wound healing.
INTRODUCTION
Low power laser irradiation has been shown to have positive bio-stimulation effects on living organisms both in vitro and in vivo, and various applications in the medical field (1-4). During the past two decades many reports have indicated that laser irradiation can promote wound healing, but the mechanism remains unclear (5-9). In recent years the key effects of mast cells on the process of wound healing have been revealed (10). Mast cells are known to be the main effector cells in allergic reaction and innate immunity (11,12). Under various stimuli, mast cells can be activated to degranulate and release mediators including histamine. The biological effects of histamine include vasodilatation, increased vessel permeability, endothelial cell proliferation and nervous stimulation (13-17). This may suggest that activation of mast cells may be required for cutaneous wound healing (18).
In the present study the effects of laser irradiation on the mast cell line (RBL-2H3) in vitro including intracellular calcium concentration ([Ca^sup 2+^ ]^sub i^) and histamine release were examined to explore the mechanism of laser irradiation in wound healing.
MATERIALS AND METHODS
[provided by backspace... thanks!]
Cell culture and preparation. Rat basophilic leukemia (RBL-2H3) cells were obtained from the cell bank of Shanghai Science Academe. Cells were grown in minimal essential medium (MEM) with Earle’s salts containing 10% fetal bovine serum, 2% L-glutamine (all from Gibco), in an incubator with a humidified atmosphere (5% CO2) at 37°C. Cells in exponential phase of growth were used.
Fluorescence measurements of intracellular Ca^sup 2+^ . The cells were seeded on the cover-slips in 12-well culture plates and allowed to attach for 12 h. Before the measurements, cells were washed with Hank’s buffer solution (KH^sub 2^PO^sub 4^ 0.06 g, NaCl 8.0 g, NaHCO^sub 3^ 0.35 g, KCl 0.4 g, glucose 1.0 g. Na^sub 2^HPO^sub 4^*H^sub 2^O 0.06 g. CaCl^sub 2^ 0.14 g. MgSO^sub 4^*7-H^sub 2^O 0.20 g, add H2O to 1000 mL), and then incubated with 10 µM fluo-3/AM (Dojindo, Tokyo, Japan) at 37°C for 30 min. After three washes, these stained cells were sealed on the slides for fluorescence microscopical measurements.
Fluorescence images of intracellular [Ca^sub 2+^ ]^sub i^ were made by confocal fluorescence microscopy (DSU, IX-71; Olympus, Japan). The blue light (460-490 nm) filtered from an attached xenon lamp was selected for excitation. Fluorescence signals were acquired by an attached CCD detector (Evolution QEi, monochrome; Media Cybernetics, CA) with 510-550 nm band-pass filter. A pinhole disk was assembled in the microscope with a 60×/1.20 water-immersion objective lens. Confocal fluorescence images were obtained with the resolution of 0.5 µm in the z-axis and processed with the Image-Pro Plus software (version 5.1; Media Cybernetics). With such a system, the distribution and amount of [Ca^sup 2+^]^sub i^ can be studied with a good resolution in three dimensions. After 1-min irradiation, a series of fluorescence images of [Ca^sup 2+^]^sub i^ were captured with the exposure time of 300 ms in every 10 s for 2 min. To minimize the phut o bleaching effect, an ND filter (12%) was used to decrease the intensity of excitation.
Measurements of histamine release. Histamine release from RBL-2H3 cells was measured by the method of fluorescence staining with the highly specific reagent o-phthalaldehyde (OPA) (Sigma, USA), as initially described by Shore et al. (19) and later improved by others (20-22).
Cells were seeded in 96-well flat bottom plates (5 × 10^sup 5^ cells/well). Before laser irradiation the MEM medium was replaced by Hank’s buffer to give the lowest background in fluorescence measurements.
After laser irradiation, the supernatant in each well was collected and incubated with OPA (0.1 mM, 300 µL) for 20 min. The fluorescence spectra and relative intensities of the supernatants were measured, respectively, by an F-2500 fluorescence spectrophotometer (Hitachi, Japan) with the excitation wavelength of 352 nm. The cell viability after laser irradiation was measured by the MTT assay.
Laser irradiation. To study laser-induced changes in [Ca^sup 2+^]^sub i^, a 405 nm laser (Coherent, CA) was introduced into the microscope in order to irradiate a single cell. The total laser power of 0.96 mW on the cell was measured with a power meter (laser-check; Coherent). The irradiation area was about 28 µm^sup 2^ (irradiation radius was about 3 µm) on the focus of the objective (60×). The irradiation time was 60 s.
To examine the effect of laser on histamine release, the 405 or 532 nm laser (solid laser; CrystaLaser) was used. The diameter of the beam spot on the cell dish was about 3 mm, and the irradiation power was adjusted by a polarizer from 0 to 10 mW according to the designed experiments. Cells were divided into different groups (three wells for each group) and irradiated with different light doses. One group with nonirradiation as a control was also included.
Immunocytochemical detection of TRPV4 in RBL-2H3 cells. The transient receptor potential vanilloid 4 (TRPV4) is a cation channel protein localized in the membranes of some cell lines, which can be activated by diverse physical and chemical stimuli to mediate Ca^sup 2+^ influx (23). To verify the presence of TRPV4 in RBL-2H3 cells, an immunocytochemical method was used. RBL-2H3 cells were seeded onto glass coverslips in cell dishes. After 16 h cells were washed with phosphate-buffered saline (PBS) and fixed in 4% paraformaldehyde for 20 min at room temperature followed by permeation with 0.1% Triton-X-100 for 15 min. The permeation was blocked with 10% normal goat serum in PBS for 30 min. The primary antibody used was rabbit anti-rat TRPV4 (Sigma) and diluted 1:150 in 1% blocking solution and applied for 1 h at 37°C. Then, secondary antibody (goat anti-rabbit IgG-FITC conjugated, diluted 1:400 in PBS [Sigma]) was applied for another 1 h at 37°C. Cells were imaged by the above fluorescence microscopic system with excitation at 460-490 nm and fluorescence capture at 510-540 nm. The nuclei of cells were stained with Hoechst 33342 and imaged at the region of 400-460 nm with an excitation filter of 320-360 nm.
Influence of Ruthenium red (RR) on histamine release. Ruthenium red is known to inhibit the function of TRPV4 and thus was used to examine the effect of TRPV4 on histamine release induced by laser in RBL-2H3 cells. The cell preparation and histamine assay were the same as described above. The cells were divided into four groups including nonirradiation control, laser irradiation alone, irradiation plus RR (3 µM) and RR (12 µM). The cell culture dish was irradiated with 10 mW laser (532 nm) using the beam spot of 3 mm diameter. The irradiation time was 10 min.
In some experiments with measurements of [Ca^sup 2+^]^sub i^ and histamine, the Ca^sup 2+^-free saline solution (pH 7.0) was used as comparison, as the Hank’s buffer contains a large amount of Ca^sup 2+^.
Statistical analysis. Each set of experiments was repeated at least three times. Fluorescence intensities at the peak wavelength of the OPA-histamine complex for each group were calculated and expressed as mean ± SEM. Statistical comparisons between treated groups and an untreated one (control) were determined by using one-way ANOVA and significant difference was accepted when P < 0.05.
RESULTS
Effect of laser irradiation on intracellular [Ca^sup 2+^]^sub i^
After fluo-3/AM loaded RBL-2H3 cells in Hank's buffer had been irradiated with 405 nm laser for 60 s, the intracellular Ca^sup 2+^ fluorescence intensity was significantly increased in a total of 30 cells measured. The typical sequential confocal fluorescence images of the cells are shown in Fig. 1. Figure 2 shows a time-dependent profile of the fluorescence intensity in an irradiated cell. However, when Ca^sup 2+^-free saline solution was used instead of the Hank's buffer the effect of laser irradiation on [Ca^sup 2+^]^sub i^ increment was not seen (Fig. 3), indicating that the increased [Ca^sup 2+^]^sub i^ in Fig. 2 was from the extracellular Hank's buffer.
Effect of laser irradiation on histamine release
The typical histamine release in the RBL-2H3 cells in Hank's buffer after laser irradiation with 405 or 532 nm is shown in Fig. 4. Normally, the RBL-2H3 cells can spontaneously release histamine without laser irradiation, as found by others in rat peritoneal mast cells (24). However, the level of the spontaneously released histamines was low (Fig. 4), indicating that the irradiation by 405 or 532 nm laser can remarkably enhance histamine release.
The dependence of histamine release on irradiation dose was further studied by varying the irradiation power or irradiation time. The 532 nm laser was selected for the experiments, because the light distribution of the laser spot is much more uniform for the 532 nm laser than that of the 405 nm laser. When the irradiation time (10 min) and the size (d = 3 mm) of laser spot on cell sample remained unchanged but the laser power varied the histamine release was found to be irradiation power-dependent (Fig. 5). Compared with the control group, the histamine release after a lower than 2 mW laser power irradiation was not significant (P > 0.05). However, a significant release was found with the laser power larger than 2 mW (P < 0.05 or 0.01). Even with the highest irradiation power (10 mW) used, no cell damage was found by the MTT assay.
When the laser power was kept at 10 mW but the irradiation time varied, the histamine release was found to be irradiation dose-dependent (Fig. 6). The cells were remarkably releasing histamine in the first 10 min, and then the release was slowed down and finally stopped after 20 min irradiation. Such cell response to laser irradiation indicates that histamine release has a saturation level and over-dose irradiation does not enhance such a release.
Presence of the TRPV4 protein
Immunocytochemical staining was performed to verify the expression of TRPV4 protein in RBL-2H3 cells. Positive labeling of TRPV4 in the membrane of RBL-2H3 cells (Fig. 7a) in contrast to unlabeled negative control (Fig. 7d) is shown in Fig. 7, confirming the presence of TRPV4 in the membranes of the cells.
Influence of RR on histamine release
To elucidate the degranulation mechanism of RBL-2H3 cells invoked by laser irradiation, RR was used to block the TRPV4 channel. Table 1 shows that after 10 min laser irradiation on cells in Hank's buffer, histamine release was significantly increased (292.4 ± 15.13 vs 211.43 ± 10.17, P = 0.003). When the RR (low concentration of 3 µM) was added into cell dishes prior to irradiation, no significant inhibition of histamine release was found in irradiated cells (P = 0.174). However, the high concentration (12 µM) of RR suppressed significantly histamine release (152.43 ± 9.37 vs 292.4 ± 15.13,P = 0.0001). When Ca^sup 2+^-free saline solution was used as the extracellular buffer and cells were still irradiated by the laser with the same dose, the enhanced histamine release disappeared with the histamine level comparable to that of the un-irradiated control cells. These data demonstrate that histamine release was mediated by the Ca^sup 2+^ influx gated by TRPV4.
Cell viability
In each set of experiments, the cell viability was measured with the MTT assay after various treatments. The cell viability was found to remain at 90 ± 2%. a cell survival similar to that of the control group, indicating that the enhanced histamine release was a result of laser stimulation but not due to the leakage from cell damage.
DISCUSSION
Bio-stimulation effects of laser irradiation have been studied in many types of cells in vitro such as dermal fibroblasts and chondrocytes, but not in mast cells (25,26). The RBL-2H3 mast cell line was used in the present work to study laser-induced histamine release. We found that the significantly enhanced histamine release occurred when the laser power density was over 2 mW mm^sup -2^ and the irradiation dose was higher than 0.3 J mm^sup -2^ (Figs. 5 and 6). These values of laser irradiation may be considered as the threshold to induce histamine release in the RBL-2H3 cells.
The initial response of the cells (1 min) to laser irradiation was the elevation of [Ca^sup 2+^+]^sub i^. The increased cellular Ca^sup 2+^ could result either from the influx of extracellular Ca^sup 2+^ or from the release of sequestered Ca^sup 2+^ from the intracellular stores such as mitochondria and endoplasmic reticulum. In our case, as shown in Fig. 3, [Ca^sup 2+^], did not increase when the extracellular solution was Ca^sup 2+^-free, indicating that the increased intracellular Ca^sup 2+^ was the result of the Ca^sup 2+^ influx from the extracellular Hank's buffer.
It has been reported that the elevation of [Ca^sup 2+^]^sub i^ in RBL-2H3 cells can induce cell degranulation to release histamine (27). In our additional experiments, the reagent A23187, a divalent cation transport antibiotic and well-known Ca^sup 2+^ ionophore, was used to confirm this finding. After incubation with A23187 histamine was released from the cells, and such a release was proportional to the A23187 incubation time (data not shown). Furthermore, in experiments of laser irradiation, the results showing the initial increment of [Ca^sup 2+^]^sub i^ followed by histamine release suggest that histamine release may be mediated by the influx of Ca^sup 2+^. This is supported by the finding that this release can be abolished when Ca^sup 2+^-free saline solution was used (Table 1).
Our results showed that TRPV4 channel proteins gated the Ca^sup 2+^ influx in laser-stimulated cells. RR, an inhibitor of TRPV4, suppressed histamine release effectively, demonstrating that TRPV4 plays an important role in cellular responses to laser irradiation. Activated by laser irradiation. TRPV4 opened the Ca^sup 2+^ channel resulting in an elevation of intracellular[Ca^sup 2+^]^sub i^, which, in turn, mediated an enhanced histamine release.
As the TRP proteins were first found in the eye of a Drosophila mutant and established in photo-induction experiments(31), TRPV4 can probably absorb photons directly to be activated. However, so far the absorption spectra of isolated TRP proteins have not been found. On the other hand, several in vitro studies have demonstrated that some endogenous chromophores such as cytochromes and porphyrins can absorb the irradiation photons and produce reactive oxygen species (ROS) by photosensitization (32,33). These cellular ROS may react with channel proteins resulting in an increment of [Ca^sup 2+^]^sub i^ at least partially via the L-type calcium channel (34,35). In our case the activation of TRPV4 by ROS can not be excluded. The real mechanism of TRPV4 activation by laser irradiation needs to be investigated further.
It is evident that the enhanced histamine release in RBL-2H3 mast cells in vitro takes place after laser irradiation. Because of the multi-functions of histamine, such as vasodilatation, increased vessel permeability, increased endothelial cell proliferation and nervous stimulation (13-17), and of the fact that mast cells exist in skin tissue in large extents and accumulate at the site of the wound, histamine release from mast cells is probably one of the reasons for the effect of laser irradiation on promoting wound healing.
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