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Journal of Clinical Laser Medicine & Surgery Volume 22, Number 2, 2004 © Mary Ann Liebert, Inc. Pp. 111–117 Effect of Low-Intensity (3.75–25 J/cm2) Near-Infrared (810 nm) Laser Radiation on Red Blood Cell ATPase Activities and Membrane Structure JOLANTA KUJAWA, M.D., Ph.D.,1 LEU ZAVODNIK, M.D., Ph.D.,2 ILYA ZAVODNIK, Ph.D., D.Sc.,2 VYACHESLAV BUKO, Ph.D.,2 ALENA LAPSHYNA, Ph.D.,2 and MARIA BRYSZEWSKA, Prof.3 ABSTRACT Objective: The biostimulation and therapeutic effects of low-power laser radiation of different wavelengths and light doses are well known, but the exact mechanism of action of the laser radiation with living cells is not yet understood. The aim of the present work was to investigate the effect of laser radiation (810 nm, radiant exposure 3.75–25 J/cm2) on the structure of protein and lipid components of red blood cell membranes and it functional properties. The role of membrane ATPases as possible targets of laser irradiation was analyzed. Background Data: A variety of studies both in vivo and in vitro showed significant influence of laser irradiation on cell functional state. At the same time another group of works found no detectable effects of light exposure. Some different explanations based on the light absorption by primary endogenous chromophores (mitochondrial enzymes, cytochromes, flavins, porphyrins) have been proposed to describe biological effects of laser light. It was suggested that optimization of the structural–functional organization of the erythrocyte membrane as a result of laser irradiation may be the basis for improving the cardiac function in patients under a course of laser therapy. Materials and Methods: Human red blood cells or isolated cell membranes were irradiated with low-intensity laser light (810 nm) at different radiant exposures (3.75–25 J/cm2) and light powers (fluence rate; 10–400 mW) at 37°C. As the parameters characterizing the structural and functional changes of cell membranes the activities of Na+-,K+-, and Mg2+-ATPases, tryptophan fluorescence of membrane proteins and fluorescence of pyrene incorporated into membrane lipid bilayer were used. Results: It was found that near-infrared low-intensity laser radiation changes the ATPase activities of the membrane ion pumps in the dose- and fluence rate–dependent manner. At the same time no changes of such integral parameters as cell stability, membrane lipid peroxidation level, intracellular reduced glutathione or oxyhaemoglobin level were observed. At laser power of 10 mW, an increase of the ATPase activity was observed with maximal effect at 12–15 J/cm2 of light dose (18–26% for the total ATPase activity). At laser power of 400 mW (fluence rate significantly increased), inhibition of ATPases activities mainly due to the inhibition of Na+-, K+-ATPase was observed with maximal effect at the same light dose of 12–15 J/cm2 (18–23% for the total ATPase activity). Fractionation of the light dose significantly changed the membrane response to laser radiation. Changes in tryptophan fluorescent parameters of erythrocyte membrane proteins and the increase in lipid bilayer fluidity measured by pyrene monomer/excimer fluorescence ratio were observed. Conclusions: Near-infrared laser light radiation (810 nm) induced long-term conformational transitions of red blood cell membrane which were related to the changes in the structural states of both erythrocyte membrane proteins and lipid bilayer and which manifested themselves as changes in fluorescent parameters of erythrocyte membranes and lipid bilayer fluidity. This resulted in the modulation of membrane functional properties: changes in the activity of membrane ion pumps and, thus, changes in membrane ion flows. 1Department of Rehabilitation, Medical University of Lodz, Lodz, Poland. of Biochemistry, National Academy of Sciences of Belarus, Grodno, Belarus. 3Department of General Biophysics, University of Lodz, Lodz, Poland. 2Institute 111 112 Kujawa et al. INTRODUCTION L OW-INTENSIVE LASER RADIATION as a phototherapeutic modal- ity has been in use for about 30 years.2 Low-intensity (the output power of laser devices in the mW range) radiation is characterized by its ability to induce athermic, nondestructive photobiological processes. These stimulatory effects are named “biostimulation” or “biomodulation.”2 A variety of studies both in vivo and in vitro showed influence of laser irradiation on cellular functional state. Numerous explanations based on the light absorption by primary endogenous chromophores (mitochondrial enzymes, cytochromes, flavins, porphyrins) have been proposed to describe photobiological effects of laser light.2 At the same time, other group of works found no detectable effects of light exposure.2 Laser therapy of coronary patients significantly diminished total cholesterol, low and very low density lipoproteins, triglycerides and atherogenic index persisted for 3–6 months.3 Primary changes in the structure of the lipid bilayer of a red cell membrane in response to laser irradiation and activation of antioxidant system were observed.3 It was established that laser irradiation of the blood of patients with exertion stenocardia was accompanied by the increase of the activity of erythrocyte membrane ATP-ases, an index of erythrocyte deformability and positive changes of cardial function.4 The optimization of the structural–functional organization of the cellular membrane under laser irradiation was suggested as the basis for improving cardiac function in patients under laser therapy.4 It has been shown that low-power He-Ne lasers (632.8 and 543.5 nm) had a protective effect on red cells against hypotonic hemolysis and stabilized the cell membrane.5 Thus, structural transformations of red blood cell membranes and changes in blood circulation can be considered as the mechanism of biological responses of tissues or whole body to low-intensity non-thermal irradiation. However, the exact mechanisms underlying laser irradiation-induced transformations of red blood cells and cellular processes are not well understood and need further clarification. The red blood cells were chosen as a model cellular system, lacking cell nucleus and the machinery for repairing cellular impairments. The aim of the present work was to investigate the effect of laser radiation (810 nm, radiant exposure 3.75–25 J/cm2) on the structure of protein and lipid components of red blood cell membranes and its functional properties. The role of membrane ATPases as possible targets of laser irradiation was analyzed. We selected such laser light wavelength and energies because these parameters are widely used in laser therapy treatments. Also the effects of light dose rate and fractionated dosages were studied. MATERIALS AND METHODS Blood from healthy donors was purchased from the Central Blood Bank in Lodz. Blood was drawn into 3% sodium citrate. After removing the plasma and leukocyte layer, erythrocytes were washed three times with cold (4°C) phosphate buffered saline (PBS: 0.15 M NaCl, 1.9 mM NaH2PO4, 8.1 mM Na2HPO 4, pH 7.4). Erythrocytes were used immediately after isolation. Erythrocyte membranes were isolated from washed cells according to the method of Dodge et al.6 with some modifications. The erythrocytes were hemolyzed with 20 volumes of 10 mM Tris—HCl buffer, pH 7.8, containing 1 mM EDTA and 0.5 mM PMSF as proteolytic inhibitors, and centrifuged for 20 min at 4°C at 20,000g. The ghosts were resuspended in ice-cold 5 mM Tris–HCl buffer, pH 7.4, and this process was continued until the ghosts were free of residual haemoglobin. The susceptibility of erythrocytes or erythrocyte membranes to near-infrared laser radiation To evaluate the extent of the oxidative stress produced by laser irradiation, accumulation of lipid peroxidation products, oxidation of intracellular reduced glutathione (GSH), and membrane SH-groups were assessed. To evaluate the erythrocyte stability and membrane integrity, the percent of cell hemolysis was measured. The changes of tryptophan fluorescence parameters of membrane proteins were measured to characterize the conformational alterations in proteins. The microviscosity and polarity of membrane lipid bilayer reflecting the state, order, and motions of membrane lipid components were determined. The functional state of erythrocyte membranes was assessed as sodium, potassium, and magnesium ATPase activities. The suspensions of erythrocytes in PBS (10% hematocrit) or erythrocyte membranes (1 mg protein/mL) in 5 mM Tris–HCl buffer, pH 7.4, were irradiated with near-infrared (810 nm) therapy laser CTL 1106MX (LASERINSTRUMENTS, Warsaw) in plastic tubes through uncovered surface with continuous gentle mixing in order to expose all cells to the same number of photons at 37°C. Volume of irradiated suspensions was 0.5 mL; the square of a light spot was 0.8 cm2. Power of the laser was changed from 10 to 400 mW, and radiant exposure was changed from 3.73 to 25 J/cm2. In experiments with fractionated dose of radiation, the total radiant exposure was 15 J/cm2. The exposure of membrane suspensions to light was performed in two parts with 1-h interval in several options: 1.9 J/cm2 +13.1 J/cm2; 3.75 J/cm2 + 11.25 J/cm2; 7.5 J/cm2 + 7.5 J/cm2; 11.25 J/cm2 + 3.75 J/cm2; 13.1 J/cm2 + 1.9 J/cm2. No temperature changes (±0.5°C) of cell or membrane suspensions were observed under light doses used. The measurement of TBARS Lipid peroxidation was assessed in erythrocyte membranes according to the method of Stocks and Dormandy7 by determining spectrophotometrically the amount of TBA-reactive species (TBARS) formed. Chemicals Reagents of analytical grade were from POCH (Gliwice, Poland), and 5,5’-dithiobis (2-nitrobenzoic acid) (Ellman’s reagent), 2-thiobarbituric acid (TBA), trichloroacetic acid (TCA), and the fluorescent probe pyrene were from SigmaAldrich GmbH, Germany. The concentration of the intracellular GSH The GSH level was determined by the method of Ellman.8 Briefly, 0.2 mL of 25% TCA was added to 2 mL of final red blood cell suspension and centrifuged. To 1 mL of the supernatant, 1 mL of 1 M phosphate buffer (pH 7.4) and 0.1 mL Ell- Cellular Effects of Laser Irradiation 113 man’s reagent (1023 M) were added for GSH determination. Concentration of GSH was monitored spectrophotometrically at 412 nm using the extinction coefficient 13.6 mM21cm21. Enzyme activity measurements Na+,K+-ATPase activity was determined in a medium containing 100 mM Tris-HCl (pH 7.4), 10 mM MgCl2, 15 mM KCl, 85 mM NaCl, 2 mM EDTA, and 2 mM ATP (final concentrations) by measurement of the difference in the liberation of inorganic phosphate in the absence and in the presence of 0.1 mM ouabain during 30-min incubation of membrane preparations at 37°C. The activity measured in the presence of ouabain was referred to as the basal Mg2+-ATPase. The phosphate liberated was estimated with Malachite Green.9 Protein was measured according to the method of Lowry et al.10 Measurement of haemolysis and concentration of metHb Percentage of haemolysis was calculated as the hemoglobin (Hb) release after irradiation: 0.05 mL of irradiated suspension was added to 2 mL of PBS and centrifuged at 4°C immediately after irradiation. The haemoglobin content in the supernatant was measured spectrophotometrically from absorbance at 414 nm. The concentration of metHb in treated erythrocytes was estimated from visible spectra of red blood cell hemolysates using the algorithm of Winterbourn based on the measurement of optical densities at 560, 577, and 630 nm.11 Measurements of the membrane fluorescence and SH-group oxidation Fluorescence of membrane protein tryptophan residues and probe pyrene incorporated into membrane lipid bilayer was measured using Perkin-Elmer LS-50B spectrofluorimeter. For membrane protein fluorescence measurements the membranes were suspended before or after irradiation in 0.5 mM Tris–HCl buffer, pH 7.4 (0.2 mg protein/mL), lexc = 296 nm. The microviscosity of the membrane lipid bilayer was measured by the monomer (395 nm) to excimer (465 nm) fluorescence ratio of the pyrene.12 Membranes before or after irradiation were suspended in 0.5 mM Tris–HCl buffer, pH 7.4 (0.2 mg protein/mL). Pyrene concentration was 5.10-6 M, lexc = 319 nm. The polarity of probe environment in the membrane was measured as the ratio of the vibrational band intensities of the pyrene monomer spectrum (I386/I395). The concentration of membrane SH-groups was measured in membrane suspensions (0.5 mg protein/mL) in 0.5 mM Tris– HCl buffer, pH 7.4, containing 0.3% sodium dodecyl sulphate, using Ellman’s reagent.8 Four to six independent replications of each experiment were performed. Results were expressed as mean ± SD of all measurements and statistical analysis was conducted using analysis of variance (ANOVA). RESULTS After irradiation of red blood cells with doses of 3.75–25 J/cm2 and power changing from 10 to 400 mW, no changes of either cell stability (measured as the percent of cell haemolysis or changes of cell osmotic fragility), membrane lipid peroxidation level (TBARS), intracellular reduced glutathione, oxyhaemoglobin levels (Table 1), or membraneous SH-group level (Fig. 2c) were observed. Thus, laser irradiation in the doses used did not produce impairments of cell membrane and did not induce any oxidative stress measurable by changing redox equilibrium in the cell. At the same time irradiation of isolated erythrocyte membranes induced alterations of the parameters characterizing the structure and functions of cell membranes. Figure 1 shows the dependence of the total red blood cell membrane ATPase activity on the light dose at different powers of laser (10, 200, and 400 mW). Irradiation at a power of 10 mW increased the total ATPase reaction rate with maximal effect for 12–15 J/cm2 of radiant exposure (the total ATPase activity increased by 18– 26%). Above this dose the radiation effect was smaller. The total ATPase activity increased due to activation of Na+,K+-ATPase (Table 2). The Mg2+-ATPase activity did not change significantly (Table 2). At higher light power (200 mW) the total ATPase activity also increased after irradiation, but to a smaller extent (9–17%) (Fig. 1). For the highest light power of 400 mW, the inhibition of total ATPase activity was observed: maximal effect was for 12–18 J/cm2 of radiant exposure (18–23% of the total ATPase activity) (Fig. 1). The total ATPase activity TABLE 1. THE EFFECT OF LASER LIGHT (810 NM) ON CELL STABILITY AND OXIDATIVE STRESS MARKERS IN RED BLOOD CELLS AT 10 OR 200 MW OF LIGHT POWER Radiant exposure, J/cm2 Control 3.75 7.5 15 25 Hemolysis, % TBARS level, nmol/mL packed cells 1.5 ± 0.5 1.5 ± 0.5 1.5 ± 0.5 2.0 ± 0.5 2.0 ± 0.5 2.8 ± 0.4 2.9 ± 0.4 2.6 ± 0.5 3.1 ± 0.6 3.0 ± 0.5 GSH, nM NaCl concentration corresponding to 50% osmotic haemolysis, nM MetHb, % 1.6 ± 0.3 1.7 ± 0.5 1.5 ± 0.4 1.7 ± 0.5 1.8 ± 0.6 63.5 ± 1.7 65.5 ± 2.0 64.5 ± 1.9 63.0 ± 2.0 66.0 ± 2.1 2.0 ± 0.7 1.5 ± 0.6 2.5 ± 0.7 2.0 ± 0.5 2.1 ± 0.5 114 Kujawa et al. FIG. 1. The effect of laser light (810 nm) on the total red blood cell ATPase activity (nmol Pi/mg protein/hour). Red blood cell membranes (1 mg protein/mL) in 5 mM Tris HCl–buffer, pH 7.4, were irradiated at 37°C at different light powers: 10 mW (1), 200 mW (2), and 400 (3) mW. decreased mainly due to inhibition of Na+-,K+-ATPase: for the radiant exposure of 15 J/cm2 and 400 mW of light power the Na+,K+-ATPase activity was inhibited by about 40%. The inhibitory effect of laser radiation on the Mg2+-ATPase was much smaller (Table 2). At higher radiant exposure the inhibitory effect of light on the total ATPase (Fig. 1) as well as on Na+,K+-, and Mg2+-ATPases (Table 2) decreased. It should be noted that, for radiant exposure of 15 J/cm2, the time of irradiation changes from 30 sec for 400 mW to 1200 sec for 10 mW. Because for 400 mW of light power the most pronounced influence of irradiation on the membrane enzymes was observed the effect of fractionated light doses on ATPase activities was studied at this particular dose rate. As one can see from the Table 3, when the radiant exposure (15 J/cm2) was divided into two non-equal parts and the smaller one (e.g., 1.9 J/cm2) was applied first, the inhibitory effect of light decreased. When the dose was fractionated either into two equal parts (7.5 J/cm2) or the bigger part of the split dose was applied first, the inhibitory effect significantly increased (up to 52%) (Table 3). Changes of ATPase activities induced by laser irradiation were accompanied by structural transformations of red blood cell membranes studied by the tryptophan fluorescence of membrane proteins and fluorescence of pyrene incorporated into the cell membrane lipid bilayer. The long wavelength shift and the increase of intensity of tryptophan fluorescence was observed for 400 mW of light power, whereas membrane irradiation at 200 mW induced the short wavelength shift and decreased the intensity of tryptophan fluorescence (Fig. 2). Such changes of tryptophan fluorescence reflect structural transformations of membrane proteins (or mem- FIG. 2. The dose dependences of red blood cell membrane state parameters under near-infrared laser radiation: membrane protein tryptophan fluorescence intensity registered at 334 nm (a), wavelength of the spectrum maximum (b) at light power 200 (1) and 400 mW (2); the membrane protein SH–group content, light power 200 mW (c). branes). At the same time no oxidation of membrane SH-groups after laser irradiation was noticed (Fig. 2). Also membrane lipid bilayer underwent transformations under laser light irradiation. Membrane irradiation induced the increase of membrane lipid bilayer fluidity, measured by the pyrene monomer/excimer fluorescence intensity ratio (Fig. 3). The polarity of the probe environment in the membrane did not change. Cellular Effects of Laser Irradiation 115 TABLE 2. THE EFFECT OF LASER LIGHT (810 NM) ON ATPASE ACTIVITIES (NMOL PI/MG PROTEIN/HOUR) OF RED BLOOD CELL MEMBRANE AT DIFFERENT LIGHT POWER: 10 MW, 200 MW, AND 400 MW 10 mW 200 mW Light dose Mg2+–ATPase K+,Na+–ATPase Mg2+–ATPase Control 3.75 J/cm2 7.5 J/cm2 11.25 J/cm2 15 J/cm2 18.75 J/cm2 25 J/cm2 172 ± 22 100% 177 ± 25 103% 182 ± 25 106% 182 ± 25 106% 184 ± 28 107% 187 ± 28 109% 189 ± 25 110% 258 ± 34 100% 244 ± 30 95% 274 ± 35 106% 360 ± 40 139%* 323 ± 35 125%* 286 ± 35 110% 281 ± 36 109% 172 ± 22 100% 170 ± 22 98% 167 ± 20 97% 174 ± 22 101% 186 ± 25 108% 187 ± 25 109% 182 ± 25 106% K+,Na+–ATPase 258 ± 34 100% 269 ± 35 104% 285 ± 36 110% 294 ± 36 114% 318 ± 37 123%* 247 ± 30 96% 269 ± 35 104% 400 mW Mg2+–ATPase K+,Na+–ATPase 172 ± 22 100% 182 ± 25 106% 167 ± 20 97% 151 ± 16 88%* 177 ± 25 103% 182 ± 30 106% 182 ± 30 106% 258 ± 34 100% 313 ± 41 121% 224 ± 30 87% 180 ± 25 70%* 151 ± 20 58%* 171 ± 25 66%* 205 ± 31 80% *p < 0.05 in comparison to control. Thus, measurable dose-dependent structural transitions of proteins and lipid components of erythrocyte membrane under laser irradiation were observed, correlated with changes in ATPase activities of cell membranes. DISCUSSION There are many reports showing different and pronounced effects of low-intensity laser irradiation on various types of cells.1 Some photochemical and photophysical reactions relevant in low-intensity laser therapy have been proposed, such as primary absorption of light by mitochondrial enzymes and local heating, or light absorption by flavins and cytochromes in the mitochondrial respiratory chain and influence on the electron transfer as well as light absorption by endogenous porphyrins and photodynamic production of singlet oxygen.1,2,13–15 For example, Friedman et al. suggested that low doses of light induced the formation of a transmembrane electrochemical proton gradient in mitochondria, which was followed by a calcium release from the mitochondria.14 At higher doses of light, too much calcium is released, which causes hyper-activity of Ca2+-ATPase of calcium pumps and exhausts the ATP pool of the cell.14 Bolton et al. using 860-nm diode laser showed a relationship between radiation-induced change of fibroblast proliferation and succinic dehydrogenase activity. At a dose of 2 J/cm2, both the cell proliferation and enzyme activity were significantly increased, whereas at a dose of 16 J/cm2, inhibition of both parameters was noted.16 Yu et al., using laser radiation of 632.8 nm and doses of 0.5–1.5 J/cm2, demonstrated the induction of interleukin secretion by cultured human keratinocytes.17 Grossman et al. demonstrated that irradiation at doses of 0.45–0.95 J/cm2 (780 nm) enhanced keratinocytes proliferation 1.3—1.9fold and that reactive oxygen species play a key role in laserinduced proliferation.18 Laser irradiation of lymphocytes in vitro at 2.4 and 4.8 J/cm2 induced lymphocyte proliferation and enhanced ATP synthesis.19 At the same time, in many studies no detectable biostimulative effects of light exposure could be found.20 Membrane ion permeability and activities of membrane ion pumps are of importance for the regulation of cellular processes and for the observed effects of laser irradiation. In the present work the influence of low-intensity near infrared (810 nm) laser light on the ATPase activities of ion pumps in isolated erythrocyte membranes and on the membrane structure was found. The photomodulation effect of radiation depended on the light dose, dose rate and was changed by dose fractiona- TABLE 3. T HE E FFECT OF FRACTIONATED NEAR-INFRARED (810 NM) LASER LIGHT ON ATPASE A CTIVITIES (NMOL PI/MG PROTEIN/HOUR) OF R ED B LOOD C ELL MEMBRANE, LIGHT POWER OF 400 MW, TOTAL LIGHT D OSE OF 15 J/CM2 Light dose Control 0 + 15 J/cm2 1.9 J/cm2 + 13.1 J/cm2 3.75 J/cm2 + 11.25 J/cm2 7.5 J/cm2 + 7.5 J/cm2 11.25 J/cm2 + 3.75 J/cm2 13.1 J/cm2 + 1.9 J/cm2 Total ATPase Mg2+–ATPase K +,Na+–ATPase 430 ± 45 100% 328 ± 41 77%* 346 ± 41 80% 387 ± 45 90% 271 ± 34 63%* 206 ± 33 48%* 208 ± 34 48%* 172 ± 22 100% 177 ± 25 103% 170 ± 23 99% 162 ± 20 94% 159 ± 25 93% 155 ± 24 90% 153 ± 24 89% 258 ± 34 100% 151 ± 20 58%* 176 ± 33 68%* 255 ± 32 87% 111 ± 20 43%* 51 ± 12 20%* 55 ± 14 21%* *p < 0.05 in comparison to control. 116 FIG. 3. The effect of near-infrared laser irradiation on red blood cell membrane lipid fluidity (1) and polarity (2); light power 200 mW. tion. At a smaller fluence rate (10 mW), the activation of the total ATPases was observed whereas at a higher fluence rate (400 mW) the inhibition of these enzymes was shown for the same light doses. The Na+,K+-ATPase but not Mg2+-ATPase was sensitive to near infrared light absorption. The ATPase activities of erythrocyte membranes depended on radiant exposure in a complex way. In the case of the light-sensitive Na+, K +-ATPase, the pronounced effect was observed at light doses of 12–15 J/cm2. At higher light doses, the irradiation effect decreased. There must be a different mechanism of light effect on biomolecules at different light doses. The irradiation of erythrocyte suspensions at the doses up to 25 J/cm2 neither changed the cell stability nor produced membrane lipid peroxidation or caused cellular glutathione and membrane SH-group oxidation. We can conclude that under these experimental conditions no oxidative stress in erythrocytes was observed. Changes of enzyme activity induced by infrared laser light might be due to either direct interaction of light with enzyme molecules or modification of enzyme environment in the membrane. Previously, Bryszewska et al. have shown that red laser light radiation (670 nm) changes the acetylcholinestrase activity of erythrocyte membranes, induces membrane hyperpolarisation and increases the microviscosity of the membrane polar region.21,22 These enzyme or membrane structural transitions are long-termed and did not disappear after the light switch is off. According to Olson et al. infrared light absorption by enzymes results in a local heating and enhancement of molecular vibration.15 The increase of molecular or domain movements due to local heating, that was not accompanied by the increase of suspension temperature, might result in structural transformations of the enzymes and changes of the membrane functional activities. Changes of membrane ion passive permeability and active transportation (due to the ATPase activation or inhibition) seem to be the common pathway of cell signal transduction and may be the mechanism of cell photoresponse. The other possibility of laser-induced modulation of membrane enzyme activities is accumulation of photochemical products in the Kujawa et al. membrane. Observed modulation of enzyme activities of the erythrocyte membranes (25–40% in the case of Na+,K+-ATPase) allow for a suggestion of the red blood cell irradiations (for example, intravenously) as a mode for laser therapy. Small doses of light and small powers can thus produce pronounced effects. In radiobiology the normal dose rate effect and the inverse dose effect of ionizing radiation are known. The first one is characterized by a decreasing radiation effect at a decreasing dose rate (for identical dose applied) and the latter one is characterized by an increasing radiation effect as the dose rate decreases due to protraction or fractionation of applied dose.23 In our case not only did the increased dose rate change the strength of response but also the duration of stimulation: laser light inhibited enzyme activities at a high dose rate (400 mW) and activated at a low dose rate (10 mW). Using photosensitizer it has previously been shown that fractionated laser light was more effective than continuous illumination of the same power in cell killing.24 We divided the light dose of 15 J/cm2 into two parts and irradiated membrane suspension with the time interval of 1 hour (Table 3). The first small light dose (1.9 J/cm2 or 3.75 J/cm2) adapted membrane (or enzyme) structure to the consecutive following part of light irradiation and decreased the total effect. The bigger dose (7.5 J/cm2 or more) sensitized membrane to the next level of light energy and increased the total effect. One can suggest that light energy absorption induced some conformational transformations of the enzyme (or membrane) and the next portion of light energy interacted with the new state of the enzyme and produced another response. Tryptophan or pyrene fluorescence showed light-induced transitions of protein or lipid components of the erythrocyte membranes (Figs. 2 and 3). Irradiation at the light power of 400 mW produced membrane transitions resulting in protein tryptophan residues transfer into a more polar environment (long-wavelength shift of the fluorescence) whereas irradiation at 200 mW caused protein tryptophan residues transfer into more hydrophobic environment (short-wavelength shift). Also fluidisation of membrane lipid bilayer was observed. No changes either in the redox equilibrium (levels of glutathione or membrane SHgroups) or in the accumulation of lipid peroxidation products in the cells were found. Fluidisation of membranes and a change in the structure of membrane proteins (enzymes) thus manifested themselves as changes in the activity of membrane ion pumps. CONCLUSION Near-infrared laser light radiation (810 nm, 3.75–25 J/cm2) induced long-term conformational transitions of red blood cell membrane which were related to the changes in the structural states of both membrane proteins and lipid bilayer and which manifested themselves as changes in fluorescent parameters of erythrocyte membrane and lipid bilayer fluidity. This resulted in the modulation of membrane functional properties: changes in the activity of membrane ion pumps. The same light doses activated membrane ATPases at a low dose rate and inhibited enzymes at a high dose rate. Fractionation of the light dose sig- Cellular Effects of Laser Irradiation nificantly influenced biomodulation effect of light. We suggest that local heating of the membrane components by near-infrared light absorption or accumulation of photochemical products can induce structural transitions of the red blood cell membrane or membrane components. Transformations of red blood cell membranes can be considered as a mechanism of the improvement in blood circulation under laser therapy. ACKNOWLEDGMENTS This study was supported by the University of Lodz research grant no. 434/2002. REFERENCES 1. Schindl, A., Schindl, M., Pernerstorper-Schon, H. et al. (2000). Low-intensity laser therapy: a review. J. Invest. Med. 48, 312–326. 2. Karu, T.I. (1988). Molecular mechanisms of therapeutic effect of low-intensity laser radiation. Laser Life Sci. 2, 53–74. 3. Vasil’ev, A.P., Strel’tsova, N.N., Zhikhareva A.I., et al. (1996). 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Address reprint requests to: Dr. Jolanta Kujawa Department of Rehabilitation Medical University of Lódź 75 Drewnowska St. /91–002 Lódź, Poland E-mail: [email protected]
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