J. Photochem. Photobiol. B: Biol., 15 (1992)
337
337441
Effect of light on calcium transport in bull sperm cells R. Lubar@,
H. Friedmannb,
T. Levinshal”,
R. Lavieb and H. Breitbart’
‘Department of Physics, Bar-Ran University, Ramat Gan 52900 (Israel) “Department of Chemistry, Bar-Ran U&e&y, Ramat Gan 52900 (Israel) ‘Deparhent of Life Sciences, Bar-Ran University, Ramat Gan 52900 (Israel)
(Received February 28, 1992; accepted March 12, 1992)
Abstract The effect of light on calcium transport was studied. Bull sperm cells were irradiated with an He-Ne (630 mm) laser and a 780 nm diode laser at various energy doses, and 45Caz+ uptake was measured by the filtration technique. It was found that there is an accelerated Ca*+ transport in the irradiated cells, which means that laser light can stimulate Ca’+ exchange through the cell membrane. This may cause transient changes in the cytoplasmic Ca2+ concentration which, in spermatozoa, has a regulatory role in control of motility and acrosome reaction, and in other cells can trigger mitosis.
Keywords: Ca ion transport,
bull sperm cells, lasers.
1. Introduction
The increasingly wide use of He-Ne lasers in phototherapy has led to a greater interest in the mechanism of light-biosystem interaction. In a recent paper [l] we irradiated fibroblastic cells with various light sources and found that at a specified relatively low energy dose there is an accelerated cell mitosis. We then suggested that the effect of low level laser irradiation in the visible and in the near-infrared region is due to light absorption either by endogenous porphyrins in the mitochondria or by the cytochromes. As intracellular Ca2+ movements play a vital role in cell proliferation and, in mammalian spermatozoa, have a pivotal role in the control of sperm motility [2] and acrosome reaction [3], we decided to measure Ca*+ transport by irradiated cells. It has already been shown [4,5] that Ca2+ transport in sperm cells is affected by factors acting in the mitochondria. In the present work we irradiated sperm cells with an He-Ne laser (632 nm) and a 780 nm diode laser at various energy doses and found an accelerated Ca*+ transport by the irradiated cells.
2. Materials
and methods
Frozen ejaculated bull sperm cells (from Hasherut Artificial Insemination Center) were thawed at 37 “C in a medium comprising 150 mM NaCl and 10 mM histidine *Author to whom correspondence should be addressed.
loll-1344/92/$5.00
0 1992 - Elsevier
Sequoia.
All rights reserved
338
(pH 7.4). The cells were washed by three centrifugations at 6OOg , at 25 “C for 10 min. The washed cells were resuspended in medium A containing 110 mM NaCl, 5 mM KC1 and 10 mM sodium morpholinopropanesulphonate (pH 7.4). 2.1. Calcium uptake Uptake of 4sCa was determined by the filtration technique. Cells (3 X lOs ml-‘) were incubated in a final volume of 125 ~1 of medium A, containing 10 mM lactate, 0.5 mM phosphate, 0.2 mM CaCl, and 0.5 &i”CaClz. The cells were irradiated with various light sources at different time intervals immediately after the addition of calcium, after which 0.1 ml was removed and immediately vacuum filtered on GF/C filters. The cells trapped on the filter were washed three times with 5 ml of solution composed of 150 mM NaCl, 10 mM Tris (pH 7.4) and 2 mM EGTA (ethylene glycol N,N,N’,N’-tetraacetic acid). The dry filters were counted in scintillation vials with 5 ml of Aquasol (DuPont). In some experiments the cells were incubated in 4sCaC12 for 20 min before the irradiation. The data are expressed as the percentage of 4sCa2+ uptake by irradiated cells relative to control non-irradiated ones. The experiments were performed in triplicate and were repeated at least three times. Each point in the graphs below represents an average value.
3. Irradiation The light sources were a 35 mW and 10 mW He-Ne laser (Spectra Physica), with A= 632 nm; and a diode laser, A = 780 nm, 13 mW (home made) and 40 mW (Lasotronics). Energy doses varied from 2 to 30 J cm-*. 4. Results
and discussion
The results are expressed graphically in Figs. 1 and 2. Maximum stimulation of 4sCa uptake was achieved in the energy density range 6-18 J cm-* when an He-Ne laser was used, and in the region of 3 J cm-* when the 780 nm laser was used. When the cells were incubated in 4sCa2+ prior to the irradiation, 8 J cm-* of He-Ne did not change the 4sCa2’ uptake nor did 3 J cm-* of 780 nm diode laser. (These data are not shown.) Cells are extremely sensitive to changes in their Ca*+ concentration. The systems which regulate Ca*’ concentration include the mitochondria [6,7], the plasma membrane ATP-dependent Ca*’ pump [g-lo], the Na+-Ca*+ anti-port [ll] and the voltagedependent Ca*+ channel [12]. We believe that the accelerated fibroblast proliferation obtained by Karu [13] and in our laboratory [l] after laser irradiation is due to transient changes in the Ca*+ concentration in the cytoplasm. In order to examine this assumption, we irradiated bovine sperm cells with an He-Ne (632 nm) laser and a 780 nm diode laser at various energy doses, and measured the Ca*+ uptake by the cells. Sperm cells are a good model for this purpose since mitochondrial activity is significantly involved in the regulation of intracellular Ca*+ concentrations [4,5] and mitochondrial respiration is highly affected by laser irradiation [14, 151. As the data show, there is an accelerated Ca*+ uptake into the cells at low energy doses (Figs. 1 and 2) and a decrease after high laser doses. Similar results were obtained by Young et al. [16] who studied the effect of light on Ca*+ uptake by
339
;
83-
21 k = 0 0’
63_ 43 0.0
1 30
I 9.0
69
I 12.0
I 15.0
I 18.0
I 21.0
I 24.0
1 270
_
ENERGY DENSITY (J cni*) Fig. 1. The percentage of Ca” uptake by irradiated cells at A= 630 nm, relative to control nonirradiated cells (the standard deviation is *lo%).
97 0.0
* 413
I
I 813
I
I 12.0
I
1 160
I 20.0
ENERGY DENSITY (J cni*) Fig. 2. The percentage of Ca*+ uptake by irradiated cells at A=780 nm, relative to control nonirradiated cells (the standard deviation is *lo%).
macrophages after 660,820 and 870 nm light irradiation. When the cells were incubated in 45CaC12 before irradiation, there was no change in Ca2+ transport. From previous work [14, 151 we know that He-Ne lasers generate an extra electrochemical potential in mitochondria which causes an increase in ATP synthesis. In recent papers [17, 181 we proposed possible ways by which the visible and far-red light is converted to the electrochemical energy of the PMF (proton motive force). We proposed two possible mechanisms: (1) singlet oxygen formation by endogenous porphyrins which activates the respiratory chain in the mitochondria, and (2) activation of the redox reactions in the respiratory chain by exciting the mitochondrial cytochromes.
340
We also suggested that the increased PMF releases Ca*+ from the mitochondria to the cytoplasm which triggers cell mitosis. However, as the present results indicate, there is an accelerated exchange of Ca” between the medium and the cell during irradiation. This phenomenon agrees with previous observations [4] on the rate of Ca2+ transport into bovine spermatozoa due to various oxygenixing substrates. The Ca2+ transport rate into the cells was found to be determined by the mitochondrial Ca2+ uptake which depends on its respiration rate. Mitochondria isolated from various tissues have the capacity to accumulate large amounts of Ca2’ in the matrix compartment by a transport process energetically coupled to electron transport [6]. This means that the irradiated mitochondria can pull more Ca2+ from the medium into the cytoplasm by a mechanism still unknown. When steady state is reached, Ca” is expelled according to the regular mechanisms regulating Ca” concentration in the cells, and this is the reason for the decrease in “Ca2+ uptake after some time [19]. As the data show, the rate of this decrease in enhanced by the irradiation, which means that light also accelerates the efflux of Ca2+ from the mitochondria to the medium [18]. The fact that there is no acceleration in Ca2+ uptake when the cells are incubated in 4’CaC12 to reach the steady state before the irradiation confirms our assumption that the laser actually stimulates the influx and efflux of Ca2+ through the cell membrane. The changes in membrane permeability may cause transient changes in the cytoplasmic Ca2+ concentration, which, in spermatozoa, has a regulatory role in control of motility and acrosome reaction [2, 31, and in other cells can trigger mitosis [l, 131.
Acknowledgment
We wish to thank Professor Kendric C. Smith of the School of Medicine, Stanford University, USA, for his encouragement of this work.
References 1 R. Lubart, Y. Wollman, H. Friedmann, S. Rochkind and I. Laulicht, Effects of visible and near infrared lasers on cell cultures, .I. P/r&o&em. Photubiol. B: Bid, 12 (1992) 305-310. 2 J. P. Singh, D. F. Babcock and H. A. Lardy, Motility activation, respiratory activation and alteration of CaZf transport in bovine sperm treated with amine local anesthetics and calcium transport antagonists, Arch Biochem. Biophys., 221 (1983) 291-303. 3 R. Yanagimachi and N. Usui, Calcium dependence of the acrosome reaction and activation of guinea pig spermatozoa, Qn. CelL Res., 89 (1974) 161-174. 4 H. Breitbart, R. Wehbie and H. Lardy, Calcium transport in bovine sperm mitochondria: effect of substrates and phosphate, Biochim. Biophys. Acta, 1027 (1990) 72-78. 5 H. Breitbart, R. Wehbie and H. Lardy, Regulation of calcium transport in bovine spermatozoa, Biochim. Biophys. Acta, 1027 (1990) 72-78. 6 A. L. Lehninger, Mitochondria and calcium ion transport, B&hem. J., II8 (1970) 129-138. 7 D. F. Babcock, N. L. First and H. A. Lardy, Action of Ionophore A23187 at the cellular level, J. Bid. Chem., 251 (1976) 3881-3886. 8 M. P. Bradley and I. T. Forrester, A Ca’+ +M2+-ATPase and active Ca’+ transport in the plasma membranes isolated from ram sperm flagells, Cell Calcium, I (1980) 381-390. 9 H. Breitbart and S. Rubinstein, Calcium transport in bull spermatozoa plasma membrane, Biochim. Biophys. Acta, 732 (1983) 464-468.
341 10 H. Breitbart, H. Stern and S. Rubinstein, Calcium transport Ca’+-ATPase activity in ram spermatozoa plasma membrane vesicles, Biochim. Biophys. Actu, 728 (1983) 349-355. 11 M. P. Bradley and I. T. Forrester, A sodium calcium exchange mechanism in plasma membrane vesicles isolated from ram flagella, FEBS Lett., I21 (1980) 15-18. 12 D. F. Babcock and D. R. Pfeiffer, Independent elevation of cytosolic Ca” and pH of mammalian sperm by voltage dependent and pH sensitive mechanisms, J. Biol. Chem., 262 (1987) 15041-15047. 13 T. I. Karu, Molecular mechanism of therapeutic effect of low intensity laser radiation, Lasers Life Sci., 2(l) (1988) 53-74. 14 S. Passarella, E. Casamassima, S. Molinari, D. Pastone, E. Quagliariello, I. M. Catalan0 and A. Cinyolani, Increase of proton electrochemical potential and ATP synthesis in rat liver mitochondria irradiated in vitro by HeNe laser, FEBS Lett., 175 (1984) 95-99. 15 C. Sallet, S. Passarella and E. Quagliariello, Effects of selective irradiation on mammalian mitochondria, Photo&em PhotobioL, 45 (1987) 433-438. 16 S. R. Young, M. Dyson and P. Bolton, Effect of light on calcium uptake by macrophages, Laser Zker., 2 (1990) 53-57. 17 R. Lubart, Z. Malik, S. Rochkind and T. Fosjer, A possible mechanism of low-level laserliving cell interaction, Laser Ther., 2 (1990) 65-68. 18 H. Friedmann, R. Lubart and I. Laulicht, A possible explanation of laser-induced stimulation, J. Photochem. Photobiol. B: Biol., 11 (1991) 87-95. 19 A. Zarca, S. Rubinstein and H. Breitbart, Transport mechanism for calcium and phosphate in ram spermatozoa, B&him. Biophys. Actu, 944 (1988) 351-358.
337
337441
Effect of light on calcium transport in bull sperm cells R. Lubar@,
H. Friedmannb,
T. Levinshal”,
R. Lavieb and H. Breitbart’
‘Department of Physics, Bar-Ran University, Ramat Gan 52900 (Israel) “Department of Chemistry, Bar-Ran U&e&y, Ramat Gan 52900 (Israel) ‘Deparhent of Life Sciences, Bar-Ran University, Ramat Gan 52900 (Israel)
(Received February 28, 1992; accepted March 12, 1992)
Abstract The effect of light on calcium transport was studied. Bull sperm cells were irradiated with an He-Ne (630 mm) laser and a 780 nm diode laser at various energy doses, and 45Caz+ uptake was measured by the filtration technique. It was found that there is an accelerated Ca*+ transport in the irradiated cells, which means that laser light can stimulate Ca’+ exchange through the cell membrane. This may cause transient changes in the cytoplasmic Ca2+ concentration which, in spermatozoa, has a regulatory role in control of motility and acrosome reaction, and in other cells can trigger mitosis.
Keywords: Ca ion transport,
bull sperm cells, lasers.
1. Introduction
The increasingly wide use of He-Ne lasers in phototherapy has led to a greater interest in the mechanism of light-biosystem interaction. In a recent paper [l] we irradiated fibroblastic cells with various light sources and found that at a specified relatively low energy dose there is an accelerated cell mitosis. We then suggested that the effect of low level laser irradiation in the visible and in the near-infrared region is due to light absorption either by endogenous porphyrins in the mitochondria or by the cytochromes. As intracellular Ca2+ movements play a vital role in cell proliferation and, in mammalian spermatozoa, have a pivotal role in the control of sperm motility [2] and acrosome reaction [3], we decided to measure Ca*+ transport by irradiated cells. It has already been shown [4,5] that Ca2+ transport in sperm cells is affected by factors acting in the mitochondria. In the present work we irradiated sperm cells with an He-Ne laser (632 nm) and a 780 nm diode laser at various energy doses and found an accelerated Ca*+ transport by the irradiated cells.
2. Materials
and methods
Frozen ejaculated bull sperm cells (from Hasherut Artificial Insemination Center) were thawed at 37 “C in a medium comprising 150 mM NaCl and 10 mM histidine *Author to whom correspondence should be addressed.
loll-1344/92/$5.00
0 1992 - Elsevier
Sequoia.
All rights reserved
338
(pH 7.4). The cells were washed by three centrifugations at 6OOg , at 25 “C for 10 min. The washed cells were resuspended in medium A containing 110 mM NaCl, 5 mM KC1 and 10 mM sodium morpholinopropanesulphonate (pH 7.4). 2.1. Calcium uptake Uptake of 4sCa was determined by the filtration technique. Cells (3 X lOs ml-‘) were incubated in a final volume of 125 ~1 of medium A, containing 10 mM lactate, 0.5 mM phosphate, 0.2 mM CaCl, and 0.5 &i”CaClz. The cells were irradiated with various light sources at different time intervals immediately after the addition of calcium, after which 0.1 ml was removed and immediately vacuum filtered on GF/C filters. The cells trapped on the filter were washed three times with 5 ml of solution composed of 150 mM NaCl, 10 mM Tris (pH 7.4) and 2 mM EGTA (ethylene glycol N,N,N’,N’-tetraacetic acid). The dry filters were counted in scintillation vials with 5 ml of Aquasol (DuPont). In some experiments the cells were incubated in 4sCaC12 for 20 min before the irradiation. The data are expressed as the percentage of 4sCa2+ uptake by irradiated cells relative to control non-irradiated ones. The experiments were performed in triplicate and were repeated at least three times. Each point in the graphs below represents an average value.
3. Irradiation The light sources were a 35 mW and 10 mW He-Ne laser (Spectra Physica), with A= 632 nm; and a diode laser, A = 780 nm, 13 mW (home made) and 40 mW (Lasotronics). Energy doses varied from 2 to 30 J cm-*. 4. Results
and discussion
The results are expressed graphically in Figs. 1 and 2. Maximum stimulation of 4sCa uptake was achieved in the energy density range 6-18 J cm-* when an He-Ne laser was used, and in the region of 3 J cm-* when the 780 nm laser was used. When the cells were incubated in 4sCa2+ prior to the irradiation, 8 J cm-* of He-Ne did not change the 4sCa2’ uptake nor did 3 J cm-* of 780 nm diode laser. (These data are not shown.) Cells are extremely sensitive to changes in their Ca*+ concentration. The systems which regulate Ca*’ concentration include the mitochondria [6,7], the plasma membrane ATP-dependent Ca*’ pump [g-lo], the Na+-Ca*+ anti-port [ll] and the voltagedependent Ca*+ channel [12]. We believe that the accelerated fibroblast proliferation obtained by Karu [13] and in our laboratory [l] after laser irradiation is due to transient changes in the Ca*+ concentration in the cytoplasm. In order to examine this assumption, we irradiated bovine sperm cells with an He-Ne (632 nm) laser and a 780 nm diode laser at various energy doses, and measured the Ca*+ uptake by the cells. Sperm cells are a good model for this purpose since mitochondrial activity is significantly involved in the regulation of intracellular Ca*+ concentrations [4,5] and mitochondrial respiration is highly affected by laser irradiation [14, 151. As the data show, there is an accelerated Ca*+ uptake into the cells at low energy doses (Figs. 1 and 2) and a decrease after high laser doses. Similar results were obtained by Young et al. [16] who studied the effect of light on Ca*+ uptake by
339
;
83-
21 k = 0 0’
63_ 43 0.0
1 30
I 9.0
69
I 12.0
I 15.0
I 18.0
I 21.0
I 24.0
1 270
_
ENERGY DENSITY (J cni*) Fig. 1. The percentage of Ca” uptake by irradiated cells at A= 630 nm, relative to control nonirradiated cells (the standard deviation is *lo%).
97 0.0
* 413
I
I 813
I
I 12.0
I
1 160
I 20.0
ENERGY DENSITY (J cni*) Fig. 2. The percentage of Ca*+ uptake by irradiated cells at A=780 nm, relative to control nonirradiated cells (the standard deviation is *lo%).
macrophages after 660,820 and 870 nm light irradiation. When the cells were incubated in 45CaC12 before irradiation, there was no change in Ca2+ transport. From previous work [14, 151 we know that He-Ne lasers generate an extra electrochemical potential in mitochondria which causes an increase in ATP synthesis. In recent papers [17, 181 we proposed possible ways by which the visible and far-red light is converted to the electrochemical energy of the PMF (proton motive force). We proposed two possible mechanisms: (1) singlet oxygen formation by endogenous porphyrins which activates the respiratory chain in the mitochondria, and (2) activation of the redox reactions in the respiratory chain by exciting the mitochondrial cytochromes.
340
We also suggested that the increased PMF releases Ca*+ from the mitochondria to the cytoplasm which triggers cell mitosis. However, as the present results indicate, there is an accelerated exchange of Ca” between the medium and the cell during irradiation. This phenomenon agrees with previous observations [4] on the rate of Ca2+ transport into bovine spermatozoa due to various oxygenixing substrates. The Ca2+ transport rate into the cells was found to be determined by the mitochondrial Ca2+ uptake which depends on its respiration rate. Mitochondria isolated from various tissues have the capacity to accumulate large amounts of Ca2’ in the matrix compartment by a transport process energetically coupled to electron transport [6]. This means that the irradiated mitochondria can pull more Ca2+ from the medium into the cytoplasm by a mechanism still unknown. When steady state is reached, Ca” is expelled according to the regular mechanisms regulating Ca” concentration in the cells, and this is the reason for the decrease in “Ca2+ uptake after some time [19]. As the data show, the rate of this decrease in enhanced by the irradiation, which means that light also accelerates the efflux of Ca2+ from the mitochondria to the medium [18]. The fact that there is no acceleration in Ca2+ uptake when the cells are incubated in 4’CaC12 to reach the steady state before the irradiation confirms our assumption that the laser actually stimulates the influx and efflux of Ca2+ through the cell membrane. The changes in membrane permeability may cause transient changes in the cytoplasmic Ca2+ concentration, which, in spermatozoa, has a regulatory role in control of motility and acrosome reaction [2, 31, and in other cells can trigger mitosis [l, 131.
Acknowledgment
We wish to thank Professor Kendric C. Smith of the School of Medicine, Stanford University, USA, for his encouragement of this work.
References 1 R. Lubart, Y. Wollman, H. Friedmann, S. Rochkind and I. Laulicht, Effects of visible and near infrared lasers on cell cultures, .I. P/r&o&em. Photubiol. B: Bid, 12 (1992) 305-310. 2 J. P. Singh, D. F. Babcock and H. A. Lardy, Motility activation, respiratory activation and alteration of CaZf transport in bovine sperm treated with amine local anesthetics and calcium transport antagonists, Arch Biochem. Biophys., 221 (1983) 291-303. 3 R. Yanagimachi and N. Usui, Calcium dependence of the acrosome reaction and activation of guinea pig spermatozoa, Qn. CelL Res., 89 (1974) 161-174. 4 H. Breitbart, R. Wehbie and H. Lardy, Calcium transport in bovine sperm mitochondria: effect of substrates and phosphate, Biochim. Biophys. Acta, 1027 (1990) 72-78. 5 H. Breitbart, R. Wehbie and H. Lardy, Regulation of calcium transport in bovine spermatozoa, Biochim. Biophys. Acta, 1027 (1990) 72-78. 6 A. L. Lehninger, Mitochondria and calcium ion transport, B&hem. J., II8 (1970) 129-138. 7 D. F. Babcock, N. L. First and H. A. Lardy, Action of Ionophore A23187 at the cellular level, J. Bid. Chem., 251 (1976) 3881-3886. 8 M. P. Bradley and I. T. Forrester, A Ca’+ +M2+-ATPase and active Ca’+ transport in the plasma membranes isolated from ram sperm flagells, Cell Calcium, I (1980) 381-390. 9 H. Breitbart and S. Rubinstein, Calcium transport in bull spermatozoa plasma membrane, Biochim. Biophys. Acta, 732 (1983) 464-468.
341 10 H. Breitbart, H. Stern and S. Rubinstein, Calcium transport Ca’+-ATPase activity in ram spermatozoa plasma membrane vesicles, Biochim. Biophys. Actu, 728 (1983) 349-355. 11 M. P. Bradley and I. T. Forrester, A sodium calcium exchange mechanism in plasma membrane vesicles isolated from ram flagella, FEBS Lett., I21 (1980) 15-18. 12 D. F. Babcock and D. R. Pfeiffer, Independent elevation of cytosolic Ca” and pH of mammalian sperm by voltage dependent and pH sensitive mechanisms, J. Biol. Chem., 262 (1987) 15041-15047. 13 T. I. Karu, Molecular mechanism of therapeutic effect of low intensity laser radiation, Lasers Life Sci., 2(l) (1988) 53-74. 14 S. Passarella, E. Casamassima, S. Molinari, D. Pastone, E. Quagliariello, I. M. Catalan0 and A. Cinyolani, Increase of proton electrochemical potential and ATP synthesis in rat liver mitochondria irradiated in vitro by HeNe laser, FEBS Lett., 175 (1984) 95-99. 15 C. Sallet, S. Passarella and E. Quagliariello, Effects of selective irradiation on mammalian mitochondria, Photo&em PhotobioL, 45 (1987) 433-438. 16 S. R. Young, M. Dyson and P. Bolton, Effect of light on calcium uptake by macrophages, Laser Zker., 2 (1990) 53-57. 17 R. Lubart, Z. Malik, S. Rochkind and T. Fosjer, A possible mechanism of low-level laserliving cell interaction, Laser Ther., 2 (1990) 65-68. 18 H. Friedmann, R. Lubart and I. Laulicht, A possible explanation of laser-induced stimulation, J. Photochem. Photobiol. B: Biol., 11 (1991) 87-95. 19 A. Zarca, S. Rubinstein and H. Breitbart, Transport mechanism for calcium and phosphate in ram spermatozoa, B&him. Biophys. Actu, 944 (1988) 351-358.
