Tiina I. Karu

Photobiology of low-power laser effects

Quantitative studies have been performed to determine the action of low-intensity visible monochromatic light on various cells (E. coli, yeasts, HeLa, Chinese hamster fibroblasts and human lymphocytes); also irradiation conditions (wavelength, dose and intensity) conducive to vital activity stimulation have been examined. Respiratory chain components are discussed as primary photoacceptors. The possible ways for photosignal transduction and amplification are discussed. It is proposed that enhanced wound healing due to irradiation with low-intensity visible laser light (He-Cd, He-Ne and semiconductor lasers) is due to the increasing proliferation of cells.

Irradiation with HeNe laser increases ATP level in cells cultivated in vitro

A monolayer of HeLa cells was irradiated with an He-Ne laser (632.8 nm, 100 J m-2, 10 s) and the amount of adenosine triphosphate (ATP) was measured by the luceferin-luciferase bioluminescent assay technique at different times (5-45 min) after irradiation. The amount of ATP in the log phase of cultured cells remained at the control level (0.79 +/- 0.09) x 10(-15) mol per cell) during the first 15 min after irradiation; it then increased sharply and, after reaching a maximum (170.8%) 20 min after irradiation, decreased slowly to the control level. The ability of monochromatic red light to induce an increase in the cellular ATP level was found to depend on the growth phase of the culture, being insignificant in the lag phase of cultured cells, increasing in the log phase of cultured cells and reaching a maximum (about 190%) in cells at the late logarithmic and early plateau phase.

Mitochondrial signaling in mammalian cells activated by red and near-IR radiation.

Mitochondrial signaling is an information channel between the mitochondrial respiratory chain and the nucleus for the transduction signals regarding the functional state of the mitochondria. The present review examines the question whether radiation of visible and near‐IR (IR‐A) radiation can activate this retrograde‐type cellular signaling pathway. Experimental data about modulation of elements of mitochondrial retrograde signaling by the irradiation (mitochondrial membrane potential ΔΨm, reactive oxygen species ROS, Ca2+, NO•, pHi, fission‐fusion homeostasis of mitochondria) are reviewed. The terminal enzyme of the mitochondrial respiratory chain cytochrome c oxidase is considered as the photoacceptor. Functions of cytochrome c oxidase as a signal generator as well as a signal transducer in irradiated cells are outlined.

A Novel Mitochondrial Signaling Pathway Activated by Visible-to-near Infrared Radiation¶

Abstract The number of cells attached to glass substratum increases if HeLa cell suspension is irradiated with monochromatic visible-to-near infrared radiation before plating (the action spectrum with maxima at 619, 657, 675, 700, 740, 760, 800, 820, 840 and 860 nm). Treating of cell suspension with sodium azide (2 × 10−5 M), sodium nitroprusside (5 × 10−5 M), ouabain (1 × 10−6 M) or amiloride (1.7 × 10−5 M) before irradiation significantly modifies the spectrum of cell attachment enhancement. A light-induced mitochondrial signaling pathway can be regulated by small ligands directly binding to the catalytic center of cytochrome c oxidase (N3, NO) as well as by chemicals specifically binding to plasma membrane enzymes (ouabain, amiloride). The comparative analysis of action spectra allows the conclusions that first, CuA and CuB chromophores of cytochrome c oxidase could be involved as photoacceptors and second, various signaling pathways (reaction channels) between cytochrome c oxidase and cell attachment regulation are at work.

Multiple Roles of Cytochrome c Oxidase in Mammalian Cells Under Action of Red and IR-A Radiation

This article reviews the current knowledge in photobiology and photomedicine about the influence of monochromatic, quasimonochromatic, and bread‐band radiation of red‐to‐near infrared (IR‐A) part on solar spectrum upon mammalian cells and human skin. The role of cytochrome c oxidase as the photoacceptor and photosignal transducer is underlined and its photosensitivity at certain circumstances is discussed. The role of ATP as a critical signaling molecule is discussed.

Mitochondrial Mechanisms of Photobiomodulation in Context of New Data About Multiple Roles of ATP

Various cellular responses to visible and IR-A radiation have been studied for decades in the context of molecular mechanisms of laser phototherapy [also called photobiomodulation, low-level light therapy (LLLT)]. LLLT uses monochromatic and quasimonochromatic light in the optical region of *600–1,000 nm to treat in a nondestructive and nonthermal fashion various soft-tissue and neurologic conditions. This modality also was recently used to reverse toxic effects of neurotoxins, to treat strokes and acute myocardial infarction, and to stimulate stem cell proliferation. This multiplicity of conditions treated with photobiomodulation has persuaded many unbelievers of the value of such an universal method. It is generally accepted that the mitochondria are the initial site of light action in cells, and cytochrome c oxidase (the terminal enzyme of the mitochondrial respiratory chain) is the responsible molecule. Mixed-valence copper components of cytochrome c oxidase, CuA and CuB, are believed to be the photoacceptors. The same photoacceptor molecule for different cellular responses can explain, at least partly, the versatility of low-power laser effects. The excitation of the photoacceptor molecule sets in motion cellular metabolism through cascades of reactions called cellular signaling or retrograde mitochondrial signaling. At least two reactions are starting points for monitoring cellular-signaling reactions after light action on the cytochrome c oxidase molecule. One of them is dissociation of NO from the catalytic center of cytochrome c oxidase. Spectroscopic studies of irradiated cellular monolayer show that two charge-transfer channels putatively to CuAred and CuBoxid , as well as two reaction channels putatively connected with d-d transition in CuBred and CuAoxid chromophores, are reorganized dependent on NO presence or absence. It has been suggested that the dissociation of NO (a physiologic regulator of cytochrome c oxidase activity) rearranges downstream signaling effects. Another signaling pathway starting from the mitochondria is connected with ATP. The ATP extrasynthesis in isolated mitochondria and intact cells of various types, under irradiation with light of different wavelengths, is well documented. ATP is a universal fuel inside living cells that drives all biologic reactions. It is known that even small changes in the ATP level can significantly alter cellular metabolism. Increasing the amount of this energy may improve the cellular metabolism, especially in suppressed or otherwise ill cells. In connection with the versatility of LLLT effects, I draw the readers’ attention to a comparatively new aspect of the ATP molecule. A long series of discoveries has demonstrated that ATP is not only an energy currency inside cells, but it is also a critical signaling molecule that allows cells and tissues throughout the body to communicate with one another. This new aspect of ATP as an intercellular signaling molecule allows broadening the understanding of universality phenomenon of LLLT as well. It is known now that neurons release ATP into muscle, gut, and bladder tissue as a messenger molecule. The specific receptors for ATP as the signaling molecule (P2 family) and for its final breakdown product, adenosine (P1 family), were found and identified. ATP activation of P2 receptors (subtypes P2X and P2Y) can produce different cellular effects. A recent article by Anders et al. demonstrated that P2Y2 and P2Y11 receptors were expressed in the irradiated at l1⁄4 810-nm normal human neural progenitor cells in vitro. It appeared that the irradiation could be used as a replacement for growth factors. This line of research opens a new understanding of the complicated mechanisms of LLLT. From the point of view of the topic of the present article, the role of ATP as a signaling molecule provides a new basis for explaining the versatility of LLLT effects. The second important point in connection with multiple functions of ATP and P2X and P2Y receptors is the following. When bound by ATP, P2X receptors form a channel that allows sodium and calcium ions to enter the cells. ATP binding to the extracellular surface of P2Y receptors starts a cascade of molecular interactions inside cells, with those resulting in intracellular calcium stores being released. The increase in intracellular Ca2þ ions ([Ca]i) due to the irradiation has been measured by many authors, but the mechanism of the phenomenon of [Ca]i increase in the irradiated cells has not been explained. Ca2þ is a global positive effector of mitochondrial function, and thus, any perturbation in mitochondrial or cytosolic Ca2þ homeostasis will have implications on mitochondrial functions. This concerns the regulation of [Ca]i from outside by binding ATP to P2X receptors. It is important to remember that both Ca2þ uptake and efflux from mitochondria consume DCm

Photobiological modulation of cell attachment via cytochrome c oxidase

The number of cells attached to glass substrates increases if HeLa cell suspensions are irradiated with monochromatic visible-to-near infrared radiation (600-860 nm, 52 J m(-2)) prior to plating. The well-structured relationship between this biological response and the radiation wavelength (action spectrum with maxima at 620, 680, 760, and 820 nm) suggests the existence of a photoacceptor responsible for the enhancement of attachment (presumably cytochrome c oxidase, the terminal enzyme of the respiratory chain) and, secondly, the existence of signaling pathways between the mitochondria, the plasma membrane, and the nucleus of the cell. Treating the cell suspension with ouabain (a Na(+), K(+)-ATPase inhibitor), amiloride (an inhibitor of N(+)/H(+) exchangers), or sodium azide (a cytochrome c oxidase inhibitor) prior to irradiation significantly modifies the action spectrum of cell attachment enhancement. The action of the chemicals under study also depends on their concentration and radiation fluence. Our results point to the existence of at least three signaling pathways (reaction channels) relating together the cell attachment, the respiratory chain, and the Na(+), K(+)-ATPase and N(+)/H(+) exchanger activities.

Effects of monochromatic low‐intensity light and laser irradiation on adhesion of HeLa cells in vitro

The adhesion of HeLa cells was evaluated after irradiation with monochromatic low‐intensity light or laser irradiation. It is well known that the cell‐cell and cell‐matrix adhesion changes during wound repair. For better understanding of low‐power laser light action on the wound healing process, it would be of interest to study the light action on cellular adhesion in vitro.

Ultrastructural changes in chondriome of human lymphocytes after irradiation with HeNe laser: Appearance of giant mitochondria

The structure of mitochondria was studied after the irradiation of human lymphocytes with an He-Ne laser (wavelength, 632.8 nm; dose, 56 J m-2). Ultrathin sections of the lymphocytes were studied by electron microscopy 1 h after the irradiation. The irradiation resulted in a 20% increase (p = 0.95) in the number of mitochondrial profiles on the cell section without an increase in their total area. Three-dimensional reconstruction of mitochondria from ultrathin sections trough the whole lymphocyte showed that the number of mitochondria was reduced to 9-12 in the irradiated cells compared with 40-45 in the control cells. In the irradiated lymphocytes, 2-4 giant branching mitochondria were also observed among small discrete mitochondria.

Mechanisms of low-power laser light action on cellular level

The most frequently used mechanism of photon energy conversion in laser medicine is heating. Average heating of irradiated samples occurs with all methods of tissue destruction (cutting, vaporization, coagulation, ablation). At low light intensities the photochemical conversion of the energy absorbed by a photoacceptor prevails. This type of reaction is well known for specialized photoacceptors such as rhodopsin or chlorophyll. In medicine light absorption by non-specialized photoacceptor molecules (that is, molecules that can absorb light at certain wavelengths, but that are not integral to specialized light reception organs) is used rather extensively. The absorbing molecule can transfer the energy to another molecule, and this activated molecule can then cause chemical reactions in the surrounding tissue. This type of reaction is successfully used in photodynamic therapy (PDT) of tumors. Alternatively, the absorbing molecule in a light-activated form can take part in chemical reactions, as occurs in treatment of skin diseases with psoralens and UVA radiation (PUVA). Importantly, in both PDT and PUVA therapy the photoabsorbing molecules are artificially introduced into a tissue before irradiation. Irradiation of cells at certain wavelengths can also activate some of the native components. In this way specific biochemical reactions as well as whole cellular metabolism can be altered. This type of reaction is believed to form the basis for low-power laser effects [1-41. One should note that light therapy methods based on photochemical conversion of photoabsorbing molecules are not laser-specific methods [1]: conventional sources generating the appropriate wavelength can also be used (as is done in PUVA and UV therapy). Laser sources are just handy tools providing many practical advantages (e.g., efficient fiber optic coupling to irradiate interior body parts, high monochromaticity and easy wavelength tunability, simplicity in use and electrical safety in the case of semiconductor lasers). It was shown that coherent effects in the light-cells interaction occur at intensities 2x1015 W/m2 [1]. Bear in mind that typical intensities used in low-power laser therapy are in the range 1OlO2 W/m2. The successful use of light-emitting diodes in low-power laser therapy in the last years [5] proves that the coherence of light is of no importance in low-power laser clinical effects. However, one should not forget about coherent laser-light speckles which can cause local heating of inhomogeneous tissues [6]. This possible mechanism forms a basis of laser thermotherapy [7] and supposedly can be taken into account at certain laser light parameters in low-power laser therapy [8] when irradiating inhomogeneously absorbing tissues. For cell culture, the localized transient heating of absorbing chromophores is considered as one ofpossible primary mechanisms (Section 2.2). A photobiological reaction involves the absorption of a specific wavelength of light by the functioning photoreceptor (jhotoacceptor) molecule. To distinguish specialized photoreceptor molecules such as rhodopsin, phytochrome, bacteriorhodopsin and chlorophylls from nonspecialized chromophores (molecules capable of absorbing the wavelength used for irradiation resulting in a photobiological response), below we will use the term photoacceptors to refer to the nonspecialized photoabsorbers. The photoacceptors take part in a metabolic reaction in a cell that is not connected with a light response. After absorbing the light of the wavelength used for irradiation this molecule assumes an electronically excited state from which primary molecular processes can lead to a measurable biological effect in certain circumstances. To work as a photoacceptor taking part in photobioregulation, this molecule must be part of a key structure that can regulate a metabolic pathway. Redox chains are example of this type of key structures which suit to these requirements. Possible photoacceptors (respiratory chain components) as well as chemical reactions occurring under illumination (primary reactions) or those following after the end of the irradiation (secondary reactions) are considered in Section 2: