Ludmila V. Pyatibrat

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.

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.

Absorption Measurements of Cell Monolayers Relevant to Mechanisms of Laser Phototherapy: Reduction or Oxidation of Cytochrome c Oxidase Under Laser Radiation at 632.8 nm

OBJECTIVE The objective of this work was a further investigation of redox mechanisms of laser phototherapy on the cellular level. BACKGROUND DATA Cytochrome c oxidase, the terminal enzyme of the mitochondrial respiratory chain, is believed to work as the photoacceptor to modulate cellular metabolism in laser phototherapy. MATERIALS AND METHODS The changes in the absorption spectra of HeLa-cell monolayers before and after irradiation at 632.8 nm using fast multi-channel recording were evaluated by the intensity ratio between the peaks at 770 and 670 nm (intensity ratio criterion). RESULTS By the intensity ratio criterion, the irradiation effects (reduction or oxidation of the photoacceptor) depended on the initial redox status of cytochrome c oxidase. The irradiation (three times at 632.8 nm, dose = 6.3 x 103 J/m(2), tau(irrad.) = 10 sec, tau(record.) = 600 msec) of cells initially characterized by relatively oxidized cytochrome c oxidase caused first a reduction of the photoacceptor, and then its oxidation (a bell-shaped curve). The irradiation by the same scheme of the cells with initially relatively reduced cytochrome c oxidase caused first oxidation and then a slight reduction of the enzyme (a curve opposite to the bell-shaped curve). CONCLUSION The experimental results of our work demonstrate that irradiation at 632.8 nm causes either a (transient) relative reduction of the photoacceptor, putatively cytochrome c oxidase, or its (transient) relative oxidation, depending on the initial redox status of the photoacceptor. The maximum in the bell-shaped dose-dependence curve or the minimum of the reverse curve is the turning point between the prevailing of oxidation or reduction processes. Our results are evidence that the bell-shaped dose dependences recorded for various cellular responses are characteristic also for redox changes in the photoacceptor, cytochrome c oxidase.

Nonmonotonic behavior of the dose dependence of the radiation effect on cells in vitro exposed to pulsed laser radiation at λ = 820 nm

In recent years, clinical low‐intensity laser therapy practice has used pulsed radiation, mainly from semiconductor lasers. Experimental works devoted to the study of relationships between biological and clinical effects and parameters of pulsed radiation are practically absent.

Donors of NO and pulsed radiation at λ=820 nm exert effects on cell attachment to extracellular matrices

The adhesion of HeLa cells to a glass matrix was evaluated after the irradiation of the cell suspension with a pulsed near-infrared light-emitting diode (=820 nm, frequency 10 Hz, dose 8–120 J/m 2 ) and treatment with two donors of nitric oxide, sodium nitroprusside (SNP, 5 ×10 − 4 M) and NaNO2 (4× 10 − 4 M). It was found that in an irradiated cell suspension, the cell–glass adhesion increases in a dose-dependent manner (a bell-shaped curve with a maximum at 60 J/m 2 ). The treatment of cells with SNP or NaNO2 before the irradiation eliminates the radiation-induced attachment stimulation. Pretreatment of cells with SNP not only eliminates the radiation-induced attachment stimulation but inhibits the attachment of irradiated (but not non-irradiated) cells. It is suggested that a modulation of the activity of respiratory chain (probably the alteration of the activity of cytochrome c oxidase) is involved in radiation-induced increase of cell attachment.

Gene Expression Under Laser and Light-emitting Diodes Radiation for Modulation of Cell Adhesion: Possible Applications for Biotechnology

Experimental data about the modulation of adhesion and proliferation of anchorage‐dependent HeLa cells with monochromatic or quasimonochromatic radiation in red to near‐infrared region are presented. Cell adhesion and proliferation can be increased by irradiation with light of certain wavelengths (maxima in action spectrum are 619, 675, 740, 760, and 820 nm) or decreased when the activity of photoacceptor (cytochrome c oxidase in mitochondrial respiratory chain) is inhibited by chemicals before the irradiation. This modality allows controlling the number of attached and/or proliferating cells. Possible biotechnology applications of this method are outlined.

Elementary Processes in Cells after Light Absorption Do Not Depend on the Degree of Polarization: Implications for the Mechanisms of Laser Phototherapy

OBJECTIVE The objective of this work was to evaluate the importance of the degree of light polarization in stimulation of cellular metabolism. BACKGROUND DATA Although the possible role of polarizations effects on the mechanisms of laser phototherapy is sometimes discussed in the literature, there are still no clear answers. MATERIAL AND METHODS A model system (HeLa cell suspension) was used in which the lengths of light scattering (l sc) and absorption (l a) were much larger than the thickness of the irradiated layer (L = 3 mm). The cell suspension (1 x 10(6) cells/cm3) was irradiated with a diode laser (lambda = 637 nm, D = 65.7 J/m2, tau = 10 sec, I = 6.57 W/m2). The polarization degree (99.4%, 60.9%, and 34.2%) of the beam was changed by means of optical fibers of different lengths. The irradiated suspension was incubated at 37 degrees C for 30 min, and the attached cells were counted afterwards. RESULTS The cell fraction stimulated to adhere by red light at 637 nm was nearly the same in all three experimental groups (58.1% +/- 2.5%, 57.6% +/- 3.5%, and 62.5% +/- 3.2% for degree of beam polarization of 99.4%, 66.9%, and 34.2%, respectively). There was no statistically significant difference in these results (p <0.8, <0.6, and <0.7, respectively). At the same time, all three groups had statistically significant differences (p < 0.01) in adherence from the sham-irradiated control group (39.1% +/- 2.2%). CONCLUSION The biological effect (stimulation of cell attachment) of light with lambda = 637 nm on cells in our model system was pronounced, but did not depend on the degree of light polarization. Elementary processes in cells (light absorption and photochemistry) do not appear to depend on the degree of light polarization.

Melatonin modulates the action of near infrared radiation on cell adhesion.

Abstract: The adhesion of human cervical cancer (HeLa) cells to a glass matrix is evaluated following their irradiation in a suspension with a pulsed near‐infrared (IR) light‐emitting diode (wavelength 820 nm, pulse repetition frequency 10 Hz, irradiation dose 16–120 J/m2) when melatonin (4 × 10−11 to 4 × 10−5 m) is added to cell suspension immediately before or after the irradiation. Also, the dependence of visible‐to‐near‐IR radiation (600–840 nm, 52 J/m2) on cell adhesion (action spectrum) is recorded in absence and presence of melatonin (4 × 10−6 m). It is found that melatonin in pharmacological concentrations (but not in physiological range) inhibited cell adherence. Irradiation of cells before or after melatonin treatment normalizes cell adhesion to control level. Melatonin in pharmacological concentrations eliminates stimulation of cell attachment induced by irradiation. Pre‐treatment (but not post‐treatment) with melatonin in the physiological concentration eliminates cell adhesion stimulation induced by irradiation. Melatonin modifies the light action spectrum significantly in near IR region (760–840 nm only). Thus, the peak at 820–830 nm characteristic for the light action spectrum is fully reduced.

Irradiation with a diode at 820 nm induces changes in circular dichroism spectra (250-780 nm) of living cells

A sensitive method for measuring the circular dichroism (CD) of living cells in visible-near IR region is developed. The changes in CD spectra from 250 to 780 nm of HeLa cell suspension after the first and second irradiation at 820 nm in dose 9 J/cm2 are investigated. The CD spectrum of the intact cells is well structured and characterized by a positive signal in the UV (250-290 nm) and visible-near IR (500-780 nm) regions as well as by a negative signal in 300-450 nm region. Distinct maxima in the visible-near IR region are recorded at 566, 634, 680, 712, and 741 nm. As a rule, the peak circular dichroism signals decrease in the irradiated cells except of the area 750-770 nm. Peak positions (exception: the peak at 680 nm) shift as a rule to the long-wavelength direction. The most remarkable changes in peak positions as well as in CD signals are recorded in the region 750-770 nm: an appearance of the new peak at 767 nm after the first irradiation and its shift to 752 nm after the second irradiation. The peaks at 712 and 741 nm disappear after the irradiation. A new peak appears at 601 nm. It is assumed that the changes in the degree of oxidation of the chromophores of cytochrome c oxidase caused by the irradiation are accompanied by conformational changes in their vicinity. It can be suggested that these changes are occurring in CuB environment.