Red and near-infrared light evokes Ca²⁺ influx, endoplasmic reticulum release and membrane depolarization in neurons and cancer cells

Highlights

• 650 and 808 nm light increases level of Ca²⁺ in neurons and cancer cells in vitro.

• In contrast, no effect is observed under 1064 nm irradiation.

• Intracellular influx and release of Ca²⁺ from endoplasmic reticulum are the causes.

• Ca²⁺ influx is distinctly associated with light-driven membrane depolarization.

• Crosstalk between light-induced ROS and endoplasmic reticulum Ca²⁺ release is studied.

Abstract

Low level light therapy uses light of specific wavelengths in red and near-infrared spectral range to treat various pathological conditions. This light is able to modulate biochemical cascade reactions in cells that can have important health implications. In this study, the effect of low intensity light at 650, 808 and 1064 nm on neurons and two types of cancer cells (neuroblastoma and HeLa) is reported, with focus on the photoinduced change of intracellular level of Ca²⁺ ions and corresponding signaling pathways. The obtained results show that 650 and 808 nm light promotes intracellular Ca²⁺ elevation regardless of cell type, but with different dynamics due to the specificities of Ca²⁺ regulation in neurons and cancer cells. Two origins responsible for Ca²⁺ elevation are determined to be: influx of exogenous Ca²⁺ ions into cells and Ca²⁺ release from endoplasmic reticulum. Our investigation of the related cellular processes shows that light-induced membrane depolarization is distinctly involved in the mechanism of Ca²⁺ influx. Ca²⁺ release from endoplasmic reticulum activated by reactive oxygen species generation is considered as a possible light-dependent signaling pathway. In contrast to the irradiation with 650 and 808 nm light, no effects are observed under 1064 nm irradiation. We believe that the obtained insights are of high significance and can be useful for the development of drug-free phototherapy.

Introduction

Low level light therapy, or photobiomodulation, is the application of low intensity light to the living organism for therapeutic purposes [1]. It is emerging as a promising drug-free therapeutic approach employing light with visible and near-infrared (NIR) irradiation to treat pain [2], promote reduction of inflammation and speed up wound healing [3,4], solid tumors and cancer cell inhibition [5], regeneration and restoration of various organism functions [6,7], etc. In recent years, the transcranial treatment using the irradiation in 600–1070 nm spectral range becomes increasingly introduced as a new neuroprotective approach to treat neurodegenerative diseases. Such therapy is shown to reduce β-amyloid plaques and neurofibrillary tangles of tau proteins in the brains of mice with Alzheimer's disease [[8], [9], [10]] and save dopaminergic neurons from death in animal models of Parkinson's disease [[11], [12], [13]]. It can be also employed to stabilize stroke [14], fight amyotrophic lateral sclerosis [15], and also improve memory [16] and conditions associated with traumatic brain injury, major depression, ischemic stroke and age-related macular degeneration [11,17]. Red and NIR light is usually utilized for therapy due to its effective absorption by biological tissues and the positive results of modulation or treatment, even with transcranial application [14]. One of the reasons for the effective application of NIR light possibly lies in its ability to penetrate deeper into the body, due to the reduced light scattering [18,19].

It is clear that efficient application of such treatment requires deep understanding of red/NIR light action on biological objects. In this regard, special attention is currently paid to the study of light influence on cellular processes and intracellular pathways [[20], [21], [22], [23], [24]]. The prime mechanism of light influence is considered to be associated with cytochrome c oxidase (CCO), mitochondrial protein complex that absorbs light in the range of 600–900 nm. A change in CCO redox state activates cellular stress response systems in the electron transport chain in mitochondria [25]. This induces an increase in cellular energy through adenosine triphosphate (ATP) production and a moderate burst in reactive oxygen species (ROS) [11,26]. As a consequence, downstream signaling pathways are triggered, resulting in the activation of neuroprotective mechanisms, rise in cell proliferation, formation of new synapses, changes in neural cell metabolism [26] and brain-based metabolic effects [25], or, on the other hand, in the instigation of apoptosis [21].

Visible-NIR light is also reported to affect the membrane potential (V_m), increasing ion permeability into cells, without affecting temperature [27,28]. In particular, 650 and 808 nm light is shown to evoke Ca²⁺ entry by neurons and cancer cells [20,29]. Membrane transporters of Ca²⁺ are localized in cell plasma membrane as well as in intracellular Ca²⁺ depot of endoplasmic reticulum (ER) and mitochondria [30]. Various exogenous Ca²⁺ channels and pumps are expressed in cell membrane to transport Ca²⁺ into the cytoplasm. These are ionotropic glutamate receptors (GluRs), such as N-methyl-d-aspartate receptors (NMDARs), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) and kainate receptors, as well as transient receptor potential cation channel subfamily V (TRPV) channels and different Ca²⁺ exchangers. Notably, NMDARs, which are open upon ligand binding of Glu and corresponding changes in V_m, can be activated by light, allowing for the foremost influx of Ca²⁺ into cancer cells [20]. However, not all the exogenous Ca²⁺ channels can be activated under low intensity light; for instance, temperature-dependent TRPV channels require much higher light power densities (causing increase in temperature) to be activated [31]. On the other hand, endogenous Ca²⁺ channels are energy-dependent ionotropic receptors responsible for transporting Ca²⁺ in place of further storage [32,33]. ER is considered to be the main intracellular Ca²⁺ store, with a concentration maintained in the range of 0.1–1 mM, compared with hundreds of nM in the cytosol and mitochondrial matrix [33].

Ca²⁺ have various critical functions in neurons: regulation of neurotransmitter release, synaptic plasticity, activity-dependent transcription, and others. Therefore, neurons are critically dependent on controlled Ca²⁺ influx, and its dysregulation is crucial for the brain cell vitality. The excessive influx of Ca²⁺ can lead to excitotoxicity, causing degeneration and death of neurons [34]. On the other hand, due to the role of neuronal ER in the intracellular Ca²⁺ signaling, it has been termed as a “neuron-inside-a-neuron” [35]; burst in intracellular Ca²⁺ release from ER, followed by an excessive uptake by mitochondria, is a mechanism that can trigger apoptosis [36]. It is important to note that the irradiation with low intensity NIR light is reported to reduce intracellular Ca²⁺ overload and ER stress in vitro (irradiation at 850 nm) [37] and protect cells from the destructive effect of high Ca²⁺ concentration, promoting the excretion of Ca²⁺ from cells and preventing excitotoxicity (irradiation at 808 nm) [38].

It should be emphasized that the role of ER-mediated Ca²⁺ signaling in neurons and other cells is a hot research topic [39,40]. However, a light influence on Ca²⁺ release from ER under irradiation remains unrevealed. Only the work by Kharkwal et al. [29] reported the influence of low intensity 808 nm light on intracellular Ca²⁺ level in neurons. Recently, we reported on stimulation of ion channels and elevation of intracellular Ca²⁺ level in cancer cells under low intensity light, covering the irradiation range from visible to 808 nm [20]. Though, the knowledge on origins of ion channel activation under low intensity light and relevant signaling pathways remains quite limited. We believe that the study of red/NIR light effect on the intracellular Ca²⁺ balance and other related photoinduced processes will help us to understand how to apply light effectively for treatment of neurodegenerative diseases and brain injuries.

In this work, we study the effects of low intensity red/NIR light on neurons, neuroblastoma and HeLa cells, with focus on the photoinduced elevation of intracellular Ca²⁺ level and signaling pathways leading to this phenomenon. We investigate the influence of 650 and 808 nm light in comparison with that of 1064 nm light, which is much less explored. Our results have shown that, in contrast to 1064 nm light (which produces no effect), both 650 and 808 nm light promotes elevation of intracellular Ca²⁺ regardless of cell type, but with different dynamics, apparently due to the specificities of Ca²⁺ regulation in neurons and cancer cells. Two origins of Ca²⁺ elevation have been determined: (i) influx of exogenous Ca²⁺ into cell and (ii) release of endogenous Ca²⁺ from ER. The 650 and 808 nm light-induced membrane depolarization is shown to be distinctly involved in the cellular mechanism of Ca²⁺ influx. To the best of our knowledge, this is the first time that the light effect on Ca²⁺ release from ER has been revealed.