Vladimir Shneyvays

Low Energy Visible Light Induces Reactive Oxygen Species Generation and Stimulates an Increase of Intracellular Calcium Concentration in Cardiac Cells

Low energy visible light (LEVL) irradiation has been shown to exert some beneficial effects on various cell cultures. For example, it increases the fertilizing capability of sperm cells, promotes cell proliferation, induces sprouting of neurons, and more. To learn about the mechanism of photobiostimulation, we studied the relationship between increased intracellular calcium ([Ca2+]i) and reactive oxygen species production following LEVL illumination of cardiomyocytes. We found that visible light causes the production of \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{O}_{2}^{{\bar{{\cdot}}}}\) \end{document} and H2O2 and that exogenously added H2O2 (12 μm) can mimic the effect of LEVL (3.6 J/cm2) to induce a slow and transient increase in [Ca2+]i. This [Ca2+]i elevation can be reduced by verapamil, a voltage-dependent calcium channel inhibitor. The kinetics of [Ca2+]i elevation and morphologic damage following light or addition of H2O2 were found to be dosedependent. For example, LEVL, 3.6 J/cm2, which induced a transient increase in [Ca2+]i, did not cause any cell damage, whereas visible light at 12 J/cm2 induced a linear increase in [Ca2+]i and damaged the cells. The linear increase in [Ca2+]i resulting from high energy doses of light could be attenuated into a non-linear small rise in [Ca2+]i by the presence of extracellular catalase during illumination. We suggest that the different kinetics of [Ca2+]i elevation following various light irradiation or H2O2 treatment represents correspondingly different adaptation levels to oxidative stress. The adaptive response of the cells to LEVL represented by the transient increase in [Ca2+]i can explain LEVL beneficial effects.

Cardioprotective effects of adenosine A1 and A 3 receptor activation during hypoxia in isolated rat cardiac myocytes

Adenosine (ADO) is a well-known regulator of a variety of physiological functions in the heart. In stress conditions, like hypoxia or ischemia, the concentration of adenosine in the extracellular fluid rises dramatically, mainly through the breakdown of ATP. The degradation of adenosine in the ischemic myocytes induced damage in these cells, but it may simultaneously exert protective effects in the heart by activation of the adenosine receptors. The contribution of ADO to stimulation of protective effects was reported in human and animal hearts, but not in rat hearts. The aim of this study was to evaluate the role of adenosine A1 and A3 receptors (A1R and A3R), in protection of isolated cardiac myocytes of newborn rats from ischemic injury. The hypoxic conditions were simulated by exposure of cultured rat cardiomyocytes (4–5 days in vitro), to an atmosphere of a N2 (95%) and CO2 (5%) mixture, in glucose-free medium for 90 min. The cardiotoxic and cardioprotective effects of ADO ligands were measured by the release of lactate dehydrogenase (LDH) into the medium. Morphological investigation includes immunohistochemistry, image analysis of living and fixed cells and electron microscopy were executed. Pretreatment with the adenosine deaminase considerably increased the hypoxic damage in the cardiomyocytes indicating the importance of extracellular adenosine. Blocking adenosine receptors with selective A1 and A3 receptor antagonists abolished the protective effects of adenosine. A1R and A3R activation during the hypoxic insult delays onset of irreversible cell injury and collapse of mitochondrial membrane potential as assessed using DASPMI fluorochrom. Cardioprotection induced by the A1R agonist, CCPA, was abolished by an A1R antagonist, DPCPX, and was not affected by an A3R antagonist, MRS1523. Cardioprotection caused by the A3R agonist, Cl-IB-MECA, was antagonized completely by MRS1523 and only partially by DPCPX. Activation of both A1R and A3R together was more efficient in protection against hypoxia than by each one alone. Our study indicates that activation of either A1 or A3 adenosine receptors in the rat can attenuate myocyte injury during hypoxia. Highly selective A1R and A3R agonists may have potential as cardioprotective agents against ischemia or heart surgery.

Angiotensin II-induced apoptosis in rat cardiomyocyte culture: a possible role of AT1 and AT2 receptors.

Objectives To investigate the mechanism of angiotensin II-induced apoptosis in cultured cardiomyocytes by determining which receptor subtype is involved, and what is the relationship between intracellular Ca2+ changes and apoptosis. Design and methods Neonatal rat cardiomyocytes were pretreated with either the AT1 antagonist irbesartan or the AT2 antagonist PD123319 before exposure to angiotensin II. Apoptotis was evaluated using morphological technique, staining nuclei by Feulgen and Hoechst methods followed by image analysis and by in situ terminal deoxynucleotidyl transferase nick-end (TUNEL) labelling. TUNEL-positive cardiocytes were distinguished from other cells by double staining with α-sarcomeric actin. Intracellular Ca2+ changes were assessed by indo-1 fluorescence microscopy, and the effect of Ca2+ on angiotensin II-induced apoptosis was tested using the calcium channel blocker verapamil. Results Exposure to angiotensin II (10 nmol/l) resulted in cell replication and a three-fold increase in programmed cell death (P < 0.05). Pretreatment with either irbesartan (an AT1receptor antagonist, 100 nmol/l) or PD123319 (an AT2 receptor antagonist, 1 μmol/l) prevented the angiotensin II-induced apoptosis, indicating the presence of both AT1 and AT2receptors on cardiomyocytes. Exposure of myocytes to angiotensin II caused an immediate and dose-dependent increase in the concentration of intracellular free Ca2+ that lasted 40–60 s. The effect was sustained in a Ca2+ free medium. Pretreatment of cells with irbesartan (100 nmol/l) and PD123319 (10 μmol/l) blocked Ca2+ elevation. Pretreatment with verapamil (10 μmol/l) prevented angiotensin II-induced apoptosis. Conclusions Angiotensin II-induced apoptosis in rat cardiomyocytes is mediated through activation of both AT1 and AT2 receptors. The apoptotic mechanism is not related to the immediate angiotensin II-induced Ca2+ rise from intracellular stores. However, it is accompanied by cardiomyocyte proliferation and requires Ca2+ influx through L-type channel activity.

Delta-9-tetrahydrocannabinol protects cardiac cells from hypoxia via CB2 receptor activation and nitric oxide production.

Delta-9-tetrahydrocannabinol (THC), the major active component of marijuana, has a beneficial effect on the cardiovascular system during stress conditions, but the defence mechanism is still unclear. The present study was designed to investigate the central (CB1) and the peripheral (CB2) cannabinoid receptor expression in neonatal cardiomyoctes and possible function in the cardioprotection of THC from hypoxia. Pre-treatment of cardiomyocytes that were grown in vitro with 0.1 – 10 μM THC for 24 h prevented hypoxia-induced lactate dehydrogenase (LDH) leakage and preserved the morphological distribution of α-sarcomeric actin. The antagonist for the CB2 (10 μM), but not CB1 receptor antagonist (10 μM) abolished the protective effect of THC. In agreement with these results using RT-PCR, it was shown that neonatal cardiac cells express CB2, but not CB1 receptors. Involvement of NO in the signal transduction pathway activated by THC through CB2 was examined. It was found that THC induces nitric oxide (NO) production by induction of NO synthase (iNOS) via CB2 receptors. L-NAME (NOS inhibitor, 100 μM) prevented the cardioprotection provided by THC. Taken together, our findings suggest that THC protects cardiac cells against hypoxia via CB2 receptor activation by induction of NO production. An NO mechanism occurs also in the classical pre-conditioning process; therefore, THC probably pre-trains the cardiomyocytes to hypoxic conditions.

Detailed analysis of reactive oxygen species induced by visible light in various cell types

Light in the visible and near infrared region stimulates various cellular processes, and thus has been used for therapeutic purposes. One of the proposed mechanisms is based on cellular production of reactive oxygen species (ROS) in response to illumination. In the present study, we followed visible light (VL)‐induced hydroxyl radicals in various cell types and cellular sites using the electron paramagnetic resonance (EPR) spin‐trapping technique.

Mechanism of glycogen supercompensation in rat skeletal muscle cultures

A model to study glycogen supercompensation (the significant increase in glycogen content above basal level) in primary rat skeletal muscle culture was established. Glycogen was completely depleted in differentiated myotubes by 2 h of electrical stimulation or exposure to hypoxia during incubation in medium devoid of glucose. Thereafter, cells were incubated in medium containing glucose, and glycogen supercompensation was clearly observed in treated myotubes after 72 h. Peak glycogen levels were obtained after 120 h, averaging 2.5 and 4 fold above control values in the stimulated- and hypoxia-treated cells, respectively. Glycogen synthase activity increased and phosphorylase activity decreased continuously during 120 h of recovery in the treated cells. Rates of 2-deoxyglucose uptake were significantly elevated in the treated cells at 96 and 120 h, averaging 1.4–2 fold above control values. Glycogenin content increased slightly in the treated cells after 48 h (1.2 fold vs. control) and then increased considerably, achieving peak values after 120 h (2 fold vs. control). The results demonstrate two phases of glycogen supercompensation: the first phase depends primarily on activation of glycogen synthase and inactivation of phosphorylase; the second phase includes increases in glucose uptake and glycogenin level.

Glycogen metabolism in rat heart muscle cultures after hypoxia

Elevated glycogen levels in heart have been shown to have cardioprotective effects against ischemic injury. We have therefore established a model for elevating glycogen content in primary rat cardiac cells grown in culture and examined potential mechanisms for the elevation (glycogen supercompensation). Glycogen was depleted by exposing the cells to hypoxia for 2 h in the absence of glucose in the medium. This was followed by incubating the cells with 28 mM glucose in normoxia for up to 120 h. Hypoxia decreased glycogen content to about 15% of control, oxygenated cells. This was followed by a continuous increase in glycogen in the hypoxia treated cells during the 120 h recovery period in normoxia. By 48 h after termination of hypoxia, the glycogen content had returned to baseline levels and by 120 h glycogen was about 150% of control. The increase in glycogen at 120 h was associated with comparable relative increases in glucose uptake (~ 180% of control) and the protein level of the glut-1 transporter (~ 170% of control), whereas the protein level of the glut-4 transporter was decreased to < 10% of control. By 120 h, the hypoxia-treated cells also exhibited marked increases in the total (~ 170% of control) and fractional activity of glycogen synthase (control, ~ 15%; hypoxia-treated, ~ 30%). Concomitantly, the hypoxia-treated cells also exhibited marked decreases in the total (~ 50% of control) and fractional activity of glycogen phosphorylase (control, ~ 50%; hypoxia-treated, - 25%). Thus, we have established a model of glycogen supercompensation in cultures of cardiac cells that is explained by concerted increases in glucose uptake and glycogen synthase activity and decreases in phosphorylase activity. This model should prove useful in studying the cardioprotective effects of glycogen.

Adenosine protects against angiotensin II-induced apoptosis in rat cardiocyte cultures.

Adenosine has been found to be cardioprotective during episodes of cardiac ischemia/reperfusion through activation of the A1 and possibly A3 receptors. Therefore, we have investigated whether activation of these receptors can protect also against apoptotic death induced by angiotensin II (Ang II) in neonatal rat cardiomyocyte cultures. Exposure to Ang II (10 nM) resulted in a 3-fold increase in programmed cell death (p < 0.05). Pretreatment with the A1 adenosine receptor agonist 2-chloro-N6-cyclopentyladenosine (CCPA, 1 μM), abolished the effects of Ang II on programmed cardiomyocyte death. Moreover, exposure of cells to the A1 adenosine receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (CPX) before pretreatment with CCPA, prevented the protective effect of the latter. Pretreatment with the A3 adenosine receptor agonist N6-(3-iodobenzyl) adenosine-5′-N-methyluronamide (IB-MECA, 0.1 μM), led to a partial decrease in apoptotic rate induced by Ang II. Exposure of myocytes to Ang II caused an immediate increase in the concentration of intracellular free Ca2+ that lasted 40–60 sec. Pre-treatment of cells with CCPA or IB-MECA did not block Ang II-induced Ca2+ elevation. In conclusion, activation of adenosine A1 receptors can protect the cardiac cells from apoptosis induced by Ang II, while activation of the adenosine A3 receptors confers partial cardioprotection.

Insights into adenosine A1 and A3 receptors function: Cardiotoxicity and cardioprotection

Adenosine (ADO) is a well‐known regulator of a variety of physiological functions in the heart. It exerts protective effects in the heart by activation of the adenosine receptors (AR). In stress conditions like hypoxia or ischemia, the concentration of ADO in the extracellular space rises dramatically. This elevated amount of adenosine might protect the heart from the hypoxic damage. It has been also shown that adenosine can significantly decrease doxorubicin (DOX)‐induced heart toxicity. An highly selective A1 receptor (A1R) agonist CCPA (2‐chloro‐N6‐ cyclopentyladenosine) and A3R agonists IB‐MECA or Cl‐IB‐MECA (2‐chloro‐N6‐(3‐iodobenzyl)adenosine‐5′‐N‐ methyluronamide) have been shown to protect against hypoxia or DOX. Blocking adenosine receptors with selective A1 and A3 receptor antagonists DPCPX (8‐cyclopentyl‐1‐3‐dipropylxanthine) for A1R and MRS1523 [5‐propyl‐2‐ethyl‐4‐propyl‐3‐ (ethylsulfanylcarbonyl)‐6‐phenylpyridine‐5‐ carboxylate] for A3R abolished the protective effects of adenosine. In addition the mean survival time of cardiomyocytes cultures treated with ADO together with DOX was significantly increased. However, at high concentrations A3R agonists IB‐MECA, Cl‐IB‐MECA (≥10 μM), or ADO (200 μM) induced apoptosis. Under these conditions, A1R, A2AR, and A2BR agonists did not have any detectable effect on cardiac cells. The selective antagonist MRS1523 protected the cardiocytes if briefly exposed to Cl‐IB‐MECA and only partially protected from prolonged (48 h) agonist exposure. Apoptosis induced by Cl‐IB‐MECA was not redox‐dependent, since the mitochondrial membrane potential remained constant until the terminal stages of cell death. Drug Dev. Res. 50:324–337, 2000.

Activation of Adenosine A1 and A3 Receptors Protects Mitochondria during Hypoxia in Cardiomyocytes by Distinct Mechanisms

Results of many investigations indicate that activation of adenosine (ADO) A1 and A3 receptors (A1Rs and A3Rs) elicits delayed protection against infarction, ischemia or hypoxia and that both A1R and A3R induce cardioprotection through opening of KATP channels. We suppose that opening of KATP channels may not be the only final mediator of cardioprotection. The protection of the mitochondrial respiratory chain and its impact on mitochondrial bioenergetics after ADO receptor activation may be achieved by different mechanisms. The contribution of mitochondrial and sarcolemmal KATP channels, the rate of mitochondrial ATP synthesis and redox state of mitochondria were compared in normoxic and hypoxic conditions on cultured newborn cardiomyocytes. Activation of both subtypes of ADO receptors induces certain decrease in energy supply and simultaneously promotes preservation of adequate amounts of ATP and maintenance of mitochondrial metabolism on a level sufficient for cell survival. It was found that neither diazoxide nor A1R agonist CCPA nor A3R agonist Cl-IB-MECA modified mitochondrial membrane potential in intact cells. Activation of adenosine A1 receptor slowed down the ΔΨ repolarization. Diazoxide also decreases the rate of energization capacity in living cardiomyocytes upon succinate oxidation. The A3R agonist Cl-IB-MECA did not affect mitochondrial bioenergetics in normoxic cardiomyocytes. It was shown that A3 adenosine receptor stimulation modulates the sarcoplasmic reticulum (SR) Ca2+ channel and may regulate Ca2+ overloading. In conclusion, our data establish that adenosine can mediate myocardial protection by acting on A1 and A3 adenosine receptors. However, the cascades of events involved in cardioprotection against hypoxia appear to be distinct for A1 and A3 receptor signaling.