Intracellular calcium transients evoked by pulsed infrared radiation in neonatal cardiomyocytes.

Neonatal rat ventricular cardiomyocytes were used to investigate mechanisms underlying transient changes in intracellular free Ca2+ concentration ([Ca2+]i) evoked by pulsed infrared radiation (IR, 1862 nm). Fluorescence confocal microscopy revealed IR-evoked [Ca2+]i events with each IR pulse (3-4 ms pulse⁻¹, 9.1-11.6 J cm⁻² pulse⁻¹). IR-evoked [Ca2+]i events were distinct from the relatively large spontaneous [Ca2+]i transients, with IR-evoked events exhibiting smaller amplitudes (0.88 ΔF/F0 vs. 1.99 ΔF/F0) and shorter time constants (τ =0.64 s vs. 1.19 s, respectively). Both IR-evoked [Ca2+]i events and spontaneous [Ca2+]i transients could be entrained by the IR pulse (0.2-1 pulse s⁻¹), provided the IR dose was sufficient and the radiation was applied directly to the cell. Examination of IR-evoked events during peak spontaneous [Ca2+]i periods revealed a rapid drop in [Ca2+]i, often restoring the baseline [Ca2+]i concentration, followed by a transient increase in [Ca2+]i.Cardiomyocytes were challenged with pharmacological agents to examine potential contributors to the IR-evoked [Ca2+]i events. Three compounds proved to be the most potent, reversible inhibitors: (1) CGP-37157 (20 µM, n =12), an inhibitor of the mitochondrial Na+/Ca2+ exchanger (mNCX), (2) Ruthenium Red (40 µM, n =13), an inhibitor of the mitochondrial Ca2+ uniporter (mCU), and (3) 2-aminoethoxydiphenylborane (10 µM, n =6), an IP3 channel antagonist. Ryanodine blocked the spontaneous [Ca2+]i transients but did not alter the IR-evoked events in the same cells. This pharmacological array implicates mitochondria as the major intracellular store of Ca2+ involved in IR-evoked responses reported here. Results support the hypothesis that 1862 nm pulsed IR modulates mitochondrial Ca2+ transport primarily through actions on mCU and mNCX.

Ratiometric calcium concentration estimation using LED excitation during mechanotransduction in single sensory neurons.

In a previous study using Oregon Green BAPTA-1 fluorescence we found that intracellular calcium concentration in spider mechanoreceptor neurons rose during mechanical stimulation. We also showed that calcium elevation required the opening of voltage-dependent calcium channels by action potentials, and could not be produced by the receptor potential alone. While evidence for mechanisms of calcium elevation in these neurons was clear, our estimates of actual calcium concentration depended on properties of the fluorescent dye in the neuron cytoplasm that could not be verified. We have now developed a method for ratiometric estimation of calcium concentration in these neurons using Fura Red dye, excitation by two light emitting diodes (LEDs) of different wavelengths, and an avalanche photodiode fluorescence detector. The method is simple and economical to implement, allows concentration changes to be measured in the millisecond time range, and could easily be applied to a wide range of preparations. Resting calcium concentration in these neurons was about 70nM and rose to a maximum of about 400nM at firing rates above 20 action potentials per second.

Testing the role of calmodulin in the excitation of Limulus photoreceptors.

The phototransduction cascade in Limulus ventral photoreceptors involves multiple second messengers, including Ca(2+) and cGMP. Light-induced Ca(2+) release from intracellular stores is an intermediate step, but the subsequent Ca(2+)-activated reaction remains to be determined. The possibility that Ca(2+)/calmodulin (Ca(2+)/CaM) might be involved is suggested by the high calmodulin content of the transducing lobe. To test whether CaM can excite the transduction cascade we injected a 25 microM Ca(2+)/CaM solution. This produced a rapid, brief depolarization similar to that produced by light, suggesting a role for CaM in the cascade. However, an important caveat is that Ca(2+) dissociating from the Ca(2+)/CaM complex might excite this process. Several control experiments argue against, but do not entirely eliminate this possibility. To test whether endogenous CaM has a function in excitation, trifluoperazine was pressure injected into the rhabdomeric region. The response to brief flashes was not affected, but the response to steady illumination was transiently attenuated by each injection. We conclude that calmodulin should be considered a candidate to couple intermediate and late stages of the transduction cascade.

EPR-Spin Trapping and Flow Cytometric Studies of Free Radicals Generated Using Cold Atmospheric Argon Plasma and X-Ray Irradiation in Aqueous Solutions and Intracellular Milieu.

Electron paramagnetic resonance (EPR)-spin trapping and flow cytometry were used to identify free radicals generated using argon-cold atmospheric plasma (Ar-CAP) in aqueous solutions and intracellularly in comparison with those generated by X-irradiation. Ar-CAP was generated using a high-voltage power supply unit with low-frequency excitation. The characteristics of Ar-CAP were estimated by vacuum UV absorption and emission spectra measurements. Hydroxyl (·OH) radicals and hydrogen (H) atoms in aqueous solutions were identified with the spin traps 5,5-dimethyl-1-pyrroline N-oxide (DMPO), 3,3,5,5-tetramethyl-1-pyrroline-N-oxide (M4PO), and phenyl N-t-butylnitrone (PBN). The occurrence of Ar-CAP-induced pyrolysis was evaluated using the spin trap 3,5-dibromo-4-nitrosobenzene sulfonate (DBNBS) in aqueous solutions of DNA constituents, sodium acetate, and L-alanine. Human lymphoma U937 cells were used to study intracellular oxidative stress using five fluorescent probes with different affinities to a number of reactive species. The analysis and quantification of EPR spectra revealed the formation of enormous amounts of ·OH radicals using Ar-CAP compared with that by X-irradiation. Very small amounts of H atoms were detected whereas nitric oxide was not found. The formation of ·OH radicals depended on the type of rare gas used and the yield correlated inversely with ionization energy in the order of krypton > argon = neon > helium. No pyrolysis radicals were detected in aqueous solutions exposed to Ar-CAP. Intracellularly, ·OH, H2O2, which is the recombination product of ·OH, and OCl- were the most likely formed reactive oxygen species after exposure to Ar-CAP. Intracellularly, there was no practical evidence for the formation of NO whereas very small amounts of superoxides were formed. Despite the superiority of Ar-CAP in forming ·OH radicals, the exposure to X-rays proved more lethal. The mechanism of free radical formation in aqueous solutions and an intracellular milieu is discussed.

UVB activates ERK1/2 and p38 signaling pathways via reactive oxygen species in cultured keratinocytes.

We have previously shown that hydrogen peroxide is an important mediator of ultraviolet B induced phosphorylation of the epidermal growth factor receptor in human keratinocytes. Here we demonstrate that physiologic doses of ultraviolet B and hydrogen peroxide stimulate activation of two related but distinct mitogen-activated protein kinase pathways: extracellular regulated kinase 1 and 2 (ERK1/2), as well as p38, the mammalian homolog of HOG1 in yeast which is a major kinase for a recently identified stress-induced signaling pathway. The time-dependent activation of ERK1/2 and p38 are distinct, and ultraviolet B-induced ERK1/2 activation is downregulated more rapidly than p38. Using dihydrorhodamine or Amplex as specific fluorescent dye probes, we show that ultraviolet B-induced peroxides can be inhibited by ascorbic acid. Ascorbic acid strongly blocks ERK1/2 and p38 activation by ultraviolet B and hydrogen peroxide whereas pyrrolidine dithiocarbamate and butyl hydroxyanisole are less effective. Pyrrolidine dithiocarbamate was unable to inhibit ultraviolet B-induced p38 activation. Cell death was increased after ultraviolet B when ERK1/2 activation was attenuated by the specific inhibitor PD098059. The distinct time courses and extents of activation and inhibition of ERK1/2 and p38 indicate that these pathways are separate and regulated independently in keratinocytes. Specific types of reactive oxygen species induced by ultraviolet B as well as selective activation or inhibition of specific phosphatases may mediate these responses in keratinocytes. These findings demonstrate that reactive oxygen species are important multifunctional mediators of ultraviolet B-induced ERK1/2 and p38 signaling transduction pathways and suggest that ERK1/2 may play an important part in protecting keratinocytes from cell death following oxidative stress.

Photoactivation and calcium sensitivity of the fluorescent NO indicator 4,5-diaminofluorescein (DAF-2): implications for cellular NO imaging.

The fluorescent indicator of nitric oxide (NO), 4,5-diaminofluorescein (DAF-2), and its membrane-permeable derivative (DAF-2 diacetate) have been recently developed to perform real-time biological imaging of NO. In this study, we show that DAF-2 is strongly influenced by factors other than the concentration of NO itself. Using measurements with a fluorimeter as well as fluorescence microscopy, we found that the divalent cation concentration in the medium, as well as the incident light, strongly affects the ability of DAF-2 to detect NO. Calcium, in particular, enhanced the signal detection of NO released by NO donors by up to 200 times. With multiple and longer exposures to light, no bleaching of the dye was observed but, instead, a potentiation of the fluorescence response could be measured. While these two properties will affect the use and interpretation of the hitherto acquired data with this fluorescent compound, they may also open up new possibilities for its application.

The response of the KHT sarcoma to radiotherapy as measured by water proton NMR relaxation times: relationships with tumor volume and water content.

The potential application of magnetic resonance imaging (MRI) to predict tumor response to radiotherapy is investigated. The water proton spin-lattice and spin-spin relaxation times (T2 and T2, respectively) of murine sarcomas (designated KHT) were measured shortly after excision. This study has demonstrated significantly different responses in T1 and T2 between the control and the irradiated tumors at various times following single doses of X rays. Quite generally, the changes in relaxation times correlated with the changes in tumor water content, indicating that the MR relaxation-time probes are fairly sensitive to radiation-induced edema and dehydration. The possible relationships between the T1 and T2 responses and radiobiological effects such as those on tumor blood flow, vascular permeability, physiological state of cells, and cell death are discussed. It is conceivable that the findings obtained from this investigation could be extended to in situ studies for potential applications in clinical radiotherapy.

Evaluation of in vitro effects of 50 and 60 Hz magnetic fields in regional EMF exposure facilities.

A weak association between magnetic-field exposure and increased incidences of cancer has been reported. While alterations in cellular processes after in vitro magnetic-field exposures have also been reported to provide plausibility for this association, other laboratories have been unable to repeat the findings. As part of an accelerated electric- and magnetic-field (EMF) research program, the National Institute of Environmental Health Sciences with the Department of Energy identified the replication of the published positive effects as a priority. Regional EMF exposure facilities were established to investigate major in vitro effects from the literature. These included effects on gene expression, intracellular calcium, colony growth in soft agar, and ornithine decarboxylase activity. The laboratories that first reported these effects provided experimental protocols, cell lines, and other relevant experiment details. Regional facility studies included sham/sham exposures (no applied field in either chamber) and were done in a blinded fashion to minimize investigator bias. In nearly all experiments, no effects of magnetic-field exposure were found. The effort provided insight into dealing with the difficulty of replication of subtle effects in complex biological systems. Experimental techniques provided some clues for the differences in experimental results between the regional facility and the original investigator. Studies of subtle effects require extraordinary efforts to confirm that the effect can be attributed to the applied exposure.

Hypocrellin A photosensitization involves an intracellular pH decrease in 3T3 cells.

The fluorescent pH probe carboxy-seminaphtorhodafluor-1 (C-Snarf-1) has been used for laser microspectro-fluorometric assays of intracellular pH in 3T3 mouse fibroblasts treated with hypocrellin A. These results are compared to those previously obtained with the structurally related hydroxylated polycyclic quinone, hypericin (Sureau et al., J. Am. Chem. Soc. 118, 9484-9487, 1996). A mean local intracellular pH drop of 0.6 units has been observed in the presence of 1 microM hypocrellin A after 90 s of exposure to 0.1 microW of laser irradiation at 514.5 nm. The time evolution of the cytoplasm acidification for hypocrellin A-treated cells is faster than that for cells treated by hypericin. Thus, release of protons from an excited state of hypocrellin A appears to be more efficient than that from hypericin. In addition, the pH dependence of the quenching of C-Snarf-1 fluorescence in 3T3 cells under continuous irradiation has been observed. It is shown here that under continuous illumination, a pH decrease is able to induce a modification of the intracellular binding equilibrium of C-Snarf-1 that results in an increase of C-Snarf-1 fluorescence intensity. This latter observation suggests that the protons generated upon the photoexcitation of hypericin or its analogs may be involved in the production of other photoreactive species. Finally, we suggest that, just as for hypericin, this pH drop may be involved in the antiviral and antitumor activity of hypocrellin A.

Disturbance of cellular calcium homeostasis by in vitro application of shock waves.

Extracorporeally generated shock waves used in lithotripsy of urinary and biliary stones exhibit tissue lesions in vivo and destroy or damage cells in vitro. The involvement of cavitation-generated free radicals in these harmful effects is discussed controversially. We investigated changes in cytoplasmic calcium concentration and intracellular calcium localization after shock-wave treatment of suspended cell cultures using flow cytometry and electron microscopy and present evidence for the disturbance of mitochondrial Ca2+ a sequestration and, therefore, for a chemically induced cell injury.

Modulation of the phototoxic effect of hypericin in human leukemia CEM cell line by N-ethylmaleimide, amiloride and omeprazole.

Hypericin is a photosensitizing plant pigment from Hypericum perforatum with multiple modes of light-induced biological activities due to production of singlet oxygen and/or excited-state proton transfer with consequent pH drop in the hypericin environment. In the present work, we studied the effects of three inhibitors of crucial mechanisms responsible for intracellular pH (pHi) regulation on hypericin phototoxicity: N-ethylmaleimide (NEM), an inhibitor of H+-ATPase, 5'-(N,N-dimethyl)-amiloride (DMA), an inhibitor of Na+/H+ exchanger, and omeprazole (OME), an inhibitor of H+K+-ATPase. Our experiments show that the effect of hypericin at 1 x 10(-5) and 1 x 10(-6) mol x l(-1) was significantly potentiated by NEM (1 x 10(-7)-1 x 10(--9) mol x l(-1)) and DMA (1 x 10(-6) and 1 x 10(-7) mol x l(-1)) in leukemic CEM cell line. On the other hand, OME had no significant effect on hypericin cytotoxicity. Our results support the hypothesis that the excited-state proton transfer and the consequent acidification of hypericin environment could play a role in the biological activity of hypericin.

[Effect of mild-frequency ultraviolet radiation on mice splenic lymphocytes]. / Deistvie srednevolnovogo ul'trafioletovogo izlucheniia na limfotsity selezenki myshei.

Investigation of the action of mid-frequency ultraviolet radiation (302 nm) on isolated lymphocytes of mice spleen pointed out that intracellular pH of lymphocytes starts decreasing from 0.3 J/cm2. Dose rise above 2.5 J/cm2 increases the number of cells with damaged plasmatic membrane within the lymphocyte population and aggravates the ability of cells to accumulate fluorescein fluorochrome as a product of intracellular fluorescein diacetate hydrolysis.

Increase of intracellular glutathione by low-dose gamma-ray irradiation is mediated by transcription factor AP-1 in RAW 264.7 cells.

The mechanism of the elevation of intracellular glutathione induced by low-dose gamma-rays was examined in RAW 264.7 cells. The expression of mRNA for gamma-glutamylcysteine synthetase (gamma-GCS) increased soon after gamma-ray (0.5 Gy) irradiation, and peaked between 3 h and 6 h post-irradiation. A dose of 0.25 to 0.5 Gy was optimum for induction of gamma-GCS mRNA expression at 3 h post-irradiation. The effect of inhibitors of activator protein-1 (AP-1) and nuclear factor kappaB (NF-kappaB) on the radiation-induced gamma-GCS gene expression was then examined. The induction of gamma-GCS mRNA expression was significantly suppressed when AP-1 DNA binding, but not NF-kappaB DNA binding, was inhibited. Finally, electrophoretic mobility shift assay showed that the low-dose radiation markedly increased the DNA binding of AP-1, but not NF-kappaB, soon after irradiation. These results suggest that the increase of glutathione levels in RAW 264.7 cells by low-dose gamma-ray irradiation is mediated by transcriptional regulation of the gamma-GCS gene, predominantly through the AP-1 binding site in its promoter.

Differential responses of UV-B irradiation on the viability and intracellular calcium influx in goat hepatocytes-in vitro effect.

Ultraviolet-B (UV-B) irradiation in the range of 280-320nm has shown to be a promising immunomodulatory tool in xenogenic hepatocyte transplantation. Most of the studies documenting the effect(s) of UV-B irradiation on hepatic transplantation have been carried out in small model systems with very little information available in larger animals. The aim of the present investigation was to study in vitro the effect(s) of UV-B irradiation (302 nm) at 0, 250, 500, 1250 and 2500 J/m2 on the viability and cellular responses in the isolated goat hepatocytes. The results showed that the cells irradiated at 0, 250, 500, 1250 and 2500 J/m2 demonstrated a viability of 90-95%. However, intracellular [Ca2+]i influx as quantitated by Flu 3-acetete showed a significant increase with irradiation as observed in confocal microscope. The intracellular pH (quantitated by the flourescence of BCCEF) although tend to show an increase with UV-B irradiation was not statistically significant. The present observations suggest that there is a modulation in the intracellular [Ca2+]i concentration within the hepatocytes at higher dose of UV-B irradiation without altering the viability of hepatocytes. These observations are significant for the xenotransplantation of cells.

Alterations of intracellular calcium concentration in mice neuroblastoma cells by electrical field and UVA.

By using a FURA2 ratio imaging method, the intracellular free calcium concentration was investigated in cultured mice neuroblastoma cells under the influence of an amplitude-modulated (AM) field (5 kHz sine wave AM 16 Hz sinusoidal 800 V/m and 80 V/m), as well as of electric field pulses (300-ms unipolar pulses of 1000 V/m and 800 V/m, 5 pulses during 10 s and 50 pulses during 100 s). An increase in free intracellular calcium was found in about 50% of cells after field application, whereas in control experiments only about 20% of the cells showed similar increases. However, this effect depended on the amount of UV irradiation used for excitation of FURA2 fluorescence. Experiments with 1/30 to former total illumination no longer demonstrated an increase in control cells or in cells treated with AM fields. The number of cells showing calcium increase after the application of pulsed fields was reduced significantly. Therefore, the UV light itself, applied as double flashes for the fluorescence measurement, activates the cellular calcium regulation. These findings offer a possible explanation for the low reproducibility of field effects found in different laboratories, in which investigations were performed with different equipment using different intensities of UV excitation.

Theoretical evaluation of dielectric absorption of microwave energy at the scale of nucleic acids.

A theoretical model is proposed for the evaluation of dielectric properties of the cell nucleus between 0.3 and 3 GHz, as a function of its nucleic acids (NA) concentration (CNA). It is based on literature data on dielectric properties of DNA solutions and nucleoplasm. In skeletal muscle cells, the specific absorption rate (SAR) ratio between nucleoplasm and cytoplasm is found to be larger than one for CNA above 30 mg/ml. A nearly linear relationship is found between CNA and this nucleocytoplasmic SAR ratio. Considering the nanoscale of the layer of condensed counterions and bound water molecules at the NA-solution interface, the power absorption per unit volume is evaluated at this precise location. It is found to be between one and two orders of magnitude above that in muscle tissue as a whole. Under realistic microwave (MW) exposure conditions, however, these SAR inhomogeneities do not generate any significant thermal gradient at the scale considered here. Nevertheless, the question arises of a possible biological relevance of nonnegligible and preferential heat production at the location of the cell nucleus and of the NA molecules.

Free radicals generated by ionizing radiation signal nuclear translocation of p53.

The p53 tumor suppressor is a transcription factor that regulates several pathways, which function collectively to maintain the integrity of the genome. Nuclear localization is critical for wild-type function. However, the signals that regulate subcellular localization of p53 have not been identified. Here, we examine the effect of ionizing radiation on the subcellular localization of p53 in two cell lines in which p53 is normally sequestered in the cytoplasm and found that ionizing radiation caused a biphasic translocation response. p53 entered the nucleus 1-2 h postirradiation (early response), subsequently emerged from the nucleus, and then again entered the nucleus 12-24 h after the cells had been irradiated (delayed response). These changes in subcellular localization could be completely blocked by the free radical scavenger, WR1065. By comparison, two DNA-damaging agents that do not generate free radicals, mitomycin C and doxorubicin, caused translocation only after 12-24 h of exposure to the drugs, and this effect could not be inhibited by WR1065. Hence, although all three DNA-damaging agents induced relocalization of p53 to the nucleus, only the translocation caused by radiation was sensitive to free radical scavenging. We suggest that the free radicals generated by ionizing radiation can signal p53 translocation to the nucleus.

Gamma-irradiation-induced cell cycle arrest and cell death in a human submandibular gland cell line: effect of E2F1 expression.

This study examined the effect of gamma-irradiation (5 and 10 Gy) on the human submandibular cell line (HSG). Radiation treatment (5 Gy and 10 Gy) induced a dose-dependent decrease in cell proliferation, with a G2/M arrest of the cell cycle, and an increase in cell death (cells with <2n DNA increased from 7% in control cells to 34% and 40% in 5 and 10 Gy irradiated cells, respectively). [Ca2+]i measurements demonstrated that the status of internal Ca2+ stores, and muscarinic receptor-mediated Ca2+ mobilization, in irradiated cells was comparable to that in non-irradiated cells. These data suggest that 1) irradiated HSG cells maintain normal physiology and 2) internal Ca2+ store depletion does not account for the decreased cell proliferation. To manipulate the radiation-induced cell cycle arrest, we examined the effect of the transcription factor E2F1, which has been shown to induce cell cycle progression in HSG cells (Lillibridge and O'Connell, 1997, J. Cell. Physiol., 1 72:343-350). The ability of irradiated HSG cells to express and appropriately route proteins was demonstrated by using adenovirus-mediated expression of beta-galactosidase, alpha1-antitrypsin, and aquaporin-1. Infection of HSG cells with an adenoviral vector encoding E2F1, either 12 h before or immediately following irradiation, but not post-irradiation, induced maintenance of cells in the S phase of the cell cycle, reduced the number of cells arrested at G2/M, and decreased the rate of appearance of cells with <2n DNA. While the mechanism of irradiation-induced cell death has not yet been confirmed, these data suggest that expression of the E2F1 gene product in HSG cells can be a useful strategy to manipulate cell cycle events and reduce the initial loss of cells due to radiation.