CYTOPROTECTION AGAINST MEROCYANINE 540-SENSITIZED PHOTOINACTIVATION OF THE Na+,K+-ADENOSINE TRIPHOSPHATASE IN LEUKEMIA CELLS: GLUTATHIONE AND SELENOPEROXIDASE INVOLVEMENT

When irradiated with broad-band visible light in the presence of merocyanine 540 (MC540), murine leukemia L1210 cells grown under selenium-deficient conditions (Se(-) cells) accumulated lipid hydroperoxides and lost viability more rapidly than selenium-satisfied controls (Se(+) cells). These findings suggest that cytoprotection against photoperoxidation and photokilling is mediated at least in part by selenoperoxidase (SePX) action. Similar protection against photoinactivation of an intrinsic membrane enzyme, the Na⁺,K⁺-ATPase, has been observed. Thus, irradiation of MC540-sensitized Se(-) cells resulted in an immediate and progressive inactivation of ouabain-sensitive Na⁺, K⁺-ATPase; by contrast, activity loss in Se(+) cells was preceded by a prominent lag. Enzyme photoinactivation in Se(-) cells was inhibited by ebselen, an SePX mimetic, confirming that SePX(s) is (are) involved in natural protection. Desferrioxamine treatment (iron sequestration/inactivation) resulted in higher hydroperoxide levels and slower Na⁺,K⁺-ATPase inactivation during MC540/light exposure, whereas ferric-8-hydroxyquinoline treatment (iron supplementation) had the opposite effect. Thus, iron appears to play an important role in both of these processes. In contrast, photoinactivation of another intrinsic enzyme in L1210 cells, acetylcholinesterase (AChE), was unaffected by selenium or iron manipulation. On the basis of these findings, we propose that lipid peroxidation plays an important role in the photoinactivation of Na⁺,K⁺-ATPase, but not AChE. This is consistent with the fact that Na⁺, K⁺-ATPase's active site lies within the membrane bilayer, whereas AChE's active site lies outside the bilayer.     

UV-ACTION SPECTRUM (254-405 nm) FOR INHIBITION OF A K+ -STIMULATED ADENOSINE TRIPHOSPHATASE FROM THE PLASMA MEMBRANE OF ROSA DAMASCENA

A K ⁺-stimulated ATPase from suspension-cultured rose cells was isolated and subjected to UV radiation. The characteristics of the ATPase resembled those of a plasma-membrane associated enzyme and not those of the mitochondrial enzyme. The ATPase required Mg²⁺ and was further stimulated up to 100% by K⁺. K⁺ stimulation was specific for ATP. The order of stimulation by monovalent cations was K⁺ > Na⁺ > Li⁺. The enzyme had a pH optimum of 6.5 in the presence of 50 mM K⁺. It was almost completely inhibited by diethylstilbestrol and partially inhibited by vanadate. but was not affected by azide or oligomycin. The inhibition of ATPase activity by various fluences of UV indicated that one fraction of the K⁺-stimulated activity was very sensitive to radiation, while another fraction was relatively insensitive. It is possible that UV distinguished between two enzymes. The action spectra for inhibition of both fractions showed maxima at 290 nm and significant but much lower action throughout the near-UV region, resembling spectra in the literature for the inhibition of transport processes in bacteria.     

Na+/K+ TRANSPORT AND PHOTOSENSITIVITY OF THE COLORLESS FLAGELLATE Peranema trichophorum (Euglenida)

Abstract— Perimycin, ouabain and elevation of extracellular K⁺ concentrations cause an increase in the fluence rate thresholds (white light) for the step-up photophobic response in Peranema trichophorum . Elevation of extracellular Na⁺ concentration decreases the thresholds for this response in comparison to the control level. The fluence rate threshold of perimycin-treated cells increases before the side effect of an antibiotic action appears. Removal of K⁺ ions from the medium of K⁺-treated cells to a concentration of 1 mM depresses the threshold for the step-up response to the control level. By addition of K⁺ or Na ⁺ ions to perimycin- or ouabain-treated cells the threshold returns to the control value. It is suggested that the flagellar and cell membrane are responsible for changes of P. trichophorum photosensitivity.     

PHTHALOCYANINE PHOTOSENSITIZATION OF MAMMALIAN CELLS: BIOCHEMICAL and ULTRASTRUCTURAL EFFECTS

Abstract— The concentrations of Na⁺, K⁺, Ca²⁺, Mg²⁺ and CI⁻ ions in the cytoplasm of octopus photoreceptor cells were determined before and after illumination by electron probe X-ray microanalysis. The concentrations of these elements in the dark-adapted photoreceptor cells were: Na⁺, 68.4; K⁺, 111.4; Ca²⁺, 4.0; Mg²⁺, 16.4; and CI⁻, 102.9 m M /kg of cytoplasm. Illumination raised the concentration of Na⁺ by 58 m M and that of Cl⁻ by 23 m M and reduced the K⁺ concentration by 47 m M /kg of cytoplasm. A trace increase of intracellular Ca²⁺ and a trace decrease of Mg²⁺ were observed. These results confirm the hypothesis that sodium ions flow in on illumination, and suggest the influx of chloride ions and the outflux of potassium ions during illumination. The intracellular concentrations of Na⁺, K⁺ and Cl⁺ can give the basis for calculating the ion permeability of ion channels in octopus photoreceptor cell membranes, using values of the membrane potentials obtained by electrophysiological studies     

EFFECTS OF K+ AND Ca2+ IONS ON MOTILITY AND PHOTOSENSORY RESPONSES OF STENTOR COERULEUS

Extracellular K⁺ ions above a critical concentration induce ciliary reversal in unstimulated Stentor coeruleus and suppress step-up photophobic response. This threshold concentration of K⁺ ions depends on the extracellular Ca²⁺ concentration, and the subsequent backward gyration and light-sensitivity suppression seem to depend on the relative concentrations of K⁺ and Ca²⁺. The concentration of Ca²⁺ necessary to overcome K⁺-mediated inhibition of phobic response and backward swimming increases non-linearly with increasing K⁺ concentration. The Ca²⁺-blocking agent. D-600, selectively inhibits photophobic responses of Stentor , thus further confirming the role of Ca²⁺ ions in photosensory transduction of this ciliate.     

BLUE LIGHT INDUCED, REVERSIBLE IN ACTIVATION OF THE TONOPLAST-TYPE H+-ATPase FROM CORN COLEOPTILES IN THE PRESENCE OF FLAVINS

Abstract— Irradiation (λ_max 447 nm; 58.5 W m^-2) of a microsomal membrane fraction of corn coleoptiles for 5 min in the presence of the in vivo concentration of riboflavin inactivates the tonoplast-type H⁺-ATPase. This inhibition is O₂-dependent, is enhanced in D₂O and suppressed by NaN₃, indicating participation of singlet molecular oxygen in the inactivating mechanism. Besides singlet oxygen, the superoxide anion (O₂^-) is generated during irradiation, which obviously has no effect on the H⁺-pumping activity. However, in the presence of superoxide dismutase (SOD), O₂^- is transformed into H₂O₂ which causes an additional strong inhibition of H⁺. ATPase activity. This inhibition can be increased by ethylenediaminetetraacetic acid (EDTA), which is known to be an electron donor of the excited flavin molecule. In contrast, catalase prevents the H₂O₂-mediated photoinactivation of the H⁺ -ATPase. The light dependent inactivation of H⁺-transport does not occur if reduced glutathion (GSH) is added prior to or after irradiation. These results indicate that the blue light mediated inhibition of the H⁺-ATPase is mediated by singlet oxygen and H₂O₂ which oxidize essential SH-groups of the enzyme into disulfides. Reduction of the formed disulfides by GSH restores the activity of the enzyme.     

HEMATOPORPHYRIN DERIVATIVE (PHOTOFRIN®) PHOTODYNAMIC ACTION ON Ca2+ TRANSPORT IN MONKEY KIDNEY CELLS (CV-1)

Abstract After 24 h incubation with Photofrinm^® (PF), photodynamic action has been studied on Ca²⁺ transport in CV-1 cells. A moderate increase of the cytosolic free Ca²⁺ concentration [Ca²⁺] is observed immediately after a dose of irradiation which yields a survival rate of less than 5% at 48 h. In parallel, studies on digitonin-permeabilized cells indicate that such a treatment inhibits endoplasmic reticulum Ca²⁺ uptake with few alterations of this process in mitochondria. In contrast, ADP-stimulated respiration is impeded and intracellular ATP level decreases. It is suggested that direct damage to endoplasmic reticulum as well as mitochondrial disturbance are the primary mechanisms responsible for a nontransient elevation of [Ca^2+]i preceding cell death.     

AN 125I-LABELED N6-SUBSTITUTED AZIDO ANALOG OF NAD+ FOR THE PHOTOAFFINITY LABELING OF NAD+-LINKED ENZYMES

Abstract ¹²⁵I- N ⁶-(N-[6-N-{5-iodo-4-azidosalicyl}-aminohexyl]-aminocarbamoylmethyl)-nicotinamide adenine dinucleotide (¹²⁵I- N ⁶-I-ASA-AH-NAD⁺) was synthesized by coupling N ⁶ -([6-aminohexyl]-carbamoylmethyl)-NAD⁺ with 4-azidosalicylic acid N-hydroxysuccinimide ester followed by radioiodination. The utility of ¹²⁵I-N ⁶ -I-ASA-AH-NAD⁺ as an effective site-directed photoprobe was demonstrated by the photolabeling of both glutamate dehydro-genase and 15-hydroxyprostaglandin dehydrogenase. Both enzymes can be saturated with labeled probe with apparent dissociation constants comparable to those reported for NAD⁺. Photoincorporation of the probe into both enzymes was found to be protected specifically by NAD⁺. These results indicate that ¹²⁵I- N ⁶-I-ASA-AH-NAD⁺ can be a specific photoprobe for NAD⁺-linked enzymes.     

THE EFFECT OF ULTRAVIOLET RADIATION ON WHEAT ROOT VESICLES ENRICHED IN PLASMA MEMBRANE

Abstract— The irradiation of plant cells with UV radiation (254nm) causes various solutes to leak from the cells. Vesicles enriched in plasma membranes were prepared from wheat roots. These were used to determine whether UV radiation alters membrane function by direct action on the membranes and to distinguish between the chemical effects produced by high and low fluences of UV. The plasma membrane-associated K⁺-stimulated ATPase was very sensitive to UV radiation (100% inhibition with 1.35kJ/m²). ATPase activity measured in the absence of K⁺ and K⁺-stimulated ATPase activity measured in the presence of diethylstilbestrol were much less sensitive. Lipid breakdown, as measured by malondialdehyde production, occurred only at UV fluences greater than 1.8 kJ/m².     

REGENERATION OF NAD+ AND NADP+ COFACTORS BY PHOTOSENSITIZED ELECTRON TRANSFER

Abstract The irradiation with visible light of photosensitizer dyes like methylene blue or N-methyl phenazonium methyl sulfate leads to the oxidation of reduced coenzymes such as pyridine nucleotides (NADH or NADPH). This photoredox reaction can be used to regenerate the oxidized form of these coenzymes in enzymatic reactions and total consumption of a substrate with catalytic amounts of enzyme, coenzyme and photosensitizer can be performed. The process has been studied on two common enzymatic reactions: ethanol oxidation by alcohol-NAD ⁺ -oxidoreductase and gluconate-6-phosphate oxidation by 6-phospho-D-gluconate-NADP⁺-2-oxidoreductase. In the first case, a turnover number of 1125 has been obtained for the photoregeneration of NAD ⁺ from NADH.     

Ca2+ INFLUX INDUCED BY PHOTODYNAMIC ACTION IN HUMAN CEREBRAL GLIOMA (U-87 MG) CELLS: POSSIBLE INVOLVEMENT OF A CALCIUM CHANNEL

Abstract The plasma membrane has been implicated as a critical target of photodynamic action on cells. We have observed that the photosensitization of human cerebral glioma (U-87 MG) cells by hematoporphyrin derivative (HpD) causes a large increase in intracellular calcium [Ca²⁺]. This increase in [Ca²⁺]_i was solely due to the influx of extracellular Ca²⁺ through the plasma membrane and showed a dependence on HpD concentration, light dose and concentration of calcium in the extracellular medium. The magnitude of the Ca²⁺ influx decreased with increasing postirradiation time, which suggests that the cell membrane partially recovers from the photodynamic injury. The photoinduced Ca²⁺ influx was inhibited by the Ca²⁺ channel blocker diltiazem and the reducing agent dithioerythritol. These findings are discussed in terms of possible activation of a Ca²⁺ channel as a result of photosensitization.     

LEAKAGE OF 86Rb+ AFTER ULTRAVIOLET IRRADIATION OF Escherichia coli K-12

Abstract— Stationary phase cultures of a DNA repair proficient Escherichia coli K-12 strain showed a release of intracellular material as assessed by three different methods (260 nm absorption; [methyl-³H]thymidine leakage and ⁸⁶Rb⁺ leakage) after broad-band (Black-Light Blue) near-UV radiation but not after far-UV (254 nm) radiation. As a control response for membrane damage to cells, this leakage of intracellular material was also determined by each method after mild-heat (52°C) treatment of E. coli K-12. An action spectrum for the release of ⁸⁶Rb⁺ from E. coli K-12 after irradiation with monochromatic wavelengths, from 254 to 405 nm, is also presented. The action spectrum for lethality (F₃₇ values) obtained for this strain, shows that leakage of ⁸⁶Rb⁺ occurs at fluences equivalent to or slightly less than fluences causing inactivation at wavelengths above 305 nm. In contrast, at wavelengths below 305 nm, leakage of ⁸⁶Rb⁺ from irradiated cells can be induced but only at fluences significantly greater than was required to cause cell inactivation. These results indicate, therefore, that near-UV radiation can induce a damaging effect on the cell's permeability barrier which may be significant in causing the death of the cell, whereas the effect is not significant in causing the death of cells by far-UV radiation where DNA damage is known to be the main cause of lethality.     

8-Oxodeoxyguanosine Formation in the DNA of Cultured Cells After Exposure to H2O2 Alone or with UVB or UVA Irradiation

Abstract— The purpose of the study was to determine if aluminum phthalocyanine tetrasulfonate (A1PcS₄) photodynamic therapy (PDT) induced the formation of micronuclei in vitro and whether micronuclei formation was dependent on fiuence or cell type. NIH-3T3 and EMT-6 monolayer cultures were incubated in AlPcS₄ (0 or 1 μg/mL) for 24 h, received 0.0, 0.5,1.0 or 1.5 J/cm ² of 675 nm light, then reincubated and harvested at either 24, 48 or 72 h. The micronucleus frequency was determined in binucleated cells using the cytochalasin-block method. Cytotoxicity was assessed by using the 3(4,5-dimenthylthiazoyl-2-yl)2,5(diphenyl-tetrazolium bromide) (MTT) method. The effect of treatment on cell cycle progression was determined by calculating a proliferative index.
Aluminum phthalocyanine tetrasulfonate PDT induced a fluence-dependent increase in the frequency of micro-nuclei in NIH-3T3 and EMT-6 cells. The maximal effect of PDT was obtained in both cell lines 24 h after treatment. NIH-3T3 and EMT-6 cells exposed to a low fiuence of 0.5 J/cm² had a significantly lower number of micro-nuclei per cell 48 h following PDT treatment compared to the number of micronuclei per cell observed 24 h following treatment; however, when cells were exposed to a fluence (1.0 or 1.5 J/cm²) the number of micronuclei per cell did not diminish until 72 h after PDT treatment. The results obtained from the micronucleus assay paralleled those results obtained from the MTT assay.     

Illumination with Ultraviolet or Visible Light Induces Chemical Changes in the Water-soluble Manganese Complex, [Mn4O6(bpea)4]Br4

We measured the photosensitivity of an artificial tetranuclear oxo–Mn(IV) complex, [Mn₄O₆(bpea)₄]Br₄, which has an adamantane-shaped {Mn₄O₆}⁴⁺ core. Illumination caused changes in the absorption spectrum of the compound consistent with a one-electron reduction in the compound. Bromide appears to be the most probable electron donor in the reaction system. Chemical modification of the cluster appears to destabilize it, predisposing it to reductive degradation. UV light was more efficient than visible light in causing the changes. The data support the suggestion that the natural oxygen-evolving Mn complex is photosensitive and can oxidize components of the oxygen-evolving complex in its excited state causing photoinhibition, and that photostability is an important issue in designing Mn complexes for artificial photosynthesis. Furthermore, light-induced oxidation of bromide by [Mn₄O₆(bpea)₄]⁴⁺ may suggest that oxidation of chloride is involved in natural water splitting or has been involved during the evolution of the water-splitting enzyme.     

INTERACTION OF PHOTODYNAMICALLY INDUCED CELL KILLING and DARK CYTOTOXICITY OF RHODAMINE 123

Abstract— Loss of clonogenicity of Chinese hamster ovary (CHO) cells, murine L929 fibroblasts and human bladder carcinoma T24 cells caused by photodynamic treatment (PDT) with hematoporphyrin derivative (HPD) is synergistically enhanced by subsequent incubation with rhodamine 123 in the dark. For CHO and L929 cells this synergistic interaction can be explained by an increased uptake of rhodamine 123 as the result of the photodynamic treatment. With aluminum phthalocyanine (AIPc) as photosensitizer only additive effects were observed in the three cell lines. Incubation in the dark with rhodamine 123, followed by a photodynamic treatment with HPD, resulted in an antagonistic interaction with regard to loss of colony formation. With AIPc the combination of treatments resulted in an additive effect with L929 and T24 cells, whereas with CHO cells a slight antagonistic interaction was observed. An antagonistic effect was also observed in model experiments, treating histidine photodynamically with HPD and measuring oxygen consumption. A possible explanation of these results could be an interaction or complex formation of rhodamine 123 with HPD resulting in a diminished singlet oxygen production. With AIPc this does not take place.     

REEVALUATION OF THE SPECTRAL AND KINETIC PROPERTIES OF HO2 AND O2- FREE RADICALS

Abstract— The spectra and molar absorbances of the HO₂ and O₂^- free radicals have been redetermined in aqueous formate solutions by pulse and stopped-flow radiolysis as well as by ⁶⁰Co gamma-ray studies. The extinction coefficients at the corresponding maxima and 23°C are ²²⁵= 1400 ± 80 M ^-1 cm^-1 and ²²⁵= 2350 ± 120 M ^-1 cm^-1 respectively. Reevaluation of earlier published rate data in terms of the new extinction coefficients yielded the following rate constants for the spontaneous decay of HO₂ and O₂^-: K _Ho2+HO2= (8.60 ± 0.62) × 10⁵ M ^-1 s^-1; K _Ho2+O2-= (1.02 ± 0.49) × 10⁸ M ^-1 s^-1; K _Ho2+O2- < 0.35 M ^-1 s^-1. For the equilibrium HO₂→ O₂^-+ H⁺ the dissociation constant is K _Ho2= (2.05 ± 0.39) × 10^-5 M or p K _HO2= 4.69 ± 0.08. G (O₂^-) has been evaluated as a function of formate concentration.     

CALCULATED RATES AND EQUILIBRIA OF THERMAL INTERCONVERSION OF TRIPLET (3g-) AND SINGLET (1Δg and g+) MOLECULAR OXYGEN

Abstract— From spectroscopic data and rate constants in the literature, equilibrium constants and rates of thermal formation of singlet oxygen (¹Δ_g and ¹Σ_g⁺) were calculated for a number of conditions. For the gas phase we estimate K _eq(¹Δ_g³Σ_g^-) = 1.67 exp(-94.31 KJ/RT) and K _eq(¹Σ_g⁺/³Σ_g^-) = 0.33 exp(-157.0 KJ/RT). The calculated rate constants for the ³Σ_g⁺→¹Δ_g transition of O₂ at 25°C varied from 2.5 × 10^-11 s^-1 in water to 4.8 × 10^-16 s^-1 in air, assuming equal solvent interactions with the ground and excited states. Physical quenchers for singlet oxygen are expected to be catalysts for its thermal formation. Equations are presented which allow one to estimate whether such catalysis by quenchers will result in a pro-oxidant effect.     

CHLOROPHYLL PHOTOSENSITIZED ELECTRON TRANSFER IN PHOSPHOLIPID BILAYER VESICLE SYSTEMS: SECONDARY OXIDATION OF REDUCED VIOLOGEN ACCEPTORS BY Ru(NH3)63+

Abstract— The kinetics of the oxidation of a homologous series of 4,4'-di(n-alkyl)-bipyridinium (viologen) radicals by Ru(NH₃)₆³⁺ in vesicle suspensions was studied using laser flash photolysis. The viologen radicals were produced photochemically in the bilayer membrane phase of the vesicles by electron transfer from the triplet state of chlorophyll-α. At high concentrations of Ru(NH₃)₆³⁺, the rate of oxidation of the viologen radicals in the aqueous phase was limited by the rate at which the radicals diffused from the membrane to the aqueous phase. The exit rate constant decreased from 2 × 10⁵ s⁻¹ for the methyl viologen radical to 4 × 10³ s⁻¹ for the pentyl viologen radical. Both the exit rate constants and the calculated values for the equilibrium association constants of the viologen radicals were unexpectedly insensitive to the length of their alkyl substituents. This, as well as other data, suggests that the radicals that diffused into the aqueous phase tended to remain associated with the membrane-water interface.     

SALT AND pH-DEPENDENT CHANGES OF THE PURPLE MEMBRANE ABSORPTION SPECTRUM

Abstract —Purple membrane suspensions change their color to blue and the absorption maximum shifts to 608 nm when the membrane is deionized on a cation exchange column or when it is washed first with < 2N NaCl followed by deionized water. The deionized chromophore is essentially identical with the chromophore produced by lowering the pH of the native membrane to < 4.0 (p K < 3.0). However, the deionized membrane does not aggregate and can be obtained in the pure state. The original purple color of the membrane is restored by addition of around 1 m M Na⁺, K⁺ or 10 μ M Mg²⁺, Ca²⁺, Sr²⁺, Mn²⁺, Pb²⁺ or La²⁺ when the protein concentration is 5μ M . The required salt concentrations decrease with decreasing pH. Direct measurement of bound Ca²⁺ by atomic absorption spectroscopy yields a ratio of Ca²⁺ to protein of <2 and a binding constant of 1.4 × 10⁶. Titration of the spectral change with salts at different pH values shows a linear relation between the pH and the logarithm of the salt concentration, with a 1:1 ratio for Na⁺ and 1:2 ratio for Ca²⁺. These relations are well predicted by Gouy-Chapman theory; however, the accompanying release of protons, changes of the CD spectrum, the complex kinetics of the spectral change during reconstitution with salt and preliminary X-ray diffraction results all suggest that conformational changes may be occurring in the protein.     

EFFECTS OF EXTRACELLULAR Ca2+ CONCENTRATION AND PAPAVERINE ON THE VISUAL CELL FUNCTION IN THE ISOLATED FROG RETINA

Abstract— Effects of extracellular Ca²⁺ concentration and papaverine on the PIII response of the electroretinogram and the dark adaptation process of the visual cells were studied in the isolated, aspartate-treated bullfrog retina. The amplitudes of both the fast and slow PIII responses are increased in 0.01 m M Ca²⁺ solution, but decreased in Ca²⁺-free solution containing 1 m M EDTA. The application of 0.1 m M papaverine in the presence of 1 m M Ca²⁺ led to the enhancement of the slow PIII response at lower stimulus intensity and the prolongation of the slow PIII response, but these effects of papaverine on the response were lost when Ca²⁺ was removed from the bathing fluid. The half-time of recovery of the fast PIII response amplitude after switching off the adapting light was a linear function of the amount of bleached rhodopsin. Papaverine in the absence of Ca²⁺ produced about 2-fold increase in the half-time of recovery of the response. These findings suggest that chemical reactions which are sensitive to papaverine in the absence of Ca²⁺ are implicated in the dark adaptation process of the visual cells.