PHOTOPHYSIOLOGY OF TURION GERMINATION IN Spirodela polyrhiza (L.) SCHLEIDEN–V. DEMONSTRATION OF A CALCIUM-REQUIRING PHASE DURING PHYTOCHROME-MEDIATED GERMINATION

Abstract— Etiolated turions of Spirodela polyrhiza are positively photoblastic and show a phytochrome-mediated low fiuence germination response. The far-red light (FR) reversibility decreased with the delay of FR irradiation (lag phase 1.06 ± 0.03 days after red light irradiation; half-maximal response 1.9 days). The action of the far-red-absorbing form of phytochrome (Pfr) was only realized by a germination response if exogenously applied Ca²+ was present. Calcium step-down (from 1 mM to 0.9 μ M Ca²+) and Ca²+ step-up (from 0.9 μ M to 1 m M Ca²+) experiments were carried out to determine the Ca²+-sensitive phase. There was no time gap between the two phases determined by the step-down and step-up experiments but a clear coincidence of both curves. Pulse treatments (24 h) with Ca²+ (1 m M ) showed the upper part of this common curve to represent the most Ca²+-sensitive phase. The Ca²+-sensitive phase was within the Pfr-requiring phase. After reversion of Pfr by FR pulses there was only a negligible response to the high Ca²+-concentration, independent of the delay between the red light (R) and FR pulses. These results are compatible with the assumption of Ca²+ acting as a second messenger of Pfr. However, the Ca²+-insensitivity in the first 12 h after the R pulse points against this hypothesis.     

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.     

PHOTOREVERSIBLE Ca2+-DEPENDENT AGGREGATION OF PURIFIED PHYTOCHROME FROM ETIOLATED PEA AND RYE SEEDLINGS

The aggregation of phytochrome purified from etiolated pea ( Pisum satirum cv. Alaska) and rye ( Secale cereale cv. Cougar) tissues was investigated by centrifugation and turbidimetry. Purified pea phytochrome (A₆₆₉/A₂₈₀= 0.88), if irradiated with red light, became precipitable in the presence of CaCl₂. The precipitation upon red-light irradiation was optimal at a Ca^2- or Mg²⁺ concentration of 10–20 m M , was greater at increased phytochrome concentration or lower pH values, and was inhibited by 0.1 M KG. The precipitated phytochrome slowly became soluble after far-red light exposure.
Turbidity of pea phytochrome solutions after red-light irradiation also increased rapidly in the presence of either Ca²⁺ or Mg²⁺. Far-red light exposure after the red light cancelled the turbidity increase. Rye phytochrome showed less turbidity increase than pea phytochrome and occurred only in the presence of Ca²⁺. Partially degraded pea phytochrome produced by endogenous proteases in the extract did not show the turbidity increase. Undegraded pea phytochrome also associated with microsomal fractions under conditions similar to those described above, but the partially degraded phytochrome did not.     

PHOTOMORPHOGENESIS IN Penicillium isariaeforme: EXOGENOUS CALCIUM SUBSTITUTES FOR LIGHT

Abstract— Penicillium isariaeforme is a photomorphogenic fungus which produces upright bundles of conidia-bearing mycelia (called coremia) when grown on a defined medium in visible (450–500 nm) light. We found that exogenous Ca²⁺ ions could substitute for light. In the dark 1–2 m M Ca²⁺ triggered coremia formation. Dark induction of coremia was specific for Ca²⁺ in that it could not be duplicated by 50 m M Ba²⁺, Fe²⁺, Mg²⁺, Mn²⁺, Sr²⁺, or Zn²⁺. Additionally, light-induced coremia formation was inhibited by both KI (2.5 m M ) and phenylacetic acid (0.25 m M ).     

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.     

Contribution of the cAMP-Dependent Signal Pathway to Circadian Synchrony of Motility and Resting Membrane Potential in Paramecium

It is known that the ciliated protozoan Paramecium multimicronucleatum has synchronized circadian rhythms of motility, resting membrane potential and cyclic adenosine 3',5'-monophosphate (cAMP) and cyclic guanosine 3',5'-monophosphate (cGMP) concentrations. The present study shows that (1) extracellularly added 4 m M tetraethylammonium (TEA)⁺ (a K⁺ channel blocker) almost completely abolishes the diurnal oscillation of intracellular cAMP concentrations; (2) even 32 m^M TEA⁺ fails to abolish the circadian motility rhythm; but (3) the motility rhythm is highly damped when 4 m^M TEA⁺ and 100 μ^M CdCl₂ (a Ca²⁺ channel blocker) are added simultaneously. A cAMP analogue ( N ⁶-monobutyryl-cAMP) added extracellularly accelerates swimming velocity. Both a K⁺ channel blocker ( e.g . TEA⁺) and an inhibitor (trifluoperazine) of adenylate cyclase (AC) suppress cAMP formation, supporting the hypothesis that AC in Paramecium has dual functions, as a K⁺ channel and as an enzyme for cAMP formation. It is hypothesized that the circadian synchrony is due to circadian fluctuations of AC causing separate circadian changes both in ciliary motion and membrane potential through a cAMP-dependent signal pathway that forms a sophisticated network of second messengers to govern the synchrony together with Ca²⁺- and cGMP-dependent pathways in a manner antiphasic and/or complementary to one another.     

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     

CALCIUM CONTROL OF PHOTOTACTIC ORIENTATION IN Chlamydomonas reinhardtii: SIGN AND STRENGTH OF RESPONSE

Abstract— Chlamydomonas reinhardtii responds to a blue light stimulus by an oriented swimming (phototaxis) toward or away from the stimulus source. In this study it is established that the sign and strength of the phototactic response are a complex function of extracellular [Ca²⁺], stimulus fluence rate, time of analysis after onset of stimulation and light pretreatment. At very low extracellular [Ca²⁺] the response is weak and usually negative. At [Ca²⁺] close to the preconditioning level, phototactic response becomes stronger and positive. As [Ca²⁺] is raised further, the initial (2 s) response remains positive but the long term (20 s) becomes negative and very strong. At extremely high [Ca²⁺] the cells become immobile. This bimodal behavior suggests that two different mechanisms determine the direction of the turn. Data cannot be explained in terms of a simple model. The model which accounts for most of the details of the behavior is that of Kamiya and Witman (1984), which proposes that positive response is triggered by a transient increase in intracellular [Ca²⁺] and negative response by a decrease below unstimulated level of Ca²⁺, at least in the range of 10^-9-10^-6 M [Ca²⁺]. The strong negative orientation which follows an initial positive response above this level of [Ca²⁺], in these experiments, is best explained by an adaptation of the cells due to an increased (on average) intracellular [Ca²⁺].     

A FLASH PHOTOLYSIS STUDY OF 1-METHYLINDOLE

Abstract— The conventional flash photolysis of 1-methylindole in aqueous media was studied at Λ_excitation≥290 nm. The transients observed 20 μs after excitation consisted mainly of the radical cation (R⁺). the hydrated electron (e^-_aq) and the triplet state (T). Electron counting experiments indicate that photoionization is the only source of R⁺ with e^-_aq/R⁺= 1.07±0.09 in neutral media. Quenching of the R⁺ yield with H⁺ indicates that the fluorescent state is the precursor to 80% of the photoionization events with the remainder probably arising from a prefluorescent state. The triplet decays with a lifetime of 29 μs in deaerated neutral media. This decay is unchanged by N₂O saturation, but T reacts with acrylamide with k ≥2.8 × 10⁹ M ^-1. In 2 M Br^-, R⁺ and T yields are increased by factors of 2–3. Consideration of fluorescence quenching and T enhancement by Br-permits an estimate of φ_Isc between 0.33 and 0.49. The increased R⁺yield at high Br-concentrations cannot be accounted for by induced photoionization or triplet state reactions.     

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.     

Effect of Magnesium and Calcium Complexation on the Photochemical Properties of Norfloxacin

Abstract— The fluoroquinolone antibiotics can induce skin photosensitivity in some patients and this has been ascribed to the generation of reactive oxygen species, such as singlet oxygen (O₂[¹Δ_g]). We have studied the photochemical properties of the different ionized forms of the fluoroquinolone norfloxacin upon complexation with Mg²+ and Ca²+ ions, as it is proposed that the antibiotic exists mainly as a complex in the blood plasma. We found that the norfloxacin cation (pH < 6) shows no photodegradation after UVA irradiation and has a low quantum yield of O₂(¹Δ_g) generation. The norfloxacin cation does not complex. Ca²+ or Mg²+ ions; when these ions are added to the solution, we observed no changes in the fluorescence quantum yields (φ_flu) and singlet oxygen yields (φ_Δ). In contrast, the neutral (6 ± pH > 8.5) and anionic (pH > 9) forms of norfloxacin are able to complex calcium and magnesium, and their generation of O₂ (¹Δ_g) is decreased by complexation. The neutral zwitterionic form and the anionic form also quench singlet oxygen by both chemical and physical pathways regardless of complex formation, while physical quenching is observed for the cation. At pH > 7.4, norfloxacin photobleaches and complexation to Ca²+ and Mg²+ increases the rate at which photobleaching occurs. Thus, both the pH of the medium and complexation with metal cations may affect the phototoxic potential of this antibiotic.     

CHLORIDE ENHANCEMENT OF QUENCHING OF TRIPLET METHYLENE BLUE BY Fe(II)

Abstract— The influence of chloride ion on the rate of decay of triplet methylene blue in 0.01 M acid in the absence and presence of ferrous ions was investigated by means of laser flash-photolysis monitored by kinetic spectrophotometry. Chloride weakly accelerates decay of ³MBH^2± in aqueous solution in the absence of Fe(II). Quenching of ³MBH²⁺ by Fe(II) is more strongly catalyzed by Cl^- in both water and 50 v/v% aq. CH₃CN. The uncatalyzed quenching constant, k ₅, is of the order of 1 × 10⁶ M ^-1 s^-1 while in 4.8 M aqueous chloride ( μ – 7.2 M ) k ₅= (37.2 ± 1.8) × 10⁶ M ^-1 s^-1. A possible role of chloride is as a bridging species in quenching via electron transfer between ³MBH²⁺ and Fe(II).     

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.     

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.     

EFFECT OF PARAMAGNETIC SPECIES ON 8-METHOXYPSORALEN-SENSITIZED PHOTOINACTIVATION OF BACTERIOPHAGE LAMBDA

Abstract. 8-Methoxypsoralen-sensitized photoinactivation of bacteriophage lambda (Λ_vir) was examined in the presence of various metallic ions. Paramagnetic ions, Co²⁺, Ni²⁺ or Mn²⁺, strongly repressed the reaction, whereas diamagnetic ions, Ca²⁺ or Zn²⁺, exerted no influence. Significant repression was also observed in the sample solution equilibrated with air.     

EVIDENCE FOR A MAGNESIUM DEPENDENT ATPase IN BOVINE ROD OUTER SEGMENT DISK MEMBRANES*†

Abstract—In the presence of Mg²⁺ and adenosine triphosphate (ATP), a rapid. light-induced, light-scattering transient is observed from bovine rod outer segments (ROS). This light-scattering transient we have labelled 'A'. Ca²⁺ cannot replace Mg²⁺. nor can guanosine triphosphate (GTP) replace ATP. 'A' is observed at ATP concentrations as low as a few μM.
The half-time of 'A', 60 ms at 20° and 20 ms at 37°, is consistent with a process possibly involved in visual transduction.
'A' has the action spectrum of rhodopsin bleaching and its amplitude is strictly proportional to the fraction of rhodopsin bleached per flash. 'A' can be regenerated by 11- cis retinal.
Inhibition studics with ATP analogues, which cannot be hydrolysed and fail to evoke an 'A' response, reveal that an ATP hydrolysis process has to precede illumination in order for 'A' to occur.
On the basis of the above findings. it is proposed that there is a Mg²⁺ dependent ATPase in ROS that allows the disk membrane to assume a new membrane state which, upon illumination, is altered. giving rise to the structural phenomenon monitored as light-scattering transient 'A'.     

THE ABSOLUTE VALUE OF THE REACTION RATE CONSTANT OF BILIRUBIN WITH SINGLET OXYGEN IN D2O

Abstract— The chemical reaction rate constant of bilirubin with singlet oxygen in basic aqueous solution has been redetermined to be 3.5 × 10⁸ M^-1 s^-1 by a competitive technique using a 1,3-diphenylisobenzofuran in sodium dodecyl sulfate micelles. Bilirubin also physically quenches a singlet oxygen with a rate constant of 9 × 10⁸ M ^-1 s^-1. The lifetime of singlet oxygen in D₂O solution has been determined to be 35 μ s . The absorption cross-section for the molecular oxygen ³∑_g-→¹δ _g + 1 v electronic transition at 1.06μn in aqueous solution is unexpectedly larger than the gas paase cross-section.     

ON THE SIGNAL-TRANSDUCTION CHAINS OF TWO Pfr- MEDIATED SHORT-TERM PROCESSES: INCREASE OF ANCHORAGE and MOVEMENT OF Mougeotia CHLOROPLASTS

Abstract— We examined two published hypotheses on the signal-transduction chain of the light-oriented chloroplast movements in the fresh-water alga Mougeotia. One hypothesis postulates a Ca²+-influx controlled by a tetrapolar gradient of phytochrome in its far-red light absorbing form (Pfr). The other hypothesis postulates anchorage sites for actin-filaments even at those areas of the plasmalemma where phytochrome is in its inactive form (Pr). Calmodulin and Ca²+-sequestering vesicles are assumed to be essential links of this transduction chain.
To test these hypotheses we have studied the effects of Ca²+-entry blockers, Ca²+ deficiency and calmodulin antagonists on chloroplast movements and on chloroplast anchorage. None of our results support the Ca²+/calmodulin hypotheses mentioned above. The results and their implications (with regard to the role of Ca²', calmodulin and anchorage sites) are discussed.     

EFFECTS OF IONOPHORES AND RUTHENIUM RED ON THE PHOTOTAXIS OF STENTOR COERULEUS AS MEASURED BY SIMPLE DEVICES

Abstract— Two simple methods of phototaxis measurements have been applied to study the effects of ionophores on the negative phototactic response in Stentor coeruleus. The inhibitory effects of Ca²⁺-ionophore (A23187), Ca²⁺-blocking agent (Ruthenium Red), and K⁺ -ionophore (valinomycin) on photo-taxis have been determined. Results suggest that the influx of Ca²⁺ plays a transducing role in the phototaxis of Stentor.     

APPLICATIONS OF CALCIUM-SENSITIVE PHOTOPROTEINS IN EXPERIMENTAL BIOLOGY

Abstract—Calcium-sensitive photoproteins are "precharged bioluminescent proteins that are triggered to emit light by binding Ca²⁺ or certain other inorganic ions. Neither molecular oxygen nor any organic cofactor is required. The first such protein to be described was aequorin, and for various reasons that has been the one most widely studied. Photoproteins have been used as Ca²⁺-indicators both in vitro and in living cells. Their chief advantages for this are (1) ease of signal detection, (2) high sensitivity, (3) relative specificity for Ca²⁺, and (4) lack of toxicity. Difficulties in the experimental use of the photoproteins stem from (1) their one-time reactivity, (2) their large molecular size, (3) their scarcity, (4) the influence of experimental conditions on the sensitivity to Ca²⁺, (5) the nonlinearity of the relation between [Ca²⁺] and light intensity, and (6) the limited speed with which light intensity follows sudden changes in [Ca²⁺]. Photoproteins have now been used as intracellular calcium indicators in more than two dozen types of cells, and experience with the method is rapidly growing. They are also useful in the determination of calcium binding constants for other substances in vitro , and as models for studies of receptor-ligand interactions in general.