Calcium as a modulator of photosensitized killing of H9c2 cardiac cells.

Illumination of H9c2 rat heart cells in the presence of Rose Bengal resulted in dose-dependent cell killing (assessed by trypan blue staining) and modification of ionic currents flowing through the heart cell membrane. Inhibitors of voltage-gated ionic currents were shown to have little effect on cell killing. Ionic current measurements were used to assess the increase in leak conductance of these cells, which has been suggested to be a causal factor in killing of other cell types (1). Inhibitors of voltage-gated ionic currents, including the sodium channel blocker tetrodotoxin (100 microM) and the calcium channel blocker lanthanum (10 microM) were shown to have little effect on cell killing. The potassium channel inhibitor tetraethylammonium (20 mM) inhibited cell killing, but the effect is viewed as being caused by an inhibition of leak current. The time course of block of voltage-activated ionic currents during illumination, in the presence of Rose Bengal, was rapid compared with that for induction of leak current and for cell killing. These observations are consistent with a role for leak current in photosensitized killing of cardiac cells. They are interpreted with respect to calcium influx through the leak current pathway as a trigger for the cellular response.

Photosensitization-induced calcium overload in cardiac cells: direct link to membrane permeabilization and calcium influx.

The purpose of the present study was to gain new insight regarding the role membrane permeabilization plays in the photosensitization-induced increase in intracellular calcium concentration. During continuous rose bengal photosensitization we monitored the contractile state (relaxed or hypercontracted) of isolated frog cardiac cells and assessed the photosensitization-induced membrane-leak conductance. We investigated the effects of irradiance, extracellular calcium concentration, intracellular chelation of calcium and substitution of tetraethylammonium (TEA) for extracellular sodium. We found that with 2 and 5 mM extracellular calcium cell hypercontracture occurred when leak conductance reached values on the order of 6-7 nS, independent of the illumination duration required to reach this conductance. With 0.5 mM calcium hypercontracture occurred when leak conductance reached values on the order of 11 nS. Chelation of intracellular calcium delayed the onset of cell hypercontracture and increased by two- to three-fold the leak conductance at the initiation of cell hypercontracture. Substitution of TEA for extracellular sodium did not affect the time to contracture onset but reduced leak conductance at contracture onset nearly three-fold. We discuss how our results support the conclusion that photosensitization induces an increase in intracellular calcium concentration via calcium influx through the transmembrane-permeability pathway opened by the photosensitization process.

GH3 cells, ionic currents and cell killing: photomodification sensitized by Rose Bengal.

Photosensitization using Rose Bengal (RB) modifies membrane ionic currents and kills cultured mouse pituitary, GH3, cells. Here we investigate the dose-response relationship for ionic current modification and for cell killing to assess a possible causal link. When exposed to 0.5 microM RB and 6.5 mW/cm2 of visible light, calcium current was blocked in 1.9 +/- 0.2 min (mean +/- SEM; 0.74 +/- 0.08 J/cm2; n = 18), a transient component of potassium current, tentatively identified as a delayed-rectifier potassium current, disappeared in 52 +/- 8 s (0.34 +/- 0.05 J/cm2; n = 10) and a steady-state component of potassium current, largely a calcium-activated potassium current, disappeared in 3.5 +/- 0.4 min (1.37 +/- 0.16 J/cm2; n = 11). Conversely, the background leak current increased in magnitude. At 5 min of illumination, the longest time studied here, it continued to increase nearly linearly, making it the only current component studied that is still changing after 5 min of light. Under the conditions used, cell killing increased to 100% in the exposure range of 4-10 min of illumination (1.6 J/cm2 to 3.9 J/cm2) when assessed using fluorescent markers, ethidium homodimer and calcein and required slightly longer exposure times when assessed using trypan blue. Thus, it is difficult to ascribe a causal role in cell killing by photosensitization to alterations of standard ion channels and known ionic currents. However, the increase in leak current has the correct dose-response characteristics to be involved.

Photomodification of cardiac membrane: chaotic currents and high conductance states in isolated patches.

We have demonstrated previously that photomodification permeabilizes cardiac cells as evidenced by activation of a whole-cell leak current. In this paper we report that photomodification induces in cell-attached and inside-out cardiac membrane patches a chaotic current. Unlike current recordings from many protein ion channels that show stepwise amplitude changes associated with open and closed states of the channel, the chaotic current consists of variable amplitude spike-like transitions. The amplitudes of these spikes can vary from tenths to tens of picoamperes at a constant transmembrane potential. We provide evidence that the chaotic current is transmembrane rather than trans-seal and has a voltage dependency expected for current flow through nonspecific conductance pathways. Photomodification can also induce high conductance states (greater than 500 pS) in cell-attached and inside-out patches. We present evidence that the high conductance state is also not related to seal breakdown. Our results suggest that both the chaotic current activity in and high conductance state of photomodified cardiac membrane patches result from the opening of many small conductance, nonspecific pathways through the membrane.

In vivo spectroscopic properties of the fluorescent pH indicator biscarboxyethyl carboxyfluorescein.

Transcutaneous detection of fluorescence from an injection of the pH-sensitive probe biscarboxyethyl carboxyfluorescein (BCECF) has been used to monitor plasma pH in conscious animals. The fluorescence signal must be calibrated with reference to a standard curve. This standard, calibration curve has been achieved using in vitro methods. Here it is shown that temperature influences the calibration curve determination in vitro and hence influences the pH determined from transcutaneous measurements. Two calibration curves have been obtained, one at room temperature (approximately 25 degrees C) and the other at 37 degrees C. At pH 7.01 the calibration curves intersect, so that, at more alkaline pH values, use of room temperature calibration data will lead to an overestimate of plasma pH. Below pH 7.01, plasma pH will be underestimated. Transcutaneous fluorescence spectra recorded from a mouse injected with BCECF are shown, indicating that baseline plasma pH was estimated about 0.3 pH units too high using room temperature calibration, and that the extent of acidification when the animal was allowed to breathe an atmosphere containing 15% CO2 was overestimated by 0.7 pH units. Additionally, it is shown that in vitro bovine serum albumin at concentrations comparable with albumin concentrations in vivo shifts the absorption, fluorescence excitation and emission spectra of BCECF. However, fluorescence spectra recorded in vivo show no such shift. The results indicate that in vitro calibration for transcutaneous fluorescence measurements in vivo can be misleading, and that in the case of pH measurement inattention to temperature can lead to spurious results.

Membrane ionic current photomodification by rose bengal and menadione: role of singlet oxygen.

Photosensitized modification of ionic leak current and potassium current was studied in frog cardiac atrial cells using whole cell patch clamp techniques. Rose bengal (RB) and menadione (MQ) were used as photosensitizers. Separate photophysical studies of the photosensitizers in deuterium oxide solution demonstrated that MQ did not produce singlet oxygen as evidenced by the lack of luminescence at 1270 nm, whereas RB was an efficient singlet oxygen generator. Both photosensitizers sensitized block of potassium current in atrial cells, and both sensitized an increase of ionic leak current. However, when photosensitizer concentrations and illumination intensities were adjusted to match the rate of block of potassium current by the two photosensitizers, there were dramatic differences in leak current increase, both quantitatively and qualitatively. Menadione sensitized a much slower increase in leak current than did RB. Further, the leak current sensitized by MQ had a more positive reversal potential than that sensitized by RB, suggesting a less potassium-selective leak current pathway. The results suggest that, while the effects of singlet oxygen and non-singlet oxygen modification of cell membranes may be similar, there may also be significant differences in the resulting membrane permeabilities. The results also demonstrate that MQ and RB may be useful agents to study the role of singlet oxygen versus non-singlet oxygen modification of biological systems.

Properties of cardiac I(leak) induced by photosensitizer-generated reactive oxygen.

We reported previously that photomodification of single frog cardiac cells by Rose Bengal induces a time-independent current, designated I(leak)++, having a linear current-voltage (I/V) relationship. The purpose of the present study is to better characterize the properties of I(leak)++. Initially, I(leak)++ has a reversal potential (ER) near -70 mV, but with time, ER shifts toward a final value near 0 mV. This shift in ER is accompanied by a marked increase in conductance (slope of I/V relationship). Evidence is presented that the depolarizing shift in ER with time during photomodification results from a loss of membrane selectivity allowing sodium to make an increasing contribution to I(leak)++. Potassium also contributes to I(leak)++, as indicated by marked depolarizing shifts in ER following replacement of intracellular potassium with either cesium or tetraethylammonium. Since these results occur in calcium-free external media, the depolarizing shifts in ER and increased conductance are not related to activation of a calcium-dependent nonselective cation channel. However, I(leak) does have some properties similar to nonselective cation currents recently reported to be activated by membrane breakdown products such as arachidonic acid and lysophosphoglycerides.

Continuous noninvasive measurement of in vivo pH in conscious mice.

Extracellular pH was measured continuously and noninvasively in hairless mice using a transcutaneous spectrofluorometric technique and a fluorescent pH probe, bis-carboxyethyl carboxyfluorescein (BCECF). The acid form of BCECF was injected intravenously. An optical fiber system excited the fluorescent probe at 450 and 500 nm alternately, using one branch of a bifurcated, fiber optic bundle. The other branch of the bundle collected the emitted fluorescence which was measured by a photomultiplier tube. The ratio of fluorescence intensities from the two excitation wavelengths was pH dependent. Measurements could be obtained for approximately 60 min following a single injection. Mice exposed to elevated partial pressures of CO2 demonstrated changes in the fluorescence ratio indicative of a dramatic decrease in extracellular pH. If the exposure was brief, the fluorescence signal recovered within 20 min. The fluorescence intensity ratio was calibrated against aqueous BCECF samples in vitro. Fluorescence pH values determined using the in vitro calibration were compared with measurements made on blood samples taken from seven mice. Extracellular pH in hairless mice was found to be approximately 7.5, within the expected physiological range. The variability of the pH signal derived from the fluorescence signal was approximately 0.05 units.

Membrane potential can influence the rate of membrane photomodification.

Though cellular photomodification has been shown to change cellular resting membrane potential, an effect of membrane potential on the rate of photomodification has never been reported. Here we demonstrate that the rate of photomodification of potassium channels in frog atrial cells is voltage dependent. The rate of potassium channel photomodification using negatively charged Rose Bengal as the photosensitizer is about 2.5 times greater at the resting membrane potential of -70 mV compared to +40 mV. Similar results are obtained using the positively charged photosensitizer methylene blue. On the other hand, the rate of photomodified increase of leak current in the same cells does not significantly change in this voltage range with Rose Bengal as photosensitizer, but demonstrates a voltage dependence like that of potassium current when methylene blue is the photosensitizer. These observations cannot be explained based on voltage-dependent partitioning of the sensitizer, as similar effects on potassium current were obtained using either a positively charged or negatively charged sensitizer.

Modification of cardiac ionic currents by photosensitizer-generated reactive oxygen.

The effects of reactive oxygen species (ROS) generated by light and the photosensitizer Rose Bengal on ionic currents in single frog atrial cells were investigated. The excitatory inward sodium and calcium currents were both suppressed by ROS as was the outward, delayed rectifier potassium current. The inactivation kinetics of the sodium current were slowed markedly whereas the kinetics of calcium current inactivation were much less affected and potassium current activation was not changed. The sodium current-voltage relationship was shifted in the depolarizing direction by ROS whereas the voltage-dependencies of both the calcium and potassium currents were not affected. In addition to suppressing the time- and voltage-dependent sodium, calcium, and potassium currents, ROS enhanced a time-independent current which was outwardly directed at positive membrane potentials. However, the induction of this time-independent current required longer ROS exposure than was required to significantly suppress the other currents. The rapid onset of ROS-induced suppression of calcium and potassium currents followed by a later enhancement of a time-independent current can explain ROS-induced changes in action potential duration. Brief ROS exposure increased action potential duration whereas longer exposure reduced action potential duration.

Membrane photomodification of cardiac myocytes: potassium and leakage currents.

Cardiac myocytes were isolated from the atria of frogs (Rana pipiens) and whole cell potassium (IK) and "leakage" (Ileak) currents were monitored using the patch clamp technique. Cells were photosensitized by exposure to Rose Bengal (0.125-0.5 microM). Illumination produced an exponential decrease in IK, and an increase in Ileak. Current modifications varied with light intensity and sensitizer concentration. IK stabilized when illumination ceased, while Ileak continued to increase at a slower rate after illumination ended. The exponential nature of IK modification suggests that potassium channels are photomodified with single hit kinetics. The stabilization of IK following illumination suggests (1) that the photomodification of the potassium channel does not involve long lasting (minutes) radical chain reactions and (2) that this photomodification is not repaired in the course of a few minutes.

Modification of cardiac action potential by photosensitizer-generated reactive oxygen.

Evidence implicating reactive oxygen species (ROS) in reperfusion-induced arrhythmias is accumulating rapidly [1,2]. However, surprisingly little is known about the effects of ROS on cardiac electrophysiology. Such knowledge would improve our understanding of reperfusion-induced arrhythmias. Photosensitizers and light are known to produce a variety of ROS. They might, therefore, be useful for investigating oxygen-mediated cell injury. To our knowledge, such an approach has not been used to investigate ROS-induced alterations in the electrophysiological properties of cardiac muscle. The purpose of this paper is to demonstrate (1) the feasibility of using photosensitizers for such an investigation, and (2) some advantages photosensitizers offer when combined with single cell and patch pipette methodologies. A comparison of the electrophysiological alterations produced by photosensitizer-generated ROS to the reported effects of xanthine-xanthine oxidase or organic hydroperoxides suggests that the electrophysiological alterations produced by superoxide initiated reactions and/or lipid peroxidation are similar to those produced by photosensitizers and light.

Effect of in vitro ultraviolet radiation on the binding capacity of anti-DNA and DNA in systemic lupus erythematosus.

The in vitro effects of various doses of ultra violet A (UVA) or UVB irradiation on DNA or anti-DNA as measured by their subsequent binding to anti-DNA or DNA were examined. Sera from 12 patients with active lupus were studied. UVA irradiation at 20-240 J/m2 or UVB irradiation at 2-24 J/m2 did not affect DNA or anti-DNA binding to anti-DNA or DNA, respectively. Modulation of the UV effect on DNA and anti-DNA by adding methylprednisolone, hydroxychloroquine and indomethacin at 10(-6) M did not alter the UV effect. UV light does not exert its effect directly on the binding of anti-DNA to DNA.

Photomodification of biological membranes with emphasis on singlet oxygen mechanisms.

This review discusses photomodification of biological membranes and model membrane systems. Current concepts of membrane structure are first reviewed briefly. The role of preillumination association of sensitizer with membranes as it relates to photomodification rate is discussed, as well as the role of singlet oxygen in membrane photomodification. Finally the characteristics of singlet oxygen generation in membranes are considered. The evidence clearly indicates that membrane photomodification cannot be understood based only on the properties of sensitizers and singlet oxygen in aqueous solution. Rather the properties of sensitizers in association with membranes are the determinants of membrane photomodifcation. These properties differ significantly in aqueous solution and in membranes.

Reexamination of the double sucrose gap technique for the study of lobster giant axons. Theory and experiments.

The double sucrose gap technique for the study of lobster giant axons has been reexamined. The leakage behavior of the system cannot be successfully modeled by conventional sucrose gap theory, but is accounted for by the McGuigan-Tsien model that takes into account the cable properties of membrane under sucrose. The facts of high-leakage conductance and the ability to maintain large resting potentials in the face of low sucrose gap resistance lead to a hypothesis that membrane resistance under sucrose is very low because of a large negative surface potential. Computer simulations of the leakage behavior of the conventional gap model and the McGuigan-Tsien model were compared with experimental measurements on lobster axons using normal sucrose or sucrose doped with Na+, Ca2+ or La3+ ions. As the concentration of doping ion increased, the leakage rose, but the species of doping ion had more influence on leakage than gap resistance. At equal gap resistance, leakage decreased with an increase in valence of the doping species. Leakage was even lower in La-doped sucrose at 20 M omega gap resistance than in normal sucrose at 200 M omega gap resistance. Resting potentials decreased with decreasing gap resistance and increasing valence of the doping species. Resting potential behavior was successfully simulated with a hybrid model consisting of a point node flanked by infinite cables and a shunt between ground and the voltage-measuring pool. The data support the hypothesis that the membrane resistance under sucrose is low and that it can be raised by doping the sucrose with multivalent cations, with La3+ being particularly effective. Both the leak conductance and resting potential are influenced more by membrane under sucrose than membrane in the node. The experiments also demonstrate that doping with La3+ vastly improves the stability and longevity properties of the lobster axon preparation.

A method to quantify the potency of photosensitizers that modify cell membranes.

The numerical assessment of sensitizing potency of different species of photosensitizer molecules has historically been impracticable because of the difficulty of measuring all of the factors that must be taken into account. This paper describes a new method for quantifying relative sensitizer potency that obviates most of the past difficulties. The physical data needed to assess photosensitizing potency are the absorption spectrum of a sample of medium containing sensitizer, the illumination spectrum impinging on the preparation, the duration of illumination, and the amount of light-induced modification. The measurement of these quantities is relatively straightforward, but it is easy to overlook a number of artifacts that can distort the values for potency. Avoiding error requires careful attention to the optical properties of the system, particularly in the light path through the medium containing sensitizer.