Calcium-binding protein regucalcin stimulates the uptake of Ca2+ by rat liver mitochondria

The effect of the calcium-binding protein regucalcin on the Ca2+ transport system in rat liver mitochondria was investigated. Ca2+ transport was assayed by the method of Millipore filtration to estimate mitochondrial 45Ca2+ accumulation. 45Ca2+ uptake was stimulated by the presence of regucalcin (1.0 and 2.0 microM). This stimulation was remarkable during 1.0 min after 45Ca2+ addition, while appreciable stimulation was no longer seen at 3 min. Regucalcin (2.0 microM)-induced stimulation of 45Ca2+ uptake was prevented by the presence of ruthenium red (1.0 microM) and lanthanum chloride (0.1 mM). Regucalcin (2.0 microM) did not increase the mitochondrial adenosine triphosphatase (ATPase) activity during 3.0 min after Ca2+ addition. Meanwhile, 45Ca2+, which accumulated in the mitochondria during 5.0 min after 45Ca2+ addition, was not released by the addition of regucalcin. Regucalcin may stimulate Ca2+ uptake in rat liver mitochondria independently of the energy.     

Quantitative study on La~(3+) influx mediated by sodium-calcium exchanger in human lymphocytes

Whether La3+ can enter human peripheral blood lymphocytes by the Na+/Ca2+ exchanger or not and the effect of La3+on the Na+/Ca2+ exchanger activity are examined by fura-2 technique. And that whether La3+ is sequestered by intracellular organelles (mainly endoplasmic reticulum and mitochondria) is studied by this method. La3+uptake is obviously stimulated by pre-treating the cells with ouabain and by removing extracellular Na+, and intracellular La3+concentration ([La3+]i) is directly proportional to its extracellular concentration ([La3+]o). But when [La3+]o exceeds 0.4 mmol/L, the 340/380 nm ratio of fluorescence is no longer varied and the maximum [La3+], is 1.5×10-12 mol · L-1. The higher concentration of La3+ (0.1 mmol/L) increases Na+/Ca2+ exchange-mediated calcium influx, but lower concentration (10 μmol/L) appears to block calcium influx. The results also suggest that cytosolic La3+ is transported by the ATP-dependent Ca2+ pump. Intracellular Ca2+ stores are depleted by ionomycin, and then ion     

Redox Stability Controls the Cellular Uptake and Activity of Ruthenium-Based Inhibitors of the Mitochondrial Calcium Uniporter (MCU)

The mitochondrial calcium uniporter (MCU) is the ion channel that mediates Ca²⁺ uptake in mitochondria. Inhibitors of the MCU are valuable as potential therapeutic agents and tools to study mitochondrial Ca²⁺. The best-known inhibitor of the MCU is the ruthenium compound Ru360. Although this compound is effective in permeabilized cells, it does not work in intact biological systems. We have recently reported the synthesis and characterization of Ru265, a complex that selectively inhibits the MCU in intact cells. Here, the physical and biological properties of Ru265 and Ru360 are described in detail. Using atomic absorption spectroscopy and X-ray fluorescence imaging, we show that Ru265 is transported by organic cation transporter 3 (OCT3) and taken up more effectively than Ru360. As an explanation for the poor cell uptake of Ru360, we show that Ru360 is deactivated by biological reductants. These data highlight how structural modifications in metal complexes can have profound effects on their biological activities.     

Redox Stability Controls the Cellular Uptake and Activity of Ruthenium‐Based Inhibitors of the Mitochondrial Calcium Uniporter (MCU)

The mitochondrial calcium uniporter (MCU) is the ion channel that mediates Ca²⁺ uptake in mitochondria. Inhibitors of the MCU are valuable as potential therapeutic agents and tools to study mitochondrial Ca²⁺. The best‐known inhibitor of the MCU is the ruthenium compound Ru360. Although this compound is effective in permeabilized cells, it does not work in intact biological systems. We have recently reported the synthesis and characterization of Ru265, a complex that selectively inhibits the MCU in intact cells. Here, the physical and biological properties of Ru265 and Ru360 are described in detail. Using atomic absorption spectroscopy and X‐ray fluorescence imaging, we show that Ru265 is transported by organic cation transporter 3 (OCT3) and taken up more effectively than Ru360. As an explanation for the poor cell uptake of Ru360, we show that Ru360 is deactivated by biological reductants. These data highlight how structural modifications in metal complexes can have profound effects on their biological activities.     

Interaction of Cu+ with mitochondria.

The uptake of Cu+ by rat liver mitochondria is rapid and extensive. Respiration is stimulated by 10 microM Cu+ then inhibited and the inhibition could not be relieved with uncoupling agents. Collapse of the membrane potential is induced by 5-10 microM Cu+. These effects are partially inhibited by radical scavengers indicating the involvement of radical production in these events. Reduction of the GSH content and production of peroxidation products by higher amounts of Cu+ was also demonstrated. Swelling of non-respiring rat liver and heart mitochondria in sodium or lithium acetate was used to study effects of Cu+ on the Na+/H+ exchanger. Swelling is stimulated by 5-100 microM Cu+. In the presence of a radical scavenger the swelling is reduced. In sodium nitrate media diltiazem-sensitive stimulated swelling is observed. Amiloride was found to inhibit Cu(+)-induced efflux of Ca2+. At high concentrations of Cu+, a general increase in permeability was the dominant feature.     

A kinetic study of Ca-containing ions reacting with O, O2, CO2 and H2O: implications for calcium ion chemistry in the upper atmosphere

A series of gas-phase reactions involving molecular Ca-containing ions was studied by the pulsed laser ablation of a calcite target to produce Ca(+) in a fast flow of He, followed by the addition of reagents downstream and detection of ions by quadrupole mass spectrometry. Most of the reactions that were studied are important for describing the chemistry of meteor-ablated calcium in the earth's upper atmosphere. The following rate coefficients were measured: k(CaO(+) + O --> Ca(+) + O(2)) = (4.2 +/- 2.8) x 10(-11) at 197 K and (6.3 +/- 3.0) x 10(-11) at 294 K; k(CaO(+) + CO --> Ca(+) + CO(2), 294 K) = (2.8 +/- 1.5) x 10(-10); k(Ca(+).CO(2) + O(2) --> CaO(2)(+) + CO(2), 294 K) = (1.2 +/- 0.5) x10(-10); k(Ca(+).CO(2) + H(2)O --> Ca(+).H(2)O + CO(2)) = (13.0 +/- 4.0) x 10(-10); and k(Ca(+).H(2)O + O(2) --> CaO(2)(+) + H(2)O, 294 K) = (4.0 +/- 2.5) x 10(-10) cm(3) molecule(-1) s(-1). The quoted uncertainties are a combination of the 1sigma standard errors in the kinetic data and the systematic errors in the models used to extract the rate coefficients. Rate coefficients were also obtained for the following recombination (also termed association) reactions in He bath gas: k(Ca(+).CO(2) + CO(2) --> Ca(+).(CO(2))(2), 294 K) = (2.6 +/- 1.0) x 10(-29); k(Ca(+).H(2)O + H(2)O --> Ca(+).(H(2)O)(2)) = (1.6 +/- 1.1) x 10(-27); and k(CaO(2)(+) + O(2) --> CaO(2)(+).O(2)) < 1 x 10(-31) cm(6) molecule(-2) s(-1). These recombination rate coefficients, as well as those for the ligand-switching reactions listed above, were then interpreted using a combination of high level quantum chemistry calculations and RRKM theory using an inverse Laplace transform solution of the master equation. The surprisingly slow reaction between CaO(+) and O was explained using quantum chemistry calculations on the lowest (2)A', (2)A' and (4)A' potential energy surfaces. These calculations indicate that reaction mostly occurs on the (2)A' surface, leading to production of Ca(+)((2)S) + O(2)((1)Delta(g)). The importance of this reaction for controlling the lifetime of Ca(+) in the upper mesosphere and lower thermosphere is then discussed.     

Effect of the calcium-binding protein regucalcin on the Ca2+ transport system in rat liver microsomes: the protein stimulates Ca2+ release

The effect of the calcium-binding protein regucalcin on the Ca2+ transport system in the liver microsomes from fed rats was investigated. Ca2+ transport was assayed by the method of Millipore filtration to estimate microsomal 45Ca2+ accumulation following addition of 10 mM adenosine triphosphate (ATP). 45Ca2+ uptake was retarded by the presence of regucalcin (1.0-4.0 microM). This retardation was remarkable at 1 min after regucalcin addition, while appreciable retardation was no longer seen at 5 min. Regucalcin (2.0 microM)-induced retardation of 45Ca2+ uptake was prevented by the presence of calmodulin (5 micrograms/ml). Calmodulin alone (1 and 5 micrograms/ml) caused a significant increase in 45Ca2+ uptake at 3 min after the start of incubation. Also, regucalcin (2.0 microM)-induced retardation of 45Ca2+ uptake was completely blocked by the presence of a Ca2(+)-trapping agent, oxalate (3 mM). On the other hand, 45Ca2+, which accumulated in microsomes during 5 min after ATP addition, was markedly released by the addition of regucalcin. This release was dose-dependent (0.5-4.0 microM). Guanosine triphosphate (GTP; 10-100 microM) caused a significant release of 45Ca2+ from the microsomes. The presence of regucalcin (2.0 microM) further enhanced the GTP effect. Regucalcin (2.0 microM)-induced release of 45Ca2+ was not blocked by the presence of the protein thiol-protecting agent dithiothreitol (0.1 mM). The presence of oxalate (3 mM) completely blocked the effect of regucalcin on 45Ca2+ release from the microsomes. These results indicate that regucalcin stimulates Ca2+ release from liver microsomes, and that the protein retards the microsomal Ca2+ uptake. The present study suggests that regucalcin can regulate the Ca2+ transport system in rat liver microsomes.     

Investigation of Cobalt(III) Cage Complexes as Inhibitors of the Mitochondrial Calcium Uniporter

The mitochondrial calcium uniporter (MCU) mediates uptake of calcium ions (Ca²⁺) into the mitochondria, a process that is vital for maintaining normal cellular function. Inhibitors of the MCU, the most promising of which are dinuclear ruthenium coordination compounds, have found use as both therapeutic agents and tools for studying the importance of this ion channel. In this study, six Co³⁺ cage compounds with sarcophagine-like ligands were assessed for their abilities to inhibit MCU-mediated mitochondrial Ca²⁺ uptake. These complexes were synthesized and characterized according to literature procedures and then investigated in cellular systems for their MCU-inhibitory activities. Among these six compounds, [Co(sen)]³⁺ ( 3 , sen=5-(4-amino-2-azabutyl)-5-methyl-3,7-diaza-1,9-nonanediamine) was identified to be a potent MCU inhibitor, with IC₅₀ values of inhibition of 160 and 180 nM in permeabilized HeLa and HEK293T cells, respectively. Furthermore, the cellular uptake of compound 3 was determined, revealing moderate accumulation in cells. Most notably, 3 was demonstrated to operate in intact cells as an MCU inhibitor. Collectively, this work presents the viability of using cobalt coordination complexes as MCU inhibitors, providing a new direction for researchers to investigate.     

Effect of the ionic strength and prostaglandin E2 on the free Ca2+ concentration and the Ca2+ influx in human red blood cells

Human red blood cells (RBCs) were loaded with the Ca(2+)-sensitive fluorescent dye fura-2 to investigate the effects of media ionic strength and prostaglandin E2 (PGE2) on the intracellular free Ca2+ concentration ([Ca2+]i). [Ca2+]i of intact RBCs in a Ca(2+)-containing physiological (high) ionic strength (HIS) solution was 75.1 +/- 8.3 nM after 5 min incubation, increasing to 114.9 +/- 9.6 nM after 1 h. In Ca(2+)-containing low ionic strength (LIS) solutions, [Ca2+]i was significantly lower than in the Ca(2+)-containing HIS solution (p = 0.041 or 0.0385 for LIS solutions containing 200 or 250 mM sucrose, respectively), but, as in HIS solution, an increase of [Ca2+]i was seen after 1 h. In Ca(2+)-free (0 Ca2+ plus 15 microM EGTA) media, [Ca2+]i decreased (ranging from 15 to 21 nM), but were not significantly different in HIS or LIS, and did not change following 1 h incubation. The effect of the ionic strength and PGE2 on passive Ca2+ influx was investigated on ATP-depleted RBCs. Ca2+ influx was faster during the initial 10 min in comparison with the subsequent time period (10-45 min), both in HIS and LIS media, decreasing from 20.3 +/- 1.9 to 12.9 +/- 1.3 micromol/(lcells x h) in HIS, and from 36.7 +/- 5.3 to 8.6 +/- 1.2 micromol/(lcells x h) in LIS. Prostaglandin E2 (PGE2; 10(-7)-10(-11) M), dissolved in deionised water or in ethanol, did not affect [Ca2+]i in either normal or in ATP-depleted RBCs suspended in Ca(2+)-containing HIS medium. Finally, the addition of carbachol (100 microM) did not affect [Ca2+]i. The present findings suggest that stimulation of the Ca(2+)-activated K+ channel by PGE2, reported in [J. Biol. Chem. 271 (1996) 18651], cannot be mediated via increased [Ca2+]i.     

Changes in the intracellular Ca2+ content in human red blood cells in the presence of glycerol

Changes of the intracellular Ca2+ content in human red blood cells (RBCs) in glycerol-containing solutions and after freeze-thawing the cells with glycerol and subsequent deglycerolization were investigated with the Ca2+-sensitive fluorescent dye fluo-4 using fluorescence microscopy. In the glycerol-containing solutions the Ca2+ content increased when compared with a physiological medium (Hepes buffered saline solution (HBSS)). This effect was most likely a result of an inhibition of the Ca2+ pump. After inhibiting the Ca2+ pump using o-vanadate, the Ca2+ uptake was not significantly different in the cells in glycerol-containing and physiological medium. Freeze-thawing and deglycerolization of RBCs resulted in a more pronounced increase in the Ca2+ content. Also in this case, the Ca2+ pump seemed to play a major role.     

Design, synthesis, and structure-activity relationships of 3,4-dihydropyridopyrimidin-2(1H)-one derivatives as a novel class of sodium/calcium exchanger inhibitor

Design, synthesis, and structure-activity relationships for 3,4-dihydropyridopyrimidin-2(1H)-one derivatives, which are aza-3,4-dihydro-2(1H)-quinazolinone derivatives, as the sodium/calcium (Na+/Ca2+) exchanger inhibitors are discussed. These studies based on 3,4-dihydro-2(1H)-quinazolinone derivatives led to the discovery of a structurally novel and potent Na+/Ca2+ exchanger inhibitor, 3,4-dihydropyridopyrimidin-2(1H)-one derivative (26), with an IC30 value of 0.02 microM. Compound 26 directly inhibited the Na+-dependent Ca2+ influx via the Na+/Ca2+ exchanger after Na+-free treatment in cardiomyocytes.     

Simultaneous uptake of Ni2+, NH4+ and PO4(3-) by amorphous CaO-Al2O3-SiO2 compounds

Simultaneous uptake of Ni2+, NH4+, and PO4(3-) by amorphous CaO-Al2O3-SiO2 (C-A-S) compounds was investigated using batch and column methods. Fifteen different C-A-S samples with systematically varied chemical compositions were prepared by coprecipitation from ethanol-water solutions containing Ca(NO3)2.4H2O, Al(NO3)3.9H2O, and Si(OC2H5)4, using NH4OH as the precipitating agent. The resulting precipitates were dried and heated at various temperatures to produce the C-A-S samples, which were then characterized by XRD, FTIR, solid state MAS NMR, DTA-TG, and N2 adsorption. All the C-A-S samples prepared at 600-900 degrees C were amorphous, apart from the CaO-rich samples. Simultaneous uptake of Ni2+, NH4+, and PO4(3-) was determined by a batch method using a solution with an initial concentration of 2 mM. In these experiments, the uptake abilities of the C-A-S samples for Ni2+ and PO4(3-) were high, but were relatively low for NH4+. The uptake abilities for Ni2+ and PO4(3-) increased but that for NH4+ decreased as the silica content in the C-A-S decreased, suggesting that similar uptake mechanisms (ion substitution and/or precipitation) are operating for Ni2+ and PO4(3-), but the uptake mechanism for NH4+ is different (physical adsorption). The column experiments indicate that the order of uptake ability of C-A-S for the three ions is NH4+ < PO4(3-) < Ni2+. Although the silica content of the C-A-S does not have the expected influence on the uptake of these three ions, for NH4+ it plays an important role in the formation of the amorphous phase and also in the suppression of Ca2+ and/or Al3+ release from the C-A-S during the uptake experiments. The optimum uptake properties of the C-A-S can thus be controlled by adjusting the chemical compositions and heating conditions under which the samples are prepared.     

Ruthenium metallopharmaceuticals

The well-developed synthetic chemistry of ruthenium, particularly with ammine, amine and imine ligands, provides for many approaches to innovative new metallopharmaceuticals. Advantages of utilizing ruthenium am(m)ine complexes in drug development include, (1) reliable preparations of stable complexes with predictable structures; (2) the ability to tune ligand affinities, electron transfer and substitution rates, and reduction potentials; and (3) an increasing knowledge of the biological effects of ruthenium complexes. Many Ru(II) and Ru(III) am(m)ine complexes selectively bind to imine sites in biomolecules. Collectively, these lend ruthenium complexes to redox-activation and photodynamic approaches to therapy as well as the development of radiopharmaceuticals containing one of several radionuclides of ruthenium. Ruthenium red and the related Ru360 strongly inhibit calcium ion uptake in the mitochondria. A number of ruthenium compounds with anticancer activity appear to penetrate tumors through a transferrin-mediated process and bind to cellular DNA following intracellular activation by reduction. Ruthenium complexes exhibit both nitric oxide release and scavenging functions that can affect vasodilation and synapse firing. Simple ruthenium complexes are unusually effective in suppressing the immune response by inhibiting T cell proliferation.     

Hepatic calcium-binding protein regucalcin decreases Ca2+/calmodulin-dependent protein kinase activity in rat liver cytosol

The effect of regucalcin, a calcium-binding protein isolated from rat liver cytosol, on cytosolic Ca2+/calmodulin-dependent protein kinase activity was investigated. The increase in cytosolic Ca2+/calmodulin-dependent protein kinase activity with passage of incubation time was clearly prevented by the presence of regucalcin (1.0 microM). An appreciable effect of regucalcin was seen at 0.5 microM. The cytosolic Ca2+/calmodulin-dependent protein kinase activity was fairly increased by increasing concentrations of added Ca2+ (0.25-1.0 mM). This increase was clearly blocked by the presence of regucalcin (1.0 microM). The inhibitory effect of regucalcin on the protein kinase activity was also seen with varying concentrations of calmodulin (2.5-15 micrograms). In the presence of regucalcin (1.0 microM), trifluoperazine (50 microM), an antagonist of calmodulin, significantly decreased the cytosolic Ca2+/calmodulin-dependent protein kinase activity. These results suggest that regucalcin can regulate the Ca2(+)-calmodulin effect in liver cytosol.     

Regulation of GTP-binding state in RalA through Ca2+ and calmodulin

RalA GTPase, a member of Ras superfamily proteins, shows alternative forms between the active GTP-binding and the inactive GDP-binding states. Ral-specific guanine nucleotide exchange factor such as RalGDS interacts with activated Ras and cooperates with Ras indicating that Ral can be activated through Ras signaling pathway. Another activation path for Ral are through Ca2+-dependent but Ras-independent manner. In this study, studies were carried out to examine possible effects of Ca2+ and calmodulin, Ca2+-binding protein, directly on the GTP/GDP-binding state to recombinant unprenylated GST-RalA proteins. The results showed that Ca2+ stimulated the binding of GTP to RalA, whereas it reduced the binding of GDP to RalA. However, it does not involve a high affinity association of Ca2+ with RalA. Ca2+/calmodulin stimulated the GTPase activity of RalA. These results indicate that Ca2+ alone activates RalA by stimulating GTP-binding to RalA and Ca2+/calmodulin inactivates RalA by increasing the activity of RalGTPase.     

Vanadate-stimulated release of hepatic lipase activity from liver.

Vanadate stimulated the release of rat hepatic lipase activity from liver slices into an incubation medium in a time- and dose-dependent manner. Insulin, however, failed to have this stimulatory action, and the release by heparin was recognized, but was not additive to that by vanadate. Amiloride, an inhibitor of tyrosine kinase in some receptors and of the Na+/H+ exchange system suppressed the vanadate-stimulated release. Biochanin A, a different type of tyrosine kinase inhibitor than amiloride, also suppressed the effect of vanadate. The stimulation by vanadate was clearly preserved in Na(+)-, K(+)-, or Ca(2+)-free medium, suggesting that neither the Na+/H+ exchange system, Na+, K(+)-adenosine triphosphatase, nor Ca(2+)-influx into cells is involved in the action of this substance. These results suggest that vanadate-stimulated release of the enzyme activity is associated with the activation of the tyrosine kinase activity.     

Modulator binding protein antagonizes activation of (Ca2+ + Mg2+)-ATPase and Ca2+ transport of red blood cell membranes.

Red blood cells contain a protein that activates membrane-bound (Ca2+ + Mg2+)-ATPase and Ca2+ transport. The red blood cell activator protein is similar to a modulator protein that stimulates cyclic AMP phosphodiesterase. Wang and Desai [Journal of Biological Chemistry 252:4175--4184, 1977] described a modulator-binding protein that antagonizes the activation of cyclic AMP phosphodiesterase by modulator protein. In the present work, modulator-binding protein was shown to antagonize the activation of (Ca2+ + Mg2+)-ATPase and Ca2+ transport by red blood cell activator protein. The results further demonstrate the similarity between the activator protein from human red blood cells and the modulator protein from bovine brain.     

重稀碱金属Cs~+跨人红细胞膜行为的研究(英文)

采用原子吸收光谱法检测体外人红细胞摄取Cs+的含量,系统讨论了胞外Cs+浓度,温育时间、温育温度、介质pH值对人红细胞摄取Cs+过程的影响。选用不同离子通道或离子载体的特异性抑制剂进一步探讨Cs+的跨膜途径和机理。结果显示,各实验参数对人红细胞摄取Cs+均有一定的促进作用。Cs+主要借助Na+/K+-泵的主动运输方式跨膜;少量的Cs+能"漏入"细胞,微量的Cs+可以模拟Na+/Li+-反向协同运输的方式跨膜;在允许HCO3-存在的pH环境下,少量Cs+以Cl-/CsCO3-交换的形式通过膜上带3蛋白进入人红细胞;Ca2+通道对Cs+没有通透作用。     

(Ca1-x-yLuy)MoO4∶xEu3+红色荧光粉的化学共沉淀合成与光致发光

使用NH4HCO3-NH3.H2O混合沉淀剂,采用化学共沉淀法合成(Ca1-x-yLuy)MoO4:xEu3+红色荧光粉,通过XRD、EDS、荧光光谱和CIE色度图研究该荧光粉的晶体结构、成分组成及发光性能。结果表明,实验按照理论化学计量比成功合成了(Ca1-x-yLuy)MoO4:xEu3+红色荧光粉,该荧光粉为CaMoO4白钨矿结构;(Ca1-x-yLuy)MoO4:xEu3+具有7F0→5L6(394 nm)和7F0→5D2(465 nm)的强电子吸收,且在613 nm处可发射高强度红光,其色坐标为(0.666 5,0.332 9),明显优于传统的Y2O2S:Eu3+红色荧光粉;此外,当Lu含量为30mol%时,荧光粉发光强度最佳。     

Reduction of Intracellular pH Is Not the Mechanism for the Synergistic Interaction Between Photodynamic Therapy and Nigericin

Abstract— Previous studies showed that photodynamic therapy (PDT) sensitized by aluminum phthalocyanine can be dramatically potentiated by the K+/H+ ionophore nigericin. Nigericin equilibrates intracellular pH (pHi) and extracellular pH (pHe) and is most effective in potentiating PDT damage when cells are in an acidic environment (pH 6.5-6.7). We therefore hypothesized that the ability of nigericin to lower pHi is causally related to its ability to potentiate PDT. To test this, the pHi of A549 cells was reduced using pHe-adjusted growth medium, with or without addition of amiloride and 4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid, inhibitors of the membrane-based exchangers responsible for regulating pHi. Using fluorescence ratio imaging, we found that pHi can be equilibrated to within ± 0.05 pH unit, in the pH range of 6.0-6.8, for up to 1 h after pHe adjustment. Cells equilibrated to various pHi were subjected to PDT at various light fluences, then plated for clonogenic survival immediately after PDT treatment. There is no significant effect of lowering pHi, to values as low as 6.23, on the toxicity of PDT, regardless of whether pHi is lowered by adjustment of the medium alone or by addition of exchange inhibitors. However, cells equilibrated to pHi 6.0 are more sensitive to PDT, with survival reduced by 1 log at 20 kJ/m² and 1.5 log at 30 kJ/m², relative to cells treated at a pHi of 6.8 (controls). In contrast, 20 μM nigericin in medium at pHe 6.7 reduces pHi to 6.55, but reduces the surviving fraction at 20 kJ/m² by nearly 3 logs relative to controls. These data conclusively demonstrate that the ability of nigericin to potentiate PDT is not directly related to its ability to lower pHi. Furthermore, they show that the expression of PDT damage is independent of pHi, except at the very low value of 6.0. Photodynamic therapy does not induce apoptosis in A549 cells, at surviving fractions of 0.1 to 0.01, under any of the treatment conditions used in this study.