Libusa Sikurova

Solvent effects on photophysical properties of merocyanine 540

Abstract Absorption and corrected fluorescence emission spectra of merocyanine 540 (MC 540) were recorded in solvents with different physico-chemical parameters – dielectric constant e r , refractive index n , dipolarity/polarizability π * , hydrogen-bonding ability α . Hypsochromic shifts of their maxima positions did not obey Lippert–Mataga equation. However, a correlation with π * and α was observed. Relative quantum yields φ f and rate constants of nonradiative deactivation processes k nr of the dye in different solvents based on steady-state measurements were estimated. Dependence on hydrogen-bonding ability was indicated – with increasing α , φ f decreased and k nr increased. The interaction between MC 540 and solvent molecules is discussed in terms of the different interactions contributing to the solvent stabilization of merocyanine dyes. Our results indicate that hydrogen bonding may contribute to solvent stabilization of MC 540.

Mitochondrial membrane fluidity, potential, and calcium transients in the myocardium from acute diabetic rats

In this study, we report for the first time concurrent measurements of membrane potential and dynamics and respiratory chain activities in rat heart mitochondria, as well as calcium transients in the hearts of rats in an early phase of streptozotocin diabetes, not yet accompanied with diabetes-induced complications. Quantitative relationships among these variables were assessed. The mitochondria from diabetic rats exhibited decreased fluorescence anisotropy values of diphenylhexatriene. This indicates that hydrophobic core of the membranes was more fluid compared with controls (p<0.05). We discuss the changes in fluidity as having been associated with augmented energy transduction through the diabetic membranes. Reduced ratio of JC-1 fluorescence (aggregates to monomers) in the mitochondria from diabetic hearts reflected descendent transmembrane potential. A significant negative association between membrane fluidity and potential in the diabetic group was found (p<0.05; r=0.67). Further, we observed an increase in calcium transient amplitude (CTA) in the diabetic cardiomyocytes (p=0.048). We conclude that some of the calcium-induced regulatory events that dictate fuel selection and capacity for ATP production in diabetic heart occur at the membrane level. Our findings offer new insight into acute diabetes-induced changes in cardiac mitochondria.

Interaction of merocyanine 540 with charged membranes

In this work, phospholipid liposomes were used to investigate the influence of lipid negative charge on the interaction of merocyanine 540 (MC540) with model membranes. Liposomes were prepared from a mixture of neutral dimyristoyl lecithin (DMPC) and negatively charged dimyristoyl phosphatidic acid (DMPA). A strong dependence between the presence of charges on the membrane and dye association was found. The affinity of the dye to liposomes was decreased with an increasing content of DMPA in liposomes. Changes in absorption spectra of MC540 suggest that the decrease in affinity of MC540 to charged membranes is accompanied by a hypsochromic solvatochromic shift and changes in monomer/dimer equilibrium of MC540 incorporated in the membrane.

Remodelling of the sarcolemma in diabetic rat hearts: The role of membrane fluidity

The hyperglycaemia and oxidative stress, that occur in diabetes mellitus, cause impairment of membrane functions in cardiomyocytes. Also reduced sensitivity to Ca-overload was reported in diabetic hearts (D). This enhanced calcium resistance is based on remodelling of the sarcolemmal membranes (SL) with down-regulated, but from the point of view of kinetics relatively well preserved Na,K-ATPase and abnormal Mg- and Ca-ATPase (Mg/Ca-ATPase) activities. It was hypothesised that in these changes may also participate the non-enzymatic glycation of proteins (NEG) and the related free radical formation (FRF), that decrease the membrane fluidity (SLMF), which is in reversal relationship to the fluorescence anisotropy (D 0.235 ± 0.022; controls (C) 0.185 ± 0.009; p 0.05). On the other hand, RAG increased considerably the vulnerability of the diabetic heart to overload with external Ca2+ (C 100% of hearts failed, D 83.3%, D + RAG 46.7% of hearts survived). So we may conclude, that: (i) The NEG and FRF caused alterations in SLMF, that accompanied the diabetes-induced remodelling of SL, also seem to participate in the protection of diabetic heart against Ca2+-overload; (ii) Although, the changes in SLMF were shown to influence considerably the ATPase activities in cells of diverse tissues, they seem to be little responsible for changes in ATPases-mediated processes in the SL of chronic diabetic hearts. (Mol Cell Biochem 249: 175–182, 2003)

Interaction of merocyanine 540 with cations of physiological solutions

Abstract The effect of H+, Na+, Ca2+, Mg2+ on merocyanine 540 (MC 540) absorption and fluorescence spectra was determined. While MC 540 showed two absorption peaks in water (at 535 nm, the monomer peak; at 503 nm, the dimer peak) the addition of salts to MC 540 aqueous solutions resulted in the formation of a new absorption peak at 517 nm and the disappearance of both MC 540 “water” bands. MC 540 fluorescence spectra showed only one peak in pure water (at 573 nm) as well as in salt and HCl aqueous solutions (at 571 nm). The addition of salts did not change the shape of the fluorescence spectra but substantially quenched the fluorescence intensity. The effect of salts reflected principally differences in their cationic charge. These spectral changes were discussed in terms of interaction between the dye anion MC 540− and the aqueous proton H+ followed by dimerization of the protonated merocyanine dye. This assumption was supported by the low pH form of MC 540. A pK value of 1.6 between the neutral protonated form and the anionic form of MC 540 was determined.

Fluidity gradient of erythrocyte membranes in diabetics: the effect of resorcylidene aminoguanidine.

We estimated in vitro membrane fluidity gradient in erythrocytes (RBC) from diabetic patients, using a fluorescent dye 1,6-diphenyl-1,3,5-hexatriene (DPH). The rate constant of DPH incorporation (k) into the membranes was determined by fitting experimental data to an exponential equation. Four important findings were made. First, membrane fluidity in the hydrocarbon region of RBC from diabetic patients is decreased compared with control cells (P<0.01). Second, the rate constant k of DPH incorporation into the membranes of RBC from diabetic patients was lower (P<0.01), which indicates an altered fluidity gradient in the membranes. Third, resorcylidene aminoguanidine (RAG) decreased significantly (P<0.001) the anisotropy values in RBC membranes from diabetic patients, which means that it apparently acted as a fluidizing agent. Lastly, no significant differences in the rate constants k were found between the control membranes (from RAG untreated RBC) and the membranes isolated from RAG pretreated blood from diabetic patients, as well as between the control membranes and those from RAG pretreated control blood. In conclusion, RAG affects lipid-protein interactions in RBC membranes, which results in membrane lipid bilayer fluidization and leads to the restoration of natural physiological membrane dynamic parameters in RBC from diabetic patients.

pH dependence of merocyanine 540 absorption and fluorescence spectra

Abstract The effect of pH on the absorption and fluorescence spectra of merocyanine 540 has been determined. At neutral pH, a double peaked (at 500 and 534 nm) absorption band and a single fluorescence peak (at 577 nm) of anionic merocyanine 540 were recorded. The spectral shape and the peak positions were almost unchanged over the pH range 1.7–7.6 at a temperature of 23 ± 2δC. Decreasing the pH from 5.5 to 1.7 caused only a reversible diminution of the absorption spectra and quenching of the fluorescence intensity without the appearance of any other new spectral band. The dye was seen to precipitate with an increase in acidity. Increasing the pH above 7.6 in aqueous solutions of merocyanine 540 resulted in the irreversible formation of a new, broad band in both the absorption (at 390 nm) and fluorescence (at 500 nm) spectra. These irreversible changes could be due to an attack on the merocyanine molecules by hydroxyl ions.

Calcium signaling-mediated endogenous protection of cell energetics in the acutely diabetic myocardium.

In acute diabetic myocardium, calcium signals propagated by intracellular calcium transients participate in the protection of cell energetics via upregulating the formation of mitochondrial energy transition pores (ETP). Mechanisms coupling ETP formation with an increase in membrane fluidity and a decrease in transmembrane potential of the mitochondria are discussed. Our results indicate that the amplification of calcium transients in the diabetic heart is associated with an increase in their amplitude. Moreover, the signals transferred by calcium transients also regulated ETP formation in nondiabetic myocardium. Evidence for the indispensable role of calcium in the regulation of transition pore formation is provided whereby an exchange of cadmium for calcium ions led to a rapid and dramatic decrease in the amount of ETP. Another possible regulatory factor of the mitochondrial function may be radical-induced damage to the diabetic heart. Nevertheless, our data indicate that radical-induced changes in mitochondria predominantly concern the respiratory chain and have no appreciable effect on the fluidity of the mitochondrial membranes. The residual mitochondrial production of ATP owing to its augmented transfer to the cytosol proved to be adequate to preserve sufficient levels of adenine nucleotides in the acute diabetic myocardium.

Penetration of laser light through red blood cell ghosts.

Hemoglobin is the main absorber of visible light in blood and blood-perfused tissues. However, hemoglobin is released from a red blood cell (RBC) during hemolysis. Hemolysis may be caused by a large number of medical conditions, including photodynamic therapy (PDT) and this subsequently can affect passage of light through the treated biological structures. The purpose of the present study was to determine the penetration of a laser beam through a suspension of hemoglobin-free human red blood cells (RBCs) - ghosts. Although hemoglobin has been efficiently removed from the samples used in our experiments, our measurements show that the samples still effectively attenuate the radiant power of penetrating laser light. We established penetration depths of 12.6mm and 15.4mm for two different laser light wavelengths, 532nm and 630nm, respectively. The penetration depth of laser light was about one order of magnitude higher for hemoglobin-free RBC ghosts as compared to intact RBCs [8,10,12]. These results can be important in case of phototherapy or biostimulation, since all photons that penetrate in a biological object may interact with it and evoke biological response.

pH, serum proteins and ionic strength influence the uptake of merocyanine 540 by WiDr cells and its interaction with membrane structures

It has been suggested that selective uptake of photosensitizers is due to significantly lower pH of the interstitial fluid in tumors compared to normal tissue. Therefore, the cellular uptake of merocyanine 540 (MC 540) was examined at two pH values: 6.8+/-0.1 and 7.4+/-0.1. There was no difference in spectral properties (absorption and fluorescence maxima positions, fluorescence intensity) of the drug in the presence of increasing amounts of either human blood plasma or FCS (0-2%) at the two pH values investigated. Nevertheless, significantly higher amounts of the drug were taken up by WiDr cells at pH 6.8+/-0.1, both in the presence of 10% FCS and in the absence of FCS. The absorption spectra of MC 540 in the presence of egg phosphatidylcholine (PC) liposomes turned out to be NaCl concentration-dependent (0.00-0.30 mol l(-1)). Membrane fluidity, as measured by fluorescence anisotropy of diphenylhexatriene (DPH), was unchanged within the experimental error in the NaCl concentration range 0.01-0.30 mol l(-1). The spectral changes indicated an enhancement of the incorporation of MC 540 into lipid membranes with increasing ionic strength. Such a salt concentration dependence suggests a possible involvement of the surface potential in the interaction of MC 540 with lipid membranes. The results might provide an explanation of the pH dependency of the cellular uptake of MC 540 observed in this study.