carbocyanines and rhod-2

Fanconi Anemia (FA) is a rare and complex inherited blood disorder associated with bone marrow failure and malignancies. Many alterations in FA physiology appear linked to red-ox unbalance including alterations in the morphology and structure of nuclei, intermediate filaments and mitochondria, defective respiration, reduced ATP production and altered ATP/AMP ratio. These defects are consistently associated with impaired oxygen metabolism indeed treatment with antioxidants N-acetylcysteine (NAC) and resveratrol (RV) does rescue FA physiology. Due to the importance of the intracellular calcium signaling and its key function in the control of intracellular functions we were interested to study calcium homeostasis in FA. We found that FANCA cells display a dramatically low intracellular calcium concentration ([Ca(2+)]i) in resting conditions. This condition affects cellular responses to stress. The flux of Ca(2+) mobilized by H2O2 from internal stores is significantly lower in FANCA cells in comparison to controls. The low basal [Ca(2+)]i in FANCA appears to be an actively maintained process controlled by a finely tuned interplay between different intracellular Ca(2+) stores. The defects associated with the altered Ca(2+) homeostasis appear consistently overlapping those related to the unbalanced oxidative metabolism in FA cells underlining a contiguity between oxidative stress and calcium homeostasis.

Topics: Acetylcysteine; Antioxidants; Calcium; Calcium-Transporting ATPases; Carbocyanines; Cell Line; Fanconi Anemia; Fanconi Anemia Complementation Group Proteins; Fibroblasts; Heterocyclic Compounds, 3-Ring; Homeostasis; Humans; Hydrogen Peroxide; Kinetics; Microscopy, Confocal; Mitochondria; Models, Biological; Resveratrol; Stilbenes; Thapsigargin

The large-conductance Ca2+-activated K+ (BK(Ca)) channel in the cardiac inner mitochondrial membrane (mitoK(Ca) channel) has been shown to protect the heart against ischemic injury. However, questions about the cardioprotective mechanism and the kinase-mediated regulation of mitoK(Ca) channels remain to be answered.. Flavoprotein fluorescence in guinea pig ventricular myocytes was measured to assay mitoK(Ca) channel activity. The mitochondrial Ca2+ concentration ([Ca2+]m) and membrane potential (DeltaPsi(m)) were measured by loading cells with rhod-2 and JC-1, respectively. Cell death was assessed by trypan blue permeability. The BK(Ca) channel opener NS1619 reversibly increased the flavoprotein oxidation in a concentration-dependent manner. NS1619 (30 micromol/L) attenuated the ouabain (1 mmol/L)-induced elevation of [Ca2+]m with accompanying depolarization of DeltaPsi(m). These effects of NS1619 were completely antagonized by the BK(Ca) channel blocker paxilline (2 micromol/L) but not by the mitochondrial ATP-sensitive K+ (mitoK(ATP)) channel blocker 5-hydroxydecanoate (500 micromol/L). Paxilline, however, failed to block the oxidative effect of diazoxide (100 micromol/L), a mitoK(ATP) channel opener. The combined application of submaximally effective concentrations of NS1619 (10 micromol/L) and diazoxide (30 micromol/L) produced additive effects. NS1619 (30 micromol/L) blunted the rate of cell death during exposure to ouabain; this cardioprotective effect was prevented by paxilline. Activation of cAMP-dependent protein kinase by 8-bromoadenosine 3'5'-cyclic monophosphate (0.5 mmol/L) and forskolin (10 micromol/L) potentiated the NS1619-induced flavoprotein oxidation.. Opening of mitoK(Ca) channels, which is modulated by cAMP-dependent protein kinase, depolarizes the DeltaPsi(m) and attenuates the mitochondrial Ca2+ overload. Our study further indicates that mitoK(Ca) channel activation confers cardioprotection in a manner similar to but independent of mitoK(ATP) channel activation.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Animals; Benzimidazoles; Carbocyanines; Cell Death; Colforsin; Cyclic AMP-Dependent Protein Kinases; Decanoic Acids; Diazoxide; Fluorescent Dyes; Guinea Pigs; Heart Ventricles; Heterocyclic Compounds, 3-Ring; Hydroxy Acids; Indoles; Intracellular Membranes; Ion Transport; Membrane Potentials; Mitochondria, Heart; Myocytes, Cardiac; Ouabain; Potassium; Potassium Channels, Calcium-Activated; Second Messenger Systems; Tetradecanoylphorbol Acetate

Apoptosis contributes to the regulation of cell growth and regeneration and to the development of neoplasia. Mcl-1 is an anti-apoptotic protein that is particularly important for the development of hematological and biliary malignancies, but the mechanism of action of Mcl-1 is unknown. A number of pro- and anti-apoptotic proteins exhibit their effects by modulating Ca2+ signals, so we examined the effects of Mcl-1 on components of the Ca2+ signaling pathway that are known to regulate apoptosis. Expression of Mcl-1 did not affect expression of the inositol 1,4,5-trisphosphate receptor or the size of endoplasmic reticulum Ca2+ stores. However, mitochondrial Ca2+ signals induced by either Ca2+ agonists or apoptotic stimuli were decreased in cells overexpressing Mcl-1 and increased in cells in which Mcl-1 expression was inhibited. These findings provide evidence that Mcl-1 directly inhibits Ca2+ signals within mitochondria, which may provide a novel mechanism to inhibit apoptosis and thereby promote neoplasia.

Topics: Adenocarcinoma; Aniline Compounds; Antibodies, Monoclonal; Apoptosis; Bile Duct Neoplasms; Calcium Signaling; Carbocyanines; Cell Line; Cell Line, Tumor; Cell Nucleus; Fluorescent Antibody Technique, Indirect; Fluorescent Dyes; Heterocyclic Compounds, 3-Ring; Humans; Hydrazines; Immunohistochemistry; Microscopy, Confocal; Mitochondria; Models, Biological; Myeloid Cell Leukemia Sequence 1 Protein; Neoplasm Proteins; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; Tissue Distribution; Xanthenes