thapsigargin 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

High-K(+)-cardioplegia (CPG) and pyruvate (Pyr) are used as cardioprotective agents. Considering that mitochondria play a critical role in cardiac dysfunction, we investigated the effect of CPG on mitochondrial Ca(2+) uptake and sarcorreticular (SR) calcium handling. Cytosolic and mitochondrial Ca(2+), as well as mitochondrial membrane potential (ΔΨm) were assessed in rat cardiomyocytes by confocal microscopy. Mechano-calorimetrical correlation was studied in perfused hearts. CPG did not modify JC-1 (ΔΨm), but transiently increased, by up to 1.8 times, the Fura-2 (intracellular Ca concentration, [Ca(2+)]i) and Rhod-2 (mitochondrial free Ca concentration [Ca(2+)]m) fluorescence of resting cells, with exponential decays. The addition of 5 µmol·L(-1) thapsigargin (Tpg) increased the Rhod-2 fluorescence in a group of cells without any effect on the Fura-2 signal. In rat hearts perfused with CPG, 1 µmol·L(-1) Tpg decreased resting heat rate (ΔH(r): -0.44 ± 0.07 mW·g(-1)), while the addition of 5 µmol·L(-1) KB-R7943 increased resting pressure (ΔrLVP by +5.26 ± 1.10 mm Hg; 1 mm Hg = 133.322 Pa). The addition of 10 mmol·L(-1) Pyr to CPG increased H(r) (+3.30 ± 0.24 mW·g(-1)) and ΔrLVP (+2.2 ± 0.4 mm Hg), which are effects potentiated by KB-R7943. The results suggest that under CPG, (i) there was an increase in [Ca(2+)]i and [Ca(2+)]m (without changing ΔΨm) that decayed by exothermic removal mechanisms; (ii) mitochondrial Ca(2+) uptake contributed to the removal of cytosolic Ca(2+), in a process that was potentiated by inhibition of sarco-endoplasmic reticulum Ca(2+)-ATPase (SERCA), and reduced by KB-R7943; (iii) under these conditions, SERCA represents the main energetic consumer; (iv) Pyr increased the energetic performance of hearts,mainly by inducing mitochondrial metabolism.

Topics: Animals; Calcium; Cytosol; Fura-2; Heart; Heart Arrest, Induced; Heterocyclic Compounds, 3-Ring; Membrane Potential, Mitochondrial; Mitochondria, Heart; Myocardium; Potassium; Pyruvic Acid; Rats; Rats, Wistar; Sarcoplasmic Reticulum; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Thapsigargin; Thiourea

Transforming growth factor-beta (TGF-beta) has been implicated as a key factor in mediating many cellular processes germane to disease pathogenesis, including diabetic vascular complications. TGF-beta alters cytosolic [Ca2+] ([Ca2+]c) signals, which in some cases may result from the downregulation of the IP3 receptor Ca2+ channels (IP3R). Ca2+ released by IP3Rs is effectively transferred from endoplasmic reticulum (ER) to the mitochondria to stimulate ATP production and to allow feedback control of the Ca2+ mobilization. To assess the effect of TGF-beta on the ER-mitochondrial Ca2+ transfer, we first studied the [Ca2+]c and mitochondrial matrix Ca2+ ([Ca2+]m) signals in single preglomerular afferent arteriolar smooth muscle cells (PGASMC). TGF-beta pretreatment (24 h) decreased both the [Ca2+]c and [Ca2+]m responses evoked by angiotensin II or endothelin. Strikingly, the [Ca2+]m signal was more depressed than the [Ca2+]c signal and was delayed. In permeabilized cells, TGF-beta pretreatment attenuated the rate but not the magnitude of the IP(3)-induced [Ca2+]c rise, yet caused massive depression of the [Ca2+]m responses. ER Ca2+ storage and mitochondrial uptake of added Ca2+ were not affected by TGF-beta. Also, TGF-beta had no effect on mitochondrial distribution and on the ER-mitochondrial contacts assessed by two-photon NAD(P)H imaging and electron microscopy. Downregulation of both IP3R1 and IP3R3 was found in TGF-beta-treated PGASMC. Thus, TGF-beta causes uncoupling of mitochondria from the ER Ca2+ release. The sole source of this would be suppression of the IP3R-mediated Ca2+ efflux, indicating that the ER-mitochondrial Ca2+ transfer depends on the maximal rate of Ca2+ release. The impaired ER-mitochondrial coupling may contribute to the vascular pathophysiology associated with TGF-beta production.

Topics: Angiotensin II; Animals; Calcium; Calcium Signaling; Cells, Cultured; Cytosol; Endoplasmic Reticulum; Endothelins; Fluorescent Dyes; Heterocyclic Compounds, 3-Ring; Inositol 1,4,5-Trisphosphate; Inositol 1,4,5-Trisphosphate Receptors; Kidney Glomerulus; Kinetics; Male; Microscopy, Confocal; Microscopy, Electron, Transmission; Microscopy, Fluorescence; Mitochondria; Myocytes, Smooth Muscle; Rats; Rats, Sprague-Dawley; Thapsigargin; Transforming Growth Factor beta

In previous work, variation in sperm mobility phenotype was attributed to the proportion of ejaculated fowl sperm containing dysfunctional mitochondria. In the present work, latent mitochondrial dysfunction was inferred from patterns of sperm egress from the oviduct's sperm-storage tubules. In addition, experiments were performed to help explain how mitochondrial function could be compromised in viable sperm cells. Confocal microscopy demonstrated that sperm Ca2+ content differed between low and high sperm-mobility phenotypes when sperm were stained with rhod-2 AM, a Ca2+ -specific dye. Fluorescence was associated with the nuclear envelope, a variant of the endoplasmic reticulum, and greater fluorescence was observed in sperm from low sperm-mobility males. Fluorescence was reduced by 50% when motile sperm were rendered immotile by incubation with a Ca2+ chelator. Thus, a relationship was established between a dynamic intracellular Ca2+ pool and sperm motility. Sperm N-methy-D-aspartic acid (NMDA) receptors were inferred by the action of D-homocysteinesulfinic acid, a potent NMDA receptor agonist. Seminal plasma from low sperm mobility males was characterized by an elevated glutamate concentration. Thapsigargin, which inhibits the smooth endoplasmic reticulum Ca2+ pump and thereby promotes Ca2+ efflux, rendered sperm immotile. This effect was blocked by cyclosporin A, which prevents the formation of the mitochondrial permeability transition pore (PTP) in response to elevated mitochondrial Ca2+ content. In summary, we propose that 1) glutamate enables Ca2+ uptake into sperm before ejaculation, 2) excessive Ca2+ uptake triggers formation of the PTP in a subpopulation of sperm, and 3) sperm mobility is decreased in proportion.

Topics: Animals; Calcium; Chickens; Endoplasmic Reticulum; Enzyme Inhibitors; Glutamic Acid; Heterocyclic Compounds, 3-Ring; Homocysteine; Magnesium; Male; Microscopy, Confocal; Mitochondria; Nuclear Envelope; Phenotype; Sperm Motility; Spermatozoa; Thapsigargin

1. Mitochondrial regulation of the cytosolic Ca2+ concentration ([Ca2+]c) in guinea-pig single colonic myocytes has been examined, using whole-cell recording, flash photolysis of caged InsP3 and microfluorimetry. 2. Depolarization increased [Ca2+]c and triggered contraction. Resting [Ca2+]c was virtually restored some 4 s after the end of depolarization, a time when the muscle had shortened to 50 % of its fully relaxed length. The muscle then slowly relaxed (t = 17 s). 3. The decline in the Ca2+ transient was monophasic but often undershot or overshot resting levels, depending on resting [Ca2+]c. The extent of the overshoot or undershoot increased with increasing peak [Ca2+]c. 4. Carbonyl cyanide m-chlorophenyl hydrazone (CCCP; 5 microM), which dissipates the mitochondrial proton electrochemical gradient and therefore prevents mitochondrial Ca2+ accumulation, slowed Ca2+ removal at high ( > 300 nM) but not at lower [Ca2+]c and abolished [Ca2+]c overshoots. Oligomycin B (5 microM), which prevents mitchondrial ATP production, affected neither the rate of decline nor the magnitude of the overshoot. 5. During depolarization, the global rhod-2 signal (which represents the mitochondrial matrix Ca2+ concentration, [Ca2+]m) rose slowly in a CCCP-sensitive manner during and for about 3 s after depolarization had ended. [Ca2+]m then slowly decreased over tens of seconds. 6. Inhibition of sarcoplasmic reticulum Ca2+ uptake with thapsigargin (100 nM) reduced the undershoot and increased the overshoot. 7. Flash photolysis of caged InsP3 (20 microM) evoked reproducible increases in [Ca2+]c. CCCP (5 microM) reduced the magnitude of the [Ca2+]c transients evoked by flash photolysis of caged InsP3. Oligomycin B (5 microM) did not reduce the inhibition of the InsP3-induced Ca2+ transient by CCCP thus minimizing the possibility that CCCP lowered ATP levels by reversing the mitochondrial ATP synthase and so reducing SR Ca2+ refilling. 8. While CCCP reduced the magnitude of the InsP3-evoked Ca2+ signal, the internal Ca2+ store content, as assessed by the magnitude of ionomycin-evoked Ca2+ release, did not decrease significantly. 9. [Ca2+]c decline in smooth muscle, following depolarization, may involve mitochondrial Ca2+ uptake. Following InsP3-evoked Ca2+ release, mitochondrial uptake of Ca2+ may regulate the local [Ca2+]c near the InsP3 receptor so maintaining the sensitivity of the InsP3 receptor to release Ca2+ from the SR.

Topics: Adenosine Triphosphatases; Animals; Antimetabolites; Calcium; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Colon; Cytosol; Enzyme Inhibitors; Fluorescent Dyes; Guinea Pigs; Heterocyclic Compounds, 3-Ring; In Vitro Techniques; Inositol 1,4,5-Trisphosphate; Ionophores; Male; Membrane Potentials; Mitochondria, Muscle; Muscle, Smooth; Oligomycins; Patch-Clamp Techniques; Thapsigargin

1. The free mitochondrial Ca2+ concentration ([Ca2+]m) in rat heart mitochondria was measured quantitatively by loading the mitochondria with fura-2 and then injecting them into Xenopus laevis oocytes. 2. When oocytes were incubated with a physiological solution, the free cytosolic Ca2+ concentration ([Ca2+]c) in the oocytes was 82 +/- 11 nM (n = 20, mean +/- s.e.m.) and the [Ca2+]m of the injected rat heart mitochondria was 116 +/- 10 nM (n = 18, mean +/- s.e.m.). 3. Inhibition of the oocyte endoplasmic reticular Ca2+-ATPase with thapsigargin produced a transient increase in averaged [Ca2+]c at sub-micromolar concentrations. 4. Injection of cardiac mitochondria blunted the peak and prolonged the duration of thapsigargin-induced [Ca2+]c transients as a result of Ca2+ sequestration by the cardiac mitochondria. 5. These results demonstrate that the present technique provides a new approach for studying [Ca2+]m regulation quantitatively under physiological environments. Furthermore, it clearly shows that cardiac mitochondria can modify the shape of thapsigargin-induced cytosolic Ca2+ pulses in Xenopus oocytes.

Topics: Animals; Calcium; Calcium-Transporting ATPases; Endoplasmic Reticulum; Enzyme Inhibitors; Female; Fluorescent Dyes; Fura-2; Heterocyclic Compounds, 3-Ring; In Vitro Techniques; Microscopy, Confocal; Mitochondria, Heart; Oocytes; Rats; Ruthenium Red; Thapsigargin; Xenopus laevis