Cyanine dye mediated mitochondrial targeting enhances the anti-cancer activity of small-molecule cargoes.

Organelle-specific delivery systems are of significant clinical interest. We demonstrate the use of common cyanine dyes Cy3 and Cy5 as vectors for targeting and delivering cargoes to mitochondria in cancer cells. Specifically, conjugation to the dyes can increase cytotoxicity by up to 1000-fold.

Targeting intracellular Staphylococcus aureus to lower recurrence of orthopaedic infection.

Staphylococcus aureus is often found in orthopaedic infections and may be protected from commonly prescribed antibiotics by forming biofilms or growing intracellularly within osteoblasts. To investigate the effect of non-antibiotic compounds in conjunction with antibiotics to clear intracellular and biofilm forming S. aureus causing osteomyelitis. SAOS-2 osteoblast-like cell lines were infected with S. aureus BB1279. Antibiotics (vancomycin, VAN; and dicloxacillin, DICLOX), bacterial efflux pump inhibitors (piperine, PIP; carbonyl cyanide m-chlorophenyl hydrazone, CCCP), and bone morphogenetic protein (BMP-2) were evaluated individually and in combination to kill intracellular bacteria. We present direct evidence that after gentamicin killed extracellular planktonic bacteria and antibiotics had been stopped, seeding from the infected osteoblasts grew as biofilms. VAN was ineffective in treating the intracellular bacteria even at 10× MIC; however in presence of PIP or CCCP the intracellular S. aureus was significantly reduced. Bacterial efflux pump inhibitors (PIP and CCCP) were effective in enhancing permeability of antibiotics within the osteoblasts and facilitated killing of intracellular S. aureus. Confocal laser scanning microscopy (CLSM) showed increased uptake of propidium iodide within osteoblasts in presence of PIP and CCCP. BMP-2 had no effect on growth of S. aureus either alone or in combination with antibiotics. Combined application of antibiotics and natural agents could help in the treatment of osteoblast infected intracellular bacteria and biofilms associated with osteomyelitis. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1086-1092, 2018.

Differential effects of mitochondrial inhibitors on porcine granulosa cells and oocytes.

Oocytes and granulosa cells rely primarily on mitochondrial respiration and glycolysis for energy production, respectively. The present study examined the effect of mitochondrial inhibitors on the ATP contents of oocytes and granulosa cells. Cumulus cell-oocyte complexes (COCs) and granulosa cells (GCs) were collected from the antral follicles of porcine ovaries. Treatment of denuded oocytes with either carbonyl cyanide m-chlorophenyl hydrazine (CCCP), antimycin, or oligomycin significantly reduced ATP content to very low levels (CCCP, 0.12 pM; antimycin, 0.07 pM; and oligomycin, 0.25 pM; P < 0.05), whereas treatment with a glycolysis inhibitor (bromopyruvic acid, BA) had no effect. Conversely, the ATP content of granulosa cells was significantly reduced by treatment with the glycolysis inhibitor but was not affected by the mitochondrial inhibitors (ATP/10,000 cells; control, 1.78 pM and BA, 0.32 pM; P < 0.05). Reactive oxygen species (ROS) generation after CCCP treatment was greater in oocytes (1.6-fold) than that seen in granulosa cells (1.08-fold). Oocytes surrounded by granulosa cells had higher ATP levels than denuded oocytes. Treatment of COCs with CCCP reduced, but did not completely abolish, ATP content in oocytes (control, 3.15 pM and CCCP, 0.52 pM; P < 0.05), whereas treatment with CCCP plus a gap junction inhibitor, 18α-glycyrrhetinic acid, and CCCP decreased the ATP content to even lower levels (0.29 pM; P < 0.05). These results suggest that granulosa cells are dependent on glycolysis and provide energy to oocytes through gap junctions, even after treatment with CCCP.

Impaired presynaptic cytosolic and mitochondrial calcium dynamics in aged compared to young adult hippocampal CA1 synapses ameliorated by calcium chelation.

Impaired regulation of presynaptic intracellular calcium is thought to adversely affect synaptic plasticity and cognition in the aged brain. We studied presynaptic cytosolic and mitochondrial calcium (Ca) dynamics using axonally loaded Calcium Green-AM and Rhod-2 AM fluorescence respectively in young (2-3 months) and aged (23-26 months) CA3 to CA1 Schaffer collateral excitatory synapses in hippocampal brain slices from Fisher 344 rats. After a tetanus (100 Hz, 200 ms), the presynaptic cytosolic Ca peaked at approximately 10 s in the young and approximately 12 s in the aged synapses. Administration of the membrane permeant Ca chelator, bis (O-aminophenoxy)-ethane-N,N,N,N-tetraacetic acid (BAPTA-AM), significantly attenuated the Ca response in the aged slices, but not in the young slices. The presynaptic mitochondrial Ca signal was much slower, peaking at approximately 90 s in both young and aged synapses, returning to baseline by 300 s. BAPTA-AM significantly attenuated the mitochondrial calcium signal only in the young synapses. Uncoupling mitochondrial respiration by carbonyl cyanide m-chlorophenylhydrazone (CCCP) application evoked a massive intracellular cytosolic Ca increase and a significant drop of mitochondrial Ca, especially in aged slices wherein the cytosolic Ca signal disappeared after approximately 150 s of washout and the mitochondrial Ca signal disappeared after 25 s of washout. These signals were preserved in aged slices by BAPTA-AM. Five minutes of oxygen glucose deprivation (OGD) was associated with a significant increase in cytosolic Ca in both young and aged synapses, which was irreversible in the aged synapses. These responses were significantly attenuated by BAPTA-AM in both the young and aged synapses. These results support the hypothesis that increasing intracellular calcium neuronal buffering in aged rats ameliorates age-related impaired presynaptic Ca regulation.

Sevoflurane depolarizes pre-synaptic mitochondria in the central nervous system.

BACKGROUND: Volatile anaesthetics protect the heart from ischaemic injury by activating mitochondrial signalling pathways. The aim of this study was to test whether sevoflurane, which is increasingly used in neuroanaesthesia, affects mitochondrial function in the central nervous system by altering the mitochondrial membrane potential (DeltaPsi(m)). METHODS: In order to correlate free cytosolic Ca(2+) ([Ca(2+)](i)) and DeltaPsi(m), rat neural presynaptic terminals (synaptosomes) were loaded with the fluorescent probes fura-2 and JC-1. During sevoflurane exposure, 4-aminopyridine (4-AP) 500 micro M to induce pre-synaptic membrane depolarization or carbonylcyanide-p-(trifluoromethoxy)-phenylhydrazone (FCCP) 1 micro M to induce maximum mitochondrial depolarization was added. In order to block mitochondrial ATP-regulated K(+)-channels (mitoK(ATP)), the antagonist 5-hydroxydecanoate (5-HD) 500 micro M was added. RESULTS: In Ca(2+)-containing medium, both sevoflurane 1 and 2 MAC gradually decreased the normalized JC-1 ratio from 0.96 +/- 0.01 in control to 0.92 +/- 0.01 and 0.89 +/- 0.01, representing a depolarization of DeltaPsi(m) (n = 9, P < 0.05). Sevoflurane 2 MAC increased [Ca(2+)](i). In Ca(2+)-depleted medium, sevoflurane 1 and 2 MAC depolarized DeltaPsi(m), while [Ca(2+)](i) remained unaltered. Sevoflurane 2 MAC attenuated the 4-AP-induced depolarization of DeltaPsi(m). When mitoK(ATP) was blocked, the sevoflurane-induced depolarization of DeltaPsi(m) was attenuated, but not blocked. The depolarizing effect of sevoflurane on DeltaPsi(m) compared with FCCP was calculated to 13.2 +/- 1.3% in Ca(2+)-containing and 15.1 +/- 1.2% in Ca(2+)-depleted medium (n = 7). CONCLUSIONS: Sevoflurane depolarizes DeltaPsi(m) in rat synaptosomes, and the effect is not dependent on Ca(2+)-influx to the cytosol. Opening of mitoK(ATP) is partly responsible for the depolarizing effect of sevoflurane.

Swelling-induced Ca2+ release from intracellular calcium stores in rat submandibular gland acinar cells.

The effects of osmotically-induced cell swelling on cytoplasmic free Ca2+ concentration ([Ca2+]i) were studied in acinar cells from rat submandibular gland using microspectrofluorimetry. Video-imaging techniques were also used to measure cell volume. Hypotonic stress (78% control tonicity) caused rapid cell swelling reaching a maximum relative volume of 1.78 +/- 0.05 (n = 5) compared to control. This swelling was followed by regulatory volume decrease, since relative cell volume decreased significantly to 1.61 +/- 0.08 (n = 5) after 10 min exposure to hypotonic medium. Osmotically induced cell swelling evoked by medium of either 78% or 66% tonicity caused a biphasic increase of [Ca2+]i. The rapid phase of this increase in [Ca2+]i was due to release of Ca2 + from intracellular stores, since it was also observed in cells bathed in Ca2+-free solution. The peak increase of [Ca2+]i induced by cell swelling was 3.40 +/- 0.49 (Fura-2 F340/F380 fluorescence ratio, n = 11) and 3.17 +/- 0.43 (n = 17) in the presence and the absence of extracellular Ca2+, respectively, corresponding to an absolute [Ca2+]i of around 1 microm. We found that around two-thirds of cells tested still showed some swelling-induced Ca2+ release (SICR) even after maximal concentrations (10(-5) M - 10(-4) M) of carbachol had been applied to empty agonist-sensitive intracellular Ca2+ stores. This result was confirmed and extended using thapsigargin to deplete intracellular Ca2+ pools. Hypotonic shock still raised [Ca2+]i in cells pretreated with thapsigargin, confirming that at least some SICR occurred from agonist-insensitive stores. Furthermore, SICR was largely inhibited by pretreatment of cells with carbonyl cyanide m-cholorophenyl hydrazone (CCCP) or ruthenium red, inhibitors of mitochondrial Ca2+ uptake. Our results suggest that the increase in [Ca2+]i, which underlies regulatory volume decrease in submandibular acinar cells, results from release of Ca2+ from both agonist-sensitive and mitochondrial Ca2+ stores.

Calcium in the in situ mitochondria of rested and stimulated myocardium.

Calcium is believed to provide feedback between myocardial energy consumption and production. Calcium content was proved to increase in mitochondria (MT) isolated from (1) stimulated hearts, and (2) hearts of increased contractility. In this work we compared Ca2+ content in the intact MT of skinned strips excised at 0 degrees C from previously stimulated or rested guinea-pig ventricles equilibrated with 45Ca and in single rested or stimulated myocytes. In both preparations Ca2+ was released from MT by means of CCCP (carbonyl cyanide m-chlorophenyl-hydrazone; 100 microM). CCCP released 1.58 +/- 0.55 nmol Ca2+/mg of MT protein from the strips of rested hearts and 3.86 +/- 1.12 nmol Ca2+/mg of MT protein from the stimulated muscles. Stimulated myocytes responded to the close micro-injection of CCCP with transient contracture which was not inhibited by caffeine (10 mM) or ryanodine (0.1 microM, 45 min), although the time-course of the contracture was changed. Contracture could not be initiated in rested cells. It is suggested that in rested myocytes MT contain much less Ca2+ than in stimulated ones.