Rapid uptake of lipophilic triphenylphosphonium cations by mitochondria in vivo following intravenous injection: Implications for mitochondria-specific therapies and probes

Background

Mitochondrial dysfunction contributes to a range of pathologies, consequently there is a need to monitor mitochondrial function and to intervene pharmacologically to prevent mitochondrial damage. One approach to this is to deliver antioxidants, probes and pharmacophores to mitochondria by conjugation to the lipophilic triphenylphosphonium (TPP) cation that is taken up selectively by mitochondria driven by the membrane potential.

Conclusions

Oral administration of TPP-conjugated antioxidants protects against mitochondrial damage in vivo. However, there is also a need to deliver molecules rapidly to mitochondria to respond quickly to pathologies and for the real-time assessment of mitochondrial function.

Methods

To see if this was possible we investigated how rapidly TPP cations were taken up by mitochondria in vivo following intravenous (iv) administration.

Results

AlkylTPP cations were accumulated selectively by mitochondria within mice within 5 min of iv injection. The extent of uptake was enhanced 10–30-fold relative to simple alkylTPP cations by attaching functional groups to the TPP cation via long, hydrophobic alkyl chains. Conclusions: Mitochondria-targeted antioxidants, probes and pharmacophores can be delivered into mitochondria within minutes of iv administration.

General significance

These findings greatly extend the utility of mitochondria-targeted lipophilic cations as therapies and probes.     

P-glycoprotein (Mdr1a/1b) and breast cancer resistance protein (Bcrp) decrease the uptake of hydrophobic alkyl triphenylphosphonium cations by the brain

Background

Mitochondrial dysfunction contributes to degenerative neurological disorders, consequently there is a need for mitochondria-targeted therapies that are effective within the brain. One approach to deliver pharmacophores is by conjugation to the lipophilic triphenylphosphonium (TPP) cation that accumulates in mitochondria driven by the membrane potential. While this approach has delivered TPP-conjugated compounds to the brain, the amounts taken up are lower than by other organs.

Methods

To discover why uptake of hydrophobic TPP compounds by the brain is relatively poor, we assessed the role of the P-glycoprotein (Mdr1a/b) and breast cancer resistance protein (Bcrp) ATP binding cassette (ABC) transporters, which drive the efflux of lipophilic compounds from the brain thereby restricting the uptake of lipophilic drugs. We used a triple transgenic mouse model lacking two isoforms of P-glycoprotein (Mdr1a/1b) and the Bcrp.

Results

There was a significant increase in the uptake into the brain of two hydrophobic TPP compounds, MitoQ and MitoF, in the triple transgenics following intra venous (IV) administration compared to control mice. Greater amounts of the hydrophobic TPP compounds were also retained in the liver of transgenic mice compared to controls. The uptake into the heart, white fat, muscle and kidneys was comparable between the transgenic mice and controls.

Conclusion

Efflux of hydrophobic TPP compounds by ABC transporters contributes to their lowered uptake into the brain and liver.

General significance

These findings suggest that strategies to bypass ABC transporters in the BBB will enhance delivery of mitochondria-targeted antioxidants, probes and pharmacophores to the brain.     

Lipophilic triphenylphosphonium cations as tools in mitochondrial bioenergetics and free radical biology

Lipophilic phosphonium cations were first used to investigate mitochondrial biology by Vladimir Skulachev and colleagues in the late 1960s. Since then, these molecules have become important tools for exploring mitochondrial bioenergetics and free radical biology. Here we review why these molecules are useful in mitochondrial research and outline some of the ways in which they are now being utilized.Translated from Biokhimiya, Vol. 70, No. 2, 2005, pp. 273–283.Original Russian Text Copyright ¢ 2005 by Ross, Kelso, Blaikie, James, Cochemé, Filipovska, Da Ros, Hurd, Smith, Murphy.This revised version was published online in April 2005 with corrections to the post codes.     

Rapid and extensive lethal action of clofibrate on hepatoma cells in vitro

Clofibrate, for a long time in use as a hypolipidemic drug, is a well known peroxisomal proliferator (PP) and hepatocarcinogen in rodents. We show here that in vitro 1 mM clofibrate induces a rapid and massive death of rat AH-130 hepatoma cells. Cell death was prominent already after 4 h of treatment, with a characteristic ;apoptotic' pattern by conventional microscopy. This was further supported by the pronounced chromatin condensation detectable on 4',6-diamine-2'-phenylindole dihydrochloride (DAPI) staining, the clearcut internucleosomal DNA fragmentation on agarose-gel electrophoresis (ladder pattern), and the accumulation of markedly hypochromic cells observed in flow cytometric DNA histograms. Consistently with the apoptotic features of the process, some parameters commonly used to detect cell death, such as plasma membrane permeabilization to trypan blue or propidium iodide, lack of mitochondrial retention of rhodamine 123, or extracellular release of lactate dehydrogenase, were all virtually negative. However, these same parameters became markedly positive after 24 h of treatment, which was suggestive for the occurrence of ;secondary' necrosis among AH-130 cells. By a combination of flow cytometric parameters, after 4 h on 1 mM clofibrate only 41% of the AH-130 cells could still be categorized as viable (i.e., non-apoptotic and non-necrotic), while 46% of cells appeared apoptotic and 13% necrotic. At 24 h, 67% of cells were necrotic, 20% apoptotic and only 13% non-apoptotic and non-necrotic. Apoptosis was also extensive in AH-130 cells treated with another PP such as nafenopin at 1 mM concentration and in human hepatoma HepG2 cells treated with clofibrate. By contrast, clofibrate did not cause apoptosis on primary rat hepatocyte cultures. These observations indicate that: (i) apart from their well-known cell growth-promoting action, PPs such as clofibrate or nafenopin may exert a substantial cytotoxic action on targets such as the AH-130 or HepG2 hepatoma cells; (ii) this cell death evolves from an initial 'apoptotic' to an eventual ;necrotic' pattern; (iii) detection of cell death requires the adoption of a full panel of tests, adequate to cover the whole evolving death pattern, while such tests may even be substantially misleading whenever applied individually; (iv) the cytotoxicity of clofibrate and similar agents on normal and, particularly, tumoural cells may deserve careful reevaluation.     

The ring of the rhodopsin chromophore in a hydrophobic activation switch within the binding pocket

The current view that the beta-ionone ring of the rhodopsin chromophore vacates its binding pocket within the protein early in the photocascade has been adopted in efforts to provide structural models of photoreceptor activation. This event casts doubt on the ability of this covalently bonded ligand to participate directly in later stages involving activation of the photoreceptor and it is difficult to translate into predictions for the activation of related G protein-coupled receptors by diffusable ligands (e.g. neurotransmitters). The binding pocket fixes the formally equivalent pair of ring methyl groups (C16/C17) in different orientations that can be distinguished easily by (13)C NMR. Solid-state NMR observations on C16 and C17 are reported here that show instead that the ring is retained with strong selective interactions within the binding site into the activated state. We further show how increased steric interactions for this segment in the activated receptor can be explained by adjustment in the protein structure around the ring whilst it remains in its original location. This describes a plausible role for the ring in operating a hydrophobic switch from within the aromatic cluster of helix 6 of rhodopsin, which is coupled to electronic changes within the receptor through water-mediated, hydrogen-bonded networks between the conserved residues in G protein-coupled receptors.     

Effects of nonsteroidal anti-inflammatory drugs on the uptake of various cations by lymphoid cells.

Several acidic nonsteroidal anti-inflammatory drugs (NSAID) as well as their corresponding alcohol molecules which are known to induce swelling of isolated lymphocytes by changing cell membrane permeability to water, are demonstrated also to induce changes of membrane permeability of lymphoid cells to one divalent cation, calcium, and to three monovalent cations, rubidium, cesium and sodium. According to the cells ionic environment, they increase or decrease the cellular uptake of cation which is itself also closely dependent on the ionic composition of the incubation medium. This drug-effect is very rapid, directly related to the medium NSAID concentration and almost totally reversible except to the most potent drugs such as flufenamic acid. Changes in intracellular ionic balance could have important catalytic effects on the metabolism of normal as well as of pathological cells. This fact could explain side-effects of these drugs as well as some of their therapeutic effects.     

Osthole activates glucose uptake but blocks full activation in L929 fibroblast cells,and inhibits uptake in HCLE cells

Aims

Osthole, a coumarin derivative, has been used in Chinese medicine and studies have suggested a potential use in treatment of diabetes and cancers. Therefore, we investigated the effects of osthole and other coumarins on GLUT1 activity in two cell lines that exclusively express GLUT1.

Main methods

We measured the magnitude and time frame of the effects of osthole and related coumarins on glucose uptake in two cells lines; L929 fibroblast cells which have low GLUT1 expression levels and low basal glucose uptake and HCLE cells which have high GLUT1 concentrations and high basal uptake. We also explored the effects of these coumarins in combination with other GLUT1 activators.

Key findings

Osthole activates glucose uptake in L929 cells with a modest maximum 1.7-fold activation achieved by 50 μM with both activation and recovery occurring within minutes. However, osthole blocks full acute activation of glucose uptake by other, more robust activators. This behavior mimics the effects of other thiol reactive compounds and suggests that osthole is interacting with cysteine residues, possibly within GLUT1 itself. Coumarin, 7-hydroxycoumarin, and 7-methoxycoumarin, do not affect glucose uptake, which is consistent with the notion that the isoprenoid structure in osthole may be important to gain membrane access to GLUT1. In contrast to its effects in L929 cells, osthole inhibits basal glucose uptake in the more active HCLE cells.

Significance

The differential effects of osthole in L929 and HCLE cells indicated that regulation of GLUT1 varies, likely depending on its membrane concentration.     

Rapid changes in specificity within single clones of cytolytic effector cells

We find rapid changes in the specificity of the cytolytic effector cells in a mixed lymphocyte culture. The lysis patterns produced by cytolytic effector cells generated near limiting dilution in murine mixed lymphocyte reactions of three types, F₁ anti-parent (F₁(A × B) anti-A), allogeneic (C anti-F₁(A × B)), and F₁ anti-modified parent (F₁(A × B) anti-A-TNP), were investigated. Cultures were characterized by their ability or inability to lyse a panel of target cells (e.g., A, B, F₁). When individual cultures were tested at two different times, changes in lytic pattern were routinely seen, with some patterns reproducibly increasing in frequency and others reproducibly decreasing (e.g., patterns involving lysis of F₁ decreased in an F₁ anti-A response but increased in a C anti-F₁ response). X-linked isoenzyme analysis showed that changes can occur within a single clone of effector cells. These results imply that the T cell specificity repertoire continues to evolve during an ongoing immune response, a conclusion incompatible with clonal selection theory.     

Mitochondria-targeted penetrating cations as carriers of hydrophobic anions through lipid membranes

High negative electric potential inside mitochondria provides a driving force for mitochondria-targeted delivery of cargo molecules linked to hydrophobic penetrating cations. This principle is utilized in construction of mitochondria-targeted antioxidants (MTA) carrying quinone moieties which produce a number of health benefitting effects by protecting cells and organisms from oxidative stress. Here, a series of penetrating cations including MTA were shown to induce the release of the liposome-entrapped carboxyfluorescein anion (CF), but not of glucose or ATP. The ability to induce the leakage of CF from liposomes strongly depended on the number of carbon atoms in alkyl chain (n) of alkyltriphenylphosphonium and alkylrhodamine derivatives. In particular, the leakage of CF was maximal at n about 10-12 and substantially decreased at n = 16. Organic anions (palmitate, oleate, laurylsulfate) competed with CF for the penetrating cation-induced efflux. The reduced activity of alkylrhodamines with n = 16 or n = 18 as compared to that with n = 12 was ascribed to a lower rate of partitioning of the former into liposomal membranes, because electrical current relaxation studies on planar bilayer lipid membranes showed rather close translocation rate constants for alkylrhodamines with n = 18 and n = 12. Changes in the alkylrhodamine absorption spectra upon anion addition confirmed direct interaction between alkylrhodamines and the anion. Thus, mitochondria-targeted penetrating cations can serve as carriers of hydrophobic anions across bilayer lipid membranes.     

The activation of the sodium pump in pig red blood cells by internal and external cations

A study has been made with pig red blood cells of the activation of the sodium pump by internal and external cations. Cell Na and K concentrations were altered using a PCMBS cation loading procedure. The procedure was characterised for resultant ionic conditions, maintenance of ATP levels and fragility. The activation of the sodium pump by external K was measured in cells suspended in choline (Na-free) solutions. External Cs was used as a substitute for K and elicited lower rates of pump activity. Both the Vmax and apparent Km for 42K influx and 134Cs influx increased as internal Na concentration was raised (within the non-saturating range). Vmax/apparent Km ratios for cation influx were constant. Raising external Cs concentration exerted a similar influence on pump activation by internal Na: both the maximum pump velocity and the apparent Na-site dissociation constant (K'Na) increased. The results provide evidence for a transmembrane connection between cation binding sites on opposite faces of the membrane and are consistent with a consecutive model for the sodium pump in pig red blood cells.     

Involvement of autophagy in trypsinogen activation within the pancreatic acinar cells

Autophagy is mostly a nonselective bulk degradation system within cells. Recent reports indicate that autophagy can act both as a protector and killer of the cell depending on the stage of the disease or the surrounding cellular environment (for review see Cuervo, A.M. 2004. Trends Cell Biol. 14:70-77). We found that cytoplasmic vacuoles induced in pancreatic acinar cells by experimental pancreatitis were autophagic in origin, as demonstrated by microtubule-associated protein 1 light chain 3 expression and electron microscopy experiments. To analyze the role of macroautophagy in acute pancreatitis, we produced conditional knockout mice lacking the autophagy-related 5 gene in acinar cells. Acute pancreatitis was not observed, except for very mild edema in a restricted area, in conditional knockout mice. Unexpectedly, trypsinogen activation was greatly reduced in the absence of autophagy. These results suggest that autophagy exerts devastating effects in pancreatic acinar cells by activation of trypsinogen to trypsin in the early stage of acute pancreatitis through delivering trypsinogen to the lysosome.     

Rhamnolipid stimulates uptake of hydrophobic compounds by Pseudomonas aeruginosa

The biodegradation of hexadecane by five biosurfactant-producing bacterial strains (Pseudomonas aeruginosa UG2, Acinetobacter calcoaceticus RAG1, Rhodococcus erythropolis DSM 43066, R. erythropolis ATCC 19558, and strain BCG112) was determined in the presence and absence of exogenously added biosurfactants. The degradation of hexadecane by P. aeruginosa was stimulated only by the rhamnolipid biosurfactant produced by the same organism. This rhamnolipid did not stimulate the biodegradation of hexadecane by the four other strains to the same extent, nor was degradation of hexadecane by these strains stimulated by addition of their own biosurfactants. This suggests that P. aeruginosa has a mode of hexadecane uptake different from those of the other organisms. Rhamnolipid also enhanced the rate of epoxidation of the aliphatic hydrocarbon alpha,omega-tetradecadiene by a cell suspension of P. aeruginosa. Furthermore, the uptake of the hydrophobic probe 1-naphthylphenylamine by cells of P. aeruginosa was enhanced by rhamnolipid, as indicated by stopped-flow fluorescence experiments. Rhamnolipid did not stimulate the uptake rate of this probe in de-energized cells. These results indicate that an energy-dependent system is present in P. aeruginosa strain UG2 that mediates fast uptake of hydrophobic compounds in the presence of rhamnolipid.     

Rapid activation of phosphatidate phosphohydrolase in mesangial cells by lipid A

Knowledge of rapid events in cell signaling initiated by lipid A, the core moiety of bacterial lipopolysaccharide, is limited. In the present study we have demonstrated that cis-parinaric acid (cis-PnA) rapidly labels 1,2-sn-diacylglycerol (DAG) subsequent to labeling of phosphatidic acid (PA). Stimulation of microsomal membranes with lipid A decreased the level of PA labeled with cis-PnA within 5 s and increased the proportion of fluorescent label in DAG. Lipid A stimulation of DAG synthesis at 5-15 s was inhibited by incubation of mesangial cells with pertussis toxin prior to isolation of microsomal membranes. Inhibition of DAG formation was accompanied by an accumulation of the mass and fluorescent label in the cis-PnA-labeled phosphatidic acid pool. GTP gamma S caused a decrease in labeled PA and an increase in labeled 1,2-DAG. We conclude that the PA pool was enlarged via the lipid A sensitive lyso-PA acyl transferase (lyso-PA-AT) and was decreased by a phosphatidate phosphohydrolase to form DAG. The phosphatidate phosphohydrolase was at least partly regulated by a pertussis-sensitive G-protein. Lipid A or 1,2-dilinoleyl-PA, a product of lyso-PA-AT, induced cell activation as monitored by actin reorganization and cellular shape changes. Pretreatment of cells with pertussis toxin prevented the morphological changes normally induced by lipid A or 1,2-dilinoleyl-PA. In contrast, 1-oleoyl-2-acetylglycerol induced rapid actin reorganization and shape change, presumably bypassing the pertussis blockade. We propose that specific pools of PA and PA-derived DAG are key elements in rapid signaling in mesangial cells and are independent of the PI cycle and phospholipase C.     

The uptake and extrusion of monovalent cations by isolated heart mitochondria

Summary The factors involved in the movement of monovalent cations across the inner membrane of the isolated heart mitochondrion are reviewed. The evidence suggests that the energy-dependent uptake of K⁺ and Na⁺ which results in swelling of the matrix is an electrophoretic response to a negative internal potential. There are no clear cut indications that this electrophoretic cation movement is carrier-mediated and possible modes of entry which do not require a carrier are examined. The evidence also suggests that the monovalent cation for proton exchanger (Na⁺ > K⁺) present in the membrane may participate in the energy-dependent extrusion of accumulated ions. The two processes, electrophoretic cation uptake (swelling) and exchange-dependent cation extrusion (contraction) may represent a means of controlling the volume of the mitochondrion within the functioning cell. A number of indications point to the possibility that the volume control process may be mediated by the divalent cations Ca⁺² and Mg⁺². Studies with mercurial reagents also implicate certain membrane thiol groups in the postulated volume control process.An invited article.     

Potassium uptake by rice plants and interaction with other cations

Summary Rice plants were grown in nutrient solutions in which the K supply was varied by stepwise substitution of Na, Mg, or Ca. Curves relating concentrations in the tissues to those in the root medium showed that K uptake depressed the transfer of Na, Mg, and Ca into the shoots even when supplies of the latter were raised at the expense of K. Only when K was depleted did the other cations substitute for K in the shoots with little change in the total cation concentration in the tissue. Uptake appears to be controlled by a single system in which the 4 cations compete for uptake sites and K was the most effective competitor. re]19730507     

Rapid expression of human immunodeficiency virus following activation of latently infected cells

The host cell activation state impacts the nature of human immunodeficiency virus infection. Activated cells facilitate productive infections; quiescent cells enable the virus to enter a latent state, the major obstacle to viral clearance. We wanted to understand how these differences affected viral gene expression. In quiescent cells activated prior to infection, viral RNA was seen 12 h postinfection; when cells were stimulated postinfection, viral RNA was not seen until 36 h postinfection. Up-regulation of viral RNA in latently infected cells occurred within 2 h poststimulation. This hierarchy also held true for viral protein production. These results may explain the rapid reemergence of viremia following termination of therapy.