Membrane Potential-dependent Uptake Research Articles

Structurally optimised BODIPY derivatives for imaging of mitochondrial dysfunction in cancer and heart cells.

The structural features required for mitochondrial uptake of BODIPY-based optical imaging agents have been explored. The first derivatives of this class of dyes shown to have mitochondrial membrane potential-dependent uptake in both cancer and heart cells have been developed.

Membrane potential-dependent uptake of 18F-triphenylphosphonium–a new voltage sensor as an imaging agent for detecting burn-induced apoptosis

BackgroundMitochondrial dysfunction has been closely related to many pathologic processes, such as cellular apoptosis. Alterations in organelle membrane potential are associated with mitochondrial dysfunction. A fluorine-18 labeled phosphonium compound: 18F-triphenylphosphonium (18F-TPP) was prepared to determine its potential use as a mitochondria-targeting radiopharmaceutical to evaluate cellular apoptosis. MethodsStudies were conducted in both ex vivo cell lines and in vivo using a burned animal model. Uptake of 18F-TPP was assessed in PC-3 cells by gamma counting under the following conditions: graded levels of extracellular potassium concentrations, incubation with carbonyl cyanide m-chlorophenylhydrazone and staurosporine. Apoptosis was studied in a burn animal model using terminal deoxynucleotidyl transferase dUTP nick end labeling staining and simultaneous assessment of 18F-TPP uptake by biodistribution. ResultsWe found that stepwise membrane depolarization by potassium (K) resulted in a linear decrease in 18F-TPP uptake, with a slope of 0.62 ± 0.08 and a correlation coefficient of 0.936 ± 0.11. Gradually increased concentrations of m-chlorophenylhydrazone lead to decreased uptake of 18F-TPP. Staurosporine significantly decreased the uptake of 18F-TPP in PC-3 cells from 14.2 ± 3.8% to 5.6 ± 1.3% (P < 0.001). Burn-induced significant apoptosis (sham: 4.4 ± 1.8% versus burn: 24.6 ± 6.7 %; P < 0.005) and a reduced uptake of tracer in the spleens of burn-injured animals as compared with sham burn controls (burn: 1.13 ± 0.24% versus sham: 3.28 ± 0.67%; P < 0.005). Biodistribution studies demonstrated that burn-induced significant reduction in 18F-TPP uptake in spleen, heart, lung, and liver, which were associated with significantly increased apoptosis. Conclusions18F-TPP is a promising new voltage sensor for detecting mitochondrial dysfunction and apoptosis in various tissues.

A Mitochondria-Targeted Macrocyclic Mn(II) Superoxide Dismutase Mimetic

Superoxide (O(2)(·-)) is the proximal mitochondrial reactive oxygen species underlying pathology and redox signaling. This central role prioritizes development of a mitochondria-targeted reagent selective for controlling O(2)(·-). We have conjugated a mitochondria-targeting triphenylphosphonium (TPP) cation to a O(2)(·-)-selective pentaaza macrocyclic Mn(II) superoxide dismutase (SOD) mimetic to make MitoSOD, a mitochondria-targeted SOD mimetic. MitoSOD showed rapid and extensive membrane potential-dependent uptake into mitochondria without loss of Mn and retained SOD activity. Pulse radiolysis measurements confirmed that MitoSOD was a very effective catalytic SOD mimetic. MitoSOD also catalyzes the ascorbate-dependent reduction of O(2)(·-). The combination of mitochondrial uptake and O(2)(·-) scavenging by MitoSOD decreased inactivation of the matrix enzyme aconitase caused by O(2)(·-). MitoSOD is an effective mitochondria-targeted macrocyclic SOD mimetic that selectively protects mitochondria from O(2)(·-) damage.

Complex I Is the Major Site of Mitochondrial Superoxide Production by Paraquat

Paraquat (1,1'-dimethyl-4,4'-bipyridinium dichloride) is widely used as a redox cycler to stimulate superoxide production in organisms, cells, and mitochondria. This superoxide production causes extensive mitochondrial oxidative damage, however, there is considerable uncertainty over the mitochondrial sites of paraquat reduction and superoxide formation. Here we show that in yeast and mammalian mitochondria, superoxide production by paraquat occurs in the mitochondrial matrix, as inferred from manganese superoxide dismutase-sensitive mitochondrial DNA damage, as well as from superoxide assays in isolated mitochondria, which were unaffected by exogenous superoxide dismutase. This paraquat-induced superoxide production in the mitochondrial matrix required a membrane potential that was essential for paraquat uptake into mitochondria. This uptake was of the paraquat dication, not the radical monocation, and was carrier-mediated. Experiments with disrupted mitochondria showed that once in the matrix paraquat was principally reduced by complex I (mammals) or by NADPH dehydrogenases (yeast) to form the paraquat radical cation that then reacted with oxygen to form superoxide. Together this membrane potential-dependent uptake across the mitochondrial inner membrane and the subsequent rapid reduction to the paraquat radical cation explain the toxicity of paraquat to mitochondria.

Characterization of membrane potential-dependent uptake of the novel PET tracer 18F-fluorobenzyl triphenylphosphonium cation

Mitochondrial dysfunction has been attributed a critical role in the etiology and pathogenesis of numerous diseases, and is manifested by alterations of the organelle's membrane potential (Deltapsi(m)). This suggests that Deltapsi(m) measurement can be highly useful for diagnostic purposes. In the current study, we characterized the capability of the novel PET agent (18)F-fluorobenzyl triphenylphosphonium ((18)F-FBnTP) to assess Deltapsi(m), compared with the well-established voltage sensor (3)H-tetraphenylphosphonium ((3)H-TPP). (18)F-FBnTP and (3)H-TPP uptake under conditions known to alter Deltapsi(m) and plasma membrane potential (Deltapsi(p)) was assayed in the H345 lung carcinoma cell line. (18)F-FBnTP biodistribution was assessed in CD1 mice using dynamic PET and ex vivo gamma well counting. (18)F-FBnTP and (3)H-TPP demonstrated similar uptake kinetics and plateau concentrations in H345 cells. Stepwise membrane depolarization resulted in a linear decrease in (18)F-FBnTP cellular uptake, with a slope (-0.58+/-0.06) and correlation coefficient (0.94+/-0.07) similar (p>0.17) to those measured for (3)H-TPP (-0.63+/-0.06 and 0.96+/-0.05, respectively). Selective collapse of Deltapsi(m) caused a substantial decrease in cellular uptake for (18)F-FBnTP (81.6+/-8.1%) and (3)H-TPP (85.4+/-6.7%), compared with control. Exposure to the proapoptotic staurosporine, known to collapse Deltapsi(m), resulted in a decrease of 68.7+/-10.1% and 71.5+/-8.4% in (18)F-FBnTP and (3)H-TPP cellular uptake, respectively. (18)F-FBnTP accumulated mainly in kidney, heart and liver. (18)F-FBnTP is a mitochondria-targeting PET radiopharmaceutical responsive to alterations in membrane potential with voltage-dependent performance similar to that of (3)H-TPP. (18)F-FBnTP is a promising new voltage sensor for detection of physiological and pathological processes associated with mitochondrial dysfunction, such as apoptosis, using PET.

The intestinal transport mechanism of fluoroquinolones: inhibitory effect of ciprofloxacin, an enoxacin derivative, on the membrane potential-dependent uptake of enoxacin.

To clarify the absorption-structure relationship for the fluoroquinolones from the point of view of inhibitory behavior. The inhibitory effects of ciprofloxacin on the transport process of enoxacin across the rat intestinal brush-border membrane was examined. Ciprofloxacin, which has a similar structure to enoxacin, exhibited a pH-dependent interference with enoxacin absorption from rat jejunal loops. The uptake experiments using BBM vesicles showed that ciprofloxacin significantly reduced not only the initial binding of enoxacin to the membrane surface, but also the K(+)- or H(+)-diffusion potential-dependent transport across the membrane. Furthermore, an H(+)-diffusion potential (interior negative) also exhibited a stimulative uptake of ciprofloxacin. These results suggest that the inhibition behavior of ciprofloxacin from the jejunal loop was closely related to the ionic diffusion potential-dependent uptake of enoxacin across the brush-border membrane.

The inhibition of Ca uptake in cardiac membrane vesicles by verapamil

Cardiac membrane vesicles take up Ca 2+ in response to Na + gradient (high inside) and negative inside membrane potential. Both components of the Ca 2+ uptake, the Na + gradient dependent uptake and the membrane potential dependent uptake are inhibited by verapamil; the action is dose-dependent and the concentrations of verapamil required to inhibit the Ca 2+ uptake to 50% of its maximal value are 50 and 60 μM respectively. In the concentration ranges tested (50–750 μM Ca 2+), the inhibitory effect of verapamil could not be antagonized by increasing the Ca 2+ concentration of the medium. Introducing verapamil into the vesicles by rapid freezing and slow thawing of the vesicles had the same inhibitory effect as adding the same concentration of verapamil on the outside of the vesicles. Adding verapamil to both sides of the vesicle membrane led to higher inhibition of Ca 2+ uptake. It is proposed that addition of verapamil can cause a change in cardiac membranes which is manifested by a decrease in the driving membrane potential and Ca 2+ transport.

Activation of potassium-dependent H+ efflux from mitochondria by cadmium and phenylarsine oxide.

Addition of Cd2+ or phenylarsine oxide (PhAsO) to respiring rat liver mitochondria results first in acidification of the medium (H+ efflux) followed by disappearance of H+ (discharge of the pH gradient or uncoupling). The first phase of H+ efflux is dependent upon the presence of K+ in the medium, and is not seen in the presence of valinomycin, which is consistent with the conclusion that H+ efflux is linked to membrane potential-dependent uptake of K+. These effects are abolished by low levels of 2,3-dimercaptopropanol but potentiated by excess of 2-mercaptoethanol, showing involvement of a dithiol type of group in the response. Mersalyl produces only the H+ efflux, and subsequent addition of Cd2+ of PhAsO produces collapse of the delta pH.