Hypotonic Sucrose Medium Research Articles

Enhancement of Lysosomal Osmotic Sensitivity Induced by the Photooxidation of Membrane Thiol Groups¶

The osmotic lysis of photodamaged lysosomes is a critical event for killing tumor cells. How the photodamage increases lysosomal osmotic sensitivity is still unclear. In this work, the effect of the photooxidation of membrane thiol groups on the lysosomal osmotic sensitivity was studied by measuring the thiol groups with 5,5′-dithio-bis(2-nitrobenzoic acid) and examining the lysosomal β-hexosaminidase latency loss in a hypotonic sucrose medium. The results show that methylene blue–mediated photooxidation of lysosomes decreased their membrane thiol groups and produced cross-linkage of membrane proteins (molecular weight ranging from 75 000 to 125 000), which was visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Simultaneously, the lysosomal osmotic sensitivity increased. These photoinduced alterations of the lysosomes could be recovered by reducing the oxidized thiol groups with dithiothreitol. It indicates that the photooxidation of membrane thiol groups can increase the lysosomal osmotic sensitivity and therefore provides a new explanation for the photoinduced lysosomal lysis.

Mechanism of Lysophosphatidylcholine-Induced Lysosome Destabilization

Lysosomal destabilization is critical for the organelle and living cells. Phospholipase A(2 )(PLA(2)) was shown to be able to destabilize lysosomes under some conditions. By what mechanism the enzyme affects lysosomal stability is not fully studied. In this study, we investigated the effects of lysophosphatidylcholine (lysoPC), a PLA(2)-produced lipid metabolite, on lysosomal ion permeability, osmotic sensitivity and stability. By measuring lysosomal beta-hexosaminidase free activity, membrane potential, proton leakage and their enzyme latency loss in hypotonic sucrose medium, we established that lysoPC could increase the lysosomal permeability to both potassium ions and protons and enhance lysosomal osmotic sensitivity. These changes in lysosomal membrane properties promoted entry of potassium ions into lysosomes via K(+)/H(+) exchange. The resultant osmotic imbalance across the membranes led to losses of lysosomal integrity. The enhancement of lysosomal osmotic sensitivity caused the lysosomes to become more liable to destabilization in osmotic shock. These results suggest that lysoPC may play a key role in PLA(2)-induced lysosomal destabilization.

Effects of phospholipase A 2 on the lysosomal ion permeability and osmotic sensitivity

In this study, we investigated the mechanism of PLA 2-induced lysosomal destabilization. Through the measurements of lysosomal β-hexosaminidase free activity, their membrane potential, the intra-lysosomal pH and the lysosomal latency loss in hypotonic sucrose medium, we established that PLA 2 could increase the lysosomal membrane permeability to both potassium ions and protons. The enzyme could also enhance the organelle osmotic sensitivity. The increases in the lysosomal ion permeability promoted influx of potassium ions into the lysosomes via K +/H + exchange. The resulted osmotic imbalance across the lysosomal membranes osmotically destabilized the lysosomes. In addition, the enhancement of the lysosomal osmotic sensitivity caused the lysosomes to become more liable to destabilization in the osmotic stress. The results explain how PLA 2 destabilized the lysosomes.

Guanosine 5′-[γ-thio]triphosphate-Mediated Activation of Cytosol Phospholipase C Caused Lysosomal Destabilization

Lysosomal disintegration is critical for the organelle functions and cellular viability. In this study, we established that guanosine 5'-[gamma-thio]triphosphate (GTP-gamma-S)-activated cytosol of rat hepatocytes could increase lysosomal permeability to both potassium ions and protons and osmotically destabilize the lysosomes via K(+)/H(+) exchange. These results were obtained through measurements of lysosomal beta-hexosaminidase-free activity, membrane potential and intralysosomal pH. Assays of phospholipase C (PLC) activity show that cytosolic PLC was activated upon addition of GTP-gamma-S to the cytosol. The effects of cytosol on the lysosomes could be abolished by D609, an inhibitor of PLC, but not by the inhibitors of phospholipase A(2). The cytosol-treated lysosomes disintegrated markedly in hypotonic sucrose medium, reflecting that the lysosomal osmotic sensitivity increased. Microscopic observations showed that the lysosomes became more swollen in hypotonic sucrose medium. This indicates that the cytosol treatment induced osmotic shock to the lysosomes and an influx of water into the organelle.

Effects of Arachidonic Acid on the Lysosomal Ion Permeability and Osmotic Stability

In this study, we investigated the effects of arachidonic acid, a PLA2-produced lipid metabolite, on the lysosomal permeability, osmotic sensitivity and stability. Through the measurements of lysosomal beta-hexosaminidase free activity, membrane potential, intralysosomal pH, and lysosomal latency loss in hypotonic sucrose medium, we established that arachidonic acid could increase the lysosomal permeability to both potassium ions and protons, and enhance the lysosomal osmotic sensitivity. As a result, the fatty-acid-promoted entry of potassium ions into the lysosomes via K+/H+ exchange, which could produce osmotic imbalance across their membranes and osmotically destabilize the lysosomes. In addition, the enhancement of lysosomal osmotic sensitivity caused the lysosomes to become more liable to destabilization in osmotic shock. The results suggest that arachidonic acid may play a role in the lysosomal destabilization.

Lysosome destabilization by cytosolic extracts, putative involvement of Ca 2+/phospholipase C

Lysosomal disintegration is a crucial event for living cells, but mechanisms for the event are still unclear. In this study, we established that the cytosolic extracts could enhance lysosomal osmotic sensitivity and osmotically destabilize the lysosomes. The cytosol also caused the lysosomes to become more swollen in the hypotonic sucrose medium. The results indicate that the cytosol induced an osmotic shock to the lysosomes and an influx of water into the organelle. Since the effects of cytosol on the lysosomes could be abolished by O-tricyclo[5.2.1.0 2,6]dec-9-yl dithiocarbonate potassium salt (D609), a specific inhibitor of cytosolic phospholipase C (PLC), the PLC might play an important role in the lysosomal osmotic destabilization. The activity of cytosolic PLC and the extent of enzyme latency loss of the cytosol-treated lysosomes exhibited a similar biphasic dependence on the cytosolic Ca 2+ concentration. In addition, the cytosol did not osmotically destabilize the lysosomes until the cytosolic calcium ions rose above 100 nM. It suggests that the destabilization effect of cytosol on the lysosomes is Ca 2+-dependent.

Enhancement of lysosomal osmotic sensitivity induced by the photooxidation of membrane thiol groups.

The osmotic lysis of photodamaged lysosomes is a critical event for killing tumor cells. How the photodamage increases lysosomal osmotic sensitivity is still unclear. In this work, the effect of the photooxidation of membrane thiol groups on the lysosomal osmotic sensitivity was studied by measuring the thiol groups with 5,5'-dithiobis(2-nitrobenzoic acid) and examining the lysosomal beta-hexosaminidase latency loss in a hypotonic sucrose medium. The results show that methylene blue-mediated photooxidation of lysosomes decreased their membrane thiol groups and produced cross-linkage of membrane proteins (molecular weight ranging from 75000 to 125000), which was visualized by sodium dodecyl sulfatepolyacrylamide gel electrophoresis. Simultaneously, the lysosomal osmotic sensitivity increased. These photoinduced alterations of the lysosomes could be recovered by reducing the oxidized thiol groups with dithiothreitol. It indicates that the photooxidation of membrane thiol groups can increase the lysosomal osmotic sensitivity and therefore provides a new explanation for the photoinduced lysosomal lysis.

INFLUENCE OF MEMBRANE FLUIDITY MODIFIERS ON LYSOSOMAL OSMOTIC SENSITIVITY

Since lysosomes are prone to osmotic lysis, we have examined the correlation between their physical state and sensitivity to osmotic challenge, using agents which modify membrane fluidity. The latency loss of β-hexosaminidase after an incubation in hypotonic sucrose medium was followed under different conditions of membrane fluidity, recorded by steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene. Increasing fluidity of the lysosomal membranes with benzyl alcohol (BA) and greater rigidity caused by cholesteryl hemisuccinate (CHS) increased and decreased the enzyme latency loss, respectively. The effects of BA and CHS treatments on osmotic sensitivity were reversible subsequently by reciprocal treatments of the lysosomes with CHS and BA, respectively. The results indicate that the physical state of the membrane does indeed affect lysosomal osmotic stability.

Regulation of oxidative activity and Δψ of liver mitochondria of active and hibernating gophers. The role of phospholipase A 2

1. 1. In the present work the initially lowered oxidase activity of liver mitochondria of hibernating gophers is shown to increase upon Ca 2+-loading, after freezing-thawing repeated three times and after swelling in a medium containing potassium acetate as well as in a hypotonic sucrose medium. 2. 2. In all cases the inhibition of phospholipase A 2 hindered the increase of the oxidase activity of mitochondria. 3. 3. Mitochondria of hibernating gophers have a lowered Δψ in comparison with active animals, which is restored in the hypotonic medium.

The effect of exogenous cytochrome c on pyruvate oxidation by rat skeletal muscle mitochondria isolated in sucrose or KCl medium

Rat skeletal muscle mitochondria were isolated in three different isolation media: isotonic sucrose, isotonic KCl, and hypotonic sucrose medium. The use of isotonic KCl medium leads to mitochondrial suspensions which are more contaminated with protein of nonmitochondrial origin than the use of sucrose media. This could be concluded from electron microscopy and the specific activity of cytochrome oxidase. Furthermore, respiratory control index values and cytochrome content were lower in these mitochondria. The ratio between cytochrome c + c 1 and cytochrome aa 3 was similar after isolation in both isotonic media but was markedly lower after isolation in hypotonic sucrose. The highest rate of pyruvate oxidation was obtained with isotonic sucrose-isolated mitochondria when this rate was expressed on a protein base. When expressed on base of spectrally measured cytochrome aa 3, reduced with succinate plus KCN, or on base of cytochrome oxidase activity, the rate of pyruvate oxidation was the same in isotonic sucrose-and isotonic KCl-isolated mitochondria. Exogenous cytochrome c stimulated pyruvate oxidation about 20% in mitochondria isolated in isotonic sucrose or KCl and about 100% in mitochondria isolated in hypotonic sucrose. In the latter case, oxidation rates in the presence of exogenous cytochrome c approached the values found with mitochondria isolated in isotonic sucrose. A clear inverse relationship between the content of endogenous cytochrome c + c 1 and the stimulatory effect of exogenous cytochrome c is established. Implications of these results for clinical studies of oxidative capacity of skeletal muscle mitochondria are discussed.

Oxidative properties of swollen rat liver mitochondria

Rat liver mitochondria undergo extensive swelling when they are incubated in hypotonic sucrose medium containing 5 m m P i. After 30 min of swelling at 25 °C, a three- to fourfold increase in volume has occurred, accompanied by gross disorganization of the matrix as observed by electron microscopy. Succinate-supported respiration was unchanged, but the respiration of NAD-linked substrates was reduced and there was a complete and irreversible loss of phosphorylation in both cases. β-Hydroxybutyrate-supported respiration was regained completely on addition of NAD to the swollen mitochondria. α-Ketoglutarate- and malate + pyruvate-supported respiration was only partially restored by the addition of NAD. This inhibition of respiration in swollen mitochondria may be due to a disorganization of a putative complex of Krebs cycle enzymes on the inner surface of the inner membrane.