Increased acridine orange uptake and lysosomal enzyme levels in rat liver after steroid administration

A steroid which stabilizes lysosomes $\text{in vitro}$ and a pyrogenic steroid which labilizes lysosomes $\text{in vitro}$ were compared with respect to their ability to modify lysosomal uptake and lysosomal enzyme levels $\text{in vivo}$. Cortisone acetate increased the uptake of acridine orange by rat liver lysosomes when the dye was administered by intrathoracic injection. The steroid increased and accelerated the uptake of acridine orange so that, in liver lysosomes from treated rats, the maximum uptake was double that of controls and was reached at 2h, whereas in controls the maximum uptake was at 4h after the injection of the dye. This large elevation of uptake is specific to the lysosomal fraction and is not seen in other subcellular fractions of rat liver. The specific activities of a lysosomal enzyme β-N-acetylglucosaminidase were increased in lysosomal fractions from cortisone acetate-treated rats. Etiocholanolone, a steroid which labilizes lysosome $\text{in vitro}$, similarly accelerated and increased acridine orange uptake by lysosomes but had little effect on lysosomal β-N-acetylglucosaminidase levels. Thus the ability of steroids to stabilize or labilize lysosomes $\text{in vitro}$ does not correlate with their effect on lysosomal uptake of injected substances $\text{in vivo}$, or with their ability to induce increased specific activities of lysosomal enzymes.     

Stabilization of rat liver lysosomes by new anti-inflammatory steroids in vitro

Steroid acid esters, synthesized by modifying the 17-ketol side chain of prednisolone, were tested for their in vitro ability to stabilize heavy mitochondrial lysosomes prepared from rat liver. Membrane stabilization was determined by assessing capability of steroids to decrease extrusion of the marker enzymes (acid phosphatase, beta-glucuronidase and aryl sulfatase) from lysosomes incubated in hypo-osmotic sucrose-Tris acetate buffer. Results indicated that prednisolone (1) significantly inhibited the lysosomal release of acid phosphatase as did the new anti-inflammatory steroid, methyl 20-dihydroprednisolonate. Methyl prednisolonate exhibited weak membrane stabilization capacities and 20-dihydroprednisolonic acid, a metabolic product of methyl 20-dihydroprednisolonate, showed virtually no membrane stabilization.     

The effect of inflammation on rat liver beta-galactosidase and beta-N-acetylglucosaminidase.

Reduced activities of β-galactosidase and β-N-acetylglucosaminidase were found in rat livers following experimentally induced inflammation. The greatest reduction in glycosidase activities were found 24 h after inflammation. The lysosomal fraction accounted for the bulk of both glycosidase activities in experimental and control rats. Kinetic experiments showed that inflammation did not affect K_m values, but did result in a significant reduction in V_max values. One suggestion to explain the results is that inflammation causes a reduction in biosynthesis or activation of the two glycosidases in rat liver.     

Turnover studies on proteins of rat liver lysosomes.

The turnover of rat liver lysosomal proteins was studied by a double isotope-labeling technique. The cellular fractions investigated included soluble lysosomal proteins, lysosomal membrane proteins, highly purified lysosomal beta-glucuronidase, and for comparison, microsomal proteins and soluble cytoplasmic proteins. Both "normal" lysosomes and Triton WR-1339-filled lysosomes (tritosomes) were studied, with similar results. It was found that (a) the turnover rate of lysosomal proteins, of both the soluble and membranous compartments, was very similar to that of the proteins of the microsomal and soluble cytoplasmic fractions, and (b) the turnover rate of lysosomal proteins was asynchronous. The latter conclusion was based on two lines of evidence: (a) lysosomal beta-glucuronidase had a distinctly slower turnover rate than the average rate of the soluble lysosomal proteins, and (b) subunits of the proteins of the soluble lysosomal fraction as separated by sodium dodecyl sulfate. Sephadex G-200 gel filtration showed different rates of degradation.     

Cardiolipin degradation by rat liver lysosomes.

The lysosomal subcellular fraction of rat liver contains acid hydrolases which can carry out the degradation of cardiolipin to yield water-soluble products and free fatty acids. The time course of appearance of the products indicates that the major catabolic route involves the sequential removal of three of the fatty acids, followed by hydrolysis to acylglycerophosphoryl glycerol (from which the fatty acid is subsequently removed) and d-glycerophosphate (which is hydrolysed to give free phosphate and glycerol). The phospholipase A activity responsible for removal of the first fatty acid is located in the lysosomal fraction.     

Neutral-sugar transport by rat liver lysosomes.

Transport of D-glucose was studied in Percoll-gradient-purified rat liver lysosomes. D-Glucose uptake had a Km of 22 mM and a t1/2 of approx. 30 s. D-Fucose, 2-deoxyglucose and methyl alpha-glucoside were the most effective competitors for uptake of D-glucose, although D-galactose, D-mannose, D-xylose and L-fucose also appeared to compete for uptake. L-Glucose was a poor competitor for uptake. No competition was observed with N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, D-glucuronic acid, N-acetylneuraminic acid, D-glucosamine or the amino acids L-glycine, L-lysine and L-proline. Uptake was unaffected by N-ethylmaleimide, dithiothreitol, KCl, NaCl, ATP/Mg or alteration of buffer pH. D-Glucose efflux from lysosomes was temperature-dependent, with a Q10 of 2.3, and was inhibited by cytochalasin B. Counter-transport could not be demonstrated. In contrast, L-fucose uptake had a Km of 65 mM and was largely unaffected by 5 M excess of neutral D-sugars. Both uptake and efflux of L-fucose were inhibited by cytochalasin B. It appears that lysosomes possess a facilitated transport system for D-glucose and perhaps other neutral D-sugars that is discrete from transport systems for acetylated and acidic sugars.     

5'-Nucleotidase in rat liver lysosomes

5'-Nucleotidase was found in purified rat liver tritosomes. When tritosomes were subfractionated into the membrane and soluble contents fractions, 73% of the total 5'-nucleotidase activity was found in the membrane fraction and 24% in the soluble contents fraction. Immunoblotting using specific polyclonal antibodies against the rat liver plasma membrane 5'-nucleotidase showed that the mobilities on SDS-polyacrylamide gel electrophoresis of both 5'-nucleotidases in the membrane and contents fractions were identical to that of the enzyme in the plasma membranes (Mr = 72,000). 5'-Nucleotidases in the membrane and contents fractions were sensitive to neuraminidase and converted into a form that was 4 kDa smaller after digestion, as observed in the case of plasma membrane enzyme. 5'-Nucleotidases, both from the membrane and contents fractions, were purified using immunoaffinity chromatography, and the isoelectric points, heat stability, and oligomeric structure of the purified enzymes were compared. Isoelectric focusing and the heat stability test indicated the resemblance of the soluble enzyme to the membrane-bound enzyme. However, the membrane-bound enzyme aggregated in the absence of Triton X-100, whereas the soluble enzyme behaved as a dimer. The topography of 5'-nucleotidase in the tritosomal membranes was studied using antibodies against 5'-nucleotidase and neuraminidase treatment. The inhibition of 5'-nucleotidase were not observed in the intact tritosomal fraction until the tritosomes had been disrupted by osmotic shock. These results show that the active sites and the oligosaccharide chains of 5'-nucleotidase are located on the inside surface of the tritosomal membranes.     

Diacylglycerol hydrolysis in rat liver lysosomes

The matrix of rat liver lysosomes exhibits high hydrolytic activity towards 1,2-diacylglycerol with an optimum at pH 4.0. The lipolytic reaction follows Michaelis-Menten kinetics (apparent Vmax 470 nmol hydrolysed/min per mg protein; apparent Km 71 microM 1,2-dioleoylglycerol). Formation of 1- and 2-monooleoylglycerols indicates an initial attack at both the primary and secondary ester bonds. The lysosomal matrix also catalyses (re)acylation reactions, i.e. the formation of 1,2-diacylglycerol from 2-monoacylglycerol and free fatty acid. However, (re)acylation proceeds at a far lower rate than deacylation of diacylglycerol. Lysosomal diacylglycerol hydrolysis is sensitive towards non-ionic detergents, cationic amphiphilic drugs and the lipase inhibitor RHC 80267.     

The capacity of lysosomes of cultured mammalian cells to accumulate acridine orange is destroyed by hyperthermia

Summary Lysosomes of cultured mammalian cells, derived from a transplantable murine mammary adenocarcinoma, irreversibly lose their capacity to accumulate the fluorescent dye acridine orange after hyperthermia. As acridine orange may be regarded as a fluorescent probe of the internal pH of the lysosomes, we may conclude that the ability of lysosomes to maintain a low internal pH is destroyed by hyperthermia.The effects of hyperthermia on lysosome fluorescence and on cell survival show several similarities: in both cases hyperthermia is more effective at low pH, below pH 7.0, and CCP (carbonylcyanide-m-chlorophenylhydrazone) enhances effects at low pH, but has no clear effect at pH 8.0. This leads to the conclusion that effects on lysosomes are an important and early event in cellular injury caused by hyperthermia. The activation energy, however, obtained for the effects of hyperthermia on lysosome fluorescence is about a factor of two lower than the activation energy reported for cell survival after hyperthermia. This suggests that the effect on lysosomes is not directly caused by hyperthermia but is triggered by some other hyperthermia-induced cellular damage.Abbreviations CCP carbonylcyanide-m-chlorophenylhydrazone - MES 2-(N-morpholino)ethanesulfonic acid - MOPS 2-(N-morpholino)propanesulfonic acid - Tricine N-tris(hydroxymethyl)methylglycine Supported in part by grants from the KWF (Koningin Wilhelmina Fonds) and the IRS (Interuniversitair Instituut voor Radiopathologie en Stralenbescherming)     

Evidence for an ATP-driven "proton pump" in rat liver lysosomes by basic dyes uptake.

When rat liver lysosomes are suspended in a medium containing acridine orange at neutral pH, accumulation of the dye may be observed within the vesicles. The uptake appears driven by a pH gradient between the external medium and the interior of the lysosomes since it is inhibited by NH₄⁺, nigericin and other electroneutral proton-cation exchangers. FCCP is ineffective in inhibiting the uptake. In the presence of Mg⁺⁺ and anions such as Cl^?, ATP promoted a further and more extensive but slower oligomycin and ouabain-insensitive dye uptake, which was also inhibited by FCCP. Very similar results were obtained with neutral red and atebrin. When the rate of the ATP-induced acridine uptake in preparations of different purification grade was compared, it was observed that the uptake rate increased in parallel with lysosomal enzymatic activity. These results suggest that an electrogenic ATP-driven-Mg⁺⁺ dependent “proton pump” is operating in the lysosomal membrane, as previously proposed.     

Latency of some glycosidases of rat liver lysosomes.

The latency of the alpha-glucosidase activity of intact rat liver lysosomes was studied by using four substrates (glycogen, maltose, p-nitrophenyl, alpha-glucoside, alpha-fluoroglucoside) at a range of substrate concentrations. The results indicate that the entire lysosome population is impermeable to glycogen and maltose, but a proportion of lysosomes are permeable to alpha-fluoroglucoside and a still higher proportion permeable to p-nitrophenyl alpha-glucoside. Incubation at 37 degrees C in an osmotically protected buffer of of pH 5.0 caused lysosomes to become permeable to previously impermeant substrates and ultimately to release their alpha-glucosidase into the medium. The latencies of lysosomal beta-glucosidase and beta-galactosidase were examined by using p-nitrophenyl beta-glucoside and beta-galactoside as substrates. The results indicate permeability properties to these substrates similar to that to p-nitrophenyl alpha-glucoside. On incubation in an osmotically protected buffer of pH 5, lysosomes progressively released their beta-galactosidase in soluble form, but beta-glucosidase remained attached to sedimentable material. Lysosomal beta-glucosidase was inhibited by 0.1% Triton X-100; alpha-glucosidase and beta-galactosidase were not inhibited.     

Effects of glucagon and insulin on liver lysosomes

Recent experimental evidence has been obtained, principally in the laboratory of Glenn Mortimore, that hepatic lysosomes can act as a pool of amino acids during fasting. This pool is generated through $\text{autophagy}$, whereby intracellular proteins are somehow captured by the lysosomes and then rapidly hydrolyzed to free amino acids by the lysosomal proteinases. Two important metabolic fates of these lysosomal digestive products can be: 1) conversion of the glucogenic amino acids into glucose, and 2) conversion of trimethyl-lysine into carnitine. The latter metabolite is required to transfer fatty acids to the mitochondrial site of β-oxidation. Most interesting is the observation that glucagon appears to induce lysosomal autophagy and the resulting degradation of intracellular proteins by decreasing the size of amino acid pools in the perfused liver. This effect of the hormone may be directed at the single amino acid glutamine, since adding it alone to the perfusate can prevent the increase in autophagy caused by glucagon. Insulin also rapidly inactivates hepatic autophagy and its ensuing proteolysis. The $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ for the rate of los of autophagic vocuoles from the insulin-treated liver (or animal) is approximately 8 min. Thus, glucagon and insulin actively control intracellular protein catabolism that takes place within hepatic lysosomes, and this regulation by the two hormones may be one of their major molecular effects on gluconegenesis in the liver.     

Effects of phenobarbital on protein synthesis and polysome levels in rat liver.

Administration of phenobarbital to rats increases the rate of synthesis of certain microsomal drug-metabolizing enzymes in a selective manner and promotes proliferation of smooth endoplasmic reticulum in the liver. Phenobarbital increased a number of factors by which protein synthesis could be enhanced in the liver. It produced a 30% increase in the amount of ribosomes and mRNA per cell. The proportion of ribosomes associated with polysomes was increased by 5-10% over normal liver. There was a 10-30% increase in the rate of ploypeptide elongation and a small increase or no change in polysome size, indicating that the rate of polypeptide initiation was increased proportionately. The product of these effects accounts for the 1.5-fold increase in the rate of total protein synthesis previously reported. The average polysome size, and the size of free polysomes in particular, was maintained when actinomycin D was administered to phenobarbital-pretreated rats, suggesting that the rate of mRNA degradation was decreased selectively. Phenobarbital did not, however, affect the distribution of ribosomes between the free and membrane-bound states or the activity of ribonucleases associated with isolated free and bound polysomes. Thus, we conclude that phenobarbital stimulates protein synthesis by expanding the mRNA pool, at least partially through effects on mRNA degradation, and by augmenting the rate of mRNA translation.