[14C]methylamine accumulation in cultured human skin fibroblasts — a biochemical test for lysosomal storage and lysosomal diseases

Kopitz, Jürgen; Gerhard, Christoph; Höfler, Petra; Cantz, Michael

doi:10.1016/0009-8981(94)90141-4

Cited by 16 publications

(11 citation statements)

References 19 publications

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“…Most (88%) of the excess endocytosed cargo retained in the three NP-C fibroblast lines studied is predicted to accumulate in compartment 3 (lysosomes). The [ 14 C]sucrose mass predicted to accumulate in lysosomes of NP-C cells is about 2-fold greater than normal cells, consistent with the previously reported 2-fold increase in the fluid-phase volume (29,30) and endocytosed sterol content (6,7,10) In order to identify the cellular compartments potentially targeted by this cellular sterol enrichment, we applied the same compartmental model (Fig. 9).…”

Section: Compartmental Modeling Predicts That Np-c Fibroblasts Are Dementioning

confidence: 49%

The Niemann-Pick C1 Protein Resides in a Vesicular Compartment Linked to Retrograde Transport of Multiple Lysosomal Cargo

Neufeld¹,

Wastney²,

Patel³

et al. 1999

Journal of Biological Chemistry

356

311

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Section: Compartmental Modeling Predicts That Np-c Fibroblasts Are Dementioning

confidence: 49%

The Niemann-Pick C1 Protein Resides in a Vesicular Compartment Linked to Retrograde Transport of Multiple Lysosomal Cargo

Neufeld¹,

Wastney²,

Patel³

et al. 1999

Journal of Biological Chemistry

356

311

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“…Further studies found that imipramine causes a dramatic increase in the accumulation of the hydrophilic marker of aqueous lysosomal volume, methylamine (Supplemental Figure 1), which would not be predicted to be a substrate for phospholipid binding based on its hydrophilicity 33 and has been previously used to indirectly measure the aqueous lysosomal storage volume of cells. 16, 34 Together, these results suggest that accumulation of the amine-containing compounds isn’t caused by the binding to accumulated drug-binding sites, such as phospholipids, but rather suggests that it results from enhanced ion trapping in an expanded lysosomal compartment.…”

Section: Resultsmentioning

confidence: 96%

Cationic Amphiphilic Drugs Cause a Marked Expansion of Apparent Lysosomal Volume: Implications for an Intracellular Distribution-Based Drug Interaction

Funk

Krise

2012

Mol. Pharmaceutics

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How a drug distributes within highly compartmentalized mammalian cells can affect both the activity and pharmacokinetic behavior. Many commercially available drugs are considered to be lysosomotropic, meaning they are extensively sequestered in lysosomes by an ion trapping-type mechanism. Lysosomotropic drugs typically have a very large apparent volume of distribution and a prolonged half-life in vivo, despite minimal association with adipose tissue. In this report we tested the prediction that the accumulation of one drug (perpetrator) in lysosomes could influence the accumulation of a secondarily administered one (victim), resulting in an intracellular distribution-based drug interaction. To test this hypothesis cells were exposed to nine different hydrophobic amine-containing drugs, which included imipramine, chlorpromazine and amiodarone, at a 10 µM concentration for 24 to 48 hours. After exposure to the perpetrators the cellular accumulation of LysoTracker Red (LTR), a model lysosomotropic probe, was evaluated both quantitatively and microscopically. We found that all of the tested perpetrators caused a significant increase in the cellular accumulation of LTR. Exposure of cells to imipramine caused an increase in the cellular accumulation of other lysosomotropic probes and drugs including LyosTracker Green, daunorubicin, propranolol and methylamine; however, imipramine did not alter the cellular accumulation of non-lysosomotropic amine-containing molecules including MitoTracker Red and sulforhodamine 101. In studies using ionophores to abolish intracellular pH gradients we were able to resolve ion trapping-based cellular accumulation from residual pH-gradient independent accumulation. Results from these evaluations in conjunction with lysosomal pH measurements enabled us to estimate the relative aqueous volume of lysosomes of cells before and after imipramine treatment. Our results suggest that imipramine exposure caused a 4-fold expansion in the lysosomal volume, which provides the basis for the observed drug interaction. The imipramine-induced lysosomal volume expansion was shown to be both time- and temperature-dependent and reversed by exposing cells to hydroxypropyl-β-cyclodextrin, which reduced lysosomal cholesterol burden. This suggests that the expansion of lysosomal volume occurs secondary to perpetrator-induced elevations in lysosomal cholesterol content. In support of this claim, the cellular accumulation of LTR was shown to be higher in cells isolated from patients with Niemann-Pick Type C disease, which are known to hyper-accumulate cholesterol in lysosomes.

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“…To measure the proton-translocating activity in lysosomal membranes the ATP-dependent acidification of lysosomal vesicles was assayed by the determination of the incorporation of the lysosomotropic compound [methyl-3 H]-methylamine into the vesicle (31,32). Isolated lysosomes (100 µg protein) incubated in the presence of an ATP-regenerating system (1mM ATP, 2mM MgCl 2 , 9mM phosphoenolpyruvate, 2.5mM NADH, 3.5U pyruvate kinase from rabbit muscle, 5U lactic dehydrogenase from rabbit muscle), 20 mM MOPS, pH 7.0, 100 mM KCl, 25 mM NaCl, 100 µg/ml bovine serum albumin, 0.5 mM EGTA, 18.5 kBq [methyl-3H]methylamine (Hartmann Analytic, Braunschweig, Germany), 7.5 µg/ml phospatidylserine.…”

Section: Measurement Of Proton-translocating Activitymentioning

confidence: 99%

Inhibition of the ATP‐driven proton pump in RPE lysosomes by the major lipofuscin fluorophore A2‐E may contribute to the pathogenesis of age‐related macular degeneration

Bergmann¹,

Schütt²,

Holz³

et al. 2004

FASEB j.

199

126

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Lipofuscin accumulation in the retinal pigment epithelium (RPE) is associated with various blinding retinal diseases, including age-related macular degeneration (AMD). The major lipofuscin fluorophor A2-E is thought to play an important pathogenetic role. In previous studies A2-E was shown to severely impair lysosomal function of RPE cells. However, the underlying molecular mechanism remained obscure. Using purified lysosomes from RPE cells we now demonstrate that A2-E is a potent inhibitor of the ATP-driven proton pump located in the lysosomal membrane. Such inhibition of proton transport to the lysosomal lumen results in an increase of the lysosomal pH with subsequent inhibition of lysosomal hydrolases. An essential task of the lysosomal apparatus of postmitotic RPE for normal photoreceptor function is phagocytosis and degradation of membranous discs shed from photoreceptor outer segments (POS) and of biomolecules from autophagy. When the lysosomes of cultured RPE cells were experimentally loaded with A2-E, we observed intracellular accumulation of exogenously added POS with subsequent congestion of the phagocytic process. Moreover, the autophagic sequestration of cytoplasmic material was also markedly reduced after A2-E loading. These data support the hypothesis that A2-E-induced lysosomal dysfunction contributes to the pathogenesis of AMD and other retinal diseases associated with excessive lipofuscin accumulation.

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[14C]methylamine accumulation in cultured human skin fibroblasts — a biochemical test for lysosomal storage and lysosomal diseases

Cited by 16 publications

References 19 publications

The Niemann-Pick C1 Protein Resides in a Vesicular Compartment Linked to Retrograde Transport of Multiple Lysosomal Cargo

The Niemann-Pick C1 Protein Resides in a Vesicular Compartment Linked to Retrograde Transport of Multiple Lysosomal Cargo

Cationic Amphiphilic Drugs Cause a Marked Expansion of Apparent Lysosomal Volume: Implications for an Intracellular Distribution-Based Drug Interaction

Inhibition of the ATP‐driven proton pump in RPE lysosomes by the major lipofuscin fluorophore A2‐E may contribute to the pathogenesis of age‐related macular degeneration

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