Dataset Information

In Vitro Assessment of Uptake and Lysosomal Sequestration of Respiratory Drugs in Alveolar Macrophage Cell Line NR8383.

ABSTRACT:

Purpose

To assess accumulation and lysosomal sequestration of 9 drugs used in respiratory indications (plus imipramine as positive control) in the alveolar macrophage (AM) cell line NR8383.

Methods

For all drugs, uptake at 5 μM was investigated at 37 and 4°C to delineate active uptake and passive diffusion processes. Accumulation of basic clarithromycin, formoterol and imipramine was also assessed over 0.1-100 μM concentration range. Lysosomal sequestration was investigated using ammonium chloride (NH4Cl), monensin and nigericin. Impact of lysosomal sequestration on clarithromycin accumulation kinetics was investigated.

Results

Both cell-to-medium concentration ratio (Kp) and uptake clearance (CLuptake) ranged > 400-fold for the drugs investigated. The greatest Kp was observed for imipramine (391) and clarithromycin (82), in contrast to no accumulation seen for terbutaline. A concentration-dependent accumulation was evident for the basic drugs investigated. Imipramine and clarithromycin Kp and CLuptake were reduced by 59-85% in the presence of NH4Cl and monensin/nigericin, indicating lysosomal accumulation, whereas lysosomal sequestration was not pronounced for the other 8 respiratory drugs. Clarithromycin uptake rate was altered by NH4Cl, highlighting the impact of subcellular distribution on accumulation kinetics.

Conclusions

This study provides novel evidence of the utility of NR8383 for investigating accumulation and lysosomal sequestration of respiratory drugs in AMs.

SUBMITTER: Ufuk A

PROVIDER: S-EPMC4628094 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Rationale: Serial measurements of alveolar macrophage (AM) transcriptional changes in patients with acute respiratory distress syndrome (ARDS) could identify cell-specific biological programs that are associated with clinical outcomes.Objectives: To determine whether AM transcriptional programs are associated with prolonged mechanical ventilation and 28-day mortality in individuals with ARDS.Methods: We performed genome-wide transcriptional profiling of AMs purified from BAL fluid collected from 35 subjects with ARDS. Cells were obtained at baseline (Day 1), Day 4, and Day 8 after ARDS onset (N = 68 total samples). We identified biological pathways that were enriched at each time point in subjects alive and extubated within 28 days after ARDS onset (alive/extubatedDay28) versus those dead or persistently supported on mechanical ventilation at Day 28 (dead/intubatedDay28).Measurements and Main Results: "M1-like" (classically activated) and proinflammatory gene sets such as IL-6/JAK/STAT5 (Janus kinase/signal transducer and activator of transcription 5) signaling were significantly enriched in AMs isolated on Day 1 in alive/extubatedDay28 versus dead/intubatedDay28 subjects. In contrast, by Day 8, many of these same proinflammatory gene sets were enriched in AMs collected from dead/intubatedDay28 compared with alive/extubatedDay28 subjects. Serially sampled alive/extubatedDay28 subjects were characterized by an AM temporal expression pattern of Day 1 enrichment of innate immune programs followed by prompt downregulation on Days 4 and 8. Dead/intubatedDay28 subjects exhibited an opposite pattern, characterized by progressive upregulation of proinflammatory programs over the course of ARDS. The relationship between AM expression profiles and 28-day clinical status was distinct in subjects with direct (pulmonary) versus indirect (extrapulmonary) ARDS.Conclusions: Clinical outcomes in ARDS are associated with highly distinct AM transcriptional programs.