Lipopolysaccharide internalization activates endotoxin-dependent signal transduction in cardiomyocytes.
We tested the hypothesis that bacterial lipopolysaccharide (LPS) must be internalized to facilitate endotoxin-dependent signal activation in cardiac myocytes. Fluorescently labeled LPS was used to treat primary cardiomyocyte cultures, perfused heart preparations, and the RAW264.7 macrophage cell line. Using confocal microscopy and spectrofluorometry, we found that LPS was rapidly internalized in cardiomyocyte cultures and Langendorff-perfused hearts. Although LPS uptake was also observed in macrophages, only a fraction of these cells were found to internalize endotoxin to the extent seen in cardiomyocytes. Colocalization experiments with organelle or structure-specific fluorophores showed that LPS was concentrated in the Golgi apparatus, lysosomes, and sarcomeres. Similar intracellular localization was demonstrated in cardiomyocytes by transmission electron microscopy using gold-labeled LPS. The internalization of LPS was dependent on endosomal trafficking, because an inhibitor of microfilament reorganization prevented uptake in both cardiomyocytes and whole hearts. Inhibition of endocytosis specifically restricted early activation of extracellular signal-regulated kinase proteins and nuclear factor-kappaB as well as later tumor necrosis factor-alpha production and inducible nitric oxide synthase expression. In conclusion, we have demonstrated that bacterial endotoxin is internalized and transported to specific intracellular sites in heart cells and that these events are obligatory for activation of LPS-dependent signal transduction.
Department of Anesthesia, Children's Hospital and Harvard Medical School, Boston, Massachusetts, USA. douglas.cowan@tch.harvard.edu
D B
DB
Cowan
S
S
Noria
C
C
Stamm
L M
LM
Garcia
D N
DN
Poutias
P J
PJ
del Nido
F X
FX
McGowan
D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D001896:Boron Compounds / Q000737:chemistry; D002460:Cell Line; D015638:Cytochalasin D / Q000494:pharmacology; D004705:Endocytosis / Q000187:drug effects / Q000502:physiology; D005456:Fluorescent Dyes / Q000737:chemistry; D006056:Golgi Apparatus / Q000378:metabolism; D008070:Lipopolysaccharides / Q000737:chemistry / Q000378:metabolism*; D008247:Lysosomes / Q000378:metabolism; D018613:Microscopy, Confocal; D008854:Microscopy, Electron; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D009206:Myocardium / Q000166:cytology / Q000378:metabolism* / Q000648:ultrastructure; D016328:NF-kappa B / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II; D010766:Phosphorylation; D051381:Rats; D017208:Rats, Wistar; D015398:Signal Transduction / Q000502:physiology*; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D001896:Boron Compounds; D005456:Fluorescent Dyes; D008070:Lipopolysaccharides; D016328:NF-kappa B; D014409:Tumor Necrosis Factor-alpha; D015638:Cytochalasin D; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496318:Nos2 protein, rat; D020928:Mitogen-Activated Protein Kinases