© 2001 American Heart Association, Inc.
Cellular Biology |
Lipopolysaccharide Internalization Activates Endotoxin-Dependent Signal Transduction in Cardiomyocytes
From the Departments of Anesthesia (D.B.C., D.N.P., F.X.M.) and Cardiac Surgery (L.M.G., C.S., P.J.D.), Children’s Hospital and Harvard Medical School, Boston, Mass, and the Department of Laboratory Medicine and Pathobiology (S.N.), University of Toronto, Toronto, Ontario, Canada.
Correspondence to Douglas B. Cowan, PhD, Department of Anesthesia, Enders Room 1355, Children’s Hospital, 300 Longwood Ave, Boston, MA 02115. E-mail douglas.cowan{at}tch.harvard.edu
Abstract—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-
B as well as later tumor necrosis factor-
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.
Key Words: Golgi apparatus • microfilaments • endocytosis • lysosomes • signal transduction
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