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in the Adult Mammalian Cardiac Myocyte
(Received for publication, March 25, 1996, and in revised form, November 26, 1996)
and
From the Cardiology Section, Department of Medicine, Veterans
Administration Medical Center, Baylor College of Medicine,
Houston, Texas 77030 and the To determine whether activation of the neutral
sphingomyelinase pathway was responsible for the immediate (<30 min)
negative inotropic effects of tumor necrosis factor-
Division of Cardiology,
Department of Medicine, University of Cincinnati,
Cincinnati, Ohio 45267
(TNF-), we
examined sphingosine levels in diluent and TNF--stimulated cardiac
myocytes. TNF- stimulation of adult feline cardiac myocytes provoked
a rapid (<15 min) increase in the hydrolysis of
[14C]sphingomyelin in cell-free extracts, as well as an
increase in ceramide mass, consistent with cytokine-induced activation of the neutral sphingomyelinase pathway. High performance liquid chromatographic analysis of lipid extracts from TNF--stimulated cardiac myocytes showed that TNF- stimulation produced a rapid (<30
min) increase in free sphingosine levels. Moreover, exogenous D-sphingosine mimicked the effects of TNF- on
intracellular calcium homeostasis, as well as the negative inotropic
effects of TNF- in isolated contracting myocytes; time course
studies showed that exogenous D-sphingosine produced
abnormalities in cell shortening that were maximal at 5 min. Finally,
blocking sphingosine production using an inhibitor of ceramidase,
n-oleoylethanolamine, completely abrogated the negative
inotropic effects of TNF- in isolated contracting cardiac myocytes.
Additional studies employing biologically active ceramide analogs and
sphingosine 1-phosphate suggested that neither the immediate precursor
of sphingosine nor the immediate metabolite of sphingosine,
respectively, were likely to be responsible for the immediate negative
inotropic effects of TNF-. Thus, these studies suggest that
sphingosine mediates the immediate negative inotropic effects of
TNF- in isolated cardiac myocytes.
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