Sphingosine Mediates the Immediate Negative Inotropic Effects of Tumor Necrosis Factor-α in the Adult Mammalian Cardiac Myocyte

doi:10.1074/jbc.272.8.4836

Sphingosine Mediates the Immediate Negative Inotropic Effects of Tumor Necrosis Factor-α in the Adult Mammalian Cardiac Myocyte*

From the Cardiology Section, Department of Medicine, Veterans Administration Medical Center, Baylor College of Medicine, Houston, Texas 77030 and
the ^‡ Division of Cardiology, Department of Medicine, University of Cincinnati, Cincinnati, Ohio 45267

^§ To whom correspondence and reprint requests should be addressed:
Cardiology Research (151C), VA Medical Center, 2002 Holcombe Blvd., Houston, TX 77030.
Tel.: 713-794-7949; Fax: 713-794-7770; E-mail: dmann{at}bcm.tmc.edu

Abstract

To determine whether activation of the neutral sphingomyelinase pathway was responsible for the immediate (<30 min) negative inotropic effects of tumor necrosis factor-α (TNF-α), we examined sphingosine levels in diluent and TNF-α-stimulated cardiac myocytes. TNF-α 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-α-stimulated cardiac myocytes showed that TNF-α stimulation produced a rapid (<30 min) increase in free sphingosine levels. Moreover, exogenous D-sphingosine mimicked the effects of TNF-α on intracellular calcium homeostasis, as well as the negative inotropic effects of TNF-α 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-α 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-α. Thus, these studies suggest that sphingosine mediates the immediate negative inotropic effects of TNF-α in isolated cardiac myocytes.

Footnotes

↵* This work was supported by research funds from the Department of Veterans Affairs and the National Institutes of Health Grant P50 HL-O6H. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

TNF-α

tumor necrosis factor-α

NO

nitric oxide

NOS

nitric oxide synthase

TNFR

TNF receptor

NOE

n-oleoylethanolamine

PC-PLC

phosphatidylcholine-dependent phospholipase C

PKC

protein kinase C

HPLC

high performance liquid chromatography

PMA

phorbol 12-myristate 13-acetate

TDA

tetradecylamine.
- Received March 25, 1996.
- Revision received November 26, 1996.