ABSTRACT
Recent evidence indicates that sphingolipids are produced by the heart during hypoxic stress and by blood platelets during thrombus formation. It is therefore possible that sphingolipids may influence heart cell function by interacting with G-protein-coupled receptors of the Edg family. In the present study, it was found that sphingosine 1-phosphate (Sph1P), the prototypical ligand for Edg receptors, produced calcium overload in rat cardiomyocytes. The cDNA for Edg-1 was cloned from rat cardiomyocytes and, when transfected in an antisense orientation, effectively blocked Edg-1 protein expression and reduced the Sph1P-mediated calcium deregulation. Taken together, these results demonstrate that cardiomyocytes express an extracellular lipid-sensitive receptorsystem that can respond to sphingolipid mediators. Because the major source of Sph1P is from blood platelets, we speculate that Edg-mediated Sph1P negative inotropic and cardiotoxic effects may play important roles in acute myocardial ischemia where Sph1P levels are probably elevated in response to thrombus.
Subject(s)
Calcium/metabolism , Immediate-Early Proteins/biosynthesis , Immediate-Early Proteins/genetics , Lysophospholipids , Myocardium/metabolism , Receptors, Cell Surface , Receptors, G-Protein-Coupled , Sphingosine/analogs & derivatives , Sphingosine/metabolism , Sphingosine/pharmacology , Animals , Blood Platelets/metabolism , Blotting, Western , Cardiac Pacing, Artificial , Cells, Cultured , Cloning, Molecular , DNA, Complementary/metabolism , Green Fluorescent Proteins , Ligands , Luminescent Proteins/metabolism , Oligonucleotides, Antisense/genetics , Plasmids/metabolism , Rats , Receptors, Lysophospholipid , Reverse Transcriptase Polymerase Chain Reaction , Sarcoplasmic Reticulum/metabolism , Signal Transduction , Sphingosine/genetics , Time Factors , TransfectionABSTRACT
A highly specific radioimmunoassay for one of the putative adrenomedullary [Met]enkephalin precursors, BAM-12P (Tyr-Gly-Gly-Phe-Met-Arg-Arg-Val-Gly-Arg-Pro-Glu-OH), has been developed. The BAM-12P antibodies recognize the COOH-terminal fragment of the peptide from Arg7 to Glu12 and do not crossreact with [Met5]- or [Leu5]enkephalin or any of their COOH-terminal lysine or arginine extended analogs. Specificity for the COOH-terminal Glu-OH is suggested by the 100% crossreactivity with BAM-12P5-12 and 0.3% crossreactivity with BAM-12P5-12 amide. Using these antibodies, we have measured three forms of BAM-12P-like immunoreactivity in extracts of bovine adrenal medulla, of which the major form (greater than 90%) corresponds to BAM-12P by molecular weight. Extracts of bovine adrenal cortex contain 1% the amount of a BAM-12P-like material (Mr approximately 1400; 20 ng per gland), possibly due to crosscontamination with adrenomedullary tissue. The major form of BAM-12P-like material in extracts of bovine neurointermediate pituitaries is of higher molecular weight than authentic BAM-12P (Mr approximately 4000); the remaining material (10%) corresponds to BAM-12P by molecular weight. There is no detectable BAM-12P-like immunoreactivity in crude or purified extracts of bovine anterior pituitaries. Extracts of bovine hypothalamic tissues contain small amounts of BAM-12P immunoreactivity (approximately 2 ng per fragment) which can be detected as one molecular form corresponding to a 1400-dalton molecule. The results indicate that the enkephalin precursor found in the adrenal medulla also may be present in the pituitary and hypothalamus. Furthermore, the processing of this molecule appears to be tissue-specific.