Occurrence of two molecular forms of human acid sphingomyelinase

Human acid sphingomyelinase (ASM) hydrolyses sphingomyelin to ceramide and phosphocholine. Metabolic studies on COS-1 cells transfected with ASM cDNA revealed the occurrence of an enzymically inactive precursor which is differentially processed to two predominant native glycoprotein forms: a 70 kDa polypeptide corresponding to human urinary protein and a 57 kDa form. Formation of these potentially active forms was shown to be restricted to distinct compartments. Maturation of the ASM precursor to a predominant 70 kDa form occurs exclusively inside acidic organelles, whereas variable amounts of 57 kDa ASM are detectable immediately after biosynthesis. Metabolic labelling of transfected COS-1 cells with [32P]Pi further suggests that this form obviously does not carry oligomannosylphosphate residues, in contrast with the mature lysosomal ASM. In order to verify that this early form of active ASM results from co-post-translational proteolysis of the ASM precursor and not from the use of different translation-initiation sites on the ASM mRNA, appropriate 5'-mutagenized cDNA constructs were transiently expressed. These results clearly indicate that the first potential in-frame AUG is exclusively used for translation initiation in vivo and that deletion of the proposed signal sequence for endoplasmic reticulum import completely eliminates the ability of the translation product to enter the vacuolar apparatus. As there are two different subcellular sites of maturation of the ASM precursor, and intracellular targeting of the two processed forms appears to be different, the two ASM proteins may contribute to distinct physiological functions

Acid Sphingomyelinase Regulates Platelet Cell Membrane Scrambling, Secretion, and Thrombus Formation

Objective-Platelet activation is essential for primary hemostasis and acute thrombotic vascular occlusions. On activation, platelets release their prothrombotic granules and expose phosphatidylserine, thus fostering thrombin generation and thrombus formation. In other cell types, both degranulation and phosphatidylserine exposure are modified by sphingomyelinase-dependent formation of ceramide. The present study thus explored whether acid sphingomyelinase participates in the regulation of platelet secretion, phosphatidylserine exposure, and thrombus formation. Approach and Results-Collagen-related peptide-induced or thrombin-induced ATP release and P-selectin exposure were significantly blunted in platelets from Asm-deficient mice (Smpd1(-/-)) when compared with platelets from wild-type mice (Smpd1(+/+)). Moreover, phosphatidylserine exposure and thrombin generation were significantly less pronounced in Smpd1(-/-) platelets than in Smpd1(+/+) platelets. In contrast, platelet integrin alpha(IIb)beta(3) activation and aggregation, as well as activation-dependent Ca2+ flux, were not significantly different between Smpd1(-/-) and Smpd1(+/+) platelets. In vitro thrombus formation at shear rates of 1700 s(-1) and in vivo thrombus formation after FeCl3 injury were significantly blunted in Smpd1(-/-) mice while bleeding time was unaffected. Asm-deficient platelets showed significantly reduced activation-dependent ceramide formation, whereas exogenous ceramide rescued diminished platelet secretion and thrombus formation caused by Asm deficiency. Treatment of Smpd1(+/+) platelets with bacterial sphingomyelinase (0.01 U/mL) increased, whereas treatment with functional acid sphingomyelinase-inhibitors, amitriptyline or fluoxetine (5 mu mol/L), blunted activation-dependent platelet degranulation, phosphatidylserine exposure, and thrombus formation. Impaired degranulation and thrombus formation of Smpd1(-/-) platelets were again overcome by exogenous bacterial sphingomyelinase. Conclusions-Acid sphingomyelinase is a completely novel element in the regulation of platelet plasma membrane properties, secretion, and thrombus formation

Functional Implications of Novel Human Acid Sphingomyelinase Splice Variants

BACKGROUND: Acid sphingomyelinase (ASM) hydrolyses sphingomyelin and generates the lipid messenger ceramide, which mediates a variety of stress-related cellular processes. The pathological effects of dysregulated ASM activity are evident in several human diseases and indicate an important functional role for ASM regulation. We investigated alternative splicing as a possible mechanism for regulating cellular ASM activity. METHODOLOGY/PRINCIPAL FINDINGS: We identified three novel ASM splice variants in human cells, termed ASM-5, -6 and -7, which lack portions of the catalytic- and/or carboxy-terminal domains in comparison to full-length ASM-1. Differential expression patterns in primary blood cells indicated that ASM splicing might be subject to regulatory processes. The newly identified ASM splice variants were catalytically inactive in biochemical in vitro assays, but they decreased the relative cellular ceramide content in overexpression studies and exerted a dominant-negative effect on ASM activity in physiological cell models. CONCLUSIONS/SIGNIFICANCE: These findings indicate that alternative splicing of ASM is of functional significance for the cellular stress response, possibly representing a mechanism for maintaining constant levels of cellular ASM enzyme activity

An Introduction to Sphingolipid Metabolism and Analysis by New Technologies

Sphingolipids (SP) are a complex class of molecules found in essentially all eukaryotes and some prokaryotes and viruses where they influence membrane structure, intracellular signaling, and interactions with the extracellular environment. Because of the combinatorial nature of their biosynthesis, there are thousands of SP subspecies varying in the lipid backbones and complex phospho- and glycoheadgroups. Therefore, comprehensive or “sphingolipidomic” analyses (structure-specific, quantitative analyses of all SP, or at least all members of a critical subset) are needed to know which and how much of these subspecies are present in a system as a step toward understanding their functions. Mass spectrometry and related novel techniques are able to quantify a small fraction, but nonetheless a substantial number, of SP and are beginning to provide information about their localization. This review summarizes the basic metabolism of SP and state-of-art mass spectrometric techniques that are producing insights into SP structure, metabolism, functions, and some of the dysfunctions of relevance to neuromedicine

Advances in the discovery of N-acylethanolamine acid amidase inhibitors

N-acylethanolamine acid amidase (NAAA) is a cysteine amidase that hydrolyzes saturated or monounsaturated fatty acid ethanolamides, such as palmitoylethanolamide (PEA) and oleoylethanolamide (OEA). PEA has been shown to exert analgesic and anti-inflammatory effects by engaging peroxisome proliferator-activated receptor-α. Like other fatty acid ethanolamides, PEA is not stored in cells, but produced on demand from cell membrane precursors, and its actions are terminated by intracellular hydrolysis by either fatty acid amide hydrolase or NAAA. Endogenous levels of PEA and OEA have been shown to decrease during inflammation. Modulation of the tissue levels of PEA by inhibition of enzymes responsible for the breakdown of this lipid mediator may represent therefore a new therapeutic strategy for the treatment of pain and inflammation. While a large number of inhibitors of fatty acid amide hydrolase have been discovered, few compounds have been reported to inhibit NAAA activity. Here, we describe the most representative NAAA inhibitors and briefly highlight their pharmacological profile. A recent study has shown that a NAAA inhibitor attenuated heat hyperalgesia and mechanical allodynia caused by local inflammation or nerve damage in animal models of pain and inflammation. This finding encourages further exploration of the pharmacology of NAAA inhibitors

Lung-restricted activation of the alveolar macrophage/monocyte system in pulmonary sarcoidosis

An activation of T-cells that is restricted to the lung has been demonstrated in pulmonary sarcoidosis. The role of blood monocytes (MO) and alveolar macrophages (AM) in this concept of compartmentalized inflammation has not yet been evaluated. In order to elucidate this question, we measured the release of tumor necrosis factor alpha (TNF alpha) and interleukin-1 (IL-1) by peripheral blood mononuclear cells (PBMNC) and AM in 43 patients with sarcoidosis (32 with active, 11 with inactive disease) without therapy and correlated the spontaneous monokine release to parameters of the T-cell alveolitis and the course of the disease. TNF alpha as well as IL-1 were spontaneously released by AM of the active group, i.e., 2,385 +/- 735 pg/ml/10(8) cells/24 h and 7/12 (IL-1+/total), respectively. Autologous PBMNC were quiescent, releasing only baseline levels of any monokine. AM were not activated in the inactive group, releasing 500 +/- 212 pg/ml/10(6) cells/24 h TNF alpha, whereas 1/5 were IL-1-positive (p less than 0.05 in both comparisons), which is within the range of the control group. Kinetic experiments revealed that the TNF alpha gene of AM is activated in vivo, resulting in TNF alpha mRNA-positive, TNF alpha-releasing cells that, cultured in vitro, regulate the TNF alpha gene transcription down and cease to release TNF alpha. Interestingly, there is no stringent correlation between the spontaneous release of TNF alpha by AM and signs of T-cell activation as soluble interleukin-2 (IL-2) receptor serum concentration, release of IL-2, and expression of IL-2 receptor by alveolar T-cells.(ABSTRACT TRUNCATED AT 250 WORDS