Blood lymphocyte numbers, essential for the development of efficient immune responses, are maintained by recirculation through secondary lymphoid organs. We show that lymphocyte trafficking is altered by the lysophospholipid sphingosine-1-phosphate (S1P) and by a phosphoryl metabolite of the immunosuppressive agent FTY720. Both species were high-affinity agonists of at least four of the five S1P receptors. These agonists produce lymphopenia in blood and thoracic duct lymph by sequestration of lymphocytes in lymph nodes, but not spleen. S1P receptor agonists induced emptying of lymphoid sinuses by retention of lymphocytes on the abluminal side of sinus-lining endothelium and inhibition of egress into lymph. Inhibition of lymphocyte recirculation by activation of S1P receptors may result in therapeutically useful immunosuppression.

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Sphingosine 1-phosphate (S1P) is a bioactive lysolipid with pleiotropic functions mediated through a family of G proteincoupled receptors, S1P 1,2,3,4,5 . Physiological effects of S1P receptor agonists include regulation of cardiovascular function and immunosuppression via redistribution of lymphocytes from blood to secondary lymphoid organs. The phosphorylated metabolite of the immunosuppressant agent FTY720 (2-amino-2-(2-[4-octylphenyl]ethyl)-1,3-propanediol) and other phosphonate analogs with differential receptor selectivity were investigated. No significant species differences in compound potency or rank order of activity on receptors cloned from human, murine, and rat sources were observed. All synthetic analogs were high-affinity agonists on S1P 1 , with IC 50 values for ligand binding between 0.3 and 14 nM. The correlation between S1P 1 receptor activation and the ED 50 for lymphocyte reduction was highly significant (p Ͻ 0.001) and lower for the other receptors. In contrast to S1P 1 -mediated effects on lymphocyte recirculation, three lines of evidence link S1P 3 receptor activity with acute toxicity and cardiovascular regulation: compound potency on S1P 3 correlated with toxicity and bradycardia; the shift in potency of phosphorylated-FTY720 for inducing lymphopenia versus bradycardia and hypertension was consistent with affinity for S1P 1 relative to S1P 3 ; and toxicity, bradycardia, and hypertension were absent in S1P 3 Ϫ/Ϫ mice. Blood pressure effects of agonists in anesthetized rats were complex, whereas hypertension was the predominant effect in conscious rats and mice. Immunolocalization of S1P 3 in rodent heart revealed abundant expression on myocytes and perivascular smooth muscle cells consistent with regulation of bradycardia and hypertension, whereas S1P 1 expression was restricted to the vascular endothelium.

Neuron, astrocyte, and oligodendrocyte cultures which were established from developing rat brain were examined for their utilization of glucose, ketone bodies, and free fatty acids by oxidative processes. 14CO2 production was measured in these cells from [1-14C] or [6-14C]glucose; [1-14C]octanoate and [1-14C], [6-14C], or [16-14C]palmitate; and [3-14C]acetoacetate and D(-)-3-hydroxy[3-14C]butyrate. Pyruvate dehydrogenase (EC 1.2.4.1.) and 3-oxoacid-CoA transferase (EC 2.8.3.5) activities were found at high levels in each of the cell populations. Astrocytes and oligodendrocytes produced much more 14CO2 from [1-14C]glucose than from [6-14C]glucose, indicating substantial hexose monophosphate shunt activity. This process was not as active in neurons. All three cell populations readily utilized the ketone bodies for oxidative metabolism at rates 7-9 times greater than they utilized glucose. Only astrocytes were able to utilize fatty acids for 14CO2 production, and the rate of utilization was greater than that of the ketone bodies. We found that the metabolic patterns of these brain cells which were derived from the developing brain complement the nature of the diet of the suckling animal which is rich in fat and low in carbohydrate. They readily utilized the ketone bodies or fatty acids and spared glucose for processes that metabolites of fat cannot fulfill.

Three closely related fungal metabolites, The mammalian isoprenoid pathway not only produces sterols but also produces dolichol, ubiquinone, the farnesyl group of heme A, the farnesyl and geranylgeranyl groups of prenylated proteins, and the isopentenyl side chain of isopentenyl adenine. The pathways for the synthesis of these other isoprenoids diverge from the synthesis ofcholesterol either at or before the farnesyl diphosphate (FPP) branch point. Thus, squalene synthase, which catalyzes the reductive dimerization of 2 mol of FPP to 1 mol of squalene (2, 3), is the first committed step in sterol synthesis. A specific inhibitor of squalene synthase should serve to inhibit cholesterol synthesis and not adversely affect the synthesis of other isoprenoids. FPP, the substrate for squalene synthase, is water soluble and may be readily metabolized (4). Thus, squalene synthase offers a potential target for the safe and specific inhibition of cholesterol synthesis.In this report we describe the isolation, structure, physical characterization, and biological properties of three structurally similar fungal metabolites that are potent inhibitors of squalene synthase. These metabolites, zaragozic acid A (5-7), zaragozic acid B (8, 9), and zaragozic acid C (10-12), had been reported previously only in the patent literature; however, during the review process of this manuscript, three manuscripts (13-15) appeared on the squalestatins: squalestatin I is identical with zaragozic acid A, squalestatin II is des-4'-acetylzaragozic acid A, and squalestatin III is des-6-acylzaragozic acid A. This class of squalene synthase inhibitors has potential utility as cholesterol-lowering agents. MATERIALS AND METHODSZaragozic Acid A, Cultures, and Media. An unidentified sterile fungal culture, ATCC 20986, isolated from a water sample taken from the Jalon river in Zaragoza, Spain (hence the name zaragozic acids), was used to produce zaragozic acid A. The culture was maintained at 25°C on medium B agar slants composed of 4 g of yeast extract, 10 g of malt extract, 4 g of dextrose, and 20 g of agar per liter at pH 7.0.Zaragozic acid A was produced in a two-tiered fermentation process consisting of mycelial growth and development in medium A of ref. 1 and product formation in medium C. Medium C contained 5 g of malt extract, 1 g of peptone, 15 g of dextrose, 1 g of KH2PO4, and 0.5 g of MgSO4 7H20 per liter. Fermentations consisted of mycelial growth in medium A for 72 hr at 250C with agitation, followed by inoculation (5-10%) of medium C. Maximum product was obtained from 14-day agitated fermentations at 250C.Isolation of Zaragozic Acid A. To isolate zaragozic acid A, 23 liters of harvested broth was filtered through Celite, and the mycelial cake was extracted twice with 7 liters of 50%o aqueous methanol. The filtrate was combined with the extracts, diluted with water to a final composition of 25% methanol, and adsorbed on a 1.5-liter column of Mitsubishi HP-20 resin. After a column wash with 6 liters of4:6 (vol/vol) methanol/water, crud...

Sphingosine and sphingosine-1-phosphate (SPP) are interconvertible sphingolipid metabolites with opposing effects on cell growth and apoptosis. Based on sequence homology with LBP1, a lipid phosphohydrolase that regulates the levels of phosphorylated sphingoid bases in yeast, we report here the cloning, identification, and characterization of a mammalian SPP phosphatase (mSPP1). This hydrophobic enzyme, which contains the type 2 lipid phosphohydrolase conserved sequence motif, shows substrate specificity for SPP. Partially purified Myc-tagged mSPP1 was also highly active at dephosphorylating SPP. When expressed in yeast, mSPP1 can partially substitute for the function of LBP1. Membrane fractions from human embryonic kidney HEK293 cells transfected with mSPP1 markedly degraded SPP but not lysophosphatidic acid, phosphatidic acid, or ceramide-1-phosphate. Enforced expression of mSPP1 in NIH 3T3 fibroblasts not only decreased SPP and enhanced ceramide levels, it also markedly diminished survival and induced the characteristic traits of apoptosis. Collectively, our results suggest that SPP phosphohydrolase may regulate the dynamic balance between sphingolipid metabolite levels in mammalian cells and consequently influence cell fate.S phingosine-1-phosphate (SPP) is a bioactive sphingolipid metabolite that regulates diverse biological processes (reviewed in refs. 1 and 2). Many of its pleiotropic actions appear to be mediated by a family of specific cell surface G proteincoupled receptors (GPCR), known as EDG (endothelial differentiation genes) receptors. Binding of SPP to EDG-1 expressed on endothelial cells enhances survival (3), chemotaxis, and in vitro angiogenesis (4), and adherens junction assembly leading to morphogenetic differentiation (5), whereas binding of SPP to EDG-5 and EDG-3 induces neurite retraction and soma rounding (6, 7). SPP induces activation of G i -gated inward rectifying K ϩ -channels in atrial myocytes (8) and inhibits motility of melanoma cells (9) through as yet uncharacterized GPCRs.SPP also plays important roles inside cells. In response to diverse external stimuli, sphingosine kinase, the enzyme that catalyzes the phosphorylation of sphingosine to SPP, is activated (10-15). Intracellular SPP, in turn, mobilizes calcium from internal stores independently of inositol triphosphate (15, 16), as well as eliciting diverse signaling pathways leading to proliferation (17, 18) and suppression of apoptosis (19)(20)(21)(22).Because of its dual function as a ligand and second messenger and its pivotal role in cell growth and survival, the synthesis and degradation of SPP should be tightly regulated in a spatialtemporal manner. Until recently, however, little was known of the enzymes involved in SPP metabolism. We have purified sphingosine kinase to apparent homogeneity from rat kidney (23) and subsequently cloned and characterized a mammalian sphingosine kinase (24), which belongs to a highly conserved gene family (24,25). Enforced expression of sphingosine kinase markedly enhanced proliferation a...

Inducible NO synthase (iNOS) present in human atherosclerotic plaques could contribute to the inflammatory process of plaque development. The role of iNOS in atherosclerosis was tested directly by evaluating the development of lesions in atherosclerosis-susceptible apolipoprotein E (apoE)−/− mice that were also deficient in iNOS. ApoE−/− and iNOS−/− mice were cross-bred to produce apoE−/−/iNOS−/− mice and apoE−/−/iNOS+/+ controls. Males and females were placed on a high fat diet at the time of weaning, and atherosclerosis was evaluated at two time points by different methods. The deficiency in iNOS had no effect on plasma cholesterol, triglyceride, or nitrate levels. Morphometric measurement of lesion area in the aortic root at 16 wk showed a 30–50% reduction in apoE−/−/iNOS−/− mice compared with apoE−/−/iNOS+/+ mice. Although the size of the lesions in apoE−/−/iNOS−/− mice was reduced, the lesions maintained a ratio of fibrotic:foam cell-rich:necrotic areas that was similar to controls. Biochemical measurements of aortic cholesterol in additional groups of mice at 22 wk revealed significant 45–70% reductions in both male and female apoE−/−/iNOS−/− mice compared with control mice. The results indicate that iNOS contributes to the size of atherosclerotic lesions in apoE-deficient mice, perhaps through a direct effect at the site of the lesion.

Recent work has revealed correlations between bacterial or viral infections and atherosclerotic disease. One particular bacterium, Chlamydia pneumoniae, has been observed at high frequency in human atherosclerotic lesions, prompting the hypothesis that infectious agents may be necessary for the initiation or progression of atherosclerosis. To determine if responses to gram-negative bacteria are necessary for atherogenesis, we first bred atherosclerosis-prone apolipoprotein (apo) E−/− (deficient) mice with animals incapable of responding to bacterial lipopolysaccharide. Atherogenesis was unaffected in doubly deficient animals. We further tested the role of infectious agents by creating a colony of germ-free apo E−/− mice. These animals are free of all microbial agents (bacterial, viral, and fungal). Atherosclerosis in germ-free animals was not measurably different from that in animals raised with ambient levels of microbial challenge. These studies show that infection is not necessary for murine atherosclerosis and that, unlike peptic ulcer, Koch's postulates cannot be fulfilled for any infectious agent in atherosclerosis.