Sphingosine reverses growth inhibition caused by activation of protein kinase C in vascular smooth muscle cells.

In certain cell systems, including neonatal vascular smooth muscle (VSM) cells, phorbol esters are growth inhibitory. Here we show that 1,2-dioctanoyl-sn-glycerol (DiC8), when added 2 h after alpha-thrombin, reverses by greater than 95% the induction of DNA synthesis in VSM cells by alpha-thrombin. Sphingosine, a naturally occurring lysosphingolipid inhibitor of protein kinase C, and its synthetic analogues N-acetylsphingosine and C11-sphingosine were used to investigate this phenomenon further. Neither phorbol 12-myristate 13-acetate (PMA;200 ng/ml) nor sphingosine (up to 10 microM) alone had any effect upon basal DNA synthesis in VSM cells. Like DiC8, PMA totally blocked the induction of DNA synthesis by alpha-thrombin. This inhibitory effect of PMA was reversed by sphingosine in a dose-dependent manner with complete reversal at 10 microM. Neither N-acetylsphingosine nor C11-sphingosine exhibited any effect on DNA synthesis in VSM cells. The effect of sphingosine and its analogues on the activity of protein kinase C extracted from VSM cells was measured by histone III-S phosphorylation. Protein kinase C activity was inhibited 50% by 300 microM sphingosine, but less than 15% by similar concentrations of N-acetylsphingosine and C11-sphingosine. To assess the effects of sphingosine and analogues on protein kinase C in intact cells, we examined the effect of the lipids on [3H]phorbol dibutyrate binding. Sphingosine (at greater than 5 microM), but not N-acetylsphingosine or C11-sphingosine, blocked [3H]phorbol dibutyrate binding in a dose- and time-dependent fashion. Thus the mechanism of growth inhibition by DiC8 and PMA in neonatal VSM cells appears to be through activation of protein kinase C by these compounds. Sphingosine reverses this growth inhibition through interference with the binding to protein kinase C of phorbol esters or other activators of this enzyme.     

Mechanism involved in the mobilization of neutrophil calcium by 5-hydroxyeicosatetraenoate

5-Hydroxyeicosatetraenoate (5-HETE), like leukotriene B4 and platelet-activating factor, stimulated human polymorphonuclear neutrophils to mobilize intracellular calcium. The three compounds acted through mechanisms that were inhibited by pertussis toxin, cholera toxin, and PMA. Each agonist, furthermore, desensitized (or down-regulated) the neutrophil's calcium mobilization response to a second challenge with the same agonist. However, 5-HETE and leukotriene B4 had little or no activity in cross-desensitizing neutrophil responses to each other or to platelet-activating factor. Furthermore, 5-HETE interfered minimally or not at all with the binding of radiolabeled leukotriene B4 and platelet-activating factor to their respective receptors on neutrophils. Thus, 5-HETE mobilizes neutrophil calcium by a mechanism different from those used by leukotriene B4 and platelet-activating factor. This mechanism appears to involve specific 5-HETE receptors that couple to pertussis toxin-inhibitable, GTP-binding proteins.     

Chemistry and biological activities of constituents from Morus australis.

A novel constituent named australone B (1) was further isolated from the cortex of Morus australis (Moraceae). The structure of 1 has been elucidated by one- and two-dimension spectra. In human citrated platelet-rich plasma, 1 showed strong inhibition of aggregation induced by adrenaline in a concentration-dependent manner with an IC(50) value of about 33.3 microM. Compound 1 (30 microM) also showed inhibitory effects on superoxide anion formation from rat neutrophils stimulated with formyl-Met-Leu-Phe (fMLP)/cytochalasin B (CB). Morusin (2) inhibited superoxide anion formation from rat neutrophils stimulated with phorbol myristate acetate (PMA) in a concentration-dependent manner with an IC(50) value of 66.9+/-2.5 microM.     

The chymotrypsin inhibitor carbobenzyloxy-leucine-tyrosine-chloromethylketone interferes with the neutrophil respiratory burst mediated by a signaling pathway independent of PtdInsP2 breakdown and cytosolic free calcium.

The effects of carbobenzyloxy-leucine-tyrosine-chloromethylketone (zLYCK), an inhibitor of chymotrypsin, were investigated on the activation pathways of the human neutrophil respiratory burst. At 10 microM zLYCK, a parallel inhibition was observed of superoxide production stimulated with the chemo-attractant FMLP and of chymotrypsin-like activity of human neutrophils. By contrast, superoxide production induced by PMA was minimally affected by zLYCK. The known transduction pathways triggered by FMLP were analyzed. zLYCK did not affect either the FMLP-induced cytosolic free calcium transient, inositol 1,4,5 trisphosphate formation, nor the PMA-induced phosphorylation of the 47-kDa substrate of protein kinase C. zLYCK did not affect the activity of protein kinase C extracted from neutrophils. In Ca(2+)-depleted cells, in which phosphatidylinositol 4,5-biphosphate breakdown does not occur, zLYCK inhibited the FMLP-induced respiratory burst in cells primed by low doses of PMA. The activity of the NADPH oxidase tested with active membranes from stimulated neutrophils or in a cell-free system was not inhibited by zLYCK. We conclude that: 1) zLYCK inhibits superoxide production through the inhibition of a chymotrypsin-like protease of the neutrophil, 2) zLYCK inhibits FMLP-induced activation of NADPH oxidase through a pathway independent of PtdInsP2 breakdown and cytosolic free calcium, and 3) zLYCK may prove a useful probe for the characterization of its target protease in neutrophil activation.     

Regulation of phosphoinositide hydrolysis in cultured astrocytes by sphingosine and psychosine.

The effects of sphingosine and psychosine on phosphoinositide hydrolysis in primary cultured astrocytes were determined. Exposure to sphingosine produced a dose-dependent stimulation of phosphoinositide hydrolysis requiring the presence of external Ca++ for optimal activity. The addition of 10 microM norepinephrine resulted in a stimulation additional to that with sphingosine. The alpha 1-antagonist prazosin completely inhibited norepinephrine-induced phosphoinositide hydrolysis but had no effect on that produced by sphingosine. Psychosine (108 microM), when co-incubated with sphingosine, produced complete inhibition of sphingosine-induced phosphoinositide hydrolysis at all doses of sphingosine tested (33-668 microM). Likewise, psychosine totally inhibited norepinephrine-induced phosphoinositide hydrolysis. The protein kinase C inhibitor staurosporine (1 microM) had no effect on sphingosine-induced phosphoinositide hydrolysis. These findings suggest that lysosphingolipids such as sphingosine and psychosine may play an important role in the regulation of phosphoinositide turnover in astrocytes by a mechanism dependent on extracellular Ca++ and independent of the alpha 1-adrenergic receptor and protein kinase C.     

Protein kinase C inhibition by sphingoid long-chain bases: effects on secretion in human neutrophils

Sphingoid long-chain bases (sphinganine and sphingosine) have recently been shown to inhibit protein kinase C both in vitro [Y. Hannun et al. (1986) J. Biol. Chem. 261, 12604-12609] and in intact human neutrophils, in which they block activation of the superoxide-generating respiratory burst [E. Wilson et al. (1986) J. Biol. Chem. 261, 12616-12623]. In the present study we have used sphingosine to investigate the pathways for agonist-induced secretion of neutrophil granule contents. Induction of secretion of the specific granule component lactoferrin by a variety of agonists [phorbol 12-myristate-13-acetate (PMA), formyl-methionyl-leucyl-phenylalanine (fMLP), and calcium ionophore A23187] was completely inhibited by sphingosine with an ED50 of 6 to 10 microM. PMA-induced secretion of lysozyme (present in both the azurophilic and specific granules) was completely blocked with an ED50 of 10 microM, whereas fMLP-induced secretion was only about 50% inhibited. Secretion of the azurophilic granule proteins beta-glucuronidase and myeloperoxidase was activated by fMLP and A23187, but not by PMA, and was not affected by sphingosine. The use of A23187 in the presence of sphingosine allowed differentiation between calcium activation of protein kinase C-dependent versus-independent pathways. The effect of sphingosine was not mediated by neutralizing intracellular acidic compartments, since treatment of neutrophils with inhibitory concentrations of sphingosine did not significantly alter the uptake of labeled methylamine. We conclude that at least two mechanisms participate in the regulation of specific and azurophilic granule secretion, respectively: a protein kinase C-dependent pathway and a calcium-dependent pathway which does not involve protein kinase C.     

Cilostazol suppresses adhesion of human neutrophils to HUVECs stimulated by FMLP and its mechanisms

The interaction between neutrophils and endothelial cells (ECs) is of great importance in many physiological and pathological progresses. Although cilostazol (CLZ), a novel selective phosphodiesterase (PDE) type 3 inhibitor, has been proved to be useful in vasodilatation and inhibition of platelet aggregation, its effect on adhesion is not clearly known. In this study, we examined the effects and investigated the mechanisms of cilostazol on neutrophil adhesion to human umbilical endothelial cells (HUVECs) triggered by N-formyl-methionyl-leucyl-phenylal-anine (FMLP), a chemotactic peptide. The soluble vascular cell adhesive molecule-1 (sVCAM-1) release from FMLP (10 microM)-stimulated HUVECs was determined by ELISA kits. Fluo-2, a fluorescent indicator, was used to investigate intracellular free calcium concentration ([Ca2+]i) in HUVECs. HL-60 cells were induced to be neutrophilic by DMSO and loaded with Fluo-3, another fluorescent indicator, to detect [Ca2+]i, and CLA was used as a chemiluminescent indicator to determine superoxide production in neutrophilic cells. The result showed that CLZ (1-100 microM) significantly inhibited neutrophil adhesion to FMLP-stimulated HUVECs. In HUVECs, CLZ obviously downregulated sVCAM-1 level, while it had no meaningful influence [Ca2)]i. But in neutrophils, FMLP-activated superoxide generation and [Ca2+]i increase were found being inhibited by exposure to CLZ . Furthermore, we also demonstrated that Ca2+ increase was preceded to the superoxide generation in neutrophils. The results suggest that CLZ involves in adhesion reactions between neutrophil and ECs, partly via VCAM-1 expression in ECs, and decreasing [Ca2+]i induced activation of neutrophils, which means a lot to prevent atherosclerosis and other cardiovascular diseases.     

Tumor necrosis factor-alpha-mediated signal transduction in human neutrophils: involvement of sphingomyelin metabolites in the priming effect of TNF-alpha on the fMLP-stimulated superoxide production

We investigated the mechanism underlying the priming effect of TNF-alpha on fMLP-stimulated superoxide production in human neutrophils. TNF-alpha enhanced fMLP-stimulated superoxide production in a concentration-dependent manner. TNF-alpha also induced sphingomyelin (SM) hydrolysis and increased the formation of its metabolite, sphingosine-1-phosphate (SP-1-P). The treatment of neutrophils with sphingomyelinase also resulted in a similar priming effect. C2 ceramide produced a concentration-dependent inhibition of fMLP-stimulated superoxide production within the concentration range of 1-30 microM. Sphingosine had a dual effect on fMLP-stimulated superoxide generation, exhibiting a priming effect at lower concentrations (0.2-1 microM), but an inhibitory effect at higher concentrations (1-30 microM). SP-1-P (1-30 microM), showed a concentration-dependent enhancement of fMLP stimulated superoxide production. Furthermore, after treating neutrophils with DL-threo-dihydro-sphingosine, a competitive inhibitor of sphingosine kinase, TNF-alpha produced a similar dual effect as observed with sphingosine. These results strongly suggest that SM hydrolysis plays a key role in the intracellular signal transduction mediating the TNF-alpha-mediated priming effect.     

ONO-4057, a novel, orally active leukotriene B4 antagonist: effects on LTB4-induced neutrophil functions.

ONO-4057(5-[2-(2-Carboxyethyl)-3-[6-(4-methoxyphenyl)-5E- hexenyl]oxyphenoxy]valeric acid), an orally active leukotriene B4(LTB4) antagonist, displaced the binding of [3H] LTB4 to the LTB4 receptor in human neutrophil (Ki = 3.7 +/- 0.9 nM). ONO-4057 inhibited the LTB4-induced rise in cytosolic free calcium (the concentration causing 50% inhibition (IC50) = 0.7 +/- 0.3 microM) and inhibited human neutrophil aggregation, chemotaxis or degranulation induced by LTB4 (IC50 = 3.0 +/- 0.1, 0.9 +/- 0.1 and 1.6 +/- 0.1 microM) without showing any agonist activity at concentration up to 30 microM. ONO-4057 did not inhibit fMLP or C5a-induced neutrophil activation at concentrations up to 30 microM. In the in vivo study, ONO-4057 given orally, prevented LTB4-induced transient neutropenia or intradermal neutrophil migration in guinea pig (the dose causing 50% efficacy (ED50) = 25.6mg/kg or 5.3mg/kg). Furthermore, ONO-4057 given topically, suppressed phorbol-12-myristate-13-acetate (PMA)-induced neutrophil infiltration in guinea pig ear (the effective dose = 1 mg/ear). These results indicate that ONO-4057 is a selective and orally active LTB4 antagonist and may be a potential candidate for the treatment of various inflammatory diseases.     

Auranofin affects early events in human polymorphonuclear neutrophil activation by receptor-mediated stimuli

Auranofin, a new oral antirheumatic gold compound, in concentrations achieved therapeutically, inhibits neutrophil phagocytosis, chemotaxis, chemiluminescence, reduction of cytochrome c, and release of lysosomal enzymes. To further characterize the mechanism by which auranofin affects neutrophils, we studied the effects of auranofin on unstimulated properties and functions of neutrophils as well as on rapidly stimulated functions. When examined by electron microscopy, 4 micrograms/ml of auranofin significantly decreased the number of visualized centriole-associated microtubules in resting cells. Furthermore, auranofin inhibited neutrophil spreading on glass and caused a decrease in negative surface charge (electrophoretic mobility). In addition, auranofin inhibited several fmet-leu-phe-stimulated responses such as shape change, increases in centriole-associated microtubules, decreases in surface charge, and elicited membrane potential changes (di-O-C5(3) dye response). Auranofin (1 micrograms/ml) inhibited fmet-leu-phe-stimulated superoxide and hydrogen peroxide production by 80% (p less than 0.05), and also increased the affinity of receptors for fmet-leu-phe (from Ka 0.035 to Ka 0.48, p less than 0.001). Auranofin also affected neutrophil responses to phorbol myristic acetate (PMA). The total amount of PMA-stimulated superoxide production was suppressed by as little as 0.4 micrograms/ml of auranofin, but the lag time for activation was shortened by low concentrations of auranofin (0.5 to 1 microgram/ml). Four micrograms per milliliter of auranofin suppressed the decrease in surface charge induced by PMA. However, auranofin did not influence superoxide production elicited by the ionophore A23187. The results indicate that auranofin affects the earliest detected responses in neutrophil activation by certain receptor-mediated stimuli.     

Evidence that a receptor-operated event on the neutrophil mediates neutrophil accumulation in vivo. Pretreatment of 111In-neutrophils with pertussis toxin in vitro inhibits their accumulation in vivo

The role of neutrophil chemoattractant receptors in neutrophil stimulation in vitro is well established, however, the precise mechanisms underlying local neutrophil accumulation at inflammatory sites in vivo have not been defined. A fundamental question that remains open is whether chemoattractants act on the endothelial cell or the neutrophil to initiate the process of neutrophil migration in vivo. To address this question we have investigated whether neutrophil accumulation in vivo can occur if chemoattractant receptor occupancy is uncoupled from neutrophil stimulation. For this purpose we have used pertussis toxin (PT) as the pharmacologic tool. We have investigated the effect of in vitro pretreatment of rabbit neutrophils with PT on their responses in vitro and on their accumulation in vivo. Pretreatment of rabbit neutrophils with PT inhibited FMLP- and C5a-, but not PMA- induced increases in CD18 expression, neutrophil adherence, and degranulation in vitro. This pretreatment procedure with PT inhibited the accumulation of radiolabeled neutrophils in vivo in response to intradermally injected FMLP, C5a, C5a des Arg, leukotriene B4, IL-8, and zymosan in rabbit skin. Further, in contrast to the in vitro results, PT inhibited the PMA-induced 111In-neutrophil accumulation in vivo. Interestingly, pretreatment of neutrophils with PT also inhibited accumulation in response to intradermally injected IL-1, despite the reports that IL-1 lacks neutrophil chemoattractant activity in vitro. Although the experimental techniques used cannot distinguish the different stages of neutrophil migration involved, these results suggest that the accumulation of neutrophils induced by local extravascular chemoattractants in vivo depends on a pertussis toxin-sensitive receptor operated event on the neutrophil itself. Further, PMA and IL-1 may release secondary chemoattractants in vivo.     

Sphingosine Toxicity in EAE and MS: Evidence for Ceramide Generation via Serine-Palmitoyltransferase Activation

Multiple sclerosis (MS) is a demyelinating disorder characterized by massive neurodegeneration and profound axonal loss. Since myelin is enriched with sphingolipids and some of them display toxicity, biological function of sphingolipids in demyelination has been investigated in MS brain tissues. An elevation of sphingosine with a decrease in monoglycosylceramide and psychosine (myelin markers) was observed in MS white matter and plaque compared to normal brain tissue. This indicated that sphingosine toxicity might mediate oligodendrocyte degeneration. To explain the source of sphingosine accumulation, total sphingolipid profile was investigated in Lewis rats after inducing experimental autoimmune encephalomyelitis (EAE) and also in human oligodendrocytes in culture. An intermittent increase in ceramide followed by sphingosine accumulation in EAE spinal cord along with a stimulation of serine-palmitoyltransferase (SPT) activity was observed. Apoptosis was identified in the lumbar spinal cord, the most prominent demyelinating area, in the EAE rats. TNFα and IFNγ stimulation of oligodendrocytes in culture also led to an accumulation of ceramide with an elevation of sphingosine. Ceramide elevation was drastically blocked by myriocin, an inhibitor of SPT, and also by FTY720. Myriocin treatment also protected oligodendrocytes from cytokine mediated apoptosis or programmed cell death. Hence, we propose that sphingosine toxicity may contribute to demyelination in both EAE and MS, and the intermittent ceramide accumulation in EAE may, at least partly, be mediated via SPT activation, which is a novel observation that has not been previously reported.     

Synthetic, non-natural sphingolipid analogs inhibit the biosynthesis of cellular sphingolipids, elevate ceramide and induce apoptotic cell death

Numerous studies have demonstrated the participation of sphingolipids in signal transduction and regulation of cell growth. Several cellular stress agents have been shown to elevate ceramide, the basic precursor of all sphingolipids, initiating a cascade of events leading to arrest of the cell cycle, apoptosis and cell death. Aiming at inhibiting metabolic pathways of sphingolipid metabolism that might lead to an increase of cellular ceramide, we have synthesized non-natural analogs of ceramide, sphingosine and trimethylsphingosine. When the respective analogs were applied to HL60 human myeloid leukemic cells they inhibited the biosynthesis of sphingomyelin (SPM) and glycosphingolipids and induced apoptosis that led to cell death. A fluorescent procedure which has been developed for quantifying the biosynthesis of cellular ceramide indicated an increase in the ceramide content following an incubation with the synthetic analogs. These results suggest that the newly synthesized sphingolipid analogs might be valuable for potential application as a therapeutic modality in leukemia and other malignancies.     

Activation of human neutrophils by Mycobacterium tuberculosis-derived sulfolipid-1.

The principal sulfatide of a group of acidic lipids from virulent Mycobacterium tuberculosis, sulfolipid-1 (SL-1), stimulates neutrophil superoxide (O2-) generation and, at lower concentrations, primes neutrophil response to several other metabolic agonists including FMLP, and PMA. These responses to SL-1 were examined in relation to diacylglycerol (DAG) generation, Ca2+ availability and activation of guanine nucleotide binding proteins to clarify the signal transduction pathways involved. Pertussis toxin inhibited the ability of SL-1 to both stimulate neutrophils directly and to prime neutrophils for subsequent responses induced by PMA, suggesting a role for one or more guanine nucleotide regulating proteins in both responses. SL-1 induced a rise in neutrophil DAG levels. DAG generation was inhibited by pretreatment of cells with pertussis toxin. Depletion of extracellular Ca2+ ablated O2- release induced by stimulatory levels of SL-1 but did not inhibit the priming effect induced by substimulatory concentrations of the lipid. Investigation of the activation of the neutrophil NADPH oxidase in a cell-free system revealed that the SL-1-priming effect was associated with translocation of the soluble cytosolic factors required for activation of the enzyme. Cytosolic factor translocation was not observed in pertussis toxin pretreated cells. Our results provide evidence for the role of a guanine nucleotide binding protein in both priming and direct activation of neutrophils by SL-1. This G protein regulates both SL-1-induced DAG generation and cytosolic cofactor translocation involved in neutrophil activation and priming. The multiplicity of effects of SL-1 on signal transduction pathways leading to phagocyte activation and priming may exert a profound influence on the pathogenicity of M. tuberculosis.     

Inhibition of sphingolipid biosynthesis by fumonisins. Implications for diseases associated with Fusarium moniliforme

Culture materials and grains contaminated with certain isolates of Fusarium moniliforme cause equine leucoencephalomalacia, porcine pulmonary edema syndrome, and liver cancer in rats. The causative agents are thought to be a family of compounds called fumonisins, which bear considerable structural similarity to the long-chain (sphingoid) base backbones of sphingolipids. Incubation of rat hepatocytes with fumonisins inhibited incorporation of [14C]serine into the sphingosine moiety of cellular sphingolipids with an IC50 of 0.1 microM for fumonisin B1. In contrast, fumonisin B1 increased the amount of the biosynthetic intermediate sphinganine, which suggests that fumonisins inhibit the conversion of [14C]sphinganine to N-acyl-[14C]sphinganines, a step that is thought to precede introduction of the 4,5-trans double bond of sphingosine (Merrill, A.H., Jr. and Wang, E. (1986) J. Biol. Chem. 261, 3764-3769). In agreement with this mechanism, fumonisin B1 inhibited the activity of sphingosine N-acyltransferase (ceramide synthase) in rat liver microsomes with 50% inhibition at approximately 0.1 microM and reduced the conversion of [3H]sphingosine to [3H]ceramide by intact hepatocytes. As far as we are aware, this is the first discovery of a naturally occurring inhibitor of this step of sphingolipid metabolism. These findings suggest that disruption of the de novo pathway of sphingolipid biosynthesis may be a critical event in the diseases that have been associated with consumption of fumonisins.     

Effect of six compounds isolated from rhizome of Anemone raddeana on the superoxide generation in human neutrophil

The effect of six compounds isolated from rhizome of Anemone raddeana on the superoxide generation in human neutrophils was investigated. The six compounds examined were 3-acetyloleanolic acid (AOA), oleanolic acid (OA), eleutheroside K (EK), oleanolic acid-3-O-alpha-L-rhamnopyranosyl-(1 --> 2)-[beta-D-glucopyranosyl-(1 --> 4)]-alpha-L-arabinopyranoside (Rd10), raddeanoside 12 (Rd12) and raddeanoside 13 (Rd13). AOA, OA, Rd12 and Rd13 suppressed the superoxide generation induced by N-formyl-methionyl-leucyl-phenylalanine (fMLP) in a concentration-dependent manner. EK and Rd10 significantly enhanced the fMLP-induced superoxide generation in a specific narrow range of low concentration (0.5-0.75 microM), while these compounds more efficiently suppressed the superoxide generation than the other four compounds in other concentrations. In the case of superoxide generation induced by phorbol 12-myristate 13-acetate (PMA), Rd12, OA, EK and Rd10 dose-dependently suppressed the superoxide generation but AOA and Rd13 gave no effect. Arachidonic acid-induced superoxide generation was suppressed by EK, Rd10, Rd12 and Rd13, but was weakly enhanced by AOA and OA. Rd12 dose-dependently inhibited fMLP-induced tyrosyl phosphorylation of 123.0, 79.4, 60.3, 56.2 and 50.1 kDa proteins in human neutrophil. On the other hand, RD10 and EK enhanced the tyrosyl phosphorylation of these proteins in a low concentration range. These phenomena were parallel to the suppression of the fMLP-induced superoxide generations.     

Weak inhibition of protein kinase C coupled with various non-specific effects make sphingosine an unsuitable tool in platelet signal transduction studies

The effects of sphingosine, the newly described inhibitor of the enzyme protein kinase C, on human platelet activation, were studied in order to gain further information on the role of protein kinase in platelet responses. Concentrations of the drug (5-20 microM) which had little effect on protein kinase C activation as measured by the phosphorylation of the 45 kDa and 20 kDa protein substrates induced by phorbol 12-myristate 13-acetate (PMA) and thrombin, strongly inhibited platelet aggregation induced by these agonists, as well as aggregation induced by ADP and ionomycin, which caused no detectable protein kinase C activation or 5-hydroxy[14C]tryptamine[( 14C]5HT) secretion. At approx. 10-fold higher concentrations (150-200 microM), sphingosine had significant inhibitory effects on PMA and thrombin-induced 45 kDa and 20 kDa protein phosphorylation. However, at these high concentrations, the drug caused extensive membrane damage/leakiness as suggested by the substantial release of [14C]5HT and [3H]adenine from pre-loaded platelets (50-70% release of both markers), and the total quenching of quin2 fluorescence by Mn2+ in the presence of the drug. Due to the increased membrane leakiness in the presence of the drug, an apparent potentiation of agonist-induced intracellular Ca2+ elevations in quin2-loaded platelets, as well as an increase in quin2 fluorescence with the drug alone (more than 50 microM) were also observed. Despite this, however, thrombin-induced [3H]arachidonate release was severely reduced in the presence of sphingosine, underlining the inhibitory effects at the membrane level. It is concluded that the weak, if any, inhibitory effects on protein kinase C at concentrations not affecting membrane integrity, as well as the inhibitory effects of sphingosine on platelet aggregation, make it an unsuitable compound as a tool for studies on platelet stimulus-response coupling.     

SC-41930: an inhibitor of leukotriene B4-stimulated human neutrophil functions

SC-41930 was evaluated for effects on human neutrophil chemotaxis and degranulation. At concentrations up to 100 microM, SC-41930 alone exhibited no effect on neutrophil migration, but dose-dependently inhibited neutrophil chemotaxis induced by leukotriene B4 (LTB4) in a modified Boyden chamber. Concentrations of SC-41930 from 0.3 microM to 3 microM competitively inhibited LTB4-induced chemotaxis with a pA2 value of 6.35. While inactive at 10 microM against C5a-induced chemotaxis, SC-41930 inhibited N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced chemotaxis, with 10 times less potency than against LTB4-induced chemotaxis. SC-41930 inhibited [3H]LTB4 and [3H]fMLP binding to their receptor sites on human neutrophils with KD values of 0.2 microM and 2 microM, respectively. SC-41930 also inhibited neutrophil chemotaxis induced by 20-OH LTB or 12(R)-HETE. At concentrations up to 10 microM, SC-41930 alone did not cause neutrophil degranulation, but inhibited LTB4-induced degranulation in a noncompetitive manner. SC-41930 also inhibited fMLP- or C5a-induced degranulation, but was about 8 and 10 times less effective for fMLP and C5a, respectively. The results indicate that SC-41930 is a human neutrophil LTB4 receptor antagonist with greater specificity for LTB4 than for fMLP or C5a receptors.     

Involvement of guanine nucleotides in superoxide release by fluoride-treated neutrophils. Implications for a role of a guanine nucleotide regulatory protein

Previous studies demonstrating hydrolysis of phosphatidylinositol bisphosphate (PIP2) and generation of inositol phosphates in neutrophils exposed to 20.0 mM NaF provide indirect evidence that activation of phospholipase-associated guanine nucleotide regulatory protein, a guanine nucleotide binding protein which regulates the activation of a membrane inositol-specific phospholipase C, is an early event in the neutrophil stimulus-response pathway triggered by fluoride. Consistent with this hypothesis, exposure of a plasma membrane rich preparation isolated from 32P labeled neutrophils to 20.0 mM NaF resulted in hydrolysis of labeled PIP2. Levels of other phospholipids were not affected. Inositol bisphosphate and inositol trisphosphate were detected in extracts of neutrophil plasma membranes exposed to fluoride. To further explore the involvement of guanine nucleotides in functional responses of intact neutrophils triggered by fluoride, we preincubated cells with 2-beta-D-ribofuranosylthiazole-4-carboxamide (tiazofurin), a selective inhibitor of inosine monophosphate dehydrogenase, to diminish guanine nucleotide synthesis and then compared superoxide generation induced by FMLP, PMA, digitonin, and 20.0 mM NaF to intracellular levels of guanine nucleotides. Preincubation of neutrophils for 2.5 h at 37 degrees C with tiazofurin resulted in dose-dependent depletion of GTP and GDP. Maximal depletion of guanine nucleotides required relatively high levels of tiazofurin (200 to 400 microM) and resulted in a 55 to 60% reduction of GTP and GDP. The effects of tiazofurin on guanine nucleotides levels were not observed when neutrophils were preincubated at 4 degrees C. AT 37 degrees C, tiazofurin also decreased intracellular ATP and ADP levels but adenine nucleotide depletion was less pronounced than guanine nucleotide depletion for each concentration of tiazofurin used. When tiazofurin was removed by washing cells after incubation, adenine nucleotide quickly returned to preincubation values but guanine nucleotide levels remained depressed. Addition of exogenous guanosine (200 microM) prevented tiazofurin-dependent depletion of guanine nucleotides but had no influence on adenine nucleotide depletion. Superoxide released triggered by FMLP and F- was inhibited to an extent similar to that of guanine nucleotide depletion under different conditions of preincubation. Inhibition of superoxide release was not observed if cells were preincubated at 4 degrees C, was not rapidly reversible, and was not observed when guanosine was added with tiazofurin.(ABSTRACT TRUNCATED AT 400 WORDS)