Localization of neutral ceramidase in caveolin-enriched light membranes of murine endothelial cells.

Sphingomyelinase (SMase) and ceramidase (CDase) activities participate in sphingomyelin (SM) metabolism and have a role in the signal transduction of a variety of ligands. In this study evidence is presented that caveolin-enriched light membranes (CELMs) of murine endothelial cells, characterized by high SM, ceramide (Cer) and cholesterol content, bear acid and neutral SMase as well as neutral CDase activities. Localization of neutral CDase in CELMs was confirmed by Western analysis. Notably, cell treatment with cyclodextrin, which depleted cell cholesterol, did not affect acid or neutral SMase activities but significantly enhanced neutral CDase activity in CELMs, indicating a negative role for cholesterol in CDase regulation. These findings suggest that neutral CDase is implicated, together with SMase activities, in the control of caveolar Cer content that may be critical for caveola dynamics.

Dual regulation of sphingosine 1-phosphate-induced phospholipase D activity through RhoA and protein kinase C-alpha in C2C12 myoblasts.

Previous studies showed that in C2C12 cells, phospholipase D (PLD) and its known regulators, RhoA and protein kinase Calpha (PKCalpha), were downstream effectors in sphingosine 1-phosphate (SPP) signalling. Moreover, the role of PKC for SPP-mediated PLD activation and the requirement of PKCalpha for RhoA translocation were reported. The present results demonstrated that inactivation of RhoA, by overexpression of RhoGDP dissociation inhibitor (RhoGDI) as well as treatment with C3 exotoxin, attenuated SPP-stimulated PLD activity, supporting the involvement of RhoA in the stimulation of PLD activity by the bioactive lipid in C2C12 myoblasts. In addition, the effect of PKCalpha inhibitor Gö6976 on the SPP-induced PLD activation in myoblasts, where RhoA function was inactivated, was consistent with a dual regulation of the enzyme through RhoA and PKCalpha. Interestingly, the subcellular distribution of PLD isoforms, RhoA and PKCalpha, in SPP-stimulated cells supported the view that the functional relationship between the two PLD regulators, demonstrated to occur in SPP signalling, represents a novel mechanism of regulation of specifically localized PLD.

Neutral/alkaline and acid ceramidase activities are actively released by murine endothelial cells.

Ceramidases (CDase(s)) play a key role in sphingolipid metabolism by hydrolyzing ceramide into sphingosine. Here we report that murine endothelial cells, macrophages, and human fibroblasts are all able to release acid as well as neutral/alkaline CDase activities in the culture medium. Endothelial cells were characterized by the highest specific activity of cellular as well as secreted CDases. The release of both enzymatic activities was reduced by protein synthesis inhibitor cycloheximide but was unaffected by the blocking of RNA transcription with actinomycin D. The discharge of acid and neutral/alkaline CDases was also diminished by brefeldin A, a fungal metabolite which disrupts Golgi apparatus. Remarkably, treatment of endothelial cells with bradykinin resulted in a significant increase of neutral/alkaline but not acid CDase release. This report represents the first evidence for the existence of constitutive and regulated release of CDase activities by endothelial cells. In view of the known ability of these cells to secrete sphingomyelinase, this finding suggests that CDase may participate in extracellular sphingomyelin metabolism which is presently known to have a role in atherogenesis and could be involved in other physiological or pathological events.

Characterization of sphingomyelinase activity released by thrombin-stimulated platelets.

In this study we report that human platelets display neutral (nSMase) and acid sphingomyelinase (aSMase) as well as acid ceramidase (aCerase) activity. Cell activation by thrombin resulted in a marked decrease of intracellular aSMase activity, accompanied by the release of enzyme into the medium. In contrast, thrombin treatment did not affect aCerase activity. Two major protein bands of 73 and 70 kDa were recognized by aSMase antibodies in resting platelet lysates and in the medium of stimulated cells. Phorbol esters together with the calcium ionophore A23187 fully reproduced thrombin action on aSMase release. The secreted enzymatic activity was insensitive to digestion with endoglycosidase H but it was stimulated by Zn2+, although to a limited extent compared to aSMase constitutively released by murine endothelial cells. Taken together, these data suggest that secreted aSMase does not originate from the lysosomal compartment but rather from other platelet vesicles.

Receptor-activated phospholipase D is present in caveolin-3-enriched light membranes of C2C12 myotubes.

Caveolin-3 (cav-3) is a key structural component of caveolar membrane in skeletal muscle. Cav-3-enriched light membrane (CELM) fractions obtained from C2C12 myotubes contain phospholipase D1 (PLD1) and its major regulators, RhoA and protein kinase Calpha (PKCalpha). All these proteins were found bound to cav-3. An in vivo assay of PLD activity, which allows to localize the reaction product in CELMs, indicated that the enzyme associated to this membrane microdomain was active. Moreover, bradykinin (BK), thrombin and phorbol 12-myristate 13-acetate induced rapid stimulation of PLD activity in CELMs. The cav-3-PLD1 complex was not affected by BK treatment, whereas the agonist induced a marked increase of RhoA association with cav-3. Furthermore, BK-induced PLD activation in CELMs was dependent, at least in part, on PKCalpha.

Sphingosine 1-phosphate induces arachidonic acid mobilization in A549 human lung adenocarcinoma cells.

In the present paper, the effect of sphingosine 1-phosphate (Sph-1-P) on arachidonic acid mobilization in A549 human lung adenocarcinoma cells was investigated. Sph-1-P provoked a rapid and relevant release of arachidonic acid which was similar to that elicited by bradykinin, well-known pro-inflammatory agonist. The Sph-1-P-induced release of arachidonic acid involved Ca(2+)-independent phospholipase A(2) (iPLA2) activity, as suggested by the dose-dependent inhibition exerted by the rather specific inhibitor bromoenol lactone. The Sph-1-P-induced release of arachidonic acid was pertussis toxin-sensitive, pointing at a receptor-mediated mechanism, which involves heterotrimeric Gi proteins. The action of Sph-1-P was totally dependent on protein kinase C (PKC) catalytic activity and seemed to involve agonist-stimulated phospholipase D (PLD) activity. This study represents the first evidence for Sph-1-P-induced release of arachidonic acid which occurs through a specific signaling pathway involving Gi protein-coupled receptor(s), PKC, PLD and iPLA2 activities.

Receptor-mediated activation of phospholipase D by sphingosine 1-phosphate in skeletal muscle C2C12 cells. A role for protein kinase C.

The present study showed that sphingosine 1-phosphate (SPP) induced rapid stimulation of phospholipase D (PLD) in skeletal muscle C2C12 cells. The effect was receptor-mediated since it was fully inhibited by pertussis toxin. All known SPP-specific receptors, Edg-1, Edg-3 and AGR16/H218, resulted to be expressed in C2C12 myoblasts, although at a different extent. SPP-induced PLD activation did not involve membrane translocation of PLD1 or PLD2 and appeared to be fully dependent on protein kinase C (PKC) catalytic activity. SPP increased membrane association of PKCalpha, PKCdelta and PKClambda, however, only PKCalpha and PKCdelta played a role in PLD activation since low concentrations of GF109203X and rottlerin, a selective inhibitor of PKCdelta, prevented PLD stimulation.

A role for phospholipase D activation in the lipid signalling cascade generated by bradykinin and thrombin in C2C12 myoblasts.

In the present study evidence is provided for a rapid activation of lipid signalling pathways induced by thrombin and bradykinin (BK) in C2C12 myoblasts. Both agonists were able to increase [3H]inositol phosphates (InsP), 1,2-[3H]diacylglycerol (DAG) and [3H]phosphatidic acid (PtdOH) levels. In particular [3H]PtdOH values were rapidly increased and maintained at significantly high values at prolonged times of incubation. BK and thrombin were able to activate phospholipase D (PLD) in vivo as demonstrated by the accumulation of [3H]phosphatidylethanol (PtdEtOH) through the transphoshatidylation reaction catalyzed by the enzyme in the presence of ethanol. The observation that ethanol could significantly reduce [3H]PtdOH formation in myoblasts stimulated with BK and thrombin indicates that stimulation of PLD has a major role. The two agonists appear to stimulate PLD activity through a common molecular mechanism, involving the activation of protein kinase C (PKC). In addition, BK and thrombin appear able to activate DAG kinase at early times of incubation and also this pathway may contribute to determine the increase in [3H]PtdOH levels. This is the first report which describes activation of lipid signalling pathways by BK and thrombin in myoblast cells and it is possible that these early signals may have an important role in mediating the biological effects of the two agonists.

Sphingomyelinase increases 2-deoxyglucose uptake and glucose metabolism of human platelets.

Recent studies have shown that the sphingomyelinase (SMase) catalyzed hydrolysis of sphingomyelin (SM) represents an important cell signalling pathway. Control of SMase activity appears to be crucial for the regulation of multiple biological events in different cell systems; in particular, SMase activity appears to be involved in the control of vascular functions and in atherogenic events. Here we report that SMase treatment of human platelets significantly increases 2-deoxyglucose uptake by decreasing K(m) value of sugar transport and increasing sugar diffusion. In addition SMase treatment enhances basal glycolytic flux of platelets as well as the stimulation of the flux induced by suboptimal concentration of thrombin. The present study demonstrates that exposure of platelets to SMase, which may take place in vivo in physiological and/or in pathological conditions, modifies biochemical parameters of resting and stimulated platelets which are essential for cell physiological role.

Activation of phospholipase D in human fibroblasts by ceramide and sphingosine: evaluation of their modulatory role in bradykinin stimulation of phospholipase D.

In the present study the modulatory action of exogenous short-chain ceramide and sphingosine on phospholipase D (PLD) activity in young and old human fibroblasts was examined. Sphingosine and also ceramide, thus far described as a negative modulator of PLD, were able to activate PLD. The stimulatory action of the exogenous lipid molecules was mimicked by cell treatment with S.aureus sphingomyelinase (SMase). Similar response was elicited by the sphingoid molecules in young and old cells. Altered levels of sphingosine and ceramide were detected in old fibroblasts confirming that a defect in sphingolipid metabolism occurs in cellular senescence. The modulatory role of sphingoid molecules on the action of bradykinin (BK) in PLD activation was then evaluated in young and old fibroblasts. C6-ceramide or SMase treatment did not affect the action of BK on PLD either in young or in old cells, whereas sphingosine further increased PLD activity stimulated by BK in young but not in old cells. In addition, preincubation with N-oleoylethanolamine, a specific inhibitor of ceramidase, did not affect BK action on PLD in young fibroblasts but significantly decreased the effect of the peptide in old fibroblasts. These results suggest that a specific alteration of BK segnalatory pathway occurs in old fibroblasts, likely involving sphingosine formation which may account for the potentiated PLD activity induced by the peptide in these cells.

Bradykinin increases ceramide and sphingosine content in human fibroblasts: possible involvement of glycosphingolipids.

Sphingolipid-derived products are recognized as second messengers able to mediate the action of extracellular signals such as cytokines and growth factors. In the present study it is shown that also bradykinin (BK), a pro-inflammatory peptide acting through G-protein-coupled receptors, is able to activate sphingolipid metabolism. Fibroblast treatment with the peptide provokes a rapid and significant increase in ceramide followed by a transient rise in sphingosine content. Sphingomyelin does not appear to be the source of ceramide since BK was unable to decrease the [3H]sphingomyelin pool and no increase in neutral or acidic sphingomyelinase activities could be detected after treatment with the peptide. The observation that the labeled glycosphingolipid pool is decreased upon BK stimulation would rather suggest that the peptide increases ceramide cellular content by rapidly mobilizing neutral glycolipids. Even though the physiological relevance of ceramide and sphingosine increase induced by BK is not known, it is noteworthy that glycosphingolipids may participate in this lipid signalling pathway.

Agonist-regulated L-arginine uptake in human platelets. Evidence against intracellular utilization of the amino acid.

L-arginine uptake takes place in human platelets through a saturable high affinity Na(+)-independent process mediated by the y(+) carrier for cationic amino acids. In the present study the effect of thrombin and collagen on L-arginine transport in human platelets was investigated. Thrombin significantly affected L-arginine uptake whereas collagen was uneffective. In particular, thrombin increased Vmax of the uptake by 77%, while it reduced the affinity of the carrier for L-arginine. The effect of thrombin on the transport process did not result in any increase in L-arginine metabolism since no conversion of the amino acid, either to L-citrulline (indicative of the presence of the L-arginine/nitric oxide pathway) or to any other metabolite, could be detected in resting or stimulated platelets.

Fructose 2,6-bisphosphate metabolism during megakaryocytic differentiation of K562 and MEG-01 cells.

Fructose 2,6-bisphosphate (Fru-2, 6-P2) represents the most powerful activator of 6-phosphofructo-1-kinase, rate-limiting enzyme of glycolysis. Fru-2,6-P2 content is tightly regulated and appears to be under the control of different hormones and growth factors, acting either through covalent modification of isoenzymatic forms of 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase (PFK-2/FBPase-2), the bifunctional enzyme responsible for the synthesis and the degradation of the compound, or through changes in transcription rate and/or in the expression of different isoforms of the enzyme. In the present study the metabolism of Fru-2,6-P2 was investigated during the differentiation toward megakaryocytes induced by phorbol 12-myristate 13-acetate (PMA) treatment of human leukemia K562 and MEG-01 cell lines. Fru-2,6-P2 content as well as PFK-2 activity were increased in a dose-dependent manner after 4 days of incubation with PMA. MEG-01 cells resulted more sensitive to the effect of the inducer, anyway in both cell types cytostatic concentrations of the phorbol ester were able to affect Fru-2,6-P2 metabolism. The effect of PMA was maximal at 4 days of incubation in both examined cell lines. Interestingly, the effect induced by the phorbol ester at 4 days was still appreciable subculturing K562 and MEG-01 cells for 3 days in the absence of the inducer and was associated with relevant changes in the molecular properties of PFK-2: namely increased Vmax and K(m). This latter finding suggests that the rise in Fru-2,6-P2 content during the differentiation process toward megakaryocytes might result from the expression of a novel PFK-2 isoform.

Potentiated bradykinin-induced increase of 1,2-diacylglycerol generation and phospholipase D activity in human senescent fibroblasts.

1. The comparative study of the effect of bradykinin (BK) in young and old IMR-90 human fibroblasts shows that old cells are characterized by a reduced increase in 1,2-diacylglycerol (1,2-DAG) generation upon stimulation after short-term treatment and a significant higher increase after long-term agonist treatment. BK-induced activation of phospholipase D (PLD), the major enzyme involved in sustained 1,2-DAG generation, was 2.5-fold higher in old cells, strongly suggesting that it is involved in the potentiated increase of 1,2-DAG formation. The increased activation of PLD by BK in old cells was specific, since in parallel experiments the effect of thrombin was not significantly different in young and old cells. PLD activity in old cells was only reduced by down-regulation of protein kinase C (PKC) activity, in contrast to what was observed in young cells where it was completely abolished. This indicates that the enzyme activity in old cells was partially PKC-independent. BK was also able to increase the release of [14C]ethanolamine, a water-soluble product of hydrolysis of phosphatidylethanolamine (PtdEtn), through PLD activation in young and old cells. The BK effect was significantly higher in old cells and, very likely, PKC-independent, since phorbol 12-myristate 13-acetate failed to induce PtdEtn hydrolysis. 2. The present results indicate that the PLD/1,2-DAG pathway is specifically potentiated by BK in old fibroblasts, demonstrating that the formation of positive effectors of PKC activation is not necessarily decreased in cellular senescence. It remains to be established whether the increased generation of DAG upon BK stimulation plays any role in the altered PKC signalling pathway which characterizes old fibroblasts.

Identification of a specific transport system for L-arginine in human platelets.

The present study demonstrates that human platelets possess a specific L-arginine transport system able to provide adequate amounts of L-arginine for endogenous nitric oxide production. L-arginine uptake takes place through a saturable high affinity carrier-mediated Na(+)-independent process which is significantly inhibited by L-ornithine, L-lysine and N omega-methyl-L-arginine. The high affinity of the transport process and the pattern of inhibition are consistent with mediation of L-arginine transport via the Na(+)-independent y+ system. The data on the kinetic parameters of the transporter suggest a possible role for arginine plasma levels in the regulation of platelet nitric oxide production.

Glutamine transport and enzymatic activities involved in glutaminolysis in human platelets.

Glutamine is actively metabolized in human platelets, representing a preferential mitochondrial oxidative substrate in these cells. The primary importance of this metabolic route of glutamine is further confirmed here by the observation that platelet glutaminase activity is entirely represented by the phosphate dependent glutaminase or glutaminase I, most probably localized in the mitochondrial platelet fraction and classified by kinetic analysis as a kidney-type form. The following step of the glutamine metabolizing pathway, allowing the entrance of the amino acid skeleton carbons in the Krebs cycle, might be catalyzed by both glutamate dehydrogenase and aspartate transaminase, the first being entirely mitochondrial and the latter 65% mitochondrial. We also investigated platelets for the presence of one or more specific transport systems involved in glutamine uptake and we present the first evidence for two glutamine transport systems in human platelets that by inhibition analysis appear to share characteristics with the Na(+)-dependent ASC system and the Na(+)-independent L system for dipolar amino acid uptake. Both systems display affinity characteristics for glutamine in the range of plasma glutamine concentration and may thus have physiological relevance for the uptake of the amino acid in these cells.

Involvement of protein kinase C and arachidonate signaling pathways in the alteration of proliferative response of senescent IMR-90 human fibroblasts.

The proliferative response of IMR-90 fibroblasts at low and high population doubling level (PDL) to protein kinase C activation has been investigated to clarify whether the reduced mitogenic responsiveness of senescent cells can be ascribed to an alteration in protein kinase C signal transduction pathway. The results show that the signaling pathway leading to DNA synthesis through protein kinase C activation, appears to be modified in senescent IMR-90 human fibroblasts. High PDL fibroblasts exhibit a different sensitivity to phorbol 12-myristate 13-acetate (PMA) and dioctanoylglycerol (diC8); high glucose reduced responsiveness to PMA only in these cells. In addition, high PDL fibroblasts are characterized by an increase in diacylglycerol (DAG) cellular mass that could contribute to the different regulatory properties of the signaling pathway. On the other hand, the ability of the cyclooxygenase inhibitor indomethacin to strikingly improve the proliferative response of high PDL cells to PMA indicates that an altered overall metabolism of arachidonate may represent a crucial step in the reduced mitogenic response involving protein kinase C activation.

Bradykinin stimulates fructose 2,6-bisphosphate metabolism in human fibroblasts.

Bradykinin (BK), a peptide released during inflammatory response, has been investigated for its ability to regulate glucose metabolism in human fibroblasts. The peptide is able to significantly increase glycolytic flux in these cells. The strict relationship between the glycolytic rate and the levels of fructose 2,6-bisphosphate (Fru-2,6-P2) strongly suggests that the metabolite plays a key role in the regulation of glucose metabolism by bradykinin. The mechanism by which bradykinin increases Fru-2,6-P2 content involves the activation of 6-phosphofructo-2-kinase (PFK-2), the enzyme responsible for the synthesis of the metabolite. The study of the multiple signalling systems triggered by bradykinin demonstrates the involvement of the rise in intracellular Ca2+ concentration and of protein kinase C mediated pathway in the mechanism by which bradykinin increases Fru-2,6-P2 content and PFK-2 activity.

Glutamine utilization in resting and stimulated platelets.

Exogenous glutamine was metabolized by platelets to glutamate, aspartate, and CO2; whereas no lactate was formed. The amount of aspartate and CO2 produced from glutamine was reduced by the presence of glucose. On the other hand the rate of oxidation of glucose was reduced by the presence of glutamine. The potential energetic value of glutamine was lower than that of glucose, but the amino acid appeared to be a preferential respiratory substrate. The energy provision from glutamine also plays a role in the process of platelet activation, known to require an extra supply of ATP, since thrombin increased the rate of glutamine utilization in platelets. Upon cell activation an increase in aspartate and CO2 formation was observed and the stimulatory effect on glutamine oxidation by thrombin was also maintained in the presence of glucose.