Valeria Vasta

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 PKCα, PKCδ and PKCλ, however, only PKCα and PKCδ played a role in PLD activation since low concentrations of GF109203X and rottlerin, a selective inhibitor of PKCδ, prevented PLD stimulation.

Single-step affinity purification, partial structure and properties of human platelet cGMP inhibited cAMP phosphodiesterase

The human platelet cilostamide- and cGMP-inhibited cAMP phosphodiesterase (cGI-PDE) was rapidly purified approximately 19,000-fold to apparent homogeneity using single step affinity chromatography on the isothiocyanate derivative of cilostamide coupled to aminoethyl agarose. Within 24 h, 30 micrograms of enzyme protein was obtained from 20 ml of packed platelets. Vmax for cAMP and cGMP was 6.1 and 0.9 mumol/min per mg protein, respectively. Several polypeptides (110/105, 79, 62, 55/53 kDa) were identified after SDS-PAGE, all of which were immunologically related to cGI-PDE and represented approx. 5, 20, 50 and 20% of the total protein, respectively. Limited proteolysis of the cGI-PDE with chymotrypsin produced a major fragment of approximately 47 kDa (and at least two smaller peptides) with catalytic activity and sensitivity to cGMP and OPC 3911 similar to controls. Phosphorylation of the cGI-PDE by cAMP-dependent protein kinase (A-kinase) resulted in maximal incorporation of 0.6-1.8 mol of 32P/mol 110/105 and 79 kDa polypeptides; much lower and variable amounts of phosphate were incorporated into the 62 and 55/53 kDa polypeptides. After digestion of cGI-PDE with several proteinases a number of peptides were isolated and sequenced. Most of the peptide sequences obtained could be aligned within the carboxy terminal domain of the deduced sequence of the human cardiac cGI-PDE. These and other results suggest that the subunit size of the intact platelet cGI-PDE is 110 kDa and that proteolytic fragments of 79, 62 and 55/53 kDa are produced during purification. The smaller fragments (62 and 55/53 kDa) contain the catalytic domain; the larger fragments (110 and 79 kDa) also contain the regulatory domain with phosphorylation sites for A-kinase.

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.

Increase of the glycolytic rate in human resting fibroblasts following serum stimulation: The possible role of the fructose-2,6-bisphosphate

We report that glycolysis in human quiescent fibroblasts stimulated by serum addition is increased, and that the changes of the metabolic route reflect the activity of the phosphofructokinase. A possible role of fructose‐2,6‐bisphosphate as a positive modulator of the key enzyme is proposed.

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.

Adenylate cyclase stimulating agents and mitogens raise fructose 2,6-bisphosphate levels in human fibroblasts Evidence for a dual control of the metabolite

Fructose 2,6‐bisphosphate, the most potent activator of 6‐phosphofructo‐1‐kinase, has been demonstrated to mediate the increase of glycolytic flux induced by mitogens human fibroblasts. In the present work the molecular basis of transmembrane control of fructose 2,6‐bisphosphate has been investigated. Prostacyclin and isoprenaline, known to activate adenylate cyclase, are able to increase fructose 2,6‐bisphosphate levels, indicating that in human fibroblasts cyclic AMP plays a positive role in the control of the metabolite concentration, opposite to that exerted in hepatocytes. Substances known to activate protein kinase C such as phorbol 12‐myristate 13‐acetate, or to stimulate phosphoinositide turnover such as thrombin and bradykinin are also effective in raising fructose 2,6‐bisphosphate. Therefore, we conclude that cyclic AMP and protein kinase C are likely involved in the control of fructose 2,6‐bisphosphate levels in human fibroblasts.

The effect of insulin on Fru-2,6-P2 levels in human fibroblasts.

We report that fructose 2,6‐bisphosphate (Fru‐2,6‐P2) levels in quiescent human fibroblasts stimulated with insulin treatment increase in a time‐ and dose‐dependent manner. The biochemical role of the sugar phosphate in the cellular response to the hormone which controls carbohydrate metabolism and acts as mitogenic factor is discussed.

An endpoint enzymatic assay for fructose 2,6-bisphosphate performed in 96-well plates☆

An endpoint enzymatic assay for fructose 2,6-bisphosphate based on the ability of the compound to stimulate pyrophosphate 6-phosphofructo-1-kinase and performed in a 96-well plate is reported here. The method presents a low detection limit and a high sensitivity that could be further improved; moreover, the use of 96-well plates greatly increases the number of samples that can be simultaneously assayed.

A phospho-oligosaccharide can reproduce the stimulatory effect of insulin on glycolytic flux in human fibroblasts

It has been recently demonstrated that insulin promotes the hydrolysis of a glycosyl-phosphatidylinositol, stimulating the release of a phospho-oligosaccharide which displays several insulin-like effects. In the present study we have investigated whether the compound is able to mimic insulin action on glucose metabolism in human fibroblasts. Similarly to the hormone, the phospho-oligosaccharide elicited a dose dependent increase in lactate output and fructose 2,6-bisphosphate content. The effect of the compound was time dependent with a progressive increase starting from 2 hours of incubation. 1 microM phospho-oligosaccharide had half maximal effect on both parameters, increasing glycolytic flux by approximately 30% and fructose 2,6-bisphosphate content by 70%. Therefore the phospho-oligosaccharide appears to be able to strictly reproduce insulin action on glucose metabolism in human fibroblasts.

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.