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 Nω-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.
Cytohesin-1, a protein abundant in cells of the immune system, has been proposed to be a human homolog of the Saccharomyces cerevisiae Sec7 gene product, which is crucial in protein transport. More ...recently, the same protein has been reported to be a regulatory factor for the α Lβ 2 integrin in lymphocytes. Overexpression of human or yeast ADP-ribosylation factor (ARF) genes rescues yeast with Sec7 defects, restoring secretory pathway function. ARFs, 20-kDa guanine nucleotide-binding proteins initially identified by their ability to stimulate cholera toxin ADP-ribosyltransferase activity and now recognized as critical components in intracellular vesicular transport, exist in an inactive cytosolic form with GDP bound (ARF-GDP). Interaction with a guanine nucleotide-exchange protein (GEP) accelerates exchange of GDP for GTP, producing the active ARF-GTP. Both soluble and particulate GEPs have been described. To define better the interaction between ARF and Sec7-related proteins, effects of cytohesin-1, synthesized in Escherichia coli, on ARF activity were evaluated. Cytohesin-1 enhanced binding of 35S-labeled guanosine 5′-γ -thiotriphosphate 35SGTPγ S or 3HGDP to ARF purified from bovine brain (i.e., it appeared to function as an ARF-GEP). Addition of cytohesin-1 to ARF3 with 35SGTPγ S bound, accelerated 35SGTPγ S release to a similar degree in the presence of unlabeled GDP or GTPγ S and to a lesser degree with GDPβ S; release was negligible without added nucleotide. Cytohesin-1 also increased ARF1 binding to a Golgi fraction, but its effect was not inhibited by brefeldin A (BFA), a drug that reversibly inhibits Golgi function. In this regard, it differs from a recently reported BFA-sensitive ARF-GEP that contains a Sec7 domain.
Objective: The prognosis of non-small cell lung cancer (NSCLC) with brain metastasis is very poor, with median survival rate below 6 months, even if treated with palliative radio and/or chemotherapy. ...To assess the effectiveness of surgical treatment for this kind of patients we reviewed our experience. Methods: From January 1989 to October 1999, 30 patients (26 males and four females; mean age: 58.7 years) with NSCLC and single brain metastasis underwent surgical treatment of both primary lung cancer and secondary cerebral lesion. Patients (pts) were divided into two major groups. In group 1 (G1) 20 pts (18 males and two females) presented a synchronous brain metastasis. In group 2 (G2) 10 pts (eight males and two females) presented a metachronous brain metastasis during the follow-up period (range 3–24 months since the primary tumor). Patients selected in G1 had T1–2, N0–1 clinical staging, good ‘performance status’ (ECOG:0–1; Karnofsky index ≫70%), age≪75 years. Craniotomy has always been the first approach. In G2 also patients with locally advanced tumors (T3 and/or N2) were included. Whole brain radiotherapy and/or chemotherapy was the post-operative choice treatment. Results: Histologic findings have shown: adenocarcinoma in 17 cases (12 in G1; five in G2), squamous cell carcinoma in 10 cases (six in G1; four in G2), large cell carcinoma in 2 (one in G1; one in G2) and large cell neuroendocrine carcinoma in one (G1). Survival analysis (Kaplan–Meier method) has shown an overall value of 80% at 1 year (95% in G1; 50% in G2), 41% at 2 years (47% in G1; 30% in G2) and 17% at 3 years (14% in G1; 20% in G2). Overall median survival is 23 months (23 in G1; 11 in G2); mean survival 27.8 months (30.3 months in G1; 22.8 months in G2). According to univariate analysis prognosis is definitively better in N0 tumors compared to N1–2 tumors and in adenocarcinoma cases compared to other histotypes (P < 0.05). Conclusions: We can conclude that combined surgical therapy is, nowadays, the choice treatment for this kind of patients, even though restricted to selected cases. The knowledge of prognostic factors may optimize indications for surgery.
This study demonstrates for the first time that sphingosine 1-phosphate (S1P) increases H
2O
2 production in NIH3T3 fibroblasts through NADPH oxidase activation, confirming the involvement of ...phosphoinositide-3-kinase and protein kinase C in the activation of this enzyme in non-phagocyte mammalian cells. The results demonstrate also that both platelet-derived growth factor (PDGF) and S1P-mediated NADPH oxidase activation and H
2O
2 production by Gi-protein coupled receptors (GPCRs) and c-Src kinase. Moreover, both PDGF and S1P activate c-Src kinase through GPCRs, indicating that this kinase can constitute a connection factor between PDGF and S1P signaling, confirming the cross-talk previously found between their receptors. Thus, Gi-protein-mediated NADPH oxidase activation with the consequent H
2O
2 increase constitutes an early event in the PDGF and S1P pathways. However, a different time course of H
2O
2 production in S1P-stimulated cells compared to that obtained in PDGF-stimulated cells has been observed, and this seems to be related to the different activation behavior of c-Src kinase induced after S1P or PDGF stimulation. Finally, these data demonstrate that S1P-induced H
2O
2 production is necessary to maximize c-Src kinase activation, confirming that this is a redox regulated kinase. After which, c-Src plays an important role both upstream and downstream from NADPH oxidase activation.
Rho GTPases participate in various important signaling pathways and have been implicated in myogenic differentiation. Here the first evidence is provided that in C2C12 myoblasts sphingosine ...1-phosphate (SPP) rapidly and transiently induced membrane association of RhoA in a pertussis toxin-insensitive manner. The bioactive lipid preferentially relocalized the GTPase to Golgi-enriched membrane. Translocation of RhoA was abolished by inhibition or down-regulation of protein kinase C (PKC). Notably, treatment with Gö6976, an inhibitor of conventional PKCs, which selectively blocked PKCα in these cells, prevented SPP-induced RhoA translocation. Conversely rottlerin, a selective inhibitor of PKCδ, was without effect, demonstrating that SPP signaling to RhoA involves PKCα but not PKCδ activation. This novel functional relationship between the two proteins may have a role in SPP-mediated regulation of downstream effectors.
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.
Phospholipase D (PLD) has been implicated as a crucial signaling enzyme in secretory pathways. Two 20-kDa guanine nucleotide-binding
proteins, Rho and ADP-ribosylation factor (ARF), are involved in ...the regulation of secretion and can activate PLD in vitro . We investigated in intact (human adenocarcinoma A549 cells) the role of RhoA and ARF in activation of PLD by phorbol 12-myristate
13-acetate, bradykinin, and/or sphingosine 1-phosphate. To express recombinant Clostridium botulinum C3 exoenzyme (using double subgenomic recombinant Sindbis virus C3), an ADP-ribosyltransferase that inactivates Rho, or dominant-negative
Rho containing asparagine at position 19 (using double subgenomic recombinant Sindbis virus Rho19N), cells were infected with
Sindbis virus, a novel vector that allows rapid, high level expression of heterologous proteins. Expression of C3 toxin or
Rho19N increased basal and decreased phorbol 12-myristate 13-acetate-stimulated PLD activity. Bradykinin or sphingosine 1-phosphate
increased PLD activity with additive effects that were abolished in cells expressing C3 exoenzyme or Rho19N. In cells expressing
C3, modification of Rho appeared to be incomplete, suggesting the existence of pools that differed in their accessibility
to the enzyme. Similar results were obtained with cells scrape-loaded in the presence of C3; however, results with virus infection
were more reproducible. To assess the role of ARF, cells were incubated with brefeldin A (BFA), a fungal metabolite that disrupts
Golgi structure and inhibits enzymes that catalyze ARF activation by accelerating guanine nucleotide exchange. BFA disrupted
Golgi structure, but did not affect basal or agonist-stimulated PLD activity, i.e. it did not alter a rate-limiting step in PLD activation. It also had no effect on Rho-stimulated PLD activity, indicating
that RhoA action did not involve a BFA-sensitive pathway. A novel PLD activation mechanism, not sensitive to BFA and involving
RhoA, was identified in human airway epithelial cells by use of a viral infection technique that preserves cell responsiveness.
Sphingosine 1-phosphate (SPP) is a bioactive lipid that exerts multiple biological effects in a large variety of cell types, acting as either an intracellular messenger or an extracellular ligand ...coupled to Edg-family receptors (where Edg stands for endothelial differentiation gene). Here we report that in C(2)C(12) myoblasts SPP elicited significant Ca(2+) mobilization. Analysis of the process using a confocal laser-scanning microscope showed that the Ca(2+) response occurred in a high percentage of cells, despite variations in amplitude and kinetics. Quantitative analysis of SPP-induced Ca(2+) transients performed with a spectrophotofluorimeter showed that the rise in Ca(2+) was strictly dependent on availability of extracellular Ca(2+). Cell treatment with pertussis toxin partially prevented the Ca(2+) response induced by SPP, indicating that G(i)-coupled-receptors were involved. Indeed, SPP action was shown to be mediated by agonist-specific Edg receptors. In particular, suramin, an antagonist of the SPP-specific receptor Edg3, as well as down-regulation of Edg3 by cell transfection with antisense oligodeoxyribonucleotides (ODN), significantly reduced agonist-mediated Ca(2+) mobilization. Moreover, an antisense ODN designed to inhibit Edg5 expression also decreased the SPP-induced rise in Ca(2+), although to a lesser extent than that observed by inhibiting Edg3. On the contrary, the SPP response was unaffected in myoblasts loaded with antisense ODN specific for Edg1. Remarkably, the concomitant inhibition of Edg3 and Edg5 receptors abolished the SPP-induced Ca(2+) increase, supporting the notion that Ca(2+) mobilization in C(2)C(12) cells induced by SPP is a receptor-mediated process that involves Edg3 and Edg5, but not Edg1.
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.
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