Platelet-activating factor mediates an autocrine proliferative loop in the endometrial adenocarcinoma cell line HEC-1A.

We investigated the synthesis and biological effects of platelet-activating factor (PAF) in the human endometrial cancer cell line HEC-1A. We found that HEC-1A cells actively synthesize and release PAF, as demonstrated by both [3H]acetate incorporation into PAF and gas chromatography-mass spectrometry studies. HEC-1A cells not only synthesize but also respond to PAF. Indeed, in fura-2-loaded cells, PAF stimulates [Ca2+]i increase with a median effective concentration of 5.6 nM. Furthermore, PAF induces a time-dependent expression increase of the nuclear protooncogene c-fos with a median effective concentration of 130 nM and stimulates DNA synthesis (median effective concentration, 700 nM). All of these effects are inhibited by the PAF receptor antagonist L659,989. Radioligand binding studies indicated the presence of two populations of PAF receptors with affinity constants in the nanomolar and micromolar range. Since the PAF antagonist per se inhibits DNA synthesis and cell proliferation, we suggest that PAF supports an autocrine growth circuit in HEC-1A cells. On the contrary, in the uterine leiomyosarcoma cell line SK-UT-1, which does not express specific binding sites for PAF, neither this phospholipid nor its receptor antagonist affect DNA synthesis. Our results provide evidence for the existence of an autocrine proliferative loop involving PAF in the endometrial cancer cell line HEC-1A.

Androgen receptor expression in prostate carcinoma cells suppresses alpha6beta4 integrin-mediated invasive phenotype.

Prostate cancer cells may lose androgen-sensitivity after androgen ablation therapy, becoming highly invasive and metastatic. The biological mechanisms responsible for higher tumurogenicity of androgen-independent prostate carcinomas are not entirely known. We demonstrate that androgen receptor regulation of adhesion and invasion of prostate cancer cells through modulation of alpha6beta4 integrin expression may be one of the molecular mechanisms responsible of this phenomenon. We found that protein and gene expressions of alpha6 and beta4 subunits were strongly reduced in the androgen-sensitive cell line LNCaP respect to the androgen-independent PC3 and that transfection of PC3 cells with a full-length androgen receptor expression vector resulted in a decreased expression of alpha6beta4 integrin, reduced adhesion on laminin, and suppressed Matrigel invasion. Growth in soft agar was also suppressed in androgen receptor-positive PC3 clones. Treatment of androgen receptor positive clones with the synthetic androgen R1881 further reduced alpha6 and beta4 messenger RNA expression as well as adhesion on laminin and Matrigel invasion. Our results indicate that androgens regulate cell-extracellular matrix adhesion and invasion by modulation of integrin expression and function, thus keeping a low invasive phenotype of prostate cancer cells.

Integrin-mediated stimulation of monocyte chemotactic protein-1 expression.

We investigated whether activation of integrin receptors could modulate the expression of monocyte chemotactic protein-1 (MCP-1) in human hepatic stellate cells (HSC), mesenchymal cells responsible for extracellular matrix synthesis within the liver. When compared to non-adherent cells, HSC plated on collagen types I or IV, or fibronectin, showed increased MCP-1 gene expression and protein secretion in the conditioned medium. Increased MCP-1 secretion was also observed when cells were plated on dishes coated with a monoclonal antibody directed against the beta1-integrin subunit, demonstrating that ligation of beta1-integrins is sufficient to stimulate MCP-1 expression. Conversely, integrin-independent cell adhesion on poly-L-lysine did not modify MCP-1 secretion. Disruption of the actin cytoskeleton by cytochalasin D blocked the collagen-dependent increase in MCP-1 secretion. Chemotactic assay of HSC-conditioned medium showed that HSC plated on collagen secrete higher amounts of chemotactic factors for lymphomonocytes, and that MCP-1 accounts for the great majority of this effect. These findings indicate a novel mechanism of MCP-1 regulation possibly relevant in those conditions where HSC interact with an altered extracellular matrix.

Effect of pentoxifylline on the degradation of procollagen type I produced by human hepatic stellate cells in response to transforming growth factor-beta 1.

1. Pentoxifylline (PTF) may act as a potential antifibrogenic agent by inhibiting cell proliferation and/or collagen deposition in cell type(s) responsible for the accumulation of extracellular matrix. The aim of the present study was to investigate at which level PTF may affect synthesis and degradation of type I collagen in human hepatic stellate cells (HSCs), a key source of connective tissue in fibrotic liver. 2. Procollagen type I synthesis and release were evaluated in cells maintained in serum free/insulin free medium for 48 h and then stimulated with transforming growth factor-beta 1 (TGF-beta 1) for different time periods in the presence or absence of PTF. TGF-beta 1 caused an upregulation of procollagen I mRNA levels with a peak increase after 3-6 h of stimulation. This effect was followed by an increase in both the cell associated and the extracellular levels of the corresponding protein, with a peak effect at 9-12 h after the addition of TGF-beta 1. Co-incubation with PTF slightly but consistently reduced basal as well as stimulated procollagen I mRNA levels, with negligible effects on the cell-associated expression of the corresponding protein. Conversely, PTF dose-dependently reduced procollagen type I levels detected in supernatants from unstimulated and stimulated cells. 3. Pulse-chase experiments employing L-[3H]-proline revealed that PTF was able to induce significantly the degradation of procollagen, mainly in the extracellular compartment. We next analysed the effect of PTF on the major pathway involved in type I collagen degradation. PTF did not affect the expression of metalloproteinase 1 (MMP-1) mRNA both in basal and stimulated conditions, whereas it markedly reduced the expression of tissue inhibitor of metalloproteinase 1 (TIMP-1) mRNA. Accordingly incubation with PTF increased the levels of 'activated MMP-1' in cell supernatants in both basal and stimulated conditions. 4. These results suggest that the antifibrogenic action of PTF on human HSCs is mainly mediated by extracellular collagen degradation rather than by a reduction of collagen synthesis.

Signaling mechanisms that mediate invasion in prostate cancer cells.

Recent evidence indicates that androgen-sensitive prostate cancer cells have a less malignant phenotype characterized by reduced migration and invasion. We investigated whether the presence of the androgen receptor could affect EGFR-mediated signaling by evaluating autotransphosphorylation of the receptor as well as activation of the downstream signaling pathway PI3K/AKT. Immunoprecipitation studies demonstrated a reduction of EGF-induced tyrosine phosphorylation of EGFR in PC3-AR cells. In addition, EGF-stimulated PI3K activity, a key signaling pathway for invasion of these cells, was decreased in PC3-AR cells and further reduced by treatment with R1881, indicating decreased functionality of EGFR. Our results suggest that the expression of androgen receptors by transfection in PC3 cells confers a less malignant phenotype by interfering with EGFR autophosphorylation and signaling leading to invasion in response to EGF. We used the selective tyrosine kinase inhibitor of the EGFR gefitinib (also known as Iressa or ZD1839) to further investigate the role of EGFR in the invasion and growth of PC cells. We demonstrate that in the androgen-insensitive cell lines PC3 and DU145 this compound was able to decrease in vitro invasion of Matrigel by inhibiting EGFR autotransphosphorylation and subsequent PI3K activation. Gefitinib may be useful in the treatment of androgen-independent prostate cancer to limit not only the proliferation but also the invasion of these tumors.

The androgen receptor associates with the epidermal growth factor receptor in androgen-sensitive prostate cancer cells.

Many recent evidences indicate that androgen-sensitive prostate cancer cells have a lower malignant phenotype that is in particular characterized by a reduced migration and invasion. We previously demonstrated that expression of androgen receptor (AR) by transfection of the androgen-independent prostate cancer cell line PC3 decreases invasion and adhesion of these cells (PC3-AR) through modulation of alpha6beta4 integrin expression. The treatment with the synthetic androgen R1881 further reduced invasion of the cells without, however, modifying alpha6beta4 expression on the cell surface, suggesting an interference with the invasion process in response to EGF. We investigated whether the presence of the AR could affect EGF receptor (EGFR)-mediated signaling in response to EGF by evaluating autotransphosphorylation of the receptor as well as activation of downstream signalling pathways. Immunoprecipitation studies demonstrated a reduction of EGF-induced tyrosine phosphorylation of EGFR in PC3-AR cells. In addition, EGF-stimulated PI3K activity, a key signalling pathway for invasion of these cells, was decreased in PC3-AR cells and further reduced by treatment with R1881, indicating decreased functionality of EGFR. An interaction between EGFR and AR has been demonstrated by immunoconfocal and co-immunoprecipitation analysis in PC3-AR cells, suggesting a possible interference of AR on EGFR signalling by interaction of the two proteins. In conclusion, our results suggest that the expression of AR by transfection in PC3 cells confers a less malignant phenotype by interfering with EGFR autophosphorylation and signalling in response to EGF leading to invasion through a mechanism involving an interaction between AR and EGFR.

Refractory ascites: definition, pathogenesis and treatment.

Refractory ascites, that is ascites which cannot be mobilized by low sodium diet and maximal doses of diuretics (up to 400 mg spironolactone or potassium canrenoate and 160 mg furosemide per day), occurs in 5% of cirrhotic patients with ascites. The development of refractory ascites is mainly related to the progression of arterial vasodilation-mediated vascular underfilling and to the imbalance between reduced synthesis of renal vasodilating factors (especially renal prostaglandins) and extreme activation of vasoconstricting systems. Further features include increased sodium reabsorption in the proximal tubule and altered pharmacokinetics and pharmacodynamics of diuretics. In patients with impaired renal function (as is the case for most patients with refractory ascites), the marked reduction of renal perfusion and glomerular filtration rate, with the consequent decrease of filtered sodium load, becomes the main pathogenetic factor. The principal therapeutic options for refractory ascites include repeated paracentesis and implantation of the LeVeen shunt. Paracentesis is a rapid and safe procedure to remove ascites, but it does not correct sodium retention. Ascites recurrence, therefore, may occur after a brief interval. The LeVeen shunt allows for better long-term control of ascites, but severe complications may supervene, and shunt occlusion is common. Neither therapeutic procedure improves survival. Different experimental therapeutic procedures have been proposed. Administration of ornipressin corrects hyperdynamic circulation and improves renal function. Thromboxane synthase inhibitors, by reducing renal synthesis of thromboxane A2, potentiate the diuretic and natriuretic response to furosemide. More invasive procedures, including portosystemic shunt and transjugular intrahepatic stent, are rarely used in the treatment of refractory ascites.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell fusion promotes chemoresistance in metastatic colon carcinoma.

Chemoresistance is an important concern in the treatment of metastatic colon cancer. It may emerge through selection of clones that are inherently resistant from the outset or through mechanisms acquired during treatment. Cell fusion represents an efficient means of rapid phenotypic evolution that make cells with new properties at a rate exceeding that achievable by random mutagenesis. Here, we first identified a number of proteins involved in cell fusion using a shotgun proteomics approach, then we investigated the role of these proteins namely tetraspanin CD81/CD9, ADAM10, GTP-binding protein α13, radixin, myosin regulatory light chain and RhoA in the regulation of colon cancer cell fusion. We also found a previously unrecognized role of ADAM10, Gα13 and RhoA in promoting cell fusion. Finally, we show that the occurrence of cell fusion in a metastatic model of colon carcinoma causes the appearance of cells resistant to both 5-fluorouracil and oxaliplatin. These findings highlight the importance of cell fusion in cancer progression and raise significant implications for overcoming chemoresistance in metastatic colon cancer.

Signal transduction in hepatic stellate cells.

Hepatic stellate cells (HSC) are presently regarded as one of the key cell types involved in the progression of liver fibrosis and in the related pathophysiological and clinical complications. Following acute or chronic liver tissue damage, HSC undergo a process of activation towards a phenotype characterised by increased proliferation, motility, contractility and synthesis of extracellular matrix (ECM) components. Several factors have been shown to play a key role in the promotion of the full-blown picture of activated HSC. These include extensive changes in the composition and organisation of the ECM, the secretion of several growth factors, cytokines, chemokines, products of oxidative stress and other soluble factors. It is evident that each cellular response to extracellular stimuli must be framed in a scenario where different forces modulate one another and result in a prevalent biological effect. Along these lines, the identification and characterisation of intracellular signalling pathways activated by different stimuli in HSC represent a mandatory step. In this review article we have made an attempt to summarise recent acquisitions to our knowledge of the involvement of different intracellular signalling pathways in key aspects of HSC biology.

Focal adhesion kinase and phospholipase C gamma involvement in adhesion and migration of human hepatic stellate cells.

BACKGROUND & AIMS: Hepatic stellate cells (HSCs) play a key role in the development of liver fibrosis. Integrin receptors contribute to the regulation cell adhesion and migration. The aim of this study was to evaluate the interaction between focal adhesion kinase (FAK) and phospholipase C gamma (PLC gamma) potentially involved in HSC integrin-mediated signaling pathways. METHODS: Interaction between FAK and PLC gamma was determined by immunoprecipitation and immunoblotting. HSC chemotactic activity was evaluated using the Boyden chamber technique. RESULTS: HSC adhesion to extracellular matrix components (collagen type I and IV, laminin, and fibronectin) and antibody-mediated beta 1 ligation elicited increased tyrosine phosphorylation of FAK. HSC adhesion to different extracellular matrix components did not result in PLC gamma tyrosine phosphorylation. However, HSC adhesion induced association between PLC gamma and FAK. All extracellular matrix components tested stimulated HSC chemotactic activity only at high concentrations. On the contrary, platelet-derived growth factor, homodimer BB (PDGF-BB), was able to stimulate HSC migration in a dose-dependent manner; this event, occurring in the presence of FAK phosphorylation, was associated to a dose-dependent PLC gamma tyrosine phosphorylation. CONCLUSIONS: These findings provide the first evidence that PLC gamma recruitment by FAK during HSC adhesion is an important process implicating a link between integrin and PDGF-mediated signaling pathways to regulate HSC adhesion and motility.

Biosynthesis of platelet-activating factor and its 1O-acyl analogue by liver fat-storing cells.

BACKGROUND/AIMS: Platelet-activating factor (PAF) is an important mediator of proinflammatory cell-to-cell interactions with powerful vasoactive properties. We evaluated the biosynthesis of PAF by cultured human fat-storing cells (FSC), liver-specific pericytes involved in the inflammatory and fibrogenic process of liver tissue. METHODS: PAF synthesis was evaluated by measuring [3H]acetate incorporation under basal conditions and upon stimulation with A23187, thrombin, and lipopolysaccharide. Further analysis of PAF species synthesized by FSC was performed using gas chromatography/mass spectrometry. RESULTS: All stimuli induced a significant increase of basal PAF synthesis by FSC. Further analysis showed that > 50% of the newly synthesized PAF species was secreted whereas the remaining fraction was cell-associated. PAF species produced by FSC were able to induce aggregation of rabbit washed platelets with an effectiveness correspondent to 10(-9) mol/L authentic PAF. Gas chromatography/mass spectrometry analysis revealed that a large percentage (74%) of PAF-like lipids synthesized by FSC consisted of 1O-acyl PAF. Finally, stimulation of FSC with PAF caused an increase in cytosolic free calcium, thus suggesting a possible involvement of this pericyte in the well-known effects of PAF on portal pressure. CONCLUSIONS: These results expand the available knowledge concerning the role of PAF in conditions characterized by extensive activation and damage of the liver sinusoidal endothelium and decreased hepatic scavenger activity.

The integrin, alpha6beta1, is necessary for the matrix-dependent activation of FAK and MAP kinase and the migration of human hepatocarcinoma cells.

Expression of the integrin, alpha6beta1, a receptor for laminins, is associated with the progression of hepatocellular carcinoma (HCC). The approach to investigating the alpha6beta1 integrin signaling in HCC cells was to express a deletion mutant of the beta4 integrin cytoplasmic domain (beta4-Deltacyt) in 2 HCC cell lines, HepG2 and Huh7. Expression of this mutant prevents formation of the alpha6beta1 heterodimer. As expected, adhesion of both the HepG2/beta4-Deltacyt and Huh7/beta4-Deltacyt transfectants to laminin, but not to collagen, was reduced compared with the mock transfectants. However, migration of the beta4-Deltacyt transfectants toward both collagen and laminin was inhibited, suggesting a role for alpha6beta1 in the signaling of migration. Migration of HCC cells requires mitogen-activated protein (MAP) kinase. The adhesion of the beta4-Deltacyt transfectants to collagen resulted in a substantial reduction in MAP kinase activation in comparison with the mock transfectants, although their ability to activate MAP kinase in response to epidermal growth factor (EGF) stimulation was not impaired. In addition, matrix adhesion of the beta4-Deltacyt transfectants did not stimulate the tyrosine phosphorylation of focal adhesion kinase (FAK), and this defect correlated with reduced binding of adaptor protein Grb2 to FAK. These results suggest that FAK tyrosine phosphorylation is dependent on alpha6beta1 expression, and that FAK-Grb2 association plays a central role in alpha6beta1-mediated activation of MAP kinase. Moreover, the expression of alpha6beta1 in HCC cells is necessary for FAK/MAP kinase-dependent migration.

Expression and function of integrin receptors for collagen and laminin in cultured human hepatic stellate cells.

BACKGROUND & AIMS: Human hepatic stellate cells (HSCs), liver-specific pericytes, are currently considered major producers of extracellular matrix (ECM) components and key elements in the development of liver fibrogenesis. However, little is known about the possible functional interactions between HSCs and the various ECM components. Therefore, the present study was designed to evaluate the expression of integrins, the major family of extracellular matrix receptors. METHODS: Integrin expression was evaluated by immunoprecipitation and confirmed by immunocytochemistry and flow cytometry. RESULTS: Human HSCs were shown to express alpha1beta1, alpha2beta1, alpha(v)beta1. Adhesion to type IV collagen, type I collagen, fibronectin, and laminin 1 was inhibited by anti-beta1 antibody identifying beta1-containing integrins as possible receptors for these components. In addition, we showed that HSCs express alpha6beta4, a heterodimer known to mediate adhesion of epithelial cells to laminin and not previously characterized in mesenchymal cells. Adhesion to laminin 1 was not inhibited by antibodies specific for alpha6 or beta4, thus establishing that laminin 1 is not a ligand for alpha6beta4 in this cell type. CONCLUSIONS: These findings represent the first description of integrin receptors in HSC and provide an attempt to cover the gap of information in the field of HSC-ECM interactions.

[The determination of myocardial necrosis in unstable angina by the immunoradiometric measurement of circulating myosin]. / Individuazione della necrosi miocardica nell'angina instabile mediante dosaggio immunoradiometrico della miosina circolante.

Ventricular myosin heavy chains serum levels are a new marker of myocardial necrosis. We have studied plasma levels of myosin in 30 patients with unstable angina, 30 patients with acute myocardial infarction and 25 healthy subjects. The myosin peak level was 317 +/- 101 microU/L in angina patients, 2510 +/- 433 microU/L in infarcted patients and 62.3 +/- 17 microU/L in the controls. In both groups, the increase in serum myosin was more marked in those with larger infarction and in those with more severe angina. These data suggest that the measurement of serum myosin can identify the presence of micronecrosis in patients with unstable angina, according to what has been found using other markers of myocellular necrosis.

Stimulation of platelet-activating factor synthesis by progesterone and A23187 in human spermatozoa.

The presence of platelet-activating factor (PAF) has been demonstrated recently in mammalian spermatozoa, together with evidence for a role of this phospholipid in enhancing sperm motility and fertilizing ability. To investigate whether PAF synthesis and release occurs in human spermatozoa following incubation with stimuli that induce acrosome reaction, spermatozoa were incubated with progesterone and A23187, two known inducers of the exocytotic event. PAF synthesis (remodelling pathway) was assessed by [3H]acetate incorporation into PAF. Treatment of spermatozoa with progesterone and A23187 resulted in an increase of [3H]acetate incorporation into PAF. Most of the newly synthesized [3H]PAF formed in response to acrosome reaction was found in the supernatant, suggesting a release of the phospholipid from spermatozoa. PAF-like material extracted from human spermatozoa was able to induce aggregation of rabbit platelets and showed identical retention time and the same ion m/e values as authentic PAF when analysed with g.c.-m.s. Lyso-PAF:acetyl-CoA acetyltransferase (EC 2.3.1.67) activity in human spermatozoa was also studied and showed similar kinetic parameters to those described for other cell systems. Stimulation of spermatozoa with progesterone and A23187 induced an increase of [3H]arachidonic acid release, suggesting an activation of phospholipase A. In conclusion, our results demonstrated increased production and release of PAF in human sperm following stimulation with progesterone and A23187 and suggest a role for this phospholipid in the activation of spermatozoa.

Loop diuretic therapy in liver cirrhosis with ascites.

Medical treatment of ascites is aimed at reverting sodium retention, that is, at creating a negative sodium balance to relieve ascites. Bed rest and low-sodium diet induce the disappearance of ascites in about 10% of patients. Loop diuretics and aldosterone antagonists must be administered to the patients not responding to the previous regimen. Available evidence indicates that aldosterone antagonists are the first-choice drugs, as these substances are more effective than furosemide. Nevertheless, loop diuretics potentiate the effects of aldosterone antagonists. The reduced efficacy of furosemide in these patients, when compared with that of spironolactone, may be related to an impairment of both pharmacodynamics and pharmacokinetics. In fact, most sodium not reabsorbed in Henle's loop, due to the action of furosemide, is subsequently taken up in the distal nephron because of hyperaldosteronism. A further mechanism of resistance may be related to an impaired excretion of furosemide into the tubular lumen. The use of diuretics in the treatment of ascites is associated with several side effects, including prerenal azotemia, hepatic encephalopathy, and electrolyte and acid-base disorders. A stepped-care approach, together with careful monitoring of patients, is the best way to reduce the incidence of these complications. Ethacrynic acid has been shown to be highly effective in the treatment of ascites, even in patients refractory to other diuretics, but its use is associated with a high incidence of hypokalemia and hypochloremic alkalosis. Bumetanide and piretanide are comparable to furosemide, in terms of both efficacy and side effects.(ABSTRACT TRUNCATED AT 250 WORDS)

Knockout of alpha6 beta1-integrin expression reverses the transformed phenotype of hepatocarcinoma cells.

BACKGROUND & AIMS: Hepatocellular carcinoma is a common complication in liver cirrhosis. The integrin alpha6 beta1, a receptor for the laminin family of extracellular matrix proteins, has been found to be overexpressed in hepatocarcinoma. In an effort to further characterize the involvement of alpha6 beta1-integrin in hepatocarcinoma progression and to study alpha6 beta1-mediated functions, a human hepatocarcinoma cell line, HepG2, that express high surface levels of alpha6 beta1 and uses only this integrin to mediate adhesion on laminin was identified. METHODS: To assess the role of alpha6 beta1 in these cells, a cytoplasmic domain deletion mutant of the beta4-integrin subunit by complementary DNA transfection was expressed. The expression of the mutant beta4 subunit in association with endogenous alpha6 showed a dominant-negative effect on alpha6 beta1 expression. RESULTS: Stable transfectants of HepG2 that expressed the mutant beta4 subunit showed a reduced ability to adhere and migrate on laminin matrices and to invade Matrigel. Furthermore, transfected cells showed significantly lower growth rates and reduced anchorage-independent growth compared with mock-transfected cells. CONCLUSIONS: These findings on the expression and function of alpha6 beta1 in hepatocarcinoma cells emphasize the potential contribution of this laminin receptor in the neoplastic transformation of hepatocytes.

Torasemide in the treatment of patients with cirrhosis and ascites.

The effects of torasemide (20 mg/day) and furosemide (50 mg/day), each given over 4 days, were compared in a randomized and crossover study carried out in seven patients with cirrhosis and tense ascites. Patients also received a low-sodium (40 mmol/day) diet and the aldosterone antagonist, potassium canrenoate (100 mg b.i.d.). Torasemide induced a remarkably higher natriuretic (120 +/- 15 vs. 33 +/- 6 mmol/day, p < 0.02) and diuretic (1450 +/- 63 vs. 900 +/- 58 ml, p < 0.005) effect than furosemide. Body weight loss was also significantly higher (2.5 +/- 1.6 vs. 0.2 +/- 1.3 kg, p < 0.01) during the torasemide period. Kaliuresis was similar during the two treatment periods, despite the striking differences observed in natriuresis. Neither torasemide nor furosemide induced any significant change in serum electrolyte or creatinine concentrations, or in ammonia levels. The results of this study indicate that torasemide is suitable for the treatment of sodium retention in patients with cirrhosis and ascites.

Defective signal transduction in platelets from cirrhotics is associated with increased cyclic nucleotides.

BACKGROUND: Patients with advanced cirrhosis show defective platelet aggregation, which is dependent, at least in part, on intrinsic platelet abnormalities. The aim of this study was to evaluate the activating and inhibitory pathways of platelet signal transduction in cirrhotic patients. METHODS: Twelve cirrhotic patients and 12 control subjects participated in this study. Measurements were performed on washed platelets. RESULTS: Thrombin-stimulated inositol 1,4,5-trisphosphate production was reduced fivefold, and the increase in cytosolic calcium concentration was significantly lower in platelets from cirrhotic patients following stimulation with thrombin, platelet activating factor, or U-46619. In addition, the activity of the platelet Na+/H+ antiporter, evaluated after an acid load, was significantly lower in platelets from cirrhotic patients (0.90 +/- 0.19 vs. 1.37 +/- 0.16 delta pHi/min, P = 0.07). Cirrhotic patients also showed a significantly increased basal intraplatelet content of both 5'-cyclic adenosine monophosphate (cAMP) (2724 +/- 330 vs. 1561 +/- 258 fmol/10(8) platelets, P < 0.05) and 5'-cyclic guanosine monophosphate (cGMP) (217 +/- 18 vs. 159 +/- 29 fmol/10(8) platelets, P < 0.05). CONCLUSIONS: Our results indicate that in platelets from cirrhotic patients, defective early signal transduction is associated with an increase in platelet cAMP and cGMP, thus revealing new mechanisms contributing to the defective platelet function in this disease.

Impaired superoxide anion, platelet-activating factor, and leukotriene B4 synthesis by neutrophils in cirrhosis.

BACKGROUND: Several alterations of polymorphonuclear leukocyte (PMN) function were found in alcoholic cirrhotics that may contribute to augmented susceptibility to infections. We evaluated function and synthesis of lipid mediators in PMN obtained from nonalcoholic cirrhotics. METHODS: We evaluated the phagocytic and chemotactic response together with superoxide anion (O2-), leukotriene B4, (LTB4) and platelet-activating factor (PAF) production in response to different stimuli in PMN from nonalcoholic cirrhotics as compared with controls. RESULTS: PMN from cirrhotics showed, after stimulation with opsonized zymosan (STZ) and phorbol-12-myristate-13-acetate, a reduced capacity to produce O2- when compared with controls. [3H]acetate incorporation into PAF was significantly higher in PMN obtained from controls in respect to cirrhotics. Gas chromatography/mass spectrometry analysis confirmed a reduced PAF synthesis by PMN obtained from cirrhotics. LTB4 production from PMN, after stimulation with calcium ionophore (A23187) and STZ, was significantly reduced in cirrhotics. [3H]arachidonic acid release from prelabeled PMN, measured upon stimulation with A23187 and STZ, was higher in controls than in cirrhotics. CONCLUSIONS: An altered synthesis of LTB4 and PAF is associated with an impaired O2- production by PMN in nonalcoholic cirrhosis. Reduced synthesis of lipid mediators may be related to an altered phospholipase A, activity.