Mass spectrometry analysis of IgA1 hinge region in patients with IgA nephropathy.

BACKGROUND: Physicochemical alterations of the IgA molecule are supposed to play a pathogenetic role in IgA nephropathy (IgAN). The present study was carried out to analyze the structural variety of O-glycans on the IgA1 hinge region in IgAN. Sera from 9 IgAN patients and 9 healthy controls were individually examined to evaluate the IgA1 content and binding lectins (jacalin and Helix aspersa), using enzyme-linked immunosorbent assay (ELISA) techniques. The IgA1 from pooled sera were separated by affinity chromatography (jacalin), and the fragment containing the hinge region was prepared by pyridylethylation and trypsin treatment. The IgA fragments containing the hinge glycopeptide (33-mer hinge peptide core (HP) + O-glycans) were separated by jacalin affinity chromatography. Because we used jacalin, we only analyzed the Gal-3GalNAc residue containing IgA. The molecular weight (MW) of the IgA1 fragments was estimated using an ion trap mass spectrometer equipped with an electrospray ion source (ESI/MS). RESULTS: IgA1 concentration in pathological sera was higher than in the control serum (p<0.01). Compared with controls, serum IgA1 from IgAN patients showed significantly greater binding to the 2 lectins, jacalin (p<0.01) and Helix aspersa (HA, p<0.001), which are specific for O-linked Gal-beta1,3-GalNAc and GalNAc, respectively. Analyses of pooled sera showed that the number of O-glycosidic chains was comparable in IgAN and normal sera. With regards to the individual residues, we found that IgAN sera contained less sugar and galactose and sialic acid moieties than sera from control subjects, was reduced in IgAN sera, while terminal N-acetylgalactosamine levels were higher when compared with normal serum. CONCLUSIONS: Abnormalities of hinge region O-linked glycans were confirmed using advanced spectrometry technology. The pathogenetic implications for aggregation and defective removal of IgA1 are discussed.

Effects of angiotensin II blockade on nitric oxide blood levels in IgA nephropathy.

BACKGROUND: The effects of renin-angiotensin system blockade on nitric oxide (NO), especially in pathological conditions, are far from being established. The influence of kinins and angiotensin type 2 receptor are largely speculative and based mainly on animal studies. This study was aimed to address these aspects in humans. METHODS: Eight IgA nephropathy patients with documented clinical and histological indicators of poor prognosis were given 50 mg of losartan, 10 mg of enalapril, and 40 mg of the NO donor isosorbide 5 mononitrate (as a control of NO generation) in randomized order for 7 days each. Treatment periods were separated by washout periods of 7 days each. Laboratory investigations were performed before and after each study period. Seven healthy controls received losartan and enalapril according to the same study design. RESULTS: Glomerular filtration rate remained stable while effective renal plasma flow increased with each treatment (P<0.05). Under losartan and enalapril, filtration fraction fell (P=0.02), plasma renin activity increased (P<0.05) and urinary aldosterone concentration decreased (P=0.02). Angiotensin-converting enzyme activity was reduced to the limit of detection under enalapril (P<0.001). Blood NO, detected as nitrosylhaemoglobin by a recently developed technique of spin-trap electron paramagnetic resonance, increased significantly, as expected, during treatment with isosorbide 5 mononitrate (P=0.01), with enalapril (P<0.05), and also with losartan (P<0.05). Unlike losartan, enalapril significantly reduced albuminuria (P=0.01) in this short-term period. In the seven healthy controls, neither enalapril nor losartan were able to increase blood NO levels significantly. CONCLUSIONS: Blood levels of nitrosylhaemoglobin, a surrogate marker of NO, increased under blockade of the renin-angiotensin system in patients with IgA nephropathy, but not in healthy volunteers. This increase could contribute to changes of effective renal plasma flow in renal disease states.

Early increase in blood nitric oxide, detected by electron paramagnetic resonance as nitrosylhaemoglobin, in haemodialysis.

The objective of this study was to determine intradialytic blood levels of nitric oxide (NO), in patients undergoing chronic haemodialysis. This was done by detection of nitrosylhaemoglobin by a sensitive technique of spin trap electron paramagnetic resonance at 0, 5, 15, 60, 180 and 240 min of a 4-h standard bicarbonate dialysis, using the same dose (6000 U) of heparin and different dialysis membranes. The study group included 12 patients treated with cellulose-derived dialysis membranes (nine with cuprophan and three with cellulose triacetate) and 10 patients treated with synthetic membranes (five with polysulfone and five with polymethylmethacrylate). Control groups included 11 normal subjects and six patients with end-stage renal failure who were receiving intermittent peritoneal dialysis. Basal blood levels of nitrosylhaemoglobin in haemodialysis patients were significantly higher than normals, but similar to peritoneal dialysis patients. A significant increase (P < 0.01) in nitrosylhaemoglobin level was detected at 15 min of haemodialysis irrespective of the membrane used. A decrease to basal levels at 180 min was observed in all but two cuprophan-treated patients who, in contrast to the others, had a symptomatic hypotension at the end of the session and a further increase in blood nitric oxide. Patients undergoing peritoneal dialysis did not show any change in blood levels of nitrosylhaemoglobin during the first 180 min of the procedure. Thus, a constant increase in nitrosylhaemoglobin levels was observed early in haemodialysis, but not in peritoneal dialysis patients. Very preliminary evidence was obtained for a role of nitric oxide in the vascular instability at the end of haemodialysis in a few patients who had hypotensive episodes.

Nitric oxide, endothelin-1, and transcranial Doppler in migraine. Findings in interictal conditions and during migraine attack.

The role of vascular phenomena taking place during an attack of migraine are poorly understood. The aim of this study was to measure systemic levels of nitric oxide and endothelin-1, two of the most potent vasoactive mediators known, and to assess vasomotor responses through transcranial Doppler ultrasound monitoring in patients suffering from migraine without aura, both during the headache event and in headache-free periods as well as after pharmacologically induced pain relief. Seven patients (mean age 31.3 years, range 24 to 49 years), five women and two men, were enrolled in the pilot study. Transcranial Doppler recordings were performed according to conventional procedure. Endothelin-1 concentrations were measured by means of radioimmunoassay, whereas nitric oxide levels were estimated using electron paramagnetic resonance spectroscopy. Ultrasound evaluation did not show significant changes during migraine attacks compared to the interictal condition. Nitric oxide levels showed only slight differences between basal and attack conditions (0.85 +/- 0.46 versus 1.56 +/- 0.88, expressed as arbitrary units), and were raised after pharmacological intervention (2.91 +/- 1.93, P < 0.05). Plasma endothelin-1 concentrations decreased during migraine attacks with respect to interictal conditions (3.99 +/- 1.21 pg/mL versus 4.23 +/- 1.19), and returned to basal values (4.44 +/- 1.08 pg/mL) after relief of pain. Coupling the measurements of systemic levels of nitric oxide and endothelin-1 with transcranial Doppler velocity results will provide useful information on the hemodynamic changes of cerebral blood flow regulation in migraineurs, thereby adding new insights into the mechanisms of the migraine attack.

[The L-arginine/nitric oxide metabolic pathway. Its physiopathology and clinical implications]. / La via metabolica L-arginina/ossido di azoto. Fisiopatologia ed implicazioni cliniche.

The L-arginine/nitric oxide (NO) pathway plays a key role in a number of biological processes within most organs and systems. Increasing attention has been addressed to its involvement in the pathogenesis of various human diseases. In this review we examine the enzymology of different NO-synthase isoforms, the major NO detection techniques as well as the possible clinical and pharmacological implications of this new metabolic pathway.

4-Hydroxynonenal triggers Ca2+ influx in isolated rat hepatocytes.

Addition of micromolar concentrations of 4-hydroxynonenal (4-HNE), a reactive end-product of lipid peroxidation, to isolated rat hepatocytes was found to cause an early and transient increase in cytosolic Ca2+ concentration followed by a more pronounced and progressive elevation. Such a late effect of 4-HNE was prevented by chelation of extracellular Ca2+ with EGTA or by the addition of GdCl3, which is known to block the activity of store operated Ca2+ channels in the hepatocyte plasma membrane. Moreover, the preincubation of isolated hepatocytes with the phospholipase C inhibitor U73122 resulted in a complete inhibition of both the early increase of cytosolic Ca2+ and the subsequent Ca2+ inflow. When 4-HNE was added to the hepatocytes 5 min after the emptying of intracellular Ca2+ pools by thapsigargin, the aldehyde caused a further increase in the accumulation of Ca2+ which was prevented in the presence of GdCl3. Taken together these results indicate that in hepatocytes 4-HNE causes Ca2+ inflow across GdCl3-sensitive Ca2+ channels. The mechanism responsible for such an effect is triggered by the emptying of intracellular Ca2+ pools likely resulting from 4-HNE mediated stimulation of phospholypase C, but 4-HNE also appears to interfere with the channel protein(s) or with the mechanism(s) regulating capacitative Ca2+ inflow.

Enzymatic, biophysical and ultrastructural changes of plasma membranes in chemical-induced rat hepatoma.

Plasma membranes from liver of control rats or from chemical-induced hepatoma were prepared. The basal activity of adenylate cyclase was increased significantly in the rat plasma membranes of DEN-induced hepatoma compared to normal tissue. The glucagon-induced response on the cellular effector systems via guanine nucleotide-binding regulatory proteins (G proteins) was inhibited in hepatoma plasma membranes. These findings suggest that in hepatoma membranes, unlike normal hepatic membranes, the response to hormonal stimuli through regulatory G proteins results in a loss of response to glucagon, as well as to GTP plus glucagon or to GTP gamma S. However, the activating effects of forskolin, which catalyses the formation of cyclic AMP from ATP acting on the catalytic subunit, were to some extent retained. The methyltransferase-I behaved in the opposite direction to the adenylate cyclase, showing a decreased activity in hepatoma plasma membranes compared to control membranes. In contrast, the activity of the ecto-5'-nucleotidase was significantly increased in hepatoma. These enzymatic changes have been found to influence the membrane fluidity and to be responsible for the ultrastructural modifications of hepatoma plasma membranes which are induced by chemical carcinogens.

Isosorbide 5 mononitrate administration increases nitric oxide blood levels and reduces proteinuria in IgA glomerulonephritis patients with abnormal urinary endothelin/cyclic GMP ratio.

An endothelin urinary hyperexcretion, which is not counterbalanced by an adequate increase in cGMP biosynthesis, was previously detected in some patients with IgA Nephropathy (IgAN). Since this imbalance might potentiate local ET1-mediated hemodynamics effects, 9 IgAN patients with an increased (> or = 0.1) urinary ET1/cGMP ratio (group 1) and 5 IgAN patients with comparable renal function and reduced ET1/cGMP ratio (group 2) were given standard doses of isosorbide 5 mononitrate (as a nitric oxide source). Blood nitric oxide (NO) levels, as detected by electron paramagnetic resonance, significantly increased after isosorbide administration (p < 0.01) and decreased after drug discontinuation in both groups. Nitric oxide levels were significantly related with those of the effective renal plasma flow (p < 0.02), but not with the glomerular filtration rate. Proteinuria levels significantly decreased after drug administration (p < 0.009) in group 1 and returned to baseline levels thereafter, except two cases showing persisting low levels. Values of filtration fraction in the same group decreased after iso5M administration (p < 0.02 compared to basal levels). These results may possibly be related to the counterbalancing effects of nitric oxide on endothelin-mediated mesangial contraction.

Changes of adenylate cyclase activity in AH-130 ascites hepatoma of Yoshida induced by enrichment with fatty acids.

Enrichment of hepatoma cells with arachidonic acid increases fluidity of plasma membranes, unstimulated lipid peroxidation and basal adenylate cyclase activity, whereas enrichment with stearic acid decreases fluidity and does not cause any variation in lipid peroxidation or adenylate cyclase. The increase in adenylate cyclase activity may be due to the increase not only in fluidity, but also in lipid peroxidation products. Indeed, adenylate cyclase is stimulated by 4-hydroxynonenal, one important product of lipid peroxidation, when added to plasma membranes. 4-hydroxynonenal increases adenylate cyclase two-fold in unenriched plasma membranes and three-fold in plasma membranes enriched with arachidonic acid in comparison with basal activity.

Evidences of 4-hydroxynonenal involvement in modulation of phagocyte activities.

The peroxidative breakdown of membrane polyunsaturated fatty acids leads to the production of various carbonylic compounds: among these, 4-hydroxynonenal (HNE) displays many biological properties related to neutrophil functions. It stimulates rat and human polymorphonuclear (PMN) cell migration and has been detected during inflammation. The aim of this study was to elucidate and well characterize the mechanism of action of HNE. We observed that micromolar HNE concentrations that influence migration do not stimulate differently from many other chemoattractants the human PMN chemiluminescence (CL) induced by opsonized zymosan or phorbol 12-myristate 13-acetate (PMA). Higher HNE concentrations inhibit the light emission of stimulated PMN. Addition of 0.5 mM L-arginine (L-arg), the substrate of nitric oxide synthase, into the incubation medium had the effect of modifying human CL. In fact, HNE at 10-6 M, a concentration which is ineffective in absence of L-Arg, at 10-5 M reduces CL emission of PMA-stimulated human PMN. These observations have been confirmed by electron-spin resonance (ESR) analysis. HNE, according to other stimuli, induced PMN phosphoinositide-specific phospholipase C (PL-C). All these results considered together suggest the conclusion that HNE represents an interesting endogenous molecule that plays a role as an inflammatory mediator involved a) in the recruitment of phagocytic cells at the inflamed area, and b) in the modulation of respiratory burst and of nitric oxide (NO) production.

Induction of differentiation in human HL-60 cells by 4-hydroxynonenal, a product of lipid peroxidation.

4-Hydroxynonenal (HNE) is the major diffusible toxic product generated by lipid peroxidation of cellular membranes. The level of lipid peroxidation and, consequently, the concentration of its products are inversely related to the rate of cell proliferation and directly related to the level of cell differentiation. In the present paper the effects of HNE on the proliferation and differentiation of the HL-60 human promyelocytic cell line have been investigated. Repeated treatment at 45-min intervals with HNE (1 microM) was performed to maintain the cells in the presence of the aldehyde for 7 1/2 or 9 h. The effect of HNE on cell proliferation and differentiation was compared with dimethyl sulfoxide (DMSO)-treated cells. HNE causes a strong inhibition of cell growth without affecting cell viability. Moreover, HL-60 cells acquire the capability to produce chemiluminescence after soluble (phorbol myristate acetate) or corpuscolate (zymosan) stimulation. The phagocytic ability has also been calculated by counting the number of cells that phagocytize opsonized zymosan. Values were 43 and 55% after 10 or 12 HNE treatments, respectively, and 88% in DMSO-treated cells. Myeloperoxidase activity, 5 days after treatment, decreased by 85% in either HNE- or DMSO-treated cells while acid phosphatase activity increased with respect to untreated cells. Results obtained indicate that HNE at concentrations close to those found in the normal tissues can induce inhibition of proliferation and induction of differentiation in the HL-60 cell line.

Influence of 4-hydroxynonenal on bombesin-induced stimulation of phospholipase C activity in rat liver.

The influence of 4-hydroxynonenal (HNE) on bombesin-induced PIP2-phospholipase C activity (PL-C) was studied in vitro on isolated liver membranes. Both bombesin and HNE stimulated the enzymatic activity at micromolar concentrations and their effect was enhanced by the nucleotide GTPgammaS. An additive synergism was observed with 1 microM bombesin and 0.1 microM HNE. When GTPgammaS was added to the reaction mixture, the degree of PL-C stimulation in the presence of both compounds was not different from the activity value induced by bombesin alone. Since such micromolar amounts of HNE can be actually found in normal liver cells, these results support the hypothesis of a physiological importance of lipid peroxidation in the regulation of phospholipase-C activity.

Effects of 4-hydroxynonenal, a product of lipid peroxidation, on cell proliferation and ornithine decarboxylase activity.

4-hydroxynonenal (HNE) is one of the major breakdown products of cellular lipid peroxidation. Its effects on proliferation, ornithine decarboxylase (ODC) activity and DNA synthesis have been investigated in leukemic cell lines. The cells were incubated for 1 hour with different aldehyde concentrations, then washed and resuspended in medium with fresh foetal calf serum. HNE concentrations ranging from 10(-5) to 10(-6) M significantly inhibited ODC activity when induced by addition of fresh foetal calf serum both in K562 and HL-60 cells. 3H-Thymidine incorporation in K562 cells was also inhibited from 6 to 12 hours after the treatment. The same HNE concentrations did not inhibit ODC activity when added to cytosol, thus a direct action on the enzyme can be excluded. Moreover, HNE did not affect the half-life of ODC, so that a specific effect on ODC synthesis may be supposed. These data indicate a reduction of proliferative capacity of the cells and are consistent with the possibility that HNE, at concentrations close to those found in normal cells, plays a role in the control of cell proliferation.

Influence of 4-hydroxynonenal on chemiluminescence production by unstimulated and opsonized zymosan-stimulated human neutrophils.

The lipid peroxidation product 4-hydroxy-2, 3-trans-nonenal (HNE) has a spectrum of biological effects on different cell types depending on the concentrations tested. In particular micromolar HNE concentrations stimulate neutrophil migration and polarization whereas higher doses inhibit. In our experimental conditions, fMet-Leu-Phe (fMLP) increased CL production of both unstimulated and zymosan-stimulated neutrophils, whereas cell stimulation with low HNE concentrations as well as zymosan addition to HNE incubated cells did not enhance light emission. In contrast 10(-4) M HNE reduced CL emission by unstimulated cells nearly to background values, completely depressed CL production by zymosan-stimulated cells and reduced phagocytosis. Cysteine was found to be able to counteract the HNE effect by about 70 per cent. The possibility that this aldehyde could exert its inhibitory effect through the alkylation of NADPH-oxidase SH-groups is postulated. Moreover, our present data on differences observed between fMLP and HNE indicate a different chemotactic mechanism induced by these two classes of compounds and lead to the conclusion that the local functional features of the attracted cells may be different.

Inhibition of the high affinity Ca2(+)-ATPase activity in rat liver plasma membranes following carbon tetrachloride intoxication.

In vivo administration of CCl4 (2.5 ml/kg, body wt.) to rats results in an early and then progressive inhibition of the high affinity Ca2(+)-ATPase activity in rat liver plasma membranes. The derangement to the Ca2(+)-ATPase seems to be independent on a 'solvent effect' of the agent since the in vitro addition of increasing concentrations of either CCl4 or ethanol to control plasma membranes does not affect the enzymatic activity. By using the technique of vitamin E pretreatment of experimental animals we show that the damage to the Ca2(+)-ATPase seems to follow a two-step kinetics. The early inhibition of the enzyme is not prevented by alpha-tocopherol supplementation and seems then unrelated to lipid peroxidative processes. The same procedure is however able to affort a significant protection against the exacerbation of the damage to the Ca2(+)-ATPase becoming evident late during the course of CCl4 intoxication. The high affinity Ca2(+)-ATPase is affected in vitro by 4-hydroxy-nonenal (HNE), a major end-product of lipid peroxidation interacting with -SH groups. Similar results were obtained after the addition to the incubation medium of sulphydryl reagents. The possible mechanisms involved in Ca2(+)-ATPase inhibition are discussed in relation to the development of CCl4 toxicity and to the role of lipid peroxidative processes.

Changes of serum iron transferrin and copper ceruloplasmin in rats given Cu(II)2 (acetylsalicylate)4 during acute inflammation.

Modifications of serum levels of iron transferrin and copper ceruloplasmin after acute inflammation by carrageenan and treatment with acetyl salicylic acid [ASA] or Cu(II)2(acetylsalicylate)4 [Cu(II)2(AS)4] were studied in the rat by EPR spectroscopy. Furthermore, the ulcerogenic potential of the two drugs was investigated after a single high oral dose. Our results indicate that Cu(II)2(AS)4 is more effective than ASA in limiting the inflammation provoked by the phlogogen. In these conditions the iron(III) non-heme and copper(II) ceruloplasmin concentration in serum was modified either during inflammation or after treatment with antiphlogistic agents: in carrageenan-injected rats the level of serum iron(III) non-heme was found to be very low, while the copper(II) ceruloplasmin concentration was partially reduced. On the other hand, after the pharmacological treatments, no changes of iron transferrin were observed and the concentration of copper ceruloplasmin was increased. With regard to their ulcerogenic effect, ASA appeared to be more irritating for gastric mucosa than Cu(II)2(AS)4.

Subcellular calmodulin distribution in rat liver after CCl4 poisoning.

Disturbed cellular calcium homeostasis has been observed during CCl4 poisoning, with an increase in calcium content 1 h after administration. Intracellular increase of calcium may be expected to alter membrane/cytosol distribution of calmodulin (CaM). This paper investigates changes in rat liver subcellular CaM distribution 30 min, 1 h and 2 h after CCl4 intoxication. The whole liver value remained unchanged, whereas the nuclear fraction increased and the microsomal and cytosolic fraction decreased. This may suggest that CaM is involved in the several liver cell alterations caused by CCl4 poisoning.

Effect of 4-hydroxynonenal on c-myc expression.

The 4-hydroxynonenal aldehyde (HNE), a product of lipid peroxidation with high biological activity, inhibits cancerous growths in vivo and in vitro. The mechanism by which this aldehyde acts is not yet understood. The c-myc oncogene seems to be involved in the regulation of cellular multiplication and transformation. We evaluated the c-myc expression and the RNA, DNA and protein synthesis in K562 cells. These cells were incubated for 1 hour in presence of several aldehyde concentrations (range 5.10(-7) to 10(-4)), then washed and kept for 20 hours in a growth medium until used. HNE inhibited both the nucleic acids and protein synthesis in a dose dependent manner, and c-myc expression was evaluated in the K562 cells after incubation with 10(-4) M or 10(-6) M HNE. HNE inhibited c-myc expression only at the highest dose. These preliminary results may suggest that the inhibition of c-myc expression is related to nucleic acid synthesis inhibition following HNE exposure.