腹膜透析引流液离心法和大网膜消化法原代培养人腹膜间皮细胞

[目的]建立和改善两种人腹膜间皮原代细胞培养的方法.[方法]分别采用离心法从腹膜透析（PD）引流液中及胰蛋白酶-EDTA消化法从人的腹膜组织中分离培养人腹膜间皮细胞（HPMC）,并分别对两种原代培养的HPMC采用相差倒置显微镜和免疫组化染色对培养的细胞进行鉴定.[结果]①两种方法均在体外原代培养成活,在相差倒置显微镜下观察细胞均呈典型的铺路石样外观,免疫组化显示两种方法培养的HPMC胞浆角蛋白、波形蛋白表达阳性,提示为腹膜间皮细胞.②经大网膜消化法获得的HPMC可传至第四代,保持原有铺路石样外观,PD引流液离心法获得的HPMC传代后生长缓慢,长期接受PD后HPMC可呈现成纤维细胞样改变.[结论]成功应用离心法和消化法进行HPMC原代培养,有助于PD研究体外实验的开展.     

Diminished L-arginine bioavailability in hypertension

L-Arginine is the precursor of NO (nitric oxide), a key endogenous mediator involved in endothelium-dependent vascular relaxation and platelet function. Although the concentration of intracellular L-arginine is well above the Km for NO synthesis, in many cells and pathological conditions the transport of L-arginine is essential for NO production (L-arginine paradox). The present study was designed to investigate the modulation of L-arginine/NO pathway in systemic arterial hypertension. Transport of L-arginine into RBCs (red blood cells) and platelets, NOS (NO synthase) activity and amino acid profiles in plasma were analysed in hypertensive patients and in an animal model of hypertension. Influx of L-arginine into RBCs was mediated by the cationic amino acid transport systems y+ and y+L, whereas, in platelets, influx was mediated only via system y+L. Chromatographic analyses revealed higher plasma levels of L-arginine in hypertensive patients (175+/-19 micromol/l) compared with control subjects (137+/-8 micromol/l). L-Arginine transport via system y+L, but not y+, was significantly reduced in RBCs from hypertensive patients (60+/-7 micromol.l(-1).cells(-1).h(-1); n=16) compared with controls (90+/-17 micromol.l(-1).cells(-1).h(-1); n=18). In human platelets, the Vmax for L-arginine transport via system y+L was 86+/-17 pmol.10(9) cells(-1).min(-1) in controls compared with 36+/-9 pmol.10(9) cells(-1).min(-1) in hypertensive patients (n=10; P<0.05). Basal NOS activity was decreased in platelets from hypertensive patients (0.12+/-0.02 pmol/10(8) cells; n=8) compared with controls (0.22+/-0.01 pmol/10(8) cells; n=8; P<0.05). Studies with spontaneously hypertensive rats demonstrated that transport of L-arginine via system y+L was also inhibited in RBCs. Our findings provide the first evidence that hypertension is associated with an inhibition of L-arginine transport via system y+L in both humans and animals, with reduced availability of L-arginine limiting NO synthesis in blood cells.     

Nitric oxide synthase inhibitors affect nitric oxide synthesis in normoxic but not in ischemic organs during intestinal ischemia and early reperfusion

Inhibition of endogenous nitric oxide (NO) synthesis during early intestinal ischemia/reperfusion (I/R(i)) enhances remote organ damage related to I/R(i). However, the effects of NO synthase (NOS) inhibitors on NO formation in various organs have not yet been specified. We therefore investigated the effects of N-G-monomethyl-L-arginine (L-NMMA), a nonspecific NOS inhibitor, and L-arginine, the NOS substrate, on NO formed in ischemic intestine versus normoxic remote organs (lung and liver). We used electron paramagnetic resonance spectroscopy and a specific NO trap to assay NO in blood, intestine, lung, and liver of rats subjected to local I/R(i), with and without L-NMMA and L-arginine supplementation. We found that I/R(i) increased NO levels in the intestine and blood, but not in the remote organs lung and liver. Administration of L-NMMA before I/R(i) decreased I/R(i)-independent basal NO levels in normoxic lung and liver without influencing I/R(i)-induced increase in NO levels in intestinal tissue or in blood. L-arginine supplementation increased circulating levels of NO, with sensitivity to L-NMMA, without affecting NO levels in normoxic or ischemic tissue. Our data suggest that NOS activity controls the NO generated in normally perfused remote organs during early I/R(i). Hence NOS inhibitors, when administered during I/R(i), decrease physiological NO levels in normoxic remote organs without affecting increased NO levels originating from ischemic intestine. This may explain the harmful effect of nonspecific NOS inhibitors during early I/R(i). In addition, the generation of NO in remote organs is not limited by tissue L-arginine concentrations and, therefore, not influenced by exogenous L-arginine. The protective effect of L-arginine supplementation during I/R(i) is probably related to increasing intravascular NO formation.     

人参总皂甙抑制乳酸盐腹膜透析液对人腹膜间皮细胞的损伤

目的观察人参总皂甙（TSPG）对乳酸盐腹膜透析液（0PDS）致人腹膜间皮细胞（HPMC）损伤程度的影响,为其保护HPMC提供实验依据。方法采用酶消化法从人腹膜组织中分离间皮细胞,建立稳定的体外培养模型后分5组,F12完全培养液对照组、腹膜透析液组（2．5％L-PDS组）、2．5％L-PDS＋TSPG组（终浓度为500μh／ml）、2．5％LPDS＋TSPG组（终浓度为50μg／ml）、单纯TSPG组。经各种因素处理后的细胞在培养60min和300min后用MTT法检测细胞的增殖,用自动生化分析仪检测各组培养液中乳酸脱氢酶的水平,来反映细胞的损伤程度。结果HPMC在终浓度为500μg／ml的TSPG和2．5％的乳酸盐腹膜透析液培养体系中培养1h后,和对照组相比,HPMC增殖能力明显增强（P〈0．05）,培养体系中的LDH含量明显下降（P〈0．05）。结论人参总皂甙能抑制乳酸盐腹透液导致的HPMC的损伤。     

Chronic peritoneal inflammation by cyanate in rats.

OBJECTIVE: During peritoneal dialysis, the peritoneum is exposed to waste products, including urea. Urea forms cyanate spontaneously at body temperature and pH, and cyanate carbamylates amino acids, peptides, and proteins. Cyanate may contribute to peritoneal injury with morphological changes in the peritoneum. To test this hypothesis, we injected cyanate into rats. METHODS: Experiments were performed in two groups of 7 rats each. In the cyanate group, each rat received 1 mL of 1.5 micromol/L potassium cyanate dissolved in 40 mmol/L sodium bicarbonate solution intraperitoneally each experiment day. In the control group, each rat received 1 mL of 1.5 micromol/L potassium bicarbonate instead of potassium cyanate. The rats in both groups were anesthetized and killed at the 85th day after the first injection. After formalin fixation, tissue samples from abdominal walls and livers were sliced, embedded in a standard manner, and stained with hematoxylin and eosin. RESULTS: Parietal peritoneum from rats in the cyanate group showed a mild increase in the number of fibroblasts, with collagen deposits, infiltration by mononuclear cells, vascular congestion, round-shaped transformation of mesothelial cells, widening of submesothelial spaces, and abundant denudation of mesothelial cells. The visceral peritoneum from rats in the cyanate group showed collagen deposits with fibroblastic proliferation. CONCLUSIONS: Cyanate can induce chronic inflammation in the peritoneum, and exposure of the peritoneum to cyanate may contribute to peritoneal injury in patients being treated with peritoneal dialysis.     

Nifedipine and diltiazem but not verapamil up-regulate endothelial nitric-oxide synthase expression

We have recently shown that felodipine, a long-acting dihydropyridine L-type calcium channel blocker (CCB), up-regulates nitric oxide (NO) production and endothelial NO synthase (eNOS) expression and activity in cultured endothelial cells as well as in animals with chronic renal failure. This study was intended to compare the effects of prototypes of the three classes of L-type CCBs on the NO system in cultured human coronary artery endothelial cells. Thus, cultured endothelial cells were incubated either with nifedipine, diltiazem, or verapamil for 24 h at 10(-5) to 10(-7) M concentrations. Cells incubated with inactive vehicle served as controls. NO production, as discerned from total nitrate plus nitrite recovered in the medium, was significantly increased by nifedipine (P <.03) and by diltiazem (P <.05). However, NO production remained unchanged with verapamil (P = NS). Similarly, eNOS protein abundance was increased significantly by nifedipine (P <.05) and diltiazem (P <.05). In contrast, eNOS expression was not changed by verapamil (P = NS). Likewise, NOS activity, as measured from [(3)H]L-arginine to [(3)H]L-citrulline conversion, significantly increased with nifedipine (P <.01) and diltiazem (P <.01). However, incubation with verapamil failed to alter NOS activity of the cultured endothelial cells (P = NS). We concluded that prototypes of dihydropyridine and benzothiazepine classes, but not phenylalkylamine class of CCBs, up-regulate the NO system. This may, in part, account for the different biological properties of these agents.     

The role of the glyoxalase pathway in reducing mesothelial toxicity of glucose degradation products.

BACKGROUND: The glucose degradation products (GDP) presentin conventional peritoneal dialysis fluids (PDF) may exert adverse effects toward human peritoneal mesothelial cells (HPMC). Some GDP can be detoxified by the glyoxalase/ glutathione pathway. It has been shown that the addition of glyoxalase I (GLO-I) and reduced glutathione (GSH) to PDF effectively eliminates GDP. We have therefore examined the GLO-I/GSH system in HPMC and assessed the impact of GLO-I/ GSH-treated PDF on the viability and function of HPMC. METHODS: Heat-sterilized PDF (H-PDF) was incubated in the presence or absence of GLO-I and GSH for 1 hour at 37 degrees C, and then mixed with an equal volume of serum-free M199 medium and applied to HPMCin culture. After 24 hours, HPMC were assessed for viability, the release of interleukin-6, GLO-I activity, and cellular glutathione. The effects were compared to those exerted by filter-sterilized PDF (F-PDF), which was devoid of GDP. RESULTS: Exposure of HPMC to H-PDF resulted in reduced GLO-I activity, GSH depletion, and a decrease in cell viability. Pretreatment of H-PDF with either a combination of GLO-I and GSH or GSH alone markedly reduced inhibitory effects of H-PDF toward HPMC, as measured by cell viability and inter-Leukin-6 generation. Exposure of HPMC to the GSH precursor L-2-oxothiazolidine-carboxylic acid increased cellular GSH and prevented the loss of GLO-I activity in response to H-PDF. CONCLUSIONS: Exposure to conventional GDP-rich PDF impairs the activity of the glyoxalase/glutathione system in HPMC. Pretreatment of PDF with GSH or replenishment of cellular GSH protects HPMC against GDP-mediated toxicity.     

A study on the effects of endogenous nitric oxide on coronary blood flow, myocardial oxygen extraction and cardiac contractility

The purpose of the present study was to clarify how endogenous nitric oxide (NO) affects cardiac contractility and myocardial oxygen consumption (MVO2) in vivo. alpha-Chloralose-anesthetized dogs (n = 18) were instrumented to perform continuous and simultaneous measurements of coronary blood flow (CBF), anterior interventricular vein oxygen saturation (with the use of a fiberoptic catheter), aortic pressure, left ventricular pressure, and left ventricular volume. CBF, myocardial oxygen extraction (O2-extract), MVO2, the relationship between CBF and O2-extract during direct vasodilation induced by intracoronary papaverine (0.1, 0.2, 0.4 mg/kg), and cardiac contractility (Emax) were examined at control, after intracoronary infusion of NG-monomethyl-L-arginine (L-NMMA, 2 mg/kg) and after antagonization of NO by L-arginine (20 mg/kg). L-NMMA decreased CBF from 62.0 +/- 1.7 to 59.7 +/- 2.4 (mL/min/100 g, P < 0.05) and increased O2-extract from 68.2 +/- 1.7 to 79.0 +/- 1.7% (P < 0.05). Emax was increased after L-NMMA from 3.2 +/- 0.2 to 3.7 +/- 0.1 (mmHg/mL/100 g, P < 0.05). These effects of L-NMMA were antagonized by L-arginine (P < 0.05 vs. after L-NMMA, P = NS vs. before L-NMMA). L-NMMA shifted CBF and O2-extract relationship determined by papaverine injection upward and L-arginine antagonized it to its baseline level. Endogenous NO reduces cardiac contractility and decreases MVO2, while increasing CBF.     

Abnormal contractile function due to induction of nitric oxide synthesis in rat cardiac myocytes follows exposure to activated macrophage-conditioned medium.

The mechanism by which soluble mediators of immune cell origin depress myocardial contractility, either globally as in systemic sepsis, or regionally in areas of inflammatory myocardial infiltrates, remains unclear. When freshly isolated ventricular myocytes from adult rat hearts were preincubated for at least 24 h in medium conditioned by endotoxin (LPS)-activated rat alveolar macrophages, their subsequent inotropic response to the beta-adrenergic agonist isoproterenol was reduced from 225 +/- 19% to 155 +/- 10% of the baseline amplitude of shortening (mean +/- SEM, P < 0.05). Neither baseline contractile function nor the contractile response to high extracellular calcium were affected. To determine whether an endogenous nitric-oxide (NO)-signaling pathway within ventricular myocytes was responsible for their decreased responsiveness to isoproterenol, the L-arginine analogue L-NMMA was added to the preincubation medium. While L-NMMA did not affect baseline contractile function or the response of control myocytes to isoproterenol, it completely restored the positive inotropic response to isoproterenol in myocytes preincubated in LPS-activated macrophage medium. Release of NO by ventricular myocytes following exposure to activated macrophage medium was detected as an increase in cGMP content in a reporter-cell (RFL-6) bioassay and also as increased nitrite content in myocyte-conditioned medium. Thus, the depressed contractile response of adult rat ventricular myocytes to beta-adrenergic agonists by a 24-h exposure to soluble inflammatory mediators is mediated at least in party by induction of an autocrine NO signaling pathway.     

ACE inhibitor and AT1-receptor blocker attenuate the production of VEGF in mesothelial cells.

OBJECTIVE: Human mesothelial cells (HMC) are a major source of intraperitoneal vascular endothelial growth factor (VEGF) and by that are presumably involved in complications of long-term peritoneal dialysis (PD), such as ultrafiltration failure. This prompted us to look for agents that reduce basic mesothelial VEGF production and abrogate VEGF-overproduction induced by proinflammatory cytokines such as tumor necrosis factor alpha (TNF-alpha) and interleukin-1alpha (IL-1alpha). Angiotensin-converting enzyme (ACE) inhibition was found to preserve peritoneal function and ameliorate morphologic changes in a rat PD model. The present in vitro study was designed to investigate the effect of the ACE inhibitors captopril and enalapril, and the angiotensin II type 1-receptor (AT1) antagonist losartan on mesothelial VEGF synthesis. METHODS: HMC were isolated from omental tissue and taken into culture. VEGF antigen concentrations were measured in the cell supernatant by ELISA. VEGF mRNA levels were determined by real-time polymerase chain reaction. RESULTS: Incubation of HMC with captopril (100-1000 micromol/L) resulted in a concentration-dependent attenuation of VEGF synthesis. Incubation with captopril (500-1000 micromol/L), enalapril (100-1000 micromol/L), and losartan (1-100 micromol/L) significantly decreased inflammatory mediator (TNF-alpha, IL-1alpha)-induced mesothelial VEGF overproduction. CONCLUSION: The results indicate that ACE inhibitors and AT1-receptor blockers are capable of effectively attenuating the overproduction of VEGF due to proinflammatory cytokine stimuli. These data suggest that locally produced angiotensin II in the peritoneal cavity may be a potential therapeutic target in ultrafiltration failure during longterm PD.     

Effect of iron sucrose on human peritoneal mesothelial cells

BACKGROUND: Iron supplementation is often required in uraemic patients with anaemia. Peritoneal cavity was proposed as an alternative intravenous route for iron infusion in patients treated with peritoneal dialysis. We studied the effect of iron sucrose (Venofer) on the function of human peritoneal mesothelial cells maintained in in vitro culture. MATERIALS AND METHODS: In in vitro experiments on human peritoneal, the mesothelial effect of elemental iron (in conc. 0.0001-1 mg mL-1) present in Venofer on their viability, growth and synthesis of IL-6 was studied. Additionally we evaluated with a fluorescent probe (2',7'-dichlorodihydro-fluorescein diacatate) generation of reactive oxygen species in cells exposed to iron sucrose. We also measured accumulation of iron in the cytoplasm of mesothelial cells after their in vitro exposure to Venofer. RESULTS: In in vitro conditions iron induces a dose-dependent inhibition of viability of the mesothelial cells as reflected by inhibition of the cells growth by 34% at Fe 0.1 mg mL-1 vs. control (P < 0.05) increased release of lactate dehydrogenase (LDH) from the cytosol: 67.1 +/- 30.3 mU mL-1 at Fe 1 mg mL-1 vs. 7.9 +/- 6.4 in control group (P < 0.001), and reduced synthesis of IL-6: 209 +/- 378 pg mg-1 cell protein at Fe 1 mg mL-1 vs. 38674 +/- 4146 pg mg-1 cell protein in controls (P < 0.001). Cytotoxicity of iron towards mesothelial cells was enhanced in vitro when it was tested in presence of the dialysis fluid. Iron used in vitro at concentration 0.0001 mg mL-1 and greater induces generation of oxygen-derived free radicals in mesothelial cells. Furthermore, iron is taken by these cells and stored in their cytosol, resulting in stimulation of the intracellular generation of free radicals. CONCLUSIONS: We conclude that iron used in the form of iron sucrose is cytotoxic to human peritoneal mesothelial cells. Accumulation of iron sucrose within cytoplasm of these cells may lead to induction of its chronic cytotoxic effect.     

Electrophysiology and glucose transport of human peritoneal mesothelial cells: implications for peritoneal dialysis.

OBJECTIVE: To elucidate ionic and glucose transport across human peritoneal mesothelium, we utilized an Ussing chamber setup and studied the electrophysiological characteristics and tissue permeabilities of human peritoneal mesothelial cells (HPMC) to L- and D-glucose. METHODS: Human mesothelial cells were grown on polyester filters (snapwell; Costar, Cambridge, MA, U.S.A.) that, upon confluence, were fitted into Ussing chambers. Transmesothelial resistance and resting potential were determined using electrophysiological techniques. Radiolabeled glucose was added to one side of the chamber and the permeabilities determined by serial sampling in the receptive compartment. RESULTS: The transmesothelial potential and resistance were 0.54 +/- 0.07 mV (apical positive) and 20.4 +/- 3.2 ohms x cm2 respectively (mean +/- SEM, n = 36). The course of overall transfer of D- and L-glucose was examined using L-glucose as a positive diffusion-plus-leak marker. The permeabilities of HPMC to D-glucose were 3.00 +/- 0.26 cm/sec (apical-to-basolateral) and 3.25 +/- 0.27 cm/sec (basolateral-to-apical) [n = 6 experiments, p = not significant (NS)], which were not different from those of L-glucose: 3.00 +/- 0.30 cm/sec (apical-to-basolateral) and 2.71 +/- 0.24 (basolateral-to-apical) (n = 6 experiments, p = NS). CONCLUSIONS: The transepithelial resistance of HPMC is low and the ionic gradient, although it exists, is small and inconsequential. Passive paracellular flow accounts for the majority of transmesothelial glucose transport. The existence of a large paracellular shunt precludes the mesothelial membrane as a clinically relevant osmotic barrier.     

Cytotoxic effects of commercial continuous ambulatory peritoneal dialysis (CAPD) fluids and of bacterial exoproducts on human mesothelial cells in vitro

Cultured human mesothelial cells were exposed to peritoneal dialysis fluids, supernatants from cultures of Staphylococcus aureus and S. epidermidis, and antibiotics. Mesothelial cell monolayer cultures were derived from surgically removed omentum. The cytotoxicity of various agents for the cultured mesothelial cells was measured by a 51 Cr-release assay. All brands of fresh peritoneal dialysis fluids induced a more than 50% 51 Cr-release after 18 h. Morphological changes observed included retraction and shrinking of cells, pyknosis of the nuclei and, finally, detachment of cells over an 18-h period. Neutralization of the acid (pH 5.2-5.5) fluids to pH 7.3 did not abolish the cytotoxicity. In contrast, effluent dialysis fluids were not toxic for mesothelial cells; neither was acid (pH 5.5) culture medium nor culture medium with glucose up to 2%. However, higher glucose concentrations induced increasing 51 Cr-release. Furthermore, filter-sterilized supernatants of S. aureus were cytotoxic for mesothelial cell monolayers in 4/7 (57%) strains of S. aureus tested. In contrast, only 4/29 (14%) strains of S. epidermidis produced cytotoxic exoproducts (p = 0.03). Antibiotics were not found to be cytotoxic, with the possible exception of erythromycin. We conclude that currently available peritoneal dialysis fluids are cytotoxic for mesothelial cells in vitro and that during episodes of peritonitis exoproducts of some bacterial strains may further reduce mesothelial cell viability.     

Glucose degradation products and peritoneal membrane function.

BACKGROUND: The bioincompatibility of peritoneal dialysis fluids (PDF) in current use has been partially attributed to the presence of glucose degradation products (GDPs), which are generated during heat sterilization of PDF. Several of the GDPs have been identified and we have recently demonstrated that these GDPs per se may impair the viability and function of human peritoneal mesothelial cells (HPMC) in vitro. It is also possible that GDP-related toxicity is further exacerbated by the milieu of PDF. We review the current literature on GDP and present the results of experiments comparing the impact of heat- and filter-sterilized PDF on the viability and function of HPMC. METHODS: Peritoneal dialysis fluids with low (1.5%) and high (4.25%) glucose concentrations were laboratory prepared according to the standard formula and sterilized either by heat (H-PDF; 121 degrees C, 0.2 MPa, 20 minutes) or filtration (F-PDF; 0.2 microns). The buildup of GDP was confirmed by UV absorbance at 284 nm. Confluent HPMC monolayers were exposed to these solutions mixed 1:1 with standard M199 culture medium. After 24 hours, cell viability was assessed with the MTT assay, and interleukin-1beta-stimulated monocyte chemotactic protein-1 (MCP-1) release with specific immunoassay. RESULTS: Exposure of HPMC to H-PDF resulted in a significant decrease in cell viability, with solutions containing 4.25% glucose being more toxic than 1.5% glucose-based PDF (27.4% +/- 3.4% and 53.4% +/- 11.0% of control values, respectively). In contrast, viability of HPMC exposed to F-PDF was not different from that of control cells. Moreover, treatment with H-PDF impaired the release of MCP-1 from HPMC to a significantly greater degree compared to F-PDF (17.4% and 24.9% difference for low and high glucose PDF, respectively). CONCLUSIONS: Exposure of HPMC to H-PDF significantly impairs cell viability and the capacity for generating MCP-1 compared to F-PDF. This effect is likely to be mediated by GDPs present in H-PDF but not in F-PDF.     

NO-synthase-/NO-independent regulation of human and murine platelet soluble guanylyl cyclase activity

Summary.  Objectives: Platelets, specialized adhesive cells, play key roles in normal and pathological hemostasis through their ability to rapidly adhere to subendothelial matrix proteins (adhesion) and to other activated platelets (aggregation), functions which are inhibited by nitric oxide (NO). Platelets have been reported to be regulated not only by exogenous endothelium-derived NO, but also by two isoforms of NO synthase, endothelial (eNOS) and inducible (iNOS), endogenously expressed in platelets. However, data concerning expression, regulation and function of eNOS and iNOS in platelets remain controversial. Methods and results: Using important positive (endothelial cells, stimulated macrophages) and negative (eNOS/iNOS knock-out mouse) controls, as well as human platelets highly purified by a newly developed protocol, we now demonstrate that human and mouse platelets do not contain eNOS/iNOS proteins or mRNA. NOS substrate ( l -arginine), NOS inhibitors (L-NAME, L-NMMA), and eNOS/iNOS deficiency did not produce detectable functional effects on human and mouse platelets. von Willebrand factor (VWF)/ristocetin treatment of platelets increased cGMP by NO-independent activation of soluble guanylyl cyclase (sGC) which correlated with Src kinase-dependent phosphorylation of sGC β₁-subunit-Tyr¹⁹². Conclusions: Human and mouse platelets do not express eNOS/iNOS. VWF/ristocetin-mediated activation of the sGC/cGMP signaling pathway may contribute to feedback platelet inhibition.     

Nitric oxide synthase inhibition increases venular leukocyte rolling and adhesion in septic rats

OBJECTIVE: Excess production of nitric oxide (NO) has been implicated in hypotension and blood flow abnormalities in sepsis, but NO is also an important inhibitor of leukocyte rolling and adhesion. Leukocyte adhesion is increased in sepsis despite elevated NO production. We hypothesized that inhibition of NO synthase (NOS) could increase leukocyte adhesion in sepsis. DESIGN: Prospective animal study. SETTING: Experimental animal laboratory. SUBJECTS: Twenty-five male rats, anesthetized with ketamine and acepromazine. INTERVENTIONS: Topical superfusion of the nonselective NOS inhibitor N(G)-monomethyl-L-arginine (NMA) on skeletal muscle postcapillary venules. MEASUREMENTS AND MAIN RESULTS: Rats made septic by cecal ligation and puncture were compared with controls that underwent sham ligation. Leukocyte rolling and adhesion were measured in cremasteric postcapillary venules of septic and control rats using in vivo videomicroscopy. The effects of NOS inhibition on leukocyte rolling and adhesion were also measured. After a stable baseline was reached, 1 microM of the nonselective NOS inhibitor NMA was suffused topically followed by physiologic buffer. The effects of L-arginine on leukocyte rolling and adhesion were also measured, both before and after suffusion of NMA. Leukocyte rolling and adhesion was increased in septic rats as compared with controls (control 5.5+/-0.9 rolling cells/min, 1.0+/-0.3 adherent cells/min; septic 13.7+/-2.0 rolling cells/min, 3.1+/-0.6 adherent cells/min; p < .001), and NOS inhibition further increased leukocyte rolling and adhesion in both septic and control rats (control 14.0+/-1.7 rolling cells/min, 2.8+/-0.5 adherent cells/min; septic 25+/-2.1 rolling cells/min, 5.4+/-0.5 adherent cells/min; both p < .001 vs. baseline). Prior suffusion of excess L-arginine prevented the increase in leukocyte adhesion with NMA in septic rats (2.6+/-0.4 adherent cells/min vs. 3.0+/-0.6 adherent cells/min; n = 3; p > .05). When administered after NMA, excess L-arginine partially reversed leukocyte adhesion in septic rats (5.4+/-0.7 adherent cells/min, with NMA vs. 4.3+/-0.7 adherent cells/min, after L-arginine; n = 5; p < .05). Venular shear did not differ between septic and control rats (600+/-109 (sec(-1)) vs. 620+/-37 (sec(-1)); p > .05). CONCLUSIONS: Although NOS inhibition may ameliorate hypotension in sepsis, such therapy may be deleterious by increasing leukocyte adhesion.     

Immune system-mediated endothelial damage is associated with NO and antioxidant system disorders.

Two distinct systems of different origin are involved in the pathogenesis of both infectious and immunological vasculitis syndrome: nitric oxide (NO) from endothelial cells and granulocyte NADPH oxidase. In this study, in 31 children with immune system dysfunction, NO, NO synthase (NOS) and antioxidant enzyme activities [catalase, superoxide dismutase (SOD), glutathione peroxidase (GPx)], as well as immunological parameters, were investigated. On the basis of the clinical findings, all children were divided into three groups: group I, 8 children clinically showing macular skin manifestations; group II, 11 children with maculo-papulous changes; and group III, 12 children with clinical findings of papulous changes. Plasma NO values in groups II and III were significantly elevated (79.14+/-30.13 and 65.32+/-6.70 micromol/l), compared to the control group (41.24+/-3.65 micromol/l), while group I showed statistically lower values (32.38+/-3.37 micromol/l). In children with the highest level of NO (group II) NOS activity was two-fold higher (1.77+/-0.59 nmol/ml/min; p<0.01) than in controls (0.98+/-0.23 nmol/ml/min). Catalase activity showed a significant increase and SOD activity a significant decrease in all experimental groups, while GPx was not significantly changed. The results show that immune system dysfunction manifested as vasculitis is associated with significant disturbances in the NO system and free radicals scavengers.