Glomerulonephritis in autopsy cases with hepatitis C virus infection

1. The inducible isoform of nitric oxide synthase (iNOS) is expressed in human and experimental cardiac allografts and is localized to infiltrating macrophages, cardiac myocytes, endothelial cells and smooth muscle cells. A recent clinical report proposes a causal link between myocardial expression of iNOS and ventricular contractile dysfunction, a potentially graft- and life-threatening post-transplant complication. 2. Coronary blood flow is elevated in human graft recipients with biopsy proven cellular rejection, indicating that vasodilation accompanies graft rejection. In Lewis-to-F-344 coronary resistance vessels, which show intimal expression of iNOS, pressure-induced myogenic tone is significantly inhibited. Selective iNOS inhibition partially reverses the inhibition of myogenic tone, confirming that iNOS produces vasoactive nitric oxide (NO) and may mediate the rejection-induced vasodilation seen clinically. 3. Endothelial dysfunction, identified as loss of endothelium-dependent dilation, has tremendous prognostic significance in vascular diseases of multiple aetiologies. In transplantation, endothelial dysfunction predicts early cardiac allograft vasculopathy and poor clinical outcome. Lewis-to-F-344 coronary vessels develop endothelial dysfunction at 1 week post-transplantation, but this is preceded by a transient state of endothelial cell hyperfunction, with enhanced endothelial production of NO. 4. The normal interaction between endothelial and smooth muscle cells in coronary resistance vessels is critical for the regulation of coronary blood flow and the maintenance of fluid homeostasis. With allospecific expression of iNOS, the inhibition of vascular tone predicts greatly enhanced intravascular pressure in precapillary arterioles and capillaries; this would be expected to cause a net movement of fluid from the intravascular compartment into the myocardial interstitium, resulting in ventricular oedema, non-compliance and poor contractile performance.     

The co-culture of dermal fibroblasts with human epidermal keratinocytes induces increased prostaglandin E2 production and cyclooxygenase 2 activity in fibroblasts

BACKGROUND: We recently demonstrated that inhibition of nitric oxide (NO) production ameliorated acute pulmonary allograft rejection. This study examined whether inducible NO synthase (iNOS) was expressed in the transplanted lung during acute rejection. METHODS: With a rat left lung transplant model, tissue from syngeneic (Fischer 344 to Fischer 344) and allogeneic (Brown Norway to Fischer 344) transplants were harvested on postoperative day 4 and analyzed for iNOS mRNA expression (ribonuclease protection assay), iNOS enzyme activity (conversion of L-[3H]-arginine to NO and L-[3H]-citrulline), and serum nitrite/nitrate levels. RESULTS: The iNOS mRNA was expressed in allograft lungs but was not detected in isografts or controls. The iNOS protein was present in allograft lungs, as demonstrated by high levels of L-[3H]-citrulline production compared with minimal iNOS enzyme activity in isograft and control lungs (10.1 +/- 2.4 vs 0.6 +/- 0.2 and 0.7 +/- 0.2 pmol L-[3H]-citrulline.mg-1.min-1, respectively; n = 6, p < 0.001). Allografts had significantly elevated systemic serum nitrite/nitrate levels compared with isografts and controls (38 +/- 6 vs 18 +/- 2 and 16 +/- 1 mumol/L, respectively; n = 6; p < 0.005). CONCLUSIONS: These results, together with our previous demonstration that iNOS inhibition ameliorated lung allograft rejection, suggest that (1) iNOS expression and increased NO production contributed to acute rejection of the transplanted lung, (2) iNOS inhibition may offer an alternative in management of acute lung allograft rejection, and (3) increased NO production, detected by the presence of iNOS mRNA or protein or noninvasively by measuring serum nitrite/nitrate levels, may serve as an early marker of acute allograft rejection.     

Cyclic GMP and cGMP-binding phosphodiesterase are required for interleukin-1-induced nitric oxide synthesis in human articular chondrocytes

This study addressed the role of guanylyl cyclase (GC) and phosphodiesterase (PDE) in interleukin (IL)-1 activation of human articular chondrocytes. The GC inhibitors LY83583 and methylene blue dose-dependently inhibited IL-1-induced nitric oxide (NO) production, inducible NO synthase (iNOS) protein, and mRNA expression. These effects of GC inhibition were consistent with the rapid induction of cGMP by IL-1, which reached maximal levels after 5 min. The effects of GC inhibitors were selective as they did not reduce IL-1-induced cyclooxygenase II protein and mRNA. An inhibitor specific for soluble GC did not affect IL-1-induced NO production, and activators of soluble GC did not induce NO. However, the expression of iNOS mRNA was induced by atrial natriuretic peptide (ANP) and C-type natriuretic peptide (CNP), activators of particulate GC, indicating that particulate rather than soluble guanylyl cyclases were involved in iNOS induction. The expression of iNOS mRNA and the production of NO were induced by a slowly hydrolyzable analog of cGMP, 8-bromo-cGMP, but not by nonhydrolyzable analog, dibutyryl cGMP, suggesting that PDE rather than cGMP-dependent protein kinase mediates the cGMP effects. Chondrocytes contained extensive cGMP PDE activity. This had PDE5 biochemical features and an inhibitor profile consistent with PDE5. Furthermore, the nonisoformspecific PDE inhibitor IBMX and PDE5-specific inhibitors suppressed IL-1-induced NO release and iNOS mRNA expression. PDE5 mRNA was constitutively expressed in chondrocytes. In addition to increasing PDE5 activities, IL-1 treatment reduced the sensitivity of PDE5 to several pharmacological inhibitors by up to 50-fold. In summary, inhibitors of either GC or PDE5 prevented IL-1 induction of iNOS; IL-1 increased the rates of both cGMP generation and hydrolysis; and exogenous PDE hydrolyzable cGMP analog induced iNOS and NO. These results suggest that increased cGMP metabolic flux is sufficient to induce iNOS, and GC and PDE5 activities are required for IL-1 induction of iNOS expression via increases in coupled cGMP synthesis and hydrolysis.     

Regulation of cardiac myocyte contractile function by inducible nitric oxide synthase (iNOS): mechanisms of contractile depression by nitric oxide

Inflammatory cytokines have been implicated in the reversible depression of cardiac contractile function accompanying local or systemic immune stimulation. Incubation of cardiac myocytes with soluble components in the supernatant from cultured rat lung macrophages activated with endotoxin decreases their contractile response to beta-adrenergic stimulation through the induction of iNOS and the subsequent production of nitric oxide by these cells. In the present study, we characterize the mechanisms underlying NO's attenuation of adrenergic responsiveness in cardiac myocytes. iNOS was induced in cultured ventricular myocytes from adult rats by incubation for 20 h with conditioned medium from lipopolysaccharide (LPS)-activated macrophages. iNOS induction did not induce any alteration in beta-adrenergic receptor density or affinity, Galphai protein abundance, or adenylyl cyclase activity in cultured myocytes. Myocyte exposure to activated macrophage-conditioned medium markedly attenuated the elevation of cAMP in response to isoproterenol (Iso, 2 nM). Induction of iNOS with the macrophage-conditioned medium also potentiated the Iso-induced increase in myocyte cGMP. This cGMP increase was totally abolished by NOS inhibitors. NOS inhibition also returned the attenuated cAMP response to 2 nM Iso to levels observed in control cells. Pre-incubation of the cells in isobutylmethylxanthine (IBMX), a phosphodiesterase inhibitor, also partly reversed the attenuation of cAMP increase with 2 nM Iso in cells expressing iNOS. Brief (15 min) exposure of myocytes to the NO donor, S-nitrosoacetylcysteine (SNAC, 100 micro M) which produced a three-fold increase in intracellular cGMP, also decreased by half the contractile response of cardiac myocytes to Iso (2 nM). We conclude that NO endogenously produced by iNOS decreases the intracellular levels of cAMP in response to beta-adrenergic stimulation in isolated cardiac myocytes, in part through a cGMP-mediated mechanism. This effect may participate in the NO-dependent depression of cardiac function following cytokine exposure.     

Beta-adrenergic signal transduction following carvedilol treatment in hypertensive cardiac hypertrophy

Cytokines and cytotoxic agents, including nitric oxide (NO) released by macrophages, play important roles during cardiac allograft rejection. In contrast to agents that suppress T-lymphocyte function, CNI-1493 is a multivalent guanylhydrazone compound that inhibits the synthesis and release of proinflammatory cytokines and NO from macrophages. This study investigated the effects of CNI-1493 on rejecting rat cardiac allografts by using Lewis to Wistar-Furth heterotopic cardiac transplants. CNI-1493 (2 mg/kg i.p., b.i.d.) or vehicle (water) was administered beginning the day before surgery. Rat cardiac allograft survival to cessation of heart beat, apoptosis of cardiac myocytes, degree of myocardial inflammation, and inducible nitric oxide synthase (iNOS) messenger RNA (mRNA), protein, and enzyme activity were studied at days 1, 3, 5, and 7 after transplantation. Allograft survival was increased significantly by 26% from 7.5 +/- 0.8 days in vehicle-treated rats (n = 6) to 9.5 +/- 1.2 days in CNI-1493-treated rats (n = 8, p < 0.05). Apoptotic cells per mm2 myocardium decreased from 2.25 +/- 1.25 to 0.84 +/- 0.49 at day 3 and 31.2 +/- 2.9 to 17.6 +/- 5.43 at day 5 after transplantation with CNI-1493 treatment (p < 0.05). The number of apoptotic myocytes and loss of cardiac muscle cells also decreased significantly at day 5 in the treated animals (p < 0.05). The reduction of myocyte loss at day 5 coincided with a significant decrease of the inflammatory response and reduced macrophage influx (p < 0.05). Myocardial iNOS mRNA, protein, and enzyme levels increased during the course of allograft rejection, and CNI-1493 did not significantly reduce iNOS expression in the rejecting rat allograft. CNI-1493 prolongs allograft survival and reduces myocyte loss, apoptosis, and inflammation during rat cardiac allograft rejection. These effects of CNI-1493 appear to be unrelated to altered NO synthesis but may be related to effects of the drug to inhibit macrophage synthesis of cytokines.     

Nitric oxide is overproduced by peritoneal macrophages in rat taurocholate pancreatitis: the mechanism of inducible nitric oxide synthase expression

To investigate the pathobiology of severe acute pancreatitis, we studied the expression of inducible nitric oxide synthase (iNOS) in peritoneal macrophages of experimental pancreatitis. Taurocholate (TCA) pancreatitis and cerulein (CE) pancreatitis were used as models of lethal and self-limited pancreatitis, respectively, and the mechanism of iNOS expression in peritoneal macrophages was studied. Serum nitrate and nitrite (NOx) concentrations increased during the course of TCA pancreatitis, and iNOS-immunoreactivity was detected in the peritoneal macrophages 12 h after the induction of TCA pancreatitis, but these phenomena were not observed in CE pancreatitis. Despite the difference in the iNOS expression, the iNOS messenger RNA (mRNA) and the activation of nuclear factor-kappa B (NF-kappa B) were detected in the peritoneal macrophages of both pancreatitis models. The supernatant of TCA pancreatitis ascites could induce iNOS in the peritoneal macrophages of normal rats in vitro, but the peritoneal lavage fluid of CE pancreatitis rats could not. The results indicated that there may be qualitative or quantitative differences in the macrophage activation between the two types of experimental pancreatitis and suggested that the ascites of rats with lethal acute pancreatitis contains some soluble factors that activate the macrophage/monocyte system and cause an overproduction of NO by the iNOS expression.     

Failure of L-NAME to cause inhibition of nitric oxide synthesis: role of inducible nitric oxide synthase

We addressed the hypothesis that administration of nitric oxide synthase inhibitor, NG -nitro-L-arginine methyl ester (L-NAME) does not result in a sustained suppression of nitric oxide (NO) synthesis, because of a compensatory expression of inducible nitric oxide synthase (iNOS). L-NAME was administered in the drinking water (0.1-1.0 mg/ml) for 7 days to guinea pigs and rats. Nitric oxide synthesis was assessed by [1] ex vivo formation of nitrite in blood vessels and intestine [2] tissue levels of cGMP [3] iNOS gene expression by RT-PCR [4] NADPH diaphorase staining [5] direct assessment of NO release in tissue explants using a microelectrode/electrochemical detection system. Chronic L-NAME administration elevated intestinal cGMP and nitrite levels in guinea pigs (p < 0.05). In rats, intestinal nitrite levels were comparable in control and L-NAME treatment groups, whereas direct assessment of NO release defined a marked increase in the L-NAME group. Chronic L-NAME resulted in an induction of iNOS gene expression in rats and guinea pigs and novel sites of NADPH diaphorase staining in the intestine. We conclude that iNOS expression is responsible for a compensatory increase or normalization of NO synthesis during sustained administration of L-NAME.     

Localization of inducible nitric oxide synthase in acute renal allograft rejection in the rat

There is increasing evidence for a role for nitric oxide (NO) in the alloimmune response and induction of NO synthesis occurs during allograft rejection. The aim of this study was to investigate the source of NO synthesis in rejecting allografts. Localization of inducible nitric oxide synthase (iNOS) was studied by immunohistochemistry, in a rat model of acute renal allograft rejection, in unmodified Lewis recipients in which rejection is complete 7 days after transplantation of F1 hybrid Lewis-Brown Norway kidneys. High levels of iNOS expression were found in infiltrating mononuclear cells in glomeruli and interstitium of rejecting kidneys; there was no expression in parenchymal renal cells, or in control isografts of either rat strain. Expression of iNOS in the cortex was present from 4 to 6 days posttransplantation, and had declined by the 7th day, where expression was principally in the medulla. The pattern of iNOS staining was similar to ED1 staining, a marker for rat macrophages. These findings suggest that infiltrating macrophages in the graft reaction are a prominent source of NO; this iNOS expression supports a role for NO in the modulation of local allogeneic responses, and possibly as a mediator of cytotoxic graft damage.     

Calcium channel blockade enhances nitric oxide synthase expression by cultured endothelial cells

In a recent study, we found marked increases in nitric oxide (NO) production and endothelial and inducible NO synthase (eNOS and iNOS) expressions with calcium channel blockade in rats with chronic renal failure. This study was undertaken to determine whether enhanced NO production with calcium channel blockade is a direct effect of this therapy or a consequence of the associated hemodynamic and humoral changes. We tested the effects of a calcium channel blocker, felodipine (10(-5), 10(-6), and 10(-7) mol/L), on nitrate and nitrite (NOx) generation, Ca2+-dependent and -independent NOS activity, and eNOS and iNOS protein masses in proliferating and quiescent rat aortic endothelial cells in culture. Compared with vehicle alone, felodipine significantly increased NOx generation, Ca2+-dependent NOS activity, and eNOS protein mass in proliferating and quiescent endothelial cells. Felodipine did not modify the stimulatory action of 10% fetal calf serum on DNA synthesis (thymidine incorporation) and cell proliferation. Ca2+-independent NOS activity and iNOS protein expression were negligible and unaffected by calcium channel blockade. NOx production and NOS expression were greater in proliferating cells than in quiescent cells. Thus, calcium channel blockade upregulates endothelial NO production in vitro, confirming our previous in vivo study. This observation indicates that the reductions in cytosolic [Ca2+] and vasodilation with calcium channel blockade are not only due to inhibition of Ca2+ entry but also to an NO-cGMP mediated mechanism.     

Incidence of primary malignancies other than breast cancer among women treated with radiation therapy for benign breast disease

In addition to left ventricular pump failure and low cardiac output, structural and metabolic alterations of skeletal muscle are thought to contribute to exercise intolerance seen in patients with CHF. Studies using cardiac myocytes have implicated nitric oxide elaborated by inducible nitric oxide synthase (iNOS) as a potential agent associated with the genesis of dilated cardiomyopathy. The present study was designed to locate iNOS in the working skeletal muscle of patients with congestive heart failure. Specific antibodies were used to detect iNOS by immunohistochemistry in skeletal muscle biopsies (m. vastus lateralis) of 37 patients with left ventricular pump failure and 8 normal controls. The expression was restricted to skeletal muscle myocytes and was increased five- to ninefold in patients with chronic heart failure. There was no statistically significant difference in iNOS expression between patients with dilated cardiomyopathy and those with ischemic cardiomyopathy. The finding of a locally increased expression of iNOS and the experimental evidence that NO attenuates the contractile performance of the skeletal muscle suggest that the expression of iNOS may be responsible for the exercise intolerance seen in patients with chronic heart failure.     

Increased endogenous nitric oxide synthase inhibitor in patients with congestive heart failure

Nitric oxide (NO) plays a role in controlling vascular tone and regulates the contractile properties of cardiac myocytes. Patients with heart failure exhibit high plasma levels of nitrite/nitrate (NOx), a stable metabolite of NO, and of cytokines such as tumor necrosis factor-alpha, a potent inducer of NO synthase. An increase in inducible NO synthase activity has been found in cardiac tissue from patients with dilated cardiomyopathy. These findings raise the possibility that local or systemic overproduction of NO induced by cytokines exerts a chronic negative inotropic effect on the myocardium and may have detrimental effects on systemic hemodynamics in patients with heart failure. Plasma levels of NG,NG-dimethylarginine (asymmetric dimethylarginine; ADMA), a circulating endogenous NO synthase inhibitor, were measured in control subjects and patients with valvular, hypertensive, or ischemic heart diseases or idiopathic cardiomyopathy. The plasma levels of NOx and ADMA were assessed by high performance liquid chromatography. The plasma levels of NOx and ADMA were significantly elevated in patients with heart failure. Both NOx and ADMA were positively correlated with New York Heart Association functional class. There was a significant inverse correlation between plasma NOx and ejection fraction, as estimated by echocardiography. A significant relationship between plasma NOx and ADMA was found only in patients with moderate to severe heart failure (r=0.41, p=0.01). Findings suggest a compensatory role of a circulating endogenous NO synthase inhibitor against induced NO synthase activity in patients with heart failure.     

Corneal penetration of diclofenac from a fixed combination of diclofenac-gentamicin eyedrops

Recent studies suggest a dual role for nitric oxide (NO) in tumour biology. High concentrations of NO can mediate tumouricidal activity, whereas lower concentrations have been shown to promote tumour growth. In this study, NO synthase (NOS) activity was investigated in cells that were prepared from tissue from primary and metastatic sites and from malignant effusions in 41 cases of suspected ovarian cancer. NO biosynthesis, determined by nitrite + nitrate (NOx) accumulation in medium from cultured cells prepared from disaggregated tumours or effusions and indicative of the inducible NO synthase isoform, was detected in 37% of the cases investigated (range 10.2-114 microM). There was a significant relationship between NOx and tumour differentiation (P = 0.014), with NOx being significantly higher for the more differentiated tumours. NOS activity, determined by the conversion of radiolabelled L-arginine to citrulline by tissue or cell extracts, was detected in 29% of cases (range 0.9-6.9 pmol/min per mg of protein), with all samples tested being moderately or poorly differentiated. Seventy percent of this activity was calcium dependent, indicative of constitutive NOS isoforms. Morphological and immunohistochemical assessment of tumour samples indicated a significant relationship between high macrophage content and NOS activity (as NOx biosynthesis) (rs = 0.726, N = 16, P < 0.01). The relationship between NOS expression, immune response, and disease progression is complex and not simply dependent on the differentiation status of ovarian cancer.     

Involvement of nitric oxide synthesis in hepatic perturbations induced in rats by a necrogenic dose of thioacetamide

1. The biological actions of nitric oxide (NO), a highly diffusible and short-lived radical, range from signal transduction to cytotoxicity. The present study investigated whether NO is released in the course of liver necrosis and regeneration induced by a single necrogenic dose of thioacetamide (6.6 mmol kg-1 body wt) to rats. Samples of liver were obtained at 0, 3, 12, 24, 48, 72 and 96 h after thioacetamide administration. 2. Inducible nitric oxide synthase (iNOS) activity was determined in purified liver homogenates and a sharp 6 fold increase (P < 0.001) in iNOS activity was recorded at 48 h of intoxication, followed by a slight but progressive increase at 72 and 96 h. Changes in the expression of iNOS, as detected by its mRNA levels, were parallel to the NOS enzyme activity. Hepatocyte NO synthesis showed a progressive increase at 24, 48 and 72 h, to 8 (P < 0.001), 13 (P < 0.001) and 13 (P < 0.001) times the initial values, respectively. 3. In isolated Kupffer cells, where initial NO release was ten fold higher than in hepatocytes, a progressive increase was detected from 48 h which reached two fold of initial at 72 h of intoxication (192%; P < 0.001). Hepatic cyclic GMP concentration did not change significantly. However, mitochondrial aconitase activity decreased markedly at 12 and 24 h of intoxication showing a sharp increase towards normal values at 48 h which was maintained at 72 and 96 h. 4. As protein kinase C (PKC) is one of the likely candidates to mediate iNOS expression, translocation (activation) of PKC was assayed in hepatocytes, and a significant two fold increase (P < 0.001) between 48 and 96 h after thioacetamide intoxication was observed. When peritoneal macrophages from control rats were incubated with serum from thioacetamide-treated rats, a sharp increase in NO release was detected with serum obtained at 48 h, reaching at 96 h a value four fold (P < 0.001) that of the control. 5. These results suggest that iNOS activity and NO release play a role in the pathophysiological mechanisms that trigger post-necrotic hepatocellular regeneration following thioacetamide administration.