John A. Corbett

Selective inhibition of the inducible nitric oxide synthase by aminoguanidine

Abstract Overproduction of the free radical nitric oxide (NO) has been implicated in the pathogenesis of a variety of inflammatory and immunologically mediated diseases as well as complications of diabetes. In the present study we have demonstrated that aminoguanidine selectively inhibits the cytokine-inducible isoform of NO synthase which appears to be responsible for the excess production of NO linked to these disease states. By using organ, cell and enzyme-based measurements we have shown that aminoguanidine is equipotent to NG-monomethyl-L-arginine (L-NMA) as an inhibitor of the cytokine-induced isoform of NO synthase but is 10 to 100-fold less potent as an inhibitor of the constitutive isoform. Thus, aminoguanidine may be useful as a selective inhibitor of the inducible NO synthase in the treatment of disease states characterized by the pathological overproduction of NO.

Aminoguanidine, a Novel Inhibitor of Nitric Oxide Formation, Prevents Diabetic Vascular Dysfunction

Increased blood flow and vascular leakage of proteins preferentially affect tissues that are sites of diabetic complications in humans and animals. These vascular changes in diabetic rats are largely prevented by aminoguanidine. Glucose-induced vascular changes in nondiabetic rats are also prevented by aminoguanidine and by NG-monomethyl-L-arginine (NMMA), an established inhibitor of nitric oxide (NO·) formation from L-arginine. Aminoguanidine and NMMA are equipotent inhibitors of interleukin-1 β-induced 1) nitrite formation (an oxidation product of NO·) and cGMP accumulation by the rat β-cell insulinoma cell line RINm5F, and 2) inhibition of glucose-stimulated insulin secretion and formation of iron-nitrosyl complexes by islets of Langerhans. In contrast, NMMA is ∼ 40 times more potent than aminoquanidine in elevating blood pressure in nondiabetic rats. These results demonstrate that aminoguanidine inhibits NO. production and suggest a role for NO· in the pathogenesis of diabetic vascular complications.

Prevention of Diabetic Vascular Dysfunction by Guanidines: Inhibition of Nitric Oxide Synthase Versus Advanced Glycation End-Product Formation

This study was undertaken to compare the ability of two guanidine compounds (aminoguanidine and methylguanidine), with different in vitro effects on NO synthase activity and AGE formation, to inhibit diabetic vascular dysfunction developing early after the onset of diabetes. In rats with STZ-induced diabetes of 5-wk duration, regional vascular [125I]albumin permeation was increased about two- to threefold in ocular tissues, sciatic nerve, and aorta; in general, both guanidine compounds normalized albumin permeation in diabetic rats without affecting it in controls. Methylguanidine was only ∼7% as effective as aminoguanidine as an inhibitor of AGE formation from L-lysine and G6P; both compounds were poor inhibitors of AR. Methylguanidine was ∼1–5% as potent as aminoguanidine and L-NMMA as an inhibitor of the cytokine- and endotoxin-inducible isoform of NO synthase. In contrast, the potency of methylguanidine as an inhibitor of the constitutive isoform of NO synthase was comparable to that of aminoguanidine, and both guanidine compounds were much less effective than L-NMMA. These observations suggest a role for a relative or absolute increase in NO production in the pathogenesis of early diabetic vascular dysfunction and raise the possibility that inhibition of diabetic vascular functional changes by aminoguanidine may reflect inhibition of NO synthase activity rather than, or in addition to, prevention of AGE formation.

Does Nitric Oxide Mediate Autoimmune Destruction of β-Cells?: Possible Therapeutic Interventions in IDDM

Cytokines have been implicated as immunological effector molecules that induce dysfunction and destruction of the pancreatic β-cell. The mechanisms of cytokine action on the β-cell are unknown; however, nitric oxide, resulting from cytokine-induced expression of nitric oxide synthase, has been implicated as the cellular effector molecule mediating β-cell dysfunction. Nitric oxide is a free radical that targets intracellular iron-containing enzymes, which results in the loss of their function. The cytokine IL-1β induces the formation of nitric oxide in isolated rat islets and the insulinoma cell line, Rin-m5F. NMMA and NAME, both inhibitors of nitric oxide synthase, completely protect islets from the deleterious effects of IL-1β. These inhibitors are competitive in nature and inhibit both the cytokine-inducible and constitutive isoforms of nitric oxide synthase with nearly identical kinetics. This may preclude their use as therapeutic agents because of increases in blood pressure which result from the inhibition of constitutive nitric oxide synthase activity. Aminoguanidine, an inhibitor of nonenzymatic glycosylation of cellular and extracellular constituents associated with diabetic complications, recently has been reported to inhibit nitric oxide synthase. Aminoguanidine is ∼40-fold more effective in inhibiting the inducible isoform of nitric oxide synthase, suggesting that aminoguanidine or analogues may serve as potential therapeutic agents to block diseases associated with nitric oxide production by the inducible isoform of nitric oxide synthase. In vivo administration of TNF IL-1 has been shown to induce antidiabetogenic effects in the NOD mouse. This anti-diabetogenic effect of cytokines appears to conflict with evidence suggesting that cytokines mediate β-cell dysfunction. The role of nitric oxide in both cytokine-mediated β-cell dysfunction, and the antidiabetogenic effects of cytokines, as well as the potential therapeutic use of aminoguanidine, are evaluated in this study.

Interleukin 1 beta induces the formation of nitric oxide by beta-cells purified from rodent islets of Langerhans. Evidence for the beta-cell as a source and site of action of nitric oxide.

Nitric oxide has recently been implicated as the effector molecule that mediates IL-1 beta-induced inhibition of glucose-stimulated insulin secretion and beta-cell specific destruction. The pancreatic islet represents a heterogeneous cell population containing both endocrine cells (beta-[insulin], alpha-]glucagon], gamma[somatostatin], and PP-[polypeptide] secreting cells) and non-endocrine cells (fibroblast, macrophage, endothelial, and dendritic cells). The purpose of this investigation was to determine if the beta-cell, which is selectively destroyed during insulin-dependent diabetes mellitus, is both a source of IL-1 beta-induced nitric oxide production and also a site of action of this free radical. Pretreatment of beta-cells, purified by FACS with IL-1 beta results in a 40% inhibition of glucose-stimulated insulin secretion that is prevented by the nitric oxide synthase inhibitor, NG-monomethyl-L-arginine (NMMA). IL-1 beta induces the formation of nitric oxide by purified beta-cells as evidenced by the accumulation of cGMP, which is blocked by NMMA. IL-1 beta also induces the accumulation of cGMP by the insulinoma cell line Rin-m5F, and both NMMA as well as the protein synthesis inhibitor cycloheximide prevent this cGMP accumulation. Iron-sulfur proteins appear to be intracellular targets of nitric oxide. IL-1 beta induces the formation of an iron-dinitrosyl complex by Rin-m5F cells indicating that nitric oxide mediates the destruction of iron-sulfur clusters of iron containing enzymes. This is further demonstrated by IL-1 beta-induced inhibition of glucose oxidation by purified beta-cells, mitochondrial aconitase activity of dispersed islet cells, and mitochondrial aconitase activity of Rin-m5F cells, all of which are prevented by NMMA. IL-1 beta does not appear to affect FACS-purified alpha-cell metabolic activity or intracellular cGMP levels, suggesting that IL-1 beta does not exert any effect on alpha-cells. These results demonstrate that the islet beta-cell is a source of IL-1 beta-induced nitric oxide production, and that beta-cell mitochondrial iron-sulfur containing enzymes are one site of action of nitric oxide.

Cytokines and Nitric Oxide in Islet Inflammation and Diabetes

Abstract Cytokines released by both T lymphocytes and activated macrophages, in particular interleukin-1 (IL-1), have been implicated as immunological effector molecules that both inhibit insulin secretion from the pancreatic β cell and induce β-cell destruction. Recent findings have demonstrated that production of the free radical nitric oxide (NO), resulting from the expression of the cytokine-inducible isoform of NO synthase (iNOS), mediates these deleterious effects. The cellular mechanism responsible for inhibition of β-cell function and destruction by NO involves, in part, inactivation of enzymes specifically localized to the β-cell mitochondria that contain iron-sulfur centers or clusters. Intraislet release of IL-1 also inhibits β-cell function by this same cellular mechanism involving the overproduction of NO. In addition, the cytokine, IL-1, induces the co-expression of both iNOS and the cytokine-inducible isoform of cyclooxygenase, COX-2. The expression of COX-2 results in the overproduction of the proinflammatory prostaglandins and thromboxanes. Furthermore, NO produced by iNOS directly stimulates the activities of both constitutive and inducible isoforms of COX, further augmenting the overproduction of these proinflammatory mediators, NO and prostaglandins, which may be important in initiating or maintaining the inflammatory response and destruction of the β cell associated with autoimmune diabetes. [P.S.E.B.M. 1996, Vol 211]

IL-1 produced and released endogenously within human islets inhibits beta cell function.

Resident macrophages have been suggested to participate in the initiation of beta cell damage during the development of autoimmune diabetes. The purpose of this study was to determine if the endogenous production and release of interleukin 1 (IL-1) in human islets of Langerhans by resident macrophages results in the inhibition of beta cell function. Treatment of human islets with a combination of tumor necrosis factor (TNF) + lipopolysaccharide (LPS) + interferon-gamma (IFN-gamma) stimulates inducible nitric oxide synthase (iNOS) expression, nitric oxide production, and inhibits glucose-stimulated insulin secretion. The IL-1 receptor antagonist protein (IRAP) prevents TNF + LPS + IFN-gamma-induced iNOS expression and nitrite production, and attenuates the inhibitory effects on glucose-stimulated insulin secretion by human islets. Inhibition of iNOS activity by aminoguanidine also attenuates TNF + LPS + IFN-gamma-induced inhibition of insulin secretion by human islets. These results indicate that the inhibitory effects of TNF + LPS + IFN-gamma are mediated by nitric oxide, produced by the actions of IL-1 released endogenously within human islets. Reverse transcriptase polymerase chain reaction was used to confirm that TNF + LPS + IFN-gamma stimulates the expression of both IL-1alpha and IL-1beta in human islets. Two forms of evidence indicate that resident macrophages are the human islet cellular source of IL-1: culture conditions that deplete islet lymphoid cells prevent TNF + LPS + IFN-gamma-induced iNOS expression, nitric oxide production, and IL-1 mRNA expression by human islets; and IL-1 and the macrophage surface marker CD69 colocalize in human islets treated with TNF + LPS + IFN-gamma as determined by immunohistochemical analysis. Lastly, nitric oxide production is not required for TNF + LPS + IFN-gamma-induced IL-1 release in human islets. However, cellular damage stimulates IL-1 release by islet macrophages. These findings support the hypothesis that activated islet macrophages may mediate beta cell damage during the development of insulin-dependent diabetes by releasing IL-1 in human islets followed by cytokine-induced iNOS expression by beta cells.

Experimental allergic encephalomyelitis in the rat is inhibited by aminoguanidine, an inhibitor of nitric oxide synthase

This study assessed the role of de novo nitric oxide (NO) production in the pathogenesis of experimental allergic encephalomyelitis (EAE) by using aminoguanidine (AG), an inhibitor of nitric oxide synthase (NOS), which preferentially inhibits the cytokine- and endotoxin-inducible isoform of NOS versus the constitutive isoforms consisting of endothelial and neuronal NOS. The maximum clinical severity of EAE and the duration of illness were significantly reduced or totally inhibited by twice daily subcutaneous injection of 100 mg/kg body weight AG. Histochemical staining for NADPH diaphorase, which detects enzymatic activity of NOS, revealed positive reactivity in untreated EAE rats both in parenchymal blood vessel walls and in anterior horn cell neurons, while normal rats and rats with EAE treated with AG showed predominantly the neuronal positivity. Moreover, this NADPH staining pattern was further supported by the immunohistochemical findings that endothelial NOS (eNOS) expression was increased in blood vessels in the inflamed lesions of untreated EAE rats and that inducible NOS (iNOS) was detected in some inflammatory cells, while treatment with AG could significantly reduce both iNOS and eNOS production. These results suggest that: (i) both iNOS and eNOS are upregulated in inflamed areas of the rat central nervous system in EAE; (ii) increased NO production plays a role in the development of clinical signs in EAE; and (iii) selective inhibitors of iNOS and/or eNOS may have therapeutic potential for the treatment of certain autoimmune diseases.

The role of reactive oxygen species and proinflammatory cytokines in type 1 diabetes pathogenesis

Type 1 diabetes (T1D) is a T cell–mediated autoimmune disease characterized by the destruction of insulin‐secreting pancreatic β cells. In humans with T1D and in nonobese diabetic (NOD) mice (a murine model for human T1D), autoreactive T cells cause β‐cell destruction, as transfer or deletion of these cells induces or prevents disease, respectively. CD4+ and CD8+ T cells use distinct effector mechanisms and act at different stages throughout T1D to fuel pancreatic β‐cell destruction and disease pathogenesis. While these adaptive immune cells employ distinct mechanisms for β‐cell destruction, one central means for enhancing their autoreactivity is by the secretion of proinflammatory cytokines, such as IFN‐γ, TNF‐α, and IL‐1. In addition to their production by diabetogenic T cells, proinflammatory cytokines are induced by reactive oxygen species (ROS) via redox‐dependent signaling pathways. Highly reactive molecules, proinflammatory cytokines are produced upon lymphocyte infiltration into pancreatic islets and induce disease pathogenicity by directly killing β cells, which characteristically possess low levels of antioxidant defense enzymes. In addition to β‐cell destruction, proinflammatory cytokines are necessary for efficient adaptive immune maturation, and in the context of T1D they exacerbate autoimmunity by intensifying adaptive immune responses. The first half of this review discusses the mechanisms by which autoreactive T cells induce T1D pathogenesis and the importance of ROS for efficient adaptive immune activation, which, in the context of T1D, exacerbates autoimmunity. The second half provides a comprehensive and detailed analysis of (1) the mechanisms by which cytokines such as IL‐1 and IFN‐γ influence islet insulin secretion and apoptosis and (2) the key free radicals and transcription factors that control these processes.

Nitric oxide mediates interleukin-1-induced cellular cytotoxicity in the rat ovary. A potential role for nitric oxide in the ovulatory process.

Treatment of primary cultures of rat ovarian dispersates with IL-1 beta results in morphologic and cytotoxic changes, thought to reflect tissue remodeling events associated with ovulation. We examined the role that the free radical nitric oxide plays in this process and report that IL-1 beta induces expression of the inducible isoform of nitric oxide synthase in ovarian cells as demonstrated by immunoprecipitation. We show that IL-1 beta treatment results in the formation of nitric oxide (as measured by accumulation of nitrite and cGMP) in both a time- and concentration-dependent manner that is prevented by aminoguanidine, a selective inhibitor of the inducible isoform of nitric oxide synthase. Aminoguanidine also inhibits IL-1-induced ovarian cellular cytotoxicity. These results suggest that nitric oxide is an important mediator of cell death and may act as a physiologically significant mediator of tissue remodeling events that occur in vivo during the ovulatory process.