The distinction between metabolic and structural changes occurring in autonomic neurons during diabetes has not been fully clarified. Here we demonstrate that nitric oxide synthase-containing (nitrergic) neurons innervating the penis and gastric pylorus of streptozotocin-induced diabetic rats undergo a selective degenerative process in two phases. In the first phase, nitrergic nerve fibers lose some of their neuronal nitric oxide synthase content and function. In the second phase, nitrergic degeneration takes place in the cell bodies in the ganglia, leading to complete loss of nitrergic function. The changes in the first phase are reversible with insulin replacement; however, the neurodegeneration in the second phase is irreversible. Neurodegeneration is due to apoptotic cell death in the ganglia, which is selective for the nitrergic neurones. Diabetes 52:2353-2362, 2003 D iabetes is a common disorder that leads to complications affecting the retina, kidney, vascular, gastrointestinal, peripheral, and autonomic nervous systems. The mechanism through which diabetic complications develop is unclear. The generally accepted view for most diabetic complications is that the disease is accompanied by metabolic changes in the affected organ that, in the long term, result in structural alterations. Thus, if insulin replacement is started before the structural lesions occur, then it should reverse the metabolic changes and prevent the development of the complication. Once the structural lesions occur, however, the process should become less reversible with insulin replacement. Although from early studies in vivo "a point of no return" had been suggested during the course of diabetic peripheral neuropathy, cardiomyopathy, and nephropathy (1-3), most of the subsequent work has concentrated either on cellular dysfunction or on cell death without addressing the distinction between metabolic and structural alterations.Nitric oxide (NO) is a well-characterized neurotransmitter in the central and peripheral nervous systems. In many tissues of the urogenital and gastrointestinal tract, NO mediates nonadrenergic noncholinergic (NANC) relaxant responses (4). Nerves that release NO are now known as nitrergic (5). NO is generated in these nerves by activation of the neuronal NO synthase (nNOS) and diffuses into the smooth muscle to activate the soluble guanylyl cyclase (sGC), producing an increase in the intracellular cyclic guanosine-3Ј, 5Ј-monophosphate concentration, leading to relaxation (6).Impaired nitrergic transmission has been shown to be responsible for erectile dysfunction and gastropathy in diabetes (7-12). To investigate the mechanisms by which the metabolic changes lead to structural disturbances in autonomic neuropathy, we studied the structure and function of nitrergic nerve fibers and neuronal cell bodies in the gastric pylorus, penis, and major pelvic ganglia in streptozotocin (STZ)-induced diabetic rats. In addition, we investigated the effect of different schedules of insulin treatment on this process. RESEARCH DE...

Two enzymes, soluble guanylyl cyclase and cytochrome c oxidase, have been shown to be exquisitely sensitive to nitric oxide (NO) at low physiological concentrations. Activation of the soluble guanylyl cyclase by endogenous NO and the consequent increase in the second messenger cyclic GMP are now known to control a variety of biological functions. Cytochrome c oxidase, the terminal enzyme of the mitochondrial respiratory chain, is inhibited by NO. However, it is not clear whether NO produced by the constitutive NO synthase interacts with cytochrome c oxidase, nor is it known what the biological consequences of such an interaction might be. We now show that NO generated by vascular endothelial cells under basal and stimulated conditions modulates the respiration of these cells in response to acute changes in oxygen concentration. This action occurs at the cytochrome c oxidase and depends on inf lux of calcium. Thus, NO plays a physiological role in adjusting the capacity of this enzyme to use oxygen, allowing endothelial cells to adapt to acute changes in their environment.Evidence in favor of a role of endogenous nitric oxide (NO) as a modulator of cell respiration has been derived from experiments in cells activated with cytokines and bacterial products in which NO is generated continuously in large quantities by the inducible NO synthase (NOS). In these conditions, NOinduced inhibition of cell respiration is persistent and attributable to nonselective inhibition of various mitochondrial enzymes, including complexes I-IV in the respiratory chain. Such inhibition contributes to the pathological actions of NO (1). On the other hand, experiments in animals have suggested that inhibition of endogenous generation of NO increases whole body oxygen consumption (2, 3), and bradykinin (Bk) and carbachol have been shown to reduce oxygen consumption in skeletal and cardiac muscle in a manner that could be prevented by an inhibitor of NOS (3-5).Exogenous administration of low concentrations of NO inhibits cytochrome c oxidase (complex IV in the mitochondrial respiratory chain) in a variety of cells and isolated mitochondria. Such inhibition is competitive with oxygen and is fully reversible (6-9) even after several hours (10). These findings suggest that endogenous NO may regulate cell respiration. To test this hypothesis, we have analyzed the effect of endogenous NO, generated under basal conditions and after stimulation with Bk and ATP, on respiration in porcine aortic endothelial cells. Our results show that endogenously released NO, by acting on cytochrome c oxidase, is responsible for the physiological regulation of respiration in these cells. MATERIALS AND METHODSMaterials. Culture media and fetal calf serum were from GIBCO. Fura-2 acetoxymethylester was from Calbiochem. Other reagents were from Sigma.Cell Culture and Preparation. Endothelial cells were prepared from fresh porcine thoracic aortae obtained from the abattoir, were cultured overnight in DMEM 20% fetal calf serum, and then were grown to confluence...

SummarySalmonella infections in naturally susceptible mice grow rapidly, with death occurring only after bacterial numbers in vivo have reached a high threshold level, commonly called the lethal load. Despite much speculation, no direct evidence has been available to substantiate a role for any candidate bacterial components in causing death. One of the most likely candidates for the lethal toxin in salmonellosis is endotoxin, specifically the lipid A domain of the lipopolysaccharide (LPS) molecule. Consequently, we have constructed a Salmonella mutant with a deletion-insertion in its waaN gene, which encodes the enzyme that catalyses one of the two secondary acylation reactions that complete lipid A biosynthesis. The mutant biosynthesizes a lipid A molecule lacking a single fatty acyl chain and is consequently less able to induce cytokine and inducible nitric oxide synthase (iNOS) responses both in vivo and in vitro. The mutant bacteria appear healthy, are not sensitive to increased growth temperature and synthesize a full-length Oantigen-containing LPS molecule lacking only the expected secondary acyl chain. On intravenous inoculation into susceptible BALB/c mice, wild-type salmonellae grew at the expected rate of approximately 10-fold per day in livers and spleens and caused the death of the infected mice when lethal loads of approximately 10 8 were attained in these organs. Somewhat unexpectedly, waaN mutant bacteria grew at exactly the same rate as wild-type bacteria in BALB/c mice but, when counts reached 10 8 per organ, mice infected with mutant bacteria survived. Bacterial growth continued until unprecedentedly high counts of 10 9 per organ were attained, when approximately 10% of the mice died. Most of the animals carrying these high bacterial loads survived, and the bacteria were slowly cleared from the organs. These experiments provide the first direct evidence that death in a mouse typhoid infection is directly dependent on the toxicity of lipid A and suggest that this may be mediated via proinflammatory cytokine and/or iNOS responses.

Inhibition of NO synthesis by L-NMMA potentiates platelet adhesion to unstimulated SGHEC-7 cells, showing that basally released NO regulates platelet adhesion. Stimulation of SGHEC-7 cells by cytokines increases their adhesive properties but at the same time causes them to express the inducible NO synthase. Nitric oxide generated by this enzyme contributes to the modulation of the adhesive properties of the endothelial cells. Thus both constitutive and inducible NO synthases modulate endothelial cell thrombogenicity.