The neuronal nitric oxide synthase inhibitor NANT blocks acetaminophen toxicity and protein nitration in freshly isolated hepatocytes

3-Nitrotyrosine (3NT) in liver proteins of mice treated with hepatotoxic doses of acetaminophen (APAP) has been postulated to be causative in toxicity. Nitration is by a reactive nitrogen species formed from nitric oxide (NO). The source of the NO is unclear. iNOS knockout mice were previously found to be equally susceptible to APAP toxicity as wildtype mice and iNOS inhibitors did not decrease toxicity in mice or in hepatocytes. In this work we examined the potential role of nNOS in APAP toxicity in hepatocytes using the specific nNOS inhibitor NANT (10 µM)(N-[(4S)-4-amino-5-[(2-aminoethyl)amino]pentyl]-N′-nitroguanidinetris (trifluoroacetate)). Primary hepatocytes (1 million/ml) from male B6C3F1 mice were incubated with APAP (1 mM). Cells were removed and assayed spectrofluorometrically for reactive nitrogen and oxygen species using diaminofluorescein (DAF) and Mitosox red, respectively. Cytotoxicity was determined by LDH release into media. Glutathione (GSH, GSSG), 3NT, GSNO, acetaminophen-cysteine adducts, NAD, and NADH were measured by HPLC. APAP significantly increased cytotoxicity at 1.5–3.0 h. The increase was blocked by NANT. NANT did not alter APAP mediated GSH depletion or acetaminophen-cysteine adducts in proteins which indicated that NANT did not inhibit metabolism. APAP significantly increased spectroflurometric evidence of reactive nitrogen and oxygen formation at 0.5 and 1.0 h, respectively, and increased 3NT and GSNO at 1.5–3.0 h. These increases were blocked by NANT. APAP dramatically increased NADH from 0.5–3.0 h and this increase was blocked by NANT. Also, APAP decreased the Oxygen Consumption Rate (OCR), decreased ATP production, and caused a loss of mitochondrial membrane potential, which were all blocked by NANT.     

Beneficial effect of gaseous nitric oxide on the healing of skin wounds

Intermittent daily exposures (60 s) to NO-containing gas flow (NO dose of 500 ppm) generated by air-plasma unit "Plason" improves healing of skin wounds in rats. The gas flow treatment shortened the recovery time of both aseptic and purulent wounds (300 mm2 area) by nearly a third. The treatment allows to achieve a marked improvement in the histological, histochemical, and electron-microscopic characteristics of the affected tissue. The mechanism of this phenomenon was studied by spin trapping method. The NO status of the wound tissue was investigated with EPR by following the formation of paramagnetic mononitrosyl complexes with iron-diethyldithiocarbamate, or with the heme groups in hemoglobin or myoglobin. For the first 5 min after a gas treatment with the exposure of 60s, detectable NO levels in the affected tissue were slightly lowered with respect to untreated controls. At subsequent times, treated tissues showed the formation of large quantities of nitroso-iron complexes: At 30-40 min after gas exposure, their levels were nearly two orders of magnitude higher than soon after (15 s-5 min) the exposure. The data demonstrate that the accumulation of nitrosyl-iron complexes reflects a sharp rise in endogenous NO production inside the affected tissue. Paradoxically, the beneficial effect of gaseous NO treatment can be mediated by the formation of limited quantities of peroxynitrite due to the reaction between exogenous NO and superoxide anions generated in high amount in wound tissue. This peroxynitrite has a strong prooxidant effect and can activate various antioxidant systems which diminish the amount of superoxide anions in wound tissue. The reduced superoxide levels allow to increase the contents of endogenous NO in gas-treated tissues. Therefore, the beneficial action of the treatment is attributed to enhanced NO bioavailability.     

Protective effect of aminoguanidine, a nitric oxide synthase inhibitor, against carbon tetrachloride induced hepatotoxicity in mice

The present study was undertaken to evaluate the effect of aminoguanidine (AG) on carbon tetrachloride (CCl4)-induced hepatotoxicity. Treatment of mice with CCl4 (20 microl/kg, i.p.) resulted in damage to centrilobular regions of the liver, increase in serum aminotransferase and rise in lipid peroxides level 24 hours after CCl4 administration. Pretreatment of mice with AG (50 mg/kg, i.p.) 30 minutes before CCl4 was found to protect mice from the CCl4-induced hepatic toxicity. This protection was evident from the significant reduction in serum aminotransferase, inhibition of lipid peroxidation and prevention of CCl4-induced hepatic necrosis revealed by histopathology. Aminoguanidine, a relatively specific inhibitor of inducible nitric oxide synthase, did not inhibit the in vitro lipid peroxidation. Taken together, these data suggest a potential role of nitric oxide as an important mediator of CCl4-induced hepatotoxicity.     

The protein inhibitor of neuronal nitric oxide synthase (PIN): characterization of its action on pure nitric oxide synthases

Neuronal NO synthase (nNOS) was discovered recently to interact specifically with the protein PIN (protein inhibitor of nNOS) [Jaffrey, S.R. and Snyder, S.H. (1996) Science 274, 774–777]. We have studied the effects on pure NOS enzymes of the same GST-tagged PIN used in the original paper. Unexpectedly, all NOS isoenzymes were inhibited. The IC₅₀ for nNOS was 18±6 μM GST-PIN with 63 nM nNOS after 30 min at 37°C. Uncoupled NADPH oxidation was inhibited similarly, whereas cytochrome c reductase activity, the K_M for l-arginine, and dimerization were unaffected. We reconsider the physiological role of PIN in the light of these results.     

Mediation by nitric oxide of the stimulatory effects of ethanol on blood flow

The contribution of nitric oxide (NO) to the hemodynamic effects associated with alcohol oxidation was assessed in rats given either ethanol or water by gastric tube, with and without pre-treatment with either the NO synthase inhibitor N(omega)-monomethyl-L-arginine (L-NMMA; 15 mg/Kg i.p.) or the alcohol dehydrogenase inhibitor 4-methylpyrazole (4-MP; 82 mg/Kg i.p.). Alcohol increased plasma NO (measured with chemiluminescence) by 63%. This was prevented by either L-NMMA or 4-MP. Cardiac output and regional blood flows were determined with 57Cobalt-labeled microspheres. Alcohol markedly enhanced portal blood flow (130 +/- 6 ml/min/Kg vs. 62 +/- 4, in controls; p < 0.01) with no changes in the hepatic, splenic or pancreatic arterial blood flows, indicating that the vasodilatation is mainly mesenteric. In addition, it quadrupled the coronary blood flow, doubled the renal flow and increased cardiac output by 38%, with no significant changes in pulmonary, cerebral or testicular flows. All the stimulatory effects of ethanol on flow, as well as the rise in NO levels, were prevented by L-NMMA, incriminating NO as the mediator of the hemodynamic effects of ethanol oxidation, acting probably via acetate and adenosine.     

Effect of subchronic acrylamide exposure on the expression of neuronal and inducible nitric oxide synthase in rat brain

Acrylamide (ACR) is a known industrial neurotoxic chemical. Evidence suggests that ACR neurotoxic effect is related to brain neurotransmission disturbances. Since nitric oxide (NO) acts as a neurotransmission modulator and is produced by nitric oxide synthase (NOS), the neuronal NOS (nNOS) and inducible NOS (iNOS) expression pattern were determined in rat cerebral cortex and striatum after subchronic exposure to ACR. Using immunocytochemistry, the neuronal count of nNOS or optical density of iNOS from sections at three coronal levels, bregma 1.0, -0.4, and -2.3 mm, were compared between ACR-treated and control rats. At all three levels, nNOS expressions were uniformly decreased in most of the neocortical subregions following the treatment of ACR. At bregma level 1.0 mm, total numbers of nNOS expressing neurons were significantly decreased to 58.7% and 64.7% of the control in the cortex and striatum of ACR-treated rats, respectively. However, at the bregma level -2.3 mm, ACR treatment did not produce a significant difference in the numbers of nNOS expressing neurons both in the cortex and striatum. Contrary to nNOS, iNOS expressions were consistently increased to approximately 32% in the neocortex and 25% in the striatum, following the subchronic ACR treatment. These data suggest that subchronic ACR exposure involves compensatory mechanism on nNOS and iNOS expression to maintain the homeostasis of NO at the rostral part of the neocortex and the striatum. However, in the caudal brain, increased iNOS expression did not suppress nNOS expression. Therefore, the present study is consistent with the hypothesis that ACR toxicity is mediated through the disturbance to the NO signaling pathway and exhibits a rostrocaudal difference through the differential expressions of nNOS and iNOS in the neocortex and the striatum.     

Detection of nitrous oxide in the neuronal nitric oxide synthase reaction by gas chromatography-mass spectrometry

Using headspace gas chromatography-mass spectrometry, we detected significant amounts of nitrous oxide in the reaction products of the monooxygenase reaction catalyzed by neuronal nitric oxide synthase. Nitrous oxide is a dimerization product of nitroxyl anion; its presence in the reaction products indicates that the nitroxyl anion is a product of the neuronal nitric oxide synthase-catalyzed reaction.     

Inhibition of nitric oxide synthase by cobalamins and cobinamides

Cobalamins are important cofactors for methionine synthase and methylmalonyl-CoA mutase. Certain corrins also bind nitric oxide (NO), quenching its bioactivity. To determine if corrins would inhibit NO synthase (NOS), we measured their effects on -l-[¹⁴C]arginine-to-l-[¹⁴C]citrulline conversion by NOS1, NOS2, and NOS3. Hydroxocobalamin (OH-Cbl), cobinamide, and dicyanocobinamide (CN₂-Cbi) potently inhibited all isoforms, whereas cyanocobalamin, methylcobalamin, and adenosylcobalamin had much less effect. OH-Cbl and CN₂-Cbi prevented binding of the oxygen analog carbon monoxide (CO) to the reduced NOS1 and NOS2 heme active site. CN₂-Cbi did not react directly with NO or CO. Spectral perturbation analysis showed that CN₂-Cbi interacted directly with the purified NOS1 oxygenase domain. NOS inhibition by corrins was rapid and not reversed by dialysis with l-arginine or tetrahydrobiopterin. Molecular modeling indicated that corrins could access the unusually large heme- and substrate-binding pocket of NOS. Best fits were obtained in the “base-off” conformation of the lower axial dimethylbenzimidazole ligand. CN₂-Cbi inhibited interferon-γ-activated Raw264.7 mouse macrophage NO production. We show for the first time that certain corrins directly inhibit NOS, suggesting that these agents (or their derivatives) may have pharmacological utility. Endogenous cobalamins and cobinamides might play important roles in regulating NOS activity under normal and pathological conditions.     

Gas-phase disproportionation of nitric oxide at elevated pressures

T.P. Melia's chemical kinetics study of the disproportionation of nitric oxide (NO), 3NO →NO₂ + N₂O , (Melia, T.P. (1965) J. Inorg. Nucl. Chem., 27, 95-98), which is the most quoted quantitative investigation presently available, revealed a rather strong dependence of the effective rate constant, Kk ', of Melia's third-order rate law,- d[NO]/d t = Kk'[NO]₃, on the initial pressure of NO. In order to estimate extent of accumulation of NO₂ and N₂O as a function of time by integration of the rate law, we have evaluated the dependence of the effective rate constant as a function of pressure and thus as a function of time on the basis of the non-ideality of NO gas. Although our approach is crude in that the non-idealities of NO₂ and N₂O and other NO_x products and a probable deviation of the gas mixture from the Dalton's law have not been considered, it provides a means for approximately estimating the rate of accumulation of NO₂ and N₂O based on Melia's data. According to these calculations, the extent of the disproportionation is generally negligible at low initial pressures, e.g. 5 atm or less, while at 200 atm, the mole fractions of NO₂ and N₂O can become as high as 12-13% only after 10 days. These values are alarmingly high for handling pressured NO- in N₂-mixture in either research or clinical settings. This information must be borne in mind when compressed NO in commercial cylinders is employed in high precision experiments. Disproportionation of NO under pressure also deserves attention in inhalation of low doses of NO in the treatment of diseases characterized by pulmonary hypertension and hypoxemia.     

Pharmacological inhibition of inducible nitric oxide synthase attenuates the development of seizures in mice

The present study has been designed to pharmacologically expound the significance of inducible nitric oxide synthase in the pathophysiological progression of seizures using mouse models of chemically induced kindled epilepsy and status epilepticus induced spontaneous recurrent seizures. Pentylenetetrazole (40 mg kg⁻¹) (PTZ) administration every second day for a period of 15 days was used to elicit kindled seizure activity in mice. Severity of kindled seizures was assessed in terms of a composite kindled seizure severity score (KSSS). Pilocarpine (100 mg kg⁻¹) was injected every 20 min until the onset of status epilepticus. A spontaneous recurrent seizure severity score (SRSSS) was recorded as a measure of quantitative assessment of the progressive development of spontaneous recurrent seizures induced after pilocarpine status epilepticus. Sub-acute PTZ administration induced the development of severe form of kindled seizures in mice. Further, pharmacological status epilepticus elicited a progressive evolution of spontaneous recurrent seizures in the animals. However, treatment of aminoguanidine, a relatively selective inhibitor of inducible nitric oxide synthase, markedly and dose dependently suppressed the development of both PTZ induced kindled seizures as well as pilocarpine induced spontaneous recurrent seizures. Therefore inducible nitric oxide synthase may be implicated in the development of seizures.     

Preemptive effects of intrathecal cyclooxygenase inhibitor or nitric oxide synthase inhibitor on thermal hypersensitivity following peripheral nerve injury

The present study provides an important implication for the management of chronic neuropathic pain, focusing on prostaglandin (PG) and nitric oxide (NO) in the spinal cord. To determine if spinally administered cyclooxygenase (COX) inhibitor or nitric oxide synthase (NOS) inhibitor had preemptive analgesia on thermal hypersensitivity induced by chronic constrictive nerve injury, Sprague-Dawley rats were chronically implanted with an intrathecal (i.t.) catheter. The left sciatic nerve was loosely ligated with 2-mm polyethylene tubing to produce painful mononeuropathy. Animals received tenoxicam (7.5, 15 or 30 micromol/10 microl, i.t.), NS-398 (15 or 30 micromol), or L-NAME (30, 150 or 300 micromol) immediately before the nerve injury, followed by daily injection extending into the four postoperative days. The hindpaw was immersed into a hot (42 degrees C, 44 degrees C and 46 degrees C) or cold (10 degrees C) water bath. The paw immersion test revealed significant thermal hyperalgesia and allodynia 5 day after nerve injury in vehicle control animals. Tenoxicam (7.5, 15 or 30 micromol) or L-NAME (30, 150 or 300 micromol) dose-dependently attenuated hyperalgesia and allodynia. Equimolar dose of NS-398 (15 or 30 micromol) also diminished these nociceptive behaviors. Higher dose of either drug primarily produced longer duration of inhibition. The inhibitory effect of tenoxicam (30 micromol) on hyperalgesia was more effective than that of an equimolar dose of NS-398 or L-NAME. These results demonstrated that intrathecally administered COX inhibitor or NOS inhibitor provides preemptive analgesia on thermal hypersensitivity following chronic constrictive nerve injury in rats.     

Intracellular nitric oxide mediates neuroproliferative effect of neuropeptide y on postnatal hippocampal precursor cells

Neuropeptide Y (NPY) is widely expressed in the central and peripheral nervous systems and is proliferative for a range of cells types in vitro. NPY plays a key role in regulating adult hippocampal neurogenesis in vivo under both basal and pathological conditions, although the underlying mechanisms are largely unknown. We have investigated the role of nitric oxide (NO) on the neurogenic effects of NPY. Using postnatal rat hippocampal cultures, we show that the proliferative effect of NPY on nestin(+) precursor cells is NO-dependent. As well as the involvement of neuronal nitric-oxide synthase, the proliferative effect is mediated via an NO/cyclic guanosine monophosphate (cGMP)/cGMP-dependent protein kinase (PKG) and extracellular signal-regulated kinase (ERK) 1/2 signaling pathway. We show that NPY-mediated intracellular NO signaling results in an increase in neuroproliferation. By contrast, extracellular NO had an opposite, inhibitory effect on proliferation. The importance of the NO-cGMP-PKG signaling pathway in ERK1/2 activation was confirmed using Western blotting. This work unites two significant modulators of hippocampal neurogenesis within a common signaling framework and provides a mechanism for the independent extra- and intracellular regulation of postnatal neural precursors by NO.     

Neither nitrite nor nitric oxide mediate toxic effects of nitroglycerin on mitochondria

It is commonly accepted that the major effect of nitroglycerin (NG) is realized through the release of nitric oxide (NO) catalyzed by aldehyde dehydrogenase-2 (ALDH2). In addition, it has been shown that NG inhibits mitochondrial respiration. The aim of this study was to clarify whether NG-mediated inhibition of mitochondrial respiration is mediated by NO. In rat liver mitochondria, NG inhibited complex-I-dependent respiration and induced reactive oxygen species (ROS) production, preferentially at complex I. Both effects were insensitive to chloral hydrate, an ALDH2 inhibitor. Nitrite, an NG intermediate, had no influence on either mitochondrial respiration or the production of ROS. NO inhibited preferentially complex I but did not elevate ROS production. Hemoglobin, an NO scavenger, and blue light had contrary effects on mitochondria inhibited by NO or NG. In summary, our data suggest that although NG induces vasodilatation via NO release, it causes mitochondrial dysfunction via an NO-independent pathway.     

Effect of nitric oxide on phagocytic activity of lipopolysaccharide-induced macrophages: possible role of exogenous L-arginine

Among the antimicrobial mechanisms associated with macrophages, NO produced by iNOS plays a major role in intracellular killing, but the relationship between NO and phagocytic activity after injection of inflammatory agents into the peritoneal cavity is not clear. The aim of the present study was to investigate the effect of nitric oxide (NO) on macrophage function after treatment with intraperitoneal lipopolysaccharide (LPS) and the role of exogenous L-arginine administration in this event. Six experimental groups and one control group, each consisting of seven Wistar rats were used: Group I: Control; Group II: LPS; Group III: LPS+L-arginine; Group IV: LPS+L-arginine+Aminoguanidine; Group V: LPS+Aminoguanidine; Group VI: L-arginine; Group VII: Aminoguanidine. Macrophage phagocytic activity and total plasma nitrite levels were increased in the LPS group. In the LPS+L-arginine group, both the phagocytic activity and total plasma nitrite levels showed large increases. Administration of aminoguanidine (AG), a specific iNOS inhibitor, abolished macrophage phagocytic activity and total plasma nitrite levels in the LPS and LPS+L-arginine groups. As a result, we showed that NO produced by macrophages has a role not only in intracellular killing, but also in phagocytic activity.     

Hepatitis B virus X protein regulates hepatic glucose homeostasis via activation of inducible nitric oxide synthase

Dysregulation of liver functions leads to insulin resistance causing type 2 diabetes mellitus and is often found in chronic liver diseases. However, the mechanisms of hepatic dysfunction leading to hepatic metabolic disorder are still poorly understood in chronic liver diseases. The current work investigated the role of hepatitis B virus X protein (HBx) in regulating glucose metabolism. We studied HBx-overexpressing (HBxTg) mice and HBxTg mice lacking inducible nitric oxide synthase (iNOS). Here we show that gene expressions of the key gluconeogenic enzymes were significantly increased in HepG2 cells expressing HBx (HepG2-HBx) and in non-tumor liver tissues of hepatitis B virus patients with high levels of HBx expression. In the liver of HBxTg mice, the expressions of gluconeogenic genes were also elevated, leading to hyperglycemia by increasing hepatic glucose production. However, this effect was insufficient to cause systemic insulin resistance. Importantly, the actions of HBx on hepatic glucose metabolism are thought to be mediated via iNOS signaling, as evidenced by the fact that deficiency of iNOS restored HBx-induced hyperglycemia by suppressing the gene expression of gluconeogenic enzymes. Treatment of HepG2-HBx cells with nitric oxide (NO) caused a significant increase in the expression of gluconeogenic genes, but JNK1 inhibition was completely normalized. Furthermore, hyperactivation of JNK1 in the liver of HBxTg mice was also suppressed in the absence of iNOS, indicating the critical role for JNK in the mutual regulation of HBx- and iNOS-mediated glucose metabolism. These findings establish a novel mechanism of HBx-driven hepatic metabolic disorder that is modulated by iNOS-mediated activation of JNK.     

A microtiter-plate assay of nitric oxide synthase activity

We describe here a microtiter-plate assay for measuring nitric oxide synthase (NOS) activity by utilizing the spectral shift in optical absorbence between the wavelengths 405 and 420 nm on conversion of oxyhemoglobin to methemoglobin by nitric oxide (NO). This is a high-throughput assay permitting 96 or 384 simultaneous kinetic measurements and is ideal for the study of NOS inhibitors and their time dependence. It is also possible to measure enzyme rates under different conditions simultaneously for the study of the cofactor and substrate dependence of NOS preparations. The assay requires approximately 10 pmol/min of NOS activity to achieve a 1moD/min rate.     

Aged garlic extract enhances production of nitric oxide

Nitric oxide (NO) controls several physiological functions of the cardiovascular system. Three kinds of NO synthases (NOSs), neuronal constitutive NOS (ncNOS), inducible NOS (iNOS) and endothelial constitutive NOS (ecNOS), were responsible for NO biosynthesis. This study investigated the effect of aged garlic extract (AGE) on NO production by measuring the NO metabolites nitrite and nitrate in the plasma of mice. AGE (2.86 g/kg, p.o.) temporarily increased NO production by 30-40% from 15 to 60 min after administration. The time course of the fluctuation in NO levels in the AGE-treated group was clearly different to that in a group of mice treated with lipopolysaccharides, a typical iNOS inducer. Arginine (63 mg/kg, p.o.) at the equivalent dose of AGE did not increase NO production. However diphenyleneiodonium chloride (1 mg/kg, i.p.), a selective cNOS inhibitor, administered prior to AGE, overcame the effect of AGE. These results indicate that AGE increased NO production by activating cNOS, but not iNOS. The arginine contained in AGE was not responsible for the effect. AGE may be a useful tool for the prevention of cardiovascular disease.     

Relationship between structure and inhibitory effect of arginine analogues on neuronal nitric oxide synthase activity

Since nitric oxide (NO) is synthesized by nitric oxide synthase (NOS) froml-arginine (Arg) which has an amidino group in its molecule, we, examined the effect of 29 kinds of Arg analogues on neuronal NOS (nNOS) activity in the rat brain. None of the Arg analogues acted as a substrate for nNOS. Diamidinocystamine, hirudonine, and guanethidine inhibited nNOS activity to 67.3%, 64.2% and 74.1%, respectively, but their inhibitory efficiency was lower than N^G-monomethyl-l-arginine (to 36.5%) which is a well known NOS inhibitor. Dimethylguanidine and N-benzoylguanidine also significantly inhibited nNOS activity to 88.0% and 90.7%, respectively. Whereas almost all of the NOS inhibitors previously reported were synthesizdd by substituting the amidino nitrogen of Arg, none of these new inhibitors were substituted at this position. Furthermore, hirudonine, which is a naturally occurring compound, was thought to act as an agonist at polyamine binding site of the N-methyl-d-aspartate type of glutamate receptor complex. It is also interesting that guanethidine, an antihypertensive agent, inhibit nNOS activity. These new drugs are useful for the investigation not only of the chemical nature of nNOS but also of the physiologic function of NO.     

Analysis of the effects of nitric oxide and oxygen on nitric oxide production by macrophages

The interactions between NO and O(2) in activated macrophages were analysed by incorporating previous cell culture and enzyme kinetic results into a novel reaction-diffusion model for plate cultures. The kinetic factors considered were: (i) the effect of O(2) on NO production by inducible NO synthase (iNOS); (ii) the effect of NO on NO synthesis by iNOS; (iii) the effect of NO on respiratory and other O(2) consumption; and (iv) the effects of NO and O(2) on NO consumption by a possible NO dioxygenase (NOD). Published data obtained by varying the liquid depth in macrophage cultures provided a revealing test of the model, because varying the depth should perturb both the O(2) and the NO concentrations at the level of the cells. The model predicted that the rate of NO(2)(-) production should be nearly constant, and that the net rate of NO production should decline sharply with increases in liquid depth, in excellent agreement with the experimental findings. In further agreement with available results for macrophage cultures, the model predicted that net NO synthesis should be more sensitive to liquid depth than to the O(2) concentration in the headspace. The main reason for the decrease in NO production with increasing liquid depth was the modulation of NO synthesis by NO, with O(2) availability playing only a minor role. The model suggests that it is the ability of iNOS to consume NO, as well as to synthesize it, that creates very sensitive feedback control, setting an upper bound on the NO concentration of approximately 1 microM. The effect of NO consumption by other possible pathways (e.g., NOD) would be similar to that of iNOS, in that it would help limit net NO production. The O(2) utilized during enzymatic NO consumption is predicted to make the O(2) demands of activated macrophages much larger than those of unactivated ones (where iNOS is absent); this remains to be tested experimentally.