Mechanism of superoxide generation by neuronal nitric-oxide synthase.

Neuronal nitric-oxide synthase (NOS I) in the absence of L-arginine has previously been shown to generate superoxide (O-2) (Pou, S., Pou, W. S., Bredt, D. S., Snyder, S. H., and Rosen, G. M. (1992) J. Biol. Chem. 267, 24173-24176). In the presence of L-arginine, NOS I produces nitric oxide (NO.). Yet the competition between O2 and L-arginine for electrons, and by implication formation of O-2, has until recently remained undefined. Herein, we investigated this relationship, observing O-2 generation even at saturating levels of L-arginine. Of interest was the finding that the frequently used NOS inhibitor NG-monomethyl L-arginine enhanced O-2 production in the presence of L-arginine because this antagonist attenuated NO. formation. Whereas diphenyliodonium chloride inhibited O-2, blockers of heme such as NaCN, 1-phenylimidazole, and imidazole likewise prevented the formation of O-2 at concentrations that inhibited NO. formation from L-arginine. Taken together these data demonstrate that NOS I generates O-2 and the formation of this free radical occurs at the heme domain.

Effect of trifluoromethyl ketone-based elastase inhibitors on neutrophil function in vitro.

Neutrophils release elastase, which is known secondarily to cause tissue damage. However, it is rapidly inactivated by the endogenous alpha1-proteinase inhibitor (alpha1Pi). Nevertheless, under pathological conditions, alpha1i is inactivated by oxidants released from neutrophils, resulting in an excess of elastase at the site of inflammation. This elastase/alpha1Pi imbalance has been implicated as a pathogenic factor in cystic fibrosis, acute respiratory distress syndrome, and emphysema. Elastase inhibitors, which do not interfere with the microbicidal activity of neutrophils and are resistant to neutrophil-released oxidants, would undoubtedly represent an important advance in the management of neutrophil-mediated tissue injury. We report that a new family of elastase inhibitors ICI200355 and ZD0892 was found to be resistant toward superoxide, hypochlorous acid, hydrogen peroxide, hydroxyl radical, and peroxynitrite mediated degradation as well as having no effect on the formation of these oxidants by activated neutrophils. More importantly, we found that these inhibitors did not interfere with the ability of human neutrophils to phagocytose and to kill Staphylococcus aureus. In conclusion, a new potent class of elastase inhibitors, while blocking the effects of neutrophil elastase, was found not to impede various physiological functions of human neutrophils, in particular the ability of these phagocytic cells to phagocytose and kill bacteria.

Biological studies of a nitroso compound that releases nitric oxide upon illumination.

2-Methyl-2-nitrosopropane (MNP) has long been known to undergo photochemical and thermal decomposition, generating di-tert-butyl nitroxide, in organic solvent. The present study was undertaken to demonstrate that MNP can be used as a caged-nitric oxide (NO), which can liberate NO upon illumination. Photolysis of MNP leads to the generation of tert-butyl radical and NO, as detected by spin-trapping/ESR spectroscopy and by oxyhemoglobin/visible spectroscopy, respectively. Using soluble guanylate cyclase in neuroblastoma N1E-115 cells as an NO target, we found that MNP in the presence of light caused a dose- and time-dependent increase in cGMP. Finally, illumination of a solution of MNP was also found to induce relaxation of preconstricted isolated rat pulmonary artery rings. These studies demonstrated that MNP can be useful biochemical research tool for delivering NO in a controlled manner, by using light.

Can nitric oxide be spin trapped by nitrone and nitroso compounds?

Increasing interest in the study of nitric oxide (NO.) in many facets of biological research necessitates a search for accurate techniques to directly identify the free radical. One recently employed strategy for NO. detection is the method of electron spin resonance (ESR) used in combination with nitrone and nitroso spin traps. Applying this technique to our studies with nitric oxide synthase (NOS), we found that NO. generated directly from the enzyme system could not be detected. Further investigation revealed that 3,5-dibromo-4-nitrosobenzenesulfonic acid (DBNBS) inhibited NO. generation by NOS at concentrations used for spin trapping. Reexamining the ability of various nitrones and DBNBS to spin trap authentic NO. dissolved in buffer, we obtained ESR spectra similar to those previously reported for the spin trap DBNBS. However, continuing our studies with 15NO. and N-hydroxylamine, we found these spectra to be artifactual. Our results emphasize the need to synthesize new spin traps, since currently available compounds are not capable of spin trapping NO. generated by NOS.

Role of intracellular Ca2+ mobilization in muscarinic and histamine receptor-mediated activation of guanylate cyclase in N1E-115 neuroblastoma cells: assessment of the arachidonic acid release hypothesis.

This study evaluates the role of intracellular levels of Ca2+ [Ca2+]i in cyclic GMP formation mediated by muscarinic and histamine receptors in the mouse neuroblastoma clone N1E-115. Muscarinic agonists activated the turnover of phosphoinositides with a relative maximal response similar to that observed previously for cyclic GMP formation. Carbamylcholine induced a transient increase in inositol trisphosphate with a time course similar to that of cyclic GMP formation. In cells loaded with the fluorescent Ca2+ probe fura-2/acetoxymethyl ester, carbamylcholine as well as histamine induced a rapid and transient rise in [Ca2+]i. The time course of the changes in [Ca2+]i induced by agonists as well as by ionomycin closely paralleled that of cyclic GMP formation. Chelation of [Ca2+]i by loading of N1E-115 cells with quin 2/acetoxymethyl ester inhibited cyclic GMP formation induced by agonists in a dose-dependent manner. When cyclic GMP formation induced by agonists was assayed after the cells were exposed to 3 mM ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) for 2 min, the formation of cyclic GMP was not inhibited significantly; however, it was completely abolished after 30-min exposure to EGTA. Treatment of cells with phospholipase A2 had no effect on resting [Ca2+]i and only slightly increased cyclic GMP formation, in spite of the induction of a marked release of [3H]arachidonate. Moreover, the formation of cyclic GMP induced by ionomycin was inhibited by the addition of phospholipase A2. Melittin contaminated with phospholipase A2 activity induced a rapid and sustained increase in cyclic GMP formation, as well as unesterified [3H]arachidonate release. However, after inactivation of the phospholipase A2 activity of melittin, its ability to stimulate cyclic GMP formation was enhanced. Our data indicate that receptor agonists stimulate cyclic GMP formation in N1E-115 cells by activating the formation of inositol trisphosphate, which is followed by the release of Ca2+ from intracellular stores. The evidence obtained does not support a major role for arachidonate release in receptor-mediated activation of guanylate cyclase. Conversely, it is consistent with an inhibitory role for arachidonic acid or its metabolites in this process.

Aging does not alter brain muscarinic receptor-mediated phosphoinositide hydrolysis and its inhibition by phorbol esters, tetrodotoxin and receptor desensitization.

The effects of aging on muscarinic receptor-mediated phosphoinositide (PI) hydrolysis in the brain were investigated in Fischer-344 rats. Oxotremorine-M stimulated this response to the same magnitude in young and old rats in the cerebral cortex, striatum, hippocampus, thalamus, hypothalamus and cerebellum, although maximal stimulation varied among brain regions within each age group. The PI response was also equally potentiated by elevated K+ or suppressed by tetrodotoxin in both age groups. In addition, a phorbol ester attenuated muscarinic receptor-mediated PI hydrolysis in the cerebral cortex to the same extent in young and aged rats. Moreover, preincubation with oxotremorine-M resulted in a similar down-regulation of cell-surface receptors and desensitization of receptor function regardless of age. Therefore, under our experimental conditions, PI hydrolysis in response to activation of brain muscarinic receptors does not appear to be sensitive to aging-related alterations.

Inhibition of cyclic AMP formation in N1E-115 neuroblastoma cells is mediated by a non-cardiac M2 muscarinic receptor subtype.

The cardioselective muscarinic antagonist, AF-DX 116 [11[2-[(diethyl-amino)-methyl]-O-1-piperidinyl]-5,11-dihydro-6H-pyrido- [2,3-b][1,4]-benzodiazepine-6-one), was weak at blocking the M2 muscarinic receptor-mediated inhibition of cyclic adenosine monophosphate (cAMP) formation in mouse neuroblastoma cells (clone N1E-115). In contrast, the glandular-selective antagonists, hexahydro-sila-difenidol (HHSiD) and 4-diphenylacetoxy-N-methyl-piperidine methiodide (4-DAMP), were quite potent at inhibiting this response, being 14- and 318-fold more potent than AF-DX 116 in this regard, respectively. According to the rank order of potency of these two classes of antagonists, these data provide the first pharmacological evidence that inhibition of cAMP formation in a neuronal tissue is mediated by a non-cardiac M2 muscarinic receptor subtype.

Effects of aging on the interaction of quinuclidinyl benzilate, N-methylscopolamine, pirenzepine, and gallamine with brain muscarinic receptors.

The objective of the present study was to investigate the effects of senescence on the binding characteristics of muscarinic receptors by using [3H]quinuclidinyl benzilate ([3H]QNB) and [3H]N-methylscopolamine ([3H]NMS) as ligands in young (3 months), middle-age (10 months) and old (24 months) male Fischer 344 rats. Muscarinic receptor density was found to decrease significantly with aging in certain brain regions, depending on the ligand employed. Moreover, the relative proportions of M1 and M2 muscarinic receptor subtypes was not significantly altered by aging, except in the aged striatum. Furthermore, the dissociation kinetics of [3H]NMS in the cerebral cortex and their allosteric modulation by gallamine were only slightly influenced by age.

Pseudo-noncompetitive antagonism of muscarinic receptor-mediated cyclic GMP formation and phosphoinositide hydrolysis by pirenzepine.

Pirenzepine selectively antagonized muscarinic receptor-mediated cyclic GMP formation in a noncompetitive fashion in mouse neuroblastoma cells (clone N1E-115). These effects of pirenzepine were time- and concentration-dependent and they were also reversible. Interestingly, whereas atropine elicited competitive antagonism of the cyclic GMP response at low concentrations, it also behaved like a noncompetitive antagonist at higher concentrations and its effects were partially reversible. Using additional approaches to study the mechanisms underlying this anomalous antagonistic profile of pirenzepine, we investigated whether this deviation from competition could be due to the short time of exposure to muscarinic agonists (30 sec) used in cyclic GMP measurements. Our data indicated that the mode of pirenzepine-induced antagonism of ligand binding to muscarinic receptors was different when assessed using nonequilibrium (30 sec) or equilibrium (1 hr) incubations. Thus, pirenzepine appeared to be noncompetitive and competitive under these two conditions, respectively. Furthermore, although pirenzepine blocked receptor-mediated phosphoinositide hydrolysis competitively when the response was measured at 20 min, it was clearly noncompetitive using 5-min incubations. Therefore, the noncompetitive antagonism by pirenzepine detected in cyclic GMP measurements might be only apparent and might be attributed, at least in part, to a lack of an equilibrium state under the specific conditions of these assays.