Nitric oxide synthesis by tracheal smooth muscle cells by a nitric oxide synthase-independent pathway

Yanlin Jia, Mary Zacour, Barbara Tolloczko, and James G. Martin

Meakins-Christie Laboratories, McGill University, and Cystic Fibrosis Laboratory, Montreal Chest Hospital, Montreal, Quebec, Canada H2X 2P2

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

Nitric oxide (NO) is known to be synthesized from L-arginine in a reaction catalyzed by NO synthase. Liver cytochrome P-450 enzymes also catalyze the oxidative cleavage of C==N bonds of compounds containing a -C(NH2)==NOH function, producing NO in vitro. The present study was designed to investigate whether there was evidence of a similar pathway for the production of NO in tracheal smooth muscle cells. Formamidoxime (10-2 to 10-4 M), a compound containing -C(NH2)==NOH, relaxed carbachol-contracted tracheal rings and increased intracellular cGMP in cultured tracheal smooth muscle cells, whereas L-arginine had no such effect. NO was detectable in the medium containing cultured tracheal smooth muscle cells when incubated with formamidoxime. Ethoxyresorufin (10-7 to 10-4 M), an alternate cytochrome P-450 substrate, inhibited formamidoxime-induced cGMP accumulation as well as tracheal ring relaxation in cultured tracheal smooth muscle cells. The NO synthase inhibitors Nomega -nitro-L-arginine (10-3 M) and NG-monomethyl-L-arginine (10-3 M) had no effect on formamidoxime-induced cGMP accumulation. These results suggest that NO can be synthesized from formamidoxime in tracheal smooth muscle cells, presumably by a reaction catalyzed by cytochrome P-450.

guanosine 3',5'-cyclic monophosphate; formamidoxime; airway relaxation; N-hydroxy-L-arginine

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

IT HAS BEEN WELL ESTABLISHED that endogenous nitric oxide (NO) is synthesized from L-arginine catalyzed by NO synthase (NOS). NOS catalyzes the formation of NO from L-arginine in two steps. First, it catalyzes the N-oxygenation of L-arginine to form Nomega -hydroxy-L-arginine (NOHA), which contains a -C(NH2)==NOH function. Second, the oxidative cleavage of the C==N bonds of NOHA produces NO and L-citrulline (29). Liver cytochrome P-450s have also been found to catalyze the oxidative cleavage of C==N bonds of compounds containing a -C(NH2)==NOH function, producing the corresponding derivatives containing a -C(NH2)==O function and NO in vitro (1).

NO appears to play an important role in regulating several biological functions in the lung, including modulation of airway smooth muscle tone (4, 11, 16, 22). NO relaxes airway smooth muscle by activating soluble guanylate cyclase, leading to the accumulation of intracellular cGMP. In the lung, NO can be synthesized in a number of cell types including macrophages, neutrophils, mast cells, nonadrenergic noncholinergic inhibitory neurons, fibroblasts, vascular smooth muscle cells, pulmonary arterial and venous endothelial cells, and airway epithelial cells (reviewed in Ref. 12). However, no constitutive NOS activity has been found in airway smooth muscle cells. Because cytochrome P-450 isoenzymes have been identified in the rat lung (30), the present study was designed to test whether evidence for the presence of a cytochrome P-450-catalyzed pathway for NO production could be obtained for airway smooth muscle cells. We chose formamidoxime [HC(NH2)==NOH], a compound containing -C(NH2)==NOH, as the substrate for this pathway. Therefore, the effects of formamidoxime on airway relaxation and intracellular cGMP accumulation in cultured tracheal smooth muscle cells were investigated. The role of cytochrome P-450 enzymes in the production of cGMP was explored indirectly with ethoxyresorufin (ER) and miconazole, alternate substrates for cytochrome P-450, to inhibit the action of cytochrome P-450 on formamidoxime (24, 25). In addition, we explored the possibility that cytochrome P-4503A1 might be present in tracheal smooth muscle cells in culture because of its action on -C(NH2)==NOH to produce NO in liver cells (27).

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Animals. Lewis rats (male, 7-9 wk old) were purchased from Harlan Sprague Dawley (Indianapolis, IN) and housed in a conventional animal care facility at McGill University (Montreal, PQ) before experimentation. The protocol was approved by an Animal Ethics Committee.

Mechanical responses of tracheal rings. Rats were killed by an overdose of pentobarbital sodium, and their tracheae were immediately excised and incubated in a physiological saline solution [containing (in mM) 118 NaCl, 4.5 KCl, 2.5 CaCl2, 1.2 MgSO4, 1.2 KH2PO4, 25.5 NaHCO3, and 5.6 glucose] bubbled with 95% O2-5% CO2. The tracheae were dissected from surrounding tissues and cut into ~3-mm rings. Only those rings from the lower end of the trachea were used to measure the mechanical responses. The tracheal rings were mounted on hooks, connected to force transducers (Grass FT03, Grass Instruments, Quincy, MA), and incubated in physiological saline solution bubbled with 95% O2-5% CO2 in 25-ml organ baths at 37°C. The passive tension was set at 1 g, and the tissue was equilibrated for 60 min. The isometric force of the tracheal rings in response to carbachol (Sigma, St. Louis, MO) was recorded. The magnitude of relaxation induced by formamidoxime [HC(NH2)==NOH; Aldrich, Milwaukee, WI] was measured on rings that were preconstricted with 10-6 M carbachol and calculated as the percent decrease in the isometric force developed with carbachol. The effects of LY-83583 (Calbiochem, San Diego, CA), ER (Molecular Probes Eugene, OR), Nomega -nitro-L-arginine (L-NNA; Sigma), and NG-monomethyl-L-arginine (L-NMMA; Calbiochem) on formamidoxime-induced relaxation of tracheal rings were also recorded.

Tracheal smooth muscle cell cultures. Rat tracheal smooth muscle cells were cultured as previously described (8, 10). Briefly, the tracheae were dissected rapidly and rinsed with ice-cold Hanks' balanced salt solution (HBSS). All extraneous tissues were carefully stripped from the tracheae. The anterior aspect of the trachea was cut longitudinally through the cartilage and incubated in HBSS containing 0.05% elastase (type IV; Sigma) and 0.2% collagenase (type IV; Sigma) for 30 min at 37°C with gentle shaking. The solution was centrifuged at room temperature at 1,200 rpm for 6 min. The pellet was resuspended in a 1:1 mixture of Dulbecco's modified Eagle's medium (DMEM; GIBCO, Grand Island, NY) and Ham's F-12 nutrient mixture (ICN Biomedicals, Costa Mesa, CA) containing 10% fetal bovine serum and 1% penicillin-streptomycin (GIBCO) and cultured in 25-cm2 cell culture flasks at 37°C in humidified air containing 5% CO2. When confluent, cells were detached from the flasks by incubation with 0.25% trypsin in HBSS containing 0.02% EDTA and subcultured in 24- or 6-well plates. Only confluent cells from the first passage were used for experiments.

Immunohistochemical staining for smooth muscle-specific alpha -actin and cytokeratin was done with immunofluorescence and alkaline phosphatase-anti-alkaline phosphatase methods, respectively, to confirm that the cells obtained were smooth muscle cells. Rat epithelial cell cultures were used as a positive control for cytokeratin staining, and human lung fibroblasts were used as a negative control for smooth muscle-specific alpha -actin.

Cyclic nucleotide measurements. Cultured tracheal smooth muscle cells were incubated in 24-well plates with 1 ml of HEPES-buffered culture medium containing 2% fetal bovine serum for 30 min at 37°C as an initial period of equilibration, followed by a 15-min incubation with the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX; 0.5 mM; Aldrich). Test agents or vehicle was added in the presence of 0.5 mM IBMX and incubated for 10 min at 37°C. The reactions were stopped by replacing the medium with 1 ml of ice-cold 0.5 N hydrochloric acid. The cells were then sonicated, and cAMP and cGMP (after acetylation of cGMP) (6) were measured by radioimmunoassay (RIA) (14). The experiments were repeated at least three times in quadruplicate. The cytochrome P-450 inhibitor miconazole was obtained from Janssen Biotech (Cedarlane Laboratories, Hornby, ON).

NO measurement. NO levels were measured with an NO chemiluminescence analyzer (Sievers Research, Boulder, CO). Cultured tracheal smooth muscle cells in six-well plates were incubated with formamidoxime for 10 min at 37°C in 1 ml of HBSS. The incubation buffer was subsequently transferred to test tubes for the measurement of NO as follows. Samples (100 ml) were injected into a modified purging chamber containing 5 ml of sodium iodide (1% in glacial acetic acid), which was continuously being purged by a stream of argon (30-40 ml/min). Any NO that may have been transformed to nitrite by interaction with O2 was reconverted to NO by sodium azide (2). The argon stream was drawn into the analyzer and mixed with internally generated ozone (by electrostatic discharge). The light emission was detected at an integration time of 0.25 s by a cooled Hamamatsu red-sensitive photomultiplier tube after the light passed through a red filter interposed to eliminate chemiluminescence due to volatile sulfides (2). The detection limit of this technique is 1 pmol. The background signal produced by the control HBSS was subtracted from the signal obtained from the formamidoxime-treated samples. The standard was constructed with potassium nitrite at the same integration time.

Western blotting for P-4503A1. We chose to examine the smooth muscle cultures for evidence of P-4503A1 by Western blotting because this subfamily has been found to liberate NO from NOHA (27). To do this, first-passage tracheal smooth muscle cells from three separate male Lewis animals were grown to confluence in 150 × 25-mm tissue culture dishes (Becton Dickinson). The cells were rinsed twice with ice-cold PBS, then lysed by rocking the plates at 4°C for 40 min with 0.75 ml of lysis buffer of the following composition: 1% Nonidet P-40 detergent, 20 mM Tris (pH 8.0), 137 mM NaCl, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, 0.15 U aprotinin/ml, and 1 mM sodium orthovanadate. The plates were scraped with a cell scraper, and the lysates were centrifuged at 4°C for 10 min at 14,000 rpm (Eppendorf centrifuge 5402). The supernatants were concentrated in Centricon tubes (Amicon), divided into aliquots, and stored at -40°C. SDS-PAGE was performed with a Bio-Rad Protean Mini II apparatus. One hundred milligrams of sample protein were loaded onto a 10% SDS-polyacrylamide minigel. Male rat liver microsomes (Oxford Biomedical Research) served as a positive control. The separated proteins were electroblotted onto nitrocellulose filters for 18 h at 30-V constant voltage, and the gel was stained with Coomassie blue to verify efficiency of the transfer. The filters were blocked for 5 h at room temperature with 3% milk powder in Tris-buffered saline with 0.05% Tween 20, then incubated overnight at 4°C with monoclonal mouse IgG anti-rat cytochrome P-4503A1 (Oxford Biomedical Research). The secondary antibody was biotinylated goat anti-mouse IgG adsorbed with rat serum proteins (Sigma Immunochemicals). The blots were incubated with streptavidin-horseradish peroxidase, then developed on Amersham hyperfilm with Amersham electrochemiluminescence reagents. Biotinylated molecular-mass markers were run, as well as nonbiotinylated markers as a control for nonspecificity.

Statistics. Data are expressed as means ± SE. Differences among several means were tested by ANOVA followed by the Newman-Keuls test for multiple comparisons. Comparisons between several means and a common control were tested by Dunnett's t-test. Comparisons of two means were tested by Student's t-test. P < 0.05 was set as the level of significance.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

Relaxant effect of formamidoxime on rat tracheal rings. Isolated tracheal rings were precontracted with 10-6 M carbachol. This concentration of carbachol evoked a force of contraction of 2.39 ± 0.19 g. The addition of formamidoxime to the organ bath induced a progressive relaxation of the precontracted rings (Fig. 1). A relaxation of 32% was induced by 10-2 M formamidoxime.


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Fig. 1.   Formamidoxime-induced relaxation of carbachol-contracted rat tracheal rings. Rat tracheal rings were precontracted with 10-6 M carbachol (n = 6). Relaxation was calculated as decrease in isometric force and is expressed as percent maximal isometric force induced by carbachol. Data are means ± SE. * P < 0.05 and ** P < 0.01 compared with control.

Immunocytochemical characterization of cultured tracheal smooth muscle cells. Cultured rat tracheal smooth muscle cells stained positively for smooth muscle-specific alpha -actin. Between 85 and 90% of the cells showed varying degrees of staining. There was no evidence that any of these cells were of epithelial origin because no cytokeratin staining was detected.

Effect of formamidoxime on intracellular cyclic nucleotide production. cGMP levels ranged from 50 to 100 fmol/well in various experiments. There was a lower coefficient of variation for replicate measurements during a single experiment (in 4 out of 5 experiments, it was within 10%) than the coefficient of variation for the control values among different experiments (20%) so that the data were normalized for baseline values for comparison purposes.

Figure 2 shows the levels of intracellular cyclic nucleotides in cultured tracheal smooth muscle cells exposed to formamidoxime. The baseline cGMP level was 90.3 ± 7.14 fmol/well, whereas the cAMP level was 7.15 ± 0.91 pmol/well. With increasing concentrations of formamidoxime, there was a progressive accumulation of cGMP in tracheal smooth muscle cells. In contrast, L-arginine (10-4 to 10-2 M) had no effect on intracellular cGMP levels under the same experimental conditions. Intracellular cAMP levels in tracheal smooth muscle cells were unaffected by formamidoxime.


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Fig. 2.   Formamidoxime-induced cGMP accumulation in cultured tracheal smooth muscle cells. Tracheal smooth muscle cells were incubated with formamidoxime or L-arginine for 10 min at 37°C. cAMP and cGMP levels in cells were measured. open circle , Formamidoxime; bullet , L-arginine; solid lines, cGMP; dashed line, cAMP. C, control (vehicle) for formamidoxime or L-arginine. Data are means ± SE from 3 experiments in quadruplicate. * P < 0.05 compared with control.

Formamidoxime, cGMP and relaxant responses. To further investigate whether the formamidoximeinduced relaxation was mediated through a cGMP-dependent mechanism, the effects of LY-83583, a selective suppressor of cGMP formation (21, 28), on the formamidoxime-induced intracellular cGMP accumulation in cultured tracheal smooth muscle cells and on the relaxation of tracheal rings were tested. Increasing concentrations of LY-83583 progressively inhibited formamidoxime-induced cGMP accumulation in cultured tracheal smooth muscle cells (Fig. 3). LY-83583 (10-5 M) also inhibited formamidoxime-induced tracheal ring relaxation significantly as shown in Fig. 4 (P < 0.01).


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Fig. 3.   LY-83583 inhibited formamidoxime-induced cGMP accumulation in tracheal smooth muscle cells. Cultured tracheal smooth muscle cells were preincubated with increasing concentrations of LY-83583 for 15 min, followed by a 10-min incubation with formamidoxime (10-2 M). C, control (vehicle) for LY-83583 in presence of formamidoxime. Data are means ± SE from 3 experiments in quadruplicate. * P < 0.05 compared with control.


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Fig. 4.   Effects of ethoxyresorufin (ER) and LY-83583 on formamidoxime-induced relaxation. Isolated tracheal rings (n = 6) were preincubated with vehicle (open bars) and ER or LY-83583 (hatched bars) for 15 min. Relaxant effects of formamidoxime (10-2 M) on carbachol (10-6 M)-contracted trachea were recorded. Data are means ± SE. * P < 0.05 compared with vehicle.

Effect of NOS inhibitors on formamidoxime-induced cGMP accumulation in cultured tracheal smooth muscle cells. When cultured tracheal smooth muscle cells were preincubated with the NOS inhibitors L-NNA (10-4 M) and L-NMMA (10-4 M), formamidoxime (10-2 M)-induced cGMP accumulation in the cells was not affected (Fig. 5).


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Fig. 5.   Effect of nitric oxide synthase inhibitors on formamidoxime-induced cGMP accumulation in tracheal smooth muscle cells. Experiments were performed in the same way as in Fig. 3. 1, Control; 2, formamidoxime (10-2 M); 3, formamidoxime+Nomega -nitro-L-arginine (10-4 M); 4, formamidoxime+NG-monomethyl-L-arginine (10-4 M). Data are means ± SE from 3 experiments in quadruplicate. * P < 0.05 compared with control. P < 0.05 for groups 3 and 4 compared with group 2 (tested by ANOVA and Newman-Keuls test).

Effect of cytochrome P-450 inhibitors on formamidoxime-induced cGMP accumulation in cultured tracheal smooth muscle cells. ER (10-7 to 10-4 M), a cytochrome P-450 substrate, inhibited formamidoxime (10-2 M)-induced cGMP accumulation in cultured tracheal smooth muscle cells in a concentration-dependent manner (Fig. 6). ER (10-5 M) also inhibited formamidoxime (10-2 M)-induced relaxation in carbachol-contracted tracheal rings (Fig. 4). Miconazole, another cytochrome P-450 substrate, had a similar inhibitory effect on formamidoxime-induced cGMP accumulation in tracheal smooth muscle cells in culture (Fig. 6).


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Fig. 6.   Cytochrome P-450 inhibitors [ER (A) and miconazole (B)] inhibited formamidoxime-induced cGMP accumulation in cultured tracheal smooth muscle cells. Experiments were performed as described in Fig. 3. C, control (vehicle) for ER or miconazole in presence of formamidoxime (10-2 M). Data are means ± SE from 3 experiments in quadruplicate. * P < 0.05 compared with control.

NO production from formamidoxime. A concentration-dependent production of NO was detected in the culture medium when tracheal smooth muscle cells were incubated with increasing concentrations of formamidoxime (10-4 to 10-2 M; Fig. 7). The maximal NO produced at 3 mM formamidoxime was ~110 pmol/well. The same concentration of formamidoxime in HBSS without cells did not produce NO. At higher concentrations, NO was detectable in the medium in the absence of cells.


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Fig. 7.   Formamidoxime-induced NO production in cultured tracheal smooth muscle cells. Cultured tracheal smooth muscle cells were incubated with formamidoxime in Hanks' balanced salt solution for 10 min at 37°C. NO in Hanks' balanced salt solution was measured by NO chemiluminescence analyzer. Data are means ± SE from 3 experiments in quadruplicate. * P < 0.05 and ** P < 0.01 compared with control.

Western blotting. Airway smooth muscle cell extracts from three separate male Lewis rats showed clear bands of immunoreactivity at ~70 and 115 kDa, with a weaker band at ~80 kDa; however, there was no immunoreactivity at the expected molecular mass for cytochrome P-4503A1 (50-52 kDa). This was in contrast to the positive control, rat liver microsomes, which showed a marked immunoreactivity at a 50-kDa band (Fig. 8).


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Fig. 8.   Western blots of smooth muscle extract (100 µg of protein; lane 1), positive control microsomal preparation (5 µg of protein; lane 2), and molecular-mass standards (lane 3). Note absence of any immunoreactive band corresponding to 52-kDa cytochrome P-4503A1 standard.

    DISCUSSION
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Abstract
Introduction
Methods
Results
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References

We have shown that formamidoxime was able to induce tracheal ring relaxation and accumulation of cGMP in cultured tracheal smooth muscle cells, whereas L-arginine had no effect on intracellular cGMP levels in similar cells. Formamidoxime also produced measurable levels of NO in the culture medium of tracheal smooth muscle cells. The effects of formamidoxime were inhibited by alternate cytochrome P-450 substrates, which presumably acted by competitive inhibition, but not by NOS inhibitors, strongly suggesting that a cytochrome P-450-catalyzed pathway for the production of NO was present in tracheal smooth muscle cells. Any possible contribution by epithelial-derived cytochrome P-450 enzymes was excluded by demonstrating positive immunocytochemical staining for smooth muscle-specific alpha -actin and the absence of staining for cytokeratin.

NO is known to relax airway smooth muscle by activating guanylate cyclase and increasing intracellular cGMP. Formamidoxime is a convenient commercially available compound containing a -C(NH2)==NOH function in which oxidative cleavage of the C==N bond is able to produce NO. In this study, we found that formamidoxime induced relaxation of carbachol-contracted tracheal rings. To investigate whether this relaxation was potentially caused by the production of NO, we measured the intracellular cGMP and cAMP levels in cultured tracheal smooth muscle cells after exposure to formamidoxime. Formamidoxime stimulated cGMP but not cAMP accumulation in cultured tracheal smooth muscle cells, which is consistent with the production of NO. To further investigate the link between the formamidoxime-induced relaxation and cGMP, we also evaluated the effect of LY-83583, an agent that decreases intracellular cGMP (21, 28), on both formamidoxime-induced tracheal relaxation and cGMP accumulation in cultured tracheal smooth muscle cells. LY-83583 inhibited both formamidoxime-induced cGMP accumulation in the tracheal smooth muscle cells in culture and the relaxation of isolated tracheal rings. These findings confirm that formamidoxime-induced relaxation is induced through a cGMP-dependent mechanism, presumably stimulated by NO. Furthermore, we measured NO production by a chemiluminescence assay that confirmed the production of NO by cultured tracheal smooth muscle cells from formamidoxime. This NO production was a cell-dependent process because 3 mM formamidoxime in the absence of cells did not produce NO. This concentration of formamidoxime was sufficient to cause the maximal NO production in cultured tracheal smooth muscle cells. These results provide direct evidence that NO is produced from formamidoxime and that it is likely responsible for the observed relaxation of tracheal smooth muscle. The relaxant effect of NO on tracheal smooth muscle has been confirmed in rats in vivo and in tracheal rings in vitro with the NO donor sodium nitroprusside (17).

NOS has been found in many cell types in the lung. However, so far, no direct evidence of the constitutive form of NOS has been found in airway smooth muscle cells. However, NO is produced in the adjacent cells such as epithelium (23) and/or neurons (3, 18, 20, 21) and may diffuse to airway smooth muscle cells to regulate smooth muscle tone. Interestingly, in the present study, we found that formamidoxime stimulated NO production and cGMP accumulation in cultured tracheal smooth muscle cells, implying that the site of production of NO from formamidoxime was within the airway smooth muscle cells. However, the lack of effect of NOS inhibitors on cGMP levels after formamidoxime treatment indicates that NO production from formamidoxime is catalyzed by another enzymatic pathway.

Both NOS and cytochrome P-450 (1) are able to catalyze the oxidative cleavage of C==N bonds of -C(NO2)==NOH and produce NO. The endogenous compound containing -C(NH2)==NOH, namely NOHA, is produced by N-oxidation of L-arginine catalyzed by NOS. Normally, NOS is the only enzyme for which L-arginine is the substrate that is capable of producing NO in vivo. However, in the absence of NOS, formamidoxime is a compound that may be catalyzed by cytochrome P-450 to produce NO. To explore the possibility that cytochrome P-450 but not NOS was involved in catalyzing the NO production from formamidoxime, we evaluated the effect of inhibitors of NOS and cytochrome P-450 on formamidoxime-induced tracheal ring relaxation and cGMP accumulation in cultured smooth muscle cells. The cytochrome P-450 substrate ER inhibited formamidoxime-induced cGMP accumulation as well as tracheal ring relaxation in airway smooth muscle cells, whereas the NOS inhibitors L-NNA and L-NMMA had no effect on formamidoxime-induced cGMP accumulation in cultured tracheal smooth muscle cells. This observation provides strong evidence that NO production from formamidoxime was catalyzed by cytochrome P-450. Although some cytochrome P-450 inhibitors such as ER may also inhibit NOS activity (5), an inhibitory effect on NOS seems unlikely in the present study in view of the lack of other evidence of constitutive NOS activity and the lack of the effect of L-arginine on cGMP levels in tracheal smooth muscle. We believe that these results provide evidence that NO production from formamidoxime is catalyzed by cytochrome P-450 and that the findings are consistent with the previous observations in vitro by Andronik-Lion et al. (1) that liver microsomal cytochrome P-450 is able to catalyze the cleavage of the C==N bonds of compounds containing a -C(NH2)==NOH function and produce NO.

Although some isoenzymes of cytochrome P-450 have been identified in the lung (7, 9, 15, 19, 26, 30,), the one that may be responsible for NO production in airway smooth muscle cells from formamidoxime is not known. It is believed that the cytochrome P-4503A subfamily is involved in catalyzing the production of NO from NOHA in vivo (27). However, cytochrome P-4503A does not appear to be responsible for NO production from formamidoxime. To our knowledge, the cytochrome P-4503A subfamily has not been identified in the lung, and in our experiments, Western blotting also failed to reveal any evidence of cytochrome P-4503A1 in rat cultured smooth muscle cells. On the other hand, NO production from formamidoxime could be inhibited by ER, a high-affinity substrate for cytochrome P-4501A1. Because cytochrome P-4501A1 can be induced in the lung (7), the NO produced from formamidoxime might be catalyzed by the activity of this subfamily of cytochrome P-450, namely cytochrome P-4501A1.

In summary, tracheal smooth muscle cells from Lewis rats may produce NO from formamidoxime, a compound containing a -C(NH2)==NOH function, by a pathway independent of NOS. Although direct confirmation of a cytochrome P-450 pathway for NO production has not been provided, the evidence supports such a pathway. The significance of the current findings for airway smooth muscle function in vivo is uncertain, but it is possible that other substrates such as NOHA may serve to stimulate cytochrome P-450-induced NO synthesis in certain circumstances. Perhaps transcellular metabolism of NOHA or increases in its circulating levels such as those reported after an injection of endotoxin in rats (13) may lead to NO production by airway smooth muscle by cytochrome P-450 enzymes.

    ACKNOWLEDGEMENTS

We thank Liz Milne for assistance in the preparation of the manuscript.

    FOOTNOTES

The study was supported by Medical Research Council (MRC) of Canada Grant 7852 and a grant from the Respiratory Health Network of Centres of Excellence.

Y. L. Jia was the recipient of a Fellowship Award from the MRC.

Address for reprint requests: J. G. Martin, Meakins-Christie Laboratories, McGill Univ., 3626 St. Urbain St., Montreal, Quebec, Canada H2X 2P2.

Received 23 June 1995; accepted in final form 5 August 1998.

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Abstract
Introduction
Methods
Results
Discussion
References

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Am J Physiol Lung Cell Mol Physiol 275(5):L895-L901
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