Meakins-Christie Laboratories, McGill University, and Cystic Fibrosis Laboratory, Montreal Chest Hospital, Montreal, Quebec, Canada H2X 2P2
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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
(102 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
N
-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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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
N-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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 106 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),
N
-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 -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
-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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Relaxant effect of formamidoxime on rat tracheal
rings. Isolated tracheal rings were precontracted with
106 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.
|
Immunocytochemical characterization of cultured
tracheal smooth muscle cells. Cultured rat tracheal
smooth muscle cells stained positively for smooth muscle-specific
-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
(104 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.
|
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
(105
M) also inhibited formamidoxime-induced tracheal ring
relaxation significantly as shown in Fig. 4
(P < 0.01).
|
|
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
(104 M) and
L-NMMA
(10
4 M), formamidoxime
(10
2 M)-induced cGMP
accumulation in the cells was not affected (Fig. 5).
|
Effect of cytochrome P-450 inhibitors on
formamidoxime-induced cGMP accumulation in cultured tracheal smooth
muscle cells. ER
(107 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).
|
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
(104 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.
|
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).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 -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.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Andronik-Lion, V.,
J. L. Boucher,
M. Delaforge,
Y. Henry,
and
D. Mansuy.
Formation of nitric oxide by cytochrome P-450-catalyzed oxidation of aromatic amidoximes.
Biochem. Biophys. Res. Commun.
185:
452-458,
1992[Medline].
2.
Archer, S.
Measurement of nitric oxide in biological models.
FASEB J.
7:
349-360,
1993
3.
Bai, T. R.,
and
A. M. Bramley.
Effect of an inhibitor of nitric oxide synthase on neural relaxation of human bronchi.
Am. J. Physiol.
264 (Lung Cell. Mol. Physiol. 8):
L425-L430,
1993
4.
Belvisi, M. G.,
C. D. Stretton,
M. Yacoub,
and
P. J. Barnes.
Nitric oxide is the endogenous neurotransmitter of bronchodilator nerves in humans.
Eur. J. Pharmacol.
210:
221-222,
1992[Medline].
5.
Bennett, B. M.,
B. J. McDonald,
R. Nigam,
P. G. Long,
and
W. C. Simon.
Inhibition of nitrovasodilator- and acetylcholine-induced relaxation and cGMP accumulation by the cytochrome P-450 substrate, 7-ethoxyresorufin.
Can. J. Physiol. Pharmacol.
70:
1297-1303,
1992[Medline].
6.
Brooker, G.,
J. F. Harper,
W. L. Terasaki,
and
R. D. Moylan.
Radioimmunoassay of cAMP and cGMP.
Adv. Cyclic Nucleotide Res.
10:
1-33,
1979[Medline].
7.
Chen, R. M.,
and
T. H. Ueng.
Induction of cytochromes P-450 1A, 2B and 2E in hamster tissues by acetone.
Arch. Toxicol.
71:
489-495,
1997[Medline].
8.
Devore-Carter, D.,
P. F. Morway,
and
E. B. Weiss.
Isolation and characterization of guinea-pig tracheal smooth muscle cells that retain differentiated function in long-term subculture.
Cell Tissue Res.
251:
325-331,
1988[Medline].
9.
De Waziers, I.,
P. H. Cugnenc,
C. S. Yang,
J. P. Leroux,
and
P. H. Beaune.
Cytochrome P 450 isoenzymes, epoxide hydrolase and glutathione transferases in rat and human hepatic and extrahepatic tissues.
J. Pharmacol. Exp. Ther.
253:
387-394,
1990[Abstract].
10.
Florio, C.,
M. Flezar,
J. G. Martin,
and
S. Heisler.
Identification of adenylate cyclase-coupled histamine H2 receptors in guinea pig tracheal smooth muscle cells in culture and the effect of dexamethasone.
Am. J. Respir. Cell Mol. Biol.
7:
582-589,
1992[Medline].
11.
Fratacci, M. D.,
C. G. Frostell,
T. Y. Chen,
J. C. Wain, Jr.,
D. R. Robinson,
and
W. M. Zapol.
Inhaled nitric oxide. A selective pulmonary vasodilator of heparin-protamine vasoconstriction in sheep.
Anesthesiology
75:
990-999,
1991[Medline].
12.
Gaston, B.,
J. M. Drazen,
J. Loscalzo,
and
J. S. Stamler.
The biology of nitrogen oxides in the airways.
Am. J. Respir. Crit. Care Med.
149:
538-551,
1994[Abstract].
13.
Hecker, M.,
C. Scott,
B. Bucher,
R. Busse,
and
J.-C. Stoclet.
Increase in serum N-hydroxy-L-arginine in rats treated with bacterial lipopolysaccharide.
Eur. J. Pharmacol.
275:
1-3,
1995[Medline].
14.
Heisler, S.
Stimulation of adrenocorticotropin secretion from AtT-20 cells by the calcium channel activator, BAY-K-8644, and its inhibition by somatostatin and carbachol.
J. Pharmacol. Exp. Ther.
235:
741-748,
1985[Abstract].
15.
Hellmold, H.,
E. Overvik,
M. Stromstedt,
and
J. A. Gustafsson.
Cytochrome P-450 forms in the rodent lung involved in the metabolic activation of food-derived heterocyclic amines.
Carcinogenesis
14:
1751-1757,
1993[Abstract].
16.
Hogman, M.,
C. G. Frostell,
H. Hedenstrom,
and
G. Hedenstierna.
Inhalation of nitric oxide modulates adult human bronchial tone.
Am. Rev. Respir. Dis.
148:
1474-1478,
1993[Medline].
17.
Jia, Y.,
L. Xu,
S. Heisler,
and
J. G. Martin.
Airways of hyperresponsive rats show decreased relaxant response to sodium nitroprusside.
Am. J. Physiol.
269 (Lung Cell. Mol. Physiol. 13):
L85-L91,
1995
18.
Kannan, M. S.,
and
D. E. Johnson.
Nitric oxide mediates the neural nonadrenergic, noncholinergic relaxation of pig tracheal smooth muscle.
Am. J. Physiol.
262 (Lung Cell. Mol. Physiol. 6):
L511-L514,
1992
19.
Kimura, S.,
C. A. Kozak,
and
F. J. Gonzalez.
Identification of a novel P-450 expressed in rat lung: cDNA cloning and sequence, chromosome mapping, and induction by 3-methylcholanthrene.
Biochemistry
28:
3798-3803,
1989[Medline].
20.
Li, C. G.,
and
M. J. Rand.
Evidence that part of the NANC relaxant response of guinea-pig trachea to electrical field stimulation is mediated by nitric oxide.
Br. J. Pharmacol.
102:
91-94,
1991[Abstract].
21.
Luond, R. M.,
J. H. McKie,
and
K. T. Douglas.
A direct link between LY-83583, a selective repressor of cGMP formation, and glutathione metabolism.
Biochem. Pharmacol.
45:
2547-2549,
1993[Medline].
22.
Morrison, K. J.,
Y. Gao,
and
P. M. Vanhoutte.
Epithelial modulation of airway smooth muscle.
Am. J. Physiol.
258 (Lung Cell. Mol. Physiol. 2):
L254-L262,
1990
23.
Nijkamp, F. P.,
H. J. van der Linde,
and
G. Folkerts.
Nitric oxide synthesis inhibitors induce airway hyperresponsiveness in the guinea pig in vivo and in vitro. Role of the epithelium.
Am. Rev. Respir. Dis.
148:
727-734,
1993[Medline].
24.
Ortiz de Montellano, P. R.
Cytochrome P-450: Structure, Mechanism, and Biochemistry. New York: Plenum, 1986, p. 273-314.
25.
Oyekan, A. O.,
J. C. McGiff,
and
J. Quilley.
Cytochrome P-450-dependent vasodilator responses to arachidonic acid in the isolated, perfused kidney of the rat.
Circ. Res.
68:
958-965,
1991[Abstract].
26.
Philpot, R. M.,
B. A. Domin,
T. R. Devereux,
C. Harris,
M. W. Anderson,
J. R. Fouts,
and
J. R. Bend.
Cytochrome P-450-dependent monooxygenase systems of lungs: relationships to pulmonary toxicity.
In: Microsomes and Drug Oxidations, edited by A. R. Boobis,
J. Caldwell,
F. de Matteis,
and C. R. Elcombe. London: Taylor and Francis, 1985, p. 248-255.
27.
Renaud, J. P.,
J. L. Boucher,
S. Vadon,
M. Delaforge,
and
D. Mansuy.
Particular ability of liver P-450s3A to catalyze the oxidation of NX omega-hydroxyarginine to citrulline and nitrogen oxides and occurrence in NO synthases of a sequence very similar to the heme-binding sequence in P-450s.
Biochem. Biophys. Res. Commun.
192:
53-60,
1993[Medline].
28.
Schmidt, M. J.,
B. D. Sawyer,
L. L. Truex,
W. S. Marshall,
and
J. H. Fleisch.
LY-83583: an agent that lowers intracellular levels of cyclic guanosine 3',5'-monophosphate.
J. Pharmacol. Exp. Ther.
232:
764-769,
1985[Abstract].
29.
Stuehr, D. J.,
N. S. Kwon,
C. F. Nathan,
O. W. Griffith,
P. L. Feldman,
and
J. Wiseman.
N omega-hydroxy-L-arginine is an intermediate in the biosynthesis of nitric oxide from L-arginine.
J. Biol. Chem.
266:
6259-6263,
1991
30.
Verschoyle, R. D.,
D. Dinsdale,
and
C. R. Wolf.
Inhibition and induction of cytochrome P-450 isoenzymes in rat lung.
J. Pharmacol. Exp. Ther.
265:
386-391,
1993[Abstract].