Departments of Pediatrics and Medicine, Duke University Medical Center, Durham, North Carolina 27710
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ABSTRACT |
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Nitric oxide (NO) functions as an
endothelium-derived relaxing factor by activating guanylate cyclase to
increase cGMP levels. However, NO and related species may also regulate
vascular tone by cGMP-independent mechanisms. We hypothesized that
naturally occurring NO donors could decrease the pulmonary vascular
response to serotonin (5-HT) in the intact lung through chemical
interactions with 5-HT2 receptors. In isolated rabbit lung
preparations and isolated pulmonary artery (PA) rings, 50-250 µM
S-nitrosoglutathione (GSNO) inhibited the response to
0.01-10 µM 5-HT. The vasoconstrictor response to 5-HT was
mediated by 5-HT2 receptors in the lung, since it could be
blocked completely by the selective inhibitor ketanserin (10 µM).
GSNO inhibited the response to 5-HT by 77% in intact lung and 82% in
PA rings. In PA rings, inhibition by GSNO could be reversed by
treatment with the thiol reductant dithiothreitol (10 mM).
3-Morpholinosydnonimine (100-500 µM), which releases NO and
O
nitric oxide; G protein-coupled receptor
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INTRODUCTION |
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NITRIC OXIDE (NO) is an endothelium-derived relaxing factor that counteracts the effects of vasoconstrictors under physiological conditions to maintain low pulmonary vascular tone. NO produces vasodilation by activating guanylate cyclase to increase cGMP levels (30). There is substantial evidence that NO also relaxes vascular smooth muscle by cGMP-independent pathways. cGMP-independent effects of NO on vascular smooth muscle can be mediated by stimulating Na+-K+-ATPase activity, modifying K+ channels, or decreasing sensitivity to or release of vasoconstrictors (5, 8, 10, 14, 18, 19, 21, 36).
The vasoconstrictor effect of serotonin [5-hydroxytryptamine (5-HT)] in the lung can be inhibited with NO. In the pulmonary vasculature, 5-HT binds to G protein-coupled receptors (GPCR), including 5-HT1B and 5-HT2A receptors, to produce vasoconstriction (23, 27, 28, 32, 45). NO inhibits the response to 5-HT in several vascular tissues, including human umbilical arteries (35, 42), bovine coronary arteries (11), and rat pulmonary artery (44). Although the vascular response to 5-HT is altered by NO, the mechanisms by which NO modifies the responses to 5-HT and their importance in the lung are not known.
The effects of NO on cellular function are mediated by its reactions
with specific molecular targets. NO can exist in several oxidation-reduction states (e.g., NO, NO·,
NO+), which promote its reactions with protein thiols,
transition metals such as iron in heme proteins, O2, and
reactive oxygen species. The biochemical reactions of NO have been
shown to activate and inactivate protein function and alter receptor
activity (20, 37, 38).
GPCR are known targets of NO via cGMP-independent pathways. Studies of
2-adrenergic receptors in cultured cells have shown that
NO donors reduce signaling by
2-adrenergic agonists and promote depalmitoylation of the receptor (1, 40, 43). In the M2 muscarinic GPCR and angiotensin (AT1)
GPCR, treatment with NO inhibits receptor-ligand binding (2,
8). The role of NO in modifying the response to 5-HT receptor
function has not been investigated.
We hypothesized that naturally occurring NO donors could decrease the pulmonary vascular response to 5-HT in the intact lung through chemical interactions involving the receptor pathway. In this study, we characterized the response to 5-HT in intact rabbit lungs and isolated pulmonary artery rings and determined that vasoconstriction in our model was mediated by the 5-HT2 receptor. We studied the effects of the NO donor S-nitrosoglutathione (GSNO) on the 5-HT response and questioned the mechanism of 5-HT inhibition. Our findings are consistent with chemical modification by NO of the 5-HT2 GPCR system to inhibit vasoconstriction by a reversible thiol-based mechanism.
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MATERIALS AND METHODS |
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Reagents and Pharmaceuticals
All chemicals were obtained from Sigma Chemical (St. Louis, MO) unless otherwise specified.Isolated Lung Preparation
The experiments were performed in isolated buffer-perfused lungs (IPL) of rabbits, as previously described (34). The buffer was Krebs-Henseleit (KH) solution containing final concentrations of 82.8 mM sodium chloride, 4.7 mM potassium chloride, 2.4 mM monobasic potassium phosphate, 25 mM sodium bicarbonate, 1.2 mM magnesium sulfate, 2.7 mM calcium chloride, and 11.1 mM dextrose, pH 7.4. New Zealand White rabbits (May's Farm, NC) weighing 2.5-3.5 kg were anticoagulated with sodium heparin (5,000 U) and anesthetized with pentobarbital sodium (25 mg/kg) by ear vein. An incision was made in the left chest wall, exposing the heart. The animal was bled via the left ventricle, and the thorax was entered by excising the rib cage. Stainless steel cannulas were placed in the trachea, main pulmonary artery, and left atrium. The aorta also was tied with the pulmonary artery to prevent loss of perfusate to systemic circulation. The lungs were inflated with 80 ml of air and ventilated with 21% O2-5% CO2-balance N2 with an animal respirator (Harvard Apparatus, S. Natick, MA) at a rate of 30 breaths/min. The tidal volume was adjusted to maintain a peak tracheal pressure of 8-10 Torr with a positive end-expiratory pressure of 2-3 Torr.The perfusion circuit contained a reservoir suspended freely from a force transducer (model FT100, Grass Instrument, Quincy, MA) and a water heater set at 37°C. Perfusate was circulated by a roller pump (Sarns, Ann Arbor, MI) and passed through a bubble trap before entering the pulmonary artery. The perfusate returned to the left atrium and then to the reservoir, which was set at the lowest portion of the lung to provide a left atrial pressure of zero. Perfusion began slowly and was gradually increased to 100 ml/min. After the lungs were rinsed free of blood with 500 ml buffer, a recirculating system was established. The total volume of KH buffer in the circuit was ~250 ml. Mean pulmonary arterial pressure (Ppa) and tracheal pressure were measured using pressure transducers (model P231D, Gould Statham Instruments, Hato Rey, PR). The weight gain of the lung as an index of pulmonary edema formation was measured as the loss of perfusate from the reservoir connected to the force transducer. Ppa, tracheal pressure, and weight gain were continuously recorded on a four-channel recorder (model 2450S, Gould, Cleveland, OH). The preparation was considered successful if the Ppa was stable between 10 and 20 Torr and there was <0.15 g/min weight gain during a 10-min stabilization period.
Pulmonary Artery Ring Preparation
Pulmonary artery rings (3 mm) were harvested from New Zealand White rabbits and mounted in 25-ml tissue baths filled with KH buffer and bubbled with 21% O2-5% CO2-balance N2. Isometric tension was measured. All rings in the study were suspended with similar baseline levels of tension (~2 g). Tissue baths were thoroughly rinsed with fresh buffer between interventions.Synthesis of GSNO
GSNO was synthesized as previously described (7). Briefly, GSNO was prepared by reacting reduced glutathione (GSH) dissolved in 0.5 N HCl with equimolar sodium nitrite dissolved in water. GSNO used in IPL experiments was precipitated with acetone, filtered, and washed sequentially with water, acetone, and ether. Solutions of GSNO in KH buffer were prepared daily from precipitated samples, and concentration was confirmed by absorption spectroscopy at 335 nm using an extinction coefficient of 0.92 optical density units · mMMeasurements of NOx Level
Perfusate was assayed for NOx (nitrite and nitrate) using a catalytic method for reduction of NO oxidation products to NO gas. Perfusate samples were injected into a refluxing glass reaction chamber containing 0.1 M vanadium(III) chloride and carried in nitrogen gas to a chemiluminescence detector. Measurements of known concentrations of nitrite and nitrate were quantitatively linear between 25 and 500 pmol (22).Experimental Protocols
Dose-response curve for 5-HT. A dose-response curve for 5-HT was performed to determine an optimal dose of 5-HT for later experiments. Perfused lungs were treated with three consecutive doses of 5-HT to provide final perfusate concentrations of 0.1, 1.0, and 10.0 µM. Lungs were monitored until the Ppa returned to baseline (typically <20 min) before administration of the next dose. A similar dose-response curve for 5-HT (0.01-10 µM) was established in the pulmonary artery ring bioassay.
5-HT in isolated perfused lungs. Experiments in isolated lungs were preceded by a 60-min perfusion period before administration of 1.0 µM 5-HT. After 5-HT, lungs were monitored for another 30 min and Ppa and weight gain were recorded continuously. One group of lungs was pretreated with the 5-HT2 receptor antagonist ketanserin to confirm that the 5-HT2 receptor was responsible for the vasoconstrictor response to 5-HT. These lungs were pretreated with 10 µM ketanserin infused via the pulmonary artery 5 min before 5-HT administration. This dose of ketanserin was chosen because it selectively inhibits the 5-HT2 receptor (27, 28).
In the next series of experiments, lungs were perfused for 60 min with or without an NO donor before administration of 5-HT. After 5-HT, Ppa and weight gain were monitored for an additional 30 min. NO-treated lungs received 50 µM GSNO or 100 µM 3-morpholinosydnonimine (SIN-1; Alexis, San Diego, CA), which simultaneously releases NO and superoxide (OEndothelin in IPL. To evaluate whether the effects of GSNO on 5-HT responses were specific for this vasoconstrictor, lungs were treated with endothelin-1 (ET-1) alone or ET-1 after pretreatment with GSNO or SIN-1. ET-1 was selected because it produces vasoconstriction by the intracellular signaling pathway used by 5-HT, in which the GPCR in smooth muscle is linked to phospholipase C (PLC), inositol trisphosphate (IP3), and diacylglycerol (DAG) (23). Lungs were pretreated with 50 µM GSNO or 100 µM SIN-1 for 60 min before administration of 10 nM ET-1. Ppa and weight gain were monitored for 30 min after ET-1.
5-HT and ET-1 in pulmonary artery rings. A dose-response curve for 0.01-10 µM 5-HT and 0.1-100 nM ET-1 in isolated rabbit pulmonary artery rings was compared before and after treatment for 60 min with 250 µM GSNO. In subsequent experiments, rings were treated with a single dose of 1.0 µM 5-HT before and after each intervention. The vasoconstrictor response to 1 µM 5-HT in the pulmonary artery bioassay was studied before and after treatment for 60 min with 250 µM GSNO or 500 µM SIN-1. The baths were flushed three times thoroughly with buffer to remove residual GSNO and SIN-1 before second doses of 5-HT. Isometric tension was measured, and the response was expressed as the percent change in tension after the second dose of 5-HT compared with the initial 5-HT response. Control experiments for GSNO were performed with 100 µM GSH or oxidized glutathione (GSSG). GSH or GSSG was added to the baths for 60 min before the second 5-HT dose. The dose-response curve to 0.01-1 µM 5-HT was tested in the presence of the guanylate cyclase inhibitor LY-83583 (10 µM; Cayman Chemical, Ann Arbor, MI) with and without 250 µM GSNO.
The thiol reducing agent dithiothreitol (DTT, 10 mM) was administered in some experiments 5 min before the second 5-HT dose to determine whether the 5-HT response could be restored. Experiments were performed in rings incubated with buffer alone, 250 µM GSNO, or 500 µM SIN-1. Data are expressed as the percent change in tension after the second dose of 5-HT after DTT compared with the initial 5-HT response.Statistical Analysis
Data were analyzed by ANOVA with repeated measures for experiments in isolated lungs and dose-response curves in rings. All other data were analyzed by ANOVA followed by Fisher's protected least-square difference test using a commercially available software program (Statview 512+, Brain Power, Calabasas, CA). Values are means ± SE. P values are provided where statistical tests were performed. ![]() |
RESULTS |
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Dose-Dependent Response to 5-HT in IPL and Pulmonary Artery Rings
5-HT transiently increased Ppa in rabbit IPL to a maximum at 5 min, and Ppa returned to baseline by 20 min. Dose-dependent vasoconstriction occurred with 0.1 and 1.0 µM 5-HT, but 10.0 µM 5-HT did not produce significant further vasoconstriction compared with 1.0 µM (Fig. 1A). Lungs treated with 5-HT showed no evidence of pulmonary weight gain throughout the experiments. On the basis of the results of the dose-response studies, the remaining IPL experiments were performed with 1.0 µM 5-HT. A dose-dependent response was also observed for 0.01-1 µM 5-HT in a pulmonary artery ring bioassay with no further increase in ring tension with 10 µM 5-HT (n = 4, P < 0.05; Fig. 1B).
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Effects of the 5-HT2 Receptor Antagonist Ketanserin on the Response to 5-HT
Ketanserin (10 µM) virtually abolished the vasoconstrictor response to 1 µM 5-HT in the rabbit IPL (Fig. 2). The change in Ppa with 5-HT at 5 min was 0.5 ± 0.2 mmHg for lungs pretreated with ketanserin compared with 11.5 ± 3.9 mmHg in control lungs (P < 0.05). These results indicate that the constrictor response to 5-HT is mediated by the 5-HT2A receptor, which is present in the pulmonary vasculature (27).
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Effects of NO Donors on the Response to 5-HT in IPL
Pretreatment with GSNO markedly inhibited the vasoconstrictor response to 5-HT in IPL (Fig. 3). A qualitatively similar result was found using SIN-1. The change in Ppa with 5-HT after 5 min was 2.6 ± 1.1 mmHg for lungs pretreated with 50 µM GSNO (23% of control 5-HT response) and 1.6 ± 0.7 mmHg for lungs pretreated with 100 µM SIN-1 (14% of control 5-HT response, P < 0.05 for each NO donor vs. control). There was no statistical difference in baseline Ppa in IPL pretreated with GSNO or SIN-1. NOx levels in the perfusate measured by chemiluminescence were higher in both NO donor groups compared with control but similar to each other (Table 1). Treatment with 25,000 U of Cu,Zn SOD did not prevent the inhibitory effects of GSNO or SIN-1 on the vasoconstrictor response to 5-HT (Fig. 4; change in Ppa at 5 min = 1.0 ± 1.0 and 0.3 ± 0.2 mmHg in GSNO- and SIN-1-treated lungs, respectively), indicating that the effect was not due to O
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Effects of In Vitro Treatment of 5-HT With NO Donors in IPL
5-HT was treated in vitro with 50 µM GSNO or 100 µM SIN-1 to evaluate whether NO functionally modified the 5-HT peptide directly. The vasoconstrictor activity of NO donor-treated 5-HT (1 µM) was similar to that of untreated 5-HT (Fig. 5). With GSNO-treated 5-HT (n = 3), the change in Ppa at 5 min was 7.5 ± 1.3 mmHg, and with SIN-1-treated 5-HT (n = 4), the change in Ppa was 7.0 ± 0.5 mmHg (P = 0.30 vs. control 5-HT).
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Effects of GSNO on the Response to ET-1 in IPL and Pulmonary Artery Rings
GSNO-pretreated or untreated lungs were treated with ET-1 to determine whether GSNO attenuated the response to ET-1. At the same concentrations that inhibited responses to 5-HT, GSNO failed to inhibit vasoconstriction to ET-1 (Fig. 6). A similar result was obtained using SIN-1. The change in Ppa 30 min after ET-1 was 33.0 ± 5.8 mmHg in control lungs, 29.4 ± 7.0 mmHg in GSNO-treated lungs, and 30.0 ± 3.8 mmHg in SIN-1-treated lungs. In pulmonary artery rings, the dose-response curve for 0.1-100 nM ET-1 was not shifted to the right by 250 µM GSNO. There was no increase in ring tension with 0.1-10 nM ET-1, while 100 nM ET-1 produced an increase of 0.85 ± 0.02 g in control rings and 0.63 ± 0.04 g in GSNO-treated rings (n = 4).
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Effects of NO Donors on the Response to 5-HT in Pulmonary Artery Rings
In rabbit pulmonary artery rings, the increase in ring tension was the same with two sequential doses of 1 µM 5-HT. Similar to IPL, the response to 5-HT in rings was nearly eliminated by 10 µM ketanserin, a 5-HT2 receptor antagonist (data not shown). As in the intact lung, the response to 5-HT also was significantly reduced after pretreatment with 250 µM GSNO (18 ± 3% of initial 5-HT response, n = 5, P < 0.05 vs. control rings) or 500 µM SIN-1 (27 ± 5% of initial 5-HT response, n = 8, P < 0.05 vs. control rings; Fig. 7A). The dose-response curve to 0.01-10 µM 5-HT for control rings was significantly attenuated in GSNO-treated rings (n = 4, P < 0.05; Fig. 7B). GSH or GSSG at the equivalent concentrations (250 and 125 µM, respectively) had no effect on the response to 5-HT (data not shown). The dose-response curve to 0.01-1 µM 5-HT was shifted to the left in pulmonary artery rings treated with the guanylate cyclase inhibitor LY-83583 (10 µM). GSNO (250 µM) prevented 90% of the response to 0.1 µM 5-HT and 40% of the response to 1.0 µM 5-HT in rings treated with LY-83583, indicating an effect of GSNO that was independent of guanylate cyclase activity (n = 3, P < 0.05; Fig. 8).
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Ability of DTT to Reverse the Inhibitory Effects of GSNO on the 5-HT Response
The thiol reducing agent DTT (10 mM) partially restored the response to 1 µM 5-HT in isolated rings treated with 250 µM GSNO (38 ± 8% of initial 5-HT response, n = 7, P < 0.05 vs. GSNO-treated rings; Fig. 9). In SIN-1 (500 µM)-treated rings, there was a trend toward restoration of the response, but the effect was not statistically significant (32 ± 6% of initial 5-HT response, n = 9, P = 0.2). DTT did not change the response in control rings (95 ± 9% of initial 5-HT response, n = 6). Overall, the response to 5-HT after DTT in GSNO-treated rings increased 2.6-fold vs. a 0.5-fold increase after DTT in SIN-1-treated rings.
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DISCUSSION |
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NO binds to thiol and metal centers in proteins to regulate protein function (37). NO may function as an endothelium-derived relaxing factor by activating guanylate cyclase in smooth muscle to decrease vascular tone or by chemically modifying vasoconstrictor agents, their receptor proteins, or contractile components in smooth muscle to prevent vasoconstriction. We examined the effects of NO donors on vasoconstriction induced by two potent pulmonary vasoconstrictors of physiological and pathophysiological relevance, 5-HT and ET-1, which bind to distinct receptors within the superfamily of GPCRs but operate in vascular smooth muscle using the same intracellular transduction mechanisms (23).
In this study, 5-HT produced vasoconstriction in isolated rabbit lungs and pulmonary artery rings. The effect of 5-HT was inhibited by ketanserin, a specific antagonist of the 5-HT2A receptor. These findings are consistent with earlier studies demonstrating that ketanserin-sensitive 5-HT2A receptors in pulmonary artery smooth muscle mediate vasoconstriction (28, 41). The lung also contains 5-HT1B receptors, which have an important role in pulmonary artery vasoconstriction under physiological conditions, as well as receptors, including 5-HT1 receptor subtypes, implicated in vasodilation (27, 31, 33). Our observation that constriction does not increase further in rabbit lungs with 10 µM 5-HT may indicate that 5-HT2 receptors are saturated or that higher doses preferentially bind to vasodilating 5-HT1 receptors to counterbalance vasoconstriction. The contributions of other 5-HT receptor subtypes involved in the vasoconstrictor response were not evaluated because of their apparent minor role under these experimental conditions.
Vasoconstriction by 5-HT was inhibited in isolated lungs and pulmonary artery rings by pretreatment with NO donors. The doses of GSNO and SIN-1 administered to isolated rabbit lungs did not measurably decrease Ppa. Because the pulmonary artery vessels were not fully dilated in our perfused lung preparations, this finding suggested that cGMP-mediated vasodilation by NO could have only partially counteracted 5-HT-mediated constriction. In GSNO-treated vascular rings, an inhibitor of guanylate cyclase restored 10-60% of the 5-HT activity. Therefore, cGMP-independent pathways were responsible for part of the effect of GSNO on inhibition of 5-HT-mediated constriction in the lung. Although LY-83583 inhibits guanylate cyclase, it may also affect S-nitrosylation and cAMP level; therefore, these data may underestimate the cGMP-independent contribution of NO donor on the 5-HT response (15). Other investigators have also shown that tissue cGMP levels do not always correlate well with pulmonary artery relaxation. Pulmonary artery relaxation by GSNO has been inhibited only partially when cGMP production is completely blocked by guanylate cyclase inhibitors (6, 18, 19). The 5-HT response inhibition by GSNO or SIN-1 in isolated lungs was reproducible in rings in which the chamber was replaced with fresh buffer to remove unreacted donor. This suggested that the NO donors had chemically modified a vascular target. These data are consistent with other studies where NO donors were found to inhibit the response to 5-HT. In rat pulmonary artery smooth muscle cells, NO-containing solutions inhibited the rise in intracellular calcium after 5-HT and inhibited vasoconstriction by 5-HT in pulmonary artery rings (44). In human umbilical artery rings, endogenous NO decreased the response to 5-HT (35). In bovine coronary artery rings, exogenous NO suppressed 5-HT-mediated contractions (11). These findings also indicate that NO modifies the pulmonary vasoconstriction response to 5-HT but do not provide a molecular mechanism or assess reversibility.
SIN-1 releases NO and also O). To examine possible involvement of
ONOO
in the 5-HT response inhibition by NO donors, lungs
were pretreated with Cu,Zn SOD or an SOD mimetic, T2E, before the NO
donors. Neither Cu,Zn SOD nor T2E restored the 5-HT response in lungs
pretreated with GSNO. These findings suggest that GSNO acted as an
S-NO donor and did not produce its effect by reacting with
O
. These data differ from
studies in coronary arteries in which scavenging O
(11). Cu,Zn SOD also did not restore the 5-HT response
after pretreatment with SIN-1. In fact, lungs had a smaller response to
5-HT when pretreated with SIN-1 and Cu,Zn SOD than with SIN-1 alone.
This indicates that scavenging of O
Signaling for the vascular responses to 5-HT includes potential targets for NO within the GPCR system, the 5-HT uptake system that inactivates 5-HT, or the vasoconstrictor itself. Treating 5-HT with GSNO or SIN-1 in vitro produced vasoconstriction in the lung similar to native 5-HT, which indicates that modification of the 5-HT molecule by NO donors was not important under these conditions (4).
Another potential target for NO is the 5-HTT, which is necessary for uptake and metabolism of 5-HT by the lung and is involved in pulmonary vascular smooth muscle cell proliferation (12, 13, 26). In our study, inhibition of the 5-HTT with fluoxetine did not restore 5-HT vasoconstriction after treatment with GSNO. Therefore, the inhibitory effects of the NO donors were not attributable to enhancement of 5-HT uptake by the lung.
The pulmonary vasoconstrictor effects of ET-1, which are also mediated through a GPCR linked to PLC, were not inhibited by GSNO or SIN-1. GSNO therefore inhibits vasoconstriction by 5-HT by modifying the response at a target(s) proximal to PLC, most likely the 5-HT2 receptor. Activation of PLC liberates two second messengers, DAG and IP3, which stimulate calcium influx and mobilize intracellular calcium from the sarcoplasmic reticulum to produce vasoconstriction (23). In pulmonary artery smooth muscle cells, NO inhibited 5-HT-induced calcium release by IP3 and DAG, supporting our conclusion that the site of action for NO in the 5-HT pathway is proximal to PLC (44).
The present data strongly suggest that NO modifies the
5-HT2A GPCR. NO may prevent ligand-receptor binding or
coupling of the receptor to the G protein. Studies of other GPCR
demonstrate that NO can modify receptor function (24,
25). For example, NO reduced signaling by
2-adrenergic agonists in vascular models (1, 40,
43) and inhibited receptor-ligand binding in M2 muscarinic GPCR and angiotensin type 1 receptors (2,
8). In aortic endothelial cells, GSNO, but not glutathione or
nitrite, inhibited bradykinin type 2 receptor-G protein coupling
(29). NO may also directly modify the G protein. NO
inhibits Go protein in synaptosomes, which prevents
pertussis toxin-catalyzed ADP-ribosylation of the Go
protein (16). Future experiments are needed to
discriminate effects of NO on 5-HT2A receptor-ligand
binding, G protein coupling, or G protein activity.
In pulmonary artery rings, GSNO inhibited the constrictor response to 5-HT, and the inhibitory effect of GSNO was largely reversible by the thiol reductant DTT. Nitrosothiol compounds such as GSNO have an NO+ character that promotes transnitrosylation of other protein thiols. Reversal by DTT indicates that GSNO probably modified the 5-HT response by thiol nitrosylation. Specific acidic and/or basic amino acid sequences surrounding a given Cys favor S-nitrosylation (3, 39), and the 5-HT2 receptor contains several Cys predicted to be susceptible to S-nitrosylation (39).
In contrast to GSNO, the NO donor SIN-1 modified the 5-HT response by a
DTT-resistant mechanism. SIN-1 produces chemical modifications different from GSNO, e.g., nitration, rather than
S-nitrosylation. Thus the fate of NO released by SIN-1 may
better represent chemical reactions associated with increased oxidative
stress and pathological ONOO formation. We did not
determine, however, whether a specific S-nitrosylation event
is responsible for the effects of GSNO. An alternative chemical
explanation, that S-thiolation, e.g., glutathiolation, was
responsible for the more potent effects of GSNO than SIN-1, cannot be
distinguished from S-nitrosylation in these studies.
However, lack of effect of GSSG argues against S-thiolation.
In summary, GSNO reversibly inhibits the response to 5-HT in the intact lung. The experimental data narrow the target of NO to the level of thiol residues on the G protein-coupled 5-HT2 receptor. 5-HT2 receptor density is low in the lung and difficult to study. The chemical modification produced by GSNO is different from that produced by SIN-1, which may reflect the specific redox chemistry of NO and the chemical properties of the receptor. Further investigations are necessary to identify specific molecular targets in the receptor-G protein complex and determine whether NO regulates other receptors in this superfamily.
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ACKNOWLEDGEMENTS |
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This work was supported by a Duke Children's Miracle Network Telethon Grant (E. Nozik-Grayck) and National Heart, Lung, and Blood Institute Grant K08 HL-04014 (T. J. McMahon).
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FOOTNOTES |
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Address for reprint requests and other correspondence: E. Nozik-Grayck, Box 3046, Dept. of Pediatrics, Duke University Medical Center, Durham, NC 27710 (E-mail: grayc001{at}mc.duke.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published November 30, 2001;10.1152/ajplung.00081.2001
Received 13 March 2001; accepted in final form 28 November 2001.
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