Pulmonary vasoconstriction by serotonin is inhibited by S-nitrosoglutathione

Eva Nozik-Grayck, Timothy J. McMahon, Yuh-Chin T. Huang, Christine S. Dieterle, Jonathan S. Stamler, and Claude A. Piantadosi

Departments of Pediatrics and Medicine, Duke University Medical Center, Durham, North Carolina 27710


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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<UP><SUB>2</SUB><SUP>−</SUP></UP> simultaneously, also blocked the response to 5-HT. Its chemical effects, however, were distinct from those of GSNO, because 5-HT-mediated vasoconstriction was not restored in isolated rings by dithiothreitol. In the intact lung, neither NO donor altered the vascular response to endothelin, which activates the same second-messenger vasoconstrictor system as 5-HT. These findings, which did not depend on guanylate cyclase, are consistent with chemical modification by NO of the 5-HT2 G protein-coupled receptor system to inhibit vasoconstriction, possibly by S-nitrosylation of the receptor or a related protein. This study demonstrates that GSNO can regulate vascular tone in the intact lung by a reversible mechanism involving inhibition of the response to 5-HT.

nitric oxide; G protein-coupled receptor


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 beta 2-adrenergic receptors in cultured cells have shown that NO donors reduce signaling by beta 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.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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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 · mM-1 · cm-1. In isolated ring experiments, GSNO was prepared from stock solutions of GSH and sodium nitrite immediately before use in tissue baths.

Measurements 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 (O<UP><SUB>2</SUB><SUP>−</SUP></UP>) (9, 17). The responses were compared with those of control lungs perfused for 1 h without administration of an NO donor. Experiments were also done in lungs treated with 25,000 U of Cu,Zn superoxide dismutase (SOD) or the intracellular superoxide scavenger T2E (gift of James D. Crapo) to determine whether scavenging O<UP><SUB>2</SUB><SUP>−</SUP></UP> could alter the effects of GSNO or SIN-1. The initial response to 1.0 µM 5-HT was compared with the second dose of 5-HT after treatment for 60 min with Cu,Zn SOD along with 50 µM GSNO or 100 µM SIN-1. The sequential response to 5-HT was also examined after a 60-min treatment with T2E and GSNO. To determine whether NO donors modified 5-HT directly, lungs treated with 120 µM 5-HT were pretreated for 1 h with 50 µM GSNO or 100 µM SIN-1. 5-HT (1.0 µM) pretreated with NO donor was administered to IPL, and Ppa and weight gain were monitored for 30 min. The final concentration of GSNO or SIN-1 in the perfusate in these experiments was 2 or 5 nM, respectively.

Uptake of 5-HT in pulmonary vascular smooth muscle cells by a 5-HT transporter (5-HTT) contributes to inactivation of 5-HT in the lung (12, 26). To assess the role of 5-HT uptake in the acute vasoconstrictor response to 5-HT, lungs were treated with 1.0 µM 5-HT and then incubated for 30 min with the 5-HTT inhibitor fluoxetine (10 µM) and a second dose of 5-HT. The response to 5-HT in each lung after fluoxetine was compared with its baseline 5-HT response. In other experiments, the 5-HT responses in control lungs were compared with responses after 10 µM fluoxetine for 10 min followed by GSNO (50 µM) for another 30 min.

Endothelin 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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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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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Fig. 1.   Dose response curves to serotonin [5-hydroxytryptamine (5-HT)]. A: in isolated perfused lungs (IPL), 0.1 µM 5-HT (n = 3) produced minimal vascular [pulmonary arterial pressure (Ppa)] response, while 1.0 µM 5-HT (n = 6) produced transient vasoconstriction, which was maximal at 5 min. 5-HT at 10 µM (n = 3) did not produce any additional vasoconstriction compared with 1.0 µM 5-HT. Lungs did not develop edema with any dose of 5-HT. B: in isolated pulmonary artery rings, 0.01-1.0 µM 5-HT produced a dose-dependent increase in ring tension, which did not increase further with 10 µM 5-HT (n = 4, P < 0.05 by ANOVA).

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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Fig. 2.   Effect of the 5-HT2 receptor antagonist ketanserin on the response to 5-HT in IPL. Pretreatment with 10 µM ketanserin (black-lozenge , n = 6) blocks the increase in Ppa produced by 1.0 µM 5-HT (, n = 6) in IPL during the 30-min experimental period. * P < 0.05 vs. 1.0 µM 5-HT (by ANOVA).

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<UP><SUB>2</SUB><SUP>−</SUP></UP> generation. This result was confirmed by pretreatment of the lungs with 1.3 mg of T2E to scavenge extra- and intracellular O<UP><SUB>2</SUB><SUP>−</SUP></UP>. T2E did not prevent the inhibitory effects of GSNO on 5-HT. In SIN-1-treated lungs, Cu,Zn SOD prevented even the small increase in Ppa with 5-HT compared with that produced by SIN-1 alone. Blocking 5-HT uptake with 10 µM fluoxetine had no effect on the peak vasoconstrictor response to 5-HT, but it delayed the return of Ppa to baseline (n = 2, data not shown). In addition, GSNO-mediated inhibition of the constrictor response to 5-HT was not prevented by fluoxetine (change in Ppa at 5 min with 5-HT = 3.3 ± 1.4 mmHg, n = 3).


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Fig. 3.   Effects of nitric oxide (NO) donors S-nitrosoglutathione (GSNO) and 3-morpholinosydnonimine (SIN-1) on the response to 5-HT in IPL. Pretreatment of IPL with 50 µM GSNO (black-triangle, n = 6) or 100 µM SIN-1 (, n = 7) for 1 h blocked the increase in Ppa produced with 1.0 µM 5-HT (). * P < 0.05 vs. 1.0 µM 5-HT (by ANOVA).


                              
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Table 1.   NOx levels in perfusate before administration of 5-HT increased to similar levels after 50 min of treatment with GSNO or SIN-1



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Fig. 4.   Cu,Zn superoxide dismutase (SOD) did not prevent the effects of GSNO or SIN-1 on the response to 5-HT in IPL. Treatment with 25,000 U of Cu,Zn SOD did not prevent the inhibitory effects of 50 µM GSNO (n = 3) or 100 µM SIN-1 (n = 6) on the vasoconstrictor response to 5-HT. A: change in Ppa at 5 min in response to 1.0 µM 5-HT after pretreatment with GSNO or SIN-1 with or without Cu,Zn SOD. B: change in Ppa over 30 min in control lungs treated with 1.0 µM 5-HT (), lungs pretreated with SIN-1 (), or lungs pretreated with Cu,Zn SOD and SIN-1 (black-lozenge ). * P < 0.05 vs. control; # P < 0.05 vs. SIN-1-treated lung.

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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Fig. 5.   Effects of in vitro treatment of 5-HT with NO donors. Vasoconstrictor activity of 5-HT treated in vitro with 50 µM GSNO (black-triangle, n = 3) or 100 µM SIN-1 (, n = 4) was similar to that of untreated 5-HT (). P >=  0.3 vs. control (by ANOVA).

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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Fig. 6.   Effects of NO donors on the response to endothelin-1 (ET-1). Pretreatment of IPL with 50 µM GSNO (black-triangle, n = 6) or 100 µM SIN-1 (, n = 7) for 1 h did not block ET-1 (10 nM)-mediated vasoconstriction (, n = 6). Doses of GSNO or SIN-1 were the same as those used for the 5-HT studies. P >=  0.3 vs. control (by ANOVA).

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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Fig. 7.   Effect of NO donors on the response to 5-HT in pulmonary artery rings. A: pretreatment of isolated pulmonary artery rings with 250 µM GSNO (n = 5) or 500 µM SIN-1 (n = 8) for 1 h blocked the increase in ring tension produced with 1.0 µM 5-HT. Values are expressed as change in ring tension standardized to initial control 5-HT response. * P < 0.05 vs. control (by ANOVA). B: dose-response curve for 0.01-10.0 µM 5-HT () was diminished after 1 h of pretreatment with 250 µM GSNO (black-triangle). * P < 0.05 (by ANOVA); n = 4.



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Fig. 8.   Effect of GSNO on the response to 5-HT in rings pretreated with the guanylate cyclase inhibitor LY-83583. Dose-response curve to 0.01-1 µM 5-HT () was shifted to the left in pulmonary artery rings treated with 10 µM LY-83583 (black-lozenge ). GSNO (250 µM) attenuated the response to 5-HT in rings treated with LY-83583 (black-triangle), indicating effects of GSNO that were independent of guanylate cyclase activity (n = 3). * P < 0.05 compared with 5-HT dose-response curve in rings treated with LY-83583.

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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Fig. 9.   Ability of dithiothreitol (DTT) to reverse effects of NO donors on the response to 5-HT in pulmonary artery rings. The thiol reducing agent DTT (10 mM) partially restored the response to 1 µM 5-HT in isolated rings treated with 250 µM GSNO (n = 4). In SIN-1-treated rings (500 µM, n = 5), there was a slight trend toward restoration of the response, which was not statistically significant. Values are expressed as change in ring tension after treatment with DTT standardized to control 5-HT response. * P < 0.05 vs. GSNO (by ANOVA).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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<UP><SUB>2</SUB><SUP>−</SUP></UP>, which reacts rapidly with NO to form the strong nitrating species peroxynitrite (ONOO-). 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<UP><SUB>2</SUB><SUP>−</SUP></UP> to form ONOO-. These data differ from studies in coronary arteries in which scavenging O<UP><SUB>2</SUB><SUP>−</SUP></UP> blocked the response to NO, implicating ONOO- (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<UP><SUB>2</SUB><SUP>−</SUP></UP> by SOD increases the bioactivity of NO and that SIN-1 had acted primarily as an NO donor.

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 beta 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.


    ACKNOWLEDGEMENTS

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).


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Adam, L, Bouvier M, and Jones TL. Nitric oxide modulates beta 2-adrenergic receptor palmitoylation and signaling. J Biol Chem 274: 26337-26343, 1999[Abstract/Free Full Text].

2.   Aronstam, RS, Martin DC, Dennison RL, and Cooley HG. S-nitrosylation of M2 muscarinic receptor thiols disrupts receptor-G protein coupling. Ann NY Acad Sci 757: 215-217, 1995[ISI][Medline].

3.   Ascenzi, P, Colasanti M, Persichini T, Muolo M, Polticelli F, Venturini G, Bordo D, and Bolognesi M. Re-evaluation of amino acid sequence and structural consensus rules for cysteine-nitric oxide reactivity. J Biol Chem 381: 623-627, 2000.

4.   Blanchard, B, Dendane M, Gallard JF, Houee-Levin C, Karim A, Payen D, Launay JM, and Ducrocq C. Oxidation, nitrosation, and nitration of serotonin by nitric oxide-derived nitrogen oxides: biological implications in the rat vascular system. Nitric Oxide 1: 442-452, 1997[ISI][Medline].

5.   Brunner, F, Leonhard B, Kukovetz WR, and Mayer B. Role of endothelin, nitric oxide and L-arginine release in ischaemia/reperfusion injury of rat heart. Cardiovasc Res 36: 60-66, 1997[ISI][Medline].

6.   Brunner, F, Schmidt K, Nielsen EB, and Mayer B. Novel guanylyl cyclase inhibitor potently inhibits cyclic GMP accumulation in endothelial cells and relaxation of bovine pulmonary artery. J Pharmacol Exp Ther 277: 48-53, 1996[Abstract].

7.   Buckley, BJ, Mirza Z, and Whorton AR. Regulation of Ca2+-dependent nitric oxide synthase in bovine aortic endothelial cells. Am J Physiol Cell Physiol 269: C575-C765, 1995.

8.   Cahill, PA, Redmond EM, Foster C, and Sitzmann JV. Nitric oxide regulates angiotensin II receptors in vascular smooth muscle cells. Eur J Pharmacol 288: 219-229, 1995[Medline].

9.   Campbell, DL, Stamler JS, and Strauss HC. Redox modulation of L-type calcium channels in ferret ventricular myocytes. Dual mechanism regulation by nitric oxide and S-nitrosothiols. J Gen Physiol 108: 277-293, 1996[Abstract].

10.   Ceron, PI, Cremonez DC, Bendhack LM, and Tedesco AC. The relaxation induced by S-nitroso-glutathione and S-nitroso-N-acetylcysteine in rat aorta is not related to nitric oxide production. J Pharmacol Exp Ther 298: 686-694, 2001[Abstract/Free Full Text].

11.   Davidson, CA, Kaminski PM, and Wolin MS. Endogenous peroxynitrite generation causes a subsequent suppression of coronary arterial contraction to serotonin. Nitric Oxide 1: 244-253, 1997[ISI][Medline].

12.   Eddahibi, S, Hanoun N, Lanfumey L, Lesch KP, Raffestin B, Hamon M, and Adnot S. Attenuated hypoxic pulmonary hypertension in mice lacking the 5-hydroxytryptamine transporter gene. J Clin Invest 105: 1555-1562, 2000[Abstract/Free Full Text].

13.   Fanburg, BL, and Lee SL. A new role for an old molecule: serotonin as a mitogen. Am J Physiol Lung Cell Mol Physiol 272: L795-L806, 1997[Abstract/Free Full Text].

14.   Feelisch, M, Kotsonis P, Siebe J, Clement B, and Schmidt HH. The soluble guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one is a nonselective heme protein inhibitor of nitric oxide synthase and other cytochrome P-450 enzymes involved in nitric oxide donor bioactivation. Mol Pharmacol 56: 243-253, 1999[Abstract/Free Full Text].

15.   Hausladen, A, and Stamler JS. Nitric oxide in plant immunity. Proc Natl Acad Sci USA 95: 10345-10347, 1998[Free Full Text].

16.   Hess, DT, Lin LH, Freeman JA, and Norden JJ. Modification of cysteine residues within Go and other neuronal proteins by exposure to nitric oxide. Neuropharmacology 33: 1283-1292, 1994[ISI][Medline].

17.   Holm, P, Kankaanranta H, Metsa-Ketela T, and Moilanen E. Radical releasing properties of nitric oxide donors GEA 3162, SIN-1 and S-nitroso-N-acetylpenicillamine. Eur J Pharmacol 346: 97-102, 1998[ISI][Medline].

18.   Homer, KL, Fiore SA, and Wanstall JC. Inhibition by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) of responses to nitric oxide donors in rat pulmonary artery: influence of the mechanism of nitric oxide generation. J Pharm Pharmacol 51: 135-139, 1999[ISI][Medline].

19.   Homer, KL, and Wanstall JC. Cyclic GMP-independent relaxation of rat pulmonary artery by spermine NONOate, a diazeniumdiolate nitric oxide donor. Br J Pharmacol 131: 673-682, 2000[Abstract/Free Full Text].

20.   Jourd'heuil, D, Miranda KM, Kim SM, Espey MG, Vodovotz Y, Laroux S, Mai CT, Miles AM, Grisham MB, and Wink DA. The oxidative and nitrosative chemistry of the nitric oxide/superoxide reaction in the presence of bicarbonate. Arch Biochem Biophys 365: 92-100, 1999[ISI][Medline].

21.   Kanagy, NL, Charpie JR, Dananberg J, and Webb RC. Decreased sensitivity to vasoconstrictors in aortic rings after acute exposure to nitric oxide. Am J Physiol Heart Circ Physiol 271: H253-H260, 1996[Abstract/Free Full Text].

22.   Kantrow, SP, Huang YC, Whorton AR, Grayck EN, Knight JM, Millington DS, and Piantadosi CA. Hypoxia inhibits nitric oxide synthesis in isolated rabbit lung. Am J Physiol Lung Cell Mol Physiol 272: L1167-L1173, 1997[Abstract/Free Full Text].

23.   Lameh, J, Cone RI, Maeda S, Philip M, Corbani M, Nadasdi L, Ramachandran J, Smith GM, and Sadee W. Structure and function of G protein coupled receptors. Pharm Res 7: 1213-1221, 1990[ISI][Medline].

24.   Lander, HM, Ogiste JS, Pearce SF, Levi R, and Novogrodsky A. Nitric oxide-stimulated guanine nucleotide exchange on p21ras. J Biol Chem 270: 7017-7020, 1995[Abstract/Free Full Text].

25.   Lander, HM, Sehajpal PK, and Novogrodsky A. Nitric oxide signaling: a possible role for G proteins. J Immunol 151: 7182-7187, 1993[Abstract/Free Full Text].

26.   Lee, SL, Wang WW, Lanzillo JJ, and Fanburg BL. Serotonin produces both hyperplasia and hypertrophy of bovine pulmonary artery smooth muscle cells in culture. Am J Physiol Lung Cell Mol Physiol 266: L46-L52, 1994[Abstract/Free Full Text].

27.   Martin, GR. Vascular receptors for 5-hydroxytryptamine: distribution, function and classification. Pharmacol Ther 62: 283-324, 1994[ISI][Medline].

28.   McMahon, TJ, Hood JS, Nossaman BD, and Kadowitz PJ. Analysis of responses to serotonin in the pulmonary vascular bed of the cat. J Appl Physiol 75: 93-102, 1993[Abstract].

29.   Miyamoto, A, Laufs U, Pardo C, and Liao JK. Modulation of bradykinin receptor ligand binding affinity and its coupled G-proteins by nitric oxide. J Biol Chem 272: 19601-19608, 1997[Abstract/Free Full Text].

30.   Moncada, S, Palmer RMJ, and Higgs EA. Nitric oxide: physiology, pathophysiology, and pharmacology. Pharmocol Rev 43: 109-142, 1991[ISI][Medline].

31.   Morecroft, I, Heeley RP, Prentice HM, Kirk A, and MacLean MR. 5-Hydroxytryptamine receptors mediating contraction in human small muscular pulmonary arteries: importance of the 5-HT1B receptor. Br J Pharmacol 128: 730-734, 1999[Abstract/Free Full Text].

32.   Morecroft, I, and MacLean MR. 5-Hydroxytryptamine receptors mediating vasoconstriction and vasodilation in perinatal and adult rabbit small pulmonary arteries. Br J Pharmacol 125: 69-78, 1998[Abstract].

33.   Neely, CF, Haile D, and Matot I. Tone-dependent responses of 5-hydroxytryptamine in the feline pulmonary vascular bed are mediated by two different 5-hydroxytryptamine receptors. J Pharmacol Exp Ther 264: 1315-1326, 1993[Abstract].

34.   Nozik-Grayck, E, Piantadosi CA, van Adelsberg J, Alper SL, and Huang YC. Protection of perfused lung from oxidant injury by inhibitors of anion exchange. Am J Physiol Lung Cell Mol Physiol 273: L296-L304, 1997[Abstract/Free Full Text].

35.   Okatani, Y, Watanabe K, Nakano Y, and Sagara Y. Relaxant effect of nitric oxide and prostacyclin on serotonin-induced vasocontraction of human umbilical artery. Acta Obstet Gynecol Scand 75: 108-112, 1996[ISI][Medline].

36.   Paolocci, N, Ekelund UE, Isoda T, Ozaki M, Vandegaer K, Georgakopoulos D, Harrison RW, Kass DA, and Hare JM. cGMP-independent inotropic effects of nitric oxide and peroxynitrite donors: potential role for nitrosylation. Am J Physiol Heart Circ Physiol 279: H1982-H1988, 2000[Abstract/Free Full Text].

37.   Stamler, JS. Redox signaling: nitrosylation and related target interactions of nitric oxide. Cell 78: 931-936, 1994[ISI][Medline].

38.   Stamler, JS, Singel DJ, and Loscalzo J. Biochemistry of nitric oxide and its redox-activated forms. Science 258: 1898-1902, 1992[ISI][Medline].

39.   Stamler, JS, Toone EJ, Lipton SA, and Sucher NJ. (S)NO signals: translocation, regulation, and a consensus motif. Neuron 18: 691-696, 1997[ISI][Medline].

40.   Travis, MD, Whalen EJ, and Lewis SJ. Heterologous desensitization of beta -adrenoceptor signal transduction in vivo. Eur J Pharmacol 328: R1-R3, 1997[ISI][Medline].

41.   Van Nueten, JM, Janseens WJ, and Vanhoutte PM. Serotonin and vascular reactivity. Pharmacol Res Commun 17: 585-608, 1985[ISI][Medline].

42.   Watanabe, K, Okatani Y, and Sagara Y. Potentiating effect of hydrogen peroxide on the serotonin-induced vasocontraction in human umbilical artery. Acta Obstet Gynecol Scand 75: 783-789, 1996[ISI][Medline].

43.   Whalen, EJ, Johnson AK, and Lewis SJ. beta -Adrenoceptor dysfunction after inhibition of NO synthesis. Hypertension 36: 376-382, 2000[Abstract/Free Full Text].

44.   Yuan, XJ, Tod ML, Rubin LJ, and Blaustein MP. NO hyperpolarizes pulmonary artery smooth muscle cells and decreases the intracellular Ca2+ concentration by activating voltage-gated K+ channels. Proc Natl Acad Sci USA 93: 10489-10494, 1996[Abstract/Free Full Text].

45.   Zifa, E, and Fillion G. 5-Hydroxytryptamine receptors. Pharmacol Rev 44: 401-458, 1992[ISI][Medline].


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