cGMP-independent mechanism of airway smooth muscle relaxation induced by S-nitrosoglutathione

William J. Perkins, Christina Pabelick, David O. Warner, and Keith A. Jones

Departments of Anesthesiology and of Physiology and Biophysics, Mayo Clinic and Foundation, Rochester, Minnesota 55906

    ABSTRACT
Top
Abstract
Introduction
Materials
Results
Discussion
References

This study tested the hypothesis that the NO donor S-nitrosoglutathione (GSNO) relaxes canine tracheal smooth muscle (CTSM) in part by a cGMP-independent process that involves reversible oxidation of intracellular thiols. GSNO caused a concentration-dependent relaxation in ACh-contracted strips (EC50 ~1.2 µM) accompanied by a concentration-dependent increase in cytosolic cGMP concentration ([cGMP]i). The soluble guanylate cyclase inhibitor methylene blue prevented the increase in [cGMP]i induced by 1 and 10 µM GSNO, but isometric force decreased by 10 ± 4 and 55 ± 3%, respectively. After recovery of [cGMP]i to baseline, GSNO-induced relaxation persisted during continuous ACh stimulation. Dithiothreitol caused a rapid recovery of isometric force to values similar to those obtained with ACh alone in these strips. We conclude that GSNO relaxes CTSM contracted by ACh in part by oxidation of intracellular protein thiols.

nitric oxide; canine lung; canine trachea; guanosine 3',5'-cyclic monophosphate; sulfhydryl reagents; DL-dithiothreitol

    INTRODUCTION
Top
Abstract
Introduction
Materials
Results
Discussion
References

NITRIC OXIDE PLAYS A significant role in a number of physiological processes, including regulation of smooth muscle tone, neurotransmission, and platelet function, and pathophysiological states such as sepsis (21). In smooth muscle, NO is thought to mediate relaxation largely by activation of soluble guanylate cyclase (10, 14), which increases levels of cGMP. cGMP subsequently activates cGMP-dependent protein kinases (PKG), which ultimately decrease both the cytosolic Ca2+ concentration ([Ca2+]i) and the amount of isometric force produced for a given [Ca2+]i (i.e., the Ca2+ sensitivity) (28) by phosphorylation of unidentified intracellular proteins (6). Although the role of NO in regulation of airway tone is unclear, airway smooth muscle (ASM) contains soluble guanylate cyclase (14) and is responsive to NO (32) and a variety of NO donors, including nitrovasodilators (34), 3-morpholinosydnonimine (SIN-1) (13), NO-nucleophile adducts (9), and S-nitrosothiols (11, 34).

The mechanisms by which NO donors relax ASM vary. For example, whereas the degree of relaxation of ASM induced by diethylaminodiazen-1-ium-1,2-diolate (DEA-NO) or SIN-1 is correlated with an increase in cytosolic cGMP concentration ([cGMP]i), sodium nitroprusside (SNP)-induced relaxation is not and is thus thought to relax ASM by cGMP-independent mechanisms (32, 34). Although little is known regarding these cGMP-independent mechanisms, there are at least two plausible mechanisms by which this could occur, both of which involve intracellular thiol oxidation. The first is S-nitrosylation of protein or other intracellular thiols, a mechanism that has been implicated in the NO-dependent regulation of proteins such as protein kinase C (PKC) (8) and glyceraldehyde-3-phosphate dehydrogenase (5). The second is oxidation of protein thiols without S-nitrosylation (31). The contribution of either of these thiol-related, cGMP-independent mechanisms to smooth muscle relaxation may be determined using a thiol reducing reagent, such as DL-dithiothreitol (DTT), which should reverse thiol oxidation-mediated effects.

S-nitrosothiols such as S-nitrosoglutathione (GSNO) are stable at 37°C and pH 7.4 in the presence of transition metal ion chelators (27). However, the presence of trace free transition metal ions, such as Cu2+/Cu+, stimulates the catalytic breakdown of GSNO to NO and glutathione disulfide (7, 27). S-nitrosothiols can also undergo nitrosonium (NO+) transfer to other intracellular thiols. S-nitrosothiols increase [cGMP]i and cause relaxation of agonist-induced contractions in ASM (11, 32, 34). The extent to which S-nitrosothiol-induced ASM relaxation is contingent on increased [cGMP]i is unknown. The following hypotheses were tested: 1) GSNO relaxes canine tracheal smooth muscle (CTSM) by both cGMP-dependent and cGMP-independent mechanisms and 2) the cGMP-independent mechanism of relaxation is mediated by reversible oxidation of intracellular thiols.

    METHODS AND MATERIALS
Top
Abstract
Introduction
Materials
Results
Discussion
References

Experimental Techniques

Tissue preparation. Mongrel dogs (15-23 kg) of either sex were anesthetized with an intravenous injection of pentobarbital (30 mg/kg) and exsanguinated. A 5- to 10-cm portion of extrathoracic trachea was excised and immersed in chilled physiological salt solution (PSS) with a composition (in mM) of 110.5 NaCl, 25.7 NaHCO3, 5.6 dextrose, 3.4 KCl, 2.4 CaCl2, 1.2 KH2PO4, and 0.8 MgSO4. Fat, connective tissue, and the epithelium were removed with tissue forceps and scissors.

Mechanical responses. For cyclic nucleotide measurements, CTSM strips (width 2-3 mm, length 1.0-1.5 cm, and weight 25-67 mg) were suspended in 25-ml water-jacketed tissue baths that were filled with PSS (37°C) aerated with 94% O2 and 6% CO2 (pH ~7.4, PO2 ~550 mmHg, and PCO2 ~36 mmHg in the PSS). One end of the strip was anchored to a metal hook at the bottom of the tissue bath; the other end was attached to a calibrated force transducer (model FT03D, Grass Instrument, Quincy, MA). During a 3-h equilibration period, the strips were repeatedly contracted isometrically for 30 s every 5 min by supramaximal electrical field stimulation (400 mA, 25 Hz, and pulse duration of 0.5 ms). Electrical field stimulation was triggered by stimulator (model S88D, Grass Instrument) and delivered by a direct current amplifier (Section of Engineering, Mayo Foundation). The length of the strips was increased after each stimulation until active force was maximal (optimal length). Each strip was maintained at this optimal length for the remainder of the experiment. During this equilibration period, the strips were washed with fresh PSS every 10 min.

Cyclic nucleotide measurements. CTSM strips were homogenized in 4 ml of cold (2°C) 95% ethanol using a ground glass pestle and homogenizing tube. The precipitated pellet was separated from the soluble extract by centrifugation at 4,000 g for 10 min. The soluble extract was evaporated to dryness at ~55°C under a stream of nitrogen and then suspended in 0.3 ml of 4 mM EDTA (pH 7.5). [3H]cGMP (0.4 µCi) or [3H]cAMP (1.25 µCi) was added as a tracer for cGMP or cAMP recovery determinations, respectively. Commercially available RIA kits were used to determine the concentrations of cGMP and cAMP in the soluble extract (3). The protein content of the precipitated pellet was determined by the method described by Lowry et al. (17), using BSA dissolved in 1 N NaOH as the standard. [cGMP]i and cytosolic cAMP concentration ([cAMP]i) were expressed as picomoles per milligram protein.

Experimental Protocols

Five experimental protocols were conducted, each on separate sets of CTSM strips. All strips were incubated with 10 µM indomethacin to prevent the formation of prostanoids (12, 34). In previous studies, contraction of CTSM with ACh during incubation with indomethacin had no effect on [cGMP]i or [cAMP]i compared with unstimulated tissue (11).

Concentration-dependent effect of GSNO on isometric force. For determination of the concentration dependence of GSNO-induced relaxation, seven strips obtained from a single dog were contracted with 0.1 µM ACh (approximately equal to the EC50) for 15 min, the time required for contractions to stabilize. Six strips were then exposed to 0.3, 1.0, 3.0, 10, 30, or 100 µM GSNO for 40 min; the seventh strip was not exposed to GSNO and served as a control for the effect of time on the ACh-induced contraction.

Time course for the effects of GSNO and DTT on isometric force, [cGMP]i, and [cAMP]i. Eight strips were contracted with 0.1 µM ACh for 15 min (see Fig. 2A). Two strips were not treated with GSNO and were flash frozen for cyclic nucleotide measurement after contraction with ACh for 15 min (baseline) and 75 min (time control for baseline). Five strips were then treated with 100 µM GSNO and flash frozen at 30 s, 1, 5, 10, and 40 min for cyclic nucleotide concentration measurements. To determine the extent to which thiol oxidation was responsible for the GSNO-mediated relaxation of ACh-induced contractions, the eighth strip was treated with 100 µM GSNO for 40 min, then was treated with 1 mM DTT for 10 min, and was flash frozen for cyclic nucleotide measurement. Isometric force was measured continuously up to the time the muscle strips were flash frozen.

Effect of methylene blue on GSNO-induced relaxation and increase in [cGMP]i. To determine the extent to which GSNO-induced relaxation is contingent on increased [cGMP]i, strips were pretreated with the soluble guanylate cyclase inhibitor methylene blue. Four pairs of strips from a single dog were prepared. One strip from each pair was treated with 10 µM methylene blue for 10 min before the onset of contraction. All strips were contracted with 0.1 µM ACh for 15 min. Then each pair of strips was exposed to either 0 (baseline), 1, 10, or 100 µM GSNO for 5 min (time required to reach peak increase in [cGMP]i). Methylene blue was selected as the soluble guanylate cyclase inhibitor in this study because it has previously been used in this laboratory to demonstrate that NO donor-mediated relaxation with DEA-NO and SIN-1 is cGMP contingent in this tissue (9, 13). The concentration of methylene blue used in this study is the highest concentration that does not cause spontaneous contractions and has no effect on ACh-induced contractions in this tissue (13).

Effect of GSNO washout on isometric force and [cGMP]i recovery. In the tissue bath experiments described in the first two protocols, GSNO was present for the duration of the experiment. In the following two protocols, the use of superfusion wells (for [cGMP]i measurements) and of a tissue superfusion chamber (for isometric force measurements) permitted rapid washout of residual GSNO and determination of whether GSNO effects on isometric force and [cGMP]i persisted after washout. In addition, this protocol permitted testing whether GSNO-mediated relaxation persisted after recovery of [cGMP]i to baseline values. Seven strips obtained from a single dog were placed in superfusion wells (see Fig. 4A). All strips were first stimulated with 0.1 µM ACh for 15 min. One of these strips was not treated with GSNO (strip 1, baseline). Two strips were then superfused with 100 µM GSNO for 1 or 5 min (strips 2 and 3, respectively), and the remaining four strips were superfused with 100 µM GSNO for 5 min and then with 0.1 µM ACh only for 1, 3, 5, or 15 min (strips 4-7, respectively) to wash out GSNO. Strips were flash frozen at the times described for [cGMP]i measurements. Isometric force was measured in a strip obtained from the same animals using the same protocol. The half time for superfusate exchange was ~19 s, resulting in >99% equilibration with superfusate within 95 s.

Effect of GSNO and DTT on isometric force. Three strips in a set were contracted with 0.1 µM ACh for 15 min. Two strips were then relaxed by addition of 100 µM GSNO to the superfusate for 5 min, and the third strip continued to be superfused with ACh alone. Thereafter, GSNO was discontinued for 15 min and 1 mM DTT was added to the superfusate of one of the GSNO-treated strips to determine whether reversible thiol oxidation played a role in relaxation that persisted after washout of GSNO. DTT was added to the strip superfused with ACh alone at the same time as the GSNO-treated strip to determine the effect of DTT alone on ACh-induced contraction. The second GSNO-treated strip was not treated with DTT for measurement of spontaneous force recovery. In a separate set of experiments, strips were contracted as described above and then relaxed by addition of 1, 10, or 100 µM GSNO for 5 min. GSNO was then discontinued for 15 min, at which time 1 mM DTT was added to the superfusate. Changes in isometric force induced by GSNO were expressed as a percent change from the stable ACh-induced responses before addition of GSNO. For each experiment, a strip from a different animal was used.

Materials

RIA kits for cGMP and cAMP measurements were purchased from Calbiochem (Arlington Heights, IL). GSNO and all other drugs and chemicals were purchased from Sigma Chemical (St. Louis, MO). All drugs and chemicals were dissolved in distilled water.

Statistical Analysis

Data are expressed as means ± SD; n represents the number of dogs. Initial forces of strips contracted with ACh during incubation with or without methylene blue were compared by unpaired Student's t-test. The effects of GSNO and DTT on isometric force, [cGMP]i, and [cAMP]i were assessed by repeated measures ANOVA with post hoc analysis using Duncan's multiple-range test. Concentration-response curves were compared by nonlinear regression analysis as described by Meddings et al. (19). In this method, force (F) at any concentration of drug (C) was given by the equation F = FmC/(EC50 + C), where Fm represents the maximal (or minimal) isometric force and EC50 represents the concentration that produces half-maximal (or half-minimal) isometric force for that drug. Nonlinear regression analysis was used to fit values of Fm and EC50 to data for F and C for each condition studied. This method allows comparison of curves to determine whether they are significantly different and whether this overall difference can be attributed to differences in Fm, EC50, or both parameters. A P value < 0.05 was considered statistically significant.

    RESULTS
Top
Abstract
Introduction
Materials
Results
Discussion
References

Concentration-Dependent Effect of GSNO on Isometric Force

Contractions induced by 0.1 µM ACh were stable within 15 min. GSNO added to the tissue baths decreased isometric force in a concentration-dependent manner (Fig. 1, A and B). GSNO-induced relaxation consisted of a rapid onset followed by a partial recovery with GSNO concentrations >0.3 µM (Fig. 1A). Peak relaxation was attained within 2-5 min, taking longer at lower concentrations. The sustained relaxation was defined as that remaining 20 min after addition of GSNO to the tissue baths. The EC50 values for GSNO-induced peak and sustained relaxation of ACh-induced contraction were 1.2 ± 0.2 and 1.2 ± 0.6 µM, respectively (Fig. 1B).


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 1.   Concentration-dependent effect of S-nitrosoglutathione (GSNO) on ACh-induced contraction of canine tracheal smooth muscle (CTSM). Seven strips were contracted with 0.1 µM ACh for 15 min and then relaxed by noncumulative addition of 0.3, 1, 3, 10, 30, or 100 µM GSNO to tissue bath. A: representative isometric force tracings. B: means ± SD for isometric force (n = 8 experiments).

Time Course for the Effect of GSNO and DTT on Isometric Force, [cGMP]i, and [cAMP]i

In addition to relaxation (Fig. 2A), 100 µM GSNO significantly increased [cGMP]i, which was maximal at 5 min but then slowly decreased to sustained levels greater than baseline (Fig. 2B). In contrast, GSNO had no effect on [cAMP]i (Fig. 2B). The baseline and time control [cGMP]i were not significantly different (0.73 ± 0.14 and 0.69 ± 0.12 pmol/mg protein, respectively).


View larger version (23K):
[in this window]
[in a new window]
 
Fig. 2.   Time course for effect of 100 µM GSNO and 1 mM DL-dithiothreitol (DTT) on isometric force (A; dotted line is time control isometric force tracing) and on cytosolic cyclic nucleotide concentrations ([cAMP]i and [cGMP]i; B) in CTSM strips contracted with 0.1 µM ACh for 15 min (n = 8 experiments). A: filled circles show time points at which tissue was flash frozen for determination of cyclic nucleotide concentrations. * Significantly different from baseline (P < 0.05). # Significantly different from preceding value (P < 0.05). PSS, physiological salt solution.

Subsequent addition of 1 mM DTT to strips relaxed with 100 µM GSNO caused a transient decrease in isometric force, followed thereafter by a rapid recovery to 91 ± 6% of the time control isometric force (Fig. 2A). The peak isometric force reduction with 100 µM GSNO was 93 ± 2% and the sustained isometric force reduction was 57 ± 6% (Figs. 1B and 2A). Addition of DTT resulted in recovery of [cGMP]i to levels not significantly different from either baseline or time control but had no effect on [cAMP]i (Fig. 2B). The transient decrease in isometric force following addition of DTT was associated with a transient increase in [cGMP]i (data not shown). Addition of 1 mM DTT to strips contracted with 0.1 µM ACh and not treated with GSNO had no effect on isometric force (Fig. 2A).

Effect of Methylene Blue on GSNO-Induced Relaxation and Increase in [cGMP]i

GSNO caused a concentration-dependent decrease in isometric force and concentration-dependent increase in [cGMP]i. Methylene blue had no effect on baseline force or on initial isometric force induced by 0.1 µM ACh. Methylene blue significantly increased the EC50 for GSNO from 2.5 ± 0.7 to 5.2 ± 0.2 µM (Fig. 3B). Methylene blue abolished the increase in [cGMP]i induced by 1 and 10 µM GSNO (Fig. 3A) but only partially inhibited GSNO-induced relaxation; 1 and 10 µM GSNO decreased isometric force by 10 ± 4 and 55 ± 3%, respectively, in the presence of methylene blue (Fig. 3B). Methylene blue significantly attenuated, but did not abolish, the increase in [cGMP]i induced by 100 µM GSNO (Fig. 3A).


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 3.   Effect of 10 µM methylene blue (MB) on increase in [cGMP]i (A) and relaxation (B) induced by 1, 10, and 100 µM GSNO in CTSM contracted with 0.1 µM ACh for 15 min (n = 8). * Significantly different (P < 0.05) from strips not treated with GSNO. # Significantly different (P < 0.05) from strips not treated with MB.

Effect of GSNO Washout on Isometric Force and [cGMP]i Recovery

Addition of 100 µM GSNO to the superfusate containing 0.1 µM ACh resulted in nearly complete relaxation (Fig. 4A) and a significant increase in [cGMP]i (Fig. 4B). After washout of GSNO during continuous superfusion with 0.1 µM ACh, isometric force slowly recovered, with only 45% isometric force recovery after 15 min. By contrast, [cGMP]i recovered to baseline values within 5 min of GSNO washout (Fig. 4B).


View larger version (20K):
[in this window]
[in a new window]
 
Fig. 4.   Effect of GSNO washout on recovery of isometric force (A; representative tracing) and [cGMP]i in CTSM strips (n = 6 experiments) during continuous stimulation with 0.1 µM ACh. Location of numbers above isometric force tracing correspond to times at which tissues were flash frozen for determination of cyclic nucleotide concentrations (B). * Significantly different from baseline (P < 0.05).

Effect of GSNO and DTT on Isometric Force

Superfusion with 0.1 µM ACh caused a sustained increase in isometric force (Fig. 5). Subsequent treatment with 100 µM GSNO for 5 min resulted in a 95.3 ± 1.1% reduction in isometric force (n = 11) that only slowly recovered over 15 min (Fig. 5). Addition of DTT after 15-min washout of GSNO resulted in a rapid recovery in isometric force to 85.7 ± 7.5% of that initially induced by ACh. The recovery of isometric force was greater in GSNO-treated strips following exposure to DTT than in strips allowed to spontaneously recover over the same time interval (46.9 ± 3.9 vs. 6.4 ± 1.2%, respectively, P = 0.011). Addition of 1 mM DTT to strips superfused with ACh alone resulted in a 4.6 ± 1.1% reduction in isometric force.


View larger version (21K):
[in this window]
[in a new window]
 
Fig. 5.   Effect of GSNO on isometric force in CTSM strips during continuous stimulation with 0.1 µM ACh. Strips were continuously superfused with 0.1 µM ACh and treated with 100 µM GSNO for 5 min. GSNO was washed out for 15 min, and strips were then either left untreated (Control) or treated with 1 mM DTT. Results were reproduced in strips obtained from 6 animals.

Whereas 1, 10, and 100 µM GSNO each caused a decrease in isometric force, the effects of GSNO washout during continuous superfusion with 0.1 µM ACh varied with GSNO concentration (Fig. 6). Whereas the recovery of isometric force was complete during washout of 1 µM GSNO, the recovery of isometric force during washout of 10 or 100 µM GSNO was not. After 15-min washout of 1, 10, or 100 µM GSNO during continuous superfusion with 0.1 µM ACh, 1 mM DTT caused a transient decrease in isometric force (Fig. 6). In strips treated with 1 µM GSNO, DTT had no effects on isometric force aside from the transient effects described above (Fig. 6A). However, in strips treated with 10 or 100 µM GSNO, DTT caused a rapid recovery of isometric force to that initially induced by 0.1 µM ACh before superfusion with GSNO (Fig. 6, B and C).


View larger version (13K):
[in this window]
[in a new window]
 
Fig. 6.   Representative tracings for effect of 1 (A), 10 (B), and 100 (C) µM GSNO on isometric force in CTSM strips contracted with 0.1 µM ACh. Strips were continuously superfused with 0.1 µM ACh and treated with GSNO for 5 min. GSNO was washed out for 15 min, and strips were then treated with 1 mM DTT for 10 min. These results were reproduced in strips obtained from 10 animals.

    DISCUSSION
Top
Abstract
Introduction
Materials
Results
Discussion
References

The major findings of this in vitro study are that during ACh-induced contraction of CTSM, the S-nitrosothiol GSNO caused relaxation mediated by both cGMP-dependent and cGMP-independent mechanisms. The cGMP-independent mechanism involves reversible oxidation of intracellular thiols on proteins that regulate smooth muscle contraction or on the contractile proteins.

The formation and decay of S-nitrosothiols, such as GSNO, may represent a mechanism for the storage or transport of NO (24). According to this hypothesis, S-nitrosothiols are produced in vivo by reaction of NO with a thiol (30) and subsequently diffuse to the site of action. S-nitrosothiols then mediate their effects either by decomposition to yield free NO or by transnitrosylation of specific protein thiols (29, 31). Although the physiological relevance of S-nitrosothiols remains to be established, these agents have been used as NO donors in smooth muscle and have been proposed as therapy for diseases such as hypertension, asthma, and uterine hypertonia. Synthetic S-nitrosothiols relax agonist-induced contraction in both vascular smooth muscle and ASM (11, 15).

In vascular smooth muscle, cGMP appears to mediate the relaxant effects of both NO (23) and S-nitrosothiols, although cGMP-independent mechanisms have not been ruled out (22). The mechanisms by which S-nitrosothiols relax ASM are not fully known. In CTSM contracted with 1 µM methacholine, S-nitroso-N-acetyl-penicillamine (SNAP) caused a relaxation that was accompanied by an increase in [cGMP]i (34). The increase in [cGMP]i and relaxation were significantly inhibited by methylene blue, suggesting that ASM relaxation induced by SNAP is in part caused by increased [cGMP]i. In the same study, methylene blue had no effect on the relaxation induced by the nitrovasodilator SNP. A similar result has been reported with SNP and SIN-1 in porcine ASM (32). On the basis of these results, it has been suggested that some NO donors may relax ASM by mechanisms that do not involve activation of soluble guanylate cyclase and increases in [cGMP]i. Similar observations have been made in vascular smooth muscle (2).

To investigate the relative importance of cGMP in mediating GSNO-induced relaxation of CTSM, we determined 1) whether the relaxation induced by GSNO was accompanied by a concentration- and time-dependent increase in [cGMP]i and 2) the effect of methylene blue, a putative soluble guanylate cyclase inhibitor, on both the GSNO-mediated increase in [cGMP]i and relaxation. GSNO relaxed CTSM strips that had been contracted with ACh in a concentration-dependent manner, with an EC50 comparable to that reported in guinea pig trachea, ~1 µM (12). The relaxation was accompanied by both a time- and a concentration-dependent increase in [cGMP]i and no change in [cAMP]i, a result consistent with those obtained in CTSM using the NO donors SIN-1 and DEA-NO (9, 13). Although methylene blue attenuated both the amount of relaxation and the increase in [cGMP]i induced by GSNO, significant relaxation was observed at 1 and 10 µM GSNO, concentrations at which methylene blue completely inhibited the increase in [cGMP]i (Fig. 3). These data indicate that, in general, CTSM relaxation induced by GSNO is mediated in part by both cGMP-dependent and cGMP-independent mechanisms. At approximately the EC50 for GSNO, cGMP-independent mechanisms account for less than one-half of the observed relaxation, but, at higher GSNO concentrations (greater than or equal to the 90% effective concentration), cGMP-independent mechanisms account for the majority of the observed relaxation. The tissues were frozen for determination of cyclic nucleotides at the time peak increases in [cGMP]i were observed in the time course studies, making it unlikely that these results were due to the kinetics of cGMP production. Further evidence supporting a role for cGMP-independent relaxation for GSNO was provided by the superfusion chamber experiments, in which washout of 100 µM GSNO resulted in relaxation that persisted after recovery of [cGMP]i to baseline (Fig. 4). However, small or highly localized increases in [cGMP]i cannot be ruled out by these experiments.

The rate-limiting step in cGMP-mediated relaxation in smooth muscle ultimately involves PKG phosphorylation and phosphatase-mediated dephosphorylation of largely uncharacterized protein targets (28). It is therefore possible that [cGMP]i does not perfectly correlate with the degree of relaxation observed in smooth muscle. A direct comparison of the kinetics of [cGMP]i vs. PKG target phosphorylation or of dephosphorylation vs. isometric force has not to our knowledge been performed. The rapid recovery of isometric force following administration of NO or NO donors to contracted vascular smooth muscle and CTSM (9), however, suggests that the phosphorylation status of the PKG targets involved in regulating smooth muscle contractile state is relatively fast.

To determine whether reversible thiol oxidation was involved in mediating GSNO-induced relaxation, the ability of the thiol reducing agent DTT to reverse GSNO-induced relaxation was investigated. In the tissue bath studies, the addition of DTT to strips relaxed by GSNO resulted in a transient additional relaxation, which was associated with a transient increase in [cGMP]i and probably results from thiol-induced release of NO from GSNO in the presence of metal ion contaminant in the buffer (18, 27). These effects were followed by rapid recovery of [cGMP]i to baseline and isometric force to time control levels (Fig. 2).

In the tissue bath studies (Fig. 2), in which GSNO was continuously present, addition of DTT to strips caused a transient additional relaxation followed by a rapid recovery of both isometric force to time control levels and [cGMP]i to baseline. These results may have been due to one of three causes: 1) DTT both stimulated and scavenged NO released from GSNO, thereby decreasing or eliminating NO transport to the heme of soluble guanylate cyclase, 2) DTT served as a competitive source of free thiol in transnitrosation reactions, or 3) DTT reduced reversibly oxidized thiols, possibly nitrosylated by NO+ from GSNO. However, in superfusion chamber experiments, following GSNO washout and recovery of [cGMP]i to baseline, addition of DTT in the absence of GSNO in the tissue bath also caused a rapid recovery of isometric force. The absence of GSNO in the superfusion experiment eliminates the possibility of a direct interaction between DTT and GSNO. This rules out the first two possible causes described above and indicates that the cGMP-independent, DTT-reversible component of GSNO-mediated relaxation involves reversible oxidation of thiols.

In the current study, addition of DTT to ACh-contracted strips that had not been relaxed by GSNO had only a small effect on isometric force. In contrast, addition of DTT to ACh-contracted strips following washout of 1 µM GSNO and complete recovery of isometric force caused a transient, much greater relaxation. Similar transient effects were observed following washout of 10 and 100 µM GSNO. Two plausible explanations for these observations are 1) that there is residual GSNO in the cell even though it has been completely removed from the superfusion chamber and 2) that NO from GSNO is stored in the tissue at functionally silent sites, possibly protein thiols, and is released via thiol-mediated release of NO from S-nitrosothiols in the presence of metal ions. The finding that [cGMP]i had completely recovered to baseline following washout of GSNO suggests that the first possible explanation is unlikely. However, conclusive distinction between the two possibilities is beyond the scope of the current investigation.

The specific proteins that underwent thiol oxidation by GSNO are unknown but may include proteins involved in the PKG pathway, the contractile proteins (actin or myosin), proteins involved in the regulation of [Ca2+]i, or proteins that regulate the amount of isometric force produced for a given [Ca2+]i, such as smooth muscle myosin light chain kinase or phosphatase. There are several recent examples of NO donor-induced reversible thiol oxidation of intracellular proteins, with resultant changes in the activity of the protein, including PKC (8), creatine kinase (33), and the ryanodine receptor Ca2+-release channel (1, 20). The common theme in each of these instances is that the protein targets have reactive thiols that are critical for their activity. In each of the examples cited, an NO donor decreased activity and the effect was reversed by treatment with a thiol-reducing agent such as DTT. The NO donor may modify the protein either by S-nitrosylation or by driving the formation of an intramolecular disulfide between vicinal thiols, as reported for the N-methyl-D-aspartate receptor (16). An attractive potential protein target that could explain the results is myosin. The smooth muscle myosin head contains reactive cysteine thiols, which, when oxidized or covalently modified, inhibit myosin ATPase activity (4). The NO donor SNP inhibits the actomyosin ATPase in rabbit psoas single fibers, an effect that is also reversible with the thiol reducing agent DTT (26). Such a modification would decrease isometric force independently of [Ca2+]i. Yet another possible molecular target is the smooth muscle myosin light chain kinase, a protein that is critical in inducing force development and that contains multiple cysteines (25). The ultimate molecular targets of GSNO in CTSM are, however, unclear at this time.

Another potential cGMP-independent GSNO effect would be to decrease ACh activation of muscarinic receptors, thereby decreasing the amount of isometric force developed at a given ACh concentration. This possibility was tested by determining the effect of GSNO on contractions induced by membrane depolarization with isotonic KCl, which does not result in activation of muscarinic receptors. As with ACh-contracted CTSM, GSNO resulted in a rapid reduction in KCl-induced contraction, an effect that persisted following washout of the NO donor (Fig. 7). It is thus unlikely that the cGMP-independent relaxation observed in CTSM is due to decreased ACh activation of muscarinic receptors.


View larger version (18K):
[in this window]
[in a new window]
 
Fig. 7.   Representative tracing for effect of 100 µM GSNO on isometric force in CTSM strips contracted with 28 mM isotonic KCl. Strips were continuously superfused with 30 mM KCl and treated with GSNO for 5 min. GSNO was washed out for 15 min, and strips were then either left untreated or treated with 10 mM DTT. Results were reproduced in strips from 4 animals.

In summary, the results of this study demonstrate that GSNO relaxes CTSM contracted by ACh. This relaxation is only partially mediated by cGMP-dependent mechanisms. The cGMP-independent mechanism of relaxation involves reversible oxidation of thiols on proteins that regulate smooth muscle contraction or on the contractile proteins, such as the myosin head. The results indicate a previously undescribed mechanism for regulation of CTSM contraction that requires further investigation.

    ACKNOWLEDGEMENTS

We thank Kathy Street and Robert Lorenz for expert technical assistance.

    FOOTNOTES

This study was supported in part by National Heart, Lung, and Blood Institute Grants HL-54757 and HL-45532 and Deutsche Forschungsgemeinschaft Research Training Grant Pa 668-1.

Address for reprint requests: W. J. Perkins, Mayo Clinic and Mayo Foundation, 200 First St. SW, Rochester, MN 55905.

Received 12 September 1997; accepted in final form 8 May 1998.

    REFERENCES
Top
Abstract
Introduction
Materials
Results
Discussion
References

1.   Aghdasi, B., M. B. Reid, and S. L. Hamilton. Nitric oxide protects the skeletal muscle Ca2+ release channel from oxidation induced activation. J. Biol. Chem. 272: 25462-25467, 1997[Abstract/Free Full Text].

2.   Bennett, B. M., L. D. Hayward, and F. Murad. Effects of the D and L stereoisomers of isosorbide dinitrate on relaxation and cyclic GMP accumulation in rat aorta and comparison to glyceryl trinitrate. J. Appl. Cardiol. 1: 203-209, 1986.

3.   Brooker, G., J. F. Harper, W. L. Terasaki, and R. D. Moylan. Radioimmunoassay of cyclic AMP and cyclic GMP. Adv. Cyclic Nucleotide Res. 10: 1-33, 1979[Medline].

4.   Chandra, T. S., N. Nath, H. Suzuki, and J. C. Seidel. Modification of thiols of gizzard myosin alters ATPase activity, stability of myosin filaments, and the 6-10 S conformational transition. J. Biol. Chem. 260: 202-207, 1985[Abstract/Free Full Text].

5.   Clancy, R. M., D. Levartovsky, J. Leszczynska-Piziak, J. Yegudin, and S. B. Abramson. Nitric oxide reacts with intracellular glutathione and activates the hexose monophosphate shunt in human neutrophils: evidence for S-nitrosoglutathione as a bioactive intermediary. Proc. Natl. Acad. Sci. USA 91: 3680-3684, 1994[Abstract].

6.   Cornwell, T. L., and T. M. Lincoln. Regulation of intracellular Ca2+ levels in cultured vascular smooth muscle cells. Reduction of Ca2+ by atriopeptin and 8-bromo-cyclic GMP is mediated by cyclic GMP-dependent protein kinase. J. Biol. Chem. 264: 1146-1155, 1989[Abstract/Free Full Text].

7.  Dicks, A. P., H. R. Swift, D. L. H. Williams, A. R. Butler, H. H. Alsadoni, and B. G. Cox. Identification of Cu+ as the effective reagent in nitric oxide formation from S-nitrosothiols (RSNO). J. Chem. Soc.-Perkin Trans II: 481-487, 1996.

8.   Gopalakrishna, R., Z. H. Chen, and U. Gundimeda. Nitric oxide and nitric oxide-generating agents induce a reversible inactivation of protein kinase C activity and phorbol ester binding. J. Biol. Chem. 268: 27180-27185, 1993[Abstract/Free Full Text].

9.   Hirasaki, A., K. A. Jones, W. J. Perkins, and D. O. Warner. Use of nitric oxide nucleophile adducts as biological sources of nitric oxide: effects on airway smooth muscle. J. Pharmacol. Exp. Ther. 278: 1269-1275, 1996[Abstract].

10.   Ignarro, L. J., J. N. Degnan, W. H. Baricos, P. J. Kadowitz, and M. S. Wolin. Activation of purified guanylate cyclase by nitric oxide requires heme. Comparison of heme-deficient, heme-reconstituted and heme-containing forms of soluble enzyme from bovine lung. Biochim. Biophys. Acta 718: 49-59, 1982[Medline].

11.   Jansen, A., J. Drazen, J. A. Osborne, R. Brown, J. Loscalzo, and J. S. Stamler. The relaxant properties in guinea pig airways of S-nitrosothiols. J. Pharmacol. Exp. Ther. 261: 154-160, 1992[Abstract].

12.   Jones, K. A., R. R. Lorenz, N. Morimoto, G. C. Sieck, and D. O. Warner. Halothane reduces force and intracellular Ca2+ in airway smooth muscle independently of cyclic nucleotides. Am. J. Physiol. 268 (Lung Cell. Mol. Physiol. 12): L166-L172, 1995[Abstract/Free Full Text].

13.   Jones, K. A., R. R. Lorenz, D. O. Warner, Z. S. Katusic, and G. C. Sieck. Changes in cytosolic cGMP and calcium in airway smooth muscle relaxed by 3-morpholinosydnonimine. Am. J. Physiol. 266 (Lung Cell. Mol. Physiol. 10): L9-L16, 1994[Abstract/Free Full Text].

14.   Katsuki, S., W. Arnold, C. K. Mittal, and F. Murad. Stimulation of guanylate cyclase by sodium nitroprusside, nitroglycerin and nitric oxide in various tissue preparations and comparison to the effects of sodium azide and hydroxylamine. J. Cyclic Nucleotide Res. 3: 23-35, 1977[Medline].

15.   Kowaluk, E. A., and H. L. Fung. Spontaneous liberation of nitric oxide cannot account for in vitro vascular relaxation by S-nitrosothiols. J. Pharmacol. Exp. Ther. 255: 1256-1264, 1990[Abstract].

16.   Lei, S. Z., Z. H. Pan, S. K. Aggarwal, H. S. Chen, J. Hartman, N. J. Sucher, and S. A. Lipton. Effect of nitric oxide production on the redox modulatory site of the NMDA receptor-channel complex. Neuron 8: 1087-1099, 1992[Medline].

17.   Lowry, O. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265-275, 1951[Free Full Text].

18.   Mayer, B., A. Schrammel, P. Klatt, D. Koesling, and K. Schmidt. Peroxynitrite-induced accumulation of cyclic GMP in endothelial cells and stimulation of purified soluble guanylyl cyclase. Dependence on glutathione and possible role of S-nitrosation. J. Biol. Chem. 270: 17355-17360, 1995[Abstract/Free Full Text].

19.   Meddings, J. B., R. B. Scott, and G. H. Fick. Analysis and comparison of sigmoidal curves: application to dose-response data. Am. J. Physiol. 257 (Gastrointest. Liver Physiol. 20): G982-G989, 1989[Abstract/Free Full Text].

20.   Meszaros, L. G., I. Minarovic, and A. Zahradnikova. Inhibition of the skeletal muscle ryanodine receptor calcium release channel by nitric oxide. FEBS Lett. 380: 49-52, 1996[Medline].

21.   Moncada, S., R. M. Palmer, and E. A. Higgs. Nitric oxide: physiology, pathophysiology, and pharmacology. Pharmacol. Rev. 43: 109-142, 1991[Medline].

22.   Moro, M. A., R. J. Russell, S. Cellek, I. Lizasoain, Y. C. Su, V. M. Darleyusmar, M. W. Radomski, and S. Moncada. cGMP mediates the vascular and platelet actions of nitric oxide: confirmation using an inhibitor of the soluble guanylyl cyclase. Proc. Natl. Acad. Sci. USA 93: 1480-1485, 1996[Abstract/Free Full Text].

23.   Murad, F. Cyclic guanosine monophosphate as a mediator of vasodilation. J. Clin. Invest. 78: 1-5, 1986[Medline].

24.   Myers, P. R., R. L. Minor, Jr., R. Guerra, Jr., J. N. Bates, and D. G. Harrison. Vasorelaxant properties of the endothelium-derived relaxing factor more closely resemble S-nitrosocysteine than nitric oxide. Nature 345: 161-163, 1990[Medline].

25.   Olson, N. J., R. B. Pearson, D. S. Needleman, M. Y. Hurwitz, B. E. Kemp, and A. R. Means. Regulatory and structural motifs of chicken gizzard myosin light chain kinase. Proc. Natl. Acad. Sci. USA 87: 2284-2288, 1990[Abstract].

26.   Perkins, W. J., Y. S. Han, and G. C. Sieck. Skeletal muscle force and actomyosin ATPase activity reduced by nitric oxide donor. J. Appl. Physiol. 83: 1326-1332, 1997[Abstract/Free Full Text].

27.   Singh, R. J., N. Hogg, J. Joseph, and B. Kalyanaraman. Mechanism of nitric oxide release from S-nitrosothiols. J. Biol. Chem. 271: 18596-18603, 1996[Abstract/Free Full Text].

28.   Somlyo, A. P., and A. V. Somlyo. Signal transduction and regulation in smooth muscle. Nature 372: 231-236, 1994[Medline].

29.   Stamler, J. S. Redox signaling: nitrosylation and related target interactions of nitric oxide. Cell 78: 931-936, 1994[Medline].

30.   Stamler, J. S., O. Jaraki, J. Osborne, D. I. Simon, J. Keaney, J. Vita, D. Singel, C. R. Valeri, and J. Loscalzo. Nitric oxide circulates in mammalian plasma primarily as an S-nitroso adduct of serum albumin. Proc. Natl. Acad. Sci. USA 89: 7674-7677, 1992[Abstract].

31.   Stamler, J. S., D. J. Singel, and J. Loscalzo. Biochemistry of nitric oxide and its redox-activated forms. Science 258: 1898-1902, 1992[Medline].

32.   Stuart-Smith, K., T. C. Bynoe, K. S. Lindeman, and C. A. Hirshman. Differential effects of nitrovasodilators and nitric oxide on porcine tracheal and bronchial muscle in vitro. J. Appl. Physiol. 77: 1142-1147, 1994[Abstract/Free Full Text].

33.   Wolosker, H., R. Panizzutti, and S. Engelender. Inhibition of creatine kinase by S-nitrosoglutathione. FEBS Lett. 392: 274-276, 1996[Medline].

34.   Zhou, H. L., and T. J. Torphy. Relationship between cyclic guanosine monophosphate accumulation and relaxation of canine trachealis induced by nitrovasodilators. J. Pharmacol. Exp. Ther. 258: 972-978, 1991[Abstract].


Am J Physiol Cell Physiol 275(2):C468-C474
0002-9513/98 $5.00 Copyright © 1998 the American Physiological Society