Br. J. Pharmacol. (1989), 98, 757-766

S-nitrosothiols as vasodilators: implications regardingtolerance to nitric oxide-containing vasodilators'Peter J. Henry, Olaf H. Drummer & 2*John D. Horowitz

Clinical Pharmacology Unit, Department of Medicine, University of Melbourne and *Department ofCardiology, Austin Hospital, Heidelberg, Melbourne, 3084, Australia

1 The formation of an S-nitrosothiol compound, S-nitroso-N-acetylcysteine (SNAC) has recentlybeen proposed to mediate the augmentation of the anti-aggregatory and haemodynamic effects ofglyceryl trinitrate observed in the presence of N-acetylcysteine. This study investigated the effects onan isolated coronary artery preparation of acute and prolonged exposure to S-nitrosothiol com-pounds and nitric oxide (NO).2 Single doses of NO and of the S-nitrosothiol compounds, SNAC and S-nitroso-N-acetyl-penicillamine (SNAP), induced rapid, but transient, relaxations in U46619-contracted bovine iso-lated coronary artery rings. Peak relaxation responses to SNAP and NO were attenuated in thepresence of N-acetylcysteine, cysteine, ascorbic acid and methylene blue. The duration of the relax-ation responses to SNAC was two to three times longer than those to SNAP and NO. In thepresence of N-acetylcysteine (but not cysteine, ascorbic acid or methylene blue) the duration of therelaxation responses to SNAP and NO (but not to SNAC) was markedly increased. H.p.l.c. assayconfirmed that, in the presence of N-acetylcysteine, SNAP and, to a lesser degree, NO were con-verted to the relatively more stable and longer acting vasodilator, SNAC.3 When compared to control rings, coronary artery rings superfused with glyceryl trinitrate weresubsequently markedly less responsive to the vasodilator actions of glyceryl trinitrate, whereasresponsiveness to SNAC or NO was only marginally reduced. On the other hand, coronary arteryrings superfused with SNAC or NO were subsequently less responsive to glyceryl trinitrate, SNACand NO. Thus prolonged vascular exposure to SNAC or NO induced a form of tolerance differentfrom that induced with glyceryl trinitrate and which is possibly associated with impaired guanylatecyclase activity.4 Coronary artery rings superfused with NO were markedly less responsive to glyceryl trinitrateand NO, whereas responses to the endothelium-dependent vasodilator A23187 and to theophyllinewere not significantly attenuated.5 It is concluded that formation of the more stable vasodilator SNAC occurs on incubation ofN-acetylcysteine with SNAP or NO. While coronary artery responsiveness to SNAC and NO isvirtually unchanged in the presence of glyceryl trinitrate-induced tolerance, after prolonged expo-sure to SNAC or NO tolerance may develop to these vasodilators with cross-tolerance to glyceryltrinitrate but not A23187. Thus, formation or therapeutic utilization of SNAC may acutely circum-vent the problem of glyceryl trinitrate-induced tolerance but, during prolonged vascular exposure toSNAC, attenuation of vascular responsiveness may occur to a wide range of vasodilators.

Introduction

Tolerance to glyceryl trinitrate and related organic these agents in the management of patients withnitrate vasodilators poses a significant clinical angina pectoris or congestive heart failure (Abrams,problem which may limit the long-term efficacy of 1986; Horowitz & Henry, 1987; Packer et al., 1987).

Previous studies have indicated that glyceryl tri-1 Author for correspondence at present address: Depart- nitrate, as well as other nitric oxide (NO)-containingment of Pharmacology, University of Western Australia, vasodilators, activate soluble guanylate cyclase viaPerth, Nedlands, 6009, Australia. an extensive cascade of biochemical events (Ignarro2 Present address: Department of Cardiology, The Queen et al., 1981; 1984) and that the resultant increase inElizabeth Hospital, Adelaide, Woodville, 5011, Australia. intracellular cyclic guanosine 3',5'-monophosphate

© The Macmillan Press Ltd 1989

758 P.J. HENRY et al.

(cyclic GMP) concentrations induces vasodilatation.Tolerance to glyceryl trinitrate is associated withreduced metabolic bioconversion, possibly reflectingreduced conversion to dinitrates and nitrite, theinitial metabolic step (Brien et al., 1986).As a number of other agents have been shown to

activate guanylate cyclase without undergoing thisinitial bioconversion (Ignarro et al., 1984), it mightbe anticipated that the development of pharmaco-logical tolerance to these agents might be limited oravoided. However, little information is availablein this regard, with virtually no clinical studies cur-rently published.

Several investigations have suggested that glyceryltrinitrate may combine with tissue sulphydryls, indu-cing the formation of S-nitrosothiols (Ignarro et al.,1981), which combine vasodilator (Kowaluk et al.,1987) and anti-platelet effects (Loscalzo, 1985). In anumber of clinical investigations, the short-term effi-cacy of intravenously infused glyceryl trinitrate hasbeen increased and glyceryl trinitrate-induced toler-ance reversed in the presence of the sulphydryldonors N-acetylcysteine and methionine (Horowitzet al., 1983; Packer et al., 1987; Levy et al., 1988).This change has been attributed to formation of S-nitroso-N-acetylcysteine (SNAC; Loscalzo, 1985;Fung et al., 1988). The question arises as to whetherSNAC or other S-nitrosothiols might be useful ther-apeutically and, in particular, whether or not thephenomenon of haemodynamic tolerance to organicnitrate vasodilators might be ameliorated by theiruse.

In the current experiments, we describe someaspects of the vasodilator activity of two S-nitrosothiols, SNAC and S-nitroso-N-acetyl-penicillamine (SNAP). In particular, we examinedthe acute vasodilator effects of SNAP, SNAC andNO and their modulation by agents including N-acetylcysteine, cysteine, ascorbic acid and methyleneblue. In addition the effects of continuous vascularexposure to SNAC or NO on subsequent vascularresponses to NO-containing and endothelium-dependent vasodilators were compared to the effectsof tolerance induced by prolonged exposure to glyc-eryl trinitrate. The results of these investigations,while supporting a potential therapeutic role for S-nitrosothiols, reveal the existence of a further site oftolerance in the organic nitrate bioconversioncascade, potentially inhibiting the vasodilatoractions of these, and related, agents.

Methods

Bovine isolated coronary artery rings

Hearts from freshly slaughtered cattle were placed incold Krebs-bicarbonate solution and transferred to

the laboratory (within 30 min). The left anteriordescending coronary artery was removed from sur-rounding myocardium and cut into 3 mm long seg-ments. Bovine isolated coronary artery rings weresuspended under 4 g resting tension in a water-jacketed organ bath containing 10 ml Krebs-bicarbonate solution (pH 7.4) bubbled with 95% 02plus 5% CO2. The composition of the Krebs-bicarbonate solution was (in mM): NaCI 117, KCI5.36, MgSO4 0.57, KH2PO4 1.03, NaHCO3 25.0,CaCl2 2.5 and glucose 11.1. Changes in coronaryartery ring tension were measured by Grass FTO3-Cforce-displacement transducers connected to a GrassModel 7D Polygraph. Rings were equilibrated for atleast 45 min before any drug addition.

Unless otherwise stated, effects of vasodilatorswere determined in coronary artery rings contractedwith the thromboxane analogue U46619(EC70-EC80 concentration, range 0.05-0.2yM).Within 15 min, U46619-induced contractionsreached a plateau level of elevated tone that wasmaintained for several hours in the absence of vaso-dilator drugs.

Single dose experiments

Addition of a single dose of either SNAP, SNAC orNO induced a rapid relaxation response which, afterattaining a peak response, returned spontaneously tothe previous level of elevated tone. The peakresponse was expressed as percentage reversal of theU46619-induced contraction. The period betweenthe attainment of, and recovery to, 50% of the peakresponse was used as the measure of the 'duration ofrelaxation' induced by the vasodilator. We examinedthe effect of several agents (N-acetylcysteine, cyste-ine, ascorbic acid and methylene blue) on the peakresponse and the 'duration of relaxation' induced bythe vasodilators. In these experiments, coronaryartery rings were exposed to a single dose of vasodil-ator at a concentration which induced a peakresponse of between 30 and 70%. On recovery to theprevious level of elevated tone, rings were exposed toN-acetylcysteine (100MM), cysteine (100M), ascorbicacid (30uM) or methylene blue (3 yM) and 2 min laterre-exposed to the same single dose of vasodilator.The peak response was variably reduced in the pre-sence of each of these agents. To overcome this inhi-bition (such that the peak response induced by thevasodilator in the presence of inhibitor was compar-able to peak response observed in its absence) ringswere re-exposed to a second, higher concentration ofvasodilator. A comparison of this relaxationresponse with the initial relaxation response (i.e.when no inhibitor was present) was used to deter-mine the effect of these agents on the 'duration ofrelaxation' induced by the vasodilators. A control

S-NITROSOTHIOLS AND TOLERANCE 759

preparation was repeatedly exposed to a single doseof vasodilator to monitor any spontaneous changesin coronary artery responsiveness.

Superfusion experiments

We examined the effects of continuous coronaryartery exposure to SNAC, NO and glyceryl trinitrateon subsequent vascular responses to a series of vaso-dilators. To minimize the potential loss of activity ofthese agents that may occur during prolonged incu-bation with vascular tissue bathed in oxygenatedKrebs-bicarbonate solution, coronary artery ringswere superfused for 60 min with Krebs-bicarbonatesolution (4 ml min- 1, 370C, carbogenated) into whichSNAC, NO or glyceryl trinitrate was infused(20 ylmin-') immediately before contact with therings. A paired control ring was concomitantlysuperfused with Krebs-bicarbonate solution that didnot contain SNAC, NO or glyceryl trinitrate. Theconcentrations of glyceryl trinitrate (10pM), SNAC(3 pM) and NO (5 y1 NO stock solution ml -1 inKrebs-bicarbonate solution) in the superfusate werechosen on the basis of data obtained from prelim-inary experiments. In these preliminary experiments,the EC50 values (95% confidence limits, number ofpreparations) of glyceryl trinitrate, SNAC and NOfor relaxation of coronary artery rings superfusedwith a Krebs-bicarbonate solution containing 30mmKC1 were respectively: 0.13 gM (0.086-0.19, n = 12),0.042 UM (0.015-0.12, n = 6) and 0.049p1 (0.034-0.072, n = 6) NO stock solution ml-' in Krebs-bicarbonate solution. Thus, the concentrations ofglyceryl trinitrate, SNAC and NO chosen for toler-ance induction in the superfusion experiments wereapproximately 100 times greater than their respec-tive EC50 values obtained for relaxation of controlcoronary artery rings. Following the 60 min super-fusion, coronary artery rings were placed in an organbath containing Krebs-bicarbonate solution andrepeatedly washed for 15min. Rings were thenexposed to U46619 and upon attainment of aplateau level of contraction (15 min) a single cumula-tive concentration-effect curve to glyceryl trinitrate,NO, SNAC, A23187 or theophylline was completed.

Formation ofSNAC; h.p.l.c. studies

Solutions of N-acetylcysteine (0, 10, 100 gM) andascorbic acid (100pM) prepared in Krebs-bicarbonate solution were bubbled with 95% 02plus 5% CO2 and maintained at 37°C. To these sol-utions, SNAP (22.7 pM) was added, and aliquotswere taken at 0.5 and 2 min for the determination ofSNAP, SNAC and N-acetylpenicillamine concentra-tions. These aliquots (40p1) were analysed by h.p.l.c.

by use of a C18-yBondapak column (WatersAssociates) and a Model 450 variable wavelengthdetector (Waters Associates) set to detect absorbanceat 214 nm. The mobile phase consisted of 30% meth-anol and 70% ammonium phosphate buffer (10mM,pH 2.5) with a flow rate of 0.8 ml min- . The reten-tion times for SNAC, N-acetylpenicillamine andSNAP were respectively 2.5, 3.5 and 8 min. Standardcurves were derived for SNAC, N-acetylpenicillamine and SNAP based on peakheights. For h.p.l.c. determination of SNAC forma-tion from reaction mixtures of N-acetylcysteine(100pM) and NO at 37°C in Krebs-bicarbonate solu-tion, a mobile phase consisting of 12% methanol and88% ammonium phosphate buffer (10 mm, pH 2.5)was used. The retention time for SNAC under theseconditions was 6 min. All other conditions for h.p.l.c.assay of SNAC were as described above.

Preparation ofsolutions ofSNAC and NO

SNAC was prepared by reacting NO with a solutionof N-acetylcysteine under acidic, 02-free conditions.Gas-tight vials containing 10mm N-acetylcysteine inHCI (pH 2.0) were evacuated and then bubbled withN2 for 30min at room temperature (approximately20°C). The N-acetylcysteine solution was cooled to4°C and bubbled with 50ml NO gas over a 3minperiod, during which time the solution turned fromcolourless to rose-pink. The vial was placed on icefor 20 min and then bubbled with N2 for 20min toremove NO. Dilutions of the SNAC solution weremade in gas-tight vials containing HC1 (pH 2.0) thathad been evacuated and bubbled with N2 for 30minat room temperature. Saturated solutions of NOwere prepared in gas-tight vials under O2-free condi-tions. Vials containing 10 mm K2HPO4 (pH 6.0)were bubbled with N2 for 30min at room tem-perature and then cooled to 4°C. Solutions werethen bubbled for 4min with NO gas (50 ml).

Drugs

SNAP was a gift from Professor L.J. Ignarro(UCLA). Solutions of SNAP were prepared inethanol (bubbled with N2) and stored in gas-tightvials on ice. Glyceryl trinitrate (Merck, Darmstadt)was stored as a 10mm solution in ethanol, and dilu-tions were made into saline. Solutions of A23187(Sigma) were stored -and diluted in ethanol. Theo-phylline, methylene blue, cysteine, N-acetylcysteine,N-acetylpenicillamine were purchased from Sigmaand dilutions were made into water, unless otherwisestated. U46619 ((15S)-hydroxy-1 1a,9a-(epoxyme-thano)prosta-5Z,13-dienoic acid) was purchasedfrom Caymen Chemical Company (Denver);

760 P.J. HENRY et al.

1 mg ml - l stock solutions of U46619 in ethanol werestored at - 20'C and dilutions to 1 JUM were made in2.4mM NaHCO3.

Statistics

Results are expressed as mean + s.e.mean. For sta-tistical evaluation of the effects of drugs or pretreat-ment on vasodilator concentration-effect curves,one-way analysis of variance with repeated measureswas completed by means of the programmeBMDP2V of the BMDP series (Dixon & Brown,1977). For comparison of means, analysis of variancewas performed followed by Student's t test forunpaired observations. The Bonferroni method wasused for multiple comparisons (Wallenstein et al.,1980). P values less than 0.05 were regarded assignificant.

Results

Effects of N-acetylcysteine, cysteine, ascorbic acid andmethylene blue on vascular responses toS-nitrosothiols and NO

In U46619-contracted bovine isolated coronaryartery rings, single doses of SNAP induced repro-ducible, concentration-dependent relaxations thatwere of rapid onset and transient duration (Figurela and b). SNAP-induced relaxations were signifi-cantly attenuated (P < 0.05) in the presence of eitherN-acetylcysteine or ascorbic acid (Figures ic, d and2b) whereas neither N-acetylcysteine nor ascorbicacid had any significant effect on concentration-effectcurves to glyceryl trinitrate or to the endothelium-dependent vasodilator, A23187 (Figure 2a and c).Although N-acetylcysteine reduced the peak relax-ation response produced by single, submaximal con-centrations of SNAP, the 'duration of relaxation'was significantly enhanced in the presence of N-acetylcysteine (Figure 3a and c). Similarly, N-acetylcysteine inhibited peak relaxation responses toNO but prolonged its 'duration of relaxation'(P < 0.05), although to a lesser extent than thatobserved to SNAP (Figure 3b). On the other hand,inhibition of peak responses to SNAP by ascorbicacid, cysteine or methylene blue was not associatedwith any significant change in the 'duration of relax-ation' produced by SNAP (Figure 3a). As shown inFigure 3b, the 'duration of relaxation' produced bySNAC was significantly greater (P < 0.001) than thatproduced by either SNAP or NO alone and more-over, was similar to that produced by SNAP in thepresence of N-acetylcysteine. In the presence of100pM N-acetylcysteine, peak relaxation responses

a

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20

Figure 1 Isometric tension recordings of S-nitroso-N-acetylpenicillamine (SNAP)-induced relaxations inU46619-contracted bovine isolated coronary arteryrings. Single doses of SNAP (0) induced rapid relax-ation responses that returned to the previous level ofinduced tone without washout of the coronary arterypreparation. SNAP-induced relaxations wereconcentration-dependent (a), reproducible (b) and wereinhibited by ascorbic acid (c) and N-acetylcysteine (d).Numbers refer to SNAP concentrations in nM.

induced by SNAC were slightly inhibited. However,the 'duration of relaxation' was not affected (Figure3b and c).

Formation ofSNACfrom N-acetylcysteine

As shown in Figure 4, N-acetylcysteine and ascorbicacid markedly enhanced the breakdown of SNAPwith the concomitant formation of N-acetylpenicillamine. In the presence of N-acetylcysteine, but not ascorbic acid, there was alsorapid formation of SNAC. Generation of SNAC wasalso observed during the co-incubation of N-acetylcysteine and NO, although the extent of SNACgeneration was considerably less than that observedduring incubation of SNAP with N-acetylcysteine.

Effect ofprolonged vascular exposure to glyceryltrinitrate, SNAC and NO on subsequent vascularresponses to selected vasodilators

Bovine isolated coronary artery rings superfused for60min with glyceryl trinitrate, SNAC or NO weresignificantly less responsive (P < 0.05) to subse-

S-NITROSOTHIOLS AND TOLERANCE 761

aSNAP

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+ Cysteine

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Gyr. tInr M)lo-8 10-7 10-6 1o-5 10-4

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Figure 2 Mean cumulative concentration-effect curvesto glyceryl trinitrate (a), S-nitroso-N-acetylpenicillamine(SNAP) (b) and A23187 (c) in the absence (-) and pre-sence of 10yM N-acetylcysteine (0), 100yM N-acetylcysteine (0), 30OM ascorbic acid (A) and 3 pmmethylene blue (El) in U46619-contracted bovine iso-lated artery rings. Shown are the mean responses from6-12 coronary artery rings. Vertical lines indicates.e.mean. In these experiments, N-acetylcysteine, ascor-

bic acid or methylene blue were added 5 min before thestart of the vasodilator concentration-effect curve.

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Figure 3 (a) Effect of N-acetylcysteine (100pM), cyste-ine (100pM), ascorbic acid (301M) and methylene blue(3 FM) on the 'duration of S-nitroso-N-acetyl-penicillamine (SNAP)-induced relaxations'. The 'dura-tion of relaxation' was estimated from SNAP-inducedrelaxations that produced, on average, 52% relaxationof the U46619-induced contraction. Shown are themean of responses from 9-15 experiments. (b) 'Durationof relaxations' induced by SNAP, NO and SNAC in theabsence (open columns) and presence (solid columns) of100pM N-acetylcysteine. Shown are the mean ofresponses from 6-10 experiments. (c) Isometric tensionrecording in a single coronary artery showing responsesto S-nitroso-N-acetylcysteine (SNAC), and to SNAP inthe presence and absence of 100pM N-acetylcysteine(V). Numbers refer to SNAP and SNAC concentrationsin nm. In (a) and (b) bars show s.e.mean.

quently administered glyceryl trinitrate than werepaired control rings (Figure 5). Relaxations inducedby SNAC and NO were markedly attenuated(P < 0.01) in rings superfused with SNAC or NO,respectively, but only marginally attenuated in ringssuperfused with glyceryl trinitrate (Figure 6).Although rings superfused with NO were subse-quently significantly less responsive to NO than were

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762 P.J. HENRY et al.

SNAP

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Figure 4 Concentrations of S-nitroso-N-acetyl-penicillamine (SNAP) (open columns), N-acetylpenicillamine (solid columns) and SNAC (cross-hatched columns) determined from aliquots of reactionmixtures of SNAP alone (22.7pM), and SNAP in thepresence of either N-acetylcysteine (10 or 100pM) orascorbic acid (100pM). Shown are the mean concentra-tions (n = 4) determined after a 2min incubation inKrebs-bicarbonate solution bubbled with 95% 02 plus5% CO2 at 37°C. Bars shows s.e.mean.

0

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Figure 5 Mean concentration-effect curves to glyceryltrinitrate in control coronary artery rings (El) and inrings superfused for 60min with a Krebs-bicarbonatesolution containing glyceryl trinitrate (10pM, 0), S-nitroso-N-acetylcysteine (SNAG) (3 pM, *) or NO (5.p1NO stock ml-1 in Krebs-bicarbonate solution, 0).Shown are the mean responses from 8-20 experiments.Vertical lines indicate s.e.mean.

control rings (P < 0.01), relaxations induced byA23187 were only marginally attenuated in ringspre-exposed to either NO or glyceryl trinitrate(Figure 7). Similarly, theophylline was not signifi-cantly less potent in coronary artery rings superfusedwith glyceryl trinitrate, SNAC or NO than in controlrings (Figure 7).

Discussion

S-nitrosothiol compounds have previously beenshown to be potent activators of guanylate cyclase(Ignarro et al., 1981) and have been postulated to beintermediates of the vasodilator and anti-aggregatory effects produced by NO-containingcompounds (Loscalzo, 1985; Fung et al., 1988;Stamler et al., 1988a). Two S-nitrosothiols of particu-lar interest are SNAP and SNAC. Ignarro et al.(1981) showed that SNAP has a relatively greaterstability than S-nitrosothiols derived from cysteine,mercaptoethylamine and 3-mercaptopropanoic acidand is a potent vasodilator in vitro and in vivo. Inaddition, we have previously shown that, whereasthe vasodilator activities of glyceryl trinitrate, iso-sorbide dinitrate, sodium nitroprusside and 3-morpholinosydnonimine are markedly reduced incoronary arteries pre-exposed to glyceryl trinitrate,the vasodilator activity of SNAP is essentially unim-paired (Henry et al., 1989). More recent attention hasfocussed on the actions of SNAC. The extracellularformation of SNAC has been postulated to beresponsible for the enhanced vasodilator and anti-aggregatory effects observed for glyceryl trinitrate inthe presence of N-acetylcysteine (Loscalzo, 1985;Fung et al., 1988). We were therefore interested inexamining further the vasodilator effects of SNAPand SNAC, with particular emphasis on their forma-tion and interconversion, and also on the effects ofprolonged vascular exposure to S-nitrosothiol com-pounds.

Administration of a single dose of SNAC and,more particularly, SNAP to a constricted coronaryartery ring induced a rapid, but transient, relaxationresponse. The brief duration of relaxation was mostprobably due to the decomposition of SNAP andSNAC rather than to the desensitization of thevascular tissue to their vasodilator actions because,firstly, re-exposure of the tissue to subsequent dosesof S-nitrosothiol produces similar vasodilatoractions, and, secondly, S-nitrosothiol compounds aregenerally unstable chemically and often undergothermal decomposition at room temperature (Oae &Shinhama, 1983).Peak relaxation responses induced by SNAP were

significantly inhibited not only in the presence of thethiol compounds N-acetylcysteine and cysteine, but

S-NITROSOTHIOLS AND TOLERANCE 763

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Figure 6 Mean concentration-effect curves to (a, b) S-nitroso-N-acetylcysteine (SNAC) and (c, d) NO in controlrings (open symbols) and in rings superfused for 60min with Krebs-bicarbonate solution containing (a, c) glyceryltrinitrate (10Mm, *), (b) SNAC (3 pM, 0) or (c) NO (5 yl NO stock il-' in Krebs-bicarbonate solution, A). Shownare the mean responses from 6-7 experiments. Vertical lines indicate s.e.mean.

also in the presence of the non-thiol anti-oxidantascorbic acid. Although co-incubation of SNAP withN-acetylcysteine markedly inhibited peak relaxationresponses, the 'duration of relaxation' to SNAP wasprolonged two to three fold. No such prolongationwas observed during the co-incubation of SNAPwith either the related thiol cysteine or with ascorbicacid. Similarly, methylene blue, an inhibitor ofsoluble guanylate cyclase, also inhibited peak relax-ation responses to SNAP without affecting its 'dura-tion of relaxation'. In view of these results, weinvestigated the possibility that N-acetylcysteine,cysteine and ascorbic acid inhibited the peak relax-ation responses to SNAP by promoting SNAPdecomposition, and that the prolongation of SNAP-induced relaxations by N-acetylcysteine was due tothe formation of a significantly longer acting vasodil-

ator, SNAC. Consistent with this proposal was theinitial observation that relaxations induced bySNAC were of significantly greater duration thanthose induced by SNAP and, moreover, were ofsimilar duration to those induced by SNAP in thepresence of N-acetylcysteine. With the use of theh.p.l.c. assay we confirmed that co-incubation ofSNAP and N-acetylcysteine increased the rate ofbreakdown of SNAP, with the concomitant forma-tion of N-acetylpenicillamine and SNAC. Under theexperimental conditions studied, the interconversionof SNAP to SNAC was almost complete within 30s.At present the transfer of the NO moiety from anS-nitrosothiol to a thiol compound is not well docu-mented and, in general, reactions between S-nitrosothiol and thiol compounds have been used asconvenient methods for preparing mixed disulphides

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764 P.J. HENRY et al.

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Figure 7 Mean concentration-effect curves to (a)A23187 and (b) theophylline in control coronary arteryrings (0) and in rings superfused for 60min withKrebs-bicarbonate solution containing glyceryl tri-nitrate (10pM, *), SNAC (3pM, El) or NO (5p1 NOstock ml-' in Krebs-bicarbonate solution, 0). Shownare the mean responses from 5-7 experiments. Verticallines indicate s.e.mean.

rather than S-nitrosothiols (Oae & Shinhama, 1983).In the current study we have not examined thepotential formation of SNAC from the incubation ofN-acetylcysteine with S-nitrosothiols prepared fromthiols of physiological significance, such as cysteineor glutathione, although such a reaction may beexpected with S-nitrosocysteine given its relativelygreater chemical instability than SNAP (Ignarro etal., 1981).

In view of the recent findings that endothelium-derived relaxing factor (EDRF) is probably NO(Palmer et al., 1987), we examined the possibility thatincubation of N-acetylcysteine with NO also pro-duced SNAC. Whereas N-acetylcysteine and NOreact rapidly in an acidic, anaerobic environment toform SNAC, its formation was significantly less atneutral pH in an oxygenated aqueous buffer.However, in view of the recent findings of Fung et al.(1988), that SNAC is readily generated during co-incubation of glyceryl trinitrate and N-acetylcysteinein rat and human plasma but not in aqueous buffer,

we cannot exclude the possibility that significantamounts of SNAC would not be produced from NOin the presence of N-acetylcysteine in vivo. Further-more, such SNAC generation might contribute tothe recently described augmentation of anti-aggregatory effects of EDRF in the presence of N-acetylcysteine (Stamler et al., 1988b).

In addition to investigating some acute effects ofS-nitrosothiol compounds and NO on an isolatedcoronary artery preparation, we also examined someeffects of prolonged exposure to these agents on thevasculature. When compared to control coronaryartery rings, those superfused with a physiologicalsolution containing SNAC or NO were subsequentlymarkedly less responsive to the vasodilator actionsof these compounds and of glyceryl trinitrate. On theother hand, coronary artery ring responses to SNACand NO were not impaired by prolonged super-fusion with glyceryl trinitrate. These findings indicatethat the tolerances induced by sustained vascularexposure to glyceryl trinitrate or to either SNAC orNO were unlikely to have occurred by similarmechanisms. It has been proposed that NO-containing vasodilators share all, or parts of, a bio-conversion cascade that culminates in guanylatecyclase activation, cyclic GMP generation andsmooth muscle relaxation (Murad et al., 1978;Ignarro et al., 1981; 1984). Organic nitrate vasodila-tors, such as glyceryl trinitrate, are denitrated andthe liberated inorganic nitrite converted to NOwhich directly, or via formation of S-nitrosothiols,stimulates guanylate cyclase. Sustained vascularexposure to glyceryl trinitrate has been shown to beassociated with reduced denitration of glyceryl tri-nitrate (Brien et al., 1986) and probably impairedNO formation (Henry et al., 1989), but capacity forguanylate cyclase activation itself was, at most, onlymarginally reduced (Keith et al., 1982; Schroder etal., 1988; Henry et al., 1989). On the other hand, sus-tained exposure of the vasculature to SNAC or NOhas not previously been described, but in the currentstudy it was associated with marked attenuation ofresponses to glyceryl trinitrate, SNAC and NO,while responses to a guanylate cyclase-independentvasodilator such as theophylline were not reduced.These results were consistent with sustained vascularexposure to SNAC or NO producing impaired gua-nylate cyclase activity, as has previously been report-ed for NO actions on soluble guanylate cyclase invitro (Braughler, 1983). These findings were of partic-ular interest for several reasons. Firstly, if NO or anS-nitrosothiol compound is an active intermediate ofglyceryl trinitrate-induced vasodilatation, then thecurrent studies indicate that the development of tol-erance by glyceryl trinitrate at the proximal sites inthe bioconversion cascade may act to protect gua-nylate cyclase from continuous exposure to high

S-NITROSOTHIOLS AND TOLERANCE 765

concentrations of NO, S-nitrosothiols or both, whichmight otherwise impair its activity. Furthermore,these findings indicate that tolerance developed atthe more distal sites in the bioconversion cascade,such as guanylate cyclase activation, will impairvascular responsiveness to a greater range of vasodil-ators than will glyceryl trinitrate-induced toleranceand thus, may be potentially more difficult to cir-cumvent with agents that share this cascade ofevents. Secondly, the results may have some implica-tions as regards the possible identity of EDRF asNO (Palmer et al., 1987; Vanhoutte, 1987; Ignarro etal., 1988), which mediates the vasodilator actions ofthe calcium ionophore A23187 (Zawadzki et al.,1980; Furchgott, 1984). The current experimentsindicated that pre-exposure of coronary artery ringsto NO markedly attenuated subsequent vascularresponsiveness to NO, while responsiveness toA23187 was, at most, marginally impaired. Further-more, vascular responses to NO, more so than toA23187, were more attenuated by sustained vascularexposure to NO than by sustained exposure to glyc-eryl trinitrate. At present the reasons for these differ-ences are unclear. On face value, these differencesbetween the vasodilator actions of NO and A23187in the face of tolerance induced by NO or glyceryltrinitrate are not entirely consistent with NO beingthe mediator of A23187-induced, endothelium-dependent relaxation and possibly support findingsby other investigators suggesting that EDRF can bedistinguished from NO (Shikano et al., 1987;Dusting et al., 1988; Loeb & Peach, 1988). However,other mechanisms may be involved and at presentthe observed differences cannot be taken asunequivocal evidence that the relaxant actions ofA23187 were not mediated by release of EDRF asNO. For example, we cannot be certain that theextent of tolerance induced by the superfusion

methods is the same throughout the coronary arteryring preparation, or that the NO released from thevascular endothelium by the action of A23187 andthe NO added exogenously to the organ bath, stimu-late identical sites to induce relaxation of the cor-onary arteries. The observed differences could beexplained if a lesser degree of tolerance was inducedat particular sites in the coronary artery that wererelatively more important for mediating relaxationresponses to the NO released by the action ofA23187.

Finally, there are potentially important implica-tions as regards the therapeutic application of S-nitrosothiols or of co-administered glyceryl trinitrateand N-acetylcysteine (May et al., 1987; Packer et al.,1987; Horowitz et al., 1988) in efforts to optimizelong-term vasodilator and anti-platelet effects. Whileseveral studies indicate that short-term co-administration of glyceryl trinitrate and N-acetylcysteine may partially reverse nitrate tolerance(May et al., 1987; Packer et al., 1987; Fung et al.,1988), there is little clinical experience to date withlong-term co-administration of these agents.Although it is possible that administration of sul-phydryl compounds such as N-acetylcysteine willinitially potentiate the haemodynamic effects of glyc-eryl trinitrate by generating S-nitrosothiol com-pounds (Fung et al., 1988), the results of the currentstudy indicate that during long-term vascular expo-sure to S-nitrosothiols the initial beneficial effectsmay not be maintained and indeed vascularresponses to a wide range of NO-containing vasodil-ators may be markedly attenuated.

This work was supported by a Programme Grant from theNational Health and Medical Research Council of Aus-tralia.

References

ABRAMS, J. (1986). Tolerance to organic nitrates. Circula-tion, 74, 1181-1185.

BRIEN, J.F., MCLAUGHLIN, B.E., BREEDON, T.H.,BENNETT, B.M., NAKATSU, K. & MARKS, G.S. (1986).Biotransformation of glyceryl trinitrate occurs concur-rently with relaxation of rabbit aorta. J. Pharmacol.Exp. Ther., 237, 608-614.

BRAUGHLER, J.M. (1983). Soluble guanylate cyclase activa-tion by nitric oxide and its reversal. Involvement of sul-phydryl group oxidation and reduction. Biochem.Pharmacol., 32, 811-818.

DIXON, W.J. & BROWN, M.G. (1977). Biomedical ComputingPrograms, P series. Berkeley: University of CaliforniaPress.

DUSTING, G.J., READ, M.A. & STEWART, A.G. (1988).Endothelium-derived relaxing factor released from

cultured cells: differentiation from nitric oxide. Clin.Exp. Pharmacol. Physiol., 15, 83-92.

FUNG, H-L., CHONG, S., KOWALUK, E., HOUGH, K. &KAKEMI, M. (1988). Mechanism for the pharmaco-logical interaction of organic nitrates with thiols. Exis-tence of an extracellular pathway for the reversal ofnitrate vascular tolerance by N-acetylcysteine. J. Phar-macol. Exp. Ther., 245, 524-530.

FURCHGOTT, R.F. (1984). The role of endothelium in theresponses of vascular smooth muscle to drugs. Ann. Rev.Pharmacol. Toxicol., 24, 175-197.

HENRY, P.J., HOROWITZ, J.D. & LOUIS, W.J. (1989).Nitroglycerin-induced tolerance affects multiple sites inthe organic nitrate bioconversion cascade. J. Pharmacol.Exp. Ther., 248, 762-768.

HOROWITZ, J.D., ANTMAN, E.M., LORELL, B.H., BARRY,

766 P.J. HENRY et al.

W.H. & SMITH, T.W. (1983). Potentiation of the cardio-vascular effects of nitroglycerin by N-acetylcysteine.Circulation, 68, 1247-1253.

HOROWITZ, J.D. & HENRY, P.J. (1987). Recent develop-ments in nitrate therapy of ischaemic heart disease.Med. J. Aust., 146, 93-96.

HOROWITZ, J.D., HENRY, C.A., SYRJANEN, M.L., LOUIS,WJ., FISH, R.D., SMITH, T.W. & ANTMAN, E.M. (1988).Combined use of nitroglycerin and N-acetylcysteine inthe management of unstable angina pectoris. Circula-tion, 77, 787-794.

IGNARRO, L.J., LIPPTON, H., EDWARDS, J.C., BARICOS,W.H., HYMAN, A.L., KADOWITZ, P.J. & GRUETTER, C.A.(1981). Mechanism of vascular smooth muscle relax-ation by organic nitrates, nitrites, nitroprusside andnitric oxide: Evidence for the involvement of S-nitrosothiols as active intermediates. J. Pharmacol. Exp.Ther., 218, 739-749.

IGNARRO, L.J., GRUETTER, C.A., HYMAN, A.L. & KADO-WITZ, P.J. (1984). Molecular mechanisms of vasodilata-tion. In Dopamine Receptor Agonists. ed. Poste, G. &Grooke, S.T. pp. 259-288. New York: Plenum Pub-lishing Corporation.

IGNARRO, L.J., BUGA, G.M., BYRNS, R.E., WOOD, K.S. &CHAUDHURI, G. (1988). Endothelium-derived relaxingfactor and nitric oxide possess identical pharmaco-logical properties as relaxants of bovine arterial andvenous smooth muscle. J. Pharnacol. Exp. Ther., 246,218-226.

KEITH, R.A., BURKMAN, A.M., SOKOLOSKI, T.D. &FERTEL, R.H. (1982). Vascular tolerance to nitroglycerinand cyclic GMP generation in rat aortic smoothmuscle. J. Pharmacol. Exp. Ther., 221, 525-531.

KOWALUK, E.K., POLISZCZUK, R. & FUNG, H-L. (1987).Tolerance to relaxation in rat aorta: comparison of anS-nitrosothiol with nitroglycerin. Eur. J. Pharmacol.,144, 379-383.

LEVY, W.S., KATZ, R.J., RUFFALO, R.L., LEIBOFF, R.H. &WASSERMAN, A.G. (1988). Potentiation of the hemo-dynamic effects of acutely administered nitroglycerin bymethionine. Circulation, 78, 640-645.

LOEB, A.L. & PEACH, M.J. (1988). EDRF released from cul-tured endothelium can be distinguished from nitricoxide. Hypertension, 12, 334.

LOSCALZO, J. (1985). N-acetylcysteine potentiates inhibi-tion of platelet aggregation by nitroglycerin. J. Clin.Invest., 76, 703-708.

MAY, D.C., POPMA, J.J., BLACK, W.H., SCHAEFFER, S., LEE,H.R., LEVINE, B.D. & HILLIS, L.D. (1987). In vivo induc-tion and reversal of nitroglycerin tolerance in humancoronary arteries. N. Engl. J. Med., 317, 805-809.

MURAD, F., MITTAL, C.K., ARNOLD, W.P., KUTUKI, S. &KIMERA, H. (1978). Guanylate cyclase: activation byazide, nitro compounds, nitric oxide, and hydroxylradical and inhibition by haemoglobin and myoglobin.Adv. Cyclic Nucleotide Res., 9, 145-158.

OAE, S. & SHINHAMA, K. (1983). Organic thionitrites andrelated substances. A review. Organ. Prep. Proc. Intern.,15, 165-198.

PACKER, M., LEE, W.H., KESSLER, W.H., GOTTLIEB, S.S.,MEDINA, N. & YUSHAK, M. (1987). Prevention andreversal of nitrate tolerance in patients with congestiveheart failure. N. Engl. J. Med., 317, 799-804.

PALMER, R.M.J., FERRIGE, A.G. & MONCADA, S. (1987).Nitric oxide release accounts for the biological activityof endothelium-derived relaxing factor. Nature, 327,524-526.

SCHRODER, H., LEITMAN, D.C., BENNETT, B.M.,WALDMAN, S.A. & MURAD, F. (1988). Glyceryltrinitrate-induced desensitization of guanylate cyclase incultured rat lung fibroblasts. J. Pharmacol. Exp. Ther.,245, 413-418.

SHIKANO, K., OHLSTEIN, E.H. & BERKOWITZ, B.A. (1987).Differential sensitivity of endothelium-derived relaxingfactor and nitric oxide in smooth muscle. Br. J. Phar-macol., 92, 483-485.

STAMLER, J., CUNNINGHAM, M. & LOSCALZO, J. (1988a).Reduced thiols and the effect of intravenous nitro-glycerin on platelet aggregation. Am. J. Cardiol., 62,377-380.

STAMLER, J., MENDELSOHN, M., COOKE, J., DAVIES, P.F.& LOSCALZO, J. (1988b). Inhibition of platelet aggre-gation by endothelium-derived relaxing factor is poten-tiated by reduced thiols. Clin. Res., 36, 320A.

VANHOUETTE, P.M. (1987). The end of the quest? Nature,327, 459-460.

WALLENSTEIN, S., ZUCKER, C.L. & FLEISS, J.L. (1980).Some statistical methods useful in circulation research.Circ. Res., 47, 1-9.

ZAWADZKI, J.V., CHERRY, P.D. & FURCHGOTT, R.F. (1980).Comparison of endothelium-dependent relaxation ofrabbit aorta by A23187 and by acetylcholine. Pharma-cologist, 22, 271.

(Received January 25, 1989Revised July 4, 1989

Accepted July 17, 1989)