Clinical and Experimental Pharmacology and Physiology (2003) 30, 709–711
SHORT COMMUNICATION
DIETHYLENETRIAMINE NITRIC OXIDE ADDUCT RELAXES PRECONTRACTED MOUSE TRACHEAL SMOOTH MUSCLE* Chen Fuh Lam,† Rommel S Lan,† Peter V van Heerden,†‡ Kenneth F Ilett† and Peter J Henry† †
Pharmacology Unit, School of Medicine and Pharmacology, University of Western Australia, Crawley and ‡ Department of Intensive Care, Sir Charles Gairdner Hospital, Nedlands, Western Australia, Australia
SUMMARY 1. Inhaled diethylenetriamine nitric oxide adduct (DETA/ NO) has been shown to be a selective pulmonary vasodilator in animal and human studies. The aims of the present study were to investigate the effect of DETA/NO on mouse precontracted isolated tracheal smooth muscle preparations and to determine the active component of this compound. 2. Mouse isolated tracheal smooth muscle rings were precontracted with carbachol (10–7 mol/L). Changes in isometric tension were recorded after cumulative addition of DETA (30–300 mol/L; n = 6), DETA/NO (30–300 mol/L; n = 9) or diluent control (n = 3). In addition, some preparations (n = 5) were pretreated with the soluble guanylyl cyclase (sGC) inhibitor 1H-[1,2,4]-oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ; 30 mol/L) before precontraction and exposure to DETA/NO. 3. Addition of DETA/NO caused a concentration-dependent relaxation of tracheal smooth muscle at 100 and 300 mol/L, with an EC25%R of 109 mol/L (95% confidence interval 72.6– 164 mol/L). The nucleophile amine carrier DETA had no effect on isometric tension. However, the relaxant effect of DETA/NO was completely abolished by pretreatment with ODQ. 4. We conclude that DETA/NO induces a concentrationdependent relaxation of mouse carbachol-contracted isolated tracheal smooth muscle that is mediated by NO released from DETA/NO via the activation of sGC. Key words: diethylenetriamine nitric oxide adduct, nitric oxide, soluble guanylyl cyclase, trachea.
dependent and diminishes quickly when NO administration is ceased.2 However, because gaseous NO is highly lipophilic, its effect is very unpredictable when delivered to the airways, which are covered in mucus.3 The NONOates or diazeniumdiolates (1-substituted diazen-1ium-1,2-diolates) are novel compounds that spontaneously release NO in vivo and in vitro at predictable rates. Because NONOates are highly water soluble, they could be ideal substitutes for gaseous NO in the treatment of airway diseases, such as asthma.3 Diethylenetriamine nitric oxide adduct (DETA/NO) has the longest in vitro NO-generating half-life (of 20 h) from the available NONOates.4 Inhaled DETA/NO has been shown to be a selective pulmonary vasodilator in animals5 and humans.6 However, the direct effects of DETA/NO on airways, where the majority of the drug is deposited after inhalation, have not been investigated previously. The present study was designed to investigate the effect of DETA/NO on precontracted mouse tracheal smooth muscle preparations in vitro and to determine the active component of DETA/NO.
METHODS Mouse isolated tracheal preparations Eight-week-old male CBA/CaH mice (Animal Resource Centre, Murdoch, WA, Australia) were used. The experiments were approved by the Animal Experimentation and Ethics Committee of the University of Western Australia. Mice were killed with pentobarbitone sodium (250 mg/kg, i.p.). A section of the trachea was dissected free from the surrounding tissue and bisected into two tracheal rings, each approximately 2 mm long.
Materials INTRODUCTION Nitric oxide (NO) mediates a direct smooth muscle relaxant effect via the formation of cGMP.1 The effect is of rapid onset, dose
Carbachol, DETA and DETA/NO were obtained from Sigma Aldrich (Castle Hill, NSW, Australia) and 1H-[1,2,4]-oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ) was obtained from Tocris Cookson (Bristol, UK). All other chemicals were of analytical reagent grade.
Isometric tension measurements Correspondence: Dr PV van Heerden, Department of Intensive Care, Sir Charles Gairdner Hospital, Hospital Avenue, Nedlands, WA 6009, Australia. Email:
[email protected] *This study was presented at the 27th Australian and New Zealand Intensive Care Society Annual Scientific Meeting, Perth, Australia, October 2002. Received 16 December 2002; revision 16 April 2003; accepted 27 April 2003.
Tracheal rings were mounted in an organ bath containing 2 mL Krebs’ bicarbonate solution (composition (in mmol/L): NaCl 117; KCl 5.36; NaHCO3 25; KH2PO4 1.03; MgSO4·7H2O 0.57; CaCl2 2.5; D-glucose 11.1), as described previously.7 The bath was maintained at 37C and preparations were aerated continuously with 5% CO2 in oxygen. Changes in muscle tension were recorded using an isometric force transducer (Model FTO3; Grass Telefactor, West Warwick, RI, USA) connected to a custom-built preamplifier and data-acquisition system. Preparations were washed every 15 min during a 45 min equilibration period and tension was adjusted to
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500 mg. After conditioning by exposure to sequential concentrations of carbachol (0.2 and 10 mol/L, yielding submaximal and supramaximal (Cmax) responses, respectively),7 preparations were washed with fresh Krebs’ solution for approximately 20 min to restore a steady baseline.
Effects of DETA/NO and DETA Tracheal preparations were precontracted with 0.1 mol/L carbachol producing a resting tension of approximately 60–70% of the carbachol Cmax. Cumulative dose–response curves (30, 100 and 300 mol/L) were constructed for DETA/NO (n = 9), DETA (n = 6) and diluent control (same volume of 0.9% w/v NaCl as used to add DETA/NO or DETA; n = 3). The extent of relaxation was expressed as a percentage of the starting level of carbachol-induced tension.
Effects of ODQ on response to DETA/NO Tracheal preparations (n = 5) were pre-incubated for 30 min with 30 mol/L ODQ (dissolved using dimethylsulphoxide at a final concentration of 0.1% v/v in the organ bath). After precontraction with carbachol as above, cumulative dose–response curves to DETA/NO (30, 100 and 300 mol/L) were constructed again as above.
Statistical analyses Data are presented as the mean±SEM. The effects of the various treatments on isometric tension in smooth muscle preparations were examined by analysis of variance (ANOVA; SigmaStat; SPSS, Chicago, IL, USA). The concentration of DETA/NO causing a 25% relaxation response in isometric tension (EC25%R) was interpolated from the log-transformed dose–response data. The EC25%R is presented as a geometric mean with associated 95% confidence intervals (CI).
RESULTS
the nucleophile and can vary from 1 min to 1 day under physiological conditions.4 Among the NONOates available, DETA/NO has the longest NO generation half-life of approximately 20 h. Interest in these compounds centres on their ability to relax smooth muscle in both vascular and extravascular tissues. Potential pulmonary toxicity from DETA/NO administration may arise from either the NO or the nucleophile parts of the molecule. Leung et al.8 fed rats a diet containing high concentrations of DETA (1000–15 000 p.p.m. for 90 days) and found a stunting of growth but no significant macroscopic or microscopic changes in the major organs at autopsy. Diethylenetriamine NO adduct also inhibits DNA synthesis in rat aorta smooth muscle cells (IC50 = 40 mmol/L).9 However, cell viability was > 95% and, hence, this antiproliferative effect did not result from direct cytotoxicity. In another study, aerosolized DETA/NO did not cause an increase in lung wet to dry weight ratio after 4 days treatment in rats.10 We have recently investigated the potential toxicity of aerosolized DETA/NO after single dose (60 mol in pigs and rats) and multiple dose (60 mol/day for up to 14 days in rats) exposure.11 In both species, serum NO2– was significantly increased after DETA/NO, but arterial methaemoglobin was unchanged. In addition, polymorphonuclear leucocytes infiltration and lung wet to dry weight ratio were not altered by DETA/NO. Thus, a limited number of previous studies suggest a low toxicity profile for both DETA and DETA/NO. Our results showed that DETA/NO induced a concentrationdependent relaxation on precontracted mouse tracheal smooth muscle. Because others have shown previously that the carrier amine in NONOates can sometimes contribute to smooth muscle relaxation,12 we also studied the effects of DETA. However, DETA had no effects over the same molar concentration range used for
Diethylenetriamine NO adduct induced concentration-dependent relaxation in mouse isolated tracheal smooth muscle rings precontracted with carbachol to 60–70% Cmax (Fig. 1). The mean concentration of DETA/NO that produced 25% relaxation (EC25%R) was 109 mol/L (95% CI 72.6–164 mol/L). The nucleophile amine carrier DETA (n = 6) had no effect on smooth muscle tension (data not shown). In the presence of 30 mol/L ODQ, DETA/NO did not induce any significant relaxation in precontracted tracheal smooth muscle rings (Fig. 1).
DISCUSSION Nitric oxide induces smooth muscle relaxation directly by activating sGC, which, in turn catalyses the production of intracellular cGMP.1 The accumulation of cGMP alters the activity of intracellular kinases that decrease intracellular Ca2+, resulting in smooth muscle relaxation. However, being highly lipophilic, the effect of gaseous NO is very unpredictable when delivered to tissues covered with mucus, such as the airways.3 In addition, due to its extremely short biological half-life and rapid inactivation, gaseous NO has to be delivered continuously, using a complex delivery system, to be clinically useful as a smooth muscle relaxant.4 The NONOates are a novel group of compounds that are highly water soluble and carry NO in the [N(O)NO]– functional group; under physiological conditions each mol NONOate releases 2 mol NO.4 The decomposition time or half-life of NO generation for NONOates depends on pH, temperature and the chemical nature of
Fig. 1 Peak relaxation responses induced by control diluent (; n = 3), diethylenetriamine nitric oxide adduct (DETA/NO; ; n = 9) and 1H[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one plus DETA/NO (; n = 5) in mouse isolated tracheal smooth muscle preparations precontracted by carbachol (10–7 mol/L). *P < 0.05, †P < 0.001 for control versus DETA/ NO.
DETA/NO relaxes mouse tracheal smooth muscle DETA/NO. As expected, ODQ, an inhibitor of guanylyl cyclase, inhibited the NO-mediated relaxant effects of DETA/NO. Our findings are consistent with those of Hirasaki et al., who have reported previously that other NONOates, namely diethylamine/NO (DEA/NO) and spermine/NO, caused a concentrationdependent increase in intracellular cGMP and relaxation in canine airway smooth muscle.3 The EC25%R for DETA/NO for inducing relaxation was 109 mol/L. This was much higher than mean EC50%R values reported by Hirasaki et al. for DEA/NO (0.13 mol/L) and spermine/NO (4.1 mol/L) for relaxing canine airway smooth muscle.3 Because each mol of a NONOate releases 1.5–2 mol NO, these differences in potency are most likely a result of different NO generation half-lives (2 min for DEA/NO, 39 min for spermine/NO and 20 h for DETA/NO)4 and/or direct smooth muscle relaxation effects that have been demonstrated, at least for spermine.12 The use of different animal species and differences in tissue distribution and metabolism of the various NONOates may also contribute to their differing potencies. In conclusion, DETA/NO induced a concentration-dependent relaxant response in mouse isolated tracheal smooth muscle and this effect was mediated by the NO component of the drug via the activation of sGC. A recent study from our laboratory has shown that DETA/NO has significant potential as a therapy for acute pulmonary hypertension in humans.6 Because of its demonstrated effects on respiratory smooth muscle and long NO-generating halflife, we suggest that DETA/NO may offer therapeutic benefits in a variety of lung diseases.
ACKNOWLEDGEMENT This study was supported, in part, by a grant from the Sir Charles Gairdner Hospital Research Fund.
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