Discrepancies Between Nitroglycerin and NO-Releasing Drugs on Mitochondrial Oxygen Consumption, Vasoactivity, and the Release of NO
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循环研究杂志 2005年第11期
the Departamento de Farmacologe (J.V.E.) and Unidad Mixta Universidad de ValenciaeCFundacie Centro Nacional de Investigaciones Cardiovasculares Carlos III (C.N., V.M.V., R.T., P.D.), Facultad de Medicina, Universidad de Valencia, Blasco Ibez, Valencia, Spain
Unidad de Citometria, Fundacie Centro Nacional de Investigaciones Cardiovasculares Carlos III (A.A.-B.), C/Melchor Fernedez Almagro, Madrid, Spain
The Wolfson Institute for Biomedical Research (S.M.), University College London, United Kingdom.
Abstract
It has been generally acknowledged that the actions of glyceryl trinitrate (GTN) are a result of its bioconversion into NO. However, recent observations have thrown this idea into doubt, with many studies demonstrating that NO is present only when there are high concentrations of GTN. We have explored this discrepancy by developing a new approach that uses confocal microscopy to directly detect NO. Intracellular levels of NO in the rat aortic vascular wall have been compared with those present after incubation with 3 different NO donors (DETA-NO, 3-morpholinosydnonimine, and S-nitroso-N-acetylpenicillamine), endothelial activation with acetylcholine, or administration of GTN. We have also evaluated the relaxant effects of these treatments on isolated rings of aorta following activation of the enzyme soluble guanylyl cyclase and their inhibitory action on mitochondrial respiration, which is an index of the interaction of NO with the enzyme of the electron transport chain cytochrome C oxidase. In the case of the various NO donors and acetylcholine, we detected a concentration-dependent relationship in the intensity of vascular relaxation and degree of NO fluorescence and an increase in the Michaelis constant (Km) for O2. GTN did not produce similar effects, and although clinically relevant concentrations of this compound caused clear, concentration-related relaxations, there was neither any increase in NO-related fluorescence nor an augmented Km for O2. The nature of these differences suggests that these concentrations of GTN do not release free NO but probably a different species that, although it interacts with soluble guanylyl cyclase in vascular smooth muscle, does not inhibit O2 consumption by vascular mitochondria.
Key Words: glyceryl trinitrate nitric oxide mitochondria vascular relaxation NO donors
Introduction
It is generally acknowledged that the action of glyceryl trinitrate (GTN) is a result of its bioconversion into the vasorelaxant agent NO.1,2 This idea is based on reports, both in vitro and in vivo,3eC7 that suggest that exposure to GTN leads to the formation of NO. However, in most of these studies, NO was observed only when GTN concentrations considerably exceeded the plasma levels reached during clinical dosing, whereas a number of further reports question the direct formation of NO.8eC10 Furthermore, the precise mechanism by which GTN is biotransformed is also currently a subject of controversy.11
Using confocal microscopy to observe the cells of vessels, we detected endothelium-synthesized NO and compared it with that released by different NO donors and with GTN. Furthermore, the vasodilatatory effects of NO and its consequences for mitochondrial respiration have also been evaluated. By studying these 2 targets of NO,12,13 we identified inconsistencies between the effects of NO, produced both endogenously and pharmacologically, and those obtained with GTN used at clinically relevant concentrations. The nature of these discrepancies suggests that the actions of GTN on the vasculature are indeed unrelated to its bioconversion to NO.
Materials and Methods
Animals
Male SpragueeCDawley rats (200 to 250 g; Harlan Laboratories, Barcelona) were decapitated and their thoracic aortas removed, cleaned of adhering tissues in Krebs solution, and cut into rings of 1 mm (for image analysis) or 5 mm (all other experiments). Unless otherwise stated, all results are mean±SEM of at least 5 experiments, each in a different animal. All protocols complied with the European Community guidelines for the use of experimental animals and were approved by the Ethics Committee of the University of Valencia.
Contractility Studies
Aortic rings were suspended in a 5-mL organ bath containing Krebs solution at 37°C. The solution was constantly gassed with 12% O2, 5% CO2, and 83% N2, producing an O2 concentration of 12 to 13x10eC5 mol/L in the bath, which was monitored with a dissolved O2 meter (ISO2; World Precision Instruments, Stevenage, Herts, UK). This level of O2 is similar to that present in the aortic blood.13 Following an equilibration period of 75 to 90 minutes, rings were submaximally (75% to 85% of maximal response) contracted with 10eC6 mol/L phenylephrine (Phe), and the presence of a functional endothelium was confirmed by eliciting a relaxant response (>90%) to 10eC5 mol/L acetylcholine (ACh). Only those vessels exhibiting this response were included in what remained of the experiment.
Following a further equilibration period of 30 to 45 minutes, rings were contracted with a depolarizing solution (6x10eC2 mol/L KCl). After being washed, the vessels returned to the baseline and a further Phe concentration of 10eC6 mol/L was added, giving a sustained contraction equivalent to the response to depolarization. Once such a stable tone was achieved, relaxation-response curves were obtained by adding cumulative concentrations of the NO donors (Z)-1-([2-aminoethyl]-N-[2-ammonioethyl]amino)diazen-1-IM1,2-diolate (DETA-NO) (10eC8 to 10eC5 mol/L), S-nitroso-N-acetylpenicillamine (SNAP) (10eC10 to 10eC5 mol/L), or 3-morpholinosydnonimine (SIN-1) (10eC11 to 10eC6 mol/L); and ACh (10eC9 to 10eC5 mol/L) or GTN (10eC10 to 10eC6 mol/L). In some experiments, the soluble guanylyl cyclase (sGC) inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) (5x10eC6 mol/L), the scavenger of NO and related species oxyhemoglobin (oxyHb) (10eC5 mol/L), or the NO synthase inhibitor N--nitro-L-arginine (L-NNA) (10eC4 mol/L) was added before the NO-producing drugs. The adequate concentration (eClog[M]) for producing 50% relaxation (pEC50) was obtained from a nonlinear regression analysis with Graph Pad Software. The percentage of relaxation obtained with the highest concentration of each agent in the absence (control) or presence of ODQ (5x10eC6 mol/L) or oxyHb (10eC5 mol/L) is represented by Emax.
Confocal Microscopy Imaging of NO Release
NO was visualized with diaminorhodamine-4M (DAR-4M AM), a cell permeable, fluorescent precursor with a convenient profile for NO bioimaging. It exhibits low background fluorescence with longer wavelength excitation, allows detection throughout a wider pH range, and is more photostable than standard fluorochromes that have been used previously for NO imaging.14 Inside the cell, the relatively nonfluorescent DAR-4M AM is converted into its triazole form, DAR-4M T, the fluorescence intensity of which is directly proportional to the concentration of NO, with a detection limit of 7x10eC9 mol/L.14
Aortic rings were incubated in the dark for 60 minutes in a Krebs solution that was constantly gassed (12% O2, 5% CO2, and 83% N2) and that contained DAR-4M AM (2.5x10eC6 mol/L). The vital cell nuclei staining fluorochrome Hoechst 33342 (10eC6 mol/L) was added to this solution after 30 minutes. Thereafter, the rings were mounted in a vertical position on 24x50-mm coverslips, fixed with a grid in Krebs solution, and placed in a heated chamber (37°C, 2 mL; Warner Instrument Corp, Hamden, Conn). Following a 10-minute stabilization period, which was considered basal, cumulative concentrations of DETA-NO (10eC7 to 10eC4 mol/L), SNAP (10eC9 to 10eC5 mol/L), SIN-1 (10eC9 to 10eC6 mol/L), ACh (10eC6 to 10eC4 mol/L), or GTN (10eC9 to 10eC6 mol/L) were added every 10 minutes. In some experiments, before addition of the agents, tissues were preincubated (10 minutes) with L-NNA (10eC4 mol/L). Images were obtained every 2 minutes throughout the entire experiment, which lasted a maximum of 60 minutes. A concentration of DETA-NO (10eC4 mol/L) was added at the end of each experiment to monitor the sensitivity of our system, and, to avoid variability, the response obtained was considered a reference for normalizing the signal elicited in each experimental series. To ensure that the fluorochromes had no influence on the viability of the tissues, parallel experiments were performed in which rings were incubated with these compounds under the same experimental circumstances that confocal analysis and contractility studies were performed. No significant differences were found either in the response to a single concentration of Phe (10eC6 mol/L) or in the concentration-response curves of relaxation to DETA-NO (pEC50: 5.98±0.09 in control versus 5.90±0.08, n=4 and 5, respectively), ACh (pEC50: 7.33±0.10 in control versus 7.23±0.21, n=5), and GTN (8.08±0.06 in control versus 8.40±0.13, n=5 and 6, respectively).
Images were observed in water immersion (x60 magnification) and studied with a Radiance 2100 confocal microscope (Bio-Rad, Hempel Hempstead, UK) using a 405-nm diode laser to excite Hoechst 33342 and a 546-nm He-Ne laser to excite DAR-4M AM. Fluorescence was detected through 440/20 band pass (BP) and 570 long pass (LP) filters for Hoechst 33342 and DAR-4M AM, respectively. DAR-4M AM fluorescence was analyzed (Laserpix software; Bio-Rad) in portions of aortic ring comprising the entire width of the vessel, from the adventitia to the endothelium. Confocal microscope settings were adjusted to produce the optimum signal/noise ratio. The vessel wall exhibited basal red fluorescence, representing the autofluorescence of the elastin fibers, which was deducted from the total quantification of fluorescence in each specific measurement.
Further experiments were performed to exclude the interference on DAR-4M AM fluorescence by GTN or some of its main intermediate metabolites. Different concentrations (10eC9 to 10eC4 mol/L) of GTN, its metabolites 1,2 glyceryl dinitrate (1,2-GDN) and 1,3 glyceryl dinitrate (1,3-GDN), and sodium nitrate (NaNO3) or sodium nitrite (NaNO2) were incubated in the presence of DAR-4M AM (2.5x10eC6 mol/L) in a cell-free Krebs solution. Fluorescence was measured with a Fluoroskan plate reader (Thermo Labsystems) and 10eC4 mol/L DETA-NO was added at the end of each experiment. No modification of the fluorochrome basal fluorescence was obtained after incubation with GTN or any of the compounds tested for 60 minutes. Furthermore, the increase in fluorescence observed after addition of DETA-NO was similar in the presence or absence of these agents (n=4 to 5 experiments; results not shown).
Electrochemical Measurement of O2 Consumption
The aorta of 2 animals (97.65±1.04 mg total wet weight) were cut in rings and placed in gas-tight chambers containing 1 mL of Krebs solution and gently agitated at 37°C. The O2 consumption by the tissue was measured with a Clark-type O2 electrode (Rank Brothers, Bottisham, UK) calibrated with an air-saturated Krebs solution, assuming an O2 concentration of 2x10eC4 mol/L. Experiments were performed in the presence of DETA-NO (10eC8 to 10eC4 mol/L), SNAP (10eC9 to 10eC5 mol/L), SIN-1 (10eC9 to 10eC6 mol/L), ACh (10eC7 to 10eC4 mol/L), or GTN (10eC9 to 10eC6 mol/L). Sodium cyanide (10eC3 mol/L) was used to confirm that O2 consumption was mainly mitochondrial (&95% to 99%). In some experiments, the reversibility of NO-induced inhibition of respiration was assessed by adding oxyHb (10eC5 mol/L) when the concentration of O2 in the chamber was 10eC4 mol/L. Measurements were collected using a data-acquisition device, Duo.18 (World Precision Instruments, Stevenage, UK). A hyperbolic function was used to describe the relationship between O2 concentration and the rate of O2 consumption (VO2).15 The maximal rate of O2 consumption (VO2max) and the apparent O2 affinity Michaelis constant (Km) (10eC6 mol/L) were calculated according to their analogy with the MichaeliseCMenten constant. The rate of O2 consumption at the interval between 3 to 2x10eC5 mol/L of O2 (similar to conditions during hypoxia16,17) was calculated from the same experiments. To avoid variability, the rate of O2 consumption was expressed as 10eC9 mol O2/min per 10eC6 g protein. Proteins were determined by the BCA protein assay kit (Pierce, Rockford, Ill) using BSA as the standard. In some cases, the viability of the rings was later assessed by confirming that their contractile response to Phe (10eC6 mol/L) and ACh (10eC5 mol/L) did not differ from that of nonmanipulated vessels.
Data Analysis
Values are expressed as mean±SEM. Statistical analysis was performed by 1-way ANOVA followed by the Student t test for unpaired samples (Graph Pad Software). Significance was defined as P<0.05.
Drugs and Solutions
The composition of the Krebs solution was as follows (10eC3 mol/L): 118 NaCl, 4.75 KCl, 1.9 CaCl2, 1.2 MgSO4, 1.2 KH2PO4, 25 NaHCO3, and 10.1 glucose. NaNO3 and NaNO2 were obtained from Panreac (Barcelona, Spain). 1,2-GDN and 1,3-GDN were purchased from LGC Promochem (Barcelona, Spain). Phe, ACh, L-NNA, sodium cyanide, ODQ, meth(a)emoglobin, and Hoechst 33342 were acquired from Sigma Aldrich (St Louis, Mo). DETA-NO was obtained from Alexis (Lausen, Switzerland). A clinically available preparation of GTN was used (Solinitrina from Allmirall Prodesfarma, Barcelona, Spain), and DAR-4M AM was obtained from Calbiochem (San Diego). OxyHb was prepared by reduction of human meth(a)emoglobin with a 10-fold molar excess of sodium dithionite, followed by dialysis against PBS. All drugs were dissolved in ultrapure water, except GTN, DAR-4M AM, and Hoechst 33342, which were dissolved in Krebs solution, and ODQ, which was dissolved in dimethyl sulfoxide (1:5000 [vol/vol]).
Results
Contractility Studies
Table 1 shows pEC50 and Emax of the relaxation curves induced in vessels precontracted in vitro with Phe (10eC6 mol/L) by cumulative concentrations of DETA-NO (10eC8 to 10eC5 mol/L), SNAP (10eC10 to 10eC5 mol/L), SIN-1 (10eC11 to 10eC6 mol/L), ACh (10eC9 to 10eC5 mol/L), and GTN (10eC10 to 10eC6 mol/L), respectively. The presence in the organ bath of the sGC inhibitor ODQ (5x10eC6 mol/L) prevented the vasorelaxant activity of all 5 substances. Likewise, incubation with the NO scavenger oxyHb inhibited the effects of DETA-NO, ACh, and GTN. The effects of ACh were inhibited by prior incubation with the NO synthesis inhibitor L-NNA (10eC4 mol/L) (data not shown).
Confocal Microscopy Imaging of NO Release
Figure 1A shows representative confocal images of the rat aorta following incubation (60 minutes) with DAR-4M AM and concentration-dependent increases in the red fluorescent signal induced by addition of cumulative concentrations of DETA-NO (10eC7 to 10eC4 mol/L) every 10 minutes. An analysis of the total changes in red fluorescence is presented in Figure 1B. Figure 1 also shows significant and concentration-dependent increases in NO-related DAR-4M AM fluorescence obtained with the other 2 NO donors used, SNAP (10eC9 to 10eC5 mol/L) and SIN-1 (10eC9 to 10eC6 mol/L). Addition of ACh at concentrations that produced maximal vasodilatation (10eC6 to 10eC4 mol/L) increased DAR-4M AM fluorescence in a concentration-dependent manner (Figure 2A). This red fluorescence, although particularly abundant in the endothelial layer, permeated through the entire thickness of the vessel and was prevented by preincubation with L-NNA (10eC4 mol/L) (Figure 2B). Figure 2C summarizes the quantitative analysis of the total changes in red fluorescence. Incubation with L-NNA induced a slight but significant decrease in the signal, which could be interpreted as an inhibition of the basal release of NO. Incubation with GTN (10eC9 to 10eC6 mol/L) did not increase DAR-4M AM fluorescence. Indeed, each increase in the concentration of GTN was accompanied by a significant reduction in red fluorescence, as represented in Figure 3A and quantified in Figure 3B.
To discard the possibility that such a reduction in the fluorescence was attributable to a direct interference on DAR-4M AM by GTN or any of its main intermediary metabolites, 10eC4 mol/L DETA was added at the end of each experiment and, in all cases, produced a significant increase in the fluorescence observed (Figure 3A). The magnitude of this fluorescence increase in the presence of 10eC4 mol/L GTN was 71.27±17.95% (n=4), and it was similar to that observed when 10eC4 mol/L DETA was added in the absence of GTN or in the presence of 10eC5 mol/L SNAP or 10eC6 mol/L SIN-1 (n=5 to 7 in each case).
Effect on the Rate of O2 Consumption
Table 2 represents the effects of DETA-NO (10eC8 to 10eC5 mol/L), SNAP (10eC9 to 10eC5 mol/L), SIN-1 (10eC9 to 10eC6 mol/L), ACh (10eC7 to 10eC4 mol/L), and GTN (10eC9 to 10eC6 mol/L). DETA-NO, SNAP, SIN-1, and ACh did not modify (data not shown) the VO2max of controls (36.50±1.1x10eC9 mol O2/min/10eC6 g protein) but significantly and concentration-dependently increased the apparent Km for O2 of isolated rings of rat thoracic aorta. Concentrations of GTN (10eC9 to 10eC6 mol/L), which produced maximal relaxation in the aortic rings, neither modified the VO2max nor increased the apparent Km for O2. However, higher concentrations of GTN (10eC5 and 10eC4 mol/L) produced a significant decrease in the VO2max (22.67±1.01 and 12.85±1.12x10eC9 mol O2/min per 10eC6 g protein, respectively) and the apparent Km for O2 (20.5±3.93 and 14.71±6.47, respectively).
The implication of NO in the effects of DETA-NO, ACh, and GTN on mitochondrial O2 consumption is shown by the representative traces of respiring rings in Figure 4. As suggested by the change in the curve slopes, the rate of O2 consumption was decreased by incubation with concentrations of DETA-NO (10eC5 mol/L) and ACh (10eC5 mol/L), which produced maximal relaxation in the aortic rings. Scavenging of NO by addition of oxyHb reversed the inhibitory effects of both drugs on respiration. This effect was not shared by GTN (10eC6 mol/L). Presence of the sGC inhibitor ODQ (5x10eC6 mol/L) did not modify the inhibitory effect on mitochondrial O2 consumption observed with the 3 agents (data not shown). Figure 5 shows the effects of the various treatments on the O2 consumption rate (VO2) of rat aortic rings in the interval during which O2 concentrations in the bath were similar to those present in hypoxic conditions, between 2 to 3x10eC5 mol/L. Within the same range of concentrations that completely relax precontracted vessels, DETA-NO, SNAP, SIN-1, and ACh reduced O2 consumption concentration dependently, whereas GTN did not modify VO2.
Discussion
GTN has been used since 1879 in the treatment of ischemic syndromes related to coronary heart disease. Its principal action has been accredited to the release of NO and subsequent activation of guanylyl cyclase, which should induce the relaxation of vessels, both coronary and peripheral. However, most studies have demonstrated that NO is released from GTN only at elevated pharmacological concentrations, beyond those achieved with therapeutic concentrations of the drug.3eC7 In our vascular relaxation experiments, the effects of GTN were similar to those of the 3 structurally different NO donors evaluated or the induction of endogenous production of NO with ACh. They all produced a potent and concentration-dependent relaxation of vascular rings, which was blocked by the presence of the NO scavenger oxyHb or the sGC inhibitor ODQ. However, this similitude of profiles disappeared when the NO was determined.
Using confocal microscopy, we performed a new technique for real-time detection of intracellular NO in isolated intact vessels. Incubation of the rat aorta with vasoactive concentrations of DETA-NO, SNAP, and SIN-1 produced concentration-dependent augmentations in DAR-4M AM fluorescence. Similarly, stimulation of the endothelium with maximal vasoactive concentrations of ACh induced an immediate increase in NO-related fluorescence, which was prevented by inhibition of NO synthase. This increase in a NO-related signal was not observed following incubation with GTN. Indeed, there was a small decrease in the intensity of DAR-4M AM fluorescence for which we presently have no explanation.
A similar absence of an NO signal produced by GTN has recently been reported with electron paramagnetic resonance spin trapping in a variety of rodent vessel.11 This technique is also highly sensitive to NO but requires the use of frozen tissue and, therefore, yields single, static measurements. By comparison, our approach allows repetitive real-time assessments of the effects of drugs in a biologically active vessel at various concentrations and throughout a prolonged period of time, thus facilitating a clearer picture of the physiological implications.
The relaxant effects of GTN on the isolated vessels are compatible with an effect mediated by NO. However, our results, considered together with the measurement of NO levels by confocal microscopy, raise the possibility that these effects result from the release of another species that shares with NO the characteristics of being scavenged by oxyHb and of stimulating sGC. This is not a new proposal, and results with other nitrates have shown that they could oxidize oxyHb at a rate that correlates with their vasodilatory potency but not with the release of NO,18,19 thus suggesting the release of a species different from NO.
The idea that the effects of GTN are not a consequence of the release of NO is reinforced further by the analysis of mitochondrial oxygen consumption. The interaction of this mediator with the terminal enzyme of the electron transport chain, cytochrome C oxidase, is emerging as a major pathway through which NO exerts important physiological/pathological functions.13,20 NO reduces the affinity of the enzyme for O2, increasing its Km and, consequently, inhibiting mitochondrial O2 consumption with an intensity that is inversely proportional to the concentration of O2.21eC24 Our results with DETA-NO, SNAP, SIN-1, and ACh confirm this mechanism of action. They reduced the rate of O2 consumption by the rat aorta in a concentration-dependent manner within the same concentration range that produced NO-mediated vasorelaxations and increased the fluorescent signals in confocal microscopy. Furthermore, they followed a pattern of competitiveness and reversibility previously described for the interaction between NO and cytochrome C oxidase: (1) competitive, because the affinity for O2 is decreased, as shown by the concentration-dependent increase in the Km that occurs without a significant change in the VO2max25,26; and (2) reversible by oxyHb.16,27
The effects of GTN differ and are not compatible with the concept that this compound releases NO, which inhibits mitochondrial O2 consumption in a competitive manner. Clinically used concentrations of GTN, which significantly relax isolated vessels by activation of sGC, do not modify VO2max or the apparent affinity for O2 represented by the Km. Higher, supratherapeutic concentrations of GTN reduce VO2max and decrease the Km in a way that is indicative of a noncompetitive action and probably related to the known GTN capacity to produce reactive oxygen species, such as superoxide and peroxynitrite,28eC30 that might compromise the function of different mitochondrial enzymes.31,32 The differences between NO donors, ACh, and GTN are particularly evident if analyzed in a hypoxic context, which highlights the inhibition by NO of mitochondrial O2 consumption. With a concentration of O2 between 2 and 3x10eC5 mol/L, equivalent to hypoxic conditions,17 all 3 NO donors and ACh, but not GTN, produce a significant and concentration-dependent inhibition of the mitochondrial O2 consumption. It has recently been suggested that the bioactivation of GTN is mediated by the mitochondrial enzyme aldehyde dehydrogenase.33 Our results do not support the idea that NO is 1 of the species formed as a product of GTN biotransformation, as it would be difficult to explain how such a NO diffuses from the mitochondria to activate the cytosolic sGC without inhibiting mitochondrial complex IV or being detected by confocal microscopy.
We conclude that the biotransformation of GTN does not release free NO. It remains to be investigated whether this leads to the release of another species that directly interacts with sGC in vascular smooth muscle without inhibiting O2 consumption by vascular mitochondria. This variation in the bioactivity of GTN and NO donors is interesting insofar as its therapeutic implications. NO-producing agents produce vasodilatation in concentrations that also significantly inhibit O2 consumption, but GTN does not. GTN is used to improve the blood flow in ischemic tissues, and the fact that this is achieved without any further reduction in the bioenergetic activity of the mitochondria could lie behind its long clinical success.
Acknowledgments
This work was supported by grants from Red Cardiovascular (RECAVA), from Spanish Comisie Interministerial de Ciencia y Tecnologe (SAF2001-0763 and SAF2004-02600), from Instituto Salud Carlos III (CP03/0024), and from Generalitat Valenciana (GV2004-A-147). C.N. is a recipient of a Centro Nacional de Investigaciones Cardiovasculare CNIC-BANCAJA fellowship, and V.M.V. is contracted with a Contrato-Investigador Fondo de Investigacie Sanitaria from Instituto Salud Carlos III. We are grateful to Raquel Nieto, Elvira Arza, and Pilar Torralbo for technical assistance with confocal microscope.
Both authors contributed equally to this work.
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Brown GC. Regulation of mitochondrial respiration by nitric oxide inhibition of cytochrome c oxidase. Biochim Biophys Acta. 2001; 1504: 46eC57.
Parker JD. Nitrate tolerance, oxidative stress, and mitochondrial function: another worrisome chapter on the effects of organic nitrates. J Clin Invest. 2004; 113: 352eC354.
Chen Z, Zhang J, Stamler S. Identification of the enzymatic mechanism of nitroglycerin bioactivation. Proc Natl Acad Sci U S A. 2002; 99: 8306eC8311.(Cristina Nez, Vetor M. Ve)
Unidad de Citometria, Fundacie Centro Nacional de Investigaciones Cardiovasculares Carlos III (A.A.-B.), C/Melchor Fernedez Almagro, Madrid, Spain
The Wolfson Institute for Biomedical Research (S.M.), University College London, United Kingdom.
Abstract
It has been generally acknowledged that the actions of glyceryl trinitrate (GTN) are a result of its bioconversion into NO. However, recent observations have thrown this idea into doubt, with many studies demonstrating that NO is present only when there are high concentrations of GTN. We have explored this discrepancy by developing a new approach that uses confocal microscopy to directly detect NO. Intracellular levels of NO in the rat aortic vascular wall have been compared with those present after incubation with 3 different NO donors (DETA-NO, 3-morpholinosydnonimine, and S-nitroso-N-acetylpenicillamine), endothelial activation with acetylcholine, or administration of GTN. We have also evaluated the relaxant effects of these treatments on isolated rings of aorta following activation of the enzyme soluble guanylyl cyclase and their inhibitory action on mitochondrial respiration, which is an index of the interaction of NO with the enzyme of the electron transport chain cytochrome C oxidase. In the case of the various NO donors and acetylcholine, we detected a concentration-dependent relationship in the intensity of vascular relaxation and degree of NO fluorescence and an increase in the Michaelis constant (Km) for O2. GTN did not produce similar effects, and although clinically relevant concentrations of this compound caused clear, concentration-related relaxations, there was neither any increase in NO-related fluorescence nor an augmented Km for O2. The nature of these differences suggests that these concentrations of GTN do not release free NO but probably a different species that, although it interacts with soluble guanylyl cyclase in vascular smooth muscle, does not inhibit O2 consumption by vascular mitochondria.
Key Words: glyceryl trinitrate nitric oxide mitochondria vascular relaxation NO donors
Introduction
It is generally acknowledged that the action of glyceryl trinitrate (GTN) is a result of its bioconversion into the vasorelaxant agent NO.1,2 This idea is based on reports, both in vitro and in vivo,3eC7 that suggest that exposure to GTN leads to the formation of NO. However, in most of these studies, NO was observed only when GTN concentrations considerably exceeded the plasma levels reached during clinical dosing, whereas a number of further reports question the direct formation of NO.8eC10 Furthermore, the precise mechanism by which GTN is biotransformed is also currently a subject of controversy.11
Using confocal microscopy to observe the cells of vessels, we detected endothelium-synthesized NO and compared it with that released by different NO donors and with GTN. Furthermore, the vasodilatatory effects of NO and its consequences for mitochondrial respiration have also been evaluated. By studying these 2 targets of NO,12,13 we identified inconsistencies between the effects of NO, produced both endogenously and pharmacologically, and those obtained with GTN used at clinically relevant concentrations. The nature of these discrepancies suggests that the actions of GTN on the vasculature are indeed unrelated to its bioconversion to NO.
Materials and Methods
Animals
Male SpragueeCDawley rats (200 to 250 g; Harlan Laboratories, Barcelona) were decapitated and their thoracic aortas removed, cleaned of adhering tissues in Krebs solution, and cut into rings of 1 mm (for image analysis) or 5 mm (all other experiments). Unless otherwise stated, all results are mean±SEM of at least 5 experiments, each in a different animal. All protocols complied with the European Community guidelines for the use of experimental animals and were approved by the Ethics Committee of the University of Valencia.
Contractility Studies
Aortic rings were suspended in a 5-mL organ bath containing Krebs solution at 37°C. The solution was constantly gassed with 12% O2, 5% CO2, and 83% N2, producing an O2 concentration of 12 to 13x10eC5 mol/L in the bath, which was monitored with a dissolved O2 meter (ISO2; World Precision Instruments, Stevenage, Herts, UK). This level of O2 is similar to that present in the aortic blood.13 Following an equilibration period of 75 to 90 minutes, rings were submaximally (75% to 85% of maximal response) contracted with 10eC6 mol/L phenylephrine (Phe), and the presence of a functional endothelium was confirmed by eliciting a relaxant response (>90%) to 10eC5 mol/L acetylcholine (ACh). Only those vessels exhibiting this response were included in what remained of the experiment.
Following a further equilibration period of 30 to 45 minutes, rings were contracted with a depolarizing solution (6x10eC2 mol/L KCl). After being washed, the vessels returned to the baseline and a further Phe concentration of 10eC6 mol/L was added, giving a sustained contraction equivalent to the response to depolarization. Once such a stable tone was achieved, relaxation-response curves were obtained by adding cumulative concentrations of the NO donors (Z)-1-([2-aminoethyl]-N-[2-ammonioethyl]amino)diazen-1-IM1,2-diolate (DETA-NO) (10eC8 to 10eC5 mol/L), S-nitroso-N-acetylpenicillamine (SNAP) (10eC10 to 10eC5 mol/L), or 3-morpholinosydnonimine (SIN-1) (10eC11 to 10eC6 mol/L); and ACh (10eC9 to 10eC5 mol/L) or GTN (10eC10 to 10eC6 mol/L). In some experiments, the soluble guanylyl cyclase (sGC) inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) (5x10eC6 mol/L), the scavenger of NO and related species oxyhemoglobin (oxyHb) (10eC5 mol/L), or the NO synthase inhibitor N--nitro-L-arginine (L-NNA) (10eC4 mol/L) was added before the NO-producing drugs. The adequate concentration (eClog[M]) for producing 50% relaxation (pEC50) was obtained from a nonlinear regression analysis with Graph Pad Software. The percentage of relaxation obtained with the highest concentration of each agent in the absence (control) or presence of ODQ (5x10eC6 mol/L) or oxyHb (10eC5 mol/L) is represented by Emax.
Confocal Microscopy Imaging of NO Release
NO was visualized with diaminorhodamine-4M (DAR-4M AM), a cell permeable, fluorescent precursor with a convenient profile for NO bioimaging. It exhibits low background fluorescence with longer wavelength excitation, allows detection throughout a wider pH range, and is more photostable than standard fluorochromes that have been used previously for NO imaging.14 Inside the cell, the relatively nonfluorescent DAR-4M AM is converted into its triazole form, DAR-4M T, the fluorescence intensity of which is directly proportional to the concentration of NO, with a detection limit of 7x10eC9 mol/L.14
Aortic rings were incubated in the dark for 60 minutes in a Krebs solution that was constantly gassed (12% O2, 5% CO2, and 83% N2) and that contained DAR-4M AM (2.5x10eC6 mol/L). The vital cell nuclei staining fluorochrome Hoechst 33342 (10eC6 mol/L) was added to this solution after 30 minutes. Thereafter, the rings were mounted in a vertical position on 24x50-mm coverslips, fixed with a grid in Krebs solution, and placed in a heated chamber (37°C, 2 mL; Warner Instrument Corp, Hamden, Conn). Following a 10-minute stabilization period, which was considered basal, cumulative concentrations of DETA-NO (10eC7 to 10eC4 mol/L), SNAP (10eC9 to 10eC5 mol/L), SIN-1 (10eC9 to 10eC6 mol/L), ACh (10eC6 to 10eC4 mol/L), or GTN (10eC9 to 10eC6 mol/L) were added every 10 minutes. In some experiments, before addition of the agents, tissues were preincubated (10 minutes) with L-NNA (10eC4 mol/L). Images were obtained every 2 minutes throughout the entire experiment, which lasted a maximum of 60 minutes. A concentration of DETA-NO (10eC4 mol/L) was added at the end of each experiment to monitor the sensitivity of our system, and, to avoid variability, the response obtained was considered a reference for normalizing the signal elicited in each experimental series. To ensure that the fluorochromes had no influence on the viability of the tissues, parallel experiments were performed in which rings were incubated with these compounds under the same experimental circumstances that confocal analysis and contractility studies were performed. No significant differences were found either in the response to a single concentration of Phe (10eC6 mol/L) or in the concentration-response curves of relaxation to DETA-NO (pEC50: 5.98±0.09 in control versus 5.90±0.08, n=4 and 5, respectively), ACh (pEC50: 7.33±0.10 in control versus 7.23±0.21, n=5), and GTN (8.08±0.06 in control versus 8.40±0.13, n=5 and 6, respectively).
Images were observed in water immersion (x60 magnification) and studied with a Radiance 2100 confocal microscope (Bio-Rad, Hempel Hempstead, UK) using a 405-nm diode laser to excite Hoechst 33342 and a 546-nm He-Ne laser to excite DAR-4M AM. Fluorescence was detected through 440/20 band pass (BP) and 570 long pass (LP) filters for Hoechst 33342 and DAR-4M AM, respectively. DAR-4M AM fluorescence was analyzed (Laserpix software; Bio-Rad) in portions of aortic ring comprising the entire width of the vessel, from the adventitia to the endothelium. Confocal microscope settings were adjusted to produce the optimum signal/noise ratio. The vessel wall exhibited basal red fluorescence, representing the autofluorescence of the elastin fibers, which was deducted from the total quantification of fluorescence in each specific measurement.
Further experiments were performed to exclude the interference on DAR-4M AM fluorescence by GTN or some of its main intermediate metabolites. Different concentrations (10eC9 to 10eC4 mol/L) of GTN, its metabolites 1,2 glyceryl dinitrate (1,2-GDN) and 1,3 glyceryl dinitrate (1,3-GDN), and sodium nitrate (NaNO3) or sodium nitrite (NaNO2) were incubated in the presence of DAR-4M AM (2.5x10eC6 mol/L) in a cell-free Krebs solution. Fluorescence was measured with a Fluoroskan plate reader (Thermo Labsystems) and 10eC4 mol/L DETA-NO was added at the end of each experiment. No modification of the fluorochrome basal fluorescence was obtained after incubation with GTN or any of the compounds tested for 60 minutes. Furthermore, the increase in fluorescence observed after addition of DETA-NO was similar in the presence or absence of these agents (n=4 to 5 experiments; results not shown).
Electrochemical Measurement of O2 Consumption
The aorta of 2 animals (97.65±1.04 mg total wet weight) were cut in rings and placed in gas-tight chambers containing 1 mL of Krebs solution and gently agitated at 37°C. The O2 consumption by the tissue was measured with a Clark-type O2 electrode (Rank Brothers, Bottisham, UK) calibrated with an air-saturated Krebs solution, assuming an O2 concentration of 2x10eC4 mol/L. Experiments were performed in the presence of DETA-NO (10eC8 to 10eC4 mol/L), SNAP (10eC9 to 10eC5 mol/L), SIN-1 (10eC9 to 10eC6 mol/L), ACh (10eC7 to 10eC4 mol/L), or GTN (10eC9 to 10eC6 mol/L). Sodium cyanide (10eC3 mol/L) was used to confirm that O2 consumption was mainly mitochondrial (&95% to 99%). In some experiments, the reversibility of NO-induced inhibition of respiration was assessed by adding oxyHb (10eC5 mol/L) when the concentration of O2 in the chamber was 10eC4 mol/L. Measurements were collected using a data-acquisition device, Duo.18 (World Precision Instruments, Stevenage, UK). A hyperbolic function was used to describe the relationship between O2 concentration and the rate of O2 consumption (VO2).15 The maximal rate of O2 consumption (VO2max) and the apparent O2 affinity Michaelis constant (Km) (10eC6 mol/L) were calculated according to their analogy with the MichaeliseCMenten constant. The rate of O2 consumption at the interval between 3 to 2x10eC5 mol/L of O2 (similar to conditions during hypoxia16,17) was calculated from the same experiments. To avoid variability, the rate of O2 consumption was expressed as 10eC9 mol O2/min per 10eC6 g protein. Proteins were determined by the BCA protein assay kit (Pierce, Rockford, Ill) using BSA as the standard. In some cases, the viability of the rings was later assessed by confirming that their contractile response to Phe (10eC6 mol/L) and ACh (10eC5 mol/L) did not differ from that of nonmanipulated vessels.
Data Analysis
Values are expressed as mean±SEM. Statistical analysis was performed by 1-way ANOVA followed by the Student t test for unpaired samples (Graph Pad Software). Significance was defined as P<0.05.
Drugs and Solutions
The composition of the Krebs solution was as follows (10eC3 mol/L): 118 NaCl, 4.75 KCl, 1.9 CaCl2, 1.2 MgSO4, 1.2 KH2PO4, 25 NaHCO3, and 10.1 glucose. NaNO3 and NaNO2 were obtained from Panreac (Barcelona, Spain). 1,2-GDN and 1,3-GDN were purchased from LGC Promochem (Barcelona, Spain). Phe, ACh, L-NNA, sodium cyanide, ODQ, meth(a)emoglobin, and Hoechst 33342 were acquired from Sigma Aldrich (St Louis, Mo). DETA-NO was obtained from Alexis (Lausen, Switzerland). A clinically available preparation of GTN was used (Solinitrina from Allmirall Prodesfarma, Barcelona, Spain), and DAR-4M AM was obtained from Calbiochem (San Diego). OxyHb was prepared by reduction of human meth(a)emoglobin with a 10-fold molar excess of sodium dithionite, followed by dialysis against PBS. All drugs were dissolved in ultrapure water, except GTN, DAR-4M AM, and Hoechst 33342, which were dissolved in Krebs solution, and ODQ, which was dissolved in dimethyl sulfoxide (1:5000 [vol/vol]).
Results
Contractility Studies
Table 1 shows pEC50 and Emax of the relaxation curves induced in vessels precontracted in vitro with Phe (10eC6 mol/L) by cumulative concentrations of DETA-NO (10eC8 to 10eC5 mol/L), SNAP (10eC10 to 10eC5 mol/L), SIN-1 (10eC11 to 10eC6 mol/L), ACh (10eC9 to 10eC5 mol/L), and GTN (10eC10 to 10eC6 mol/L), respectively. The presence in the organ bath of the sGC inhibitor ODQ (5x10eC6 mol/L) prevented the vasorelaxant activity of all 5 substances. Likewise, incubation with the NO scavenger oxyHb inhibited the effects of DETA-NO, ACh, and GTN. The effects of ACh were inhibited by prior incubation with the NO synthesis inhibitor L-NNA (10eC4 mol/L) (data not shown).
Confocal Microscopy Imaging of NO Release
Figure 1A shows representative confocal images of the rat aorta following incubation (60 minutes) with DAR-4M AM and concentration-dependent increases in the red fluorescent signal induced by addition of cumulative concentrations of DETA-NO (10eC7 to 10eC4 mol/L) every 10 minutes. An analysis of the total changes in red fluorescence is presented in Figure 1B. Figure 1 also shows significant and concentration-dependent increases in NO-related DAR-4M AM fluorescence obtained with the other 2 NO donors used, SNAP (10eC9 to 10eC5 mol/L) and SIN-1 (10eC9 to 10eC6 mol/L). Addition of ACh at concentrations that produced maximal vasodilatation (10eC6 to 10eC4 mol/L) increased DAR-4M AM fluorescence in a concentration-dependent manner (Figure 2A). This red fluorescence, although particularly abundant in the endothelial layer, permeated through the entire thickness of the vessel and was prevented by preincubation with L-NNA (10eC4 mol/L) (Figure 2B). Figure 2C summarizes the quantitative analysis of the total changes in red fluorescence. Incubation with L-NNA induced a slight but significant decrease in the signal, which could be interpreted as an inhibition of the basal release of NO. Incubation with GTN (10eC9 to 10eC6 mol/L) did not increase DAR-4M AM fluorescence. Indeed, each increase in the concentration of GTN was accompanied by a significant reduction in red fluorescence, as represented in Figure 3A and quantified in Figure 3B.
To discard the possibility that such a reduction in the fluorescence was attributable to a direct interference on DAR-4M AM by GTN or any of its main intermediary metabolites, 10eC4 mol/L DETA was added at the end of each experiment and, in all cases, produced a significant increase in the fluorescence observed (Figure 3A). The magnitude of this fluorescence increase in the presence of 10eC4 mol/L GTN was 71.27±17.95% (n=4), and it was similar to that observed when 10eC4 mol/L DETA was added in the absence of GTN or in the presence of 10eC5 mol/L SNAP or 10eC6 mol/L SIN-1 (n=5 to 7 in each case).
Effect on the Rate of O2 Consumption
Table 2 represents the effects of DETA-NO (10eC8 to 10eC5 mol/L), SNAP (10eC9 to 10eC5 mol/L), SIN-1 (10eC9 to 10eC6 mol/L), ACh (10eC7 to 10eC4 mol/L), and GTN (10eC9 to 10eC6 mol/L). DETA-NO, SNAP, SIN-1, and ACh did not modify (data not shown) the VO2max of controls (36.50±1.1x10eC9 mol O2/min/10eC6 g protein) but significantly and concentration-dependently increased the apparent Km for O2 of isolated rings of rat thoracic aorta. Concentrations of GTN (10eC9 to 10eC6 mol/L), which produced maximal relaxation in the aortic rings, neither modified the VO2max nor increased the apparent Km for O2. However, higher concentrations of GTN (10eC5 and 10eC4 mol/L) produced a significant decrease in the VO2max (22.67±1.01 and 12.85±1.12x10eC9 mol O2/min per 10eC6 g protein, respectively) and the apparent Km for O2 (20.5±3.93 and 14.71±6.47, respectively).
The implication of NO in the effects of DETA-NO, ACh, and GTN on mitochondrial O2 consumption is shown by the representative traces of respiring rings in Figure 4. As suggested by the change in the curve slopes, the rate of O2 consumption was decreased by incubation with concentrations of DETA-NO (10eC5 mol/L) and ACh (10eC5 mol/L), which produced maximal relaxation in the aortic rings. Scavenging of NO by addition of oxyHb reversed the inhibitory effects of both drugs on respiration. This effect was not shared by GTN (10eC6 mol/L). Presence of the sGC inhibitor ODQ (5x10eC6 mol/L) did not modify the inhibitory effect on mitochondrial O2 consumption observed with the 3 agents (data not shown). Figure 5 shows the effects of the various treatments on the O2 consumption rate (VO2) of rat aortic rings in the interval during which O2 concentrations in the bath were similar to those present in hypoxic conditions, between 2 to 3x10eC5 mol/L. Within the same range of concentrations that completely relax precontracted vessels, DETA-NO, SNAP, SIN-1, and ACh reduced O2 consumption concentration dependently, whereas GTN did not modify VO2.
Discussion
GTN has been used since 1879 in the treatment of ischemic syndromes related to coronary heart disease. Its principal action has been accredited to the release of NO and subsequent activation of guanylyl cyclase, which should induce the relaxation of vessels, both coronary and peripheral. However, most studies have demonstrated that NO is released from GTN only at elevated pharmacological concentrations, beyond those achieved with therapeutic concentrations of the drug.3eC7 In our vascular relaxation experiments, the effects of GTN were similar to those of the 3 structurally different NO donors evaluated or the induction of endogenous production of NO with ACh. They all produced a potent and concentration-dependent relaxation of vascular rings, which was blocked by the presence of the NO scavenger oxyHb or the sGC inhibitor ODQ. However, this similitude of profiles disappeared when the NO was determined.
Using confocal microscopy, we performed a new technique for real-time detection of intracellular NO in isolated intact vessels. Incubation of the rat aorta with vasoactive concentrations of DETA-NO, SNAP, and SIN-1 produced concentration-dependent augmentations in DAR-4M AM fluorescence. Similarly, stimulation of the endothelium with maximal vasoactive concentrations of ACh induced an immediate increase in NO-related fluorescence, which was prevented by inhibition of NO synthase. This increase in a NO-related signal was not observed following incubation with GTN. Indeed, there was a small decrease in the intensity of DAR-4M AM fluorescence for which we presently have no explanation.
A similar absence of an NO signal produced by GTN has recently been reported with electron paramagnetic resonance spin trapping in a variety of rodent vessel.11 This technique is also highly sensitive to NO but requires the use of frozen tissue and, therefore, yields single, static measurements. By comparison, our approach allows repetitive real-time assessments of the effects of drugs in a biologically active vessel at various concentrations and throughout a prolonged period of time, thus facilitating a clearer picture of the physiological implications.
The relaxant effects of GTN on the isolated vessels are compatible with an effect mediated by NO. However, our results, considered together with the measurement of NO levels by confocal microscopy, raise the possibility that these effects result from the release of another species that shares with NO the characteristics of being scavenged by oxyHb and of stimulating sGC. This is not a new proposal, and results with other nitrates have shown that they could oxidize oxyHb at a rate that correlates with their vasodilatory potency but not with the release of NO,18,19 thus suggesting the release of a species different from NO.
The idea that the effects of GTN are not a consequence of the release of NO is reinforced further by the analysis of mitochondrial oxygen consumption. The interaction of this mediator with the terminal enzyme of the electron transport chain, cytochrome C oxidase, is emerging as a major pathway through which NO exerts important physiological/pathological functions.13,20 NO reduces the affinity of the enzyme for O2, increasing its Km and, consequently, inhibiting mitochondrial O2 consumption with an intensity that is inversely proportional to the concentration of O2.21eC24 Our results with DETA-NO, SNAP, SIN-1, and ACh confirm this mechanism of action. They reduced the rate of O2 consumption by the rat aorta in a concentration-dependent manner within the same concentration range that produced NO-mediated vasorelaxations and increased the fluorescent signals in confocal microscopy. Furthermore, they followed a pattern of competitiveness and reversibility previously described for the interaction between NO and cytochrome C oxidase: (1) competitive, because the affinity for O2 is decreased, as shown by the concentration-dependent increase in the Km that occurs without a significant change in the VO2max25,26; and (2) reversible by oxyHb.16,27
The effects of GTN differ and are not compatible with the concept that this compound releases NO, which inhibits mitochondrial O2 consumption in a competitive manner. Clinically used concentrations of GTN, which significantly relax isolated vessels by activation of sGC, do not modify VO2max or the apparent affinity for O2 represented by the Km. Higher, supratherapeutic concentrations of GTN reduce VO2max and decrease the Km in a way that is indicative of a noncompetitive action and probably related to the known GTN capacity to produce reactive oxygen species, such as superoxide and peroxynitrite,28eC30 that might compromise the function of different mitochondrial enzymes.31,32 The differences between NO donors, ACh, and GTN are particularly evident if analyzed in a hypoxic context, which highlights the inhibition by NO of mitochondrial O2 consumption. With a concentration of O2 between 2 and 3x10eC5 mol/L, equivalent to hypoxic conditions,17 all 3 NO donors and ACh, but not GTN, produce a significant and concentration-dependent inhibition of the mitochondrial O2 consumption. It has recently been suggested that the bioactivation of GTN is mediated by the mitochondrial enzyme aldehyde dehydrogenase.33 Our results do not support the idea that NO is 1 of the species formed as a product of GTN biotransformation, as it would be difficult to explain how such a NO diffuses from the mitochondria to activate the cytosolic sGC without inhibiting mitochondrial complex IV or being detected by confocal microscopy.
We conclude that the biotransformation of GTN does not release free NO. It remains to be investigated whether this leads to the release of another species that directly interacts with sGC in vascular smooth muscle without inhibiting O2 consumption by vascular mitochondria. This variation in the bioactivity of GTN and NO donors is interesting insofar as its therapeutic implications. NO-producing agents produce vasodilatation in concentrations that also significantly inhibit O2 consumption, but GTN does not. GTN is used to improve the blood flow in ischemic tissues, and the fact that this is achieved without any further reduction in the bioenergetic activity of the mitochondria could lie behind its long clinical success.
Acknowledgments
This work was supported by grants from Red Cardiovascular (RECAVA), from Spanish Comisie Interministerial de Ciencia y Tecnologe (SAF2001-0763 and SAF2004-02600), from Instituto Salud Carlos III (CP03/0024), and from Generalitat Valenciana (GV2004-A-147). C.N. is a recipient of a Centro Nacional de Investigaciones Cardiovasculare CNIC-BANCAJA fellowship, and V.M.V. is contracted with a Contrato-Investigador Fondo de Investigacie Sanitaria from Instituto Salud Carlos III. We are grateful to Raquel Nieto, Elvira Arza, and Pilar Torralbo for technical assistance with confocal microscope.
Both authors contributed equally to this work.
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