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The Wolfson Institute for Biomedical Research, University College London, London, United Kingdom (C.G., V.W., J.G.); and Division of Neurophysiology, National Institute for Medical Research, Mill Hill, London, United Kingdom (T.B.)
Received June 25, 2003; accepted August 27, 2003
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1 and 21 isoforms were 0.9 nM and 0.5 nM, respectively. The slopes of the concentration-response curves were more shallow than before (Hill coefficient of 1 rather than 2), questioning the need to consider the binding of more than one NO molecule for receptor activation. The discrepancies are ascribable to limitations of the earlier method. Other biological problems can readily be addressed by adaptations of the new method.
; Koesling and Friebe, 2000). Depending on its concentration and/or other experimental conditions, however, biological effects can be exerted through other pathways, including the binding of NO to cytochrome c oxidase, resulting in the inhibition of mitochondrial respiration (Brown, 1999), reaction with other radicals (e.g., with superoxide ions to give reactive peroxynitrite anions), and the production of nitrosating species after the reaction of NO with oxygen (Beckman and Koppenol, 1996; Augusto et al., 2002). The extent to which these other reactions contribute to the biology of NO in vivo remains uncertain. Nevertheless, the spectrum of possible effects of NO in vitro can make the interpretation of experimental findings difficult, particularly when NO is applied in constantly changing concentrations, as happens with the current methods of delivery.
NO can be dispensed from concentrated anaerobic solutions, but on dilution into aerobic solutions used in the laboratory, it is consumed by reaction with oxygen (autoxidation) at a rate proportional to the square of its concentration (Ford et al., 1993). Alternatively, NO can be provided by donors, of which the so-called NONOates are preferred because they degrade to release NO with predictable kinetics (Morley and Keefer, 1993), and different NONOates with widely differing half-lives (1.8 s to 20 h) are commercially available. When added to biological media, however, the NO concentration increases (at a rate governed by the half-life) and then decreases as the autoxidation rate exceeds the NO release rate (Schmidt et al., 1997). Moreover, autoxidation itself creates problems because it leads to the generation of reactive nitrosating agents (e.g., NO2 and N2O3), and the rate of autoxidation at a given NO concentration will be approximately 10-fold higher in the hyperoxic environment in which cells are maintained in vitro than it would be in vivo (Ford et al., 1993; Augusto et al., 2002).
Clearly, methods are needed for applying NO in the controlled manner that would be axiomatic for meaningful studies of the biology of other signaling molecules. To try to address this problem, an apparatus for maintaining "clamped" NO concentrations has been designed (Zhelyaskov and Godwin, 1999), but it is complex, expensive to construct, and unsuited to most biological applications. In principle, steady NO concentrations can be achieved by marrying a constant rate of NO production with an appropriate rate of inactivation. We recently exploited this concept by using red blood cells as biological NO sinks, which allowed for the determination of the absolute and relative sensitivities of the purified lung GC-coupled receptor and mitochondrial respiration to NO (Bellamy et al., 2002). Nevertheless, the method has several limitations, including the following: 1) having to prepare a washed red blood cell suspension for each experiment; 2) a slow increase of the NO level to plateau concentrations (60 s) so that, for rapid kinetics experiments, additions need to be made to a pre-equilibrated mixture in a small enough volume not to disturb the equilibrium, which limits the scope of the technique; 3) even a small leakage of free hemoglobin could compromise the experiment, because free hemoglobin inactivates NO at a much higher rate than when the protein is packaged in red blood cells (Liu et al., 1998); and 4) possible interference from bioactive substances taken up into, or released from, the red blood cells.
To address these and other limitations we sought to devise a cell-free method for generating steady NO concentrations covering the presumed physiological range (0-100 nM). This was achieved satisfactorily using the combination of a NONOate donor and the chemical NO scavenger CPTIO. When used to address one key issue in NO biology, namely the sensitivity of the GC-coupled receptors to NO, the new method gave results that differed in important ways from those obtained previously using red blood cells to provide the clamped NO concentration.
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). Measurement of NO. NO concentrations were recorded in buffer (1 ml) incubated in a sealed, stirred vessel (at 37°C) equipped with an NO electrode (Iso-NO; World Precision Instruments, Stevenage, Hertfordshire, UK). The rate of NO release from SPER/NO was measured by the addition of 10 µM of the donor to 50 mM Tris-HCl buffer containing 1000 U/ml superoxide dismutase (pH 7.4 at 37°C). After the 15 to 30 s required for the electrode response to settle (see Results), the measured NO concentration increased linearly for approximately another 60 s until autoxidation became significant as the NO concentration exceeded 250 nM. The NO release rate was obtained by measuring the gradient between 30 and 60 s after the addition of the donor.
Measurement of NO-Evoked GC Activity. Experiments were carried out in 50 mM Tris-HCl buffer supplemented with 1000 U/ml superoxide dismutase, 300 µM uric acid, 3 mM MgCl2, 0.1 mM EGTA, 0.01 mM DTT, 0.05% bovine serum albumin, and 1 mM GTP, pH 7.4 at 37°C, and, except when DEA/NO was used, CPTIO (200 µM unless specified otherwise). When cell extracts were assayed, the buffer also contained 5 mM creatine phosphate and 200 µg/ml of creatine kinase. Receptor protein purified from bovine lung (soluble guanylyl cyclase; Alexis) was diluted in a buffer, pH 7.4, containing 50 mM Tris-HCl, 1 mM DTT, and 0.5% bovine serum albumin to give a stock concentration of 5 µg/ml, which was stored on ice and subsequently diluted 1:100 into assay buffer maintained at 37°C. NO donor was added, and aliquots of the reaction mix were removed at intervals and inactivated in boiling buffer (50 mM Tris, 4 mM EDTA). To examine individual receptor isoforms, COS-7 cells were transfected with combinations of either the 1 and 1 subunits or the 2 and 1 subunits (both rat) as described previously (Gibb et al., 2003) and maintained for 48 h before harvesting by trypsinization. The cells were pooled, pelleted at 1500g for 5 min, and resuspended at 3 mg protein/ml in ice-cold lysis buffer, pH 7.4, containing 50 mM Tris-HCl, 0.1 mM DTT, and a protease inhibitor cocktail (complete mini EDTA-free; Roche Diagnostics, East Sussex, UK). After the addition of glycerol (to give 5%), the homogenate was frozen until use. The NO-evoked GC activity of the two isoforms was compared directly. The homogenates were thawed, stored on ice, and diluted 1:10 into assay buffer pre-equilibrated at 37°C. SPER/NO was added to achieve varying steady-state NO concentrations, and 2 min later, aliquots of the reaction mixture were removed and inactivated as described above. cGMP levels were quantified by radioimmunoassay. Data are given as means ± S.E.M., and results were analyzed using an unpaired Student's t test (two-tailed).
Mathematical Modeling. The chemical reactions on which the NO delivery method depends were incorporated into a mathematical model using the rate constants listed in Table 1. The model consisted of the following differential equations, where x is the number of moles of NO released per mole of SPER/NO:
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The equations were solved numerically using the adaptive Runge-Kutta algorithm in Mathcad (version 2001i; Adept Scientific, Letch-worth, Herts, UK). Calculation of the NO concentrations registered by the electrode was carried out by multiplying the derived NO concentration by the factor [1 - exp(-ket)], where ke is the rate constant of the electrode (0.116 s-1) (Griffiths and Garthwaite, 2001).
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The equation describing the change in NO concentration over time is
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where x represents the number of moles of NO released per mole of donor. For an effectively constant rate of NO release (required to achieve steady NO concentrations), the donor must decompose slowly relative to the duration of the experiment. For the present purposes, NO concentrations that were steady over the time scale of a few minutes were desired, so SPER/NO, which has a half-life of 39 min at 37°C (Keefer et al., 1996), was selected. The corresponding rate constant for NO release from SPER/NO (ka) is 2.96 x 10-4s-1.
To achieve rapid steady-state, the kb value needs to be much greater than ka, but not too large or the resulting NO concentrations would be too low to exert biological effects. When kb»ka, the steady-state NO concentration is given by
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A kb value on the order of 1 s-1 (imposing on NO a half-life of 0.7 s) would give rapid attainment of steady state (roughly within the mixing time of an experiment conducted manually) and would provide NO concentrations in the nanomolar range when micromolar concentrations of SPER/NO are added. Such a value of kb renders insignificant any loss of NO through autoxidation (e.g., at 100 nM NO, autoxidation would consume NO at a rate of only 25 pM/s, giving an NO half-life of 66 min). The sink needs to have the capacity to consume NO over the requisite time scale without significant exhaustion. With 100 µM SPER/NO and assuming x = 1 (see below), the initial rate of NO release (and consumption) would be approximately 1.8 µM/min, so sink concentrations in the 100 µM range are required. These considerations indicate that the sink needs to consume NO with a bimolecular rate constant of around 10-4M-1s-1.
The nitronyl nitroxides 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide and its carboxylated derivative (CPTIO) are stable radicals that scavenge NO at approximately the required rate (Akaike et al., 1993). We chose to use CPTIO, the less cell-permeant of the two, to reduce possible unwanted intracellular effects when used with intact cells (although, to our knowledge, none has yet been described). Despite the NO being largely consumed extracellularly, the resulting extracellular and intracellular NO concentrations would be in dynamic equilibrium because of the fast rate of NO diffusion in lipid and aqueous environments. The reaction between NO and CPTIO forms the NO2 radical, which is undesirable because it is a reactive oxidizing species that undergoes various reactions, including rapid combination with other radicals such as NO (Augusto et al., 2002). Therefore, NO2 needs to be scavenged. Urate, an endogenous antioxidant (Becker, 1993) that converts NO2 into NO2- (k = 2 x 107 M-1s-1) (Ford et al., 2002), was used for the purpose.
To analyze the reactions quantitatively, a more complex mathematical model was constructed using the rate constants given in Table 1. According to the model, with an initial CPTIO concentration of 200 µM, the addition of 300 µM SPER/NO results in a rapid increase of the NO concentration to 15 nM and the NO2 concentration to 3 nM, both being stable over several minutes (Fig. 1). The inclusion of urate at the concentration found in plasma (300 µM) (Becker, 1993) leads to a doubling of the NO concentration (eliminating loss caused by a reaction with NO2) and a reduction in the NO2 concentration to 16 pM. Although included in the model for the sake of completion, the reaction of NO with O2 is negligible compared with the reaction with CPTIO. In the presence of urate, therefore, the complex model reduces to the simple scheme outlined above (eqs. 1 and 2), providing that there is no significant depletion of NO donor or CPTIO. The mixture of urate, CPTIO, and SPER/NO (with the addition of 1000 U/ml of superoxide dismutase to scavenge any superoxide ions that would otherwise react with NO) was used for the experimental tests.
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Evaluation of the Method for Delivering Clamped NO Concentrations. By measuring the initial rate of increase of the NO concentration after the addition of SPER/NO, the NO release rate was found to be 19.1 ± 0.8 nM NO/min/µM SPER/NO (n = 3). Assuming a SPER/NO half-life of 39 min, this value signifies 1.08 mol of NO released per mole of SPER/NO (x in eqs. 1 and 2 and in the more complex model). It is important to note that the rate of NO release from NONOates (including SPER/NO) can vary between different batches and between different suppliers (results not shown). Equation 2 predicts a linear relationship between the steady-state NO concentration and the donor concentration. To test this, SPER/NO was added in a range of concentrations, and the resulting profile of the NO concentration was measured using an electrochemical probe. The probe responds too slowly to register the rising phase accurately (see below), but increasing the SPER/NO concentration between 100 and 1000 µM produced graded increases in steady-state NO concentration from 10 to 100 nM (Fig. 2a). The amplitude of the plateau NO concentration (measured as the average recorded between 45 and 75 s after addition of SPER/NO) was directly proportional to the SPER/NO concentration (Fig. 2b). From the gradient of the line (10-4 M NO/M SPER/NO) and an x value of 1.08, the value of kb comes to 3.2 s-1. Dividing this pseudo-first-order rate constant by the CPTIO concentration (200 µM) gives a rate constant for the reaction of CPTIO with NO of 1.6 x 104 M-1s-1 at 37°C, a value compatible with the published value of 1.01 x 104 M-1s-1 at 25°C (Akaike et al., 1993).
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) was incorporated into the model (see Materials and Methods), the shape of the recorded response was well approximated (Fig. 2c), suggesting that the sluggishness of the electrode accounts for the slow recorded rise time. To explore the limitation of the method with respect to the time over which clamped NO concentrations can be maintained, recordings were made for more prolonged periods. With 100 µM SPER/NO, the NO concentration (initially approximately 10 nM) remained low for at least 50 min (Fig. 2d). With 300 µM SPER/NO, NO remained fairly steady for 10 to 15 min (at 30-40 nM) but then rose at a progressively increasing rate. With 1000 µM SPER/NO, the secondary increase in NO concentration was accelerated to the extent that there was initially more of a shoulder than a plateau (Fig. 2d). The recorded profiles of the NO concentration resemble those predicted by the more complex model (Fig. 3a), which suggests that the time over which NO can be maintained is a function both of the CPTIO concentration, which declines as it is used up (Fig. 3b), and the NO release rate, which reduces as the donor decomposes (Fig. 3c). At high SPER/NO concentrations, the former predominates, and as the CPTIO becomes depleted, the NO concentration eventually increases steeply (until curtailed by autoxidation to the low micromolar range) (data not shown). At low SPER/NO concentrations, on the other hand, the decreasing rate of NO release results in a gradually diminishing NO concentration. For example, at 100 µM SPER/NO, NO is predicted to decrease from 10 to 5.5 nM over 1 h. The measurement of such a change, however, is beyond the capability of the recording apparatus (Fig. 2d) whose quantifiable limit of detection is approximately 10 nM, depending on the particular electrode being used.
Application of the Method to Activation of GC-Coupled NO Receptors. To explore the usefulness of the method for biological purposes, we first investigated the kinetics of activation by NO of its GC-coupled receptor purified from bovine lung. Despite the more complex reaction mixture used for measuring GC activity (see Materials and Methods), the SPER/NO-CPTIO couple generated stable NO concentrations over a 2-min period that were linearly related to the SPER/NO concentrations (Fig. 4a). The slight difference in the slope compared with the simple buffer used previously (Fig. 2b) is probably attributable to the use of different batches of both SPER/NO and CPTIO. NO concentrations in the GC reaction mixture were unaffected by the addition of receptor protein (data not shown).
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Possible untoward effects of the new approach were investigated by comparing the time course of GC activity over 2 min at a maximally effective NO concentration produced by the SPER/NO-CPTIO couple (50 nM; see below) with that occurring on the addition of a supramaximal concentration of a donor used frequently in the past, DEA/NO (1 µM). The GC activity in each case was linear with time, and the slopes were not significantly different (approximately 10 µmol of cGMP/mg of protein/min; P > 0.05) (Fig. 4b). With DEA/NO, receptor activity cannot be monitored usefully at submaximal concentrations because the NO concentration changes rapidly and continuously (Bellamy et al., 2002). In contrast, using the SPER/NO-CPTIO couple, GC activity remained linear with time at the low NO concentration of 2 nM (Fig. 4b). The addition of a further 2 nM NO after 1 min increased the rate from 4.4 to 6.4 µmol/mg of protein/min, whereas addition of hemoglobin to scavenge NO led to an immediate cessation of GC activity (Fig. 4c), indicating that if any biologically significant variation in the NO concentration had occurred over time, it would have been detected.
The concentration-response relationship was studied using 2-min exposures. The curve had a threshold of approximately 0.1 nM NO and displayed maximal activity at approximately 20 nM (Fig. 4d). It was well fitted by the Hill equation, with an EC50 of 1.7 nM and a Hill coefficient of 1.0.
With such low NO concentrations being effective, it is necessary to question whether the depletion of ligand through receptor binding could have distorted the results. Assuming a molecular mass of 150 kDa for GC and a single heme binding site on each protein (Denninger and Marletta, 1999; Koesling and Friebe, 2000), the total concentration of available binding sites at the protein concentration used (50 ng/ml) amounts to 0.33 nM, which is approximately 3-fold higher than the lowest effective NO concentration for GC activation (Fig. 4d). With normal methods of ligand application, therefore, ligand depletion would be significant and would have to be accounted for. To examine this issue using the present method of NO delivery, we incorporated reversible receptor binding into the model and assumed that the resultant GC activity was dependent on the concentration of the NO-bound species (see Fig. 5 legend for parameters). At 0.33 nM receptor, there would be negligible ligand depletion because the amount bound to the receptor is rapidly restored by NO release from the donor (Fig. 5a). Significant slowing of the attainment of the steady-state NO concentration and a resulting underestimate of GC activity is predicted to occur only with the receptor at concentrations 100-fold higher or more (Fig. 5, a and b).
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To check that the concentration-response curve obtained for the purified receptor (Fig. 4b) was not peculiar to the use of SPER/NO as the donor, we evaluated the combination of diethylenetriamine/NO (DETA/NO; half-life = 20 h) and CPTIO for the same purpose. To avoid the use of excessive DETA/NO concentrations, the concentration of CPTIO was decreased to 50 µM, allowing steady-state NO concentrations to be achieved in 4 s. As with SPER/NO, there was a linear relationship between the concentrations of DETA/NO and NO, the gradient being 4.6 x 10-5 M NO/M DETA/NO (data not illustrated). When the mixture was used to investigate the concentration-response curve for NO on purified lung GC (2-min exposure), the results (EC50 = 1.4 nM, Hill coefficient = 1.0; data not illustrated) were indistinguishable from those obtained using SPER/NO.
The established GC-coupled NO receptors are heterodimers, and the lung may contain both known isoforms, 11 and 21, with the former predominating (Mergia et al., 2003). Accordingly, the response of the purified lung protein may be a composite one. To examine the sensitivity of the separate isoforms to NO, they were expressed in COS-7 cells, and the NO-evoked GC activity was followed in cell lysates. The resultant maximal activity of the two isoforms was similar (Fig. 6, a and b). Moreover, the EC50 values for NO were also comparable (0.9 nM for 11 and 0.5 nM for 21). The slopes of both curves were described by a Hill coefficient of 1.1.
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First, it is necessary to consider the biological reactivity of the ingredients and products other than NO. CPTIO has frequently been used as an NO scavenger to test for its participation in various biological phenomena, and we are unaware of any unrelated side effects. SPER/NO belongs to the much-used class of NONOate donor, but the carrier molecule, spermine, is an endogenous polyamine with biological activity (Bachrach et al., 2001). The rate of NO release from SPER/NO may also depend on constituents of the medium and on donor concentration (Davies et al., 2001), although we have not yet observed such inconsistencies (Figs. 2 and 4) and have obtained identical results with another donor (DETA/NO) not reported to exhibit this anomalous behavior (Davies et al., 2001). Other NONOates such as NOC-5 or NOC-12 with half-lives of 25 and 100 min, respectively, could be used instead (http://www.dojindo.com/newprod/1/no/nodonors/nocsb.html). To deplete NO2, urate was used at the concentration found in plasma (Becker, 1993) and so it can be regarded as a physiological ingredient. The fate and reactivity of the resulting urate radicals, however, are unclear, and it seems wise to limit their production. Finally NO2-, produced by the reaction of NO2 with urate, will be formed at the same rate at which CPTIO is consumed. The NO2- concentration range found in human bodily fluids is 0.5 to 210 µM (Augusto et al., 2002), and it is relatively unreactive at neutral pH and therefore unlikely to create problems. Because of an overall lack of anticipated side effects, we recently used the method to analyze the kinetics of the NO-cGMP-phosphorylation pathway in suspensions of intact platelets (E. Mo, H. Amin, and J. Garthwaite., unpublished results) without encountering any problems.
Second, the method is limited in the range and duration of the NO concentrations obtainable. These two parameters are linked to the concentration and half-life of the donor and to the capacity of the sink. The immediate aim was to have a method that delivers fixed NO concentrations rapidly and maintains them over a time scale of minutes. With the combination of the donor and sink concentrations chosen, this objective was met for NO concentrations up to 100 nM. At this upper limit, the NO concentration was not constant but increased at a sufficiently slow rate to remain usable. For other types of experiments, exposures to NO of longer than a few minutes may be desirable. In this case, a donor with a longer half-life, such as DETA/NO (half-life 20 h), would be preferred. In this scenario, it is unlikely that the final NO concentration needs to be attained as rapidly as described here, so both donor and sink can be diluted to reduce the chemical flux. To illustrate the scope of such an application, Fig. 7 displays the predicted profile of the NO concentration obtained with a combination of 2 µM CPTIO and 3 to 10 µM DETA/NO. The time required for the initial equilibration of the NO concentration is approximately 2 min. With 3 µM DETA/NO, NO can be maintained at nearly 1 nM for at least 10 h, whereas at lower DETA/NO concentrations, the NO concentration decreases slowly as the donor decays. With 6 µM DETA/NO, the NO concentration is reasonably stable (2-3 nM) for approximately 5 h, but with 10 µM, the usable duration falls to approximately 1 h (3-4 nM NO). The duration can be extended quite easily by timely supplementation with fresh CPTIO, as illustrated for 6 µM DETA/NO in Fig. 7. Using essentially the same method, therefore, long exposures to physiological NO concentrations could be achieved.
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The third methodological issue is variability. The precise value of the NO concentration obtained, and its duration, relies critically on the purity of the CPTIO and the release rate of NO from the donor. As mentioned earlier (see Results), we have noticed significant variation in the rate of NO released from NONOates depending on the particular batch and supplier used. In addition, the rate of decomposition of the NONOates depends on both temperature and pH (Davies et al., 2001). For this reason, it is essential that the pH of the buffers is adjusted at the temperature used for the experiment and that the buffers have a sufficient capacity to tolerate the addition of alkaline solutions (used to dissolve uric acid and the NONOates) or the production of protons (by the reaction of NO2 with urate) without a change in pH. Finally, it should be noted that some cells can avidly consume NO (Griffiths and Garthwaite, 2001), which may necessitate the calibration of the NO delivery system in the presence of the cells under study.
Activity of GC-coupled NO Receptors Under Steady-State Conditions. In the past, a lack of control over NO concentrations has led to widely differing estimates of the potency of NO on its GC-coupled receptors. An initial estimate of the EC50 value, derived from the addition of NO from concentrated solutions, was 
250 nM (Stone and Marletta, 1996), and the similar potency found for the NONOate DEA/NO in standard GC assays (approximately 300 nM) (Russwurm et al., 1998) sustained the concept that physiological NO signaling involved NO concentrations in the 100 nM range. By monitoring the profile of NO release during the course of such assays, however, we found that a measured EC50 value of 300 nM for DEA/NO is compatible with the true potency of NO being in the low nanomolar range that had been suggested by a series of studies on intact cells from the brain (Bellamy et al., 2002). Furthermore, by using red blood cells to maintain constant NO concentrations, we obtained an EC50 of 4 nM for the purified lung receptor (Bellamy et al., 2002), suggesting that the high potency of NO in cells did not reflect some peculiarity of the protein in an intracellular environment. In addition, the slope of the concentration-response curve was unexpectedly steep (Hill coefficient of 2) which, if correct, would have important mechanistic implications for receptor activation in that it implies cooperative binding of two or more molecules of NO to each receptor.
Re-examination of this issue in the present study using the new NO delivery system supports the potency of NO for its receptor in lung being in the low nM range, although the actual EC50 value (
1.5 nM) was approximately 2-fold lower than that obtained using the red blood cell method (Bellamy et al., 2002). More importantly, the Hill coefficient was of the value (1) predicted for a single NO binding site. The discrepancy is best attributed to differences in the methodology and, in particular, to the former use of red blood cells. Any lysis of cells in the suspension would result in the release of free hemoglobin, which binds and inactivates NO far more avidly than when encapsulated in red blood cells (Liu et al., 1998). Although there was no evidence for free hemoglobin at the NO concentrations that were measurable (
5-10 nM) significant cell lysis (calculated to be 0.1% or greater) would preferentially impact the lower NO concentrations that could not be measured. Should this occur, the lower half of the concentration-response curve, which relied on predicted NO concentrations, would be artificially steepened, giving rise to an overestimate of the Hill coefficient. Such an effect would also explain the higher EC50 value obtained beforehand.
There had been no previous examination of the relative or absolute NO sensitivity of the individual 11 and 21 receptor isoforms in cell-free preparations. Concentration-response curves to DEA/NO were reported to be similar (Russwurm et al., 1998), but this result is equivocal (Bellamy et al., 2002). Nevertheless, a direct comparison using the new method indicated that the EC50 values for NO are closely comparable with each other (approximately 1 nM) and with the value obtained for the purified receptor protein from lung. We had previously found similar absolute potencies of NO toward the two isoforms when expressed in COS-7 cells (Gibb et al., 2003), but these estimates were complicated by receptor desensitization and bell-shaped concentration-response curves observed with the receptors in intact cells.
In conclusion, the kinetic parameters for activation of the GC-coupled NO receptor derived in this study are likely to be more reliable than those determined previously using the red blood cell method. The modified parameters obtained will simplify the development of models of receptor activation because they eliminate the need for incorporating cooperative binding of NO to its receptor. More generally, the results support the usefulness of the new method for delivering physiological concentrations of NO to biological preparations in a reliable and reproducible manner, which should assist the analysis of NO signal transduction.
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V.W. is a University College London M.B.Ph.D. student.
ABBREVIATIONS: NO, nitric oxide; CPTIO, 2-4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide; DEA/NO, diethylamine/nitric oxide adduct; DETA/NO, diethylenetriamine/nitric oxide adduct; GC guanylyl cyclase; SPER/NO, spermine/nitric oxide adduct; DTT, dithiothreitol; NOC, nitric oxide-amine complex.
Address correspondence to: Dr. John Garthwaite, The Wolfson Institute for Biomedical Research, University College London, Gower Street, London, WC1E 6BT, United Kingdom. E-mail: john.garthwaite{at}ucl.ac.uk
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) and 50 nM (
) or to 1 µM DEA/NO (
). Data are fit with a linear function and are the means ± S.E.M. of three independent runs. c, cGMP accumulation after exposure of the receptor protein to 2 nM NO without further addition (
) or a further 2 nM NO (
