Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

Nitric oxide (NO) donors such as nitroglycerin (NTG), S-nitroso-N-acetylpenicillamine (SNAP), and linsidomine (SIN-1) are generally believed to elicit their vasodilatory effects through a common mediator, NO (Feelisch, 1993), that can bind to the heme site of soluble guanylate cyclase (sGC), activating the enzyme and catalyzing the conversion of GTP to cGMP (McDonald and Murad, 1995). Accumulated cellular cGMP then lowers intracellular calcium, leading to vasodilation (Murad, 1986; McDonald and Murad, 1996). Therefore, the vasodilatory action of NO donors is generally thought to occur primarily through the activation of the heme site of sGC.

Recently, a heme site-specific inhibitor for sGC, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) (Schrammel et al., 1996), has been used to examine the specificity of sGC activation induced by NO donors (Brunner et al., 1996). It was shown that the sensitivity of ODQ-mediated inhibition of vascular relaxation appeared to be different among NO donors, although the mechanism for this phenomenon was not examined (Brunner et al., 1996). More recently, Homer et al. (1999) showed that, with the rat pulmonary artery, ODQ was more inhibitory to the vasodilatory effects of organic nitrates such as nitroglycerin (NTG) than to those of SIN-1 and two different nucleophilic NO donors (the NONOates). These results strongly indicate that not all NO donors elicit vasodilation through exclusive activation of the heme site of sGC.

Indeed, S-nitrosothiols, a subgroup of NO donors, have been shown to activate heme-deficient sGC through modulation of its thiol group (Ignarro et al., 1981a). Furthermore, S-nitrosothiols were able to trigger sGC-independent signal transduction pathways, such as the activation of L-type calcium channels in ferret ventricular myocytes through covalent modification of proteins (Campbell et al., 1996). We also showed that although NTG induced hemodynamic tolerance in rats with congestive heart failure, other NO donors such as S-nitroso-N-acetylpenicillamine (SNAP) and organic nitrites did not (Bauer et al., 1997). These results indicate further that the vasodilatory mechanisms of NO donors may be diverse.

In an attempt to better understand the vascular pharmacology of NO donors, we examined the susceptibility of various NO donors toward the heme-site inhibition of sGC mediated by ODQ. We used NO donors that represent a range of chemical classes and purported donors of additional specific NO redox forms in addition to NO^· (Sokolovsky et al., 1966; Feelisch et al., 1989; Wink et al., 1991; Fukuto et al., 1992; Stamler et al., 1992). Thus, S-nitrosothiols such as SNAP and S-nitrosoglutathione (GSNO), and sodium nitroprusside (SNP) have been classified as NO⁺ donors; Piloty's acid as a NO donor; tetranitromethane (TNM) as a NO₂⁺ donor; SIN-1 as a concurrent O₂ donor; NONOates such as diethylenetriamine-NO (DETA-NO) as a predominant NO^· donor; and NTG as a mixed NO donor (Feelisch and Stamler, 1996). Our objective was to determine whether these NO donors exhibit different sensitivities toward ODQ inhibition, and if so, the underlying mechanism(s) responsible for such a phenomenon. We used a phosphoesterase inhibitor specific for cGMP, zaprinast (ZAP), and a thiol donor, N-acetylpenicillamine (NAP) as mechanistic probes.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Chemicals. Sources for chemicals were as follows: ODQ (Tocris, Ballwin, MO); C-type natriuretic peptide (CNP), SNP, ZAP, and NAP (Sigma, St. Louis, MO); NTG (Schwarz Pharma, Monheim, Germany); SNAP, GSNO, and SIN-1 (Alexis, San Diego, CA); DETA-NO (Research Biochemical, Natick, MA); Piloty's acid (Fluka, Milwaukee, WI); and TNM (Aldrich, Milwaukee, WI). These chemicals were used without further purification. All drug solutions were made immediately before experiments. ODQ and TNM were dissolved in ethyl alcohol and the remaining compounds were dissolved in 5% dextrose.

Preparation of Rat Aortic Rings. All procedures were approved by the University at Buffalo Institutional Animal Care and Use Committee. Male Sprague-Dawley rats (300-350 g) were anesthetized with ketamine/xylazine (90/10 mg/kg). Loss of consciousness was indicated by the lack of the foot reflex response. The abdomen was then exposed, and the rat was exsanguinated via the abdominal aorta. The thoracic cavity was quickly opened, and the thoracic aorta was excised and placed in ice-cold Krebs' buffer, of the following composition: 120 mM NaCl, 5.6 mM KCl, 1.2 mM MgCl, 1.2 mM NaH₂PO₄, 10 mM dextrose, 25 mM NaHCO₃, 2.5 mM CaCl₂, (pH 7.4). The fascia was carefully removed from the thoracic aorta. The ends (5 mm each) of the vessel were discarded, and the vessel was then cut into transverse rings, approximately 2 mm wide. The rings were immediately mounted in 10-ml tissue baths containing Krebs' buffer and gassed continuously with 5% CO₂ in O₂. The aortic rings were washed with Krebs' buffer every 15 min and adjusted to maintain a tension of 1 g over a period of 1 h. The tension of the rings was measured using a force displacement transducer (FTO3C; Grass Instruments, Quincy, MA) and displayed on a Grass model 79D polygraph. Only rings showing stable tension >1 g at the end of the adjustment period were used in this study.

Vasorelaxation Studies. After an equilibration period of 1 h as described above, the rings were incubated with ODQ (0.1-10 µM) for 15 min and then precontracted with phenylephrine (PE; 2 µM). PE addition produced submaximal (50-80% of 2 g) contraction of the aorta preparation. After the contractile response of the rings to PE reached a plateau (approximately 15 min), incremental doses of CNP (10¹¹1.51 × 10⁶ M) or NO donors (2.2 × 10¹⁰5.87 × 10⁵ M) were added. Each dose was added at an interval of 4 min or until a plateau of the effect was reached. Appropriate vehicle control experiments were also conducted; vehicle effects were not observed. In separate studies, either ZAP (10 µM) or NAP (100 µM) was added to the tissue baths before NO donors in the presence of ODQ at a concentration of 1 µM.

Data Analysis. Relaxation effect was expressed as a percentage of the tone induced by the addition of PE (100%). The negative log molar EC₅₀ (pEC₅₀) was calculated through curve fitting, assuming a sigmoid E_max model (GraphPad, version 2; GraphPad Software, Inc., San Diego, CA). The area under the percentage of relaxation versus log concentration (M) curve (AURCC) was calculated with the trapezoidal rule (Gibaldi and Perrier, 1982), using the range of concentration studied (2.2 × 10¹⁰-5.87 × 10⁵ M) for all NO donors examined. Percentage of inhibition was estimated as (1  AURCC_ODQ/AURCC_C) × 100, where AURCC_ODQ was calculated from the ODQ treatment groups, and AURCC_C was the mean value calculated from the control group. The data were expressed as means ± S.D. The effects of different ODQ concentrations on the relaxation response of a NO donor were evaluated by one-way ANOVA followed by Dunnett's test. Reversal effects of ZAP and NAP on ODQ-mediated inhibition were also evaluated by a similar statistical test. ODQ-mediated inhibitions by NO donors were evaluated by two-way ANOVA with post hoc Duncan's multiple range test. Statistical significance was accepted at the 5% level.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

The NO donors studied exhibited a wide range of vasodilating potency toward the isolated rat aorta. Table 1 lists the pEC₅₀ and AURCC values of the eight NO donors studied. The least potent NO donor studied, DETA-NO, exhibited an EC₅₀ that was 300-fold less than the most potent compounds in the group, viz., SNP and NTG. A plot of the pEC₅₀ versus AURCC values obtained shows an excellent correlation between these two indices of vasorelaxant potencies (Fig. 1, r² = 0.997); the higher the AURCC value, the more potent (smaller EC₅₀) the vasodilator.

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Relationship between pEC50 and AURCC, two indices of vasorelaxant potencies used in this study for NO donors. See Table 1 for number of replicates for each NO donor.

Addition of ODQ to untreated blood vessels caused a slight contraction before the addition of PE. No correction for this slight effect was made. As expected, the vasorelaxant effect of CNP, a known activator of particulate GC (Schulz et al., 1991), was not affected by ODQ (Fig. 2A). The pEC₅₀ values for CNP were 6.65 ± 0.23, 6.48 ± 0.24, and 6.76 ± 0.11, respectively, in the presence of 0, 1, and 10 µM ODQ (ANOVA, P > .05), corresponding to AURCC values (from 1 × 10¹¹ to 1.51 × 10⁶ M of the vasodilator) of 87.9 ± 21.7, 70.6 ± 25.0, and 101 ± 15 (with a unit of % relaxation × log M; ANOVA, P > .05).

View larger version (28K): [in this window] [in a new window]   Fig. 2.   The vasodilatory effects of CNP (A), SNAP (B), SNP (C), and NTG (D) under control conditions (), or in the presence of 10 µM (), 1 µM (), 0.1 µM () ODQ. Data represent means ± S.D. of 3 to 12 experiments. *P < .05, compared with 10 µM ODQ; #P < .05, compared with 1 µM ODQ.

Preincubation of ODQ for 15 min before adding the NO donors caused significant rightward shift of the concentration-response curves of all compounds studied, but to varying degrees. For illustration, the relaxation curves of SNAP, SNP, and NTG in the presence of ODQ are shown (Fig. 2, B-D).

When the percentage of inhibition was analyzed for each of the NO donors as a function of ODQ concentration (Fig. 3), it was observed that the NO donors exhibited a varying degree of sensitivity toward this inhibitor. For example, at 0.1 µM, the lowest inhibitor concentration studied, ODQ inhibited S-nitrosothiol relaxation (SNAP and GSNO) only to 60 to 70%, whereas inhibition was almost complete for NTG, even though their pEC₅₀ values in control conditions were comparable (Table 1). A two-way ANOVA indicated that ODQ-mediated inhibition of NO donor vasorelaxation was dependent on both ODQ concentration and the NO donor used. Resistance to ODQ inhibition was the weakest for NTG, which was significantly more inhibited by ODQ compared with all NO donors studied except TNM. The degree of resistance toward ODQ inhibition generally can be grouped in the following order: SNAP, GSNO > DETA-NO > SNP, Piloty's acid, SIN-1 > TNM, NTG. The order of ODQ resistance was not related to the potency of the NO donor (see Table 1).

View larger version (44K): [in this window] [in a new window]   Fig. 3.   Comparison of sensitivity toward ODQ inhibition by various NO donors. Two-way ANOVA indicated that the degree of resistance toward ODQ inhibition among these compounds were dependent on both NO donors and ODQ concentration (P < .05). The order of ODQ resistance was SNAP, GSNO > DETA-NO > SNP, Piloty's acid, SIN-1 > TNM, NTG. Data represent 3 to 12 experiments for each NO donor.

The nature of the differential resistance toward ODQ inhibition on the vasodilatory effects mediated by SNAP, SNP, and NTG was further investigated. These compounds represent NO donors with a range of resistance toward ODQ inhibition: SNAP (most), SNP (intermediate), and NTG (least). Table 2 shows the effect of 10 µM ZAP or 100 µM NAP on the reversal of ODQ inhibition of these three NO donors. Analysis of the AURCC showed that the reversal effect of ZAP against ODQ was significant for SNAP and SNP, but not for NTG (Table 2). The same conclusion can be drawn when comparing the data based on percentage of inhibition of relaxation of control blood vessels (Table 2). Each of the experiments involving ZAP and NAP was run against a concurrent ODQ control. The minor differences in AURCC values of ODQ controls between the two series of studies (ZAP versus NAP) were likely due to variabilities among animals and in the preparation of aortic rings.

Addition of the thiol NAP caused a significant leftward shift of the concentration-relaxation curve for SNAP and SNP. NAP also enhanced the vasodilatory activity of NTG in the presence of ODQ, albeit to a lesser extent than those seen for SNAP and SNP. Table 2 compares the AURCC values obtained from experiments. Based on this parameter, it is observed that 100 µM NAP statistically dampened the inhibitory effect of 1 µM ODQ for SNAP and SNP, but not for NTG. When the data were presented as percentage of inhibition of control relaxation, the same statistical difference was seen (Table 2).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

ODQ, a recently discovered inhibitor of sGC, is an oxadiazoloquinoxaline derivative and a heme oxidant that is specific for sGC (Schrammel et al., 1996). We found that the vasodilatory effect of CNP, an activator for particulate GC, was not affected by ODQ, which is consistent with literature data (Olson et al., 1997). The vasorelaxation mediated through the -adrenoreceptor or ATP-sensitive K⁺ channel also was not affected by ODQ (Olson et al., 1997). Moreover, ODQ was shown not to affect the total amount of NO released from NONOates (Olson et al., 1997) and not to interfere with the production of cAMP induced by forskolin (Garthwaite et al., 1995). Thus, sensitivity toward ODQ inhibition of vascular relaxation would indicate a predominant mechanism of heme site-mediated activation of sGC, whereas resistance toward ODQ would suggest the possible presence of alternate mechanisms of vasodilatation.

It is interesting to observe, therefore, that SNAP and NTG represented the extremes, among the NO donors examined, in terms of their sensitivity toward ODQ inhibition. NTG was completely inhibited by ODQ, even at the lowest concentration studied (0.1 µM). In contrast, SNAP exhibited the most resistance toward ODQ inhibition and, at 0.1 µM ODQ, manifested about one-third of the vasorelaxation potency when compared with control (in the absence of ODQ). This result would suggest that SNAP can elicit vascular relaxation, at least partially, through a secondary pathway independent of activation of the heme site of sGC. It is important to note that the additional mechanism elicited by S-nitrosothiol was also sensitive to ODQ-mediated inhibition, because 10 µM ODQ almost totally abolished the vasodilatory effects of all NO donors examined. Thus, it is unlikely that the secondary mechanism involves either particulate GC, adenylate cyclase, the ATP-sensitive K⁺ channel, or -adrenergic receptor, because it has been shown that these mechanisms are insensitive to ODQ inhibition at 10 µM (Garthwaite et al., 1995; Olson et al., 1997).

Another possible cause for the differential sensitivity of NO donors toward ODQ inhibition may arise from their dissimilar dependence on heme-related metabolic activation. It is well known that NTG requires cellular metabolism to produce NO (Chung and Fung, 1990), using a variety of enzymes such as glutathione S-transferase, cytochrome P-450, and a microsomal enzyme (Bennett et al., 1994). Among these enzymes, at least one, namely cytochrome P-450, is a heme-containing enzyme, and it has been suggested that a reduced heme molecule is required for the biotransformation of NTG (Bennett et al., 1986). Thus, the presence of ODQ may have inhibited NTG bioactivation in addition to specific inhibition of sGC. This view has been proposed by Homer et al. (1999), but supportive data were not available. Our data indicate that other NO donors, such as DETA-NO, Piloty's acid, and TNM, which are known to produce NO spontaneously (Sokolovsky et al., 1966; Morley and Keefer, 1993; Zamora et al., 1995), were significantly more inhibited by ODQ than the S-nitrosothiols, which also require metabolic activation to produce NO (Kowaluk and Fung, 1990a). Thus, ODQ-mediated inhibition of heme-dependent NO bioactivation may not play a substantial role in the observed phenomenon.

Ignarro et al. (1981a) observed that S-nitrosothiol still activated sGC and generated cGMP in a preparation of isolated sGC deprived of heme. Moreover, the basal enzyme activity and the activity stimulated by S-nitrosothiol were abolished by various disulfides, sulfhydryl oxidants, and thiol alkylating agents, suggesting that the thiol group is involved in the mechanism of activation. The present experiments provide evidence that S-nitrosothiols can functionally elicit partial vasorelaxation independent of the heme site of sGC, and this is achieved probably via the sulfhydryl site of the enzyme.

Our studies show that both ZAP and NAP can significantly dampen (or reverse) the inhibitory activity of ODQ for some NO donors (SNAP and SNP) but not for another (NTG). The observation that ZAP enhanced SNAP and SNP vasodilation in the presence of ODQ inhibition of the heme site of sGC indicates that cyclic nucleotide(s) is involved in a secondary mechanism of vasodilation for these NO donors. This pathway of action however is not available to NTG, which appeared to rely on heme site activation of sGC as its exclusive mechanism of vasodilation. ZAP has been shown to have a 15-fold selectivity for cGMP-specific phosphodiesterase versus its cAMP counterpart (Coste and Grondin, 1995). These results indicate that, in the presence of complete inhibition of the heme site of sGC (as shown by the results with NTG), SNAP and SNP can still activate a cellular enzyme/site that catalyzes the production of cGMP, possibly through activation of the sulfhydryl site of sGC.

Our conclusion, which is summarized in Fig. 4, is supported by the following findings in the literature. First, it has been demonstrated that NTG could not activate heme-deficient sGC in the absence of thiol, indicating that NTG cannot interact with the thiol site of sGC to produce cGMP (Ignarro and Gruetter, 1980). This finding is consistent with the present observation that, when the heme site of sGC is completely blocked by 1 µM ODQ, NTG cannot elicit any additional vasodilatory activity in the presence of ZAP, because significant amounts of cGMP are not produced by NTG under these conditions. The presence of NAP did lead to a slight enhancement of NTG-induced relaxation in the presence of ODQ. This finding can nevertheless be explained by the chemical formation of S-nitrosothiol between NTG and thiol in this system (Ignarro et al., 1981b).

View larger version (22K): [in this window] [in a new window]   Fig. 4.   A proposed mechanistic scheme showing the heme-mediated and sulfhydryl-mediated activation of sGC by NO donors.

Second, S-nitrosothiols can activate heme-deficient sGC, albeit in much reduced activity (Ignarro et al., 1982). Thus, during ODQ inhibition, in which the catalytic heme site is no longer available, SNAP may still be able to act on the sulfhydryl site to produce a limited quantity of cGMP. Given the steep concentration-response curve of cGMP, such accumulation may be functionally sufficient to produce partial reversal of inhibition of vascular relaxation.

Third, the behavior of SNP is also consistent with this interpretation. cGMP production by SNP has been shown to be much enhanced in the presence of thiols (Ignarro et al., 1980), which is consistent with our observation (Table 2) that NAP significantly reversed ODQ inhibition of SNP-induced relaxation, possibly through the in vitro formation of S-nitrosothiols. SNP is presumed to contain some NO⁺ characteristics (Stamler et al., 1992), therefore a direct reaction of this nitrosylating species with the sulfhydryl site of sGC is also feasible.

It is recognized that other mechanisms of action have been described for S-nitrosothiols, e.g., activation of L-type Ca²⁺ channel (Campbell et al., 1996). The present study did not address the specific applicability of these alternative mechanisms, and therefore their possible involvement cannot be ruled out at this time.

The use of a phosphodiesterase inhibitor in this study to probe the possible involvement of cGMP in the signaling pathway is in contrast with the approach of several other groups, which used the direct measurement of cGMP in vascular tissues. For example, Onoue and Katusic (1998) also found that, with isolated canine cerebral arteries, diethylamine-NONOate, a spontaneous NO donor, caused complete relaxation at 3 × 10⁶ M ODQ, whereas increase in cGMP was completely abolished. These authors favored a cGMP-independent mechanism to explain their data. Brunner et al. (1996) found that changes in cGMP accumulation were not qualitatively associated with those of relaxation when bovine pulmonary artery strips were exposed to GSNO in three different concentrations of ODQ. These authors therefore concluded that a cGMP-independent mechanism may be responsible for the resistance of GSNO toward ODQ inhibition.

However, in the latter study (Brunner et al., 1996), even at 10 µM ODQ, limited amounts of cGMP were produced in response to higher concentrations of GSNO, indicating that some activation of sGC still occurred. It should be noted that the concentration-response curve of cGMP in blood vessels is quite steep. Kenkare and Benet (1996) showed that in rabbit aorta a change from 30 to 40 pmol cGMP/g wet wt. of tissue produced a change in percentage of relaxation from 30 to 80%. Tissue cGMP also has an extremely short half-life, i.e., 23 s (Tzeng and Fung, 1992), therefore the accuracy of determining tissue cGMP may be limited. We have also shown that a quantitative relationship between vascular cGMP and relaxation could not be obtained for NTG and SNAP (Kowaluk and Fung, 1990b). The advantage of using a phosphodiesterase inhibitor to define a mechanistic role for cyclic nucleotide(s) is that such an inhibitor amplifies the presence of these signaling molecules by decreasing and delaying their degradation.

A limitation of this study is that it was not possible to examine possible changes in E_max of the NO donors in the presence of significant ODQ inhibition. Therefore, it is not known whether this parameter is also affected. In the absence of a definitive E_max, the determination of EC₅₀ may be highly inaccurate (Dutta et al., 1996). In this study, therefore, we used the AURCC as a primary parameter to assess the degree of inhibition. We have shown in Fig. 1 that, in the absence of ODQ inhibition, this parameter is well correlated with changes in pEC₅₀ values observed for the various NO donors.

Kowaluk and Fung (1990a) have shown previously that the vasorelaxant response of SNAP was decreased in the presence of NAP. In contrast, we showed that NAP enhanced the relaxation response of SNAP in the presence of ODQ. Kowaluk and Fung (1990a) have suggested that the presence of an exogenous thiol might compete for NO transfer from SNAP to sulfhydryl sites on the vascular surface, thus diminishing the effectiveness of SNAP. However, there are multiple points of interactions between NAP and the cascade of mechanism of action for SNAP. It has been reported that reduced sulfhydryl sites enhance the activation of sGC through the heme site (Hobbs, 1997). There is, however, no literature reporting how the status of oxidation of the heme site might affect the activity of sulfhydryl site. It is possible that, in the presence of inhibition of the heme site of sGC by ODQ, production of cGMP from the sulfhydryl site of sGC is more sensitized, resulting in a net positive effect from the exogenous thiol. However, additional experiments need to be conducted to fully explain the apparent discrepancy among these observations.

In conclusion, this study showed that NO donors do not behave identically toward ODQ inhibition of vascular relaxation. This finding would suggest that the biochemical mechanism(s) of vasorelaxation of NO donors is not necessarily the same. Specifically, S-nitrosothiols, such as SNAP, may activate cellular vasodilating mechanisms through interaction with sulfhydryl site(s) and such mechanisms may not be available to (or feasible for) NTG.