As described earlier, the chemical stability of the 5-nitrosothiols was monitored spectrophotometrically in the presence of different copper concentrations or in the presence of the corresponding thiols. The synthetic 5-nitrosothiols (1-13) were tested for biological activity in two assay systems, an isolated smooth muscle preparation and a platelet aggregation assay. All the 5-nitrosothiols tested were active in each assay. They relaxed smooth muscle preparations (rat tail artery), and inhibited collagen-induced platelet aggregation. Although the structures and chemical properties of the synthetic 5- nitrosothiols varied considerably, all the compounds were active in both bioassay systems. Coupled with the fact that the starting thiols and the product disulphide were inactive, this indicates that the -S-N=0 functional group is responsible for the biological activity of these compounds. The R- group, however, significantly influences the biological activity. We have found that 5-nitrosated dipeptides are potent vasodilators and suitable inhibitors for platelet aggregation but are chemically very stable in the absence of copper ions (SNAP < 5-nitrosated dipeptides < GSNO; Table 2.8). The potency of the 5-nitrosothiols varied as a function of the structure by as much as 0.8 - 45 |lM orders of magnitude in the isolated smooth muscle preparation, and 1.3 - 7.1 jxM in the platelet aggregation assay. It is clear that the platelet aggregation assay is more sensitive to 5-nitrosothiols than the isolated smooth muscle preparation. The effect of the R- group on biological activity was different in each assay; this is reflected by the different rank orders of activity observed in the systems. We found that the solution stability did not correlate with inhibition of platelet aggregation or relaxation of vascular smooth muscle. The effect of the structure on activity is pronounced; similar structures had different activity profiles (Table 2. 8).
A close inspection of Table 2.8 indicates that 2-12 (except 6 which was impure) are substantially more potent than SNAP in the smooth muscle relaxation bioassay, although the chemical environments of the -SNO groups are almost identical. There is an inverse correlation between chemical reactivity and biological activity only in the smooth muscle relaxation bioassay, a result which provides further evidence that extracellular decomposition of 5-nitrosothiols (2-12) to give NO cannot account for all their vasodilator effects. It suggests, rather, that the 5-nitrosothiol enters the cell intact, a process controlled partly by the lipophilicity of the complete molecule, before decomposition occurs or 5- nitrosated dipeptides interact with tissue components. Also the biological activity of the 5- nitrosothiols (1-13) in the platelet aggregation assay does not coiTelate with their chemical stability.
Unlike acidified nitrite, where biological activity is due to NO (Furchgott, 1988), the activity of 5-nitrosothiols is not due to the generation of NO in solution. These results are consistent with previous reports suggesting that 5-nitrosothiols do not act by releasing NO in solution. For example, the relaxation of smooth muscle by 5-nitrosothiols was shown to be enhanced by cysteine (Askew et al, 1995a&b) and superoxide dismutase and inhibited by A-acetyl-D,L-penicillamine, agents that had the opposite effect on 5-nitrosothiol degradation (Kowaluk and Fung, 1990a&b), indicating that spontaneous liberation of NO was not responsible for the vascular activity of these compounds.
TABLE 2. 8
The correlation between structure, chemical stability, and physiological activity
Compound Number Relative Rate of Decomposition (^5-Nitrosothio/^GSNo) * Smooth Muscle Relaxation ED50 (|X M) Inhibition of Platelet Aggregation IC50 (p M) SNAP (1) 2000 2 0 4.4 2 132 4 1.8 3 64 3.5 4.1 4 64 3 4.1 5 64 1.5 4.6 6 68 45 7.1 7 248 10 5.3 8 68 4.5 6.2
9 insoluble in the buffer 9.5 2.7**
1 0 100 5 1.9
1 1 112 0.8 2 . 8
1 2 172 2 3.2
GSNO (13)
* roriTAi — To/ 1 0.9 1.3
[Cu] = [added Cu] + [Cu present in the buffer]
** The stock solution was the drug dissolved in DMSO, because the drug is insoluble in the buffer.
Relaxation of vascular smooth muscle by a variety of agents, including 5-nitrosothiols, is believed to be due to stimulation of soluble GC (Ignarro and Kodowitz, 1995). Elevated cGMP levels may also regulate tone in nonvascular trached smooth muscle (Katsuki and Murad, 1977b) and may be involved in platelet aggregation (Mellion et al., 1981). Inhibition of GC using methylene blue (Ignarro et al., 1981 a&b) and N- methylhydroxylamine (Gibson et al., 1992) blocks the activity of 5-nitrosothiols. Thus, all of the actions of 5-nitrosothiols (1-13) that we have reported here, could be explained by stimulation of soluble GC. However, the biological activity of the 5-nitrosothiols in the smooth muscle relaxation assays and in the platelet aggregation assay does not correlate with the ability to stimulate platelet soluble GC. This suggests that additional factors are important in determining activity in these systems.
The stimulation of soluble GC in these systems could occur either directly or indirectly via
NO release. Direct stimulation would require that the intact 5-nitrosothiols enter the cell and activate GC, perhaps by transnitrosation of the active site haem moiety to form catalytically active NO haem (Ignarro, 1990). Alternatively, once inside the cell, the 5-nitrosothiols could liberate NO to stimulate GC. Although the ability of 5-nitrosothiols to cross membranes is not known, some of the 5-nitrosothiols examined here, such as SNAP (1) and GSNO (13), would not be expected to be able to enter cells easily. From our results we suggest that the ability of 5-nitrosothiols to cross membranes and enter cells is required for activity.