Effects of nitroglycerin/L-cysteine on soluble guanylate cyclase: evidence for an activation/inactivation equilibrium controlled by nitric oxide binding and haem oxidation

Abstract

GTN (nitroglycerin; glycerol trinitrate) causes dilation of blood vessels via activation of nitric oxide (NO)-sensitive sGC (soluble guanylate cyclase), a heterodimeric haem protein that catalyses the conversion of GTP into cGMP. Activation of sGC by GTN requires enzymatic or non-enzymatic bioactivation of the nitrate. Based on insufficient NO release and lack of spectroscopic evidence for formation of NO-sGC, the cysteine (Cys)-dependent activation of sGC by GTN was proposed to occur in an NO-independent manner. This extraordinary claim is questioned by the present findings. First, the effect of GTN/Cys was blocked by the NO scavenger oxyhaemoglobin, the superoxide-generating compound flavin mononucleotide and the haem-site sGC inhibitor ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one). Secondly, at equi-effective concentrations, GTN/Cys and the NO donor 2,2-diethyl-1-nitroso-oxyhydrazine released identical amounts of NO. Finally, at sufficiently high rates of NO release, activation of sGC by GTN/Cys was accompanied by a shift of the Soret band from 431 to 399 nm, indicating formation of NO-sGC. In the absence of Cys, GTN caused haem oxidation, apparent as a shift of the Soret band to 392 nm, which was accompanied by inactivation of the NO-stimulated enzyme. These results suggest that the effect of GTN/Cys is the result of an activation/inactivation equilibrium that is controlled by the rate of NO release and haem oxidation.

Figure 1. Effects of (A) DEA/NO and (B) GTN/Cys on sGC activity in the absence and presence of YC-1

Purified sGC (50 ng) was incubated at 37 °C for 10 min in a final volume of 0.1 ml with the indicated concentrations of DEA/NO (A) and GTN (B) in the absence (○) or presence (●) of 0.2 mM YC-1. Assay mixtures contained 50 mM TEA/HCl, pH 7.4, [α-³²P]GTP (0.5 mM, ≈150000 c.p.m.), 3 mM MgCl₂, 1 mM cGMP and 2 mM GSH or Cys. Samples were analysed for [³²P]cGMP as described in the Materials and methods section. Results are shown as the means±S.E.M. for three to five experiments.

Figure 2. Effects of GTN/Cys, DEA/NO and ferricyanide on light absorbance of sGC

Spectroscopy was performed with 2.5 μM sGC in 50 mM TEA/HCl, pH 7.4, 75 mM NaCl, 2 mM GSH, 1 mM EDTA and 20% glycerol at 18 °C. When GTN was present, the final concentrations of these agents were slightly lower: 37 mM TEA/HCl, pH 7.4, 56 mM NaCl, 1.5 mM GSH, 0.7 mM EDTA, 15% glycerol and 36–50 mM glucose. The spectra are representative of three (DEA/NO), five (ferricyanide), eight (GTN/Cys), and 19 (no additions) experiments, performed with two different enzyme preparations. (A) Light absorption spectra of sGC (2.5 μM) measured in the absence (thick continuous line) or presence of 125 μM DEA/NO (dotted line), 0.88 mM GTN/2 mM Cys (thin continuous line) or 25 μM ferricyanide (broken line). The spectra obtained with DEA/NO and GTN/Cys were recorded after 30 min, the spectrum obtained with ferricyanide after ≈1 min. Raw spectra were processed as described in the Materials and methods section. (B) Corresponding absorbance difference spectra illustrating the effects of addition of DEA/NO (dotted line), GTN/Cys (thin continuous line), and ferricyanide (broken line) to native (ferrous) sGC (thick continuous line at ΔΔA=0). (C) The kinetics of the reaction between sGC (2.5 μM) and 0. 7 mM GTN alone (○) and 0.67 mM GTN/1.5 mM Cys (●) is presented as the change in the absorbance difference (ΔΔA) between 472 and 432 nm over time. For reasons of clarity, the absorbance difference at the zero time point was arbitrarily set to 0.02 and 0 for the results obtained with GTN alone and GTN/Cys respectively. The lines drawn through the data points are best fits to single exponentials, with first-order rate constants of 0.138±0.007 and 0.126±0.007 min⁻¹ for GTN alone and GTN/Cys respectively. Note that the fits intersect the y-axis well above the expected points for both reactions, indicating the presence of a fast phase that contributes approx. 25% to the total absorbance change.

Figure 3. Effects of FMN, oxyhaemoglobin and ODQ on GTN/Cys-activated sGC in the absence and presence of YC-1

Purified sGC (50 ng) was incubated at 37 °C for 10 min in a final volume of 0.1 ml with 0.1 mM GTN and 2 mM Cys in the absence (○) or presence (●) of 0.2 mM YC-1 and 0.1 mM FMN, 10 μM oxyhaemoglobin (OxyHb) or 30 μM ODQ. Results are the means±S.E.M. for three experiments.

Figure 4. Correlation of sGC activity and NO release from DEA/NO and GTN/Cys

(A) Re-plot of the control curves from Figures 1(A) and 1(B). (B) DEA/NO (0.03, 0.1, 0.3, 1 and 3 μM) or GTN (0.1, 0.3 and 1 mM) were incubated at 37 °C for 10 min in the presence of 2 mM Cys, followed by determination of NO by chemiluminescence, as described in the Materials and methods section. NO concentrations were plotted against the corresponding sGC activities shown in (A). Results are the means±S.E.M. for four to six measurements.

Figure 5. Simultaneous determination of sGC activity and light absorbance in the presence of DEA/NO and GTN/Cys

GTN (1 mM) was pre-incubated for 10 min at 37 °C in 5 mM [α-³²P]GTP, 15 mM MgCl₂, 1 mM cGMP, 50 mM TEA/HCl, pH 7.4, in the presence of 10 mM Cys (A–C) or GSH (D–F) in a final volume of 450 μl, followed by addition of 30 μg of GC in 50 μl of 50 TEA/HCl, pH 7.4, containing 10 mM Cys (A–C) or GSH (D, E), at the zero time point. Light absorbance spectra were recorded every 30 s using the 8453 diode array device from Agilent Technologies. For determination of sGC activity, 0.1 ml aliquots were removed 30, 60, 90 and 120 s after addition of the enzyme and quenched in 450 μl of zinc acetate (120 μM), followed by isolation of [³²P]cGMP. When indicated, DEA/NO (0.1 mM final) was added 1 min before addition of sGC. Results are means±S.E.M. for four experiments performed in duplicate. (A) sGC activity in the presence of 1 mM GTN and 10 mM Cys alone (○) or in combination with 0.1 mM DEA/NO (●). (B) Light absorbance spectra of GTN/Cys-treated sGC recorded at the indicated time points after addition of the enzyme. (C) Light absorbance spectra of sGC in the additional presence of DEA/NO. (D) sGC activity in the presence of 1 mM GTN and 10 mM GSH alone (○) or in combination with 0.1 mM DEA/NO (●). (E) Light absorbance spectra of GTN/GSH-treated sGC recorded at the indicated time points after addition of the enzyme. (F) Light absorbance spectra of sGC in the additional presence of DEA/NO.

Figure 6. Effect of GTN/Cys on sGC activity measured after 2 min of incubation

GTN (1 μM to 1 mM) was pre-incubated with 10 mM Cys for 10 min at 37 °C in the sGC assay mixture described in the legend to Figure 1, followed by 2 min of incubation with 0.1 μg of sGC in a final volume of 0.1 ml and determination of [³²P]cGMP, as described in the Materials and methods section. Where indicated, DEA/NO (0.1 mM final) was added 1 min before addition of sGC. Results are the means±S.E.M. for three experiments.

Figure 7. Inactivation of DEA/NO-stimulated sGC by GTN

(A) Purified sGC (0.5 μg) was pre-incubated with 0.3 mM GTN or vehicle in 0.1 ml of TEA/HCl, pH 7.4, for 10 min at 37 °C. Subsequently, samples were put on ice for 5 min, and 10 μl aliquots were incubated for 10 min at 37 °C in the presence of [α-³²P]GTP (0.5 mM, ≈150000 c.p.m.), 3 mM MgCl₂, 1 mM cGMP, 2 mM GSH and the indicated concentrations of DEA/NO. (B) Purified sGC (0.5 μg) was pre-incubated with 0.3 mM GTN or vehicle in 0.1 ml of TEA/HCl, pH 7.4, at 37 °C for 0–10 min. At the indicated time points, 10 μl aliquots were removed and incubated for 1 min at 37 °C in the presence of [α-³²P]GTP (0.5 mM, ≈150000 c.p.m.), 3 mM MgCl₂, 1 mM cGMP, 2 mM GSH and 1 μM DEA/NO. Results are expressed as percentages of the activity of sGC pre-incubated with vehicle, and represent means±S.E.M. for three experiments.