There is evidence for an adverse

There is evidence for an adverse sympatho-stimulatory effect in the medulla oblongata mediated by the α1/β1 isoform, implying a possible benefit of selective α2/β1 activation [19]. While the stimulator BAY 41–2272 increases NO-sensitivity of both isoforms [20], a prevalence of the α1/β1 isoform compared to α2/β1 was suggested for cinaciguat [21]. The response of the α2/β1 isoform towards BAY 60–2770 has not been evaluated to date. In the current study, we investigated the effects of the activator drugs cinaciguat and BAY 60–2770 on the NOsGC isoforms α1/β1 and α2/β1 expressed in Sf9 cells. For both isoforms, we show a higher efficacy of BAY 60–2770 compared to cinaciguat. We introduce a new method of NOsGC expression in the presence of activator drugs. This new approach shows I) stable insertion of activator into the enzyme during protein biosynthesis and maturation independent of the heme redox state and II) a decrease in overexpressed NOsGC subunits by activator drugs that is dependent on an intact catalytic site of the enzyme.
Materials and methods
Results
Discussion The activators BAY 60–2770 and cinaciguat (BAY 58–2667) are heme-mimicking compounds, which stimulate heme-free NOsGC. To obtain a heme-free NOsGC fraction, various methods are known: incubation with the detergent Tween 20 [30], [31], incubation with the NOsGC inhibitor ODQ which oxidizes the heme group [32], [33] and mutation of histidine 105, the proximal ligand of ferrous heme iron [34], [35]. All of these approaches have the disadvantage of enzyme manipulation, either by incubation with detergents or unspecific oxidants or by the introduction of a mutation. In the present study, we introduce a new and simple experimental strategy to remove heme and insert activator by addition of the drug to the expression system. Cytosolic preparations of Sf9 Cy5 TSA expressing NOsGC in the presence of activator showed strongly increased activity, although most of the free activator is removed (see Fig. 1C, D). Purification and UV VIS spectroscopic analysis revealed that the NOsGC heme peak is not detectable in preparations where activator was added during expression, demonstrating a loss of heme and suggesting an insertion of activator. The strongly increased activity of these preparations even after a two-step purification by affinity and subsequent size exclusion chromatography indicates a stable insertion of activator into the enzyme. Stasch et al. [13] reported that α1/β1 activation by cinaciguat is lower than activation by NO. This is consistent with our findings generated by post-expression activator application. However, with the new expression with activator approach, the cinaciguat-induced activity increases up to the level of NO-induced activity. For BAY 60–2770, this activity is even considerably higher than NO-induced activity (see Fig. 6). As there is evidence for an adverse sympatho-stimulatory effect mediated by the α1/β1 isoform [19], a preferential activation of the α2/β1 isoform could be beneficial. Matching previous results of our group [21], the purified α2/β1 isoform is only weakly activated by cinaciguat with the conventional route of activator application. However, using the expression with activator approach, we could show significant stimulation of α2/β1 by activator. As reported by Kumar et al. [14], the BAY 60–2770 efficacy on the α1/β1 isoform is higher compared to cinaciguat. In the current study, we demonstrate that this is also true for the α2/β1 isoform (see Fig. 1B, D, F; Fig. 6B). To investigate whether NO-stimulation of NOsGC still occurs following the expression in the presence of cinaciguat or BAY 60–2770, DEA/NO activation was measured at concentrations ranging from 1 nM to 10 mM. Between low concentrations (1 nM to 100 µM) and high concentrations (1 and 10 mM), marked differences in the DEA/NO activation profiles were detectable. At the low concentration range, activities of the enzymes expressed without activator result in a typical concentration-response curve. As expected for an enzyme preparation considered heme-free, the enzymes expressed in the presence of sGC activator showed no response to DEA/NO. Taken together, the low-concentration DEA/NO activation shows the expected differences between the heme-containing enzymes expressed without sGC activator and the heme-free enzymes expressed in the presence of sGC activator. In contrast, at very high and likely unphysiological concentrations of DEA/NO, the activities deviate upwards – for the enzymes expressed without activator upwards from the concentration-response curves, for the enzymes expressed with sGC activator upwards from the constant activity level. The first explanation for the DEA/NO-activation of the enzymes expressed with sGC activator would be a residual heme content. However, as there is no Soret peak detectable in UV VIS spectroscopy, the heme content of these enzyme preparations could only be low. The response to DEA/NO should thus be much smaller than the response of the high-heme content enzymes expressed without activator. However, as shown in Fig. 7C and D, the activation at the high DEA/NO concentrations increases by similar factors for the enzymes expressed in the absence and presence of sGC activator. We conclude that the observed effects of DEA/NO at 1 and 10 mM must be heme-independent, mediated by NO binding outside the heme moiety. Such a heme-independent effect of NO has been described by several groups. Marletta et al. describe a stimulatory effect of NO binding outside the heme domain [36]. The opposite effect may be expected due to S-nitrosylation of cysteines inside NOsGC that were shown to decrease enzyme activity [37]. As the reducing agent DTT was included in all activity measurements in the current study, the latter effect is likely prevented. This could explain that the activating influence of NO with very high NO concentrations prevails in our experimental setting. Preparation of sGC activator-bound enzymes as described in the current study might be a useful tool to investigate heme-independent effects of NO.