Stimulation of GC A by CNP was further corroborated by

Stimulation of GC-A by CNP was further corroborated by using a cGMP enzyme immunoassay. We could show that CNP significantly stimulated cGMP synthesis in the GC-A reporter cell line. CNP stimulation resulted in similar maximal cGMP levels compared to stimulation by ANP and BNP. Therefore, by luminescence measurements and cGMP determination we could clearly show that CNP is a full agonist of rat GC-A . A similar finding has recently been published (Dickey et al., 2008). We also characterized the activity of the commercially available synthetic receptor ligands mini-ANP, Cys-ANF(4-18) and Arg-ANF(6-18), which have been described as full ANP receptor agonist, clearance receptor ligand and ANP receptor antagonist, respectively (Maack et al., 1987, von Geldern et al., 1990, Li et al., 1995). As expected, mini-ANP was characterized as a potent GC-A agonist. Unexpectedly, Cys-ANF(4-18) and Arg-ANF(6-18) could be characterized as partial ANP receptor agonists. Whereas Cys-ANF(4-18) nearly fully stimulated the GC-A reporter cell line, Arg-ANF(6-18) showed rather low efficacy. Therefore, the observed biological effects of Cys-ANF(4-18) might be due to a combined effect related to clearance receptor binding and direct ANP receptor stimulation. Recently, different ligands have been proposed as GC-D agonists, namely guanylin and uroguanylin (Leinders-Zufall et al., 2007), uroguanylin but not guanylin (Duda and Sharma, 2008) and Fludara australia (Hu et al., 2007, Sun et al., 2009). Up to now, the proposed GC-D ligands are controversely discussed and should be confirmed independently. In this respect, our novel pGC reporter assay is an ideal tool to verify the putative GC-D ligands. However, we have not been able to show significant stimulation of GC-D by guanylin, uroguanylin or bicarbonate ions (F. Wunder, unpublished data). In summary, the results imply that our pGC reporter cell lines are well suited for the characterization of receptor pharmacology. Like our previously described cell-based sGC, nitric oxide and PDE reporter assays, the receptor guanylyl cyclase assays described in this report were also successfully transferred to the 1536-well MTP format (data not shown). This opens up the possibility to identify novel pGC ligands and modulators by uHTS (Wunder et al., 2005a, Wunder et al., 2005b, Wunder et al., 2007, Wunder et al., 2009). This novel reporter system may, therefore, be used for drug discovery and for natural ligand characterization of guanylyl cyclase orphan receptors like GC-D and GC-G .
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Acknowledgements
Introduction Nitric oxide sensitive guanylyl cyclase (NOsGC), the major physiological receptor for nitric oxide (NO), is a heterodimer consisting of an α and a heme-containing β subunit. The α1/β1 and the α2/β1 isoforms are known to be important for NO-signaling in humans. Activated by the endogenous ligand NO, both isoforms catalyze the conversion from GTP to cGMP. The two isoforms co-exist in a wide range of tissues, such as lung and vascular smooth muscle cells. The expression level of the α2/β1 isoform is generally lower, with the exception of brain tissue where both isoforms are present in similar amounts [1]. The two isoforms differ in their intracellular distribution. In the central nervous system the α2/β1 isoform is directed to the postsynaptic density via interaction with postsynaptic density protein 95 (PSD-95) [2]. We could recently show that the α2β1 isoform also interacts with Lin7a and is directed to cell-cell contacts upon overexpression in human embryonic kidney cells [3]. NO/cGMP signaling is protective in different disease pathomechanisms including angina pectoris [4], pulmonary hypertension [5], [6], congestive heart failure [7], [8] and fibrotic diseases [9], [10], [11]. Organic nitrates as NO-liberating drugs have been used for decades [4]. During the last 20 years, two classes of compounds modulating the NO/cGMP signaling pathway have been discovered: the heme-dependent stimulators such as BAY 41–2272 [12], riociguat [6] and vericiguat [8], and heme-independent activators such as cinaciguat [13] and BAY 60–2770 [14] (for excellent review, see [15], [16]). Riociguat is approved as therapeutic for chronic thromboembolic pulmonary hypertension (CTEPH) and pulmonary arterial hypertension (PAH) [5]. A phase II study investigating the effects on Systemic Sclerosis (SSc) is currently enrolling patients [10]. In a recent phase IIb study, another sGC stimulator vericiguat with a once daily kinetic profile in humans was well tolerated and showed beneficial effects in patients with chronic heart failure and reduced ejection fraction (HFrEF) [8], [17]. A phase III outcome study is ongoing in HFrEF patients [8]. While the sGC stimulators stimulate the enzyme and synergize with NO activation in a heme-dependent manner, the effect of the activators is heme-independent and is increased after removal of heme [13]. The prevailing view is that activator binding is restricted to heme-free NOsGC or oxidized NOsGC that has low affinity towards the ferric heme [15], [16], [18].