Receptor guanylyl cyclases play essential roles in cardiovas

Receptor guanylyl cyclases play essential roles in cardiovascular and gastrointestinal (patho-)physiology, reproduction biology, cell proliferation, bone growth and sensory signal transduction and, therefore, are important pharmacological targets. Accordingly, the receptor guanylyl cyclase agonists ANP and BNP (carperitide and neseritide) have been approved for the treatment of heart failure. Additional natural peptides (e.g., CNP, DNP and urodilatin) as well as designer peptides (e.g., CD-NP) are currently evaluated as potential therapeutics. As a drawback, all these approaches require parenteral application of the respective peptides. Therefore, small molecule pGC modulators with oral bioavailability would be very desirable as both pharmacological tools and novel therapeutics (Forte, 2004, Walther and Stepan, 2004, Dickey et al., 2008, Woodard and Rosado, 2008, Boerrigter et al., 2009, Kuhn, 2009, Potter et al., 2006, Potter et al., 2009). In addition, guanylin and its derivatives (e.g., linaclotide and guanilib) are explored for the treatment of GI disorders and colon cancer (Forte, 2004, Harris and Crowell, 2008; www.callistopharma.com).
Materials and methods
Results
Discussion We have shown previously that reporter cell lines expressing the homomeric, cGMP-sensitive CNGA2 channel and the calcium-sensitive photoprotein aequorin are well suited for the identification and characterization of modulators of the NO/cGMP signaling pathway. Using this cGMP reporter assay platform, we have identified and characterized sGC activators and stimulators, PDE inhibitors and modulators of NO synthesis (Wunder et al., 2005a, Wunder et al., 2005b, Wunder et al., 2007, Wunder et al., 2009, Mittendorf et al., 2009). In this report, we describe an additional application of the cGMP reporter assay: the functional Debio-1347 mg and pharmacological characterization of membrane-bound guanylyl cyclases. In the literature, EC50 values in the range of 0.4–100nM for the activation of GC-A by ANP or of GC-B by CNP, and of 150–300 nM for STa-mediated GC-C activation have been reported. These results were obtained by classical cGMP RIA measurements (Schulz et al., 1990, de Sauvage et al., 1991, Lowe and Fendly, 1992, Ohyama et al., 1992, Suga et al., 1992, Li et al., 1995, Schoenfeld et al., 1995, Dickey et al., 2008). Using our novel pGC reporter cell lines, corresponding EC50 values for GC-A and GC-B activation of ∼0.02–0.04nM and for GC-C activation of ∼0.2nM were determined. Therefore, the sensitivity of our reporter assay is at least 10 to 100-fold higher compared to cGMP RIA measurements with respect to the detection of pGC activity. The unexpectedly high sensitivity of this novel assay format is likely due to the direct plasma membrane colocalization of the membrane-bound guanylyl cyclases with the CNG channel used as the cGMP biosensor. The high assay sensitivity might also be due to signal amplification caused by calcium influx through the CNG channel and to cGMP compartmentation. As has been shown in cardiomyocytes, cGMP synthesized by the ANP receptor is located in subsarcolemmal microdomains and shows only limited diffusion into the cytosol (Castro et al., 2006, Fischmeister et al., 2006). In addition, due to the necessary cell lysis step, conventional cGMP assays only allow the determination of total intracellular cGMP, which impaires the detection of pGC activity. Using our novel GC-A and GC-B reporter cell lines, we could show that ANP, BNP and CNP significantly stimulate GC-A as well as GC-B receptors. This is an important finding, since the significance of activation of GC-A by CNP and of GC-B by ANP and BNP is under controversial discussion in the literature (Koller et al., 1991, Ohyama et al., 1992, Suga et al., 1992, Schoenfeld et al., 1995, Dickey et al., 2008, Woodard and Rosado, 2008). However, since in these studies receptor stimulation has been determined by RIA measurements, the observed discrepancies are likely due to the much lower sensitivity of the RIA assay for the detection of pGC activity. In addition, differences in receptor activation may also be observed when using receptors from different species (Dickey et al., 2008).