Two Opposite Signal Transducing Mechanisms Regulate a G-Protein-Coupled Guanylyl Cyclase

Membrane-bound guanylyl cyclase (GC) is regulated by muscarinic receptors (mAChRs). Carbamylcholine (CC) induces a “dual” biological response on GC activity. Thus, an activation is observed at 0.1 nM and a maximal response at 1 nM CC. However, at higher agonist concentration (>100 nM), there is an agonist-dependent inhibition of GC. This CC dual response is affected by 4-DAMP and HDD (M3antagonists), which produce a right-shift of the CC curve; the maximal CC dose response with 4-DAMP is more potent than that with HDD. Moreover, AFDX-DS (an M2antagonist) increases basal activity and decreases the agonist-dependent inhibition. Neither the CC response nor the CC maximal dose responses are affected by pirenzepine (PZ, M1antagonist). The agonist-dependent stimulation of GC activity is inhibited by 4-DAMP showing a −log IC50= 8.4 ± 0.4, while AFDX116 DS poorly inhibits such activity with a −log IC50= 5.0 ± 0.2. The agonist-independent (basal) GC activity also was inhibited by 4-DAMP, in a dose-dependent manner, with an IC50= 8.5 ± 0.2. Nonetheless, other muscarinic antagonists (PZ and HDD) were not able to inhibit this basal GC. Pertussis toxin treatment produces a complete blockade of the agonist-dependent inhibition of GC with a full expression of the agonist-dependent activation of membrane-bound GC. These results indicate that membrane-bound GC is regulated by muscarinic agents through two opposite signaling pathways; one involves the activation of GC via an M3mAchR coupled to a PTX-insensitive G protein, while the GC inhibition is mediated through a PTX-sensitive Gi/oprotein possibly coupled to an M2mAChR.