Gi Proteins Regulate Adenylyl Cyclase Activity Independent of Receptor Activation

Abstract

Background and purposeDespite the view that only β2- as opposed to β1-adrenoceptors (βARs) couple to Gi, some data indicate that the β1AR-evoked inotropic response is also influenced by the inhibition of Gi. Therefore, we wanted to determine if Gi exerts tonic receptor-independent inhibition upon basal adenylyl cyclase (AC) activity in cardiomyocytes.Experimental approachWe used the Gs-selective (R,R)- and the Gs- and Gi-activating (R,S)-fenoterol to selectively activate β2ARs (β1AR blockade present) in combination with Gi inactivation with pertussis toxin (PTX). We also determined the effect of PTX upon basal and forskolin-mediated responses. Contractility was measured ex vivo in left ventricular strips and cAMP accumulation was measured in isolated ventricular cardiomyocytes from adult Wistar rats.Key resultsPTX amplified both the (R,R)- and (R,S)-fenoterol-evoked maximal inotropic response and concentration-dependent increases in cAMP accumulation. The EC50 values of fenoterol matched published binding affinities. The PTX enhancement of the Gs-selective (R,R)-fenoterol-mediated responses suggests that Gi regulates AC activity independent of receptor coupling to Gi protein. Consistent with this hypothesis, forskolin-evoked cAMP accumulation was increased and inotropic responses to forskolin were potentiated by PTX treatment. In non-PTX-treated tissue, phosphodiesterase (PDE) 3 and 4 inhibition or removal of either constitutive muscarinic receptor activation of Gi with atropine or removal of constitutive adenosine receptor activation with CGS 15943 had no effect upon contractility. However, in PTX-treated tissue, PDE3 and 4 inhibition alone increased basal levels of cAMP and accordingly evoked a large inotropic response.Conclusions and implicationsTogether, these data indicate that Gi exerts intrinsic receptor-independent inhibitory activity upon AC. We propose that PTX treatment shifts the balance of intrinsic Gi and Gs activity upon AC towards Gs, enhancing the effect of all cAMP-mediated inotropic agents.

Atropine augments cardiac contractility by inhibiting cAMP-specific phosphodiesterase type 4

“…1e,f ). We also observed a well-documented increase of sensitivity to ISO after PTX treatment which might be due to off-target effects of this toxin 16 . To further test the hypothesis that atropine can mediate cardiac effects independent of muscarinic receptor blockade, we isolated ventricular cardiomyocytes from M 2 - or M 1/3 -receptor knockout mice 17 , 18 and expressed the cAMP-FRET sensor in these cells by adenoviral gene transfer.…”

Nuclear translocation of nuclear factor kappa B is regulated by G protein signaling pathway in arsenite-induced apoptosis in HBE cell line

“…The possible explanation for this phenomenon is that the mammals express nine membranous AC isoforms (ACs 1–9) and a soluble AC (sAC). Among these AC isoforms and sAC, only ACs‐5 and ACs‐6 were found to be regulated by Gα protein (Hanoune and Defer, ; Melsom et al, ). Thus, the changes on the contents of AC in response to arsenite exposure may not be more sensitive than the alteration of Gα protein as well as the PKA or cAMP contents.…”

Monitoring Cannabinoid CB2 -Receptor Mediated cAMP Dynamics by FRET-Based Live Cell Imaging

“…The faster response to 1 µM FSK in all Epac1-CB2-HEK cell response types as compared to Epac1-HEK cells might indicate that heterologous CB2 expression alters the available G-protein pool. It has been reported that free Gαi subunits can cause effects at ACs that are independent of receptor activation [ 32 ]. Through the presence of, e.g., spontaneously inactive CB2 that couple Gαi subunits [ 33 ], but do not elicit a signaling response, the pool of free Gαi might be reduced, leading to a faster cAMP production after direct AC activation with FSK in Epac1-CB2-HEK cells.…”