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.
Highlights
According to conventional understanding, G proteins transduce signals from activated G protein-coupled receptors (GPCRs) via second messengers to regulate numerous downstream signalling targets in the cell [1,2]
We propose that pertussis toxin (PTX) treatment shifts the balance of intrinsic Gi and Gs activity upon adenylyl cyclase (AC) towards Gs, enhancing the effect of all cAMP-mediated inotropic agents
Gi was essentially inactivated by pertussis toxin The effectiveness of in vivo PTX treatment to inhibit Gi was assessed by measuring PTX-catalysed incorporation of [32P]ADPribose from [32P]NAD into available Gi in ventricular tissue
Summary
G proteins transduce signals from activated G protein-coupled receptors (GPCRs) via second messengers to regulate numerous downstream signalling targets in the cell [1,2]. Upon GPCR activation, GDP is exchanged for GTP on Ga, and both the GTPliganded Ga and the Gbc dimer regulate downstream targets. The predominant Gi-coupled receptor in the heart ventricle is the M2 muscarinic acetylcholine receptor [4]. Constitutive activity of this receptor exerts a mild, continuous inhibition of AC activity in normal rat ventricular cardiomyocyte membranes [5,6,7]. Despite the view that only b2- as opposed to b1-adrenoceptors (bARs) couple to Gi, some data indicate that the b1AR-evoked inotropic response is influenced by the inhibition of Gi. we wanted to determine if Gi exerts tonic receptor-independent inhibition upon basal adenylyl cyclase (AC) activity in cardiomyocytes. Contractility was measured ex vivo in left ventricular strips and cAMP accumulation was measured in isolated ventricular cardiomyocytes from adult Wistar rats
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