Dokument

Summary:
A wide variety of signaling substances such as hormones, neurotransmitters, odorants and chemokines control intracellular signaling by regulating the production of the second messenger cAMP. By activating Epac, PKA and cyclic nucleotide-gated ion channels, the production of cAMP alters a wide range of biological processes including cell division and metabolism. A number of GPCRs controls intracellular cAMP levels via stimulatory or inhibitory G proteins via adenylyl cyclases. The function of the broadly expressed AC5 and AC6 is enhanced by stimulatory (Gαs) or attenuated by inhibitory (Gαi) G proteins. Mechanistically both inhibition and stimulation is mediated via a direct protein-protein interaction. In addition to this direct regulation, several previous studies reported a cAMP rebound stimulation after withdrawal of Gi stimulation in cardiac myocytes for which the mechanism is debated (Hartzell, 1988; Wang & Lipsius, 1995). A similar cAMP rebound response was observed previously in our lab after termination of α2A-AR adrenergic receptor activation in HEK293T cells (Markus et al., 2013). The present study was aimed at investigating mechanisms underlying Gi-induced cAMP rebound effects. Many genetically encoded biosensors have been developed based on fluorescence resonance energy transfer (FRET) to visualize the spatiotemporal dynamics of various intracellular signals including second messengers. FRET-based cAMP biosensor (Epac1-camps) as well as heterologous overexpression system was used to investigate the mechanisms underlying Gi-mediated cAMP rebound stimulation in cardiac myocytes and also in heterologous expression system. When studying the mechanism of the long-known phenomenon of cAMP rebound stimulation after withdrawal of Gi stimulation in cardiac myocytes, we observed a PTX-sensitive/Gi-mediated/ adenylyl cyclase (type 5/6)/ cAMP-dependent pathway for this cAMP rebound stimulation. In addition, we observed that inhibition of Gβγ by gallein led to an attenuation of the AC5- mediated cAMP rebound response, although, overexpression of AC4 did not produce additional cAMP stimulation. This implies that different Gβγ-mediated signaling pathways may exist. Interestingly, we observed that PI3K inhibitor attenuates AC5/6-dependent cAMP rebound effects. This indicated that Gi-mediated cAMP rebound response was mediated via the PI3K-dependent pathway. Indeed, overexpression of PIP3-specific phosphatase PTEN confirmed that PIP3 itself either directly or indirectly mediated Gi-dependent cAMP rebound responses. Additionally, inhibition of PIP2-specific phosphatase SHIP and downstream events of PIP3-dependent regulation of Akt further confirm the influence of PIP3 on cAMP rebound levels. Indeed, surpassing Gi-mediated PI3K activation through PDGF-receptor stimulation strengthens this pathway. In addition, we confirmed that inhibition of PI3K also prevented cAMP rebound response after withdrawal of ACh in atrial myocytes. We suppose that the novel PIP3 dependent regulation of AC5/6 might represent a missing mechanism that explains physiological phenomena such as post vagal tachycardia.

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