cAMP microdomains and L‐type Ca²⁺ channel regulation in guinea‐pig ventricular myocytes

Abstract

Many different receptors can stimulate cAMP synthesis in the heart, but not all elicit the same functional responses. For example, it has been recognized for some time that prostaglandins such as PGE1 increase cAMP production and activate PKA, but they do not elicit responses like those produced by β-adrenergic receptor (βAR) agonists such as isoproterenol (isoprenaline), even though both stimulate the same signalling pathway. In the present study, we confirm that isoproterenol, but not PGE1, is able to produce cAMP-dependent stimulation of the L-type Ca 2+ current in guinea pig ventricular myocytes. This is despite finding evidence that these cells express EP 4 prostaglandin receptors, which are known to activate G s -dependent signalling pathways. Using fluorescence resonance energy transfer-based biosensors that are either freely diffusible or bound to A kinase anchoring proteins, we demonstrate that the difference is due to the ability of isoproterenol to stimulate cAMP production in cytosolic and caveolar compartments of intact cardiac myocytes, while PGE1 only stimulates cAMP production in the cytosolic compartment. Unlike other receptor-mediated responses, compartmentation of PGE1 responses was not due to concurrent activation of a G i -dependent signalling pathway or phosphodiesterase activity. Instead, compartmentation of the PGE1 response in cardiac myocytes appears to be due to transient stimulation of cAMP in a microdomain that can communicate directly with the bulk cytosolic compartment but not the caveolar compartment associated with βAR regulation of L-type Ca 2+ channel function.

Spatiotemporal Dynamics of β-Adrenergic cAMP Signals and L-Type Ca 2+ Channel Regulation in Adult Rat Ventricular Myocytes

“…7 Thus, cAMP diffusion from the membrane to the bulk cytosol must be restricted either by an enzymatic or a physical barrier. 12,22 The rate of I Ca,L stimulation reported here is similar to those reported earlier for I Ca,L in frog ventricular myocytes, 23 for neuronal potassium channels, 24 and for the membraneassociated PKA sensor AKAR2. 4 Activation of Ca v 1.2 developed Ͼ2-times slower than the rise of cAMP at the membrane, suggesting that the rate-limiting step in the ␤-AR stimulation of I Ca,L occurs after cAMP accumulation.…”

Cytoplasmic cAMP concentrations in intact cardiac myocytes

“…However, only the Epac2-camps responds to prostaglandin receptor activation, which indicates that it is able to detect changes in cAMP occurring in subcellular locations outside of those where type II PKA is found (35). This raises the question of whether or not ␤ 1 AR stimulation elicits a uniform increase in cAMP throughout the cell.…”

“…The Epac2-camps biosensor used in this study consists of the cAMP binding domain of Epac2 with yellow fluorescent protein (YFP) and cyan fluorescent protein (CFP) attached to the amino and carboxy terminal ends, respectively (18). An adenovirus expressing this biosensor (Ad-YFP-Epac2-CFP) was created by use of the Clonetech Adeno-X-expression system (BD Biosciences), as described previously (35). Myocytes were exposed to virus-containing media (MOI 10 to 50) for up to 48 h. Epac2-camps expression was monitored by measuring CFP fluorescence.…”

“…When expressed in adult ventricular myocytes, the PKAand Epac2-based probes both respond to ␤ 1 AR stimulation (35). However, only the Epac2-camps responds to prostaglandin receptor activation, which indicates that it is able to detect changes in cAMP occurring in subcellular locations outside of those where type II PKA is found (35).…”

“…This includes the FRET-based biosensor constructed with the nucleotide binding domain of the type 2 exchange protein activated by cAMP (Epac2-camps). The absence of anchoring sequences found in the full-length protein results in homogeneous expression of this probe throughout the cytoplasm of cells (19,35). Consequently, this probe is expected to respond to changes in cAMP occurring anywhere in the cell.…”