Stimulus Specific or Localized cAmppka Signaling in Cardiomyocytes

cAMP constitutes an important intracellular second messenger molecule that is formed in response to the activation of many cell-surface G-protein-coupled receptors (GPCRs). In theory, cAMP is a freely diffusible second messenger that can act over long distances in cells. However, experimental results from over 25 years ago provided the first clues that cAMP/protein kinase A (PKA) pathways are compartmentalized in cells. Recognizing that the PKA regulatory subunit is expressed as structurally and functionally distinct RI and RII isoforms (forming the PKA-RI and PKA-RII holoenzymes), Brunton and colleagues showed that 0-adrenergic receptors (0ARs) activate particulate PKA-RII and induce changes in cardiac contractility, whereas prostaglandin E2 (which stimulates a different GPCR) activates soluble PKA-RI and regulates glycogen metabolism, but does not influence cardiac contractile function. Subsequent studies identified specificity at each step of the signal transduction pathway, including evidence that: (1) Gs-cou-pled GPCRs differ drastically in their ability to increase cAMP accumulation in cardiomyocytes. Certain GPCRs (such as the 02AR) increase cAMP accumulation when heterologously overexpressed in model cell types, but do not detectably elevate cAMP in certain cardiomyocyte preparations (Steinberg and Brunton 2001). Gs-coupled 0ARs also differ in their susceptibility to inhibitory input from muscarinic acetylcholine receptors (Aprigliano et al. 1997). (2) cAMP generated in response to GPCR activation does not necessarily gain access to all available PKA. (3) PKA tends to act in an agonist-specific manner to phosphorylate selective cellular targets. (4) cAMP signals can be terminated by phosphodiesterase (PDE) enzymes in a stimulus-specific manner.

Direct proof that cAMP signals are compartmentalized in cells is relatively recent and has resulted from technological advances that permit real-time imaging of cAMP gradients and compartments in cells. The Fischmeister laboratory used whole-cell patch-clamp recordings of Ca2+ transients at two physically distinct sites on a single isolated frog ventricular myocyte as a strategy to discriminate calcium channel regulation by local versus global pools of cAMP (Jurevicius and Fischmeister 1996). They showed that 0ARs activate a local population of calcium channels, whereas forskolin (a pharmacologic activator of adenylyl cyclase) activates calcium channels at both local and distal sites in the cell. Zaccolo and Pozzan (2002) took advantage of live cell imaging techniques to show that 0AR-dependent cAMP signals are localized by PDE enzymes, leading to the activation of a specific sub-population of PKA molecules in cardiomyocytes.

While cAMP can act through several cellular effectors, including PKA, cyclic-nucleotide-gated ion channels, phosphodiesterases, and guanine nucleotide-exchange proteins activated by cAMP (EPACs), cAMP signaling via PKA (historically viewed as the primary cAMP effector) has been studied most intensively. PKA exists as a tetramer consisting of two catalytic subunits that are held inactive by two regulatory (R) subunits. While PKA activation in theory could lead to the phosphorylation of a wide array of cellular proteins, PKA activity is regulated by A-kinase anchoring proteins (AKAPs) in cells. AKAPs position the PKA holoenzyme at specific subcellular microdomains where it can sense localized spikes in cAMP and gain access to specific cellular target substrates. AKAPs also function as multivalent scaffolds to assemble PKA in multi-protein complexes with other signaling kinases, phosphatases, and phosphodiesterases. By exerting bi-directional controls on both the propagation and termination of cAMP-initiated signals, AKAPs ensure a high degree of spatial and temporal control of cAMP signaling. This section summarizes current notions regarding mechanisms (involving AKAPs and other cellular scaffolds) that localize Gs-coupled receptors and the cAMP/PKA signaling pathway in cardiomyocytes.

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