Role of cyclic nucleotide phosphodiesterase isoforms in cAMP compartmentation following beta2-adrenergic stimulation of ICa,L in frog ventricular myocytes

Abstract

The role of cyclic nucleotide phosphodiesterase (PDE) isoforms in the b 2 -adrenergic stimulation of the L-type Ca 2+ current (I Ca,L ) was investigated in frog ventricular myocytes using double patchclamp and double-barrelled microperfusion techniques. Isoprenaline (ISO, 1 nM to 10 mM) was applied on one half of the cell, either alone or in the presence of PDE inhibitors, and the local and distant responses of I Ca,L were used to determine the gradient of local vs. distant cAMP concentration (a). IBMX (100 mM), a non-selective PDE inhibitor, reduced a from 40 to 4.4 indicating a 9-fold reduction in intracellular cAMP compartmentation when all PDE activity was blocked. While PDE1 and PDE2 inhibition had no effect, PDE3 inhibition by milrinone (3 mM) or PDE4 inhibition by Ro 20-1724 (3 mM) reduced a by 6-and 4-fold, respectively. A simultaneous application of milrinone and Ro 20-1724 produced a similar effect to IBMX, showing that PDE3 and PDE4 were the major PDEs accounting for cAMP compartmentation. Okadaic acid (3 mM), a non-selective phosphatase inhibitor, or H89 (1 mM), an inhibitor of cAMP-dependent protein kinase (PKA), had no effect on the distant response of I Ca,L to ISO indicating that PDE activation by PKA played a minor role in cAMP compartmentation. Our results demonstrate that PDE activity determines the degree of cAMP compartmentation in frog ventricular cells upon b 2 -adrenergic stimulation. PDE3 and PDE4 subtypes play a major role in this process, and contribute equally to ensure a functional coupling of b 2 -adrenergic receptors with nearby Ca 2+ channels via local elevations of cAMP.

Spectrin-anchored phosphodiesterase 4D4 restricts cAMP from disrupting microtubules and inducing endothelial cell gap formation

“…However, more recent studies indicate colocalization of receptors, signaling intermediates, modulatory proteins and effectors to specialized microdomains, such as caveolae-enriched lipid rafts, is necessary for appropriate signal transduction (Head et al, 2005;Jurevicius et al, 2003;Ostrom et al, 2001;Rybin et al, 2000). Rich and co-workers (Rich et al, 2000;Rich et al, 2001a) have indicated that cells maintain tenfold greater cAMP levels at the membrane compared with the bulk cytosol.…”

“…Although all PDE4 isozymes have closely related kinetic properties and are sensitive to rolipram inhibition, they vary in subcellular localization (Beard et al, 1999;Brunton, 2003;Georget et al, 2003;Houslay and Adams, 2003;Jin et al, 1998;Jurevicius et al, 2003;Karpen and Rich, 2001;Willoughby et al, 2006). Our next studies sought to identify the specific PDE4 responsible for localizing cAMP hydrolyzing activity to PMVEC membranes.…”

cGMP-Hydrolytic Activity and Its Inhibition by Sildenafil in Normal and Failing Human and Mouse Myocardium

“…With respect to cAMP-mediated signaling, different cellular responses can be elicited depending on the G-protein-coupled receptors through which adenylyl cyclase activity is stimulated and intracellular cAMP content is increased (Hayes et al, 1980;Xiao et al, 1994;Kuschel et al, 1999;Xiao et al, 1999;Leroy et al, 2008). There are also several examples of differences in the potentiation of individual cAMP-mediated responses depending on the particular phosphodiesterase that is inhibited (Jurevicius et al, 2003;Mongillo et al, 2006;Nikolaev et al, 2006;Rochais et al, 2006). cGMP-mediated signaling in cardiac myocytes has been less thoroughly studied in this regard, but different cellular responses are elicited depending on whether intracellular cGMP levels are increased by stimulating soluble or membrane-associated forms of guanylyl cyclase and depending on which cGMP phosphodiesterases are inhibited Piggott et al, 2006).…”

Alveolar Epithelial β2-Adrenergic Receptors

“…There are nine types of adenylyl cyclases, which are all transmembrane proteins, synthesizing cAMP at the plasma membrane (73,74). This results in a gradient in which higher concentrations of cAMP are found at the membrane and lower concentrations in the cytosol (73,(75)(76)(77)(78). This gradient may allow localization of the physiologic effect of bAR and resultant production of cAMP to microenvironments, which in turn activate specific target proteins.…”