Sinoatrial nodal cells (SANC) express Ca2+-activated adenylate cyclase (AC) isoforms that generate a high basal level of cAMP-mediated, protein kinase A (PKA)-dependent Ca2+ cycling protein phosphorylation, resulting in spontaneous rhythmic Ca2+ oscillations that ignite the surface membrane to generate rhythmic APs, i.e. pacemaker automaticity. Differences in basal phosphodiesterase (PDE) and AC activities and in PDE:AC activity within protein microenvironments in SANC are a potential mechanism for compartmental regulation of cAMP levels, leading to local differences in the effectiveness of cAMP signaling within these microdomains. The present study measured the AC activity and PDE activity in SANC in detergent-resistant microdomains (DRM) of SANC lysates to determine the PDE and AC activities in lipid raft gradients indexed by GM-1 and caveolin-3 immunolabeling. The microdomain Ca2+ dependence of AC activity, and the relative abundance of microdomain PDE types, based upon the effects of specific PDE inhibitors, were also determined. Under the conditions of our assay, PDE and AC activities are nearly identically matched in the fractions that contain higher densities of lipid raft markers. As the lipid raft density decreases below a threshold the PDE:AC activity ratio becomes increased: from 10 to more then 200 fold , depending upon the Ca2+ milieu, while neither the relative extent of AC activation by different Ca2+ significantly varied among the different microenvironments, the Ca2+ milieu in lipid raft-rich fractions affects the matching of PDE to AC activities. The most optimal milieu for cAMP production and survival is when the Ca2+ is 1 microM, and the lipid raft marker density is high;when Ca2+ is reduced to 200 nM or heavily buffered by BAPTA, however, PDE activity exceeds that of AC, even in lipid raft-rich fractions. The very high PDE activity and lower AC activity in non-lipid raft domains favors degradation of cAMP, not only maintaining a reduced local level of cAMP production, but also creating a functional barrier for diffusion of cAMP from lipid rafts into non-lipid raft domains or into the cytosol. Most of the transcripts coding PDE catalytic subunits demonstrated significant difference in level of expression in SANC and LV. mRNA level of PDE 1A, 3B, 4B and 5A was higher in SANC cells then in LV cells. Moreover, expression level of PDE1A, 3B and 4B was significantly different not only between SANC and LV but also between RA and SANC (p<0.0001, p<0.05 and p<0.05 respectively). For the rest of the transcripts expression level in SANC was lower than in LV cells (PDE1C, 3A, 4A, 4D, 7A). Biochemical experiments on cell lysates demonstrated that from cAMP-hydrolyzing subtypes of PDE, Calmodulin / Ca+ - activated PDE1 (Lukyanenko new results with Vinpocetine and Nimodipine) and PDE4 are present in SANC. We are trying to detect impact of each type of PDEs in the total c-AMP regulating PDE activity in the cells and their distribution within different microenvironment. Immunoprecipitation of mAKAP scaffolding protein revealed co-precipitated PDE activity in rabbit VM lysates. Because PDE activity also can be regulated by phosphorylation, currently we study the importance of PDE phosphorylation status on PDE activity in SANC and VM lysates.

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Lukyanenko, Yevgeniya O; Younes, Antoine; Lyashkov, Alexey E et al. (2016) Ca(2+)/calmodulin-activated phosphodiesterase 1A is highly expressed in rabbit cardiac sinoatrial nodal cells and regulates pacemaker function. J Mol Cell Cardiol 98:73-82
Younès, Antoine; Lukyanenko, Yevgeniya O; Lyashkov, Alexey E et al. (2011) A bioluminescence method for direct measurement of phosphodiesterase activity. Anal Biochem 417:36-40
Lakatta, Edward G; Maltsev, Victor A; Vinogradova, Tatiana M (2010) A coupled SYSTEM of intracellular Ca2+ clocks and surface membrane voltage clocks controls the timekeeping mechanism of the heart's pacemaker. Circ Res 106:659-73
Maltsev, Victor A; Lakatta, Edward G (2010) Funny current provides a relatively modest contribution to spontaneous beating rate regulation of human and rabbit sinoatrial node cells. J Mol Cell Cardiol 48:804-6
Lakatta, Edward G (2010) A paradigm shift for the heart's pacemaker. Heart Rhythm 7:559-64
Lakatta, Edward G; Vinogradova, Tatiana M; Maltsev, Victor A (2008) The missing link in the mystery of normal automaticity of cardiac pacemaker cells. Ann N Y Acad Sci 1123:41-57
Younes, Antoine; Lyashkov, Alexey E; Graham, David et al. (2008) Ca(2+) -stimulated basal adenylyl cyclase activity localization in membrane lipid microdomains of cardiac sinoatrial nodal pacemaker cells. J Biol Chem 283:14461-8