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 PDEs, PDE1, PDE2 and PDE4 are present in SANC. PDE1 represents the highest activity (43%) in our conditions. The most important PDE in LV are PDE3 and PDE4. PDE4 represents up to 50% of total PDE activity in LV. We found that inhibition of protein phosphatases in our experiments and increase in phosphorylation of cellular proteins causes activation of total PDE activity in both cell types and PDE1 and PDE4 in SANC. Addition of calmodulin dramatically increased PDE activity in the lysates of both cell types, revealing previously undetectable PDE1 in LV. We tried to elucidate microenvironment of PDEs within cells. In order to do this we performed several experiments using immunoprecipitation techniques. We found that mAKAP scaffolding protein co-precipitated PDE activity in rabbit VM lysates.
|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|