Phosphorylation site-specific antibodies, quantification of 32 P incorporation into phospholamban, and simultaneous measurements of mechanical activity were used in Langendorff-perfused rat hearts to provide further insights into the underlying mechanisms of phospholamban phosphorylation. Immunological detection of phospholamban phosphorylation sites showed that the isoproterenol concentration-dependent increase in phospholamban phosphorylation was due to increases in phosphorylation of both 16 and Thr 17 , respectively (3). These phosphorylations are independent of each other, and when both are operating, they appear to have an additive action (4). In the intact heart, -adrenergic stimulation phosphorylates phospholamban at both sites (5), which indicates that PKA-and CaMKII-dependent pathways are also working in the functioning heart. Whether these phosphorylation mechanisms are independent of each other and additive, as described in the isolated SR membranes, remains unknown. Different attempts to phosphorylate phospholamban by CaMKII in the intact heart have systematically failed unless cAMP levels within the cell increase (6 -11). This consistent finding strongly suggests an interaction between PKA and CaMKII pathways of phospholamban phosphorylation in the intact heart. The nature of this interaction as well as the cause for the difference between the in vivo and in vitro results have never been explored.The availability of phosphorylation-site specific antibodies to phospholamban, which precisely discriminate between Ser 16 and Thr 17 phosphorylation sites (12), prompted us to reexamine the issue. Combination of this technique with the quantitative assessment of phospholamban phosphorylation by radiochemical labeling of ATP pools and simultaneous measurements of mechanical parameters allowed us to characterize the PKA and CaMKII-dependent mechanisms of phospholamban phosphorylation in the intact heart and their relative physiological roles on cardiac performance. EXPERIMENTAL PROCEDURESHeart Perfusions-Experiments were performed in isolated hearts from male Wistar rats (250 -350 g body wt) perfused according to the Langendorff technique as described previously (8). The composition of the physiological salt solution (PSS) was (in mM): 128.3 NaCl, 4.7 KCl, 1.35 CaCl 2 , 20.2 NaHCO 3 , 0.4 NaH 2 PO 4 , 1.1 MgCl 2 , 11.1 glucose, and 0.04 Na 2 EDTA. This solution was equilibrated with 95% O 2 , 5% CO 2 to give a pH of 7.4. The mechanical activity of the heart was assessed by either sewing an isometric strain gauge arch (Micro Measurements, type MA-06 -030LB-120) to the left ventricular wall or passing into the left ventricle a latex balloon connected to a pressure transducer (Namic, perceptor DT disposable transducer). The initial length of the gauge was set by stretching the segment attached by approximately 30%. The balloon was filled with aqueous solution to achieve a left ventricular
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.