Immunodetection of phosphorylation sites gives new insights into the mechanisms underlying phospholamban phosphorylation in the intact heart
Phosphorylation site-specific antibodies, quantification of <sup>32</sup>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 phosp...
Guardado en:
| Autores principales: | , , , , |
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| Formato: | Articulo |
| Lenguaje: | Inglés |
| Publicado: |
1996
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| Materias: | |
| Acceso en línea: | http://sedici.unlp.edu.ar/handle/10915/123153 |
| Aporte de: |
| Sumario: | Phosphorylation site-specific antibodies, quantification of <sup>32</sup>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 Ser<sup>16</sup> and Thr<sup>17</sup> residues. When isoproterenol concentration was increased at extremely low Ca²⁺ supply to the myocardium, phosphorylation of Thr<sup>17</sup> was virtually absent. Under these conditions, <sup>32</sup>P incorporation into phospholamban, due to Ser<sup>16</sup>, decreased by 50%. Changes in Ca²⁺ supply to the myocardium either at constant β-adrenergic stimulation or in the presence of okadaic acid, a phosphatase inhibitor, exclusively modified Thr<sup>17</sup> phosphorylation. Changes in phospholamban phosphorylation due to either Ser<sup>16</sup> and/or Thr<sup>17</sup> were paralleled by changes in myocardial relaxation. The results indicate that cAMP- (Ser<sup>16</sup>) and Ca²⁺-calmodulin (Thr<sup>17</sup>)-dependent pathways of phospholamban phosphorylation can occur independently of each other. However, in the absence of β-adrenergic stimulation, phosphorylation of Thr<sup>17</sup> could only be detected after simultaneous activation of Ca²⁺-calmodulin-dependent protein kinase and inactivation of phosphatase. It is suggested that under physiological conditions, this requisite is only filled by cAMP-dependent mechanisms. |
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