Said Matilde, Mundiña-Weilenmann Cecilia, Vittone Leticia, Mattiazzi Alicia
Centro de Investigaciones Cardiovasculares, Facultad de Ciencias Médicas, Universidad Nacional de La Plata, 60 y 120, 1900 La Plata, Argentina.
Pflugers Arch. 2002 Sep;444(6):801-9. doi: 10.1007/s00424-002-0885-y. Epub 2002 Aug 1.
Contractility and relaxation measurements were combined with the determination of total phospholamban (PLB) phosphorylation and the immunodetection of PLB-phosphorylation sites in the intact, beating rat heart to identify the contributions of PLB phosphorylation at the Thr(17) and Ser(16) residues at different levels of beta-adrenoceptor stimulation. Whereas with 30-300 nM isoproterenol, phosphorylation of Thr(17), the Ca(2+)-calmodulin-dependent protein kinase-II (CaMKII) site and Ser(16), the protein kinase A (PKA) site, contributed approximately 50% each to PLB phosphorylation, and both participated in the relaxant action of isoproterenol, at lower a level of beta-adrenoceptor stimulation (isoproterenol 0.3-3 nM), both effects were exclusively due to Ser(16) phosphorylation. Increasing Ca at 3 nM isoproterenol, to obtain an increase in contractility comparable to that produced by 30 nM isoproterenol, significantly increased Thr(17) phosphorylation and the relaxant effect produced by 3 nM isoproterenol. An increase in Thr(17) phosphorylation and in the relaxant effect of 3 nM isoproterenol was also obtained by phosphatase inhibition (okadaic acid). In this case, Ser(16) phosphorylation was also increased. Moreover, perfusion with 30 nM isoproterenol in the presence of the PKA inhibitor H-89 decreased phosphorylation at both PLB residues and diminished the inotropic and relaxant responses to the beta-agonist. The relative contribution of Thr(17) phosphorylation to the isoproterenol-induced phosphorylation of PLB and relaxation thus increased with the level of beta-adrenoceptor stimulation and the consequent increase in PKA activity. The lack of Thr(17) phosphorylation at low isoproterenol concentrations might therefore be attributed to a level of PKA activity insufficient to increase Ca to activate the CaMKII system and/or to inhibit the phosphatase that dephosphorylates PLB
收缩性和舒张性测量与完整跳动大鼠心脏中总受磷蛋白（PLB）磷酸化的测定以及PLB磷酸化位点的免疫检测相结合，以确定在不同水平的β-肾上腺素能受体刺激下，苏氨酸（Thr）17和丝氨酸（Ser）16残基处PLB磷酸化的作用。在30 - 300 nM异丙肾上腺素作用下，Thr(17)（钙调蛋白依赖性蛋白激酶-II（CaMKII）位点）和Ser(16)（蛋白激酶A（PKA）位点）的磷酸化对PLB磷酸化的贡献各约为50%，且二者均参与了异丙肾上腺素的舒张作用；而在较低水平的β-肾上腺素能受体刺激（0.3 - 3 nM异丙肾上腺素）下，两种效应均仅归因于Ser(16)磷酸化。在3 nM异丙肾上腺素时增加细胞外钙浓度（Ca），以使收缩性增加至与30 nM异丙肾上腺素产生的收缩性相当，显著增加了Thr(17)磷酸化以及3 nM异丙肾上腺素产生的舒张效应。通过磷酸酶抑制（冈田酸）也可使3 nM异丙肾上腺素的Thr(17)磷酸化及舒张效应增加。在这种情况下，Ser(16)磷酸化也增加。此外，在PKA抑制剂H - 89存在下用30 nM异丙肾上腺素灌注，降低了两个PLB残基的磷酸化，并减弱了对β-激动剂的变力性和舒张反应。因此，Thr(17)磷酸化对异丙肾上腺素诱导的PLB磷酸化和舒张的相对贡献随着β-肾上腺素能受体刺激水平以及随之而来的PKA活性增加而增加。因此，在低异丙肾上腺素浓度下缺乏Thr(17)磷酸化可能归因于PKA活性水平不足以增加细胞内钙浓度（Ca）以激活CaMKII系统和/或抑制使PLB去磷酸化的磷酸酶
