A Functional Relationship between Specific NOS Isozymes and Beta-adrenergic Recep

描述(申请人提供)：心脏收缩能力的一个重要调节因素是肾上腺素能受体(AR)信号。(受体1和受体2是心脏表达的主要亚型。？1受体通过调节L钙通道、磷蛋白(PLB)和肌钙蛋白I(TnL)产生正性变力和趋化作用。？2受体的独特之处在于它们只起到正性肌力的作用；这些受体通过调节L类型的钙通道来促进收缩。AR信号本身受一氧化氮合酶(NOS)产生的一氧化氮合酶(NO)调节。在心肌细胞内，有两种一氧化氮合酶亚型的结构性表达：NOS1和NOS3。NOS1信号被证明增强了对非特异性AR刺激的功能反应，而NOS3信号则降低了它。然而，特定的一氧化氮合酶亚型在调节单个AR受体中有什么作用仍有待确定。有趣的是，NOS1和NOS1受体通过相似的蛋白靶点(即PLB)调节收缩，而NOS3和NOS2受体通过L型钙通道调节收缩。最近，NOS3和NOS2受体被证明在心肌细胞内被划分到相似的位置。因此，我们假设NOS1将增加由于1受体激活而引起的功能反应，而不调节2受体信号(Aim1)。此外，我们假设NOS3将降低由于没有调节1受体信号(AIM2)的2受体激活而引起的功能反应。我们将在暴露于不同AR刺激的野生型(对照)、NOS1基因敲除(KO)和NOS3KO小鼠分离的心肌细胞中进行功能实验(即钙瞬变、缩短幅度和L钙通道电流的测量)和蛋白质印迹(pLB-磷酸化)来研究这一功能相互作用。我们的研究认为这与健康有关，因为当AR信号发生变化时，这一信号通路会促进收缩功能障碍和心脏重构。因此，确定AR信号如何被调节是至关重要的。随着对这一现象的深入了解，我们的研究将使开发新的治疗方法成为可能。

DESCRIPTION (provided by applicant): An essential regulator of cardiac contractility is ¿-adrenergic receptor (¿AR) signaling. (¿1 and ¿2 receptors are the primary subtypes expressed in the heart. ¿1 receptors produce positive inotropic and lusitropic effects through modulation of the L-type Ca2+ channel, phospholamban (PLB) and troponin I (Tnl). ¿2 receptors are unique in that they only function as positive inotropes; these receptors promote contractility through regulation of the L-type Ca2+ channel. ¿AR signaling itself can be regulated by nitric oxide (NO) produced via NO synthase (NOS). Within the myocyte, two NOS isoforms are constitutively expressed: NOS1 and NOS3. NOS1 signaling has been shown to augment the functional response to non-specific ¿AR stimulation while NOS3 signaling reduces it. However, it remains to be determined what effect specific NOS isoforms have in the regulation of individual ¿AR receptors. Interestingly, NOS1 and ¿1 receptors have been shown to regulate contractility via similar protein targets (i.e.PLB), while NOS3 and ¿2 receptors regulate contractility through the L-type Ca2+ channel. Recently, NOS3 and ¿2 receptors have been shown to compartmentalize to similar locations within the myocyte. Therefore, we hypothesize NOS1 will increase the functional response due to ¿1 receptor activation with no modulation of ¿2 receptor signaling (Aim1). In addition, we hypothesize NOS3 will decrease the functional response due to ¿2 receptor activation with no modulation of ¿1 receptor signaling (Aim2). Functional experiments (i.e. measurement of Ca2+ transient, shortening amplitude, and L-type Ca2+channel current) and western blotting (PLB-phosphorylation) to investigate this proposed functional interaction will be conducted in isolated myocytes from wild type (WT, control), NOS1 knockout (KO) and NOS3 KO mice exposed to various ¿AR stimuli. Our study holds health-relatedness due to the fact that when ¿AR signaling is altered, this signaling pathway promotes contractile dysfunction and cardiac remodeling. Thus, it is crucial to determine how ¿AR signaling is regulated. With a deeper understanding of this phenomena, our study will make possible the development of new therapeutic treatments.