Sodi R, Dubuis E, Shenkin A, Hart G
Department of Clinical Biochemistry & Metabolic Medicine, Royal Liverpool & Broadgreen University Hospital, Prescot street, Liverpool L7 8XP, United Kingdom.
Regul Pept. 2008 Nov 29;151(1-3):95-105. doi: 10.1016/j.regpep.2008.06.006. Epub 2008 Jun 20.
A fundamental question in physiology is how hormones regulate the functioning of a cell or organ. It was therefore the aim of this study to investigate the effect(s) of BNP-32 on calcium handling by ventricular myocytes obtained from the rat left ventricle. We specifically tested the hypothesis that BNP-32 decreased the L-type calcium current (I(Ca,L)). Perforated patch clamp technique was used to record I(Ca,L) and action potential (AP) in voltage and current clamp mode, respectively. Myocyte shortening was measured using a photodiode array edge-detection system and intracellular calcium transients were measured by fluorescence photometry. Western blotting was used to determine the relative change in the expression of proteins. At the concentrations tested, BNP-32 significantly decreased cell shortening in a dose-dependent manner; increased the phase II slope of the AP by 53.0%; increased the APD(50) by 16.9%; reduced the I(Ca,L) amplitude with a 22.9% decrease in the peak amplitude and reduced Ca(2+)-dependent inactivation; increased the V(1/2) activation of the L-type calcium channel by 51.1% and decreased V(1/2) inactivation by 31.8%; and, intracellular calcium transient amplitude was significantly decreased by 32.0%, whereas the time to peak amplitude and T(1/2) were both significantly increased by 38.7% and 89.4% respectively. Sarcoplasmic reticulum Ca(2+)-ATPase (SERCA2a) protein expression was reduced by BNP-32. These data suggest that BNP-32 regulates ventricular myocyte function by attenuating I(Ca,L), altering the AP and reducing SERCA2a activity and/or expression. This study suggests a novel constitutive mechanism for the autocrine action of BNP on the L-type calcium channel in ventricular myocytes.
生理学中的一个基本问题是激素如何调节细胞或器官的功能。因此，本研究的目的是探讨BNP-32对从大鼠左心室获取的心室肌细胞钙处理的影响。我们特别检验了BNP-32降低L型钙电流（I(Ca,L)）的假设。采用穿孔膜片钳技术分别在电压钳和电流钳模式下记录I(Ca,L)和动作电位（AP）。使用光电二极管阵列边缘检测系统测量肌细胞缩短情况，并通过荧光光度法测量细胞内钙瞬变。采用蛋白质印迹法测定蛋白质表达的相对变化。在所测试的浓度下，BNP-32以剂量依赖性方式显著降低细胞缩短；使AP的II期斜率增加53.0%；使动作电位时程（APD(50)）增加16.9%；降低I(Ca,L)幅度，峰值幅度降低22.9%，并减少钙依赖性失活；使L型钙通道的V(1/2)激活增加51.1%，V(1/2)失活降低31.8%；细胞内钙瞬变幅度显著降低32.0%，而达到峰值幅度的时间和半衰期（T(1/2)）分别显著增加38.7%和89.4%。BNP-32降低了肌浆网钙ATP酶（SERCA2a）蛋白表达。这些数据表明，BNP-32通过减弱I(Ca,L)、改变AP以及降低SERCA2a活性和/或表达来调节心室肌细胞功能。本研究提示了BNP对心室肌细胞L型钙通道自分泌作用的一种新的组成机制。
