Protas Lev, Barbuti Andrea, Qu Jihong, Rybin Vitalyi O, Palmiter Richard D, Steinberg Susan F, Robinson Richard B
Department of Pharmacology, Columbia University, 630 W 168th St, New York, NY 10032, USA.
Circ Res. 2003 Nov 14;93(10):972-9. doi: 10.1161/01.RES.0000099244.01926.56. Epub 2003 Oct 2.
Cell culture studies demonstrate an increase in cardiac L-type Ca2+ current (ICa,L) density on sympathetic innervation in vitro and suggest the effect depends on neurally released neuropeptide Y (NPY). To determine if a similar mechanism contributes to the postnatal increase in ICa,L in vivo, we prepared isolated ventricular myocytes from neonatal and adult mice with targeted deletion of the NPY gene (Npy-/-) and matched controls (Npy+/+). Whole-cell voltage clamp demonstrates ICa,L density increases postnatally in Npy+/+ (by 56%), but is unchanged in Npy-/-. Both ICa,L density and action potential duration are significantly greater in adult Npy+/+ than Npy-/- myocytes, whereas ICa,L density is equivalent in neonatal Npy+/+ and Npy-/- myocytes. These data indicate NPY does not influence ICa,L prenatally, but the postnatal increase in ICa,L density is entirely NPY-dependent. In contrast, there is a similar postnatal negative voltage shift in the I-V relation in Npy+/+ and Npy-/-, indicating NPY does not influence the developmental change in ICa,L voltage-dependence. Immunoblot analyses and measurements of maximally activated ICa,L (in presence of forskolin or BayK 8644) show that the differences in current density between Npy+/+ and Npy-/- cannot be attributed to altered Ca2+ channel alpha1C subunit protein expression. Rather, these results suggest that the in vivo NPY-dependent postnatal increase in ICa,L density in cardiac myocytes results from regulation ICa,L properties by NPY.
细胞培养研究表明，在体外交感神经支配下，心脏L型钙电流（ICa,L）密度增加，且提示该效应依赖于神经释放的神经肽Y（NPY）。为了确定类似机制是否有助于体内出生后ICa,L的增加，我们从NPY基因靶向缺失的新生和成年小鼠（Npy-/-）以及配对对照（Npy+/+）中制备了分离的心室肌细胞。全细胞膜片钳显示，Npy+/+中ICa,L密度在出生后增加（增加56%），但在Npy-/-中无变化。成年Npy+/+心肌细胞的ICa,L密度和动作电位时程均显著大于Npy-/-心肌细胞，而新生Npy+/+和Npy-/-心肌细胞的ICa,L密度相当。这些数据表明，NPY在产前不影响ICa,L，但出生后ICa,L密度的增加完全依赖于NPY。相反，Npy+/+和Npy-/-的I-V关系在出生后有类似的负电压偏移，表明NPY不影响ICa,L电压依赖性的发育变化。免疫印迹分析和最大激活ICa,L的测量（在福斯可林或BayK 8644存在下）表明，Npy+/+和Npy-/-之间电流密度的差异不能归因于Ca2+通道α1C亚基蛋白表达的改变。相反，这些结果提示，心肌细胞中体内依赖NPY的出生后ICa,L密度增加是由NPY对ICa,L特性的调节所致。
