Alternative splicing of cGMP-dependent protein kinase I in angiotensin-hypertension - Novel mechanism for nitrate tolerance in vascular smooth muscle
Alternative splicing of cGMP-dependent protein kinase I in angiotensin-hypertension - Novel mechanism for nitrate tolerance in vascular smooth muscle
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DOI：
10.1161/01.res.0000097872.69043.a0
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发表时间：
2003-10-31
影响因子：
20.1
通讯作者：
Simard, JM
中科院分区：
文献类型：
--
作者：
Gerzanich, V;Ivanov, A;Simard, JM
高血压患者的硝酸盐耐受(NT)是由于可溶性鸟苷酸环化酶(SGC)活性降低所致。我们检测了正常血压大鼠、高血压大鼠(AHR)和血管紧张素II(Ang)组大鼠基底动脉血管平滑肌细胞(VSMCs)中NT的含量。NO供体对AHR VSMCs的钙激活K+(Maxi-K)通道有抑制作用，这与NT的表现一致。8-BR-cGMP的量效关系右移2.5倍，提示sGC异常不能单独解释NT。AHR由内向外的斑块显示外源性cGMP依赖的蛋白激酶I(CGKI)正常激活，提示cGKI下游没有异常。我们推测，8-BR-cGMP对AHR中cGKI的表观亲和力降低可能是由于cGKIpha与cGKIbeta的相对量的变化，因为cGKIbeta对cGMP激活剂的敏感性低于cGKIbeta。这在AHR中表现为：(1)cGKIα选择性激活剂8-APT-cGMP的作用降低；(2)定量免疫荧光和Western blotts中总cGKI蛋白(两种异构体)减少，但cGKIbeta蛋白增加；(3)与Maxi-K通道分离的cGKI免疫异构体类似的变化；以及(4)实时荧光PCR和Northern blotts中cGKIbeta mRNA的大幅增加和cGKIpha mRNA的减少。细胞内cGKIβ在血管紧张素转换酶治疗后4天，即高血压发生前明显上调。我们的数据确定了cGKIbeta在VSMC中的功能作用，此前仅归因于cGKIpha。血管紧张素转换酶诱导的cGKI选择性剪接是降低NO/cGMP敏感性的一种新机制。
Nitrate tolerance (NT) in hypertension is attributed to reduced activity of soluble guanylyl cyclase (sGC). We examined NT in basilar artery vascular smooth muscle cells (VSMCs) from control rats, rats infused with angiotensin II (Ang; 240 mug/kg per hour for 4 days), which were normotensive, and Ang-hypertensive rats (AHR; 240 mug/kg per hour for 28 days). Ca2+-activated K+ (Maxi-K) channels in VSMCs from AHR showed reduced activation by NO donor, consistent with NT. The concentration-response relationship for 8-Br-cGMP was shifted 2.5-fold to the right, indicating that abnormal sGC alone could not account for NT. Inside-out patches from AHR showed normal activation with exogenous cGMP-dependent protein kinase I (cGKI), suggesting no abnormality downstream of cGKI. We hypothesized that the reduction in apparent affinity of 8-Br-cGMP for cGKI in AHR might be due to a change in relative amounts of cGKIalpha versus cGKIbeta, since cGKIbeta is less sensitive to cGMP activators than cGKIbeta. This was substantiated by showing the following in AHR: (1) reduced effect of the cGKIalpha-selective activator 8-APT-cGMP; (2) reduced total cGKI protein (both isoforms), but an increase in cGKIbeta protein in quantitative immunofluorescence and Western blots; (3) similar changes in cGKI isoforms immunoisolated with Maxi-K channels; and (4) a large increase in cGKIbeta mRNA and a decrease in cGKIalpha mRNA in real-time PCR and Northern blots. Upregulation of cytosolic cGKIbeta was evident 4 days after Ang infusion, before development of hypertension. Our data identify a functional role for cGKIbeta in VSMCs previously ascribed exclusively to cGKIalpha. Ang-induced alternative splicing of cGKI represents a novel mechanism for reducing sensitivity to NO/cGMP.