低氧对大鼠远端肺静脉平滑肌细胞内钙浓度的影响及机制
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摘要
慢性低氧性肺动脉高压(CHPH)是大部分慢性阻塞性肺疾病(COPD)患者最终发展成为肺心病而死亡的主要原因,关于其发病机制已经进行了大量的研究,临床以此为基础的治疗也取得了一定的疗效,然而,临床治疗CHPH的手段仍然有限,疗效依然欠佳。肺血管低氧早期(急性低氧)的收缩反应和慢性低氧性肺血管结构重建是CHPH发生的主要机制,造成CHPH的这两个主要环节不仅发生在肺动脉,而且包括肺静脉。尽管关于肺动脉急性低氧性收缩反应和慢性低氧性肺血管结构重建的机制已经进行了大量的研究,然而,关于肺静脉的此类研究却少有报道。大量的研究证明:细胞内Ca~(2+)在血管平滑肌细胞的收缩和增生的复杂机制中起至关重要的作用。但是,关于肺静脉平滑肌细胞(PVSMC)Ca~(2+)信号转导以及低氧对PVSMC细胞内Ca~(2+)浓度([Ca~(2+)]_i)作用的研究却鲜有报道,我们推测:低氧可能通过某些机制使PVSMC的[Ca~(2+)]_i改变,参与CHPH的发生、发展过程。因此,我们建立了大鼠远端PVSMC原代培养模型及CHPH大鼠模型,对急慢性低氧诱导大鼠远端肺静脉平滑肌[Ca~(2+)]_i改变的作用及机理进行了研究。从以往研究甚少的肺静脉方面来阐述CHPH发生的可能机制,为寻找有效的防治肺动脉高压的方法打下坚实的理论基础。
方法
一、急性低氧对大鼠远端PVSMC[Ca~(2+)]_i的影响:首先利用胶原酶消化法分离、原代培养大鼠远端PVSMC,通过细胞形态学观察、平滑肌细胞α-actin免疫荧光和western-blot免疫印迹分析以及[Ca~(2+)]_i对高钾KRBS溶液的反应来鉴定PVSMC,从而建立大鼠远端PVSMC原代培养模型。然后利用InCyte细胞内钙浓度检测系统,观测急性低氧对大鼠远端PVSMC[Ca~(2+)]_i的影响
二、急性低氧对大鼠远端PVSMC[Ca~(2+)]_i影响的机制:1、利用InCyte细胞内钙浓度检测系统,观测电压依赖性钙通道(VDCC)拮抗剂硝苯地平对急性低氧诱导大鼠远端PVSMC[Ca~(2+)]_i改变的干预作用,明确是否有VDCC以外的钙通道参与急性低氧诱导的大鼠远端PVSMC[Ca~(2+)]_i改变过程。2、分离大鼠远端肺静脉平滑肌,原代培养大鼠远端PVSMC,通过荧光定量PCR和western-blot免疫印迹分析来检测经典瞬时受体电位(TRPC)蛋白在大鼠远端肺静脉平滑肌和培养的大鼠远端PVSMC的mRNA水平和蛋白水平的表达,明确大鼠远端肺静脉平滑肌是否表达TRPC蛋白。3、利用InCyte细胞内钙浓度检测系统,通过恢复细胞外钙法、锰荧光焠灭法,并利用储量操纵性钙通道(SOCC)拮抗剂SKF-96365和NiCl2,在大鼠远端PVSMC上检测Ca~(2+)通过由TRPC蛋白组成的SOCC而形成的储量操作性钙内流(SOCE),明确大鼠远端肺静脉平滑肌是否存在SOCE。4、利用InCyte细胞内钙浓度检测系统,通过恢复细胞外钙法、锰荧光焠灭法,并利用SOCC拮抗剂SKF-96365和NiCl2观测急性低氧对大鼠远端PVSMC的SOCE的影响,明确SOCE在急性低氧引起的大鼠远端PVSMC[Ca~(2+)]_i改变中的作用。
三、慢性低氧对大鼠远端PVSMC的[Ca~(2+)]_i的影响:首先,通过慢性持续低氧(10%O2)条件下饲养大鼠3周建立CHPH大鼠模型;然后,分离正常大鼠和CHPH大鼠远端PVSMC;最后,利用InCyte细胞内钙浓度检测系统,分别检测正常大鼠和CHPH大鼠远端PVSMC的[Ca~(2+)]_i,从而明确慢性低氧对大鼠远端PVSMC的[Ca~(2+)]_i的影响。
四、慢性低氧对大鼠远端PVSMC的[Ca~(2+)]_i的影响的机制:1、分离正常大鼠和CHPH大鼠远端肺静脉平滑肌,通过荧光定量PCR和Western-blot免疫印迹分析来检测TRPC在正常大鼠和CHPH大鼠远端肺静脉平滑肌的表达,明确慢性低氧对大鼠远端肺静脉平滑肌TRPC表达的影响。2、分离正常大鼠和CHPH大鼠远端PVSMC,利用InCyte细胞内钙浓度检测系统,首先分别检测由环匹阿尼酸(CPA)诱导的正常大鼠和CHPH大鼠远端PVSMC的[Ca~(2+)]_i变化,然后分别检测由CPA诱导的正常大鼠和CHPH大鼠远端PVSMC的锰荧光焠灭,从而明确慢性低氧对大鼠远端肺静脉平滑肌SOCE的影响;接着分别检测由油酰基乙酰甘油(OAG)诱导的正常大鼠和CHPH大鼠远端PVSMC的[Ca~(2+)]_i变化,最后分别检测由OAG诱导的正常大鼠和CHPH大鼠远端PVSMC的锰荧光焠灭,从而明确慢性低氧对大鼠远端肺静脉平滑肌由受体操纵性钙通道(ROCC)介导的Ca~(2+)内流的影响。
结果
一、急性低氧对大鼠远端PVSMC[Ca~(2+)]_i的影响:
1、成功建立了大鼠远端PVSMC原代培养模型,原代培养的大鼠远端PVSMC不仅表现出典型的血管平滑肌细胞的形态学和免疫学特征,而且具有完好的生理学功能。
2、急性低氧使大鼠远端PVSMC[Ca~(2+)]_i升高。
二、急性低氧对大鼠远端PVSMC[Ca~(2+)]_i影响的机制:
1、作为VDCC拮抗剂,硝苯地平能完全阻断高钾KRBS溶液引起的大鼠远端PVSMC [Ca~(2+)]_i升高,却只能部分抑制急性低氧引起的大鼠远端PVSMC的[Ca~(2+)]_i升高,提示VDCC以外的钙通道参与急性低氧引起的大鼠远端PVSMC[Ca~(2+)]_i升高。
2、在大鼠远端肺静脉平滑肌组织和培养的PVSMC都有TRPC的mRNA表达,其中以TRPC-1、TRPC-4和TRPC-6,尤其是TRPC-1和TRPC-6的mRNA表达最强。Western-blot免疫印迹分析进一步表明,在大鼠远端肺静脉平滑肌组织和培养的PVSMC都有TRPC蛋白的表达,且尤其以TRPC-1和TRPC-6的蛋白表达最强。
3、在去除细胞外Ca~(2+)、用硝苯地平阻断VDCC并用CPA使Ca~(2+)从PVSMC肌浆网完全清空的条件下,恢复PVSMC细胞外Ca~(2+)后, PVSMC [Ca~(2+)]_i可迅速显著升高;在去除细胞外Ca~(2+)并用硝苯地平阻断VDCC的条件下,CPA使Mn~(2+)引起的PVSMC的荧光焠灭增强;作为SOCC拮抗剂,SKF-96365和NiCl2均能明显抑制由CPA诱导的PVSMC [Ca~(2+)]_i升高和锰荧光焠灭增强,但对高钾KRBS溶液引起的PVSMC [Ca~(2+)]_i升高没有影响,提示大鼠远端PVSMC存在SOCE。
4、急性低氧能显著增加由CPA诱导的、恢复细胞外钙后所引起的大鼠远端PVSMC[Ca~(2+)]_i升高,并能增强由CPA诱导的大鼠远端PVSMC的锰荧光焠灭; SKF-96365和NiCl2均能明显抑制急性低氧引起的大鼠远端PVSMC的SOCE增加;在常氧情况下,用不含CPA但含有硝苯地平的无钙溶液孵育PVSMC,锰荧光焠灭与不加Mn~(2+)的细胞的荧光自发衰减量没有差异;而在急性低氧情况下,用不含CPA但含有硝苯地平的无钙溶液孵育PVSMC,锰荧光焠灭显著增加,而且此反应能被SKF-96365和NiCl2明显抑制,提示急性低氧自身能诱发大鼠远端PVSMC的SOCE,而且能够通放大SOCE,引起PVSMC [Ca~(2+)]_i升高。
三、慢性低氧对大鼠远端PVSMC的[Ca~(2+)]_i的影响:
1、成功建立CHPH大鼠模型,慢性持续性低氧3周大鼠的MPAP、RVSP、RV/(LV+S)都明显增高,肺内血管壁明显增厚。
2、CHPH大鼠远端PVSMC[Ca~(2+)]_i较正常大鼠明显增高,提示慢性低氧使大鼠远端PVSMC的[Ca~(2+)]_i升高。
四、慢性低氧对大鼠远端PVSMC[Ca~(2+)]_i影响的机制:
1、荧光定量PCR和western-blot免疫印迹分析证实:在CHPH大鼠远端肺静脉平滑肌组织TRPC-6蛋白的表达较正常大鼠明显增强,而TRPC-1和TRPC-4蛋白的表达较正常大鼠没有明显改变,提示慢性低氧上调大鼠远端肺静脉平滑肌组织TRPC-6蛋白的表达。
2、用硝苯地平阻断VDCC并恢复细胞外Ca~(2+)后,CPA不仅能导致CHPH大鼠远端PVSMC [Ca~(2+)]_i迅速升高,而且能使正常大鼠远端PVSMC的[Ca~(2+)]_i也迅速升高,两者升高幅度无差别;用含CPA和硝苯地平的无钙溶液孵育细胞,CHPH大鼠远端PVSMC的锰荧光焠灭与正常大鼠的也无差别。然而,用硝苯地平阻断VDCC并恢复细胞外Ca~(2+)后,OAG能使正常大鼠远端PVSMC的[Ca~(2+)]_i升高,而CHPH大鼠远端PVSMC由OAG诱导的[Ca~(2+)]_i升高较正常大鼠更明显;同时,OAG诱导的锰荧光焠灭在CHPH大鼠远端PVSMC也比正常大鼠表现的更强,提示慢性低氧通过增加由ROCC介导的Ca~(2+)内流使大鼠远端PVSMC的[Ca~(2+)]_i升高。
结论
1、急性和慢性低氧都能使大鼠远端肺静脉平滑肌细胞内钙浓度升高。
2、大鼠远端肺静脉平滑肌主要表达TRPC-1、TRPC-4和TRPC-6,尤以TRPC-1和TRPC-6为主;大鼠远端肺静脉平滑肌存在SOCE。
3、急性低氧不仅自身能够诱发大鼠远端肺静脉平滑肌细胞的SOCE,而且能够放大SOCE,从而引起细胞内钙浓度升高。
4、慢性低氧能够上调大鼠远端肺静脉平滑肌TRPC-6的表达,使主要由TRPC-6蛋白组成的ROCC增加,导致由ROCC介导的Ca~(2+)内流增加,从而引起细胞内钙浓度升高。
Objective
Hypoxia causes remodeling and alters contractile responses in both pulmonary arteries and pulmonary veins (PV). Although pulmonary arteries have been studied extensively in this disorder, the mechanisms by which pulmonary veins respond to hypoxia and whether these responses contribute to chronic hypoxic pulmonary hypertension (CHPH) remain poorly understood. In pulmonary arterial smooth muscle, influx of Ca~(2+) through store-operated calcium channels thought to be composed of canonical transient receptor potential (TRPC) proteins is likely to play an important role in development of CHPH. The aim of this study is to determine whether this mechanism could also be operative in pulmonary venous smooth muscle.
Methods
The rat distal PVSMC were isolated and cultured by collagenase digestion and characterized by morphological activity, by immunoblotting and immunofluorescence of smooth muscle -actin and by response of intracellular Ca~(2+) concentration ([Ca~(2+)]_i) to KCL.
The effect of acute hypoxia on [Ca~(2+)]_i in PVSMC and the inhibition of nifedipine were measured by Incyte intracellular Ca~(2+) concentration system. The expression of TRPC in distal pulmonary venous smooth muscle or PVSMC was detected by real-time PCR and western blotting. Store-operated Ca~(2+) entry (SOCE) and the effect of acute hypoxia on SOCE were assessed by measurement of increase in [Ca~(2+)]_i caused by restoration of extracellular Ca~(2+) and the rate at which extracellular Mn~(2+) quenched fura-2 fluorescence.
CHPH rats were obtained using a hypoxic (10 % O2) chamber for 21 days. PVSMC were isolated from CHPH rats or normal rats. The effect of chronic hypoxia on [Ca~(2+)]_i in PVSMC was assessed by comparison of [Ca~(2+)]_i in PVSMC from CHPH rats with that from normal rats.
The effect of chronic hypoxia on TRPC expression was measured by comparison of TRPC mRNA and protein expression in pulmonary venous smooth muscle from CHPH rats with that from normal rats using real-time PCR and western blotting. PVSMC from CHPH rats or normal rats were isolated. The mechanisms by which [Ca~(2+)]_i in PVSMC respond to chronic hypoxia were assessed by comparison of CPA-induced Ca~(2+) influx and Mn~(2+) quenching in PVSMC isolated from CHPH rats with that from normal rats and by comparison of OAG-induced Ca~(2+) influx and Mn~(2+) quenching in PVSMC isolated from CHPH rats with that from normal rats.
Results
Cells isolated from rat distal pulmonary veins displayed morphological characteristic of vascular smooth muscle cells and expressed smooth muscle -actin and the functional voltage-depended Ca~(2+) channels (VDCC). Acute hypoxia could increase [Ca~(2+)]_i in rat distal PVSMC.
VDCC antagonist nifedipine could block KCL-induced increase of [Ca~(2+)]_i in rat distal PVSMC. However, nifedipine partially prevented the effect of acute hypoxia on [Ca~(2+)]_i , indicating acute hypoxia actived influx of Ca~(2+) through VDCC and another independent pathway. Real-time PCR and western blotting both indicated that TRPC were expressed in rat distal pulmonary venous smooth muscle or PVSMC with most abundant expression of TRPC1, TRPC6 and TRPC4.
Restoration of extracellular Ca~(2+) caused a large increase of [Ca~(2+)]_i in PVSMC perfused with Ca~(2+)-free KRB solution containing CPA and nifedipine. In the presence of CPA, fura-2 fluorescence decreased more than that in the absence of CPA after administration of Mn~(2+) in PVSMC perfused with Ca~(2+)-free KRB solution containing nifedipine. The store-operated Ca~(2+) channels (SOCC) antagonists SKF-96365 and NiCl2 both inhibited CPA-induced Ca~(2+) influx and Mn~(2+) quenching, but had no effect on KCL-induced increase of [Ca~(2+)]_i in PVSMC. These data indicated SOCE in rat distal PVSMC.
Acute hypoxia caused enhancement of CPA-induced increase in [Ca~(2+)]_i elicited by Ca~(2+) restoration and the rate of Mn~(2+) quenching. SKF-96365 and NiCl2 blocked hypoxic enhancement of both the [Ca~(2+)]_i response to extracellular Ca~(2+) restoration and the rate of Mn~(2+) quenching. In normoxic PVSMC treated with nifedipine but not CPA, Mn~(2+) quenching did not differ from the spontaneous decline of fluorescence in cells not exposed to Mn~(2+). During acute hypoxia, however, the rate of Mn~(2+) quenching in the absence of CPA increased, and this increase was blocked by SKF-96365 or NiCl2.
The MPAP, RVSP and RV/(LV+S) were all increased in rats exposed to chronic hypoxia. The pulmonary vessels from CHPH rats were thicker than that from normal rats. [Ca~(2+)]_i in PVSMC from CHPH rats was higher than that from normal rats, indicating chronic hypoxia could increase [Ca~(2+)]_i in rat distal PVSMC.
Both TRPC6 mRNA and protein expression in pulmonary venous smooth muscle from CHPH rats were stronger than that from normal rats. But neither TRPC1 nor TRPC4 mRNA or protein expression in pulmonary venous smooth muscle from CHPH rats differed from that from normal rats.
In the presence of nifedipine and CPA, neither the increase of [Ca~(2+)]_i elicited by Ca~(2+) restoration nor the rate of Mn~(2+) quenching in PVSMC from CHPH rats differed from that from normal rats. In the presence of nifedipine and OAG, however, restoration of extracellular Ca~(2+) caused a larger increase of [Ca~(2+)]_i in PVSMC from CHPH rats than that from normal rats. Otherwise, in the presence of nifedipine and OAG, the rate of Mn~(2+) quenching in PVSMC from CHPH rats was also larger than that from normal rats.
Conclusion
1. Both acute and chronic hypoxia could increase [Ca~(2+)]_i in rat distal PVSMC.
2. SOCE and TRPC proteins were expressed in rat distal pulmonary venous smooth muscle with most abundant expression of TRPC1, TRPC6 and TRPC4.
3. Acute hypoxia was able to evoke SOCE in rat distal PVSMC on its own, as well as amplify SOCE.
4. Chronic hypoxia could increase Ca~(2+) influx through receptor-operated Ca~(2+) channels (ROCC) thought to be composed of TRPC6 protein by upregulation of TRPC6 expression in rat distal PVSMC.
方法
一、急性低氧对大鼠远端PVSMC[Ca~(2+)]_i的影响:首先利用胶原酶消化法分离、原代培养大鼠远端PVSMC,通过细胞形态学观察、平滑肌细胞α-actin免疫荧光和western-blot免疫印迹分析以及[Ca~(2+)]_i对高钾KRBS溶液的反应来鉴定PVSMC,从而建立大鼠远端PVSMC原代培养模型。然后利用InCyte细胞内钙浓度检测系统,观测急性低氧对大鼠远端PVSMC[Ca~(2+)]_i的影响
二、急性低氧对大鼠远端PVSMC[Ca~(2+)]_i影响的机制:1、利用InCyte细胞内钙浓度检测系统,观测电压依赖性钙通道(VDCC)拮抗剂硝苯地平对急性低氧诱导大鼠远端PVSMC[Ca~(2+)]_i改变的干预作用,明确是否有VDCC以外的钙通道参与急性低氧诱导的大鼠远端PVSMC[Ca~(2+)]_i改变过程。2、分离大鼠远端肺静脉平滑肌,原代培养大鼠远端PVSMC,通过荧光定量PCR和western-blot免疫印迹分析来检测经典瞬时受体电位(TRPC)蛋白在大鼠远端肺静脉平滑肌和培养的大鼠远端PVSMC的mRNA水平和蛋白水平的表达,明确大鼠远端肺静脉平滑肌是否表达TRPC蛋白。3、利用InCyte细胞内钙浓度检测系统,通过恢复细胞外钙法、锰荧光焠灭法,并利用储量操纵性钙通道(SOCC)拮抗剂SKF-96365和NiCl2,在大鼠远端PVSMC上检测Ca~(2+)通过由TRPC蛋白组成的SOCC而形成的储量操作性钙内流(SOCE),明确大鼠远端肺静脉平滑肌是否存在SOCE。4、利用InCyte细胞内钙浓度检测系统,通过恢复细胞外钙法、锰荧光焠灭法,并利用SOCC拮抗剂SKF-96365和NiCl2观测急性低氧对大鼠远端PVSMC的SOCE的影响,明确SOCE在急性低氧引起的大鼠远端PVSMC[Ca~(2+)]_i改变中的作用。
三、慢性低氧对大鼠远端PVSMC的[Ca~(2+)]_i的影响:首先,通过慢性持续低氧(10%O2)条件下饲养大鼠3周建立CHPH大鼠模型;然后,分离正常大鼠和CHPH大鼠远端PVSMC;最后,利用InCyte细胞内钙浓度检测系统,分别检测正常大鼠和CHPH大鼠远端PVSMC的[Ca~(2+)]_i,从而明确慢性低氧对大鼠远端PVSMC的[Ca~(2+)]_i的影响。
四、慢性低氧对大鼠远端PVSMC的[Ca~(2+)]_i的影响的机制:1、分离正常大鼠和CHPH大鼠远端肺静脉平滑肌,通过荧光定量PCR和Western-blot免疫印迹分析来检测TRPC在正常大鼠和CHPH大鼠远端肺静脉平滑肌的表达,明确慢性低氧对大鼠远端肺静脉平滑肌TRPC表达的影响。2、分离正常大鼠和CHPH大鼠远端PVSMC,利用InCyte细胞内钙浓度检测系统,首先分别检测由环匹阿尼酸(CPA)诱导的正常大鼠和CHPH大鼠远端PVSMC的[Ca~(2+)]_i变化,然后分别检测由CPA诱导的正常大鼠和CHPH大鼠远端PVSMC的锰荧光焠灭,从而明确慢性低氧对大鼠远端肺静脉平滑肌SOCE的影响;接着分别检测由油酰基乙酰甘油(OAG)诱导的正常大鼠和CHPH大鼠远端PVSMC的[Ca~(2+)]_i变化,最后分别检测由OAG诱导的正常大鼠和CHPH大鼠远端PVSMC的锰荧光焠灭,从而明确慢性低氧对大鼠远端肺静脉平滑肌由受体操纵性钙通道(ROCC)介导的Ca~(2+)内流的影响。
结果
一、急性低氧对大鼠远端PVSMC[Ca~(2+)]_i的影响:
1、成功建立了大鼠远端PVSMC原代培养模型,原代培养的大鼠远端PVSMC不仅表现出典型的血管平滑肌细胞的形态学和免疫学特征,而且具有完好的生理学功能。
2、急性低氧使大鼠远端PVSMC[Ca~(2+)]_i升高。
二、急性低氧对大鼠远端PVSMC[Ca~(2+)]_i影响的机制:
1、作为VDCC拮抗剂,硝苯地平能完全阻断高钾KRBS溶液引起的大鼠远端PVSMC [Ca~(2+)]_i升高,却只能部分抑制急性低氧引起的大鼠远端PVSMC的[Ca~(2+)]_i升高,提示VDCC以外的钙通道参与急性低氧引起的大鼠远端PVSMC[Ca~(2+)]_i升高。
2、在大鼠远端肺静脉平滑肌组织和培养的PVSMC都有TRPC的mRNA表达,其中以TRPC-1、TRPC-4和TRPC-6,尤其是TRPC-1和TRPC-6的mRNA表达最强。Western-blot免疫印迹分析进一步表明,在大鼠远端肺静脉平滑肌组织和培养的PVSMC都有TRPC蛋白的表达,且尤其以TRPC-1和TRPC-6的蛋白表达最强。
3、在去除细胞外Ca~(2+)、用硝苯地平阻断VDCC并用CPA使Ca~(2+)从PVSMC肌浆网完全清空的条件下,恢复PVSMC细胞外Ca~(2+)后, PVSMC [Ca~(2+)]_i可迅速显著升高;在去除细胞外Ca~(2+)并用硝苯地平阻断VDCC的条件下,CPA使Mn~(2+)引起的PVSMC的荧光焠灭增强;作为SOCC拮抗剂,SKF-96365和NiCl2均能明显抑制由CPA诱导的PVSMC [Ca~(2+)]_i升高和锰荧光焠灭增强,但对高钾KRBS溶液引起的PVSMC [Ca~(2+)]_i升高没有影响,提示大鼠远端PVSMC存在SOCE。
4、急性低氧能显著增加由CPA诱导的、恢复细胞外钙后所引起的大鼠远端PVSMC[Ca~(2+)]_i升高,并能增强由CPA诱导的大鼠远端PVSMC的锰荧光焠灭; SKF-96365和NiCl2均能明显抑制急性低氧引起的大鼠远端PVSMC的SOCE增加;在常氧情况下,用不含CPA但含有硝苯地平的无钙溶液孵育PVSMC,锰荧光焠灭与不加Mn~(2+)的细胞的荧光自发衰减量没有差异;而在急性低氧情况下,用不含CPA但含有硝苯地平的无钙溶液孵育PVSMC,锰荧光焠灭显著增加,而且此反应能被SKF-96365和NiCl2明显抑制,提示急性低氧自身能诱发大鼠远端PVSMC的SOCE,而且能够通放大SOCE,引起PVSMC [Ca~(2+)]_i升高。
三、慢性低氧对大鼠远端PVSMC的[Ca~(2+)]_i的影响:
1、成功建立CHPH大鼠模型,慢性持续性低氧3周大鼠的MPAP、RVSP、RV/(LV+S)都明显增高,肺内血管壁明显增厚。
2、CHPH大鼠远端PVSMC[Ca~(2+)]_i较正常大鼠明显增高,提示慢性低氧使大鼠远端PVSMC的[Ca~(2+)]_i升高。
四、慢性低氧对大鼠远端PVSMC[Ca~(2+)]_i影响的机制:
1、荧光定量PCR和western-blot免疫印迹分析证实:在CHPH大鼠远端肺静脉平滑肌组织TRPC-6蛋白的表达较正常大鼠明显增强,而TRPC-1和TRPC-4蛋白的表达较正常大鼠没有明显改变,提示慢性低氧上调大鼠远端肺静脉平滑肌组织TRPC-6蛋白的表达。
2、用硝苯地平阻断VDCC并恢复细胞外Ca~(2+)后,CPA不仅能导致CHPH大鼠远端PVSMC [Ca~(2+)]_i迅速升高,而且能使正常大鼠远端PVSMC的[Ca~(2+)]_i也迅速升高,两者升高幅度无差别;用含CPA和硝苯地平的无钙溶液孵育细胞,CHPH大鼠远端PVSMC的锰荧光焠灭与正常大鼠的也无差别。然而,用硝苯地平阻断VDCC并恢复细胞外Ca~(2+)后,OAG能使正常大鼠远端PVSMC的[Ca~(2+)]_i升高,而CHPH大鼠远端PVSMC由OAG诱导的[Ca~(2+)]_i升高较正常大鼠更明显;同时,OAG诱导的锰荧光焠灭在CHPH大鼠远端PVSMC也比正常大鼠表现的更强,提示慢性低氧通过增加由ROCC介导的Ca~(2+)内流使大鼠远端PVSMC的[Ca~(2+)]_i升高。
结论
1、急性和慢性低氧都能使大鼠远端肺静脉平滑肌细胞内钙浓度升高。
2、大鼠远端肺静脉平滑肌主要表达TRPC-1、TRPC-4和TRPC-6,尤以TRPC-1和TRPC-6为主;大鼠远端肺静脉平滑肌存在SOCE。
3、急性低氧不仅自身能够诱发大鼠远端肺静脉平滑肌细胞的SOCE,而且能够放大SOCE,从而引起细胞内钙浓度升高。
4、慢性低氧能够上调大鼠远端肺静脉平滑肌TRPC-6的表达,使主要由TRPC-6蛋白组成的ROCC增加,导致由ROCC介导的Ca~(2+)内流增加,从而引起细胞内钙浓度升高。
Objective
Hypoxia causes remodeling and alters contractile responses in both pulmonary arteries and pulmonary veins (PV). Although pulmonary arteries have been studied extensively in this disorder, the mechanisms by which pulmonary veins respond to hypoxia and whether these responses contribute to chronic hypoxic pulmonary hypertension (CHPH) remain poorly understood. In pulmonary arterial smooth muscle, influx of Ca~(2+) through store-operated calcium channels thought to be composed of canonical transient receptor potential (TRPC) proteins is likely to play an important role in development of CHPH. The aim of this study is to determine whether this mechanism could also be operative in pulmonary venous smooth muscle.
Methods
The rat distal PVSMC were isolated and cultured by collagenase digestion and characterized by morphological activity, by immunoblotting and immunofluorescence of smooth muscle -actin and by response of intracellular Ca~(2+) concentration ([Ca~(2+)]_i) to KCL.
The effect of acute hypoxia on [Ca~(2+)]_i in PVSMC and the inhibition of nifedipine were measured by Incyte intracellular Ca~(2+) concentration system. The expression of TRPC in distal pulmonary venous smooth muscle or PVSMC was detected by real-time PCR and western blotting. Store-operated Ca~(2+) entry (SOCE) and the effect of acute hypoxia on SOCE were assessed by measurement of increase in [Ca~(2+)]_i caused by restoration of extracellular Ca~(2+) and the rate at which extracellular Mn~(2+) quenched fura-2 fluorescence.
CHPH rats were obtained using a hypoxic (10 % O2) chamber for 21 days. PVSMC were isolated from CHPH rats or normal rats. The effect of chronic hypoxia on [Ca~(2+)]_i in PVSMC was assessed by comparison of [Ca~(2+)]_i in PVSMC from CHPH rats with that from normal rats.
The effect of chronic hypoxia on TRPC expression was measured by comparison of TRPC mRNA and protein expression in pulmonary venous smooth muscle from CHPH rats with that from normal rats using real-time PCR and western blotting. PVSMC from CHPH rats or normal rats were isolated. The mechanisms by which [Ca~(2+)]_i in PVSMC respond to chronic hypoxia were assessed by comparison of CPA-induced Ca~(2+) influx and Mn~(2+) quenching in PVSMC isolated from CHPH rats with that from normal rats and by comparison of OAG-induced Ca~(2+) influx and Mn~(2+) quenching in PVSMC isolated from CHPH rats with that from normal rats.
Results
Cells isolated from rat distal pulmonary veins displayed morphological characteristic of vascular smooth muscle cells and expressed smooth muscle -actin and the functional voltage-depended Ca~(2+) channels (VDCC). Acute hypoxia could increase [Ca~(2+)]_i in rat distal PVSMC.
VDCC antagonist nifedipine could block KCL-induced increase of [Ca~(2+)]_i in rat distal PVSMC. However, nifedipine partially prevented the effect of acute hypoxia on [Ca~(2+)]_i , indicating acute hypoxia actived influx of Ca~(2+) through VDCC and another independent pathway. Real-time PCR and western blotting both indicated that TRPC were expressed in rat distal pulmonary venous smooth muscle or PVSMC with most abundant expression of TRPC1, TRPC6 and TRPC4.
Restoration of extracellular Ca~(2+) caused a large increase of [Ca~(2+)]_i in PVSMC perfused with Ca~(2+)-free KRB solution containing CPA and nifedipine. In the presence of CPA, fura-2 fluorescence decreased more than that in the absence of CPA after administration of Mn~(2+) in PVSMC perfused with Ca~(2+)-free KRB solution containing nifedipine. The store-operated Ca~(2+) channels (SOCC) antagonists SKF-96365 and NiCl2 both inhibited CPA-induced Ca~(2+) influx and Mn~(2+) quenching, but had no effect on KCL-induced increase of [Ca~(2+)]_i in PVSMC. These data indicated SOCE in rat distal PVSMC.
Acute hypoxia caused enhancement of CPA-induced increase in [Ca~(2+)]_i elicited by Ca~(2+) restoration and the rate of Mn~(2+) quenching. SKF-96365 and NiCl2 blocked hypoxic enhancement of both the [Ca~(2+)]_i response to extracellular Ca~(2+) restoration and the rate of Mn~(2+) quenching. In normoxic PVSMC treated with nifedipine but not CPA, Mn~(2+) quenching did not differ from the spontaneous decline of fluorescence in cells not exposed to Mn~(2+). During acute hypoxia, however, the rate of Mn~(2+) quenching in the absence of CPA increased, and this increase was blocked by SKF-96365 or NiCl2.
The MPAP, RVSP and RV/(LV+S) were all increased in rats exposed to chronic hypoxia. The pulmonary vessels from CHPH rats were thicker than that from normal rats. [Ca~(2+)]_i in PVSMC from CHPH rats was higher than that from normal rats, indicating chronic hypoxia could increase [Ca~(2+)]_i in rat distal PVSMC.
Both TRPC6 mRNA and protein expression in pulmonary venous smooth muscle from CHPH rats were stronger than that from normal rats. But neither TRPC1 nor TRPC4 mRNA or protein expression in pulmonary venous smooth muscle from CHPH rats differed from that from normal rats.
In the presence of nifedipine and CPA, neither the increase of [Ca~(2+)]_i elicited by Ca~(2+) restoration nor the rate of Mn~(2+) quenching in PVSMC from CHPH rats differed from that from normal rats. In the presence of nifedipine and OAG, however, restoration of extracellular Ca~(2+) caused a larger increase of [Ca~(2+)]_i in PVSMC from CHPH rats than that from normal rats. Otherwise, in the presence of nifedipine and OAG, the rate of Mn~(2+) quenching in PVSMC from CHPH rats was also larger than that from normal rats.
Conclusion
1. Both acute and chronic hypoxia could increase [Ca~(2+)]_i in rat distal PVSMC.
2. SOCE and TRPC proteins were expressed in rat distal pulmonary venous smooth muscle with most abundant expression of TRPC1, TRPC6 and TRPC4.
3. Acute hypoxia was able to evoke SOCE in rat distal PVSMC on its own, as well as amplify SOCE.
4. Chronic hypoxia could increase Ca~(2+) influx through receptor-operated Ca~(2+) channels (ROCC) thought to be composed of TRPC6 protein by upregulation of TRPC6 expression in rat distal PVSMC.
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