Identification and its functional analysis of ion channels involving in pathophysiologic remodeling of vascular smooth muscle

电压门控Na^+通道电流（I(Na)）在几种类型的平滑肌细胞中表达。我们评估了I（Na）在培养的人（hASMCs）和兔主动脉平滑肌细胞（rASMCs）中的表达、功能作用、病理生理以及它与血管内膜增生的关系。在全细胞电压箝位下，I（Na）电位为正至-40 mV，被河蟹毒素（TTX）阻断，并以n -甲基-d-氨基葡萄糖替代细胞外Na+。通过定量RT-PCR检测，与天然主动脉相比，培养的hASMCs强烈表达编码Na(V)1.7的SCN9A。SCN9A小干扰（si）RNA对I（Na）有抑制作用（P<0.01）。TTX和SCN9A siRNA显著抑制了细胞迁移（P<0.01）和辣根过氧化物酶摄取（P<0.01）。TTX在处理后6和12 h显著降低了基质金属蛋白酶-2的分泌（P<0.01和P<0.05）。TTX和siRNA对细胞增殖均无影响。在新分离的rASMCs中记录到l型Ca^<2+>通道电流，未观察到I(Na)，而在培养的rASMCs中记录到ttx敏感的I（Na）。定量RT-PCR和Na(V)1.7免疫染色显示，SCN9A在球囊损伤48小时后在培养的rASMCs和主动脉中显著表达，而在天然主动脉中不表达。这些研究表明，I（Na）在培养和病变条件下表达，而在正常主动脉中不表达。Na(V)1.7在细胞迁移、内吞和分泌中起重要作用。Na(V)1.7也在球囊损伤后的主动脉中表达，提示Na(V)1.7在内膜增生的进展中可能起作用。此外，血清淀粉样蛋白A (SAA)（一种急性期蛋白）和溶血磷脂酰胆碱（LPC）（一种氧化的LDL成分）参与动脉粥样硬化和心血管疾病的生理过程。然而，SAA/LPC对人冠状动脉平滑肌细胞（hCASMCs）的影响尚未得到充分的研究。因此，我研究了SAA/LPC对hCASMCs中Ca^<2+>/Mg^<2+>动员的影响及其潜在机制。我们发现SAA/LPC激活hCASMCs中的Ca^<2+>内流；SAA通过ptx敏感的g蛋白、PLC和TRPC途径激活它，其中TRPC4可能参与其中。另一方面，LPC可能独立于这些途径通过TRPM7激活它。因此，在SAA/LPC引发的hCASMCs中，TRP蛋白可能是Ca^<2+>信号的靶分子，可能在动脉粥样硬化等病理生理和炎症条件下冠状动脉肌肉功能障碍中发挥作用。这些结果提供了离子通道阻滞剂作为治疗动脉粥样硬化性疾病如冠状动脉疾病的可能性。少

Voltage-gated Na^+ channel currents (I(Na)) are expressed in several types of smooth muscle cells. We evaluated the expression of I(Na), its functional role, pathophysiology in cultured human (hASMCs) and rabbit aortic smooth muscle cells (rASMCs), and its association with vascular intimal hyperplasia. In whole cell voltage clamp, I(Na) was observed at potential positive to -40 mV, was blocked by tetrodotoxin (TTX), and replacing extracellular Na+ with N-methyl-d-glucamine in cultured hASMCs. In contrast to native aorta, cultured hASMCs strongly expressed SCN9A encoding Na(V)1.7, as determined by quantitative RT-PCR. I(Na) was abolished by the treatment with SCN9A small-interfering (si)RNA (P<0.01). TTX and SCN9A siRNA significantly inhibited cell migration (P<0.01, respectively) and horseradish peroxidase uptake (P<0.01, respectively). TTX also significantly reduced the secretion of matrix metalloproteinase-2 6 and 12 h after the treatment (P<0.01 and P<0.05, respectively). However, n … More either TTX nor siRNA had any effect on cell proliferation. L-type Ca^<2+> channel current was recorded, and I(Na) was not observed in freshly isolated rASMCs, whereas TTX-sensitive I(Na) was recorded in cultured rASMCs. Quantitative RT-PCR and immunostaining for Na(V)1.7 revealed the prominent expression of SCN9A in cultured rASMCs and aorta 48 h after balloon injury but not in native aorta. These studies showed that I(Na) is expressed in cultured and diseased conditions but not in normal aorta. The Na(V)1.7 plays an important role in cell migration, endocytosis, and secretion. Na(V)1.7 is also expressed in aorta after balloon injury, suggesting a potential role for Na(V)1.7 in the progression of intimal hyperplasia. In addition, serum amyloid A (SAA), an acute-phase protein, and lysophosphatidylcholine (LPC), an oxidized LDL component, contribute to physiological processes of atherosclerosis and cardiovascular disease. However, the effects of SAA/LPC on human coronary artery smooth muscle cells (hCASMCs) have not been fully investigated. Therefore, I examined the effects of SAA/LPC on Ca^<2+>/Mg^<2+> mobilization and its underlying mechanisms in hCASMCs. We showed that SAA/LPC activate Ca^<2+> influx in hCASMCs; SAA activates it via PTX-sensitive G-protein, PLC and TRPC pathways, where TRPC4 may be involved. On the other hand, LPC may activate it via TRPM7 independently of these pathways. Thus, TRP protein appears to be a target molecule of Ca^<2+> signaling in hCASMCs elicited by SAA/LPC, which may play roles in coronary muscle dysfunction under the pathophysiological and inflammatory conditions such as atherosclerosis. These results provide a possibility of ion channel blockers as a therapy for atherosclerotic diseases such as coronary artery diseases. Less