Ion channels, Ca2+ & exercise: Regulating myogenic tone

该项目将专注于离子通道对肌源性张力调节的适应。这一信息是所有项目的核心，因为肌源性反应在建立基础血管阻力、血流、毛细血管压力和血管活动机制的双向调节方面至关重要。本研究旨在探讨生理(运动训练；EX)和病理(饮食高脂；HF)对冠状动脉平滑肌L型电压门控钙通道(VGCC)电流密度(I/Ca)的影响及其机制。总的假设是，EX和HF在VGCC的表达和活性上产生相反的变化，VGCC通过随后的肌浆[Ca~(2+)](CaM)调节和K+通道激活的变化而改变肌源性张力。EX和HF对CSM的相互作用将在遭受EX的HF动物身上进行检测。通道蛋白和表达水平将通过免疫印迹和RT-PCR确定，通道活性和特性由全细胞电压钳和膜片钳技术确定。我们的假设是，无论是VGCC的表达还是PKC依赖的活性，EX增加，HF减少，而对单通道电导没有影响。HF期间的EX将减弱单独使用HF时观察到的衰减。2)测定ICa密度变化对肌浆钙和K+电流的影响。同时应用电压钳和微量荧光法测定Ia变化对钙、钾电流的影响。同时应用电压钳和显微荧光法测定在长时间去极化过程中，I/Ca变化对CaM和K+通道活性的影响。假设：1)在延长的去极化过程中，肌浆网(VGCC)和钾/钙离子通道(K/Ca)的肌浆网(SR)钙摄取和释放过程中，肌浆网(VGCC)和钾/钙(K/Ca)的SR偶联会减弱钙通道(CaM)的活性，而不是在强去极化过程中，VGCC和K/Ca的SR偶联会减弱钙调素的活性。3)确定I/Ca浓度改变对肌源性张力调节的影响。分离微血管的膜电位(Vm)、CaM和直径测量将决定肌张力过程中Vm和CaM之间的关系。假说是肌源性反应与I/Ca成正比，VGCC和K/Ca通道的功能耦合调节了这一关系。这个项目的意义将是提供新的信息，描述在Ex和HF中肌张力的适应性调节的细胞/分子基础。鉴于VGCC在冠脉张力调节中的核心作用，基础和临床相关信息都应该来自这些研究。

The project will focus on adaptations in ion channel regulation of myogenic tone. This information is central to all projects as the myogenic response is critical in establishing basal vascular resistance, blood flow, capillary pressure and bidirectional modulation by vasoactive mechanisms. The general aim of the proposed research is to determine the mechanism and functional impact of physiological (exercise training; EX) and pathological (dietary high fat; HF) changes in L-type voltage-gated Ca2+ channel (VGCC) current density (I/ca) in coronary smooth muscle (CSM). The overall hypothesis is that EX and HF produce opposing changes in the expression and activity of VGCCs which alter myogenic tone through consequent changes in myoplasmic [Ca2+] (Cam) regulation and K+ channel activation. The interaction of EX and HF on CSM will be examined in HF animals subjected to EX. Channel protein and expression levels will be determined by immunoblot and RT-PCR with channel activity and characteristics determined by whole cell voltage clamp and patch clamp techniques. The hypothesis is that EX increases, and HF decreases, both VGCC expression and PKC-dependent activity, with no effect on single channel conductance. EX during HF will attenuate the decrement observed with HF alone. 2) Determine effect of changes in Ica density on mycoplasmic Ca2) and K+ current. Simultaneous voltage- clamp and microfluorometry will determine the effect of changes in Ia on Ca2+ and K+ current. Simultaneous voltage-clamp and microfluorometry will determine the effect of changes in I/Ca on Cam and K+ channel activity during prolonged depolarization. The hypotheses are that: 1) EX will increase, and HF decrease, Ca2+-activity K+ channel (KCa) activity during prolonged depolarization, 2) the enhanced KCA activity in EX will be dihydropyridine-sensitive (i.e. functionally coupled to I/Ca) and dependent upon sarcoplasmic reticulum (SR) Ca2+ uptake and release and 3) in EX, SR coupling of VGCC and K/Ca will attenuate Cam during mild, but not strong depolarization. 3) Determine the effect of altered I/Ca density on regulation of myogenic tone. Membrane potential (Vm), Cam and diameter measures in isolated microvessels will determine the relationship between Vm and Cam during myogenic tone. The hypotheses are that myogenic responsiveness is proportional to I/Ca and functional coupling of VGCC's and K/Ca channels modulates this relationship. The significance of this project will be to provide novel information describing the cellular/molecular basis for adaptive regulation of myogenic tone in both Ex and HF. Given the central role of VGCCs in regulation of coronary tone, both basic and clinically relevant information should results from these studies.