Ion channels, Ca2+ & exercise: Regulating myogenic tone

该项目将重点关注离子通道调节肌原性张力的适应性。这一信息是所有项目的核心，因为肌源性反应对于建立基础血管阻力、血流、毛细血管压力和血管活性机制的双向调节至关重要。拟议研究的总体目的是确定生理（运动训练；EX）和病理（饮食高脂肪；HF）改变冠状动脉平滑肌（CSM）中l型电压门控Ca2+通道（VGCC）电流密度（I/ca）的机制和功能影响。总的假设是，EX和HF在vgc的表达和活性方面产生相反的变化，通过随后的肌浆[Ca2+] （Cam）调节和K+通道激活的变化来改变肌原性张力。将在接受EX治疗的HF动物中检测EX和HF对CSM的相互作用。通道蛋白和表达水平将通过免疫印迹和RT-PCR测定，通道活性和特征将通过全细胞电压钳和膜片钳技术测定。假设VGCC表达和pkc依赖性活性均增加EX，降低HF，但对单通道电导没有影响。HF期间的EX会减弱单独HF时观察到的衰减。2)确定Ica密度变化对支原体Ca2)和K+电流的影响。同时电压箝位和微荧光测定法将确定变化的Ia对Ca2+和K+电流的影响。同时电压钳和微荧光测定法将确定在长时间去极化期间I/Ca变化对Cam和K+通道活性的影响。假设是：1)在长时间的去极化过程中，EX会增加Ca2+活性K+通道（KCa）活性，HF会降低；2)EX中KCa活性的增强将是二氢吡啶敏感的（即功能上偶联于I/Ca），并依赖于肌浆网（SR） Ca2+的摄取和释放；3)在EX中，VGCC和K/Ca的SR偶联将在轻度但不强烈的去极化过程中减弱Cam。3)确定I/Ca密度改变对肌原性张力调节的影响。膜电位（Vm）， Cam和离体微血管直径测量将决定Vm和Cam在肌张力期间的关系。假设肌源性反应与I/Ca成正比，VGCC和K/Ca通道的功能耦合调节了这种关系。该项目的意义在于提供描述Ex和HF中肌原性张力适应性调节的细胞/分子基础的新信息。鉴于vgc在调节冠状动脉舒张中的核心作用，这些研究应该得出基础和临床相关的信息。

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.