Ion channels, Ca2+ & exercise: Regulating myogenic tone

该项目将集中在肌源性张力的离子通道调节的适应性。这一信息对所有项目都至关重要，因为肌源性反应在建立基础血管阻力、血流量、毛细血管压力和血管活性机制的双向调节中至关重要。拟议研究的总体目的是确定冠状动脉平滑肌（CSM）L型电压门控钙通道（VGCC）电流密度（I/ca）的生理（运动训练; EX）和病理（饮食高脂肪; HF）变化的机制和功能影响。总体假设是EX和HF在VGCC的表达和活性中产生相反的变化，其通过随后的肌浆[Ca 2 +]（Cam）调节和K+通道激活的变化来改变肌源性张力。将在接受EX的HF动物中检查EX和HF对CSM的相互作用。通过免疫印迹和RT-PCR测定通道蛋白和表达水平，通过全细胞电压钳和膜片钳技术测定通道活性和特征。假设是EX增加，HF降低，VGCC表达和PKC依赖性活性，对单通道电导没有影响。HF期间的EX将减弱单独HF时观察到的减量。2)测定伊卡浓度变化对支原体Ca 2+和K+电流的影响。同时电压钳和显微荧光测定法将确定Ia的变化对Ca 2+和K+电流的影响。同时电压钳和显微荧光测定法将确定在I/Ca的变化对凸轮和K+通道活性在延长的去极化的影响。假设是：1）在延长的去极化期间，EX将增加Ca 2+活性K+通道（KCa）活性，而HF降低Ca 2+活性K+通道（KCa）活性，2）EX中增强的KCA活性将是二氢吡啶敏感的（即功能上与I/Ca偶联）并且依赖于肌浆网（SR）Ca 2+摄取和释放，以及3）在EX中，VGCC和K/Ca的SR偶联将在轻度但不强的去极化期间减弱Cam。3)确定改变的I/Ca密度对肌源性张力调节的影响。膜电位（Vm），凸轮和直径的措施，在孤立的微血管将确定之间的关系Vm和凸轮在肌张力。假设是肌源性反应是成比例的 VGCC和K/Ca通道的功能偶联调节了这种关系。该项目的意义将是提供新的信息描述的细胞/分子基础的适应性调节肌张力在Ex和HF。鉴于VGCC在冠状动脉张力调节中的核心作用，这些研究应提供基础和临床相关信息。

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.