Ion channels, Ca2+ & exercise: Regulating myogenic tone

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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.