Development of a flexible image-based approach for studying signal transduction in isolated arterioles

Abstract

Myogenic constriction in arterioles occurs when membrane tension leads to smooth muscle depolarization and voltage-gated calcium channel activation. A barrier to understanding the exact signaling events is the limited ability of glass electrodes to follow temporal changes in membrane potential (Em). This study aimed to develop an imaging station to measure Em, [Ca²+]i and diameter in isolated arterioles. A Forster Resonance Enery Transfer (FRET) approach was proposed for Em meaurement due to enhanced sensitivity and temporal responsiveness. FRET indicators used were CC2-DMPE (Coumarin-labelled Phospholipid, Donor, ex: 405nm) and DisBAC₄(3) (Oxonol, acceptor). For measurement of [Ca²+]i Fluo-4 AM (ex 488nm) was used. Initial studies examined toxicity of the multiple fluorophores. Isolated cells remained viable after loading with the fluorophores. Arterioles maintained myogenic, and agonist, responsiveness pre and post dye loading suggesting a lack of toxicity. Spectral characteristics of the fluorophores used in the project required specific optical filter units and development of a. custom built optical system. Using the system, FRET was demonstrated, as was the ability to detect changes in fluorescence signal to known depolarizing stimuli. The developed system is now ready for translation to the study of myogenic signaling in intact and functional arterioles.