title: A new method to detect rapid oxygen changes around cells: how quickly do calcium channels sense oxygen in cardiomyocytes?
abstract: Acute hypoxia is thought to trigger protective responses that, in tissues like heart and carotid body, include rapid (5-10 s) suppression of Ca(2+) and K(+) channels. To gain insight into the mechanism for the suppression of the cardiac l-type Ca(2+) channel, we measured O2-dependent fluorescence in the immediate vicinity of voltage-clamped cardiac cells subjected to rapid exchange of solutions with different O2 tensions. This was accomplished with an experimental chamber with a glass bottom that was used as a light guide for excitation of a thin ruthenium-based O2-sensitive ORMOSIL coating. Fluorescence imaging showed that steady-state Po2 was well controlled within the entire stream from an electromagnetically controlled solution "puffer" but that changes were slower at the periphery of the stream (τ1/2 ∼ 500 ms) than immediately around the voltage-clamped myocyte (τ1/2 ∼ 225 ms) where, in turn, firmly attached cells produced an additional local delay of 50-100 ms. Performing simultaneous voltage clamp and O2 measurements, we found that acute hypoxia gradually and reversibly suppressed the Ca(2+) channel (CaV1.2). Using Ba(2+) as charge carrier, the suppression was significant after 1.5 s, reached ∼10% after 2.5 s, and was nearly completely reversible in 5 s. The described fluorescence measurements provide the means to check and fine tune solution puffers and suggest that changes in Po2 can be accomplished within ∼200 ms. The rapid and reversible suppression of barium current under hypoxia is consistent with the notion that the cardiac Ca(2+) channel is directly modulated by O2.
authors.affiliation: Cardiac Signaling Center of University of South Carolina, Medical University of South Carolina, and Clemson University, Charleston, South Carolina.
authors.forename: John A
authors.initials: JA
authors.lastname: Scaringi
authors.forename: Angelo Oscar
authors.initials: AO
authors.lastname: Rosa
authors.forename: Martin
authors.initials: M
authors.lastname: Morad
authors.forename: Lars
authors.initials: L
authors.lastname: Cleemann
mesh_terms: D000818:Animals; D020746:Calcium Channels, L-Type / Q000378:metabolism*; D000860:Hypoxia / Q000378:metabolism; D008297:Male; D032383:Myocytes, Cardiac / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D020746:Calcium Channels, L-Type; D010100:Oxygen
keywords: dissolved oxygen sensing; hypoxic suppression of Ca2+ current; ormosil