Pulsed laser imaging of rapid Ca2+ gradients in excitable cells.
Excitable cells are thought to respond to action potentials by forming short lived and highly localized Ca2+ gradients near sites of Ca2+ entry or near the site of Ca2+ release by intracellular stores. However, conventional imaging techniques lack the spatial and temporal resolution to capture these gradients. Here we demonstrate the use of pulsed-laser microscopy to measure Ca2+ gradients with submicron spatial resolution and millisecond time resolution in two preparations where the Ca2+ signal is thought to be fast and highly localized: adrenal chromaffin cells, where the entry of Ca2+ through voltage dependent Ca2+ channels triggers exocytotic fusion; and skeletal muscle fibers, where intracellular Ca2+ release from the sarcoplasmic reticulum initiates contraction. In chromaffin cells, Ca2+ gradients developed over 10-100 ms and were initially restricted to discrete submembrane domains, or hot spots, before developing into complete rings of elevated Ca2+ concentration. In frog skeletal muscle large, short-lived (approximately 6 ms) Ca2+ gradients were observed within individual sarcomeres following induction of action potentials. The pulsed laser imaging approach permits, for the first time, the capture and critical examination of rapid Ca2+ signaling events such as those underlying excitation-secretion and excitation-contraction coupling.
Monck
J R
JR
Mayo Clinic, Department of Physiology and Biophysics, Rochester, Minnesota 55905.
Robinson
I M
IM
Escobar
A L
AL
Vergara
J L
JL
Fernandez
J M
JM
D000200:Action Potentials; D000313:Adrenal Medulla / Q000166:cytology / Q000502:physiology*; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002417:Cattle; D002478:Cells, Cultured; D066298:In Vitro Techniques; D007834:Lasers; D008856:Microscopy, Fluorescence / Q000295:instrumentation / Q000379:methods; D009132:Muscles / Q000502:physiology*; D011892:Rana catesbeiana; D012518:Sarcomeres / Q000502:physiology*; D015398:Signal Transduction
D002118:Calcium