Calcium gradients in single smooth muscle cells revealed by the digital imaging microscope using Fura-2

Abstract

Calcium is believed to control a variety of cellular processes, often with a high degree of spatial and temporal precision. For a cell to use Ca²⁺ in this manner, mechanisms must exist for controlling the ion in a localized fashion. We have now gained insight into such mechanisms from studies which measured Ca²⁺ in single living cells with high resolution using a digital imaging microscope and the highly fluorescent Ca²⁺-sensitive dye, Fura-2. Levels of Ca²⁺ in the cytoplasm, nucleus and sarcoplasmic reticulum (SR) are clearly different. Free [Ca²⁺] in the nucleus and SR was greater than in the cytoplasm and these gradients were abolished by Ca²⁺ ionophores. When external Ca²⁺ was raised above normal in the absence of ionophores, free cytoplasmic Ca²⁺ increased but nuclear Ca²⁺ did not. Thus, nuclear [Ca²⁺] appears to be regulated independently of cytoplasmic [Ca²⁺] by gating mechanisms in the nuclear envelope. The observed regulation of intranuclear Ca²⁺ in these contractile cells may thus be seen as a way to prevent fluctuation in Ca²⁺-linked nuclear processes during the rise in cytoplasmic [Ca²⁺] which triggers contraction. The approach described here offers the opportunity of following changes in Ca²⁺ in cellular compartments in response to a wide range of stimuli, allowing new insights into the role of local changes in Ca²⁺ in the regulation of cell function.