Measurement of [Ca²⁺]i in smooth muscle strips using front-surface fluorimetry

Changes in the cytosolic Ca(2+) concentrations ([Ca(2+)](i)) play a primary role in the regulation of the contraction of smooth muscle cells. However, the relationship between [Ca(2+)](i) and tension exhibits a temporal change during the time course of contraction or relaxation. The extent of the tension development for a given change in [Ca(2+)](i) also varies depending on the type of contraction and relaxation. Therefore, it is essential to measure [Ca(2+)](i) and tension simultaneously in order to determine the molecular and cellular mechanisms in both the regulation of contraction and relaxation of smooth muscle. This chapter provides the basic principles of the technique of front-surface fluorimetry as well as the protocols and tips for the simultaneous measurement of [Ca(2+)](i) and tension in the smooth muscle tissues with use of fura-2 or Fura-PE3 as fluorescent Ca(2+) indicators. The loading of sufficient amount of the Ca(2+) indicators in smooth muscles is essential for the successful measurement of [Ca(2+)](i) with minimum optical artifacts. The protocol gives our practice for the loading of the Ca(2+) indicators in various smooth muscle tissues.