Figure 2.

Dissociation of agonist-stimulated [Ca²⁺]_i transients into IP3-induced release and CCE-mediated replenishment components. The first rapid rise does not require extracellular Ca²⁺ and returns rapidly to the starting [Ca²⁺]_i, due to extrusion of Ca²⁺ from the cells by plasma membrane Ca-ATPases and reuptake of Ca²⁺ into stores by sarcoplasmic endoplasmic reticulum Ca-ATPases. The second component, stimulation of CCE, is seen as influx of Ca²⁺ from the extracellular milieu that occurs upon Ca²⁺ addition after completion of the first Ca²⁺ transient. This form of Ca²⁺ entry is due to stimulation of CCE channels by a signal generated by the IP3-induced store depletion and/or by some other agonist-induced signaling process. [Ca²⁺]_i changes were measured in murine L cells (Lc4 cells) expressing in stable form the M5 muscarinic acetylcholine receptor. Bars on top depict changes in composition of the medium. The lower bars indicate Ca²⁺ additions; open bar indicates addition of 0.5 mM EGTA to medium without added Ca²⁺, and the black bar and gray area depicts change to a balanced salt solution containing 1.8 mM CaCl₂. The upper bars indicate agonist additions. When added, concentration of CCh was 20 μM and that of ATP was 90 μM. Note that the initial rise of [Ca²⁺]_i after agonist addition in the absence of extracellular Ca²⁺ returns spontaneously to baseline levels, and also note that addition of extracellular Ca²⁺ uncovers the presence of an activated influx pathway that causes a robust increase in [Ca²⁺]_i. This influx is referred to as CCE. Note further that in these cells the release of Ca²⁺ from intracellular stores occurs as a concerted event without hint of oscillations.