Activation and co-ordination of InsP3-mediated elementary Ca2+ events during global Ca2+ signals in Xenopus oocytes.

The second messenger inositol 1,4,5-trisphosphate (InsP3) functions primarily by liberating calcium ions from intracellular stores, thereby causing elevations in cytosolic free calcium concentration that are frequently observed as repetitive waves propagating throughout the cell (Berridge, 1993). Recent experiments have suggested that these waves may arise through recruitment of functionally discrete intracellular release units, which have been proposed to comprise the elementary building blocks for global calcium signals (Lipp & Niggli, 1996; Parker, Choi & Yao, 1996a; Berridge, 1997). The first visualization of such elementary calcium signals was obtained in Xenopus oocytes, in which stimulation by low concentrations of InsP3 evoked transient, localized calcium elevations (calcium ‘puffs’), whereas higher concentrations evoked propagating waves (Parker & Yao, 1991; Yao, Choi & Parker, 1995). Similar findings have subsequently been obtained in a variety of other excitable and non-excitable cell types, including HeLa cells (Bootman, Niggli, Berridge & Lipp, 1997B), rat RBL tumour cells (Horne & Meyer, 1997), glia (Yagodin, Holtzclaw, Sheppard & Russell, 1994) and PC12 cells (Reber & Schindelholz, 1996), suggesting that a hierarchical arrangement of elementary units may be a ubiquitous feature of InsP3-mediated calcium signalling. The appearance of calcium signals as either localized, transient elevations or actively propagating waves is thought to arise through two key properties of InsP3-evoked calcium liberation. Firstly, opening of the integral release channel associated with the InsP3 receptor (InsP3R) is promoted by moderate elevations of cytosolic calcium as well as by InsP3 (Iino, 1990; Finch, Turner & Goldin, 1991; Bezprozvanny, Watras & Ehrlich, 1991). This positive feedback imparts a regenerative characteristic to calcium liberation giving rise to a mechanism of calcium-induced calcium release (CICR). Secondly, the InsP3R appear to be distributed in clusters, forming discrete functional release sites spaced a few micrometres apart (Yagodin et al. 1994; Yao et al. 1995; Bootman et al. 1997b; Horne & Meyer, 1997). Depending upon the concentration of InsP3, which is a major factor in determining the sensitivity of the system, CICR may either be restricted to individual sites or involve successive triggering of multiple sites mediated by diffusion of calcium ions in the cytosol. Thus, calcium puffs probably arise through calcium feedback recruiting several InsP3R within a tight cluster (Yao et al. 1995; Bootman et al. 1997b;Sun, Callamaras, Marchant & Parker, 1998), but the puff will remain localized if neighbouring receptor clusters are insufficiently sensitive to respond to calcium diffusing across the much larger distances between release sites. At higher concentrations of InsP3, however, such triggering is enabled, turning the cytoplasm into an excitable medium capable of supporting propagating waves (Lechleiter & Clapham, 1992). In the preceding paper (Sun et al. 1998), we used a high-resolution linescan confocal microscope to examine mechanisms underlying the generation of individual elementary calcium events (puffs and blips) evoked in Xenopus oocytes by photorelease of constant, low concentrations of InsP3. Here we extend these studies on a more global scale within the cell by exploring the recruitment of puffs by increasing concentrations of InsP3 and their co-ordination to generate calcium waves.