A hierarchical concept of cellular and subcellular Ca2+-signalling

8.1. A General Concept of Cellular and Subcellular Ca2+ -Signalling. The rapid evolution of techniques to access the subcellular distribution of the second messenger Ca2+ has unveiled a completely new insight into subcellular Ca2+ -signalling. These techniques, especially the different types of confocal microscopy, have enabled us to visualize processes during signal transduction cascades with spatial and temporal resolution at a level that we have not been able to realize before. Changes in the distribution or the concentration of Ca2+ ions have been investigated on the 'μs'-time and the 'sub-μm'-spatial domain. The intention of this review was to give the readers a brief overview of different variants of these techniques in modern cellular physiology. Then we presented a brief summary of the status of the current research progress made on the different cell types and we introduced the cardiac myocyte as a prototype for cellular and subcellular Ca2+- signalling. Despite the great heterogeneity of the cell types and there specific variants of cellular and subcellular Ca2+-signalling, a hierarchical Ca2+-signalling system appears to be evolved during evolution of Ca2+ signal transduction systems in cells that utilize Ca2+ as the second messenger. In the following paragraphs we would like to extend the notion of such a putative hierarchical system. For reasons of simplicity we will only concentrate on Ca2+-signalling events that strictly rely on the occurrence of Ca2+-release from intracellular stores, such as SR or ER, thereby mediated by InsP3- and/or ryanodine receptors. 8.2. The Fundamental Level of Ca2+-Signalling: The Ca2+-Quark. The fundamental level of Ca2+ signals is believed to be represented by Ca2+ transients originating from the direct gating of Ca2+-release channels located in the membrane of the SR or ER. Here, the two basic types of Ca2+-release channels are represented either by the InsP3- ('Ca2+ -blips') or the ryanodine receptor ('Ca2+ -quark'). Direct visualization of these fundamental events in subcellular Ca2+-signalling is still missing. Although indirect evidence for such events was derived from the homogeneity of SR-Ca2+-release transients in cardiac myocytes (Lipp and Niggli, 1996), stronger evidence can be presented for the existence of such basic elements, the Ca2+-blip (Parker and Yao, 1996). Since Parker and Yao used a confocal point detection system neither spatial characteristics nor the relation of Ca2+-blips to the Ca2+-puff (i.e. Ca2+-spark) or Ca2+-waves could be demonstrated. In principal the direct visualization of these Ca2+-quarks should be easier in InsP3-mediated Ca2+-release than in release triggered by Ca2+ itself since for the InsP3 trigger the contrast between background [Ca2+] and the change in [Ca2+] due to the opening of the channel triggered by InsP3 can be expected to be much higher. The only attempts to directly visualize Ca2+-quarks has been carried out in skeletal muscle fibres by Tsugorka and coworkers (Tsugorka et al., 1995). They described Ca2+ transients with an amplitude of ≃30 nM but were able to estimate lateral spreading of the signals. For these events an underlying ionic current of ≃0.1 pA was calculated, indicating that such small events may arise from the opening of single ryanodine receptors (usual ionic current associated with the opening of a ryanodine receptor was ≃1-3 pA; see above). Nevertheless, the definite identity of these Ca2+ transients remains unclear. 8.3. The Intermediate Level of Ca2+-Signalling: The Ca2+-Spark. While Ca2+-quarks represent the fundamental, indivisible entity of cellular Ca2+-signalling, groups or clusters of these channels (either InsP3R, i.e. 'Ca2+-puff'; or ryanodine receptor, i.e. 'Ca2+-spark') are noted to generate microscopically visible Ca2+ transients. In contrast to Ca2+-quarks that are still not widely accepted, the existence of Ca2+-sparks or analogue signals and their functional role in cellular Ca2+-signalling is quite established. These intermediate, elementary events have been characterized extensively in cardiac muscle cells by a rapid growing number of research groups. In other muscle cells (i.e. smooth and skeletal muscle) Ca2+-sparks have been less extensively characterized. Ca2+-puffs most likely correspond to Ca2+- sparks since their functional role for cellular Ca2+-signalling in the electrically non-excitable cells is almost identical to that in the muscle cells. Although there might be an obvious relation between Ca2+-quarks and Ca2+-sparks (i.e. individual channel and cluster of channels), direct experimental evidence for the Ca2+-quark being the building block for Ca2+-quarks is still missing.