Unravelling the role of the ryanodine receptor type 3 in smooth muscle.

Abstract

Calcium signalling in smooth muscle is complex, intimately involved in the regulation of a diverse range of cellular processes including cell differentiation, proliferation, gene expression, contraction and apoptosis. Intracellular calcium signals are initiated via calcium entry from the extracellular space and calcium mobilisation from intracellular sarcoplasmic reticulum calcium stores by inositol 1,4,5-trisphosphate and opening of ryanodine receptors (RYRs). The spatio-temporal pattern of these signals is thought to facilitate the diversity of cellular responses to calcium. Heterogeneity amongst ion channels, calcium pump distribution and the spatial organisation of other elements such as contractile filaments, mitochondria and sarcoplasmic reticulum are thought to play an important part in determining local and global transmission of the calcium signal, as well as the frequency of the signals, within the cell (Pabelick et al. 2001). It is important therefore to understand the expression patterns/profiles of calcium regulatory proteins and relate them to cell function. RYRs, the focus of the paper by Mironneau et al. in this issue of The Journal of Physiology, are proposed to play an essential role in the generation of elementary calcium signals and the larger events of calcium ‘sparks’ and ‘puffs’. In many cells, calcium sparks and puffs contribute to the generation of calcium waves and oscillations and also provide a mode of signalling within highly defined cellular domains to regulate membrane potential and cell excitability via the activation of calcium-sensitive potassium and/or chloride channels. The endogenous activators of RYR receptor activity are calcium itself (via calcium-induced calcium release, CICR) and cyclic ADPribose (Berridge et al. 2000). Over recent years, three genomically distinct RYR isoforms have been identified (RYR1–3). RYR1 is predominantly found in skeletal muscle, RYR2 in cardiac muscle and RYR3 is ubiquitously expressed in many cells. RYR3 has two splice variants which may differ in sensitivity to calcium. The question then arises as to why three RYR isoforms exist and what their functional role is in co-ordinating calcium signalling in smooth muscle. Of the three RYRs, the physiological role of RYR3 remains elusive. The relation between RYRs and the generation of calcium oscillations has been of particular interest for those studying myometrial smooth muscle, a spontaneously active tissue. However, reports vary as to the functional role of RYR and the mRNA expression pattern of RYR receptor isoforms in myometrium in the pregnant and non-pregnant state. The thorough study by Mironneau et al. (2002) clarifies RYR mRNA and protein expression in non-pregnant mouse myometrium and provides initial experimental evidence regarding the function of RYR3 in mouse myometrial cells outside pregnancy. Two particularly interesting aspects of the paper are that (i) RYR3 does not appear to be involved in the generation of calcium sparks (supported by the homogeneous expression of RYR3 protein throughout mouse myometrial cells) and (ii) RYR3 is only active when SR luminal calcium concentrations are raised. Previously, RYR3 knockout mice studies had reported that RYR3 acts in synergy with RYR1 in neonatal skeletal muscle to elicit calcium sparks (Bertocchini et al. 1997). The decreased sensitivity of RYR3 to luminal calcium provides an elegant system for the regulation of RYR3 and cell responses by the state of calcium loading of the SR. Indeed, it is proposed that RYR3 in vascular smooth muscle is responsible for maintaining calcium release after RYR1 and RYR2 are inactivated. This may be pertinent to recent observations that other pathways contributing to calcium signalling (e.g. SERCA isoforms and store-operated calcium entry) associated with the SR calcium store are increased in human myometrium during late pregnancy/labour (Tribe, 2001). Indeed, it would be interesting to extend the present study to pregnant mouse myometrium. Perhaps, even more importantly, Mironneau et al. (2002) describe a smooth muscle cell model in which only the RYR3 receptor is expressed and hence can be studied in isolation from the other RYR isoforms. This has advantages over the classical pharmacological approaches to determine function of a particular protein, as the inhibition of one protein may cause the upregulation of a compensatory protein and mask the role of the protein of interest. Similarly, data can be open to misinterpretation when one or more subtypes of a protein are present and its function cannot adequately be isolated from the others. More recently, antisense- and antibody-based techniques, overexpression studies and gene-knockout mice have been applied to the study of RYR3 function, but again there are associated drawbacks to these approaches. This new myometrial cell model described by Mironneau et al. (2002) therefore has the potential, in combination with the other techniques, to enhance our understanding of the physiological function and regulation of this receptor in the wider context of calcium signalling per se.