1058 Conclusion The application of Ca2+-imaging technology has radically altered the way we have to think about signalling at the cellular level. The complex and highly dynamic responses evoked in smooth muscle by agonists, for example, were not obvious in microﬂuorimetry recordings of average [Ca2+], and suggest that frequency modulation of phasic Ca2+ waves and oscillations plays an important role in determining the contractile response. Subcellular detail has also been revealed, with brief, spatially localized Ca2+- release events. These Ca2+ sparks can contribute directly to the elevation of cell [Ca2+] and contraction, as they do in cardiac excitation–contraction coupling and in some smooth muscles, or indirectly through activation of Ca2+- sensitive membrane conductances, altering membrane potential and thus regulating Ca2+ inﬂux via changes in activation of voltage-operated channels. As is usual in science, the ability to look at Ca2+ signals in more detail has generated as many new questions as answers, and the challenge of integrating the relevant detail into functional models at the cell and tissue level will be considerable. Future developments will probably include increased use of genetically coded Ca2+ indicators, in which proteins with Ca2+-sensitive ﬂuorescent or luminescent properties are expressed in speciﬁc organelles or tissues (Alvarez & Montero, 2002; Kotlikoff, 2007). This reﬂects the same basic strategy that underpinned the initial use of aequorin, but with molecules genetically engineered to increase Figure 10. Contraction of bronchiolar smooth muscle is a function of Ca2+ oscillation frequency The results were obtained by using Ca2+ imaging in pulmonary slices and applying different concentrations of 5-hydroxytryptamine (5-HT) or acetylcholine (ACH), or by using K+ to depolarize the smooth muscle (KCl). Reproduced with permission from Perez & Sanderson (2005a). Qualitatively similar results were obtained for pulmonary arterioles (Perez & Sanderson, 2005b). J. G. McGeown Exp Physiol 95.11 pp 1049–1060 the range of possible applications and target indicator expression to speciﬁc cell types or organelles (Eglen & Reisine, 2008). References Alvarez J & Montero M (2002). Measuring [Ca2+] in the endoplasmic reticulum with aequorin. Cell Calcium 32, 251–260. Ashley CC & Ridgway EB (1968). Simultaneous recording of membrane potential, calcium transient and tension in single muscle ﬁbers. Nature 219, 1168–1169. Ashley CC & Ridgway EB (1970). On the relationships between membrane potential, calcium transient and tension in single barnacle muscle ﬁbres. J Physiol 209, 105–130. Bao R, Lifshitz LM, Tuft RA, Bellve K, Fogarty KE & ZhuGe R (2008). A close association of RyRs with highly dense clusters of Ca2+-activated Cl– channels underlies the activation of STICs by Ca2+ sparks in mouse airway smooth muscle. J Gen Physiol 132, 145–160. Benham CD & Bolton TB (1986). Spontaneous transient outward currents in single visceral and vascular smooth muscle cells of the rabbit. J Physiol 381, 385–406. Benham CD, Bolton TB, Lang RJ & Takewaki T (1986). Calcium-activated potassium channels in single smooth muscle cells of rabbit jejunum and guinea-pig mesenteric artery. J Physiol 371, 45–67. Berridge MJ, Lipp P & Bootman MD (2000). The versatility and universality of calcium signalling. Nat Rev Mol Cell Biol 1, 11–21. Bolton TB, Gordienko DV, Povstyan OV, Harhun MI & Pucovsky V (2004). Smooth muscle cells and interstitial cells of blood vessels. Cell Calcium 35, 643–657. Brakenhoff GJ, van Spronsen EA, Van Der Voort HT & Nanninga N (1989). Three-dimensional confocal ﬂuorescence microscopy. Methods Cell Biol 30, 379–398. Burdyga T & Wray S (2005). Action potential refractory period in ureter smooth muscle is set by Ca sparks and BK channels. Nature 436, 559–562. Chalﬁe M (2009). GFP: lighting up life. Proc Natl Acad Sci U S A 106, 10073–10080. Chalﬁe M, Tu Y, Euskirchen G, Ward WW & Prasher DC (1994). Green ﬂuorescent protein as a marker for gene expression. Science 263, 802–805. Cheng H & Lederer WJ (2008). Calcium sparks. Physiol Rev 88, 1491–1545. Cheng H, Lederer WJ & Cannell MB (1993). Calcium sparks: elementary events underlying excitation-contraction coupling in heart muscle. Science 262, 740–744. Curtis TM, Scholﬁeld C & McGeown DJ (2007). Calcium signaling in ocular arterioles. Crit Rev Eukaryot Gene Expr 17, 1–12. Curtis TM, Tumelty J, Dawicki J, Scholﬁeld CN & McGeown JG (2004). Identiﬁcation and spatiotemporal characterization of spontaneous Ca2+ sparks and global Ca2+ oscillations in retinal arteriolar smooth muscle cells. Invest Ophthalmol Vis Sci 45, 4409–4414. Eglen RM & Reisine T (2008). Photoproteins: important new tools in drug discovery. Assay Drug Dev Technol 6, 659–671. C 2010 The Author. Journal compilation C 2010 The Physiological Society