1056 J. G. McGeown Exp Physiol 95.11 pp 1049–1060 again suggesting an excitatory rather than an inhibitory role (Tumelty et al. 2007). Pharmacological interventions that reduce spark frequency also reduce spontaneous oscillations, and constrictor agonists increase both sparks and oscillations (Tumelty et al. 2007; Jeffries et al. 2010). Similar observations have been made by other groups, with intracellular sites that demonstrate high levels of spontaneous spark activity (termed ‘frequent discharge sites’) acting as points of initiation for cell- wide spontaneous Ca2+ waves and evoked Ca2+ transients. This suggests that, as in cardiac muscle, sparks in smooth muscle can also act as ‘elementary building blocks’, from which excitatory global Ca2+ signals are constructed (Bolton et al. 2004). It should also be noted that, although sparks can exert negative feedback by activating outward I KCa, the presence of Ca2+-activated Cl– channels in the plasma membrane also opens up the possibility of positive feedback following a rise in [Ca2+]i (Fig. 7; Large & Wang, 1996; Jackson, 2000; McGahon et al. 2009). In smooth muscle, Cl− currents are Figure 8. Calcium sparks and oscillations recorded from retinal arteriole myocytes in situ Arterioles were loaded with the Ca2+ indicator ﬂuo-4, and scanned transversely across their diameter along a single scanline 500 s−1 using a confocal laser microscope (see Fig. 9B). Normalized ﬂuorescence (F/F0) is a measure of local [Ca2+]. A, two spontaneous Ca2+ sparks, with a rapid rising phase and slower decay. The entire event typically lasts <150 ms. B, a more prolonged Ca2+ oscillation lasting several seconds. These are sometimes associated with myocyte contraction. Spark activity is clearly seen near the upper cell margin, and sparks appear to summate during the rising phase of the oscillation. For more detail see Curtis et al. (2004) and Tumelty et al. (2007). Image courtesy of Dr Tim Curtis. inwardly directed at normal resting potentials (outward movement of negatively charged Cl− ions equates to inward conventional current). The resulting spontaneous transient inward currents (STICs) tend to depolarize the plasma membrane, increasing the steady-state activation of voltage-operated Ca2+ channels and so raising cell [Ca2+]i. Calcium sparks have been shown to generate STICs in a number of different smooth muscle types (Bao et al. 2008). Indeed, depending on the conditions, a single spark can activate a STOC followed by a STIC. The resulting biphasic current has been rather philosophically dubbed a STOIC (ZhuGe et al. 1998). The functional consequences of such complex signals are only just beginning to be explored (ZhuGe et al. 2010). What you see depends on how you look at things: agonist stimulation and Ca2+ oscillations Another aspect of Ca2+ signalling in smooth muscle of considerable interest relates to its role in control of contraction by neurotransmitters and endocrine or paracrine messenger molecules. We have been interested in how endothelin-1 (Et-1), a highly potent endothelium- derived vasoconstrictor, regulates Ca2+ signals in retinal arterioles. Recording the average smooth muscle [Ca2+]i using microﬂuorimetry results in a relatively simple response, with an initial Ca2+ transient lasting approximately 100 s, which then falls back towards the control level but remains elevated above baseline (Fig. 9A). This triggers a sustained vessel constriction (Curtis et al. 2007). The intuitive model of stimulus– contraction coupling suggested by this result is one in which muscle contraction tracks the increase in [Ca2+]i, during the rising phase at least, consistent with the data from skinned muscle showing that tension is a function of the steady-state [Ca2+]i (Fig. 3). Calcium imaging reveals a very different picture, however, in which endothelin-1 stimulates phasic Ca2+ oscillations with a much shorter period (<5 s) than the transient seen at the whole vessel level (Fig. 9B; Curtis et al. 2007). Presumably, the asynchronous nature of these oscillations accounts for the much slower and relatively tonic increase in [Ca2+]i recorded using microﬂuorimetry. Importantly, no individual cell experiences the temporal proﬁle of [Ca2+]i changes recorded in the spatially averaged recording. Any realistic model of cell signalling in response to Et-1 must, therefore, be able to account for the more complex pattern revealed by high-speed imaging. Similar oscillatory responses to constricting stimuli, ﬁrst reported in vascular tissue in 1994 (Iino et al. 1994), have subsequently been described in a variety of other smooth muscles. Studies on pulmonary arteriolar and bronchiolar smooth muscle, for example, have demonstrated that increasing concentrations of C 2010 The Author. Journal compilation C 2010 The Physiological Society