Calcium Mobilization via Intracellular Ion Channels, Store Organization and Mitochondria in Smooth Muscle
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In smooth muscle, Ca2+ release from the internal store into the cytoplasm occurs via inositol trisphosphate (IP3R) and ryanodine receptors (RyR). The internal Ca2+ stores containing IP3R and RyR may be arranged as multiple separate compartments with various IP3R and RyR arrangements, or there may be a single structure containing both receptors. The existence of multiple stores is proposed to explain several physiological responses which include the progression of Ca2+ waves, graded Ca2+ release from the store and various local responses and sensitivities. We suggest that, rather than multiple stores, a single luminally-continuous store exists in which Ca2+ is in free diffusional equilibrium throughout. Regulation of Ca2+ release via IP3R and RyR by the local Ca2+ concentration within the stores explains the apparent existence of multiple stores and physiological processes such as graded Ca2+ release and Ca2+ waves. Close positioning of IP3R on the store with mitochondria or with receptors on the plasma membrane creates ‘IP3 junctions’ to generate local responses on the luminally-continuous store.
KeywordsSmooth muscle Calcium signalling Calcium stores IP3 receptors Ryanodine receptors Quantal calcium release Mitochondria
Physiological Functions Proposed to Be Explained by the Structure of the Store
A discontinuous structure of the store has also been proposed to explain the graded IP3 concentration-dependent Ca2+ release process [17, 18]. Low concentrations of IP3 release only part of the overall available Ca2+ content of the store [17, 19, 20, 21, 22]. As the IP3 concentration increases, a further release of Ca2+ occurs [reviewed 23]. Such a graded release seems incompatible with the positive feedback CICR-like facility at IP3R , which would be anticipated to fully deplete the store when activated. To explain graded Ca2+ release, the store has been proposed to assemble in multiple separate units, each endowed with a finite Ca2+ storage capacity and sensitivity to IP3 (Fig. 11.4b). At any given concentration of IP3 only some stores will be activated to release Ca2+ [17, 18, 25] (Fig. 11.4b). This same feature of the store may also explain the reported variations in sensitivity different parts of the cell to IP3 [19, 26, 27].
Structure of the Ca2+ Stores
Thus, data from various functional studies suggest there may be structural discontinuities in the store and that different types of receptor arrangements on those stores exist. Proposals for stores which contain only IP3R or RyR exist as do proposals for stores with RyR and IP3R together and in combination with additional separate stores in the same cells containing only either IP3R or RyR. The questions arise, why is such a diversity of stores and receptor arrangement required and do functional experiments unambiguously reveal structural discontinuities in the store?
Methods Used to Investigate Stores May Create the Appearance of Multiple Stores
It could be the case that the experimental conditions used to investigate the stores may contribute to the diversity of proposals on arrangement. In native cells, methods for studying Ca2+ store subcompartments are limited. The main experimental approach is to define the structural organisation of the Ca2+ stores from functional (Ca2+ response) data. To do this, the store is depleted typically via one receptor (RyR or IP3R) by repeated activation with a single concentration of either IP3 or caffeine under conditions which prevent store refilling with Ca2+. After depletion via one receptor (e.g. RyR), whether or not Ca2+ is available to be released via the other receptor (e.g. IP3R) is then determined. If depletion via one receptor abolishes Ca2+ release from the other, the receptors are suggested to be co-localized on a single store and access a common Ca2+ source. However, if depletion of the stores from one receptor leaves the other receptor’s response largely unaffected, the two channels are suggested to be localized on different stores. With this approach, some investigations (e.g. on portal vein and pulmonary artery) have shown a single store containing both RyR and IP3R, since depletion of the Ca2+ store by caffeine (which activates RyR) prevented IP3-mediated Ca2+ release [31, 32, 39, 40]. On the other hand, other studies on pulmonary artery have suggested there may be separate stores for each receptor since depletion of the RyR-containing store did not abolish agonist-evoked IP3-mediated Ca2+ release and vice versa . In yet other studies (e.g. portal vein, pulmonary artery and taenia caeci), one store may express RyR and IP3R and other stores, in the same cell, only IP3R [11, 35, 42]. This conclusion came from the finding that depletion of the IP3R-containing store abolished Ca2+ release via RyR, while depletion of the RyR-containing store did not abolish Ca2+ release via IP3R. In further studies in other cell types (mesenteric artery) and in our own investigations in colonic smooth muscle , some stores may express both RyR and IP3R while others only RyR [30, 43]. In this case, depletion of the RyR-containing store abolished Ca2+ release via IP3R, while depletion of the IP3R-containing store did not abolish Ca2+ release via RyR—a result apparently consistent with there being a store which contained RyR alone.
Depletion of the RyR-sensitive store at one site also depleted the entire store [44, 46]. In this case the RyR-containing store was depleted by attaching a pipette containing ryanodine to one small site of the cell to deplete the store there. Caffeine was applied to the entire cell. If the RyR containing store comprised separate elements, depletion of one aspect of the store should not affect the Ca2+ available to be released in another area of the store. However, caffeine-evoked Ca2+ transients decreased uniformly throughout the cell [44, 46] suggesting that ryanodine, acting at one part of the cell, had depleted the entire store i.e. a single luminally-continuous store exists.
The question of whether there is a single store with luminal continuity or multiple stores has also been addressed in other cell types (HeLa, RBL, CHO) using a Ca2+ store-located green fluorescent protein (GFP) [47, 48]. Prolonged GFP photobleaching in a small restricted region of the cell resulted in the disappearance of fluorescence throughout store, suggesting GFP could move freely around the store to be eventually photobleached. Short periods of photobleaching were followed by a rapid restoration of fluorescence by the diffusion of GFP from sites neighbouring the photobleached region [47, 49]. A single store with luminal continuity throughout was also suggested by the diffusion of Ca2+ in pancreatic acinar cells . The Ca2+ store in the apical region was refilled with Ca2+ originating from a pipette attached to the opposite side of the cell on the basolateral membrane [see also 9]. Together, these experiments suggest the store is a luminally-continuous entity in which Ca2+ can diffuse freely throughout. How then does the appearance of multiple stores  occur on a single luminally-continuous store structure?
Complex RyR and IP3R Regulation Characteristics and Apparent Store Configuration
Interpreting the amplitude of a Ca2+ response to a single repeatedly applied concentration of either IP3 or caffeine as the store content declines is problematic as the amplitude of the response depends (1) on the position of the activator concentration on the concentration-response relationship curve and (2) the store luminal Ca2+ concentration. The absence of a response to a single concentration of IP3 or caffeine, therefore, may not reflect an absence of available Ca2+ within the store but rather termination of channel activity by luminal regulation of the store release channels as the store Ca2+ content declines.
Rather than there being various separate stores with different receptor arrangements, these results suggests that partial depletion of the store terminates activity of the channels by luminal channel regulation by [Ca2+] within the store.
These results (Figs. 11.6, 11.8, 11.9) do not dispute the existence of multiple stores but suggest that care is required when interpreting results from functional data in terms of store structure. In some cells, multiple stores do exist unequivocally. Different Ca2+concentrations have been measured in various regions of the store using recombinant aequorin , electron microscopic determination of Ca2+ content  or fluorescent indicators loaded into the cell , suggesting that discontinuities exist within the structures surrounding the lumen itself. The store  may adopt different configurations within the cell and components may even detach and reattach, so influencing the pattern and distribution of Ca2+ release channel . In Purkinje neurons, for example, IP3R-expressing regions may separate off from other internal store elements . Store compartments exist which accumulate and release Ca2+ but are luminally-discontinuous from the bulk of the store have been observed in cultured hippocampal dendrites . Life cycle stage or prior experimental conditions of the cell may influence the appearance of subcompartments. [Ca2+]c increases which persisted for at least 10 min, led to the breakdown of the Ca2+ store into subcompartments in rat basophilic leukaemia cells . Store structural changes are also associated with fertilization and mitosis . Fertilization leads to a reorganization of the store, measured as a slowing of the diffusion of membrane probes and luminal proteins, in sea urchin eggs [55, 56]. In mitosis, significant Ca2+ store changes also occur, which include the structure itself fragmenting into subcompartments [57, 58].
Other structures within the cell such as Golgi, mitochondria, granules and the nucleus may also contribute to Ca2+ storage [59, 60, 61, 62, 63] and generate subregions which appear to have various Ca2+ concentrations, especially when lipophilic Ca2+ indicators are used to image the distribution of [Ca2+] through the cell.
Graded Ca2+ Release, Ca2+ Waves and Local Ca2+ Events from a Luminally-Continuous Store
If the Ca2+ store in smooth muscle is indeed a single, luminally-continuous entity, how do the various physiological events (waves, graded release, local responses) previously explained with multiple separate stores occur?
Ca 2+ waves: Ca2+ waves are the progressive movement of Ca2+ through the cell following Ca2+ release from the internal store. Using localized activation of IP3R, the forward movement of the Ca2+ wave was shown to arise from CICR at the IP3R [13, 16]. The decline in [Ca2+]c—the back of the wave—occurred not because of depletion of separate stores but from a functional compartmentalization of the store which rendered the site of IP3-mediated Ca2+ release—and only this site—refractory to IP3 after Ca2+ release . A localized feedback deactivation of IP3R produced by an increased [Ca2+]c caused the functional compartmentalization . The deactivation of the IP3R was delayed in onset, compared with the time of the rise in [Ca2+]c and persisted (>30 s) even when [Ca2+]c had been restored to resting levels [13, 16]. This feedback deactivation ensures the wave’s progressive movement in a single direction .
Graded Ca 2+ release: There are several proposals for graded IP3-mediated Ca2+ release that do not require the presence of numerous stores with various sensitivities to IP3. Rather, at any given [IP3] the entire Ca2+ store is activated and releases a fraction of its content, becoming partially depleted. Partial depletion may deactivate Ca2+ release [64, 65]. Raising the [IP3] reactivates IP3R to renew the Ca2+ release process. This proposal does not require multiple stores but a complex adaptive change in IP3R activity. Negative feedback processes operating either at the cytoplasmic or the luminal aspects of IP3R may explain the adaptive behaviour. In one proposal the binding of IP3 to IP3R may initially activate, then partially inactivate IP3R in a concentration-dependent way to produce graded Ca2+ release [66, 67, 68]. To test this proposal we examined the time course of IP3R activation at a constant [IP3] but under conditions in which there was varying amplitude of Ca2+ release . The latter was achieved by buffering the cytoplasmic Ca2+ concentration (BAPTA) or partial depletion of the store (Ca2+ free bath solution). If IP3 inactivated IP3R to prevent release, then at constant [IP3], release should stop at approximately the same time regardless of the amplitude of the [Ca2+]c rise. However, as the amplitude of the [Ca2+]c rise declined (in either BAPTA or in Ca2+-free solution) the time course of release became more prolonged . This result suggests that mechanisms other than IP3 inactivation of IP3R would appear responsible for terminating IP3-mediated Ca2+ release.
In another proposal, the sensitivity of IP3R to IP3 is controlled by the luminal [Ca2+] so that as the concentration of the ion within the store lumen falls so does IP3R activity [e.g. 65, 69]. For example, decreasing the store [Ca2+] to below 80 % of the steady-state level abolished IP3-mediated Ca2+release in rat uterine myoctes  [see also 65, 69]. However, it is unclear whether or not the control of IP3R activity by luminal Ca2+ operates over the store’s physiological Ca2+ concentration range. The threshold for luminal regulation to begin altering the activity of IP3R is depletion of the store by >70 % of the steady-state luminal Ca2+ concentration (500–600 μM; ) in HeLa cells. The store [Ca2+] must also be substantially depleted in hepatocytes (>45 or 95 %) [72, 73] and in A7r5 cells by >70 %  before IP3R sensitivity changes are detected. In each case, control of IP3R activity by Ca2+ binding to the luminal aspect of the receptor, is unlikely to explain ‘quantal’ Ca2+ release when store [Ca2+] exceeds 55, 5, or 30 % of the normal steady-state value respectively in these cells [72, 73, 74].
On the other hand, IP3R might not be controlled by luminal Ca2+ at all. Single channel IP3R activity, measured in planar lipid bilayers, increased when the [Ca2+] at the luminal aspect of the channel declined . In the latter study a luminal [Ca2+] exceeding 1 mM inhibited IP3R activity  (see also ). In other studies in permeabilized cells (e.g. portal vein;  or hepatocytes; ), decreases in store [Ca2+] failed to reduce the sensitivity of IP3-mediated Ca2+ release or alter Ca2+ leak when pumps were blocked in permeabilized avian supraorbital nasal gland cells . Together, these results suggest that regulation of IP3R by Ca2+ at the luminal aspect of the channel may, at best, operate over a limited range of store [Ca2+].
Our results (Fig. 11.8) [44, 45, 46] suggest that as the store content falls IP3R become less responsive to IP3. However, rather than luminal regulation being expressed from within the store at the luminal aspect of IP3R, detection of [Ca2+] within the store may lie at the cytoplasmic aspect of IP 3 R . The Ca2+ current flowing through IP3R evokes further release by a positive feedback effect of the ion at the cytoplasmic aspect of the channel, i.e. a Ca2+-dependent positive feedback loop. Reduction of the store Ca2+ content reduces the Ca2+ current flowing through IP3R and will result in a falling positive feedback at the cytoplasmic aspect of IP3R until release eventually stops. Ca2+ release is renewed by an increased [IP3]. In this case, the co-incidental activation of several neighboring IP3Rs within a cluster offsets the declining IP3R Ca2+ current to renew positive feedback and Ca2+ release and accounts for graded IP3-mediated Ca2+ release.
Alternatively, the rise in cytoplasmic [Ca2+]c, which derives from the activity of IP3R, may itself inactivate the receptor [79, 80, 81]. However, if Ca2+-dependent inactivation terminated release [16, 79] to explain the graded IP3-mediated Ca2+ release, the Ca2+ chelator BAPTA, would have been expected to have potentiated IP3-evoked [Ca2+]c increase; BAPTA decreased IP3-mediated Ca2+ release .
Localized Ca 2+ responses IP3 is a rapidly diffusing messenger and IP3R are subject to positive feedback CICR on a single luminally-continuous entity, so how do highly-localized Ca2+ changes occur? In heart cells, the store is also a continuous network  in which Ca2+ can rapidly redistribute [83, 84] and positive feedback CICR occurs at RyR, yet highly localized Ca2+ release events occur. The highly localized responses arise in specialized domains formed by a junction of the store with the plasmalemma (‘peripheral couplings’) or the store and transverse (T)-tubules (‘Dyads’). A number of proteins accrue at these specialized store domains: the L-type channel dihydropyridine receptors of the plasmalemma and T-tubules; the RyRs of store; triadin and junctin, of the store membrane; and calsequestrin (CSQ), the internal calcium binding protein . The close coupling of dihydropyridine receptors and RyR provides control of Ca2+ release by Ca2+ influx. The quaternary complexes between triadin, junctin, RyR, and CSQ provides the luminal Ca2+ sensing capabilities that regulates RyR activity.
IP3-mediated Ca2+ signaling may also generate highly localized responses even though IP3 is a messenger that can diffuse quickly to evoke activity throughout the cell. To do this, certain receptors co-localize with IP3R to form a local signalling complex [86, 87, 88, 89]. In cultured sympathetic neurons, although muscarinic and bradykinin receptors each stimulate phospholipase C, only bradykinin receptors co-immunoprecipitate with, and activate, IP3R to evoke Ca2+ release . The arrangement enables PLC activation by muscarinic and bradykinin receptors to evoke different cellular responses. In SH-SY5Y cells the positioning of IP3R near the plasma membrane provides a mechanism which may enable agonist activation, acting via IP3, to target specific types of cellular response i.e. by generating Ca2+ rises in specific regions of the cell . The clustering of agonist-activated surface receptors in certain regions on the plasma membrane (e.g. the Escherichia coli chemotaxis receptor) may contribute further, by providing areas with increased sensitivity to extracellular stimuli .
Smooth muscle also assembles IP3 Ca2+ release components into specialized Ca2+ domains  (Fig. 11.1). This conclusion came initially from the observation that Ca2+ waves, triggered by agonists applied to the entire cell, began consistently at the same site on successive activations in smooth muscle i.e. there appeared to be regions with preferential IP3-mediated Ca2+ release. Using centre of mass co-localization analysis of the distribution of the surface membrane receptors (for ACh) and IP3R, a small percentage (~10 %) of sites showed co-localization. Significantly, the extent of co-localization was greatest at the Ca2+ wave initiation site. At these sites of co-localization, wave initiation may arise from a preferential delivery of IP3 from mAChR3 activity to particular IP3R clusters to generate faster local [Ca2+]c increases. When the Ca2+ rise at the initiation site was rapidly and selectively attenuated (using photolysis of the caged Ca2+ buffer diazo-2) the Ca2+ wave shifted and initiated at a new site. Conversely, when a localized subthreshold ‘priming’ IP3 concentration was applied rapidly to regions distant from the initiation site, the wave initiation site shifted to the site of priming IP3 release. These results indicate that Ca2+ waves initiate where the most rapid Ca2+ change occurs at sites in which there is a structural and functional coupling of ACh receptors and IP3R (Fig. 11.1). The coupling generates junctions in which IP3 acts as a highly localized signal by being rapidly and selectively delivered to IP3R.
Role of Mitochondria in Modulating Ca2+Signals
Away from the plasma membrane, IP3R activity in smooth muscle is also tightly regulated by mitochondria. Mitochondria have a well-developed Ca2+ uptake facility and may modulate bulk cytoplasmic Ca2+ signals [93, 94, 95, 96] derived from Ca2+ entry and release . Mitochondria also provide tight local control of Ca2+ release via IP3R [93, 94, 98] but Ca2+ influx via voltage-dependent Ca2+ channels or release via RyR appears to be less tightly controlled at a local level by mitochondria [93, 94].
Mitochondrial control of IP3R arises at IP3-mediated release sites. IP3-sensitive Ca2+ release initiates at discrete sites on the store that contain a few tens of IP3R from which the local increase in [Ca2+] is called a ‘puff’. Ca2+ puffs are spatially restricted events and of short duration but may interact and coalesce to generate a global release in Ca2+. Mitochondria are positioned close to IP3R and regulate activity of the channels ; inhibition of mitochondrial Ca2+ uptake attenuated the magnitude of Ca2+ puffs . Indeed mitochondrial Ca2+ uptake was rapid enough to influence Ca2+ communication within an IP3R cluster. Mitochondrial Ca2+ uptake appears to prevent the negative feedback effect of high [Ca2+]c on IP3R activity within a cluster to prolong Ca2+ release from the store . As a consequence of the control at IP3R, mitochondrial Ca2+ uptake exerts a pronounced effect on IP3-mediated Ca2+ release throughout the cell [93, 94, 98, 101].
Mitochondria and IP3R appear to be close, and perhaps tethered, to allow mitochondrial Ca2+ uptake, ATP supply, ROS production and or redox/antioxidant control to influence IP3R activity. Conversely, mitochondrial division (required to maintain mitochondrial population health and allow cell proliferation) involves encircling of the dividing mitochondria by a store membrane tubule at the point of mitochondrial constriction . During smooth muscle proliferation IP3R expression and activity are increased [103, 104, 105] and there is a marked switch in mitochondrial phenotype from stationary to highly motile . Inhibiting either IP3R activity [104, 107] or mitochondrial motility and division [106, 108] inhibits smooth muscle proliferation. The interplay between mitochondria and IP3R in smooth muscle thus presents an interesting potential therapeutic avenue by which pathological smooth muscle proliferation in vascular disease may be targeted.
This work was funded by the Wellcome Trust (092292/Z/10/Z) and British Heart Foundation (PG/11/70/29086)
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