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Calcium Signaling in

David C. Hill-Eubanks1, Matthias E. Werner2, Thomas J. Heppner1, and Mark T. Nelson1,2

1Department of Pharmacology, College of Medicine, University of Vermont, Burlington, Vermont 05405 2Cardiovascular Medicine, Faculty of Medical and Human Sciences, University of Manchester, Manchester M13 9NT, United Kingdom Correspondence: [email protected]

Changes in intracellular Ca2þ are central to the function of smooth muscle, which lines the walls of all hollow organs. These changes take a variety of forms, from sustained, -wide increases to temporally varying, localized changes. The nature of the Ca2þ signal is a reflec- tion of the source of Ca2þ (extracellular or intracellular) and the molecular entity responsible for generating it. Depending on the specific channel involved and the detection technology employed, extracellular Ca2þ entry may be detected optically as graded elevations in intra- cellular Ca2þ, junctional Ca2þ transients, Ca2þ flashes, or Ca2þ sparklets, whereas release of Ca2þ from intracellular stores may manifest as Ca2þ sparks, Ca2þ puffs, or Ca2þ waves. These diverse Ca2þ signals collectively regulate a variety of functions. Some functions, such as con- tractility, are unique to smooth muscle; others are common to other excitable cells (e.g., modulation of membrane potential) and nonexcitable cells (e.g., regulation of gene expression).

SMOOTH MUSCLE changes may regulate the translational move- ment of the organ’s contents, such as in the mooth muscle cells form a continuous layer , where the peristaltic Sthat lines the walls of the hollow organs of action caused by sequential contraction of the body, such as blood vessels, intestines, uri- smooth muscle segments is responsible for the nary bladder, airways, lymphatics, penis, and movement of food, and the , uterus. A defining feature of smooth muscle where smooth muscle in the wall relaxes during cells is their ability to contract. This property filling and contracts forcefully to expel urine reflects the excitable nature of these cells, which during micturition. Uterine smooth muscle allows for membrane potential-dependent plays a similar role, relaxing during gestation influx of calcium (Ca2þ) and the Ca2þ-depend- to accommodate fetal growth and contracting ent formation of cross-bridges between vigorously during parturition. In the vascula- and —the two major contractile ture, the contractility of smooth muscle in the that drive contraction. The contractile property vessel wall is a primary determinant of blood of smooth muscle plays an important func- pressure, which in turn controls blood flow tional role in these organs, notably by allowing and the distribution of nutrients and oxygen dynamic changes in luminal volume. These throughout the body.

Editors: Martin Bootman, Michael J. Berridge, James W. Putney, and H. Llewelyn Roderick Additional Perspectives on Calcium Signaling available at www.cshperspectives.org Copyright # 2011 Cold Spring Harbor Laboratory Press; all rights reserved; doi: 10.1101/cshperspect.a004549 Cite this article as Cold Spring Harb Perspect Biol 2011;3:a004549

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D.C. Hill-Eubanks et al.

Ca2þ SIGNALS IN SMOOTH MUSCLE indicators change when Ca2þ is bound; thus, such dyes can be used to detect intracellular Smooth muscle contractility, and therefore Ca2þ levels. Ion fluxes in smooth muscle can hollow organ function, is regulated by changes occur very rapidly—often in the millisecond in intracellular Ca2þ concentration ([Ca2þ] ). i range. This property has motivated the develop- These changes may take a variety of forms, ment of a number of very rapid fluorescent dyes the simplest of which is a “global” (cell-wide) that enable detection of such changes in ion increase of the type associated with Ca2þ-trig- concentrations with high temporal resolution. gered actin-myosin cross-bridge formation Fluorescent dyes can be loaded into cells by and contraction. However, changes in intracel- microinjection, but are more commonly intro- lular Ca2þ may also be highly localized and duced by incubating isolated smooth muscle often include a temporal component. Thus, cells or intact tissue with the membrane- Ca2þ changes may be sustained or transient, permeant acetoxymethyl (AM) ester of the stationary or moving, or regularly repeating dye. The AM form is readily taken up by cells, (Berridge 1997; Sanders 2001). but is acted on by intracellular esterases that The nature of the Ca2þ signal depends to cleave the ester bond to release the free anion, a large extent on the molecular mechanism which is not membrane permeant and is thus responsible for generating it. Broadly speaking, retained within the cell. Most fluorescent Ca2þ there are two major mechanisms by which indicators are based on fluorophore-conjugated [Ca2þ] is raised in smooth muscle: (1) entry i derivatives of the Ca2þ chelator, BAPTA(bis-[o- of Ca2þ from the extracellular space, and (2) amino-phenoxy]-ethane-N,N,N0N0-tetraacetic release of Ca2þ from intracellular stores. acid), which is used for both ratiometric Influx of extracellular Ca2þ is mediated by ion and nonratiometric Ca2þ applications (Tsien channels in the plasmalemmal membrane, 1980). Ratiometric dyes are used to measure the most prominent of which is the voltage- the intracellular concentration of Ca2þ. These dependent Ca2þ channel (VDCC). Nonselective dyes show a shift in the excitation (e.g., cation channels, such as transient Fura-2) or emission (e.g., indole-1) spectrum potential (TRP) channels and ionotropic puri- according to the concentration of free or nergic (P2X) receptors, are also potentially unbound Ca2þ. From the ratio of bound and important extracellular Ca2þ entry pathways in unbound Ca2þ, the free intracellular Ca2þ con- smooth muscle cells. Although a number of centration can be determined. Nonratiometric intracellular take up and release dyes, such as those in the Fluo family, show an Ca2þ, the (SR) repre- increase in fluorescence quantum yield or sents the largest pool of releasable Ca2þ in intensity on binding Ca2þ and are useful for smooth muscle cells. In response to a variety of detecting qualitative changes in Ca2þ levels. stimuli, Ca2þ-release channels in the SR, namely Although nonratiometric dyes are not usually ryanodine receptors (RyR) and inositol tri- used for quantitative Ca2þ measurements, sphosphate receptors (IP Rs), mediate efflux of 3 methods have been developed to calculate Ca2þ from the SR into the of the cell. Ca2þ concentrations using single-wavelength fluorescence signals (Jaggar et al. 1998a; Mara- vall et al. 2000). One advantage of ratiometric IMAGING INTRACELLULAR Ca2þ dyes is that the ratio normalizes fluorescence Fluorescence, a property that allows certain variations caused by uneven cell thickness, dye molecules to absorb specific wavelengths of distribution, dye leakage, or photobleaching— light and release energy in the form of light problems that are common to nonratiometric at a longer wavelength, has been exploited to dyes. A disadvantage of ratiometric dyes is produce numerous fluorescent indicators, that they require excitation in the UV range. including Ca2þ-sensitive fluorescent dyes. Some parameters to consider when selecting The fluorescence properties of Ca2þ-sensitive a fluorescent dye include (1) ion specificity,

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Calcium Signaling in Smooth Muscle

2þ (2) dissociation constant (Kd), (3) hardware Ca -sensitive dyes allows laser-scanning con- suitability (excitation and emission spectra), focal systems to routinely achieve detailed spa- (4) fluorescence intensity, (5) availability as an tial and temporal resolution, which is crucial AM ester, and (6) sensitivity to photobleaching. for determining the origin of a Ca2þ event. Ca2þ signals are typically imaged using Confocal microscopy systems can also be laser-scanning confocal microsopes. In its used to measure changes in cellular Ca2þ caused simplest form, a confocal microscope system by activation of photoprotected (“caged”) com- comprises three main components: (1) a light pounds. In this technique, the concentration source; (2) optical and electronic components of ions, signaling molecules, or other biologi- to manipulate, display, and analyze signals; cally active compounds can be instantaneously and (3) a microscope. Lasers, coupled to either changed by UV stimulation of cells loaded upright or inverted microscopes, are commonly with their caged equivalent. The resulting used as the light source for confocal micro- changes in intracellular Ca2þ can be monitored scopes. Although gas lasers (e.g., Ar-ion, Kr- using the same system. By employing caged 2þ 2þ ion, HeNe) provide numerous lasing lines Ca or Ca -releasing compounds (e.g., IP3), from UV to red, and are well suited for opti- this approach can also be used to directly elevate mal excitation of fluorescent probes, solid-state Ca2þ within a cell. This uncaging strategy facil- lasers are increasingly being used because they itates the study of a particular signaling step offer the advantages of longer lifetime, lower independent of preceding steps in the signaling power consumption, and compact size. pathway. To record very rapid events or determine Specialized techniques have also been devel- the kinetics of a Ca2þ event by confocal micros- oped for measuring Ca2þ signals in specific sub- copy, researchers have typically measured Ca2þ cellular compartments. One such technique is fluxes using a line-scanning procedure. In line- total internal reflection fluorescence (TIRF) scan mode, a single line is repeatedly scanned microscopy, which is used to selectively visual- across the cell for a period of time. Each line ize the area immediately beneath the plasma- is then aligned to form an image that is a plot lemma. In TIRF microscopy, an evanescent of fluorescence along the scanned line versus wave is created by the reflection of a laser time (Fig. 1). Although line scans are still beam at the interface between a glass coverslip used, the development of very sensitive CCD and the cytoplasm of cells attached to the cov- (charge-coupled device) cameras and rapid erslip (Fig. 2). Because TIRF measurements

Cell t 5 μm o F / 3.0

Scan 3.0 1.0 F/Fo line 1.0 200 msec

Figure 1. Line-scan imaging. The image shown demonstrates the time course of fractional fluorescence (F/F0; bottom) and spatial distribution of the Ca2þ spark (left) fitted to a Gaussian distribution (red line). Gray bar labeled “t” indicates the region over which the fluorescence time course was averaged. Scan lines are displayed vertically in a continuous manner. (Inset) Orientation of scanning line. (Adapted from Bonev et al. 1997; reprinted with permission from The American Physiological Society # 1997.)

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D.C. Hill-Eubanks et al.

B

~ Membrane – 200 nm Aqueous medium A Cover glass Cell Oil

Cover glass Oil Objective C A lens

Laser DM

BE FL BF

Image intensifier

CCD camera

Figure 2. TIRF microscopy. (A) A schematic of the TIRF imaging system. A, adjustable rectangular knife-blade aperture; BE, beam expander; FL, focusing lens; BF, barrier filter; DM, dichroic mirror; CCD, charge-coupled device. (B) Imaging membrane-proximate fluorescent Ca2þ signals near an open channel by TIRF microscopy. (C) A single frame of a TIRF microscopy video image illustrating Ca2þ signals generated by three channels within an 80 80-mm patch of membrane. Increasing Ca2þ concentrations are indicated by both “warmer” colors and height. (Adapted from Demuro and Parker 2004; reprinted with permission from Elsevier # 2004.)

usually use nonratiometric dyes, they have organization of the a1 subunit creates a pseu- the same limitations noted above for these dotetrameric structure in which the four repeat fluorophores. domains (I, II, III, IV), each composed of six transmembrane segments (S1–6) and intracel- lular amino- and carboxyl-termini (Catterall Ca2þ SIGNALS FROM OUTER SPACE: 2000; Jurkat-Rott and Lehmann-Horn 2004), Ca2þ INFLUX are analogous to the individual subunits of structurally similar, tetrameric voltage-depend- Signals Mediated by VDCCs ent potassium (Kþ) channels. The S4 trans- Membrane potential depolarization activates membrane segments of each domain serve as VDCCs—the major contributors to increases voltage sensors; in response to changes in mem- 2þ in [Ca ]i. VDCCs are multisubunit complexes brane potential, they move outward and rotate, comprising a pore-forming a1 subunit and reg- producing a conformational change that opens ulatory b, a2d, and g subunits (Curtis and Cat- the pore (Catterall 2000). terall 1984; Hosey et al. 1987; Leung et al. 1987; The pore-forming a1 subunit is expressed Vaghy et al. 1987). Most of the functional prop- as multiple splice variants with different regula- erties of the VDCC channel, including voltage tory and biophysical properties. Additional sensitivity, Ca2þ permeability, Ca2þ-dependent molecular diversity is provided by four differ- inactivation, and sensitivity to pharmacological ent, variably spliced b subunits (Birnbaumer block by organic Ca2þ channel blockers, are et al. 1998), which further modify VDCC attributable to the a1 subunit. The domain biophysical properties and regulate surface

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Calcium Signaling in Smooth Muscle

expression of the a1 subunit; properties of the It also has been suggested that voltage-depend- VDCC complex may be additionally modulated ent Ca2þ currents in smooth muscle with a by splice variants of the a2d regulatory subunit T-type pharmacology may arise because of a (Angelotti and Hofmann 1996). CaV3.1 (and/or CaV3.2) splice variant with VDCC-mediated currents are characterized a more depolarized activation voltage (Kuo by high voltage of activation, large single-chan- et al. 2010). nel conductance, and slow voltage-dependent Three different Ca2þ signals mediated by inactivation. VDCC-mediated currents also VDCCs have been identified in smooth muscle: display a characteristic sensitivity to dihydro- (1) global elevations in intracellular Ca2þ, (2) pyridines (Reuter 1983), a class of drugs used Ca2þ “flashes,” and (3) Ca2þ “sparklets.” clinically in the treatment of hypertension (Nelson et al. 1990; Snutch et al. 2001). In Global Ca21 Signals vascular and visceral smooth muscle, these dihydropyridine-sensitive currents are attribut- As the name suggests, global Ca2þ signals reflect able to the expression of the L-type VDCC changes in Ca2þ concentration that are essen- pore-forming a1C subunit (CaV1.2) (Keef tially uniform throughout the cell. In smooth et al. 2001; Moosmang et al. 2003; Wegener muscle, depolarization of the membrane by et al. 2004); but in some smooth muscle 15 mV from its resting potential (approxi- 2þ types, the a1D (CaV1.3) subunit is expressed mately –50 to –40 mV) elevates global Ca as well (Nikitina et al. 2007). Of the four b to 300–400 nM, whereas hyperpolarization subunits, only two—b2 and b3—are clearly by 15 mV lowers Ca2þ to 100 nM. These detected at the level in smooth muscle, signals are typically monitored using ratiomet- although mRNA for all four isoforms is ric dyes (e.g., Fura-2), which are ideally suited expressed (Hullin et al. 1992; Murakami et al. to measuring Ca2þ concentration over this 2003). range (Kd 300 nM). Thus, by modulating There is also evidence for the expression of the steady-state open probability of VDCCs, a dihydropyridine-insensitive Ca2þ current in slow changes in membrane potential can have some smooth muscle types. This T-type (transi- profound, sustained effects on global intracellu- 2þ ent) current is mediated by CaV3 pore-forming lar Ca . This is illustrated in Figure 3, which a subunits—primarily the CaV3.1 isoform in shows that the membrane potential depolariza- smooth muscle (Bielefeldt 1999; Perez-Reyes tion that accompanies elevation of intraluminal 2003). Expression of T-type channels varies pressure from 60 mmHg (Fig. 3A) to 100 mmHg between different smooth muscle types, but (Fig. 3B) increases the global intracellular their presence often goes undetected because Ca2þ concentration in the vascular wall. In- of their low levels of expression or because hibition of VDCC channels with nisoldipine 2þ they are obscured by the specific recording decreases global [Ca ]i (Fig. 3C). Changes in conditions used. Moreover, the negative steady- global Ca2þ are accompanied by changes in vas- state inactivation property of these channels cular diameter, underscoring the importance of results in their being half-inactivated at about global Ca2þ in regulating the contractile state of –70 mV. Therefore, over the range of smooth smooth muscle (see below). muscle resting membrane potential (–50 to –30 mV), T-type channels may be completely Ca21 Flashes inactivated. Because of these gating properties, the importance of T-type currents in the regu- Some types of smooth muscle (e.g., urinary lation of smooth muscle membrane potential bladder, gallbladder, ) show action poten- is a matter of controversy. However, there is tials. These rapid, transient changes in mem- evidence from the rabbit urethra that, at least brane potential, which show a characteristic in this tissue, T-type channels regulate ac- temporal profile, are unique to excitable cells. tion potential frequency (Bradley et al. 2004). However, unlike other excitable cells, such as

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D.C. Hill-Eubanks et al.

ABC 2 2 2

1 1 1

0 0 0

189 245 68

Figure 3. Global Ca2þ.Ca2þ images obtained from a rat basilar pressurized to (A) 60 mmHg, (B) 100 mmHg, and (C) 100 mmHg in the presence of nisoldipine. The numbers below each panel correspond to the Ca2þ concentration in the smooth muscle of the vascular wall, calculated from the ratio of Fura-2 fluo- rescence at 340 and 380 nm. Note contraction and dilation in B and C, respectively, relative to A. (Adapted from Knot and Nelson 1998; reprinted with permission from The Journal of Physiology # 1998.)

and cardiac myocytes, where action of an individual channel or cluster of channels potentials are initiated by activation of channels (Navedo et al. 2005). The average area of a that predominantly mediate sodium (Naþ) Ca2þ sparklet is 0.8 mm2 or 0.08% of the influx, the upstroke of the in surface membrane of a typical arterial smooth smooth muscle reflects massive Ca2þ entry (Santana et al. 2008). Ca2þ influx through VDCCs. The resulting rapid elevation through Ca2þ sparklet sites is quantal, and the of global intracellular Ca2þ can be detected size of a given Ca2þ sparklet depends on the optically as a Ca2þ flash that brightly and briefly number of quanta activated. One quantal unit 2þ 2þ 2þ lights up cells loaded with fluorescent Ca of Ca release elevates [Ca ]i by about 35 nM. indicators. A recording of a Ca2þ flash in uri- The specific locations of Ca2þ sparklets vary nary bladder smooth muscle is shown in Fig- between cells, but within a cell, Ca2þ sparklets ure 4. Ca2þ flashes can be evoked by electrical are predominantly stationary events that occur field stimulation, but also occur spontaneously in specific regions of the sarcolemmal mem- (as in the example shown), likely reflecting the brane (Fig. 5A–C). Both low- and high-activity spontaneous release of from Ca2þ sparklet sites have been described. The fre- sympathetic (e.g., mesenteric ) or para- quency of the latter type of event, termed a per- sympathetic (e.g., urinary bladder) nerve termi- sistent sparklet, is increased by activation of nals (Klockner and Isenberg 1985; Heppneret al. protein C (PKC), which recruits previ- 2005). Note that in this example, Ca2þ was ously silent sites and increases the frequency of simultaneously elevated in two adjacent cells, low-activity sites (Fig. 5D). indicating that these events may be coupled. In heart muscle, where local coupling of Ca2þ influx through single VDCCs to RyRs is central to excitation-contraction coupling Ca21 Sparklets (Cannell et al. 1995; Lopez-Lopez et al. 1995), Cheng and colleagues first measured the local aCa2þ sparklet can activate four to six nearby Ca2þ signal caused by the opening of a single RyRs to cause a Ca2þ spark (see below). How- L-type VDCC in (Wang et al. ever, there is no evidence for this direct 2001b), and referred to these events as Ca2þ VDCC-to-RyR communication in smooth sparklets. Ca2þ sparklets are also present in vas- muscle. Instead, Ca2þ currents through VDCCs cular smooth muscle (Fig. 5), where they are appear to activate RyRs indirectly through ele- detected by TIRF microscopy as highly local- vations in global Ca2þ and SRCa2þ load (Collier ized, dihydropyridine-sensitive, subplasmalem- et al. 2000; Herrera and Nelson 2002; Wellman mal Ca2þ-release events reflecting the activity and Nelson 2003; Essin and Gollasch 2009).

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Calcium Signaling in Smooth Muscle

A (a) (b) (c) and members of the Orai family (Feske et al. 2006; Vig et al. 2006; Zhang et al. 2006) of trans- membrane proteins as the entities responsible for mediating Ca2þ entry (reviewed in Varnai et al. 2009). These researchers showed that, in response to a decrease in ER Ca2þ concentra- 2þ 25 μm tions, the low-affinity Ca -binding STIM B proteins aggregate to form discrete plasmalem- (b) mal-proximate clusters that tether Orai pro-

0.5 F/Fo teins. This physical coupling activates Orai, 2þ 2 sec (a) (c) which is a highly selective Ca channel, thereby promoting extracellular Ca2þ entry. The identi- fication of the STIM-Orai mechanism has 10 mV sparked renewed interest in investigating SOCE in smooth muscle (reviewed in Wang et al. 2008). These studies, most of which have –50 mV been performed using cultured smooth muscle 2þ cells, have consistently shown that STIM and Figure 4. Ca flashes. (A) Selected images recorded before (a), during (b), and after (c) a spontaneous Orai family members are expressed in smooth action potential in a smooth muscle bundle loaded muscle, and, under the conditions tested, are with Fluo-4 and impaled with a microelectrode capable of functionally coupling store depletion (green rectangle). (B) Simultaneous recordings of to extracellular Ca2þ entry (Peel et al. 2006, 2þ changes in Ca -activated fluorescence (upper trace) 2008; Takahashi et al. 2007; Ng et al. 2010; and voltage (lower trace) from a single bundle of uri- Park Hopson et al. 2011). It has also been sug- nary bladder smooth muscle. Changes in Ca2þ- activated fluorescence were measured from the red gested that, in addition to promoting Orai box in A; the letters a, b, and c denote the times at activity, STIM1 negatively regulates VDCCs which the correspondingly labeled images in A were (Wang et al. 2010). Some recent studies have acquired. Note that each of the three action potentials provided evidence for STIM-Orai coupling in induced a simultaneous increase in Ca2þ-activated native smooth muscle preparations, and have fluorescence. (Adapted from Heppner et al. 2005; suggested a role for this mechanism in hyper- reprinted with permission from The Journal of Phys- tension (Giachini et al. 2009, 2010). An optical iology # 2005.) signature of Orai-mediated Ca2þ influx has not been defined and additional research will be required to definitively establish the physiological relevance of this pathway in native Signals Mediated by Store-Operated smooth muscle tissues. Ca2þ Channels Extracellular Ca2þ influx in response to deple- Signals Mediated by Nonselective Cation tion of intracellular Ca2þ stores, a process Channels termed store-operated Ca2þ entry (SOCE), is known to play an important role in a number In contrast to VDCCs, which show a high selec- of cell types, notably nonexcitable cells. How- tivity for Ca2þ ions over monovalent cations, ever, the molecular mechanism underlying nonselective cation channels typically also allow this coupling long remained elusive. Recent influx of extracellular Naþ. Although channels seminal work by a number of independent of this type are permeable to Ca2þ and thus di- 2þ groups effectively resolved this question, clearly rectly increase [Ca ]i to some degree, in many 2þ identifying ubiquitously expressed STIM pro- if not most cases, their major impact on [Ca ]i teins (Liou et al. 2005; Roos et al. 2005) as is indirect through Naþ-dependent membrane (ER) Ca2þ sensors, potential depolarization and activation of

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D.C. Hill-Eubanks et al.

A C 80

) 5 μm 140 M 60 120 ] (m 100 2+ 40 a 80 20

Δ [Ca 60 0 40 20 d Number of events 0 200150100500 >300300250 2+ Δ [Ca ] (mM) c b D

2 mM 100 B Control 75 a PDBu b 50

c 25 Number of events

M 0 m d 200150100500 300250 >300 40 2+ Δ [Ca ] (mM) 2 sec

Figure 5. Ca2þ sparklets. (A) Surface plot of Ca2þ imaged in a freshly isolated arterial myocyte. (Inset) Higher 2þ 2þ magnification view of boxed area showing three active Ca sparklet sites (2 mM extracellular Ca ). (B)Traces 2þ 2þ showing time course of changes in Ca at sites a–d. (C) Amplitude histogram of Ca sparklets (20 mM extra- 2þ 2þ 2þ cellular Ca ). (D) Amplitude histogram of Ca sparklets (2 mM extracellular Ca ) in the absence and pres- ence of the PKC activator phorbol 12,13-dibutyrate (PDBu). Solid lines in C and D are best fits to a Gaussian function. (Adapted from Navedo et al. 2005; reprinted with permission from The National Academy of Sciences # 2005.)

VDCCs. Several types of Ca2þ-permeable, non- connected by a large extracellular domain selective cation channels are present in smooth (Khakh 2001; North 2002). Binding of ATP muscle, including receptor-activated channels, (three molecules per complex) to a site in the mechanosensitive channels, tonically active large extracellular domain induces a conforma- channels, and channels activated by SR Ca2þ tional change that results in rapid (millisec- store depletion. onds) opening of the pore. These Ca2þ- and Naþ-permeable channels (Benham and Tsien 1987; Schneider et al. Receptor-Activated Cation Channels 1991) mediate a rapid local influx of Naþ and Of the various nonselective cation channels Ca2þ at nerve–muscle junctions following acti- expressed in smooth muscle, only the ATP- vation by neurally released ATP (Lamont and gated P2X receptor (P2XR) is clearly associated Wier 2002; Lamont et al. 2006). The influx of with an optically identifiable Ca2þ signal. The Naþ and Ca2þ creates an excitatory junction functional P2XR complex is thought to be a potential (EJP) that contributes directly to the trimer (Aschrafi et al. 2004)—an unusual struc- increase in postjunctional excitability. Multiple tural arrangement in ion-channel space where lines of evidence, including studies using tetramers dominate. Each subunit contains knockout mice, indicate that the P2X1 receptor intracellular amino- and carboxyl-termini and is the predominant P2X receptor isoform two membrane-spanning domains that are expressed in smooth muscle (Mulryan et al.

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Calcium Signaling in Smooth Muscle

2000; Vial and Evans 2002; Lamont et al. 2006; been reported in the literature, given the relative Heppner et al. 2009). selectivity of these channels for Ca2þ (and the Although most of the excitatory junction high Ca2þ permeability and single-channel current (EJC) associated with P2X1 receptor ac- conductance of some TRP family members, tivation is carried by the more abundant Naþ notably TRPV), imaging methods that have ions, Ca2þ influx is substantial. This influx can been used to examine jCaT-like events (confocal be detected optically in the form of local elemen- microscopy) and/or VDCC-mediated spark- tary purinergic-induced Ca2þ transients (Fig. 6). lets (TIRF) may ultimately provide a means These events have been described in vas deferens to optically detect Ca2þ influx through these (Brain et al. 2002), mesenteric arteries (Lamont channels. and Wier 2002), and urinary bladder (Heppner

et al. 2005), where they have been termed neuro- 2þ 2þ 2þ Ca SIGNALS FROM INNER SPACE: RELEASE effector Ca transients (NCTs), junctional Ca 2þ transients ( jCaTs), and nerve-evoked element- OF Ca FROM INTRACELLULAR STORES 2þ ary purinergic Ca transients, respectively. The The most important intracellular Ca2þ store in 2 kinetic properties of these purinergic Ca þ signals smooth muscle is the SR. Cytosolic Ca2þ is aresimilartooneanotherandareclearlydistinct transported into the SR by the action of the 2þ from those of other local Ca transients (Hill- SR/ER Ca2þ ATPase (SERCA). SERCA activity Eubanks et al. 2010). is negatively regulated by the protein phospho- lamban, a target of A (PKA) and protein kinase G (PKG). On phosphorylation, Other Nonselective Cation Channels the SERCA inhibitory activity of phospholam- Smooth muscle cells express a number of non- ban is lost, increasing Ca2þ uptake and SR selective cation channels of the TRP family. Ca2þ load. Free Ca2þ taken up by the SR is buf- Although no signature signaling event associated fered by Ca2þ-binding proteins, which retain with Ca2þ influx through TRP channels has transported Ca2þ and reduce the free Ca2þ

10 μm

2.0 2.0 3.0 F/Fo F/Fo F/Fo 1.0 1.0 1.0

Figure 6. Elementary purinergic Ca2þ transients recorded from urinary bladder smooth muscle. Transient events were evoked by electrical field stimulation (2-sec train, 5 Hz; 378C) in bladder strips loaded with Fluo-4 and scanned at a rate of 30 images/sec. The top three images illustrate nerve processes before (top panel, left, indicated by arrows) and after stimulation (middle and right panels). The three bottom panels illustrate 2þ color-coded ratios (F/F0) of the images above. Intracellular Ca increases first in the nerve fibers, and then two local Ca2þ transients are detected (right panel, bottom right quadrant, indicated by arrows). The activity of Ca2þ transients continued throughout the duration of field stimulation. (Unpublished data from Mark Nelson.)

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concentration gradient, facilitating continued spread is 12.6 mm, and this spread corre- Ca2þ uptake from the cytoplasm (Pozzan et al. sponds to 1% of the surface membrane. The 1994). duration (half-time) of these events is 50– Ca2þ sequestered in the SR may be delivered 60 msec; this contrasts with nerve-evoked puri- 2þ to the through RyRs and IP3 receptors in nergic Ca transients, which have half-times the SR membrane. Although these two channel of 110–145 msec (Brain et al. 2002; Lamont types are phenotypically similar on a superficial and Wier 2002; Heppner et al. 2005). Current level (both function to release Ca2þ from SR evidence indicates that smooth muscle Ca2þ stores) they are very different molecular entities sparks are attributable to activation of RyR2, with distinctive regulatory features and charac- although both RyR1 and RyR3 may influence teristic Ca2þ-release signatures. spark activity (Vaithianathan et al. 2010). In smooth muscle, localized increases in Ca2þ associated with Ca2þ sparks activate Signals Mediated by RyRs: Ca2þ Sparks closely juxtaposed large-conductance, Ca2þ- þ There are three RyR subtypes (RyR1-3), each of activated K (BKCa) channels in the plasma 2þ which is expressed at varying levels in different membrane. The Kd of BKCa channels for Ca smooth muscle tissues (Neylon et al. 1995; is 20 mM at the physiological membrane Yang et al. 2005; Prinz and Diener 2008). RyRs potential of –40 mV. A single spark causes a 2þ are large tetrameric complexes formed from local increase of [Ca ]i of 10–30 mM and acti- 560-kDa subunits. Each subunit contains vates about 30 nearby BKCa channels, increasing four membrane-spanning domains, a large their open probability by approximately cytosol-facing amino-terminal region contain- 100-fold (Jaggar et al. 2000; Perez et al. 2001). ing the Ca2þ-binding site as well as binding In smooth muscle from adult animals, there is sites for numerous accessory proteins, and a a one-to-one relationship between sparks and short SR-luminal carboxy-terminal domain BKCa channel–mediated transient outward (reviewed in Zalk et al. 2007; Lanner et al. 2010). currents, indicating that all spark sites are func- 2þ Ca flux through ryanodine receptors is tionally coupled to BKCa channel clusters. In detectable in the form of elementary release current-clamp mode, activation of BKCa chan- events termed Ca2þ sparks. First discovered in nels by a single spark causes about a 20-mV cardiac muscle (Cheng et al. 1993) and subse- hyperpolarization (Jaggar et al. 1998b). quently identified in skeletal (Klein et al. Stimuli that increase SR Ca2þ load increase 1996) and smooth (Nelson et al. 1995) muscle, the frequency of Ca2þ sparks, but sparks also aCa2þ spark represents the opening of a few occur spontaneously. The outward currents (likely four to six) RyR channels in the SR mem- associated with this latter activity are termed brane (Cheng and Lederer 2008). Ca2þ sparks “spontaneous transient outward currents” or have been detected in a wide variety of smooth “STOCs” (Benham and Bolton 1986). An muscle types, including those from arteries example depicting the time course and decay (Nelson et al. 1995), portal vein (Mironneau kinetics of a single Ca2þ spark event is presented et al. 1996; Gordienko and Bolton 2002), uri- in Figure 7A. Simultaneous electrophysiological nary bladder (Herrera et al. 2001), ureter (Bur- recordings and traces showing analyzed Ca2þ dyga and Wray 2005), airway (Sieck et al. 1997), signals (Fig. 7B) highlight the one-to-one rela- and the gastrointestinal tract (Gordienko et al. tionship between sparks and STOCs. 2þ 1998). Ca sparks in arterial smooth muscle In addition to activating BKCa channels can be detected in isolated myocytes as well as to produce STOCs, Ca2þ sparks can also acti- 2þ 2þ in intact pressurized arteries. Ca sparks are vate Ca -sensitive chloride (ClCa) channels rapid, transient, stationary events. The rise time to produce spontaneous transient inward cur- of sparks in vascular smooth muscle and urinary rents (STICs) (Hogg et al. 1993). Where BKCa bladder smooth muscle is 20–40 msec (Nel- and ClCa channels coexist, spontaneous tran- son et al. 1995; Herrera et al. 2001); their spatial sient outward/inward currents (STOICs) are

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Calcium Signaling in Smooth Muscle

A Grayson et al. 2004). IP3Rs are large homo- or heterotetrameric complexes formed from ap- 0 msec 8.3 msec 16.7 msec 25.0 msec proximately 2700 to 2800 amino acid subunits. Each subunit contains six membrane-spanning domains, a large cytosol-facing amino-terminal region containing the IP3 binding site, and a

33.3 41.6 50.0 58.3 66.7 75.0 83.3 91.7 short cytosolic carboxy-terminal domain. The pore of the channel is formed by the coassocia- 100.0 108.3 116.7 125.0 133.3 141.7 150.0 158.3 tion of transmembrane domains 5 and 6 from each of the four subunits (Foskett et al. 2007). 2þ 5 μm Ca release by IP3Rs is regulated by two F/Fo 1.0 2.0 3.0 4.0 second messengers: IP and Ca2þ. The proto- B 3 typical signaling pathway that leads to elevation STOCs 20 pA of IP3 is activation of Gaq/11-type G protein- coupled receptors. Among the most prominent agonists of this pathway in smooth muscle are neurohumoral vasoconstrictors. Activation of Sparks 0.5 F/Fo this pathway stimulates C, re- sulting in hydrolysis of membrane-associated phosphoinositide 4,5-bisphosphate (PIP2)into 500 msec IP3 and diacyclglycerol. The IP3 generated by Figure 7. Ca2þ sparks in an isolated rat basilar artery this pathway binds IP3Rs and promotes channel 2þ myocyte. (A)(Top) Two-dimensional confocal gating, releasing Ca into the cytosol. The 2þ images of an entire smooth muscle cell showing the Ca released by IP3Rs can reciprocally modu- 2þ time course of the fractional increase in Fluo-3 fluo- late IP3R activity in two ways: as Ca rises 2þ rescence (F/F0) of a typical Ca spark. (Bottom) from low nanomolar basal levels to low micro- Images obtained from the region of interest in the molar levels in the vicinity of the channel, it top-right panel (dotted box) depicting spark decay. Pseudocolor denotes relative Ca2þ levels as indicated activates IP3Rs; at higher local levels, the chan- by the bar. (B) Simultaneous measurements of nel becomes inactivated. These activation/inac- STOCs and sparks at –40 mV highlighting the tem- tivation properties together with regulation of poral association between the events. The pink bar channel function by multiple interacting fac- below the sparks trace corresponds to the time period tors, allow IP3Rs to generate a large variety of imaged in A. (Adapted from Perez et al. 1999; temporally and spatially modulated Ca2þ- reprinted with permission from The Rockefeller Uni- signaling patterns within the cell (Foskett et al. versity Press # 1999.) 2007). In some smooth muscle types, IP3R- produced (ZhuGe et al. 1998; Jaggar et al. 2000; mediated Ca2þ release is transient and local- Wellman and Nelson 2003). Both hyperpolariz- ized. For example, smooth muscle cells from ing (STOCs) and depolarizing currents (STICs) colon and portal vein show spontaneous Ca2þ modulate membrane potential and excitability, spark-like events that are enhanced by IP3 pro- with STOCs being inhibitory and STICs being duction and eliminated by IP3R blockade (Bay- excitatory. guinov et al. 2000; Gordienko and Bolton 2002). These Ca2þ release events, termed Ca2þ “puffs,” have a biophysical signature (e.g., kinetics, Signals Mediated by IP Rs: Ca2þ Waves 3 magnitude, spatial spread) that distinguishes 2þ There are three IP3 receptor subtypes (IP3R1-3), them from the RyR-mediated Ca sparks that each of which is expressed at varying levels in are prominent in most other smooth muscle different smooth muscle tissues (Newton et al. types (e.g., vascular smooth muscle, gallblad- 1994; Tasker et al. 1999; Boittin et al. 2000; der, and urinary bladder) (Nelson et al. 1995;

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D.C. Hill-Eubanks et al.

Herrera et al. 2001; Pozo et al. 2002). Although each other out because of depletion of SR Ca2þ puffs are unitary events, they can act as Ca2þ stores on either side of the collision site initiation sites for intracellular Ca2þ waves and (Stevens et al. 1999). Therefore, a continuous thereby contribute to global Ca2þ signals (Boot- cycling of SR Ca2þ release and reuptake main- man and Berridge 1996; Thomas et al. 1998). tains the Ca2þ waveform. Ca2þ released from ACa2þ wave, defined as an increase in SR stores is sufficient for further release and 2þ 2þ [Ca ]i that propagates across the entire smooth maintenance of Ca waves, indicating that muscle cell from an initial site of release, is the mechanism of smooth muscle Ca2þ wave 2þ perhaps the most studied IP3R-mediated Ca - propagation is independent of extracellular signaling event. First described by Iino in rat Ca2þ entry (Boittin et al. 1999; Jaggar and Nel- tail arteries (Fig. 8), Ca2þ waves are a common son 2000; Peng et al. 2001; Heppner et al. 2002). 2þ feature of vascular smooth muscle cells exposed Repeating IP3R-dependent Ca signals may to Gaq/11-coupled vasoconstrictor agonists, also take the form of whole-cell oscillations in 2þ such as UTP (Jaggar and Nelson 2000) and [Ca ]i. 2þ norepinephrine (Iino et al. 1994; Boittin et al. The view of Ca waves as strictly IP3R- 1999; Miriel et al. 1999; Ruehlmann et al. mediated events oversimplifies the true situa- 2000), or electrical field stimulation of perivas- tion. In actuality, Ca2þ waves arise in response cular nerves. to activation of IP3Rs and/or RyRs (Iino et al. In the current view, Ca2þ waves reflect the 1994; Boittin et al. 1999; Hirose et al. 1999; Jag- activation/inactivation properties of IP3Rs, gar and Nelson 2000; Lee et al. 2002), and their and arise through a regenerative Ca2þ-induced properties as well as the relative contributions of 2þ 2þ Ca release (CICR) mechanism. Ca released IP3Rs and RyRs may differ depending on the from the SR acts on successive adjacent IP3Rs or nature of the stimulus or tissue context. In IP3R clusters in a cascading fashion, creating a some cases, these events appear to exclusively leading edge of Ca2þ elevation that traverses reflect the activity of RyRs. One such example the length of the cell. When Ca2þ waves propa- is provided by rat cerebral arteries, where gate toward each other and collide, they cancel Nelson and colleagues (Heppner et al. 2002)

A a 4 b 5 c 15 d 26 e 54 f 61 1– 2– –3

B 5.0 c d e 012345 4.0 b f 0 3.0 1 3 F / 2.0 2 a 1.0 5 Hz, 300 pulses 0.0 0204060 80 100 Frame number/time (sec)

Figure 8. Ca2þ waves. (A) Two-dimensional pseudocolor confocal microscopic images of rat tail artery smooth muscle showing dynamic, recurrent changes in intracellular Ca2þ (measured by changes in the intensity of Fluo-3 fluorescence) following electrical stimulation of perivascular sympathetic nerves. Six of 96 consecutive frames collected at a rate of one frame per second are shown. (B) Red, blue, and green lines depict changes in fluorescence intensity as a function of time (a–f in A) in three selected regions of interest (white boxes in a). (Adapted from Iino et al. 1994; reprinted with permission from The EMBO Journal # 1994.)

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Calcium Signaling in Smooth Muscle

have found that Ca2þ waves are induced by chain phosphatase (Somlyo and Somlyo 2003; caffeine, which acts on RyRs but not IP3Rs. Mizuno et al. 2008). Moreover, these events are insensitive to the nominally selective IP R blockers, xestospongin 3 Global Ca21 C and 2-aminoethoxydiphenyl borate (2-APB), but are completely eliminated by ryanodine. Membrane potential plays an important role in Interestingly, RyR-mediated Ca2þ signaling in all excitable cells, including smooth muscle, 2þ this preparation is sharply dependent on pH. where it regulates [Ca ]i and thereby smooth Increasing the pH of the bathing solution . The resting membrane from 7.4 to 7.5 increased Ca2þ spark frequency potential of smooth muscle is approximately by 50%; above this pH, Ca2þ waves came to –50 to –40 mV, which is positive to the equi- þ predominate, increasing approximately three- librium potential for K (EK). In arterial fold between pH 7.5 and pH 7.6 and doubling smooth muscle, this membrane potential is again at pH 7.7–7.8. sufficient to increase the steady-state open pro- bability of VDCCs, elevate global intracellular 2þ Ca from 100 nM to 200 nM, and cause a FUNCTIONAL CORRELATES OF Ca2þ tonic constriction (Knot and Nelson 1998). As SIGNALS noted above, membrane potential hyperpo- larization to –60 mV lowers Ca2þ to about Contractility 100 nM, and depolarization to about –30 mV 2þ 2þ Smooth muscle contraction is driven by Ca - elevates global Ca to 300–400 nM; these activation of myosin light chain changes in global intracellular Ca2þ are suffi- kinase, which has a Ca2þ half-activation of cient to cause maximal dilation and constric- 400 nM (Stull et al. 1998). The gain of tion, respectively. The fundamental relationship smooth muscle contraction to Ca2þ can be between global Ca2þ and smooth muscle mem- adjusted through regulation of myosin light brane potential is depicted in Figure 9, which

400 PSS

PSS with 10 nM Nisoldipine

300 M

P100 200 P80 P40 P20 P60 P10

Arterial (calcium), n wall 100

0 –70 –60 –50 –40 –30 –20 –10 Membrane potential, mV

2þ Figure 9. Intravascular pressure-membrane potential-[Ca ]i relationships. Fundamental relationships among intravascular pressure (P10–P100, in mm Hg), membrane potential, and arterial wall Ca2þ. (Adapted from Knot and Nelson 1998; reprinted with permission from The Journal of Physiology # 1998.)

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D.C. Hill-Eubanks et al.

also highlights the role of intravascular pressure additional increment of Ca2þ may sum with 2þ as a physiological driver of changes in mem- P2X1R and VDCC-mediated Ca to augment brane potential. Oscillations in membrane the transient contraction. Alternatively, CICR- potential caused by fluctuations in Ca2þ entry activated RyRs could, in theory, couple to through VDCCs lead to vasomotion of the arte- BKCa channels to oppose contraction. Depend- rial wall. ing on the relative speed of IP3 production by concurrent activation of adrenergic or musca- rinic receptors, it is also possible that Ca2þ in- Junctional Ca21 Transients and flux through P2X Rs could amplify local IP R Ca21 Flashes 1 3 activation by IP3, a possibility that has not yet Nerve stimulation–evoked localized Ca2þ in- been explored experimentally. flux through P2X1R channels causes a de- polarizing current carried by Naþ and Ca2þ Ca21 Sparks ions that activates VDCCs; in urinary bladder smooth muscle, this manifests as a Ca2þ flash. In cardiac and , local Ca2þ entry Coordinated flash activity among smooth through VDCCs activates proximate RyRs, muscle cells in a bundle leads to a transient con- producing Ca2þ sparks that summate to create traction. Thus, junctional Ca2þ transients a substantial increase in global Ca2þ; thus, 2þ mediated by P2X1Rs may indirectly modulate Ca sparks play a dominant role in contrac- contraction by triggering VDCC activity. tion in these tissues. In smooth muscle, the mo- The bursts of local elevations in intracellular lecular architecture is much different, resulting Ca2þ provided by junctional Ca2þ transients in unique linkages that create a phenotypically also have the potential to trigger activation of opposite functional outcome. In particular, proximate RyRs in the SR through a CICR the close physical coupling between VDCCs mechanism. The best evidence for the existence and RyRs that characterizes striated muscle cells of such a mechanism comes from studies of the is absent in smooth muscle cells. In its place is vas deferens by Cunnane and coworkers (Brain a close linkage between the RyR and plasma et al. 2003). In this preparation, the magnitude membrane BKCa channels, which are not ex- of neurally evoked, ATP-induced Ca2þ tran- pressed in cardiac or skeletal muscle cells. As a sients was reduced by 45% by inhibition of result of this unique architecture, Ca2þ released RyRs with ryanodine. Moreover, treatment by RyRs in the SR in the form of sparks activ- with caffeine to increase RyR activity produced ates juxtaposed BKCa channels, promoting an a 16-fold increase in the frequency of neurally outward Kþ current that hyperpolarizes the evoked junctional Ca2þ transients. Collectively, smooth muscle membrane and reduces VDCC these results argue that the neurallyevoked Ca2þ activity. The resulting decrease in Ca2þ influx signal associated with extracellular Ca2þ influx thus opposes VDCC-mediated smooth muscle triggers—and merges with—a RyR-mediated contraction (Nelson et al. 1995; Perez et al. Ca2þ signal, creating an optically detectable 1999; Jaggar et al. 2000). Under this scenario, 2þ signal that reflects a summation of the two sepa- Ca influx through VDCCs initiates the BKCa rate transient release events. The more modest channel-mediated feedback mechanism by inhibitory effect of ryanodine on jCaTs in mes- enhancing RyR activity, by increasing global enteric arteries (13%) (Lamont and Wier Ca2þ and SR Ca2þ stores (Collier et al. 2000; 2002) and the apparent absence of an effect of Herrera and Nelson 2002; Wellman and Nelson ryanodine on purinergic Ca2þ transients in rat 2003; Essin and Gollasch 2009). urinary bladder (Heppner et al. 2005) suggest As is observed with P2X1 agonists, simulta- a degree of variability among tissues and spe- neous activation of RyRs by rapid addition of cies. How (or if ) this communication from high levels of the RyR activator, caffeine, can 2þ P2X1Rs to RyRs influences the contractile cause global Ca transients and a transient behavior of smooth muscle is not clear. The contraction (Wellman and Nelson 2003).

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Calcium Signaling in Smooth Muscle

Ca21 Waves a mechanism for phenotypic switching to a “synthetic” phenotype characterized by expres- Ca2þ waves normally occur asynchronously in sion of genes that promote proliferation, matrix smooth muscle cells. However, in some vascular deposition, and other functions that come into beds, Ca2þ waves may synchronize in neighbor- play under pathological conditions and in the ing arterial myocytes to initiate vasomotion context of vessel repair and new vessel forma- (Peng et al. 2001), supporting the idea that tion. Recent work from Owens and colleagues Ca2þ waves can supply the Ca2þ needed for (Wamhoff et al. 2004) has shown that VDCC- smooth muscle contraction (Kasai et al. 1997; mediated elevations in Ca2þ act through two Boittin et al. 1999; Mufti et al. 2010). Alterna- distinct mechanisms to regulate the contractile tively, Ca2þ waves can influence contractility and synthetic/proliferative phenotypes. In the indirectly through activation of Ca2þ-dependent first, depolarization-induced Ca2þ elevation ion channels located in the plasmalemmal mem- induces SRF (serum response factor)-regulated brane. For example, Ca2þ waves can activate Cl Ca smooth muscle–specific genes (e.g., myosin channels to promote membrane depolarization heavy chain, smooth muscle a-actin) through leading to enhanced Ca2þ entry through VDCCs activation of Rho/Rho kinase and stimulation (Mironneau et al. 1996). Ca2þ waves can also of myocardin, a potent coactivator of SRF first activate BK channels, thereby promoting Ca identified by Olson and colleagues (Wang et al. membrane potential hyperpolarization, closure 2001a). In the second mechanism, elevated intra- of VDCCs, and induction of smooth muscle cellular Ca2þ acts through calmodulin-depen- relaxation (Young et al. 2001). Thus, while infor- dent kinase (CaMK) to activate CREB (cAMP mation on Ca2þ waves in smooth muscle contin- responsive element binding protein) and the ues to accumulate, the physiological function of immediately early gene, c-fos, which is involved these Ca2þ signals remains uncertain. in proliferative responses. A similar CaMK/ CREB-dependent mechanism has been impli- Ca2þ-DEPENDENT TRANSCRIPTION cated in the VDCC-mediated induction of FACTOR ACTIVATION Egr-1 (Pulver-Kaste et al. 2006) and c-fos (Cartin et al. 2000) in native cerebral arteries, and TRP Global Ca2þ: Excitation-Transcription channels in gall bladder smooth muscle (Morales Coupling et al. 2007). In this latter study, a role for the Excitation-contraction coupling, in which de- phosphatase calcineurin was also suggested. polarization induces contraction through VDCC-mediated increases in intracellular Spatially and Temporally Modulated Ca2þ, is paralleled by a conceptually similar Ca2þ Signals mechanism that links depolarization-induced 2þ 2þ 2þ increases in [Ca ]i to activation of Ca - Studies on the effects of Ca signal modulation sensitive transcription factors. This process, on activation in smooth which has been termed excitation-transcription muscle cells are limited, but seminal work by coupling, translates short-term Ca2þ-signaling Lewis and Tsien and colleagues (Dolmetsch and contractile events into long-term regula- et al. 1998; Li et al. 1998) in nonexcitable cells tion of the smooth muscle cell transcriptome. has shown a role for amplitude and frequency Unlike cardiac and skeletal muscle cells, smooth modulation of Ca2þ signals in differentially muscle cells are highly plastic; their phenotype regulating the activity of Ca2þ-sensitive tran- is maintained through dynamic regulation scription factors. These researchers showed 2þ of gene expression in response to environmental that large transient increases in [Ca ]i are suf- cues (Owens 1995). Thus, excitation-transcrip- ficient to robustly activate NF-kB and c-Jun ter- tion coupling serves to maintain the contractile minal kinase (JNK) but not NFAT (nuclear phenotype by promoting the expression of factor of activated T-cells), which is effectively smooth muscle-specific genes. It also provides activated by a sustained, graded increase in

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global intracellular Ca2þ. It has been further the Totman Trust for Medical Research; Re- shown that activation of Ca2þ-sensitive tran- search into Ageing (P332); The Royal Society scription factors is modulated by oscillatory ele- (RG080197); and the British Heart Foundation vations in intracellular Ca2þ: for all transcription (PG/07/115). factors tested (NFAT, Oct/OAP, and NF-kB), high-frequencyoscillations enhanced the efficacy REFERENCES of a given increase in Ca2þ,whereaslow- frequency oscillations activated only NF-kB. Amberg GC, Rossow CF, Navedo MF, Santana LF. 2004. NFATc3 regulates Kv2.1 expression in arterial smooth muscle. J Biol Chem 279: 47326–47334. 2þ Ca -Signaling Microdomains Angelotti T, Hofmann F. 1996. Tissue-specific expression of splice variants of the mouse voltage-gated calcium chan- Recent studies by Santana and coworkers sug- nel a2d subunit. FEBS Lett 397: 331–337. gest a model in which the scaffolding protein Aschrafi A, Sadtler S, Niculescu C, Rettinger J, Schmalzing AKAP250 targets PKC and calcineurin to G. 2004. Trimeric architecture of homomeric P2X2 and caveolin-containing membrane microdomains, heteromeric P2X1þ2 receptor subtypes. J Mol Biol 342: 333–343. where they associated with VDCCs to form a Bayguinov O, Hagen B, Bonev AD, Nelson MT, Sanders signaling unit capable of mediating persistent KM. 2000. Intracellular calcium events activated by ATP Ca2þ sparklets (Santana and Navedo 2009). in murine colonic myocytes. Am J Physiol Cell Physiol These studies further indicate that persistent 279: C126–C135. 2þ Benham CD, Bolton TB. 1986. Spontaneous transient out- VDCC-mediated Ca sparklets activate the ward currents in single visceral and vascular smooth 2þ Ca -calmodulin-dependent transcription fac- muscle cells of the rabbit. J Physiol 381: 385–406. tor NFATc3 (Nieves-Cintron et al. 2008), which Benham CD, Tsien RW. 1987. A novel receptor-operated 2þ modulates expression of the Kv2.1 voltage- Ca -permeable channel activated by ATP in smooth þ muscle. Nature 328: 275–278. dependent K channel and the b1 subunit of Berridge MJ. 1997. Elementary and global aspects of cal- the BKCa channel in these cells (Amberg et al. cium signalling. J Physiol 499: 291–306. 2004). By extension, similar complexes of Bielefeldt K. 1999. Molecular diversity of voltage-sensitive P2X Rs with and phosphatases might calcium channels in smooth muscle cells. J Lab Clin 1 Med 133: 469–477. form Ca2þ-signaling microdomains in post- Birnbaumer L, Qin N, Olcese R, Tareilus E, Platano D, junctional smooth muscle cell membranes, Costantin J, Stefani E. 1998. Structures and functions of enabling nerve-evoked purinergic transients to b subunits. J Bioenerg Biomembr 30: regulate activation of NFAT or other Ca2þ- 357–375. Boittin FX, Macrez N, Halet G, Mironneau J. 1999. Norepi- sensitive transcription factors. nephrine-induced Ca2þ waves depend on InsP(3) and activation in vascular myocytes. Am CONCLUSIONS J Physiol 277: C139–C151. Boittin FX, Coussin F, Morel JL, Halet G, Macrez N, Miron- In addition to global elevations in intracellu- neau J. 2000. Ca2þ signals mediated by Ins(1,4,5)P(3)- lar Ca2þ mediated by VDCCs, smooth muscle gated channels in rat ureteric myocytes. Biochem J 349: shows a variety of local Ca2þ signals, including 323–332. 2þ 2þ 2þ Bonev AD, Jaggar JH, Rubart M, Nelson MT. 1997. Activa- Ca sparks (RyRs), Ca puffs (IP3Rs), Ca 2þ 2þ tors of protein kinase C decrease Ca spark frequency in waves (IP3Rs/RyRs), junctional Ca transients smooth muscle cells from cerebral arteries. Am J Physiol 2þ 2þ 273: C2090–C2095. (P2X1Rs), Ca flashes (VDCCs), and Ca 2þ sparklets (VDCCs). Each signal represents the Bootman MD, Berridge MJ. 1996. Subcellular Ca signals underlying waves and graded responses in HeLa cells. manifestation of different molecular circuits, Curr Biol 6: 855–865. which collectively serve to modulate membrane Bradley JE, Anderson UA, Woolsey SM, Thornbury KD, potential, contractility, and gene expression. McHale NG, Hollywood MA. 2004. Characterization of T-type calcium current and its contribution to electrical activity in rabbit urethra. Am J Physiol Cell Physiol 286: ACKNOWLEDGMENTS C1078–C1088. The work was supported by NIH grants R37DK Brain KL, Jackson VM, Trout SJ, Cunnane TC. 2002. Inter- mittent ATP release from nerve terminals elicits focal 053832, RO1 DK065947, RO1 HL44455, PO1 smooth muscle Ca2þ transients in mouse vas deferens. HL077378, P20 R016435, and RO1 HL098243; J Physiol 541: 849–862.

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Calcium Signaling in Smooth Muscle

David C. Hill-Eubanks, Matthias E. Werner, Thomas J. Heppner and Mark T. Nelson

Cold Spring Harb Perspect Biol 2011; doi: 10.1101/cshperspect.a004549 originally published online June 27, 2011

Subject Collection Calcium Signaling

The Endoplasmic Reticulum−Plasma Membrane Primary Active Ca2+ Transport Systems in Health Junction: A Hub for Agonist Regulation of Ca 2+ and Disease Entry Jialin Chen, Aljona Sitsel, Veronick Benoy, et al. Hwei Ling Ong and Indu Suresh Ambudkar Calcium-Handling Defects and Neurodegenerative Signaling through Ca2+ Microdomains from Disease Store-Operated CRAC Channels Sean Schrank, Nikki Barrington and Grace E. Pradeep Barak and Anant B. Parekh Stutzmann Lysosomal Ca2+ Homeostasis and Signaling in Structural Insights into the Regulation of Ca2+ Health and Disease /Calmodulin-Dependent Protein Kinase II (CaMKII) Emyr Lloyd-Evans and Helen Waller-Evans Moitrayee Bhattacharyya, Deepti Karandur and John Kuriyan Ca2+ Signaling in Exocrine Cells Store-Operated Calcium Channels: From Function Malini Ahuja, Woo Young Chung, Wei-Yin Lin, et al. to Structure and Back Again Richard S. Lewis Functional Consequences of Calcium-Dependent Bcl-2-Protein Family as Modulators of IP3 Synapse-to-Nucleus Communication: Focus on Receptors and Other Organellar Ca 2+ Channels Transcription-Dependent Metabolic Plasticity Hristina Ivanova, Tim Vervliet, Giovanni Monaco, et Anna M. Hagenston, Hilmar Bading and Carlos al. Bas-Orth Identifying New Substrates and Functions for an Calcium Signaling in Cardiomyocyte Function Old : Calcineurin Guillaume Gilbert, Kateryna Demydenko, Eef Dries, Jagoree Roy and Martha S. Cyert et al. Fundamentals of Cellular Calcium Signaling: A Cytosolic Ca2+ Buffers Are Inherently Ca2+ Signal Primer Modulators Martin D. Bootman and Geert Bultynck Beat Schwaller

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Role of Two-Pore Channels in Embryonic Organellar Calcium Handling in the Cellular Development and Cellular Differentiation Reticular Network Sarah E. Webb, Jeffrey J. Kelu and Andrew L. Wen-An Wang, Luis B. Agellon and Marek Michalak Miller

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