Smooth muscle cell calcium activation mechanisms

Abstract

Smooth muscle cell (SMC) contraction is controlled by the Ca²⁺ and Rho kinase signalling pathways. While the SMC Rho kinase system seems to be reasonably constant, there is enormous variation with regard to the mechanisms responsible for generating Ca²⁺ signals. One way of dealing with this diversity is to consider how this system has been adapted to control different SMC functions. Phasic SMCs (vas deferens, uterus and bladder) rely on membrane depolarization to drive Ca²⁺ influx across the plasma membrane. This depolarization can be induced by neurotransmitters or through the operation of a membrane oscillator. Many tonic SMCs (vascular, airway and corpus cavernosum) are driven by a cytosolic Ca²⁺ oscillator that generates periodic pulses of Ca²⁺. A similar oscillator is present in pacemaker cells such as the interstitial cells of Cajal (ICCs) and atypical SMCs that control other tonic SMCs (gastrointestinal, urethra, ureter). The changes in membrane potential induced by these cytosolic oscillators does not drive contraction directly but it functions to couple together individual oscillators to provide the synchronization that is a characteristic feature of many tonic SMCs.

Smooth muscle cells (SMCs) are characterized by their phenotypic plasticity and diversity. The activation mechanisms that control contraction display a similar diversity in that each SMC type has a signalling system that is uniquely adapted to control its particular function. The aim of this perspective is to confront this diversity by considering how the activation mechanisms for generating Ca²⁺ signals have been adapted to control different SMC functions.

Classification of smooth muscle Ca²⁺ activation mechanisms

Excitation–contraction coupling in SMCs occurs through two main mechanisms. Many SMCs are activated by Ca²⁺ signalling cascades (Haddock & Hill, 2005; Wray et al. 2005). In addition, there is a Rho/Rho kinase signalling pathway that acts by altering the Ca²⁺ sensitivity of the contractile system (Somlyo & Somlyo, 2003). Since the latter appears to have more of a modulatory function, most attention will focus on how Ca²⁺ signalling is activated. Since membrane depolarization is a key element for the activation of many SMCs, much attention will focus on the mechanisms responsible for depolarizing the membrane. However, there are other SMCs where activation depends on the periodic release of Ca²⁺ from internal stores. These different Ca²⁺ activation mechanisms fall into the following three main groups (Fig. 1).

Figure 1. The three main mechanisms responsible for generating the Ca²⁺ transients that trigger smooth muscle cell (SMC) contraction.

A, receptor-operated channels (ROCs) or a membrane oscillator induces the membrane depolarization (ΔV) that triggers Ca²⁺ entry and contraction. B, a cytosolic Ca²⁺ oscillator induces the Ca²⁺ signal that drives contraction. C, a cytosolic Ca²⁺ oscillator in interstitial cells of Cajal (ICCs) or atypical SMCs induces the membrane depolarization that spreads through the gap junctions to activate neighbouring SMCs. Reproduced from Berridge (2008), with permission.

Mechanism A

Many SMCs are activated by membrane depolarization (ΔV) that opens L-type voltage-operated channels (VOCs) allowing external Ca²⁺ to flood into the cell to trigger contraction. This depolarization is induced either by ionotropic receptors (vas deferens) or a membrane oscillator (bladder and uterus). The membrane oscillator, which resides in the plasma membrane, generates the periodic pacemaker depolarizations responsible for the action potentials that drive contraction. The depolarizing signal that activates gastrointestinal, urethral and ureter SMCs is described in mechanism C.

Mechanism B

The rhythmical contractions of vascular, lymphatic, airway and corpus cavernosum SMCs depend on an endogenous pacemaker driven by a cytosolic Ca²⁺ oscillator that is responsible for the periodic release of Ca²⁺ from the endoplasmic reticulum. The periodic pulses of Ca²⁺ often cause membrane depolarization, but this is not part of the primary activation mechanism but has a secondary role to synchronize and amplify the oscillatory mechanism. Neurotransmitters and hormones act by modulating the frequency of the cytosolic oscillator.

Mechanism C

A number of SMCs are activated by pacemaker cells such as the interstitial cells of Cajal (ICCs) (gastrointestinal and urethral SMCs) or atypical SMCs (ureter). These pacemaker cells have a cytosolic oscillator that generates the repetitive Ca²⁺ transients that activate inward currents that spread through the gap junctions to provide the depolarizing signal (ΔV) that triggers contraction through mechanism A.

In the following sections, some selected SMC types will illustrate how these signalling mechanisms have been adapted to control different contractile functions with particular emphasis on how Ca²⁺ signals are activated.

Vas deferens

Sympathetic neurons activate the rapid peristaltic contractions of the vas deferens responsible for inducing sperm transfer during the brief period of ejaculation. The ATP and noradrenaline (NA) released during each burst of neural activity induce rapid contractions using different signalling mechanisms (Mulryan et al. 2000). ATP acting on P2X₁ receptors stimulates an influx of Ca²⁺ to produce the excitatory junctional potentials (EJPs) that sum to depolarize the membrane sufficiently to activate the VOCs (Brain et al. 2002) (Fig. 2). The entry of Ca²⁺ then activates ryanodine receptors (RYRs) to release Ca²⁺ from the internal store. The initial release occurs close to the plasma membrane and then spreads into the cell through the regenerative release of Ca²⁺ by the RYRs and/or the inositol 1,4,5-trisphosphate (InsP₃) receptors in the form of an intracellular Ca²⁺ wave (Brain et al. 2003). The Ca²⁺ near the membrane activates large-conductance Ca²⁺-sensitive K⁺ (BK) channels and the resulting spontaneous transient outward currents (STOCs) hyperpolarize the membrane and help to terminate the activation process (Ohi et al. 2001; White & McGeown, 2003).

Figure 2. Neural activation of vas deferens smooth muscle cells.

Sympathetic neurons have numerous varicosities that release ATP and noradrenaline (NA) that activate contraction through separate signalling pathways. See text for further details. BK, large conductance Ca²⁺-sensitive K⁺ channels; EJP, excitatory junction potential; STOC, spontaneous transient outward currents; I, inositol 1,4,5-trisphosphate (InsP₃) receptor; R, ryanodine receptor. Reproduced from Berridge (2008), with permission.

Noradrenaline (NA) acts by stimulating α₁-adrenoreceptors to produce InsP₃, which then releases Ca²⁺ that may induce an intracellular Ca²⁺ wave similar to that triggered by the ATP-dependent entry of external Ca²⁺. In addition, the α₁-adrenoreceptors also activate the smooth muscle Rho/Rho kinase signalling pathway that serves to increase the Ca²⁺ sensitivity of the contractile machinery.

Detrusor smooth muscle cells

The bladder, which functions to store and expel urine, is surrounded by layers of detrusor SMCs. The latter have two operational modes: during bladder filling they remain relaxed but contract vigorously to expel urine during micturition. The switch from relaxation to contraction, which is triggered by neurotransmitters released from parasympathetic nerves, depends on the acceleration of an endogenous membrane oscillator that produces the repetitive trains of action potentials (mechanism A in Fig. 1) that drive contraction (Hashitani et al. 2004).

The main components of the membrane oscillator are the Ca²⁺ and K⁺ channels that sequentially depolarize and hyperpolarize the membrane, respectively. This oscillator generates the periodic pacemaker depolarizations that trigger each action potential (Fig. 3). The resulting Ca²⁺ signal lags behind the action potential because it spreads into the cell as a slower Ca²⁺ wave (Morimura et al. 2005) mediated by the type 2 RYRs (Hotta et al. 2007). About 10 ms after the depolarization, Ca²⁺ appears in the form of sparks near the membrane. These sparks then develop into waves that spread into the cell to produce a global signal after about 200 ms. Not all depolarization-induced Ca²⁺ sparks trigger a regenerative wave and this ‘loose coupling’ implies that the signalling mechanism has a low pass filter capable of integrating information from high-frequency electrical signals (Collier et al. 2000). If the RYR2s are blocked by 100 μm ryanodine, the Ca²⁺ signal is restricted to a smaller elevation immediately below the membrane, which might represent Ca²⁺ sparklets resulting from the opening of the VOCs (Fig. 3). The L-type VOC, which is responsible for the upstroke of the action potential, is terminated by Ca²⁺-induced desensitization of the VOCs and by the activation of the BK and SK channels that hyperpolarize the membrane (Hashitani & Brading, 2003a,b; Meredith et al. 2004). As Ca²⁺ is pumped out of the cell, the hyperpolarizing influence of these channels declines and this contributes to the gradual pacemaker depolarization. Oscillator frequency is acutely sensitive to changes in membrane potential being accelerated by depolarization and slowed or stopped when the membrane is hyperpolarized.

Figure 3. Bladder smooth muscle cells (SMCs) have a membrane oscillator that generates the periodic action potentials that initiate the process of excitation–contraction coupling.

Neurotransmitters such as ATP and acetylcholine (ACh), which are released from parasympathetic axonal varicosities that innervate the bladder, activate or accelerate the oscillator by inducing membrane depolarization (ΔV) through two separate pathways. Reproduced from Berridge (2008), with permission.

During the resting phase when the bladder is filling, electrical activity generated by this membrane oscillator is restricted to small areas of the bladder. During bladder emptying, neural stimulation induces action potentials throughout the bladder causing most of the bladder cells to contract in unison. The parasympathetic neurons that innervate the bladder release both acetylcholine (ACh) and ATP, which then induce a more widespread activation of this membrane oscillator by increasing membrane depolarization using different mechanisms. ATP acts through a P2X receptor to increase the entry of Ca²⁺, which produces an excitatory junction potential (EJP) and this depolarization (ΔV) then activates the VOCs (Fig. 3) (Hashitani et al. 2000,2004; Heppner et al. 2005). ACh stimulates an increase in InsP₃ that acts through the InsP₃ receptors (I, shown in green) to release Ca²⁺ that can also accelerate the membrane oscillator through a mechanism that remains to be determined. One possibility is that InsP₃ depletes stores near the membrane to activate store-operated channels (SOCs) to provide the inward current necessary to depolarize the membrane.

The depolarizing drive provided by NA and ACh induces all the detrusor SMCs to enter an excitable state such that a spontaneous action potential in one cell spreads rapidly through the gap junctions at a rate of 40 mm s⁻¹ to provide the near-synchronous contraction of the bladder (Hashitani et al. 2001,2004).

Uterine smooth muscle

During early pregnancy, the uterus is relatively quiescent. There are a few weak twitches resulting from localized SMC contractions. With the onset of labour, the uterus begins to develop stronger and more frequent contractions that appear first in the fundus and then spread rapidly through the body of the uterus (Young, 2007; Wray & Noble, 2008). This remarkable switch from quiescence to oscillatory cycles of strong contractions is an example of phenotypic plasticity that depends on cells being able to remodel their signalling systems (Berridge et al. 2003). In the case of the uterus, this remodelling results in the emergence of a membrane oscillator, which is an endogenous pacemaker mechanism located in the plasma membrane that generates the repetitive action potentials responsible for driving contractions during labour (Young, 2007).

The uterine contractions that occur during labour are driven by periodic action potentials that are preceded by a characteristic pacemaker depolarization (Fig. 4). Synchronous uterine contractions depend on action potentials spreading through the smooth muscle cell layer with conduction velocities of approximately 4 cm s⁻¹. Even though all the SMCs are capable of generating a rhythm, those in the fundus are inherently more rhythmical and thus initiate each contraction wave. Since there appear to be no ICCs and there is little neural innervation, the uterine myometrium is driven by an endogenous membrane oscillator (horizontal box in Fig. 4) that generates a pacemaker depolarization (ΔV) to trigger the excitation–contraction coupling mechanism (vertical green box). The Ca²⁺ that enters through the VOCs then activates contraction. There appears to be little direct role for Ca²⁺ release from the internal stores since contractions continue when these stores have been depleted (Kupittayanant et al. 2002; Wray & Shmygol, 2007). However, as part of the remodelling process during the end of pregnancy, there is an increased expression of RYR2s and these may open in response to the entry of external Ca²⁺ to provide an additional boost of Ca²⁺ to increase the force of contraction.

Figure 4. A model for the activation mechanism of human uterus contractility.

Contractions of the uterus during labour are driven by an endogenous membrane oscillator (horizontal green to pink box) that induces the slow pacemaker depolarization responsible for triggering the excitation–contraction coupling mechanism (vertical green box). The oscillator depends on an interaction between ion channels, pumps and exchangers that are colour coordinated to indicate their contribution to either depolarization (green) or hyperpolarization (red). When the pacemaker depolarization (ΔV) reaches the threshold for the activation of the L-type voltage-operated channels (VOCs) (dashed line in the membrane potential trace at the top), excitation–contraction coupling is triggered and the muscle contracts. See text for further details. Reproduced from Berridge (2008), with permission.

While the nature of this excitation–contraction coupling mechanism is fairly well established (Shmygol et al. 2007), there is less information on the nature of the membrane oscillator that generates the pacemaker depolarization that triggers the periodic action potentials. The following membrane oscillator model attempts to explain how the slow pacemaker depolarization is generated (Fig. 4). At any moment in time, membrane potential depends upon the coordinated activity of ion channels, pumps and exchangers that can either depolarize or hyperpolarize the membrane. At the end of an action potential, the hyperpolarizing components (shown in red) are dominant but as their influence wanes the depolarizing components (shown in green) begin to take over to produce the pacemaker depolarization. A number of candidates have been implicated in this periodic switching from hyperpolarization to depolarization.

Hyperpolarizing components BK and SK channel

The Ca²⁺-sensitive BK and SK channels are activated during the repolarizing phase of the action potential and thus contribute to the membrane hyperpolarization at the start of the pacemaker phase. As Ca²⁺ is pumped out of the cell, their hyperpolarizing influence will wane and this will contribute to the gradual pacemaker depolarization. Inhibition of the SK3 channel with apamin results in an acceleration of the oscillator. Conversely, if SK3 is overexpressed in rat uterus, there is a drastic reduction in oscillator frequency (Brown et al. 2007).

In addition to contributing to the membrane oscillator during labour, the BK channel maintains the quiescent state of the uterus during pregnancy. These BK channels are concentrated in caveolae, which are rich in cholesterol that enhances BK channel activity to reduce membrane excitability and thus contributes to the quiescent state. The high cholesterol levels that occur in obesity may increase the risk of complications in pregnancy by reducing uterine contractility during labour (Wray, 2007).

Na⁺–K⁺-ATPase pump

During the course of the action potential, opening of the L-type VOC results in Na⁺ entering the cell together with Ca²⁺. A hyperpolarization is generated when the electrogenic Na⁺ pump extrudes 2Na⁺ for 3K⁺. The pump will be activated immediately after an action potential to extrude Na⁺ and this will contribute to the early hyperpolarizing phase. As the Na⁺ concentration returns to resting levels, the resulting decline in this hyperpolarizing effect will contribute to the depolarizing pacemaker.

Na⁺/Ca²⁺ exchanger (NCX)

The Na⁺/Ca²⁺ exchanger (NCX) has a somewhat ambiguous role because it can operate in both forward and reverse modes and thus can contribute to either depolarization or hyperpolarization. When functioning in its forward mode to extrude Ca²⁺ it will hyperpolarize the membrane. When the uterus begins to oscillate, the resting membrane potential is approximately −50 mV, which is probably more positive than the equilibrium potential for Na⁺, so it is likely that the exchanger will be operating in its reverse mode during which 1Ca²⁺ is exchanged for 3Na⁺ to induce depolarization as indicated in Fig. 4.

Depolarizing components

Less is known about the channels responsible for providing inward current during the pacemaker depolarization.

Store-operated Ca²⁺ channel (SOC)

SMCs are known to express SOCs that may contribute inward current. The ability of oxytocin to accelerate the membrane oscillator in the human myometrium (Nakao et al. 1997) may be explained by activation of such a SOC mechanism. The suggestion is that oxytocin stimulates the formation of InsP₃ that then empties part of the endoplasmic reticulum (ER) store that lies near the membrane to promote the opening of the SOCs and this increase in inward current will steepen the pacemaker depolarization to accelerate the oscillator. The Ca²⁺ that enters the cell may also switch on channels such as the Ca²⁺-sensitive chloride channels (CLCA) to induce the efflux of Cl⁻ and membrane depolarization (Fig. 4). Ca²⁺ may also activate TRPM4 or TRPM5 channels that gate inward monovalent currents, which have been implicated in the operation of other membrane oscillators.

Ca_V3 T-type channels

Uterine smooth muscle cells express Ca_V3.1 (Blanks et al. 2007), which is one of the T-type channel isoforms that have been implicated in the pacemaker activity of other excitable cells. At the low membrane potential of activated uterine SMCs, these T-type channels are probably inactivated. However, a small population may still contribute to a pacemaker depolarization by gating a small inward flux of Ca²⁺. Such a role for the T-type channel is consistent with the observation that low concentrations of Ni²⁺ slow the oscillator (Blanks et al. 2007). The entry of Ca²⁺ through these T-type channels may also contribute to the depolarization by activating other inward currents such as those gated by TRPM4 or TRPM5 mentioned above.

In summary, uterine contraction is driven by a membrane oscillator that provides the pacemaker depolarization to generate the periodic action potentials that trigger contractions. Many of the hormones that modulate contractility are likely to influence some of these oscillatory components. For example, the model outlined in Fig. 4 suggests that oxytocin may enhance contractions by stimulating the formation of InsP₃ which then switches on a SOC to provide an inward Ca²⁺ current resulting in a depolarizing signal. Oxytocin may have additional actions because it can increase the force of contraction in muscle cells where the internal stores have been depleted of Ca²⁺ following treatment with cyclopiazonic acid (CPA) (Kupittayanant et al. 2002). In this case, it seems likely that oxytocin may switch on the smooth muscle Rho/Rho kinase signalling pathway to enhance the sensitivity of the contractile mechanism.

Vascular, lymphatic and airway smooth muscle cells

Vascular, lymphatic and airway smooth muscle, which generate rhythmical contractions over an extended period of time, have an endogenous pacemaker mechanism driven by a cytosolic Ca²⁺ oscillator. In addition, these SMCs also respond to neurotransmitters released from the neural innervation. In the case of mesenteric arteries, the perivascular nerves release both ATP and noradrenaline (NA). The ATP acts first to produce a small initial contraction that is then followed by a much larger contraction when NA initiates a series of Ca²⁺ transients (Lamont et al. 2003). Such agonist-induced Ca²⁺ oscillations are a characteristic feature of the activation mechanisms of vascular (Iino et al. 1994; Lee et al. 2001; Peng et al. 2001; Perez & Sanderson, 2005b; Shaw et al. 2004) and airway SMCs (Kuo et al. 2003; Perez & Sanderson, 2005a; Sanderson et al. 2008). In some blood vessels, a specific tone is maintained by the spatial averaging of asynchronous oscillations. However, there are some vessels where the oscillations in groups of cells are synchronized resulting in the pulsatile contractions known as vasomotion (Mauban et al. 2001; Peng et al. 2001; Lamboley et al. 2003; Haddock & Hill, 2005). Such vasomotion is also a feature of lymphatic vessels (Imtiaz et al. 2007). Another feature of this oscillatory activity is that variations in transmitter concentration are translated into a change in contractile tone through a mechanism of frequency modulation (Iino et al. 1994; Kuo et al. 2003; Perez & Sanderson, 2005a,b). Frequency modulation is one of the mechanisms used for encoding and decoding signalling information through Ca²⁺ oscillations (Berridge, 2007).

The periodic pulses of Ca²⁺ that drive these rhythmical SMCs are derived from the internal stores through the operation of a cytosolic Ca²⁺ oscillator (Haddock & Hill, 2005; Imtiaz et al. 2007; Sanderson et al. 2008). The following general model, which applies to vascular, lymphatic, airway and perhaps also to corpus cavernosum SMCs, attempts to describe the nature of this oscillator and how it can be induced or modulated by neurotransmitters. A luminal loading Ca²⁺ oscillation mechanism (Berridge & Dupont, 1994; Berridge, 2007) forms the basis of this cytosolic oscillator model that depends upon the following sequential series of events (Fig. 5).

Figure 5. Vascular or airway SMCs are driven by a cytosolic oscillator that generates a periodic release of Ca²⁺ from the endoplasmic reticulum that usually appears as a propagating Ca²⁺ wave.

The oscillator is induced/modulated by neurotransmitters such as acetylcholine (ACh), 5-hydroxytryptamine (5-HT), noradrenaline (NA) and endothelin-1 (ET-1), which act through inositol 1,4,5-trisphosphate (InsP₃) that initiates the oscillatory mechanism. The sequence of steps 1–9 is described in the text. Reproduced from Berridge (2008), with permission.

(1) The initiation and/or modulation of this oscillator depends upon the action of transmitters and hormones such as ACh, 5-HT, NA and endothelin-1 (ET-1) that increase the formation of InsP₃ and diacylglycerol (DAG), both of which promote oscillatory activity.

(2) The oscillator is very dependent on Ca²⁺ entry to provide the Ca²⁺ necessary to charge up the stores for each oscillatory cycle. The nature of these entry mechanisms (yellow panel in Fig. 5) vary between cell types and are still being characterized. Some of the main contenders are the Na⁺/Ca²⁺ exchanger (NCX) operating in its reverse mode (Lee et al. 2001; Dai et al. 2006; Rebolledo et al. 2006), the ATP-sensitive P2X receptor responsible for generating junctional Ca²⁺ transients (jCaTs) (Lamont & Wier, 2002) or the L-type Ca_V1.2 channel operating as clusters in ‘a high open probability mode’ to produce persistent Ca²⁺ sparklets (Amberg et al. 2007). In the case of the latter, entry of Ca²⁺ through these L-type channels does not activate RYRs directly to produce sparks as occurs in cardiac myocytes. Rather, the persistent sparklets provide much of the background entry of Ca²⁺ necessary to charge up the ER to sensitize the RYRs (Essin et al. 2007). Other possibilities are the various isoforms of the transient receptor potential (TRP) ion channel family (Saleh et al. 2008). For example, TRPC3 and TRPC7 are of interest because they are sensitive to DAG that is formed together with InsP₃ during the action of the transmitters that drive the cytosolic oscillator (Peppiatt-Wildman et al. 2007). Members of the Ca_V3 family of T-type channels have also been implicated as an entry mechanism in airway SMCs (Perez & Sanderson, 2005a). The ability of hormones and neurotransmitters to accelerate oscillator frequency may depend on their ability to increase some of these Ca²⁺ influx mechanisms.

(3) The entry of external Ca²⁺ charges up the ER to sensitize the RYRs and InsP₃ receptors prior to the next phase of release. An important determinant of this sensitivity is the luminal concentration of Ca²⁺ and as this builds up the release channels become sensitive to Ca²⁺ and can participate in the process of Ca²⁺-induced Ca²⁺ release (CICR), which is responsible for orchestrating the regenerative release of Ca²⁺ from the ER. The proposed role of cyclic ADP-ribose (cADPR) in airway SMCs (Deshpande et al. 2005) is consistent with this aspect of the model on the basis of its proposed action of stimulating the SERCA pump to enhance store loading (Berridge et al. 2003) and such a mechanism has been described in colonic SMCs (Bradley et al. 2003).

(4) The mechanism responsible for initiating Ca²⁺ release may depend either on the RYRs or the InsP₃ receptors (I). RYR channels are sensitive to store loading and the InsP₃ receptors will be sensitized by the agonist-dependent formation of InsP₃.

(5) This initial release of Ca²⁺ is then amplified by regenerative Ca²⁺ release by either the RYRs or InsP₃ receptors, depending on the cell type (Dai et al. 2006; Sanderson et al. 2008). The resulting global Ca²⁺ signal often appears as a wave travelling down the length of the cell (Iino et al. 1994; Shaw et al. 2004).

(6) The global Ca²⁺ signal then activates contraction.

(7) The recovery phase depends on the sarco-endoplasmic reticulum Ca²⁺-ATPase (SERCA), that pumps some of the Ca²⁺ back into the ER, and the plasma membrane Ca²⁺-ATPase (PMCA), that pumps Ca²⁺ out of the cell.

(8) One of the effects of the released Ca²⁺ is to stimulate Ca²⁺-sensitive K⁺ channels such as the BK and SK channels that will lead to membrane hyperpolarization. The BK channels are activated by Ca²⁺ sparks resulting from the opening of RYRs.

(9) Another action of Ca²⁺ is to stimulate Ca²⁺-sensitive chloride channels (CLCA) (Liu & Farley, 1996; Haddock & Hill, 2002), which result in membrane depolarization to activate the Ca_V1.2 channels that introduce Ca²⁺ into the cell resulting in further membrane depolarization (ΔV). The entry of further Ca²⁺ completes a positive feedback loop that helps to speed up and complete the release of internal Ca²⁺.

(10) This depolarization can spread to neighbouring cells by current flow through the gap junctions to provide a synchronization mechanism in those cases where the oscillators are coupled together to provide vasomotion (Haddock & Hill, 2002,2005; Imtiaz et al. 2007). The membrane depolarization will activate Ca_V1.2 channels in neighbouring cells to provide a Ca²⁺ pulse that will help to initiate their Ca²⁺ transients thus bringing them into phase with each other. This synchronization mechanism is explored more fully later in a section on the slow waves generated by the ICCs.

Corpus cavernosum smooth muscle cells

The corpora cavernosum, which are two elongated bodies that lie on either side of the penis, fill up with blood during an erection. The sponge-like cavernous spaces are connected to the penile arteries that are lined with SMCs, which regulate the flow of blood into the corpora cavernosum. The penis is flaccid when these muscles are contracted, but becomes rigid when these muscles relax to allow blood to flow into the cavernous spaces. Contraction is activated by a cytosolic Ca²⁺ oscillator similar to that found in vascular and airway SMCs (Fig. 5). This oscillator generates pulses of Ca²⁺ that spread through the muscle cells as fast waves travelling at about 800 μm s⁻¹. This wave may spread to neighbouring cells by current flow through the gap junctions to synchronize the oscillators of neighbouring cells as described for the ICC phase waves (see below).

A large number of neural stimuli control the switch between muscle contraction and relaxation (Fig. 6). One of the primary regulators of contraction is the NA released from sympathetic nerves. The ICCs that have been described in the corpus cavernosum do not appear to play a direct role in regulating contraction but they express large amounts of cyclooxygenase-2 (COX-2) that functions in the synthesis of prostaglandin F_2α (PGF_2α), which diffuses across to activate the corpus cavernosum SMCs (Hashitani, 2006). NA and PGF_2α have two main actions. Firstly, they generate InsP₃, which activates the cytosolic Ca²⁺ oscillator. Secondly, they can activate the Rho/Rho kinase signalling pathway to increase the Ca²⁺ sensitivity of the contractile machinery (Wang et al. 2002). The Ca²⁺ transients also activate Ca²⁺-sensitive chloride channels (CLCAs) that result in membrane depolarization to activate voltage-operated channels (VOCs) (Craven et al. 2004; Hashitani et al. 2005), which not only introduce Ca²⁺ to modulate the oscillator but also create a flow of current to entrain the oscillatory activity of neighbouring cells to account for the way these corpora cavernosa cells contract in near-unison with each other.

Figure 6. The corpus cavernosum SMCs, which are poised between contraction and relaxation, are controlled by multiple stimuli released from neurons, endothelial cells and the interstitial cells of Cajal.

Some of the stimuli such as noradrenaline (NA) and prostaglandin F_2α (PGF_2α) drive contraction, whereas nitric oxide (NO), prostaglandin E₂ (PGE₂) and vasoactive intestinal peptide (VIP) control relaxation. See text for details of their signalling pathways. Reproduced from Berridge (2008), with permission.

A different set of signalling systems control the relaxation responsible for penile erection. One of the major stimuli is nitric oxide (NO) derived from non-adrenergic non-cholinergic (NANC) neurons. NO is also synthesized by the endothelial cells following stimulation by ACh released from the parasympathetic neurons. The ACh acts through muscarinic M3 receptors to stimulate InsP₃ formation and the release of Ca²⁺, which then activates endothelial nitric oxide synthase (eNOS) to produce NO. The NO causes relaxation by increasing the formation of cyclic GMP, which acts on various targets such as the BK channels and the Rho/Rho kinase systems, and it may also reduce the formation and action of InsP₃ to slow down the cytosolic oscillator. Prostaglandin E₂ (PGE₂), which is also synthesized in the endothelial cells in response to cholinergic stimulation and vasoactive intestinal peptide (VIP) released from neurons act by increasing the formation of cyclic AMP that can also promote relaxation, but the mechanisms are less well understood than those mediated by cyclic GMP.

Gastrointestinal and urethral smooth muscle cells

Gastrointestinal and urethral SMCs share a common activation mechanism in that they are driven by a pacemaker signal received from the ICCs (see mechanism C in Fig. 1) (Sergeant et al. 2000; Hirst & Ward, 2003; McHale et al. 2006; Sanders et al. 2006). These ICCs are long thin cells connected together through gap junctions to form a network (Komuro, 2006) that coordinates the activity of large populations of SMCs to form the peristaltic waves of contraction. In the urethra, the contractions close off the sphincter that prevents urine leaving the bladder during urine storage, but they relax when urine is voided. The neural control of these two functions depends on the ability of transmitters to modulate the pacemaker activity of the ICCs (Ward & Sanders, 2006). The ICCs are thus key regulators of gastrointestinal and urethral SMC activation.

ICC cytosolic Ca²⁺ oscillator model

The ICCs generate pulses of Ca²⁺, which are responsible for the periodic depolarizations known as slow waves that activate the inward currents that spread through the gap junctions providing the depolarization that activates the SMCs (see mechanism C in Fig. 1). Since the ICCs do not produce action potentials, excitation is propagated through the network using a potential-dependent mechanism to synchronize the individual oscillators. This ICC oscillator resembles that found in various SMCs (Fig. 5) but there are enough differences for them to be described separately. These differences are related to how the ICCs function to activate the SMCs. The endogenous muscle oscillator of the vascular and airway SMCs provides the internal Ca²⁺ signal that triggers contraction (Fig. 5), whereas the ICC oscillator generates the depolarizing signal that activates the local population of muscle cells (Fig. 7). The following hypothesis describes how the ICC pacemaker mechanism might be generated and synchronized (Fig. 7).

Figure 7. The cytosolic Ca²⁺ oscillator responsible for pacemaker activity in interstitial cells of Cajal releases periodic pulses of Ca²⁺ that form a Ca²⁺ wave.

The increase in Ca²⁺ activates Cl⁻ channels (CLCA) to give the spontaneous transient inward currents (STICs) that sum to form the spontaneous transient depolarizations (STD) resulting in the slow waves of membrane depolarization (see inset). Current flow through gap junctions allows these waves to spread into neighbouring smooth muscle cells to activate contraction. See text for a description of the oscillator that drives this activation process. Reproduced from Berridge (2008), with permission.

(1) The onset of each transient is probably a stochastic process that is primed by loading up the store with Ca²⁺ that then sensitizes the release channels, for example the ryanodine receptor 3 (RYR3), which is strongly expressed in ICCs (Aoyama et al. 2004), may also initiate transients. This early release appears as elementary events such as the Ca²⁺ puffs and sparks that activate the spontaneous transient inward currents (STICs) that occur during the pacemaker phase preceding each transient (Sergeant et al. 2006a).

(2) Once the elementary events reach a particular amplitude and/or frequency, they initiate a Ca²⁺ wave that spreads along the cell through the process of Ca²⁺-induced Ca²⁺ release (CICR) to form a Ca²⁺ wave. The wave terminates when release stops, perhaps because the decrease in Ca²⁺ concentration within the store inactivates the release channels.

(3) During the recovery period, Ca²⁺ is removed from the cytoplasm by three processes: it is extruded from the cell by the plasma membrane Ca²⁺-ATPase (PMCA); it is pumped back into the ER by the sarco-endoplasmic reticulum Ca²⁺-ATPase (SERCA) to begin reloading the store; and it is rapidly taken up by the mitochondria. The ICCs are tightly packed with mitochondria that are closely associated with the ER to form ‘pacemaker units’ that are a central feature of an alternative ICC oscillator model (for details see Sanders et al. 2006). In the model depicted in Fig. 7, the mitochondria have a more passive role as a temporary store of Ca²⁺, which is taken up rapidly during the recovery period and is then released slowly during the interburst period to help reload the store.

(4) Such store reloading is facilitated by entry of external Ca²⁺ and this accounts for why ICC Ca²⁺ oscillations are critically dependent on the external concentration of Ca²⁺ (Johnstone et al. 2005; Hashitani & Suzuki, 2007). When the concentration of external Ca²⁺ was either elevated or lowered, the baseline level of Ca²⁺ increased or decreased, respectively. Such marked changes in the resting level of Ca²⁺ are indicative of a high resting level of Ca²⁺ entry, which is essential for maintaining oscillatory activity. The nature of the ICC Ca²⁺ entry mechanisms is still being determined. In the case of the urethral ICCs, entry might be maintained by the Na⁺/Ca²⁺ exchanger (NCX) operating in its reverse mode (Bradley et al. 2006; McHale et al. 2006), but there may be other entry mechanisms such as T-type channels (Kito et al. 2005).

(5) The Ca²⁺ that is released during each wave activates Ca²⁺-sensitive chloride channels that set up the slow waves of membrane depolarization (see inset in Fig. 7). The early Ca²⁺ puffs generate the STICs that occur during the pacemaker depolarization (McHale et al. 2006).

(6) Summation of these STICs then results in the spontaneous transient depolarization (STD) that initiates each slow wave.

(7) In many cases, this initial pacemaker depolarization provided by the STD is enhanced by the opening of a VOC, which accounts for the rapid rising phase observed for many slow waves. Opening of the VOCs is not essential for the operation of this ICC oscillator, but the additional depolarization and entry of external Ca²⁺ have two important functions. Firstly, entry of Ca²⁺ helps release Ca²⁺ from the internal stores distributed down the length of the long thin ICC. If the recruitment of all the stores depended solely on CICR, the rate of rise of the slow wave would be much slower. This is evident when the opening of the VOCs is prevented by raising the external concentration of potassium to depolarize the membrane (Kito & Suzuki, 2003). The slow rate of rise can be explained by the fact that it takes time for the Ca²⁺ wave to travel down the long thin ICC cell when the VOCs are disabled. The Ca²⁺ coming from the VOCs thus serves as an entrainment signal to sharpen the wave front by speeding up the recruitment of the intracellular release channels located down the length of the long ER store. The second role for this rapid depolarization is to drive the phase wave that synchronizes the oscillatory activity of individual cells throughout large regions of the ICC network (see next section).

(8) The ICC pacemaker slow wave provides the depolarizing stimulus to activate the SMCs. Transfer of the activation stimulus from ICC to SMC is achieved through a passive flow of current through the gap junctions (Fig. 7). During the pacemaker slow wave, therefore, the inward current flow that discharges the membrane of the ICC simultaneously draws current from the SMCs that also begin to depolarize.

(9) SMC contraction begins when this depolarization reaches the activation threshold for the L-type Ca²⁺ channels that open to initiate excitation–contraction coupling as occurs in colonic SMCs (McCarron et al. 2004). There is a close relationship between the Ca²⁺ transient in the ICC, which initiates the slow wave, and the resulting Ca²⁺ transient in the SMC (Yamazawa & Iino, 2002; Hashitani & Suzuki, 2007). However, there is a striking difference in the time course of the two responses. The ICC Ca²⁺ responses last very much longer than the brief Ca²⁺ transient in the SMCs. The functional significance of this temporal difference may relate to the way in which the ICCs transmit their pacemaker signal to the SMCs. Since the ICC network does not produce action potentials, it has to transmit its message through a passive electrotonic mechanism that decays rapidly as it spreads through the gap junctions into the large population of SMCs. By generating a prolonged Ca²⁺ transient, the ICC maximizes its chances of spreading a meaningful signal to the SMCs. Another way of maximizing pacemaker activity is for all the ICCs within the network to operate together and this synchronization of oscillatory activity creates a phase wave as described below.

(10) Agonists such as ACh, NA and nitric oxide (NO) modulate ICC pacemaker activity (van Helden & Imtiaz, 2003). For example, ACh and NA accelerate the oscillator by increasing the formation of InsP₃ that then enhances the sensitivity of the InsP₃ receptor to the stimulatory action of Ca²⁺ (step 10 in Fig. 7). Conversely, inhibitory nerves that release NO have the opposite effect of reducing slow wave amplitude and frequency. This action of NO, which reduces the pacemaking drive, is mediated through cyclic GMP that acts through cyclic GMP-dependent protein kinase Iβ (cGKIβ) that phosphorylates the InsP₃ receptors to reduce their Ca²⁺ sensitivity (Sergeant et al. 2006b).

ICC phase waves

This ICC network coordinates the contractile activity of large groups of smooth muscle cells by synchronizing its individual oscillators (van Helden & Imtiaz, 2003; Park et al. 2006). This synchronicity, which has been referred to as a phase wave, is achieved by coupling oscillators together through a depolarizing signal spreading through the gap junctions (Kim et al. 2002; van Helden & Imtiaz, 2003; Kito et al. 2005; Imtiaz et al. 2006; Sanders et al. 2006). If slow waves are recorded from two cells widely separated from each other by distances of 4.5 mm, the regular waves of membrane depolarization occur almost exactly in phase with each other (van Helden & Imtiaz, 2003). In reality, there are small phase shifts that result from the fact that excitation spreads quickly down the network at 5–40 mm s⁻¹ to account for the waves of peristalsis. Phase waves can also be observed when the Ca²⁺ transients responsible for these slow waves are simultaneously recorded from separate ICCs (Park et al. 2006). This phase locking of ICC Ca²⁺ oscillators depends on a signal being passed through the gap junctions.

The phase wave spreads too fast to be explained by an intercellular Ca²⁺ wave, which has an upper propagation rate of about 100 μm s⁻¹. Most attention is now focused on the idea that the oscillatory activities of neighbouring cells are coupled together by a depolarization signal that spreads through the gap junctions. Since this oscillatory activity depends upon the spontaneous activation of InsP₃ receptors (Fig. 7), depolarization may entrain oscillations by enhancing the local supply of either the Ca²⁺ or InsP₃ necessary to trigger release. It seems most likely that the coupling mechanism is the depolarization-induced influx of Ca²⁺, which can rapidly initiate the release of Ca²⁺ from primed InsP₃ receptors. Since all the cells within the network have Ca²⁺ oscillators that pulse at similar frequencies, the cell that generates a Ca²⁺ signal first will provide the depolarization to entrain its neighbours. The importance of this depolarization is that it spreads electronically very rapidly to provide an influx of external Ca²⁺, which entrains neighbouring cells to release their pulse of Ca²⁺ in phase with everyone else.

The critical feature of this mechanism is that all the oscillators in the network are coupled together through a voltage-dependent mechanism. At any moment in time, an individual cell is either functioning as a pacemaker to initiate a Ca²⁺ transient or it is responding to the depolarization emanating from a neighbouring pacemaker cell. It seems that these two functions are not fixed, because cells can switch roles on a beat by beat basis (van Helden & Imtiaz, 2003). This system of coupled oscillators is highly dynamic in that each cell is not only capable of initiating each wave but it is also finely tuned to respond to an entrainment signal coming from any of its neighbours and this ensures that each beat is synchronous within a large population of cells.

Ureter smooth muscle cell

Transfer of urine from the kidney to the bladder is carried out by the pelviureteric system that has two components, the renal pelvis and the ureter. Spontaneous action potentials that begin in the proximal pelvis propel urine through the distal pelvis and ureter. Within the renal pelvis, the propagating action potentials are associated with a wave of Ca²⁺ that sweeps through the typical SMCs at a rate of approximately 1 mm s⁻¹ (Lang et al. 2007). Activation of ureter SMC contraction resembles most closely mechanism C in Fig. 1. The pelviureteric pacemaker mechanism responsible for triggering the periodic action potentials seems to be located in the atypical SMCs, which are long thin cells that form a loose network lying on top of the smooth muscle layer.

The pacemaker mechanism in these atypical SMCs is similar to that found in the ICCs (Fig. 7). They have a cytosolic Ca²⁺ oscillator that produces spontaneous transient depolarizations (STDs) to provide the depolarizing drive to trigger the action potentials that spread to the layer of typical SMCs (Lang et al. 2007). One important difference is that the oscillations in cytosolic Ca²⁺ activate an inward Na⁺ current carried by channels that remain to be determined rather than the Ca²⁺-sensitive Cl⁻ channels found in many ICCs. Like the ICCs, the L-type Ca²⁺ channels synchronize the STDs into large enough pacemaker events capable of triggering the action potentials. Despite the fact that the atypical smooth muscle cells generate STDs at a high frequency (10–40 min⁻¹), the action potentials that they initiate in the typical SMCs occur at a much lower frequency (6–15 min⁻¹) and this difference is caused by a prolonged refractory period induced by the Ca²⁺ that enters the cell during the action potential. During the recovery period, much of the Ca²⁺ released during the action potential is sucked up by the ER and this loading of the store sensitizes the RYR2s, which immediately begin to generate Ca²⁺ sparks (Burdyga & Wray, 2005). These sparks then activate BK channels and the resulting hyperpolarization inhibits action potentials thus resulting in a refractory period. Once the Ca²⁺ level in the stores returns to normal, the sparks decline and the membrane can now respond to the depolarizing drive coming from the atypical smooth muscle cells to initiate another action potential. This action potential spreads down the ureter to drive the peristaltic waves of contraction that propel urine into the bladder.

Conclusion

SMCs employ a great variety of Ca²⁺ signalling systems that are adapted to control their different contractile functions. Those that have to respond quickly use transmitters to induce the membrane depolarization and rapid influx of Ca²⁺ as occurs in the vas deferens. A membrane oscillator provides the periodic depolarizations necessary to drive contractions in bladder and uterine SMCs. A model for the uterus oscillator attempts to provide a mechanism to account for the pacemaker depolarization and how it might be modulated by oxytocin. Tonic SMCs, which are less reliant on membrane depolarization to initiate Ca²⁺ signals, are driven by a cytosolic Ca²⁺ oscillator. In some cases (vascular, airways and corpus cavernosum), this oscillator is located within the SMC whereas in others (gastrointestinal, urethra) it is located in specialized pacemaker cells such as the interstitial cells of Cajal (ICCs) that are connected to the SMCs through gap junctions. A luminal loading Ca²⁺ oscillator model can explain this spontaneous release of stored Ca²⁺ and how such oscillations can be accelerated by neurotransmitters and hormones. The periodic pulses of Ca²⁺ not only drive contraction but they also induce the membrane depolarization that couples the individual oscillators together to provide the characteristic synchronization of many tonic SMCs.