Regulation of gastrointestinal motility by Ca²⁺/calmodulin-stimulated protein kinase II

Abstract

Gastrointestinal (GI) motility ultimately depends upon the contractile activity of the smooth muscle cells of the tunica muscularis. Integrated functioning of multiple tissues and cell types, including enteric neurons and interstitial cells of Cajal (ICC) is necessary to generate coordinated patterns of motor activity that control the movement of material through the digestive tract. The neurogenic mechanisms that govern GI motility patterns are superimposed upon intrinsic myogenic mechanisms regulating smooth muscle cell excitability. Several mechanisms regulate smooth muscle cell responses to neurogenic inputs, including the multifunctional Ca²⁺/calmodulin-stimulated protein kinase II (CaMKII). CaMKII can be activated by Ca²⁺ transients from both extracellular and intracellular sources. Prolonging the activities of Ca²⁺-sensitive K⁺ channels in the plasma membrane of GI smooth muscle cells is an important regulatory mechanism carried out by CaMKII. Phospholamban (PLN) phosphorylation by CaMKII activates the sarcoplasmic reticulum (SR) Ca²⁺-ATPase (SERCA), increasing both the rate of Ca²⁺clearance from the myoplasm and the frequency of localized Ca²⁺ release events from intracellular stores. Overall, CaMKII appears to moderate GI smooth muscle cell excitability. Finally, transcription factor activities may be facilitated by the neutralization of HDAC4 by CaMKII phosphorylation, which may contribute to the phenotypic plasticity of GI smooth muscle cells.

Introduction

The digestive tract is a muscular tube in which food is ingested, nutrients are absorbed, and waste is eliminated. Processing of nutrients requires movement of materials through the digestive tract, which is accomplished by the coordinated contractions and relaxations of the smooth muscles (tunica muscularis) comprising the various organs of the digestive tract. GI motility refers to the movement, or lack thereof, of material through the digestive tract. Motility patterns encompass the propulsive, mixing, and reservoir functions of the digestive tract necessary for the spatio-temporal processing of ingested food and elimination of waste products. Functional integration of multiple tissues and cell types is necessary to generate the various motility patterns characteristic of each organ in the digestive tract [1]. The smooth muscles of the digestive tract organs generate the force and perform the work; receiving regulatory input from various control systems, including motor neurons, ICC, hormones, paracrine substances and inflammatory mediators. GI smooth muscle cells also exhibit autonomous (myogenic) behavior arising from intrinsic regulatory pathways that can amplify or oppose signaling from the higher regulatory systems, depending upon the state of the resting membrane potential of the smooth muscle cells [2]. The modern definition of intrinsic (i.e. non-neural, non-hormonal) or myogenic regulation of gut motility now includes, in addition to smooth muscle mechanisms, the actions of ICC. The ICC affect the resting membrane potential of smooth muscle cells via gap junctions and generate the pacemaker activity that is the basis for electrical slow wave activity in phasic GI smooth muscles [3]. It is now recognized that the neurogenic mechanisms regulating GI motility patterns are superimposed upon the myogenic mechanisms. The enteric nervous system is of fundamental importance for generating major motor patterns, and regulating the amplitudes of contractions, but the smooth muscle response to external signals ultimately depends upon the excitability of the ICC-smooth muscle syncytium [4].

Regulation of smooth muscle excitability by membrane potential

Regulation of resting membrane potential is an important intrinsic mechanism that controls smooth muscle cell excitability by establishing the point from which smooth muscle cells respond to depolarizations from pacemaker cells and agonists [2]. Resting membrane potentials of GI smooth muscle cells vary widely in different regions of the gut (−85 mV to −40 mV), due to different ion channel expression patterns and open probabilities [5]. Membrane potential is coupled to opening of voltage-dependent Ca²⁺ channels, so depolarization of GI smooth muscle cells leads to Ca²⁺ influx and initiation of contraction. In regions with more negative membrane potentials, the open probability of Ca²⁺ channels is very low at the resting potential, and Ca²⁺ influx mainly occurs during excitable events such as slow waves and action potentials. These areas display phasic contractions generally timed by the frequency of slow waves. Phasic contractions contribute to organ level motility patterns such as peristalsis and segmentation. In regions with less negative potentials, there is low but regular openings of Ca²⁺ channels and continuous influx of Ca²⁺ (window current). These regions of muscle are characterized by tonic contractions and relaxation and contribute to motility patterns such as sphincter regulation and storage functions.

The wide range of resting potentials and electrical patterns of GI smooth muscles is mostly due to variable K⁺ channel expression of smooth muscle cells [6]. However, non-selective cation channels also contribute to the diversity in resting membrane potentials by shifting membrane potentials positive to the equilibrium potential of K⁺ ions. Several Ca²⁺-sensitive K⁺ channels expressed on smooth muscle cells are important determinants of GI smooth muscle excitability [2]. Investigations of the molecular basis of the Ca²⁺ sensitivity of these channels revealed a component regulated by phosphorylation [7–9]. CaMKII, a Ca²⁺/CaM-stimulated, multifunctional Ser/Thr protein kinase, modulates the activities of several Ca²⁺-sensitive proteins, enzymes, and ion channels [10–12]. Several studies have now clearly demonstrated the contribution of CaMKII in regulating the activities of Ca²⁺ sensitive K⁺ channels expressed by GI smooth muscles [8,9,13–15].

Structure-function of the multifunctional Ca²⁺/calmodulin-stimulated protein kinase II

It is now evident that the amplitude and frequency of cytosolic Ca²⁺ oscillations encodes information that is translated into physiological responses [16–18]. CaMKII has the physical, catalytic, and regulatory properties required of a molecular decoder of Ca²⁺ oscillations [19]. Understanding how CaMKII responds to cytosolic Ca²⁺ transients is critical to understanding how it modulates GI smooth muscle contractile activity. The present review briefly summarizes our current knowledge of the physiological roles of CaMKII in regulating GI motility. Considering the extensive studies demonstrating the importance of CaMKII to cardiac muscle and vascular smooth muscle physiology, and the well established and critical roles of CaMKII for the function of central nervous system neurons, there is a lack of corresponding comprehensive and focused studies of CaMKII in GI smooth muscles and the enteric nervous system. However, recent studies have begun to delineate the various proteins and ion channels regulated by this Ca²⁺ sensitive multifunctional Ser/Thr protein kinase in GI smooth muscles.

CaMKII is ubiquitously expressed. It is a family of the closely related α, β, γ, δ isoforms, which are the products of four separate genes [20]. At the mRNA level, different tissues express different patterns of CaMKII subunit isoforms [21–28]. The α isoform is restricted to neural tissues, while the β, γ and δ isoforms are widely distributed [29]. The CaMKII holoenzyme is a dodecamer of 12 kinase subunits [30–32]. Each kinase subunit consists of an N-terminal catalytic/regulatory domain and a C-terminal association domain, connected by a linker region containing a number of discrete variable domains (Figure 1). The variable domains undergo alternative splicing, giving rise to the 38 known mammalian isozymes [29]. The variable domains confer unique regulatory properties to the holoenzyme. For example, variable domain 3, which is only found in γ and δ splice variants, contains a nuclear import signal, allowing CaMKII holoenzymes containing these splice variants to enter the nucleus and regulate transcription factor activity [12].

Figure 1. Structure of the CaMKII holoenzyme.

A. Domain organization of the kinase subunits. B. Surface view from above depicts the 12 kinase subunits arranged as dimers in a ring that is coplanar with the central hub as revealed by small-angle X-ray scattering and X-ray diffraction data [25].

The neuronal αCaMKII gene encodes three splice variants [33]. αCaMK-II is important in learning and memory, but is not necessary during embryonic development [33].αCaMKII knockout mice display defects in specific fear responses, spatial learning and spatial memory [34–36]. The expression of αCaMKII in the enteric nervous system has not been examined.

βCaMKII is encoded as seven splice variants, none of which possess nuclear targeting sequences [29]. βCaMKII knockout mice are embryonic lethal [37]. βCaMKII expression is strongest in the adult brain, and is enriched in the cerebellum [38]. βCaMKII is also expressed in skeletal muscle, small intestine, and in endocrine tissues, such as the pancreas, adrenal, and pituitary [20,39]. We found weak expression of α and β CaMKII mRNA in gastric fundus and proximal colon smooth muscle tissues, but not in pure smooth muscle cells, suggesting that these two isoforms may be expressed in other cell types, such as enteric neurons [40].

The γCaMKII gene encodes 13 known splice variants [29]. γCaMKII is ubiquitously expressed, with highest expression in brain, oocytes and skeletal muscle and lowest in liver, testis, lung, thymus, and kidney [20,41]. It is essential for oocyte activation by regulating cell cycle resumption [41]. Multiple roles have been attributed to γCaMKII isozymes in the immune and endocrine systems [29].

The δCaMKII gene encodes at least 15 splice variants [29]. δCaMKIIs are important in embryogenesis and in cardiac and neuronal morphogenesis [33,42]. δCaMKII is highly expressed in brain, skeletal, vascular, and cardiac muscle, with significant quantities in intestine, liver, and lung [20,25,33,42,43]. In the heart δCaMKII phosphorylates several important Ca²⁺ handling proteins and channels with multiple functional consequences for cardiac myocytes [10].δCaMKII activity and expression are increased in cardiac hypertrophy, human heart failure, contributing to cardiac disease through excitation-transcription coupling [44–46].

The different CaMKII isoforms can co-assemble into heteromeric holoenzymes in vitro, raising the possibility that both homomultimers and heteromultimers of CaMKII exist in vivo [47–50]. However, except for brain, the subunit composition of CaMKII has not been determined in the tissues where its isoform expression has been investigated. Since the different subunit isoforms have different affinities for Ca²⁺/CaM, the holoenzyme composition also modulates the response of CaMKII to Ca²⁺ oscillations [19,23,51]. For example, homomeric CaMKII composed of either the α or β isoforms exhibit distinct variations in autophosphorylation and substrate phosphorylation rates [31]. These results suggest that different tissues express different subunit isoforms in order to assemble heteromeric CaMKII holoenzymes that optimally respond to specific frequencies and amplitudes of Ca²⁺ oscillations. However, except for brain, the subunit composition of CaMKII holoenzymes has not been determined in the tissues where its isoform expression has been investigated. We found that murine GI smooth muscle cells express γB, γC, γI, γJ, δA, δB, δC, δD, and δE [40]. The γ:δ CaMKII ratios are different in fundus and proximal colon smooth muscles, suggesting that these two functionally distinct GI smooth muscles express CaMKII holoenzymes with different subunit compositions and functional properties [40].

Early studies of the CaMKII three-dimensional structure suggested that the CaMKII holoenzyme consisted of two ‘wheels’ of six catalytic and regulatory domains tethered together by the twelve association domains (hub-and-spoke model) [31,52]. Other analyses suggested that there is actually an outer ring of 12 catalytic and regulatory domains, rather than two ‘wheels’ of six [53]. Higher resolution SAXS (small angle X-ray scattering) resolved this controversy, which showed that the CaMKII three-dimensional structure is highly unusual, with the 12 catalytic and regulatory domains organized into dimers, which are arranged in an outer ring that is coplanar with the ring formed by the association domains [54] (Figure 1). Another feature of the paired catalytic domain structure is that the ATP-binding site is occluded, and the T286 autophosphorylation site that confers calcium-independence to the enzyme, is blocked by the dimer interface [32,54]. Thus, catalytic domains dimers must separate to allow ATP access for kinase activity and to allow autophosphorylation for persistent activation by T286 autophosphorylation [32,54]. This structure also suggests that T286 autophosphorylation is not required for kinase activation, but subsequently prevents the reformation of inactive catalytic domain dimers [30,32]. This structural arrangement could allow for a circular array of switches around the holoenzyme with each of the six catalytic domain pairs acts as a separate “switch” [30,32]. The number of paired catalytic domains activated by Ca²⁺/CaM in each holoenzyme could potentially encode graded information [19,32]. In addition, the pattern of paired and unpaired catalytic dimers within a holoenzyme could have physiological significance by regulating subcellular targeting [30,32].

Following an increase in Ca²⁺, CaMKII is activated and autophosphorylated [55]. The holoenzyme kinase subunits are activated by Ca²⁺/CaM binding, resulting in phosphorylation of target substrates, and rapid intersubunit autophosphorylation of several sites [18,55]. Autophosphorylation of Thr286 (numbering according to α isoform, Thr287 for β, γ and δ) by adjacent subunits disrupts the interaction of the autoinhibitory domain with the catalytic domain, and converts the holoenzyme into a Ca²⁺/CaM independent kinase (autonomous activity) that allows CaMKII to continue to phosphorylate its substrates after cytosolic Ca²⁺ levels decrease [11,55]. In addition, the affinity of CaMKII for CaM increases about 1000-fold, a phenomenon termed ‘CaM trapping’ [56]. The ability of CaMKII to retain activity when Ca²⁺ levels decrease following prior activation by Ca²⁺ provides a form of ‘molecular memory’ that has been linked, for the α and β isoforms, to the processes of synaptic plasticity, learning, and memory [18,19]. The role of CaMKII in learning and memory is strongly supported by the αCaMKII knockout mouse model [57]. A modified form of memory has been linked to a prolongation of vascular tone in vascular smooth muscle [58]. Ca²⁺/CaM dissociation from the Thr286 phosphorylated protein allows subsequent autophosphorylation at Thr305, Thr306 and Ser314 to occur [59]. Autophosphorylation of Thr305 and Thr306 are inhibitory as they prevent further CaM binding [59]. However, Ser314 autophosphorylation has no effect on CaM binding [59]. Thus, the Thr286, and Thr305/306 autophosphorylation status further influences the response of the holoenzyme to Ca²⁺/CaM [18,19]. The physiological roles of autonomous CaMKII activity in GI smooth muscle cells is unclear, but may provide a mechanism for triggering changes in gene expression involved in the phenotypic remodeling of GI smooth muscle cells [60,61].

Ion channel regulation by CaMKII in GI smooth muscle cells

A component of the 4-aminopyridine (4-AP)-sensitive, tetraethylammonium (TEA)-insensitive, rapidly inactivating delayed rectifier current plays an important role in regulating the rhythmic electrical activity of the murine proximal colon [62]. This ‘A-like’ K⁺ current has properties similar to members of the Kv4 family of K⁺ channels [62]. Inhibition of this A-like current changes the pattern of electrical activity and induces repetitive Ca²⁺ action potentials [8]. CaMKII has been shown to regulate this delayed rectifier K⁺ current in murine colonic myocytes [8]. Whole cell studies revealed that the rate of inactivation of the delayed rectifier K⁺ current in murine colonic myocytes was increased by stronger Ca²⁺ buffering, and regulated by Ca²⁺ in the range of 10–100 nM. The CaMKII inhibitors KN-62 or KN-93 (but not the inactive KN-92 analog or PKC inhibitors) increased the rate of inactivation and slowed recovery from inactivation of the TEA-insensitive outward current, while dialysis of myocytes with constitutively active autothiophosphorylated CaMKII slowed the inactivation. The finding that CaMKII is a potent regulator of the TEA-insensitive current in colonic myocytes suggests a mechanism by which CaMKII is involved in regulating the pattern of electrical activity and thus the pattern of phasic contractions. CaMKII would be activated by the rise in cytosolic Ca²⁺ during bursts of action potentials, and phosphorylate the channel responsible for the A-type K⁺ current, resulting in a slowing of A-type current inactivation and reduced smooth muscle excitability [8].

A number of studies have suggested that members of the Kv4 family mediate the native A-type current in murine colonic myocytes [7,63]. Kv4.1 and Kv4.3 have consensus CaMKII phosphorylation sequences near the N-terminus of the channel protein which could mediate direct regulation of the Kv4 channels by CaMKII as has been suggested for Kv1.4 channels [64]. These channels undergo N-type inactivation similar to that shown by Kv4 channels and Kv1.4 phosphorylation slows inactivation and accelerates recovery from inactivation [64]. A similar mechanism contributing to the regulation of N-type inactivation of cloned Kv channels and the native GI smooth muscle delayed rectifier K⁺ currents was provided in a study by Sergeant et al., in which HEK-293 cells stably expressing the K_v4.3 channel were used to investigate the regulation of K_v4.3 currents by CaMKII [15]. Dialysis of cells with autothiophosphorylated CaMKII decreased the rateof inactivation and increased the rate of recovery from inactivation, while dialysis of cells with the CaMKII inhibitory peptide increased the rate of inactivation of K_v4.3 currents and decreased the recovery time from inactivation. KN-93 also increased the rate of inactivation of K_v4.3 currents and slowed the recovery from inactivation. CaMKII regulation of K_v4.3 currents appears to be mediated by direct phosphorylation of the pore-forming α-subunit. Site-directed mutation at Ser⁵⁵⁰ (i.e., S550A), a CaMKII consensus site near the COOH terminus, resulted in currents that were unaffected by dialysis with either autothiophosphorylated CaMKII or the CaMKII inhibitory peptide, suggesting that following activation by Ca²⁺/CaM, CaMKII slows the inactivation and accelerates the rate of recovery from inactivation of K_v4.3 currents by phosphorylation of Ser550. In addition, these currents inactivated more rapidly and recovered from inactivation at a slower rate than that of wild-type controls, suggesting that basal phosphorylation by autonomous CaMKII regulates the kinetics of wild-type K_v4.3 channels [15]. Studies have demonstrated that K_v4.3 is likely the major molecular determinant of the A-type current in GI smooth muscle cells and that this conductance participates in the maintenance of the resting potential [7,63]. Activation of K_v4.3 generates outward currents that oppose membrane depolarization and tend to stabilize membrane potential or reduce excitability [15]. Thus, CaMKII activation by elevations in cytosolic Ca²⁺ would slow K_v4.3 current inactivation and shift the window current range toward more positive potentials. CaMKII phosphorylation of the Kv4.3 pore-forming α-subunit would be expected to moderate membrane potential depolarizations in GI smooth muscle cells that use K_v4.3 currents to regulate membrane potential.

Another ion channel regulated by CaMKII is the small conductance Ca²⁺-activated K⁺ (SK) channel [9]. The inhibitory neurotransmitter ATP mediates a significant portion of the inhibitory regulation of GI smooth muscles [65]. Post-junctional responses to purinergic inhibition occur via stimulation of P_2Y receptors and activation of SK channels [66,67]. SK channel activation leads to outward current and reduced membrane excitability [9]. Localized Ca²⁺ release from IP₃ receptor-operated (IP₃R) stores in GI smooth muscle cells activates SK channels in response to ATP release [9]. SK channels transduce intracellular Ca²⁺ transients into changes in membrane potential to regulate membrane excitability and the open probability of voltage-dependent Ca²⁺ channels [68]. Under whole-cell patch-clamp conditions of colonic myocytes, SK channel openings are resolved as charybdotoxin-insensitive spontaneous transient outward currents (STOCs) [9]. In excised patches, autothiophosphorylated CaMKII increased the open probability of SK channels while boiled CaMKII had no effect. KN-93 reduced the frequency of charybdotoxin-insensitive STOCs, but did not affect spontaneous Ca²⁺-release events. SK2, the predominant SK channel isoform expressed by GI smooth muscles, has four potential CaMKII phosphorylation sites suggesting that CaMKII affects SK channel by direct phosphorylation [9]. CaMKII regulation of SK channel activity in GI smooth muscle cells indicates its role in the neurogenic regulation of colonic motility, since these channels are typically activated by purines, which serve as enteric inhibitory motor neurotransmitters. Regulation of SK channel open probability by CaMKII provides a mechanism for regulation complementary to local changes in Ca²⁺ concentration, increasing the effect of a given Ca²⁺ transient on SK channel open probability, and leading to enhanced SK channel open probability and facilitation of channel openings and inhibitory responses with repetitive stimulation [9].

Regulation of intracellular Ca²⁺ transport by CaMKII

While extracellular Ca²⁺ ions enter smooth muscle cells mainly through L-type Ca²⁺ channels during muscle action potentials, intracellular storage and release of Ca²⁺ can occur during and between action potentials [69,70]. The sarcoplasmic reticulum (SR) is the main organelle in smooth muscle cells that stores and releases Ca²⁺ [71]. In GI smooth muscles, the uptake of Ca²⁺ by intracellular stores tends to reduce excitability and contractility by removing Ca²⁺ from the myoplasm [72]. Ca²⁺ release from the SR, however, can also reduce excitability by activating Ca²⁺-sensitive K⁺ channels in the plasma membrane, hyperpolarizing cells and reducing voltage-dependent Ca²⁺ entry [9]. Ca²⁺ release events occur from either IP₃Rs (Ca²⁺ puffs), ryanodine receptors (RyR) (Ca²⁺ sparks), or from both types of receptors due to their Ca²⁺-sensitive properties and expression profiles [73,74]. Ca²⁺ puffs and sparks initiate STOCs in GI smooth muscles, affecting membrane potential and excitability [75,76]. Both SK and BK channels contribute to STOCs [77]. Ca²⁺ transients originating from the SR can also develop into intracellular Ca²⁺ waves that appear to propagate along a peripheral cellular compartment without activating contractile proteins [78]. Instead, the intracellular Ca²⁺ waves arising from Ca²⁺ puffs or sparks can hyperpolarize cells by increasing the open probability of Ca²⁺ sensitive K⁺ channels [14].

The level of luminal SR Ca²⁺ depends on the balance between Ca²⁺ efflux through Ca²⁺ release channels and Ca²⁺ influx through SERCA [71,79]. SERCA contributes to relaxation by lowering the [Ca²⁺]_i and decreasing the amount of Ca²⁺ available for contraction [80,81]. In addition, elevations in luminal SR Ca²⁺ load by SERCA increases the frequency of Ca²⁺ sparks and or puffs, which also contributes to relaxation by increasing the frequency of STOCs [82]. SERCA activity is under dynamic control by the SR membrane protein PLN to continuously vary the rate of Ca²⁺ influx into the SR via its phosphorylation levels, from maximal SERCA inhibition of 50% by dephospho-PLN to maximal SERCA activity by phospho-PLN [83,84]. Phosphorylation of PLN by cAMP-dependent protein kinase (PKA) or cGMP-dependent protein kinase (PKG) at Ser16, or by CaMKII at Thr17 removes its inhibitory effects on SERCA, thereby increasing Ca²⁺ uptake into the SR and contributing to relaxation [85–87]. Because the frequency and amplitudes of localized Ca²⁺ release events through ryanodine- or IP3-sensitive Ca²⁺ channels is in the range that will activate CaMKII [88], a number of investigations have examined the regulation of CaMKII activity and PLN phosphorylation by localized Ca²⁺ release events from internal Ca²⁺ stores of GI smooth muscles.

Gastric accommodation is a postprandial, vagally mediated reflex resulting in reduced tone of the proximal stomach that allows the proximal stomach to expand during food intake with no change in pressure [89]. Insufficient gastric accommodation is a frequent pathophysiological mechanism underlying functional dyspepsia [90]. Investigating the intrinsic excitation-contraction mechanisms of gastric fundus smooth muscles is necessary to enhance our understanding of stomach smooth muscle physiology and the pathophysiology of gastric motility disorders [91]. In gastric fundus smooth muscles, caffeine increases the frequency of ryanodine-sensitive Ca²⁺ release and causes relaxation that is sensitive to the Ca²⁺-sensitive K⁺ channel (K_Ca) blockers TEA and iberiotoxin, suggesting that membrane hyperpolarization and STOCs induced by large-conductance Ca²⁺-activated K⁺ channels contribute to fundus smooth muscle relaxation and gastric accommodation [92–94]. Increasing the luminal SR Ca²⁺ content increases RyR open probability, resulting in increased Ca²⁺ spark frequency [77,82,87,95]. SERCA activity and luminal SR Ca²⁺ content are increased by cAMP-dependent protein kinase (PKA) or CaMKII phosphorylation of PLN, suggesting that CaMKII activation and PLN phosphorylation are involved in gastric fundus smooth muscle relaxation by ryanodine-sensitive Ca²⁺ release [85–87].

In murine gastric fundus smooth muscles, caffeine was found to activate CaMKII in a tetracaine- and ryanodine-sensitive manner, suggesting that CaMKII activation involves increased Ca²⁺ spark activity [96]. Caffeine also increased PLN Thr17 phosphorylation, indicating that PLN is a substrate of CaMKII. The SERCA inhibitor cyclopiazonic acid (CPA) prevented caffeine-induced CaMKII activation and PLN phosphorylation, suggesting that SR luminal Ca²⁺ levels were lowered and insufficient to support caffeine-induced Ca²⁺ spark activity and CaMKII activation [96–98]. In addition, the caffeine-induced relaxation and PLN Thr17 phosphorylation were inhibited by KN-93 [96]. These findings suggest that KN-93 inhibition of CaMKII and PLN Thr17 phosphorylation mimics the effects of SERCA inhibition. These results suggest that the inhibition of caffeine-induced relaxation of gastric fundus smooth muscle by KN-93 involves inhibition of PLN Thr17 phosphorylation by CaMKII, resulting in SERCA inhibition and decreased Ca²⁺ spark activity. The findings that relaxation of gastric fundus smooth muscles by caffeine is ryanodine sensitive, and involves PLN Thr17 phosphorylation by CaMKII point to a role for both increased Ca²⁺ clearance from the cytosol and increased Ca²⁺ spark activity in gastric fundus smooth muscle relaxation. These findings provide further insights into the Ca²⁺-sensitive processes that modulate excitation-contraction of tonic GI smooth muscles.

It is well established that the relaxant effects of nitric oxide (NO) and NO donors on smooth muscles are mediated by cGMP, but the mechanisms of NO-induced relaxation appear to differ between different smooth muscle types [87,99]. The NO donor sodium nitroprusside (SNP) increases Ca²⁺ spark frequency and activates STOCs via an NO-soluble guanylyl cyclase (sGC)-cGMP pathway in cerebral and coronary artery myocytes [95]. Similarly, SNP-induced relaxation of the rat gastric fundus is partially ryanodine sensitive and involves small-conductance K_Ca channels, while in guinea pig gastric antral myocytes, SNP increases K_Ca current and enhances STOCs, which are sensitive to IP₃R inhibitors [100,101]. However, studies have also provided evidence that NO-induced smooth muscle relaxation involves uptake of myoplasmic Ca²⁺ into SR stores by increasing SERCA activity [102–105]. PLN phosphorylation by PKG, PKA, or calmodulin (CaM) kinase II eliminates its inhibitory effects on SERCA, thereby activating Ca²⁺ uptake into the SR and lowering cytosolic Ca²⁺ levels, causing relaxation [81,106]. However, SR Ca²⁺ load is also an important modulator of the frequency of Ca²⁺ release events from the SR [77,107]. If SR Ca²⁺ load is increased by SERCA activation, Ca²⁺ spark frequency is increased, leading to increased STOC frequency and membrane potential hyperpolarization and relaxation [77]. Removal of Ca²⁺ from the myoplasm and increasing the SR Ca²⁺ load and the frequency of Ca²⁺ release events from the SR may both contribute to relaxation in a complementary manner [72].

The NO donor SNP has been used to address the role of PLN phosphorylation by CaMKII in NO-induced relaxation of the functionally distinct proximal (fundus) and distal (antrum) stomach [108,109]. SNP activated CaMKII, hyperpolarized the cells, and relaxed gastric fundus and antrum circular smooth muscles. Western blot analysis showed that SNP increased PLN Ser16 and Thr17 phosphorylation, suggesting that SNP activated both PKG and CaMKII. The membrane-permeablec GMP analog 8-bromo-cGMP relaxed gastric fundus and antrum smooth muscles and increased PLNSer16 and Thr17 phosphorylation. PLNThr17 phosphorylation, but not PLNSer16 phosphorylation, was inhibited by KN-93 and CPA, suggesting that PLNThr17 phosphorylation by CaMKII involves Ca²⁺ release from the SR. In contrast, both PLNser16 and Thr17 phosphorylation were inhibited by the soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo-[4,3-α]quinoxalin-1-one (ODQ), suggesting that CaMKII activation by SR Ca²⁺ release is mediated by an NO-sGC-cGMP pathway [108,109]. These findings revealed a novel link between nitrergic and Ca²⁺/CaM-dependent signaling pathways in stomach smooth muscles. In response to cGMP production by sGC, PLNSer16 phosphorylation by PKG activates SERCA, increasing SR Ca²⁺ uptake and elevating SR Ca²⁺ levels. Higher SR Ca²⁺ levels increase the frequency SRCa²⁺ release events, activating CaMKII and increasing PLNThr17 phosphorylation. In addition, an increased frequency of Ca²⁺ release events through RyRs and IP₃Rs would likely contribute to smooth muscle relaxation by activating K_Ca channels, resulting in hyperpolarization of the membrane potential. The SNP-induced release of SR Ca²⁺ may also potentiate the direct regulation of K_Ca channels by PKG and CaMKII [110,111]. For example, as noted earlier, CaMKII increases A-type current activity in GI smooth muscles by slowing its inactivation, suggesting that SR Ca²⁺ release could oppose depolarization by activating CaMKII to slow A-type current inactivation [96,108,109]. The physiological significance of CaMKII activation by NO remains unclear; however, its Ca²⁺-independent activity may provide a mechanism to prolong the effects of NO/cGMPon SERCA and K_Ca channel activities after the cGMP and Ca²⁺ signals have ceased.

CaMKII in ulcerative colitis

Ulcerative colitis and Crohn’s disease are inflammatory bowel diseases characterized by chronic intestinal inflammation associated with dysmotility, resulting in symptoms that include abdominal pain, weight loss and bloody diarrhea [112–114]. Inflammation is associated with reduced colonic mixing patterns and impaired haustra formation, but increased propulsive motility from propagating contractions, causing rapid movement of the colonic contents to the rectum, accentuating the diarrhea [115–118]. Multiple mechanisms involving changes in enteric neurotransmission, afferent sensory input, and intrinsic smooth muscle contractility underlie the dysmotility [116,118].

Improper calcium mobilization by the smooth muscles has been implicated in the development of colonic dysmotility in inflammatory bowel disease. Impaired Ca²⁺ influx in smooth muscles from the inflamed colon has been reported to occur by reduced L-type Ca²⁺ channel expression or by decreased L-type Ca²⁺ channel activity with no change in expression [119,120]. In addition, SERCA expression in rat colon smooth muscle is decreased in experimental colitis, suggesting that intracellular Ca²⁺ mobilization in smooth muscles is disrupted due to reduced SR Ca²⁺ uptake [121]. Understanding the molecular mechanisms underlying the disruptions in Ca²⁺ mobilization and the impaired contractile responses of colon smooth muscles to intestinal inflammation is important for understanding the myriad factors underlying the myogenic basis of the dysmotility that is associated with colitis.

The activities and expression of proteins involved with intracellular Ca²⁺ mobilization in colon smooth muscle cells are altered by dextran sulfate sodium (DSS)-induced colitis [61]. Functional disruptions are evident by impairments in spontaneous and acetylcholine-evoked contractions of distal colon smooth muscles from mice with DSS-colitis. DSS-colitis also increased the amplitudes, but not the frequency, of intracellular Ca²⁺ waves in colonic myocytes. The expression of both SERCA and PLN are reduced, suggesting that SERCA and PLN down-regulation lead to disruptions in intracellular Ca²⁺ mobilization and the altered intracellular Ca²⁺ wave patterns. In contrast, γCaMKII expression and activity are increased in distal colon smooth muscles from mice with DSS-colitis. Along with the increase in CaMKII activity, cytosolic levels of HDAC4 are increased, and IκBα levels are decreased in distal colon smooth muscles from DSS-treated mice [61]. IκBα degradation is necessary for NF-kB translocation into the nucleus and activation of NF-κB-dependent gene expression [122]. Additional transcriptional co-activators, including histone acetyltransferases (HATs) and histone deacetylases (HDACs) are required for NF-kB activity [122]. Histone acetylation enhances gene expression, while HDACs repress gene transcription by deacetylating histones to allow DNA to re-wind around the histone octamer [122]. HDAC4 repression of gene transcription is regulated, in part, by nucleo-cytoplasmic shuttling [123]. HDAC4 gene repression is neutralized by Ca²⁺ signals that activate CaMKII to phosphorylate HDAC4 and sequester it in the cytoplasm, allowing its target genes to be de-repressed and transcribed [123]. These findings link alterations in intracellular Ca²⁺ mobilization and contractile responses with changes in SERCA, PLN, and γCaMKII protein expression in distal colon smooth muscles from mice with DSS-colitis. The findings that IκBα levels are reduced, and cytosolic HDAC4 levels and binding to CaMKII were increased, suggest that CaMKII activation and HDAC4 phosphorylation may facilitate NF-κB-dependent gene transcription that contributes to the reduced contractility of colon smooth muscles as a result of colitis [61].

Summary

Higher regulatory systems (enteric neurons, ICC, and hormones) control and coordinate gut motility. However, GI smooth muscle cells are equipped with intrinsic regulatory pathways that can amplify or oppose signaling from these higher regulatory systems. The smooth muscle cells of the gut wall generate the force and carry out the work of the digestive tract organs; thus it is important to investigate the intrinsic excitation-contraction mechanisms of GI smooth muscles to advance our understanding of GI smooth muscle physiology and the pathophysiology of GI motility disorders.

Calcium sensitive K⁺ channels involved in setting membrane potentials and the excitability of GI smooth muscle cells are regulated by CaMKII. Kv4.3 is responsible for the A-type K⁺ current in GI smooth muscle cells with an important role in the maintenance of resting membrane potential [15]. In murine colonic myocytes, phosphorylation of the Kv4.3 pore-forming α-subunit by CaMKII slows the rate of inactivation and accelerates the rate of recovery from inactivation. Thus, during depolarization-induced Ca²⁺ influx, CaMKII activation would tend to limit the extent of depolarization and moderate smooth muscle excitability. The inhibitory neurotransmitter ATP contributes to the inhibitory regulation of GI smooth muscles. Stimulation of P2Y receptors activates SK channels via localized Ca²⁺ release from intracellular Ca²⁺ stores, leading to outward currents and decreased membrane excitability. Activation of CaMKII increases the open probability of SK channels, enhancing the impact of localized Ca²⁺ transients on SK currents and facilitating the inhibitory effects of purinergic inputs to reduce smooth muscle cell excitability [9].

In gastric fundus and antrum smooth muscles, the NO donor SNP increases PLN phosphorylation and activates CaMKII, revealing a novel contribution of Ca²⁺/CaM-dependent signaling pathways to nitrergic relaxation in stomach smooth muscles [109]. PLN phosphorylation by CaMKII at Thr17 stimulates SERCA, increasing Ca²⁺ uptake into the SR [85–87]. Ca²⁺ clearance from the myoplasm into intracellular stores by SERCA tends to reduce excitability and contractility by reducing the availability of Ca²⁺ for contraction [72]. Elevations in luminal SR Ca²⁺ load also increase the frequency of Ca²⁺ sparks and or puffs, resulting in an increase in STOCs which hyperpolarizes the cells and reduces voltage-dependent Ca²⁺ entry [9,14].

Impaired Ca²⁺ mobilization by colonic smooth muscles has been implicated in the development of colonic dysmotility in inflammatory bowel disease. The findings that HDAC4 levels and binding to CaMKII are increased, and IκBα levels are decreased in an experimental colitis model suggest that CaMKII activation and HDAC4 phosphorylation may facilitate NF-κB-dependent gene transcription that contributes to the reduced contractility of colon smooth muscles as a result of colitis [61].

Overall, these studies suggest that the effects of CaMKII on the activities of K⁺ channels, intracellular Ca²⁺ handling proteins, and transcriptional activity, tend to reduce GI smooth muscle cell excitability. These findings are consistent with the additional regulation of MLCK by CaMKII in smooth muscle cells, in which MLCK phosphorylation by CaMKII decreases the Ca²⁺ sensitivity of myosin light phosphorylation, as has been reviewed elsewhere [124]. More work is needed to identify other targets and determine how spatio-temporal Ca²⁺ signaling regulates CaMKII expression, activity, and localization in GI smooth muscle cells. In addition, the expression and roles of CaMKII in ICC has not been studied. These cells are linked to the smooth muscle syncytium, so changes in membrane conductances, gap junction coupling, and pacemaker mechanisms could also be regulated by CaMKII. Finally, while CaMKII isoforms are expressed in enteric neurons little is known about how ionic conductances, Ca²⁺ handling mechanisms and reflex pathways are regulated by CaMKII in these cells. Sustained and focused studies of CaMKII in GI smooth muscles, ICC, and the enteric nervous system, are needed to obtain a more complete picture of its roles in the gut to more fully evaluate and appreciate its impact on GI motility, and to identify novel regulatory pathways for improved and innovative therapeutic strategies for GI motility disorders.