Calcium-associated mechanisms in gut pacemaker activity

Abstract A considerable body of evidence has revealed that interstitial cells of Cajal (ICC), identified with c-Kit-immunoreactivity, act as gut pacemaker cells, with spontaneous Ca2+ activity in ICC as the probable primary mechanism. Namely, intracellular (cytosolic) Ca2+ oscillations in ICC periodically activate plasmalemmal Ca2+-dependent ion channels and thereby generate pacemaker potentials. This review will, thus, focus on Ca2+-associated mechanisms in ICC in the gastrointestinal (GI) tract, including auxiliary organs.


Introduction
Research has shown that Ca 2+ -dependent plasmalemmal ion channels are responsible for interstitial cells of Cajal (ICC) pacemaker potentials [1][2][3], and spontaneous Ca 2+ activity in ICC is considered the primary mechanism. Namely, oscillations of the intracellular (cytosolic) Ca 2+ concentration ([Ca 2+ ]i) in ICC periodically activate plasmalemmal Ca 2+dependent ion channels, thereby generating pacemaker potentials. This review will, thus, focus on Ca 2+ -associated mechanisms in ICC in the gastrointestinal (GI) tract including auxiliary organs.
Numerous preparations and methods are used in studies of various types of ICC contained in the GI tract. In the following sections, types of ICC are identified in descriptions of most tissue-level experiments; the term ICC represents interstitial cells expressing c-Kit or other ICC markers in experiments using isolated cells and cultured preparations.

Voltage-gated Ca 2+ channels
Voltage-gated Ca 2+ channels (VGCC) are thought to play a central role in E-C coupling in smooth muscle  2+ antagonists, such as nifedipine and nicardipine largely depress contractile activity. It has been shown that the ␣1-subunit of the smooth muscle L-type Ca 2+ channel (Cav1.2b) is a splice variant of the cardiac one (Cav1.2a) and has a higher sensitivity to DHP Ca 2+ antagonists [4].
Guinea-pig stomach smooth muscle, frequently used to investigate mechanisms underlying spontaneous electrical activity, referred to as slow waves [5], provides a good example with which we can assess the role of VGCC in smooth muscle tissues showing spontaneous phasic contractions. DHP Ca 2+ antagonists completely abolish spontaneous contractile activity, with little effect, however, on pacemaker potentials (and electrical activity recorded from smooth muscle cells, that is, slow waves) [6,7]. Similar spontaneous electrical activities resistant to the DHP-Ca 2+ antagonist have been reported in several other GI smooth muscle tissues [8,9]. It is thus considered that DHP-sensitive L-type Ca 2+ channels play an essential role in E-C coupling in GI smooth muscle cells, although these channels are not involved in the generation of pacemaker electrical activity in ICC (Fig. 1). For this reason, DHP-Ca 2+ antagonists are frequently used to differentiate pacemaker electrical activity by suppressing smooth muscle activity. However, pacemaker cells in some tissues, for example, sub-mucosal ICC (ICC-SM) in the colon, produce different responses to DHP Ca 2+ antagonists: 1 µM nifedipine completely abolishes the spontaneous plateau potentials [10]. Furthermore, in the guinea-pig stomach, a small inhibitory effect was observed when nifedipine was greater than 10 µM [7]. In cardiac pacemaker cells, T-type (low voltageactivated [LVA]) Ca 2+ channels, known to play an important role in pacemaking, are suppressed with low concentrations (~40 µM) of Ni 2+ [11]. Applications of similar concentrations of Ni 2+ to guinea-pig stomach smooth muscle tissue (including the smooth muscle layer and myenteric plexus) have little effect on spontaneous electrical activity [7]. On the other hand, in the isolated circular smooth muscle layer, which does not contain myenteric ICC (ICC-MY) but contains only intramuscular ICC (ICC-IM), very low concentrations of Ni 2+ (1-10 µM) significantly suppress spontaneous electrical activity. The inhibitory effect is more potent in the plateau phase than in the initial upstroke of pacemaker electrical activity, agreeing well with the notion that ICC-IM produces re-generative potentials forming the plateau phase [12,13]. However, the discrepancy in the Ni 2+ concentration range may suggest the existence of Ni 2+ -sensitive mechanisms other than T-type Ca 2+ channels involved in ICC-IM. Recent reverse transcriptase-polymerase chain reaction (RT-PCR) examinations have provided supporting evidence that neither the L-nor the T-type Ca 2+ channel gene was detected in ICC-DMP (deep muscular plexus) and ICC-IM of the murine and human small intestine [14]. The existence of VGCC may differ depending on the ICC types, locations of the gut and species. Ward and Sanders [15,16] [19,20].

Non-selective cation channels
Non-selective cation channels (NSCC) can carry an electric charge for ICC pacemaking current, and many of these channels can permeate Ca 2+ . It is well-known that spontaneous electrical activities in GI smooth muscle tissues require extracellular Ca 2+ [5]. Therefore, NSCC may make a significant contribution to ICC pacemaking.
Under a voltage clamp condition, Thomsen et al. [21] and Koh et al. [22] recorded oscillating inward currents from cultured ICC of the murine small intestine. Removal of extracellular Na + abolishes the oscillatory inward currents [22], suggesting that oscillating inward currents are produced by the periodic activation of NSCC. Nakayama and Torihashi [23] showed that high concentrations (100-120 µM) of Cd 2+ and Ni 2+ suppress oscillatory inward currents in cell cluster preparations isolated from the murine small intestine, which contains ICC. Using a special thin muscle layer preparation made by enzymatic treatment under hydrostatic pressure, Goto et al. [24] demonstrated that depolarization steps can evoke large inward currents through NSCC in ICC showing spontaneous electrical activity. Transient receptor potential (TRP) homologues form NSCC. Epperson et al. [25] and Liu et al. [26] detected mRNA of classical (or canonical) TRP (TRPC), such as TRPC2, TRPC4 and TRPC6 in ICC, using RT-PCR. Torihashi et al. [27] showed immunohistochemical evidence for the expression of TRPC4 in the caveolae where numerous cellular signals interact. Walker et al. [3] recorded oscillatory inward currents similar to TRPC4: a NSCC inward current inhibited by Ca 2+ (see also Note added in proof).
Melastatin-type TRP (TRPM) homologues are channel/enzyme fusion proteins. TRPM6 and TRPM7 (formerly referred to as <TRPC>), which contain a kinase domain in the C-terminus, are well-known to act as Mg 2+ -permeable channels [28][29][30]. Kim et al. [31,32] showed mRNA of TRPM2, TRPM4, TRPM7 and TRPM8 in cultured ICC from the murine small intestine, and they reported that TRPM7 channels play an essential role in generating oscillatory currents in ICC; that is, the ionic selectivity and pharmacological properties are essentially the same between TRPM7 and ICC oscillatory currents. The authors also showed that the knockdown of TRPM7 by the use of siRNA suppressed spontaneous electrical activity in ICC. However, the reduction of TRPM7 expression may affect ICC pacemaking through intracellular Mg 2+ homeostasis and cell viability [30,33]. The regulation of intracellular Mg 2+ via TRPM-like Mg 2+ -permeable channels has been shown in intestinal [34,35] and vascular smooth muscle cells [36][37][38].
The frequency and duration of GI pacemaker activity are largely modulated by temperature and energy metabolism [23,39,40]. Nakamura et al. [41] suggested that in such modulations of pacemaker activity, several pathways are operating in parallel. Although the mRNA expression of TRPM4 and TRPM8 has been shown in cultured murine ICC [31,32], the existence of vanilloid type (TRPV) and ankyrin-like TRP (TRPA) channels has not yet been  assessed. TRPV1, TRPV2, TRPV3, TRPV4, TRPM2,  TRPM4 and TRPM5 are heat activated, whereas  TRPM8 and TRPA1 are cold activated [42]. Further investigation into TRP homologue channels may, therefore, clarify the mechanisms underlying the characteristic features of GI pacemaker activity. In addition, mitochondria [43] and sulfonylurea receptors (SUR) [44,45] may also contribute to the temperature-and energy-dependence of ICC pacemaking. ]i oscillations in ICC are thought to be a primary mechanism for the generation of pacemaker potentials, which may account for characteristic features of GI pacemaker activity, such as the low voltage sensitivity of the frequency. Publicover et al. [52]  ]i activity was observed in W/W v mice lacking ICC.

Cl channels
Using cell cluster preparations from the murine small intestine, Torihashi et al. [27] and Nakayama et al. [23] recorded [Ca 2+ ]i oscillations synchronized with spontaneous electrical and mechanical activities (Fig. 2). These results agree well with the hypothesis that [Ca 2+ ]i oscillations in ICC generate pacemaker electrical activity by periodically activating Ca 2+ -activated ion channels in the plasma membrane (Scenario 1 in Fig. 3) ]i oscillation in ICC follows the upstroke of the electrical activity recorded from a near-by cell, with a short delay (~60 ms). VGCC insensitive to DHP or VGSC [18][19][20]  ]i (Scenario 2 in Fig. 3). Further investigation is necessary to elucidate the details of mechanisms that link [Ca 2+ ]i oscillations and pacemaker potentials and to comprehensively address the cell-to-cell coupling among pacemaker cells and smooth muscle cells [58,59] ]i increase. Suzuki et al. [68] and Takano et al. [69] showed that spontaneous electrical and mechanical activities are greatly impaired in the stomach smooth muscle of mice lacking the type-1 inositol trisphosphate receptor (InsP3R1). Liu et al. [26] showed that among three InsP3R isoforms InsP3R1 and InsP3R2 are predominant in ICC in the murine stomach. Aoyama et al. [62] reported that InsP3R2 and InsP3R3 are predominant in the small intestine. Taken together, these findings suggest that InsP3R1 expressed in stomach ICC plays an important role in generating pacemaker activity on its own without using intercellular mechanisms, while the role of InsP3R1 may be substituted by InsP3R2 and/or InsP3R3 in small intestine ICC. Therefore, it is of interest to check whether spontaneous activity is preserved in the small intestine of mutant mice lacking InsP3R1.
There is an increasing body of pharmacological evidence for the involvement of InsP3R in ICC pacemaker activity. The applications of 2-aminoethoxydiphenyl borate (2-APB) and xestospongin C (Xe C), membrane-permeable blockers for InsP3R, suppress or terminate ICC electrical and [Ca 2+ ]i activities in numerous GI preparations (Table 1) [26,55,57,58,60,62,[70][71][72][73]. It has also been shown that the application of heparin with a reversible permeabilization loading procedure suppressed depolarizationinduced electrical activities reflecting ICC-IM activity [74]. On the other hand, 2-APB affects TRP homologue channels, including TRPM7 [75]. The inhibitory effect of 2-APB on [Ca 2+ ]i oscillations might involve the blockage of TRPM7 because this channel reportedly plays an essential role in generating ICC pacemaker activity [31,32].
Another important Ca 2+ release channel is the ryanodine receptor (RyR). Using cell cluster preparations from the murine small intestine, Aoyama et al. [62] showed that in addition to blockers for InsP3R and Ca 2+ influx, RyR blockers and FK506, which modulates RyR activity through FK506-binding pro- ]i oscillations in ICC-like cells of gut-like organ formed from mouse embryonic stem (ES) cells [76]. These results suggest that the coordination of the two families of Ca 2+ release channels, that is, RyR and  oscillations after the transfection of RyR3. This is also true for RyR2 (Aoyama et al. unpublished observation). Mice lacking RyR3 show apparently normal growth and reproduction [77]. In these mice, RyR2 may compensate for the role of RyR3.
In ICC-like interstitial cells of the rabbit portal vein and urethra, Harhun et al. [78] and Johnston et al. [79], respectively showed essentially the same pharmacological profiles of ]i activity of the GI tract [57,60,70,80]. Gastrointestinal stromal tumours (GIST), the most common mesenchymal tumours of the human GI tract, are thought to derive from ICC by gain-of-function mutations of c-Kit [81]. The application of the selective c-Kit-receptor inhibitor, imatinib mesylate, which is used to treat advanced GIST, suppresses myogenic activity of the human small intestine [82]. Furuzono et al. [83] reported that isolated ICC-like tumour cells from a human duodenal GIST with the most frequent type of gain-of-function mutation only occasionally produced spontaneous [Ca 2+ ]i activity. These ICC-like GIST cells expressed InsP3R1 and InsP3R2, but RyR2/3 were below detectable levels (Furuzono et al., unpublished observation [84,85]) are required.