- Top of page
- Conflict of interest
Interstitial cells of Cajal (ICC) have a fundamental role in both the generation of electrical rhythmicity and neuromuscular transmission in the gastrointestinal tract (Sanders et al., 2006). Myenteric ICC (ICC-MY) form an extensive network in the myenteric layers between the circular and longitudinal muscle layers, which generate driving potentials that propagate to adjacent smooth muscle layers to initiate slow waves (Dickens et al., 1999). Another population of ICC (intramuscular ICC; ICC-IM), distributed between the smooth muscle cells within the circular layers, mediate signal transmission from both cholinergic excitatory and nitrergic inhibitory neurons to smooth muscle, and thus modulate spontaneous activity (Hirst and Ward, 2003).
ICC-like cells (ICC-LCs) have also been identified in non-gastrointestinal tissues, including the urinary tract and male genital system by their immunoreactivity against Kit receptor tyrosine kinase, a cell surface marker specific for ICC, or vimentin filament, a marker for cells of mesenchymal origin (Hashitani, 2006; Lang et al., 2006). However, the physiological function of ICC-LCs in non-gastrointestinal tissue still remains to be elucidated, with the exception of those in the rabbit urethra, which may act as electrical pacemakers such as ICC in the gastrointestinal tract (Hashitani et al., 1996; Sergeant et al., 2000; Johnston et al., 2005; Hashitani and Suzuki, 2007).
In general, blockers of L-type Ca2+ channels either abolish or largely diminish the electrical discharge of smooth muscle, although having little effect on ICC or ICC-LC activity. Thus, Ca2+ channel blockade has allowed researchers to differentiate ICC or ICC-LC activity from smooth muscle activity in situ (Yamazawa and Iino, 2002; Hashitani et al., 2003; Hennig et al., 2004; Hashitani and Suzuki, 2007). On the other hand, although ICC activity generally depends on Ca2+ handling by intracellular Ca2+ stores and mitochondria (Sanders et al., 2006), blockers of the function of these organelles would also be affecting neighbouring smooth muscle cells. Thus, insights into the role of ICC or ICC-LC in smooth muscle activity are not readily available in situ, except when genetically modified mouse models with loss-of-function mutations in the Kit signalling pathway are studied (Sanders and Ward, 2007). The identification of specific blockers for ICC or ICC-LC function should boost our understanding of the physiological importance of these cells, which are being discovered in many smooth muscle systems.
As both development and maintenance of ICC require Kit (Beckett et al., 2007), an inhibitor of Kit signalling could be used as a specific blocker for the function of ICC or ICC-LCs. Recently, imatinib mesylate, a Kit tyrosin kinase inhibitor has been shown to suppress contractile activity in various phasic smooth muscles, including the guinea-pig bladder (Kubota et al., 2004, 2006), human uterus and small intestine (Popescu et al., 2006) and guinea-pig gall bladder (Lavoie et al., 2007). Imatinib alters the pattern of action potential generation in the bladder (Kubota et al., 2004) and gall bladder (Lavoie et al., 2007), and exhibits comparable effects on spontaneous Ca2+ transients in both smooth muscle and ICC-LCs in the gall bladder (Lavoie et al., 2007). Relatively high concentrations of imatinib (50–100 μM) also suppress L-type Ca2+ channel and K-selective outward currents in single detrusor smooth muscle cells, indicating a nonspecific action (Kubota et al., 2004). However, none of the studies has demonstrated that imatinib, by inhibiting Kit signalling, specifically inhibits ICC or ICC-LCs.
In the present study, we have investigated the effects of imatinib on electrical, Ca2+ and mechanical activity of smooth muscles of the guinea-pig stomach, where the function of two distinct populations of ICC, ICC-MY and ICC-IM, has been well established (Dickens et al., 1999; Hirst and Ward, 2003).
- Top of page
- Conflict of interest
In the present study, imatinib (1–10 μM) suppressed spontaneous phasic contractions in antral smooth muscles of the guinea-pig as it does in other types of smooth muscle. Simultaneous recordings of electrical and mechanical activity from antral muscle strips revealed that the suppression of spontaneous contractions with imatinib (1 μM) was not associated with a reduction in the amplitude of their corresponding slow waves, suggesting that imatinib suppresses spontaneous contractions by inhibiting pathways that increase [Ca2+]i in smooth muscles rather than by specifically acting on Kit signalling in ICC. This was supported by the finding that imatinib (1 μM) reduced the amplitude of spontaneous Ca2+ transients by about 25%. In preparations where L-type Ca2+ channels had been blocked with nicardipine, imatinib (1 μM) was capable of further suppressing the residual Ca2+ transients by about 20%, thus it was not possible to determine if imatinib acts exclusively on either L-type Ca2+ channels or the release of Ca2+ from intracellular stores.
In the stomach and other smooth muscles, ICC determine the rhythmicity of spontaneous contractile and electrical activity (Dickens et al., 1999), whereas contraction amplitude is largely attributed to Ca influx through L-type Ca channels on the smooth muscle cells (Dickens et al., 2000). Thus, inhibition of ICC with imatinib would be expected largely to reduce the frequency of spontaneous activity. However, in various other smooth muscles, imatinib predominantly reduces the amplitude of spontaneous contractions without altering their frequency much (Kubota et al., 2004; Popescu et al., 2006), in a manner similar to the effects of nifedipine (Dickens et al., 2000). We have preliminary data demonstrating that imatinib (10 μM) reduces the amplitude of Ca2+ transients in detrusor smooth muscle of the guinea-pig bladder by about 20% (H Hashitani et al., personal observations). As detrusor smooth muscles generate spontaneous action potentials, which exclusively rely on the opening of L-type Ca2+ channels (Hashitani and Brading, 2003) and associated Ca2+ transients (Hashitani et al., 2003), this suggests that imatinib (10 μM) could inhibit Ca2+ influx through L-type Ca2+ channels, although previous studies showed an apparent lack of inhibition on action potentials with this concentration of imatinib (Kubota et al, 2004, 2006). After all, interpretation of the effects of imatinib in in vitro experiments should be treated with caution, particularly for contractile studies.
Imatinib (1–10 μM) increased the frequency of antral slow waves and corresponding contractions. As the rhythmicity of antral slow waves is determined by ICC-MY, which generate driving potentials to compose the first components of slow waves (Dickens et al., 1999), this suggests that imatinib may act on ICC-MY. This was consistent with its effect on the frequency of follower potentials recorded from longitudinal smooth muscles, which more directly reflect driving potentials generated by ICC-MY (Dickens et al., 1999). The rising phase of driving potentials results from the opening of T-type-like, voltage-dependent Ca2+ channels (Kito et al., 2002) that are sensitive to nickel or mibefradil (Kito and Suzuki, 2003; Kito et al., 2005). Imatinib has been shown to block recombinant T-type Ca2+ channels expressed in human embryonic kidney-293 cells by a protein tyrosine kinase-independent mechanism (Cataldi et al., 2004). However, imatinib did not alter the maximum rise slope of follower potentials, although their duration was reduced upon inhibition of a sustained component. Both depolarization and Ca2+ influx during the initial components evoke following sustained components by opening Ca2+-activated chloride channels (Kito et al., 2002). Thus, the sustained components are selectively diminished by blockers of Ca2+-activated chloride channels or SERCA, or by membrane depolarizations (Kito et al., 2002). Imatinib did not affect either the initial components of driving potentials or electrical coupling between ICC-MY and adjacent circular or longitudinal smooth muscles. Rather, imatinib might inhibit either SERCA or the opening of Ca2+-activated chloride channels to shorten the duration of pacemaker potentials, as does cycliopazonic acid (Kito et al, 2002). Such an inhibition of Ca2+ release is consistent with the suppression of Ca2+ transients by imatinib in the presence of nicardipine. Alternatively, imatinib might cause the release of neurohumoral factors to accelerate the generation of slow waves and therefore follower potentials.
If imatinib has an inhibitory action on the Ca2+ release from intracellular stores and the subsequent activation of Ca-activated chloride channels, it would be expected to suppress ICC-IM activity, which contributes to the secondary, regenerative components of slow waves (Suzuki and Hirst, 1999). Although inhibition of the regenerative components with imatinib was not obvious in whole layer antral preparations, both antral slow potentials and corporal slow waves were clearly suppressed. As these electrical events are exclusively generated by ICC-IM (Suzuki and Hirst, 1999; Hashitani et al., 2005), our results suggest that imatinib inhibits ICC-IM activity selectively. However, imatinib did not alter the frequency of corporal slow waves. Therefore, imatinib may nonspecifically inhibit intracellular Ca2+ handling and associated activation of Ca-activated chloride channels rather than inhibit ICC activity through its action on the Kit signalling pathway. In isolated detrusor smooth muscle cells, imatinib inhibits outward K current in a Ca2+-dependent manner (Kubota et al., 2004), suggesting that it inhibits intracellular Ca2+ handling rather than Ca2+-activated chloride channels.
The effects of imatinib on slow waves are small; therefore, the Kit receptors, although necessary for maintaining the ICC phenotype, are not directly required for the pacemaker mechanism. This result is consistent with data from previous studies where neither imatinib (Beckett et al., 2007) nor ACK-2, an anti-Kit antibody (Rich et al., 2002), had an effect on slow wave activity. Although this insensitivity of ICC to imatinib (1–10 μM) seems to argue against the usefulness of imatinib as a specific inhibitor of Kit-dependent ICC activity, this effect may be beneficial for its clinical applications. A lower concentration of imatinib (0.1 μM), which does not affect the function of differentiated ICC or ICC-LCs distributed in many types of smooth muscle, effectively suppressed proliferation of the GIST cell line in culture (Mukaisho et al., 2001; Tuveson et al., 2001). This suggests that imatinib used at these low concentrations is unlikely to have unwanted effects on smooth muscle activity. In addition, imatinib could well be used for the treatment of overactivity in smooth muscles, a condition that has been correlated with pathological differentiations of ICC or ICC-LCs. In the guinea-pig bladder, outlet obstruction increases the population of ICC-LCs and their Kit immunoreactivity (Kubota et al., 2007), suggesting that the altered distribution of ICC-LCs may contribute to the pathophysiology of bladder overactivity. This may well explain why imatinib was more effective at inhibiting spontaneous and evoked contractions in overactive bladders than in normal bladders (Biers et al., 2006). In conclusion, acute application of imatinib in vitro is unlikely to suppress ICC activity specifically through its inhibition of Kit signalling, instead it probably induces this effect by inhibiting intracellular Ca2+ handling. As imatinib is able to inhibit pathways that increase [Ca2+]i in smooth muscle at concentrations that do not affect ICC activity, it seems inappropriate to infer the effects of imatinib on ICC or ICC-LC activity after monitoring only smooth muscle function. Rather the effects of imatanib on identified ICC or ICC-LC activity need to be investigated directly.