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Trimethyltin chloride induced chloride secretion across rat distal colon
Haijie Yu*, Siliang Chen*, Zihuan Yang*, Ao Pan*, Geng Zhang*, Jiajie Shan*, Xiaojiang Tang† and Wenliang Zhou*1
*The School of Life Science, Sun Yat-sen University, Guangzhou 510275, China, and †Department of Toxicology, Guangdong Provincial Center for Occupational Disease Prevention and Treatment, Guangzhou, China
TMT (trimethyltin chloride), an organotin, is ubiquitous in the environment. The consumption of contaminated food may cause exposure of the human diet to this toxic compound. The present study was to investigate the effects of TMT on the regulation of ion transport across the rat distal colon. The rat colonic mucosa was mounted in Ussing chambers. The effects of TMT were assessed using the Isc (short-circuit current). Both apical and basolateral TMT induced, dose-dependently, an increase in Isc, which was due to a stimulation of Cl− secretion as measured using ion substitution experiments and pharmacological manoeuvres. The secretion was also inhibited by several K+ channel blockers administrated at the basolateral side. When the apical side was permeabilized by nystatin, the TMT-induced K+ conductance was effectively blocked by tetrapentylammonium, a Ca2+-sensitive K+ channel blocker. The response of TMT was sensitive to the basolateral Ca2+ and the intracellular Ca2+ store, which could be disclosed by applying the inhibitors of ryanodine receptors and inositol 1,4,5-trisphosphate receptors. In conclusion, TMT led to Cl− secretion, which was essentially regulated by basolateral Ca2+-sensitive K+ channels. These results suggest the importance of K+ channels in the toxicity hazard of TMT.
Key words: Cl− secretion, cystic fibrosis transmembrane conductance regulator (CFTR), K+ channel, rat distal colon, short circuit current, trimethyltin chloride
Abbreviations: 2-APB, 2-aminoethoxydiphenylborate, CA, carbonic anhydrase, (Ca2+)i, intracellular Ca2+, CF, cystic fibrosis, CFTR, CF transmembrane conductance regulator, COX, cyclo-oxygenase, CRAC, Ca2+ release-activated channel, DIDS, 4,4′-diisothiocyanato-stilbene-2, 2′-disulfonic acid, DPC, diphenylamine-2-carboxylate, ENaC, epithelial Na+ channel, IP3, inositol triphosphate, Isc, short-circuit current, KH, Krebs–Henseleit, NKCC, Na+-K+-2Cl− symport, PD, potential electrical difference, Rt, resistance, RyR, ryanodine receptor, TEA, tetraethylammonium, TMT, trimethyltin chloride, TPeA, tetrapentylammonium
1To whom correspondence should be addressed (email email@example.com).
Organotin compounds, ubiquitous environmental toxins, are industrially produced in large quantities for applications such as PVC stabilizers, glass coatings, biocides, additives for wood preservers and anti-fouling paints (Snoeij et al., 1987; Winship, 1988; Fent, 1996). TMT (trimethyltin chloride) is among the most toxic organotin compounds. It possesses both lipophilic and ionic properties. Therefore it is readily absorbed via the skin, lung and gastrointestine (Barnes et al., 1958). In marmosets, the first signs of poisoning included fine tremor, diarrhoea and salivation; later, these signs deteriorated to whole-body tremor, ataxia, agitation, aggression and loss of appetite (Brown et al., 1984). After injecting TMT into rats, a high concentration was found in the blood, liver, kidney and brain (Hasan et al., 1984). Accidental virulent exposures of humans to TMT have been documented (Saary et al., 2002). Although progress of research in the biological activities of TMT has been made, its specific mechanism still needs to be clearly investigated. The main mechanisms of the toxic effects of TMT were focused on oxidative stress (Skarning et al., 2002; Yoneyama et al., 2008), induction of apoptosis (Buck-Koehntop et al., 2005) and (Ca2+)i (intracellular Ca2+) (Yu et al., 2000; Florea et al., 2005a, 2005b).
The mammalian colon is a typical electrolyte-transporting epithelium and it has the ability to modify the electrolyte contents of the faeces with balance between absorption and secretion. Under physiological conditions, it absorbs water, sodium and chloride while secreting NaCl, as well as potassium and bicarbonate, through a number of ion channels, cotransporters and pumps located on the epithelial polar membrane (Barrett et al., 2000; Greger, 2000; Kunzelmann and Mall, 2002). All of these membrane proteins are subtly organized in an integrated system. They are elaborately modulated by the cytoplastic second message to form a successful chain-reacting function. For example, the CFTR [CF (cystic fibrosis) transmembrane conductance regulator], which can be regulated by intracellular cAMP, functions as an ion channel in epithelial cells. The cholera toxin causes diarrhoea and CF causes constipation, both of which result from abnormally activating and blocking the CFTR in colonic epithelium respectively (Littlewood, 1992; Ma et al., 2002). Therefore attempts to search for activators and inhibitors of CFTR are being made to find new therapies for ion-channel disease (Duszyk et al., 2001; Cuthbert, 2003).
In the present study, the poisonous effects of TMT and possible mechanisms underlying its actions were investigated in rat distal colonic epithelium. Our results demonstrated that TMT could lead to a Cl− secretion through the apical CFTR Cl-channel, and the secretion was essentially regulated by a basolateral TPeA (tetrapentylammonium)-sensitive K+ channel, which is dependent on the mobilization of (Ca2+)i.
2. Materials and methods
KH (Krebs–Henseleit) solution contained 117 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO4, 1.2 mM KH2PO4, 25 mM NaHCO3, 2.56 mM CaCl2 and 11.1 mM glucose, and it was bubbled with 95% O2 and 5% CO2 to attain a pH of 7.4 at 37°C kept by heated water jackets. In Cl−-free KH solution, NaCl, KCl and CaCl2 were replaced with the respective salts of gluconate, other compositions and conditions were not changed. In HCO3−-free solution, NaHCO3 was replaced by the same amount of NaCl and 10 mM Hepes was added. The gas contained 100% O2. In Cl−-free/HCO3−-free solution, the components were the same as the Cl−-free solution, except NaHCO3 was replaced by 25 mM sodium gluconate and 10 mM Hepes, and the gas was 100% O2. 2-APB (2-aminoethoxydiphenylborate), acetazolamide, amiloride, bumetanide, DIDS (4,4′-diisothiocyanato-stilbene-2, 2′-disulfonic acid), DPC (diphenylamine-2-carboxylate), forskolin, glibenclamide, indomethacin, nystatin, piraxicam, quinidine, Ruthenium Red, TEA (tetraethylammonium) and TPeA were purchased from Sigma–Aldrich. TMT was obtained from J & K Chemicals. TEA, TPeA and Ruthenium Red were dissolved in aqueous stock solutions. All of the chemicals were dissolved in DMSO. The final DMSO concentration in the experiments never exceeded 0.1% (v/v). The solvent was tested in the control tissue to exclude contamination by the solvent itself. For permeabilization studies, nystatin was used at a concentration of 200 μg/ml (Cermak et al., 2002), and was kept from light and ultrasonified before use.
2.2. Animals and tissue preparation
All animal procedures were approved by the University Ethics Committee. Male Sprague–Dawley rats (200–250 g of body weight) were obtained from Guangdong Medicine Experimental Animal Centre (Guangzhou, China). The animals were maintained on a 12h/12 h light/dark cycle, and had free access to normal water and food until they were killed by CO2 asphyxiation. A segment of the distal colon (∼5 cm) was removed and rinsed with ice-cold PBS saturated with air. The longitudinal and circular muscles were carefully separated by dissection. The left epithelial sample was then cut to an appropriate size.
2.3. Isc (short circuit current) measurement
The appropriate fresh colonic mucosa was mounted between the two halves of modified Ussing chambers with an internal area of 0.5 cm2. The chambers were filled with 5 ml of identical modified KH solution on both sides of the mucosa. Electrodes for measuring the transepthelial PD (potential electrical difference) and the passing current were connected to the chamber. The transepithlial PD was then clamped to 0 mV. The Isc was recorded by a VCC MC6 voltage–current clamp amplifier (Physiologic Instruments) and displayed using a signal collection and analysis system (BL-420S; Chengdu Technology & Market Company). Four identical pipette electrodes were made of sintered Ag-AgCl wire (World Precision Instruments) in agar bridges filled with a solution of 3% (by weight) agarose in 3 M KCl solution. One pair of pipette electrodes measuring the PD was called the voltage-sensing electrodes, and the other pair was called the current-passing electrodes, which recorded the electrical current across the mucosa. The change in Isc was defined as the maximal rise or fall in the Isc following the stimulus, and it was normalized to the current change per unit area of the epithelial monolayer (μA/cm2). The Isc value was expressed as positive when the current flowed from mucosal side to serosal side. Before the experiments were conducted, the samples were allowed to sit for 90 min to obtain equilibration. The transepithelial Rt (resistance) could be gained through Ohm's law by calculating the deviation when the tissue was stimulated by a series of given pulses.
The K+ current across the basolateral membrane was measured in the tissues apically permeabilized with 200 μg/ml nystatin. Also, an apical-to-basolateral K+ concentration gradient was maintained by the application of asymmetric buffers. The NaCl of the apical side was replaced by equimolar potassium gluconate, whereas basolateral NaCl was substituted with equimolar sodium gluconate (Cermak et al., 2002).
2.4. Data analysis
The changes in ion transport (ΔIsc) were given as the peak values by subtracting its respective baseline value before drug administration from the peak of a current response. Concentration–response curves in the present study were obtained using the computer program Origin 7 (OriginLab Corporation). The results are expressed as means±S.E.M. The difference between the two different groups was analysed using a Student's t test. A P value of less than 0.05 was considered to indicate statistical significance.
3.1. The effects of TMT on Isc
The effects of TMT on the Isc in the rat distal colonic epithelium were examined. In the Sprague–Dawley rat, the basal Isc in isolated distal colonic mucosa was 41.6±4.4 μA/cm2 (n = 26), and the tansepithelial Rt was 142.3±9.3 Ω·cm2. The changes in Isc were calculated as the current change over 30 min. The apical applications of TMT caused a concentration-dependent increase in the Isc. The waveforms with the different doses of TMT varied (Figure 1a). At a low concentration, TMT produced a persistent increase in current, whereas the response to higher concentrations was not maintained at its peak values, but eventually attenuated in a short time. The peaks at each concentration were used to depict the dose-dependent curves. The EC50 value was 36.4 μM (Figure 1b). Addition of TMT to the basolateral side also caused a similar change in the current (data not shown), which yielded an EC50 value of 39.7 μM (Figure 1b). Further Isc experiments were performed using TMT (50 μM) at the apical side only.
3.2. The effects of ionic replacement and inhibitors on TMT-induced ion transport
In order to study the ion species involved in mediating the TMT-induced Isc, a series of ion-substitution experiments were conducted (Figure 2). The removal of Cl− from the bathing solution by gluconate inhibited the TMT-induced Isc increase by 94.7% (n = 5, P<0.05; Figure 2a). In the experiments with epithelia incubated in the HCO3−-free solution, the Isc of TMT was suppressed by 58.0% (n = 5, P<0.01; Figure 2b). When both extracellular Cl− and HCO3− were removed, the TMT-induced Isc was reduced by 98.8% (n = 7, P<0.05; Figure 2c). All of the responses above were significantly smaller than that in the normal KH solution (ΔIsc was 88.1±5.6 μA/cm2, n = 6; Figures 2d and 2e).
The different Cl− channel blockers, such as DIDS and DPC were studied (Figure 2f). DIDS (100 μM, apically), a type of Ca2+-activated Cl− channel blocker, had no clear inhibitory effect on the TMT-induced current. In contrast, the subsequent application of DPC (1 mM, apically), a non-specific Cl− channel blocker, completely inhibited the TMT-induced Isc increase and a large part of the basal Isc as well, suggesting that a primary proportion of the basal Isc was due to Cl− secretion (Figure 2f). The same inhibitory response could also be observed in the basolateral stimulus of TMT (data not shown). However, glibenclamide, a blocker of CFTR, failed to inhibit the TMT-induced secretion in the present study (data not shown).
Bumetanide (100 μM, basolaterally), an inhibitor of NKCC (Na+-K+-2Cl− symport), and acetazolamide (100 μM, basolaterally), an inhibitor of CA (carbonic anhydrase), inhibited the TMT-induced Isc by various degrees. This proved that NKCC was in charge of Cl− uptake, and that CA was involved in the reaction of TMT (Figure 2g). After pretreating the bumetanide to exclude most Cl− secretion, the phenomena in which the acetazolamide had a partial depressive impact on the current induced by TMT, implied that CA had some effects on the secretion by TMT.
Consequently, these results indicated that the current evoked by TMT was mainly due to Cl− secretion, and CA was involved in this secretion. In this TMT-induced Cl− secretion, the apical Cl− channel and the basolateral NKCC played an important role.
3.3. The effects of a Na+ channel blocker
ENaCs (epithelial Na+ channels) were investigated because the Na+ reabsorption process was needed to maintain liquid transport in some epithelia, such as renal epithelia (Guan et al., 2005). The addition of amiloride (100 μM, apically), an ENaC blocker, had no evident effect after the tissue was stimulated with TMT. Also, the apical pretreatment of amiloride (100 μM, apically), had no significant suppressive influence on the TMT-induced Isc (ΔIsc was 80.5±11.6 μA/cm2, n = 5; Figure 3b) compared with the control group (ΔIsc was 89.1±10.6 μA/cm2, n = 5; Figure 3b). The two individual applications of TMT excluded the mediation of ENaC in this response.
3.4. The effects of a COX (cyclo-oxygenase) inhibitor
Previous work has shown that bradykinin (Cuthbert, 1999), angiotensin (Hosoda et al., 2000), substance P (Koon et al., 2006) and rutaecarpine (Wu and Hu, 2008), stimulated Cl− secretion by forming prostaglandins, which are able to increase intracellular cAMP to open the CFTR channel. The experiments in the present study were performed in epithelia pretreated with indomethacin (10 μM, basolaterally) or piroxicam (10 μM, basolaterally), COX inhibitors which could inhibit prostaglandin synthesis. It was observed that a considerable part of the basal Isc was suppressed by the pretreatment with either of these two inhibitors (Figure 4a), suggesting that a primary proportion of current was maintained by prostaglandin release which may be through the autocrine and paracrine pathways (Carew et al., 2000). A significant inhibition of the effects of TMT was found in the presence of indomethacin or piroxicam (Figure 4a). The inhibitory ratios were 80.1% (n = 5, P<0.05) and 69.8% (n = 5, P<0.05) respectively (Figure 4b). However, when the tissue was stimulated with indomethacin combined with a low concentration of forskolin (50 nM, basolaterally), which could enhance intracellular cAMP, the TMT-induced Isc response was restored (Figure 4c). In contrast, TMT did not have any effect on the tissue when the tissue had been treated with 10 mM forskolin (Figure 4d). These results indicated that the CFTR channel was the apical pathway for TMT-induced Cl− secretion. Secondly, compared with the opening state of the CFTR channel, prostaglandin was not needed to sustain the response of TMT. Otherwise, TMT could not induce any action in the presence of an appropriate concentration of forskolin because TMT was assumed to stimulate the prostaglandin release. However, it is necessary to have persistent epithelium-derived prostaglandin to maintain the normal function of the CFTR (Strabel et al., 1995; Schultheiss et al., 2008), thus other factors could exercise their special functions on this background. In the high concentration of forskolin experiment, TMT lost its action on the tissue, which also suggested that the CFTR was the pathway for the TMT-induced Cl− secretion, because forskolin and TMT shared the same pathway.
3.5. The effects of K+ channel blockers
K+ channels of the basolateral membrane were important for maintaining the driving force in Cl− secretion. In the rat colon, there appeared to be several distinct K+ channels (Schultheiss and Diener, 1997; Warth et al., 1999; Schroeder et al., 2000; Kunzelmann et al., 2001; Joiner et al., 2003). They could be activated by (Ca2+)i or by cAMP. In order to differentiate the K+ channels possibly involved in the secretory activity of TMT, we added diverse K+ channel blockers to the basolateral side of rat mucosa. When added before TMT, Ba2+ (5 mM, basolaterally) reduced the TMT-induced Isc by 52.1% (from 93.9±10.1 μA/cm2 to 45.0±7.8 μA/cm2, n = 5, P<0.05; Figure 5a). Pre-incubation with quinidine (100 μM, basolaterally), another non-specific K+ channel blocker, depressed the subsequent TMT-evoked Isc to 23.6 μA/cm2 (n = 5), which was 28.5% of the control response (94.9 μA/cm2, n = 5, P<0.05; Figure 5a). The effect of TMT on Isc was blocked in the presence of TPeA (50 μM, basolaterally), an inhibitor of the Ca2+-activated K+ channel. Its inhibitory ratio was 58.4% (from 103.8±9.2 μA/cm2 to 43.2±8.1 μA/cm2, n = 4, P<0.05; Figure 5a), suggesting an involvement of the Ca2+-activated K+ channel in the response of TMT. However, pre-incubation with TEA (5 mM, basolaterally), another K+ channel blocker, did not have an effect on the TMT-induced Isc (89.6±6.6 μA/cm2, n = 5; Figure 5a) compared with the control value (97.1±8.4 μA/cm2, n = 5; Figure 5a). The application of Ba2+ (5 mM, basolaterally), quinidine (100 μM, basolaterally) or TPeA (50 μM, basolaterally) after TMT significantly inhibited the Isc (Figures 5b–5d), also confirming the inhibitory effects of these K+ channel blockers.
To further determine whether or not TMT was activating a basolateral K+ conductance in colonic epithelia, the poreforming antibiotic nystatin (200 μg/ml) was used to bypass the apical membranes. After the addition of nystatin, the transepithelial Rt was changed from 156.4±11.4 Ω·cm2 to 77.8±9.1 Ω·cm2 (n = 5). The asymmetrical transepithelial ion gradients were also established as described under the Materials and methods section. Under these conditions, the monolayer was exposed to an apical-to-basolateral K+ gradient. When the nystatin-evoked basal K+ current had reached the semi-steady-state condition, TMT stimulated an increase in outward current, consistent with activation of basolateral membrane K+ conductance. This current increase could be inhibited by TPeA (50 μM, basolaterally), an inhibitor of the Ca2+-activated K+ channel, which indicated the involvement of the K+ channel in TMT-induced Cl− secretion.
3.5. The effects of TMT on the Ca2+ signalling pathway
It has been previously found that TMT could elevate (Ca2+)i in different cells, such as HeLa S3 cells (Florea et al., 2005a), SY5Y cells (Florea et al., 2005b), spiral ganglion cells and outer hair cells (Liu and Fechter, 1996). To investigate a possible role for Ca2+ in the response of TMT in rat colon, the effects of extracellular Ca2+ and the intracellular Ca2+ store were examined. The TMT-induced Isc increase was dependent on the extracellular Ca2+ because removing Ca2+ from a basolateral bath solution led to a significant reduction of the TMT response by approx. 55.9% (from 93.4±12.4 μA/cm2 to 41.3±5.5 μA/cm2, n = 5, P<0.05; Figures 6a and 6d). Intracellular membrane receptors such as RyRs (ryanodine receptors) or IP3 (inositol triphosphate) receptors were related to the release from the Ca2+ stores and CRACs (Ca2+ release-activated channels) that control the Ca2+ uptake (Siefjediers et al., 2007; Prinz and Diener, 2008). The presence of Ruthenium Red (50 μM, basolaterally), an inhibitor of RyRs, abolished the response of TMT by 89.0% (from 94.4±18.4 μA/cm2 to 10.4±3.1 μA/cm2, n = 5, P<0.05; Figures 6b and 6d). 2-APB (100 μM, apically and basolaterally), an inhibitor of IP3 receptors, inhibited the response of TMT by 78.4 μA/cm2, which was 79.8% of the control response (98.3 μA/cm2, n = 4, P<0.05; Figures 6c and 6d), suggesting that IP3 receptors were involved, as well as RyRs.
Both the apical and the basolateral application of TMT induced an Isc response across the rat distal colon in a dose-dependent pattern because TMT is not only water-soluble but also lipid-soluble. The similar dose-dependent curve suggested that TMT may pass through the cellular membrane easily to act at the intracellular site of the cell. The apical administration of TMT was fixed for the duration of the experiment in the present study because of the possible contamination of TMT in the gastrointestinal tract. Such a contamination may lead to an occurrence of acute diarrhoea. The waveforms of TMT varied in the different doses, which may be due to the diverse feedback mechanisms or the feedback speed when the tissue was faced with the various concentrations of TMT. Although the dose-dependent curve based on the change in the quantity of electricity would give more information, the curve based on the amplitude of the current change has helped us to fix the proper concentration of TMT and to tell the significant difference.
The TMT-induced Isc was mainly due to Cl− secretion. The TMT-evoked Isc was inhibited by substituting the Cl− ion with its counterpart of gluconate. Secondly, bumetanide caused an evident reduction in the current of TMT because of the inhibitory effect on the NKCC, which was mainly in charge of the entrance of Cl− into the epithelium from the serosal side. The increase in Isc was also abrogated by the non-specific Cl− channel blocker DPC. These results show that Cl− entered into the colonic epithelial cells through the basolateral NKCC, and the ions exited via the apical Cl− channel, to complete Cl− secretion. HCO3− had a reinforced action in the TMT-induced Cl− secretion, because of the descending current in the HCO3−-free KH solution. Furthermore, the current induced by TMT was sensitive to acetazolamide, an inhibitor of CA. In the colonic epithelium, the current of Na+ absorption and Cl− secretion had the same direction. The ENaC blocker, amiloride, was used to exclude the contamination of Na+ absorption. The TMT-induced Isc was not influenced by the amiloride. Accordingly, the results indicated that TMT stimulated Cl− secretion and fluid transport in rat colonic mucosa.
It has been reported that the toxic effects after the exposure to TMT might be due to an elevation of (Ca2+)i. However, DIDS, a Ca2+-activated Cl− channel blocker, could not suppress the TMT-induced current. On the other hand, DPC, which was able to block the CFTR at the concentrations used in the present study (Schultz et al., 1999), caused a cessation of the effects of TMT. These responses were unexpected, as the opening of the CFTR was mediated by the intracellular cAMP level rather than by (Ca2+)i. In the mammalian colon, epithelial cells were actively engaged in the transport of electrolytes and water. The CFTR located at the apical membrane played a predominant role in the process of harmonizing the digestion and absorption. The prostaglandins, as a kind of epithelium-derived secretagogue, were released though autocrine or paracrine ways to elevate the intracellular cAMP content, which could activate the apical CFTR as a result. Our results have shown that the prostaglandins were essential to support the TMT-induced Cl− secretion, because indomethacin and piroxicam, COX inhibitors, strongly inhibited the TMT-induced Isc for their putative inhibitory impact on the CFTR. However, the inhibited current was restored by increasing the intracellular cAMP level with forskolin, an adenylate cyclase stimulator, at a low concentration. The results suggested that the prostaglandins did not mediate the response of TMT, even though the forskolin was added. TMT still might not be able to activate the Isc response. However, the prostaglandins were necessary to keep the unremitting opening of CFTR to sustain the TMT-induced Isc increase. The persistent release of prostaglandins was crucial for other neurotransmitters and hormonal agents to function effectively. In a later experiment, TMT failed to potentiate any Isc increase when a high dose of forskolin elicited the Cl− secretion, indicating that both events occurred through the CFTR in the apical membrance of the rat distal colon. However, glibenclamide, another blocker of CFTR, did not inhibit the TMT-induced Cl− secretion. We do not have an explanation for the latter observation. A similar discovery was also reported in the Sprague–Dawley rat colonic mucosa treated with the flavonol quercetin (Cermak et al., 2002).
The activation of the basolateral K+ channel was required to maintain K+ circulation in the basolateral membrane. It could co-operate with NKCC to promote the driving force for apical Cl− secretion. There were two types of K+ channels located at the basolateral membrane of rat colon mucosa: Ca2+-dependent K+ channels and cAMP-activated KCNQ1/KCNE3 channels (Warth et al., 1999; Schroeder et al., 2000; Flores et al., 2007). Several K+ channel blockers were also utilized to examine the roles of K+ channels involved in Cl− secretion induced by TMT in rat intact colon. The response activated by TMT was effectively diminished by the non-selective K+ channel blockers Ba2+ and qunindine, but not by TEA. TPeA, an effective Ca2+-activated K+ channel blocker in human distal colon (Lam et al., 2004), had a strong inhibitory effect on the TMT-induced current. By using apical-permeabilized epithelium and the application of a K+ gradient, the K+ conductance was established. The K+ conductance activated by TMT was abolished by TPeA. These results indicate that the Ca2+-activated K+ channel took part in the TMT-induced Cl− secretion. In Figure 4(c), the low concentration of forskolin could recruit the intracellular cAMP to renerve CFTR, which had been inhibited by the pretreatment with indomethacin. In these conditions, the ability of CFTR may not be fully exerted, because the basolateral pathway for Cl− entry could be a hindrance to Cl− secretion. When the basolateral K+ channel was opened by the subsequent administration of TMT, the cask effect of Cl− secretion was shifted; this may also happen under physiological conditions. Besides the apical Cl− channel, the basolateral K+, including the Ca2+-activated K+ channel and cAMP-activated K+ channels both contributed to transepithelial Cl− secretion. However, the K+ channel could not have any action when the Cl− channel had been exhausted by a strong stimulator, such as a high dose of forskolin.
Our results demonstrated that Ca2+ played a predominant role in the Cl− secretion evoked by TMT in rat colonic mucosa. The manipulations of interference in Ca2+-dependent signalling pathways interrupted the response of TMT. This was indicated by the results that removing Ca2+ from basolateral KH solution abolished the TMT-induced Isc, which agreed with the discovery that the depletion of intracellular Ca2+ stores initiates store-operated Ca2+ entry (Yeromin et al., 2006). We then studied the mechanisms of Ca2+ stores underlying the TMT-evoked Cl− secretion. There were two main release channels reported at the intracellular Ca2+ store in rat colonic epithelia: RyRs and the IP3 receptors (Kocks et al., 2002; Siefjediers et al., 2007). Both RyRs, as well as the IP3 receptor, were involved in regulation of the response to TMT because of the depressive contribution of Ruthenium Red on RyR and the effective inhibition of the IP3 receptor by 2-APB. Redox regulation had much influence on Ca2+-dependent signal transduction, for example, the redox state of hydrosulfide groups could alter the protein character, as well as the IP3 receptor (Joseph et al., 2006) and RyR (Xia et al., 2000). In addition, the effects of oxidants on activating PKC (protein kinase C) and SERCA (sarco/endoplasmic reticulum Ca2+-ATPase) have also been described (Zima and Blatter, 2006). The oxidative stress induced by TMT was reported to be one of the harmful causes of neuronal damage and other tissue pathological changes (Skarning et al., 2002; Yoneyama et al., 2008). Consequently, in the present study, the enhanced oxidative stress induced by TMT might cause the disorder in intracellular Ca2+ homoeostasis, or interact with a key point in the Ca2+ signalling pathway. As a result, TMT activated the basolateral Ca2+-activated K+ channel to induce Cl− secretion across the colonic epithelium. All of these may provide the reason for TMT-related pathological diarrhoea.
The results derived from the present study demonstrated that, in rat colonic mucosa, TMT could increase intracellular Ca2+ which in turn stimulated the basolateral K+ channel to enhance Cl− secreted via the apical CFTR, whose opening state was sustained by prostaglandins. It might make a contribution to the oxidant-induced secretion under pathological conditions or it might prompt the use of a more potent agent, synthesized to increase Cl− secretion.
Haijie Yu designed the experiment and took part in the whole study. Siliang Chen performed the dose experiment, ion species experiment and cAMP signal experiment. Zihuan Yang took part in the cAMP signal and K+ channel experiments. Ao Pan, Geng Zhang and Jiajie Shan all prepared the colonic tissue. Ao Pan analysed the data and edited the Figures. Geng Zhang performed the dose-dependent experiments. Jiajie Shan performed the permeability experiments. Xiaojiang Tang took part in the Ca2+ signalling experiment and edited the paper. Wenliang Zhou supervised the experiment and wrote the paper.
This work was supported by the
Brown, AW, Verschoyle, RD, Street, BW, Aldridge, WN and Grindley, H (1984) The neurotoxicity of trimethyltin chloride in hamsters, gerbils and marmosets. J Appl Toxicol 4, 12-21
Buck-Koehntop, BA, Mascioni, A, Buffy, JJ and Veglia, G (2005) Structure, dynamics, and membrane topology of stannin: a mediator of neuronal cell apoptosis induced by trimethyltin chloride. J Mol Biol 354, 652-65
Florea, AM, Dopp, E and Busselberg, D (2005a) Elevated Ca2+i transients induced by trimethyltin chloride in HeLa cells: types and levels of response. Cell Calcium 37, 251-8
Florea, AM, Splettstoesser, F, Dopp, E, Rettenmeier, AW and Busselberg, D (2005b) Modulation of intracellular calcium homeostasis by trimethyltin chloride in human tumour cells: neuroblastoma SY5Y and cervix adenocarcinoma HeLa S3. Toxicology 216, 1-8
Flores, CA, Melvin, JE, Figueroa, CD and Sepulveda, FV (2007) Abolition of Ca2+-mediated intestinal anion secretion and increased stool dehydration in mice lacking the intermediate conductance Ca2+-dependent K+ channel Kcnn4. J Physiol 583, 705-17
Guan, Y, Hao, C, Cha, DR, Rao, R, Lu, W and Kohan, DE (2005) Thiazolidinediones expand body fluid volume through PPARγ stimulation of ENaC-mediated renal salt absorption. Nat Med 11, 861-6
Hasan, Z, Zimmer, L and Woolley, D (1984) Time course of the effects of trimethyltin on limbic evoked potentials and distribution of tin in blood and brain in the rat. Neurotoxicology 5, 217-44
Hosoda, Y, Winarto, A, Iwanaga, T and Kuwahara, A (2000) Mode of action of ANG II on ion transport in guinea pig distal colon. Am J Physiol Gastrointest Liver Physiol 278, G625-34
Joiner, WJ, Basavappa, S, Vidyasagar, S, Nehrke, K, Krishnan, S and Binder, HJ (2003) Active K+ secretion through multiple KCa-type channels and regulation by IKCa channels in rat proximal colon. Am J Physiol Gastrointest Liver Physiol 285, G185-96
Koon, HW, Zhao, D, Zhan, Y, Rhee, SH, Moyer, MP and Pothoulakis, C (2006) Substance P stimulates cyclooxygenase-2 and prostaglandin E2 expression through JAK-STAT activation in human colonic epithelial cells. J Immunol 176, 5050-9
Kunzelmann, K, Hubner, M, Schreiber, R, Levy-Holzman, R, Garty, H and Bleich, M (2001) Cloning and function of the rat colonic epithelial K+ channel KVLQT1. J Membr Biol 179, 155-64
Liu, Y and Fechter, LD (1996) Comparison of the effects of trimethyltin on the intracellular calcium levels in spiral ganglion cells and outer hair cells. Acta Otolaryngol 116, 417-21
Ma, T, Thiagarajah, JR, Yang, H, Sonawane, ND, Folli, C, Galietta, LJ and Verkman, AS (2002) Thiazolidinone CFTR inhibitor identified by high-throughput screening blocks cholera toxin-induced intestinal fluid secretion. J Clin Invest 110, 1651-8
Schroeder, BC, Waldegger, S, Fehr, S, Bleich, M, Warth, R, Greger, R and Jentsch, TJ (2000) A constitutively open potassium channel formed by KCNQ1 and KCNE3. Nature 403, 196-9
Siefjediers, A, Hardt, M, Prinz, G and Diener, M (2007) Characterization of inositol 1,4,5-trisphosphate (IP3) receptor subtypes at rat colonic epithelium. Cell Calcium 41, 303-15
Skarning, CR, Varhaug, LN, Fonnum, F and Osmundsen, H (2002) Effects of in vivo treatment of rats with trimethyltin chloride on respiratory properties of rat liver mitochondria. Biochem Pharmacol 64, 657-67
Warth, R, Hamm, K, Bleich, M, Kunzelmann, K, von Hahn, T and Schreiber, R (1999) Molecular and functional characterization of the small Ca2+–regulated K+ channel (rSK4) of colonic crypts. Pflugers Arch 438, 437-44
Xia, R, Stangler, T and Abramson, JJ (2000) Skeletal muscle ryanodine receptor is a redox sensor with a well defined redox potential that is sensitive to channel modulators. J Biol Chem 275, 36556-61
Yeromin, AV, Zhang, SL, Jiang, W, Yu, Y, Safrina, O and Cahalan, MD (2006) Molecular identification of the CRAC channel by altered ion selectivity in a mutant of Orai. Nature 443, 226-9
Yoneyama, M, Nishiyama, N, Shuto, M, Sugiyama, C, Kawada, K and Seko, K (2008) In vivo depletion of endogenous glutathione facilitates trimethyltin-induced neuronal damage in the dentate gyrus of mice by enhancing oxidative stress. Neurochem Int 52, 761-9
Yu, ZP, Matsuoka, M, Wispriyono, B, Iryo, Y and Igisu, H (2000) Activation of mitogen-activated protein kinases by tributyltin in CCRF-CEM cells: role of intracellular Ca2+. Toxicol Appl Pharmacol 168, 200-7
Received 18 June 2009/11 August 2009; accepted 16 September 2009
Published as Cell Biology International Immediate Publication 16 September 2009, doi:10.1042/CBI20090022
© 2010 The Author(s) Journal compilation. © 2010 Portland Press Ltd
ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
Published by Portland Press Limited on behalf of the International Federation for Cell Biology (IFCB)