Activating of ATP-Dependent K+ Channels Comprised of Kir 6.2 and SUR 2B by PGE2 Through EP2 Receptor in Cultured Interstitial Cells of Cajal from Murine Small Intestine

The interstitial cells of Cajal (ICC) are pacemaker cells in gastrointestinal tract and generate an electrical rhythm in gastrointestinal muscles. We investigated the possibility that PGE<sub>2</sub> might affect the electrical properties of cultured ICC by activating ATPdependent K<sup>+</sup> channels and, the EP receptor subtypes and the subunits of ATP-dependent K<sup>+</sup> channels involved in these activities were identified. In addition, the regulation of intracellular Ca<sup>2+</sup> ([Ca<sup>2+</sup>]i) mobilization may be involved the action of PGE<sub>2</sub> on ICC. Treatments of ICC with PGE<sub>2</sub> inhibited electrical pacemaker activities in the same manner as pinacidil, an ATPdependent K<sup>+</sup> channel opener and PGE<sub>2</sub> had only a dose-dependent effect. Using RT-PCR technique, we found that ATP-dependent K<sup>+</sup> channels exist in ICC and that these are composed of K<sub>ir</sub> 6.2 and SUR 2B subunits. To characterize the specific membrane EP receptor subtypes in ICC, EP receptor agonists and RT-PCR were used: Butaprost (an EP<sub>2</sub> receptor agonist) showed the actions on pacemaker currents in the same manner as PGE<sub>2</sub>. However sulprostone (a mixed EP<sub>1</sub> and EP<sub>3</sub> agonist) had no effects. In addition, RT-PCR results indicated the presence of the EP<sub>2</sub> receptor in ICC. To investigate cAMP involvement in the effects of PGE<sub>2</sub> on ICCs, SQ-22536 (an inhibitor of adenylate cyclase) and cAMP assays were used. SQ-22536 did not affect the effect of PGE<sub>2</sub> on pacemaker currents, and PGE<sub>2</sub> did not stimulate cAMP production. Also, we found PGE<sub>2</sub> inhibited the spontaneous [Ca<sup>2+</sup>]i oscillations in cultured ICC. These observations indicate that PGE<sub>2</sub> alters pacemaker currents by activating the ATP-dependent K<sup>+</sup> channels comprised of K<sub>ir</sub> 6.2-SUR 2B in ICC and this action of PGE<sub>2</sub> are through EP<sub>2</sub> receptor subtype and also the activation of ATP-dependent K<sup>+</sup> channels involves intracellular Ca<sup>2+</sup> mobilization.


Introduction
Prostaglandins (PGs) are widely distributed throughout the gastrointestinal tract and play a significant role in its physiology and pathophysiology [1][2][3]. In particular, PGE 2 is known to contract longitudinal muscle and to relax circular muscle in humans and in various animal species [4,5]. However, this action varies greatly, and depends on PGE 2 concentration, the organ, the species, and even the muscle layer studied [5][6][7]. Previous studies demonstrated that PGE 2 exerts its biological actions through binding to four specific membrane receptor subtypes known as EP 1 , EP 2 , EP 3 and EP 4 [8,9]. These subdividing are the basis of the relative potency of selective agonists and antagonists in both functional and binding studies.
Recent studies have shown that the interstitial cells of Cajal (ICC) act as pacemakers and conductors of electrical slow waves in gastrointestinal smooth muscles [10][11][12][13][14]. Although the precise mechanisms underlying these events remain unclear, there are many evidences that spontaneous intracellular Ca 2+ activities in ICC involve the producing of pacemaker action [15][16][17]. Also, several studies have suggested that endogenous agents such as neurotransmitters, hormones, and paracrine substances may modulate gastrointestinal tract motility by influencing ICC ion channels. In particular, recent reports have suggested that deoxycholic acid inhibits pacemaker currents by activating ATP-dependent K + channels through PGE 2 in ICC of the murine small intestine [18]. In addition, several molecular studies have shown that functional ATP-dependent K + channels are formed by a combination of a sulfonylurea receptor (SUR) and an inward rectifier K + channel subunit of the K ir 6 family [19][20][21][22][23].
Although previous studies have shown that PGE 2 influences motility in the small intestine [7,24] and that it also modulates ATP-dependent K + channels in ICC, the make up of ATP-dependent K + channels, the subtypes of the PGE 2 receptor and the regulation of intracellular Ca 2+ oscillations involved in PGE 2 action are unknown. Therefore, in this study, we investigated the possibility that PGE 2 affects the electrical properties of cultured ICC by activating ATP-dependent K + channels and the action of PGE 2 involves the mobilization of intracellular Ca 2+ oscillation in ICC. In addition, EP receptor subtypes and subunits of the ATP-dependent of K + channels involved in these effects were investigated.

Materials
Butaprost, sulprostone, and SC19220 were purchased from Cayman Chemicals (Ann Arbor, MI, USA); glibenclamide from RBI (Natick, NA, USA); and prostaglandin E 2 and SQ-22536 from Sigma (St. Louis, MO, USA). For stock solutions, all drugs were dissolved in distilled water or dimethylsulfoxide and stored at -20 °C.

Preparation of cells and tissues
All experiments were carried out according to the guiding principles for the care and use of animals approved by the ethics committee in Chosun University and the National Institutes of Health Guide for the care and Use of Laboratory Animals, and every effort was made to minimize both the number of animals used and their suffering. Balb/C mice (8-13 days old) of either sex were anesthetized with ether and sacrificed by cervical dislocation. Small intestines from 1 cm below the pyloric ring to the cecum were removed and opened along the mesenteric border. Luminal contents were removed by washing with Krebs-Ringer bicarbonate solution, tissues were pinned to the base of a Sylgard dish, and mucosa were removed by sharp dissection. Small tissue stripes of intestinal muscle (contained both circular and longitudinal muscles) were equilibrated in Ca 2+ -free Hanks solution (containing in mM: KCl 5.36, NaCl 125, NaOH 0.34, Na 2 HCO 3 0.44, glucose 10, sucrose 2.9 and HEPES 11) for 30 min. The cells were then dispersed in an enzyme solution containing collagenase (Worthington Biochemical Co, Lakewood, NJ, USA) 1.3 mg/ml, bovine serum albumin (Sigma) 2 mg/ml, trypsin inhibitor (Sigma) 2 mg/ml and ATP 0.27 mg/ml. Cells were plated onto sterile glass coverslips coated with murine collagen (2.5 ìg/ml, Falcon/ BD) in a 35 mm culture dish-, and cultured at 37 °C in a 95 % O 2 -5 % CO 2 incubator in SMGM (smooth muscle growth medium, Clonetics Corp., San Diego, CA, USA) supplemented with 2 % antibiotics/antimycotics (Gibco, Grand Island, NY, USA) and murine stem cell factor (SCF, 5 ng/ml, Sigma). Interstitial cells of Cajal (ICC) were identified immunologically with a monoclonal antibody for Kit protein (ACK 2 ) labelled with Alexa Fluor 488 (Molecular Probes, Eugene, OR, USA).

Patch clamp experiments
The whole-cell configuration patch-clamp technique was used to record cultured ICC membrane currents (voltage clamp) and potentials (current clamp, and Axopatch 1-D (Axon Instruments, Foster, CA, USA) amplified membrane currents and potentials. Command pulses were applied using an IBMcompatible personal computer and pClamp software (version 7.2; Axon Instruments). Data were filtered at 5 kHz and displayed on an oscilloscope, a computer monitor, and a pen recorder (Gould 2200, Gould, Valley View, OH, USA). The cells were bathed in a solution containing (in mM): KCl 5, NaCl 135, CaCl 2 2, glucose 10, MgCl 2 1.2 and HEPES 10, adjusted to pH 7.4 with tris. The pipette solution contained (in mM) KCl 140, MgCl 2 5, K 2 ATP 2.7, Na 2 GTP 0.1, creatine phosphate disodium 2.5, HEPES 5 and EGTA 0.1, adjusted to pH 7.2 with tris. Results were analyzed using pClamp and Graph Pad Prism (version 2.01) software. All experiments were performed at 30°C.

RT-PCR with c-Kit positive cell
First, we identified the c-Kit positive cells with under a confocal laser scanning microscope and then single cell with c-Kit positive was collected by applying negative pressure to a cell in contact with recording pipette, lifting the cell out of the bath, and immediately single cell was expelled from the pipette into PCR tube, which contained lysis buffer. Total RNA was isolated from c-Kit positive single cell using a RNeasy micro kit (Qiagen, catalog no. 74004). cDNAs were produced from the total RNA using Sensiscript reverse transcriptase kit (Qiagen, catalog no. 205213). Primers used as follows : EP 1   cAMP assay ICCs were preincubated with 100 µM IBMX for 30 min at 37 °C to inhibit cAMP degradation and then incubated with PGE 2 (5 µM) for 10 min. The sample was extracted by homogenization in buffer containing 4 mM EDTA to prevent enzymatic cAMP degradation, followed by heating for 5 min in a boiling water bath to coagulate protein. After centrifugation at 3,000 rpm for 5 min, the cAMP in the supernatants was transferred into a new tube and stored at 4°C. The samples were assayed for cAMP using the [ 3 H]-cAMP assay system (Amersham Pharmacia Biotech, Little Chalfont, UK).

Measurement of intracellular Ca 2+ concentration
Changes intracellular Ca 2+ concentration ([Ca 2+ ] i ) were monitored by using fluo-3/AM, which was initially dissolved in dimethyl sulfoxide and stored at -20 °C. The cultured ICC on coverslip (25 mm) were rinsed twice with a bath solution [in mM: KCl 5, NaCl 135, CaCl 2 2, glucose 10, MgCl 2 1.2 and HEPES 10, adjusted to pH 7.4 with tris], incubated in the bath solution containing 5 µM fluo-3/AM with 5% CO 2 -95% O 2 at 37 °C for 20 min, rinsed two more times with the bath solution, mounted on a perfusion chamber, and scanned every second with a confocal microscope (×200; fluoviews 300, Olympus). Fluorescence was excited at 488 nm, and emitted light was observed at 515 nm. During scanning of Ca 2+ imaging, the temperature of the perfusion chamber containing the cultured ICC was kept at 30 °C. The variations of intracellular Ca 2+ fluorescence emission intensity were expressed as F1/F0 that F0 means the intensity of first imaging.

Statistical analysis
Data were expressed as means ± standard errors. Differences were evaluated using the Student's t test. P values of < 0.05 were taken to be statistically significant. The n values reported in the text refer to the number of cells used in patchclamp experiments.

Effect of PGE 2 on pacemaker currents in ICC
Previous reports have described that the naturally occurring prostaglandins (PGs) comprise PGs D 2 , E 2 , F 2α , I 2 and TXA 2 [25]. In this study, because of their diverse action on the gastrointestinal tract, we checked the action of PGE 2 on pacemaker currents in cultured ICC. Under control conditions at a holding potential of -70 mV, the frequency, amplitude, and resting current levels were 13 ± 1.4 cycles/min, -360 ± 42 pA, and -27 ± 9 pA, respectively. When PGE 2 (1 µM) was applied to ICC, both the frequencies and the amplitudes of the pacemaker currents decreased, and the resting currents increased in the outward direction under voltage-clamp conditions (98 ± 11.4 pA) ( Fig. 1A and B). In addition, the corresponding frequencies and amplitude under these conditions were and D), respectively. Also, in current clamp mode (I=0), we examined the effect of PGE 2 on membrane potentials and pacemaker potentials of ICC. PGE 2 produced membrane hyperpolarization and decreased the amplitude of the pacemaker potentials (Fig. 1E). These results suggested that PGE 2 modulates pacemaker currents in cultured ICC. Furthermore, we found that the PGE 2induced effects were returned to the base-line level by treating with glibenclamide (10 µM), the ATP-sensitive K + channels blocker ( Fig. 1A and 1E).

Dose-dependency of the action of PGE 2 on pacemaker currents in ICC
In previous studies, we found that PGE 2 (1 µM) had an inhibitory effect on pacemaker currents in cultured ICC. In the present study, we tested that whether PGE 2 has a dose-dependent inhibitory effect on pacemaker currents in cultured ICC. Under a voltage clamp at a holding potential of -70 mV, ICC generated spontaneous inward currents. The mean frequency of these pacemaker currents was 13 ± 1.4 cycles/min and their mean amplitude and mean resting current level were -360 ± 42 pA and -27 ± 9 pA, respectively (n = 6). The addition of 10 or 100 nM PGE 2 slightly reduced the amplitude and frequency of these pacemaker currents; frequencies were 10 ± 2.6 cycles/min at 10 nM and 8.3 ± 3.2 cycles/min at 100 nM, and the resting currents and amplitudes were -24 ± 7 pA and -196 ± 32 pA at 10 nM and -20 ± 8 pA and -127 ± 26 pA at 100 nM (n = 7) (Fig. 2E, F and G), respectively. The presence of 10 or 100 nM PGE 2 slightly increased resting currents in the outward direction ( Fig. 2A and B). In the presence of 1 or 10 µM PGE 2 under voltage-clamp conditions, pacemaker currents were largely inhibited and resting currents were increased in the outward direction ( Fig. 2C and D). The inhibitory frequencies and amplitudes by PGE 2 were 2.1 ± 1.8 cycles/min and -20.9 ± 16 pA at 1 µM PGE 2 and 1.8 ± 1.4 cycles/min and -16 ± 19 pA at 10 µM PGE 2 , respectively. The resting current levels were 95 ± 5.9 pA D C E B A significantly different from the control (p < 0.05). Dotted lines indicate zero current levels. (E) Pacemaker potentials from ICC exposed to PGE 2 (1 µM) in current clamp mode (I=0). PGE 2 caused membrane hyperpo-larization and decreased amplitude of pacemaker potentials. Glibenclamide (10 µM) also blocked the effects of PGE 2 on pacemaker potentials in ICC. at 1 µM PGE 2 and 90 ± 7.2 pA at 10 µM PGE 2 (n = 7) (Fig. 2E, F and G). These results suggest that PGE 2 inhibits pacemaker currents in a dose-dependent manner in cultured ICC.

Localization of and the molecular expression of ATP-dependent K + channels in ICC
Recently, we showed that pinacidil inhibits pacemaker currents, and activates outward currents, which are antagonized by glibenclamide, suggesting that ATP-dependent K + channels exist in ICC, and that the activity of ATP-dependent K + channels in ICC may be involved in the action of pacemaker currents [18]. In this study, K ir subunits and SURs were identified by RT-PCR method. To determine the presence of K ir subunits and SURs, RT-PCR with c-kit positive single cell was performed using K ir 6.1, K ir 6.2, SUR1, and SUR2B genespecific primers in ICC. In case of smooth muscle cell, the expression of K ir 6.2 and SUR2B was predominant (Fig. 3A). Also, in ICC, RT-PCR detected transcripts for K ir 6.2 and SUR2B (Fig. 3B), but the specific primers K ir 6.1 and SUR1 did not produce cDNA fragments of the appropriate size in smooth muscle and ICC cell (Fig. 3A  and B). This finding indicate that ATP-dependent K + channels exist in cultured ICC and that they are composed of K ir 6.2 and SUR 2B. Characterization of the EP receptor subtypes, involved in the effects of PGE 2 on pacemaker currents in ICC Four subtypes of EP receptor have been identified to date, and have been arbitrarily named EP 1 , EP 2 , EP 3 , and EP 4 . In the present study, we attempted to discern which of these EP receptor subtypes mediate the inhibitory actions of PGE 2 on pacemaker currents in cultured ICC. First, we examined the effects of butaprost, a specific agonist for the EP 2 receptor subtype, on pacemaker currents in cultured ICC. The addition of butaprost (1 µM) caused a reduction in spontaneous inward currents frequencies and amplitudes in cultured ICC (Fig. 4A) and increased resting currents in the outward direction (n = 5) (control vs. butaprost; resting currents, -56 ± 5 pA vs. 86 ± 5.4 pA; amplitudes -370 ± 39 pA vs. -25 ± 12 pA; and frequencies, 15 ± 1.9 cycles/min vs. 3 ± 2.7 cycles/ min) (bar graph not shown). These results are similar to those of PGE 2 treatments (as shown in figure1) and also glibenclamide pre-or co-treatment (1 µM, an ATPdependent K + channel blocker) blocked the effects of butaprost on pacemaker currents ( Fig. 4A and B). Sulprostone (an EP 3 and EP 1 receptor agonist; 1 µM) had no effects on the frequency or amplitude of pacemaker currents in ICC, and had no effects on resting pacemaker currents in cultured ICC (data not shown). In addition, the pretreatment of ICC with either SC19220, an EP 1 receptor antagonist, (1 µM) or co-treatment with SC-19220 (1 µM) and sulprostone (1 µM) did not have any effects on pacemaker currents (Fig. 4C). Also, PCR Fig. 3. Agarose gels of the RT-PCR products of the subunits of ATPdependent K + channels using single smooth muscle and ICC cell. This representative 1.2 % agarose gel was loaded with 5 ml of PCR product and stained with ethi-dium bromide. The markers shown in lane indicate bp. (A) K ir 6.2 and SUR 2B primers produ-ced the expected pro-ducts in single smooth muscle cell (lanes 5 and 7) (K ir 6.2: 340 bp, SUR 2B: 347 bp). However, K ir 6.1 and SUR 1 primers failed to produce their respective product bands. (B) K ir 6.2 and SUR 2B primers produced the expected products in single ICC (lanes 5 and 7) (K ir 6.2: 340 bp, SUR 2B: 347 bp). However, K ir 6.1 and SUR 1 primers failed to produce their respective product bands. assays with c-kit positive single cell using EP 2 receptors primers yielded a product of the appropriate size (245 bp). We found that the EP 2 PCR product was produced from c-kit positive single cell, but that EP 1 , EP 3 , and EP 4 were never amplified (Fig. 4D). These results suggest that PGE 2 may affects ATP-dependent K + channels in ICC by stimulating EP 2 subtype receptors.

PGE 2 -induced pacemaker currents inhibition is not mediated via cAMP pathway
To investigate the involvement of cAMP on the effects of PGE 2 in ICCs, we used SQ-22536, an inhibitor of adenylate cyclase, cell-permeable 8-bromo-cAMP, and cAMP assays. The preincubation of ICCs with SQ-22536 (100 µM) for 10 min had no effects on the control states of the pacemaker currents, and co-treatment with SQ-22536 (100 µM) and PGE 2 (1 µM) inhibited the pacemaker currents (n = 5) (Fig. 5A), thus indicating that SQ-22536 had no influence on PGE 2 -induced pacemaker currents inhibition. In addition, the cell-permeable 8-bromo-Camp (100 µM) had no effect on the generation of pacemaker currents (n = 4) (Fig. 5B). Moreover, to evaluate whether a change in cAMP content is involved in the effect of PGE 2 on ICCs, intracellular cAMP contents were measured under basal and PGE 2 -stimulated conditions. 5 µM of PGE 2 did not stimulate cAMP production (control: 12.5 ± 1.5 vs. PGE 2 : 13.1 ± 2.9 pmol/mg -1 protein) (Fig. 5C). These results indicate that cyclic AMP does not mediate the actions of pacemaker currents induced by PGE 2 .

Involvement of [Ca 2+ ] i on PGE 2 -induced action in ICC
Because many reports suggested [Ca 2+ ] i oscillations in ICC are considered to be the primary mechanism for the pacemaker activity in gastrointestinal activity, we examined the effect of PGE 2 on [Ca 2+ ] i oscillations in ICC. In this study, we measured spontaneous [Ca 2+ ] i oscillations of ICC which are connected with cell clusters. Spontaneous [Ca 2+ ] i oscillations observed in many ICC (Fig. 6A) which was loaded with fluo3-AM. And in the presence of 1 µM PGE 2 , [Ca 2+ ] i oscillations in ICC rapidly was declined (Fig. 6C). Also, spontaneous [Ca 2+ ] i oscillations inhibited by PGE 2 in ICC was recovered by co-treatment of 10 µM glibenclamide (Fig. 6E). The data of time series are summarized in Fig. 6B, D, and F). These results suggest that the action of PGE 2 on ICC may involve the regulation of spontaneous [Ca 2+ ] i oscillations.

Discussion
Prostaglandins (PGs) act as local regulatory agents, and control smooth muscle contractile activity. Moreover, PGE 2 has been shown to contract intestinal longitudinal smooth muscle and relax circular smooth muscle [26,27], implying that PGE 2 may regulate gastrointestinal motility. Furthermore, since ICC generate electrical slow waves that are the basic determinants of gastrointestinal motility, PGE 2 may have effects on ICC slow waves and control gastrointestinal motility. In the present study, we investigated pacemaker currents inhibition by PGE 2 by modulating ATP-dependent K + channels composed of K ir 6.2 and SUR 2B in ICC and characterized the EP 2 receptor subtypes involved in the inhibition of pacemaker currents by PGE 2 . We also showed the action of PGE 2 on pacemaker currents in ICC was not mediate via cAMP pathway and involved the regulation of spontaneous [Ca 2+ ] i oscillations generated by ICC.
Fatty acid cyclooxygenase in the gastrointestinal tract converts eicosateraenoic acid (arachidonic acid) primarily to prostacyclin (prostaglandin I 2 ), and, to a lesser extent, to PGE 2 , PGF 2 α, and thromboxane A 2 [28][29][30][31]. Previous studies on gastrointestinal tract motility have shown that PGE 2 generally contracts the longitudinal smooth muscle layer of the small intestine and relaxes the circular muscle layer [26,27]. In the present study, we found that PGE 2 promotes outward pacemaker currents in ICC (Fig. 1A). Moreover, recent reports have shown that deoxycholic acid inhibits ICC pacemaker currents by activating ATPdependent K + channels by inducing PGE 2 production [18]. Specifically, this report suggests that PGE 2 affects pacemaker currents by modulating ATP-dependent K + channels in ICC. Taken together, PGE 2 does modulate gastrointestinal tract motility, and this action depends on species and PGE 2 concentration [5][6][7].
In terms of its concentration, PGE 2 has been shown to have dual effects on the colonic motility of rabbit in vivo and in vitro; i.e., a suppressive effects at low concentration, and an activating effect at high concentration [32] and similar effects on stomach activity in a guinea-pig model [33]. Those results mean that PGE 2 have various actions depending on concentration, the organ, and the species. In ICC from murine small intestine, we found that PGE 2 have only a dose-dependent inhibitory effects on pacemaker currents (Fig. 2) and, a slight or no inhibitory effect on pacemaker currents at 1 nM or 100 pM (data not shown).
Many studies have found that ATP-dependent K + channels play important roles in regulating resting membrane potential and membrane excitability in a variety of tissues. In addition, Jun et al [18] in an electrophysiological study reported on the role of ATP-dependent K + channels in ICC. ATP-dependent K + channels are heteromultimers comprised of inwardly rectifying K + channel subunits (K ir 6.x) and sulfonylurea receptors (SURs). Various combinations of these two subunits convey the heterogeneity of channel properties observed in native cells, such as K ir 6.2-SUR 1 in pancreatic βcells, K ir 6.2-SUR 2A in cardiac and skeletal muscles, and K ir 6.1-SUR 2B or K ir 6.2-SUR 2B in smooth muscle [34]. In this study, we identified ATP-dependent K + channels subunits using RT-PCR with c-kit positive single cell. By using single cell RT-PCR assay, we observed K ir 6.2 and SUR 2B mRNA transcripts (Fig. 3B) in ICC. Also, in smooth muscle cell, we found K ir 6.2 and SUR 2B mRNA transcripts (Fig. 3A). Interestingly, there are many reports that give rise to much controversy. Especially, Koh et al (1998) [35] suggested the presence of K ir 6.2 and SUR 2B subunits in colonic smooth muscle cells same as our results but Nakayama et al (2005) [36] showed that RT-PCR examinations revealed predominant expression of K ir 6.1 and SUB 2B in smooth muscle, with predominant expression of K ir 6.1 and SUR 1 in ICC. While we could not make clear the difference our and previous suggestions, we thought that our results that was verified using single cell are more exact contrary to previous reports. Therefore, our results indicate that the effects of PGE 2 on pacemaker currents in ICC occur via ATP-dependent K + channels comprised of K ir 6.2-SUR 2B.
The recent cloning and expression of PG receptors has confirmed not only the existence of at least four of frequencies in the murine gastric antrum [40]. However, in the murine small intestine, only butaprost was found to have an inhibitory effect on cultured ICC pacemaker currents, and this effect was similar to that of PGE 2 (Fig.  4A). Without sulprostone, the pretreatment of ICC with SC19920 or co-treatment with SC19920 and sulprostone (to block the EP 1 receptor) had no effects on spontaneous inward currents (Fig. 4C). Furthermore, RT-PCR assays using single cell only amplified the EP 2 primer (Fig. 4D). As described above, the action of PGE 2 varies greatly, and depends on its concentration, the organ, the species, and even the muscle layer studied [5][6][7]. Therefore, we suggest that only the EP 2 receptor subtypes affect five classes of prostaglandin receptor (IP for PGI 2 binding, FP for PGF 2 α binding, EP for PGE 2 binding and, TP for TXA 2 binding), but also support the logical subdivision of EP receptors into at least three subtypes, including EP 1 , EP 2 (or EP 4 ), and EP 3 . Several agonists and antagonists are known for the EP receptor. To date, butaprost appears to be the most selective EP 2 receptor subtype agonist [37], and sulprostone is known to agonist the EP 1 and EP 3 receptors [38]. Moreover, ONO-AE-248 antagonizes the EP 4 receptor, and SC19920 the EP 1 receptor [39], but as yet no antagonists of the EP 2 and EP 3 receptors have been identified. Interestingly, it was reported that EP 2 , EP 3 , and EP 4 agonists affect pacemaker current pacemaker currents in murine small intestine, and that this differs from the situation in the gastric antrum. Also, in as shown in fig. 4A and B, we found that pre-or cotreatment with glibenclamide (ATP-dependent K + channel blocker) blocked the effects of butaprost on pacemaker currents in ICC. This result suggests that the stimulation of EP 2 receptors in ICC may regulate the activities ATPdependent K + channels. Almost all of the studies of PG and second messengers until the late 1980s were concerned with cyclic nucleotides, particularly cAMP. Butcher and colleagues were the first to demonstrate an association between PGs and cAMP [41,42], and although their observation made little initial impact, in became increasingly accepted that E-series PGs at least were capable of stimulating adenylyl cyclase to cause increases in intracellular cAMP [43,44]. Several reports suggested the participation of cAMP on PGE 2 actions, especially the EP2 receptor. The results by Hardcastle et al. (1982) provide direct evidence for positive coupling of an EP receptor to adenylate cyclase in their demonstration of an association between EP2 receptors and cAMP generation in enterocytes [45] and similarly it was found an association between EP2 receptor stimulation and cAMP generation in corneal endothelial cells [46]. Furthermore, in cells expressing the recombinant murine EP2 receptor, PGE 2 increased the intracellular cAMP level without any change in inositol phosphate content [47]. These several reports predict that, on pacemaker currents in ICC, PGE 2 may have the actions of cyclic nucleotides signaling pathway. Namely, in ICC, the generation of pacemaker currents and the regulation of ATP-sensitive K + channels on this may involve the cAMP signaling. However, in a recent study and figure 5B, the treatment 8-bromo-cAMP (a cell-permeable cAMP analog) in ICC had no effects on pacemaker currents [48]. Also, pretreatment with SQ-22536, an adenylate cyclase inhibitor, does not influence PGE 2 actions on pacemaker currents, and in a cAMP assay, PGE 2 did not stimulated the production of cAMP. Taken together, PGE 2 appears to function in diverse cells and tissues by modulating a cAMP-dependent pathway. However, in ICC, PGE 2 has an inhibitory effect on pacemaker currents that is independent of the cAMP pathway. Further studies on the actions of PGE 2 in ICC are needed, especially on second messenger.
Recent studies have suggested that pacemaker activity depends on a link between Ca 2+ release from cellular stores, oxidative metabolism, and the pacemaker conductance in the plasma membrane [49]. Especially, we noted the inositol 1,4,5-triphosphate receptor plays a role in generating spontaneous electrical activity in gastrointestinal pacemaker cells [15] and previous suggestion that periodic Ca 2+ release from intracellular Ca 2+ stores produces [Ca 2+ ] i oscillations in ICC, using cell cluster preparations isolated from mouse ileum [50] and these actions seen in ICC are considered to be the primary pacemaker activity in the gut. But, we thought that small cell clusters that show stable spontaneous rhythmicity in terms of mechanical, electrical and intracellular Ca 2+ activities contain c-kit immunopositive ICC, smooth muscle cells and enteric neurons. So, in this study we examined [Ca 2+ ] i oscillations of ICC that are branched with cluster. In fig. 6 Taken together, we conjecture that the opening of ATPsensitive K + channels by PGE 2 hyperpolarize membrane potentials of ICC and this action evokes inhibition of periodic Ca 2+ release from intracellular Ca 2+ stores that are considered to be the primary pacemaker activity in ICC.
In conclusion, the present results indicate that PGE 2 directly alters pacemaker currents by modulating ATPdependent K + channels comprised of K ir 6.2-and SUR 2B in ICC. Moreover, this affects of PGE 2 on pacemaker currents is not mediated via cyclic nucleotides-dependent pathway and involved by the activation of EP 2 receptors and mobilization of [Ca 2+ ] i .