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Journal of Physiology (2002), 538.1, pp. 145-157
© Copyright 2002 The Physiological Society
DOI: 10.1113/jphysiol.2001.012765
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ABSTRACT |
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PGE2 has been linked to the production of gastric arrhythmias such as tachygastria. The interstitial cells of Cajal (ICC) generate electrical rhythmicity in gastrointestinal muscles, and may therefore be a target for PGE2 in gastric muscles. We cultured ICC from the murine gastric antrum, verified that cells were Kit immunoreactive, and measured spontaneous slow waves. These events were caused by spontaneous inward (pacemaker) currents that were not blocked by nifedipine. Forskolin and 8-bromoadenosine 3':5'-cyclic monophosphate (8-Br-cAMP) reduced the frequency of pacemaker currents in ICC and of slow waves in intact antral muscles. The effects of forskolin and 8-Br-cAMP were not blocked by inhibitors of protein kinase A, suggesting that cAMP has direct effects on pacemaker activity. PGE2 mimicked the effects of forskolin and 8-Br-cAMP on ICC, but increased slow-wave frequency in intact muscles. Therefore, the chronotropic effects of specific prostaglandin EP receptor agonists were examined. Butaprost and ONO-AE1-329, EP2 and EP4 receptor agonists, mimicked the effects of forskolin and 8-Br-cAMP on ICC and intact muscles. Sulprostone (EP3>EP1 agonist), GR63799, and ONO-AE-248 (EP3 agonists) enhanced the frequencies of pacemaker currents in ICC and slow waves in intact muscles. The effects of sulprostone were not blocked by SC-19220, an EP1 receptor antagonist. These observations suggest that the positive chronotropic effects of PGE2 in intact muscles are mediated by EP3 receptor stimulation. The effects of PGE2 in intact muscles may be dependent upon the relative expression of EP receptors and/or proximity of receptors to sources of PGE2.
(Received 21 May 2001; accepted after revision 24 September 2001)
Corresponding author K. M. Sanders: Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, NV 89557, USA. Email: kent{at}physio.unr.edu
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INTRODUCTION |
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Current evidence suggests that the interstitial cells of Cajal (ICC) generate the electrical pacemaker activity (slow waves) in gastrointestinal (GI) muscles (cf. Langton et al. 1989; Ward et al. 1994; Huizinga et al. 1995; Sanders, 1996; Dickens et al. 1999; Ördög et al. 1999). Smooth muscle cells, which are electrically coupled to ICC, respond to slow-wave depolarizations with activation of a variety of voltage-dependent ionic conductances (Dickens et al. 1999; Horowitz et al. 1999), including L-type Ca2+ channels. Entry of Ca2+ via voltage-dependent Ca2+ channels links the slow waves to phasic contractions (see Ozaki et al. 1991).
Freshly isolated ICC from the canine colon are spontaneously active, generating slow-wave-like depolarizations (Langton et al. 1989). Cultured ICC from the murine intestine also demonstrate spontaneous rhythmicity, and this preparation has been used to deduce that the spontaneous inward current is due to periodic activation of a non-selective cation conductance (Thomsen et al. 1998; Koh et al. 1998). Activation of the spontaneous inward current depends upon the release of Ca2+ from IP3 receptor-operated stores and uptake of Ca2+ by mitochondria (Ward et al. 2000). The same drugs that interfere with spontaneous inward currents in murine ICC and intact small intestine also block slow waves in muscles from the guinea pig stomach and canine colon (Ward et al. 2000), suggesting that a common pacemaker mechanism exists in the GI muscles of various organs and species.
Although the mechanism underlying pacemaker activity, and the current responsible for slow waves may be common in different organs of the GI tract, slow waves occur at a wide range of frequencies (i.e. from < 1 to > 30 min-1; see Szurszewski, 1987). Endogenous agents such as neurotransmitters, hormones and paracrine substances can alter slow-wave frequency. In general, slow-wave frequency is fairly regular in the intestine and corpus of the stomach, but the gastric antral region has unique properties of slow-wave regulation. Several agonists dramatically alter the frequency of slow waves (cf. El-Sharkawy et al. 1978; Sanders, 1984; Ozaki et al. 1992a, b). This unique property of antral slow-wave generation may be why this region of the GI tract is susceptible to arrhythmias such as tachygastria and bradygastria (Code & Marlett, 1974; Kim et al. 1986). Arrhythmias in the terminal stomach can interfere with the normal propagation of slow waves (i.e. from corpus to pylorus) and cause pathological delays or defects in gastric empyting (Telander et al. 1978; You et al. 1981; You & Chey, 1984; Koch et al. 1989; Chen et al. 1995; Walsh et al. 1996).
At present, the cellular signals responsible for the development of gastric arrhythmias are unknown. Excitatory neurotransmitters and hormones substantially increase slow-wave frequency (El-Sharkawy & Szurszewski, 1978; El-Sharkawy et al. 1978), but inhibitory agonists have variable effects. For example, many agonists that reduce frequency are linked to the production of cyclic nucleotides (Ozaki et al. 1992b; Tsugeno et al. 1995), but prostaglandin E2 (PGE2), often responsible for increasing levels of cAMP, enhances slow-wave frequency (Sanders, 1984; Kim et al. 1985). Past studies performed on animals or on isolated muscle strips have failed to determine the direct effects of cAMP-dependent agonists on antral pacemaker activity, due to the multicellular complexity of these preparations. We have developed a preparation of cultured ICC from the murine gastric antrum and recorded spontaneous inward currents from these cells. We characterized the effects of drugs with cAMP-dependent actions on pacemaker frequency, and compared these effects to the responses of intact muscle. The unique properties of PGE2 in regulating pacemaker activity were also investigated.
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METHODS |
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Preparation of cells and tissues
Balb/C mice (0-30 days old) of either sex were anaesthetized with CO2 and killed by cervical dislocation. Stomachs were removed and opened along the lesser curvature. Luminal contents were washed away with Krebs-Ringer bicarbonate solution (KRB). Tissues were pinned to the base of a Sylgard dish and the mucosa of the antral region was removed by sharp dissection. The antral muscles were cut into small strips to isolate cells, or used as a sheet in experiments on intact muscles.
Isolation and culturing of ICC
Small strips of antral muscle were equilibrated in calcium-free Hanks' solution for 10 min. Cells were dispersed, as described previously (Koh et al. 1998), with an enzyme solution containing: collagenase (Worthington Type II), 1.3 mg ml-1; bovine serum albumin (Sigma, St Louis, MO, USA), 2 mg ml-1; trypsin inhibitor (Sigma), 2 mg ml-1; and ATP, 0.27 mg ml-1. Cells were plated onto sterile glass coverslips coated with murine collagen (2.5 µg ml-1, Falcon/BD) in 35 mm culture dishes. The cells were cultured in smooth muscle growth medium (Clonetics, San Diego, CA, USA) supplemented with 2 % antibiotic/antimycotic (Gibco, Grand Island NY, USA) and murine stem cell factor (5 ng ml-1, Sigma), at 37 °C in an incubator supplied with 95 % O2-5 % CO2.
ICC were identified in acetone-fixed gastric cell cultures (4 °C, 2 min) with antibodies to the receptor tyrosine kinase, Kit (ACK2, monoclonal rat anti-mouse IgG; see Torihashi et al. 1995; 5 µg ml-1, overnight at 4 °C), as described previously (Koh et al. 1998). Immunoreactivity was detected using a secondary antibody conjugated to Alexa Fluor 488 (anti-rat IgG; Molecular Probes, Eugene, OR, USA; 10 µg ml-1). As controls, the primary or the secondary antibodies were omitted from the incubation solutions. Phase-contrast and wide-field fluorescence images were acquired with a Leitz Wetzlar Diaplan microscope equipped with a PL Fluotar 
40, 0.70 NA objective, a mercury arc lamp and a Leica LEI-750 colour camera. Images were digitized with Matrox Meteor Driver and MetaMorph 3.0 software (Universal Imaging, West Chester, PA, USA). Confocal microscopy was performed using a BioRad MRC 600 system (Hercules, CA, USA) coupled to a Nikon Diaphot microscope and a PlanApo 60, 1.4 NA oil-immersion lens; the fluorochrome was excited with the 488 nm line of an ArKr laser. Combined differential interference contrast (DIC) and fluorescent images were acquired with an Olympus Fluoview FV500 confocal system equipped with an argon ion laser and coupled to an Olympus IX microscope and a 60, DIC objective lens. The morphology of Kit-immunopositive gastric ICC was distinct from other cell types present in these cultures, so it was possible to identify them for subsequent electrophysiological recordings with phase-contrast microscopy alone (Fig. 1).
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Figure 1. Cultured interstitial cells of Cajal (ICC) from the murine gastric antrum A, phase-contrast micrograph of a small ICC network. Note the characteristic fusiform cell bodies interconnected by multiple, slender processes. Kit expression in same cells is shown in a fluorescent image in panel B. C and D, additional examples of Kit-immunopositive ICC networks as imaged by confocal microscopy (C) or by differential interference contrast microscopy with a confocal fluorescent image superimposed (D). Scale bars are 20 µm in all panels. | ||
Patch-clamp experiments
The whole-cell configuration of the patch-clamp technique was used to record inward currents (voltage clamp) from cultured ICC after 2 or 3 days in culture. Typically, recordings were made from small clusters of ICC (< 10 cells) because, as reported previously in other studies of intestinal ICC, the spontaneous inward currents from small groups of cells are more robust and more regular than from single cells (e.g. Koh et al. 1998). Currents were amplified with an Axopatch 200B patch-clamp amplifier (Axon Instruments, Foster City, CA, USA) and digitized with a 12 bit A/D converter (Axon Instruments). Recording, storage and analyses were performed with Axoscope software (Axon Instruments). All recordings were performed at 29 °C.
The cells were bathed in a solution containing (mM): KCl 5, NaCl 135, CaCl2 2, glucose 10, MgCl2 1.2 and Hepes 10; adjusted to pH 7.4 with Tris. The pipette solution contained (mM): KCl 140, MgCl2 5, K2ATP 2.7, Na2GTP 0.1, creatine phosphate disodium 2.5, Hepes 5, and EGTA 0.1; adjusted to pH 7.2 with Tris.
Electrophysiological experiments on intact gastric muscles
For intact tissue electrophysiological experiments, the entire antral region of the murine stomach (5 mm 8 mm), devoid of mucosa, was pinned to the floor of a recording chamber lined with Sylgard elastomer (Dow Corning) with the mucosal aspect of the circular muscle layer facing upwards. Circular muscle cells were impaled with glass microelectrodes filled with 3 M KCl that had a resistance of 70-100 M
. Transmembrane potentials were measured with a high-impedance electrometer (WPI duo 773, World Precision Instruments, Sarasota, FL, USA) and outputs were displayed on a Tektronix 2224 oscilloscope (Wilsonville, OR, USA). Electrical signals were recorded on video tape (Vetter, Rebersburg, PA, USA). Durations of slow waves were measured at half of their maximum amplitudes, and periods between slow waves were measured as the time between peak depolarizations.
Mechanical experiments on intact gastric muscle strips
A separate series of mechanical experiments was performed using standard organ bath techniques. Strips of muscle (approximately 3 mm 6 mm) were cut parallel to the circular muscle and attached to a fixed mount and to a Fort 10 isometric strain gauge (World Precision Instruments). The muscles were immersed in organ baths maintained at 37 ± 0.5 °C with oxygenated KRB. A resting force of 300 mg was applied, which was found to set the muscles to an optimum length for maximal spontaneous contraction. This was followed by an equilibration period of 1 h, during which time the bath was continuously perfused with oxygenated KRB. Signals were recorded using Acqknowledge software (Biopac Systems, Santa Barbara, CA, USA).
Solutions and drugs
The standard KRB solution used in studies of intact muscles included (in mM): NaCl 118.5, KCl 4.5, MgCl2 1.2, NaHCO3 23.8, KH2PO4 1.2, dextrose 11.0 and CaCl2 2.4. The pH of the KRB was 7.3-7.4 when bubbled with 97 % O2 -3 % CO2 at 37 ± 0.5 °C.
8-Bromoadenosine 3':5'-cyclic monophosphate (8-Br-cAMP), nicardipine, and PGE2 were purchased from Sigma. Forskolin (FSK), dideoxy-FSK, myristoylated protein kinase A inhibitor (mPKI) and KT5720 were purchased from Calbiochem (San Diego, CA, USA). Butaprost, sulprostone and SC-19220 were purchased from Cayman Chemicals (Ann Arbor, MI, USA). GR 63799X was a gift from Glaxo Welcome, and ONO-AE-248 and ONO-AG1-329 were gifts from ONO Pharmaceuticals (Osaka, Japan). 8-Br-cAMP and mPKI were dissolved in water. PGE2 and nicardipine were dissolved in ethanol. The other drugs tested were dissolved in DMSO. The final concentration of DMSO was less than 0.1 %, and DMSO had no effect at this concentration. All drugs tested were applied via bath perfusion for 10-20 min.
Statistical analyses
Data are expressed as means ± S.E.M. Differences in the data were evaluated by ANOVA or Student's t test. P values less than 0.05 were taken as a statistically significant difference. The 'n' values reported in the text refer to the number of cells used in patch-clamp experiments or the number of animals used in intracellular electrophysiological experiments.
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RESULTS |
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Spontaneous activity of ICC in antral cell cultures
ICC cultured from the murine antrum were identified with the aid of Kit immunofluroescence. Cells with Kit-like immunoreactivity (Kit-LI) had a distinctive morphology that was easily recognized in cultures. Cell bodies with Kit-LI were fusiform and had multiple thin processes that often appeared to form a network with adjacent cells. Figure 1 shows phase-contrast, DIC, and Kit immunofluorescence images of gastric antral ICC.
Recording from cultured ICC under current-clamp conditions showed that these cells were spontaneously active, generating slow-wave-like activity at an average frequency of 1.0 ± 0.2 min-1 (n = 8; Fig. 2A). The spontaneous depolarizations recorded from cultured ICC were similar in waveform to events recorded from intact strips of antral muscle (Ördög et al. 1999; and see below). Switching the amplifier to voltage-clamp mode and holding cells at -60 mV, showed that the slow-wave-like activity was mirrored by spontaneous inward currents. The spontaneous inward currents were characterized by a single rapid phase of inward current followed by a sustained 'plateau' inward current. The average frequency of the currents was 1.0 ± 0.1 min-1 (n = 42), and the amplitude and duration averaged -339 ± 42 pA and 24.3 ± 1.4 s, respectively, at a holding potential of -60 mV. Since murine electrical slow waves are not blocked by dihydropyridines (Ward et al. 1994), we investigated the effects of nicardipine (1 µM) on spontaneous inward currents. Nicardipine (1 µM) had no significant effect on the spontaneous inward currents (n = 7, Fig. 2C).
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Figure 2. Spontaneous membrane potential and currents from antral ICC A, typical slow-wave-like oscillations in membrane potential from ICC under current-clamp conditions (I = 0). Note the similarity in waveform to slow waves recorded from intact antral muscles (see Figs 9 and 11). B, spontaneous membrane currents from the same cell as in A after turning the amplifier to voltage-clamp mode (holding potential -60 mV). The currents consisted of cells with this single sharp inward current and a sustained 'plateau' current. C, nicardipine (10-6 M) had no significant effect on spontaneous inward currents in antral ICC. D, voltage steps (10 min) were applied to small networks of cells (-80 to +20 mV). The current-voltage relationship for the peak inward currents is shown. E, the frequency of the spontaneous inward currents is plotted as a function of holding potential. Data in D and E summarize the results of four experiments. | ||
Voltage-clamp experiments were performed on small networks, and the holding potential was stepped for 10 min step-1 from -80 to +20 mV. In these experiments, the inward currents averaged -206 ± 54 pA and -63 ± 18 pA at holding potentials of -40 and 0 mV, respectively (n = 4). At a holding potential of +20 mV, the spontaneous inward currents were not resolved (Fig. 2D). The frequency of the spontaneous inward currents was independent of holding potentials at potentials from -80 to 0 mV (e.g. the frequencies were 1.1 ± 0.1 and 1.1 ± 0.2 cycles min-1 at -40 and 0 mV, respectively; Fig. 2E). These data suggest that voltage-dependent channels may not be involved in the generation of spontaneous inward currents, and, as in the small intestine, non-selective cation currents may be responsible for the spontaneous inward currents. In the remainder of the experiments we used a constant holding potential of -60 mV.
Effects of FSK on the pacemaker activities of ICC and intact antral muscles
After establishing basic spontaneous activity, we characterized the responses to agonists known to elevate levels of cAMP. In the first series of experiments we tested the effects of FSK (10-10-10-7 M; Fig. 3) on spontaneous inward currents. Under control conditions at a holding potential of -60 mV, the frequency and duration of spontaneous inward currents in the cells used for these experiments were 1.2 ± 0.4 min-1 and 23.7 ± 0.7 s, respectively. FSK (10-8 M) decreased spontaneous inward current frequency and duration (e.g. to 0.7 ± 0.2 min-1 and 10 ± 1.5 s, n = 5; P < 0.05) without significant effect on the amplitude of the spontaneous inward currents (P > 0.05). At 10-7 M, FSK blocked spontaneous inward currents in five out of the five cells tested. This concentration also resulted in the generation of a net outward current (42 ± 22 pA; the control was 9.7 ± 2.4 pA; n = 5), but this did not reach a level of significance in the experiments performed (P > 0.05). A similar current was activated by FSK in small intestinal ICC, and it was blocked by glibenclamide, suggesting that it was due to the ATP-dependent K+ (KATP) current (Koh et al. 2000). A high concentration of an inactive analogue of FSK, dideoxy FSK (10-6 M), had no effects on pacemaker or resting current (n = 4, Fig. 3D). Data from these experiments are summarized in Fig. 3E-F.
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Figure 3. Effects of forskolin (FSK) on spontaneous inward currents from cultured ICC from the murine antrum A-C, spontaneous inward currents from the same cell exposed to FSK (10-9-10-7 M). FSK decreased the duration, frequency and amplitude of slow waves in a dose-dependent manner. D, dideoxy-FSK (10-6 M) had no effect on spontaneous inward currents. The effects of FSK on spontaneous inward currents are summarized in E (duration) and F (frequency). | ||
FSK is a potent activator of adenylyl cyclase, and increased concentrations of cAMP are known to activate cAMP-dependent protein kinase (PKA). To examine the intracellular signalling mechanism effected by FSK, cells were treated with PKA inhibitor (mPKI). Administration of mPKI (10-7 M) did not affect spontaneous inward currents, and after pretreatment with mPKI, the effects of FSK (10-8 M) were unchanged (i.e. after FSK, the frequency was 0.13 ± 0.03 min-1, n = 5; Fig. 4A-B). Another PKA inhibitor, KT5720, similarly did not affect the response to FSK. After incubation with KT5720 (10-7 M), FSK (10-8 M) reduced the frequency of spontaneous inward currents (i.e. to 0.18 ± 0.04 min-1; P > 0.05 for both as compared to the FSK response in the absence of PKA inhibitors, n = 4). These data suggest that the effects of FSK, if mediated via cAMP, were not due to activation of PKA.
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Figure 4. Effects of protein kinase A (PKA) inhibitors on the effects of FSK A, FSK (10-8 M) reduced the frequency of spontaneous inward currents. B, pretreatment of myristoylated PKA inhibitor (mPKI, 10-7 M) did not inhibit the effects of FSK on the same cell. C, another PKA inhibitor (KT5720, 10-7 M) also had no effect on the response to FSK. The effects of PKA inhibitors on FSK frequency responses are summarized in D (mPKI) and E (KT5720). | ||
In intact antral muscles, FSK (10-8-10-7 M) produced a concentration-dependent hyperpolarization of the resting membrane potential (RMP) and reduced slow-wave frequency. Under control conditions, the RMP averaged -69.2 ± 2.9 mV, and slow waves of 31.6 ± 2.2 mV in amplitude and 3.9 ± 0.4 s in duration occurred at a frequency of 3.8 ± 0.5 min-1 (n = 5). The addition of FSK (10-8 M) did not change the RMP (-69.8 ± 3.6 mV) or the amplitude and duration of slow waves (i.e. amplitude 33.2 ± 2.1 mV, duration 3.4 ± 0.5 s; P > 0.05). However, FSK reduced the frequency of slow waves to 2.2 ± 0.6 min-1 (P < 0.005 compared to control). At higher concentrations (5 10-8 M), FSK completely abolished slow-wave activity in four out of five preparations, and hyperpolarized membrane potential from -64.8 ± 2.6 mV to -67.1 ± 2.0 (P < 0.05). In one preparation, normal slow-wave activity was disrupted by FSK into a pattern where several slow-wave cycles were grouped together with an increased frequency, and the clusters were interspersed with periods of electrical quiescence. At a concentration of 10-7 M, FSK inhibited slow waves in five out of six preparations and hyperpolarized membrane potentials from -62.0 ± 1.2 to -68.4 ± 0.7 mV (P < 0.01). Typical responses to FSK are illustrated in Fig. 5A-F.
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Figure 5. Effects of FSK and 8-bromoadenosine 3':5'-cyclic monophosphate (8-Br-cAMP) on the electrical slow waves of intact gastric antral muscles Control slow waves (A) were reduced in frequency by FSK (10-8 M; B), and the effects were fully reversible upon washout (C). A-C are excerpts of a continuous record. The dotted line denotes the control resting potential. D-E, excerpts of a continuous record showing the effects of FSK at a higher concentration. FSK (5 | ||
Effects of 8-Br-cAMP on spontaneous inward currents in ICC and slow waves in antral muscles
The membrane-permeable cAMP analogue, 8-Br-cAMP, mimicked the effects of FSK on the frequency of spontaneous inward currents in ICC. Under voltage-clamp conditions (holding potential -60 mV), control frequency, resting current and spontaneous inward current averaged 1.0 ± 0.3 min-1, 1.0 ± 11 pA, and -197 ± 9 pA, respectively (n = 4). Application of 8-Br-cAMP decreased pacemaker frequency in a dose-dependent manner (Fig. 6). High concentrations of 8-Br-cAMP (e.g. 10-3 M) inhibited spontaneous inward currents. Pretreatment of cells with mPKI had no effect on spontaneous inward currents (Fig. 6D), and 15-20 min incubation with mPKI had no effect on responses to 8-Br-cAMP. These data confirm observations with FSK and suggest that the effects of 8-Br-cAMP are a direct action of cAMP, and do not occur via activation of PKA and protein phosphorylation. Experiments testing 8-Br-cAMP are summarized in Fig. 6E and F.
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Figure 6. Effects of 8-Br-cAMP and PKA inhibitors on spontaneous inward currents of cultured ICC A-C, spontaneous inward currents from the same cell exposed to 8-Br-cAMP (10-5-10-3 M). 8-Br-cAMP decreased the frequency of spontaneous inward currents in a dose-dependent manner. D, pretreatment with mPKI (10-7 M) did not block the effects of 8-Br-cAMP. The responses to 8-Br-cAMP on spontaneous inward currents are summarized in E (duration) and F (frequency). | ||
The addition of 8-Br-cAMP (10-3 M) affected slow waves in a manner similar to FSK. In experiments to test 8-Br-cAMP, RMP averaged -64.5 ± 3.3 mV and slow waves, 34.6 ± 2.1 mV in amplitude and 5.1 ± 0.6 s in duration, occurred at a frequency of 3.5 ± 0.4 min-1 in control conditions. 8-Br-cAMP (10-3 M) hyperpolarized membrane potential to -69.3 ± 3.2 mV (P < 0.001, n = 5). Slow-wave amplitude was not significantly affected (i.e. 37.2 ± 1.8 mV; P > 0.05). Slow-wave duration and frequency were reduced to 4.5 ± 0.6 s (P < 0.05) and 2.0 ± 0.2 min-1 (P < 0.01, Fig. 5G-I), respectively, by 8-Br-cAMP. During the initial exposure to 8-Br-cAMP, slow waves developed an irregular pattern similar to that described earlier in two preparations exposed to FSK.
Effects of PGE2 on spontaneous inward currents and slow waves
We also tested the effects of PGE2 on spontaneous inward currents, because the effects of this naturally occurring eicosanoid might be mediated via cAMP. Under control conditions at a holding potential of -60 mV, the frequency and duration of spontaneous inward currents were 1.0 ± 0.2 min-1 and 28 ± 4 s, respectively (n = 4). PGE2 (10-10 M) did not affect the frequency and duration of spontaneous inward currents (Fig. 7A, 0.9 ± 0.4 min-1 and 29 ± 6 s). PGE2 (10-9 M, n = 4) reduced the frequency of slow waves, and completely inhibited spontaneous inward currents (10-8 M, n = 4). A higher concentration of PGE2 (10-8 M) generated a sustained outward current averaging 43 ± 27 pA (P < 0.05; Fig. 7B-C). These effects were similar to those of FSK (see above).
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Figure 7. Effects of PGE2 on spontaneous inward currents A-C, spontaneous inward currents from the same cell exposed to PGE2 (10-10-10-8 M). PGE2 caused a concentration-dependent decrease in frequency. Spontaneous inward currents were abolished at 10-8 M. The effects of PGE2 on spontaneous inward currents are summarized in D (duration) and E (frequency). | ||
Previous studies have shown that PGE2 increases slow-wave frequency in intact canine and feline antral muscles (Sanders & Szurszewski, 1981; Sanders, 1984; Kim et al. 1985). Therefore, we tested PGE2 on intact murine antral muscles to determine whether the effects observed in ICC differed from whole muscle responses. Membrane potential was not altered significantly by PGE2 (10-7 M; i.e. -65.3 ± 1.4 mV in control and -67.8 ± 2.4 mV in the presence of PGE2; n = 7; P > 0.05). At this concentration, PGE2 decreased slow-wave amplitude from 33.8 ± 1.6 mV to 24.3 ± 2.0 mV (Fig. 8A-C; P < 0.001) and reduced the half-maximal duration of slow waves from 4.0 ± 0.5 to 2.1 ± 0.4 s. Slow waves occurred at a frequency of 3.0 ± 0.3 min-1 under control conditions, and the frequency was not significantly changed (e.g. 2.9 ± 0.6 min-1) in the presence of PGE2. Higher concentrations of PGE2 (10-6 M; n = 7) did not significantly affect membrane potential (i.e. -68.7 ± 1.6 mV before and -67.9 ± 2.6 mV after PGE2). Slow-wave amplitude, however, was reduced from 32.7 ± 2.3 mV to 15.7 ± 2.6 mV (P < 0.001). Before PGE2, slow-wave duration was 5.0 ± 0.5 s, and this decreased to 3.6 ± 0.6 s in the presence of PGE2 (P < 0.05). Slow-wave frequency was increased from 3.4 ± 0.2 to 7.0 ± 0.6 min-1 in the presence of PGE2 (Fig. 8D-F; P < 0.001).
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Figure 8. Effects of PGE2 on the electrical and contractile activity of antral muscles A-C, the effects of PGE2 (10-7 M) on gastric slow waves. PGE2 produced membrane hyperpolarization and reduced the amplitude of slow waves (B). These effects were reversible upon washout (C). A-C are excerpts from a continuous recording. At 10-6 M, PGE2 further decreased the amplitude and increased the frequency of slow waves (D-E), effects that were reversible upon washout (F). The lower panel shows the effect of 10-6 M PGE2 on mechanical activity. PGE2 increased contractile frequency but decreased the amplitude of phasic contractions and was fully reversible upon washout. The time scale bar shown in F also applies to A-E. The time scale bar under the mechanical record applies to that trace only. | ||
In all previous experiments, the effects of agonists on ICC and intact smooth muscle strips was equivalent. The response to PGE2 was opposite. Therefore, we performed two additional control experiments to test whether the temperature differences between cell and tissue experiments could be responsible for the different response to PGE2. In these experiments tissues were perfused with a bath solution held at 29 °C. Application of PGE2 under these conditions produced equivalent responses to those observed at 37 °C.
We also tested the effects of PGE2 on contractile activity in separate experiments on eight antral muscles. PGE2 (10-7 M) increased contractile frequency from a control value of 2.1 ± 0.2 to 3.1 ± 0.2 contractions min-1 (n = 8; P < 0.01) and decreased the average amplitude of contractions from 4.8 ± 1.0 to 0.6 ± 0.1 mN (P < 0.01; i.e. contractile amplitude was decreased to 16.6 ± 5.1 % of the original value). The bottom trace in Fig. 8 shows the mechanical effects of PGE2.
Basis of the chronotropic effects of PGE2
It is possible that the difference in cell and tissue responses to PGE2 could be due to differences in prostaglandin receptor dominance in the two preparations. Four receptors for PGE2 have been cloned (see classifications in Coleman et al. 1994), and these receptors are coupled via G proteins to different cellular signalling pathways. We tested butaprost, an EP2 receptor agonist that is coupled via Gs to stimulate cAMP production (Bastien et al. 1994). Butaprost caused a concentration-dependent reduction in the spontaneous inward current frequency in ICC, mimicking the effects of PGE2 on spontaneous inward currents (Fig. 9, n = 4). The duration of spontaneous inward currents also decreased as a function of butaprost concentration (Fig. 9E). Butaprost (10-8 M) induced outward currents (50 ± 34 pA) and inhibited spontaneous inward currents. The EP4 receptor agonist ONO-AE1-329 had similar effects to butaprost (Fig. 9D; n = 5). ONO-AE1-329 (10-8 M) caused a reduction in the frequency of spontaneous inward currents (e.g. from 1.2 ± 0.2 to 0.4 ± 0.2 min-1, n = 6). A higher concentration of ONO-AE1-329 (10-7 M), blocked spontaneous inward currents (n = 3).
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Figure 9. Effects of EP2 (butaprost) and EP4 (ONO-AE1-329) agonists on spontaneous inward currents from cultured ICC A-C, spontaneous inward currents from the same cell exposed to butaprost (10-10-10-8 M). Butaprost caused a concentration-dependent decrease in spontaneous inward current frequency and activated an outward current. These effects are similar to the effects of PGE2 and FSK. D, ONO-AE1-329 (10-8 M) also decreased the frequency of spontaneous inward currents. The effects of butaprost on spontaneous inward currents are summarized in E (duration) and F (frequency). | ||
Butaprost was tested on intact muscles at concentrations of 10-7-10-6 M. At 10-7 M, butaprost did not produce significant effects on the electrical activity of the circular muscle layer (n = 6; not shown). At 10-6 M (n = 6), butaprost hyperpolarized the membrane potential from -61.4 ± 1.9 mV to -70.3 ± 1.7 mV (P < 0.005). In four out of six preparations, slow-wave activity was completely inhibited by this concentration of butaprost. In the remaining preparations, slow-wave amplitude, duration and frequency were reduced from 35 to 31 mV, from 4.6 to 4.4 s, and from 4.2 to 3.1 min-1, respectively. Responses to butaprost are shown in Fig. 10A-F.
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Figure 10. Effects of EP receptor agonists on the electrical slow waves of intact antral muscles A-F, the effects of butaprost at 10-7 M and 10-6 M on slow-wave activity. Butaprost caused a concentration-dependent hyperpolarization of membrane potential and decreased the frequency of slow waves. A, control slow waves. B, the effects of butaprost (10-6 M). Membrane hyperpolarization was reversed upon washout of the drug (C). At a higher concentrations, butaprost (10-6 M) hyperpolarized tissues and completely blocked slow waves (E) in a reversible manner (F). Sulprostone (10-7 M; G-I), an EP3 > EP1 agonist, reduced the amplitude of slow waves and increased slow-wave frequency by shortening the inter-slow-wave period (H). J-L, the EP1 receptor antagonist, SC-19220 (10-5 M) did not alter slow-wave activity (J), and in the continued presence of SC-19220, the positive chronotrophic effects of sulprostone were not blocked (K) but were reversed upon wash out (L). M-O, the effects of an EP3 receptor agonist, ONO-AE-248 (10-9 M). ONO-AE-248 mimicked the effects of sulprostone, causing membrane depolarization and increased slow-wave frequency (N), which were reversible upon washout of the drug (O). The time scale bar in L applies to all other panels on this figure. | ||
Since the stimulation of EP2 and EP4 receptors could not explain the positive chronotropic effect of PGE2 on slow waves in gastric muscles, we tested sulprostone (a mixed EP1 and EP3 agonist). Sulprostone (10-9-10-8 M) increased the frequency of spontaneous inward currents in ICC and induced a net inward current (Fig. 11A). Pretreatment with a blocker of EP1 receptors (SC-19220, 10-5 M) did not affect these responses (n = 5; Fig. 11B). Two additional EP3 agonists, GR63799X (10-9 M, n = 7) and ONO-AE-248 (10-7 M; n = 4) also enhanced spontaneous inward current frequency and induced a net inward current (Fig. 11C and D). Data from these experiments are summarized in Fig. 11E-G. These data suggest that the positive chronotropic effect of PGE2 is mediated by EP3 receptors.
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Figure 11. Effects of EP3 receptor agonists on spontaneous inward currents of cultured ICC A, sulprostone (an EP3 > EP1 agonist, 10-9 M) increased the frequency of spontaneous inward currents. B, pretreatment with an EP1 antagonist (SC-19220, 10-5 M) did not inhibit responses to sulprostone. C-D, EP3 agonists (GR 63799, 10-9 M and ONO-AE-248, 10-7 M) increased the frequency of spontaneous inward currents. The effects of sulprostone (Sul; | ||
We tested the effects of sulprostone (10-7 M; n = 6) on the electrical activity of intact antral muscles. Sulprostone depolarized the RMP from -64.0 ± 2.4 mV to -56.2 ± 2.0 mV (P < 0.01) and reduced the amplitude of slow waves from a control value of 30.6 ± 3.1 mV to 15.5 ± 4.3 mV (P < 0.01). The duration of slow waves was not significantly affected by sulprostone, but the period between slow waves was reduced from 20.6 ± 3.5 to 8.9 ± 0.5 s (P < 0.05). The frequency of slow waves almost doubled, from 3.4 ± 0.4 to 6.7 ± 0.7 min-1 (P < 0.05; Fig. 10G-I). In order to determine if sulprostone was acting via EP1 or EP3 receptors, we also tested its effects after pretreatment with a specific EP1 receptor antagonist SC-19220 (10-5 M). SC-19220 (n = 5) did not alter spontaneous activity and did not block the effects of sulprostone (Fig. 10J-L). In the presence of SC-19220, RMP averaged -60.4 ± 2.3 mV, and slow waves (27.6 ± 3.0 mV in amplitude) occurred at a frequency of 3.1 ± 0.4 min-1. In the presence of SC-19220, sulprostone depolarized the membrane potential to -46.8 ± 2.9 mV, reduced slow-wave amplitude to 9.3 ± 2.4 mV, and increased their frequency to 6.8 ± 0.6 min-1. In the presence of SC-19220, sulprostone dramatically reduced the inter-slow-wave period from 23.8 ± 5.8 to 8.6 ± 0.8 s (P < 0.05).
We also studied the effects of a specific EP3 agonist on intact muscles. ONO-AE-248 (10-9-10-8 M) had similar effects on electrical slow waves as sulprostone. ONO-AE-248 produced a dose-dependent depolarization in membrane potential. At 10-9 M ONO-AE-248, the RMP was depolarized from -65.4 ± 1.9 mV to -58.5 ± 2.6 mV (n = 7, P < 0.001). At a concentration of 3 10-9 M, ONO-AE-248 depolarized circular muscle cells from -66.7 ± 1.1 to -57.4 ± 2.4 mV (P < 0.05), and at 10-8 M, the membrane potential depolarized from -66.3 ± 1.5 to -52.0 ± 2.0 mV (P < 0.001). Slow-wave amplitude was also reduced from 26.7 ± 2.1 to 17.7 ± 3.3 mV (P < 0.01), from 29.9 ± 1.8 to 15.2 ± 3.6 mV (P < 0.05), and from 30.5 ± 2.5 to 10.9 ± 3.2 mV (P < 0.01) at 10-9, 3 10-9 and 10-8 M ONO-AE-248, respectively. The frequency of slow waves increased in a dose-dependent manner, from 4.1 ± 0.5 to 6.0 ± 0.7 min-1 with 10-9 M (P < 0.01), from 4.1 ± 0.7 to 7.4 ± 0.4 min-1 with 3 10-9 M (P < 0.01), and from 3.9 ± 0.6 to 9.2 ± 0.9 min-1 with 10-8 M ONO-AE-248 (n = 4, P < 0.01; see Fig. 10M-O). ONO-AE-248 did not produce significant changes in slow-wave duration at any of the concentrations investigated; however, at all concentrations the inter-slow-wave period was reduced (i.e. from 16.0 ± 1.9 to 11.6 ± 1.5 s at 10-9 M (P < 0.01), from 16.5 ± 2.6 s to 8.0 ± 0.5 at 3 10-9 M (P < 0.05), and from 17.3 ± 3.8 to 7.0 ± 0.5 s at 10-8 M; P < 0.05).
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DISCUSSION |
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Previous studies have shown that freshly isolated ICC from the canine colon (Langton et al. 1989) and cultured ICC from the murine small intestine generate spontaneous electrical pacemaker activity (Koh et al. 1998; Thomsen et al. 1998). The present study is the first to isolate, culture, and demonstrate electrical rhythmicity in gastric ICC. Spontaneous depolarizations were generated by ICC from the murine antrum, and these events were mirrored by spontaneous inward currents in voltage-clamp mode that were characterized by a rapid phase of inward currents followed by a sustained plateau current, lasting several seconds. The waveforms of the inward currents were similar to the waveforms of slow waves recorded from antral muscles (this study and see El-Sharkaway & Szurszewski, 1978; Ozaki et al. 1991), and from ICC impaled in situ (Dickens et al. 1999). Like the slow waves of intact muscles, the spontaneous inward currents of antral ICC were not blocked by nifedipine (Ozaki et al. 1991). Thus, the preparation used in this study appears to be a cell culture model of antral pacemaker cells. These cells are useful for investigations into the mechanisms of electrical rhythmicity and the frequency regulation of gastric slow waves.
The regulation of antral slow waves is interesting because clinical studies have made a link between gastric arrhythmias, which typically occur in the distal stomach, and functional motility disorders (see You et al. 1981; Kim et al. 1986; Koch et al. 1989; Chen et al. 1995). At present there is substantial uncertainty about the pathophysiological basis of gastric arrhythmias, and therefore therapies have been difficult to develop. In a few cases, tachygastria has been monitored in vivo before surgery, and then electrical activity was recorded from isolated antral muscles with intracellular microelectrode techniques (Telander et al. 1978; You et al. 1981; Sanders, 1984). These muscles displayed abnormally fast slow waves that were coupled to extremely weak contractions. Treatment with indomethacin restored a more normal electrical pattern and normal mechanical responses (see Sanders, 1984). The effects of indomethacin were reversed by PGE2, suggesting the involvement of prostaglandins in the generation of the basic arrhythmia (Sanders, 1984). Investigations of gastric arrhythmias in animals have led to mixed ideas about the physiological causes of arrhythmias. Tachygastria can occur spontaneously in dogs (Code & Marlett, 1974; Gullikson et al. 1980), and biologically active agents can also induce arrhythmias with patterns of both tachygastria and bradygastria (Kim et al. 1986). Among these are PGE2, glucagon, and vasopressin. The present study clarifies how PGE2, depending upon the dominance of specific types of EP receptors, might yield different types of gastric arrhythmias.
The role of cAMP in regulating slow-wave frequency has been studied in vitro with intracellular electrophysiological recordings from strips of muscle. For example, in guinea-pig stomach, FSK, dibutyryl cAMP, 8-Br-cAMP, and 3-isobutyl-1-methylxanthine slowed or stopped spontaneous slow-wave activity (Tsugeno et al. 1995). Variable frequency responses were observed in canine antral muscles exposed to drugs known to enhance cAMP (e.g. slow-wave frequency was reduced by FSK and isoproterenol; vaso-intenstinal peptide enhanced the frequency of spontaneous slow-wave activity, and calcitonin-gene-related peptide had no significant effect (Ozaki et al. 1992b). Experiments in the present study showed that FSK reduced slow-wave frequency in intact muscles and in cultured ICC. Additional agonists known to enhance cAMP levels mimicked these effects. However, inhibitors of PKA did not change the effects of FSK or 8-Br-cAMP, suggesting that cAMP exerts direct actions on the pacemaker mechanism. These observations suggest that the regulation of antral spontaneous inward currents by cAMP differs from regulation of small intestinal pacemaker activity. We have shown previously that that membrane-permeable analogues of cAMP were ineffective in slowing pacemaker frequency in ICC from the small intestine (Koh et al. 2000).
Although FSK and 8-Br-cAMP reduced slow-wave frequency in intact murine antral muscles, an increase in frequency was noted when muscles were exposed to PGE2. FSK and 8-Br-cAMP also inhibited spontaneous inward currents in ICC, and PGE2 inhibited spontaneous inward currents in these cells. We reasoned that the differences between the actions of PGE2 on pacemaker cells and intact muscles might be due to stimulation of different PGE2 receptors. Butaprost, a EP2 agonist, mimicked the effects of PGE2 in ICC and inhibited spontaneous inward currents. Butaprost also inhibited the frequency of electrical slow waves in intact muscles, an effect opposite to that observed with PGE2. An EP3/EP1 agonist, sulprostone (EP3 > EP1), and the EP3 agonists GR63799X and ONO-AE-248, stimulated the frequencies of spontaneous inward currents in ICC and slow waves in intact muscles. The effects of sulprostone were not blocked in cells or tissues by SC-19220, an antagonist of EP1 receptors. Taken together, these observations suggest that the dominant positive chronotropic effects of PGE2 in intact muscles are due to the stimulation of EP3 receptors.
Mechanisms linked to EP2 receptors appear to dominate in cultured ICC. Binding of ligands to EP2 receptors activates adenylyl cyclase (Honda et al. 1993; Bastien et al. 1994), so it is consistent that butaprost mimicked the effects of FSK and 8-Br-cAMP. EP3 receptors are thought to be coupled through Gi protein to inhibit adenylyl cyclase (Namba et al. 1993), so in gastric muscles the ongoing production of cAMP may limit the spontaneous frequency of the slow waves generated by antral pacemakers. Acetylcholine, which is also known to enhance the frequency of slow waves (El-Sharkawy & Szurszewski, 1978; Ördög et al. 1999), inhibits cAMP production via M2 receptors in GI muscles (see Zhang & Buxton, 1991). Thus, cAMP-dependent mechanisms may reduce slow-wave frequency, and inhibition of cAMP production may be a stimulus for increasing the frequency of antral slow waves. Some reports have also suggested that EP3 receptors activate phospholipase C via a pertussis-toxin-insensitive pathway (i.e. these effects are not coupled via G
i, see Asboth et al. 1996). Based on the fundamental role of IP3-receptor-dependent Ca2+ release in initiating spontaneous inward currents (Ward et al. 2000; Suzuki et al. 2000; van Helden et al. 2000), it is also possible that increasing IP3 production could be a signal that mediates the positive chronotropic effects of PGE2 in intact muscles. Our results suggest that the abnormal expression of EP receptors could lead to inappropriate signalling via PGE2 in antral muscles, and depending upon how the balance of EP3/EP2/EP4 receptors shifts, arrhythmias such as tachygastria or bradygastria could occur. The site of prostanoid production and the proximity of various EP receptors to the sources of prostanoid synthesis could also alter the nature of responses to PGE2.
In summary, cultured ICC from the murine antrum generate rhythmic spontaneous inward currents. These currents appear to be the basis for gastric slow-wave activity. The cAMP-dependent regulation of spontaneous inward currents in ICC is consistent with cAMP-dependent effects on slow waves in intact antral muscles. Stimuli coupled via cAMP reduce the frequency of spontaneous inward currents and gastric slow waves. Regulation by PGE2 is dependent upon the dominant receptor type expressed by pacemaker cells. The positive chronotropic effects of PGE2 in antral muscles appear to be due to the stimulation of EP3 receptors. Antagonists to these receptors may be useful in treating some types of gastric arrhythmias and functional disorders of the stomach. The second-messenger cascade that mediates the chronotropic effects of EP3 receptors has not yet been determined.
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Acknowledgements
This study was supported by a research grant from the National Institute of Digestive Diseases and Kidney Diseases (RO1 DK40569). Morphological studies were performed in a core lab supported by PO1 DK41315. We are grateful to Dr Takayuki Maruyama, ONO Pharmaceutical, for gifts of EP receptor agonists, Dr Alan Mangel, GLAXO Welcome, for GR 63799X, and Kyle Pellett for help with the mechanical experiments.
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in G), GR 63799 (GR; 
in G) and ONO-AE-248 (ONO; 
in G) on spontaneous inward currents are summarized in E (duration), F (frequency) and G (resting current). IR denotes resting current at the holding potential. Asterisks denotes significant increases (P < 0.05).