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J Physiol (2003), 548.1, pp. 175-189
© Copyright 2003 The Physiological Society
DOI: 10.1113/jphysiol.2002.035550
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ABSTRACT |
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Modulation of spontaneous electrical activities (slow waves, pacemaker potentials and follower potentials) in response to hyperpolarization produced by the ATP-sensitive K+ channel openers (KCOs) pinacidil or nicorandil was investigated in smooth muscle tissues of the guinea-pig stomach antrum. With hyperpolarization, the amplitude of slow waves and follower potentials was reduced and that of pacemaker potentials was increased, with a minor modulation of their frequency. The attenuation of slow waves was associated with an inhibition of the 1st component and abolition of the 2nd component. All these actions of KCOs were antagonized by glibenclamide. An increase in the extracellular K+ concentration prevented the KCO-induced hyperpolarization with partial restoration of slow waves, suggesting that the inhibition was produced mainly by a decrease in membrane resistance. Exposure of tissues to KCOs for a long period of time (> 20 min) resulted in the reappearance of slow waves displaying both 1st and 2nd components. The 2nd component of the slow wave, which displayed a slower recovery, was inhibited again by 5-hydroxydecanoic acid, an inhibitor of mitochondrial ATP-sensitive K+ channels. Noradrenaline hyperpolarized the membrane by activating apamin-sensitive K+ channels and increased the amplitude and frequency of slow waves through activation of1-adrenoceptors, actions different from those of KCOs. Thus, inhibition of slow waves by KCOs may be primarily related to the decrease in amplitude of a passive electrotonic component, possibly due to a reduction of the input resistance. The hyperpolarization shifted the threshold potential for generation of the 2nd component of slow waves to negative levels, presumably due to modulation of mitochondrial functions.
(Received 5 November 2002; accepted after revision 16 January 2003; first published online 21 February 2003)
Corresponding author H. Suzuki: Department of Physiology, Nagoya City University Medical School, Mizuho-ku, Nagoya 467-8601, Japan. Email: hisuzuki{at}med.nagoya-cu.ac.jp
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INTRODUCTION |
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Gastric smooth muscles are spontaneously active, and generate rhythmic activities such as slow waves or action potentials, or both (Tomita, 1981). Thuneberg (1982) considered that the rhythmic activities might originate in the interstitial cells of Cajal (ICC) distributed in the myenteric region of gastrointestinal tracts, since these cells are rich in mitochondria and have close contact with surrounding ICC and smooth muscle cells. ICC express c-Kit immunoreactivity and form gap junctional connections with each other and with nearby smooth muscle cells (Komuro et al. 1996, 1999; Sanders, 1996, 1999; Huizinga et al. 1997). When intracellular recordings are made from the guinea-pig gastric antrum, three types of electrical responses are found: pacemaker potentials, which are recorded from myenteric interstitial cells (ICC-MY); slow waves, which are recorded from circular smooth muscles; and follower potentials, which are recorded from longitudinal smooth muscles (Dickens et al. 1999). Simultaneous recordings of electrical responses from ICC-MY and smooth muscle cells show that pacemaker potentials appear prior to slow waves or follower potentials, suggesting that the electrical signals are generated in ICC-MY and are propagated to smooth muscle cells, possibly through gap junctions (Dickens et al. 1999; Hirst & Edwards, 2001).
ATP-sensitive K+ (KATP) channels, first identified in the sarcolemma of cardiac muscle by Noma (1983), are distributed widely in many tissues including pancreatic 
-cells, neurons, skeletal muscle cells and smooth muscle cells (Kuriyama et al. 1998). In cardiac muscles, there are two subtypes of KATP channel, a sarcolemmal KATP (sarco-KATP) channel and a mitochondrial KATP (mito-KATP) channel; KATP channel openers (KCOs) are considered to protect against ischaemia-reperfusion injury through activation of mito-KATP channels (Grover & Garlid, 2000). Mito-KATP channels distributed in the mitochondrial inner membrane are a different isoform to sarco-KATP channels (Inoue et al. 1991), and may be involved in mitochondrial volume control, mitochondrial Ca2+ handling or production of reactive oxygen species (O'Rourke, 2000). In isolated gastric antrum muscle of the guinea-pig, an involvement of mito-KATP channels in the generation of spontaneous activity is suggested from the inhibition of slow potentials by glibenclamide or 5-hydroxydecanoic acid (5-HDA), known inhibitors of mito-KATP channels (Fukuta et al. 2002).
KATP channels are activated by cromakalim, diazoxide, nicorandil and pinacidil, chemicals known as K+ channel openers, and are inhibited by sulfonylurea derivatives such as glibenclamide (Kuriyama et al. 1998). In guinea-pig gastric myocytes, KATP channels are composed of Kir6.1 and sulfonylurea receptor (SUR)2B (Sim et al. 2002). Stimulation of KATP channels by cromakalim inhibits the mechanical activity of circular muscle strips isolated from guinea-pig stomach antrum, with associated hyperpolarization of the membrane (Katayama et al. 1993; Huang et al. 1999). Formation of inositol 1,4,5-trisphosphate (IP3) may be involved in the generation of slow waves in stomach (Suzuki et al. 2000; Suzuki, 2000; Hirst & Edwards, 2001; Fukuta et al. 2002), and in vascular smooth muscle hyperpolarization with KCOs inhibits formation of IP3 stimulated by agonists (Itoh et al. 1992). It is therefore expected that the hyperpolarization of the membrane with KCOs may inhibit rhythmic generation of spontaneous activity in the stomach.
Experiments were carried out to investigate the effects of hyperpolarization with KCOs on slow waves, pacemaker potentials and follower potentials recorded from the gastric antrum of the guinea-pig stomach. Gastric smooth muscles are also hyperpolarized by stimulation with -adrenoceptor agonists such as adrenaline (Chihara & Tomita, 1987). The effects of membrane hyperpolarization on waveforms were further characterized by comparing them with those induced by stimulation of -adrenoceptors with noradrenaline (NAd). The results indicate that KCOs hyperpolarize the membrane and reduce electrical responses of smooth muscles conducted passively from pacemaker cells through gap junctions, while they augment the activity of pacemaker cells. In circular muscles, generation of the 2nd component of slow waves was inhibited following the application of KCOs, because the amplitude of the 1st component decreased until it failed to reach threshold for generation of the 2nd component. Hyperpolarization produced by either KCOs or NAd shifted the threshold potential for generation of the 2nd component, presumably due to modulation of mitochondrial functions.
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METHODS |
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Albino guinea-pigs of either sex, weighing 200-300 g, were anaesthetized with fluoromethyl 2,2,2-trifluoro-1-(trifluoromethyl) ethyl ether (Sevoflurane, Maruishi Pharmaceuticals, Osaka, Japan), and exsanguinated from the femoral artery. All animals were treated according to the Guidelines for the Care and Use of Laboratory Animals of Nagoya City University Medical School, accredited by The Physiological Society of Japan. The stomach was excised, and opened in Krebs solution (see below) by cutting along the small curvature. The mucosal layers were removed by cutting with fine scissors, and smooth muscle tissues were isolated from the antral region. The serosal layer was carefully removed under a dissecting microscope. A tissue segment (about 1.5 mm wide and 3 mm long), with longitudinal and circular muscles attached, was pinned out with the serosal side uppermost on a silicone rubber plate fixed at the bottom of an organ bath (8 mm wide, 8 mm deep, 20 mm long). The tissue was superfused with warmed (35°C), oxygenated Krebs solution, at a constant flow rate of about 2 ml min-1. Experiments were carried out in the presence of 1 µM nifedipine throughout, to minimize the movement of muscles.
Conventional microelectrode techniques were used to record intracellular electrical responses of smooth muscle tissues; the glass capillary microelectrodes (outer diameter, 1.2 mm; inner diameter, 0.6 mm; Hilgenberg, Germany) filled with 3 M KCl had tip resistances ranging between 50 and 80 M
. Electrical responses recorded via a high-input impedance amplifier (Axoclamp-2B, Axon Instruments, Inc., Foster City, CA, USA) were displayed on a cathode-ray oscilloscope (SS-7602, Iwatsu, Osaka, Japan) and stored on a personal computer for later analysis.
The ionic composition of the Krebs solution was as follows (mM): Na+, 137.4; K+, 5.9; Ca2+, 2.5; Mg2+, 1.2; HCO3-, 15.5; H2PO4-, 1.2; Cl-, 134; and glucose, 11.5. Solutions containing high concentrations of K+ (high-K+ solutions) were prepared by replacing NaCl with KCl. The solutions were aerated with O2 containing 5 % CO2, and the pH of the solutions was maintained at 7.2-7.3.
Drugs used were diazoxide, glibenclamide, 5-HDA, nifedipine, noradrenaline hydrochloride (NAd), 1H-[1,2,4]-oxadiazole[4,3-a]quinoxalin-1-one (ODQ), pinacidil (all from Sigma, St Louis, MO, USA), apamin (Peptide Institute, Osaka, Japan) and phentolamine mesylate (Ciba-Geigy, Basel, Switzerland). Nicorandil was a gift from Chugai Pharmaceutical Co. Ltd. Stock solutions of diazoxide, glibenclamide, nicorandil, nifedipine, ODQ and pinacidil were made in dimethyl sulphoxide (DMSO), and added to Krebs solution to give the desired concentrations just prior to use. Other drugs tested were dissolved in distilled water. The final concentration of the solvent in Krebs solution did not exceed 1/1000. Addition of these chemicals to Krebs solution did not alter the pH of the solution.
Experimental values are expressed as the mean value ± standard deviation (S.D.). Statistical significance was tested using Student's t test, and probabilities of less than 5 % (P < 0.05) were considered significant.
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RESULTS |
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Effects of KATP channel openers on spontaneous electrical activity recorded from guinea-pig antrum
In antral tissues of the guinea-pig stomach, three distinct patterns of ongoing discharge of rhythmic electrical activity were recorded: triangular potentials with slow rates of rise (< 0.2 V s-1) and with amplitudes of 20-30 mV (slow waves), square-shaped potentials with fast rates of rise (> 0.2 V s-1) and subsequent plateau components with amplitudes of 40-50 mV (pacemaker potentials), and square-shaped potentials with slow rates of rise (< 0.2 V s-1) and subsequent plateau components with amplitudes of 20-30 mV (follower potentials). Comparison of the properties of these potentials with those reported previously (Dickens et al. 1999; Hirst & Edwards, 2001; Kito et al. 2002a; Kito & Suzuki, 2003) indicated that the slow waves, follower potentials and pacemaker potentials were recorded from circular muscle, longitudinal muscle and ICC-MY, respectively.
The effects of pinacidil and nicorandil on slow waves were investigated in the presence of 1 µM nifedipine. Application of pinacidil (0.01-100 µM) hyperpolarized the membrane in a concentration-dependent manner, and at concentrations higher than 10 µM the hyperpolarization reached a stable amplitude of about 10 mV (data not shown). Experiments were carried out to test the effects of 15 µM pinacidil on slow waves, since this concentration of pinacidil hyperpolarized the membrane by about 10 mV (mean, 9.7 ± 1.3 mV, n = 19). With hyperpolarization, the amplitude of the slow waves was reduced (control, 32.7 ± 2.8 mV; in pinacidil, 8.6 ± 4.1 mV; n = 19; P < 0.01; each n value represents a separate animal, unless indicated otherwise), with a non-significant decrease in frequency (control, 3.23 ± 0.63 min-1; in pinacidil, 2.49 ± 0.65 min-1; n = 19; P > 0.05). Glibenclamide (10 µM) added in the presence of pinacidil restored the membrane potential to the resting level (control, -67.4 ± 2.5 mV; in pinacidil, -77.4 ± 2.1 mV; n = 8; P < 0.01; co-application of pinacidil and glibenclamide, -67.9 ± 2.2 mV; n = 8; P > 0.05), and also the amplitude (control, 32.1 ± 2.1 mV; in pinacidil, 9.4 ± 5.7 mV; n = 8; P < 0.01; co-application of pinacidil and glibenclamide, 30.9 ± 3.3 mV; n = 8; P > 0.05) and frequency (control, 3.20 ± 0.89 min-1; in pinacidil, 2.12 ± 0.67 min-1; n = 8; P > 0.05; co-application of pinacidil and glibenclamide, 2.97 ± 0.74 min-1; n = 8; P > 0.05) of slow waves (Fig. 1A). The pinacidil-induced hyperpolarization was also inhibited by an increase in the extracellular K+ concentration to 10.6 mM (10.6 mM [K+]o) (control, -67.9 ± 2.4 mV; in pinacidil, -77.2 ± 2.4 mV; n = 14; P < 0.01; co-application of pinacidil and 10.6 mM [K+]o, -63.4 ± 2.5 mV; n = 14; P < 0.01); the increase in extracellular K+ concentration also resulted in an increase in the frequency of slow waves (control, 3.25 ± 0.45 min-1; in pinacidil, 2.75 ± 0.53 min-1; n = 14; P > 0.05; co-application of pinacidil and 10.6 mM [K+]o, 4.58 ± 0.86 min-1; n = 14; P < 0.01; Fig. 1B). However, in the presence of 10.6 mM [K+]o solution, the amplitude of the slow waves recovered partially in five preparations (23.7 ± 5.5 mV; P < 0.05, when compared to the control values), but still remained low in nine preparations (7.6 ± 1.1 mV; P < 0.01 when compared to the control values; Fig. 1B).
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Figure 1. Effects of KATP channel openers on slow waves recorded from guinea-pig antrum A and B, slow waves recorded during application of 15 µM pinacidil followed by 10 µM glibenclamide (A) or 10.6 mM [K+]o solution (B). C, slow waves recorded during application of 300 µM nicorandil followed by 10 µM glibenclamide. D, in the presence of 10 µM ODQ for 30 min, slow waves recorded during application of nicorandil (300 µM), followed by 10 µM glibenclamide. In this and subsequent figures, drugs were applied during the period indicated by the horizontal bar below each record. The resting membrane potentials were: A, -66 mV; B, -68 mV; C, -64 mV; D, -67 mV. All responses were recorded from different tissues. | ||
Nicorandil (300 µM) also hyperpolarized the membrane by about 10 mV (mean, 10.4 ± 1.7 mV; n = 10) and reduced the amplitude (control, 31.5 ± 2.3 mV; in nicorandil, 6.9 ± 3.3 mV; n =10; P < 0.01) and frequency (control, 3.32 ± 0.96 min-1; in nicorandil, 2.22 ± 1.16 min-1; n = 10; P < 0.05) of slow waves. Glibenclamide (10 µM) added in the presence of nicorandil restored the membrane potential to the resting level (-68.3 ± 2.4 mV; n = 10; P > 0.05 when compared to the resting potential) and restored the frequency of slow waves (3.11 ± 1.41 min-1; n = 10; P > 0.05 when compared to the control values). The amplitude of the slow waves was partially restored by application of glibenclamide in the presence of nicorandil (26.2 ± 4.0 mV; n = 10; P < 0.05 when compared to the control values; Fig. 1C). Nicorandil relaxes vascular smooth muscle by two mechanisms: a hyperpolarization of the membrane through activation of KATP channels and elevation of intracellular cGMP levels due to its role as a nitric oxide (NO) donor (Taira, 1989). The possible involvement of NO in the nicorandil-induced inhibition of slow waves was evaluated by testing the effects of ODQ, an inhibitor of guanylate cyclase. Application of 10 µM ODQ had no effect on the resting membrane potential (control, -65.2 ± 2.6 mV; in ODQ, -66.2 ± 2.4 mV; n = 8; P > 0.05) or the amplitude (control, 32.5 ± 4.4 mV; in ODQ, 31.7 ± 4.5 mV; n = 8; P > 0.05) or frequency (control, 3.21 ± 0.50 min-1; in ODQ, 3.19 ± 0.57 min-1; n = 8; P > 0.05) of slow waves, as has been reported previously (Kito & Suzuki, 2003). In the presence of ODQ, nicorandil (300 µM) hyperpolarized the membrane (10.7 ± 1.6 mV, n = 8) and decreased the amplitude (10.2 ± 3.5 mV; n = 8; P < 0.01) and frequency (2.09 ± 0.68 min-1; n = 8; P < 0.05) of slow waves; the effects were similar to those in the absence of ODQ. The restoration by glibenclamide of the nicorandil-induced hyperpolarization (-67.4 ± 2.4 mV; n = 8; P > 0.05 when compared to the resting potential) and the slow wave frequency (2.99 ± 0.68 min-1; n = 8; P > 0.05 when compared to the control values) was again similar in the absence and presence of ODQ. However, the slow wave amplitude (31.0 ± 2.7 mV; n = 8; P > 0.05 when compared to the control values) was restored completely by glibenclamide in the presence of ODQ (Fig. 1D). These results indicate that the inhibition of slow waves by nicorandil is produced by the opening of KATP channels and activation of guanylate cyclase.
In pacemaker potential-generating cells, both pinacidil (15 µM) and nicorandil (300 µM) hyperpolarized the membrane, and increased the amplitude and decreased the frequency of pacemaker potentials. The duration of pacemaker potentials measured at one-half peak amplitude (half-width) and the maximum rate of rise (dV/dtmax) of the initial component were also reduced by these KCOs (Fig. 2 and Table 1). Similar alterations were also elicited on the time course of follower potentials in the presence of pinacidil (15 µM) or nicorandil (300 µM), although not on the amplitude. Pinacidil and nicorandil hyperpolarized the membrane, and decreased the amplitude, frequency, half-width and dV/dtmax of follower potentials (Fig. 2 and Table 2). Glibenclamide (10 µM) completely antagonized the effects of KCOs on both pacemaker potentials and follower potentials (Tables 1 and 2). Application of 10.6 mM [K+]o solution depolarized the membrane to a level more positive than the resting potential, and reversed the effects of KCOs on the amplitude, frequency and dV/dtmax of pacemaker potentials (Table 1). In contrast, the amplitude and dV/dtmax of follower potentials were not restored by application of 10.6 mM [K+]o solution in the presence of KCOs (Table 2).
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Figure 2. Effects of KATP channel openers on pacemaker potentials and follower potentials recorded from guinea-pig antrum A and B, pacemaker potentials recorded during application of 15 µM pinacidil followed by 10 µM glibenclamide (A) or 10.6 mM [K+]o solution (B). C and D, follower potentials recorded during application of 15 µM pinacidil followed by 10 µM glibenclamide (C) or 10.6 mM [K+]o solution (D). The resting membrane potentials were: A, -65 mV; B, -66 mV; C, -61 mV; D, -64 mV. All responses were recorded from different tissues. | ||
Slow waves are composed of 1st and 2nd components (Tomita, 1981; Dickens et al. 1999; Kito et al. 2002a); experiments were therefore carried out to observe the changes in configuration of slow waves during hyperpolarization with 15 µM pinacidil (Fig. 3). With hyperpolarization, the amplitude of the 1st component and the duration of the 2nd component were decreased, and the threshold potential for generation of the 2nd component (equal to the peak amplitude of the 1st component) was shifted to more negative levels (control, -52.7 ± 3.6 mV; in pinacidil, -64.4 ± 2.1 mV; n = 22; P < 0.01; Fig. 3B-D). When the amplitudes of the 1st and 2nd components of slow waves were plotted as a function of hyperpolarization produced by pinacidil, the relationship indicated a linear decrease in the amplitude of the 1st component with hyperpolarization (Fig. 4A). The regression line calculated using the least squares method indicated that the change of the 1st component could be fitted by Y = 13.65 - 0.55X (where Y is the amplitude of the 1st component of the slow wave, and X is the amplitude of hyperpolarization; r = 0.61, P < 0.05). The amplitude of the 2nd component tended to increase with up to 6 mV of hyperpolarization, with a regression line of Y = 31.50 + 0.73X (where Y is the amplitude of the 2nd component of the slow wave, and X is the amplitude of hyperpolarization; r = 0.61, P < 0.05), and then decreased with further hyperpolarization (Fig. 4A). The amplitude of the pacemaker potentials increased with hyperpolarization, with a regression line of Y = 51.15 + 0.67X (where Y is the amplitude of the pacemaker potential, and X is the amplitude of hyperpolarization; r = 0.33, P < 0.05; Fig. 4B). The amplitude of the follower potentials decreased with hyperpolarization, with a regression line of Y = 28.85 - 1.68X (where Y is the amplitude of the follower potential, and X is the amplitude of hyperpolarization; r = 0.87, P < 0.05; Fig. 4C).
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Figure 3. Pinacidil-induced inhibition of slow waves in the guinea-pig antrum A, slow waves recorded during application of 15 µM pinacidil. B-D, high-speed traces of slow waves recorded in the absence (B) and presence of 15 µM pinacidil for 2 min (C) and for 3 min (D). Arrows indicate the peak amplitude of the 1st component of the slow wave. The resting membrane potential was -66 mV. | ||
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Figure 4. The relationship between pinacidil-induced hyperpolarization and the amplitude of slow waves, pacemaker potentials and follower potentials recorded from guinea-pig antrum A, the relationship between pinacidil (15 µM)-induced hyperpolarization (abscissa) and the amplitude of the 1st ( | ||
These results indicate that the amplitude of the passive component of electrical responses (i.e. slow waves, follower potentials) decreases with hyperpolarization, whereas the amplitude of active potentials (i.e. pacemaker potentials) increases with hyperpolarization induced by KCOs. The hyperpolarization modulates the amplitude of the 2nd component of slow waves in a biphasic way: an increase is followed by a decrease. The results also show that the threshold potential for generation of the 2nd component of slow waves is shifted to a negative level with hyperpolarization.
Modulation of slow waves during exposure to KATP channel openers for a long period of time
In the guinea-pig stomach, the effects on spontaneous activities of stimulation of smooth muscle tissues with KCOs for long periods were investigated. In the absence of pinacidil, slow waves were composed of two components. The 1st component started from the resting potential (dashed line in Fig. 5B) and the 2nd component appeared around -60 mV (arrow in Fig. 5Ba). Pinacidil (15 µM) initially hyperpolarized the membrane by about 9 mV; however, the membrane did not remain polarized to the same level during sustained exposure to pinacidil. The amplitude diminished gradually to reach a stable value of 3-5 mV positive to the peak hyperpolarization within 20-30 min (Fig. 5A). Associated with these changes in the membrane potential, in the presence of pinacidil, the amplitude of the slow waves increased gradually. In the presence of 15 µM pinacidil for 4 min, the membrane polarized to about -78 mV and the amplitude of the slow waves was reduced to about 5 mV. These slow waves were composed mainly of the 1st component with a trace of the 2nd component appearing close to their peak (around -73 mV; Fig. 5Bb). In slow waves that had increased to about 10 mV in amplitude in the presence of pinacidil for 19 min, the threshold for generation of the 2nd component was around -70 mV (Fig. 5Bc). In the presence of pinacidil for 22 min, slow waves with two components were generated, with the 2nd component appearing around -70 mV (Fig. 5Bd). Co-application of glibenclamide with pinacidil resulted in the recovery of slow waves to those observed before application of pinacidil, with the threshold potential for the 2nd component around -60 mV (Fig. 5Be).
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Figure 5. Effects of long-term exposure to KATP channel openers on slow waves recorded from guinea-pig antrum A, slow waves recorded during application of 15 µM pinacidil followed by 10 µM glibenclamide. B, high-speed traces of slow waves recorded before (a) and during application of 15 µM pinacidil at 4 min (b), 19 min (c), 22 min (d), and during co-application of 10 µM glibenclamide (e). Arrows indicate the peak amplitude of the 1st component of the slow wave and the dashed line indicates the resting potential. C, slow waves recorded during application of 15 µM pinacidil followed by 3 mM 5-HDA and 10 µM glibenclamide. D, high-speed traces of slow waves recorded before (a) and during application of 15 µM pinacidil for 5 min (b), 34 min (c), 45 min (d), and during co-application of 3 mM 5-HDA (e), and further application of 10 µM glibenclamide (f). Arrows indicate the level of the peak potential of the 1st component of the slow wave. The resting membrane potentials were: A, -67 mV; C, -68 mV. A and C were recorded from different tissues. | ||
The peak potentials of the 1st and 2nd components were measured in slow waves generated in the initial 3-6 min (short period), in those which had increased to a stable amplitude but with a partial 2nd component (middle period), in those with 1st and 2nd components (long period), and in those generated in the presence of both pinacidil and glibenclamide. The summarized data show that the threshold potential for generation of the 2nd component was around -52 mV in slow waves generated in the absence of pinacidil, -69 mV in those generated during the short period, -66 mV in those generated during the middle period, -64 mV in those generated during the long period, and -52 mV in those generated in the presence of both pinacidil and glibenclamide (Fig. 6C). The peak potentials of the slow waves were similar in the absence or long-term presence of pinacidil and also in the presence of both pinacidil and glibenclamide (about -33 mV), but were significantly negative in the presence of pinacidil for the short and middle periods (-60 to -65 mV). In a different series of experiments, nicorandil (300 µM) with ODQ (10 µM; Fig. 6D) or diazoxide (200 µM, data not shown; n = 4) also modulated slow waves, as in the case of pinacidil. Thus, the hyperpolarization produced by KCOs was accompanied by a shift of the threshold potential for generation of the 2nd component of slow waves to negative levels. Experiments carried out in 27 preparations indicated that the time required for generation of slow waves with two components to recommence varied widely, between 14 and 63 min (Fig. 6A).
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Figure 6. Distribution of time required for the reappearance of the 2nd component of slow waves during application of KATP channel openers and effects of long-term application of KATP channel openers on the membrane potential and amplitude of slow waves recorded from guinea-pig antrum A and B, histograms showing the time required for the reappearance of the 2nd component of slow waves during application of 15 µM pinacidil (n = 27; A) or 300 µM nicorandil (n = 14; B). KATP channel openers were applied at time zero. C and D, resting membrane potential ( | ||
In preparations in which the two components of slow waves had been generated during long-term stimulation with pinacidil, co-application of 5-HDA (3 mM) with pinacidil reduced the amplitude of the 2nd component of the slow wave from 27.2 ± 3.8 mV (n = 14) to 7.1 ± 3.1 mV (n = 14, P < 0.01), with no significant alteration in the membrane potential (in pinacidil, -75.0 ± 3.2 mV; in pinacidil and 5-HDA, -74.7 ± 2.9 mV; n = 14; P > 0.05) or frequency of the slow waves (in pinacidil, 2.75 ± 0.70 min-1; in pinacidil and 5-HDA, 2.70 ± 1.09 min-1; n = 14; P > 0.05; Fig. 5C, Dd and De). The amplitude of the 1st component of the slow waves generated in the presence of pinacidil and 5-HDA (7.7 ± 2.8 mV; n = 14; P > 0.05) was similar to that prior to the recovery of the 2nd component (7.1 ± 3.3 mV; n = 14; P > 0.05; Fig. 5Dc). In three preparations, 5-HDA partially inhibited slow waves generated in the presence of pinacidil (in pinacidil, 36.5 ± 4.5 mV; in pinacidil and 5-HDA, 26.9 ± 4.4 mV; P > 0.05). Addition of 10 µM glibenclamide together with pinacidil and 5-HDA restored the membrane potential to the resting level (-66.5 ± 1.8 mV; n = 14; P > 0.05 when compared to the resting potential); however, the amplitude of the slow wave was smaller than that in the absence of pinacidil, due to reduction of the 2nd component (14.5 ± 3.1 mV; n = 14; P < 0.05 when compared to the control values), with no alteration in the 1st component (10.9 ± 4.0 mV; n = 14; P > 0.05 when compared to the control values; Fig. 5Df). These data are summarized in Fig. 6E.
In a different series of experiments, the effects of long-term application of nicorandil on slow waves were also investigated in antrum smooth muscles. In the presence of ODQ (10 µM), nicorandil (300 µM) elicited similar changes in slow waves to those produced by pinacidil (n = 7; Fig. 6F). The time required for restarting generation of slow waves with two components varied widely between 9 and 59 min (Fig. 6B). The actions of diazoxide (200 µM), another type of KCO, on slow waves were found to be similar to those of pinacidil and nicorandil (data not shown; n = 6).
The effects of exposure to pinacidil for long periods of time on the amplitudes of pacemaker potentials and follower potentials were also studied. Application of 15 µM pinacidil for 3-5 min hyperpolarized the membrane by about 9 mV and reduced the amplitude of follower potentials (control, 30.6 ± 5.3 mV; in pinacidil, 10.0 ± 2.6 mV; n = 5; P < 0.01). During exposure to pinacidil, the amplitude of follower potentials gradually increased and in 30 min had reached a stable value of 20.6 ± 6.6 mV (n = 5; P < 0.05), this value being significantly smaller than that in the absence of pinacidil. The amplitude of the pacemaker potentials remained unaltered in the presence of 15 µM pinacidil for up to 60 min (data not shown; n =3).
Effects of noradrenaline on spontaneous electrical activity recorded from guinea-pig antrum
The effects of NAd on slow waves, pacemaker potentials and follower potentials were compared with those of KCOs, since both kinds of agent hyperpolarize the membrane in gastric smooth muscles. Application of NAd (15 µM) hyperpolarized the membrane by 7.2 ± 1.7 mV (n = 14) and increased the amplitude of slow waves (control, 30.9 ± 3.9 mV, n = 14; in NAd, 39.4 ± 2.7 mV, n = 14; P < 0.01; Fig. 7A). In the presence of apamin (0.3 µM), the NAd-induced hyperpolarization was abolished (control, -68.5 ± 2.7 mV; in apamin, -68.0 ± 2.6 mV; n = 10; P > 0.05; in apamin and NAd, -67.2 ± 2.5 mV; n = 10; P > 0.05). In this condition NAd increased the amplitude (control, 30.3 ± 3.8 mV; in apamin, 31.7 ± 4.1 mV; n = 10; P > 0.05; in apamin and NAd, 36.9 ± 3.3 mV; n = 10; P < 0.05) and frequency (control, 3.20 ± 0.37 min-1; in apamin, 3.53 ± 0.47 min-1; n = 10; P > 0.05; in apamin and NAd, 4.41 ± 0.37 min-1; n = 10; P < 0.01) of slow waves (Fig. 7B). The NAd-induced hyperpolarization was also blocked by 1 µM phentolamine, an -adrenoceptor antagonist (in phentolamine, -67.3 ± 2.2 mV; in phentolamine and NAd, -68.0 ± 2.5 mV; n = 8; P > 0.05). In the presence of phentolamine, NAd did not alter the amplitude (in phentolamine, 33.5 ± 4.9 mV; in phentolamine and NAd, 33.4 ± 4.7 mV; n = 8; P > 0.05) or frequency (in phentolamine, 3.09 ± 0.35 min-1; in phentolamine and NAd, 3.10 ± 0.38 min-1; n = 8; P > 0.05) of slow waves.
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Figure 7. Effects of NAd on slow waves, pacemaker potentials and follower potentials recorded from the guinea-pig antrum The responses of slow waves (A and B), pacemaker potentials (C and D) and follower potentials (E and F) to the application of NAd (15 µM) recorded in the absence (A, C and E) and presence (B, D and F) of apamin (0.3 µM) for 15 min. The resting membrane potential was: A, -67 mV; B, -68 mV; C, -65 mV; D, -66 mV; E, -61 mV and F, -60 mV. All responses were recorded from different tissues. | ||
The high-speed traces of slow waves generated during stimulation with 15 µM NAd indicate that the increase in amplitude of slow waves during the hyperpolarization was associated with a decrease in amplitude of the 1st component (control, 14.8 ± 4.4 mV; in NAd, 10.1 ± 2.4 mV; n = 14; P < 0.01) and an increase in the 2nd component (control, 16.1 ± 4.4 mV; in NAd, 29.3 ± 3.1 mV; n = 14; P < 0.01), with no alteration to the peak levels of potentials (control, -35.6 ± 4.4 mV; in NAd, -34.0 ± 4.1 mV; n = 14; P > 0.05; Fig. 8A and B). These changes were also accompanied by a shift of the threshold potential for generation of the 2nd component of slow waves to a more negative level (control, -51.3 ± 5.5 mV; in NAd, -63.3 ± 4.0 mV; n = 14; P < 0.01).
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Figure 8. Effects of NAd on membrane potential and configuration of slow waves recorded from guinea-pig antrum Slow waves were recorded in the absence (A, left) and presence (A, right) of 15 µM NAd. Arrows indicate the peak amplitude of the 1st component of the slow wave. The resting membrane potential (dashed line) was -65 mV. B, membrane potentials at the most negative level ( | ||
In a different series of experiments, the effects of NAd on pacemaker potentials and follower potentials were examined; the results are summarized in Table 3. In both cell types, NAd (15 µM) hyperpolarized the membrane by about 7 mV, and decreased the duration (half-width) and dV/dtmax of pacemaker potentials and follower potentials (Fig. 7C and E). However, NAd increased the amplitude of pacemaker potentials and decreased that of follower potentials. In pacemaker potential-generating cells, the NAd-induced hyperpolarization was abolished by 0.3 µM apamin (Fig. 7D) or 1 µM phentolamine (Table 3). In follower potential-generating cells, the NAd-induced hyperpolarization was reduced by about one-half by 0.3 µM apamin (Fig. 7E and F) and was abolished by 1 µM phentolamine (Table 3). Thus, although both KCOs and NAd hyperpolarize the membrane, the amplitude of slow waves is increased by NAd and decreased by KCOs, with no marked alteration to pacemaker potentials. The amplitude of follower potentials decreased in the presence of both KCOs and NAd.
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DISCUSSION |
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The present experiments show that, in antrum smooth muscle tissues, the KCOs tested (pinacidil, nicorandil, diazoxide) hyperpolarized the membrane by similar amounts and their actions were antagonized by glibenclamide, suggesting an involvement of KATP channels. With KCO-induced hyperpolarization, slow waves and follower potentials were changed in configuration and amplitude, with little change or augmentation in pacemaker potentials. Slow waves are composed of 1st and 2nd components (Tomita, 1981). The 1st component may represent the electrotonic spread of pacemaker potentials generated in ICC-MY and the 2nd component is formed as a summation of unitary potentials (Dickens et al. 1999; Edwards et al. 1999; Hirst & Edwards, 2001). The unitary potentials are produced by an increase in the conductance of Ca2+-activated Cl- channels and are considered to originate in intramuscular ICC (ICC-IM) (Hirst et al. 2002), since these potentials are absent in antrum circular muscle of W/Wv mutant mice lacking ICC-IM (Dickens et al. 2001). The pacemaker potentials propagated to the longitudinal muscle form follower potentials (Hirst & Edwards, 2001). Thus, KCO-induced inhibition appeared mainly to affect the passive component of spontaneously generating potentials with minor inhibition of pacemaker activities. This may well explain why the frequency of spontaneous activity is not markedly modulated by cromakalim, another type of KCO, in the guinea-pig stomach (Katayama et al. 1993; Huang et al. 1999).
The amplitude of pacemaker potentials was increased by similar amounts during hyperpolarization with either KCOs or NAd. The pacemaker potential consists of a primary component with a rapidly rising phase and a subsequent prolonged plateau component. The former component is produced by activation of voltage-dependent Ca2+-permeable channels and the latter component is formed by activation of Ca2+-activated Cl- channels (Hirst & Edwards, 2001; Kito et al. 2002a). The amplitude of pacemaker potentials decreases with depolarization (Kito et al. 2002a), since the level of the plateau component is close to the equilibrium potential for Cl- (equal to around -20 mV; Aickin & Brading, 1982). Thus, the increase in amplitude of pacemaker potentials during hyperpolarization with KCOs or NAd may be mainly due to the increase in potential difference between the resting potential and equilibrium potential for Cl-. Unitary potentials generated in ICC-IM could also function as a pacemaker in circular muscles with no attached ICC-MY, by forming regenerative slow potentials (Suzuki & Hirst, 1999; Edwards et al. 1999) or the 2nd component of slow waves (Dickens et al. 2001). However, the 2nd component of the slow waves was inhibited by KCOs but augmented by NAd, even though the degree of hyperpolarization was similar. The NAd-induced hyperpolarization may be related to an increase in [Ca2+]i, since the channels activated are a Ca2+-sensitive type (i.e. apamin-sensitive K+ channel), whereas KCOs activate ATP-sensitive K+ channels. These results suggest that NAd increases and KCO reduces [Ca2+]i in ICC-IM.
The present study indicates that the decrease in amplitude of the 1st component of the slow waves and of follower potentials with KCOs paralleled the hyperpolarization. These two potentials are passive components, propagated from ICC-MY to smooth muscle cells in an electrotonic manner via gap junctions (Dickens et al. 1999; Hirst & Edwards, 2001). The question arises as to how the amplitude of these passive components decreases in spite of an increase in amplitude of pacemaker potentials during the hyperpolarization. In 10.6 mM [K+]o solutions, KCOs still inhibited the amplitude of slow waves or follower potentials, with no hyperpolarization of the membrane, indicating that the hyperpolarization was not the main determinant for the amplitude of these potentials. The KCO-induced hyperpolarization is produced by activation of KATP channels, and the input resistance of the membrane may be reduced during the hyperpolarization (Nakao et al. 1988). Our preliminary experiments indicated that, in a circular muscle bundle of the guinea-pig antrum, the hyperpolarization with pinacidil resulted in a reduction in amplitude of electrotonic potentials produced by current injection to about a quarter, suggesting a reduction of the input resistance of the membrane to about one-sixteenth (Y. Kito & H. Suzuki, unpublished data). Thus, the decrease in amplitude of the 1st component of slow waves and of follower potentials may be mainly due to the reduction of membrane resistance.
The partial recovery of slow waves during exposure to KCOs for a long period of time may be due to the desensitization of sulphonylurea receptors to KCOs, as has been observed in vascular smooth muscle tissue (Nakao et al. 1988). During this recovery, the amplitude of the 1st component of the slow waves increased to some extent, and the 2nd component successively recovered to form full size slow potentials. The 2nd component of the slow waves may be formed as a summation of passively propagated unitary potentials generated in ICC-IM (Edwards et al. 1999; Dickens et al. 2001). Therefore, the inhibition of this component during the hyperpolarization with KCOs agrees with the conclusion that KCOs inhibit passively generated potentials such as the 1st component of slow waves and follower potentials. The reappearance of the 2nd component may be unrelated to the changes in conductance of intercellular gap junctions, since this component appears with no alteration to the amplitude of the 1st component. The recovered 2nd components were inhibited again by 5-HDA, a selective inhibitor of mito-KATP channels, suggesting that the generation of the 2nd component is related to mitochondrial function. In circular smooth muscle of the guinea-pig gastric antrum, spontaneously generating slow potentials are inhibited by glibenclamide or 5-HDA with no significant alteration to [Ca2+]i (Fukuta et al. 2002). The hyperpolarization produced by KCOs was inhibited by glibenclamide but not by 5-HDA, supporting the idea that the mito-KATP channels, but not sarco-KATP channels, are mainly involved. These results also suggest that the cellular mechanisms for the generation of spontaneous activity may differ between ICC-MY and ICC-IM, since glibenclamide and 5-HDA do not alter the frequency of the 1st component of slow waves.
In cardiac muscles, three possibilities are proposed to explain the protective effects of mitochondrial KCOs from ischaemia-reperfusion injury: (1) controlling mitochondrial volume, (2) regulating the mitochondrial Ca2+-handling mechanism, and (3) producing reactive oxygen species (O'Rourke, 2000). Both diazoxide and pinacidil activate Ca2+ release from isolated rat cardiac mitochondria, possibly through depolarizing the mitochondrial membrane and decreasing the driving force for Ca2+ entry (Holmuhamedov et al. 1999). In intact cardiomyocytes, diazoxide decreases mitochondrial [Ca2+]i, the action being antagonized by 5-HDA (Holmuhamedov et al. 1999). It is therefore speculated that, in gastric muscles, KCOs release Ca2+ from mitochondria in ICC-IM and increase the generation of unitary potentials, which opens Ca2+-activated Cl- channels (Hirst et al. 2002). It has been shown recently that diazoxide inhibits succinate oxidation whereas pinacidil inhibits NADH oxidation, without alteration to the mitochondrial membrane potential, and 5-HDA-CoA, synthesized from 5-HDA and CoA by acyl-CoA synthetase, compensates for the partial inhibition of the respiratory chain induced by diazoxide (Hanley et al. 2002). These results suggest that the reappearance of the 2nd component of slow waves during long exposure to KCOs is related to the mitochondrial functions in ICC-IM.
The threshold potential for generation of the 2nd component (equal to the peak amplitude of the 1st component) is voltage sensitive, and hyperpolarization with KCOs or NAd shifts the potential to more negative levels. Similar phenomena are also elicited when the membrane is hyperpolarized by electrical stimulation (Hirst et al. 2002). The generation of regenerative slow potentials has a refractory period (Nose et al. 2000; Kito et al. 2002b; Suzuki et al. 2002), and is facilitated by depolarization, through activation of protein kinase C (Kito et al. 2002b). The generation of regenerative slow potentials is also facilitated after release of hyperpolarization (Suzuki & Hirst, 1999; Hirst et al. 2002). However, the present experiments indicated that the 2nd component of the slow waves that reappears during KCO-induced hyperpolarization is inhibited by blocking mito-KATP channels with 5-HDA. In considering the importance of inositol 1,4,5-trisphosphate (IP3) in the generation of spontaneous activity (Suzuki et al. 2000), it is speculated that hyperpolarization facilitates the processes for the production of IP3 or related signalling processes. In gastric muscles, hyperpolarization reduces [Ca2+]i (Fukuta et al. 2002), and this would facilitate the release of Ca2+ from mitochondria and thus accelerate the Ca2+-handling mechanisms.
In smooth muscles, stimulation of 1-adrenoceptors increases the production of IP3 through activation of phospholipase C, and this increases the release of Ca2+ from internal stores (Minneman, 1988). In the guinea-pig stomach, the hyperpolarization produced by adrenaline is mediated through activation of 1-adrenoceptors (Chihara & Tomita, 1987). The present experiments indicated that, in circular muscles, the NAd-induced hyperpolarization was blocked by apamin and phentolamine, suggesting that it was elicited by the opening of small conductance Ca2+-activated K+ (SK) channels, through activation of -adrenoceptors. In circular smooth muscle cells and ICC-MY of the guinea-pig stomach, SK3 channels are expressed (Klemm & Lang, 2002). In contrast, in longitudinal muscles NAd-induced hyperpolarization was completely blocked by phentolamine but only partially blocked by apamin. These results raise the possibility that NAd activates not only SK channels but also other types of channel in longitudinal muscles. NAd hyperpolarized the membrane, reduced the amplitude of the component produced by electrotonic spread of pacemaker potentials (1st component of slow waves and follower potentials), increased the amplitude of pacemaker potentials, and shifted the threshold potential for the 2nd component of slow waves to more negative levels. Taken together, these results indicate that the modulation of spontaneous activities in response to hyperpolarization is similar for NAd and KCOs, irrespective of the difference in the mechanisms involved.
In conclusion, the present study indicated that the actions of KCOs differ between slow waves, pacemaker potentials and follower potentials, mainly due to the properties of individual waveforms. Hyperpolarization of the membrane has two distinct actions on slow waves: reduction of the amplitude of the 1st component and a shift of the threshold potential for generation of the 2nd component to more negative levels. The former action may cause the inhibition of the generation of the 2nd component due to insufficient amplitude of the 1st component for generating the unitary potentials in ICC-IM. The shift of the threshold potential for the 2nd component during hyperpolarization may be related to the mitochondrial Ca2+-handling mechanisms in ICC-IM. The results support the idea that mitochondrial KCOs are applicable for the improvement of delayed gastric emptying, as a new type of prokinetic agent (Chaudhuri & Fink, 1991).
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Acknowledgements
The authors are grateful to Dr Frank R. Edwards for critical reading of the manuscript. Nicorandil was a gift from Chugai Pharmaceutical Co. Ltd (Japan). The present experiments were supported partly by the Grant-in-Aid for the Scientific Research (C) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan to H.S. (No. 14570044).
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) and 2nd (
) components of slow waves (ordinate). The regression line for the amplitude of the 1st component is given by Y = 13.65 - 0.55X (Y, amplitude of the 1st component; X, hyperpolarization; r = 0.61, n = 83 from 14 tissues, P < 0.05). The regression line for the amplitude of the 2nd component is given by Y = 31.50 + 0.73X (Y, amplitude of the 2nd component; X, hyperpolarization; r = 0.61, n = 83 from 14 tissues, P < 0.05). B, the relationship between pinacidil (15 µM)-induced hyperpolarization (abscissa) and the amplitude of pacemaker potentials (ordinate). The regression line is given by Y = 51.15 + 0.67X (Y, amplitude of pacemaker potentials; X, hyperpolarization; r = 0.33, n = 38 from 9 tissues, P < 0.05). C, the relationship between pinacidil (15 µM)-induced hyperpolarization (abscissa) and the amplitude of follower potentials (ordinate). The regression line is given by Y = 28.85 - 1.68X (Y, amplitude of follower potentials; X, hyperpolarization; r = 0.87, n = 59 from 12 tissues, P < 0.05).
), and peak membrane potentials of the 1st (
) and 2nd (
) components of slow waves measured before (control) and during application of pinacidil (C) or nicorandil in the presence of 10 µM ODQ (D) for 3-6 min (short), for the period just before the reappearance of the 2nd component of the slow wave (middle), for the period during which the 2nd component of the slow wave reappeared (long), and during co-application of 10 µM glibenclamide (Glib). **P < 0.01, significant difference from control. #P < 0.05, ##P < 0.01, significant difference from short. Means ± S.D. (n = 15 for pinacidil; n = 6 for nicorandil). E, effects of 15 µM pinacidil on the amplitudes of the 1st (
) and 2nd (
) components of the slow wave. F, effects of 300 µM nicorandil with 10 µM ODQ on the amplitudes of the 1st (