Role of Ca2+-activated Clminus channels and MLCK in slow IJP in opossum esophageal smooth muscle

Yong Zhang and William G. Paterson

Gastrointestinal Disease Research Unit and Departments of Medicine, Biology, and Physiology, Queen's University, Kingston, Ontario, Canada K7L 5G2


    ABSTRACT
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INTRODUCTION
METHODS AND MATERIALS
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The possible contribution of Ca2+-activated Cl- channel [ICl(Ca)] and myosin light-chain kinase (MLCK) to nonadrenergic, noncholinergic slow inhibitory junction potentials (sIJP) was studied using conventional intracellular microelectrode recordings in circular smooth muscle of opossum esophageal body and guinea pig ileum perfused with Krebs solution containing atropine (3 µM), guanethidine (3 µM), and substance P (1 µM). In opossum esophageal circular smooth muscle, resting membrane potential (MP) was -51.9 ± 0.7 mV (n = 89) with MP fluctuations of 1-3 mV. A single square-wave nerve stimulation of 0.5 ms duration and 80 V induced a sIJP with amplitude of 6.3 ± 0.2 mV, half-amplitude duration of 635 ± 19 ms, and rebound depolarization amplitude of 2.4 ± 0.1 mV (n = 89). 9-Anthroic acid (A-9-C), niflumic acid (NFA), wortmannin, and 1-(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine (ML-9) abolished MP fluctuations, sIJP, and rebound depolarization in a concentration-dependent manner. A-9-C and NFA but not wortmannin and ML-9 hyperpolarized MP. In guinea pig ileal circular smooth muscle, nerve stimulation elicited an IJP composed of both fast (fIJP) and slow (sIJP) components, followed by rebound depolarization. NFA (200 µM) abolished sIJP and rebound depolarization but left the fIJP intact. These data suggest that in the tissues studied, activation of ICl(Ca), which requires MLCK, contributes to resting MP, and that closing of ICl(Ca) is responsible for sIJP.

nitric oxide; niflumic acid; wortmannin; intracellular microelectrode recording


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS AND MATERIALS
RESULTS
DISCUSSION
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FOUR DECADES AGO, IT WAS THOUGHT that two substances, acetylcholine and adrenaline, mediated transmission between nerves and gastrointestinal smooth muscle. However, in 1963, Burnstock et al. (13) unequivocally demonstrated that both excitatory and inhibitory junction potentials (IJP) were present between nerve endings and intestinal smooth muscle cells in which neurotransmission was effected by nonadrenergic, noncholinergic (NANC) neurotransmitters. Since then, a number of putative neurotransmitters have been proposed as mediators of the NANC IJP, including ATP (14), NO (49), vasoactive intestinal peptide (22), CO (19), and pituitary adenylate cyclase-activating peptide (23). In 1983, it was discovered that the NANC IJP consisted of both fast (fIJP) and slow (sIJP) components (39, 40). Subsequently, compelling evidence was presented that NO released from nerve terminals mediated the sIJP via activation of cGMP (16, 17, 24, 46). Recent studies in mice lacking the neuronal NO synthase gene have confirmed that NO released by nerve endings in gut circular smooth muscle is responsible for sIJP, which leads to muscle relaxation (29, 34). However, the ionic mechanisms underlying sIJP induced by NO are unclear.

It was previously proposed that the opening of K+ channels contributed to the sIJP (26, 27, 46). Indeed, NO donors were reported to activate multiple types of K+ channels and whole cell K+ currents in smooth muscle sensitive to either tetraethylammonium (TEA), apamin, quinine, or 4-aminopyridine (4-AP; Refs. 10, 25, 32, 35, 53). Unfortunately, none of the putative K+ blockers, such as TEA, apamin, 4-AP, and glibenclamide, could block the sIJP in opossum esophagus (16, 26), suggesting that the opening of previously described K+ channels may not underlie the sIJP in this tissue. Based on Cl- substitution and application of 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), an anion channel blocker, in Tomita bath experiments, Crist et al. (16, 17) proposed that the sIJP in opossum circular smooth muscle was due to the closing of Ca2+-activated Cl- channels [ICl(Ca)]. Unfortunately, this hypothesis remained somewhat speculative because of the nonselective effects of DIDS (46) and the actions of Cl- substitution on other channel activity (43).

Because specific ICl(Ca) blockers are now available, it is imperative to reevaluate the possible contribution of ICl(Ca) to NANC sIJP. It is well established that transmural nerve stimulation induces a NO-mediated sIJP in circular smooth muscle of opossum esophageal body under substance P (SP) desensitization and NANC conditions; therefore, this tissue was used in the current studies. However, contraction-induced dislodgement of intracellular microelectrodes is a significant problem in excitatory smooth muscle tissues. Therefore, wortmannin, reported to inhibit contraction of visceral smooth muscle without affecting electrical properties of the cell (11, 12), was used to facilitate stable intracellular microelectrode impalement. Surprisingly, we observed that wortmannin significantly inhibited electrical activity that linked to the niflumic acid (NFA)-sensitive ion channels. In this article, we provide evidence for the abolition of NANC sIJP by the more specific ICl(Ca) blockers, 9-anthroic acid (A-9-C) and NFA, and the myosin light-chain kinase (MLCK) inhibitors, wortmannin and 1-(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine (ML-9), suggesting that both ICl(Ca) and MLCK are involved in the generation of sIJP.


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Tissue Preparation

The protocols were approved by the Animal Care Committee of Queen's University. Opossums (Didelphis virginiana) of either sex and weighing between 2.5 and 5 kg were anesthetized by tail vein injection of phenobarbital sodium (40 mg/kg). The chest and abdominal cavities were then exposed via midline incisions, and the lower part of the esophagus and a short segment of attached stomach were removed and placed in preoxygenated Krebs solution. The opossums were then killed by intracardiac injection of phenobarbital sodium. The lower part of esophagus and esophagogastric junction were opened longitudinally and pinned out with mucosa side up in a dissecting dish. Using a binocular microscope, the mucosa and connective tissue layers were carefully removed by sharp dissection. A sheet of circular smooth muscle of esophageal body of ~5 × 15 mm from ~2 cm above the lower esophageal sphincter was excised for intracellular recordings. Circular smooth muscle of the guinea pig ileum was prepared for intracellular recordings as previously described (17).

Intracellular Microelectrode Recordings

Conventional intracellular microelectrode recording techniques were used to study electrical properties of smooth muscle. In the studies of circular smooth muscle of opossum esophageal body and guinea pig ileum, sheets of tissues were pinned mucosa-side up on the silicon-coated bottom of a 2-ml electrophysiological recording chamber mounted on the stage of an Olympus IX-70 inverted microscope (Olympus, Tokyo, Japan). Silver wire electrodes, placed on either side of the muscle strip, were used to electrically stimulate intramural nerves using square-wave pulses of 0.5-ms duration and 80 V generated by a Grass S88 Stimulator (Grass Instruments, Quincy, MA). The chamber was continuously perfused at 2.6 ml/min with prewarmed and preoxygenated Krebs solution and maintained at 35°C. Tissue was allowed to equilibrate for 1 h before the experiment. Glass microelectrodes were pulled using a vertical microelectrode puller (Sutter Instruments) and filled with 3 M KCl. Microelectrode resistance was 50-70 MOmega . The microelectrode was positioned to impale a smooth muscle cell using the inverted microscope. The criterion for acceptance of a successful impalement was a sharp voltage drop on penetration maintained for <= 2 min. Transmembrane potential was amplified and measured with an intracellular electrometer (model IE-210; Warner Instruments). An agar bridge (2% agar in 3 M KCl) was used to minimize junction potentials. Resting membrane potential (MP) was calibrated on withdrawal of the microelectrode from the cell. Output of the signal was displayed on an oscilloscope (model 5103N; Tektronics, Beaverton, OR) and coupled to the Axon Digidata-1200 acquisition system (Axon Instruments, Foster City, CA). Data were digitized at a frequency of 500 Hz and stored in a Pentium II computer for later analysis using Scope 7.0 software (Axon Instruments). Resting MP, amplitude of IJP, half-amplitude duration of IJP, and amplitude of sIJP rebound were used to quantitatively analyze the smooth muscle electrical properties.

Solutions and Drugs

Modified Krebs solution contained (in mM) 118.07 NaCl, 25.00 NaHCO3, 11.10 D(+)-glucose, 4.69 KCl, 2.52 CaCl2, 1.00 MgSO4, and 1.01 NaH2PO4. NFA was purchased from ICN Biochemicals, and all other compounds were from Sigma. NFA and wortmannin were dissolved in DMSO as stock solutions. A-9-C, nifedipine, ML-9, and LY-294002 were prepared in ethyl alcohol (100%) and the other solutions in distilled water. SP was prepared following the manufacturer's instructions (Sigma). These were diluted to final concentrations with Krebs solution. In cumulative concentration-response experiments of NFA, wortmannin, and ML-9, final concentration of solvents in Krebs solution was <= 1%. At this concentration, solvents had no effect on electrical activity. Krebs solution containing diluted drugs was fully bubbled with 5% CO2-95% O2 to restore pH before the application.

Statistical Analysis

Data are shown as means ± SE; n = number of cells impaled. One kind of drug was tested only once in one animal. Only recordings in which a full protocol was completed in the same cell are included in the statistical analysis. Pre- and postdrug comparisons were made using the paired Student's t-test, and a P value of <0.05 was considered statistically significant.


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METHODS AND MATERIALS
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Studies in Circular Smooth Muscle of Opossum Esophageal Body

Effects of atropine plus guanethidine on electrical properties. Figure 1 demonstrates the general electrical properties recorded in a cell after equilibration and perfusion with Krebs solution containing SP (1 µM) for 60 min. SP desensitization was tested by short-duration exposure (4 min) to an additional 1 µM SP to ensure that the excitatory effect of this agent was no longer present. Resting MP was -53.6 ± 2.6 mV (n = 5) with MP fluctuations of 1-3 mV. No spontaneous action potentials were observed. A single square-wave of transmural nerve stimulation (0.5 ms duration, 80 V) induced a sIJP with amplitude of 5.3 ± 0.2 mV and half-amplitude duration of 533 ± 49 ms, followed by rebound depolarization with amplitude of 2.3 ± 0.6 mV (n = 5). With increase in number of stimulating pulses (2 and 10 pulses at 20 Hz), action potentials were usually superimposed on the sIJP rebound. Figure 1C shows that in the presence of nifedipine (1 µM) to prevent contraction and loss of cell impalement, longer transmural nerve stimulation (9 s, 20 Hz) evoked a sIJP, which reached a peak of 15 mV in 2 s, recovered partially at ~5 s, and then stayed at a steady hyperpolarization state until the stimulus terminated. Application of atropine (3 µM) plus guanethidine (3 µM) expanded the half-amplitude duration of sIJP induced by the single pulse stimulation from 533 ± 49 to 611 ± 13 and 655 ± 29 ms at 5 and 10 min, respectively (n = 5, P < 0.05) (Fig. 2A). Other electrical parameters were not significantly altered. Furthermore, MP fluctuations were not affected by the application of atropine plus guanethidine, nifedipine (1 µM) (Figs. 1C and 2A), and TTX (1 µM) (Fig. 3). However, TTX abolished sIJP. Atropine, guanethidine, and SP were used in all subsequent experiments to ensure NANC conditions. In these experiments, in which a total of 89 cells were impaled, resting MP, sIJP amplitude, sIJP half-amplitude duration, and rebound amplitude were -51.9 ± 0.7 mV, 6.3 ± 0.2 mV, 635 ± 19 ms, and 2.4 ± 0.1 mV, respectively.


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Fig. 1.   Slow inhibitory junction potential (sIJP) evoked by transmural nerve stimulation in circular smooth muscle of opossum esophageal body. A square wave was used with a duration of 0.5 ms and voltage of 80 V. A: original intracellular recordings. Resting membrane potential (MP) was characterized by spontaneous fluctuations of 1-3 mV. B: snap views from A labeled as a-c at expanded time scale. Single-pulse nerve stimulation induced sIJP followed by rebound (afterdepolarization) (B, a). With increase of pulse number (at 20 Hz), amplitude of sIJP was augmented, duration of sIJP was lengthened, and spike-like action potentials were superimposed on the rebound (B, b and c). C: membrane hyperpolarization induced by long-term nerve stimulation in the presence of nifedipine (1 µM). The fluctuations of MP were unaffected by nifedipine. NS, nerve stimulation.



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Fig. 2.   Effects of atropine plus guanethidine (3 µM; A) and indomethacin (B) on sIJP induced by single pulse nerve stimulation in the presence of substance P (SP; 1 µM). A, a: control. A, b-c: 5 and 10 min after atropine and guanethidine. Atropine and guanethidine significantly expanded duration of sIJP but without any effects on the MP fluctuations. A, d: sIJPs were superimposed to compare before and after the administration of atropine and guanethidine. Because of the significant effects on duration of sIJP, atropine and guanethidine (3 µM) were included in the perfusion solution in all ensuing experiments to ensure nonadrenergic, noncholinergic (NANC) conditions. B: effects of indomethacin on sIJP. Snapshots of sIJP in control (a), 5 min (b), and 15 min (c) after application of indomethacin (10 µM). B, d: traces of sIJP were overlapped. Indomethacin did not significantly affect the measured parameters of sIJP or the MP fluctuations.



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Fig. 3.   Abolition of sIJP by TTX (1 µM) in the presence of atropine plus guanethidine (3 µM), SP (1 µM), and nifedipine (1 µM). A, ii, 5 min after TTX. B: superimposed traces before and after TTX. TTX abolished sIJP, without any effect on MP fluctuations and MP, suggesting that spontaneous MP fluctuations are myogenic.

Effects of A-9-C and NFA on NANC sIJP. The maximal A-9-C (1 mM) effect was reached within 5-10 min, at which point MP fluctuations and sIJP rebound were abolished. MP was hyperpolarized from -50.0 ± 3.2 to -55.3 ± 4.2 mV, and sIJP amplitude decreased from 7.1 ± 1.6 to 1.6 ± 0.3 mV (n = 5, P < 0.05). This effect was rapidly reversible, with recovery to baseline values occurring within 5-10 min of washout. However, 500 µM A-9-C usually inhibited ~50% of sIJP amplitude. NFA had a similar time course of action as A-9-C, i.e., the inhibitory action peaked in 10 min and recovered 10-15 min after washout. Therefore, the interval time chosen for each cumulative concentration of NFA was 10 min in concentration-response experiments. NFA hyperpolarized MP and abolished sIJP and rebound of sIJP in a concentration-dependent manner with IC50 of 25.7 ± 3.5, 76.5 ± 18.3, and 38.8 ± 5.4 µM (n = 6) (Fig. 4), respectively. The maximal effective concentration was 300 µM. However, NFA also abolished MP fluctuations at a concentration of 100 µM or above (Fig. 4A).


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Fig. 4.   Effects of niflumic acid (NFA), a Ca2+-activated Cl- channel blocker, on sIJP. A: NFA hyperpolarized MP and abolished sIJP and MP fluctuations in a concentration-dependent manner. B: superimposed sIJPs after application of NFA in different concentrations. MPs were neutralized to compare sIJPs after NFA. The maximally effective concentration of NFA is 300 µM, which completely abolishes sIJP and MP fluctuations. C: statistical analysis of effects of NFA on electrical properties. Concentration-response curves were fitted using the sigmoid equation, which yielded IC50 of 25.7 ± 3.5, 76.5 ± 18.3, and 38.8 ± 5.4 µM for MP (I), amplitude of sIJP (II), and rebound amplitude of sIJP (III), respectively; n = 6.

Effects of indomethacin on electrical properties. Because NFA is used as a nonsteroidal anti-inflammatory drug, it is possible that abolition of sIJP by NFA could be due to the blockade of prostaglandin synthesis. The effect of the putative cyclooxygenase inhibitor indomethacin was, therefore, investigated in the current studies. As shown in Fig. 2B, application of indomethacin (10 µM) to the perfused Krebs solution for <= 15 min did not affect MP fluctuations or any of the measured electrical parameters of the sIJP (n = 4).

Effects of combined application of TEA and NFA on electrical properties. Because NFA in concentrations >50 µM has been reported to activate Ca2+-activated large-conductance K+ channels (BKCa) (21), it is possible that the hyperpolarization produced by NFA could be secondary to the opening of BKCa channels. To exclude this possibility, TEA, a putative BKCa channel blocker, was used. TEA (1.5 mM) depolarized MP from -52.8 ± 2.0 to -48.0 ± 2.0 mV 4 min after the application (n = 7, P < 0.05) (Fig. 5). sIJP amplitude, half-amplitude duration, and rebound amplitude were significantly increased from 6.3 ± 0.8 mV, 697 ± 47 ms, and 1.9 ± 0.4 mV to 10.4 ± 0.3 mV, 944 ± 65 ms, and 4.0 ± 0.5 mV (n = 7, P < 0.05), respectively. MP fluctuations were not affected. However, the concomitant application of NFA (300 µM) for 10 min hyperpolarized MP by 4.8 ± 1.0 mV and abolished MP fluctuations, sIJP, and rebound (n = 7, P 0.05). The hyperpolarization in the presence of TEA (1.5 mM) was similar in magnitude to that produced by NFA alone (Figs. 4 and 5; P > 0.05).


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Fig. 5.   Effects of concomitant application of NFA on electrical properties in the presence of tetraethylammonium (TEA). A: raw recordings. B: snapshots of original recordings labeled in A. TEA (1.5 µM), a Ca2+-activated large-conductance K+ channel (BKCa) blocker, depolarized MP and potentiated sIJP but had no effects on MP fluctuations (B, I and II). However, the concomitant application of NFA (300 µM) hyperpolarized MP and abolished MP fluctuations and sIJP (B, III), suggesting that the inhibitory effect of NFA is not due to the opening of BKCa channels. B, V and VI: superimposed snapshots before and after TEA and NFA for better comparison. MP was neutralized.

Inhibitory effects of wortmannin and ML-9. The possible involvement of MLCK in generation of the sIJP was studied by the application of wortmannin, a specific inhibitor of phosphatidylinositol (PI)-3 kinase and MLCK, and ML-9, an inhibitor of MLCK and protein kinases A and C, to the perfused solution. Time course studies demonstrated that wortmannin had maximal effect in ~10 min, but the effect was not reversible. The time course of ML-9 was similar to wortmannin, but its effects were reversible (in 10-15 min). The interval time for each cumulative concentration of either wortmannin or ML-9 was 10 min in concentration-response experiments. Wortmannin and ML-9 abolished MP fluctuations, sIJP, and sIJP rebound in a concentration-dependent manner, but unlike NFA and A-9-C, wortmannin and ML-9 had no significant effect on MP (Fig. 6). IC50 for the inhibition of sIJP and amplitudes of sIJP rebound was 10.7 ± 0.8 and 4.4 ± 1.9 µM by wortmannin and 26.7 ± 3.3 and 17.3 ± 0.1 µM by ML-9, respectively, with a maximal effective concentration of 30 µM and 100 µM (n = 5) (Fig. 7). Because it has been reported that wortmannin in nanomolar concentrations specifically inhibits PI-3 kinase (5), we also used LY-294002, a putative specific PI-3 kinase inhibitor, to test whether PI-3 kinase was involved in the MP fluctuations and sIJP. However, LY-294002 (10 µM) did not produce any measurable alterations of MP fluctuations and sIJP (n = 3).


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Fig. 6.   Example of inhibition of sIJP by wortmannin and ML-9 in cumulative concentrations recorded in the same cells. A and B: sIJP recorded at different concentrations. Interval time was 10 min. C and D: overlapped tracings of sIJP recordings from A and B. The maximally effective concentration of wortmannin and ML-9 were 30 µM and 100 µM, respectively. These concentrations abolished sIJP and MP fluctuations but had no significant effect on MP.



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Fig. 7.   Statistical analysis of effects of wortmannin and ML-9 on sIJP. The concentration-response curves were fitted with sigmoid equation, which yielded IC50 for amplitude of sIJP (B) and rebound amplitude of sIJP (C) of 10.7 ± 0.8 and 4.4 ± 1.9 µM for wortmannin, and 26.7 ± 3.3 and 17.3 ± 0.1 µM for ML-9, respectively (n = 5).

Studies in circular smooth muscle of guinea pig ileum. Nerve stimulation evokes only a sIJP in circular smooth muscle of opossum esophageal body. Therefore, to exclude the possibility that abolition of the sIJP by NFA was due to nonspecific inhibition of neurotransmitter release, additional studies were performed in circular smooth muscle of guinea pig ileum where both sIJP and fIJP are present. All studies were performed under NANC conditions as described above. In addition, nifedipine (1 µM) was added to the bath to prevent spontaneous muscle contraction and thus stabilize the preparation. Figure 8 is an example of the NANC IJP induced by one and four pulses transmural nerve stimulation in circular smooth muscle of guinea pig ileum. Single pulse nerve stimulation evoked an IJP composed of both fast and slow components, followed by rebound depolarization, whereas four pulses (20 Hz) of stimulation made the sIJP more visible (Fig. 8A). Application of NFA (200 µM) abolished the sIJP and rebound depolarization, but fIJP was left intact (Fig. 8B).


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Fig. 8.   An example of blockade of sIJP by NFA (200 µM) recorded in a circular smooth muscle cell of guinea pig ileum in the presence of atropine (3 µM), quanethidine (3 µM), SP (1 µM), and nifedipine (1 µM). A: NANC IJP consists of fast and slow components, followed by rebound (afterdepolarization). With increase of nerve stimulation pulse from 1 to 4 at 20 Hz, the slow component of IJP became more visible, whereas the amplitude of IJP was not significantly affected. B: NFA abolished sIJP and left fIJP intact, implying that the release of neurotransmitters was not affected by NFA.


    DISCUSSION
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Major findings of the current study are that: 1) gastrointestinal circular smooth muscle demonstrates MP fluctuations of 1-3 mV, which are sensitive to the ICl(Ca) blockers A-9-C and NFA as well as to the MLCK inhibitors wortmannin and ML-9 but insensitive to atropine plus quanethidine, SP desensitization, nifedipine, and TTX; 2) ICl(Ca) blockers hyperpolarize MP; 3) ICl(Ca) blockers and MLCK inhibitors abolish the NANC sIJP and ensuing rebound depolarization; and 4) fIJP is not affected by NFA in guinea pig ileal circular smooth muscle. Taken together, these data strongly suggest that ICl(Ca) contributes to the resting MP of gastrointestinal circular smooth muscle and that their closing underlies the NANC sIJP. In addition, it appears that MLCK is involved in the activation of ICl(Ca).

Roles of ICl(Ca) in Control of MP and sIJP

NANC IJP has been discriminated into two components, fIJP and sIJP, by application of apamin and SP desensitization (16, 17, 39, 42, 51). It is well known that the opening of small conductance, apamin-sensitive Ca2+-activated K+ channels underlies the fIJP induced by the release of ATP from nerves (48). More recent studies (29, 34) in mice lacking neuronal NO synthase have provided convincing evidence that the sIJP is mediated by NO release from nerve terminals. So far, two hypotheses have been proposed for the ionic mechanisms underlying the NO-induced sIJP, namely opening of K+ channels (26, 27, 32, 46) or closing of ICl(Ca) (16, 17). The idea that the opening of K+ channels underlies sIJP dates back to Tomita bath experiments done in the mid-1980s in which the effects of conditioning hyperpolarization and depolarization on the sIJP were studied in opossum esophageal circular smooth muscle (26, 27). These investigators demonstrated that the amplitude of the sIJP became smaller when the conditioning potential approached the K+ equilibrium potential and that membrane input resistance decreased during sIJP, suggesting that opening of K+ conductance was responsible for the sIJP. However, no known K+ channel blocker could inhibit the sIJP. Subsequently, other investigators performed similar experiments but with opposing results, leading the authors to suggest that closing of ICl(Ca) mediated the sIJP (16, 17). An intriguing question that arises from the above publications is why two independent laboratories got such opposing results in the same tissue. In the earlier experiments, it appears that atropine plus quanethidine was not used to ensure NANC conditions, presumably due to the investigators' previous studies (47) that suggested that the circular smooth muscle of the opossum esophagus lacked functional intrinsic cholinergic innervation. However, other laboratories have reported that cholinergic nerves play a physiological role in opossum esophageal circular smooth muscle contractions (15, 18, 41). Furthermore, as early as 1983, Niel et al. (39, 40) reported in circular smooth muscle of guinea pig ileum that the amplitude of apamin-resistant IJP (i.e., what we now call sIJP) was increased when the conditioning potential approached the K+ reversal potential under SP desensitization and NANC conditions, implying that sIJP may not be due to K+ conductance. Our data are in keeping with the presence of a functional cholinergic innervation in circular smooth muscle of opossum esophagus, because atropine plus quanethidine expanded the sIJP duration (Fig. 2A). This effect is likely due to muscarinic rather than adrenergic blockade, because it is acknowledged that gut smooth muscle lacks functional adrenergic innervation (9). One possible explanation for the above controversy is that if atropine is not present in the bath, coreleased acetylcholine interferes with the membrane input resistance measurement via its ability to open nonselective cation channels (8).

It has been reported that the Cl- reversal potential is more positive than resting MP (2, 3); thus opening of Cl- channels produces inward currents at the resting state, which in turn causes membrane depolarization. We previously showed that opposing activity of Cl- and K+ channels set resting MP of circular smooth muscle of opossum lower esophageal sphincter at a relatively more positive level (54). Current studies provide further evidence that ICl(Ca) contributes to resting MP, because both A-9-C and NFA hyperpolarized MP and abolished the spontaneous MP fluctuations. The latter may be due to basal activity of ICl(Ca). The origin of basal activity of ICl(Ca) remains unclear. Recent reports (37, 55) have suggested it may result from spontaneous Ca2+ sparks caused by release of Ca2+ from the sarcoplasmic reticulum.

Our data also suggest that closing of ICl(Ca) by NO is responsible for the sIJP. This conclusion rests on the selectivity of A-9-C and NFA. In some tissues, NFA has been found to have nonspecific effects, including opening of large-conductance Ca2+-activated K+ channels and direct inhibition of contractile proteins (28, 31). However, such previously reported nonspecific effects could not explain the observed abolition of sIJP. Furthermore, it is unlikely that two structurally unrelated ICl(Ca) would both have the same effect on the sIJP via a nonspecific action. The current conclusion is further supported by evidence that NFA still hyperpolarized MP and abolished MP fluctuations and sIJP in the presence of 1.5 mM TEA (Fig. 5). It has also been reported that NFA inhibits hyperpolarization-activated cation channels in myocytes isolated from the sinoatrial node of the rabbit heart (1) and potentiates the alpha 1-adrenoceptor-activated nonselective cation channels in rabbit portal vein smooth muscle (52). However, it appears that NFA in concentrations of <= 100 µM has no inhibitory effect on cation channels in smooth muscle cells from rat cerebral arteries (38, 50). Nevertheless, we cannot completely exclude the possibility that cation channels are involved in the sIJP produced by NO. This would require patch-clamp studies in dispersed single smooth muscle cells. In addition, it is less likely that abolition of sIJP by NFA is due to presynaptic inhibition of neurotransmitter release, because NFA inhibited sIJP without affecting the fIJP in guinea pig ileum smooth muscle (Fig. 8).

Mechanisms for sIJP Rebound (Afterdepolarization)

Mechanisms responsible for the "rebound" depolarization after the sIJP are unclear. It has been proposed that this is either due to rebound after anodal-break excitation caused by the preceding IJP or to an unidentified coreleased excitatory transmitter. Indeed, our data confirmed that sIJP rebound was significantly associated with amplitude of the sIJP. On the other hand, we found that the sIJP rebound was not correlated with the fIJP (Fig. 8). In other words, the rebound occurs after sIJP but not fIJP, which is inconsistent with a mechanism of anodal break excitation. Recent publications have suggested that prostaglandins (20) acetylcholine and SP (7) may be involved in mediation of rebound depolarization. Our results do not support these possibilities because the rebound was unaffected by atropine plus quanethidine, indomethacin, or SP desensitization (Fig. 2). The blockade of rebound depolarization by ICl(Ca) blockers A-9-C and NFA suggests a role of these channels in this phenomenon. This is further supported by a previous publication in which the reversal potential of the rebound depolarization in circular smooth muscle of chicken rectum was -15.3 mV, which is close to the Cl- equilibrium potential (33).

MLCK in the Regulation of ICl(Ca)

Wortmannin, originally purified from a fungal strain, was reported to specifically and irreversibly inhibit PI-3-kinase at nanomolar concentrations, as well as MLCK at µM concentrations (5, 36, 44). Recent reports (11, 12) suggested that wortmannin may inhibit contraction of visceral smooth muscle without any effects on electrical properties. Our initial purpose in using wortmannin was to immobilize the muscle contraction and maintain stable intracellular microelectrode impalement. Surprisingly, we observed abolition of MP fluctuations within 10 min of application (Fig. 6A). Furthermore, wortmannin abolished the sIJP and rebound depolarization in a concentration-dependent manner, with a minimally effective concentration of 3 µM and maximally effective concentration 30 µM. Concentrations of wortmannin that block PI-3 kinase were ineffective in our preparation. Furthermore, LY-294002 (10 µM), a specific PI-3 kinase inhibitor, had no measurable effects on MP fluctuations and sIJP, suggesting that PI-3 kinase is not involved in the regulation of ICl(Ca). These data imply that MLCK is involved in the activation of ICl(Ca). Inhibitory effects of ML-9 on MP fluctuations and sIJP further support this. ML-9, a cell-permeable synthetic naphthalenesulfonamide derivative, has been reported to selectively inhibit MLCK purified from chicken gizzard at low concentrations (Ki = 3.8 µM), whereas at 10- to 20-fold higher concentrations it appears to also inhibit protein kinase A (45). The inhibition of MP fluctuations by both wortmannin and ML-9 suggests that MLCK has basal activity. Unlike blockers of ICl(Ca), inhibitors of MLCK had no effect on resting MP. Reasons for this are unclear, but it is consistent with these two classes of antagonists working via quite different mechanisms.

A number of previous studies have reported that MLCK inhibitors may affect activity of other ion channel currents, including reduction of the M-currents in bullfrog sympathetic neurons (4) and the nonselective cation currents in gastric myocytes (30) and rabbit portal vein (6). The current study is the first to report an interaction between MLCK and ICl(Ca). However, the mechanism(s) and physiological significance of this interaction remains unclear.

In summary, the current studies demonstrate that A-9-C and NFA, two different ICl(Ca) blockers, hyperpolarize resting MP and abolish MP fluctuations, sIJP, and rebound depolarization in a concentration-dependent manner. In addition, wortmannin and ML-9, at concentrations that inhibit MLCK, also abolished MP fluctuations, sIJP, and rebound depolarization, suggesting that ICl(Ca) cross talks with regulators of contractile proteins via MLCK. These studies suggest that activation of ICl(Ca), which requires MLCK, contributes to resting MP and that closing of ICl(Ca) is responsible for the sIJP.


    ACKNOWLEDGEMENTS

This study was supported by Grant MOP-9978 from the Canadian Institutes of Health Research.


    FOOTNOTES

Address for reprint requests and other correspondence: W. G. Paterson, Gastrointestinal Diseases Research Unit, Hotel Dieu Hospital, 166 Brock St., Kingston, Ontario, Canada, K7L 5G2 (E-mail: patersow{at}hdh.kari.net).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

First published March 28, 2002;10.1152/ajpgi.00052.2002

Received 11 February 2002; accepted in final form 22 March 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS AND MATERIALS
RESULTS
DISCUSSION
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