Gastrointestinal Disease Research Unit and Departments of Medicine, Biology, and Physiology, Queen's University, Kingston, Ontario, Canada K7L 5G2
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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.
![]() |
METHODS AND MATERIALS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 MSolutions 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 wasStatistical 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. ![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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.
|
|
|
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).
|
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).
|
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).
|
|
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).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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
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 wasMLCK 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Accili, EA,
and
DiFrancesco D.
Inhibition of the hyperpolarization-activated current (if) of rabbit SA node myocytes by niflumic acid.
Pflügers Arch
431:
757-762,
1996[ISI][Medline].
2.
Aickin, CC,
and
Brading AF.
Measurement of intracellular chloride in guinea-pig vas deferens by ion analysis, 36chloride efflux and micro-electrodes.
J Physiol
326:
139-154,
1982[ISI][Medline].
3.
Aickin, CC,
and
Brading AF.
Towards an estimate of chloride permeability in the smooth muscle of guinea-pig vas deferens.
J Physiol
336:
179-197,
1983[Abstract].
4.
Akasu, T,
Ito M,
Nakano T,
Schneider CR,
Simmons MA,
Tanaka T,
Tokimasa T,
and
Yoshida M.
Myosin light chain kinase occurs in bullfrog sympathetic neurons and may modulate voltage-dependent potassium currents.
Neuron
11:
1133-1145,
1993[ISI][Medline].
5.
Arcaro, A,
and
Wymann MP.
Wortmannin is a potent phosphatidylinositol 3-kinase inhibitor: the role of phosphatidylinositol 3,4,5-trisphosphate in neutrophil responses.
Biochem J
296:
297-301,
1993[ISI][Medline].
6.
Aromolaran, AS,
Albert AP,
and
Large WA.
Evidence for myosin light chain kinase mediating noradrenaline-evoked cation current in rabbit portal vein myocytes.
J Physiol
524:
853-863,
2000
7.
Bartho, L,
and
Lefebvre R.
Nitric oxide induces acetylcholine-mediated contractions in the guinea-pig small intestine.
Naunyn Schmiedebergs Arch Pharmacol
350:
582-584,
1994[ISI][Medline].
8.
Benham, CD,
Bolton TB,
and
Lang RJ.
Acetylcholine activates an inward current in single mammalian smooth muscle cells.
Nature
316:
345-347,
1985[ISI][Medline].
9.
Bennett, MR.
Non-adrenergic non-cholinergic (NANC) transmission to smooth muscle: 35 years on.
Prog Neurobiol
52:
159-195,
1997[ISI][Medline].
10.
Bolotina, VM,
Najibi S,
Palacino JJ,
Pagano PJ,
and
Cohen RA.
Nitric oxide directly activates calcium-dependent potassium channels in vascular smooth muscle.
Nature
368:
850-853,
1994[ISI][Medline].
11.
Burdyga, TV,
and
Wray S.
The effect of inhibition of myosin light chain kinase by Wortmannin on intracellular [Ca2+], electrical activity and force in phasic smooth muscle.
Pflügers Arch
436:
801-803,
1998[ISI][Medline].
12.
Burke, EP,
Gerthoffer WT,
Sanders KM,
and
Publicover NG.
Wortmannin inhibits contraction without altering electrical activity in canine gastric smooth muscle.
Am J Physiol Cell Physiol
270:
C1405-C1412,
1996
13.
Burnstock, G,
Campbell G,
Bennett M,
and
Holman ME.
Inhibition of the smooth muscle of the Taenia coli.
Nature
200:
581-582,
1963[ISI].
14.
Burnstock, G,
Campbell G,
Satchell D,
and
Smythe A.
Evidence that adenosine triphosphate or a related nucleotide is the transmitter substance released by non-adrenergic inhibitory nerves in the gut.
Br J Pharmacol
40:
668-688,
1970[ISI][Medline].
15.
Crist, J,
Gidda JS,
and
Goyal RK.
Intramural mechanism of esophageal peristalsis: roles of cholinergic and noncholinergic nerves.
Proc Natl Acad Sci USA
81:
3595-3599,
1984[Abstract].
16.
Crist, JR,
He XD,
and
Goyal RK.
Chloride-mediated inhibitory junction potentials in opossum esophageal circular smooth muscle.
Am J Physiol Gastrointest Liver Physiol
261:
G752-G762,
1991
17.
Crist, JR,
He XD,
and
Goyal RK.
Chloride-mediated junction potentials in circular muscle of the guinea pig ileum.
Am J Physiol Gastrointest Liver Physiol
261:
G742-G751,
1991
18.
Dodds, WJ,
Christensen J,
Dent J,
Wood JD,
and
Arndorfer RC.
Esophageal contractions induced by vagal stimulation in the opossum.
Am J Physiol Endocrinol Metab Gastrointest Physiol
235:
E392-E401,
1978
19.
Farrugia, G,
Irons WA,
Rae JL,
Sarr MG,
and
Szurszewski JH.
Activation of whole cell currents in isolated human jejunal circular smooth muscle cells by carbon monoxide.
Am J Physiol Gastrointest Liver Physiol
264:
G1184-G1189,
1993
20.
Franck, H,
Kong ID,
Shuttleworth CW,
and
Sanders KM.
Rebound excitation and alternating slow wave patterns depend upon eicosanoid production in canine proximal colon.
J Physiol
520:
885-895,
1999
21.
Greenwood, IA,
and
Large WA.
Comparison of the effects of fenamates on Ca-activated chloride and potassium currents in rabbit portal vein smooth muscle cells.
Br J Pharmacol
116:
2939-2948,
1995[Abstract].
22.
Grider, JR,
Cable MB,
Said SI,
and
Makhlouf GM.
Vasoactive intestinal peptide as a neural mediator of gastric relaxation.
Am J Physiol Gastrointest Liver Physiol
248:
G73-G78,
1985
23.
Jin, JG,
Katsoulis S,
Schmidt WE,
and
Grider JR.
Inhibitory transmission in Tenia coli mediated by distinct vasoactive intestinal peptide and apamin-sensitive pituitary adenylate cyclase activating peptide receptors.
J Pharmacol Exp Ther
270:
433-439,
1994[Abstract].
24.
Jury, J,
Ahmedzadeh N,
and
Daniel EE.
A mediator derived from arginine mediates inhibitory junction potentials and relaxations in lower esophageal sphincter: an independent role for vasoactive intestinal peptide.
Can J Physiol Pharmacol
70:
1182-1189,
1992[ISI][Medline].
25.
Jury, J,
Boev KR,
and
Daniel EE.
Nitric oxide mediates outward potassium currents in opossum esophageal circular smooth muscle.
Am J Physiol Gastrointest Liver Physiol
270:
G932-G938,
1996
26.
Jury, J,
Jager LP,
and
Daniel EE.
Unusual potassium channels mediate nonadrenergic noncholinergic nerve-mediated inhibition in opossum esophagus.
Can J Physiol Pharmacol
63:
107-112,
1985[ISI][Medline].
27.
Kannan, MS,
Jager LP,
and
Daniel EE.
Electrical properties of smooth muscle cell membrane of opossum esophagus.
Am J Physiol Gastrointest Liver Physiol
248:
G342-G346,
1985
28.
Kato, K,
Evans AM,
and
Kozlowski RZ.
Relaxation of endothelin-1-induced pulmonary arterial constriction by niflumic acid and NPPB: mechanism(s) independent of chloride channel block.
J Pharmacol Exp Ther
288:
1242-1250,
1999
29.
Kim, CD,
Goyal RK,
and
Mashimo H.
Neuronal NOS provides nitrergic inhibitory neurotransmitter in mouse lower esophageal sphincter.
Am J Physiol Gastrointest Liver Physiol
277:
G280-G284,
1999
30.
Kim, YC,
Kim SJ,
Kang TM,
Suh SH,
So I,
and
Kim KW.
Effects of myosin light chain kinase inhibitors on carbachol-activated nonselective cationic current in guinea-pig gastric myocytes.
Pflügers Arch
434:
346-353,
1997[ISI][Medline].
31.
Kirkup, AJ,
Edwards G,
Green ME,
Miller M,
Walker SD,
and
Weston AH.
Modulation of membrane currents and mechanical activity by niflumic acid in rat vascular smooth muscle.
Eur J Pharmacol
317:
165-174,
1996[ISI][Medline].
32.
Koh, SD,
Campbell JD,
Carl A,
and
Sanders KM.
Nitric oxide activates multiple potassium channels in canine colonic smooth muscle.
J Physiol
489:
735-743,
1995[Abstract].
33.
Komori, S,
and
Ohashi H.
Some membrane properties of the circular muscle of chicken rectum and its non-adrenergic non-cholinergic innervation.
J Physiol
401:
417-435,
1988[Abstract].
34.
Mashimo, H,
He XD,
Huang PL,
Fishman MC,
and
Goyal RK.
Neuronal constitutive nitric oxide synthase is involved in murine enteric inhibitory neurotransmission.
J Clin Invest
98:
8-13,
1996
35.
Murray, JA,
Shibata EF,
Buresh TL,
Picken H,
O'Meara BW,
and
Conklin JL.
Nitric oxide modulates a calcium-activated potassium current in muscle cells from opossum esophagus.
Am J Physiol Gastrointest Liver Physiol
269:
G606-G612,
1995
36.
Nakanishi, S,
Kakita S,
Takahashi I,
Kawahara K,
Tsukuda E,
Sano T,
Yamada K,
Yoshida M,
Kase H,
and
Matsuda Y.
Wortmannin, a microbial product inhibitor of myosin light chain kinase.
J Biol Chem
267:
2157-2163,
1992
37.
Nelson, MT,
Cheng H,
Rubart M,
Santana LF,
Bonev AD,
Knot HJ,
and
Lederer WJ.
Relaxation of arterial smooth muscle by calcium sparks.
Science
270:
633-637,
1995[Abstract].
38.
Nelson, MT,
Conway MA,
Knot HJ,
and
Brayden JE.
Chloride channel blockers inhibit myogenic tone in rat cerebral arteries.
J Physiol
502:
259-264,
1997[Abstract].
39.
Niel, JP,
Bywater RA,
and
Taylor GS.
Apamin-resistant post-stimulus hyperpolarization in the circular muscle of the guinea-pig ileum.
J Auton Nerv Syst
9:
565-569,
1983[ISI][Medline].
40.
Niel, JP,
Bywater RA,
and
Taylor GS.
Effect of substance P on non-cholinergic fast and slow post-stimulus depolarization in the guinea-pig ileum.
J Auton Nerv Syst
9:
573-584,
1983[ISI][Medline].
41.
Paterson, WG.
Neuromuscular mechanisms of esophageal responses at and proximal to a distending balloon.
Am J Physiol Gastrointest Liver Physiol
260:
G148-G155,
1991
42.
Rakestraw, PC,
Snyder JR,
Sanders KM,
and
Shuttleworth WC.
Intracellular microelectrode recording to characterize inhibitory neuromuscular transmission in jejunum of horses.
Am J Vet Res
61:
362-368,
2000[ISI][Medline].
43.
Reddy, MM,
Light MJ,
and
Quinton PM.
Activation of the epithelial Na+ channel (ENaC) requires CFTR Cl channel function.
Nature
402:
301-304,
1999[ISI][Medline].
44.
Reinhold, SL,
Prescott SM,
Zimmerman GA,
and
McIntyre TM.
Activation of human neutrophil phospholipase D by three separable mechanisms.
FASEB J
4:
208-214,
1990
45.
Saitoh, M,
Ishikawa T,
Matsushima S,
Naka M,
and
Hidaka H.
Selective inhibition of catalytic activity of smooth muscle myosin light chain kinase.
J Biol Chem
262:
7796-7801,
1987
46.
Sanders, KM,
and
Ozaki H.
Excitation-contraction coupling in gastrointestinal smooth muscles.
In: Pharmacology of Smooth Muscle, edited by Szekeres L,
and Papp JG.. Berlin, Germany: Springer-Verlag, 1994, p. 331-404.
47.
Serio, R,
and
Daniel EE.
Electrophysiological analysis of responses to intrinsic nerves in circular muscle of opossum esophageal muscle.
Am J Physiol Gastrointest Liver Physiol
254:
G107-G116,
1988
48.
Vogalis, F,
and
Goyal RK.
Activation of small conductance Ca2+-dependent K+ channels by purinergic agonists in smooth muscle cells of the mouse ileum.
J Physiol
502:
497-508,
1997[Abstract].
49.
Ward, SM,
Dalziel HH,
Thornbury KD,
Westfall DP,
and
Sanders KM.
Nonadrenergic, noncholinergic inhibition and rebound excitation in canine colon depend on nitric oxide.
Am J Physiol Gastrointest Liver Physiol
262:
G237-G243,
1992
50.
Welsh, DG,
Nelson MT,
Eckman DM,
and
Brayden JE.
Swelling-activated cation channels mediate depolarization of rat cerebrovascular smooth muscle by hyposmolarity and intravascular pressure.
J Physiol
527:
139-148,
2000
51.
Xue, L,
Farrugia G,
Sarr MG,
and
Szurszewski JH.
ATP is a mediator of the fast inhibitory junction potential in human jejunal circular smooth muscle.
Am J Physiol Gastrointest Liver Physiol
276:
G1373-G1379,
1999
52.
Yamada, K,
Waniishi Y,
Inoue R,
and
Ito Y.
Fenamates potentiate the alpha 1-adrenoceptor-activated nonselective cation channels in rabbit portal vein smooth muscle.
Jpn J Pharmacol
70:
81-84,
1996[ISI][Medline].
53.
Yuan, XJ,
Tod ML,
Rubin LJ,
and
Blaustein MP.
NO hyperpolarizes pulmonary artery smooth muscle cells and decreases the intracellular Ca2+ concentration by activating voltage-gated K+ channels.
Proc Natl Acad Sci USA
93:
10489-10494,
1996
54.
Zhang, Y,
Miller DV,
and
Paterson WG.
Opposing roles of K+ and Cl channels in maintenance of opossum lower esophageal sphincter tone.
Am J Physiol Gastrointest Liver Physiol
279:
G1226-G1234,
2000
55.
ZhuGe, R,
Sims SM,
Tuft RA,
Fogarty KE,
and
Walsh JVJ
Ca2+ sparks activate K+ and Cl channels, resulting in spontaneous transient currents in guinea-pig tracheal myocytes.
J Physiol
513:
711-718,
1998