Intestinal Disease Research Program and Department of Medicine, McMaster University, Hamilton, Ontario, Canada L8N 3Z5
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ABSTRACT |
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Slow waves determine
frequency and propagation characteristics of contractions in the small
intestine, yet little is known about mechanisms of slow wave
regulation. We propose a role for intracellular Ca2+,
inositol 1,4,5,-trisphosphate (IP3)-sensitive
Ca2+ release, and sarcoplasmic reticulum (SR)
Ca2+ content in the regulation of slow wave frequency
because 1)
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM,
a cytosolic Ca2+ chelator, reduced the frequency or
abolished the slow waves; 2) thapsigargin and cyclopiazonic
acid (CPA), inhibitors of SR Ca2+-ATPase, decreased slow
wave frequency; 3) xestospongin C, a reversible, membrane-permeable blocker of IP3-induced Ca2+
release, abolished slow wave activity; 4) caffeine and
phospholipase C inhibitors (U-73122, neomycin, and
2-nitro-4-carboxyphenyl-N,N-diphenylcarbamate) inhibited slow wave frequency; 5) in the presence of CPA or
thapsigargin, stimulation of IP3 synthesis with carbachol,
norepinephrine, or phenylephrine acting on
1-adrenoceptors initially increased slow wave frequency
but thereafter increased the rate of frequency decline, 6)
thimerosal, a sensitizing agent of IP3 receptors increased slow wave frequency, and 7) ryanodine, a selective modulator
of Ca2+-induced Ca2+ release, had no effect on
slow wave frequency. In summary, these data are consistent with
a role of IP3-sensitive Ca2+ release and the
rate of SR Ca2+ refilling in regulation of intestinal slow
wave frequency.
pacemaker activity; intestinal motility; cation channel; smooth muscle; interstitial cell of Cajal
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INTRODUCTION |
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PERISTALTIC CONTRACTIONS ARE generated through interplay between smooth muscle cells, the enteric nervous system, and interstitial cells of Cajal (ICC). ICC are critical in the generation of slow wave activity, which in turn sets the maximum frequency of contractions and determines propagation characteristics of the rhythmic contractions in the intestine (7, 14, 33, 38). The ionic and regulatory mechanisms responsible for slow wave generation still remain obscure. In the canine colon, slow waves were found to be relatively insensitive to changes in the membrane potential (11), insensitive to L-type Ca2+ channel blockers (12), and highly sensitive to the removal of extracellular Ca2+ and to cytosolic levels of cAMP (11). Furthermore, the canine colonic slow wave frequency could be reduced with either cyclopiazonic acid (CPA), a selective inhibitor of the sarcoplasmic reticulum (SR) Ca2+-ATPase, and 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM (BAPTA-AM), an intracellular Ca2+ chelator (21). Hence, it was suggested that an intracellular metabolic process involving Ca2+ release from the SR and sensitive to cAMP triggers the canine colonic slow waves. Much less is known about the regulatory mechanisms for the small intestinal slow waves. The temperature sensitivity of the slow waves, as observed in the small intestines of cats (5) and mice (19), suggests involvement of a metabolic process in the regulation of the slow wave. The sensitivity of the slow waves to removal of extracellular Ca2+ and their insensitivity to L-type Ca2+ channel blockers in the murine (17, 24, 25) and feline (5) small intestines point to participation of Ca2+ conductances other than those mediated by L-type Ca2+ channels. The intracellular regulatory mechanisms appear to be different in the colon and small intestine, because activation of the protein kinase A (PKA) pathway decreases slow wave activity in the former (11) and increases it in the latter (current study). It still remains to be established whether intracellular Ca2+ also plays a critical role in the regulation of the slow waves in the small intestine. Data in this study provide such evidence in the murine small intestine.
Intracellularly, the major sites for Ca2+ release are inositol 1,4,5,-trisphosphate (IP3) and ryanodine-sensitive Ca2+ stores residing in sections of the SR. Release of Ca2+ from these stores is highly regulated and involves Ca2+ channels in the SR membrane. Unique IP3-regulated Ca2+ channels have been purified from bovine aortic microsomes (3) and from membranes of rat vas deferens smooth muscle (29). In intestinal smooth muscle, existence of the IP3- and ryanodine-sensitive Ca2+ stores has been demonstrated (15, 16, 18, 44). The roles of these stores in regulation of intestinal slow waves remain undetermined.
The present study set out to determine whether intracellular Ca2+ and intracellular Ca2+ stores, particularly of the IP3 type, are involved in regulating slow wave frequency in the murine small intestine. A major obstacle that this study had to overcome was the lack of selective pharmacological agents acting on the IP3 receptor. Two of the most commonly utilized pharmacological agents known to act on IP3-induced Ca2+ release are IP3, an activator of the receptor, and heparin, a blocker of the receptor or channel. Because of their plasma membrane impermeability, neither agent could be used in this study. Instead, this study relied on a novel, membrane-permeable blocker of IP3-induced Ca2+ release, xestospongin C (8), and other agents known to act by different mechanisms on the IP3 signaling pathway, leading to the release of Ca2+ from intracellular Ca2+ stores. The approach was to test a number of agents involved in IP3 metabolism and Ca2+ release and construct a hypothesis based on the experimental findings.
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METHODS |
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Preparation and recording of electrical activities. A standard microelectrode technique, as previously described in detail elsewhere (13, 24), was used to record slow waves from the murine small intestine. Adult mice used in this study were of either sex and supplied by Charles River Laboratories (CD1, St. Constant, QC, Canada). The animals were cared for in accordance with the principles and guidelines of the Canadian Council on Animal Care.
Throughout the experiments, L-type Ca2+ blockers (1-5 µM nifedipine or verapamil) were present. As reported in detail in our (13, 24) previous studies on the murine small intestinal musculature, a major effect of the blockade is to attenuate generation of spike-like action potentials superimposed onto the plateau phases of the slow waves. In contrast, the intrinsic pacemaker activity (41) is insensitive to L-type Ca2+ channel blockade and is reflected in tissue as the nifedipine-insensitive part of the slow wave (24). Consequently, mechanisms of the regulation and generation of slow waves can be studied in the presence of the L-type Ca2+ channel blockers (verapamil or nifedipine).Solutions and drugs. The composition of Krebs solution was (in mM) 120.3 NaCl, 5.9 KCl, 2.5 CaCl2, 1.2 MgCl2, 20 NaHCO3, 1.2 NaH2PO4, and 11.5 glucose. The Krebs solution (7.30-7.35 pH) was continuously gassed with a mixture of 95% O2-5% CO2. Verapamil, nifedipine, CPA, neomycin, 2-nitro-4-carboxyphenyl-N,N-diphenylcarbamate (NCDC), caffeine, forskolin, sodium nitroprusside (SNP), norepinephrine, phenylephrine, prazosin, carbachol, thimerosal, and U-73122 were all obtained from Sigma Chemical (St. Louis, MO). BAPTA-AM was obtained from Molecular Probes (Eugene, OR). Xestospongin C was from Calbiochem (La Jolla, CA) and thapsigargin from Alomone Labs (Jerusalem, Israel).
Data analysis. Data were obtained usually from the same cells, before and in the presence of a drug. A difference was considered to be significant at P < 0.05. Data are expressed as means ± SE, with n representing the number of different animals used. When the effects of a specific drug or experimental condition are described, only one such experiment was carried out on a single tissue. Any given animal tissue on a single day was used to perform two or more separate pharmacological experiments. In experiments reporting percent increase or decrease, the percent change was obtained first for each individual experiment, and then mean and SE values were calculated based on these numbers. Paired or unpaired Student's t-tests were employed whenever appropriate for comparison of the electrical activities determining the effects of drugs and increased extracellular K+ solutions.
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RESULTS |
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Pharmacological modulation of intracellular Ca2+
homeostasis.
The murine proximal small intestine displayed slow wave activity at
45.8 ± 1.9 cycles/min, with a duration of 0.75 ± 0.03 s, an amplitude of 20.3 ± 2.1 mV, and an average rate of rise of
the upstroke of 249 ± 57 mV/s from a resting membrane potential of 62.3 ± 1.8 mV (n = 42). The membrane
permeable Ca2+ chelator BAPTA-AM (25-50 µM) led to a
decline in frequency and amplitude of the slow waves and in three of
five experiments complete abolishment of the activity
(n = 5; Fig.
1A). CPA (1-3
µM), an inhibitor of the SR Ca2+-ATPase (SERCA) pump
(9), decreased the slow wave frequency by 46.1 ± 7.3% (n = 11, P < 0.001) from
39.8 ± 2.1 to 20.9 ± 1.5 cycles/min (Fig.
1B) after at least 20 min of perfusion. During the initial 10-20 min of superfusion, CPA transiently depolarized the cells by up to 20 mV. At this time, it also became technically difficult to maintain stable impalements. After this initial period of
recording instability, stable impalements could be obtained. In
addition to the reduction in frequency, CPA also depolarized the
tissues by 6.2 ± 1.7 mV (P < 0.006) at the
~30- to 40-min perfusion recording mark. At this point, CPA (1-3
µM) significantly affected the slow wave amplitude, rate of upstroke
increase, and duration. To establish the effect of depolarization per
se, extracellular K+ was increased from 5.9 to 11.8 mM,
causing a depolarization of 6.0 ± 1.5 mV (n = 7;
P < 0.05). Similar reductions in the slow wave amplitude
and the average rate of upstroke increase were observed with CPA and
K+-induced depolarization. In contrast, reductions in the
slow wave frequency and duration with CPA were significantly different
from those identified with 11.8 mM K+. CPA decreased the
slow wave frequency by 46.1 ± 7.3% (n = 11), whereas the decrease with 11.8 mM K+ was 15.1 ± 3.6%
(n = 7, P < 0.02). The reduction in
the slow wave duration by CPA was 26.0 ± 5.1% (n = 11), significantly different from that by 11.8 mM K+,
which was 6.2 ± 0.4% (n = 7, P < 0.05). Hence, the effects of CPA on the slow wave frequency and
duration were for the most part independent from the effects on the
membrane potential. Depolarization per se with CPA likely produced the
observed reductions in the slow wave amplitude and in the average rate
of increase of the upstroke. This conclusion found confirmation in
results with thapsigargin, another SERCA inhibitor (40).
Thapsigargin (1 µM) decreased the slow wave frequency at a rate of
0.12 cycles/min [n = 15; regression coefficient
(R2) = 0.96] (Figs. 1C and
2) until elimination of the slow wave at
~400 min of perfusion. No depolarization was observed, and the membrane potentials at 0, 2, and 4 h of perfusion were not significantly different, being
57.8 ± 2.7,
61.7 ± 3.4, and
62.1 ± 3.4 mV, respectively (n = 15, P > 0.05). At the 30- to 40-min perfusion mark, the
slow wave amplitude and rate of increase were not affected at 18.0 ± 0.6 mV and 297 ± 121 mV/s (P > 0.05),
respectively.
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Effect of blockade of IP3-induced Ca2+
release.
Xestospongin C has been shown to be a blocker of
IP3-induced Ca2+ release (8, 22),
which, unlike heparin, is membrane permeable, although it has been
suggested that it can also block the SERCA pump (6). On
addition of xestospongin C (0.5 µM; n = 5), normal slow wave activity began to "wax and wane" (n = 3),
and at a higher concentration of 1 µM, slow wave activity was
abolished (n = 3) (Fig.
3).
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Effect of 1-adrenoceptor activation.
Stimulation of
1-adrenoceptors activates phospholipase C
(PLC) to generate IP3 via a G protein-dependent mechanism
(36). Norepinephrine (3 and 10 µM) and phenylephrine (1 and 10 µM) increased the slow wave frequency by 5.7 ± 1.8%
(n = 19, P < 0.005), 7.0 ± 1.8%
(n = 12, P < 0.005), 4.1 ± 1.6%
(n = 11, P < 0.04), and 5.1 ± 1.7% (n = 6, P < 0.05), respectively
(Fig. 4A). These
effects of phenylephrine and norepinephrine (and those
described below) were inhibited by pretreatment of tissues with
prazosin (5 µM). Prazosin on its own had no effects on the slow wave
(n = 8, Fig. 4B).
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Effect of carbachol.
Carbachol induces IP3 synthesis via activation of
M3 receptors in intestinal smooth muscle (2).
On addition of carbachol in the presence of CPA, a consistent transient
increase in slow wave frequency was observed of 99.0 ± 35.0%
(n = 7, P < 0.05) (Fig.
5A) followed by a progressive
reduction in frequency. In the presence of thapsigargin, an initial
increase in slow wave frequency also occurred, although less
consistently. Thereafter, a progressive decline in frequency occurred
at a rate of 1.04 cycles/min (n = 15, R2=0.91), an approximate 10-fold more rapid
decline than in the presence of thapsigargin alone (Figs. 1C
and 2).
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Effect of PLC inhibition with U-73122, neomycin, and NCDC. To further characterize a possible role of IP3-induced Ca2+ release, we carried out experiments with known inhibitors of the phosphatidylinositol-specific PLC, thus limiting the generation of IP3 (10, 39). U-73122 (1 mM), although not significantly affecting the resting membrane potential or the slow wave duration, decreased slow wave frequency from 51.4 ± 2.1 to 41.4 ± 1.5 cycles/min (n = 5, P < 0.05; Fig. 5B). The amplitude was also reduced from 20.6 ± 1.0 to 13.5 ± 2.3 mV (P < 0.05). When U-73122 (1 mM) was added in the presence of thapsigargin (1 µM), when the slow wave frequency was already reduced, complete abolishment of the slow waves occurred within ~40 min of perfusion (n = 5). Neomycin (4 mM) decreased the slow wave frequency by 27.2 ± 8.8% and in the presence of CPA completely abolished slow wave activity (n = 7; Fig. 5C). NCDC at 0.3 mM abolished the slow wave activity in four of six experiments (Fig. 5C). In the remaining two experiments, marked reductions in frequency and amplitude were observed. Effects of NCDC and neomycin were not associated with significant effects on the resting membrane potential.
Effects of thimerosal. Thimerosal reportedly sensitizes the IP3 receptors, leading to an enhancement of IP3-induced Ca2+ release (1). The addition of thimerosal (50 µM) caused increased slow wave frequency by 16.4 ± 4.2%, (n = 5, P < 0.03) with a depolarization (7.0 ± 1.2 mV, n = 5, P < 0.03) within 2-5 min of perfusion. In addition, thimerosal reduced both the amplitude (by 24.6-40.4%) and the rate of increase of slow waves (by 31.8-38.2%). These effects were similar to those obtained with 11.8 mM extracellular K+, thus suggesting their dependence on depolarization.
Effect of caffeine. Caffeine is known to block IP3-induced Ca2+ release at high concentrations (10-20 mM) (30). At 10 mM, caffeine led to a transient hyperpolarization lasting for 1-3 min followed by recovery to near original levels. At this time, the slow waves were either completely abolished or marked reductions in the slow wave frequency and amplitude were observed. Prolonged perfusions with caffeine of 10 or more minutes produced a marked depolarization of 14.0 ± 3.4 mV (n = 11, P < 0.01) and usually complete abolition of the slow wave. The effects of prolonged perfusion with caffeine on the slow wave were likely independent of depolarization because 17.7 mM extracellular K+ failed to abolish the slow waves.
Effects of forskolin and SNP. One of the known effects of caffeine is to inhibit cytosolic nucleotide phosphodiesterases (4). This leads to increases in cytosolic levels of cAMP and cGMP and subsequent activation of the PKA and PKG signaling pathways. Neither forskolin, which increases intracellular levels of cAMP, nor SNP, which increases cGMP (34, 45), mimicked the effects of caffeine on the slow wave, except for the hyperpolarization. Concentrations of 1 µM forskolin and 1 µM SNP hyperpolarized the tissues by 13.8 ± 2.9 (n = 7, P < 0.004) and 6.3 ± 1.2 mV (n = 7, P < 0.003), respectively. SNP also significantly increased slow wave amplitude (11.0 ± 3.4%, n = 7, P < 0.03) and the average rate of increase of the upstroke (24.9 ± 0.3%, n = 7, P < 0.003). Forskolin (1 µM) affected slow wave amplitude (decrease of 4.8 ± 1.9%, n = 7, P < 0.04) and frequency (increase of 10.2 ± 2.2%, n = 7, P < 0.002).
Effect of Ca2+-induced Ca2+ release.
Both caffeine and ryanodine act on the Ca2+-induced
Ca2+-release mechanism (15, 16, 18). To
examine the role of this Ca2+ store in the regulation of
slow wave activity, we tested the effects of caffeine (5 mM) and
ryanodine (10 and 50 µM, Fig. 6). Both
are expected to stimulate Ca2+-induced Ca2+
release in intestinal smooth muscle (15, 16, 18). The
effects of caffeine at 5 mM, a concentration exhibiting greater
selectivity for Ca2+-induced Ca2+ release over
IP3-induced Ca2+ release, were similar to those
of the 10 mM concentration described above, albeit with a lower
magnitude of response. An initial transient hyperpolarization was
observed, and after 10 min of perfusion, the tissues were depolarized
by 8.8 ± 2.9 mV (n = 8, P < 0.02). Ryanodine (10 µM) did not have any effect on the electrical
activity during 30 min of perfusion. The addition of higher
concentrations of ryanodine (50 µM, n = 6) caused a
10.0 ± 2.4 mV (P < 0.01) depolarization, a
55.0 ± 7.7% (P < 0.005) reduction in slow wave amplitude, and a 13.1 ± 0.2% (P < 0.005)
reduction in slow wave frequency after ~20 min of perfusion. However,
a similar depolarization caused by 17.7 mM K+ produced
similar changes in slow wave activity, suggesting that ryanodine had
negligible effects on slow wave activity that were not related to
depolarization.
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DISCUSSION |
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The data presented in this study are consistent with the hypothesis that the regulation of the slow wave frequency in the murine small intestine involves the release of Ca2+ from IP3-sensitive intracellular Ca2+ stores. Intracellular Ca2+ is critical for the generation of slow waves, as the experiments with BAPTA showed. When Ca2+ release induced by IP3 was blocked by xestospongin C, slow wave production was abolished, suggesting a critical role of IP3-induced Ca2+ release in slow wave initiation. Stimulators of IP3 synthesis or the IP3 receptor caused an increase in frequency (premature slow waves), suggesting that IP3-induced Ca2+ release may trigger the initiation of the slow wave. This may be mediated by activation of a nonselective cation channel (24, 41). Both CPA and thapsigargin, at submaximal concentrations that inhibit the SR Ca2+ pump, produced a progressively declining slow wave frequency. This is consistent with the rate of Ca2+ store refilling being an important factor in determining the frequency of the slow wave, because IP3-induced Ca2+ release is contingent on a critical level of luminal SR Ca2+. Missiaen and co-workers (26, 28) showed that in smooth muscle cells release of Ca2+ from IP3-sensitive stores is dependent on SR Ca2+ levels. As luminal SR Ca2+ concentration increases, so too does the IP3 receptor sensitivity to endogenous IP3. In the presence of CPA, increasing IP3 can increase the slow wave frequency as higher concentrations of IP3 can cause the activation of the IP3 receptor to occur at lower SR Ca2+ concentrations (27, 28, 31). Although increased levels of IP3 may cause more Ca2+ to be released from the stores, initially the SERCA pump can still compensate for the increase in release (35). As the SR pump is inhibited more strongly, refilling the store will take longer and the slow wave frequency will further decrease. This proposed role of IP3-sensitive Ca2+ release in the regulation of the slow wave finds strong support in the recent observation (37) that gastric smooth muscles that lack the IP3 receptor do not generate slow waves.
The effects of drugs with a more or less specific action on
IP3 synthesis or signaling pathway were consistent with the
decreased or increased generation of IP3 causing a decrease
or increase, respectively, in slow wave frequency. Activation
of M3 muscarinic receptors by carbachol and stimulation of
1-adrenoceptors by phenylephrine or norepinephrine
increase IP3 synthesis and release of Ca2+ from
IP3-sensitive Ca2+ stores in a variety of
cells, including smooth muscle cell types (2, 36). These
agents, in the presence of SERCA blockade, could substantially increase
the slow wave frequency. Perfusion with carbachol or phenylephrine in
the presence of SERCA blockade, however, reduced the slow wave
frequency or abolished the electrical activity consistent with
depletion of the IP3-sensitive Ca2+ stores.
U-73122, neomycin, and NCDC are inhibitors of
phosphatidylinositol-specific PLC, and they inhibit agonist-stimulated
IP3 generation as seen in both ileal longitudinal smooth
muscle cells (43) and coronary arterial smooth muscle
cells (39). In this study, these PLC blockers reduced the
slow wave frequency and amplitude or abolished the electrical activity
altogether. Thimerosal sensitizes the IP3 receptor, causing
greater Ca2+ release from SR and elevating cytosolic
Ca2+ levels (1), although other mechanisms by
which thimerosal activates intracellular Ca2+ release have
been proposed (32). Perfusion with thimerosal (for up to 5 min) increased the slow wave frequency. Additional effects of
thimerosal on the slow wave amplitude and the rate of increase of the
upstroke were likely due to the depolarization that also developed.
Caffeine at high concentrations is known to block IP3-induced Ca2+ release (30). In the present study, 10 mM caffeine inhibited the slow wave, apparently independent of the effect of depolarization. In addition to the change in frequency, caffeine caused a transient hyperpolarization followed by depolarization. The hyperpolarization was likely due to increase in cytosolic cAMP or cGMP levels similar to the action of forskolin or SNP. Effects of high concentrations of caffeine (5-10 mM) were previously studied on antral (42) and colonic slow waves (21). In these tissues, caffeine inhibited the slow waves. However, the effect of caffeine on the antral and colonic slow waves may not only be explained by the inhibition of the IP3-induced Ca2+ release. Increases in cytosolic cAMP levels may also play a role as activation of the PKA signaling pathway led to the abolition of the slow waves or marked reductions in the slow wave amplitude or frequency in the stomach and colonic musculature (11, 42). In this respect, the murine small intestine offers an advantage in interpreting the effects of caffeine. Activation of the PKA and PKG signaling pathways, judged in this study by respectively identifying responses to forskolin and SNP, did not mimic the effects of caffeine on the slow wave except for the hyperpolarization.
Another well-established effect of caffeine is on the stimulation of the Ca2+-induced Ca2+ release via ryanodine receptors (15). To determine the role of these stores in the regulation of the electrical activity in the murine small intestine, we tested the effects of ryandine as well as a lower concentration of caffeine. Both of these agents produced a depolarization, suggesting that activation of this release mechanism is linked to the regulation of the membrane potential. Further study is needed to determine the intracellular mechanisms responsible for this effect. Other observed effects of ryanodine and caffeine on the slow wave, apart from alterations in frequency (by caffeine), can be explained by depolarization, consistent with only a minor role of Ca2+-induced Ca2+ release in regulation of the slow wave. As explained above, reduction of frequency by caffeine can be attributed to inhibition of IP3-induced Ca2+ release.
Because the initiation of slow waves occurs in ICC (17, 41), it is likely that IP3-induced Ca2+ release in ICC influences the slow wave frequency. The hypothesis we would like to put forward is that cyclic release of Ca2+ from IP3-sensitive Ca2+ stores initiates the rhythmic slow wave activity. The rate of refilling of the SR Ca2+ stores would determine the slow wave frequency. Basal levels of IP3 would be sufficient to release Ca2+ when a certain level of luminal SR Ca2+ is reached. When Ca2+ is released from the SR, luminal Ca2+ concentration is decreased, thus desensitizing the IP3 receptor (26) and halting Ca2+ release until SR Ca2+ levels are restored via the SERCA pump. Indeed, we showed that interference with any of these components affects the slow wave frequency. IP3 receptor-associated cyclic Ca2+ release is common in nonexcitable cells (28). The above-described hypothesis will have to be proven in isolated ICC, which have recently been shown (17, 41) to generate rhythmic slow wave activity. Slow waves actively propagate into the smooth muscle layers (20), hence smooth muscle cells posses most of the ionic mechanisms to generate slow waves, apart from the triggering mechanism. This means that drug action on the IP3 signaling pathway in smooth muscle cells can be responsible for the observed changes in the slow wave parameters and the resting membrane potential. For example, U-73122 and NCDC, PLC inhibitors, reduced the slow wave amplitude, and adrenoceptor agonists affected the resting membrane potential, the slow wave amplitude (upstroke and plateau), and the duration.
In summary, we provide evidence for a role of intracellular Ca2+ stores, IP3-induced Ca2+ release, and cytosolic Ca2+ in the regulation of slow wave frequency and amplitude in the murine small intestine. The proposed hypothesis is that cyclic release of Ca2+ from IP3-sensitive Ca2+ stores in ICC underlies the rhythmic activation of the slow waves in the intestinal musculature. The main factors in the regulation of the slow wave frequency may be the rate of refilling of the SR stores together with the regulation of the sensitivity of the IP3 receptor.
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ACKNOWLEDGEMENTS |
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A portion of the current work was presented at the Ninth American Motility Society Meeting (Traverse City, MI, 1996) and has been published previously in abstract form (23).
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FOOTNOTES |
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Present address of J. Malysz: Abbott Laboratories, Pharmaceutical Products Division, Neurological and Urological Diseases Research, 100 Abbott Park Rd., Abbott Park, IL 60064.
This research was financially supported by the Medical Research Council of Canada through operating funds and scholarships.
Address for reprint requests and other correspondence: J. D. Huizinga, McMaster Univ., Intestinal Disease Research Programme, HSC-3N5C, 1200 Main St. West, Hamilton, ON, Canada, L8N 3Z5 (E-mail: huizinga{at}mcmaster.ca).
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.
Received 5 November 1999; accepted in final form 6 September 2000.
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