Regulation of slow wave frequency by IP3-sensitive calcium release in the murine small intestine

John Malysz, Graeme Donnelly, and Jan D. Huizinga

Intestinal Disease Research Program and Department of Medicine, McMaster University, Hamilton, Ontario, Canada L8N 3Z5


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha 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


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
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REFERENCES

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.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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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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Fig. 1.   Effects of chelation of cytosolic Ca2+ and inhibition of sarcoplasmic reticulum (SR) Ca2+-ATPase. A: 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'- tetraacetic acid-AM (BAPTA-AM) abolished slow wave activity (25 µM, 30 min). B: cyclopiazonic acid (CPA) markedly reduced slow wave frequency (2 µM, 30 min). C: thapsigargin (1 µM) markedly reduced slow wave activity. C1, control; C2: i, same tissue after 2 h of perfusion with thapsigargin; ii, same tissue after 5 min perfusion with 0.5 µM carbachol. C3, tissue from the same mouse after a 4-h perfusion with thapsigargin. Continuous trace showing elimination of the slow wave. Throughout all the experiments, L-type Ca2+ channel blockers were present (1-5 µM nifedipine or verapamil).



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Fig. 2.   Slow wave frequency over time during SR Ca2+-ATPase inhibition. Thapsigargin (1 µM) progressively decreases the slow wave frequency. Perfusion with phenylephrine (3 µM) or carbachol (0.5 µM), in the presence of thapsigargin (1 µM), enhanced the rate of decline in the slow wave frequency. Experiments were performed in the presence of L-type Ca2+ channel blockade (1 µM nifedipine).

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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Fig. 3.   Disruption of slow wave activity after blockade of inositol 1,4,5,-trisphosphate (IP3)-induced Ca2+ release. Xestospongin C (0.5 µM) progressively disrupts the slow wave pattern. Compared with the control tracing at top, observe the "waxing and waning" of the middle trace. With higher concentrations of xestospongin C (1 µM), the slow wave was abolished (bottom trace). Experiments were performed in the presence of L-type Ca2+ channel blockade (1 µM nifedipine).

Effect of alpha 1-adrenoceptor activation. Stimulation of alpha 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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Fig. 4.   Increase in slow wave frequency by stimulation of alpha 1-adrenoceptors. A: 10 µM phenylephrine affects frequency and plateau potential (top trace is control activity; activity shown after 10 min). B: 10 µM phenylephrine has no effect in the presence of prazosin (5 µM; activity shown after 10 min). C: 2 µM norepinephrine has a marked effect (after 3 min, bottom trace) on the frequency in the presence of CPA (2 µM, top trace). D: 3 µM phenylephrine markedly affects slow wave frequency (after 3 min, bottom trace) in the presence of CPA (1 µM, top trace). Throughout all the experiments, L-type Ca2+ channel blockers were present (1-5 µM nifedipine or verapamil).

In the presence of CPA (1-3 µM) with a reduction in frequency of at ~40%, norepinephrine (2 µM) and phenylephrine (3 µM) increased the slow wave frequency by 69.5 ± 28.3% (n = 6, P < 0.002) and 78.8 ± 32.4% (n = 7, P < 0.04), respectively, within the first 20 min of perfusion (Fig. 4, C and D). No other slow wave characteristics were affected. Long-term effects of alpha 1-adrenoceptor agonist perfusion in CPA were not determined due to the developing depolarization by CPA.

In the presence of thapsigargin (1 µM) with the slow wave frequency reduced by at least 20%, phenylephrine (3 µM) either increased or decreased the slow wave frequency in the first few minutes. Thereafter phenylephrine caused an increase in the rate of decline in slow wave frequency to 1.5 cycles/min (n = 5, R2=0.68) compared with thapsigargin alone (Fig. 2). The slow wave activity was completely abolished within ~30 min of perfusion with phenylephrine and thapsigargin.

In addition to an effect on frequency, when examined in the absence of SERCA blockade, phenylephrine and norepinephrine affected the slow wave amplitude, average rate of increase, and duration. To describe the effects of the adrenoceptor agonists on the amplitude, it became necessary to make a distinction between the upstroke amplitude and plateau amplitude. In other experiments described here, the plateau amplitude was affected to the same degree as the upstroke amplitude, and only the upstroke amplitude was reported. Phenylephrine (10 µM) decreased plateau amplitude but increased upstroke amplitude. The plateau amplitude was decreased from 17.0 ± 1.3 to 14.6 ± 1.5 mV (n = 19, P < 0.02) with 3 µM phenylephrine and from 17.3 ± 0.9 to 11.8 ± 0.9 mV (n = 12, P < 0.0001) with 10 µM phenylephrine. In comparison, the upstroke amplitude increased from 19.3 ± 1.0 to 20.5 ± 1.0 mV (n = 19, P < 0.05) with 3 µM phenylephrine and from 20.3 ± 1.3 to 22.1 ± 1.3 mV (n = 12, P < 0.01) with 10 µM phenylephrine. Compared with the effects of phenylephrine, norepinephrine did not increase upstroke amplitude but also decreased plateau amplitude. At 1 µM norepinephrine, plateau amplitude decreased from 17.4 ± 2.2 to 14.6 ± 2.4 mV (n = 11, P < 0.05), and at 10 µM norepinephrine, from 16.6 ± 0.7 to 11.1 ± 0.8 mV (n = 6, P < 0.01). A concentration of 1 µM norepinephrine had no effect on the upstroke amplitude, whereas 10 µM norepinephrine decreased the upstroke amplitude from 21.1 ± 1.5 to 15.2 ± 1.7 mV (n = 5, P < 0.03). Hence, the slow wave upstroke amplitude could be increased by phenylephrine but not by norepinephrine. In addition, phenylephrine decreased the slow wave duration and increased the average rate of increase of the upstroke, and both phenylephrine (3 and 10 µM) and norepinephrine (10 µM) produced significant hyperpolarizations. The respective hyperpolarizations were 2.5 ± 0.7 (n = 19, P < 0.002), 3.4 ± 0.8 (n = 12, P < 0.003), and 8.8 ± 1.0 mV (n = 6, P < 0.0006). It was unlikely that any of the observed effects, especially those of phenylephrine, were due to hyperpolarization because 1 µM cromakalim, a KATP channel opener, produced a hyperpolarization of 8.6 ± 1.4 mV (n = 11, P < 0.0001) without significantly affecting any slow wave characteristics (data not shown).

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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Fig. 5.   Effects of modulation of phospholipase C activity on slow wave activity. A: carbachol (1 µM) transiently increased the slow wave frequency in the presence of CPA (1 µM). B: U-73122 (1 mM) decreased the slow wave frequency and amplitude. C: the traces at left are controls with the middle trace (ii) being recorded in the presence of CPA (3 µM). The traces at right illustrate effects of 4 mM neomycin (i and ii) and 0.3 mM 2-nitro-4-carboxyphenyl-N,N-diphenylcarbamate (NCDC) (iii). Throughout all the experiments, L-type Ca2+ channel blockers were present (1-5 µM nifedipine or verapamil).

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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Fig. 6.   Effect of caffeine (5 mM) and ryanodine on slow wave frequency. A: caffeine (5 mM) transiently hyperpolarized, then depolarized the membrane potential. The slow wave frequency was also reduced, and the electrical activity was eventually abolished. A continuous recording shows control activity and the effects of caffeine after 1, 3, and 10 min of perfusion. The effects on the frequency were consistent with the inhibition of IP3-induced Ca2+ release. B: ryanodine (50 µM) depolarized the membrane potential and caused minor effects on the slow wave not related to the depolarization (see RESULTS). A continuous experiment showing control activity and after 10 and 20 min of perfusion with ryanodine. Throughout all the experiments, L-type Ca2+ channel blockers were present (1-5 µM nifedipine or verapamil).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha 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.


    ACKNOWLEDGEMENTS

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).


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Bird, GS, Burgess GM, and Putney JWJ Sulfhydryl reagents and cAMP-dependent kinase increase the sensitivity of the inositol 1,4,5-trisphosphate receptor in hepatocytes. J Biol Chem 268: 17917-17923, 1993[Abstract/Free Full Text].

2.   Candell, LM, Yun SH, Tran LL, and Ehlert FJ. Differential coupling of subtypes of the muscarinic receptor to adenylate cyclase and phosphoinositide hydrolysis in the longitudinal muscle of the rat ileum. Mol Pharmacol 38: 689-697, 1990[Abstract].

3.   Chadwick, CC, Saito A, and Fleischer S. Isolation and characterization of the inositol trisphosphate receptor from smooth muscle. Proc Natl Acad Sci USA 87: 2132-2136, 1990[Abstract].

4.   Cheung, WY. Properties of cyclic 3',5'-nucleotide phosphodiesterase from rat brain. Biochemistry 6: 1079-1087, 1967[ISI][Medline].

5.   Dahms, V, Prosser CL, and Suzuki N. Two types of `slow waves' in intestinal smooth muscle of cat. J Physiol (Lond) 392: 51-69, 1987[Abstract].

6.   De Smet, P, Parys JB, Callewaert G, Weidema AF, Hill E, De Smedt H, Erneux C, Sorrentino V, and Missiaen L. Xestospongin C is an equally potent inhibitor of the inositol 1,4,5-trisphosphate receptor and the endoplasmic-reticulum Ca2+ pumps. Cell Calcium 26: 9-13, 1999[ISI][Medline].

7.   Der-Silaphet, T, Malysz J, Arsenault AL, Hagel S, and Huizinga JD. Interstitial cells of Cajal direct normal propulsive contractile activity in the small intestine. Gastroenterology 114: 724-736, 1998[ISI][Medline].

8.   Gafni, J, Munsch JA, Lam TH, Catlin MC, Costa LG, Molinski TF, and Pessah IN. Xestospongins: potent membrane permeable blockers of the inositol 1,4,5-trisphosphate receptor. Neuron 19: 723-733, 1997[ISI][Medline].

9.   Goeger, DE, Riley RT, Dorner JW, and Cole RJ. Cyclopiazonic acid inhibition of the Ca2+-transport ATPase in rat skeletal muscle sarcoplasmic reticulum vesicles. Biochem Pharmacol 37: 978-981, 1988[ISI][Medline].

10.   Hattori, Y, Endou M, Shirota M, and Kanno M. Dissociation of phosphoinositide hydrolysis and positive inotropic effect of histamine mediated by H1-receptors in guinea-pig left atria. Naunyn Schmiedebergs Arch Pharmacol 340: 196-203, 1989[ISI][Medline].

11.   Huizinga, JD, Farraway L, and Den Hertog A. Effect of voltage and cyclic AMP on frequency of slow wave type action potentials in colonic smooth muscle. J Physiol (Lond) 442: 31-45, 1991[Abstract].

12.   Huizinga, JD, Farraway L, and Den Hertog A. Generation of slow-wave-type action potentials in canine colon smooth muscle involves a non-L-type Ca2+ conductance. J Physiol (Lond) 442: 15-29, 1991[Abstract].

13.   Huizinga, JD, Thuneberg L, Klüppel M, Malysz J, Mikkelsen HB, and Bernstein A. The W/kit gene required for interstitial cells of Cajal and for intestinal pacemaker activity. Nature 373: 347-349, 1995[ISI][Medline].

14.   Huizinga, JD, Thuneberg L, Vanderwinden JM, and Rumessen JJ. Interstitial cells of Cajal as pharmacological targets for gastrointestinal motility disorders. Trends Pharmacol Sci 18: 393-403, 1997[ISI][Medline].

15.   Iino, M. Calcium-induced calcium release mechanism in guinea pig taenia caeci. J Gen Physiol 94: 363-383, 1989[Abstract].

16.   Iino, M, Kobayashi T, and Endo M. Use of ryanodine for functional removal of the calcium store in smooth muscle cells of the guinea-pig. Biochem Biophys Res Commun 152: 417-422, 1988[ISI][Medline].

17.   Koh, SD, Sanders KM, and Ward SM. Spontaneous electrical rhythmicity in cultured interstitial cells of Cajal from the murine small intestine. J Physiol (Lond) 513: 203-213, 1998[Abstract/Free Full Text].

18.   Kuemmerle, JF, Murthy KS, and Makhlouf GM. Agonist-activated, ryanodine-sensitive, IP3-insensitive Ca2+ release channels in longitudinal muscle of intestine. Am J Physiol Cell Physiol 266: C1421-C1431, 1994[Abstract/Free Full Text].

19.   Lee, JCF, Thuneberg L, Berezin I, and Huizinga JD. The generation of slow waves in membrane potential is an intrinsic property of interstitial cells of Cajal. Am J Physiol Gastrointest Liver Physiol 277: G409-G423, 1999[Abstract/Free Full Text].

20.   Liu, LWC, and Huizinga JD. Electrical coupling of circular muscle to longitudinal muscle and interstitial cells of Cajal in canine colon. J Physiol (Lond) 470: 445-461, 1993[Abstract].

21.   Liu, LWC, Thuneberg L, and Huizinga JD. Cyclopiazonic acid, inhibiting the endoplasmic reticulum calcium pump, reduces the canine colon pacemaker frequency. J Pharmacol Exp Ther 275: 1058-1068, 1995[Abstract].

22.   Ma, HT, Patterson RL, van Rossum DB, Birnbaumer L, Mikoshiba K, and Gill DL. Requirement of the inositol trisphosphate receptor for activation of store-operated Ca2+ channels. Science 287: 1647-1651, 2000[Abstract/Free Full Text].

23.   Malysz, J, and Huizinga JD. Regulation of pacemaker activity in the mouse small intestine involves IP3 sensitive calcium release (Abstract). Dig Dis Sci 41: 1888, 1997.

24.   Malysz, J, Richardson D, Farraway L, Christen MO, and Huizinga JD. Generation of slow wave type action potentials in the mouse small intestine involves a non-L-type calcium channel. Can J Physiol Pharmacol 73: 1502-1511, 1995[ISI][Medline].

25.   Malysz, J, Thuneberg L, Mikkelsen HB, and Huizinga JD. Action potential generation in the small intestine of W mutant mice that lack interstitial cells of Cajal. Am J Physiol Gastrointest Liver Physiol 271: G387-G399, 1996[Abstract/Free Full Text].

26.   Missiaen, L, De Smedt H, Droogmans G, and Casteels R. Ca2+ release induced by inositol 1,4,5-trisphosphate is a steady-state phenomenon controlled by luminal Ca2+ in permeabilized cells. Nature 357: 599-602, 1992[ISI][Medline].

27.   Missiaen, L, Parys JB, De Smedt H, Oike M, and Casteels R. Partial calcium release in response to submaximal inositol 1,4,5- trisphosphate receptor activation. Mol Cell Endocrinol 98: 147-156, 1994[ISI][Medline].

28.   Missiaen, L, Taylor CW, and Berridge MJ. Spontaneous calcium release from inositol trisphosphate-sensitive calcium stores. Nature 352: 241-244, 1991[ISI][Medline].

29.   Mourey, RJ, Estevez VA, Marecek JF, Barrow RK, Prestwich GD, and Snyder SH. Inositol 1,4,5-trisphosphate receptors: labeling the inositol 1,4,5-trisphosphate binding site with photoaffinity ligands. Biochemistry 32: 1719-1726, 1993[ISI][Medline].

30.   Parker, I, and Ivorra I. Caffeine inhibits inositol trisphosphate-mediated liberation of intracellular calcium in Xenopus oocytes. J Physiol (Lond) 433: 229-240, 1991[Abstract].

31.   Parys, JB, Missiaen L, De Smedt H, and Casteels R. Loading dependence of inositol 1,4,5-trisphosphate-induced Ca 2+ release in the clonal cell line A7r5. Implications for the mechanism of quantal Ca2+ release. J Biol Chem 268: 25206-25212, 1993[Abstract/Free Full Text].

32.   Parys, JB, Missiaen L, De Smedt H, Droogmans G, and Casteels R. Bell-shaped activation of inositol-1,4,5-trisphosphate-induced Ca2+ release by thimerosal in permeabilized A7r5 smooth-muscle cells. Pflügers Arch 424: 516-522, 1993[ISI][Medline].

33.   Sanders, KM. A case for interstitial cells of Cajal as pacemakers and mediators of neurotransmission in the gastrointestinal tract. Gastroenterology 111: 492-515, 1996[ISI][Medline].

34.   Seamon, KB, and Daly JW. Forskolin: its biological and chemical properties. Adv Cyclic Nucleotide Protein Phosphorylation Res 20: 1-150, 1986[ISI][Medline].

35.   Seidler, NW, Jona I, Vegh M, and Martonosi A. Cyclopiazonic acid is a specific inhibitor of the Ca2+-ATPase of sarcoplasmic reticulum. J Biol Chem 264: 17816-17823, 1989[Abstract/Free Full Text].

36.   Summers, RJ, and McMartin LR. Adrenoceptors and their second messenger systems. J Neurochem 60: 10-23, 1993[ISI][Medline].

37.   Suzuki, H, Takano H, Yamamoto Y, Komuro T, Saito M, Kato K, and Mikoshiba K. Properties of gastric smooth muscles obtained from mice which lack inositol trisphosphate receptor. J Physiol (Lond) 525: 105-111, 2000[Abstract/Free Full Text].

38.   Szurszewski, JH. Electrophysiological basis of gastrointestinal motility. In: Physiology of the Gastrointestinal Tract, edited by Johnson LR.. New York: Raven, 1987, p. 383-422.

39.   Takei, M, Ueno M, Endo K, and Nakagawa H. Effect of NCDC, a protease inhibitor, on histamine release from rat peritoneal mast cells induced by anti-IgE. Biochem Biophys Res Commun 181: 1313-1322, 1991[ISI][Medline].

40.   Thastrup, O. Role of Ca2+- ATPases in regulation of cellular Ca2+ signalling, as studied with the selective microsomal Ca2+-ATPase inhibitor, thapsigargin. Agents Actions 29: 8-15, 1990[ISI][Medline].

41.   Thomsen, L, Robinson TL, Lee JCF, Farraway L, Hughes MJG, Andrews DW, and Huizinga JD. Interstitial cells of Cajal generate a rhythmic pacemaker current. Nat Med 4: 848-851, 1998[ISI][Medline].

42.   Tsugeno, M, Huang SM, Pang YW, Chowdhury JU, and Tomita T. Effects of phosphodiesterase inhibitors on spontaneous electrical activity (slow waves) in the guinea-pig gastric muscle. J Physiol (Lond) 485: 493-502, 1995[Abstract].

43.   Wang, XB, Osugi T, and Uchida S. Different pathways for Ca2+ influx and intracellular release of Ca2+ mediated by muscarinic receptors in ileal longitudinal smooth muscle. Jpn J Pharmacol 58: 407-415, 1992[ISI][Medline].

44.   Wibo, M, and Godfraind T. Comparative localization of inositol 1,4,5-trisphosphate and ryanodine receptors in intestinal smooth muscle: an analytical subfractionation study. Biochem J 297: 415-423, 1994[ISI][Medline].

45.   Zhou, HL, and Torphy TJ. Relationship between cyclic guanosine monophosphate accumulation and relaxation of canine trachealis induced by nitrovasodilators. J Pharmacol Exp Ther 258: 972-978, 1991[Abstract].


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