Increased calcium influx is responsible for the sustained mechanical tone in colon from dystrophic (mdx) mice

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Increased Calcium Influx Is Responsible for the Sustained Mechanical Tone in Colon From Dystrophic (mdx) Mice FLAVIA MULE`* and ROSA SERIO‡ *Dipartimento Farmaco-Biologico, Universita` della Calabria, Arcavacata di Rende (Cs), and ‡Dipartimento di Biologia cellulare e dello Sviluppo, Universita` di Palermo, Palermo, Italy

Background & Aims: Proximal colon from dystrophic mice develops spontaneous tone increment, but the mechanisms involved in its development have not been investigated. This study examined whether alterations in the properties of cell membrane calcium channels and/or sarcoplasmic reticular (SR) Ca2ⴙ-adenosine triphosphatase (ATPase) contribute to tone development. Methods: Effects of calcium-free solution, nifedipine, pinaverium (calcium channel blockers), and cyclopiazonic acid (CPA; SR Ca2ⴙ-ATPase inhibitor) on the contractile activity of colon from mdx and control mice were determined. Results: Calcium-free solution abolished spontaneous contractions in both preparations, but decreased the tone only in mdx mice. Nifedipine or pinaverium abolished phasic contractions, acting with different sensitivities on the 2 preparations. They also decreased the tone in colons of mdx mice, and Ca2ⴙ-free solution did not cause any further loss of tone. CPA, after an early contractile effect, abolished spontaneous contractions in control animals. It did not suppress the contractile activity in mdx mice. CPA inhibited the repletion of intracellular calcium stores in both tissues to the same degree. Conclusions: Increased Ca2ⴙ influx through L-type voltage-dependent Ca2ⴙ channels seems to be responsible for the sustained mechanical tone of proximal colon from mdx mice. The mechanisms for sequestering calcium appear to be unaltered.

atients with Duchenne muscular dystrophy (DMD), and mdx mice, suffer from a genetic disorder characterized by the absence of dystrophin, a cytoskeletal protein1,2 that is present mainly in skeletal muscle fibers3 but also in cardiac and smooth muscle.4 Although the exact function of dystrophin is not yet understood, this protein is believed to stabilize the sarcolemmal membrane,5 and its absence would lead to the chronic muscle degeneration characteristic of DMD. Several other functions of this protein are still being discussed, including a role in the binding of nitric oxide synthase to the inner surface of the sarcolemma6 and in the regulation of intracellular calcium.7

P

It is generally assumed that Ca2⫹-mediated pathologic reactions play an important part in the process of cell destruction, and it is likely that an excess of intracellular Ca2⫹ induces the death of dystrophic muscle fibers.8 However, the role of Ca2⫹ in the dystrophic process is controversial. An enhancement of total Ca2⫹ content has been found in cultured myotubes and in the skeletal muscles of the mdx mice.9 –12 This increase is caused mainly by an augmented influx of Ca2⫹,13,14 postulated by different reports to be attributable to enhancement of the open probability of leak channels,15 to activation of stretch-sensitive channels,16,17 and/or to a fragile and leaky membrane.18 However, other investigators were unable to show an enhanced intracellular concentration of Ca2⫹ in the dystrophin-deficient state.19 –21 Up to now, the functionality of smooth muscle in patients with DMD and mdx mice has received limited attention. However, studies on the dystrophic smooth muscle could be useful to gain new insights into the relationship between dystrophin and cytoplasmatic Ca2⫹ homeostasis. Lack of dystrophin with different degrees of dystrophic involvement has been observed in mdx smooth muscle of the digestive tract,22,23 and different clinical manifestations, including gastric dilatation and intestinal pseudo-obstruction, have been reported in patients with DMD.24 –26 Recent data indicate that mdx mice also show altered colonic motility caused by a derangement of neural coordination of motor activity,27,28 probably nitrergic in nature.28,29 In particular, our previous study28 showed that proximal colon from mdx mice, in contrast to control animals, developed a spontaneous tone increment, detected as an enhancement in intraluminal pressure, but the mechanisms involved in its development have not been investigated. The tone increment could be Abbreviations used in this paper: ATPase, adenosine triphosphatase; CPA, cyclopiazonic acid; DMD, Duchenne muscular dystrophy; EGTA, ethylene glycol-bis(␤-aminoethyl ether)-N,N,N⬘,N⬘-tetraacetic acid; SNP, sodium nitroprussiole. © 2001 by the American Gastroenterological Association 0016-5085/01/$35.00 doi:10.1053/gast.2001.24054

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related to an impairment of calcium homeostasis because it is well known that the principal regulator of the contractile activity in smooth muscle is the Ca2⫹ intracellular concentration, which in turn is determined by the sarcoplasmic reticulum on the one hand and fluxes of Ca2⫹ via cell membrane on the other. The present study was undertaken to verify if an altered Ca2⫹ handling is responsible for the increased tone observed in mdx colon. Specifically, we wished to determine if dysregulation of Ca2⫹ homeostasis is a consequence of changes which occur at the cell membrane level and/or at the sarcoplasmic reticular Ca2⫹-ATPase. To investigate alterations of the cell membrane, we compared the effects of nifedipine and pinaverium, blockers of voltage-operated calcium channels, on the contractile activity of control and mdx mice. The role of sarcoplasmic reticular Ca2⫹ stores was evaluated using the specific sarcoplasmic reticular Ca2⫹-ATPase inhibitor cyclopiazonic acid (CPA).30

Materials and Methods Experiments were authorized by the Ministero della Sanita` (Rome, Italy). Adult (12–18-month-old) male control (C57BL/10SnJ) and mdx (C57BL/10Sn-Dmd/J) mice were used. The animals were killed by cervical dislocation, the abdomens were immediately opened, and segments of proximal colon (about 2 cm long) were removed just distal to the cecum. The contents of the excised segments were gently flushed out with Krebs solution. Colonic segments were mounted horizontally in a custom-designed organ bath continuously perfused with oxygenated (95% O2 and 5% CO2) and heated (37°C) Krebs solution with the following composition: NaCl, 119 mmol/L; KCl, 4.5 mmol/L; MgSO4 , 2.5 mmol/L; NaHCO3 , 25 mmol/L; KH2PO4 , 1.2 mmol/L; CaCl2 , 2.5 mmol/L; glucose, 11.1 mmol/L.

Recording of Mechanical Activity As described previously,28 the distal end of each segment was tied around the mouth of a J-tube connected to a pressure transducer (Statham model P23XL, Grass Instruments Co., Quincy, MA). The ligated proximal end was secured with a silk thread to an isometric force transducer (Grass FT03; Grass Instruments) to preload the preparations of 0.5 g. The preparations were allowed to equilibrate for at least 30 minutes. The mechanical activity was detected as changes of intraluminal pressure, which are mainly generated by circular muscle, and recorded on ink-writer polygraph (Grass model 7D). At the end of the equilibration period, the preparations were challenged with sodium nitroprusside (100 ␮mol/L) to verify if they were able to relax.

Role of Extracellular Calcium We investigated the effect of Ca2⫹-free solution on the mechanical activity of both preparations. For these experi-

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ments, Krebs was prepared with the same composition described previously, but CaCl2 was omitted and 100 ␮mmol/L ethylene glycol-bis(␤-aminoethyl ether)-N,N,N⬘,N⬘-tetraacetic acid (EGTA) was added. We also determined the effects of blocking the voltage-dependent Ca2⫹-channels with nifedipine or pinaverium. Nifedipine (1 nmol/L to 1 ␮mol/L) or pinaverium (1–10 ␮mol/L) were tested in progressively increasing concentrations, which were introduced to the Krebs reservoir and superfused for at least 25–30 minutes. The experiments using nifedipine were conducted in a darkened laboratory because of its photosensitive nature.

Experiments Using CPA Experiments using the sarcoplasmic reticulum-specific Ca2⫹-ATPase inhibitor, CPA, were designed to determine if alterations in the intracellular handling of calcium occur in mdx colonic muscle. In a first series of experiments, the effects of perfusion with CPA on normal and dystrophic colonic segments were determined both in control conditions and after incubation in Ca2⫹-free solution. In a second series of experiments, the ability of CPA to block the repletion of intracellular store was evaluated. After the equilibration period, tissues were challenged with 10 ␮mol/L carbachol in normal Krebs. All subsequent responses were expressed as a percentage of this contraction, measured at its maximum. Tissues were washed and incubated in Ca2⫹-free Krebs solution for approximately 15 minutes. Carbachol was then added to the bath. Using the terminology of Low et al.,31 the first contraction in Ca2⫹-free solution was referred to as “release” and was considered indicative of the amount of Ca2⫹ uptake into the storage sites. Tissue exposure to carbachol was maintained until the contraction returned to or near its original baseline. Subsequent exposures to carbachol were repeated until carbachol had no contractile effect to obtain the depletion of carbachol-releasable intracellular calcium store. Repletion of the intracellular calcium stores was accomplished by incubating the tissues in normal Krebs for 30 minutes in either the absence or presence of 10 ␮mol/L CPA. Subsequent stimulation of the tissues with carbachol in Ca2⫹-free solution was used to assess the effectiveness of the refilling process and was referred to as “repletion.”

Data Analysis and Statistical Tests The spontaneous mechanical activity was evaluated as tone and amplitude of the high contractile peaks of intraluminal pressure. The tone was taken to be the difference between the pressure observed at the beginning of the experiment and the new stable level reached at the end of the equilibration time and was referred to as spontaneous tone increment. The previously described additional low contractions, present only in mdx colon, were not considered.28 All data are expressed as mean values ⫾ SEM. Statistical analysis was performed by means of the Student t test or analysis of variance when appropriate. A P value of ⬍0.05 was considered significant.

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Drugs The following drugs were used: nifedipine, CPA, carbachol, sodium nitroprusside (SNP), and EGTA, all purchased from Sigma Chemical Corp. (St. Louis, MO); and pinaverium bromide, a kind gift from Solvay Pharma (Suresnes, France). Nifedipine was dissolved in 70% ethanol, whereas CPA in dimethyl sulfoxide. The final concentration of dimethyl sulfoxide never exceeded 0.3%. Experiments using the solvent alone showed that none had effects on the tissue.

Results As previously described,28 segments of proximal colon from control or mdx mice showed spontaneous mechanical activity, consisting of phasic changes of endoluminal pressure (control animals, 10 ⫾ 4 cm H2O in amplitude and 1.9 ⫾ 0.4 cpm in frequency, n ⫽ 12; mdx animals, 11.2 ⫾ 3.7 cm H2O in amplitude and 1.7 ⫾ 0.5 cpm in frequency, n ⫽ 12; P ⬎ 0.05). Colonic segments from mdx mice, once mounted in the organ bath, developed spontaneous tone increment, detectable as an increase in the recording baseline. This increase reached a stable level of approximately 3– 4 cm H2O within 20 minutes (Figure 1). On the contrary, colonic segments from normal animals did not develop any spontaneous tone increment. However, they were able to relax in response to 100 ␮mol/L SNP (3.8 ⫾ 0.5 cm H2O, n ⫽ 12). The relaxation caused by 100 ␮mol/L SNP in mdx colon was much more consistent (6.9 ⫾ 0.6 cm H2O, n ⫽ 12; P ⬍ 0.05).

Figure 2. Time-dependent effects of Ca2⫹-free Krebs on the amplitude of the spontaneous contractions in colonic segments from control or mdx mice. (A ) Typical intraluminal pressure recordings before and 5 and 15 minutes after omission of Ca2⫹ from external solution. (B) Graphical representation of the mean amplitude of spontaneous contractions measured at different times. The contractions of normal colon were abolished within 15 minutes, and those of mdx colon were abolished within 25 minutes. Data are expressed as a percentage of the amplitude of the spontaneous contractions. All values are means ⫾ SEM of 7 experiments.

Effect of Ca2ⴙ-Free Solution Ca2⫹-free Krebs solution gradually decreased the amplitude of spontaneous contractions. The contractions from normal colon decreased in amplitude more rapidly than that of mdx colon and were abolished within 10 minutes (Figure 2). In both preparations, spontaneous mechanical activity returned to the control values 10 –15 minutes after restoration of normal Krebs solution. Moreover, omission of Ca2⫹ from external solution induced a decrease in the tone increment (⫺2.9 ⫾ 0.5 cm H2O, n ⫽ 7) of the preparations from mdx mice but did

Figure 1. Typical tracings showing the development of active tone only in the isolated colonic segments from dystrophic mouse.

not affect the spontaneous tone of control animals. In these experimental conditions, 100 ␮mol/L SNP was still able to induce relaxation in both tissues (data not shown). Effect of Ca2ⴙ Channel Blockers Nifedipine (3 nmol/L to 1 ␮mol/L) reduced the amplitude of the spontaneous contractions up to complete suppression in both control and mdx mice. These results are summarized in Figure 3. Mdx colon was significantly more sensitive to nifedipine at each concentration used. The 50% inhibitory concentration for nifedipine was 10 ⫾ 4 nmol/L (n ⫽ 6) in controls and 3 ⫾ 1 nmol/L (n ⫽ 6) in mdx (P ⬍ 0.05). Moreover, nifedipine depressed the muscle spontaneous tone increment of the mdx colon without affecting the spontaneous tone of the control preparations. In the presence of nifedipine (1 ␮mol/L), Ca2⫹-free solution did not cause any further decrease in muscle tone (Figure 4). Similar results were obtained using pinaverium, which has no voltage dependence in block-

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Figure 4. Effects of nifedipine and/or Ca2⫹-free Krebs on the spontaneous tone of colonic segments from mdx mice. Nifedipine as well as omission of Ca2⫹ from extracellular medium reduced the mechanical tone. Nifedipine and Ca2⫹-free Krebs did not cause any additive effects. All values are means ⫾ SEM of 5–7 experiments.

Figure 3. Inhibitory effects of nifedipine on the spontaneous contractions of colonic segments from control or mdx mice. (A ) Typical intraluminal pressure recordings before and after administration of nifedipine (0.1 ␮mol/L). (B) Graphical representation of the mean amplitude of spontaneous contractions measured in the presence of the different concentrations of nifedipine. The amplitude of the spontaneous contractions before addition of nifedipine was taken as 100%. The decrease in the amplitude of contractions of colon from mdx mice was significantly greater than that of control animals at every concentration of nifedipine, except at 1 ␮mol/L, which abolished all phasic activity. All values are means ⫾ SEM of 6 experiments. *P ⬍ 0.05.

ing L-type calcium channels. In fact, in colon from control animals, pinaverium (1 ␮mol/L) significantly depressed to 57% ⫾ 8 % (n ⫽ 4; P ⬍ 0.05) the amplitude of spontaneous contractions, which were abolished at 10 ␮mol/L. No effect on the spontaneous tone was observed. In mdx colon, 1 ␮mol/L pinaverium was able to abolish the spontaneous contractions. In addition, it caused a reduction in the spontaneous tone increment (⫺3.1 ⫾ 0.7 cm H2O, n ⫽ 4). Effects of CPA With and Without Calcium In control preparations, application of CPA (10 ␮mol/L) caused an initial contractile effect, consisting of a slow transient increase in muscle tone and large spontaneous contractions. After CPA application, the phasic activity decreased progressively in amplitude and disappeared within 15 minutes (Figure 5). The effect produced by CPA was reversed by washing the preparation. Also in mdx proximal colon, CPA (10 ␮mol/L) produced

Figure 5. Time course of the effect of CPA (10 ␮mol/L) on the amplitude of spontaneous contractions of colonic segments from control or mdx mice. (A ) Typical intraluminal pressure recordings before and after 20 minutes of CPA administration. (B) Graphical representation of the mean amplitude of spontaneous contractions measured at different times from the beginning of perfusion with CPA. After an early increase in amplitude of the pressure waves in both preparations, CPA abolished the contractions only in the control preparations. All values are means ⫾ SEM of 5 experiments and are expressed as percentages of the amplitude of the spontaneous contractions.

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a phasic increase in the tone as well as in the amplitude and frequency of spontaneous mechanical activity. However, a relatively slow fading of the spontaneous contractions was observed, and their amplitude equilibrated at a new level, lower than the control activity. Abolition of spontaneous contractions was never observed (Figure 5). When these experiments were performed in Ca2⫹-free solution, a condition in which the contractions were already abolished, CPA (10 ␮mol/L) did not induce any further effect on the mechanical activity of normal or mdx colonic segments. Evaluation of Intracellular Calcium Store Repletion Finally, the contraction evoked by the addition of carbachol (10 ␮mol/L) in the 2 types of preparations was considered indicative of the amount of Ca2⫹ uptake into the storage sites. In Ca2⫹-free solution, carbachol induced a contraction that was termed “release” because it is thought to be caused by release of Ca2⫹ from sarcoplasmic reticulum. Mdx preparations had a significantly greater release contraction than control (87.3% ⫾ 7.4% for mdx vs. 58.4% ⫾ 9.9% for control; Figure 6). Moreover, a second application of carbachol had virtually no contractile effect on colon from normal mice but was still able to induce an appreciable contraction in colon from mdx mice. Therefore, in mdx mice, more applications of carbachol were necessary to deplete the calcium store. After the functional depletion of intracellular calcium stores, preparations were allowed to replete intracellular stores in the absence and presence of CPA (10 ␮mol/L) for 30 minutes in normal Krebs. The subsequent contraction induced by carbachol in Ca2⫹-free solution was termed repletion (Figure 6). There was no significant difference in the repletion contraction between control and mdx colonic segments (54.15% ⫾ 8.3% for control vs. 54.8% ⫾ 13.1%; P ⬎ 0.05). The presence of CPA (10 ␮mol/L) during the repletion period significantly reduced but did not abolish the ability of both control and mdx colon to replete calcium stores (Figure 6).

Discussion The pathology of dystrophin-deprived smooth muscles has received limited attention. Recent studies in mdx mice have shown that muscular dystrophy may modify the motor function of the large intestine,27–29 and alteration of endogenous nitric oxide could explain the different motor pattern observed in mdx colon.28,29 In the present study, we found that adult mdx colonic smooth muscle shows an alteration in the homeostasis of intracellular calcium, which appears to be the consequence of

Figure 6. Effects of CPA on the contraction induced by carbachol (10 ␮mol/L) in colon from control or mdx mice. (A ) Typical tracings showing the effect of carbachol in different experimental conditions. Arrowheads indicate carbachol application. (B) Graphical representation of the mean amplitude of contractions induced by carbachol in different experimental conditions. Original refers to the contraction in normal Krebs. Release refers to the first contraction induced by carbachol (10 ␮mol/L) in Ca2⫹-free solution and is indicative of the carbachol-releasable intracellular calcium store. The release contraction of mdx colon was significantly greater (P ⬍ 0.01) than that of control, suggesting that a larger intracellular calcium store exists in mdx colon. Repletion refers to the carbachol-induced contraction after tissues previously depleted of calcium were allowed to replete intracellular calcium stores for 30 minutes in normal Krebs without or with CPA (10 ␮mol/L). The repletion contraction was recorded in Ca2⫹-free solution and thus is caused by intracellular release of calcium. There was no significant difference (P ⬎ 0.05) in the repletion contraction between control and mdx colon. The presence of CPA during the 30-minute repletion period significantly reduced the ability of both control and mdx colon to replete calcium stores. All values are means ⫾ SEM of 6 experiments and are expressed as percentages of the contraction induced by carbachol (10 ␮mol/L) in normal Krebs. The effect of carbachol was measured as maximum contraction. *Significantly different from original contraction. **Significantly different from release contraction of mdx colon. ***Significantly different from the repletion contraction.

an increased influx of calcium through the cell membrane. Under control conditions, colonic segments from C57 or mdx mice had rhythmic contractile activity. In both preparations, this spontaneous activity depends primarily on calcium influx through the L-type calcium channels because it can be completely blocked by nifedipine or removal of calcium from the extracellular solution. In contrast to control animals, colonic segments from mdx mouse developed an extra spontaneous tone increment,

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which may be related to impairment of calcium homeostasis. In fact, in resting muscles the tone depends on the steady-state value of calcium intracellular concentration, which in turn is the net result of influxes, sequestration, and extrusion of calcium. The observation that the omission of Ca2⫹ from the extracellular medium induced a reduction in the spontaneous tone increment in mdx preparations suggests that increased influx of Ca2⫹ through the sarcolemma is responsible for the sustained mechanical tone in mdx colonic muscle. However, because wild-type and mdx colon relax in response to SNP, even in Ca2⫹-free solution, both tissues show a tone that does not depend on extracellular Ca2⫹. The issue of whether membrane permeability for calcium is increased in muscular fibers of mdx mice is still debated.32 Boland et al.33 have shown that the homeostasis of the intracellular calcium concentration is normal in the mdx smooth muscle. However, they considered only smooth muscle of vas deferens. In a subsequent study, the same investigators found no morphologic modification in smooth muscle from urogenital layers but alterations in the digestive layers.34 Therefore, a possible involvement of the intestinal smooth muscle in dystrophy is related to the frequent and prolonged muscle activity. In skeletal muscle, it is not clear whether the excess of calcium enters the fibers via transient membrane lesions,5,35 through ion channels,12,14,15,17 or via both pathways. In our preparation, the excess of calcium seems to involve L-type voltage-sensitive Ca2⫹ channels because nifedipine, known to block voltage-dependent Ca2⫹ channels, reduced the mechanical tone only in mdx colon. On the other hand, Ca2⫹-free solution in the presence of nifedipine did not cause any additive effects, indicating that the increased influx of Ca2⫹ is through voltagesensitive channels. This is in contrast with our knowledge on the dystrophin-deficient skeletal muscle because the activity of voltage-sensitive channels does not appear to contribute to elevated Ca2⫹ influx.7 However, the intestinal smooth muscle works in a different way than the skeletal muscle. In fact, the opening of these channels is the consequence of a depolarization of smooth muscle cells. Intracellular electrical recordings performed in our laboratory have showed that circular muscle from mdx proximal colon has a membrane potential more depolarized than that observed in colon from normal mice.36 This depolarization could explain the opening of the Ca2⫹ channels. Mdx colon spontaneous contractions were significantly more sensitive to nifedipine than control preparations. Because nifedipine displays voltage-dependent blocking on smooth muscle cells and is more effective at blocking L-type calcium channels at more positive

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membrane potentials, we also tested pinaverium, which is reported to show no voltage dependence in the blocking of L-type calcium channels.37 Mdx colon was also more sensitive than control to pinaverium, indicating that calcium entry into the cell through L-type calcium channels plays a major role in spontaneous contractions of colon from mdx mouse. In mdx skeletal fibers, the mechanisms for sequestering calcium may be impaired38 and secondary alterations in the sarcoplasmic reticulum Ca2⫹ pump can contribute to abnormal Ca2⫹ handling.39 However, Martonosi40 reported that the sarcoplasmic reticulum Ca2⫹-ATPase is unaltered in dystrophic muscle. Therefore, experiments using the sarcoplasmic reticulum Ca2⫹-ATPase–specific inhibitor, CPA,30 were performed to determine if alterations at the sarcoplasmic reticulum level also occur in mdx colonic preparations. It has been reported that CPA is capable of selectively inhibiting the ATP-dependent Ca2⫹ uptake into intestinal smooth muscle sarcoplasmic reticulum41 and depleting Ca2⫹ stores.42 In the present study, CPA abolished the spontaneous contractions in normal colon. It is conceivable that, as reported for murine jejunum,43 the rhythmic contractions depend on influx of calcium, which secondarily triggers calcium release. Moreover, CPA caused an early increase in tone with large spontaneous contractions. This effect may be consistent with the hypothesis that Ca2⫹ pump inhibition may lead to a transient accumulation of spontaneously released calcium. However, the early contractile effect was not observed in the presence of Ca2⫹-free solution, suggesting that as in other muscle intestinal preparations,44,45 depletion of intracellular calcium stores can activate calcium influx at the cell membrane. In any case, in normal colon the Ca2⫹ influx through sarcolemma by itself is not enough to sustain the spontaneous mechanical activity. In mdx colon, the abolition of spontaneous rhythmic activity caused by CPA was not observed. This could mean that there are differences at the sarcoplasmic reticulum level between the 2 tissues or that in dystrophic conditions, the influx of extracellular calcium is enough to be able to directly sustain the spontaneous activity. Therefore, we evaluated the functionality of sarcoplasmic reticulum calcium pump using CPA. In Ca2⫹-free solution, carbachol induced a significantly smaller contraction (“release” contraction) in colon from normal mice than in mdx colon, and a single application was usually sufficient to deplete the calcium stores, whereas more applications of carbachol were necessary in mdx mice. These results suggest that mdx colonic smooth muscle contains a larger carbachol-releasable intracellular calcium store. This could mean that in

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mdx fibers, the Ca2⫹-sequestering mechanisms must be coping with an overload. The activity of the calcium sequestration mechanisms could be increased as a result of the elevated resting intracellular concentration of calcium in dystrophic muscle. However, there was no difference in the repletion response or in the ability of CPA to block the repletion in the 2 types of tissue. These observations argue against an impairment of the sarcoplasmic reticulum Ca2⫹ pump in dystrophic smooth muscle and suggest that a defect in calcium resequestration does not contribute to the increased tone generation in mdx colon. Moreover, CPA did not abolish the repletion in either tissue. This finding might be explained assuming the existence of a CPA-insensitive Ca2⫹ store, as hypothesized for other digestive smooth muscles.46 In conclusion, this is the first study to provide evidence of an altered homeostasis of calcium in intestinal smooth muscle from mdx mice. In dystrophic colonic muscle, increased Ca2⫹ influx through L-type voltagedependent channels is large enough to overcome the mechanisms that normally keep the Ca2⫹ intracellular concentration very low and is responsible for the sustained mechanical tone. The mechanisms for sequestering calcium apparently are unaltered, although a larger carbachol-releasable intracellular calcium store exists in mdx colon.

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Received April 18, 2000. Accepted January 24, 2001. Address requests for reprints to: Flavia Mule `, Dipartimento di Biologia cellulare e dello Sviluppo, Laboratorio di Fisiologia generale, Universita ` di Palermo, Viale delle Scienze, 90128 Palermo, Italy. e-mail: [email protected]; fax: (39) 91-6577501. Supported by grant 1134 from Comitato Telethon Fondazione ONLUS-Italy.