Calcium source diversity in feline lower esophageal sphincter circular and sling muscle

Ahmad Muinuddin,2,3 Leila Neshatian,2,3 Herbert Y. Gaisano,1,3 and Nicholas E. Diamant1,2,3

Departments of 1Medicine and 2Physiology, University of Toronto, Toronto, Ontario, M5S 1A8; and 3Toronto Western Research Institute, University Health Network, Toronto, Ontario, Canada M5T 2S8

Submitted 10 July 2003 ; accepted in final form 20 September 2003


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Within muscular equivalents of cat lower esophageal sphincter (LES), the circular muscle develops greater spontaneous tone, whereas the sling muscle is more responsive to cholinergic stimulation. Smooth muscle contraction involves a combination of calcium release from stores and of calcium entry via several pathways. We hypothesized that there are differences in the sources of Ca2+ used for contraction in sling and circular muscles and that these differences could contribute to functional asymmetry observed within LES. Contraction of muscle strips from circular and sling regions of LES was assessed in the presence of TTX. In Ca2+-free Krebs, tone was inhibited to a greater degree in circular than sling muscle. L-type Ca2+ channel blockade with nifedipine or verapamil inhibited tone in LES circular but not sling muscle. Sarcoplasmic reticulum (SR) Ca2+-ATPase inhibitor cyclopiazonic acid (CPA) caused greater increase in tone in sling than in circular muscle. The phospholipase C inhibitor U-73122 and the SR inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] receptor blocker 2-aminoethoxydiphenyl borate (2-APB) inhibited tone in circular and sling muscles, demonstrating that continuous release of Ca2+ from Ins(1,4,5)P3-sensitive stores is important in tone generation in both muscles. In Ca2+-free Krebs, ACh-induced contractions (AChC) were inhibited to a greater degree in sling than circular muscles. However, nifedipine and verapamil greatly inhibited AChC in the circular but not sling muscle. Depletion of SR Ca2+ stores with CPA or inhibition of Ins(1,4,5)P3-mediated store release with either U-73122 or 2-APB inhibited AChC in both muscles. We demonstrate that LES circular and sling muscles 1) use intracellular and extracellular Ca2+ sources to different degrees in the generation of spontaneous tone and AChC and 2) use different Ca2+ entry pathways. These differences hold the potential for selective modulation of LES tone in health and disease.

acetylcholine; tone; contractility; calcium handling; smooth muscle


THE LOWER ESOPHAGEAL SPHINCTER (LES) creates a zone of high pressure that prevents the reflux of gastric contents into the esophagus. Control of LES tone is essential for nutrition and health. Differences in the smooth muscle properties between the LES and esophageal body (EB) are numerous and include differences in calcium handling (3), signal transduction pathways (2, 31, 30), contractile protein isoforms (28), cholinergic sensitivity (17, 20), and length-tension relations (17, 19, 20). However, less is known about regional specialization within the gastroesophageal junction.

The LES is composed of two main muscular equivalents-the circular smooth muscle and the oblique sling fibers. In the human, the circular fibers form only a partial ring or "clasp" (14), whereas in other species such as the dog (10), opossum (6), and cat (4, 10), the LES circular muscle forms a complete ring. Studies of the circular and sling fibers from cat and human LES have demonstrated that the circular muscle has greater spontaneous tone but less cholinergic responsiveness than the sling muscle (19, 20). Like other smooth muscles, the contractile state of the LES depends on the level of intracellular Ca2+ concentration ([Ca2+]i). However, intracellular Ca2+ is not homogeneously distributed within smooth muscle cells. Ca2+ stores, in addition to being a source of Ca2+, also contribute to the sequestration of Ca2+ from the cytosol, and therefore influence the activity of ion channels and calcium-mediated processes. In LES circular muscle of all species studied, muscle tone is supported by ongoing entrance of extracellular Ca2+ through plasmalemmal Ca2+ channels. Adding L-type calcium channel (LCa) blockers or reducing extracellular calcium reduces LES tone in opossum (9), cat (3), dog (1, 23), and human (36). However, in the feline LES circular muscle, some tone persisted in Ca2+-free extracellular solution (3), a finding explained by continuous release of Ca2+ from intracellular Ca2+ stores to maintain LES tone. Biancani et al. (2) have since demonstrated that ongoing activation of phospholipase C (PLC) and production of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] resulted in Ca2+ release from intracellular stores and potentiation of diacylglycerol to activate a PKC-dependent pathway. Taken together, these results suggested that LES tone was dependent on continuous low levels of Ca2+ release from intracellular stores.

Characterization of relative contributions of Ca2+ influx pathways and release from stores is important for understanding the physiological and pathological control of LES smooth muscle. Although some studies have examined the role of calcium sources in LES circular muscle contractility, the role of calcium sources in sling muscle contractility has not yet been investigated. Therefore, the present studies in cat LES circular and sling muscle were performed to assess and compare the relative importance of extracellular calcium pathways and intracellular calcium handling on 1) myogenic tone generation and 2) agonist-induced cholinergic contractions.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Tissue Removal

The experimental protocol was approved by the University Health Network Animal Care Committee. Adult cats of either sex weighing between 2.5 and 5.0 kg were anesthetized with ketamine hydrochloride (Bimeda-MTC, Cambridge, ON, Canada) (0.15 ml/kg im) and euthanized with pentobarbital sodium (0.5 ml/kg iv; Bimeda-MTC). A midline incision was made, and the chest was opened. A length of esophagus from 10 cm above the LES to 4 cm below it was removed and immediately placed in Krebs solution equilibrated with 5% CO2-95% O2 and maintained at pH 7.4 ± 0.05.

Contractility Studies

The specimen was freed of surrounding fascia and stretched to its in situ length. The esophagus was then opened up along the greater curvature, and the mucosa was removed by sharp dissection. Circular muscle and sling fibers of the LES were readily visible (20). Muscle strips measuring 2 mm in width and 10 mm in length were obtained from the oblique sling and circular muscle regions of the LES. A silk thread was tied to each end, and the strips were transferred to 25-ml organ baths containing Krebs solution bubbled with 5% CO2-95% O2 at 37°C and maintained at pH 7.4 ± 0.05. One end of the strip was fixed to an electrode holder and the other end to an isometric force transducer (model FT-03; Grass Instruments, Quincy, MA) coupled to a chart recorder (model 79E; Grass Instruments). The force transducer was supported on a rack-and-pinion clamp (Harvard Apparatus, Holliston, MA), which facilitated accurate length adjustment of the muscle strips. Transmural electrical field stimulation (EFS) was delivered by a Grass SP9 stimulator through platinum wire electrodes placed on either side of the tissue strips. EFS consisted of 0.5-ms square-wave pulses in a 5-s train at 10 Hz and a strength of 50 V. Tissue strips were hung loosely with no tension being applied to them in the organ baths for a 1-h equilibration period before studies began. Each strip was gently stretched to an initial length (L0) that was first determined with a micrometer as the length at which a rapid stretch caused a small transient deflection of the recorder pen (~50 mg of tension) and allowed to equilibrate at L0 for 30 min. At this level of stretch, any slack in the strip or silk ties was eliminated and further stretch began to produce active tension. Muscle strips were then slowly stretched and tested at increments of 25% of their L0 until optimal response to cholinergic stimulation was achieved. TTX was then added to the bath to assess the true myogenic responses. EFS confirmed that neural responses had been abolished by TTX. At the end of all experiments, the strips were blotted on filter paper and the weight was determined. Tension of both phasic and tonic responses was then normalized and expressed as a function of cross-sectional area of the muscle strip in milliNewtons per centimeter squared.

Composition of Solutions

Krebs solution had the following composition (in mM): 115 NaCl, 4.6 KCl, 1.2 MgSO4, 1.2 NaH2PO4, 22 NaHCO3, 2 CaCl2, and 11 glucose. The Ca2+-free medium contained (in mM): 115 NaCl, 4.6 KCl, 1.2 MgSO4, 1.2 NaH2PO4, 22 NaHCO3, 11 glucose, and 0.5 EGTA.

Drugs

BAY K 8644, 2-aminoethoxydiphenyl borate (2-APB), U-73122, cyclopiazonic acid (CPA), nifedipine, ACh, TTX, and all other chemical reagents were obtained from Sigma (St. Louis, MO). BAY K 8644, nifedipine, and CPA were prepared in DMSO with a final DMSO concentration in a solution of 0.1%. TTX and ACh were prepared fresh as a 10 mM stock in double-distilled water. In the concentrations used, separate experiments were carried out to confirm that vehicles had no effect on tone or ACh-induced contractions.

Statistical Analysis

Data are presented as means ± SE, and n is the number of muscle strips studied. When assessing the amplitude of tone, baseline stretch-induced tension was subtracted from the tension measurements. When assessing the amplitude of cholinergic contractions, the initial peak amplitude in response to ACh was used. Student's t-test was used to compare data between two groups (using Instat Graph Pad software version 3). P < 0.05 was considered to be a significant difference.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Over 70 muscle strips were studied from the sling and circular muscle regions of the LES. All muscle strips examined contracted in response to ACh (10 µM), demonstrating that the muscarinic (M3) signaling pathways were intact. In the presence of the Na+ channel blocker TTX (1 µM), EFS-induced responses were abolished, ensuring that the true myogenic responses in these studies were being assessed.

Spontaneous Tone: Role of Extracellular Ca2+

LES circular muscle developed more tone than sling muscle (18.6 ± 2.2 vs. 6.8 ± 1.9 mN/cm2, n = 6 each region, P < 0.05). To determine the role of extracellular calcium in the generation of tone, the following two experimental approaches were utilized: 1) perfusion of muscle strips in Ca2+-free Krebs solution or 2) prevention of extracellular calcium entry in the cytosol by blockade of LCa.

When perfused in Ca2+-free Krebs, muscle strips from the circular smooth muscle region of the LES demonstrate a rapid decrease in tone in a time-dependent fashion (Fig. 1). The decrease in tone was greater and more rapid in circular than sling muscle. For example, when perfused in Ca2+-free Krebs for only 2 min, circular muscle exhibited a 54.6% inhibition of tone, whereas sling muscle tone was decreased by only 5.9%. By t = 10 min, tone had reduced to only 18.3% of control in LES circular muscle, whereas 44.3% of control tone in sling muscle still persisted. Moreover, by this time, the level of tone was similar in both circular and sling tissue (3.6 ± 1.7 vs. 3.8 ± 2.2 mN/cm2, P > 0.05). Subsequent perfusion with calcium-containing Krebs resulted in a return of tone to near control levels.



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Fig. 1. Effect of Ca2+-free Krebs on lower esophageal sphincter (LES) tone. LES circular smooth muscle developed more myogenic tone than sling muscle (*P < 0.05; n = 6 each region). When perfused in Ca2+-free Krebs, muscle strips from both regions of the LES demonstrate a decrease in tone in a time-dependent fashion. Decrease in tone was greater and more rapid in LES circular muscle than LES sling muscle. By t = 10 min exposure to Ca2+-free Krebs, there is no significant difference between tone in these two muscle types.

 

Addition of the dihydropyridine LCa antagonist nifedipine (1 µM) to the organ bath chamber inhibited tone in LES circular but not sling muscle (Fig. 2A). Decrease in tone was great and rapid in the LES circular muscle (36.5 ± 12% of control at time t = 10 min, n = 7 each, P < 0.05) (Fig. 2A). Higher concentrations of nifedipine (10 µM) caused further inhibition of tone in LES circular muscle with slight inhibition of tone in sling muscle. We then examined the effect of the phenylalkamine verapamil, another inhibitor of LCa, on LES tone. Verapamil (5 µM) caused significant inhibition of tone in LES circular muscle but not sling (Fig. 2B). At higher concentrations, verapamil (20 µM) caused slight but significant inhibition of tone in sling muscle (to ~85% of control). The final amplitude of tone in LES circular muscle in the presence of nifedipine (1 µM) or verapamil (5 µM) was not significantly different from sling muscle (Fig. 2).



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Fig. 2. Effect of L-type Ca2+ channel antagonists on LES tone. A: addition of the dihydropyridine nifedipine (1 µM) to the organ bath resulted in inhibition of tone in LES circular muscle but not sling muscle (*P < 0.05; n = 7 each region). B: phenylalkamine verapamil (5 µM) inhibited tone in LES circular muscle but not sling muscle (*P < 0.05; n = 5 each region).

 

Spontaneous Tone: Role of Ca2+ from Internal Stores

To examine the role of intracellular Ca2+ stores in tone generation, two approaches were examined: 1) inhibition of Ca2+ uptake into sarcoplasmic reticulum (SR) and 2) inhibition of Ca2+ release from Ins(1,4,5)P3-sensitive stores. We expected that inhibition of Ca2+ uptake into the SR would result in increased [Ca2+]i available for tonic contraction. Addition to the bath of the SR Ca2+-ATPase inhibitor CPA resulted in a gradual increase in tone in both LES circular and sling regions. After 30 min incubation with CPA (10 µM), the increase in tone had leveled off. CPA caused greater increase in tone in sling than in LES circular muscle (493.1 ± 55 vs. 240 ± 21%, n = 5 each, P < 0.05) (Fig. 3A). However, final tone in tissues after CPA treatment was similar. Spontaneous phasic contractions usually appeared in the presence of CPA in sling muscle but less frequently in LES circular muscle. CPA-induced tone was inhibited by nifedipine in LES circular muscle but not in LES sling muscle. However, phasic contractions in response to CPA in sling muscle were inhibited by nifedipine.



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Fig. 3. Effect of intracellular calcium modulation on LES tone. A: sarcoplasmic reticulum (SR) Ca2+-ATPase inhibitor cyclopiazonic acid (CPA) increased tone in both LES circular and LES sling muscles. CPA (10 µM) caused greater increase in tone in sling than in circular muscle (493.1 ± 55 vs. 240 ± 21%, n = 5 each region). B: SR inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] receptor blocker 3-aminoethoxydiphenyl borate (2-APB; 100 µM) inhibited tone in both regions of the LES (n = 5 each region). C: phospholipase C (PLC) inhibitor U-73122 (10 µM) inhibited tone in both LES circular and sling muscles (n = 4 each region) (*P < 0.05, **P < 0.02).

 

To examine the effect of Ca2+ release from Ins(1,4,5)P3-sensitive calcium stores, two different agents were employed: 1) 2-APB inhibits Ins(1,4,5)P3-dependent Ca2+ release without preventing Ins(1,4,5)P3 binding (15); and 2) the phosphoinositide-specific PLC inhibitor U-73122, which prevents the formation of Ins(1,4,5)P3 (2). 2-APB (100 µM) inhibited tone in LES circular and sling muscle in a time-dependent manner, with maximal inhibition by t = 30 min (Fig. 3B) of 59.3 ± 8.0% of control tone in sling tissue and 50.1 ± 9.4% of control tone in LES circular tissue (n = 5 each region). The PLC inhibitor U-73122 (10 µM) inhibited tone in both LES muscles to a similar degree, with maximal inhibition by t = 40 min (Fig. 3C) of 62.5 ± 7% of control in circular muscle and 55.5 ± 6.4% in sling tissue (n = 4 each region).

ACh-Induced Contractions: Role of Extracellular Ca+2

ACh-induced contractions (AChC) were of greater amplitude in LES sling than in LES circular muscle (14.3 ± 2.1 vs. 3.9 ± 1.4 mN/cm2, n = 5, P < 0.05). In control experiments, addition of ACh to the bath for 30 s at the specified times resulted in contractions of similar amplitude with no evidence of either tissue fatigue or tachyphylaxis. In Ca2+-free Krebs, AChC were inhibited in a time- and challenge-dependent manner in both LES muscles (Fig. 4), with greater inhibition in the sling (97.6 ± 0.2% in sling vs. 82.7 ± 7.0% in LES circular muscle at t = 10 min, n = 5 each region, P < 0.05). To examine the effect of exposure time alone of Ca2+-free Krebs to AChC, a separate set of experiments were performed in which tissues were bathed in Ca2+-free Krebs for exactly 10 min before they were stimulated with ACh. In these experiments, ACh-induced contractions were inhibited in both LES tissues.



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Fig. 4. Effect of Ca2+-free Krebs on ACh-induced contractions in LES. In Ca2+-free Krebs, ACh-induced contractions (AChC) were inhibited in a time- and challenge-dependent manner in both LES sling and LES circular muscles. AChC in sling muscle were inhibited to a greater degree than in LES circular muscle (*P < 0.05, n = 5 each region).

 

Addition of nifedipine (1 µM) to the organ bath chamber caused significant inhibition of AChC in the LES circular smooth muscle (33.5 ± 11.2% of control, n = 6) (Fig. 5A). However, nifedipine (1 µM) did not inhibit AChC in sling muscle. Even when sling muscles were exposed to higher concentrations of nifedipine (10 µM) and for longer durations, no significant inhibition of AChC was observed, although AChC in LES circular muscles were virtually abolished at these higher concentrations. In a separate set of experiments, LCa blockade with verapamil (5 µM) inhibited AChC in LES circular but not sling muscle (n = 5 each region) (Fig. 5B).



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Fig. 5. Effect of L-type calcium channel modulation on AChC. A: nifedipine (1 µM) inhibited AChC in LES circular but not sling muscle (*P < 0.05; n = 6 each region). B: verapamil (5 µM) inhibited AChC in LES circular muscle but not sling (*P < 0.05; n = 5 each region).

 

ACh-Induced Contractions: Role of Ca2+ from Internal Stores

Depletion of SR Ca2+ stores with CPA caused similar inhibition of AChC in both LES circular and sling muscles (37.1 ± 4.2 vs. 43.8 ± 5.2% of control, n = 5 for each region) (Fig. 6A).



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Fig. 6. Effect of intracellular calcium modulation on ACh-induced contractions. A: SR Ca2+-ATPase inhibitor CPA (10 µM) inhibited AChC in both LES circular and sling muscles to a similar degree (*P < 0.05; n = 5 for each region). B: inhibition of Ca2+ release from SR Ca2+ stores with the SR Ins(1,4,5)P3 receptor blocker 2-APB (100 µM) inhibited AChC in LES circular and sling muscles to a similar degree in a dose-dependent manner (*P < 0.05; n = 4 each region). C: PLC inhibitor U-73122 (10 µM) inhibited AChC in both LES circular and sling muscles (*P < 0.05; n = 5 each region).

 

Inhibition of the SR Ins(1,4,5)P3 receptor with 2-APB (100 µM) inhibited AChC in LES circular and sling muscles. There were no significant differences in 2-APB-induced inhibition of AChC in LES circular and sling muscles (32 ± 12 vs. 26 ± 6% of control at 200 µM, n = 4 each) (Fig. 6B). The inhibitory effect of 2-APB was not reversible even with extensive wash-out of the drug.

The PLC inhibitor U-73122 (10 µM) inhibited AChC in both regions of the LES (Fig. 6C). There were no significant differences in inhibition of AChC in LES circular and sling muscles (42 ± 6 vs. 36 ± 7% of control, n = 4 each region).


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The central role of Ca2+ in smooth muscle contraction provides a logical focus for exploring differences in muscle function. In smooth muscle, the control of [Ca2+]i involves at least two integrated membrane systems: 1) the plasmalemma, which is under the control of membrane potential and agonists, and 2) the SR, which is under the control of intracellular second messengers. Although some aspects of calcium handling have been examined in LES circular smooth muscle, to our knowledge no studies have examined the role of calcium in LES sling contractility. Findings of the present study provide evidence that fundamentally important differences exist between these muscle types in the regulation of muscle contractility. Tone is greater in LES circular muscle, whereas cholinergic sensitivity is greater in the sling. Although contraction relies heavily on release of intracellular Ca2+ from the SR, we demonstrate that influx of extracellular calcium plays a central role in the maintenance of myogenic tone and ACh-induced contractility in both LES muscles. This influx occurs through different pathways in LES circular muscle (LCa)] and sling muscle (nifedipine-insensitive pathway). Moreover, prevention of SR store refilling of intracellular calcium triggers different contraction patterns in the LES sling and LES circular muscles, again with different sensitivity to blockade of the LCa.

Calcium Dependence of LES Tone

Extracellular calcium. Early studies (3) of the cat LES circular smooth muscle suggested that spontaneous tonic contraction is supported mainly by release of Ca2+ from intracellular stores. However, the LCa channel blocker nifedipine has also been shown to inhibit tone in the LES circular muscle of several species including opossum (9), dog (23), and human (36). Hence the relative contribution of calcium sources to tone generation remains unclear. In this study, we demonstrate the central importance of extracellular calcium sources in tone generation. Perfusion of LES circular muscle strips in Ca2+-free Krebs results in rapid and dramatic reduction in tone. Moreover, nifedipine and verapamil, two different but specific LCa channel antagonists, inhibited tone in LES circular muscle. Therefore, basal feline LES tone due to the circular muscle fibers relies on a continuous supply of Ca2+ from the extracellular solution, the Ca2+ entry mainly via an LCa.

Unlike the LES circular muscle, we found that extracellular calcium contributes to myogenic tone to a much lesser degree in sling muscle. When perfused in Ca2+-free Krebs, muscle tone in the sling showed gradual and partial inhibition, demonstrating a role for extracellular calcium in maintenance of tone in sling muscle, although not as much as in the LES circular muscle. Surprisingly, neither nifedipine or verapamil significantly inhibited tone in LES sling muscle, suggesting that the limited influx of extracellular calcium for tone generation occurs primarily via a non-LCa. Several pathways for calcium entrance are possible in smooth muscle and could contribute to tone as well as agonist-induced contraction. These include a T-type Ca2+ channel (26), capacitative Ca2+ entry (CCE) (37), and other sarcolemmal Ca2+ channels, such as receptor-operated calcium channels (ROC) and nonselective cation channels (24). We did not explore these pathways or their role in generating LES muscle tone.

Intracellular calcium. To examine the contribution of intracellular stores to LES tone generation, we examined the effect of depletion of Ca2+ stores in the SR and inhibition of Ca2+ release from the SR stores via Ins(1,4,5)P3 receptors. In smooth muscles, the mycotoxin CPA, an indole tetramic acid metabolite of Aspergillus and Penicillium, prevents calcium refilling of SR stores by inhibiting the SR Ca2+-ATPase pump activity, leading to rapid store depletion of calcium via nonspecific leakage (due to diffusion) and/or release of calcium from Ins(1,4,5)P3 and ryanodine receptors (5, 7). In the present studies, depletion of Ca2+ from SR stores with CPA did not cause a reduction in tone. In fact, CPA enhanced LES tone as expected, if less influxing Ca2+ was sequestered in the CPA-sensitive stores, allowing higher [Ca2+]i levels near the contractile proteins. It is of interest that there was a significant regional difference in CPA-induced tone within the LES. LES sling muscle developed more tension and demonstrated a greater increase in tone than LES circular muscle. CPA did cause phasic contractions in sling muscle as well. CPA-induced sustained tonic contractions were inhibited by nifedipine in LES circular muscle but not sling muscle. Although nifedipine did not inhibit CPA-induced tonic contraction in sling muscle, nifedipine inhibited CPA-induced phasic contractions in this muscle. These results in the LES circular muscle are consistent with those reported in the circular muscle of the canine LES (23) and feline gastric antrum muscles (18) in which CPA caused an increase in tone. A large part of this contraction was mediated by Ca2+ influx via LCa. Regional differences observed in CPA-induced tone suggest that the Ca2+-ATPase pump may be more active and/or present in greater number in the sling muscle; however, our experiments were not designed to test this hypothesis. Alternatively, the contractile machinery may be more responsive to an increase in Ca2+ in the sling muscle. Again the difference in nifedipine sensitivity shows that Ca2+ being released from the SR is replenished via an LCa in the LES circular muscle and a nifedipine-insensitive pathway in the LES sling.

Inhibition of Ca2+ release from Ins(1,4,5)P3-sensitive SR stores with either the Ins(1,4,5)P3 receptor antagonist 2-APB or the PLC antagonist U-73122 resulted in significant inhibition of tone in both LES muscles, supporting the concept that release of calcium from SR stores is also important for spontaneous tone generation. Regional differences in the contribution of calcium release from SR stores in both muscles was not apparent by using these methodologies.

Despite the fact that a defining characteristic of LES smooth muscle is the development of sustained tonic contraction, resting [Ca2+]i was not significantly different in SMCs from the tonically contracted LES circular muscle when compared with relaxed muscle from the EB (37). Whether or not there are differences in resting [Ca2+]i between the LES circular and sling muscles is yet to be determined. One alternative to tonically elevated [Ca2+]i in LES is enhanced sensitivity of LES contractile elements to [Ca2+]i. However, when EB circular smooth muscle was compared with LES circular smooth muscle in cats, both muscles shared contractile systems and similar dependence of maximal shortening velocity and myosin light-chain phosphorylation (38). In this and in other measures, the two muscles were similar in the contractile mechanisms. On the other hand, differences in the contractile proteins in phasic and tonic smooth muscles of the opossum EB and LES, respectively, have been described (35). Whether differences exist between LES sling and LES circular muscles has not been studied. It is also possible that there is a heterogeneous distribution of calcium in the LES such that there is more calcium near or accessible to contractile proteins in the sling. Further studies utilizing high-resolution calcium imaging technology could help to resolve many of these questions.

Taken together, these findings are consistent with the view that tone in LES circular muscle is due to continuous release of Ca2+ from the Ins(1,4,5)P3-mediated stores of the SR via a PLC-dependent pathway, which in turn are replenished by continuous influx of extracellular Ca2+ into the smooth muscle primarily via LCa. Calcium released from SR stores is then available for contraction via a PKC-dependent pathway (12, 32). Continuous refilling of SR Ca2+ stores is essential to maintaining tone in LES circular muscle and occurs via LCa. Low intrinsic tone in the sling is also likely due to a combination of a small continuous release of Ca2+ from Ins(1,4,5)P3-sensitive stores and little entry of extracellular calcium. In this case, the low level of tone is less dependent on extracellular Ca2+, and the Ca2+ enters by other than an LCa.

Calcium Dependence of and Sources for Cholinergic Contractions

Extracellular calcium. Our results show significant inhibition of AChC in Ca2+-free Krebs in both LES circular and sling muscle, demonstrating the central role of extracellular calcium in cholinergic contractility. In LES circular muscle, this extracellular calcium is entering primarily via LCa channels and in LES sling enters via non-LCa influx pathway(s). Similar nifedipine sensitivity as well as reduction of contraction amplitude in Ca2+-free medium was seen in circular muscle strips of the dog LES (23). These authors also postulated the presence of a special extracellular calcium source located near the plasma membrane from which calcium enters through LCa channels when a cholinergic contraction is induced in the absence of extracellular calcium. On the other hand, some studies (11) in isolated cells have reported that calcium-free medium did not reduce AChC in isolated cells from the cat LES circular muscle. The reason for these differences is not clear. Perhaps the isolation of cells alters a calcium source located near the plasma membrane in intact muscle tissue. It is also possible that calcium requirements of muscle strips may be different from isolated cells due to the syncitial nature of the tissue. In airway smooth muscles, others have noted similar significant differences between calcium handling in whole tissues and isolated cell preparations (13). Hence caution must be exercised when comparing data not only from different species and tissue regions, but also from different levels of tissue structure.

As opposed to the reduction in contraction we observed in LES circular smooth muscle, AChC were not significantly inhibited by either nifedipine or verapamil in the sling, even at high doses. Therefore, as with tone, the cholinergic contraction in the sling muscle also relies less on entry of calcium through the LCa and uses primarily nifedipine-insensitive pathways. To what extent the decreased expression of LCa in sling muscle is associated with this difference in tone and cholinergic contraction between LES circular and sling muscles requires further study (16). The T-type Ca2+ channel, previously identified in cat esophageal smooth muscle (27), is rapidly inactivated, and it is unlikely that it can be responsible for sustained agonist-induced contractions. Another calcium entry pathway that has received considerable recent attention is CCE (21). CCE involves the regulation of plasma membrane Ca2+ channels by the filling state of IC Ca2+ stores in the SR. The source of agonist-mediated increase in IC Ca2+ is predominantly the Ca2+ released from intracellular stores. Depletion of the stores triggers CCE and thus promotes Ca2+ influx. In this model, other sarcolemmal Ca2+ channels, ROC and nonselective cation channels, are also involved in this agonist-induced influx. CCE and the expression of transient receptor potential (TRP) channels have recently been demonstratred in human esophageal circular smooth muscles from the EB and LES (37). Whether TRP or other nonselective cation channel currents allow for calcium entry in LES smooth muscles has yet to be determined. One difficulty in determining whether TRP channels are involved in CCE is the lack of specific inhibitors of these channels.

Intracellular calcium. AChC in both LES circular and sling muscles were dependent on Ca2+ influx as well as SR function. Both depletion of intracellular SR Ca2+ stores and inhibition of Ca2+ release from SR stores resulted in inhibition of AChC in the LES, demonstrating the important role of calcium release from SR stores. These results are consistent with studies in the circular muscle of the dog LES (23) and cat LES (11) where similar importance of calcium release from intracellular stores has been demonstrated. There are no previous studies of this release in sling muscle.

We expected that a greater AChC in the sling region of the LES might be due to greater release of calcium from intracellular stores. However, inhibition of Ca2+ release from the SR caused similar and marked inhibition of contraction in both LES muscles. As noted before, CPA caused greater increase in tone in sling muscle. Although this finding may suggest that the SR of the sling can store more Ca2+ for release on stimulation, alternate explanations for the finding do exist. It is of interest that although CPA caused an increase in tonic contraction in both tissues presumably by allowing accumulation of [Ca2+]i near contractile proteins, CPA inhibited AChC, demonstrating that release of Ca2+ from Ins(1,4,5)P3-sensitive stores is of prime importance for this contraction. The release mechanism is presumably activated via the M3 signaling pathway.

The fact that significant amplitude and area under the contraction remained after CPA treatment suggests that CPA-insensitive Ca2+ stores may exist. Refilling of these CPA-insensitive stores could occur via a leak channel, or a specialized pathway could exist from the plasma membrane to the SR.

Implications of these regional differences are of considerable interest. A greater LCa in the LES circular muscle region would be consistent with these muscles using this channel predominantly in some way to maintain the elevated level of myogenic tone. A lower LCa current in the sling region could indicate that smooth muscle from this region may have a greater reliance on release of calcium from intracellular Ca2+ stores for contraction as is seen in many smooth muscles (33). In this case, the modulation of the level of contraction through the M3 receptor-Ins(1,4,5)P3 link for release of calcium from the SR would provide a mechanism for greater neural excitatory cholinergic control of the LES. Because the LES pressure profile in vivo shows a higher pressure in the left lateral-posterior aspect in both the human (22, 25, 34) and the cat (20), this aspect being most sensitive to atropine, it is likely that the sling, in addition to the LES circular muscle, is an integral and important physical and physiological contributor to the LES. In the case of the sling, the neural cholinergic control becomes of more importance and gives credence to the use of cholinergic agonists in raising LES pressure in patients with gastroesophageal reflux disease (GERD) (8). Clinically, Schneider et al. (25) raised the importance of regional LES differences in patients with achalasia, and similar attention has been paid to the two muscle regions and their potential role in the pathogenesis of GERD (34). There is, therefore, good reason to further explore regional differences in calcium handling in health and disease in the search for more specific therapeutic targets.

In conclusion, in LES sling and circular smooth muscle, intracellular and extracellular Ca2+ sources are utilized to different degrees in the generation of spontaneous tone and ACh-induced contractions. Roles of these differences in the pathogenesis and treatment of disease states requires further study.


    ACKNOWLEDGMENTS
 
GRANTS

This work was supported by Grant MOP36499 from the Canadian Institutes of Health Research (to N. E. Diamant and H. Y. Gaisano). A. Muinuddin was supported by a Lipton Neurosciences Fellowship and a L'Anson Scholarship.


    FOOTNOTES
 

Address for reprint requests and other correspondence: N. E. Diamant, 12-419 McLaughlin Pavilion, Toronto Western Hospital, 399 Bathurst St. Toronto, ON, Canada M5T 2S8.

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.


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