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Peptides do not induce contractions in gastrointestinal smooth muscle in calcium-free solution
Gen. Pharmac. Vol. 22, No. 6, pp. 1135-1137,1991 Printed in Great Britain.All fights reserved
0306-3623/91 $3.00+ 0.00 Copyright © 1991PergamonPresspie
PEPTIDES DO NOT I N D U C E CONTRACTIONS IN GASTROINTESTINAL SMOOTH MUSCLE IN CALCIUM-FREE SOLUTION ALLEN W. MANGEL,* J. GREGORYFITZ and IAN L. TAYLOR Division of Gastroenterology, Department of Medicine,Duke University Medical Center and the Durham VAMC, Durham, NC 27710, U.S.A. [Tel. (919) 684--3581]
(Received 12 March 1991) Abstract--1. Smooth muscle from six sites in the cat gastrointestinal tract was evaluated with respect to its ability to generate contractions in calcium-free solutions. 2. Membrane depolarization and carbachol, but not cholecystokinin or neurotensin, increased tension in smooth muscle segments of esophagus, corpus, duodenum, ileum, proximal colon and distal colon in calcium-free solution. 3. Substance-P produced a contractile response in the absence of calcium but only in the corpus and distal colon. 4. These findings indicate that peptide mediated release of intracellular calcium plays a minimal role in activation of cat gastrointestinal smooth muscle.
INTRODUCTION A large variety of peptides are known to modulate gastrointestinal smooth muscle activity. However, the ability of these agents to elicit contractions in calcium-free solution has been evaluated in only a limited number of studies. In studies with guinea-pig and rabbit intestinal smooth muscle, cholecystokinin (CCK), motilin and substance-P have been shown to induce contractions in calcium-free solution (Grider and Maklouf, 1988; Hoizer and ThiLippe, 1984; Matthijis et al., 1989). Similar results were obtained following exposure of guinea-pig gastric and gallbladder smooth muscle to CCK or gastrin (Bitar et al., 1986; Collins, 1986; Lee et al., 1989). These observations suggest that stimulation of contraction by these peptides can occur independently of influx of extracellular calcium. In the present study we evaluated the ability of three gastrointestinal peptides to induce contractions in calcium-free solution when six different smooth muscles of the cat gastrointestinal tract were examined. Results were compared to contractions observed after depolarization of the membrane by high potassium solution or after exposure to a cholinergic agonist under identical conditions. Removal of extracellular calcium markedly reduced pcptide-induced contractions, suggesting that release of intracellular calcium induced by peptides plays a minimal role in peptide induced smooth muscle contraction in the cat. MATERIALS AND METHODS
General Segments of gastrointestinal smooth muscle were removed from adult cats anesthetized with teoazol *To whom all correspondence should be addressed at: P.O. Box 31019, Duke University Medical Center, Durham, NC 27710, U.S.A.
(0.3-0.5mg/kg-i.m.) and placed immediately into Krebs solution of the followingcomposition (in raM): NaCI 118.5, KCI 4.7, MgC12 1.2, NaHCO3 23.8, KH2PO4 1.2, CaC!22.5 and glucose 5.5. Solutions were aerated with a 95% 02-5% CO2 gas mixture. Calcium-free Krebs saline was prepared identically except that no calcium was added and 5 mM ethyleneglycolbis([3-aminoethylether)-N,N'-tetraacetic acid (EGTA) was included. All experimentswere run at 35-37°C. The regions of the gastrointestinal tract examined were: esophagus (lower third); corpus (from the cardiac to angular notch); duodenum (most proximal 6--8era); ileum (most distal 6-8 crn); proximal colon (most proximal 6-8 era); and distal colon (most distal 6-8 era).
Preparation for recording mechanical activity Cylinders of tissue were opened along the circular axis of the gut. Muscle segments were prepared as in our previous studies (Mangel et al., 1979, 1982; Mangel, 1984). After preparations were mounted in the recording chamber, tissues were attached to isometric force transducers (Grass Instruments FTO3D). Preparations were allowed 30-45 rain equilibration in calcium containing media before experimentation. The output of the transducer was connected to Gould 13-4615-50transducer amplifiers and displayed on a polygraph (Brush No. 280). Drugs Cholecystokinin octapeptide (sulfated), neurotensin, substance-P, carbachol and atropine sulfate were obtained from Sigma Chemical Company (St. Louis, MO). RESULTS
Peptide induced activity in normal saline Addition of CCK (10-12-10-7M), neurotensin (10-ILl0 -7 M) or substance-P (10-1°-10 -6 M) to isolated segments of esophagus, corpus, duodenum, ileum, proximal colon or distal colon had tissue specific effects. With CCK, a stimulation of contraction frequency, amplitude and baseline tension occurred in segments of corpus (n = 7). In the distal colon (n ---4) the primary CCK-mediated effect was
1135
ALLENW. MANGELet al.
1136
stimulation in contraction frequency (threshold 5.6 × 10 -9 M) without change in amplitude. In four duodenal segments, CCK (10-8 M) produced an inhibition of contractile activity manifested by a slowing in the frequency of spontaneous contractile activity. In separate experiments with four to five muscle segments from each region of the gastrointestinal tract, neurotensin did not stimulate contractile activity. In intestinal muscle strips, neurotensin produced inhibition of spontaneous contractile activity. In all regions examined, substance-P stimulated the phasic amplitude of contractions. Four to eight muscle segments from each region were examined and threshold values for activation occurred between 10-9-10-7M. In addition to a stimulation in the phasic amplitude of contraction, an increase in baseline tension occurred in all regions with 10-9M substance-P.
Activity on calcium free solution Spontaneous activity. Exposure to calcium-free solution resulted in abrupt termination of spontaneous contractile activity in 63% of the preparation examined from the six gastrointestinal regions studied. In one-quarter of these segments, this was followed by the development of spontaneous rhythmic contractions. This contractile activity is presumably triggered by rhythmic voltage changes which develop in gastrointestinal smooth muscles during incubation in calcium-free solution (Mangel et al., 1979, 1982b; Mangel, 1984; Prosser et al., 1977). In 25% of muscle segments studied, a transient stimulation of contractions followed the transition from normal saline to calcium-free solution. This may result from plasma membrane depolarization which occurs in calcium-free solution (Mangel et al., 1982b; Mangel, 1984). In 12% of preparations, a gradual slowing and eventual elimination of contractile activity occurred in calcium-free solution. All three patterns occurred in all regions studied. Depolarization induced activity. We examined the ability of high potassium depolarization to induce contractile activity in smooth muscle segments during incubation in calcium-free solution. After 20-30 min incubation in calcium-free solution, a stimulation of Table 1. Effects of high-potassium* and carbachol't"on muscle segmentsin calcium-freesolution % Demonstratingcontraction Tissue Esophagus Corpus Duodenum Ileum Proximal colon Distal colon
HiK 38 (n = 8) 86 (n = 7) 50 (n = 8) 72 (n = 7) 88 (n = 8) 88
Carbachol
(n = 8)
(n = 4)
Corpus 20 mln Ca-flee 3gm 30 s e c
SP Fig. 1. Stimulation of a segment of corpus smooth muscle by substance-P. Preparation was quiescent in calcium free saline for 20 min. As shown, following administration of 10-s M substance-P, a contractile response was produced. contractile activity by high-potassium was seen in all regions (Table 1). Cholinergic agonist. The ability of the cholinergic agonist, carbachol, to elicit contractions in calciumfree solution was studied next. As shown in Table 1, all regions studied produced a contractile response to carbachol in calcium-free solution. Previously, we have shown (Mangel, 1984) that carbachol stimulated activity in colonic muscle segments during incubation in calcium-free solution is atropine sensitive. Peptides. The effects of CCK (10-12-10-5 M), neurotensin (10- ~t-10- 6 M), and substance-P ( I 0 - J°-10 -6 M) were evaluated in four to eight muscle segments from each gastrointestinal region in separate experiments during incubation in calcium-free solution. Only in segments of corpus (n = 4) (Fig. 1) and distal colon (n = 4 ) (Fig. 2) did contractile activity occur and only with substance-P. Threshold for activation was with 10 -s and 10 -6 M substance-P, respectively. The substance-P induced activity was atropine (10 -4 M) insensitive in both tissues. DISCUSSION
In the present study we have evaluated and compared the ability of three gastrointestinal polypeptides, depolarization with high potassium solution, and carbachol, to induce contractions in gastrointestinal smooth muscle in calcium-free solution. In general, the effects of polypeptides, but not depolarization or carbachol, were markedly inhibited by incubation of muscle in calcium-free conditions. This observation implies that influx of calcium is necessary for activation of contraction by these peptides.
100
(n = 3) 100 (n = 5) 50 (n = 4) 100 (n = 4) I00 (n = 3) 100
*High potassium solution was prepared by substitutionof NaCl by KCl. tCarbachol 10-4M.
D. Cokm
lmin C.a-fir~ 20 rain
Fig. 2. Simulation of a segment of distal colonic smooth muscle by substance-P. Preparation was quiescent for 20rain in calcium-free saline. As shown, following the administration of l0 -~ M substance-P (at asterisk), a contraction was produced.
Peptides do not induce contractions The calcium-free saline which was used in this study contained 5 mm EGTA. We have previously observed partial depolarization of cat intestinal smooth muscle membrane potential in this solution from approx. - 7 0 m V (Mangel et aL, 1982a) to a stable value of approx. - 4 0 m v (Mangel et aL, 1982b, 1984). Electrical potential changes which trigger rhythmic tension changes are also recorded in this solution (Mangel et al., 1979, 1982b; Mangel, 1984). After return to normal saline, repolarization to control values with recovery of normal slow waves, spikes and contractions occurs. Thus, we believe the calcium-free solution used in the present study was a suitable choice although other investigators have used calcium-free saline with differing concentrations of EGTA. The cholinergic agonist carbachol and membrane depolarization were capable of inducing a contractile response in all tissues examined in the absence of extracellular calcium. Our previous studies with gastrointestinal smooth muscle are consistent with the generation of contraction by cholinergic-receptor occupation or depolarization in calcium-free solution (Mangel et al., 1979, 1982b, 1984). The effects of CCK, substance-P, and neurotensin were examined on six gastrointestinal smooth muscles in normal saline and calcium-free solution. During incubation in calcium-free solution, we found only substance-P to induce a stimulation of contractile activity, and then only in gastric and distal colonic muscle. Thus, our results do not suggest an important component of intracellular calcium release by peptides in cat gastrointestinal smooth muscle segments. Acknowledgements--We gratefully acknowledge typing of the manuscript by Rhonda Shue. Support from NIH/ NIDDK grants 1F32 DK 08452 and DK 38216 and from Veterans Administration funds are acknowledged. REFERENCES
Bitar K. N., Burgess G. M., Putney J. W. Jr and Makhlouf G. M. (1986) Source of activator calcium is isolated
1137
guinea pig and human gastric muscle cells. Am. J. Physiol. 250, G280-286. Collins S. M. (1986) Calcium utilization by dispersed canine gastric smooth muscle ceils. Am. J. Physiol. 251, G181-188. Gilder J. R. and Makhlouf G. M. (1988) Contraction mediated by Ca2+ release in circular and Ca2+ influx in longitudinal intestinal musclecells. J. Pharmac. exp. Ther. 244, 432-437. Holzer P. and ThiLippe I. (1984) Substance-P can contract the longitudinal muscle of the guinea-pig small intestine by releasing intracellular calcium. Br. J. Pharmac. 82, 259-267. Lee K. Y., Biancani P. and Behar J. (1989) Calcium sources utilized by cholecystokinin and acetylcholine in the cat gallbladder muscle. Am. J. Physiol. 256, G785-788. Mangel A. W. (1984) Voltage and receptor mediated contractile activity of colonic smooth muscle in calcium-free solution. Fur. J. Pharm. 102, 165-168. Mangel A. W., Connor J. A. and Prosser C. L. (1982a) Effects of alterations in calcium levels on cat small intestinal slow waves. Am. J. Physiol. 243, C7-13. Mangel A. W., Mangel C. P. and Sanders K. M. (1984) Modulation of intestinal electrical and mechanical activity by calcium. In Calcium Regulations in Smooth Muscles, Vol. 124, pp. 111-118. Inserm. Mangel A. W., Nelson D. O., Conner J. A. and Prosser C. L. (1979) Contractions of cat small smooth intestinal muscle in calcium-free solution. Nature 281, 582-583. Mangel A. W., Nelson D. O., Rabovsky J. L., Prosser C. L. and Conner J. A. (1982b) Depolarization induced contractile activity of smooth muscle in calcium-freesolution. Am. J. Physiol. 242, C36-40. Matthijis T. L., Petters and Vantrappen G. (1989) The role of intracellular calcium stores in motilin induced contractions of the longitudinal muscle of the rabbit duodenum. Naungn-Schmiedeberg's Arch. Pharmac. 339, 332-339. Prosser C. L., Kreulen D. L., Weigel R. J. and Yau W. (1977) Prolonged potentials in gastrointestinal muscles induced by calcium chelation. Am. J. Physiol. 233, C19-24. Renzetti L. M., Wang M. B. and Ryan J. P. (1990) Contribution of intracellular calcium to gallbladder smooth muscle contraction. Am. J. Physiol. 259, GI-5.
0306-3623/91 $3.00+ 0.00 Copyright © 1991PergamonPresspie
PEPTIDES DO NOT I N D U C E CONTRACTIONS IN GASTROINTESTINAL SMOOTH MUSCLE IN CALCIUM-FREE SOLUTION ALLEN W. MANGEL,* J. GREGORYFITZ and IAN L. TAYLOR Division of Gastroenterology, Department of Medicine,Duke University Medical Center and the Durham VAMC, Durham, NC 27710, U.S.A. [Tel. (919) 684--3581]
(Received 12 March 1991) Abstract--1. Smooth muscle from six sites in the cat gastrointestinal tract was evaluated with respect to its ability to generate contractions in calcium-free solutions. 2. Membrane depolarization and carbachol, but not cholecystokinin or neurotensin, increased tension in smooth muscle segments of esophagus, corpus, duodenum, ileum, proximal colon and distal colon in calcium-free solution. 3. Substance-P produced a contractile response in the absence of calcium but only in the corpus and distal colon. 4. These findings indicate that peptide mediated release of intracellular calcium plays a minimal role in activation of cat gastrointestinal smooth muscle.
INTRODUCTION A large variety of peptides are known to modulate gastrointestinal smooth muscle activity. However, the ability of these agents to elicit contractions in calcium-free solution has been evaluated in only a limited number of studies. In studies with guinea-pig and rabbit intestinal smooth muscle, cholecystokinin (CCK), motilin and substance-P have been shown to induce contractions in calcium-free solution (Grider and Maklouf, 1988; Hoizer and ThiLippe, 1984; Matthijis et al., 1989). Similar results were obtained following exposure of guinea-pig gastric and gallbladder smooth muscle to CCK or gastrin (Bitar et al., 1986; Collins, 1986; Lee et al., 1989). These observations suggest that stimulation of contraction by these peptides can occur independently of influx of extracellular calcium. In the present study we evaluated the ability of three gastrointestinal peptides to induce contractions in calcium-free solution when six different smooth muscles of the cat gastrointestinal tract were examined. Results were compared to contractions observed after depolarization of the membrane by high potassium solution or after exposure to a cholinergic agonist under identical conditions. Removal of extracellular calcium markedly reduced pcptide-induced contractions, suggesting that release of intracellular calcium induced by peptides plays a minimal role in peptide induced smooth muscle contraction in the cat. MATERIALS AND METHODS
General Segments of gastrointestinal smooth muscle were removed from adult cats anesthetized with teoazol *To whom all correspondence should be addressed at: P.O. Box 31019, Duke University Medical Center, Durham, NC 27710, U.S.A.
(0.3-0.5mg/kg-i.m.) and placed immediately into Krebs solution of the followingcomposition (in raM): NaCI 118.5, KCI 4.7, MgC12 1.2, NaHCO3 23.8, KH2PO4 1.2, CaC!22.5 and glucose 5.5. Solutions were aerated with a 95% 02-5% CO2 gas mixture. Calcium-free Krebs saline was prepared identically except that no calcium was added and 5 mM ethyleneglycolbis([3-aminoethylether)-N,N'-tetraacetic acid (EGTA) was included. All experimentswere run at 35-37°C. The regions of the gastrointestinal tract examined were: esophagus (lower third); corpus (from the cardiac to angular notch); duodenum (most proximal 6--8era); ileum (most distal 6-8 crn); proximal colon (most proximal 6-8 era); and distal colon (most distal 6-8 era).
Preparation for recording mechanical activity Cylinders of tissue were opened along the circular axis of the gut. Muscle segments were prepared as in our previous studies (Mangel et al., 1979, 1982; Mangel, 1984). After preparations were mounted in the recording chamber, tissues were attached to isometric force transducers (Grass Instruments FTO3D). Preparations were allowed 30-45 rain equilibration in calcium containing media before experimentation. The output of the transducer was connected to Gould 13-4615-50transducer amplifiers and displayed on a polygraph (Brush No. 280). Drugs Cholecystokinin octapeptide (sulfated), neurotensin, substance-P, carbachol and atropine sulfate were obtained from Sigma Chemical Company (St. Louis, MO). RESULTS
Peptide induced activity in normal saline Addition of CCK (10-12-10-7M), neurotensin (10-ILl0 -7 M) or substance-P (10-1°-10 -6 M) to isolated segments of esophagus, corpus, duodenum, ileum, proximal colon or distal colon had tissue specific effects. With CCK, a stimulation of contraction frequency, amplitude and baseline tension occurred in segments of corpus (n = 7). In the distal colon (n ---4) the primary CCK-mediated effect was
1135
ALLENW. MANGELet al.
1136
stimulation in contraction frequency (threshold 5.6 × 10 -9 M) without change in amplitude. In four duodenal segments, CCK (10-8 M) produced an inhibition of contractile activity manifested by a slowing in the frequency of spontaneous contractile activity. In separate experiments with four to five muscle segments from each region of the gastrointestinal tract, neurotensin did not stimulate contractile activity. In intestinal muscle strips, neurotensin produced inhibition of spontaneous contractile activity. In all regions examined, substance-P stimulated the phasic amplitude of contractions. Four to eight muscle segments from each region were examined and threshold values for activation occurred between 10-9-10-7M. In addition to a stimulation in the phasic amplitude of contraction, an increase in baseline tension occurred in all regions with 10-9M substance-P.
Activity on calcium free solution Spontaneous activity. Exposure to calcium-free solution resulted in abrupt termination of spontaneous contractile activity in 63% of the preparation examined from the six gastrointestinal regions studied. In one-quarter of these segments, this was followed by the development of spontaneous rhythmic contractions. This contractile activity is presumably triggered by rhythmic voltage changes which develop in gastrointestinal smooth muscles during incubation in calcium-free solution (Mangel et al., 1979, 1982b; Mangel, 1984; Prosser et al., 1977). In 25% of muscle segments studied, a transient stimulation of contractions followed the transition from normal saline to calcium-free solution. This may result from plasma membrane depolarization which occurs in calcium-free solution (Mangel et al., 1982b; Mangel, 1984). In 12% of preparations, a gradual slowing and eventual elimination of contractile activity occurred in calcium-free solution. All three patterns occurred in all regions studied. Depolarization induced activity. We examined the ability of high potassium depolarization to induce contractile activity in smooth muscle segments during incubation in calcium-free solution. After 20-30 min incubation in calcium-free solution, a stimulation of Table 1. Effects of high-potassium* and carbachol't"on muscle segmentsin calcium-freesolution % Demonstratingcontraction Tissue Esophagus Corpus Duodenum Ileum Proximal colon Distal colon
HiK 38 (n = 8) 86 (n = 7) 50 (n = 8) 72 (n = 7) 88 (n = 8) 88
Carbachol
(n = 8)
(n = 4)
Corpus 20 mln Ca-flee 3gm 30 s e c
SP Fig. 1. Stimulation of a segment of corpus smooth muscle by substance-P. Preparation was quiescent in calcium free saline for 20 min. As shown, following administration of 10-s M substance-P, a contractile response was produced. contractile activity by high-potassium was seen in all regions (Table 1). Cholinergic agonist. The ability of the cholinergic agonist, carbachol, to elicit contractions in calciumfree solution was studied next. As shown in Table 1, all regions studied produced a contractile response to carbachol in calcium-free solution. Previously, we have shown (Mangel, 1984) that carbachol stimulated activity in colonic muscle segments during incubation in calcium-free solution is atropine sensitive. Peptides. The effects of CCK (10-12-10-5 M), neurotensin (10- ~t-10- 6 M), and substance-P ( I 0 - J°-10 -6 M) were evaluated in four to eight muscle segments from each gastrointestinal region in separate experiments during incubation in calcium-free solution. Only in segments of corpus (n = 4) (Fig. 1) and distal colon (n = 4 ) (Fig. 2) did contractile activity occur and only with substance-P. Threshold for activation was with 10 -s and 10 -6 M substance-P, respectively. The substance-P induced activity was atropine (10 -4 M) insensitive in both tissues. DISCUSSION
In the present study we have evaluated and compared the ability of three gastrointestinal polypeptides, depolarization with high potassium solution, and carbachol, to induce contractions in gastrointestinal smooth muscle in calcium-free solution. In general, the effects of polypeptides, but not depolarization or carbachol, were markedly inhibited by incubation of muscle in calcium-free conditions. This observation implies that influx of calcium is necessary for activation of contraction by these peptides.
100
(n = 3) 100 (n = 5) 50 (n = 4) 100 (n = 4) I00 (n = 3) 100
*High potassium solution was prepared by substitutionof NaCl by KCl. tCarbachol 10-4M.
D. Cokm
lmin C.a-fir~ 20 rain
Fig. 2. Simulation of a segment of distal colonic smooth muscle by substance-P. Preparation was quiescent for 20rain in calcium-free saline. As shown, following the administration of l0 -~ M substance-P (at asterisk), a contraction was produced.
Peptides do not induce contractions The calcium-free saline which was used in this study contained 5 mm EGTA. We have previously observed partial depolarization of cat intestinal smooth muscle membrane potential in this solution from approx. - 7 0 m V (Mangel et aL, 1982a) to a stable value of approx. - 4 0 m v (Mangel et aL, 1982b, 1984). Electrical potential changes which trigger rhythmic tension changes are also recorded in this solution (Mangel et al., 1979, 1982b; Mangel, 1984). After return to normal saline, repolarization to control values with recovery of normal slow waves, spikes and contractions occurs. Thus, we believe the calcium-free solution used in the present study was a suitable choice although other investigators have used calcium-free saline with differing concentrations of EGTA. The cholinergic agonist carbachol and membrane depolarization were capable of inducing a contractile response in all tissues examined in the absence of extracellular calcium. Our previous studies with gastrointestinal smooth muscle are consistent with the generation of contraction by cholinergic-receptor occupation or depolarization in calcium-free solution (Mangel et al., 1979, 1982b, 1984). The effects of CCK, substance-P, and neurotensin were examined on six gastrointestinal smooth muscles in normal saline and calcium-free solution. During incubation in calcium-free solution, we found only substance-P to induce a stimulation of contractile activity, and then only in gastric and distal colonic muscle. Thus, our results do not suggest an important component of intracellular calcium release by peptides in cat gastrointestinal smooth muscle segments. Acknowledgements--We gratefully acknowledge typing of the manuscript by Rhonda Shue. Support from NIH/ NIDDK grants 1F32 DK 08452 and DK 38216 and from Veterans Administration funds are acknowledged. REFERENCES
Bitar K. N., Burgess G. M., Putney J. W. Jr and Makhlouf G. M. (1986) Source of activator calcium is isolated
1137
guinea pig and human gastric muscle cells. Am. J. Physiol. 250, G280-286. Collins S. M. (1986) Calcium utilization by dispersed canine gastric smooth muscle ceils. Am. J. Physiol. 251, G181-188. Gilder J. R. and Makhlouf G. M. (1988) Contraction mediated by Ca2+ release in circular and Ca2+ influx in longitudinal intestinal musclecells. J. Pharmac. exp. Ther. 244, 432-437. Holzer P. and ThiLippe I. (1984) Substance-P can contract the longitudinal muscle of the guinea-pig small intestine by releasing intracellular calcium. Br. J. Pharmac. 82, 259-267. Lee K. Y., Biancani P. and Behar J. (1989) Calcium sources utilized by cholecystokinin and acetylcholine in the cat gallbladder muscle. Am. J. Physiol. 256, G785-788. Mangel A. W. (1984) Voltage and receptor mediated contractile activity of colonic smooth muscle in calcium-free solution. Fur. J. Pharm. 102, 165-168. Mangel A. W., Connor J. A. and Prosser C. L. (1982a) Effects of alterations in calcium levels on cat small intestinal slow waves. Am. J. Physiol. 243, C7-13. Mangel A. W., Mangel C. P. and Sanders K. M. (1984) Modulation of intestinal electrical and mechanical activity by calcium. In Calcium Regulations in Smooth Muscles, Vol. 124, pp. 111-118. Inserm. Mangel A. W., Nelson D. O., Conner J. A. and Prosser C. L. (1979) Contractions of cat small smooth intestinal muscle in calcium-free solution. Nature 281, 582-583. Mangel A. W., Nelson D. O., Rabovsky J. L., Prosser C. L. and Conner J. A. (1982b) Depolarization induced contractile activity of smooth muscle in calcium-freesolution. Am. J. Physiol. 242, C36-40. Matthijis T. L., Petters and Vantrappen G. (1989) The role of intracellular calcium stores in motilin induced contractions of the longitudinal muscle of the rabbit duodenum. Naungn-Schmiedeberg's Arch. Pharmac. 339, 332-339. Prosser C. L., Kreulen D. L., Weigel R. J. and Yau W. (1977) Prolonged potentials in gastrointestinal muscles induced by calcium chelation. Am. J. Physiol. 233, C19-24. Renzetti L. M., Wang M. B. and Ryan J. P. (1990) Contribution of intracellular calcium to gallbladder smooth muscle contraction. Am. J. Physiol. 259, GI-5.