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Effect of Progesterone Pretreatment on Guinea Pig Gallbladder Motility In Vitro
GASTROENTEROLOGY 1982;83:81-3
Effect of Progesterone Pretreatment on Guinea Pig Gallbladder Motility In Vitro JAMES P. RYAN and DENNIS PELLECCHIA Department of Physiology, Temple University Health Sciences Center, Philadelphia, Pennsylvania
Experiments were designed to examine the effect of chronic pretreatment with progesterone (2 mg/ kg . day for 5 days) on the in vitro contractile response of gallbladder muscle strips from adult male guinea pigs. The muscle strips were challenged with either acetylcholine or the octapeptide of cholecystokinin and dose-response relationships were determined. The data were compared with doseresponse curves obtained from untreated control animals. Progesterone pretreatment produced rightward shifts in the acetylcholine and octapeptide of cholecystokinin curves that were characterized by significant decreases in the maximal contractile response. Serum progesterone concentrations in the pretreated animals were significantly increased over control levels. The data support the hypothesis that the sluggish behavior of the gallbladder during pregnancy and during the luteal phase of the menstrual cycle may be due, in part, to progesterone-related alterations in the contractile properties of gallbladder smooth muscle. There is increasing evidence that the steroid hormone, progesterone, can affect the electrical and mechanical properties of gastrointestinal smooth muscle (1-5). Chronic pretreatment with progesterone has been reported to disrupt the propagation characteristics of gastric and small intestinal electrical slow waves (2,3) and to decrease the in vitro contractile response of the esophagus, antrum, and colon to cholinergic stimulation (1,4,5). These data have lead to the hypothesis that progesterone-related decreases in smooth muscle activity account for the heartburn, nausea, and constipation experienced by many women during pregnancy (6). Both the fasting volume of the gallbladder and the Received July 7, 1981. Accepted March 2, 1982. Address requests for reprints to: James P. Ryan, Ph.D., Department of Physiology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140. © 1982 by the American Gastroenterological Association 0016-5085/82/070081-03$02.50
residual volume after a meal are increased during pregnancy (7,8). Although no direct proof is available, it has been proposed that progesterone also affects the motor responsiveness of gallbladder smooth muscle (7). The purpose of this study was to test this hypothesis by examining the effect of chronic pretreatment with progesterone on the in vitro contractile response of muscle strips from the guinea pig gallbladder. The muscles were examined for their contractile response to acetylcholine and the octapeptide of cholecystokinin and the results were compared with data obtained from untreated control animals.
Methods Ten adult male guinea pigs (400-600 g) were pretreated with progesterone (2 mg/kg, s.c.) in peanut oil (injection volume, 0.1 ml) once daily for 5 consecutive days. Ten paired control animals received daily injections of the vehicle only. All animals were killed 24 h after the fifth injection, blood samples were taken for radioimmunoassay analysis of progesterone levels, and the gallbladders removed. The gallbladders were immediately opened lengthwise and rinsed with Krebs-Ringer solution (composition in millimoles: Na+, 138.6; K+, 4.6; Ca 2+, 2.5; Mg2+, 2.1; Cl-, 126.2; HC0 3 -, 21.9; P0 4 '" , 1.2; glucose, 15.5). Circularly oriented muscle strips were prepared as described previously (9) and mounted in separate 20-ml tissue chambers. The tissue baths contained warmed Krebs-Ringer solution (36°-38°C) which was continuously gassed with 95% O 2 and 5% CO 2, One end of each muscle strip was attached through an inelastic wire to an external force transducer (FT -03C, Grass Instrument Co., Quincy, Mass.) while the other end was attached to a metal rod that could be raised or lowered by the adjustment of a screw micrometer. A permanent record of the isometric force developed by the muscle strips was graphed on a Grass multichannel recorder. The muscle length at which maximal tension was developed in response to acetylcholine (1 x 10- 6 M) was determined for each muscle strip and maintained for the duration of the experiment. Dose-response curves were constructed for acetylcho-
82
GASTROENTEROLOGY Vol. 83, No.1, Part 1
RYAN AND PELLECCHIA
line and the octapeptide of cholecystokinin (CCK-OP, donated by the Squibb Institute for Medical Research) on each muscle strip. Stock solutions of the agonists were prepared and added to the tissue baths in microliter amounts to achieve the final molar concentrations report~d. Individual doses were given in random order and no cumulative doses were given. One-half hour recovery periods were allowed between additions of the agonists to the baths. The data were normalized for differences in tissue size by expressing the tension per cross sectional area. This was calculated from the· equation A = M/pL, where M is mass (g), p is density (glml) and numerically equal to the specific gravity, and L is the length of the muscle (cm). Each point on the dose-response curves is expressed as a percent of the maximal response to either acetylcholine or CCK-OP as measured in the control groups. In each case the peak response to acetylcholine and CCK-OP developed at doses of 1· x 10- 4 M and 1 X 10- 8 M, respectively. This is evident from the figures which show an absence of measures of deviation at these points. The statistical significance of the differences in peak contractile response was determined using the Student's t-test for unpaired observations (10). Radioimmunoassay analysis of the serum progesterone levels was provided by the clinical laboratories of the Smith Kline Corporation, Waltham, Mass. Their assay procedure utilizes radiolabeled progesterone and progesterone antibodies obtained commercially from PANTEX, Santa Monica, Calif.
Results The gallbladder muscle strips taken from animals pretreated with progesterone showed a marked reduction in contractile response when compared
~
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:3 a:
100 80
-
CONTROL
O--OP(2 mg/Kg/Day)
...J
0 a: tz 0
(J
...J
«
w 100
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Z
a:
80
0 a: t-
60
W
CONTROL
0--0 P(2mg/Kg/Day)
0
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-
...J
Z
0
(J
...J
« 40 ~
x« ~
20
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CCK-OP(M) Figure 2. Effect of progesterone pretreatment on the in vitro contractile response of gallbladder smooth muscle to CCK-OP stimulation. The symbols are the same as in Figure 1. Each point is expressed as a percent of the maximal response to CCK-OP in the control group. The data points represent the mean and standard error of the results obtained on 10 muscle strips from 10 animals.
with tissue taken from control animals. The effect of hormone pretreatment was to produce a rightward shift in both the acetylcholine and CCK-OP doseresponse curves (Figures 1 and 2) and to significantly decrease the maximal contractile response to each agonist (Table 1). Each point on the dose-response curves represents the mean ± the standard error of results obtained on 10 muscle strips from 10 animals. Serum progesterone concentrations in the hormone-pretreated group ranged from 1.5 to 2.0 ng/ml, with a mean value of 1.7 ± 0.3 ng/m!. This value is significantly greater (p < 0.001) than the mean serum progesterone concentration of the untreated controls. These animals had a serum progesterone concentration <0.1 ng/m!.
60
Discussion 40
There is increasing evidence that gallbladder motility is impaired during pregnancy (7,8). The biliary stasis is characterized by an increase in both
~
X
« 20 ~
I.L
0
-s.
10~
108
107
106
10 5
10
4
10- 3
ACETYLCHOLINE 1M)
Figure 1. Effect of progesterone pretreatment on the in vitro contractile response of gallbladder smooth muscle to acetylcholine stimulation. Solid circles represent the response from untreated control animals and open circles represent the response from progesterone-pretreated animals. Each point is expressed as a percent of the maximal contractile response obtained from the control animals. The data points represent the mean and standard error of the results obtained on 10 muscle strips from 10 animals.
Table 1. Effect of Progesterone Pretreatment on Maximal Contractile Activity Agonist Experimental group Control Pretreated
Acetylcholine response (kg/em 2 ) 0.43 ± 0.03 0.34 ± 0.02
a
CCK-Opb
p
response (kg/cm2)
p
<0.05
0.44 ± 0.4 0.35 ± 0.03
0.05
Mean ± SEM of 10 responses from 10 animals. a Dose M. b Dose = 1 x 10- 8 M.
=
1 x 10- 4
PROGESTERONE AND GALLBLADDER MOTILITY
July 1982
the fasting and residual volumes and by a decrease in emptying capacity. It has been suggested (7) that these alterations reflect a decrease in muscle tone and a decrease in the sensitivity of the muscle to neural and hormonal stimuli. Little is known about the factors responsible for the apparent reduction in motility. Braverman et al. (7) have proposed that the hypotony develops as a consequence of the increased serum progesterone levels which occur during pregnancy. Their hypothesis is based primarily on observations made by other investigators who have shown that progesterone can reduce the contractile activity of other gastrointestinal tissues (1,3,5,11,12). Similarly, Nilsson and Stattin (13) have used the concept of progesterone inhibition of smooth muscle activity to explain the sluggish behavior of the gallbladder during the luteal phase of the menstrual cycle. The results of previous studies done in our laboratory have shown that the direct addition of progesterone into tissue chambers containing isolated gallbladder muscle strips was characterized by a decrease in the contractile responsiveness of the tissue to cholinergic and hormonal stimulation (14). In the present experiments, the chronic elevation of serum progesterone levels was associated with a significant decrease in the in vitro contractility of gallbladder in response to acetylcholine and CCKOP. The data are at odds with an earlier report by Smith et al. (15). However, uncertainty regarding the sensitivity of their recording technique and the purity of the hormone preparations they used limits the significance of their observations. Our findings, however, do support the hypothesis that the sluggish behavior of the gallbladder during pregnancy and during the luteal phase of the menstrual cycle is the result of progesterone-related decreases in the sensitivity of the muscle to neurohumoral stimulation. The possible mechanisms by which progesterone exerts this antagonistic effect have not been addressed in this study. Others have suggested, however, that the hormone may affect motility by altering transmembrane potentials, by disrupting the electrical coordination between cells, or by reducing the amount of calcium available for contraction (2,16,17). The importance of any of these mechanisms must remain speculative until appropriate experiments can be designed and performed. The results of this and previous experiments strongly suggest that progesterone can affect the contractile activity of gastrointestinal smooth muscle. A question arises, however, concerning the physiologic vs. pharmacologic significance of the observations. Although the serum progesterone levels measured in this study were within the range reported for the guinea pig estrus cycle (18), the values are considerably greater than normally occur
83
in male animals. Thus, the serum concentrations may reflect pharmacologic levels. The adult male animal also has been used by others to define the relationship between elevated serum progesterone levels and gastrointestinal motility. While the results have proved useful, more critical studies using normal females, ovariectomized females, and pregnant animals are needed to resolve the physiologic role of progesterone in affecting gastrointestinal smooth muscle.
References
I
1. Kumar D. In vitro inhibitory effect of progesterone on extrauterine human smooth muscle. Am J Obstet Gynecol 1962;84:1300-4. 2. Bortoff A, Morello E, Mistretta P. Effects of progesterone and 17-0H progesterone on intestinal slow wave propagation. In: Christensen J, ed. Gastrointestinal motility. New York: Raven Press, 1980:387-93. 3. Milenov K, Kazakov 1. Influence of ovarian hormones on electromyograms of uterus, stomach, and intestines in dogs. Endocrinol Exp 1970;7:163-70. 4. Bruce LA, Behsudi FM. Differential inhibition of regional gastrointestinal tissues. Life Sci 1979;25:729-34. 5. Bruce LA, Behsudi FM. Differential inhibition of regional gastrointestinal tissue to progesterone in the rat. Life Sci 1980;27:427-38. 6. Moir DD. Gastrointestinal function in pregnancy and labor. In: Obstetric anasthesia and analgesic. Baltimore: Williams and Wilkins Company, 1976:25-34. 7. Braverman DZ, Johnson ML, Kern F Jr. Effects of pregnancy and contraceptive steroids on gallbladder function. N Engl J Med 1980;302:362-4. 8. Gerdes MM, Boyden EA. The rate of emptying of the human gallbladder in pregnancy. Surg Gynecol Obstet 1938;66:14556. 9. Ryan J, Cohen S. Interaction of gastrin I, secretin, and cholecystokinin on gallbladder smooth muscle. Am J Physiol 230:553-6. 10. Snedecor GW, Cochran WG. Statistical methods. 6th ed. Ames Iowa: Iowa State University Press, 1967. 11. Fisher RS, Roberts GS, Grabowski q, et a1. Inhibition of lower esophageal circular smooth muscle by female sex hormones. Am J PhysioI1978;234:E243-7. 12. Schulze K, Christensen J. The lower esophageal sphincter of the opossum esophagus in pseudopregnancy. Gastroenterology 1977;73:1082-5. 13. Nilsson S, Stattin S. Gallbladder emptying during the normal menstrual cycle. Acta Chir Scand 1967;133:648-52. 14. Ryan JP, McCallion M. Influence of progesterone on guinea pig gallbladder smooth muscle. Am J Physiol (in press). 15. Smith JJ, Pomeranc MM, Ivy AC. The influence of pregnancy and sex hormones on gallbladder motility in the guinea pig. Am J PhysioI1941;132:129-40. 16. Carsten, ME. Biochemical aspects of uterine contractility: role of prostaglandins. In: Friedman EA, Noah ML, Work BA Jr, eds. Uterine physiology. Littleton, Mass.: PSG Publishing Co., 1979:3-31. 17. Csapo A. The in vivo and in vitro effects of estrogen and progesterone on the myometrium. In: Ville CA, Engel LL, eds. Mechanisms of action of steroid hormones. New York: Academic Press, 1961:68-74. 18. Challis JRG, Heap RB, Illingworth DV. Concentration of estrogen and progesterone in the plasma of non-pregnant, pregnant, and lactating guinea pigs. J Physiol 1971;51:33345.
Effect of Progesterone Pretreatment on Guinea Pig Gallbladder Motility In Vitro JAMES P. RYAN and DENNIS PELLECCHIA Department of Physiology, Temple University Health Sciences Center, Philadelphia, Pennsylvania
Experiments were designed to examine the effect of chronic pretreatment with progesterone (2 mg/ kg . day for 5 days) on the in vitro contractile response of gallbladder muscle strips from adult male guinea pigs. The muscle strips were challenged with either acetylcholine or the octapeptide of cholecystokinin and dose-response relationships were determined. The data were compared with doseresponse curves obtained from untreated control animals. Progesterone pretreatment produced rightward shifts in the acetylcholine and octapeptide of cholecystokinin curves that were characterized by significant decreases in the maximal contractile response. Serum progesterone concentrations in the pretreated animals were significantly increased over control levels. The data support the hypothesis that the sluggish behavior of the gallbladder during pregnancy and during the luteal phase of the menstrual cycle may be due, in part, to progesterone-related alterations in the contractile properties of gallbladder smooth muscle. There is increasing evidence that the steroid hormone, progesterone, can affect the electrical and mechanical properties of gastrointestinal smooth muscle (1-5). Chronic pretreatment with progesterone has been reported to disrupt the propagation characteristics of gastric and small intestinal electrical slow waves (2,3) and to decrease the in vitro contractile response of the esophagus, antrum, and colon to cholinergic stimulation (1,4,5). These data have lead to the hypothesis that progesterone-related decreases in smooth muscle activity account for the heartburn, nausea, and constipation experienced by many women during pregnancy (6). Both the fasting volume of the gallbladder and the Received July 7, 1981. Accepted March 2, 1982. Address requests for reprints to: James P. Ryan, Ph.D., Department of Physiology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140. © 1982 by the American Gastroenterological Association 0016-5085/82/070081-03$02.50
residual volume after a meal are increased during pregnancy (7,8). Although no direct proof is available, it has been proposed that progesterone also affects the motor responsiveness of gallbladder smooth muscle (7). The purpose of this study was to test this hypothesis by examining the effect of chronic pretreatment with progesterone on the in vitro contractile response of muscle strips from the guinea pig gallbladder. The muscles were examined for their contractile response to acetylcholine and the octapeptide of cholecystokinin and the results were compared with data obtained from untreated control animals.
Methods Ten adult male guinea pigs (400-600 g) were pretreated with progesterone (2 mg/kg, s.c.) in peanut oil (injection volume, 0.1 ml) once daily for 5 consecutive days. Ten paired control animals received daily injections of the vehicle only. All animals were killed 24 h after the fifth injection, blood samples were taken for radioimmunoassay analysis of progesterone levels, and the gallbladders removed. The gallbladders were immediately opened lengthwise and rinsed with Krebs-Ringer solution (composition in millimoles: Na+, 138.6; K+, 4.6; Ca 2+, 2.5; Mg2+, 2.1; Cl-, 126.2; HC0 3 -, 21.9; P0 4 '" , 1.2; glucose, 15.5). Circularly oriented muscle strips were prepared as described previously (9) and mounted in separate 20-ml tissue chambers. The tissue baths contained warmed Krebs-Ringer solution (36°-38°C) which was continuously gassed with 95% O 2 and 5% CO 2, One end of each muscle strip was attached through an inelastic wire to an external force transducer (FT -03C, Grass Instrument Co., Quincy, Mass.) while the other end was attached to a metal rod that could be raised or lowered by the adjustment of a screw micrometer. A permanent record of the isometric force developed by the muscle strips was graphed on a Grass multichannel recorder. The muscle length at which maximal tension was developed in response to acetylcholine (1 x 10- 6 M) was determined for each muscle strip and maintained for the duration of the experiment. Dose-response curves were constructed for acetylcho-
82
GASTROENTEROLOGY Vol. 83, No.1, Part 1
RYAN AND PELLECCHIA
line and the octapeptide of cholecystokinin (CCK-OP, donated by the Squibb Institute for Medical Research) on each muscle strip. Stock solutions of the agonists were prepared and added to the tissue baths in microliter amounts to achieve the final molar concentrations report~d. Individual doses were given in random order and no cumulative doses were given. One-half hour recovery periods were allowed between additions of the agonists to the baths. The data were normalized for differences in tissue size by expressing the tension per cross sectional area. This was calculated from the· equation A = M/pL, where M is mass (g), p is density (glml) and numerically equal to the specific gravity, and L is the length of the muscle (cm). Each point on the dose-response curves is expressed as a percent of the maximal response to either acetylcholine or CCK-OP as measured in the control groups. In each case the peak response to acetylcholine and CCK-OP developed at doses of 1· x 10- 4 M and 1 X 10- 8 M, respectively. This is evident from the figures which show an absence of measures of deviation at these points. The statistical significance of the differences in peak contractile response was determined using the Student's t-test for unpaired observations (10). Radioimmunoassay analysis of the serum progesterone levels was provided by the clinical laboratories of the Smith Kline Corporation, Waltham, Mass. Their assay procedure utilizes radiolabeled progesterone and progesterone antibodies obtained commercially from PANTEX, Santa Monica, Calif.
Results The gallbladder muscle strips taken from animals pretreated with progesterone showed a marked reduction in contractile response when compared
~
z ~
:3 a:
100 80
-
CONTROL
O--OP(2 mg/Kg/Day)
...J
0 a: tz 0
(J
...J
«
w 100
C/)
Z
a:
80
0 a: t-
60
W
CONTROL
0--0 P(2mg/Kg/Day)
0
C/)
-
...J
Z
0
(J
...J
« 40 ~
x« ~
20
I.L
0
~
CCK-OP(M) Figure 2. Effect of progesterone pretreatment on the in vitro contractile response of gallbladder smooth muscle to CCK-OP stimulation. The symbols are the same as in Figure 1. Each point is expressed as a percent of the maximal response to CCK-OP in the control group. The data points represent the mean and standard error of the results obtained on 10 muscle strips from 10 animals.
with tissue taken from control animals. The effect of hormone pretreatment was to produce a rightward shift in both the acetylcholine and CCK-OP doseresponse curves (Figures 1 and 2) and to significantly decrease the maximal contractile response to each agonist (Table 1). Each point on the dose-response curves represents the mean ± the standard error of results obtained on 10 muscle strips from 10 animals. Serum progesterone concentrations in the hormone-pretreated group ranged from 1.5 to 2.0 ng/ml, with a mean value of 1.7 ± 0.3 ng/m!. This value is significantly greater (p < 0.001) than the mean serum progesterone concentration of the untreated controls. These animals had a serum progesterone concentration <0.1 ng/m!.
60
Discussion 40
There is increasing evidence that gallbladder motility is impaired during pregnancy (7,8). The biliary stasis is characterized by an increase in both
~
X
« 20 ~
I.L
0
-s.
10~
108
107
106
10 5
10
4
10- 3
ACETYLCHOLINE 1M)
Figure 1. Effect of progesterone pretreatment on the in vitro contractile response of gallbladder smooth muscle to acetylcholine stimulation. Solid circles represent the response from untreated control animals and open circles represent the response from progesterone-pretreated animals. Each point is expressed as a percent of the maximal contractile response obtained from the control animals. The data points represent the mean and standard error of the results obtained on 10 muscle strips from 10 animals.
Table 1. Effect of Progesterone Pretreatment on Maximal Contractile Activity Agonist Experimental group Control Pretreated
Acetylcholine response (kg/em 2 ) 0.43 ± 0.03 0.34 ± 0.02
a
CCK-Opb
p
response (kg/cm2)
p
<0.05
0.44 ± 0.4 0.35 ± 0.03
0.05
Mean ± SEM of 10 responses from 10 animals. a Dose M. b Dose = 1 x 10- 8 M.
=
1 x 10- 4
PROGESTERONE AND GALLBLADDER MOTILITY
July 1982
the fasting and residual volumes and by a decrease in emptying capacity. It has been suggested (7) that these alterations reflect a decrease in muscle tone and a decrease in the sensitivity of the muscle to neural and hormonal stimuli. Little is known about the factors responsible for the apparent reduction in motility. Braverman et al. (7) have proposed that the hypotony develops as a consequence of the increased serum progesterone levels which occur during pregnancy. Their hypothesis is based primarily on observations made by other investigators who have shown that progesterone can reduce the contractile activity of other gastrointestinal tissues (1,3,5,11,12). Similarly, Nilsson and Stattin (13) have used the concept of progesterone inhibition of smooth muscle activity to explain the sluggish behavior of the gallbladder during the luteal phase of the menstrual cycle. The results of previous studies done in our laboratory have shown that the direct addition of progesterone into tissue chambers containing isolated gallbladder muscle strips was characterized by a decrease in the contractile responsiveness of the tissue to cholinergic and hormonal stimulation (14). In the present experiments, the chronic elevation of serum progesterone levels was associated with a significant decrease in the in vitro contractility of gallbladder in response to acetylcholine and CCKOP. The data are at odds with an earlier report by Smith et al. (15). However, uncertainty regarding the sensitivity of their recording technique and the purity of the hormone preparations they used limits the significance of their observations. Our findings, however, do support the hypothesis that the sluggish behavior of the gallbladder during pregnancy and during the luteal phase of the menstrual cycle is the result of progesterone-related decreases in the sensitivity of the muscle to neurohumoral stimulation. The possible mechanisms by which progesterone exerts this antagonistic effect have not been addressed in this study. Others have suggested, however, that the hormone may affect motility by altering transmembrane potentials, by disrupting the electrical coordination between cells, or by reducing the amount of calcium available for contraction (2,16,17). The importance of any of these mechanisms must remain speculative until appropriate experiments can be designed and performed. The results of this and previous experiments strongly suggest that progesterone can affect the contractile activity of gastrointestinal smooth muscle. A question arises, however, concerning the physiologic vs. pharmacologic significance of the observations. Although the serum progesterone levels measured in this study were within the range reported for the guinea pig estrus cycle (18), the values are considerably greater than normally occur
83
in male animals. Thus, the serum concentrations may reflect pharmacologic levels. The adult male animal also has been used by others to define the relationship between elevated serum progesterone levels and gastrointestinal motility. While the results have proved useful, more critical studies using normal females, ovariectomized females, and pregnant animals are needed to resolve the physiologic role of progesterone in affecting gastrointestinal smooth muscle.
References
I
1. Kumar D. In vitro inhibitory effect of progesterone on extrauterine human smooth muscle. Am J Obstet Gynecol 1962;84:1300-4. 2. Bortoff A, Morello E, Mistretta P. Effects of progesterone and 17-0H progesterone on intestinal slow wave propagation. In: Christensen J, ed. Gastrointestinal motility. New York: Raven Press, 1980:387-93. 3. Milenov K, Kazakov 1. Influence of ovarian hormones on electromyograms of uterus, stomach, and intestines in dogs. Endocrinol Exp 1970;7:163-70. 4. Bruce LA, Behsudi FM. Differential inhibition of regional gastrointestinal tissues. Life Sci 1979;25:729-34. 5. Bruce LA, Behsudi FM. Differential inhibition of regional gastrointestinal tissue to progesterone in the rat. Life Sci 1980;27:427-38. 6. Moir DD. Gastrointestinal function in pregnancy and labor. In: Obstetric anasthesia and analgesic. Baltimore: Williams and Wilkins Company, 1976:25-34. 7. Braverman DZ, Johnson ML, Kern F Jr. Effects of pregnancy and contraceptive steroids on gallbladder function. N Engl J Med 1980;302:362-4. 8. Gerdes MM, Boyden EA. The rate of emptying of the human gallbladder in pregnancy. Surg Gynecol Obstet 1938;66:14556. 9. Ryan J, Cohen S. Interaction of gastrin I, secretin, and cholecystokinin on gallbladder smooth muscle. Am J Physiol 230:553-6. 10. Snedecor GW, Cochran WG. Statistical methods. 6th ed. Ames Iowa: Iowa State University Press, 1967. 11. Fisher RS, Roberts GS, Grabowski q, et a1. Inhibition of lower esophageal circular smooth muscle by female sex hormones. Am J PhysioI1978;234:E243-7. 12. Schulze K, Christensen J. The lower esophageal sphincter of the opossum esophagus in pseudopregnancy. Gastroenterology 1977;73:1082-5. 13. Nilsson S, Stattin S. Gallbladder emptying during the normal menstrual cycle. Acta Chir Scand 1967;133:648-52. 14. Ryan JP, McCallion M. Influence of progesterone on guinea pig gallbladder smooth muscle. Am J Physiol (in press). 15. Smith JJ, Pomeranc MM, Ivy AC. The influence of pregnancy and sex hormones on gallbladder motility in the guinea pig. Am J PhysioI1941;132:129-40. 16. Carsten, ME. Biochemical aspects of uterine contractility: role of prostaglandins. In: Friedman EA, Noah ML, Work BA Jr, eds. Uterine physiology. Littleton, Mass.: PSG Publishing Co., 1979:3-31. 17. Csapo A. The in vivo and in vitro effects of estrogen and progesterone on the myometrium. In: Ville CA, Engel LL, eds. Mechanisms of action of steroid hormones. New York: Academic Press, 1961:68-74. 18. Challis JRG, Heap RB, Illingworth DV. Concentration of estrogen and progesterone in the plasma of non-pregnant, pregnant, and lactating guinea pigs. J Physiol 1971;51:33345.