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Progesterone effects on three regional gastrointestinal tissues
Life Sciences, Vol . 25, pp . 729-734 Printed in the U .S .A .
Pergamon Press
PROGESTERONE EFFECiS CIV THREE REGICiIVAL GASTROINrE.SfINAL TISSUES Larry A. Brucet and Faiz M. Behsudi; Department of Physiology, Southwestern Medical School, University of Texas Health Science Center, Dallas, Texas 75235 (Received
in
final form July 20, 1979)
Summary Esophageal, antral, and colonic tissues excised from progesterone pretreated males were demonstrated to exhibit a significantly reduced contractile activity compared to the corresponding regions excised from non-treated male control animals . Progesterone blood levels (measured with RLA) of the treated males were found to fall within the range reported for non-pregnant cyclic females. A common complaint issued by a large number of women during the course of pregnancy is same degree of gastrointestinal discomfort . A recent clinical study (1) indicates a decreased louver esophageal sphincter (LES) pressure in the third trimester of pregnancy when progesterone blood levels are relatively high . Additional studies in humans (2) and opossim (3) in vivo suggest that LES pressures are influenced by considerably lower progesterone blood levels than those associated with pregnancy. An in vitro study (4) indicates that a progesterone inhibition can be demonstrate~rvi~ 20 min exposure time . A significant reduction in gastric antral and gastroduodenal functional tissue motility function has been attributed to a progesteranic influence (5) . A reduced colonic activity resulting in varying degrees of constipation is a general complaint during pregnancy but the etiology is unlmown (6) . We have studied the influence of progesterone on esophageal, antral and colonic tissue activity . Methods Pro esterone Treatment In Vivo . Male Sprague-Dawley rats weighing 210 ± taneous y with progesterone (3 mg/kg) in sesame seed 20 g were pretreat oil (injection volume, 0 .1 ml) . Paired control animals received 0 .1 ml of sesame seed oil . Control and treated groups received 5 injections over a 96-h period . Isolation of Gastrointestinal Tissue . Animals were fasted 24 h before being y a aw to e abdominal cavity was immediately opened . Sections of gut tissue were excised from three regions of the tract, t(To wham reprint requests should be addressed .) Grant AM21657 . Postdoctoral Research Fellow
Supported in part by NII-I
0024-3205/79/090729-0602 .00/0 Copyright (c) 1979 Pergamon Press Ltd
730
Progesterone Effects on GI Motility
Vol . 25, No . 9,
1979
esophagus ; 1 cm proximal to stomach, antrum ; 0 .5 cm proximal to pyloric sphincter, and colon; mid-transverse colon. The tissue rings were immediately placed in oxygenated mammalian Krebs saline solution (composition in mM : NaCl, 84 .9 ; KC1, 4 .7 ; CaC1 2 2 .5 ; NaHC03 , 25 .0 ; MgSO , 1.2 ; KH 2P0 4 , 1.1 ; glucose, 7 .2) maintained at 37° ± 0 .5° C and aerate with 5~ CO - 95~ 0 throughBy incising the circular rings of tis~ue and l~gating out the experiment . both ends with suture, longitudinal preparations in the circular muscle plane were made that were placed in 30 ml baths with one end attached to a stabilization bar and the free end connected to a tension transducer (Grass, model Ft 0 .03 force displacement transducer) . The transducer was mounted on an assembly arm which could be raised or lowered by a screw-drive micromanipulator in mm increments . Three units were utilized so that the three regional tissues from one animal were tested simultaneously . erimental Protocol . A 60 min equilibration period in the muscle baths were ~ or ea preparation. Optimal basal tension levels (T ) for each type of regional tissue were obtained as previously described (5) aid placed on the tissues of the beginning of the equilibration period and maintained at 10 min intervals thrwghout the course of the experiment . Spontaneous tissue activity was quantitated for a 5 min period following the equilibration period . Chemical stimulation of the tissue preparations was achieved with arecholine (1 X 10 6M) being a~ninistered into the muscle baths with micropipets (vol 50 uls) after the 5 min control period . Tissue activity was quantitated for each preparation for 3 sequential 5 min intervals . Data antitation . A detailed description of the recording system may be ere riefly, the transducer signals were transmitted simulfound e s taneously to an integrator and a fiber optic-cathode ray tube recorder (Honeywell) . Contractile activity was integrated in terms of gt~ec to give the total force of contraction (units : g sec) for a 5 min quantitation period . Frequency of contraction for each 5 min period was also measured . The dissimilarity among the regional tissue responses necessitated presentation of Chemically challenged esophageal tissue the data in three different formats . responded with a single tonic contraction and this activity is presented as force of contraction per min. Antral tissue responses were phasic and are Phasic and tonic contracpresented as force of contraction per contraction. tions were recorded with the colonic tissue and this activity is presented as a motility index which equals frequency X force of contraction (Fig . 1) . Collection of sera for determination of ro esterone levels . Ors ml of blood was co ect y car rac puncture ea anima concurrently with the excision of gut tissue . Blood samples were centrifuged at 2000 g for 20 min and serum stored at -20°C until analyzed . Extraction . The extraction procedure and radioimmunoassay used have been A 40~ progesterone r~covery frown the extraction prodescri e in etail (7) . cedure was determined from the recoveries of H -progesterone added to the incubation medium or to "pooled sera" from control male animals . The antiserum G~i No . 331 was used at a dilution of RIA- ro esterone . 1 :500 . e concentrations of progesterone were determined from 0 .5 ml of seam . The concentrations of progesterone were determined from 0 .5 ml of Sensitivity of seem . Binding of H -progesterone to the antibody was 14~ . *The source for the progesterone antiserum was from Dr . G.D . Nilwender (Colorado State University) and was kindly supplied to us by Dr . S .R . Ojeda (Univ . of Texas Health Science Center, Dallas) .
Vol .
25, No . 9, 1979
the assay was 25 pg/tube . Pg/tube .
Progesterone Effeets on GI Motility
73 1
Linearity of the standard curve ranged from 25-400
FIG 1 A typical recording of three regional gastroint stinal tissues challenged with arecholine-HC1 (1 X 10 - ~ . This final bath concentration represented an ED for the 75for tissues . B1 . baseline - at the respective To each tissue . All statistical evaluations were performed by t tests (8) . P<0 .05 were considered significant .
Values of
Results Tissues taken from animals pretreated with progesterone revealed a marked reduction in contractile activity compared to tissue activity taken from paired control animals not receiving progesterone . The percentage inhibition seen with in vivo progesterone pretreatment on in vitro esophageal, antral, and coloni~tissue activity was 68 .7, 67 .3 and .~3 .~respectively, compared to the corresponding non-treated regional tissue responses (Fig . 2) . No significant difference in the frequency parameter could be quantitated between the progesterone pretreated and non-treated tissues from either the antral or colonic regions (Table 1) . Esophageal tissue responded with a single tonic contraction for each experimental run (15 min), therefore the esophageal frequency data canparison has been omitted from Table . 1 . Seam progesterone levels for the progesterone pretreated animals ranged from 29 .7 to 36 .3 ng/ml . Sen~m progesterone levels for the non-treated animals
732
Progesterone Effects on GI Motility
Vol . 25, No . 9,
1979
ranged from 1.9 to 2 .1 ng/ml.
Esophagus
Antrum
Colon
FIG 2 Contractile responses of three regional gastrointestinal tissues excised frown non-treated (open bars) and progesterone pretreated (stippled bars) animals . Each bar represents a mean ± SEM of 18 - 5 min quantitation periods with 6 tissues . *A : *B : *C :
force of contraction (g . sec) per minute force of contraction (g . sec) per contraction force of contraction (g . sec) X frequency per 5 minutes
Non-treated tissue responses are significantly different from treated tissue responses ; *A and *B (P< .025), *C (P< .05) . Table 1 Challenged Frequency Responses of Regional Tissues From Either Progesterone Pretreated or Non-treated Animals ~réchôline 1 uM 5 Mi nt rtes
Antr~n Control (8) Pretreatment (8) Colon Control (8) Pretreatment (8)
*N .S . - non-significant (P>0 .05)
15
21 .0 ± 3 .1 17 .9 ± 2 .5 N.S .*
21 .3 ± 3 .5 18 .5 ± 2 .3 N.S .
19 .6 ± 3 .2 19 .9 ± 2 .1 N .S .
41 .5 ± 9.0 40 .5 ± 11 .3 N.S .
52 .8 ± 6 .3 36 .9 t 9 .9 N.S .
52 .5 ± 6 .1 39 .0 ± 8 .8 N.S .
Vol . 25, No, 9, 1979
Progesterone Effects on GI Motility
73 3
Discussion In a recent study we reported a significant decrease in the force of contraction of antral and adjacent gastroduodenal junctionâl tissue excised from progesterone-pretreated animals (5) . The data in this report suggest that at least two . other regions of the gastrointestinal tract are equally infl uenced by progesterone pretreatment in the male rat . Furthermore, the progesterone blood levels measured in the pretreated male animals suggest that the progesterone blood level required to influence gastrointestinal activity is within the range attained by the cyclic female as reported from three different laboratories (9-11) . This is not to infer that these progesterone blood levels are physiological in the male but rather, that the levels in this study are within reported physiological levels for the cyclic female and an inhibition of the activity in three different regions of gastrointestinal tract has been observed . Several recent studies have addressed the question of possible influence of progesterone on lower esophageal sphincter (LES) fimction. Fisher and coworkers (4) have tested the acute effects of progesterone on IFS activity in vitro . A significant inhibition in LES challenged activity was reported wilt com~uiations of acetylcholine and progesterone, gastrin and progesterone when compared to the respective agonist challenged activity alone . A progressive reduction in LES pressure and an increase in progesterone blood levels have been quantitated in a group of normal pregnant women followed through the gestation and postpartum periods (1) . A significant reduction in LES pressure and a 20-fold increase in progesterone blood levels were noted in the 24th and 36th week of gestation compared to LES pressure and progesterone blood levels measured postpartum. Schulze and (hristensen (3) have successfully demonstrated a decrease in opossum LF5 pressure in vivo following a 7 day estrogen and a subsequent 5 day combined estrogen-progésterone treatment period . No change in LES pressure was noted on days 1 or 7 of the estrogen treatment ; however, a significant reduction in LES pressure was recorded on day 12 following 5 days of estrogen-progesterone treatment . Information concerning the influence of progesterone on colonic activity is lacking . Texts of .obstetrics and gynecology (6,12) acknowledge that a common complaint during pregnancy is reduced colonic activity or constipation ; however, little research attention has been directed toward this area. An earlier report (13) suggested that the spontaneous activity of colonic strips taken from resected fi.~an colonic tissue was inhibited with progesterone administration in vitro. The data from our study suggest that a progesterone inhibitory inflûence on colonic activity can be demonstrated in the male at progesterone blood concentrations which are within the female physiological range . The activity from three anatomically separated regions of the male gastrointestinal tract have been demonstrated to be significantly inhibited in vitro when progesterone was administered in vivo . Progesterone blood levelswere within the female progesterone bloo~l~s attained during the peak of the estrus cycle . Interpretation of these results would suggest a non-specific effect of progesterone on at least three regions of the gastrointestinal tract . More important, perhaps is the implication that physiological levels of progesterone may significantly influence gastrointestinal function in the female .
734
Progesterone Effects on GI Motility
Vol . 25, No . 9, 1979
Acknowled~ements The authors gratefully acknowledge the technical support of Michael F . Cronin and secretarial assistance of Mrs . Vicki Raukin . References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 .
T .H . VAN THIEL, J .S . GAUALER, S . N . JOSHI, R .K . SARA and J . STREMPLE, Gastroenterology 72, 666-668 (1977) . D .H . VAN THIEL, .~. GAUALER and J . STREMPLE, Gastroenterology 71, 232234 (1976) . K . SCHILZE and J . CHRISTENSEN, Gastrcenterology 73, 1082-1085 (1977) . R .S . FIS3-~R, G .S . ROBERTS, C .J . GRABOWSKI and S .~Od-LLTTf, Am . J . Physiol . 234, E 243-274 (1978) . LAC . BRUCE, F .M. BEI-iS[JDI and I .E . DAM-~F, Am . J . Physiol . _235, E 422-428 (1978) . D .H. WINSHIP, Medical Co lications Du~~ Pre ,~1an - . pp . 322-330, B .N . Borrow and i~.~erris e . , W .n . aawbaers, rniladelphia (1975) . J .P . ADVIS and S . R . QJEDA, Endocrinology 103, 924-935 (1978) . G .W. SNEDECOR and W .G . OOC[-IItAN, Statistic~tMethods , p . 59, Iowa. State Univ . Press, Ames (1967) . R .L . GOOTMAN, Endocrinology 102, 142-150 (1978) . R .L . , W . E . COLLINS a~ N .W . FUGO, Endocrinology 94, 1704-1708 (1974) . M .S . SMITH, M .E . FREIIMAN and J .D . NEILL, Endocrinology 96, 219-226 (1975) . H .A. KAMINETZKY, Medical lications ~ Pregnancy pp . 303-333, ~9 D .M . Haynes (ed .),Tc~raw,New or D . KUMAR, Am . J . Obst . and Gynec . 84, 1300-1304 (1962) .
Pergamon Press
PROGESTERONE EFFECiS CIV THREE REGICiIVAL GASTROINrE.SfINAL TISSUES Larry A. Brucet and Faiz M. Behsudi; Department of Physiology, Southwestern Medical School, University of Texas Health Science Center, Dallas, Texas 75235 (Received
in
final form July 20, 1979)
Summary Esophageal, antral, and colonic tissues excised from progesterone pretreated males were demonstrated to exhibit a significantly reduced contractile activity compared to the corresponding regions excised from non-treated male control animals . Progesterone blood levels (measured with RLA) of the treated males were found to fall within the range reported for non-pregnant cyclic females. A common complaint issued by a large number of women during the course of pregnancy is same degree of gastrointestinal discomfort . A recent clinical study (1) indicates a decreased louver esophageal sphincter (LES) pressure in the third trimester of pregnancy when progesterone blood levels are relatively high . Additional studies in humans (2) and opossim (3) in vivo suggest that LES pressures are influenced by considerably lower progesterone blood levels than those associated with pregnancy. An in vitro study (4) indicates that a progesterone inhibition can be demonstrate~rvi~ 20 min exposure time . A significant reduction in gastric antral and gastroduodenal functional tissue motility function has been attributed to a progesteranic influence (5) . A reduced colonic activity resulting in varying degrees of constipation is a general complaint during pregnancy but the etiology is unlmown (6) . We have studied the influence of progesterone on esophageal, antral and colonic tissue activity . Methods Pro esterone Treatment In Vivo . Male Sprague-Dawley rats weighing 210 ± taneous y with progesterone (3 mg/kg) in sesame seed 20 g were pretreat oil (injection volume, 0 .1 ml) . Paired control animals received 0 .1 ml of sesame seed oil . Control and treated groups received 5 injections over a 96-h period . Isolation of Gastrointestinal Tissue . Animals were fasted 24 h before being y a aw to e abdominal cavity was immediately opened . Sections of gut tissue were excised from three regions of the tract, t(To wham reprint requests should be addressed .) Grant AM21657 . Postdoctoral Research Fellow
Supported in part by NII-I
0024-3205/79/090729-0602 .00/0 Copyright (c) 1979 Pergamon Press Ltd
730
Progesterone Effects on GI Motility
Vol . 25, No . 9,
1979
esophagus ; 1 cm proximal to stomach, antrum ; 0 .5 cm proximal to pyloric sphincter, and colon; mid-transverse colon. The tissue rings were immediately placed in oxygenated mammalian Krebs saline solution (composition in mM : NaCl, 84 .9 ; KC1, 4 .7 ; CaC1 2 2 .5 ; NaHC03 , 25 .0 ; MgSO , 1.2 ; KH 2P0 4 , 1.1 ; glucose, 7 .2) maintained at 37° ± 0 .5° C and aerate with 5~ CO - 95~ 0 throughBy incising the circular rings of tis~ue and l~gating out the experiment . both ends with suture, longitudinal preparations in the circular muscle plane were made that were placed in 30 ml baths with one end attached to a stabilization bar and the free end connected to a tension transducer (Grass, model Ft 0 .03 force displacement transducer) . The transducer was mounted on an assembly arm which could be raised or lowered by a screw-drive micromanipulator in mm increments . Three units were utilized so that the three regional tissues from one animal were tested simultaneously . erimental Protocol . A 60 min equilibration period in the muscle baths were ~ or ea preparation. Optimal basal tension levels (T ) for each type of regional tissue were obtained as previously described (5) aid placed on the tissues of the beginning of the equilibration period and maintained at 10 min intervals thrwghout the course of the experiment . Spontaneous tissue activity was quantitated for a 5 min period following the equilibration period . Chemical stimulation of the tissue preparations was achieved with arecholine (1 X 10 6M) being a~ninistered into the muscle baths with micropipets (vol 50 uls) after the 5 min control period . Tissue activity was quantitated for each preparation for 3 sequential 5 min intervals . Data antitation . A detailed description of the recording system may be ere riefly, the transducer signals were transmitted simulfound e s taneously to an integrator and a fiber optic-cathode ray tube recorder (Honeywell) . Contractile activity was integrated in terms of gt~ec to give the total force of contraction (units : g sec) for a 5 min quantitation period . Frequency of contraction for each 5 min period was also measured . The dissimilarity among the regional tissue responses necessitated presentation of Chemically challenged esophageal tissue the data in three different formats . responded with a single tonic contraction and this activity is presented as force of contraction per min. Antral tissue responses were phasic and are Phasic and tonic contracpresented as force of contraction per contraction. tions were recorded with the colonic tissue and this activity is presented as a motility index which equals frequency X force of contraction (Fig . 1) . Collection of sera for determination of ro esterone levels . Ors ml of blood was co ect y car rac puncture ea anima concurrently with the excision of gut tissue . Blood samples were centrifuged at 2000 g for 20 min and serum stored at -20°C until analyzed . Extraction . The extraction procedure and radioimmunoassay used have been A 40~ progesterone r~covery frown the extraction prodescri e in etail (7) . cedure was determined from the recoveries of H -progesterone added to the incubation medium or to "pooled sera" from control male animals . The antiserum G~i No . 331 was used at a dilution of RIA- ro esterone . 1 :500 . e concentrations of progesterone were determined from 0 .5 ml of seam . The concentrations of progesterone were determined from 0 .5 ml of Sensitivity of seem . Binding of H -progesterone to the antibody was 14~ . *The source for the progesterone antiserum was from Dr . G.D . Nilwender (Colorado State University) and was kindly supplied to us by Dr . S .R . Ojeda (Univ . of Texas Health Science Center, Dallas) .
Vol .
25, No . 9, 1979
the assay was 25 pg/tube . Pg/tube .
Progesterone Effeets on GI Motility
73 1
Linearity of the standard curve ranged from 25-400
FIG 1 A typical recording of three regional gastroint stinal tissues challenged with arecholine-HC1 (1 X 10 - ~ . This final bath concentration represented an ED for the 75for tissues . B1 . baseline - at the respective To each tissue . All statistical evaluations were performed by t tests (8) . P<0 .05 were considered significant .
Values of
Results Tissues taken from animals pretreated with progesterone revealed a marked reduction in contractile activity compared to tissue activity taken from paired control animals not receiving progesterone . The percentage inhibition seen with in vivo progesterone pretreatment on in vitro esophageal, antral, and coloni~tissue activity was 68 .7, 67 .3 and .~3 .~respectively, compared to the corresponding non-treated regional tissue responses (Fig . 2) . No significant difference in the frequency parameter could be quantitated between the progesterone pretreated and non-treated tissues from either the antral or colonic regions (Table 1) . Esophageal tissue responded with a single tonic contraction for each experimental run (15 min), therefore the esophageal frequency data canparison has been omitted from Table . 1 . Seam progesterone levels for the progesterone pretreated animals ranged from 29 .7 to 36 .3 ng/ml . Sen~m progesterone levels for the non-treated animals
732
Progesterone Effects on GI Motility
Vol . 25, No . 9,
1979
ranged from 1.9 to 2 .1 ng/ml.
Esophagus
Antrum
Colon
FIG 2 Contractile responses of three regional gastrointestinal tissues excised frown non-treated (open bars) and progesterone pretreated (stippled bars) animals . Each bar represents a mean ± SEM of 18 - 5 min quantitation periods with 6 tissues . *A : *B : *C :
force of contraction (g . sec) per minute force of contraction (g . sec) per contraction force of contraction (g . sec) X frequency per 5 minutes
Non-treated tissue responses are significantly different from treated tissue responses ; *A and *B (P< .025), *C (P< .05) . Table 1 Challenged Frequency Responses of Regional Tissues From Either Progesterone Pretreated or Non-treated Animals ~réchôline 1 uM 5 Mi nt rtes
Antr~n Control (8) Pretreatment (8) Colon Control (8) Pretreatment (8)
*N .S . - non-significant (P>0 .05)
15
21 .0 ± 3 .1 17 .9 ± 2 .5 N.S .*
21 .3 ± 3 .5 18 .5 ± 2 .3 N.S .
19 .6 ± 3 .2 19 .9 ± 2 .1 N .S .
41 .5 ± 9.0 40 .5 ± 11 .3 N.S .
52 .8 ± 6 .3 36 .9 t 9 .9 N.S .
52 .5 ± 6 .1 39 .0 ± 8 .8 N.S .
Vol . 25, No, 9, 1979
Progesterone Effects on GI Motility
73 3
Discussion In a recent study we reported a significant decrease in the force of contraction of antral and adjacent gastroduodenal junctionâl tissue excised from progesterone-pretreated animals (5) . The data in this report suggest that at least two . other regions of the gastrointestinal tract are equally infl uenced by progesterone pretreatment in the male rat . Furthermore, the progesterone blood levels measured in the pretreated male animals suggest that the progesterone blood level required to influence gastrointestinal activity is within the range attained by the cyclic female as reported from three different laboratories (9-11) . This is not to infer that these progesterone blood levels are physiological in the male but rather, that the levels in this study are within reported physiological levels for the cyclic female and an inhibition of the activity in three different regions of gastrointestinal tract has been observed . Several recent studies have addressed the question of possible influence of progesterone on lower esophageal sphincter (LES) fimction. Fisher and coworkers (4) have tested the acute effects of progesterone on IFS activity in vitro . A significant inhibition in LES challenged activity was reported wilt com~uiations of acetylcholine and progesterone, gastrin and progesterone when compared to the respective agonist challenged activity alone . A progressive reduction in LES pressure and an increase in progesterone blood levels have been quantitated in a group of normal pregnant women followed through the gestation and postpartum periods (1) . A significant reduction in LES pressure and a 20-fold increase in progesterone blood levels were noted in the 24th and 36th week of gestation compared to LES pressure and progesterone blood levels measured postpartum. Schulze and (hristensen (3) have successfully demonstrated a decrease in opossum LF5 pressure in vivo following a 7 day estrogen and a subsequent 5 day combined estrogen-progésterone treatment period . No change in LES pressure was noted on days 1 or 7 of the estrogen treatment ; however, a significant reduction in LES pressure was recorded on day 12 following 5 days of estrogen-progesterone treatment . Information concerning the influence of progesterone on colonic activity is lacking . Texts of .obstetrics and gynecology (6,12) acknowledge that a common complaint during pregnancy is reduced colonic activity or constipation ; however, little research attention has been directed toward this area. An earlier report (13) suggested that the spontaneous activity of colonic strips taken from resected fi.~an colonic tissue was inhibited with progesterone administration in vitro. The data from our study suggest that a progesterone inhibitory inflûence on colonic activity can be demonstrated in the male at progesterone blood concentrations which are within the female physiological range . The activity from three anatomically separated regions of the male gastrointestinal tract have been demonstrated to be significantly inhibited in vitro when progesterone was administered in vivo . Progesterone blood levelswere within the female progesterone bloo~l~s attained during the peak of the estrus cycle . Interpretation of these results would suggest a non-specific effect of progesterone on at least three regions of the gastrointestinal tract . More important, perhaps is the implication that physiological levels of progesterone may significantly influence gastrointestinal function in the female .
734
Progesterone Effects on GI Motility
Vol . 25, No . 9, 1979
Acknowled~ements The authors gratefully acknowledge the technical support of Michael F . Cronin and secretarial assistance of Mrs . Vicki Raukin . References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 .
T .H . VAN THIEL, J .S . GAUALER, S . N . JOSHI, R .K . SARA and J . STREMPLE, Gastroenterology 72, 666-668 (1977) . D .H . VAN THIEL, .~. GAUALER and J . STREMPLE, Gastroenterology 71, 232234 (1976) . K . SCHILZE and J . CHRISTENSEN, Gastrcenterology 73, 1082-1085 (1977) . R .S . FIS3-~R, G .S . ROBERTS, C .J . GRABOWSKI and S .~Od-LLTTf, Am . J . Physiol . 234, E 243-274 (1978) . LAC . BRUCE, F .M. BEI-iS[JDI and I .E . DAM-~F, Am . J . Physiol . _235, E 422-428 (1978) . D .H. WINSHIP, Medical Co lications Du~~ Pre ,~1an - . pp . 322-330, B .N . Borrow and i~.~erris e . , W .n . aawbaers, rniladelphia (1975) . J .P . ADVIS and S . R . QJEDA, Endocrinology 103, 924-935 (1978) . G .W. SNEDECOR and W .G . OOC[-IItAN, Statistic~tMethods , p . 59, Iowa. State Univ . Press, Ames (1967) . R .L . GOOTMAN, Endocrinology 102, 142-150 (1978) . R .L . , W . E . COLLINS a~ N .W . FUGO, Endocrinology 94, 1704-1708 (1974) . M .S . SMITH, M .E . FREIIMAN and J .D . NEILL, Endocrinology 96, 219-226 (1975) . H .A. KAMINETZKY, Medical lications ~ Pregnancy pp . 303-333, ~9 D .M . Haynes (ed .),Tc~raw,New or D . KUMAR, Am . J . Obst . and Gynec . 84, 1300-1304 (1962) .