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Effect of progesterone on canine colonic smooth muscle
GASTROENTEROLOGY
1985:88:1941-7
Effect of Progesterone on Canine Colonic Smooth Muscle R. C. GILL,
K. L. BOWES,
and Y. J. KINGMA
Surgical Medical Research Institute and Departments University of Alberta, Edmonton, Alberta, Canada
This study was undertaken to ascertain the effect and mode of action of progesterone on canine colonic smooth muscle in vitro. Circularly orientated strips of smooth muscle exhibited low-amplitude contractions at the slow wave frequency. Longitudinally orientated strips exhibited larger-amplitude contractions that were associated, electrically, with a series of “spikes” superimposed on a high-frequency oscillation in membrane potential. Progesterone reduced Ihe contractile force of both the circularly and JongitudinaJJy orientated strips and the contractile frequency of the longitudinally orientated strips in a dose-dependent manner; this inhibitory effect was observed in the presence of tetrodotoxin but not atropine. The stimulatory effects of a high-potassium superfusate were antagonized by progesterone and verapamil, the effect of progesterone being reversed by increasing the calcium concentration of the superfusate. We conclude that progesterone exerts an inhibitory effect on colonic smooth muscle and this may be mediated by changes in the cytoplasmic calcium concentration. Despite the fact that constipation has so frequently been described as a complication of pregnancy (l-4), the investigation of colonic smooth muscle and the effects of steroid hormones on its electrical and mechanical properties has received little attention. An early in vitro study [5) demonstrated that the spontaneous contractile activity of human colonic smooth muscle could be completely inhibited by high concentrations of progesterone. This inhibitory
Received August Address requests
9, 1984. Accepted December 13, 1984. for reprints to: Dr. Richard C. Gill, Department
of Surgery, University of Alberta, Edmonton, Alberta, Canada T6G 2G3. Richard C. Gill was a recipient of an award from the Alberta Heritage Foundation for Medical Research. This work was supported in part by the Medical Research Council of Canada. 0 1985 by the American Gastroenterological Association 0016-5085/85/$3.30
of Surgery
and Electrical
Engineering,
effect appeared to be specific for progesterone, as estradiol and corticosterone were without effect. A later study (6) demonstrated the possible physiologic significance of this effect in that colonic smooth muscle excised from male rats pretreated with progesterone had reduced contractility in vitro. The serum progesterone levels in these rats were considered to be in the normal physiologic range of the female. Neither of these studies, however, provided any information on the effects of progesterone on the different muscle layers of the colon, their electrical activities, or on the mode of action of progesterone. In this study, the effect of progesterone on the electrical and mechanical activities of the different muscle layers of the canine colon has been determined. The mechanism of action of progesterone has also been studied.
Materials and Methods In these experiments, tissue from male animals was used to avoid the complications that could arise due to fluctuating estrogen and progesterone levels that normally occur in the female (7). Strips of circularly and longitudinally orientated colonic smooth muscle were studied in vitro. The effects of progesterone alone and in the presence of atropine, tetrodotoxin, verapamil, and high-potassium superfusate also were determined.
Tissue
Preparation
Under pentobarbital anesthesia (30 mgikg i.v.), the abdomen was opened by a midline incision. A segment of proximal colon -7 cm long was removed immediately after clamping of the blood supply. The colonic segment was opened along the mesenteric border, carefully cleaned to prevent soiling of the muscle layers with fecal content, and pinned to the Sylgard surface of a dissecting dish filled with oxygenated Krebs-Ringer solution. The mucosa was removed by sharp dissection. Strips of smooth muscle (0.5 x 4.0 cm) were then cut in either the circular or longitudinal direction. Strips were mounted in a horizontal tissue chamber maintained at 37” * 1°C and continuously superfused with oxygenated Krebs-Ringer solution.
1942
GILL ET AL.
GASTROENTEROLOGY
Solutions “‘_,~~l~~~i~,J’u”~~~r~~~f
The Krebs-Ringer
contractile
z z LL Z-
Vol. 88, No. 6
and Drugs
standard superfusate solution containing
used the
was a modified following (mM):
Nat, 139.“; ,K’. 5.4; Ca2+, 2.5; ;g2’, 1.2; Cl-, 125.1; HCOs- 22 0, H,PO,-, 1.2; glucose 10.1. When equilibrated with 98% 02-5% CO2 gas mixture at 3X, this solution had a pH of 7.3-7.4. For a high-potassium solution, NaCl was replaced with sufficient KC1 to provide a final K+ concentration of 10 mM. For a high-calcium solution, CaC12 was added directly to the high-potassium KrebsRinger solution to provide a final Ca2+ concentration of 5.0
2;
(Jo-
5,” 0; xc
4020-* 0
Drugs used throughout this study were progesterone [Sigma Chemical Co., St. Louis, MO.), atropine sulfate (BDH Pharmaceuticals Ltd., London, England), tetrodotoxin (Sigma), and verapamil hydrochloride (ISOPTIN,
-1 f16%
Of17X
0219%
I
I
I
1
1
2
3
4
TIME
(hours)
Figure 1. Spontaneous contractile and surface electrical activity recorded from circularly orientated strips of canine colonic smooth muscle (n = 8). Each of the lowamplitude (<3oO mg wt) contractions was associated with a change in electrical potential referred to as a slow wave. The mean force of the contractions in each as a 10 min of a 4-h control ueriod was exoressed percentage of that observed in the first hour of study. The variation in mean contractile force for each subsequent hour was calculated (panel A]. In the same way, variation in contractile and electrical slow wave frequency recorded from these strips during the 4-h control period was calculated (panel B).
5 LI
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-1Of13%
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15+-21%
I 4
I 3
13?20%
1
1
Recording
Techniques
Electrical activity was recorded from the serosal (longitudinally orientated strips) or mucosal (circularly orientated strips) surface with a low-noise, low-drift silver-silver chloride pressure electrode (8). The reference electrode was a large silver-silver chloride electrode placed in the superfusing fluid. Contractile activity of the muscle strip was recorded with a force displacement transducer (Grass FT03, Grass Instrument Co., Quincy, Mass.). Recordings were made of the electrical and mechanical activity on a Beckman Dynograph R411 polygraph (Beckman Instruments, Inc., Fullerton, Calif.); for the electrical activity, filters were set to give a lowfrequency cutoff at 0.16 Hz and a high-frequency cutoff at 32.0 Hz.
2 Time
;
4
(hours)
Figure 2. Spontaneous contractile and surface electrical activity recorded from longitudinally orientated strips of canine colonic smooth muscle (n = 8). Each of the prolonged-duration, large-amplitude (>l.O g wt) contractions was associated, electrically, with a series of changes in potential at a frequency of 25-35 min-‘: evidence of contractile activity at this frequency was superimposed on the prolonged-duration contractions. The mean force of the prolonged-duration contractions in each 10 min of a 4-h control period was expressed as a percentage of that observed in the first hour of study. The variation in mean contractile force for each subsequent hour was calculated (panel A). In the same way, variation in contractile frequency recorded from these strips during the 4-h control periods was calculated (panel B).
June
MOTILITY
1985
Searle, Skokie, 111.).Progesterone was solubilized in ethanol to an initial concentration of z x IO-~-Z x IO-’ M, 0.5 ml of these solutions were diluted to 5.0 ml with propylene glycol per liter of Krebs-Ringer solution to provide final progesterone concentrations in the range 1.0 x 10-7-1.0 x 10m4 M.
Experimental
Protocol
Preliminary experiments demonstrated that the contractile activity of both circularly and longitudinally orientated strips was dependent on the degree of stretch (Figures 1-3). The force of the omnipresent, spontaneous contractions recorded from the circularly orientated strips increased linearly with the degree of stretch. However, when stretched to >220% of their initial length (LJ, defined as the length at which an increase in baseline force was first recorded, these strips demonstrated occasional large-amplitude contractions. The force of these large-amplitude contractions increased in parallel with the baseline tension as the strips were stretched further. In contrast, the force of the spontaneous, prolonged-duration contractions recorded from the longitudinally orientated strips increased with the degree of stretch until -160% of their
CIRCULARLY OFHENTATEDSTRIPS
OF COLONIC
SMOOTH
MUSCLE
1943
initial length. Once these strips were stretched beyond 170% of their initial length, the contractile force decreased; contractions ceased at 190% of the initial length. This decrease in active contractile force was marked by a rise in the baseline tension recorded. The contractile frequency of the circularly orientated strips remained unchanged by stretching when compared with the control preparations (Figure 1). The contractile frequency of the longitudinally orientated strips decreased with increasing length as compared with the control strips (Figure 2). At the commencement of each study, basal tension (corresponding to -115% of initial length) was applied to all strips. Each strip was set at this length for the remainder of the experiment. After a 30-min equilibration period, during which the strips were superfused with oxygenated Krebs-Ringer solution, the electrical and mechanical activities of the muscle strips were recorded for a period of 1 h as a control. Thereafter, except for “control” strips, drugs or ions were added directly to the superfusate as indicated in Tables 1 and 2. An additional control was performed using a superfusate containing the ethanol-propylene gylco1 solvent on two circularly orientated and two longitudinally orientated strips of colonic smooth muscle. The solvent was found to have no effect on the spontaneous contractile or electrical activity of these preparations.
LONGITUDINALLY ORIENTATED STRIPS
i!$l-$$fy[;I?~l*,,y*,,*,,F*.{~\ 100
140
160
220
260
100
120
140
160
160
I
% Li
Figure
2
I
I
I
I
I
100
120
140
160
160
% Li
3. Relationship between the percentage initial length (%L,) of circularly and longitudinally orientated strips of canine colonic smooth muscle and the baseline and active contractile force and contractile frequency recorded. The initial length L, was defined as the length at which an increase in baseline force was first recorded. The force of the spontaneous, low-amplitude contractions (A) recorded from the circularly orientated strips (n = 8) increased with the degree of stretch (panel A). At lengths >220% Li, the force of the occasional, large-amplitude contractions (m; note scale change) increased in parallel with the baseline force (0). Panel B shows the change in contractile frequency of the circularly orientated strips during these studies. The force of the spontaneous, prolonged-duration contractions (A] recorded from the longitudinally orientated strips (n = 8) increased until 160% Li (panel C). At lengths >17O% Li, the active contractile force decreased and became zero at 190% L,. Marked increases in baseline force (0) were noted at lengths ~70% L,. Panel D shows a decrease in the frequency of the prolonged-duration contractions with increasing strip length; the contractile frequency fell to zero at 19Ou/, L,.
1944
Table
GILL ET AL.
1.
Effect of Progesterone on Circularly and Longitudinally Orientated Strips of Canine Colonic
Smooth
Muscle
Circularly orientated strips
Progesterone concentration (M) 0, Control (n = 8) lo-' (n = lo-" (n = 10m5 (n = lo-* (n =
GASTROENTEROLOGY Vol. 88. No. 6
A% Force 0 + 19
A% Frequency -5LlZ
Longitudinally orientated strips A% Force
A% Frequency
-10 t 13
15 ir 21
6) 6)
-15 rt 7 -12 * 2 -2lk 20 -82 3
-16k 3 -26 + 8'
-14 2 3b -21 2 7"
6) 6)
-33 -+ 5d -10 It 5 -57213d -13 + 5
-35 * 2d -82 f 5d
-26 k 4d -37 t 3d
Results shown are percentage changes (A%) in mean spontaneous contractile force and frequency between the second hour of tissue superfusion and the preceding hour (minus sign indicates reduction). Statistical significance was assessed by comparison of changes observed in tissue superfused with progesterone-containing Krebs-Ringer solution and those of control preparations. n p < 0.05;b p < 0.01;c p < 0.005;d p < 0.001.
Analysis
of Data
Recordings of electrical and mechanical activity were analyzed visually. Each hour of study was divided into six lo-min periods that were analyzed separately; mean values for each hour of study were then calculated. Time-dependent alterations in the mechanical and electrical activity of the control strips were calculated as a percentage per 1-h study period. In the same way, the effects of drugs or ions on the electromechanical properties
Table
2. Effects of Drugs and Ions on Circularly Muscle
of the preparations were expressed as a percentage change from those values recorded in the previous 1-h period. Statistical significance of these results was tested by comparison of the percentage effect observed with the drug or ion and the percentage effect observed during the same time period in the control preparations using the Student’s nonpaired t-test.
Results Control Preparations Circularly orientated strips. Spontaneous contractile activity of the circularly orientated strips was characterized by the omnipresence of low-amplitude (<300 mg wt) contractions at a frequency of 4.5-6.5 min-I. These contractions were associated with changes in the membrane potential occurring at the (slow waves) (9). No spikes were same frequency
recorded during the control studies (Figure 1). Timedependent changes in the contractile force and contractile/slow wave frequency recorded from the control strips are shown in Figure 1. Longitudinally orientated strips. Mechanical activity of the longitudinally orientated strips was marked by the occurrence of large-amplitude (>l.O g wt) contractions at a frequency of 0.4-1.4 min-‘. Electrically, a high-frequency oscillation in membrane potential was observed at a frequency of 25-35 min-’ during the majority of the recording time as
and Longitudinally Circularly orientated A% Force
Orientated
A% Frequency
of
-33 +- 5': -54 -)_5" -10 c 4
atropine lo-" M (n = 6) Tetrodotoxin lo-" M (n = 6) Progesterone 1O-5 M in presence
of
253 2 160b -31 c 90
-5k -42
of
58 k 19’ -39 2 23”
-92 -82
tetrodotoxin 10e6 M (n = 6) t [K+] 10 mM (n = 6) Progesterone
10M5 M in presence
t [K+] 10 mM (n = 6) Progesterone lo-’ M + t [K+] 10 mM + t [Ca’+] 5 mM (n = 6) t (K+] 10 mM + verapamil 5 X lo-‘M(n = 6) t [K+] 10 mM + verapami15 X 1Om6 M + t [Ca’+] 5 mM (n = 6)
-10 k 30 0
Results shown are percentage changes [A%) between the mean spontaneous Krebs-Ringer solution containing drugs or altered ion concentration, and indicates reduction). Statistical significance was assessed by comparison of solution containing drugs or altered ion concentrations, and those of control 0.005: c p < 0.001.
0
A% Force
Smooth
orientated
strips
A% Frequency -26 + 4" -51 2 9', 127
7 10
-62 " 13" -39 2 13"
-12 -49k
3 6
58 + 36’ -35 t 12b
-5 + 15 -39 2 190
0+5 -22
Colonic
-35? 2' -70 k 9'. -132 6
-1025 -1k 10 -14+ 10
105 It 34"
of Canine
Longitudinally
strips
Progesterone 10e5 M (n = 6) Atropine 10efiM (n = 6) Progesterone lo-' M in presence
Strips
512 2
-100"
0
38"
8 22"
19 2 23" -100"
0
contractile force and frequency of tissue superfused with the activity observed in the preceding hour (minus sign changes observed in tissue superfused with Krebs-Ringer preparations over the same time period. ’ p < 0.01; ’ p <
MOTILITY OF COLONIC SMOOTH MUSCLE
June 1985
previously described (10). Associated with each of the large-amplitude contractions was a characteristic electrical event that consisted of a series of largeramplitude changes in membrane potential at a frequency of 25-35 min -’ that sometimes appeared to bear spikes. In addition, there was evidence of contractile activity at a frequency of 25-35 min-’ superimposed on the large-amplitude contractions (Figure 2). Time-dependent changes in the contractile force and frequency recorded from the longitudinally orientated strips are shown in Figure 2.
Effects
of Drugs
1945
60
and Ions
Progesterone. Progesterone (10-7-10-4 M) produced a dose-dependent, reversible inhibition of the contractile force observed in both circularly and longitudinally orientated strips. The frequency of the large-amplitude contractions and their electrical correlates recorded from the longitudinally orientated strips were also decreased by progesterone in a dose-dependent manner; the high-frequency oscillation in membrane potential was still observed. In contrast, progesterone had no significant (p > 0.1) effect on the contractile or slow wave frequency recorded from the circularly orientated strips (Table 1; Figure 4). Atropine sulfate. Atropine sulfate (lo-” M) produced a significant (p < 0.001) decrease in the contractile force recorded from both circularly and longitudinally orientated muscle strips. The frequency of the large-amplitude contractions and their electrical correlates recorded from the longitudinally orientated strips was also significantly reduced (p < 0.001) by atropine at this dose; the high-frequency oscillation in membrane potential was still recorded. No significant effect (p > 0.5) of atropine on the contractile or slow wave frequency was observed. Progesterone (lop5 M), in the presence of atropine (10e6 M), exerted no significant effects (p > 0.1) on the contractile force or frequency of the circularly or longitudinally orientated strips (Table 2). Tetrodotoxin. Tetrodotoxin (lop6 M) produced a significant (p < 0.005) increase in the force of the contractions recorded from the circularly orientated muscle strips; spikes were recorded electrically phase-locked to the slow waves. In contrast, tetrodotoxin had a significant (p < 0.001) inhibitory effect on the force of contractions recorded from the longitudinally orientated strips, Tetrodotoxin had no significant effects (p > 0.1) on the frequency of contractions, or their electrical correlates, recorded from either the circularly or longitudinally orientated strips. Progesterone (10e5 M), in the presence of tetrodotoxin (lOme M), exerted inhibitory effects that
0
t--t+’ 0
1o-7
-20‘Co”t:ol (2nd hour)
,
1
10-6
Progesterone
1o-5
I
1o-4
1
M
Figure 4. The effect of progesterone on the force (@) and frequency (A] of spontaneous contractions recorded from circularly orientated (upper panel) and longitudinally orientated (lower panel] strips of canine colonic smooth muscle. The effects of superfusing progesterone-containing (lo-‘-lo-“ M) Krebs-Ringer solution for a period of 1 h are expressed as the percentage inhibition of that activity recorded from the strips during the previous 1-h control period. Comparable percentage changes in contractile force (0) and frequency (A] of control strips (n = 8) during the second hour of their superfusion with Krebs-Ringer solution are given.
were not significantly different (p > 0.05) from those observed in the absence of this neurotoxin (Table 2). High-potassium ion superfusate. High-K+ superfusate (10 mM) produced a significant (p < 0.001) increase in the contractile force recorded from both circularly and longitudinally orientated strips. Electrically, spikes were phase-locked to the slow waves recorded from the circularly orientated strips. The increased potassium concentration had no significant effect (p > 0.05) on the contractile frequency recorded from the circularly or longitudinally orientated muscle strips. In the presence of a high-K+ superfusate, the effects of progesterone (lop5 M) were not significantly different (p > 0.1) from those observed in standard Krebs-Ringer superfusate. The inhibitory effects of progesterone were completely
1946
GILL ET AL
blocked, however, by increasing the Caz+ concentration of the high-K+ superfusate to 5 mM [Table 2). Verapamil hydrochloride. Verapamil hydrochloride (5 x lop6 M) in the presence of a high-K+ superfusate completely inhibited all contractile activity in the longitudinally orientated strips. The contractile force and frequency of the circularly orientated strips were not significantly (p > 0.4) altered by verapamil at this concentration; complete inhibition of contractile, but not electrical slow wave, activity being observed in the presence of lop5 M verapamil. The effects of verapamil could not be overcome by increasing the Ca2+ concentration of the high-K+ superfusate to 5 mM (Table 2).
Discussion The concept that changes in the serum concentration of female steroid hormones may affect the contractility of gastrointestinal smooth muscle, both during pregnancy and the luteal phase of the menstrual cycle, has received considerable support particularly from recent studies of the lower esophageal sphincter (ll,12). Consistent with this concept, we have demonstrated that progesterone has a dosedependent inhibitory effect on both longitudinal and circular smooth muscle layers of the canine colon. Because a bath concentration of lop6 M progesterone was required to exert this effect, the physiologic significance of these results may be questioned. Several remarks may be made on this point. First, progesterone is generally considered to be physiologically important in the regulation of uterine muscle function in pregnancy. Yet, similar high concentrations of progesterone were necessary to affect uterine muscle strips in vitro (13). Second, variations in lower esophageal sphincter pressure observed both during pregnancy and the menstrual cycle have been correlated with the serum levels of progesterone. Moreover, similarly high concentrations of progesterone were necessary to affect lower esophageal sphincter smooth muscle strips in vitro (14). Third, there are difficulties in comparing bath concentrations of a hormone in vitro with the serum concentrations recorded in vivo. The effects of atropine on colonic smooth muscle strips suggest that the spontaneous release of acetylcholine is an important determinant not only of the force of the contractions recorded from both muscle layers but also of the frequency at which the longitudinal muscle layer contracts. In the presence of atropine, progesterone did not exert any significant inhibitory effects on either the circular or longitudinal muscle layers; there were no additive effects of these two drugs. The results of our studies with tetrodotoxin on
GASTROENTEROLOGY
Vol. 88. No. 6
colonic smooth muscle suggest that the predominant tonic neural discharge to the circular muscle layer is inhibitory, whereas that to the longitudinal muscle is excitatory. Despite these differences, in the presence of tetrodotoxin, progesterone exerted an inhibitory effect that was not different from that observed in the absence of the neurotoxin. These results suggest that progesterone may act either to decrease the spontaneous activity of tetrodotoxin-resistant excitatory nerve cells [type “2” (15)] or to affect the excitation-contraction coupling apparatus at the cell membrane or intracellular level. It was of interest that neither atropine nor tetrodotoxin appeared to affect either the electrical slow wave activity recorded from the circular smooth muscle or the high-frequency oscillation recorded from the longitudinal muscle. This confirms the findings of others (9,16,17) and supports the concept of these electrical activities being myogenic in origin. It has been suggested that changes of force development by smooth muscle may be directly related to changes in the cytoplasmic calcium concentration (18,191. Further, it is generally assumed that potassium depolarizes the cell membrane and permits the opening of voltage-dependent channels through which external calcium may enter the cells (19). The dependence of both the circular and longitudinal smooth muscle layers of the canine colon on external calcium was evidenced both by the increased contractile force observed in the presence of a highpotassium superfusate and also by the complete abolition of all contractile activity in the presence of verapamil, a calcium-entry inhibitor; this later effect could not be reversed by increasing the extracellular calcium concentration to 5 mM. It is noteworthy that the electrical slow wave activity recorded from the circular muscle layer remained unaffected by 10e5 M verapamil; others have used higher concentrations of verapamil (20) or calcium-free solutions (20,211 to abolish colonic slow waves. The use of a high-potassium superfusate provided a means of studying the effects of progesterone under circumstances in which the calcium flux across the cell membrane had been increased and the intracellular calcium concentration raised. Under these conditions, not only did progesterone exert significant inhibitory effects, but, in addition, these effects could be reversed by increasing the extracellular calcium concentration. It would seem likely that the inhibitory effects of progesterone were mediated by alterations of either the calcium flux across the cell membrane or the intracellular distribution of unsequestered calcium. In this regard, progesterone has been shown both to increase cytoplasmic adenosine triphosphatase-dependent calcium binding (22) and
June
also to hyperpolarize (23) uterine smooth muscle cells. Although this study does not provide a precise elucidation of the mechanism by which progesterone acts in canine colonic smooth muscle, the results are consistent with the concept that progesterone acts either at the cell membrane or intracellular level to modulate cytoplasmic calcium concentrations.
References 1. Barnes
2.
3.
4.
5.
6.
MOTILITY
1985
CG. Disorders of the alimentary tract. In: Barnes CG, ed. Medical disorders in obstetric practice. Oxford: Blackwell Scientific, 1974:145-56. Benson RC. Medical and surgical complications during pregnancy. In: Benson RC, ed. Current obstetric and gynecologic diagnosis and treatment. Los Altos: Lange Medical, 1980:82749. Greenhill JP, Friedman EA. Gastrointestinal disorders. In: Greenhill JP. Friedman EA. eds. Biological principles and modern practice of obstetrics. Philadelphia: WB Saunders, 1974:478-85. Winship DH. Gastrointestinal diseases. In: Burrow GN, Ferris TF, eds. Medical complications during pregnancy. Philadelphia: WB Saunders, 1975:275-350. Kumar D. In vitro inhibitory effect of progesterone on extrauterine human smooth muscle. Am J Obstet Gynecol 1962; 84:1300-4. Bruce LA, Behsudi FM. Progesterone effects on three regional gastrointestinal tissues. Life Sci 1979;25:729-34. Concannon PW, Hansel W, Visek WJ. The ovarian cycle of the bitch: plasma estrogen, LH and progesterone. Biol Reprod 1975:13:112-21. Kingma YJ. Lenhart J, Durdle NG, et al. Improved AgiAgCl pressure electrodes. Med Biol Eng Comput 1983;21:351-7. El-Sharkawy TY. Electrophysiological control of motility in canine colon. In: Duthie HL, ed. Gastrointestinal motility in health and disease. Lancaster: MTP Press. 1978:387-98.
OF COLONIC SMOOTH
MUSCLE
1947
and circular 10. Debski L, Bowes KL, Kingma YJ. Longitudinal muscle of canine colon have different and characteristic electrical and mechanical activities (abstr). Gastroenterol Clin Biol 1983;7:723. 11. Van Thiel DH, Gavaler JS, joshi SN, et al. Heartburn of pregnancy. Gastroenterology 1977;72:666-8. 12. Van Thiel DH, Gavaler JS, Stremple J. Lower esophageal sphincter pressure in women using sequential oral contraceptives. Gastroenterology 1976;71:232-4. 13. Csapo AI. The molecular basis of myometrical function and its disorders. In: Csapo AI, ed. La prophylaxie en gynecologie et obstretrique. Geneva: George, 1954. 14. Fisher RS, Roberts GS, Grabowski CJ, et al. Inhibition of lower esophageal sphincter circular smooth muscle by female sex hormones. Am J Physiol 1978;234:243-7. 15. Muruyama T, Suzuki T. Physiological properties of cultured Auerbach’s plexus cells. In: Weinbeck M. ed. Motility of the digestive tract. New York: Raven, 1982:109-14. 16. El-Sharkawy TY, MacDonald WM, Diamant NE. Electrophysiological control of motility in longitudinal (LML) and circular (CML) muscle layers of canine colon (abstr]. Gastroenterology 1980:78:1162. 17. Bulbring E, Kuriyama H. Effects of changes in the external sodium and calcium concentrations in spontaneous electrical activity in smooth muscle of guinea-pig taenia coli. J Physiol (Lond) 1963;166:29-58. 18. Somlyo AP, Somlyo AV, Shuman H, et al. Calcium and monovalent ions in smooth muscle. Fed Proc 1982;41:288390. 19. Casteels R. Droogmans G. Membrane potential and excitation-
20.
21. 22. 23.
contraction coupling in smooth muscle. Fed Proc 1982; 41:2879-82. Snape WJ Jr, Merkin S. The role of calcium in the colonic myoelectrical response to neurohumoral stimulation in the cat (abstr). Gastroenterology 1980;78:1264. Weinbeck M, Christensen J. Cationic requirements of colon slow waves in the cat. Am J Physiol 1971;220:513-9. Carsten ME. Hormonal regulation of myometrial calcium transport. Gynecol Invest 1974;5:269-75. Marshall JM. Effects of estrogen and progesterone on single uterine muscle fibres in the rat. Am J Physiol 1959:197:93542.
1985:88:1941-7
Effect of Progesterone on Canine Colonic Smooth Muscle R. C. GILL,
K. L. BOWES,
and Y. J. KINGMA
Surgical Medical Research Institute and Departments University of Alberta, Edmonton, Alberta, Canada
This study was undertaken to ascertain the effect and mode of action of progesterone on canine colonic smooth muscle in vitro. Circularly orientated strips of smooth muscle exhibited low-amplitude contractions at the slow wave frequency. Longitudinally orientated strips exhibited larger-amplitude contractions that were associated, electrically, with a series of “spikes” superimposed on a high-frequency oscillation in membrane potential. Progesterone reduced Ihe contractile force of both the circularly and JongitudinaJJy orientated strips and the contractile frequency of the longitudinally orientated strips in a dose-dependent manner; this inhibitory effect was observed in the presence of tetrodotoxin but not atropine. The stimulatory effects of a high-potassium superfusate were antagonized by progesterone and verapamil, the effect of progesterone being reversed by increasing the calcium concentration of the superfusate. We conclude that progesterone exerts an inhibitory effect on colonic smooth muscle and this may be mediated by changes in the cytoplasmic calcium concentration. Despite the fact that constipation has so frequently been described as a complication of pregnancy (l-4), the investigation of colonic smooth muscle and the effects of steroid hormones on its electrical and mechanical properties has received little attention. An early in vitro study [5) demonstrated that the spontaneous contractile activity of human colonic smooth muscle could be completely inhibited by high concentrations of progesterone. This inhibitory
Received August Address requests
9, 1984. Accepted December 13, 1984. for reprints to: Dr. Richard C. Gill, Department
of Surgery, University of Alberta, Edmonton, Alberta, Canada T6G 2G3. Richard C. Gill was a recipient of an award from the Alberta Heritage Foundation for Medical Research. This work was supported in part by the Medical Research Council of Canada. 0 1985 by the American Gastroenterological Association 0016-5085/85/$3.30
of Surgery
and Electrical
Engineering,
effect appeared to be specific for progesterone, as estradiol and corticosterone were without effect. A later study (6) demonstrated the possible physiologic significance of this effect in that colonic smooth muscle excised from male rats pretreated with progesterone had reduced contractility in vitro. The serum progesterone levels in these rats were considered to be in the normal physiologic range of the female. Neither of these studies, however, provided any information on the effects of progesterone on the different muscle layers of the colon, their electrical activities, or on the mode of action of progesterone. In this study, the effect of progesterone on the electrical and mechanical activities of the different muscle layers of the canine colon has been determined. The mechanism of action of progesterone has also been studied.
Materials and Methods In these experiments, tissue from male animals was used to avoid the complications that could arise due to fluctuating estrogen and progesterone levels that normally occur in the female (7). Strips of circularly and longitudinally orientated colonic smooth muscle were studied in vitro. The effects of progesterone alone and in the presence of atropine, tetrodotoxin, verapamil, and high-potassium superfusate also were determined.
Tissue
Preparation
Under pentobarbital anesthesia (30 mgikg i.v.), the abdomen was opened by a midline incision. A segment of proximal colon -7 cm long was removed immediately after clamping of the blood supply. The colonic segment was opened along the mesenteric border, carefully cleaned to prevent soiling of the muscle layers with fecal content, and pinned to the Sylgard surface of a dissecting dish filled with oxygenated Krebs-Ringer solution. The mucosa was removed by sharp dissection. Strips of smooth muscle (0.5 x 4.0 cm) were then cut in either the circular or longitudinal direction. Strips were mounted in a horizontal tissue chamber maintained at 37” * 1°C and continuously superfused with oxygenated Krebs-Ringer solution.
1942
GILL ET AL.
GASTROENTEROLOGY
Solutions “‘_,~~l~~~i~,J’u”~~~r~~~f
The Krebs-Ringer
contractile
z z LL Z-
Vol. 88, No. 6
and Drugs
standard superfusate solution containing
used the
was a modified following (mM):
Nat, 139.“; ,K’. 5.4; Ca2+, 2.5; ;g2’, 1.2; Cl-, 125.1; HCOs- 22 0, H,PO,-, 1.2; glucose 10.1. When equilibrated with 98% 02-5% CO2 gas mixture at 3X, this solution had a pH of 7.3-7.4. For a high-potassium solution, NaCl was replaced with sufficient KC1 to provide a final K+ concentration of 10 mM. For a high-calcium solution, CaC12 was added directly to the high-potassium KrebsRinger solution to provide a final Ca2+ concentration of 5.0
2;
(Jo-
5,” 0; xc
4020-* 0
Drugs used throughout this study were progesterone [Sigma Chemical Co., St. Louis, MO.), atropine sulfate (BDH Pharmaceuticals Ltd., London, England), tetrodotoxin (Sigma), and verapamil hydrochloride (ISOPTIN,
-1 f16%
Of17X
0219%
I
I
I
1
1
2
3
4
TIME
(hours)
Figure 1. Spontaneous contractile and surface electrical activity recorded from circularly orientated strips of canine colonic smooth muscle (n = 8). Each of the lowamplitude (<3oO mg wt) contractions was associated with a change in electrical potential referred to as a slow wave. The mean force of the contractions in each as a 10 min of a 4-h control ueriod was exoressed percentage of that observed in the first hour of study. The variation in mean contractile force for each subsequent hour was calculated (panel A]. In the same way, variation in contractile and electrical slow wave frequency recorded from these strips during the 4-h control period was calculated (panel B).
5 LI
~=@W@g#/#~&?@@ 80
?< $2
60-
;g
20-A 0 1601
I 2
I 1
-13f13%
-17?18%
-1Of13%
40-
14t23%
15+-21%
I 4
I 3
13?20%
1
1
Recording
Techniques
Electrical activity was recorded from the serosal (longitudinally orientated strips) or mucosal (circularly orientated strips) surface with a low-noise, low-drift silver-silver chloride pressure electrode (8). The reference electrode was a large silver-silver chloride electrode placed in the superfusing fluid. Contractile activity of the muscle strip was recorded with a force displacement transducer (Grass FT03, Grass Instrument Co., Quincy, Mass.). Recordings were made of the electrical and mechanical activity on a Beckman Dynograph R411 polygraph (Beckman Instruments, Inc., Fullerton, Calif.); for the electrical activity, filters were set to give a lowfrequency cutoff at 0.16 Hz and a high-frequency cutoff at 32.0 Hz.
2 Time
;
4
(hours)
Figure 2. Spontaneous contractile and surface electrical activity recorded from longitudinally orientated strips of canine colonic smooth muscle (n = 8). Each of the prolonged-duration, large-amplitude (>l.O g wt) contractions was associated, electrically, with a series of changes in potential at a frequency of 25-35 min-‘: evidence of contractile activity at this frequency was superimposed on the prolonged-duration contractions. The mean force of the prolonged-duration contractions in each 10 min of a 4-h control period was expressed as a percentage of that observed in the first hour of study. The variation in mean contractile force for each subsequent hour was calculated (panel A). In the same way, variation in contractile frequency recorded from these strips during the 4-h control periods was calculated (panel B).
June
MOTILITY
1985
Searle, Skokie, 111.).Progesterone was solubilized in ethanol to an initial concentration of z x IO-~-Z x IO-’ M, 0.5 ml of these solutions were diluted to 5.0 ml with propylene glycol per liter of Krebs-Ringer solution to provide final progesterone concentrations in the range 1.0 x 10-7-1.0 x 10m4 M.
Experimental
Protocol
Preliminary experiments demonstrated that the contractile activity of both circularly and longitudinally orientated strips was dependent on the degree of stretch (Figures 1-3). The force of the omnipresent, spontaneous contractions recorded from the circularly orientated strips increased linearly with the degree of stretch. However, when stretched to >220% of their initial length (LJ, defined as the length at which an increase in baseline force was first recorded, these strips demonstrated occasional large-amplitude contractions. The force of these large-amplitude contractions increased in parallel with the baseline tension as the strips were stretched further. In contrast, the force of the spontaneous, prolonged-duration contractions recorded from the longitudinally orientated strips increased with the degree of stretch until -160% of their
CIRCULARLY OFHENTATEDSTRIPS
OF COLONIC
SMOOTH
MUSCLE
1943
initial length. Once these strips were stretched beyond 170% of their initial length, the contractile force decreased; contractions ceased at 190% of the initial length. This decrease in active contractile force was marked by a rise in the baseline tension recorded. The contractile frequency of the circularly orientated strips remained unchanged by stretching when compared with the control preparations (Figure 1). The contractile frequency of the longitudinally orientated strips decreased with increasing length as compared with the control strips (Figure 2). At the commencement of each study, basal tension (corresponding to -115% of initial length) was applied to all strips. Each strip was set at this length for the remainder of the experiment. After a 30-min equilibration period, during which the strips were superfused with oxygenated Krebs-Ringer solution, the electrical and mechanical activities of the muscle strips were recorded for a period of 1 h as a control. Thereafter, except for “control” strips, drugs or ions were added directly to the superfusate as indicated in Tables 1 and 2. An additional control was performed using a superfusate containing the ethanol-propylene gylco1 solvent on two circularly orientated and two longitudinally orientated strips of colonic smooth muscle. The solvent was found to have no effect on the spontaneous contractile or electrical activity of these preparations.
LONGITUDINALLY ORIENTATED STRIPS
i!$l-$$fy[;I?~l*,,y*,,*,,F*.{~\ 100
140
160
220
260
100
120
140
160
160
I
% Li
Figure
2
I
I
I
I
I
100
120
140
160
160
% Li
3. Relationship between the percentage initial length (%L,) of circularly and longitudinally orientated strips of canine colonic smooth muscle and the baseline and active contractile force and contractile frequency recorded. The initial length L, was defined as the length at which an increase in baseline force was first recorded. The force of the spontaneous, low-amplitude contractions (A) recorded from the circularly orientated strips (n = 8) increased with the degree of stretch (panel A). At lengths >220% Li, the force of the occasional, large-amplitude contractions (m; note scale change) increased in parallel with the baseline force (0). Panel B shows the change in contractile frequency of the circularly orientated strips during these studies. The force of the spontaneous, prolonged-duration contractions (A] recorded from the longitudinally orientated strips (n = 8) increased until 160% Li (panel C). At lengths >17O% Li, the active contractile force decreased and became zero at 190% L,. Marked increases in baseline force (0) were noted at lengths ~70% L,. Panel D shows a decrease in the frequency of the prolonged-duration contractions with increasing strip length; the contractile frequency fell to zero at 19Ou/, L,.
1944
Table
GILL ET AL.
1.
Effect of Progesterone on Circularly and Longitudinally Orientated Strips of Canine Colonic
Smooth
Muscle
Circularly orientated strips
Progesterone concentration (M) 0, Control (n = 8) lo-' (n = lo-" (n = 10m5 (n = lo-* (n =
GASTROENTEROLOGY Vol. 88. No. 6
A% Force 0 + 19
A% Frequency -5LlZ
Longitudinally orientated strips A% Force
A% Frequency
-10 t 13
15 ir 21
6) 6)
-15 rt 7 -12 * 2 -2lk 20 -82 3
-16k 3 -26 + 8'
-14 2 3b -21 2 7"
6) 6)
-33 -+ 5d -10 It 5 -57213d -13 + 5
-35 * 2d -82 f 5d
-26 k 4d -37 t 3d
Results shown are percentage changes (A%) in mean spontaneous contractile force and frequency between the second hour of tissue superfusion and the preceding hour (minus sign indicates reduction). Statistical significance was assessed by comparison of changes observed in tissue superfused with progesterone-containing Krebs-Ringer solution and those of control preparations. n p < 0.05;b p < 0.01;c p < 0.005;d p < 0.001.
Analysis
of Data
Recordings of electrical and mechanical activity were analyzed visually. Each hour of study was divided into six lo-min periods that were analyzed separately; mean values for each hour of study were then calculated. Time-dependent alterations in the mechanical and electrical activity of the control strips were calculated as a percentage per 1-h study period. In the same way, the effects of drugs or ions on the electromechanical properties
Table
2. Effects of Drugs and Ions on Circularly Muscle
of the preparations were expressed as a percentage change from those values recorded in the previous 1-h period. Statistical significance of these results was tested by comparison of the percentage effect observed with the drug or ion and the percentage effect observed during the same time period in the control preparations using the Student’s nonpaired t-test.
Results Control Preparations Circularly orientated strips. Spontaneous contractile activity of the circularly orientated strips was characterized by the omnipresence of low-amplitude (<300 mg wt) contractions at a frequency of 4.5-6.5 min-I. These contractions were associated with changes in the membrane potential occurring at the (slow waves) (9). No spikes were same frequency
recorded during the control studies (Figure 1). Timedependent changes in the contractile force and contractile/slow wave frequency recorded from the control strips are shown in Figure 1. Longitudinally orientated strips. Mechanical activity of the longitudinally orientated strips was marked by the occurrence of large-amplitude (>l.O g wt) contractions at a frequency of 0.4-1.4 min-‘. Electrically, a high-frequency oscillation in membrane potential was observed at a frequency of 25-35 min-’ during the majority of the recording time as
and Longitudinally Circularly orientated A% Force
Orientated
A% Frequency
of
-33 +- 5': -54 -)_5" -10 c 4
atropine lo-" M (n = 6) Tetrodotoxin lo-" M (n = 6) Progesterone 1O-5 M in presence
of
253 2 160b -31 c 90
-5k -42
of
58 k 19’ -39 2 23”
-92 -82
tetrodotoxin 10e6 M (n = 6) t [K+] 10 mM (n = 6) Progesterone
10M5 M in presence
t [K+] 10 mM (n = 6) Progesterone lo-’ M + t [K+] 10 mM + t [Ca’+] 5 mM (n = 6) t (K+] 10 mM + verapamil 5 X lo-‘M(n = 6) t [K+] 10 mM + verapami15 X 1Om6 M + t [Ca’+] 5 mM (n = 6)
-10 k 30 0
Results shown are percentage changes [A%) between the mean spontaneous Krebs-Ringer solution containing drugs or altered ion concentration, and indicates reduction). Statistical significance was assessed by comparison of solution containing drugs or altered ion concentrations, and those of control 0.005: c p < 0.001.
0
A% Force
Smooth
orientated
strips
A% Frequency -26 + 4" -51 2 9', 127
7 10
-62 " 13" -39 2 13"
-12 -49k
3 6
58 + 36’ -35 t 12b
-5 + 15 -39 2 190
0+5 -22
Colonic
-35? 2' -70 k 9'. -132 6
-1025 -1k 10 -14+ 10
105 It 34"
of Canine
Longitudinally
strips
Progesterone 10e5 M (n = 6) Atropine 10efiM (n = 6) Progesterone lo-' M in presence
Strips
512 2
-100"
0
38"
8 22"
19 2 23" -100"
0
contractile force and frequency of tissue superfused with the activity observed in the preceding hour (minus sign changes observed in tissue superfused with Krebs-Ringer preparations over the same time period. ’ p < 0.01; ’ p <
MOTILITY OF COLONIC SMOOTH MUSCLE
June 1985
previously described (10). Associated with each of the large-amplitude contractions was a characteristic electrical event that consisted of a series of largeramplitude changes in membrane potential at a frequency of 25-35 min -’ that sometimes appeared to bear spikes. In addition, there was evidence of contractile activity at a frequency of 25-35 min-’ superimposed on the large-amplitude contractions (Figure 2). Time-dependent changes in the contractile force and frequency recorded from the longitudinally orientated strips are shown in Figure 2.
Effects
of Drugs
1945
60
and Ions
Progesterone. Progesterone (10-7-10-4 M) produced a dose-dependent, reversible inhibition of the contractile force observed in both circularly and longitudinally orientated strips. The frequency of the large-amplitude contractions and their electrical correlates recorded from the longitudinally orientated strips were also decreased by progesterone in a dose-dependent manner; the high-frequency oscillation in membrane potential was still observed. In contrast, progesterone had no significant (p > 0.1) effect on the contractile or slow wave frequency recorded from the circularly orientated strips (Table 1; Figure 4). Atropine sulfate. Atropine sulfate (lo-” M) produced a significant (p < 0.001) decrease in the contractile force recorded from both circularly and longitudinally orientated muscle strips. The frequency of the large-amplitude contractions and their electrical correlates recorded from the longitudinally orientated strips was also significantly reduced (p < 0.001) by atropine at this dose; the high-frequency oscillation in membrane potential was still recorded. No significant effect (p > 0.5) of atropine on the contractile or slow wave frequency was observed. Progesterone (lop5 M), in the presence of atropine (10e6 M), exerted no significant effects (p > 0.1) on the contractile force or frequency of the circularly or longitudinally orientated strips (Table 2). Tetrodotoxin. Tetrodotoxin (lop6 M) produced a significant (p < 0.005) increase in the force of the contractions recorded from the circularly orientated muscle strips; spikes were recorded electrically phase-locked to the slow waves. In contrast, tetrodotoxin had a significant (p < 0.001) inhibitory effect on the force of contractions recorded from the longitudinally orientated strips, Tetrodotoxin had no significant effects (p > 0.1) on the frequency of contractions, or their electrical correlates, recorded from either the circularly or longitudinally orientated strips. Progesterone (10e5 M), in the presence of tetrodotoxin (lOme M), exerted inhibitory effects that
0
t--t+’ 0
1o-7
-20‘Co”t:ol (2nd hour)
,
1
10-6
Progesterone
1o-5
I
1o-4
1
M
Figure 4. The effect of progesterone on the force (@) and frequency (A] of spontaneous contractions recorded from circularly orientated (upper panel) and longitudinally orientated (lower panel] strips of canine colonic smooth muscle. The effects of superfusing progesterone-containing (lo-‘-lo-“ M) Krebs-Ringer solution for a period of 1 h are expressed as the percentage inhibition of that activity recorded from the strips during the previous 1-h control period. Comparable percentage changes in contractile force (0) and frequency (A] of control strips (n = 8) during the second hour of their superfusion with Krebs-Ringer solution are given.
were not significantly different (p > 0.05) from those observed in the absence of this neurotoxin (Table 2). High-potassium ion superfusate. High-K+ superfusate (10 mM) produced a significant (p < 0.001) increase in the contractile force recorded from both circularly and longitudinally orientated strips. Electrically, spikes were phase-locked to the slow waves recorded from the circularly orientated strips. The increased potassium concentration had no significant effect (p > 0.05) on the contractile frequency recorded from the circularly or longitudinally orientated muscle strips. In the presence of a high-K+ superfusate, the effects of progesterone (lop5 M) were not significantly different (p > 0.1) from those observed in standard Krebs-Ringer superfusate. The inhibitory effects of progesterone were completely
1946
GILL ET AL
blocked, however, by increasing the Caz+ concentration of the high-K+ superfusate to 5 mM [Table 2). Verapamil hydrochloride. Verapamil hydrochloride (5 x lop6 M) in the presence of a high-K+ superfusate completely inhibited all contractile activity in the longitudinally orientated strips. The contractile force and frequency of the circularly orientated strips were not significantly (p > 0.4) altered by verapamil at this concentration; complete inhibition of contractile, but not electrical slow wave, activity being observed in the presence of lop5 M verapamil. The effects of verapamil could not be overcome by increasing the Ca2+ concentration of the high-K+ superfusate to 5 mM (Table 2).
Discussion The concept that changes in the serum concentration of female steroid hormones may affect the contractility of gastrointestinal smooth muscle, both during pregnancy and the luteal phase of the menstrual cycle, has received considerable support particularly from recent studies of the lower esophageal sphincter (ll,12). Consistent with this concept, we have demonstrated that progesterone has a dosedependent inhibitory effect on both longitudinal and circular smooth muscle layers of the canine colon. Because a bath concentration of lop6 M progesterone was required to exert this effect, the physiologic significance of these results may be questioned. Several remarks may be made on this point. First, progesterone is generally considered to be physiologically important in the regulation of uterine muscle function in pregnancy. Yet, similar high concentrations of progesterone were necessary to affect uterine muscle strips in vitro (13). Second, variations in lower esophageal sphincter pressure observed both during pregnancy and the menstrual cycle have been correlated with the serum levels of progesterone. Moreover, similarly high concentrations of progesterone were necessary to affect lower esophageal sphincter smooth muscle strips in vitro (14). Third, there are difficulties in comparing bath concentrations of a hormone in vitro with the serum concentrations recorded in vivo. The effects of atropine on colonic smooth muscle strips suggest that the spontaneous release of acetylcholine is an important determinant not only of the force of the contractions recorded from both muscle layers but also of the frequency at which the longitudinal muscle layer contracts. In the presence of atropine, progesterone did not exert any significant inhibitory effects on either the circular or longitudinal muscle layers; there were no additive effects of these two drugs. The results of our studies with tetrodotoxin on
GASTROENTEROLOGY
Vol. 88. No. 6
colonic smooth muscle suggest that the predominant tonic neural discharge to the circular muscle layer is inhibitory, whereas that to the longitudinal muscle is excitatory. Despite these differences, in the presence of tetrodotoxin, progesterone exerted an inhibitory effect that was not different from that observed in the absence of the neurotoxin. These results suggest that progesterone may act either to decrease the spontaneous activity of tetrodotoxin-resistant excitatory nerve cells [type “2” (15)] or to affect the excitation-contraction coupling apparatus at the cell membrane or intracellular level. It was of interest that neither atropine nor tetrodotoxin appeared to affect either the electrical slow wave activity recorded from the circular smooth muscle or the high-frequency oscillation recorded from the longitudinal muscle. This confirms the findings of others (9,16,17) and supports the concept of these electrical activities being myogenic in origin. It has been suggested that changes of force development by smooth muscle may be directly related to changes in the cytoplasmic calcium concentration (18,191. Further, it is generally assumed that potassium depolarizes the cell membrane and permits the opening of voltage-dependent channels through which external calcium may enter the cells (19). The dependence of both the circular and longitudinal smooth muscle layers of the canine colon on external calcium was evidenced both by the increased contractile force observed in the presence of a highpotassium superfusate and also by the complete abolition of all contractile activity in the presence of verapamil, a calcium-entry inhibitor; this later effect could not be reversed by increasing the extracellular calcium concentration to 5 mM. It is noteworthy that the electrical slow wave activity recorded from the circular muscle layer remained unaffected by 10e5 M verapamil; others have used higher concentrations of verapamil (20) or calcium-free solutions (20,211 to abolish colonic slow waves. The use of a high-potassium superfusate provided a means of studying the effects of progesterone under circumstances in which the calcium flux across the cell membrane had been increased and the intracellular calcium concentration raised. Under these conditions, not only did progesterone exert significant inhibitory effects, but, in addition, these effects could be reversed by increasing the extracellular calcium concentration. It would seem likely that the inhibitory effects of progesterone were mediated by alterations of either the calcium flux across the cell membrane or the intracellular distribution of unsequestered calcium. In this regard, progesterone has been shown both to increase cytoplasmic adenosine triphosphatase-dependent calcium binding (22) and
June
also to hyperpolarize (23) uterine smooth muscle cells. Although this study does not provide a precise elucidation of the mechanism by which progesterone acts in canine colonic smooth muscle, the results are consistent with the concept that progesterone acts either at the cell membrane or intracellular level to modulate cytoplasmic calcium concentrations.
References 1. Barnes
2.
3.
4.
5.
6.
MOTILITY
1985
CG. Disorders of the alimentary tract. In: Barnes CG, ed. Medical disorders in obstetric practice. Oxford: Blackwell Scientific, 1974:145-56. Benson RC. Medical and surgical complications during pregnancy. In: Benson RC, ed. Current obstetric and gynecologic diagnosis and treatment. Los Altos: Lange Medical, 1980:82749. Greenhill JP, Friedman EA. Gastrointestinal disorders. In: Greenhill JP. Friedman EA. eds. Biological principles and modern practice of obstetrics. Philadelphia: WB Saunders, 1974:478-85. Winship DH. Gastrointestinal diseases. In: Burrow GN, Ferris TF, eds. Medical complications during pregnancy. Philadelphia: WB Saunders, 1975:275-350. Kumar D. In vitro inhibitory effect of progesterone on extrauterine human smooth muscle. Am J Obstet Gynecol 1962; 84:1300-4. Bruce LA, Behsudi FM. Progesterone effects on three regional gastrointestinal tissues. Life Sci 1979;25:729-34. Concannon PW, Hansel W, Visek WJ. The ovarian cycle of the bitch: plasma estrogen, LH and progesterone. Biol Reprod 1975:13:112-21. Kingma YJ. Lenhart J, Durdle NG, et al. Improved AgiAgCl pressure electrodes. Med Biol Eng Comput 1983;21:351-7. El-Sharkawy TY. Electrophysiological control of motility in canine colon. In: Duthie HL, ed. Gastrointestinal motility in health and disease. Lancaster: MTP Press. 1978:387-98.
OF COLONIC SMOOTH
MUSCLE
1947
and circular 10. Debski L, Bowes KL, Kingma YJ. Longitudinal muscle of canine colon have different and characteristic electrical and mechanical activities (abstr). Gastroenterol Clin Biol 1983;7:723. 11. Van Thiel DH, Gavaler JS, joshi SN, et al. Heartburn of pregnancy. Gastroenterology 1977;72:666-8. 12. Van Thiel DH, Gavaler JS, Stremple J. Lower esophageal sphincter pressure in women using sequential oral contraceptives. Gastroenterology 1976;71:232-4. 13. Csapo AI. The molecular basis of myometrical function and its disorders. In: Csapo AI, ed. La prophylaxie en gynecologie et obstretrique. Geneva: George, 1954. 14. Fisher RS, Roberts GS, Grabowski CJ, et al. Inhibition of lower esophageal sphincter circular smooth muscle by female sex hormones. Am J Physiol 1978;234:243-7. 15. Muruyama T, Suzuki T. Physiological properties of cultured Auerbach’s plexus cells. In: Weinbeck M. ed. Motility of the digestive tract. New York: Raven, 1982:109-14. 16. El-Sharkawy TY, MacDonald WM, Diamant NE. Electrophysiological control of motility in longitudinal (LML) and circular (CML) muscle layers of canine colon (abstr]. Gastroenterology 1980:78:1162. 17. Bulbring E, Kuriyama H. Effects of changes in the external sodium and calcium concentrations in spontaneous electrical activity in smooth muscle of guinea-pig taenia coli. J Physiol (Lond) 1963;166:29-58. 18. Somlyo AP, Somlyo AV, Shuman H, et al. Calcium and monovalent ions in smooth muscle. Fed Proc 1982;41:288390. 19. Casteels R. Droogmans G. Membrane potential and excitation-
20.
21. 22. 23.
contraction coupling in smooth muscle. Fed Proc 1982; 41:2879-82. Snape WJ Jr, Merkin S. The role of calcium in the colonic myoelectrical response to neurohumoral stimulation in the cat (abstr). Gastroenterology 1980;78:1264. Weinbeck M, Christensen J. Cationic requirements of colon slow waves in the cat. Am J Physiol 1971;220:513-9. Carsten ME. Hormonal regulation of myometrial calcium transport. Gynecol Invest 1974;5:269-75. Marshall JM. Effects of estrogen and progesterone on single uterine muscle fibres in the rat. Am J Physiol 1959:197:93542.