Digestive Diseases and Sciences, Vol. 31, No. 5 (May 1986), pp. 513-518

Influence of Progesterone on Guinea Pig Gallbladder Motility in vitro

MARJORIE DAVIS, MS, and JAMES P. RYAN, PhD

Experiments were designed to examine the in vitro effect of progesterone (1.6 x I0 -s M; 8 x 10 .5 M) on the tone and contractility of guinea pig gallbladder smooth muscle. Muscle strips from animals of both sexes were stimulated with acetylcholine, cholecystokinin-octapeptide, histamine, and potassium prior to and 15 rain after exposure to progesterone. The results were as follows: (1) progesterone at all doses studied completely abolished spontaneous phasic contractions; (2) progesterone produced a concentration---dependent decrease in resting tension; (3) the hormone significantly decreased the maximal contractile response to each o f the agonists but had no effect on either the EDso of the acetylcholine and cholecystokinin dose-response curves; and (4) estrogen priming is not necessary to demon- strate an inhibitory effect o f progesterone. The data suggest that progesterone can directly affect the force-generating capacity of gallbladder smooth muscle both at rest and in response to stimulation. However, because of the relatively high concentration o f hormone used, the physiological significance of the data must remain speculative.

It has been noted that pregnancy is associated with alterations in gallbladder motility in vivo (1-6). The biliary stasis which develops during this period is characterized by an increase in the fasting and residual volumes and by a decrease in emptying capacity (7-9). It has been suggested that these changes reflect a decrease in the tone and contrac- tility of gallbladder smooth muscle (7-10). Little is known about the factors responsible for the appar- ent alterations in muscle behavior. The most com- monly held belief is that the hypotony develops as a consequence of progesterone-mediated changes in muscle function (7). The hypothesis is based on the evidence that progesterone affects the electrical and mechanical[ properties of other gastrointestinal tis-

Manuscript received September i2, 1984; revised manuscript received April 3, 1985; accepted June 27, 1985.

From the Department of Physiology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140.

This study was supported in part by grant HD 16132 from the National Institute of Child Health and Human Development.

Address for reprint requests: Dr. James P. Ryan, Department of Physiology, Temple University School of Medicine, Philadel- phia, Pennsylvania 19140.

sues (11-15) and on the fact that increases in serum progesterone are associated with reduced gallblad- der motility both in vivo and in vitro (16-20).

To date, no studies have examined the direct effect of the steroid hormone on gallbladder smooth muscle. The present study was designed to address this point by examining the effect of progesterone on the tone and contractility of isolated strips of guinea pig gallbladder smooth muscle.

MATERIALS AND METHODS

Adult guinea pigs (500-600 g) of both sexes were utilized in this study. Preliminary studies indicated that no differences existed between the sexes with respect to either the contractile responsiveness to the agonists or to the sensitivity to progesterone. All animals were fasted overnight prior to being sacrificed by cervical dislocation. The gallbladders were opened along their longitudinal axis and rinsed with a warmed, aerated Krebs-Ringer solution which contained (in mM): NaC1, 117; KC1, 4.6; MgClz'6H20, 2.1; CaClz.2H20, 2.5; NazHPO4, 1.2; NaHCO3, 15.5; glucose, 11.5, buffered at pH 7.4. Following removal of the mucosa by blunt dissection, muscle strips (5 x 10 ram) were prepared from the body of the gallbladder by cutting parallel to the long axis of the tissue.

Digestive Diseases and Sciences, Vol. 31, No. 5 (May 1986) 0163-2116/86/0500-0513505.00/0 �9 1986 Plenum Publishing Corporation

513

The muscle strips were mounted in separate 20-ml tissue baths which were filled with the buffer solution. One end of each strip was connected via fine thread to an external force transducer (Grass FT.03C) while the other end was clipped to a metal rod that could be raised or lowered by the adjustment of a screw micrometer. After a 1-hr equilibration period, the length of each muscle strip was increased in 1-mm increments until the maximal contractile response to acetylcholine (10 -5 M) was achieved. The muscle strips were maintained at their optimal length for active tension development for the duration of each experiment. Permanent records of the changes in isometric tension which occurred were ob- tained using a Grass multichannel recorder.

The tissues were examined for their contractile re- sponse to acetylcholine (ACh), the octapeptide of chole- cystokinin (CCK-OP), histamine (4.5 ~M), and potassium (25 mM) alone and in the presence of progesterone (Calbiochem, La Jolla, California). Stock solutions of the agonists were diluted with buffer and added to the tissue baths in microliter amounts to achieve the final molar concentrations reported. When the contractile response to increased extracellular potassium was evaluated, the NaC1 concentration of the buffer solution was reduced proportionately in order to maintain 0smolality. For ACh and CCK-OP, individual doses were given in random order; no cumulative dose-response curves were con- structed.

Progesterone was dissolved in propylene glycol. Initial studies Confirmed that the solvent had no independent effect on the contractile behavior of the tissues. The hormone was added to the tissue baths 15 min prior to the addition of an agonist. Preliminary studies indicated that this time period was necessary to allow for complete expression of the inhibitory effect of the hormone on basal activity. The inhibitory effects of progesterone were fully reversible upon washing of the tissues in hormone- free buffer. Both the resting tone and full contractile response to agonist stimulation returned to control Values within 10 rain. The contractile responses obtained in the presence of progesterone were expressed as a percentage of the control response. All data were analyzed statisti- cally using the Student's t test for paired observation.

Because it is well documented that the inhibitory effect of progesterone On uterine smooth muscle is augmented by prior exposure of the tissue to estrogen (21), experi- ments were performed to determine whether a similar estrogen dependency was evident for gallbladder smooth muscle. Five adult, sexually mature females were ovariectomized and two weeks after Surgery pretreated with estrogen (estradiol valerate, 400 txg/kg). Each animal received an injection once a day for seven days. Twenty- four hours following the last pretreatment, the tissues were prepared and examined for the effect of progester- one (8 • 10 -5 M) on basal tension and acetylcholine- induced contractions.

RESULTS

Effect of Progesterone on Basal Activity. Proges- terone at all concentra t ions used in this exper iment

514

DAVIS AND RYAN

~. 2 .0 Z O

z 1.0 I--

(P) a X 1 o'SM

b g ~'o T I M E ( m i n )

Fig i . Effect of progesterone on resting tone and spontaneous contractile activity. The progesterone effect reached maximum within 15 min. Estrogen pretreatment did not affect the response to progesterone.

had an inhibitory effect on the phasic contracti le activity of the muscle strips (Figure 1). Progester- one also produced a gradual reduct ion in tone that reached a max imum level of inhibition within 15 min. Figure 2 summarizes quanti tat ively the effect of progesterone on basal tension. The' decrease in tone was significant and dose-related and varied f rom 32 - 5% for proges terone at 8 x 10 -6 M to 55 -+ 10% at a proges terone concentra t ion of 8 • 10 -5 M. Increasing the hormone concentra t ion above the latter value had no additional effect on the magni- tude of the inhibition. Concentra t ions of progester- one less than 8 • 10 -6 M had no effect on either the phasic or tonic activity of the muscle strips.

Effect of Progesterone on Agonist-Induced Con- tractions. Proges terone at 1.6 • 10 -5 M had a slight inhibitory effect on the contracti le response to ACh, although the results did not reach statistical signif- icance. In contrast , p roges terone at 8 • 10 -5 M significantly decreased the contracti le responses at each point along the dose - r e sponse curve (Figure

1 0 0 - Z 2 -

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ca 6 0 - m

z

uJ r

i,iJ iv, o ir a

4 0

20

"I-

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I TI 1.6xlO "5 8x10 -5

CONCENTRATION (M)

Fig 2. Effect of progesterone on resting tension. Progesterone produced a significant and dose-related decrease in tone in aU muscle strips. Each value represents the mean +- SO of at least 15 determinations.

Digestive Diseases and Sciences, Vol. 31, No. 5 (May 1986)

PROGESTERONE AND GALLBLADDER MOTILITY

IO0 --7 ~ /~CH ~ _/#Tk~.~ ~0 ~ ACH + P(SXIO-6(M)

h , - ~ t , ACH +P(SXIO'5(M] 8 o

=~ eo

4 0

z ~ zo

r O- I I I I

iO-9 iO-8 iO-7 iO-6 iO-5 iO-4 IO-3

ACETYLCHOLINE (M)

Fig 3. Effect of proges terone on ACh- induced contractions. The ho rmone was given 15 rain prior to addition of the agonist. All resul ts are expressed as a percent of the maximal control response . Each point represents the mean - SD of at least 10 determinat ions .

I 0 0 - - H O P - C C K O z ,I,---A Op CCK+P(axIo 5M) / ~

:7 8 0 - Y

6 0 - )<

4 0 - LL 0 I"-- z 2 0 LLI s n" W ~_ -y/

iO-12 IO-,~ io-IO 10-9 iO-8 10 -7

OP-CCK (M) Fig 4. Effect of proges terone on CCK-OP- induced contract ions. The data are expressed as descr ibed in Figure 2.

3). The maximal contractile response (0.39 + 0.03 kg/cm 2) was decreased by 24.6 --- 3.2% to 0,30 -+ 0.04 kg/cm 2 in the presence of progesterone (P < 0.01). Although progesterone affected the magni- tude of the contractions, normalization of each curve to 100% of its respective maximal response yielded virtually identical dose-response relation- ships (data, not shown). The EDs0 for the control curve was 5.7 --- 0.6 x 10 -7 M, 7.0 --- 0.8 x 10 -7 M for ACh plus progesterone (1.6 x 10 -5 M), and 8.6 +-- 1.0 x 10 .7 M for ACh plus progesterone (8 x 10 -5 M).

Progesterone (8 x 10 -5 M) also significantly decreased CCK-OP-induced contractions at each point along; the dose-response curve (Figure 4). The response to a maximally effective concentration of CCK-OP (80 nM) was decreased 22.6 +- 6.1% from 0.42 +- 0.04 kg/cm 2 to 0.33 -+ 0.03 kg/cm 2. Normal- ization of the individual data revealed virtually identical dose-response relationships (data not shown).

The inhibitory effect of progesterone (8 x 10 -5 M) on the contractile responses to maximally effec- tive concentrations of potassium (25 mM) and his- tamine (4.5 ~M) is summarized in Figure 5. Potas- sium-induc, ed contractions were decreased by 61.8 --- 12.7% (mean --- so); histamine-induced contrac- tions were reduced by 35.6 - 13.2%.

Effect ot' Estrogen Pretreatment on Progesterone Inhibition. Estrogen pretreatment did not augment the inhibitory effect of progesterone on either the resting tone or the contractile response of the

tissues to ACh. The data are summarized in Table 1. The effectiveness of the hormone pretreatment was determined by comparing the uterine weights of tissues from untreated animals (1.12 -+ 0.37) with those from estrogen pretreated animals (0.98 -+ 0.15). The values do not differ significantly from one another.

DISCUSSION

Pregnancy decreases the in vivo contractile activ- ity of the gallbladder (1-9). The increase in fasting volume which occurs during this period is believed to develop as the result of a decrease in gallbladder

1 0 0 -

..J O lr ~- 80 Z 0 r

=~ 6 0 - O Ir i, nl r 4 0 -

uJ

(J ,,, 2 0 - Q #

KCL (25mM)

HISTAMINE (4.5 juM)

Fig 5. Effect of prol es terone on potass ium- and his tamine- induced contract ions. The data are expressed as the mean --- SD of at least 15 determinat ions .

Digestive Diseases and Sciences, Vol. 31, No. 5 (May 1986) 515

DAVIS AND RYAN

TABLE 1. EFFECT OF ESTROGEN PRETREATMENT ON PROGESTERONE INHIBITION

Decrease Decrease maximal Uterine Animal group tone (%) ACh response (%) weight (g)

Control 55.2 --- 10 24.6 --- 3.2 1.12 --- 0.37 Es t rogen 53.6 + 7 25.4 --- 4.8 0.98 -4- 0.15

pretreated*

*Estradiol valerate, 400 ixg/kg/day x 7 days; progesterone, 8 x 10 -5 M.

smooth muscle tone (7). The impaired emptying capacity is thought to reflect a decrease in the contractile responsiveness to neural and hormonal stimulation (7, 19, 20). It has been proposed that the biliary stasis which develops is directly related to the increase in serum progesterone during preg- nancy (7-9). This hypothesis is based on the obser- vations that pretreatment of animals with progester- one alters the electromechanical properties of several gastrointestinal smooth muscle organs, including the gallbladder (10-15, 18). Whether the progesterone- related inhibition of gallbladder motility is the result of a direct action of the steroid hormone on the contractile process or whether intermediary factors are responsible has not been examined.

In the present study, progesterone demonstrated a direct inhibitory effect on both the phasic activity and tone of the unstimulated muscle strips. The inhibitory effects were reversible despite prolonged and repeated exposure of the tissues to the hor- mone. Thus it does not seem likely that the results are due to hormone or carrier-related tissue injury. Progesterone also has been reported to inhibit the in vitro phasic contractions of the antrum, small bowel, and colon (12, 15). In contrast, progesterone has no effect on the resting tone in these tissues. The reasons for the different response of gallbladder smooth muscle are unknown. However, should a similar alteration in tone occur in vivo during preg- nancy, it might help explain the increased fasting volume which occurs. Studies which directly exam- ine the pressure-volume characteristics of the gall- bladder during pregnancy are needed to test this possibility.

This study did not address the question as to how the steroid hormone affects the basal activity of the muscle strips in vitro. We have recently shown, however, that both the tone and phasic contractions of the gallbladder in vitro are highly dependent upon the presence and influx of extracellular calcium (22). Each is markedly reduced following incuba-

tion of the tissues in a calcium-free medium, or upon addition of the calcium channel blocker Verapamil to the bathing medium. It is tempting to speculate that progesterone also affects the basal activity of the tissues by reducing the transmem- brane influx of extraceUular calcium. Calcium flux studies are needed to further investigate this point.

Progesterone also decreased the magnitude of the contractile responses to ACh and CCK-OP without affecting the sensitivity of the tissues to the ago- nists. A decrease in the efficacy of an agonist with no apparent change in tissue sensitivity would be expected if progesterone induced an alteration in the contractile process that was independent of the stimulus applied. In the present study, progesterone also affected the contractile responses to histamine and potassium. It appears, therefore, that proges- terone can antagonize the contractile responses to an agonist that utilizes a specific membrane recep- tor (ACh, CCK-OP, histamine), as well as to an agonist that acts independent of membrane recep- tors (potassium depolarization). It has recently been reported that potassium-stimulated gallblad- der contraction depends exclusively upon the influx of extracellular calcium (22), whereas the ACh-, CCK-OP-, and histamine-induced contractions ap- pear to utilize calcium from both extracellular and intracellular calcium pools (23-25). Considering these observations, it seems reasonable to suggest that progesterone may have decreased the contractile re- sponsiveness to the agonists utilized in this study by reducing the influx of extraceUular calcium. How the hormone might affect calcium influx is not known. Furthermore the data do not rule out the possibility that progesterone also interferes with the release of bound intracellular calcium. Such an effect has been reported to occur in uterine smooth muscle (26).

It is of interest that estrogen priming did not augment the inhibitory effects of progesterone. This observation is in contrast to uterine smooth muscle where it is well documented that prior exposure of the tissue to estrogen is necessary to fully demon- strate progesterone's effects on motility (21, 26). Progesterone also has been reported to decrease the contractile activity of other nonuterine smooth muscles in the absence of prior exposure of the tissues to estrogen. Bortoff et al (27) reported that the disruptive effect of progesterone on intestinal slow-wave propagation is not altered by estrogen pretreatment. Fisher et al (28) found no difference in the inhibitory effect of progesterone on lower esophageal sphincter smooth muscle when compar-

516 Digestive Diseases and Sciences, Vol. 31, No. 5 (May 1986)

P R O G E S T E R O N E AND G A L L B L A D D E R MOTILITY

ing tissues taken from male and female oppossums. In addition, Hettiaratchi and Pickford (29) reported that pretreatment of rats with estrogen did not diminish or enhance the inhibitory effect of proges- terone on the pressor action of angiotensin. The evidence suggests that nonuterine and uterine smooth muscle differ in their dependency upon estrogen priming as a requisite for demonstrating an inhibitory action of progesterone.

In summary, progesterone has been shown to have a direct inhibitory effect on both the basal and agonist-stimulated contractile activity of the guinea pig gallbladder in vitro. The data would suggest that the sluggish behavior of the gallbladder during preg- nancy is the result of progesterone-induced alter- ations in excitation-contraction coupling. How- ever, because the concentrations of hormone used in this study (16-80 txM) were higher than normally found in the plasma (I ~zM), the physiological significance of the data must remain speculative. It is possible the results reflect a pharmacological action of the hormone. This concern is further highlighted by the recent observation that preg- nancy decreases the in vitro contractile response of the guinea pig gallbladder to ACh and CCK-OP, but it has no ,effect on potassium-induced contractions (20). In the present study progesterone decreased the contractile reslbonse to each of the agonists. Thus, it appears that real differences exist between the acute, in vitro effects, and the chronic, in vivo effects of the hormone. This suggests that caution must be exercised when extrapolating in vitro ob- servation to in vivo phenomena.

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3. Gerdes MM, Boyden EA: The rate of emptying of the human gallbladder in pregnancy. Surg Gynecol Obstet 66:145-156, 1938

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5. Stauffer RA, Adams A, Wygal J, Lavery JP: Gallbladder disease in pregnancy. Am J Obstet Gynecol 144:661-664, 1982

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13. Bortoff A, Morello E, Mistretta P: Effects of progesterone and 17-OH progesterone on intestinal slow wave propaga- tion. In Gastrointestinal Motility. J Christenson (ed). New York, Raven Press, 1980, pp 387-393

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15. Yoshida T, Mori T: The effects of sex steroids upon the motility of isolated small intestine of rabbits. Acta Obstet Gynecol Jpn 16:252-257, 1969

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17. Smith JJ, Pomeranc MM, Ivy AC: The influence of preg- nancy and sex hormones on gallbladder motility in the guinea pig. Am J Physiol 132:12%140, 1941

18. Ryan JP, Pellecchia D: Effect of progesterone pretreatment on guinea pig gallbladder motility in vitro. Gastroenterology 83:81-83, 1982

19. Zhu NG, Fried GM, Greely G, Thompson JC: Effect of estrogen and progesterone on cholecystokinin stimulated gallbladder contraction in vitro. Gastroenterology 82:1218A, 1982

20. RyanJP: The effect ofpregnancy on gallbladder contractility in the guinea pig. Gastroenterology 87:674678, 1984

21. Csapo A: The in vivo and in vitro effects of estrogen and progesterone on the myometrium. In Mechanism of Action of Steroid Hormones. CA Vilee, LL Engel (eds.) New York, Academic Press, 1961, pp 68-74

22. Ryan JP: Calcium and gallbladder smooth muscle contrac- tion in the guinea pig: Effect of pregnancy. Gastroenterology 1985 (in press)

23. Brotschi EA, La Morte WW, Williams LF: The rote of extracellular calcium in gallbladder contractility. Gastroen- terology 86:1036A, 1984

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26. Carston ME: Biochemical aspects of uterine contractility: role of prostaglandins. In Uterine Physiology. EA Friedman, ML Noah, BA Work Jr (eds). Littleton, Massachusetts, PSG Publishing, 1979, pp 3-31

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27. Bortoff A, Morello E, Mistretta P: Effect of progesterone and 17-OH progesterone on intestinal slow wave propaga- tion. In Gastrointestinal Motility. J. Christensen (ed). New York, Raven Press, 1980, pp 387-393

28. Fisher RS, Roberts GS, Grabowski CJ, Cohen S: Inhibition

of lower esophageal circular smooth muscle by female sex hormones. Am J Physiol 234:E243-E247, 1978

29. Hettiaratchi ESG, Pickford M: The effect of estrogen and progesterone on the pressor action of angiotensin in the rat. J Physiol 196:447-541, 1968

518 Digestive Diseases and Sciences, Vol. 31, No. 5 (May 1986)