Effects of ethanol on contractile response of gall bladder isolated from guinea pig
H i d e h i s a Masu i a, I ch i ro W a k a b a y a s h i a, K a t s u h i k o H a t a k e b, Sach iko Y o s h i m o t o a and K u n i h i r o S a k a m o t o a
Department of Hygiene and b Department of Legal Medicine, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya, Hyogo 663, Japan
Received 7 December 1992, accepted 30 March 1993
The effects of ethanol treatment in vitro and in vivo on gall bladder contraction were investigated using gall bladder strips isolated from guinea pigs. In vitro pretreatment of the strips with ethanol at a concentration of over 50 mM significantly attenuated the reactivity and sensitivity of contractile responses to KCI, acetylcholine and histamine in a concentration-depen- dent manner. Indomethacin treatment or removal of extracellular calcium remarkably reduced gall bladder contractile response to acetylcholine. The depressive effect of ethanol in vitro on gall bladder contraction was also noted in the presence of indomethacin or absence of calcium in the medium. The concentration-response curve of calcium-induced contraction in 40 mM KCi-depolarized gall bladder strip shifted to the right on pretreatment with ethanol. In the case of strips following the chronic administration of 3% ethanol solution ad libitum for 4 weeks, contractile responses to KCI, acetylcholine and histamine did not differ, compared to those in the pair-fed group. This chronic ethanol administration induced tolerance to the acute inhibitory effect of ethanol on gall bladder contractile responses to the agonists. Ethanol is thus shown to exert direct inhibitory action on gall bladder contraction by lowering the calcium sensitivity of the contractile apparatus of smooth muscle; it is unlikely that ethanol consumption would affect gall bladder motility in vivo, owing to the tolerance produced toward the acute inhibitory action of ethanol.
Ethanol; Gall bladder; Smooth muscle; Tolerance; Ca 2+
1. Introduction
There is controversy with regard to the relationship between alcohol intake and incidence of gall stone. In a recent study, patients with alcoholic liver cirrhosis showed higher incidence of gall bladder stone com- pared to those with non-alcoholic liver cirrhosis (Conte et al., 1991). It has been demonstra ted that drinking is an independent risk factor for this incidence (Schwe- singer et al., 1985). On the other hand, according to reports, formation of cholesterol gall stone is appar- ently prevented by alcohol intake (Scragg et al., 1984; Schwesinger et al., 1988).
The prolonged retention of the gall bladder contents may be an important prerequisite for lithogenesis, in which case, gall bladder hypomotility should contribute to stone formation (Forgacs et al., 1984; Pomeranz and
Correspondence to: I. Wakabayashi, Department of Hygiene, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya, Hyogo 663, Japan. Tel. 0798-45-6562; Fax 0798-48-6261.
Shaffer, 1985). Smooth muscle tone is affected by ethanol; ethanol depresses the contractile response of isolated smooth muscle of the vascular wall (Yang et al., 1987; Altura et al., 1980), trachea (Richards et al., 1989), intestine (Wali et al., 1987) and vas deferens (Wali and Hayter, 1989), whereas it induces a contrac- tion in tracheal (Jakupi et al., 1986) and vascular smooth muscle (Altura et al., 1983; Knych et al., 1984; Knych, 1987). There is an increased incidence of gall stone under conditions associated with infrequent or impaired gall bladder emptying, such as fasting, total parenteral nutrition or pregnancy (Paumgartner and Sauerbruch, 1991). Thus, it should be established whether ethanol consumption reduces gall bladder motility and induces cholestasis in the gall bladder. However, the effects of ethanol on the tone of gall bladder smooth muscle are little understood. This study was thus conducted to elucidate the influence of in vitro ethanol t reatment and in vivo ethanol administra- tion on agonist-induced contraction of gall bladder strips from the guinea pig.
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2. Materials and methods
2.1. Animals
16 male Hartley guinea pigs weighing 400-450 g were divided into eight pairs; one member of each pair ingested 3% ethanol solution diluted with distilled water, instead of water, ad libitum (ethanol-fed group), while the other ingested a calorie- and volume-matched glucose solution instead of 3% ethanol solution ad libitum (pair-fed group). After a period of 4 weeks, isolated gall bladder contraction experiments were per- formed. Body weight was measured before and weekly after initiating administration.
2.2. Preparation of gall bladder strips
After an overnight fast, guinea pigs were anes- thetized by an intraperitoneal injection of sodium pen- tobarbital (25 mg/kg) and killed by exsanguination. The gall bladder was rapidly excised, put into Krebs- Ringer solution (millimolar composition: NaCI 122, KC1 4.5, KH2PO 4 1.2, MgSO 4 1.2, CaCI 2 2.5, NaHCO 3 25, glucose 10 at pH 7.4) and cut into longitudinal strips 4 mm wide and 7 mm long.
2.3. Measurement of isometric tension
After washing the mucosal surface with Krebs- Ringer solution, each strip was mounted vertically in an organ bath containing 10 ml of the above solution
maintained at 37°C and bubbled with 95% 02-5% CO 2. The upper end of the preparation was connected to a force-displacement transducer (Nihon Kohden Ko- gyo, Tokyo, Japan). After 1 h of equilibrium at a resting tension of 1 g, change in isometric force was recorded. The contractile response was expressed as percentage of the maximal contractile force by each agonist.
2.4. Blood ethanol determination
Arterial blood, obtained by cardiac puncture, was stored at -20°C and later assayed for ethanol content by head space gas chromatography using a flame-ioni- zation detector (Stowell, 1979).
2.5. Substances
Ethanol, acetylcholine hydrochloride and histamine dihydrochloride were purchased from Wako Pure Chemical Co., Osaka. Indomethacin and sodium pen- tobarbital were purchased from Sigma Chemical Co., St. Louis, MO, and Abbott Laboratories, North Chicago, IL, respectively. Acetylcholine and histamine were dissolved in distilled water to give stock solutions of 0.1 M. Indomethacin was dissolved in 1 mM Na2CO 3 to produce a solution of 10 txM before use.
2.6. Statistical analyses
The data were expressed as mean _+ SE. Significance was determined by Tukey's method after one-way anal-
KCI ( m M ) ACh ( - l o g M) Histamine ( - l o g M)
Fig. l. Effects of pre t rea tment with ethanol (EtOH) at various concentrations in vitro on KCI-, acetylcholine (ACh)- and histamine-induced contractions in gall bladder strips from guinea pigs orally administered calorie-matched glucose for 4 weeks. Asterisks denote statistically
significant differences with the control (without ethanol in vitro) (* p < 0.05).
ysis of variance and p values of less than 0.05 wcre considered significant. ECs0, a concentration to induce a half-maximal response, for gall bladder contraction was determined graphically after calculating the linear regression of the 20-80% region of each log concentra- tion-response curve.
3. Results
3.1. Effects of ethanol administration on body weight of guinea pig and wet weight of isolated gall bladder strip
Ethanol consumption per day by each guinea pig ranged from 3.36 to 4.21 g (3.94_4-0.15 g); i.e., the amount was essentially the same. The mean amount of glucose ingested by the control guinea pigs was 6.84 _+ 0.19 g / d a y (ranged from 6.22 to 7.24 g/day). During the administration, no significant difference in body weight was found between the ethanol-fed and pair-fed guinea pigs. At 4 weeks after ethanol administration, body weights of ethanol-fed and pair-fed guinea pigs were 693 _+ 19 g and 682_+22 g, respectively. Wet weights of the gall bladder strips were essentially the same for the two groups (8.16 _+ 0.51 mg and 8.34 _+ 0.62 mg, respectively).
3.2. Ethanol concentration in blood
Ethanol could not be detected in the blood (< 0.01%) of either the ethanol-fed or pair-fed guinea pigs.
3.3. Acute effects of ethanol on gall bladder contraction (#z t~itro effects)
In gall bladder strips from control guinea pigs, con- tractile responses to KC1, acetylcholine and histamine were significantly attenuated by pretreatment of the strips with ethanol at 50 mM and beyond for 20 min in a concentration-dependent manner (Fig. 1). ECs0 for contractions by KC1, acetylcholine and histamine signif- icantly increased after pretreatment with 50 mM and
105
100
g L~
L3
o~
j ~ Control
~ Indomethacin / r / ~ / 1 /
.... ~ ' , Indom~thacin / / tO 5 EtO. ~//= /
= ' ,
8 7 6 5 4
ACh ( - IogM)
Fig. 2. Effects of pretreatment with 200 mM ethanol (EtOH) in vitro on acetylcholine-induced contraction of the gall bladder strips in the presence of 10 ~zM indomethacin. Asterisks denote statistically sig- nificant differences with the control (*) or response in the absence
of ethanol and presence of indomethacin (#).
over of ethanol (Table 1). However, after washing out ethanol, even at a high concentration (600 mM), from the organ bath, gall bladder contraction by above ago- nists completely recovered (data not shown).
Indomethacin pretreatment for 30 min inhibited contraction of the strips by acetylcholine. In the pres- ence of indomethacin, ethanol (200 mM) pretreatment of the gall bladder strips significantly attenuated con- tractile response to acetylcholine (Fig. 2).
Fig. 3A shows the concentration-response relation- ship of calcium-induced contraction of gall bladder strip depolarized with 40 mM KCI. This curve shifted to the right and ECs0 for the calcium contraction significantly increased after ethanol pretreatment in vitro (ECs0 value, 0.9 mM (control strip); 1.7 mM (ethanol-pretreated strip)). Maximal contractile force by calcium (5 mM) in the KCl-depolarized strips also significantly diminished in the ethanol-pretreated group
TABLE 1
Effects of in vitro pretreatment with ethanol on ECs0 of KCI, acetylcholine and histamine-induced contraction of the gall bladder strips from guinea pigs orally administered calorie-matched glucose (pair-fed) or 3% ethanol (EtOH-fed) for 4 weeks
Asterisks denote statistically significant differences with values in the absence of ethanol in vitro (* p < 0.05: n.d., not determined).
EtOH(mM) in vitro KCI (raM) ACh (/.t M) Histamine (#M)
Fig. 3. Effects of pre t reatment with 200 mM ethanol (EtOH) in vitro on concentrat ion-response relationship for calcium-induced contrac- tion of the gall bladder strips, depolarized with 40 mM KCI, from guinea pigs orally administered calorie-matched glucose (A) or 3% ethanol (B) for 4 weeks. The strips were incubated in the medium without calcium chloride and with 0.1 mM E G T A for 20 rain, and then stimulated with 40 mM KCL followed by the cumulative addi- tion of calcium chloride. The contractile response was expressed as percentage of maximal contractile force by 5 mM calcium in the strips from each group. Asterisks denote statistically significant dif-
ferences with the control (without ethanol in vitro) (* p < 0.05).
compared to the control group (0.71 4- 0.02 g (ethanol- fed group); 0.99 _+ 0.10 g (pair-fed group), p < 0.05).
Figs. 4 and 5 show the effects of in vitro pretreat- ment with ethanol (200 raM) on 0.1 mM of acetyl- choline and histamine-induced contractions in the presence or absence of extracellular calcium (2.5 mM). Removal of calcium chloride from the medium reduced maximal contractile force induced by acetylcholine (0.1 raM) and histamine (0.1 mM) by 74.5% and 63.6%, respectively. Ethanol pretreatment further attenuated the contractile responses to acetylcholine and his- tamine in the absence of extracellular calcium by 64.8% and 67.9%, respectively.
3.4. Chronic effects of ethanol on gall bladder contrac- tion (in c'it'o effects)
There was no significant difference in ECs0 and maximal force of contractions by KCI, acetylcholine and histamine in gall bladder strips from the control and chronically ethanol-administered guinea pigs. In strips from ethanol-fed guinea pigs, pretreatment with ethanol at 200 mM and beyond inhibited contractile responses to acetylcholine and histamine, and that at 600 mM only inhibited the response to KC1 (Fig. 6). In the ethanol-fed group, ECs. for KC1-, acetylcholine- and histamine-induced contractions did not change on pretreatment with lower concentrations of ethanol (50, 100 and 200 mM). Pretreatment with 600 mM ethanol alone increased the ECs0 for KCI- and acetylcholine- induced contractions. In the gall bladder strips from both groups, ECs0 for histamine contraction in the presence of 600 mM ethanol could not be determined,
,oo]
~5
c 50- 0 (D
0
r ̧ - - i
T
Ca + C a + +
EtOH
Ca - Ca - +
EtOH
Fig. 4. Effects of 200 mM ethanol (EtOH) pre t rea tment on acetyl- choline (0.1 mM)-induced contraction in the medium with (Ca + ) or without ( C a - ) extracellular calcium (2.5 raM). Experiments using calcium-free medium were performed as follows: the strips were washed four times with calcium-free medium containing 0.1 mM EGTA, and then 200 mM ethanol or vehicle was added. 20 min later, the strips were stimulated with 0.1 mM acetylcholine. Asterisks denote statistically significant differences between indicated data
s e t s (* p < 0 . 0 5 ; * * p<0 .01) .
since 600 mM ethanol pretreatment almost completely abolished the histamine-induced contraction (Table 1). In strips from the ethanol-fed group, the concentra- tion-response relationship of calcium-induced contrac- tion in the presence of 40 mM KCI was not altered by
lOOq I
~5 m
50
0 J
[ ~ , I
Ca + Ca + Ca - +
EtOH
C a - +
EtOH
Fig. 5. Effect of 200 mM ethanol (EtOH) pretreatment on histamine (0.1 mM)-induced contraction in the medium with ( C a + ) or without (Ca ) extracellular calcium (2.5 raM). Experiments using the cal- cium-free medium were performed as follows: the strips were washed four times with calcium-free medium containing 0.1 mM EGTA, and then 200 mM ethanol or vehicle was added. 20 min later, the strips were stimulated with 0.1 mM histamine. Asterisks denote statistically significant differences between indicated data sets (* p < 0 . 0 5 ;
** p < 0.01).
107
r ~ > Control ~ . ~ Control .)-'~2 Control H EtOH 50mM ~ [ H EtOH 50mM H EtOH 50mM
~-H ! EtOH 600mM L'~"--~ EtOH 600mM ~i P'~ EtOH 600mM y / f
o *
50 50 50 E o
T.
0 40 80 8 7 6 5 4 7 6 5 4
KCI (mM) ACh ( - log M) Histamine ( - log M)
Fig. 6. Effects of pretreatment with ethanol (EtOH) at various concentrations in vitro on KCI-, acetylcholine (ACh)- and histamine-induced contraction in gall bladder strips from guinea pigs orally administered 3% ethanol for 4 weeks. Asterisks denote statistically significant
differences with the control (without ethanol in vitro) (* p < 0.05).
in vitro pretreatment of the strips with 200 mM of ethanol (Fig. 3B).
4. Discussion
Ethanol inhibits smooth muscle contraction of vari- ous tissues. However, the mechanism of inhibitory ac- tion of ethanol has not been determined in detail. In vascular smooth muscle, ethanol depresses transmem- branous calcium movement, resulting in decreased cal- cium uptake under basal conditions and agonist-in- duced contraction (Altura et al., 1976; Turlapaty et al., 1979a). In this study, ethanol treatment in vitro inhib- ited gall bladder contraction by KC1, acetylcholine and histamine in a concentration-dependent manner. The threshold concentration of ethanol for inhibition was 50 raM, this being essentially the same as those previ- ously reported in intestinal smooth muscle (40 mM) (Mayer et al., 1980) and microvascular arteriolar and venular smooth muscle (40 mM) (Altura, 1978; Altura et al., 1979). Ethanol attenuated maximal contractile force by agonists and increased their ECs0. Ethanol thus inhibits both reactivity and sensitivity of the ago- nist-induced contraction of gall bladder smooth muscle in vitro. This finding confirms the mode of the in- hibitory action of ethanol on smooth muscle contractil- ity, which was first demonstrated by Altura et al. (1976) using vascular smooth muscle preparations. The in- hibitory action of ethanol was not specific for contrac-
tile agonists, since ethanol inhibited contractions by receptor agonists such as acetylcholine and histamine, and KC1 which induces contraction by the direct depo- larization of plasma membrane. Ethanol (200 mM) pretreatment resulted in a rightward-shift of the con- centration-response curve for contractile response to calcium in the KCI (40 mM)-depolarized gall bladder strips. Ethanol may thus inhibit gall bladder contrac- tion by decreasing sensitivity to calcium. Elevation of the intracellular calcium concentration is a prerequisite for smooth muscle contraction. Increase in intracellu- lar calcium during smooth muscle contraction is due to the release of calcium from intracellular storages and /or influx of extracellular calcium through calcium channels. Elevated calcium binding to calmodulin greatly stimulates myosin light chain kinase, followed by phosphorylation of myosin light chain, and then activated myosin interacts with actin. In the present study, acetylcholine (0.1 mM)- and histamine (0.1 mM)-induced contractions were remarkably attenuated in the medium without calcium and containing 0.1 mM EGTA, but about 30% of the contractions due to these agonists still remained. These residual contractions de- pendent on intracellular calcium were further inhibited by 200 mM ethanol as well as contractions in presence of calcium. KCl-induced contraction in gall bladder strip was strongly reduced in the calcium-free medium (9.6% of 40 mM KCl-induced contraction in the medium containing 2.5 mM calcium) and thus mainly dependent on extracellular calcium. These data indi-
108
cate a mechanism in which ethanol mainly inhibits the pathway following elevation of intracellular calcium concentration in gall bladder contraction. Thus, ethanol may well decrease the calcium sensitivity of the intra- cellular contractile apparatus in gall bladder smooth muscle. However, the effects of ethanol on intracellu- lar calcium concentration could not be determined in the present study. Ethanol is known to be a membrane-fluidizing agent (Chin and Goldstein, 1977) and might affect plasma membrane a n d / o r sarcoplas- mic reticulum membrane. Ethanol decreases intracellu- lar calcium ions using molecular probes in single vascu- lar smooth muscle cells (Zhang et al., 1992). Therefore, it cannot be denied that ethanol inhibits contraction of gall bladder smooth muscle by suppression of calcium influx through plasma membrane a n d / o r intracellular calcium mobilization from sarcoplasmic reticulum.
A previous report showed that several prostaglandins (PGE: , PGF2, ~, 6-keto-PGFl~ ~ and thromboxanc B~) can be detected in guinea pig gall bladder in vitro (Booker and LaMorte, 1983). Exogenous PGE~, -E~ and -F2c ~ produce gall bladder contraction and in- domethacin, a cyclooxygenase inhibitor, reduces the contractile response of gall bladder strips (Nakata et al., 1981). Endogenous prostaglandins are thus in- volved to some degree in the contraction of gall blad- der smooth muscle. It has previously been shown quite clearly by Altura et al. that indomethacin and other prostaglandins synthetase inhibitors do not particularly alter the actions of ethanol in smooth muscle (Altura and Altura, 1982). In a series of earlier reports, they noted ethanol to be capable of affecting vascular smooth muscle contractility in response to prostaglan- dins in different ways: low concentrations of ethanol can markedly inhibit the contractile action of PGE~ on isolated rat arteries and veins, but enhances contrac- tion induced by PGB~ and PGA 1 on isolated rat blood vessels, while high concentrations of ethanol (greater than 100 mM) markedly attenuate contractions in- duced by all prostanoid compounds (Altura and A1- tura, 1976; Altura and Edgarian, 1976; Edgarian and Altura, 1976). In this study, indomethacin pretreatment significantly reduced acetylcholine-induced contraction of gall bladder strips in ethanol-free medium, and ethanol (200 mM) pretreatment further inhibited the contraction in the presence of indomethacin. Ethanol thus appears to inhibit gall bladder contraction, irre- spective of the action of endogenous prostaglandins.
In gall bladder strips from a chronically ethanol-ad- ministered guinea pig, contractile responses to KCI, acetylcholine and histamine were not altered in com- parison to the controls. This indicates that ethanol concentration (3%) used for its oral administration does not affect ex vivo gall bladder contraction. In the strips from the pair-fed guinea pigs, contractile re- sponses to KCI, acetylcholine and histamine were at-
tenuated by ethanol at 50 mM and over; 200 mM and over was required to attenuate the contractile re- sponses in the strips from chronically ethanol-fed guinea pigs. Thus, chronic ethanol administration pro- duces tolerance to acute inhibitory action on gall blad- der contraction. Only 3% ethanol solution given ad libitum for 4 weeks made isolated gall bladder strips tolerant to the acute inhibitory action of ethanol. Moreover, ethanol was not detected in the blood of guinea pigs on chronic ethanol administration just be- fore the experiment. It would thus appear that the smooth muscle of gall bladder easily tolerates the in- hibitory action of ethanol on contraction. Considering the low concentration (3%) of ethanol solution avail- able for free drinking, it is not surprising that ethanol concentration in blood could not be detected. Al- though to our knowledge, there is no study demonstrat- ing tolerance to the effects of ethanol on gall bladder contraction after chronic ethanol treatment, tolcrance to depressive action of ethanol on smooth muscle con- traction has been reported in vascular smooth muscle (Altura et al., 1980; Strickland and Wooles, 1988), vas deferens (DeTurck and Pohorecky, 1986; 1987a) and ileum longitudinal muscle (Mayer ct al., 1987). Ele- vated extracellular calcium reverses the inhibitory ef- fect of ethanol in ileum longitudinal muscle (Mayer et al., 1980). In vas deferens, the blocking action of cal- cium antagonist on mechanical responses of the vas in ethanol-free medium was attenuated by in vivo ethanol treatment (DeTurck and Pohorecky; 1987b). In a study by Altura et al., (1980) using aorta and portal vein preparations from rats, and in this study using gall bladder preparation from guinea pigs, calcium-induced contraction in KCl-depolarized strips was inhibited by acute in vitro ethanol and the inhibition was attenu- ated by in vivo ethanol treatment. All these findings would indicate that a change in calcium mobilization is possibly involved in the development of tolerance to- ward the inhibitory action of ethanol on smooth muscle contraction, this being consistent with the earlier study by Altura et al. (1980) using vascular smooth muscle.
Previous studies have shown that dietary ethanol inhibits the formation of cholesterol gall stones in the prairie dog; this may be due to decreased cholesterol and calcium concentrations in gall bladder bile (Schwesinger et al., 1988). On the other hand, cholesta- sis generally accelerates the formation of gall stones (Busch and Matern, 1991). Moreover, isolated gall bladder strips from patients with pigment gall stones have been noted to show augmented contractile re- sponse to cholecystokinin, acetylcholine and KCI, com- pared to those from patients with cholesterol gall stone (Behar et al., 1989). In patients with cholesterol gall stone, gall bladder emptying induced by ursodeoxy- cholic acid is significantly attenuated~Festi et al., 1990). Thus, changes in gall bladder motility may influence
the incidence of gall stone formation. The present study shows that ethanol directly inhibits gall bladder contraction. The effects of ethanol on smooth muscle contraction are observed even at lower concentrations with an increase in ethanol exposure time (Karanian and Salem, 1986). The incubation period of ethanol in the acute in vitro experiments of the present study was restricted. There is thus the possibility that ethanol affects gall bladder tone in vivo, although the threshold concentration of the inhibitory action of ethanol is above physiological levels. It is, however, unlikely that ethanol reduces the motility of gall bladder smooth muscle with consequent acceleration of cholestasis in vivo, since the chronic consumption of low-dose ethanol may produce tolerance to the acute inhibitory action of ethanol on contraction. Accordingly, the present re- sults support the hypothesis that dietary ethanol may not be a risk factor of gall stone formation based on assessment of gall bladder tone.
In conclusion, in vitro ethanol directly depresses gall bladder contraction by lowering calcium sensitivity for smooth muscle contraction and chronic ethanol admin- istration produces tolerance to this inhibitory action.
Acknowledgements
This study was supported by a Research Grant from the Hyogo College of Medicine.
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