Cholestatic action of somatostatin in the rat: Effect on the different fractions of bile secretion

GASTROENTEROLOGY

1981;81:552-82

Cholestatic Action of Somatostatin in the Rat: Effect on the Different Fractions of Bile Secretion GIOVANNI

L. RICCI

and JOHAN

FEVERY

Laboratory of Hepatology, Katholieke Universiteit Leuven, Campus Teaching and Research Building, Leuven, Belgium

Somatostatin was administered intravenously to male Wistar rats, recovered for 3 h from an anesthesia during which the common bile duct and jugular vein were cannulated. Different bile acid-secretory rates were obtained by infusion of saline, or of Na+-taurocholate (150 nmol/min/100 g body wt), or by 8-h bile depletion. At the dose of 2 pg/h/lOO g body wt, somatostatin causes a prompt decrease of bile flow (about 30%) and of bile acid secretion (32%47%). The bile acid-independent fraction of canalicular bile is more decreased than the one associated with bile acid secretion. The changes are dose dependent and show a saturation pattern, with halfmaximal saturation already at 2.2 ng/min/ZOO g body wt. Despite this cholestasis, endogenous bilirubin secretion remained unchanged, pointing to different secretory mechanisms for bilirubin and bile acids. In the isolated and perfused liver, somatostatin displays an anticholeretic effect, proportional to the amount of Na+-taurocholate present in the system. Hepatic blood flow and 0, consumption remained constant during perfusion, and were not affected by somatostatin. The hepatic transport of bile acid, and the water and electrolyte secretion are directly affected by somatostatin, and the experimentally-induced cholestasis seems a new and suitable model for studying mechanisms of bile secretion. Received June 20,198O. Accepted May 12, 1981. Address requests for reprints to: G.L. Ricci, M.D., Laboratory of Hepatology, Katholieke Universiteit Leuven, Campus Gasthuisberg, Teaching and Research Building, B-3988 Leuven, Belgium. The authors thank Professor J. De Groote and Professor K.P.M. Heirwegh for advice and critical discussions, Dr. W. Lissens (Clinical Chemistry) for help with the electrolyte determinations, Mrs. M. Crets for technical assistance, and the representatives of Ayerst Laboratories in Belgium for the generous gift of somatostatin. This work was supported by the National Foundation for Scientific Research of Belgium. Dr. Ricci is a recipient of a fellowship from the Katholieke Universiteit Leuven. 0 1981 by the American Gastroenterological Association OOlS-5085/81/090552-11$02.50

Gasthuisberg,

Several hormones given in acute or long-term experiments stimulate bile secretion. Among these, gastrointestinal polypeptides released after meals have been extensively investigated (1,2). In dogs and primates, food intake results in a significant increase of the bile acid-independent flow (3,4) possibly mediated by gut hormones. The pattern of hormonal regulation is less clear in rodents. This has been attributed to different feeding habits when compared with primates (l), as periodic feeding in the latter could be connected to a rather specialized regulatory system, triggered by food intake. Recent work demonstrated that fasting decreased the bile flow in nonanesthetized rats (5,6). Several hormones have been tested to find the potential mediator of an effect on bile flow, and several different experimental conditions and dosages have been used. Pentagastrin was reported to have an effect on bile flow in the anesthetized rat when infused with taurocholate (7) or in the nonaaesthetized animal 3 wk after gastrectomy and bile duct cannulation (8); it seemed to produce a slight increase in bile flow and bilirubin output, but it caused a decrease in bile acid output in rats equipped with gastric and biliary fistulas. When cholecystokinin and cerulein were given, bile flow and bicarbonate concentration and output increased (7); with glucagon an enhancement of the bile acid-independent canalicular bile flow was noted (9). Secretin produced only a slight (12%-17%) increase in bile flow with a nearly parallel increase in erythritol and mannitol clearance, i.e., in the hepatocytic bile (10, 11). More extensive investigation seems necessary however to reach final conclusions about the action of polypeptide hormones on bile secretion. The release and digestive effects of several of these hormones-and a consequent possible action on the liver-can be suppressed by somatostatin, and indeed somatostatin-like immunoreactive cells have been demonstrated recently in the digestive

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554

GASTROENTEROLOGY Vol. 81, No. 3

RICCI AND FEVERY

tin or sham infusion in order to establish an internal control of the experiment. Blood was immediately put in plastic tubes containing some dry heparin powder, centrifuged, and the plasma separated. DOSE-RESPONSE CURVES. As in group A, animals were investigated 3 h after surgery and different dosages of somatostatin were infused for 60 min (0.05-20 pg/h/lOO g body wt). Bile samples were collected as indicated above. At the end of each experimental infusion, animals were stunned, the abdomen quickly opened and the liver perfused in situ through the hepatic veins with chilled 0.16 M KCI. After removing the blood, the liver was taken out, wiped with filter paper, weighed, and stored at -25’C for subsequent analysis. STUDY OF THE ELECTROLYTE COMPOSITION OF BILE.In order to have suitable amounts of bile, a new se-

ries of animals was studied for this purpose. Rats (n = 6) were treated as in group A, received somatostatin 2 pg/h/ 100 g body wt, and were tested 3 h after catheterization; controls (n = 4) received only saline. The concentration of HCO,- and Cl- was measured in all bile samples; Na+,K+ and total osmolarity were checked in bile pooled from equal volumes obtained from the different rats at a given time point. Investigation

in isolated and Perfused Liver

Preparation. The surgical removal of the liver and perfusion procedures followed are in accordance with previous reports (for a review see reference 17).The perfusion medium consisted of 150 ml of Krebs-Ringer-bicarbonate solution, pH 7.45, containing 2.6 g/d1 bovine serum albumin (fraction V from bovine plasma, Armour Pharmaceutical Company Ltd., Eastbourne, England), 106 mg/dl D-glucose (BDH Chemicals, Ltd., Poole, England), and human erythrocytes, washed 3 times in physiologic saline and finally in Krebs-Ringer-bicarbonate, to a final packed cell volume of 20%, and oxygenated under a constant flow of a mixture of 95% O,-5%CO, of 300 ml/min, at atmospheric pressure. The medium also contained 10 pg/ ml ampicillin sodium salt to avoid bacterial overgrowth, and heparin (50 U/h) was constantly infused and stirred in the main reservoir. The livers were perfused at 37.5OC with a mean pressure of 11 cm H,O. Hepatic blood flow was monitored at the midpoint of each bile collection by measuring the time necessary to fill up a precalibrated cylinder (10 ml) interposed in the circuit at the liver outflow. pH was continuously recorded and adjusted at pH 7.45 with HCO,- 0.16 M, mainly during the equilibration period before the experiments. The viability of the livers was assessed by measuring PO, and PCO~ in the inflow and outflow blood, and calculating 0, consumption (16). Experiments. Different bile acid secretory rates were obtained by infusing physiologic saline or taurocholate (60 or 120 nmol/min/g liver) throughout the whole experiment. The weight of the liver was estimated as 3.5% of the whole body weight in order to calculate the amount of taurocholate and somatostatin before perfusion. Forty minutes of equilibration were allowed before starting collection of bile samples; samples were collected every 10 min, as before. After a control period of >40 min, a con-

stant-rate infusion of physiologic saline (0.95 ml/h) was added through a needle inserted in the portal cannula, for 60 min (n = 11).In the experimental perfusions (n = 9) after control bile collection, somatostatin was dissolved in the saline at the precalculated dosage of 50 ng/min/g liver.

Assays Biliary bilirubin was usually assayed after diazocleavage at pH 2.7 with ethyl-anthranilate (19). The azoderivatives were analyzed by thin-layer chromatography (TLC) as described (20). Samples from the dose-response experiments were analyzed at pH 6.6 by using modified reagent (1 mM ethyl-anthranilate) to avoid reaction of isomerized ester-glycosides with free-ethyl-anthranilate (21). The azodipyrroles, extracted in 2 ml of 2-pentanone, were applied to TLC plates and were developed in a system of chloroform/methanol (65:15, vol/vol). The ISazopigment (azodipyrrole-glucuronide) was then scraped off, collected in a glass tube, and reextracted with 2 ml of 2-pentanone. To this 1 ml of ethereal diazomethane was added at 0°C to form the methyl derivatives (21,22). Bile acids were assayed with 3-a-hydroxysteroid-dehydrogenase (EC 1: 1:1:50; Worthington Biochemicals Corp., Freehold, N.J.) according to Turley and Dietschy (23). Bicarbonate concentration was measured by titration after bubbling the sample with CO,. Chloride was measured with a silver electrode chloride meter (model 920, Corning Medical, Medfield, Mass.), sodium and potassium with a flame photometer (model 543, Instrumentation Laboratories, Milan, Italy) and the osmoiarity by the method of freezing-point depression (model 3.DI1, Advanced Instruments Inc., Needham Heights, Mass.) Oxygen pressure and PCO* were measured in an automated gas analytic system (model 175 automatic pH/blood gas system meter, Corning Medical). Uridine diphosphate-glucuronyltransferase (EC 2 : 4: 1: 17) activity in liver homogenate was assayed with bilirubin as the acceptor substance (24). Another part of the homogenate was centrifuged for 10 min at 9000 g,, at 4’C. The supernatant was diluted 1.2-1.5-fold with 0.155 M KC1 and centrifuged for 1 h at 105 000 8.“. Cytochrome P,, was measured in the resuspended microsomal fraction (25) at pH 7.4 (0.1 M HEPES buffer), and the supernatant was used to assay UDP-glucose dehydrogenase activity (EC 1: 1: 1:22), essentially with the method of Gainey and Phelps (26). To determine [“Clerythritol concentration, 100 ~1 of plasma was digested overnight at room temperature with 500 pl of Soluene 350 (Packard Instrument Co., Downers Grove, Ill.) before adding 10 ml of scintillation medium (Instagel, Packard Instrument Co). For bile, 100 pi was diluted with 500 1.11 of water and left to bleach overnight under eight 20-W Vita-lite fluorescent lamps before adding the scintillation medium. 14C radioactivity in bile and plasma were measured in a Packard 2420 liquid scintillation spectrometer. Correction for quenching was made by external standardization. Quenching was similar in vials containing bile and plasma. The clearance of [“Clerythritol was calculated according to the formula: [‘4C]erythritol clearance = bile flow x (Bile radioactivity/ Plasma radioactivity). Statistical analysis was performed

September

SOMATOSTATIN-INDUCED

1981

Student’s t-test for paired and unpaired data and linear-regression analysis. Results are expressed as mean -+ SEM.

with the

CHOLESTASIS

sham-treatment animals groups (Figure 1).

IN THE RAT

was present

555

in the three

Bile Acid Secretion

Results Effect of Somatostatin

on Bile Flow

Bile flow in the control period was 5.09 f 0.11 $/min/lOO g body wt for group A, 5.79 f 0.15 pl/ min/lOO g body wt for the taurocholate-infused animals of group B and 3.86 f 0.08 pl/min/lOO g body wt for the 8-h bile acid-depleted group C. Bile flow decreased 5%-7% over the 60-min sham-infusion period in the control animals (Figure 1). In contrast, administration of somatostatin induced a decrease of bile flow in all animals within the first lo-min sample. After 30 min a constant flow was obtained, which was 67 f 2%, 73 f l%, and 77 + 2% of the flow in the control period in group A, B, and C respectively (Figure 1, Table 1). When somatostatin was stopped, bile flow returned to pretreatment levels within 20 min. After a 30-min recovery period, a rebound of the bile flow above the mean level of the c._-_1

GROUP

*to-

In the sham-infused control animals, bile acid concentration decreased slightly over the 60-min period till 94% (group C) or 85% (group A) of the control period (Table 1); no significant changes were observed in the taurocholate-infused animals (group B). Somatostatin infusion rapidly produced a decrease in the concentration (Figure 2) and biliary output (Figure 2) in groups A and C. In the taurocholate-infused group B, bile acid concentration started to increase within the first 10 min to reach 113 f 6% in the steady-state period (Figure 2). However, due to the greater decrease in bile flow, the bile acid output (134.5 + 5.9 nmol/min/lOO g body wt) significantly decreased (Figure 2, Table 1) when compared with the control period (78%) or with the sham-infused animals (69%) and fell below the infusion rate of taurocholate (Figure 3). When somatostatin was stopped, bile acid excretion went

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Figure 1. Bile was collected in consecutive lo-min samples in rats 3 h after cannulation receiving glucose in saline (uncompensated rats, group A: 0 and ?? ) or taurocholate 150 nmol/min/lOO g body wt (group B: 0 and r) or after 8-h biliary drainage (bile acid-depleted, group C: A and A ). The percentage variation with regard to the mean of the control period (O-30 min) is given as the mean f SEM, in animals receiving somatostatin (2 pg/ min/lOO g body wt) (closed symbols: n = 7 for A and B, n = 6 for C) from 30 to 90 min and in “sham” (shaminfused) rats (n = 5 for each group) infused with glucose in saline (open symbols). *Indicates the first sample statistically different at the level p c 0.05 with regard to the preceding period or to the “sham” animals (in the recovery period).

556

RICCI AND FEVERY

GASTROENTEROLOGY

L

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GROUP

I

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GROUP

C

Vol. 81, No. 3

A

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Figure 2. Bile acid concentration and biliary output was measured in lo-min samples in rats of groups A (0, ?? ), B (0, W), and C (A, A). The change in percentage relative to the mean of the control period (o-30 min) is given as mean f SEM for saline-infused “sham” animals (each time n = 5, open symbols) and for rats receiving somatostatin, 2 ~g/min/lOO g body wt (closed symbols) from 30 to 90 min.

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up immediately, far in excess of the amount found in the sham rats, suggesting that retention in the liver or in blood had temporarily occurred.

Bile Acid&dependent Fraction and Canalicular Bile Secretion Bile flow from all sham- and somatostatintreated rats (control and test periods) are plotted as a function of the bile acid output in Figure 4. By extrapolation to a bile acid output equal to zero, the socalled bile acid-independent fraction (BAIF) of bile flow is obtained. It is clear that somatostatin significantly reduced this fraction. To obtain more information, [“Clerythritol was used in animals of group Table

2.

Changes in the Ratio of Bile to Plasma Concentrations of r’C]erythritoJ Control period

Sham infusion (n = 8) Somatostatin infusion (n = 9) No statistically significant pressed as mean f SEM.

0.92 f 0.04 0.99 f 0.02

differences

Experiment 0.97 f 0.03 1.03 f 0.04

were obtained.

Results ex-

B (taurocholate-infused) as a marker of the hepatocytic bile flow, as it seems to diffuse freely across the hepatocyte. The concentration in bile as it rises from the hepatocyte is assumed to be equal to that of plasma. Modifications of this primary bile in the ductules will be reflected by changes in the bile/ plasma ratio of the bile, as collected. During the control period the overall bile/plasma ratio was 0.96 f 0.02 (n = 17) indicating that ductular secretion of water is minimal in these nonanesthetized rats receiving taurocholate. Both in the sham- and somatostatin-infused animals, the bile/plasma ratio did not change significantly (Table 2). The decrease in total bile flow found (Figure 5) seems to be due mainly if not entirely to a decrease in hepatocytic flow. There is not enough evidence for a role played by ductular secretion as a component of somatostatin cholestasis. Thus the decreased BAIF seems entirely of canalicular origin in the rat. Dose-Response Increasing more pronounced

Curves

dosages of somatostatin had a effect on bile flow; with all dos-

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CEIXl(INI-NILVLSOIV~OS

RICCI AND FEVERY

558

A

GASTROENTEROLOGY

’ A

” i

--> 22s

I

I

1

2

tostatin infusion, thus the extent of the decrease in bile acid output closely follows the bile flow. Hepatic blood flow was the same in all the experiments, 2.1 + 0.1 ml/min/g liver. Somatostatin did not even transiently change the hepatic blood flow, which remained stable throughout the whole perfusion. Oxygen consumption was the same at the beginning and the end of the perfusion, 3.36 f 0.35 pmol of O,.min/g liver, and it was not modified by adding somatostatin.

23

3

'

10

)rg/lOOg hwl./h Somatostatin

KM N22ng/lDOgbwt/m1n

, -10

/

/+ -5

0

-1, S

I

I

5

10

Vol. 81, No. 3

I

20

1 JJ~ ST /lOOg b.wt./h

Figure 6. The relative decrease in bile flow (BF) after 10, 20, and 30 min of infusion and in the steady-state period is given for different amounts of somatostatin (given in a semilogarithmic scale). The points given are the mean of 3-7 experiments (A). From the results obtained in the steady-state period, a double reciprocal plot is given relating l/BF to l/amount of ST (somatostatin) given (B). From this, an apparent “K,” (half-maximal dosage) of 2.2 ng/min/100 g body wt is obtained.

Isolated Liver Perfusion Control bile flow rose according to increasing loads of taurocholate in a linear relationship with the bile acid output (r = 0.87, p < 0.05). In perfusions where somatostatin was not added, levels of bile flow in the experimental period did not differ significantly from that of controls (Table 4). When somatostatin was infused, a decrease in bile flow was only observed in the presence of taurocholate. It started to decrease immediately after somatostatin infusion, and generally reached a steady state after 40 min. The mean 40-60-min decrease was more pronounced with higher amounts of taurocholate (Table 4). No significant changes in bile acid concentration were seen between the control period and soma-

Discussion In the present investigation somatostatin produced a pronounced and promptly reversible cholestasis in nonanesthetized Wistar rats. The rapid onset of this action parallels the effects reported for other parts of the gastrointestinal tract. A decrease in bile flow of about 30% was obtained in all rats, A steadystate was obtained after 30-40 min of administration. The cholestasis was dose dependent, and it showed saturation properties. The decreased bile flow could be the result of a reduction in bile acid excretion or in the BAIF. In fact, the bile acid concentration in bile decreased in groups A and C (bile acid-depleted rats) as compared to the sham-treated animals (Figure 2) and the bile acid output was reduced in the three groups. The amount of bile flow linked to 1 pmol of bile acid remained the same (slope of Figure 4), and the absolute bile acid-dependent flow was decreased, to different extents in the three groups of rats studied in vivo. All the points moved toward the y-axis. Bile acids undergo a high fractional clearance by the liver, and it might be speculated that the effect of somatostatin is secondary to a decreased arrival of bile acid to the liver. A decrease of splanchnic and hepatic blood flow-which somatostatin causes in humans (27,28)-might account for this phenomenon, and it would appear to be secondary to hemodynamic changes. Moreover, somatostatin delays the absorption of nutrients (29,30), water, and electrolytes (31) by the small intestine, and it could affect in the same way absorption of bile acid still present in the gut. A potential mechanism of action could be the suppression of secretin-enhanced transport of bile acid across the intestinal wall (32). Although in vivo a reduction of splanchnic and then portal blood flow is still likely, in isolated rat liver somatostatin does not decrease hepatic blood flow, even transiently. The effect on bile flow is dependent on the presence of bile acid (Na+-taurocholate, 60 or 120 nmol/min/g liver) in the perfusion medium, as if a primary inhibition of bile acid transport is the mechanism of action. Furthermore, the abrupt

September

20

0

SOMATOSTATIN-INDUCED

1981

LO

60

Figure 7. Bilirubin output given for rats of (A; n = 8) before ter (90-150 min) g body wt). The

60

100

120

1LO InI"

in bile collected in lo-min samples is groups A (0; n = 7);B m; n = 7) and C (9-30 min), during (30-90 min) and afinfusion of somatostatin (2 pg/min/liXl mean f SEM is given.

drop of bile acid output during somatostatin infusion below the intravenous input in animals receiving taurocholate (Figure 3), and the very rapid return to normal, with a partial overshooting, suggest a direct effect of somatostatin on hepatic uptake or on secretion of taurocholate, or both, supporting the concept of a primary action in vivo on bile acid handling by the liver. In a further analysis, the bile acid-independent flow was obtained from plotting bile flow as a function of bile acid output. By extrapolation to a bile acid output equal to zero, the intercept with the yaxis yields the BAIF, which decreased from 3.8 to 2.8 ~mol/min/lOO g body wt during somatostatin infusion. This decrease in BAIF was more important than the change in bile acid-dependent flow and all points are moved down toward the x-axis (Figure 4). As both bile acid-dependent and -independent flows are decreased, in an attempt to analyze the dynamics of the phenomenon, we have introduced the ratio bile acid concentration/bile flow, which is a numeric factor with no biologic dimensions. A change in this ratio points out which of these comTable

3.

Electrolyte

Composition

IN THE RAT

559

ponents of bile secretion is more affected. During the first 20-30 min of somatostatin infusion, the ratio did not change significantly; in the steady state it increased at different extents in the three groups (17% in group A, 49% in B, and 8% in group C), implying a quantitatively more important reduction of the fraction independent from bile acid secretion. Debate still remains whether these two fractions are separable, and indeed two recent investigations did not obtain a single constant relationship (33.34). The BAIF can be of canalicular and ductular origin. The hepatic clearance of [“Clerythritol is used to measure the canalicular bile flow, whereby it is assumed that erythritol diffuses freely from the blood through the hepatocyte in bile and is not reabsorbed nor secreted in the ductular system. Although recent work in the dog (35) and rat (38) points to possible modifications, the classical view is still held. At a ratio of the bile/plasma concentration of erythritol of 0.98 f 0.05 (n = 17), <4% of the total flow seems to be due to ductular secretion in nonanesthetized Wistar rats receiving taurocholate. During the experimental period the bile/plasma ratio did not change significantly. Due to the paucity of this fraction and minor changes induced by somatostatin, possible modifications of ductular secretion or absorption do not appear of quantitative relevance and fail to identify a role of the polypeptide at this site. Therefore the source of the bile acid-independent flow decreased by somatostatin is chiefly canalicular (hepatocytic). The effect on bile flow and bile acid secretion in the isolated liver is in favor of an action of somatostatin within the organ. The mechanism is difficult to explain. The possibility of a vascular intrahepatic bypass is still possible, and the decreased perfusion of the sinusoidal plates would account for a diminished extraction of bile acids. Areas of periportal vasoconstriction or bypass such as produced by chlorpromazine (37) affect instead the BAIF and coincided with

of Bile Sham infusion

PH Na+ (mEq/L) K+(mEq/L) Cl- (mEq/L) HCO,-(mEq/L) Bile acid (mM) Bile acid osmolality (mOsmoI/kg) Anionic gap Anionic gap-bile acid

CHOLESTASIS

Somatostatin

infusion

Control

Experimental

Recovery

Control

Experimental

Recovery

8.69 170 5.1 96 24 32.5 295 55 23

8.30 185 5.1 101 23 26.4 292 47 21

8.38 169 5.1 101 23 26.2 299 50 24

8.43 164 5.8 94 30 25 291 45 20

8.53 169 5.3 103 30 20 296 32 12

8.44 161 5.0 100 30 15.4 284 36 21

560

Table

RICCI AND FEVERY

4.

Bile Flow and Biliary

GASTROENTEROLOGY

Bile Acid Output

From the Isolated

Rat-Liver

Perfusions Bile acid output (nmol/min/g

Bile flow (I.Lg/min/g liver) Na-taurocholate in perfusate (nmol/min/g Jwer) 0 nmol 60 nmol 120 nmol

Control 40-60 min Control 40-60 min Control 40-60 min

Values are given as mean f SEM. 0 Somatostatin

Sham 1.55 f 1.49 f 1.87 f 1.84 f 2.57 f 2.46 f

0.07 0.12 0.03 0.05 0.05 0.06

= 50 ng/min/g

a peak decrease of hepatic blood flow. In the present experiments no hemodynamic changes were observed at the liver outflow and there is not enough evidence to recall a mechanism of vasoconstriction. The inhibition of bile acid transport could either be decreased uptake or secretion, or both. In our study the isolated liver perfusion was designed to investigate whether the effect of somatostatin is directly on the organ. A higher concentration (about 8%) of bile acid observed in the perfusate at the end of somatostatin infusion as compared to sham perfusions is not conclusive, as a diminished biliary secretion of bile acid, with consequent intracellular retention, could affect the overall bile acid uptake as well. The mean electrolyte composition, studied in the in vivo experiments, did not show significant changes, thus failing to provide evidence of another anionic group exchanged for bile acid. The bile acidindependent flow is decreased as well, probably masking the mechanism that affects the fraction linked to bile acid excretion. In the present experiments, the endogenous bilirubin output remained unchanged in contrast to bile acid, and the bilirubin concentration increased in parallel to the decrease in bile flow. The ability of somatostatin to dissociate these two secretions is in accordance with the existence of independent secretory mechanisms for bilirubin and bile acid in nonanesthetized rats. Apparently conflicting results have been reported about the relationship between biliary bile acid and pigments excretion (38-42), explained, at least in part, by species and substrate differences and experimental conditions. When the effect of somatostatin was studied in condition of maximal secretory rate (Tm) for bilirubin (43) a similar decrease in bile flow and bile acid output was noted with an increased bilirubin concentration and no effect on the Tm. The effect of somatostatin on bile flow is saturable, with a maximal cholestatic effect displayed between a dosage of 17-33 ng/min/lOO g body wt. The “apparent half-maximal dosage” was calculated

Somatostatin” 1.56 f 1.50 f 2.02 f 1.73 f 2.40 f 1.58 *

Vol. 81. No. 3

0.04 0.06 0.04 0.12b 0.19 0.12=

Sham 2.28 f 2.25 f 42.03 f 41.42 f 91.10 f 89.57 f

0.22 0.14 1.35 2.05 1.67 2.48

liver)

Somatostatina 2.17 f 2.14 f 44.18 f 38.93 f 89.45 f 57.78 f

0.12 0.18 1.48 2.33b 3.75 3.61’

liver. b p < 0.05. ’ p < 0.01.

to be about 2.2 ng/min/lOO g body wt, or 7.5 ng/ min/rat. Assuming that the distribution volume of somatostatin corresponds to the plasma compartment, the calculated (44,45) concentration of somatostatin producing 50% of the effect would be about 0.5 ng/ml, which is in the same order of magnitude of the normal concentration in the portal vein (0.523 f 0.076 ng/ml) (46). Although the dosage and the rapid onset of effect would favor a physiologic role for somatostatin in modulating bile flow in the rat, there is no evidence at present that the actions of endogenous somatostatin are similar to those obtained with the exogenous polypeptide. A pronounced decrease in flow and bile acid excretion was also demonstrated in the dog (47-49) where an apparent K, of about loo-150 ng/min/kg body wt can be calculated (47). The action of somatostatin on the liver would favor an endocrine role for the polypeptide in the rat, in which a negative gradient is present between the portal and hepatic venous concentration of somatostatin (46), suggesting entry in, and catabolism or secretion by the liver cell. It has been shown that somatostatin can block the increase of CAMP levels in rat liver slices induced by glucagon (13) and enhance the activity of guanylate cyclase (50) at the same concentrations. However the role of CAMP with regard to choleresis has been challenged (51). Somatostatin could also activate specific receptors having a negative effect on some metabolic steps. At the moment, somatostatin can be proposed as a model to investigate integrate mechanisms of bile secretion. The absence of a gallbladder as a reservoir of biliary bile acids in the rat, suggests that the storage compartment is a dynamic, not an anatomic one, where bile acid cycling slows down with the transit velocity during the interprandial states. Somatostatin could be the endocrine modulator, changing the transit time in the liver and at the intestinal wall.

References 1. Forker

EL. Mechanisms Physiol 1977;39:323-47.

of hepatic bile formation.

Ann Rev

September

1981

2. Jones RS, Meyers WC. Regulation of hepatic biliary secretion. Ann Rev Physiol 1979;41:67-82. 3. Strasberg SM, Siminovitch KA, Ilson RG. Bile production in fasted and fed primates. Ann Surg 1974;180:356-83. 4. Austin GL, Johnson SM, Shires GT, Jones RS. The effect of feeding on the bile salt-independent canalicular secretion in dogs. Am J Surg 197&135:36-g. 5. Vonk RJ, Van Doorn ABD, Strubbe JH. Bile secretion and bile composition in the freely, moving unanesthetized rat. Influence of food intake on bile flow. Clin Sci Mol Med 1978;55:253-9. 6. Merle L, Dangoumau J, Balabaud C. Effect of food and light schedule on bile flow in the rat. Experientia 1978;34:764-5. 7. Dangoumau J, Balabaud C, Bussier-Leboeuf C, et al. Influence de la pentagastrine, de la cholecistokinine et de la caeruleine sur la cholerese chez le rat. J Pharmacol (Paris) 1977;8:197-264. 8. Kowalewski K. The effect of histamine and pentagastrin on secretion of bile in rats. Arch Int Pharmacodyn Ther 1972; 200:370-7. 9. Balabaud D, Peron-Marc D, Noel M, Dangoumau J. Influence du glucagon sur la cholerese chez le rat. J Pharmacol 1976;7:265-74. 10. Forker EL, Hicklin T, Sornson H. The clearance of mannitol and erythritol in rat bile. Proc Sot Exp Biol Med 1976;126:115-119. 11. Balabaud C, Noel M, Dangoumau J. Influence de la secretine sur la cholerese chez le rat. J Pharmacol (Paris) 1977;8:191-6. 12. Ghirlanda G, Bataille D, Dubois MP, Rosselin G. Variation of the somatostatin content of gut, pancreas and brain in the developing rat. Metabolism 1978;27(Suppl 1):1167-70. 13. Catalan RE, Avila C, Vila T, Castillon P. Somatostatin effect on cyclic-AMP levels mediated by glucagon stimulation in rat liver. Metabolism 1978;27:1359-61. 14. Sacks H, Waligora K. Mattew J, Pimstone BL. Inhibition by somatostatin of glucagon-induced glucose release from the isolated perfused rat liver. Endocrinology 1977;101:1751-9. 15. Meyers WC, Hanks JB, Jones RC. Inhibition of basal and meal-stimulated choleresis by somatostatin. Surgery 1979;86:301-6. 16. Klaassen CD. Bile flow and composition during bile acid depletion and administration. Can J Physiol Pharmacol 1974;52:334-48. 17. Meijer DKF, Keulemans K, Mulder GJ. The isolated and perfused rat liver technique. In: Jakoby WB, ed. Methods in enzymology. Detoxification and drug metabolism: conjugation and related systems. New York: Academic Press, 1980. 18. Tavoloni N, Reed JS, Boyer JL. Hemodynamic effects on determinants of bile secretion in isolated rat liver. Am J Physiol 1978;234:E584-92. 19. Van Roy FP, Heirwegh KPM. Determination of bilirubin glucuronide and assay of glucuronyltransferase with bilirubin as an acceptor. Biochem J 1968;107:507-18. 20. Heirwegh KPM, Fevery J, Michiels R, et al. Separation by thin-layer chromatography and structure elucidation of bilirubin conjugates isolated from dog bile. Biochem J 1978;145:185-99. 21. Compernolle F, Van Hees GP, Blanckaert N, Heirwegh KPM. Glucuronic acid conjugates of bilirubin IX in normal bile compared with post-obstructive bile. Transformation of the I-O-acylglucuronide into 2-, 3-, and 4-0-acylglucuronides. Biothem J 1978;171:185-201. 22. Blanckaert N, Compernolle F, Leroy P, et al. The fate of bilirubin-IX a-glucuronide in cholestasis and during storage in vitro. Biochem J 1978;171:203-14. 23. Turley SD, Dietschy JM. Re-evaluation of the 3a-hydroxysteroid dehydrogenase assay for total bile acids in bile. J Lipid Res 1978;19:924-8.

SOMATOSTATIN-INDUCED

CHOLESTASIS

IN THE RAT

561

24. Heirwegh KPM, Van de Vijver M, Fevery J. Assay and properties of digitonin-activated bilirubin UDP-glucuronyltransferase from rat liver. Biochem J 1972;129:605-18. 25. Omura T, Sato R. The carbon monoxide-binding pigment of liver microsomes. I. Evidence for its hemoprotein nature. J Biol Chem 239:2370-2378. 26. Gainey PA, Phelps CF. Uridine diphosphate glucuronic acid production and utilization in various tissues actively synthesizing glycosaminoglycans. Biochem J 1972;128:215-27. 27. Bosch J, Kravetz D, Rodes J. Effects of somatostatin on hepatic and systemic hemodynamics in patients with cirrhosis of the liver: comparison with vasopressin. Gastroenterology 1981;80:518-25. 28. Sonnenberg GE, Keller U, Perruchoud A, et al. Effect of somatostatin on splanchnic hemodynamics in patients with cirrhosis of the liver and in normal subjects. Gastroenterology 1981;80:526-32. 29. Schusdziarra V, Zyznar E, Roullier D, et al. Splanchnic somatostatin: a normal regulator of nutrient homeostasis. Science 1980;207:530-2. 30. Wilson FA, Autarson DL, Hart BL, et al. The effect of somatostatin on the intestinal transport of glucose, in vivo and in vitro in the rat. Endocrinology 1980;106:1562-7. 31. Guandalini S, Kachur JF, Smith PL, et al. In vitro effects of somatostatin on ion transport in rabbit intestine Am J Physiol 1980;238:G67-74. 32. Gardiner BN, Small DM. The effect of secretin and cholecystokinin on secretion of bile salts and biliary lipids. Clin Res 1972;20:454. 33. Balabaud C, et al. The assessment of the bile salt-nondependent fraction of canalicular bile water in the rat. J Lab Clin Med 1977;89:393-9. 34. Baker AL, Wood RAB, Moossa AR, Boyer JL. Sodium taurocholate modifies the bile acid-independent fraction of canalicular bile flow in the rhesus monkey. J Clin Invest 1979;64:312-20. 35. Barnhart JL, Combes B. Erythritol and mannitol clearances with taurocholate and secretin-induced cholereses. Am J Physiol 1978;243:E148-56. 36. Olson JR, Fujimoto JM. Evaluation of hepatobiliary function in rat by the segmented retrograde intrabiliary injection technique. Biochem Pharmacol 1980;29:205-11. 37. Tavoloni N, Reed JS, Hruban Z, Boyer JL. Effect of chlorpromazine on hepatic perfusion and bile secretory function in the isolated perfused rat liver. J Lab Clin Med 94:726-41. 38. Alpert S, Mosher M, Vhanske A, Arias IM. Multiplicity of hepatic excretory mechanisms for organic anions. J Gen Physiol 1969;53:238-47. 39. Cornelius CE. Organic anion transport in mutant sheep with congenital hyperbilirubinemia. Arch Environ Health 1969;19:852-6. 40. O’Maille ERL, Richards TG, Short AH. Factors determining the maximal rate of organic anion secretion by the liver and further evidence on the hepatic site of action of the hormone secretin. J Physiol 1966;186:424-38. 41. Goresky CA, Haddad HH, Kluger WS, et al. The enhancement of maximal bilirubin excretion with taurocholate-induced increments in bile flow. Can J Physiol Pharmacol 1974;52:389-463. 42. Van Steenbergen W, Kutz K, Fevery J. Effects of conjugation, bile flow and bile acid load on the apparent maximal secretion of bilirubin (“Tm”) 1979;3:208-12. In: The liver. Preisig R, Bircher J., eds. Aulendorf, Germany: Edit Cantor. 43. Ricci GL, Fevery J. Effect of secretin and somatostatin on the hepatic handling of bilirubin in the rat (abstr). Gastroenterology 1980;79,1121. 44. Lewis AE, Goodman RD. Schunk EA. Organ blood volume

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measurements in normal rats. J Lab Clin Med 1952;39:704-10. 45. Fingll E, Woodbury DM. In: Goodman LS, Gilman A, eds. The pharmacological bases of therapeutics. 5th ed. New York: MacMillan, 1975. 48. Berelowitz hi, Kronheim S, Pimstone B, Shapiro B. Somatostatin-like immunoreactivity in rat blood. Characteriiation, regional differences and responses to oral and intravenous glucose. J Clin Invest 1978;21:1410-5. 47. Lin T-M, Spray GF, Tust RH. Action of somatostatin (SS) on choledochal sphincter (CS), gallbladder (GB) and bile flow (BF) in dogs. Fed Proced 1978;57:557.

GASTROENTEROLOGY

Vol. 81,No. 3

48. Holm I, Thulin L. Samnegard H, et al. Anticholeretic effect of somatostatin in anesthetized dogs. Acta Physiol Stand 1978;104:241-3. 49. Hattinger S, Preisig R. Dual inhibitory effect of somatostatin on canalicular and ductular bile salt independent bile formation (abstr). Gastroenterology 1980;79:11QQ. 50. Vesely DL. The interrelationship of somatostatin and guanylate cyclase activity. Mol Cell Biochem 1980;32:131-4. 51. Poupon RE, Do1 M-L, Dumont M, Erlinger S. Evidence against a physiological role of CAMP in choleresis in dogs and rats. Biochem Pharmacol1978;27:2413-16.