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Biliary lipid output by isolated perfused rat livers in response to cholyl-lysylfluorescein
Biochi~ic~a
et Biophysica A~ta
ELSEVIER
Biochimica et Biophysica Acta 1256 (1995) 374-380
Biliary lipid output by isolated perfused rat livers in response to cholyl-lysylfluorescein Debbie J. Baxter
a,
Khalid Rahman a, Alison J. Bushell a, Charles O. Mills b Elwyn Elias b David Billington a,*
School of Biomolecular Sciences, Lieerpool John Moores University, Byrom Street, LiL~erpoolL3 3AF, UK b Department of Medicine, Queen Elizabeth Hospital, Edgbaston, Birmingham, B15 2TH, UK Received 1 September 1994; revised 23 January 1995; accepted 6 March 1995
Abstract The biliary output of bile acids and lipids is tightly coupled. The ability of the natural bile acid glycocholate to trigger biliary lipid secretion was compared with that of the fluorescent bile acid analogue cholyl-lysylfluorescein (cholyl-lys-F). When administered as a 5 min pulse of 2.5 /.~mol/min to bile acid-depleted rat livers perfused under recycling conditions, glycocholate produced well-defined peaks of phospholipid and cholesterol output, and of bile flow, which were coincident with the peak of bile acid output. Although cholyl-lys-F did trigger biliary lipid secretion, its time course of appearance was delayed and well-defined peaks of output were not observed. However, the increased biliary output of phospholipid and cholesterol was coincident with that of bile acids and, as judged by phospholipid/bile acid and cholesterol/bile acid ratios, cholyl-lys-F was as effective as glycocholate in triggering biliary lipid output. When administered to livers perfused under single pass conditions, perfusate to bile transfer of glycocholate was > 85% at infusion rates of up to 5 #tool/rain whereas transfer of cholyl-lys-F showed saturation at infusion rates of > 0.2 p.mol/min; the time course of biliary output of both bile acids was similar. Thus, under recycling conditions, cholyl-lys-F not taken up during first pass will be continually represented for transfer to bile, explaining why bile acid and lipid output did not occur as well-defined peaks. Keywords: Cholyl-lysylfluorescein; Glycocholate; Biliary lipid; Bile acid; Liver
1. Introduction In mammals most naturally occurring bile acids are conjugated to either glycine or taurine via the -COOH moiety at C-24. These are extracted with high efficiency from portal blood by hepatocytes and are rapidly secreted into bile [1,2]. Under normal circumstances, the biliary secretion of cholesterol and phospholipid is tightly coupled to that of the bile acids [3]. Rapid kinetic experiments have shown that bile acids are secreted into the canaliculus separate from, and prior to, the biliary lipids [4]. As the canalicular concentration of bile acids increases above that required to form micelles, the bile acids are believed to intercalate into fluid microdomains of the canalicular membrane [5]. Cholesterol and biliary-type phospholipid
Abbreviations: ALT, alanine aminotransferase; BSA, bovine serum albumin; cholyl-lys-F, cholyl-lysylfluorescein. * Corresponding author. Fax: +44 151 298 1014. 0005-2760/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0 0 0 5 - 2 7 6 0 ( 9 5 ) 0 0 0 5 0 - X
are then delivered into the canaliculus by outward vesiculation of these fluid microdomains [3,5]. Processing of the biliary lipids into other physical forms is thought to occur as the bile acid concentration in the biliary tract increases further [3]. A variety of fluorescent bile acid analogues are now available and have been used to study bile acid transport. One such analogue is cholyl-lysylfluorescein (cholyl-lys-F). In this compound lysine is conjugated at C-24 of cholate and fluorescein isothiocyanate is conjugated to the ~-NH 2 of the lysine [6]. Biliary excretion of cholyl-lys-F in bile fistula rats and extraction of low doses of cholyl-lys-F by perfused rat livers were similar to the natural bile acid glycocholate suggesting that both compounds are handled in a similar fashion [6]. Indeed, cholyl-lys-F has been used successfully to study bile acid transport in hepatocyte couplets [7,8]. The aim of this study was to compare the ability of cholyl-lys-F and glycocholate to stimulate biliary lipid secretion. This was achieved by infusing the bile acids as
D.J. Baxter et al. / Biochimica et Biophysica Acta 1256 (1995) 374-380
pulses of 5 min duration into perfused rat livers operating under either recycling or single pass conditions.
2. Materials and methods 2.1. Materials
Bovine serum albumin (fraction V) and 3c~-hydroxysteroid dehydrogenase were obtained from Sigma Chemical, Poole, Dorset, UK. Glycocholate (grade A, > 98% pure) was obtained from Calbiochem, Hereford, UK and [l-~4C]glycocholate (2.07 GBq/mmol) was from Amersham International, Aylesbury, UK. Cannulation tubing PP10 was from Portex, Hythe, UK and Ecoscint A was from Mensura Technology, Parbold, Wigan, UK. Cholyllys-F was synthesised and characterised as described previously [6]. Human blood was obtained through the courtesy of the Merseyside Blood Transfusion Centre and was used within 7 days of donation. Other fine chemicals were purchased from either Sigma Chemical or BDH, Poole, Dorset, UK. 2.2. LiL'er pe~usion
Male Wistar rats (250-320 g), fed a standard laboratory diet and maintained under a constant 12 h light-dark cycle, were used throughout. Perfusion of rat livers in situ was performed as described previously [9]. Briefly, bile ducts were cannulated with 80 cm of PP10 tubing (0.28 mm internal diameter). Recycling perfusion was commenced at a steady flow rate of 17 ml/min with 150 ml of KrebsRinger bicarbonate, pH 7.4 containing 1 mM CaCI 2, 5 mM glucose, a physiological amino acid mixture [10], 1% bovine serum albumin (BSA) and 20% ( v / v ) washed human erythrocytes. The perfusate was gassed continuously with O J C O 2 (19:1, v / v ) and animals were maintained in a thermostatically controlled cabinet at 37°C throughout the experiment. Viability of livers was assessed by a marked colour difference in the afferent and efferent perfusate, the liver's homogeneity and an initial bile flow rate of at least 0.8 /zl/min per g of liver. Bile was collected initially in 10 min aliquots for 30 rain to ensure that livers were depleted of endogenous bile acids. After 30 rain, either glycocholate or cholyl-lys-F were infused from a syringe pump into the livers via a side-arm port just prior to the portal cannula. The bile acids were dissolved in BSA- and erthyrocyte-free KrebsRinger bicarbonate buffer, pH 7.4 and were administered as a 5 rain pulse of 2.5 /zmol/min and at a flow rate of 0.19 ml/min. Bile was collected in 5 min aliquots for a further 40 min after commencing the pulse of bile acid. Bile flow rates were determined gravimetrically. In order to assess possible liver damage caused by the bile acids, approx. 1 ml of perfusate was taken just prior to starting the pulse of bile acid and at the end of the experiment,
375
centrifuged at 10000 X g for 1 rain to remove erythrocytes and the supernatant assayed for alanine aminotransferase (ALT) by a NAD+-coupled reaction [11] using Boehringer test kits. At the end of the experiment, the liver was removed, weighed and homogenised in BSA- and erythrocyte-free Krebs-Ringer bicarbonate buffer, pH 7.4, prior to assay for ALT. Another series of experiments were undertaken to assess the biliary recovery of exogenous bile acids. After 30 rain of recycling perfusion, livers were converted to single pass conditions using fresh medium and maintained in this mode for 10 rain. At the same time, a 5 min pulse of the bile acid was infused into the perfusion line at rates of 0.3-10/,tmol/min for glycocholate or 0.1-2.5 p, mol/min for cholyl-lys-F. The pulse of glycocholate also contained approx. 30 kBq of [1-14C]glycocholate. Livers were returned to recycling perfusion for the remaining 30 rain and bile was collected in 2 min aliquots. 2.3. Bilia O' lipid analyses
Bile was assayed for total phospholipid essentially by the method of Bartlett [12] after lipid extraction by the method of Bligh and Dyer [13]. Biliary cholesterol was measured by gas-liquid chromatography of its trimethylsilyl ester using 5a-cholestane as internal standard [14]. Total bile acids were assayed using 3o~-hydroxysteroid dehydrogenase as described previously [15] except that assays were performed in a Titertek Multiskan MCC/340 plate-reader in a final volume of 0.2 ml. In experiments to determine the biliary recovery of the exogenous bile acids, 20 p,1 of bile obtained after a pulse of [1-14C]glycocholate was mixed with 10 ml of Ecoscint A and radioactivity counted in a Canberra-Packard TR 1600 liquid scintillation counter. Aliquots of bile after a pulse of cholyl-lys-F were diluted into BSA- and erthyrocyte-free Krebs-Ringer bicarbonate, pH 7.4 and the absorbance at 490 nm read in a Cecil CE5501 spectrophotometer. Biliary output of both bile acids was expressed as nmol/min per g of liver by reference to appropriate standards and as a cumulative percentage of the infused dose.
3. Results 3.1. Recycling pe~, usions
After isolation of livers, bile flow rates (Fig. la) and the biliary output of bile acids (Fig. lb), phospholipid (Fig. lc) and cholesterol (Fig. ld) all declined substantially. When administered as a 5 rain pulse of 2.5 p, mol/min to livers operating under recycling conditions, glycocholate increased bile flow rates by 50% when compared to those just prior to the pulse, This choleresis was maximal in the 30-35 min bile sample (Fig. l a). Marked increases in biliary lipid output were also seen. Thus, total bile acid
D.J. Baxter et al. / Biochimica et Biophysica Acta 1256 (1995) 374-380
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Fig. 1. Bile flow rates (a) and biliary output of total bile acids (b), phospholipid (c) and cholesterol (d) in response to exogenous bile acids. Glycocholate ( • ) or cholyl-lys-F ( • ) were administered to isolated perfused rat livers operating under recycling conditions as a 5 min pulse (hatched bar) between 30-35 min after liver isolation. Values are means of 6 observations + S.E.
D.J. Baxter et al. / Biochimica et Biophysica Acta 1256 (1995) 374-380
(a)
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Table l ALT activity in the perfusate of isolated perfused rat livers operating under recycling conditions Pulse
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output was increased by at least 20-fold when compared to that just prior to the pulse (Fig. lb), whilst phospholipid and cholesterol output were increased by approx. 6-fold (Fig. lc,d). Peak outputs of bile acid, phospholipid and cholesterol occurred in the 35-40 min bile sample. In contrast to glycocholate, cholyl-lys-F caused only a minimal choleresis with bile flow being increased by < 10% in the 30-35 min bile sample (Fig. la). In addition, whilst cholyl-lys-F did stimulate substantial biliary lipid secretion, its time course was markedly different to that caused by glycocholate. Thus, although biliary bile acid output was increased several fold over that prior to the pulse of cholyl-lys-F, a well-defined peak was not
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The perfusate was assayed for ALT 30 min and 70 min after liver isolation (i.e., just prior to a pulse of either glycocholate or cholyl-lys-F and at the end of the perfusion). Values are mean + S.E. of 6 observations.
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TIME (MINS) Fig. 2. Cholesterol/bile acid (a) and phopholipid/bile acid (b) ratios in bile. Glycocholate ( • ) or cholyl-lys-F ( • ) were administered to isolated perfused rat livers operating under recycling conditions as a 5 min pulse (hatched bar) between 30-35 min after liver isolation, Values are means of 6 observations + S.E.
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PULSE OF BILE ACID ( p m o l l m l n ) Fig, 3. Biliary recovery of [ 1-14C]glycocholate ( • ) and cholyl-lys-F ( • ). Each bile acid was administered to isolated perfused rat livers operating under single pass conditions as a 5 min pulse at various infusion rates. Recoveries are percentages of the total infused dose.
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D.J. Baxter et al. / Biochimica et Biophysica Acta 1256 (1995) 374 380
observed and output rose to a broad maximum after 40-50 rain before gradually declining (Fig lb). However, even after 70 min of perfusion, bile acid output had not declined to values similar to those just prior to the pulse of cholyllys-F (Fig l b). Broadly similar time courses of phospholipid (Fig. lc) and cholesterol (Fig. ld) output were also observed after a pulse of cholyl-lys-F. The biliary output of phospholipid and cholesterol stimulated by cholyl-lys-F was similar to that stimulated by glycocholate. Thus, the biliary cholesterol/bile acid and phospholipid/bile acid ratios were similar after a pulse of cholyl-lys-F or glycocholate (Fig. 2). In addition, it is noteworthy that both the cholesterol/bile acid and phospholipid/bile acid ratios decreased sharply after the pulse of bile acid and then increased towards pre-pulse values throughout the rest of the perfusion (Fig 2). Minimal increases in perfusate ALT activity occurred after the pulse of either glycocholate or cholyl-lys-F and a further 40 rain of perfusion (Table 1). Indeed, when assessed by Student's t-test, these increases were not significantly different.
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Fig. 4. Time course of the biliary output of (a) glycocholate and (b) cholyl-lys-F. Each bile acid was administered to isolated peffused rat livers operating under single pass conditions as a 5 min pulse at the following infusion rates. (a): 10 /xmol/min ( • ) , 7.5 /zmol/min (filled hour glass), 5 /~mol/min ( a ) , 2.5 /.~mol/min (O), 1.25 p, mol/min (O), 0.6 /zmol/min (A); (b): 2.5 /xmol/min (filled hour glass), 1.25 /zmol/min ( • ), 0.6/zmol/min ( • ), 0.3/.zmol/min (O), 0.2 /zmol/min ( * ) , 0.1 /zmol/min (B).
3.2. Single pass pe~. usions When administered as a 5 min pulse to perfused livers operating under single pass conditions, > 85% of the infused [1-14C]glycocholate was transferred from perfusate to bile at infusion rates of 2.5 /zmol/min or less (Fig. 3). At higher infusion rates, perfusate to bile transport of [1-14C]glycocholate began to show saturation such that biliary recovery was approx. 70% at infusion rates of 10 ~mol/min. Greater than 85% of cholyl-lys-F was transported from perfusate to bile only at infusion rates of < 0.2 /zmol/min (Fig. 3). Transport of cholyl-lys-F showed saturation at much lower rates of infusion than glycocholate. Thus, when infused at 1 /J.mol/min, only approx. 40% of cholyl-lys-F was recovered in bile (Fig. 3). Peak output of [1-14C]glycocholate occurred in the 3638 min bile sample at all infusion rates whilst peak output of cholyl-lys-F varied between the 36-38 min and 40-42 rain samples (Fig. 4). Taking into account the volume of the bile duct cannula (49 /xl) and the biliary tree (approx. 35 /zl, [16]), and the bile flow rate in each experiment, there was a delay of 3-8 rain between the bile acid entering the bile canaliculi and it appearing in the collection tube. Correction of the peak output times for this delay showed that peak appearance of the bile acid in the canaliculi was the same for glycocholate and cholyl-lys-F and occurred after 34-35 rain (i.e., 4-5 min after the start of the pulse).
4. Discussion
Biliary bile acids are derived either from de novo synthesis in the liver or from the sinusoidal uptake of bile
D.J. Baxter et al. / Biochimica et Biophysica Acta 1256 (1995) 374-380
acids returning to the liver via the enterohepatic circulation. Pre-perfusion of livers for 30 rain substantially decreased the biliary output of bile acids (and hence biliary lipid and bile flow rate) due to total interruption of the enterohepatic circulation. Thus, just prior to the pulse of bile acid, biliary lipid output was almost entirely related to de novo synthesis of bile acids (see also [10,17]). Previous work has shown that 80-90% of conjugated trihydroxy bile acids are removed from the blood or perfusion medium during a single passage through the liver [18,19]. Because both glycocholate and cholyl-lys-F were believed to be handled in a similar fashion [6], they were presented to livers perfused under recycling conditions as a 5 min pulse at 2.5/xmol/min. This size of pulse was chosen so as to trigger substantial lipid release without exerting toxicity towards the liver. The lack of hepatotoxicity of both glycocholate and cholyl-lys-F was confirmed by the minimal increases in perfusate ALT activity after the pulse of bile acid and a further 40 min of perfusion (Table 1). The pulse of glycocholate produced well-defined peaks of biliary bile acid, phospholipid and cholesterol output and of flow rate; all these peaks were effectively coincident (Fig. 1). These results confirm that the biliary output of lipid and glycocholate are tightly coupled. However, collecting bile in relatively large 5 rain aliquots did not allow us to confirm that biliary bile acid secretion precedes that of phospholipid and cholesterol. Cholyl-lys-F did not produce well-defined peaks of biliary output and lipid output was considerably extended compared to that induced by glycocholate. Nevertheless, the outputs of bile acid, phospholipid and cholesterol were still coincident (Fig. 1). This implies that once in the canaliculus, cholyllys-F triggers biliary lipid output in a similar fashion to glycocholate. Individual bile acids vary in their ability to promote biliary lipid secretion. In general, lipid secretion is positively correlated to the hydrophobicity of the bile acid [3]. Thus, bile acids possessing two hydroxyl groups are more effective in promoting lipid secretion than their trihydroxy counterparts [20]. In addition, if one or more of the hydroxyls is sulfated, or replaced by a keto group, or it's orientation to the steroid nucleus changed from a to /3, biliary lipid output is decreased [20-22]. Both glycocholate and cholyl-lys-F have three c~-substituted hydroxyl groups and therefore their ability to stimulate biliary lipid secretion would be expected to be broadly similar. Indeed, as judged by the cholesterol/bile acid and phospholipid/bile acid ratios, cholyl-lys-F is as effective as glycocholate in stimulating biliary lipid output (Fig. 2). Other fluorescent bile acids such as nitrobenzoxadiazol-Ncholyltaurine (7/3-NBD-NCT) [23,24] might be expected to be less effective than natural bile acids in promoting biliary lipid secretion owing to the substitution at, and change in the orientation of, the hydroxyl group at C-7. In order to investigate why the time courses of the
379
biliary output of bile acids and lipid after a pulse of glycocholate or cholyl-lys-F were different, each bile acid was presented to perfused livers operating under single pass conditions. Fig. 3 clearly shows that rat liver has a much greater capacity to transfer glycocholate from perfusate to bile than cholyl-lys-F. At infusion rates of < 0.2 /~mol/min, > 85% of both glycocholate and cholyl-lys-F were transferred to bile. It is noteworthy that Mills et al. [6], when first comparing the extraction of glycocholate and cholyl-lys-F by perfused livers, administered each bile acid as a 1 min pulse of 0.2 p, mol. It is only at infusion rates of > 0.2 /xmol/min that differences in the ability of livers to handle glycocholate and cholyl-lys-F became apparent. After allowing for the dead volume of the bile duct cannula and the biliary tree, the transcellular transit times of glycocholate and cholyl-lys-F were shown to be similar (Fig. 4). Thus, it is likely that glycocholate and cholyl-lys-F have different affinities for their transport proteins in the sinusoidal and canalicular membranes and this causes the different biliary secretion capacities. The results presented here show that cholyl-lys-F stimulates biliary phospholipid and cholesterol output and is as effective as glycocholate. However, at high infusion rates, cholyl-lys-F is transferred to bile less efficiently than glycocholate. Thus, when presented as a 5 min pulse at 2.5 /xmol/min, < 40% of cholyl-lys-F is transferred first pass from perfusate to bile. Under recycling conditions, the remaining cholyl-lys-F will be continually presented for transfer to bile, explaining why bile acid and lipid output were only increased moderately and were extended over a long time period (Fig. 1). In contrast, > 85% of glycocholate is transferred first pass such that little is available for transfer on subsequent passages through the liver; hence biliary output occurred as well-defined peaks (Fig. 1). It would be reasonable to suggest that at low infusion rates under recycling conditions, when cholyl-lys-F is transferred to bile as efficiently as glycocholate, the time courses of biliary bile acid and lipid output would be similar. In conclusion, our studies show that cholyl-lys-F has similar properties to it's natural counterpart glycocholate, at least at low rates of infusion. The relatively easy detection and quantitation of cholyl-lys-F make it a useful tool with which to probe hepatobiliary function.
Acknowledgements DEB is in receipt of a Liverpool JMU studentship.
References [l] Levy,D. (1993) in HepaticTransportand Bile Secretion:Physiology and Pathophysiology(Travoloni, N. and Berk, P.D., eds.), pp. 245-252, Raven Press, New York.
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[2] Meier, P.J. (1993) in Hepatic Transport and Bile Secretion: Physiology and Pathophysiology (Travoloni, N. and Berk, P.D., eds.), pp. 587-596, Raven Press, New York. [3] Coleman, R. and Rahman, K. (1992) Biochim. Biophys. Acta 1125, 113-133. [4] Lowe, P.J., Barnwell, S,G. and Coleman, R. (1984) Biochem. J. 222, 631-637. [5] Coleman, R. (1987) Biochem. Soc. Trans. 15, 68S-80S. [6] Mills, C.O., Rahman, K., Coleman, R. and Elias, E. (1992) Biochim. Biophys. Acta 1115, 151-156. [7] Wilton, J.C., Coleman, R., Lankester, D.J. and Chipman, J.K, (1993) Cell Biochem. Funct. 11, 179-185. [8] Wilton, J.C., Chipman, J.K, Lawson, C.J., Strain, A.J. and Coleman, R. (1993) Biochem. J. 292, 773-779. [9] Billington, D., Chard, P.S. and Clayton, M. (1990) Biochem. Pharmacol. 39, 1624-1627. [10] Barnwell, S.G., Godfrey, P.P., Lowe, P.J. and Coleman, R. (1983) Biochem. J. 210, 549-557. [11] Bergmeyer, H.U., Scheibe, P. and Wahlefeld, A.W. (1978) Clin. Chem. 24, 58-73. [12] Bligh, E.G. and Dyer, W.J. (1959) Can. J. Biochem. Physiol. 37, 911-919.
[13] Bartlett, G.R. (1959) J. Biol. Chem. 234, 466-468. [14] Rahman, K. and Coleman, R. (1986) Biochem. J. 237, 301-304. [15] Coleman, R., Iqbal, S., Godfrey, P.P. and Billington, D. (1979) Biochem. J. 178, 201-208. [16] Olson, J.R. and Fugimoto, LM. (1979) Biochem. Pharmacol. 29, 205-211. [17] Barnwetl, S.G., Lowe, P.J. and Coleman, R. (1984) Biochem. J. 220, 723-731. [18] Hoffman, N.E., lser, J.H. and Smallwood, R.A. (1975) Am. J. Physiol. 229, 298-302. [19] Iga, T. and Klassen, C.D. (1981) Biochem. Pharmacol. 31,205-209. [20] Gurantz, D. and Hofman, A.F. (1984) Am. J. Physiol. 247, G736G748. [21] YouseL I.M., Barnwell, S.G., Tuchweber, B., Weber, A. and Roy, C.C. (1987) Hepatology 7, 535-542. [22] Bilhartz, L.E. and Dietschy, J.M. (1988) Gastroenterology 95, 771779. [23] Schramm, U., Frickert, G., Buscher, H.P., Gerok, W. and Kurz, G. (1993) J. Lipid Res. 34, 741-757. [24] Weinman, S.A., Graf, J., Veith, C. and Boyer, J.L. (1993) Am. J. Physiol. 264, G220-G230.
et Biophysica A~ta
ELSEVIER
Biochimica et Biophysica Acta 1256 (1995) 374-380
Biliary lipid output by isolated perfused rat livers in response to cholyl-lysylfluorescein Debbie J. Baxter
a,
Khalid Rahman a, Alison J. Bushell a, Charles O. Mills b Elwyn Elias b David Billington a,*
School of Biomolecular Sciences, Lieerpool John Moores University, Byrom Street, LiL~erpoolL3 3AF, UK b Department of Medicine, Queen Elizabeth Hospital, Edgbaston, Birmingham, B15 2TH, UK Received 1 September 1994; revised 23 January 1995; accepted 6 March 1995
Abstract The biliary output of bile acids and lipids is tightly coupled. The ability of the natural bile acid glycocholate to trigger biliary lipid secretion was compared with that of the fluorescent bile acid analogue cholyl-lysylfluorescein (cholyl-lys-F). When administered as a 5 min pulse of 2.5 /.~mol/min to bile acid-depleted rat livers perfused under recycling conditions, glycocholate produced well-defined peaks of phospholipid and cholesterol output, and of bile flow, which were coincident with the peak of bile acid output. Although cholyl-lys-F did trigger biliary lipid secretion, its time course of appearance was delayed and well-defined peaks of output were not observed. However, the increased biliary output of phospholipid and cholesterol was coincident with that of bile acids and, as judged by phospholipid/bile acid and cholesterol/bile acid ratios, cholyl-lys-F was as effective as glycocholate in triggering biliary lipid output. When administered to livers perfused under single pass conditions, perfusate to bile transfer of glycocholate was > 85% at infusion rates of up to 5 #tool/rain whereas transfer of cholyl-lys-F showed saturation at infusion rates of > 0.2 p.mol/min; the time course of biliary output of both bile acids was similar. Thus, under recycling conditions, cholyl-lys-F not taken up during first pass will be continually represented for transfer to bile, explaining why bile acid and lipid output did not occur as well-defined peaks. Keywords: Cholyl-lysylfluorescein; Glycocholate; Biliary lipid; Bile acid; Liver
1. Introduction In mammals most naturally occurring bile acids are conjugated to either glycine or taurine via the -COOH moiety at C-24. These are extracted with high efficiency from portal blood by hepatocytes and are rapidly secreted into bile [1,2]. Under normal circumstances, the biliary secretion of cholesterol and phospholipid is tightly coupled to that of the bile acids [3]. Rapid kinetic experiments have shown that bile acids are secreted into the canaliculus separate from, and prior to, the biliary lipids [4]. As the canalicular concentration of bile acids increases above that required to form micelles, the bile acids are believed to intercalate into fluid microdomains of the canalicular membrane [5]. Cholesterol and biliary-type phospholipid
Abbreviations: ALT, alanine aminotransferase; BSA, bovine serum albumin; cholyl-lys-F, cholyl-lysylfluorescein. * Corresponding author. Fax: +44 151 298 1014. 0005-2760/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0 0 0 5 - 2 7 6 0 ( 9 5 ) 0 0 0 5 0 - X
are then delivered into the canaliculus by outward vesiculation of these fluid microdomains [3,5]. Processing of the biliary lipids into other physical forms is thought to occur as the bile acid concentration in the biliary tract increases further [3]. A variety of fluorescent bile acid analogues are now available and have been used to study bile acid transport. One such analogue is cholyl-lysylfluorescein (cholyl-lys-F). In this compound lysine is conjugated at C-24 of cholate and fluorescein isothiocyanate is conjugated to the ~-NH 2 of the lysine [6]. Biliary excretion of cholyl-lys-F in bile fistula rats and extraction of low doses of cholyl-lys-F by perfused rat livers were similar to the natural bile acid glycocholate suggesting that both compounds are handled in a similar fashion [6]. Indeed, cholyl-lys-F has been used successfully to study bile acid transport in hepatocyte couplets [7,8]. The aim of this study was to compare the ability of cholyl-lys-F and glycocholate to stimulate biliary lipid secretion. This was achieved by infusing the bile acids as
D.J. Baxter et al. / Biochimica et Biophysica Acta 1256 (1995) 374-380
pulses of 5 min duration into perfused rat livers operating under either recycling or single pass conditions.
2. Materials and methods 2.1. Materials
Bovine serum albumin (fraction V) and 3c~-hydroxysteroid dehydrogenase were obtained from Sigma Chemical, Poole, Dorset, UK. Glycocholate (grade A, > 98% pure) was obtained from Calbiochem, Hereford, UK and [l-~4C]glycocholate (2.07 GBq/mmol) was from Amersham International, Aylesbury, UK. Cannulation tubing PP10 was from Portex, Hythe, UK and Ecoscint A was from Mensura Technology, Parbold, Wigan, UK. Cholyllys-F was synthesised and characterised as described previously [6]. Human blood was obtained through the courtesy of the Merseyside Blood Transfusion Centre and was used within 7 days of donation. Other fine chemicals were purchased from either Sigma Chemical or BDH, Poole, Dorset, UK. 2.2. LiL'er pe~usion
Male Wistar rats (250-320 g), fed a standard laboratory diet and maintained under a constant 12 h light-dark cycle, were used throughout. Perfusion of rat livers in situ was performed as described previously [9]. Briefly, bile ducts were cannulated with 80 cm of PP10 tubing (0.28 mm internal diameter). Recycling perfusion was commenced at a steady flow rate of 17 ml/min with 150 ml of KrebsRinger bicarbonate, pH 7.4 containing 1 mM CaCI 2, 5 mM glucose, a physiological amino acid mixture [10], 1% bovine serum albumin (BSA) and 20% ( v / v ) washed human erythrocytes. The perfusate was gassed continuously with O J C O 2 (19:1, v / v ) and animals were maintained in a thermostatically controlled cabinet at 37°C throughout the experiment. Viability of livers was assessed by a marked colour difference in the afferent and efferent perfusate, the liver's homogeneity and an initial bile flow rate of at least 0.8 /zl/min per g of liver. Bile was collected initially in 10 min aliquots for 30 rain to ensure that livers were depleted of endogenous bile acids. After 30 rain, either glycocholate or cholyl-lys-F were infused from a syringe pump into the livers via a side-arm port just prior to the portal cannula. The bile acids were dissolved in BSA- and erthyrocyte-free KrebsRinger bicarbonate buffer, pH 7.4 and were administered as a 5 rain pulse of 2.5 /zmol/min and at a flow rate of 0.19 ml/min. Bile was collected in 5 min aliquots for a further 40 min after commencing the pulse of bile acid. Bile flow rates were determined gravimetrically. In order to assess possible liver damage caused by the bile acids, approx. 1 ml of perfusate was taken just prior to starting the pulse of bile acid and at the end of the experiment,
375
centrifuged at 10000 X g for 1 rain to remove erythrocytes and the supernatant assayed for alanine aminotransferase (ALT) by a NAD+-coupled reaction [11] using Boehringer test kits. At the end of the experiment, the liver was removed, weighed and homogenised in BSA- and erythrocyte-free Krebs-Ringer bicarbonate buffer, pH 7.4, prior to assay for ALT. Another series of experiments were undertaken to assess the biliary recovery of exogenous bile acids. After 30 rain of recycling perfusion, livers were converted to single pass conditions using fresh medium and maintained in this mode for 10 rain. At the same time, a 5 min pulse of the bile acid was infused into the perfusion line at rates of 0.3-10/,tmol/min for glycocholate or 0.1-2.5 p, mol/min for cholyl-lys-F. The pulse of glycocholate also contained approx. 30 kBq of [1-14C]glycocholate. Livers were returned to recycling perfusion for the remaining 30 rain and bile was collected in 2 min aliquots. 2.3. Bilia O' lipid analyses
Bile was assayed for total phospholipid essentially by the method of Bartlett [12] after lipid extraction by the method of Bligh and Dyer [13]. Biliary cholesterol was measured by gas-liquid chromatography of its trimethylsilyl ester using 5a-cholestane as internal standard [14]. Total bile acids were assayed using 3o~-hydroxysteroid dehydrogenase as described previously [15] except that assays were performed in a Titertek Multiskan MCC/340 plate-reader in a final volume of 0.2 ml. In experiments to determine the biliary recovery of the exogenous bile acids, 20 p,1 of bile obtained after a pulse of [1-14C]glycocholate was mixed with 10 ml of Ecoscint A and radioactivity counted in a Canberra-Packard TR 1600 liquid scintillation counter. Aliquots of bile after a pulse of cholyl-lys-F were diluted into BSA- and erthyrocyte-free Krebs-Ringer bicarbonate, pH 7.4 and the absorbance at 490 nm read in a Cecil CE5501 spectrophotometer. Biliary output of both bile acids was expressed as nmol/min per g of liver by reference to appropriate standards and as a cumulative percentage of the infused dose.
3. Results 3.1. Recycling pe~, usions
After isolation of livers, bile flow rates (Fig. la) and the biliary output of bile acids (Fig. lb), phospholipid (Fig. lc) and cholesterol (Fig. ld) all declined substantially. When administered as a 5 rain pulse of 2.5 p, mol/min to livers operating under recycling conditions, glycocholate increased bile flow rates by 50% when compared to those just prior to the pulse, This choleresis was maximal in the 30-35 min bile sample (Fig. l a). Marked increases in biliary lipid output were also seen. Thus, total bile acid
D.J. Baxter et al. / Biochimica et Biophysica Acta 1256 (1995) 374-380
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Fig. 1. Bile flow rates (a) and biliary output of total bile acids (b), phospholipid (c) and cholesterol (d) in response to exogenous bile acids. Glycocholate ( • ) or cholyl-lys-F ( • ) were administered to isolated perfused rat livers operating under recycling conditions as a 5 min pulse (hatched bar) between 30-35 min after liver isolation. Values are means of 6 observations + S.E.
D.J. Baxter et al. / Biochimica et Biophysica Acta 1256 (1995) 374-380
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output was increased by at least 20-fold when compared to that just prior to the pulse (Fig. lb), whilst phospholipid and cholesterol output were increased by approx. 6-fold (Fig. lc,d). Peak outputs of bile acid, phospholipid and cholesterol occurred in the 35-40 min bile sample. In contrast to glycocholate, cholyl-lys-F caused only a minimal choleresis with bile flow being increased by < 10% in the 30-35 min bile sample (Fig. la). In addition, whilst cholyl-lys-F did stimulate substantial biliary lipid secretion, its time course was markedly different to that caused by glycocholate. Thus, although biliary bile acid output was increased several fold over that prior to the pulse of cholyl-lys-F, a well-defined peak was not
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TIME (MINS) Fig. 2. Cholesterol/bile acid (a) and phopholipid/bile acid (b) ratios in bile. Glycocholate ( • ) or cholyl-lys-F ( • ) were administered to isolated perfused rat livers operating under recycling conditions as a 5 min pulse (hatched bar) between 30-35 min after liver isolation, Values are means of 6 observations + S.E.
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D.J. Baxter et al. / Biochimica et Biophysica Acta 1256 (1995) 374 380
observed and output rose to a broad maximum after 40-50 rain before gradually declining (Fig lb). However, even after 70 min of perfusion, bile acid output had not declined to values similar to those just prior to the pulse of cholyllys-F (Fig l b). Broadly similar time courses of phospholipid (Fig. lc) and cholesterol (Fig. ld) output were also observed after a pulse of cholyl-lys-F. The biliary output of phospholipid and cholesterol stimulated by cholyl-lys-F was similar to that stimulated by glycocholate. Thus, the biliary cholesterol/bile acid and phospholipid/bile acid ratios were similar after a pulse of cholyl-lys-F or glycocholate (Fig. 2). In addition, it is noteworthy that both the cholesterol/bile acid and phospholipid/bile acid ratios decreased sharply after the pulse of bile acid and then increased towards pre-pulse values throughout the rest of the perfusion (Fig 2). Minimal increases in perfusate ALT activity occurred after the pulse of either glycocholate or cholyl-lys-F and a further 40 rain of perfusion (Table 1). Indeed, when assessed by Student's t-test, these increases were not significantly different.
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Fig. 4. Time course of the biliary output of (a) glycocholate and (b) cholyl-lys-F. Each bile acid was administered to isolated peffused rat livers operating under single pass conditions as a 5 min pulse at the following infusion rates. (a): 10 /xmol/min ( • ) , 7.5 /zmol/min (filled hour glass), 5 /~mol/min ( a ) , 2.5 /.~mol/min (O), 1.25 p, mol/min (O), 0.6 /zmol/min (A); (b): 2.5 /xmol/min (filled hour glass), 1.25 /zmol/min ( • ), 0.6/zmol/min ( • ), 0.3/.zmol/min (O), 0.2 /zmol/min ( * ) , 0.1 /zmol/min (B).
3.2. Single pass pe~. usions When administered as a 5 min pulse to perfused livers operating under single pass conditions, > 85% of the infused [1-14C]glycocholate was transferred from perfusate to bile at infusion rates of 2.5 /zmol/min or less (Fig. 3). At higher infusion rates, perfusate to bile transport of [1-14C]glycocholate began to show saturation such that biliary recovery was approx. 70% at infusion rates of 10 ~mol/min. Greater than 85% of cholyl-lys-F was transported from perfusate to bile only at infusion rates of < 0.2 /zmol/min (Fig. 3). Transport of cholyl-lys-F showed saturation at much lower rates of infusion than glycocholate. Thus, when infused at 1 /J.mol/min, only approx. 40% of cholyl-lys-F was recovered in bile (Fig. 3). Peak output of [1-14C]glycocholate occurred in the 3638 min bile sample at all infusion rates whilst peak output of cholyl-lys-F varied between the 36-38 min and 40-42 rain samples (Fig. 4). Taking into account the volume of the bile duct cannula (49 /xl) and the biliary tree (approx. 35 /zl, [16]), and the bile flow rate in each experiment, there was a delay of 3-8 rain between the bile acid entering the bile canaliculi and it appearing in the collection tube. Correction of the peak output times for this delay showed that peak appearance of the bile acid in the canaliculi was the same for glycocholate and cholyl-lys-F and occurred after 34-35 rain (i.e., 4-5 min after the start of the pulse).
4. Discussion
Biliary bile acids are derived either from de novo synthesis in the liver or from the sinusoidal uptake of bile
D.J. Baxter et al. / Biochimica et Biophysica Acta 1256 (1995) 374-380
acids returning to the liver via the enterohepatic circulation. Pre-perfusion of livers for 30 rain substantially decreased the biliary output of bile acids (and hence biliary lipid and bile flow rate) due to total interruption of the enterohepatic circulation. Thus, just prior to the pulse of bile acid, biliary lipid output was almost entirely related to de novo synthesis of bile acids (see also [10,17]). Previous work has shown that 80-90% of conjugated trihydroxy bile acids are removed from the blood or perfusion medium during a single passage through the liver [18,19]. Because both glycocholate and cholyl-lys-F were believed to be handled in a similar fashion [6], they were presented to livers perfused under recycling conditions as a 5 min pulse at 2.5/xmol/min. This size of pulse was chosen so as to trigger substantial lipid release without exerting toxicity towards the liver. The lack of hepatotoxicity of both glycocholate and cholyl-lys-F was confirmed by the minimal increases in perfusate ALT activity after the pulse of bile acid and a further 40 min of perfusion (Table 1). The pulse of glycocholate produced well-defined peaks of biliary bile acid, phospholipid and cholesterol output and of flow rate; all these peaks were effectively coincident (Fig. 1). These results confirm that the biliary output of lipid and glycocholate are tightly coupled. However, collecting bile in relatively large 5 rain aliquots did not allow us to confirm that biliary bile acid secretion precedes that of phospholipid and cholesterol. Cholyl-lys-F did not produce well-defined peaks of biliary output and lipid output was considerably extended compared to that induced by glycocholate. Nevertheless, the outputs of bile acid, phospholipid and cholesterol were still coincident (Fig. 1). This implies that once in the canaliculus, cholyllys-F triggers biliary lipid output in a similar fashion to glycocholate. Individual bile acids vary in their ability to promote biliary lipid secretion. In general, lipid secretion is positively correlated to the hydrophobicity of the bile acid [3]. Thus, bile acids possessing two hydroxyl groups are more effective in promoting lipid secretion than their trihydroxy counterparts [20]. In addition, if one or more of the hydroxyls is sulfated, or replaced by a keto group, or it's orientation to the steroid nucleus changed from a to /3, biliary lipid output is decreased [20-22]. Both glycocholate and cholyl-lys-F have three c~-substituted hydroxyl groups and therefore their ability to stimulate biliary lipid secretion would be expected to be broadly similar. Indeed, as judged by the cholesterol/bile acid and phospholipid/bile acid ratios, cholyl-lys-F is as effective as glycocholate in stimulating biliary lipid output (Fig. 2). Other fluorescent bile acids such as nitrobenzoxadiazol-Ncholyltaurine (7/3-NBD-NCT) [23,24] might be expected to be less effective than natural bile acids in promoting biliary lipid secretion owing to the substitution at, and change in the orientation of, the hydroxyl group at C-7. In order to investigate why the time courses of the
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biliary output of bile acids and lipid after a pulse of glycocholate or cholyl-lys-F were different, each bile acid was presented to perfused livers operating under single pass conditions. Fig. 3 clearly shows that rat liver has a much greater capacity to transfer glycocholate from perfusate to bile than cholyl-lys-F. At infusion rates of < 0.2 /~mol/min, > 85% of both glycocholate and cholyl-lys-F were transferred to bile. It is noteworthy that Mills et al. [6], when first comparing the extraction of glycocholate and cholyl-lys-F by perfused livers, administered each bile acid as a 1 min pulse of 0.2 p, mol. It is only at infusion rates of > 0.2 /xmol/min that differences in the ability of livers to handle glycocholate and cholyl-lys-F became apparent. After allowing for the dead volume of the bile duct cannula and the biliary tree, the transcellular transit times of glycocholate and cholyl-lys-F were shown to be similar (Fig. 4). Thus, it is likely that glycocholate and cholyl-lys-F have different affinities for their transport proteins in the sinusoidal and canalicular membranes and this causes the different biliary secretion capacities. The results presented here show that cholyl-lys-F stimulates biliary phospholipid and cholesterol output and is as effective as glycocholate. However, at high infusion rates, cholyl-lys-F is transferred to bile less efficiently than glycocholate. Thus, when presented as a 5 min pulse at 2.5 /xmol/min, < 40% of cholyl-lys-F is transferred first pass from perfusate to bile. Under recycling conditions, the remaining cholyl-lys-F will be continually presented for transfer to bile, explaining why bile acid and lipid output were only increased moderately and were extended over a long time period (Fig. 1). In contrast, > 85% of glycocholate is transferred first pass such that little is available for transfer on subsequent passages through the liver; hence biliary output occurred as well-defined peaks (Fig. 1). It would be reasonable to suggest that at low infusion rates under recycling conditions, when cholyl-lys-F is transferred to bile as efficiently as glycocholate, the time courses of biliary bile acid and lipid output would be similar. In conclusion, our studies show that cholyl-lys-F has similar properties to it's natural counterpart glycocholate, at least at low rates of infusion. The relatively easy detection and quantitation of cholyl-lys-F make it a useful tool with which to probe hepatobiliary function.
Acknowledgements DEB is in receipt of a Liverpool JMU studentship.
References [l] Levy,D. (1993) in HepaticTransportand Bile Secretion:Physiology and Pathophysiology(Travoloni, N. and Berk, P.D., eds.), pp. 245-252, Raven Press, New York.
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[2] Meier, P.J. (1993) in Hepatic Transport and Bile Secretion: Physiology and Pathophysiology (Travoloni, N. and Berk, P.D., eds.), pp. 587-596, Raven Press, New York. [3] Coleman, R. and Rahman, K. (1992) Biochim. Biophys. Acta 1125, 113-133. [4] Lowe, P.J., Barnwell, S,G. and Coleman, R. (1984) Biochem. J. 222, 631-637. [5] Coleman, R. (1987) Biochem. Soc. Trans. 15, 68S-80S. [6] Mills, C.O., Rahman, K., Coleman, R. and Elias, E. (1992) Biochim. Biophys. Acta 1115, 151-156. [7] Wilton, J.C., Coleman, R., Lankester, D.J. and Chipman, J.K, (1993) Cell Biochem. Funct. 11, 179-185. [8] Wilton, J.C., Chipman, J.K, Lawson, C.J., Strain, A.J. and Coleman, R. (1993) Biochem. J. 292, 773-779. [9] Billington, D., Chard, P.S. and Clayton, M. (1990) Biochem. Pharmacol. 39, 1624-1627. [10] Barnwell, S.G., Godfrey, P.P., Lowe, P.J. and Coleman, R. (1983) Biochem. J. 210, 549-557. [11] Bergmeyer, H.U., Scheibe, P. and Wahlefeld, A.W. (1978) Clin. Chem. 24, 58-73. [12] Bligh, E.G. and Dyer, W.J. (1959) Can. J. Biochem. Physiol. 37, 911-919.
[13] Bartlett, G.R. (1959) J. Biol. Chem. 234, 466-468. [14] Rahman, K. and Coleman, R. (1986) Biochem. J. 237, 301-304. [15] Coleman, R., Iqbal, S., Godfrey, P.P. and Billington, D. (1979) Biochem. J. 178, 201-208. [16] Olson, J.R. and Fugimoto, LM. (1979) Biochem. Pharmacol. 29, 205-211. [17] Barnwetl, S.G., Lowe, P.J. and Coleman, R. (1984) Biochem. J. 220, 723-731. [18] Hoffman, N.E., lser, J.H. and Smallwood, R.A. (1975) Am. J. Physiol. 229, 298-302. [19] Iga, T. and Klassen, C.D. (1981) Biochem. Pharmacol. 31,205-209. [20] Gurantz, D. and Hofman, A.F. (1984) Am. J. Physiol. 247, G736G748. [21] YouseL I.M., Barnwell, S.G., Tuchweber, B., Weber, A. and Roy, C.C. (1987) Hepatology 7, 535-542. [22] Bilhartz, L.E. and Dietschy, J.M. (1988) Gastroenterology 95, 771779. [23] Schramm, U., Frickert, G., Buscher, H.P., Gerok, W. and Kurz, G. (1993) J. Lipid Res. 34, 741-757. [24] Weinman, S.A., Graf, J., Veith, C. and Boyer, J.L. (1993) Am. J. Physiol. 264, G220-G230.