250
BIOCHEMICAL SOCIETY TRANSACTIONS
view of this finding the effect of menthol on cholesterol
synthesis was not determined as menthol and cineole have
been shown to have the same effect on hepatic cholesterol
synthesis in vivo (Clegg et al., 1980). However, the results in
Table 1 show that metyrapone is not only able to prevent the
loss of cytochrome P-450 but also produces a significant
( P < 0.05) inhibition of the incorporation of [2-I4C]acetate
into non-saponifiable lipid. Two conclusions emerge from
the present work.
Firstly, while the oral administration to rats of cineole
and cyclandelate inhibits the incorporation of [2-I4C]acetate
into non-saponifiable lipid (Clegg et al., 1980; Middleton
et al., 1983) the addition of comparable amounts to rat
hepatocytes in culture is unable to produce an inhibition of
cholesterol synthesis by the same criterion. Clegg et al.
(1980) suggest that cyclic monoterpenes exhibit their anticholeretic effects by inhibiting 3-hydroxy-3-methylglutarylCoA reductase (ECI. 1.1.34), the rate-limiting enzyme of
cholesterol synthesis. However, Middleton et al. (1979) have
shown that cyclic monoterpenes d o not inhibit 3-hydroxy3-methylglutaryl-CoA reductase in vitro. Therefore the
results of these studies taken together suggest that cineole
and cyclandelate may not be inhibitors of hepatic cholesterol synthesis per se but may be biotransformed, after oral
administration to rats, to an inhibitory species. Finally, the
finding that metyrapone can prevent the loss of cytochrome
P-450 and inhibit cholesterol synthesis in hepatocyte culture
(Table 1) may suggest a causal relationship. This hypothesis
merits further investigation, especially as the anti-choleretic
agent clofibrate (Cohen et a1 , 1974) has recently been shown
to prevent the loss of cytochrome P-450 in rat hepatocyte
culture (Lake et al., 1983).
S.G. received an M.R.C. Intercalated Award to study for the BSc
degree in Biochemistry of the University of London.
Aufenanger, J., Pill, J., Schmidt, F. H. & Stcgmeier, K . (1986)
Biochem. Phurmacol. 35, 9 1 1-9 I6
Clegg, R. J., Middleton. B., Bell, G. D. & White, D. A. (1980)
Biochem. Pharmacol. 29. 2125-2I27
Cohen, B. I., Raicht, R. F.. Shefer, S. & Mosbach. E. H. (1974)
Biochim. Biophys. Acta 369. 79-85
Crivello. J. F. (1985)Endocrinolngy 117, 1-10
Holick, M. F. & Clark, M. B. (1978)Fed. Proc. Fed. Am. SOC.Esp.
Biol. 37, 2567-2572
Hornsby, P. J., Aldern, K . A. & Harris, S. E. (1985) Biochem.
Pharmacol. 34,865-872
Lake, B. G.. Gray. T. J. B.. Stubberfield, C. R.. Beamand. J. A. &
Gangolli, S. D. (1983)Life Sci. 33, 249-254
Middleton, A., Middleton, B. White, D. A. & Bell. G. D. (1979)
Biochem. Soc. Trans. 7, 407408
Middleton, A. White, D. A., Bell, G. D. & Middleton, B. (1983)
Biochem. Pharmacol. 32, 3079 3083
Paine, A. J., Williams, L. J. & Legg, R. F. (19794in The Liver: Quantitative Aspects of Structure and Function (Preisig, R.. Bircher, J. &
Paumgartner, G . ,eds.), pp. 99-109. vol. 3, Editio Cantor, Aulendorf
Paine, A. J. Hockin, L. J. & Legg, R. F. (1979h) Biochem. J . 184.
46I463
Paine, A. J.. Villa, P. & Hockin. L. J. (1980)Biochem. J . 188. 937 939
Takemori, S.& Kominami, S. (1984)T r m h Bioc,hem. Sci. 9. 393 396
Received 26 August 1986
Bile acid secretion in isolated hepatocytes: the effect of Na+ , Ca2+ and cyclic AMP
KATHLEEN M. BOTHAM*
and KEITH E. SUCKLING?
* Depurtrnent
of Physiology, Biochemistry and
Phurrnucvlogy , The Royul Veterinary College,
Royal College Street. London N W l OTU, U . K . ,
und tSmith. KIine and French Reseurch Lid.,
The Frythe, Welwyn. Herts. AL6 9 A H , U . K .
The liver secretes newly synthesized bile acids into bile
via the bile canaliculi, as well as those recirculated in the
enterohepatic circulation. Recent experiments have shown
that incubation of dibutyryl cyclic AMP with isolated
hepatocytes causes a 90% decrease in the ratio of the
amount of bile acid found within the cells to that Sound in
the medium (Botham & Boyd, 1983; Sundaram et al., 1983),
suggesting that the cyclic nucleotide may have a stimulatory
effect on bile acid secretion. We have investigated this possibility by studying the efflux of taurocholic acid from isolated
hepatocytes under various conditions. In addition, the
effects of Na+ and Ca'+, which have been shown to cause
changes in bile acid uptake in isolated liver cells (Anwer
& Hegner, 1978; Botham & Suckling, 1986), have been
studied.
Hepatocytes were prepared and incubated as described
previously (Botham & Boyd, 1983). After I h incubation
the cells were centrifuged and resuspended in medium without bovine serum albumin, approx. 30 kBq of [c~JrhOnj+
''C]taurochloic acid (final concentration, 20 pM) was added
and the flasks were incubated for a further 15 min to allow
equilibrium between uptake and efflux to be reached
(Schwarz et al., 1976). Efflux of taurocholic acid was then
determined by rapid centrifugation through silicone oil as
t Nu'
Table 1. E j f i ~ of
, Ca-" and dibutyryl cyclic A M P on the initial rail' o f ' q f l u . ~of'
taurocholic a c i d f r o m isolated hepatocytes
Hepatocytes were prepared and incubated as described previously (Botham et al., 1980).
The initial rate of efflux of taurocholic acid from the cells was determined in the presence
and absence of Na' with the concentrations of Ca" indicated. Values are given are
means S.D. and the number of experiments is shown in parentheses. Significance limits
compared with no dibutyryl cyclic AMP ( + N a t ) : -*P < 0.05, tnot significant.
t P < 0.025.
Initial rate of taurocholic acid efflux (nmoll
min per mg of protein)
Control
++
+
0.12
0.12
0 ( + 0.I mM-EGTA)
I .2
0.12 f 0.05(4)
0.21 f 0.01 (3)*
0.12 f 0.04(4)
0.1I
0.05 (4)
Dibutyryl cyclic
AMP ( 5 0 0 1 ~ ~ )
0.22 f 0.05 (4)'
0.20 f 0.02 (3)*
0.15 f 0.05 (4)t
0.23 f 0.05 (4)f
1987
25 1
620th MEETING, DUBLIN
described by Schwarz et al. ( 1 976). The initial rate of efflux
was calculated from the values obtained from duplicate
samples incubated for 30,60,90 and 120 s before separation
of cells from the medium, using the linear regression line. All
treatments were applied only to the efflux medium to avoid
interference with uptake during the loading period.
Preliminary experiments showed that secretion of taurocholic acid from the cells was linear for up to 2min. Efflux
over the first 2min was therefore assumed to represent
secretion only without a contribution from uptake.
The initial rate of efflux of taurocholic acid was found to
be increased in hepatocytes incubated with dibutyryl cyclic
AMP (5&1000p~).The increase was statistically significant
at concentrations above 2 0 0 p ~( P < 0.05).
When hepatocytes were incubated in medium in which all
the N a + had been replaced by choline and K + the initial rate
of taurocholic acid secretion was significantly higher than
that in cells incubated in control medium. In the absence
of N a + , dibutyryl cyclic AMP did not stimulate the rate
further (Table I). As cyclic AMP is known to affect Na+
fluxes in the liver (Friedmann, 1972), our results suggest that
the effect of the cyclic nucleotide on bile acid secretion by
the hepatocytes is related to the effect of N a + .
Changing the concentration of Ca2+ from 0 ( + 0.1 mMEGTA) to 1.2 mM did not alter the initial rate of efflux of
taurocholic acid from the cells, but in the absence of Ca2+
the stimulatory effect of dibutyryl cyclic AMP was almost
abolished (Table I). I t has been shown that dibutyryl cyclic
AMP causes a net efflux of Ca2+ from hepatocytes incubated in medium low in C a 2 + ,but a net influx in medium
containing 1.8mM-CaZ+(Mauger et al., 1985; Mauger &
Claret, 1986). Our findings suggest, therefore, that the
increase in bile acid secretion caused by the cyclic nucleotide
is related to the changes it initiates in Ca2+fluxes in the cells.
As the rates of taurocholic acid efflux in the absence of
Na+ and in the presence of Ca2+and dibutyryl cyclic AMP
were raised to a similar extent the efflux of the two cations
may also be related.
In the experiments reported here we have shown that
dibutyryl cyclic AMP is able to increase the rate of secretion
of bile acids from hepatocytes and that changes in the fluxes
of Na+ and Ca'+ in the cells may be involved in bringing
about this effect. These findings raise the possibility of a role
for cyclic AMP in bile acid transport.
Anwer. M. S. & Hegner, D.(1978)Hoppe-Seylers Z. Physiol. Chem.
359, 18I- 192
Botham, K . M . & Boyd, G. S. (1983) Eur. J . Biochem. 136, 313-319
Botham, K. M . &Suckling, K. E.(1986)Biochim. Biophys. Acfa in the
press
Botham, K . M . Beckett, G. J., Percy-Robb, 1. W. & Boyd, G. S.
(1980)Eur. J . Biochem. 103,299-305
Friedmann, N. (1972)Biochim. Biophys. Acfu 274, 214 225
Mauger, J. P. & Claret, M. (1986)FEES Lerr. 195, 106 I10
Mauger, J. P., Poggioli, J. & Claret, M. (1985)J . B i d . C'hrm. 260,
11635-11642
Schwarz, L. R.. Schwenk. M., Pfaff, E. & Greim, H. (1976)Eur. J .
Biochem. 71, 369 313
Sundaram, G. S., Rothman, V. & Margolis, S. (1983) Lipid.7 18,
443447
Received 23 September 1986
Receptor-mediated endocytosis of fluorescent-probe-labelled low-density lipoprotein using human
lymphocytes, fluorimetry and flow cytometry
mined immunoturbidimetrically using antibody from Unipath Ltd. To label LDL from one normal subject (Pitas
et al., 1981), LDL was diluted at 1 mg of protein/2ml of
lipoprotein-deficient serum and 50pg of di-I was added
from stock at 3 mg/ml in dimethylsulphoxide, gently mixed
and incubated at 37°C for 8-15 h. This solution was diluted
to 12ml with 0 . 1 5 ~ - N a C at
I d 1.006, density adjusted to d
Initial developments in fluorophore-labelled lipoproteins 1.063 with KBr and purified by flotation at 140000g for
16 h, and the aspirated LDL was extensively dialysed
and flow cytometry (Krieger et al. 1979, 1983) have now
proceeded to products without major modification of low- against 0.15 M-NaCI/I mM-EDTA at pH 7.4.
Lymphocytes at 5 x 10' cells/ml were incubated at 37' C
density lipoprotein (LDL) composition and which apparently
retain the receptor specificity of native LDL (Barak & with 1&30pg/ml of di-I-LDL with or without a 20-fold
Webb, 1981). Internalized fluorophore also accumulates in excess of native LDL. At various times from 0 to 40h,
lysosomes, without the recycling evident with '251-label, duplicate aliquots were removed after gentle mixing, centrisimplifying evaluation of internalization. (Pitas et af., 1983). fuged and washed twice before determination of fluorFluorophore labelling has also allowed visualization of escence at excitation 520nm, emission > 570 nm in a
binding to high-affinity cell-surface receptors at 4°C (Pitas Perkin-Elmer 204A fluorospectrophotometer or a Coulter
Epic C or Becton-Dickinson FACS analyser.
el a/., 1981) and thus has potential for assessment of variOn assessment by fluorimetry, internalization appeared
ation in binding affinity.
We have followed endocytosis of LDL incorporating progressive for at least 20h, and this incubation period
the fluorophore 1 , l -di-octadecyl-3,3,3,3-tetramethylindo- became standard. Specific uptake was defined as the difcarbocyanine perchlorate (di-I; Molecular Probes Inc., ference between total uptake and that in the presence of
Junction City, U.S.A.) into lymphocytes from normal sub- excess unlabelled LDL. In three different experiments on
four normal and seven FH subjects, specific uptake recorded
jects and heterozygotes for familial hypercholesterolaemia
(FH) at 37°C. in the further development of methods of was 16.8 f 6.1 and 2.2
0.9 (s.E.M.) ng of apoB bound/
assessment of receptor-mediated endocytosis of lipoproteins
lohcells at 1Opg of apoB/ml initial incubation concentration
using fluorimetry and flow cytometry. LDL and lymphocytes respectively. However, although individual interassay variwere prepared as described previously (Wojciechowski ation was < lo%, results for normal and FH subjects overet al., 1987) and LDL-apolipoprotein B (apoB) was deter- lapped and some FH incubations produced no recordable
uptake. The expected difference between normal and FH
cells was therefore generally evident, but discrimination was
Abbreviations used: LDL, low-density lipoprotein; di-I, I ,I-dipoor as with other internalization assays and flow cytometry
octadecyl-3.3.3.3-tetramethylindocarbocyanine; FH, familial hypercholesterolaemia; apoB, apolipoprotein B.
was utilized in the further evaluation of the di-ILLDL
ANDREW P. WOJCIECHOWSKI,*
ANTHONY F. WINDER*
and ANDREW C. CAMPBELL?
Departments of *Chemical Pathology and
tlmmunopathology, Royal Injirmary and
Medical School. Leicester LEI SWW, U . K .
+
Vol. 15