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Glucocorticoid and catecholamine stimulation of amino acid transport in rat hepatocytes
Molecular and Cellular Endocrinology, 19 (1980) 253-261 0 EIsevier/~ortb-HoIland Scientific Pub~shers, Ltd.
GLUCOCORTICOID AND CATECHOL~INE T~NSPORT IN RAT HEPATO~~ES
253
STIMULATION OF AMINO ACID
SYNTHESIS OF A HIGH-AFFINITY COMPONENT
Bertrand CANIVET, Max FEHLMANN and Pierre FREYCHET instirut ~atio~l de Ia San?&et de la Recherche M&d&ale (r.ff.~.E.R.M.)~ Groupe U 145 and ~aborarory of ~~~erimen~a~Medicine, Faculty of Medicine, ~6~34 Nice Cedex (~&n&e) Received 2 April 1980; accepted 20 May 1980
The kinetic properties of g~ucocorticoid and catecholam~e stimulation of amino acid transport in freshly isolated rat hepatocytes were investigated. In the basal state (i.e., with hepatocytes incubated for 2 h in the absence of ghrcocorticoid or catecholamine), the saturable transport of a-aminoisobutyric acid (AIB) was accounted for mainly by a low-affinity component (Km for AIB w 5 mM). Hepatocyte exposure to cortisol (or dexamethasone), or to epinephrine (or isoproterenol), for 2 h resulted in a 3- to 4-fold increase in the Pm,, of a high-affinity component (Km for AIB = 1 mM) which was only weakly expressed in the basal state. Neither glucocorticoids nor catecholam~es exerted a detectable effect on the low-af~nity transport component. Cycloheximide prevented the emergence of the high-af~nity component in hepatocytes exposed to dexamethasone or epinephrine. The results suggest that the stimulatory effect of glucocorticoids and catecholamines on ammo acid transport in hepatocytes results from the synthesis of a high-affinity transport component. Keywords:
glucocorticoids;
catecholamines;
amino acid transport; hepatocytes.
Recent studies have established that glucocorticoids and catecholamines enhance amino acid transport in hepatocytes in vitro. Thus, epinephrine stimulates uptake of ol-aminoisobutyric acid (AIB) both in primary cultures of adult rat-liver cells (Pariza et al., 1977) and in freshly prepared suspensions of adult rat hepatocytes (Le Cam and Freychet, 1978). Dexamethasone, on the other hand, has no effect on AIB transport in primary cultures of rat hepatocytes, although in this preparation the steroid markedly enhances the stimulatory effect of glucagon (Kletzien et al., 197.5; Pariza et al., 1976) and catecholamine (Pariza et al., 1977) on AIB uptake, suggesting an indirect, permissive type of action. In freshly isolated rat hepatocytes, however, glucocorticoids per se were capable of stimulating the uptake of AIB (Le Cam and Freychet, 1977b). Freshly prepared suspensions of adult rat hepatocytes are suitable for investigating the kinetic properties of amino acid transport under basal and hormone-stimu-
254
Bertrand Can&et, Max Fehlmann,
Pierre Freychet
lated conditions, and for delineating the transport system affected by the hormonal stimulation. (For a review, see Freychet et al., 1979.) In the present study, we further characterized the effect of ~ucocorticoids and catecholam~es on AIB transport in freshly isolated rat hepatocytes. The kinetic analysis of the glucocorticoid and catecholamine-stimulated AIB uptake indicates that these agents promote the synthesis of a high-affinity transport component.
MATE~A~
AND METHODS
Animals and isolation of hepatocytes
Male Wistar rats (100-l 50 g) were maintained in a constant temperature (23’C) animal room with a fixed 12-h artificial light cycle (08.00-20.00 h) for at least a week before use. They had free access to a laboratory chow consisting of (kcal/lOO kcal) 63% carbohydrate, 26% protein and 11% fat until they were killed (lO.OO12.00 h). Hepatocytes were isolated by collagenase dissociation of the liver as previously described (Le Cam et al., 1976; Fehlmann et al., 1979b). Incubation procedure
Experiments were carried out at 37’C in Krebs-Ringer bicarbonate buffer, pH 7.4, conta~mg 1% (w/v) bovine serum albumin (Fraction V), gentiamycin (50 f.ig/ ml) and gassed with a mixture of 5% CO,--95% 0s. Cell suspensions (1 S-2.0 X lo6 cells/ml) were incubated for 120 min with or without hormones before amino acid transport assays. This duration of incubation was selected because previous studies had shown that the maximal stimulation of amino acid transport by glucocorticoids (Le Cam and Freychet, 1977b) or catechol~~es (Le Cam and Freychet, 1978) required at least a 2-h exposure of hepatocytes to these agents. Transport assays and expression of results
At the end of the 2-h incubations, hepatocytes were collected by centrifugation (500 X g for 10 set), then resuspended in the same buffer as above, except that hormone, albumin and ~tibiotic were omitted and the buffer volume was reduced in order to concentrate the cell suspension 2- to 3-fold. Transport assays were carried out in 1.5-ml tubes (Eppendorf). The assay was initiated by adding 200 r_l1of the cell suspension to 50 /J of Krebs-Ringer bicarbonate buffer containing a mixture of &amino [ l- 14C]isobutyric acid (AIB) (about 0.1 $i) and unlabelled AIB to give the desired final concentration of amino acid. The reaction was stopped by adding 1 ml of chiied buffer. Cells were ~mediately collected by a 5-see centrifugation at 2000 X g, resuspended in 1 ml of chilled buffer and centrifuged again. The cell pellet was then resuspended in 100 ~1 of water, and 1.5 ml of scintillation liquid (Unisolve, Koch-Light Laboratories) was added for 14C-radioactivity determination. Results are expressed as nmoles of AIB taken up per IO6 viable cells and per min. The dist~bution ratio (ratio of ~tra~e~ular to extracellular concent~tion of
Hormonal stimulation of amino acid transport in hepatocytes
255
AIB) was calculated as previously described (Le Cam and Freychet, 1977a; Fehlman et al., 1979a). The sodium-dependent part of AIB transport was determined at each substrate concentration by subtracting the values obtained in a sodium-free medium from the total velocities measured in the presence of sodium (Le Cam and Freychet, 1977a). The relationship between the initial rate of the saturable, sodium-dependent transport, u, and the substrate concentration [AIB] was plotted as u against u/[AIB]. When curvilinear plots were obtained, the assumption was made that 2 independent Michaelis-Menten components contributed to transport. Values of kinetic parameters were obtained by computer analysis of experimental data (Fehlmann et al., 1979a). Chemicals
Hydrocortisone, dexamethasone, L(-)-epinephrine, L(-)-norepinephrine-HCl, L(-)phenylephrine-HCl, DL(-)-propranolol, and cycloheximide were purchased from Sigma. Phentolamine-HCl was from Ciba-Geigy, and o-amino [l -‘4C]isobutyric acid from the Radiochemical Centre (Amersham, U.K.). Other reagents were of the best grade commercially available.
RESULTS The effects of glucocorticoids and catecholamines on the time course of AIB uptake by isolated hepatocytes are depicted in Figs. 1 and 2, resp. Both glucocorticoids and catecholamines increased the concentrative uptake of AIB by hepatocytes as denoted by the values of the distribution ratio at later time points (Figs. 1 and 2). With both types of agent, the extent of the stimulation of AIB uptake was
0
10 20 TIME, min
30
,
0
20 10 TIME,min
50
Fig. 1. Effect of cortisol and dexamethasone (DXM) on the time course of AIB uptake. Hepatocytes were incubated for 120 min at 37°C in the absence (basal) and presence of cortisol or DXM at 0.1 &I. The uptake of AIB (0.1 mM, left; 30 mM, right) was then measured at the times indicated. Each point is the mean of triplicate determinations.
256
Bertrand Canivet, Max ~e~l~n~,
0
10 20 Tlk(E,f?Wn
30
0
10 20 TIME, mm
Pierre Frey&et
30
Fig. 2. Effect of epinephrine and isoproterenol on the time course of AIB uptake. Hepatocytes were incubated for 120 min at 37’C in the absence (basal) and presence of epinephrine or isoproterenol at 100 PM. The uptake of AIB (0.1 mM, left; 30 mM, right) was then measured at the times indicated. Each point is the mean of triplicate determinations.
greater at low (0.1 mM) than at high (30 mM) concentrations of AIB. Thus, at 0.1 mM AIB, glucocorticoids and catecholamines increased the initial rate of uptake 2- to 3-fold (Figs. 1 and 2, left), whereas at 30 mM AIB the agents enhanced the rate of uptake by only about 30-40% (Figs. 1 and 2, right). In both basal and hormone-stimulated conditions, AIB uptake increased linearly up to 10 min (Figs. 1 and 2). Accordingly, in subsequent kinetic experiments, the uptake was measured over 5.min periods to ensure that initial rates of uptake (influx) were actually determined. The effects of ~ucocorticoids (Fig. 3) and catecholam~es (Fig. 4) on AIB influx in hepatocytes were measured at various AIB concentrations (ranging from 0.1 to 30 mM), and the data were plotted as u against uI[AIB] . In basal conditions, i.e., with hepatocytes that had not been incubated (before the transport assay) with glucocorticoid or catecholamine, this plot deviated from linearity only slightly. By contrast, with hepatocytes previously exposed to glucocorticoids (Fig. 3) or catecholamines (Fig. 4), the plots were markedly curvilinear. The plots obtained from experimental data were resolved into 2 linear components by computer analysis, assuming that 2 independent Michaelis-Menten components contributed to total saturable transport (Fehlmann et al., 1979a). The resulting kinetic parameters are indicated in Table 1. In basal conditions, the major part of AIB influx occurred through a low-affmity, incapacity component; neither glucocorticoids nor catecholamines significantly altered the kinetic parameters of this component (Table 1). By contrast, both types of agent caused a 3- to 4-fold increase in the I,max of the hip-affmity component; the K, of this component was not signiflcantly affected by any of the agents tested (Table 1). Because the stimulatory effect of glucocorticoids or catecholamines was restricted to the hid-affmity (Km = 1 mM) component of transport, doseresponse relationships were investigated with AIB at 0.1 mM. Dexamethasone
Hormonal stimulation of amino acid transport in hepatoeytes
251
.c \
\
E
2 :
i
“0 1 7
a
E 4
5
0
1
0
2
2
v/[AlB,mM]
v/[A:B,mM]
Fig. 3. Effect of cortisol and dexamethasone (DXM) on the dependence of AIB influx on AIB concentration. Hepatocytes were incubated without (basal) or with cortisol or DXM at 100 nM for 120 min at 3?‘C. Initial velocities of AIB uptake were then determined over 5-min periods at various concentrations of AIB from 0.1 to 30 mM. The sodium-independent component of AIB uptake has been subtracted from total transport at each AIB concentration. Each point is the mean of triplicate determinations.
Wso e 0.1 PM) was more potent than cortisol (ED50 x 1 .O /.N) in stimulating the influx of 0.1 mM AIB {Fig. 5). Deoxycorticosterone, a mineralocorticoid devoid of glucocorticoid activity, was without effect (not shown). The order of potency for the catecholamine stimulation of AIB (0.1 mM) influx was norepinephrine > 20-
.r E \ 2 s “0 \ ;
i
10.
c x3 2 P Q >. 0
0
I
2 1 v/[AlB,mM]
3
0
I
Fig. 4. Effect of epinephrine and isoproterenol on the dependence of AIB influx on AIB concentration. Hepatocytes were incubated without (basal) or with epinephrine or isoproterenol at 100 nM for 120 mm at 37°C. Initial velocities of AIB uptake were then determined at various concentrations of AIB, as indicated in the legend to Fig. 3. Each point is the mean of triplicate determinations.
258
Bertrand Canivet,Max Fehlmnn, Pierre Freychet
Table 1 Kinetic parameters of AIB transport in r&t kq~+tcj~~t~8 Effects of glucocorticoids
and c&&:hekti@&
Low-affinity component
High-affinity component
Vmax (nmoles/l O6 cells/min)
v (n~~es~lO6 celIs/min)
Kin (mMf
Basal (n = 6)
27.5 f 5.1
SS.8 t a,8
0%
f 0.07
0.73 f 0.17
Cortisol (n = 4)
20.8 f 1.3
48.0 f 9.8
0.77 * 0.17
0.93 + 0.26
Dexamethasone (n = 3)
28.0 f 2.1
54.0 * 6.2
1.04 f 0.15
1.12 5 0.16
Epinephrine (n = 4) Isoproterenol
34.5 f 7.0
46.2 f 6,2
1_.2”vf 0.19
1.21 f 0.21
(n = 3)
31.0 f 6.3
45.3 f 6.5
1.13 f 0.29
1.!4 2 0.31
Hepatocytes were incubated without (basal) or with glucocortl&& % catecholamines at 100 MM for 2 h at 37°C. AIB influx was then determined a8 ind&&d in Methods and in the legend to Fig. 3. Values of K, and Vm,, were obtained by computer analysis of the experlttlanttll plots. Each value represents the mean G.E. of n separate Expts.
ep~epl~e > phenylep~~e > isoproterenol (Fig. Q), ~~~~~~arn~e (75 m) inhibited the epinephrine-stimulated AIB (0.1 m&l) uptake by 90% whereas propranolol (100 PM) caused only a 15% inhibition (not shown), suggesting that the catecholamine st~ulation of the hi~~ff~ty component of AIB transport in rat h&g&o=
[GLUCOCORTICOIO],-IogM
Fig. 5. Dose responses of cortisol and dexamethasone stimulation of AIB influx. Hepatocytes were incubated for 120 min at 37°C with various concentrations of either agent, before the addition of 0.1 mM AIB. Amino acid influx was measured 5 min later. Results are the means f SEM of 3 separate Expts.
Hormonal stimulation of amino acid transport in hepatocytes
259
NOREPINEPHRINE
6 [CATECHOLAMINE]
, -log
5
A
M
Fig. 6. Dose responses of epinephrine, norepinephrine, phenylephrine and isoproterenol stimulation of AIB influx. Hepatocytes were incubated for 120 min at 37°C with various concentrations of each agent, before the addition of 0.1 mM AIB. Amino acid influx was measured 5 min later. Results are the means + SEM of 4 separate Expts.
cytes is mediated predominantly through (Yreceptors. To investigate whether the glucocorticoid or catecholamine stimulation of AIB influx, which resulted in an increase in the I’,,, of the high affinity component (Figs. 3 and 4; Table l), was dependent on protein synthesis, hepatocytes were incubated with dexamethasone or epinephrine and with or without cycloheximide (0.1 mM) * for 2 h before the transport assay. In cycloheximide-treated cells, the u over u/[AIB] plot was linear indicating that, in this condition, the high-affinity component of AIB transport was absent, and the stimulatory effect of dexamethasone or epinephrine on the emergence of this high-affinity component was completely suppressed (Fig. 7).
DISCUSSION Previous studies from this laboratory have shown that glucocorticoids (Le Cam and Freychet, 1977b) and catecholamines (Le Cam and Freychet, 1978) enhance AIB uptake in freshly prepared suspensions of adult rat hepatocytes. These effects were ascribed to an increase in the I’,,, of the A system of amino acid transport, and they were dependent on protein synthesis. In the present study we analyzed in more detail the kinetic properties of the glucocorticoid and catecholamine-stimulated AIB transport by measuring AIB influx over a broad range of AIB concentrations after exposure of hepatocytes to these * Cycloheximide
at 0.1 mM inhibited protein synthesis in isolated hepatocytes
by about 90%.
260
Bertrand Canivet, Max Fehlmann,
Pierre Freychet
!' \.~ExAME~HAS~NE
5 ;is 2
2
i
DXM 2 i 0&0H -,L. 0
l
z p
k
05
:-
*--&.._. 1 1.0 v/[AlB]
\
i EPINEPHRINEIi
f 4 ‘, > g-y.cudo".*sn , 1.5
\ A \\/EPINEPHRINE
5-
0
,
05
1.5
20
"/&I
Fig. 7. Effect of cycloheximide (Cycloh.) on the stimulation of AIB influx by dexamethasone (DXM, feft) or epinephrine (right). Hepatocytes were incubated for 120 min at 37°C with DXM (IO0 PM) or epinephrine (100 PM) in the absence or presence of cycloheximide (100 MM). Initial velocities of AIB uptake were then determined at various AIB concentrations, as indicated in the legend to Fig. 3. Each point is the mean of triplicate determinations.
hormonal agents in vitro. The kinetic characteristics revealed by this analysis are compatible with a model in which 2 components (a low-affinity, high-capacity, and a ~~-affinity, low-capacity component) contribute to total saturable transport, with the major part of AIB transport occurring through the low-affinity component in hepatocytes not treated with hormones. The effect of glucocorticoids and catecholamines was to increase the V,,, of the high-affinity component, without significantly altering the low-affinity component. For both types of agent this effect was suppressed by cycloheximide. When maximally stimulating concentrations of dexamethasone and epinephrine, or dexamethasone and glucagon, were combined, no enhancing (“permissive”) effect of the steroid was detected on the ep~eph~e or glucagon ~duction of the high-affinity transport component (not presented). Therefore, in freshly isolated hepatocytes the glucocorticoid induction of the high-affinity component of AIB transport appears to be due to glucocorticoid per se, in agreement with a previous report (Le Cam and Freychet, 1977b) but at variance with observations made in primary cultures of adult rat hepatocytes (Kletzien et al., 1975;Pariza et al., 1976, 1977). The order of potency of catecholamines, in stimulating AIB influx, and the inhibition of the ep~eph~e-educed effect by phentol~ne but not by propranolol, strongly suggest that the catecholamine induction of the high-affinity transport component is exerted predominantly through a-adrenergic mediation, in agreement with a previous study (Le Cam and Freychet, 1978). In the latter study, kinetic data did not permit one to ascribe the catecholamine stimulation of AIB uptake by isolated hepatocytes to the induction of a high-affinity transport component. Studies from this laboratory (Fehlmann et al., 1979b) have shown that glucagon, and insulin, stimulate AIB transport in freshly prepared suspensions of adult rat hepatocytes by promoting the emergence of a highafftity transport component
Hormonal stimulation of amino acid transport in hepatocytes
261
that had the properties of system A. The high-affmity component that results from glucocorticoid or catecholamine stimulation in the present study displays similar kinetic properties to those observed for the transport component induced by insulin and glucagon (system A). Therefore, the induction of a hip-affmity transport component through a process dependent on protein synthesis appears to be a general mechanism whereby glucocorticoids, catecholamines, glucagon and insulin, enhance amino acid transport in liver cells. Such a mechanism may also be operative in vivo, as suggested by our recent fmding that isolated hepatocytes from fasting ~e~rna~ et al., 1979a) or partially hepatectomized (Le Cam et al., 1979) rats exhibit an enhanced ability to take up AIB through the emergence of a high-affinity transport component. This component is similar to that induced by hormones in vitro in normally fed rat hepatocytes, as observed with glucocorticoids and catecholamines in the present study.
We thank A. Kowalski for technical assistance, G. Visciano for illustration work, and J. Duch for secretarial assistance. This work was supported by a grant from INSERM (CRL 78.5.2 16.4) and by research funds from the University of Nice.
REFERENCES Fehlmann, M., Le Cam, A., Kitabgi, P., Rey, J.F., and Freychet, P. (1979a) J. Biol. Chem. 254, 401-407. Fehlmann, M., Le Cam, A., and Freychet, P. (1979b) J. Biol. Chem. 254,10431-10437. Freychet, P., Fehlmann, M., Le Cam, A., Rey, J.F., and Canivet, B. (1979) in: Hormone Receptors in Digestion and Nutrition, Eds.: G. Rosselin, P. Fromageot and S. Bonfils (Elsevier/ North-HolIar+, Amsterdam) pp. 277-286. Kletzien, R.F., Pariza, M.W., Becker, J.E., and Potter, V.R. (1975) Nature (London) 256,4647. Le Cam, A., and Freychet, P. (1976) Biochem. Biophys. Res. Commun. 72,893-901. Le Cam, A., and Freychet, P. (1977a) J. Biol. Chem. 252,148156. Le Cam, A., and Freychet, P. (1977b) Mol. Cell. Endocrinol. 9,205-214. Le Cam, A., and Freychet, P. (1978) Endocrinology 102,379-385. Le Cam, A., Guillouzo, A., and Freychet, P. (1976) Exp. CeB Res. 98,382-395. Le Cam, A., Rey, J.F., Fehlmann, M., Kitabgi, P., and Freychet, P. (1979) Am. J. Physiol. 236, E594-E602. Pariza, M.W., Butcher, F.R., Kletzien, R.F., Becker, J.E., and Potter, V.R. (1976) Proc. Natl. Acad. Sci. (U.S.A.) 73,4511-4515. Pariza, M.W., Butcher, F.R., Becker, J.E., and Potter, V.R. (1977) Proc. Natl. Acad. Sci. (U.S.A.) 74,234-237.
GLUCOCORTICOID AND CATECHOL~INE T~NSPORT IN RAT HEPATO~~ES
253
STIMULATION OF AMINO ACID
SYNTHESIS OF A HIGH-AFFINITY COMPONENT
Bertrand CANIVET, Max FEHLMANN and Pierre FREYCHET instirut ~atio~l de Ia San?&et de la Recherche M&d&ale (r.ff.~.E.R.M.)~ Groupe U 145 and ~aborarory of ~~~erimen~a~Medicine, Faculty of Medicine, ~6~34 Nice Cedex (~&n&e) Received 2 April 1980; accepted 20 May 1980
The kinetic properties of g~ucocorticoid and catecholam~e stimulation of amino acid transport in freshly isolated rat hepatocytes were investigated. In the basal state (i.e., with hepatocytes incubated for 2 h in the absence of ghrcocorticoid or catecholamine), the saturable transport of a-aminoisobutyric acid (AIB) was accounted for mainly by a low-affinity component (Km for AIB w 5 mM). Hepatocyte exposure to cortisol (or dexamethasone), or to epinephrine (or isoproterenol), for 2 h resulted in a 3- to 4-fold increase in the Pm,, of a high-affinity component (Km for AIB = 1 mM) which was only weakly expressed in the basal state. Neither glucocorticoids nor catecholam~es exerted a detectable effect on the low-af~nity transport component. Cycloheximide prevented the emergence of the high-af~nity component in hepatocytes exposed to dexamethasone or epinephrine. The results suggest that the stimulatory effect of glucocorticoids and catecholamines on ammo acid transport in hepatocytes results from the synthesis of a high-affinity transport component. Keywords:
glucocorticoids;
catecholamines;
amino acid transport; hepatocytes.
Recent studies have established that glucocorticoids and catecholamines enhance amino acid transport in hepatocytes in vitro. Thus, epinephrine stimulates uptake of ol-aminoisobutyric acid (AIB) both in primary cultures of adult rat-liver cells (Pariza et al., 1977) and in freshly prepared suspensions of adult rat hepatocytes (Le Cam and Freychet, 1978). Dexamethasone, on the other hand, has no effect on AIB transport in primary cultures of rat hepatocytes, although in this preparation the steroid markedly enhances the stimulatory effect of glucagon (Kletzien et al., 197.5; Pariza et al., 1976) and catecholamine (Pariza et al., 1977) on AIB uptake, suggesting an indirect, permissive type of action. In freshly isolated rat hepatocytes, however, glucocorticoids per se were capable of stimulating the uptake of AIB (Le Cam and Freychet, 1977b). Freshly prepared suspensions of adult rat hepatocytes are suitable for investigating the kinetic properties of amino acid transport under basal and hormone-stimu-
254
Bertrand Can&et, Max Fehlmann,
Pierre Freychet
lated conditions, and for delineating the transport system affected by the hormonal stimulation. (For a review, see Freychet et al., 1979.) In the present study, we further characterized the effect of ~ucocorticoids and catecholam~es on AIB transport in freshly isolated rat hepatocytes. The kinetic analysis of the glucocorticoid and catecholamine-stimulated AIB uptake indicates that these agents promote the synthesis of a high-affinity transport component.
MATE~A~
AND METHODS
Animals and isolation of hepatocytes
Male Wistar rats (100-l 50 g) were maintained in a constant temperature (23’C) animal room with a fixed 12-h artificial light cycle (08.00-20.00 h) for at least a week before use. They had free access to a laboratory chow consisting of (kcal/lOO kcal) 63% carbohydrate, 26% protein and 11% fat until they were killed (lO.OO12.00 h). Hepatocytes were isolated by collagenase dissociation of the liver as previously described (Le Cam et al., 1976; Fehlmann et al., 1979b). Incubation procedure
Experiments were carried out at 37’C in Krebs-Ringer bicarbonate buffer, pH 7.4, conta~mg 1% (w/v) bovine serum albumin (Fraction V), gentiamycin (50 f.ig/ ml) and gassed with a mixture of 5% CO,--95% 0s. Cell suspensions (1 S-2.0 X lo6 cells/ml) were incubated for 120 min with or without hormones before amino acid transport assays. This duration of incubation was selected because previous studies had shown that the maximal stimulation of amino acid transport by glucocorticoids (Le Cam and Freychet, 1977b) or catechol~~es (Le Cam and Freychet, 1978) required at least a 2-h exposure of hepatocytes to these agents. Transport assays and expression of results
At the end of the 2-h incubations, hepatocytes were collected by centrifugation (500 X g for 10 set), then resuspended in the same buffer as above, except that hormone, albumin and ~tibiotic were omitted and the buffer volume was reduced in order to concentrate the cell suspension 2- to 3-fold. Transport assays were carried out in 1.5-ml tubes (Eppendorf). The assay was initiated by adding 200 r_l1of the cell suspension to 50 /J of Krebs-Ringer bicarbonate buffer containing a mixture of &amino [ l- 14C]isobutyric acid (AIB) (about 0.1 $i) and unlabelled AIB to give the desired final concentration of amino acid. The reaction was stopped by adding 1 ml of chiied buffer. Cells were ~mediately collected by a 5-see centrifugation at 2000 X g, resuspended in 1 ml of chilled buffer and centrifuged again. The cell pellet was then resuspended in 100 ~1 of water, and 1.5 ml of scintillation liquid (Unisolve, Koch-Light Laboratories) was added for 14C-radioactivity determination. Results are expressed as nmoles of AIB taken up per IO6 viable cells and per min. The dist~bution ratio (ratio of ~tra~e~ular to extracellular concent~tion of
Hormonal stimulation of amino acid transport in hepatocytes
255
AIB) was calculated as previously described (Le Cam and Freychet, 1977a; Fehlman et al., 1979a). The sodium-dependent part of AIB transport was determined at each substrate concentration by subtracting the values obtained in a sodium-free medium from the total velocities measured in the presence of sodium (Le Cam and Freychet, 1977a). The relationship between the initial rate of the saturable, sodium-dependent transport, u, and the substrate concentration [AIB] was plotted as u against u/[AIB]. When curvilinear plots were obtained, the assumption was made that 2 independent Michaelis-Menten components contributed to transport. Values of kinetic parameters were obtained by computer analysis of experimental data (Fehlmann et al., 1979a). Chemicals
Hydrocortisone, dexamethasone, L(-)-epinephrine, L(-)-norepinephrine-HCl, L(-)phenylephrine-HCl, DL(-)-propranolol, and cycloheximide were purchased from Sigma. Phentolamine-HCl was from Ciba-Geigy, and o-amino [l -‘4C]isobutyric acid from the Radiochemical Centre (Amersham, U.K.). Other reagents were of the best grade commercially available.
RESULTS The effects of glucocorticoids and catecholamines on the time course of AIB uptake by isolated hepatocytes are depicted in Figs. 1 and 2, resp. Both glucocorticoids and catecholamines increased the concentrative uptake of AIB by hepatocytes as denoted by the values of the distribution ratio at later time points (Figs. 1 and 2). With both types of agent, the extent of the stimulation of AIB uptake was
0
10 20 TIME, min
30
,
0
20 10 TIME,min
50
Fig. 1. Effect of cortisol and dexamethasone (DXM) on the time course of AIB uptake. Hepatocytes were incubated for 120 min at 37°C in the absence (basal) and presence of cortisol or DXM at 0.1 &I. The uptake of AIB (0.1 mM, left; 30 mM, right) was then measured at the times indicated. Each point is the mean of triplicate determinations.
256
Bertrand Canivet, Max ~e~l~n~,
0
10 20 Tlk(E,f?Wn
30
0
10 20 TIME, mm
Pierre Frey&et
30
Fig. 2. Effect of epinephrine and isoproterenol on the time course of AIB uptake. Hepatocytes were incubated for 120 min at 37’C in the absence (basal) and presence of epinephrine or isoproterenol at 100 PM. The uptake of AIB (0.1 mM, left; 30 mM, right) was then measured at the times indicated. Each point is the mean of triplicate determinations.
greater at low (0.1 mM) than at high (30 mM) concentrations of AIB. Thus, at 0.1 mM AIB, glucocorticoids and catecholamines increased the initial rate of uptake 2- to 3-fold (Figs. 1 and 2, left), whereas at 30 mM AIB the agents enhanced the rate of uptake by only about 30-40% (Figs. 1 and 2, right). In both basal and hormone-stimulated conditions, AIB uptake increased linearly up to 10 min (Figs. 1 and 2). Accordingly, in subsequent kinetic experiments, the uptake was measured over 5.min periods to ensure that initial rates of uptake (influx) were actually determined. The effects of ~ucocorticoids (Fig. 3) and catecholam~es (Fig. 4) on AIB influx in hepatocytes were measured at various AIB concentrations (ranging from 0.1 to 30 mM), and the data were plotted as u against uI[AIB] . In basal conditions, i.e., with hepatocytes that had not been incubated (before the transport assay) with glucocorticoid or catecholamine, this plot deviated from linearity only slightly. By contrast, with hepatocytes previously exposed to glucocorticoids (Fig. 3) or catecholamines (Fig. 4), the plots were markedly curvilinear. The plots obtained from experimental data were resolved into 2 linear components by computer analysis, assuming that 2 independent Michaelis-Menten components contributed to total saturable transport (Fehlmann et al., 1979a). The resulting kinetic parameters are indicated in Table 1. In basal conditions, the major part of AIB influx occurred through a low-affmity, incapacity component; neither glucocorticoids nor catecholamines significantly altered the kinetic parameters of this component (Table 1). By contrast, both types of agent caused a 3- to 4-fold increase in the I,max of the hip-affmity component; the K, of this component was not signiflcantly affected by any of the agents tested (Table 1). Because the stimulatory effect of glucocorticoids or catecholamines was restricted to the hid-affmity (Km = 1 mM) component of transport, doseresponse relationships were investigated with AIB at 0.1 mM. Dexamethasone
Hormonal stimulation of amino acid transport in hepatoeytes
251
.c \
\
E
2 :
i
“0 1 7
a
E 4
5
0
1
0
2
2
v/[AlB,mM]
v/[A:B,mM]
Fig. 3. Effect of cortisol and dexamethasone (DXM) on the dependence of AIB influx on AIB concentration. Hepatocytes were incubated without (basal) or with cortisol or DXM at 100 nM for 120 min at 3?‘C. Initial velocities of AIB uptake were then determined over 5-min periods at various concentrations of AIB from 0.1 to 30 mM. The sodium-independent component of AIB uptake has been subtracted from total transport at each AIB concentration. Each point is the mean of triplicate determinations.
Wso e 0.1 PM) was more potent than cortisol (ED50 x 1 .O /.N) in stimulating the influx of 0.1 mM AIB {Fig. 5). Deoxycorticosterone, a mineralocorticoid devoid of glucocorticoid activity, was without effect (not shown). The order of potency for the catecholamine stimulation of AIB (0.1 mM) influx was norepinephrine > 20-
.r E \ 2 s “0 \ ;
i
10.
c x3 2 P Q >. 0
0
I
2 1 v/[AlB,mM]
3
0
I
Fig. 4. Effect of epinephrine and isoproterenol on the dependence of AIB influx on AIB concentration. Hepatocytes were incubated without (basal) or with epinephrine or isoproterenol at 100 nM for 120 mm at 37°C. Initial velocities of AIB uptake were then determined at various concentrations of AIB, as indicated in the legend to Fig. 3. Each point is the mean of triplicate determinations.
258
Bertrand Canivet,Max Fehlmnn, Pierre Freychet
Table 1 Kinetic parameters of AIB transport in r&t kq~+tcj~~t~8 Effects of glucocorticoids
and c&&:hekti@&
Low-affinity component
High-affinity component
Vmax (nmoles/l O6 cells/min)
v (n~~es~lO6 celIs/min)
Kin (mMf
Basal (n = 6)
27.5 f 5.1
SS.8 t a,8
0%
f 0.07
0.73 f 0.17
Cortisol (n = 4)
20.8 f 1.3
48.0 f 9.8
0.77 * 0.17
0.93 + 0.26
Dexamethasone (n = 3)
28.0 f 2.1
54.0 * 6.2
1.04 f 0.15
1.12 5 0.16
Epinephrine (n = 4) Isoproterenol
34.5 f 7.0
46.2 f 6,2
1_.2”vf 0.19
1.21 f 0.21
(n = 3)
31.0 f 6.3
45.3 f 6.5
1.13 f 0.29
1.!4 2 0.31
Hepatocytes were incubated without (basal) or with glucocortl&& % catecholamines at 100 MM for 2 h at 37°C. AIB influx was then determined a8 ind&&d in Methods and in the legend to Fig. 3. Values of K, and Vm,, were obtained by computer analysis of the experlttlanttll plots. Each value represents the mean G.E. of n separate Expts.
ep~epl~e > phenylep~~e > isoproterenol (Fig. Q), ~~~~~~arn~e (75 m) inhibited the epinephrine-stimulated AIB (0.1 m&l) uptake by 90% whereas propranolol (100 PM) caused only a 15% inhibition (not shown), suggesting that the catecholamine st~ulation of the hi~~ff~ty component of AIB transport in rat h&g&o=
[GLUCOCORTICOIO],-IogM
Fig. 5. Dose responses of cortisol and dexamethasone stimulation of AIB influx. Hepatocytes were incubated for 120 min at 37°C with various concentrations of either agent, before the addition of 0.1 mM AIB. Amino acid influx was measured 5 min later. Results are the means f SEM of 3 separate Expts.
Hormonal stimulation of amino acid transport in hepatocytes
259
NOREPINEPHRINE
6 [CATECHOLAMINE]
, -log
5
A
M
Fig. 6. Dose responses of epinephrine, norepinephrine, phenylephrine and isoproterenol stimulation of AIB influx. Hepatocytes were incubated for 120 min at 37°C with various concentrations of each agent, before the addition of 0.1 mM AIB. Amino acid influx was measured 5 min later. Results are the means + SEM of 4 separate Expts.
cytes is mediated predominantly through (Yreceptors. To investigate whether the glucocorticoid or catecholamine stimulation of AIB influx, which resulted in an increase in the I’,,, of the high affinity component (Figs. 3 and 4; Table l), was dependent on protein synthesis, hepatocytes were incubated with dexamethasone or epinephrine and with or without cycloheximide (0.1 mM) * for 2 h before the transport assay. In cycloheximide-treated cells, the u over u/[AIB] plot was linear indicating that, in this condition, the high-affinity component of AIB transport was absent, and the stimulatory effect of dexamethasone or epinephrine on the emergence of this high-affinity component was completely suppressed (Fig. 7).
DISCUSSION Previous studies from this laboratory have shown that glucocorticoids (Le Cam and Freychet, 1977b) and catecholamines (Le Cam and Freychet, 1978) enhance AIB uptake in freshly prepared suspensions of adult rat hepatocytes. These effects were ascribed to an increase in the I’,,, of the A system of amino acid transport, and they were dependent on protein synthesis. In the present study we analyzed in more detail the kinetic properties of the glucocorticoid and catecholamine-stimulated AIB transport by measuring AIB influx over a broad range of AIB concentrations after exposure of hepatocytes to these * Cycloheximide
at 0.1 mM inhibited protein synthesis in isolated hepatocytes
by about 90%.
260
Bertrand Canivet, Max Fehlmann,
Pierre Freychet
!' \.~ExAME~HAS~NE
5 ;is 2
2
i
DXM 2 i 0&0H -,L. 0
l
z p
k
05
:-
*--&.._. 1 1.0 v/[AlB]
\
i EPINEPHRINEIi
f 4 ‘, > g-y.cudo".*sn , 1.5
\ A \\/EPINEPHRINE
5-
0
,
05
1.5
20
"/&I
Fig. 7. Effect of cycloheximide (Cycloh.) on the stimulation of AIB influx by dexamethasone (DXM, feft) or epinephrine (right). Hepatocytes were incubated for 120 min at 37°C with DXM (IO0 PM) or epinephrine (100 PM) in the absence or presence of cycloheximide (100 MM). Initial velocities of AIB uptake were then determined at various AIB concentrations, as indicated in the legend to Fig. 3. Each point is the mean of triplicate determinations.
hormonal agents in vitro. The kinetic characteristics revealed by this analysis are compatible with a model in which 2 components (a low-affinity, high-capacity, and a ~~-affinity, low-capacity component) contribute to total saturable transport, with the major part of AIB transport occurring through the low-affinity component in hepatocytes not treated with hormones. The effect of glucocorticoids and catecholamines was to increase the V,,, of the high-affinity component, without significantly altering the low-affinity component. For both types of agent this effect was suppressed by cycloheximide. When maximally stimulating concentrations of dexamethasone and epinephrine, or dexamethasone and glucagon, were combined, no enhancing (“permissive”) effect of the steroid was detected on the ep~eph~e or glucagon ~duction of the high-affinity transport component (not presented). Therefore, in freshly isolated hepatocytes the glucocorticoid induction of the high-affinity component of AIB transport appears to be due to glucocorticoid per se, in agreement with a previous report (Le Cam and Freychet, 1977b) but at variance with observations made in primary cultures of adult rat hepatocytes (Kletzien et al., 1975;Pariza et al., 1976, 1977). The order of potency of catecholamines, in stimulating AIB influx, and the inhibition of the ep~eph~e-educed effect by phentol~ne but not by propranolol, strongly suggest that the catecholamine induction of the high-affinity transport component is exerted predominantly through a-adrenergic mediation, in agreement with a previous study (Le Cam and Freychet, 1978). In the latter study, kinetic data did not permit one to ascribe the catecholamine stimulation of AIB uptake by isolated hepatocytes to the induction of a high-affinity transport component. Studies from this laboratory (Fehlmann et al., 1979b) have shown that glucagon, and insulin, stimulate AIB transport in freshly prepared suspensions of adult rat hepatocytes by promoting the emergence of a highafftity transport component
Hormonal stimulation of amino acid transport in hepatocytes
261
that had the properties of system A. The high-affmity component that results from glucocorticoid or catecholamine stimulation in the present study displays similar kinetic properties to those observed for the transport component induced by insulin and glucagon (system A). Therefore, the induction of a hip-affmity transport component through a process dependent on protein synthesis appears to be a general mechanism whereby glucocorticoids, catecholamines, glucagon and insulin, enhance amino acid transport in liver cells. Such a mechanism may also be operative in vivo, as suggested by our recent fmding that isolated hepatocytes from fasting ~e~rna~ et al., 1979a) or partially hepatectomized (Le Cam et al., 1979) rats exhibit an enhanced ability to take up AIB through the emergence of a high-affinity transport component. This component is similar to that induced by hormones in vitro in normally fed rat hepatocytes, as observed with glucocorticoids and catecholamines in the present study.
We thank A. Kowalski for technical assistance, G. Visciano for illustration work, and J. Duch for secretarial assistance. This work was supported by a grant from INSERM (CRL 78.5.2 16.4) and by research funds from the University of Nice.
REFERENCES Fehlmann, M., Le Cam, A., Kitabgi, P., Rey, J.F., and Freychet, P. (1979a) J. Biol. Chem. 254, 401-407. Fehlmann, M., Le Cam, A., and Freychet, P. (1979b) J. Biol. Chem. 254,10431-10437. Freychet, P., Fehlmann, M., Le Cam, A., Rey, J.F., and Canivet, B. (1979) in: Hormone Receptors in Digestion and Nutrition, Eds.: G. Rosselin, P. Fromageot and S. Bonfils (Elsevier/ North-HolIar+, Amsterdam) pp. 277-286. Kletzien, R.F., Pariza, M.W., Becker, J.E., and Potter, V.R. (1975) Nature (London) 256,4647. Le Cam, A., and Freychet, P. (1976) Biochem. Biophys. Res. Commun. 72,893-901. Le Cam, A., and Freychet, P. (1977a) J. Biol. Chem. 252,148156. Le Cam, A., and Freychet, P. (1977b) Mol. Cell. Endocrinol. 9,205-214. Le Cam, A., and Freychet, P. (1978) Endocrinology 102,379-385. Le Cam, A., Guillouzo, A., and Freychet, P. (1976) Exp. CeB Res. 98,382-395. Le Cam, A., Rey, J.F., Fehlmann, M., Kitabgi, P., and Freychet, P. (1979) Am. J. Physiol. 236, E594-E602. Pariza, M.W., Butcher, F.R., Kletzien, R.F., Becker, J.E., and Potter, V.R. (1976) Proc. Natl. Acad. Sci. (U.S.A.) 73,4511-4515. Pariza, M.W., Butcher, F.R., Becker, J.E., and Potter, V.R. (1977) Proc. Natl. Acad. Sci. (U.S.A.) 74,234-237.