Cellular Signalling Vol. 4, No. 6, pp. 709-714, 1992.

08986568/92 $5.00 + 0.00 © 1992 Pergamon Press Ltd

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PHORBOL ESTER EFFECTS ON HORMONAL RESPONSES IN FRESHLY ISOLATED SHORT-TERM INCUBATED AND CULTURED HEPATOCYTES T. HERMSDORF and D. DETTMER* Institute of Biochemistry, University Leipzig, Liebigstrasse 16, D(0)-7010 Leipzig, F.R.G.

(Received 1 May 1992; and accepted 27 July 1992) Abstraet--Beta-adrenergic, alpha-l-adrenergic and glucagon stimulation of glucose release were compared between hepatocytes which were freshly isolated, incubated for 3 h in suspension or cultivated for 4 or 24 h in plastic culture flasks in the presence and absence of the protein kinase C activator 12-O-tetradecanoylphorbol13-acetate (TPA). In contrast to the absence of an isoproterenol effect in freshly isolated hepatocytes, an increased sensitivity of glucose liberation towards isoproterenol could be observed 4 h after the start of culture, whereas the beta-receptor number was not found to be increased before 24 h. TPA has no effect on isoproterenol-stimulated glucose release at all investigated conditions. The alpha-l-adrenergic responses tested by using the alpha-l-adrenergic agonist phenylephrine is blocked completely in freshly isolated hepatocytes preincubated with 10-6 M TPA. However, after 3 h incubation of hepatocytes in suspension or in primary culture, TPA had no effect on phenylephrine-stimulated glucose release. The effect of 10-9M glucagon on glucose release from freshly isolated hepatocytes was not influenced by TPA, whereas after 90 and 180 min incubation a significant decrease could be observed. On the other hand, TPA inhibited stimulation of adenylate cyclase activity by glucagon concentrations of 10-5 M in freshly isolated hepatocytes, but no effect was found in hepatocytes incubated for 3 h in suspension or maintained for 24 h in primary culture. The different TPA effects may be an expression of changes of the accessibility of protein kinase C to TPA caused by translocation and/or intracellular activation of this enzyme at the tested experimental conditions.

Key words: Phorbol ester, hepatocytes, culture, glucagon, phenylephrine, isoproterenol.

INTRODUCTION

(TPA) can activate PKC directly, bypassing the activation of phospholipase C and the formal ~ o ~ n ~ KI~ASE C (PKC) has been considered tion o f the second messengers inositol-l',to play an essential role in the intracellular 4,5-trisphosphate (IP3) and diacylglycerol transduction and modulation of hormonal (DAG) [4]. TPA seems to substitute for D A G signals for cell function and proliferation [1, 2]. and lowers the requirement for Ca 2+ [4]. An Activation of P K C is mediated by generation of essential step of the activation process is the diacylglycerol and increase in cytosolic calcium translocation of PKC from cytosol to plasma concentration as a result of hormonal stimulamembrane where this enzyme is fixed by tion o f the phospholipase C [3]. It has been phospholipids like phosphatidylserine [5, 6]. A shown that the tumour-promoting phorbol rapid translocation and association of the ester 12-O-tetradecanoylphorbol- 13-acetate enzyme with cellular membranes was described also in TPA-treated cells [7]. Recently, it has * Author to whom correspondenceshould be addressed. been reported that within the first hours of Abbreviations: BSA--bovine serum albumin; DAG-hepatocyte culture in suspension, PKC is transdiaeylglycerol; DMEM--Dulbeceo's minimum essential medium; HEPES----[N-(2-hydroxyethyl)piperazine-N'-2- located to the plasma membrane [5]. After ethanesuiphonic acid]; IP3--inositol-l,4,5-trisphosphate; partial translocation of PKC from cytosol to KHBB---Krebs--Henseleit bicarbonate buffer; PKC--protein kinase C; TPA--12-O-tetradecanoyl-phorbol-13- the plasma membrane in hepatocytes of reacetate. generating liver, reduced TPA effects were to be 709

710

T. H~LSDORFand D. DET'r~mR

found [8]. The question arises as to whether, during or after translocation, the accessibility of P K C to TPA is diminished. Whereas T P A effects caused by P K C activation on alpha-ladrenergic [9, 10], beta-adrenergic [11] and glucagon responses [12-14] were described in freshly isolated hepatocytes, no such results exist for T P A effects in hepatocytes incubated in suspensions or maintained in primary culture with respect to a possible translocation and intracellular activation of PKC. Therefore, we have investigated the biological responsiveness in beta-adrenergic-, alpha-l-adrenergic- and glucagon-hormone-receptor systems of freshly isolated hepatocytes, in suspensions of hepatocytes incubated for 3 h and in hepatocytes cultured for 4 and 24 h in the presence and absence of TPA.

MATERIALS AND METHODS

50 min with glucose-free KHBB containing 1.25 mM CaC12 and 1% BSA at the same conditions to determine glucose release. TPA was added at the beginning and hormones added after 10min of incubation. Hepatocytes were cultured as monolayers (60,000 cells/cm 2) in Dulbecco's minimal essential medium (DMEM) with HEPES ([N-(2hydroxyethyl)piperazine-N-2-ethanesulphonic acid]), SERVA, Heidelberg, F.R.G., supplemented with 50/zg/ml gentamycin, 10 -7 M dexamethasone, 10-8 M insulin and 5% foetal calf serum in 50-ml plastic culture flasks (Greiner, F.R.G.) coated with rat-tail collagen in an atmosphere of 95% air-5% CO2. After 4 and 24 h the medium was changed and the cultures were incubated for 1 h in medium containing 0.1% BSA instead of foetal calf serum. For determination of glucose-release, cultures were washed with glucose-free KHBB bicarbonate buffer, pH 7.4, with 0.1% BSA and incubated in the same buffer at 37°C in an atmosphere of 95% air-5% CO2 for 50 min. TPA was added at the beginning and the hormone added after 10 min of incubation. For binding studies cultures were treated with 0.05% collagenase in KHBB for 15 min and the suspended cells were washed twice with KHBB.

Chemicals Glucagon was from Novo Mainz (F.R.G.), isoproterenol and phenylephrine were purchased from AWD Dresden (F.R.G.), collagenase from Boehringer Mannheim (F.R.G.), TPA from Sigma, (St Louis, MO, U.S.A.), 3H-CGP 12 177 from Amersham-Buchler Braunschweig (F.R.G.) and bovine serum albumin (BSA) from SERVA (F.R.G.).

Animals and hepatocyte preparation Male Wistar albino rats (220-300 g) were maintained at standard conditions of light and temperature and fed ad libitum. Hepatocytes were isolated using a modified collagenase perfusion method [15, 16]. Cell viability was about 90% estimated by trypan blue exclusion and increased to more than 95% by employing a Percoll centrifugation step [17] for cell incubations in suspension.

Hepatocyte incubation in suspension and primary culture Cells (1 million/ml) were suspended in Krebs-Henseleit bicarbonate buffer (KHBB) containing 1.25mM CaC12, 1% BSA and 20mM glucose at 37°C at continuous shaking (100 cycles/ rain) in an atmosphere of 95% 02-5% CO2. At indicated times, cell suspensions were washed twice with glucose-free KHBB and incubated for a further

Radioligand binding assay ~-Adrenergic receptor binding studies. Onehundred and fifty-thousand cells were incubated at saturating concentrations of 4nM 3H-CGP 12 177 at 25°C for 20 min in KHBB with 0.1% BSA (final volume 500/~1) [18]. Nonspecific binding was determined in the presence of 1/zM propranolol. The binding reaction was stopped by dilution with 4 ml ice-cold KHBB followed by rapid filtration over Whatman GF/C glass fibre filters in vacuo and washed with 15 ml stop buffer. Glucagon binding studies. Glucagon was labelled with z25I using the chloramine T method [19] and purified electrophoretically. Crude membranes (600 #g) prepared according to Hermsdorf et al. [18] were incubated in Hanks-HEPES buffer containing (in raM): NaCI 137, KC1 5.4, MgSO4 0.4, Na2HPO( 0.34, KH2PO4 0.44, HEPES 25, bacitracin 1 and 1% BSA, pH 7.4, with 1 nM12SI-glucagon for 30 rain at 25°C (500/~1 final volume). Nonspecific binding was determined in the presence of 10 #M glucagon. Stop reaction and filtration were performed as described above with Hanks-HEPES buffer containing 0.1% BSA.

Adenylate cyclase assay The adenylate cyclase was measured as previously described [20].

P h o r b o l ester effects o n h o r m o n a l responses 300

Glucose and protein determination

After centrifugation of incubated cells the supernatants were immediately deproteinized with NaOH/ZnSO4. Glucose was measured by the glucose oxidase method. Protein measurement was carried out according to Lowry et al. [21].

250

.o

O

RESULTS Incubations of hepatocytes were performed in the presence of 20 mM glucose in order to maintain a high glycogen content and ensure optimal experimental conditions to measure glucose release. Changes of the responsiveness of hepatocytes towards isoproterenol, glucagon and phenylephrine during incubation were investigated in suspension cultures with continuous shaking or in primary culture. Freshly isolated hepatocytes did not show any isoproterenol stimulation of glucose release, whereas after 4 and 24 h of cultivation at I0 -s M isoproterenol, glucose liberation was increased significantly (Fig. 1). However, phorbol ester did not affect the isoproterenol response of freshly isolated and cultured hepatocytes either (Fig. 1). Surprisingly, using the beta-adrenergic antagonist 3H-CGP 12 177 for radioligand binding studies on intact hepatocytes, it could be shown that in comparison with the results for freshly isolated bepatocytes, beta-antagonist binding increased significantly after 24h of primary culture, but not after 4h. The measured values given as means+S.D, fmol CGP 12 177 bound to 100,000 cells were 0.9+0.3 for freshly isolated hepatocytes (n = 3), 1.6 + 0.6 after 4 h suspension culture (n = 4) and 5.6 + 2.1 after 24 h of primary culture (n = 6). The stimulation of glucose release by glucagon increased from 175 to 206% and by phenylephrine from 134 to 198% after incubation of suspended hepatocytes for 90 min. However, after an incubation time of 180 min the glucagon stimulation decreased to about 150% and the phenylephrine stimulation to 135% (Table 1). TPA inhibited the phenylephrine-stimulated glucose liberation at time zero completely but did not affect alpha-l-adrenergic response significantly after an incubation

711

200

150

~t

lOO

50

I -10

I -9

I -8

I -7

I -6

L -5

Log [isoproterenol] M

FIG. 1. Dose-response curves for isoproterenol action on glucose release of freshly isolated and cultured hepatocytes without (open symbols) and with TPA (closed symbols) pretreatment. Freshly isolated hepatocytes (©, O), hepatocytes cultured for 4 h (V, V) and 24 h (l'q, ll) were preincubated for 10 min in glucose-free KHBB (0.1% BSA) followed by addition of the indicated concentrations of isoproterenol and further incubation for 50 rain. Values are given as means + S.D. of three separate experiments.

time of 180 min. Glucagon stimulation was not influenced by TPA at the beginning of incubation, whereas a significant decrease was found after 90 and 180 min incubation (Table 1). The lack of direct cell-cell contact during incubation of suspended hepatocytes over many hours may also influence the hormonal responsiveness. Therefore, we have used hepatocytes in primary cultures for comparative studies. It could be shown that stimulation of glucose release by glucagon was not changed during cultivation (Table 2), whereas glucagon binding measured as 125I-glucagon bound to milligrams membrane protein slightly decreased from 113.2+12.6 at time zero (n = 3) to 96.7+15.3 after 4h suspension culture (n = 3) and to 78.6+9.7 (n = 3) after 24 h of culturing. The phenylephrine response was increased to 180% after 24 h or cultivation. Neither the stimulation of glucose release by phenylephrine nor by glucagon were influenced by TPA (Table 2).

712

T. HEP,~SDOm~and D. DFrl"MF~

TABLE 1.

EFFECT OF TPA ON THE STIMULATION OF GLUCOSE RELEASE BY GLUCAGON AND PHENYLEPHRINE AFTER INCUBATION OF HEPATOCYTI~ IN SUSPENSION

Incubation time (min)

Glucagon 10 -9 M

TPA 10-6 M + glucagon 10-9 M Phenylephrine 10-6 M TPA 10-6 M + phenylephrine 10-6 M

0

30

90

180

175 -+ 8

226_+ 6

206 + 37

151 + 12"

173+ 18 134_+ 12

207-+ 12 174-+24

136+ 16" 198+25

120-+ 11" 135+ 13

101 + 1"

109-+9"

116-+7"

122_+ 12

Hepatocytes were incubated in suspension as described in Materials and Methods. At the indicated times, cells were washed giucose-free and preincubated in glucose-free KHBB (1% BSA) with or without TPA for 10rain. Then glucagon or phenylephrine were added and incubated for a further 50m in. Values are given as means_+S.D., expressed as percentage stimulation of basal glucose output, n = 4. * Statistically significant differences vs values without TPA (P < 0.05, t-test). Determination o f the glucagon stimulation of adenylate cyclase gives the possibility of obtaining information on whether modulation o f this signal transduction by T P A takes place. T P A reduced glucagon-stimulated adenylate cyclase in freshly isolated hepatocytes only at very high concentrations (Table 3), which were also used by Refsnes et al. [22] for desensitization studies. However, no T P A effects were observed in hepatocytes incubated for 180 min in suspension or cultured for 24 h.

DISCUSSION Characteristic changes in the alpha-l-, betaadrenergic and glucagon responses during the first hours of incubation of hepatocytes in suspension or primary culture have been described [23-27]. It could be generally observed that alpha-1-adrenergic and glucagon TABLE 3. EFFECTS OF TPA oN ADENYLATECYCLASE ACTIVITY OF FRF.SHLY ISOLATED, 3 h INCUBATED AND CULTURED HEPATOCYTES

Adenylate cyclase activity (pmol cAMP/min/mg protein)

TABLE 2. EFFECTS OF TPA ON GLUCAGON AND PHENYLEPHRINE STIMULATION OF GLUCOSE RELEASE IN PRIMARY CULTURE

Time of culturing (h)

Glucagon 10-9 M TPA 10-6 M + glucagon 10-9 M Phenylephrine 10-6 M TPA 10-6 M + phenylephrine 10-6 M

4

24

196-t-26

157-+ 10

188-1-31 155 -+24

172-+25 181 _+22

159_+34

178_+29

After 3 and 23 h, cultures were washed and incubated for I h in KHBB (0.1% BSA) with or without TPA followed by addition of giucagon or phenylephfine. Values are given as means _+S.D. expressed as

percentage stimulation of basal glucose output. n ~-- 3.

Control Freshly isolated hepatocytes None l0 -s M giucagon 3 h suspension culture of hepatocytes None l0 -5 M giucagon 24 h primary culture of hepatocytes None l0 -5 M giucagon

TPA

0.53 + 0.06 0.55 + 0.08 3.60_+0.25 2.42_+0.38 0.56_+ 0.06 2.38-+0.43

0.59 + 0.08 2.79+0.35

0.74 _+0. l I 0.66_+0.08 3.94_+0.08 3.54_+0.06

Values are given as means+S.D, of triplicate determinations of three separate preparations.

Phorbol ester effectson hormonalresponses stimulation of phosphorylase is decreased and beta-adrenergic stimulation is increased. In agreement with these reports we found an increasingly strong beta-adrenergic response of glucose release during primary culture of hepatocytes. However, there are distinct differences between changes in biological response and in receptor number. After 4 h of culture a betaresponse of glucose liberation has been clearly developed, whereas beta-antagonist binding was changed weakly. This indicates that the development of beta-response takes place in two steps. At first beta-response is realized by improved coupling between receptor and G-protein [6] and in the second step by the rise of receptor number possibly induced by the presence of foetel calf serum in the culture medium [28]. In agreement with Nakamura et al. [29] it could be found that the phenylephrine and glucagon stimulation of glycogenolysis was not decreased. On the contrary, at some conditions alpha- l-adrenergic response was increased. Alpha-l-adrenergic effects on glucose release were found to be blocked by TPA in freshly isolated hepatocytes (see also [9, 10]). However, after 180 min incubation of suspended hepatocytes the TPA effect became smaller and was lost in cultured hepatocytes. The TPA effect in freshly isolated hepatocytes has been explained by phosphorylation of the alpha-l-receptor which leads to an uncoupling of the receptor from the G-protein and inhibits phosphoinositide metabolism [30]. Obviously, after short-term incubation the PKC is translocated from the cytosol to the membrane (see [6]) and it can be considered that this activated PKC has lost its accessibility to TPA. The absence of TPA effects in foetal hepatocytes or in regenerating tissues as reported previously [8, 20] may be explained in the same way. The adenylate cyclase activity stimulated by 10-5 M glucagon was decreased by 30% only in freshly isolated hepatocytes. This effect may be an expression of different desensitization phenomena depending on time and conditions of incubation and cultivation. Moreover, inhibition of cAMP-phosphodiesterases by TPA has also to be taken into account [31]. Obviously, CELLS 4 : 6 4

713

these findings cannot explain the lack of correlation between regulation of adenylate cyclase and glucose output which was found to be maximally stimulated at glucagon concentrations of 10 -9 M and not influenced by TPA. We have found in our own experiments no stimulation of adenylate cyclase activity by 10-gM glucagon and have discussed in connection with this different sensitivities of glucagon-stimulated glucose release and glucagon-stimulated adenylate cyclase activity [20]. This supports the hypothesis of two different glucagon receptors----one high-affinity receptor and one lowaffinity receptor [32]. Only the low-affinity glucagon receptor seems to be coupled to the adenylate cyclase system and may be accessible to TPA, whereas via the high-affinity receptor --primarily not accessible to TPA--the glucose release could be regulated. In contrast, further incubation in suspension led to a decrease of glucagon-stimulated glucose release by TPA while in primary cultures TPA was not efficient. These contrary results could be caused by changes in the endogenous activation of PKC [5], or changes of the interaction between the different constituents of the tested hormonereceptor systems and PKC. Summarizing our results we conclude that short-term incubation and culturing of hepatocytes lead to changes of TPA effectiveness most likely caused by translocation of PKC in connection with its intraceUular activation. Acknowledgements--The authors wish to thank MRs BARBARA WOITHE for excellent technical assistance.

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