Effect of forskolin and cyclic AMP analog on adenosine transport in cultured chromaffin cells

Neurochem. Int. Vol. 17, No. 4, pp. 523-528, 1990 Printed in Great Britain. All rights reserved

0197-0186/9053.00+0.00 Copyright © 1990Pergamon Press plc

EFFECT OF FORSKOLIN A N D CYCLIC AMP ANALOG ON ADENOSINE TRANSPORT IN C U L T U R E D C H R O M A F F I N CELLS RAQUEL P. SEN, ESMERILDAG. DELICADO and M. TERESA MIRAS-PORTUGAL* Departamento de Bioquimica, Facultad de Veterinaria, Universidad Complutense, 28040 Madrid, Spain (Received 30 October 1989 ; accepted 13 March 1990) Abstract--The adenosine transport in cultured chromattin cells was inhibited by the presence of the adenylate cyclase activator, forskolin, and a cAMP analog. The Vmaxvalues of this transport obtained for control and in the presence of 8-(-4-chlorophenylthio)adenosine-Y : 5'-monophosphate cyclic (C1PhcAMP, 100 aM) or forskolin (0.5/~M) were 85 + 5; 45 + 1.5 and 38 __+3 pmol/106 cells/rain, respectively. The Km values were not significantly modified. The number of adenosine transporters in cultured chromaffin cells, measured by nitrobenzylthioinosine (NBTI) binding, were decreased by the above mentioned effectors. The values of binding sites per cell were 30,000_+ 3200 ; 12,000__+1000 and 21,300 + 2000 for control, CIPhcAMP and forskolin, respectively ; without changing the dissociation constant. When the binding studies were conducted with cellular homogenates, a significant decrease in the maximal binding capacity for nitrobenzylthioinosine was obtained. The values were as follows : 0.087 ___0.01 pmol/mg protein for control, 0.044 + 0.02 pmol/mg protein for CIPhcAMP; and 0.032 + 0.01 pmol/mg protein for forskolin. In this neural tissue, the adenosine transport system seems to be inhibited by stimulation of the adenylate cyclase or by the cyclic AMP analogue that enters the cells. These results suggest that this inhibition could be mediated by a molecular modification of adenosine transporters, the binding with NBTI is therefore a possible parameter of this modification.

The adenosine transport across the plasma membrane has been characterized in several m a m m a l i a n cells (Bender et al., 1981; Miras-Portugal et al., 1986; K w o n g et al., 1987). This transport can be efficiently inhibited by a wide variety o f substances, such as dipyridamole and nitrobenzylthioinosine (Jarvis, 1986; M o r g a n and Marangos, 1987). These compounds have also been used to quantify the number of binding sites (Torres et al., 1986; Marangos and Deckert, 1987 ; Lee and Jarvis, 1988). Therefore it is possible to study in parallel the adenosine transport itself and the number of transporters in any cellular model. N o research has been done so far with respect to the possibility of adenosine transport regulation by external effectors or the corresponding intracellular signals. F o r this reason, the bibliographical data on a similar transport system, such as, sodium-independent glucose, are of great interest (Simpson et al., *Author to whom all correspondence should be addressed. 523

1983; Shanahan et al., 1986; Delicado and MirasPortugal, 1987; Delicado et al., 1988). An additional working hypothesis for adenosine transport might be the possibility of changes in the transporter number at the plasma membrane or their modulation by intracellular signals. Due to the extent of this experimental problem, we limited the present study to the effects o f proteinkinase A stimulation in two ways : first by increasing the intracellular c A M P with forskolin, and second with the c A M P analog, CIPhcAMP, that enters the cell. The cellular model employed was the chromaffin cell, because the adenosine transport and their transporters have previously been characterized (MirasPortugal et al., 1986 ; Torres et al., 1988). This cellular model also presents a homogeneous neuron-like population. The studied parameters were: the adenosine transport and the nitrobenzylthioinosine binding sites in cultured cells. Binding to membrane preparations from chromaffin tissue were performed in the presence of the above mentioned effectors.

RAQUEL P. SEN et al.

524 EXPERIMENTAL PROCEDURES

Materials Adenosine, 8-(-4-chlorophenylthio) adenosine-Y: 5'monophosphate cyclic (C1PhcAMP), collagenase (EC 3.4.24.3) and phenylmethylsulfonylfluorid (PMSF) were supplied by Boehringer; adenosine-5'-tripbosphate, adenosine-5'-O-(3-thio-triphosphate) (ATP-7-S), cystosine arabinofuranoside, dipyridamole, fluorodeoxyuridine, forskolin and nitrobenzylthioinosine(NBTI) by Sigma. Culture media, fetal calf serum and antibiotics were purchased from Seromed. Culture vessels were obtained from Nuclon. The [2-8-3H]adenosine (29 Ci/mmol) and the cyclic AMP [3HI assay system were from Amersham. The [3H]nitrobenzylthioinosine (23 Ci/mmol) was from New England Nuclear. The scintillation liquid, Ready Safe, for aqueous and nonaqueous samples was purchased from Beckman. All other reagents were supplied by Merck. Methods Isolation and culture of ehromaffin cells Chromaffin cells were isolated from bovine adrenal glands according to the method of Miras-Portugal et al. (1985). The cells were isolated by collagenase action, purified through a Percoll gradient, carefully collected and washed with Ca `'+/Mg 2+ free Locke's solution. The Percoll gradient step removed most of the endothelial and cortical cells which present a lower density. The cells were then suspended in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum, penicillin (5 units per ml), streptomycin (5 #g/ml), kanamycin (100/~g/ml), amphotericin (2.5 ~g/ml), 50 #M cytosine arabinofuranoside, and 10 /~M fluorodeoxyuridine. The last two compounds were employed to avoid the cellular proliferation of the rare fibroblasts that could be present at the cellular cultures. Cells were plated in 24-well Costar cluster dishes at a density of 250,000 cells/well for the adenosine transport studies, and 500,000 cells/well for the binding studies. The purity of chromaffin cell preparations was tested by selective staining with vital dye neutral red, as described by Role and Perlman (1980), which is specific for cells containing monoamines. Adenosine transport experiments The adenosine transport was measured, as described by Miras-Portugal et al. (1986) and Torres et al. (1987) using [2-8-3H]adenosine (29 Ci/mmol). Cells were incubated with 0.5 #Ci/well in a volume of 200 pl of Locke's solution, containing non-radioactive adenosine added to reach required concentrations. Transport was only determined during the linear period, which corresponded to the first minute from the start. Transport was stopped by aspiration of the fluid. Subsequently, two washes with 1 ml of Locke's solution with 10 pM dipyridamole were performed. Cells were scraped out of the plastic wells, and the radioactivity was then measured. To study the effect of C1PhcAMP and forskolin, cultured chromaffin cells were preincubated in the presence or absence of these compounds for 10 min at required concentrations. All the incubations were done at 37°C. Trypan blue exclusion and lactate dehydrogenase activity release were routinely employed as controls of cellular integrity. Lactate dehydrogenase activity was assayed according to the Bergmeyer procedure (1974).

Nitrobenzylthioinosine binding experiments Nitrobenzylthioinosine binding studies were carried out as described by Torres et al. (1988). 500,000 cultured chromaffin cells were incubated with [3H]nitrobenzylthioinosine (23 Ci/mmol) in a final volume of 500 #1 of Locke's solution. The labeled ligand concentration range was from 0.02 to 5 nM. After 30 min of incubation at 37°C, the cells were washed twice with 1 mt of cold Locke's solution containing 10 #M nitrobenzylthioinosine, and were then scraped out of the plastic dish and the radioactivity counted. Controls for these experiments were made in the presence of the corresponding [3H]nitrobenzylthioinosine and 10 #M of non-labeled compound. Non-specific binding values were subtracted from assays. To determine the effect of C1PhcAMP and forskolin on nitrobenzylthioinosine binding, cultured chromaffin cells were preincubated as described for the adenosine transport experiments. To study the nitrobenzylthioinosine binding to chromaffin tissue homogenates, the adrenal glands were processed as follows: non-frozen adrenal glands were dissected and homogenized in 0.32 M sucrose with 50/~M of the protease inhibitor PMSF (1:4, w/vol). This homogenate was centrifuged at 800 g for 10 min and the supernatant collected. To eliminate the high levels of purine bases, nucleosides, and nucleotides present in this tissue, five cycles of dialysis with buffer phosphate 10 mM (pH 7.4) containing 50 mM KC1 were performed. The binding of [3H]nitrobenzylthioinosine to the dialyzed homogenate was carried out by incubating 1 mg protein in 500 #1 of 10 mM phosphate buffer (pH 7.4), 50 mM KC1 and 10 mM MgCI2, with graded concentrations of the labeled compound (0.02--5 nM). After a 30 min incubation period at 37°C, membranes were collected on Whatman GF/F glass fiber filters. The filters were washed twice with 3 ml ice cold buffer containing 10 /~M nitrobenzylthioinosine, dried and the radioactivity was counted. The experiments using C1PhcAMP and forskolin were effectuated with a preincubation of 10 min and in the presence of ATP (1 mM), and EGTA (0.1 mM). Controls were also carried out in the presence of the two latter compounds. lntracellular cAMP content was measured with the assay system of Amersham employing [3H]cyclic AMP. Routinely l06 cells were used in the presence or absence of 0.5 #M forskolin with 10 min preincubation time. The quantity of protein was determined according to Bradford (1976). Values are expressed as means+SD. Linear regression equations were calculated by the least-squares method, using a linear regression program.

RESULTS Effects ~ / C I P h c A M P and forskolin on adenosine transport in cultured chromaffin cells The time course o f adenosine transport was studied in the presence or absence o f forskolin (0.5 p M ) and C I P h c A M P (100 #M) as shown in Fig. 1. Both comp o u n d s inhibited the adenosine transport. The transport rate was linear during the first minute, either in the presence or absence o f these effectors. The concentration d e p e n d e n t effect o f forskolin and C l P h c A M P on adenosine transport is shown in Fig.

525

Adenosine transport regulation

60

o) u

after treatment with 0.5 #M forskolin for 10 min (means+ SD of three experiments in triplicate). In Fig. 2(B), the inhibitory pattern of CIPhcAMP on adenosine transport is shown. The 100 /~M concentration produced about 50% inhibition on the transport of this nucleoside. Adenosine transport was measured at different extracellular adenosine concentrations in the presence and absence of 0.5 #M forskolin and 100 /~M C1PhcAMP. The classical Michaelis-Menten representation is shown in Fig. 3. From these values and with the Lineweaver-Burk representation (not shown), the Km and Vmaxvalues were obtained and are summarized in Table 1. Few changes were observed for the Km values, but a significant decrease of V m a x was observed, reaching the 47 and 55% inhibition for C1PhcAMP and forskolin, respectively.

~/~

• Control

40

E o

Q.

20

1

2

time (rain)

Fig. I. Time course of adenosine transport in the presence of CIPhcAMP and forskolin. Costar wellswith 250,000 cells were preincubated with CIPhcAMP (100/aM) and forskolin (0.5/aM) for I0 min, and the transport was determined for different times at 1/aM adenosine concentration. Values are means + SD of 3 experiments performed in quadruplicate.

2. Forskolin was a good inhibitor of adenosine transport in these experimental conditions [Fig. 2(A)]. The 0.5/tM forskolin concentration produced a 37% inhibition and an increase of 2.8 times the cAMP levels. The values of this cyclic nucleotide were 20+ 1 pmol/106 cells for the control and 56 + 4 pmol/106 cells

Effects of CIPhcAMP and forskolin on nitrobenzylthioinosine bindiny to chromaffin cells [3H]Nitrobenzylthioinosine binding to cultured chromaffin cells is shown in Fig. 4. Both C1PhcAMP and forskolin decreased the number of high affinity binding sites, without changing the Kd values, as summarized in Table 1. The inhibition percentage of high affinity binding sites reached 60% when cells were in the presence of CIPhcAMP, and 30% in the presence of forskolin.

[3HI Nitrobenzylthioinosine bindin 9 to ehromaffin tissue homoyenates Nitrobenzylthioinosine binding was carried out in order to specifically label the adenosine uptake sites

100

100

s0

50

[~.._

R

0 Q

- l o g [ForskoIIn] M

6 S 4 3 - log [ClPh CAMP] M

2

Figure 2. Dose-dependent inhibition by forskolin (A) and C1PhcAMP(B) on adenosine transport. Cultured chromaffin cells (250,000 cells/well)were preincubated in the presence of graded concentrations of these compounds for 10 min. Adenosine transport was measured with 1 /aM [3H]adenosine and processed as described in Experimental Procedures. Values are the means ___SD of 4 experiments in quadruplicate. Control values are expressed as 100% (0).

526

RAQUEL

P. SEN

et al.

Table 1. Effectof forskolin and CIPhcAMP on adenosine transport in cultured chromaffincells ADO transport

Control C1PhcAMP (100 ,uM) Forskolin (0.5/~M)

NBTI binding

Km (,uM)

Vm~ (pmol/I 0~ cells/min)

K~ (riM)

Bn,,~ (binding sites per cell)

1.5+_0.2 2.0+_0.1 2.2 + 0.3

85_+.5.0 4 5 + 1.5" 38 ± 3.0*

0.50_+0.04 0.42+0.03 0.49 _+0.05

30,000+3200 12,000 ± 1000" 21,300 + 2000*

Values are the means _+SD of 3 experiments performed in quadruplicate. *P <0.001.

on cellular membranes which were recovered on fiber glass filters. The 800g supernatant presented the advantage of containing the cytosolic components that were necessary for the c A M P action on the kinase A system. The Scatchard analysis of equilibrium is shown in Fig. 5. Controls in the presence and absence of ATP showed no significant differences. In this figure the control in the presence of ATP and E G T A is shown. E G T A is necessary to avoid any possible actions of calcium. The Kd value obtained for this control was 0.4_+0.05 nM. A similar result was obtained in the presence of both C1PhcAMP (Kd = 0.31 +_0.03 nM) or forskolin (Kd = 0.26_+0.06 nM). On the contrary, C1PhcAMP and forskolin significantly decreased the binding capacity of nitrobenzylthioinosine at all studied concentrations (Fig. 5). The maximal binding capacities were 0.087 + 0.01,

0.044 + 0.02 and 0.032 _+0.01 pmol/mg protein for control, C I P h c A M P and forskolin, respectively. Similar experiments were carried out in the presence of ATP-~,-S, which can be employed as a substrate of protein kinase A, but not as a substrate for protein phosphatases. The presence of ATP-7-S did not have a significant effect on the control, but in the presence of the protein kinase A effector, C1PhcAMP, this compound significantly decreased the number of high affinity binding sites (Fig. 6). DISCUSSION

The present experimental work was initiated in order to examine the effects of protein kinase A stimu-

•

0.10 • 80

o CIPh

Control

o CI Ph

Control

"

cAMP

~

•

cAMP

ForskoIin

~, o ~ E

u~

\

\X

\

1

2

3 [Adenosine]

4

5

pM

\

~ \

\\ \\

\ 10

Fig. 3. Effects of several effectors on adenosine transport in chromaffin cells. The cells were preincubated for l0 min with CIPhcAMP (100 /~M) and forskolin (0.5 /~M) before starting the transport experiments. These studies were carried out with 250,000 cells, as described in Experimental Procedures, but in this Michaelis Menten representation, the transport capacity was expressed by pmol/106 cells/rain. Values are the means +_SD of 3 experiments in quadruplicate.

\

X \

\ 20

30

B( fmol / 5 o o , o o o c e . s )

Fig. 4. Scatchard analysis of equilibrium of [3H]NBTI binding to cultured chromaffin cells. The binding was made with 500,000 cells in the presence of C1PhcAMP (100 #M) and forskolin (0.5/iM), as described in the text. B (bound) corresponds to fmol/500,000 cells. This plot represents a typical experiment performed in quadruplicate.

Adenosine transport regulation

• Control

0.6

o CI Ph cAMP • Forskolln

0.4

0.2

50 B(frnoI

/

100 mg protein)

Fig. 5. Scatchard representation of equilibrium of [3H]NBTI binding to chromaffin tissue homogenate. These assays were made with 1 mg protein in the presence of CIPhcAMP (100 #M) and forskolin (0.5 pM), as described in Experimental Procedures. B (bound) was expressed as fmol/mg protein. Results are the means+SD of 3 experiments in quadruplicate.

e Control

0A

m

0.2

"\\\\ \

\

\ \ 30

\

\ \

\

60

\

,'o

B ( f m o l / nlg protein)

Fig. 6. Scatchard analysis of equilibrium of [3H]NBTI binding to chromaffin tissue homogenate in the presence of ATP7-S. These assays were made with 1mg protein in the presence or absence of ATP-~-S(1 mM), as described in Experimental Procedures. B (bound) was expressed as fmol/mg protein. Values are the means + SD of 3 experimentsin quadruplicate.

527

lation on the adenosine transport system in cultured chromaffin cells. As the first report in this field, the present study indicates that cAMP plays an inhibitory role in adenosine transport. A similar situation is obtained for the sodium-independent glucose transport in isolated rat cardiac myocytes (Shanahan et al., 1986) and adipocytes (Simpson and Cushman, 1986; Davies et al., 1986). In both systems a transport inhibition mediated by cAMP-dependent mechanisms exists. In chromaffin cells, the decrease in adenosine transport capability is obtained without significant changes in the affinity. In this model, insulin also enhanced the glucose transport by increasing the number of transporters at the plasma membrane level (Delicado and Miras-Portugal, 1987; Delicado et al., 1988). Thus, at least two main possibilities exist to explain the adenosine transport decrease in these cells. First, a molecular modification of the transporter which changes its capability. Second, changes of the transporter number at the plasma membrane. In order to test these main possibilities, the number of transporters was quantified with nitrobenzylthioinosine. Forskolin and C1PhcAMP decreased the number of maximal binding sites for this ligand. Apparently, the regulatory mechanism on adenosine transport was the transporter number modification. Nevertheless, it was necessary to study the action of forskolin and C1PhcAMP in vitro on the NBTI binding sites. In this way, adrenal medulla homogenates were submitted to forskolin and the direct kinase A effector, resulting in a binding decrease for NBTI. This means that nitrobenzylthioinosinedid not recognize the high affinity adenosine transporter, after they were treated with kinase A effectors, in chromarlin cell homogenates and presumably in cultured cells. The adenosine transport decrease was probably due to a modification of the transporters. Furthermore, cAMP seems to play a major role in secretion from adrenal chromaffin cells (Helle and Serck-Hanssen, 1975 ; Burgoyne, 1984 ; Wilson, 1988). A possible coordination could exist between the exocytotic process and the recovery of adenosine which is the main metabolite of the released granular ATP after the ectonucleotidases action (Keller and Zimmerman, 1983; Richardson et al., 1987). Thus, an inhibition of adenosine transport would enhance the inhibitory effects on catecholamine secretion mediated by adenosine receptors (Chern et al., 1987). The results in the present paper enable us to suggest that adenosine transport is a highly regulated process, but further work is necessary to relate it with the

528

RAQUEL P. SEN et al.

signals derived from neural function and their physiological significance. Acknowledgements We thank Dr P. Gonz~ilez and Dr M. J. Osset for the cAMP determinations and Erik Lundin for his help in the preparation of the manuscript. This work was supported by a research grant PB 86-0009 from the Spanish Comisi6n Interministerial de Ciencia y Tecnologia. Raquel P. Sen is a research fellow of "Caja Madrid".

REFERENCES

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