Jouriiul

01 Newocltrmmtry,

1976. Vol. 26, pp. 265-273. Pcrgamon Press. Printed i n Great Brltaln.

NEUROBLASTOMA CELL ADENYLATE CYCLASE: DIRECT ACTIVATION BY ADENOSINE AND PROSTAGLANDINS J. PENIT, J. HUOT' and S. JARD Laboratoire de Physiologie Cellulaire, College de France 75231 Paris Cedex 05, France (Received 1 May 1975. Accepted 2 July 1975) Abstract-Adenylate cyclase activity of permeabilized neuroblastoma cells was measured by the converM) induced a dose-dependent sion of [a3'P]ATP into labelled cyclic AMP. Adenosine increasc in cyclic AMP formation. This effect could not be accounted for either by an adenosine-induced inhibition of the phosphodiesterase activity present in the enzyme preparation, or by a direct conversion of adenosine into cyclic AMP. This indicates that the observed increase in cyclic AMP accumulation reflected an activation of adenylate cyclase. Adenosine is partially metabolized during the course of incubation with the enzyme preparation. However: none of the identified non-phosphorylated adenosine metabolites were able to induce an adenylate cyclase activation. This suggests that adenosine itself is the stimulatory agent. The apparent K , of the adenylate cyclase for adenosine was 5 x 10-6-10-5 M. Maximal activation represented 3-4 times the basal value (10-100 pmol cyclic AMP formed/lO min/mg protein). The adenosine effect was stereospecific, since structural analogues of adenosine were inactive. Adenosine increased the maximal velocity of the adenylate cyclase reaction. The stimulatory effect of adenosine was inhibited by theophylline. Prostaglandin PGE, had a stimulatory effect much more pronounced than that of adenosine (6-10-fold the basal value at M). Dopaminc and norepinephrine induced a slight adenylate cyclase activation which was not potentiated by adenosine. It is concluded that adenosine is able to activate directly neuroblastoma cell adenylate cyclase. It scems very likely that such a direct activation is also present in intact nervous tissue and account, at least partly, for the observed cyclic AMP accumulation in responsc to adenosine.

ADENOSINEin concentrations varying from to hances the cyclic AMP accumulation by bone cells from foetal rat clavaria (PECKrt al. 1974) and by M has been shown to induce cyclic AMP accumulation by brain slices (SATTIN& RALL, 1970). human platelets (MILLS& SMITH, 1971); it antaA similar action has also been demonstrated in cell gonizes the nucleotide accumulation elicited in adicultures from the nervous system: foetal brain cells pose cells by adrenergic agonists (FAIN,1973). The (SCHRIER & GILMAN,1973), cloned astrocytoma cells mode of action of adenosine in eliciting cyclic AMP (CLARK& PERKINS, 1972), and neuroblastoma cells accumulation is not yet fully understood. Three mefrom various clonal lines (SCHULTZ & HAMPRECHT, chanisms in particular have been put forward: (1) in1973; BLUMErt al., 1973). Inhibitors of cyclic nucleo- corporation of adenosine into the ATP pool utilized tide phosphodiesterases such as papaverine and iso- for the cyclic AMP formation, (2) inhibition of cyclic butylmethylxanthine markedly potentiate the effect of AMP hydrolysis by phosphodiesterases, and (3) adenosine on neuroblastoma cells (SCHULTZ & HAM- stimulation of cyclic AMP formation by adenylate cyclase. MCILWAIN PRECHT. 1972). Conversely, theophylline antagonizes & BACHELARD (1971) showed that the action o f . adenosine in brain slices (SATTIN guinea pig brain slices contain all the transport and & RALL, 197O), neuroblastoma cells (SCHULTZ& enzymatic systems needed to incorporate the extracelHAMPRECHT, 1973), and astrocytoma cells (CLAKKet lular adenosine into the intra-cellular ATP pool(s). aL, 1974). Furthermore adenosine is liberated follow- Moreover, both cyclic ['4C]AMP and cyclic ing treatment with depolarizing agents and electrical C3H]AMP were found whcn the nuclcotide pools of stimulation of isolated nervous tissues (PULL& cerebral slices were labelled with [l4qadenine and C3H]adenosine (SHIMIZU& DALY, 1970). This MCILWAIN, 1972a, b). These observations suggested, among others, that strongly supports the suggestion that adenosine might adenosine might act as a putative neurotransmitter. act as a precursor of cyclic AMP formation. ConcernAdenosine also affects the cyclic AMP content of ing the second mechanism postulated above, it should cellular systems other than the nervous tissue; it en- be mentioned that adenosine is generally regarded as a poor inhibitor of phosphodiesterases from various (HUANG & KEMP,1971; ROBERTS & SIMONSEN, ' This research was supported by funds from the Centre sources 1970). However, recent data suggested that adenosine National de la Recherche Scientifique L.A. No. 219. might block the phosphodiesterase in brain slices Fellow from the Canadian MRC. 265

J. PENT.J. HUOTand S . JARD

266

(SCHULTZ & DALY,1973h). Finally adenosine is able to activate directly the adenylate cyclase from human platelets (HASLAM & LYNHAM,1972). However, it is not yet possible to demonstrate such a direct activation of adenylate cyclase activity in brain homogenates (SATTIN& RALL,1970). Nevertheless, several indirect experimental data indicate that adenosine is able t o elicit accumulation of cyclic AMP by interacting with an extracellular site which is distinct from the transport site and which is probably connected with the membrane adenylate cyclase. (SCHULTZ & DALY.1973a: HUANC& DALY,1974; CLARKet a[., 1974; STURGILL et a!., 1975; SHIMUZU et al., 1973; MCILWAIN.1973). I n the present study we demonstrate that adenosine is able t o enhance the formation of labelled cyclic AMP from C3*P]ATP by permeabilised preparations from neuroblastoma cells.

MATERIALS AND METHODS Cell cultures The experiments were carried out on mouse cholinergic et al., 1972). The cells neuroblastoma cells NS-20 (AMANO were seeded in Falcon flasks (75 cm2) at concentrations of 5.105 cells per flask. They were cultivated as layers in 15 ml Eagle’s minimal essential medium (MEM) supplemented with 1004 foetal calf serum (GIBCO) and with 100 IU/ml penicillin G plus 100 y d m l streptomycin sulphate. The media were removed on the fourth day and were replaced by 25 ml of fresh MEM containing only 2% foetal calf serum. Two days after, the cells were harvested for the experimental procedures described below. Cultures werc incubated at 37°C in a humidified atmosphere of 10% CO2/90% air. Dye exclusion tests with Trypan blue were employed before each experiment and revealed that W 9 5 % of the cells were viable.

Preparation of cells for the enzymatic assays. The experiments were performed on two types of cellular preparations: frozen preparations and permeabilised cells. Before harvesting the cells, MEM medium was gently removed and Hank‘s solution (pH 7.4) was added to each culture flask. The cells from the various flasks were detached by manual shaking before being pooled. The pooled cells were centrifuged at 3009 and rinsed 3 times with Hank’s balanced salt solution. The pellet was submitted to an osmotic shock by being suspended (20 x lo6 cells/ml) and chilled (4°C) for 30 min. in a hypotonic medium containing 25 m~-Tris-HCl,(pH 8.0) and 5 mM-EDTA (pH 8.0). Thereafter the cells were centrifuged for 10 min at 1OOOg. The supernatant was discarded and the pellet resuspended in the above mixture to which was added 10 mM-MgC12 to avoid clumping. The preparations were stored in liquid nitrogen before being used for the cnzymatic assays. They are referred to as frozen preparations in the following description (text). Permeabilised cells were obtained as follows; after the harvesting, cells were washed and centrifuged 3 times (la0g x 3 min) and then made permeable by treatment with cold (4°C) 25 mM-Tris-HC1, (pH 8.0) and 0.1 mM-EDTATris, (pH 8.0) for 30 min. The enzymatic assays were performed immediately after the osmolysis.

Adenylate cyclase assay The activity of adenylate cyclase was measured by the conversion of [w3’P]ATP into labelled cyclic AMP. Incubations were performed for 10 min at 37°C. The incubation medium (final volume 100 yl) contained: 100 mM-Tris-HCl pH 8.0; 0 1 mM-ATP; 0.7 yCi [a3’P]ATP; 0.5 mM-MgCI,, for frozen preparations or 0.25 mM for permeabilised cells; 0 I mwpapaverine hydrochloride; 1.0 mwcyclic GMP; 20 mwcreatine phosphate; 100 yg-creatine kinase; and various concentrations o f the substances investigated. The reaction was initiated by the addition of the cellular preparation (20-40 pg of protein under 25 pl). It was stopped with an excess of by cooling and dilution of [E~~P]ATP unlabelled ATP. Cyclic AMP was separated by chromatography on dry aluminium oxide (1 g) columns according to the technique of Ramachadran (Ramachadran & Lee, 1970). The chromatographic yield was evaluated by adding cyclic c3y]AMP to the medium before filtration. Radioactivity was measured by liquid scintillation spectrometry. Details of the technique have been previously deet al., 1972). All determinations were scribed (BOCKAERT performed in duplicate.

Assay of phosphodiesterase actiuity The effectiveness of cyclic G M P and papaverine to inhibit the phosphodiesterase activities present in our cellular preparations was controlled by assaying their eventual residual hydrolytic activities. These latter were measured by the hydrolysis of labelled cyclic [H3]AMP. The incubation medium contained cyclic [H3]AMP (0.02 yCi, 30-500 nM) which was separated from its metabolites either by filtration on aluminium oxide columns or by TLC on cellulose plates. In the former case (method I), 0.1 mM-S’-AMP and 50 mM-NaF were added to the incubation medium in order to impair the further conversion of C3H]5’ AMP into compounds such as adenosine and adenine which are not retained by the column. All other conditions were otherwise identical to those described for adenylate cyclase assay. The reaction was stopped by cooling and dilution of cyclic AMP, with a large excess (1 mM) of unlabelled cyclic AMP. Just prior to filtration, cyclic [14C]AMP, jiCi was added to the samples in order to estimate the yield of the separation procedure. Radioactivity eluted from the columns was expressed as a percentage of that recovered from the control samples in which the enzymic preparation was absent from the incubation medium and added after cooling and dilution with unlabelled cyclic AMP. When cyclic AMP was separated from its metabolites by TLC on cellulose plates (method 2) the experimental conditions were identical to those employed for the adenylate cyclase assay except for the presence of cyclic [‘4C]AMP, 2.10-3 pCi, 0.4 PM. The chromatograms were developed at room temperature in the ammonium acetate/ ethanol system ( 7 3 , v/v). The radioactivity of the cyclic AMP spot was expressed as a percentage of that recovered from the control samples (see above). Ident$cation of adenosine metabolites The eventual conversion of adenosine to its metabolites during the time course of the adenylate cyclase assay was investigated by incubating the cellular preparations in presence of [2-3H]adenosine. The labelled metabolites were separated by silica gel TLC. Corresponding unlabelled metabolites were added as carriers. The development system et al. (1970): n-butanol, was that described by SHIMUZU ethyl-acetate, ammonia (sp. gr.. 0.88). methanol (7:4:3 by

Adenosine-responsivc adenylate cyclase

267

vol). The R , values calculated for thc unlabelled compounds were: inosine. 0.27; xanthine, 0.30; cyclic AMP. 0.47; hypoxanthine, 049; adenosine, 058; adenine, 0.71 ; 5’-ATP, 5’-ADP, 5’-AMP (near the origin). Chemicals

[a3’P]ATP (1 Ci/mM), cyclic C3H]AMP. (29 Ci/mM), cyclic [ 14C]AMP (52 mCi/mM) were purchased from the Commissariat a 1’Energie Atomique (Saclay) and [2-jH]adenosine (20 Ci/mM) from Amersham Radiochemical Centre, England. Radiochemical purity of adenosine was checked by TLC on silica gel plates as described above. Aluminium oxide was from Woelm and creatine kinase and phosphocreatine from Boehringer. All other reagcnts were A grade. Prostaglandins E, (PGE,), E, (PGE,) and F,a (PGF,a) were kindly furnished by Upjohn. Except for PGE, and PGE,, the various agents investigated were prepared in aqueous solution before being added in the incubation medium. The original solution of PGE, (1 mg/ml: 24. M)was in 70% cthanol and of PGE2 in 95% ethanol. They werc further diluted serially in water before being added to the incubation mixture.

RESULTS Effect of adenosine on adenylate cyclase activity Figure 1 illustrates the effects of adenosine and PGE, on the formation of cyclic AMP from [a3’P]ATP by neuroblastoma cell adenylate cyclase. In both frozen preparations and permeabilised cells, adenosine and PGE induced a concentration-dependent increase in cyclic AMP formation. Tn either preparation, adenosine exerted its effect in the same range of concentrations to M). However, the action of adenosine was greater on the permeabilised cells (3.9 f 1.6 times the basal value: 21 determinations P < 0.01) than on the frozen extracts (1.8 f 0.4 times the basal valuc: 33 determinations P < 0.05).The stimulation of cyclic AMP production by PGE, was much more pronounced and did not M. Furthermore, the enhancshow saturation at ing action of PGE, (loT4M) was about the same in the two types of preparations: being respectively

FIG. 1. Dose-dependent adenylate cyclase activation by adenosine and prostaglandin PGE,. Comparison between frozen preparations and permeabilized cells. Adenylate cyclase activity was measured as indicated under ‘Methods’. The enzyme used was either a frozen preparation (29pg protein per assay) or permeabilized cells (35 pg protein). Adenylate cyclase activity is expressed in terms of activation ratio, (i.e. activity measured in presence of adenosine or PGE,/activity measured under basal conditions). Basal adenylate cyclase activity was 56 and 32 pmol cyclic AMP formed/l0 min/mg protein for the frozen preparation (filled symbols) and the permeabilized cells (open symbols). respcctively. All determinations were performed in duplicate.

17.3 f 5.5 (14 determinations) and 16.0 6.0 (17 determinations) times the basal value for frozen and permeabilised samples. Basal cyclic AMP formation varied from experiment to experiment but remained within the same range for the two types of prcparations (2&100 pmol cyclic AMP formed/l0 min/mg protein). Unless otherwise specified, all the experiments described below were performed on permeabilised neuroblastoma cell preparations.

t ._ t 0

e

Q

F

\

a 5 Q

0 Time,

min

10

30

20 Protein,

40

pg

FIG.2. Effect of incubation time and protein content on cyclic AMP accumulation. Left: Permeabilized cells (30pg protein per assay) were incubated for increasing periods of time under adenylate cyclase assay conditions (see Methods) either alone (0)or in presence of adcnosine: 0.1 mM (A) or PGE,: 0 1 mM (0).Right: Cyclic AMP accumulation during a 10 min incubation period was measured in presence of increasing amounts of enzyme.

J. PENIT,J. HUOTand S. JARD

268

TABLE1. RESIDUAL.PHOSPHODIESTERASE

ACTIVITY UNDER ADENYLATE CYCLASE ASSAY CONDITIONS

Labelled cyclic AMP (w)

Adenosine (mM)

% radioactivity in

PGE, (mM)

cyclic AMP

Method 1 0.03 003 0.03 0.08 008 0.08 0.13 013 0.13 05 0-5 05

0.I 0.0 1 0.1 ___ 0.01 01

95 82 84 80 83 83 84 97 83

-

OQ1 0.1

Method 2

0.2

04 04 04 ~

83 88 81

~

0.01

77 76 74

~

The experiments were performed using permeabilized cells (40 pg protein). Methods 1 and 2 used for assaying phosphodiesterase activity are described under ‘Methods’. The activity was measured in the presence of the indicated amounts of cyclic AMP (substrate), adenosine or, prostaglandin PGE,. Values in the last column are the percentage of radioactivity recovered as cyclic AMP at the end of a 10 min incubation period.

Control experiments under basal conditions or after stimulation by adenosine as well as by PGE, indicated that the rate of formation of labelled cyclic AMP by permeabilised cells was linear with time for incubation periods up to 15 min and with total protein content up to 30 pg (Fig. 2). In most of the described methods for assaying adenylate cyclase activity, a large excess of unlabelled cyclic AMP is used to prevent the hydrolysis of labelled cyclic AMP formed from radioactive ATP. We did not retain this experimental procedure in order to avoid the possible liberation of adenosine from the TABLE 2. EFFECTOF ADENOSINE

Agent tested Exp. 1 Adenosine Adenine Exp. 2 Adenosine Inosine Exp. 3 Adenosine Hypoxanthine Xanthine Exp. 4 Adenosine Guanosine

) cyclic added nucleotide. Papaverine (0.25 m ~ and GMP (1 mM) which we used as phosphodiesterase inhibitors failed to completely prevent the hydrolysis of labelled cyclic AMP during the course of the adenylate cyclase assay. When tracer amounts of labelled cyclic AMP were added at the beginning of the 10 min incubation period not less than 75-80% of the radioactivity introduced was recovered as cyclic AMP (Table 1). However, it must be pointed out that this rather weak hydrolysis of cyclic AMP was unaffected either by adenosine (0.01 to 0.1 mM), or by PGE, (0.1 m ~ ) .

DERIVATIVES ON NEUROBLASTOMA CELL ADENYLATE CYCLASE

Adenylate cyclase activity relative to basal value Concentration in the incubation medium (PM) 0 0.0 1 01 1 10 100 100 100

93

117 87

140

104

105

180 100

100 100

90

91

96

96

255 81

128 114

300 99

130

240 125 125

122 120

180 108

226

100 100 100

100 100

100

89 105 115

200

98

264 96

105

104

Adenylate cyclase activity was determined as indicated under ‘Methods’. All values in the table are activities expressed as per cent of basal activity. Experiments 1 and 4 were performed using frozen preparations and experiments 2 and 3 using permeabilized cells. Values are the mean of duplicate determinations.

Adenosine-responsiveadenylate cyclase

Adenosine

[hi]

269

lites formed in these conditions were recovered in the migratory zone of inosine and xanthine. The rather important metabolism of adenosine which occurred during the course of the adenylate cyclase assay made it difficult to evaluate the apparent K , of the adenylate cyclase for adenosine. The estimated values M, whether varied from about 10- M to about the enzyme activity was plotted as a function of the concentrations of adenosine initially added or of those present at thc end of the incubation period. None of the non-phosphorylated metabolites of adenosine were able to induce adenylate cyclase activation up to a concentration of 0.2 mM (Table 2). Guanosine which might be libcrated from cyclic GMP added t o the incubation medium was also inactive. On the other hand, low concentrations of AMP induced a dose-dependent inhibition of cyclic AMP production (Fig. 4). In order to evaluate the possible formation of cyclic AMP from adenosine permeabilized cells were incubated in presence of [3H]adenosine (0.2 mM, 4 p a ) and [a3*P]ATP (0.1 m ~ 2, pCi). Cyclic AMP was purified using Dowex chromatography (Dowex AG 50 x 8, 200-400 mesh). The fraction containing cyclic AMP was concentrated and submitted to TLC on cellulose plates. It was found that the cyclic AMP spot contained negligible amounts of C3H]radioactivity. The latter expressed in terms of cyclic AMP formed from adcnosine represented less than 1% of the amount of cyclic nucleotidc formed from added [G?~P]ATP.

FIG. 3. Metabolic transformation of adenosine undcr adenylate cyclase assay conditions. Upper part: A frozen preparation of neuroblastoma cells (30pg protein) was incubated in the presence of various concentrations of adenosine. The incubation medium was identical to that used for adenylate cyclase assay except that it containcd [2-3H]adenosine (0.13pCi) but not [CL~~PI-ATP. The reaction was allowed to proceed for 10min. It was stopped by cooling and dilution with a solution containing: (0.1 to 1 mM of ATP, SAMP, xanthine, hypoxanthine, inosine, cyclic AMP, adenosine and adenine). Aliquots (20pl) were spotted on silica gel plates and the chromatogram developed as indicated under ‘Methods’. The chromatogram was divided into successive strips (1 cm width) in which C3H]radioactivity content was determined. The radioactivity present in the identified spots was expressed as a percentage of total radioactivity on the chromatogram: adenosine, (A); ATP, ADP, S’AMP, IMP (A);xanthine, inosine (0);cyclic AMP. hypoxanthine (0);adenine (0). Characteristics of adeaosine-sensitive adenylate cyclase The calculatcd values are plottcd as a function of the initial Basal and stimulated adenylate cyclase activity was adenosine concentration. Experiments conducted using enhanced by the same order of magnitude when ATP permeabilized cells gave similar results. When the enzyme concentrations were increased from 0.01 to 0.5 m~ was introduced immediately after the addition of the solution used for stopping the reaction, 3H radioactivity was recovered as adenosine. Lower part: In a parallel experiment the dose-dependent adenylate cyclase activation by adenosine was determined. Adenosine-stimulated activity is plotted as a function of adenosine concentration at the beginning of the incubation period (solid line) or as a function of the adenosine concentrations remaining at the end of the incubation (dotted line). Adenosine was metabolized during the course of the adenylate cyclase assay. The fraction of converted adenosine as well as the nature of the metabolites formed were related to the concentration of adenosine added to the medium (Fig. 3). More than 93% of adenosine was converted at the lowest and inactive concentration (10- M); the most abundant metabolites separated by TLC were found in the migration zones corresponding to thosc of inosine and xanthine ( R F :-0.27 and 0.30) and of nucleotides, ATP, ADP, AMP, I M P (near the origin). For concentrations which induced a dose-dependent activation of adenylate cyclase. the fraction of transormed adenosine was lower (lcss than 50% at M). The major metabo-

FIG.4.Inhibitory effect of 5‘-AMP on neuroblastoma cell adenylate cyclase. Adenylate cyclase activity was measured as described under ‘Methods’ in the presencc of various concentrations of either adenosine (A) or 5’-AMP(0).The enzyme used was a frozen preparation (60 p g protein).

J. PENIT, J. HUOTand

270 C

I

I

I

I

1

CI

0

50

0 01

005

010

025

050

ATP [ m ~ ]

FIG.5. Elfect of ATP concentration on basal and adeno-

sine-sensitiveadenylate cyclase. The described experiments were performed using a frozen preparation (43 pg protein). The incubation medium contained the indicated amounts of ATP. Dashed lines: 0.5 mM-Mg*+;full lines: 1 mM-Mg2+. Adenylate activity was measured under basal conditions (0, 0 ) or in presence of 0Q5mM-adenosine (A, A). (Fig. 5). Thus the r a t i e a d e n o s i n e stimulated activity/basal activity was almost independent of the concentration of ATP. This indicates that adenylate cyclase activation by adenosine resulted in an increase in the maximal velocity of the reaction. The stimulatory effect of adenosine appeared to be stereospecific as illustrated by the failure of some of its closely related structural analogues (2-deoxyadenosine; 3-deoxyadenosine; adenine p-D arabinofuranoside) to activate the adenylate cyclasc (Fig. 6). Theophylline induced a concentration-dependent inhibition of the adenylate cyclase activation by adenosine. This inhibition is characterized by both a shift in the apparent K , for adenosine and by a decrease of the maximal activation. The blocking action of theophylline was observed for concentrations M. Theophylline M) inhifrom lo-“ M to bited by 25% the stimulatory effects of adenosine M. Theophylline also induced a slight but concen-

Adenosine or analogues [ M I

6 . Effect of adenosine analogues on neuroblastoma cell adenylate cyclase. The experiment was performed using permeabilized cclls (35 [lg protein). Adenylate cyclase activity was measured as detailed under ‘Methods’ in the presence of increasing concentrations of adenosine (A), 2 deoxyadenosine (@), 3 deoxyadenosine (0) and adenine 8-D-arabinofuranoside (0).

tration-dependent decrease of the basal adenylate cyclase activity (Fig. 8).

Interactions between adenosine, prostaglandins and adrenergic agents Adenylate cyclase activity from permeabilized neuroblastoma cells was slightly enhanced by dopamine and norcpinephrine but not by isoproterenol (0.1 mM). No mutual potentialisation of the effects of adenosine and adrenergic agents could be demonstrated. However, the effect of adenosine (0.1 mM) in combination with either dopamine (0.1 mM) or norepinephrine (0-1mM) was higher than the effects of the same agents tested alone (Table 3). PGE, was the most effective (Fig. 7) among the three prostaglandins tested PGE,, PGEz and PGF,a. An apparent saturation plateau was observed at about M-PGE,.Higher concentrations led to a marked additional stimulation. The same pattern of

TABLE 3. EFFFCTS OF AURENtRGlC AGENTS ALONE AND IN COMBINATION WITH ADtNOSINt ON T H t 32P CYCLIC AMP FORMATION BY PFRMFABILIZtD NEUROBLASTOMA CELLS

Agents added (0.1 mM) None Adenosine Dopamine Norepinephrine Isoproterenol Adenosine + Dopamine Adenosine + Norepinephrine Adenosine + lsoproterenol

Cyclic AMP (pmoljmg protein/l0 min) 19-24 52-54 37-41 35-36 21 20

61-66 60-63 5657

Adenylate cyclase activity was measured as indicated under ‘Methods’. The experiment was performed using permeabilized cells (30 p g protein).

Adenosine-responsive adenylate cyclase

271

Adenosine [M]

Prostaglandins

[MI

FIG. 7. Dose-dependent adenylate cyclase activation by prostaglandins. The experiment was performed using permeabilized cells (30 p g protein). Adenylate cyclase activity was measured in the presence of the indicated amounts of PGE, (O), PGEz (0) or PGF,G((0). activation was obtained with PGE,. whereas PGF,cr was poorly active. Ethanol in concentrations (from 6x M to 2.6 M) which were equal t o those introduced with prostaglandins (from lo-’ M to 4.10-4 M) did not affect basal adenylate cyclase activity. Adenosine M) induced a slight but significant inhibition of adenylate cyclase activity measured in presence of PGE, M). Theophylline which did not affect the prostaglandin-sensitive activity failed t o

TARLF4. EFFECTSOF

FIG.8. Inhibition of adenosine-sensitive adenylate cyclase by theophylline. The experiment was performed on permeabilized cells ( I 0 0 p g protein). The concentration-dependent adenylate cyclase activation by adenosine was determined in presence of various amounts of theophylline: @, I mM; 0, 0.1 mM; 0, 0.01 mM; 0. 0001 mM, A: control curve with no theophylline added. The apparent K , values for adenosine were: 5 @I (A), 13 p~ (O), 10 p~ (O), 22 p~ (0) and 4 4 p (o), ~ respectively.

reverse the inhibitory effect of adenosine observed in the presence of prostaglandins (Table 4). DISCUSSION The experiments described above were performed using crude preparations of permeabilized neuroblast o m a cells. This procedure was retained since we previously observed that the successive centrifugation

ADENOSINE THEOPHYLLINE AND PGE, ALONE AND IN COMBINATION ON THE CYCLIC FORMATION RY PERMEABILIZED NEUROBLASTOMA CELLS

C3’P]AMP

Cyclic AMP (pmol/mg protein/lO min) Experiment I1 Experiment I

Agents added

None Adenosine 0.01 mM Adenosine 0 1 mM PGE, 0.001 mM Theophylline 0.1 mM Adenosine 0.01 mM + PGE, 0.001 mM Adenosine 0.1 mM + PGE, 0001 mM Adenosine 001 mM theophylline 0.1 mM Adenosine 0 1 mM + theophylline 0.1 mM PGE, 0001 mM theophylline 0 1 mM Adenosine 0.01 mM PGE, 0.001 mM + theophylline 0 1 mM Adenosine 01 mM PGE, 0.001 mM + theophylline 0.1 mM

+

27.6 f 0.8 54.6 3.8 89.5 & 6.7 252 -I 14 24 f 2.0 230 f 17 212 f 5

51.3 _+ 7.3 190 -I 20 14.3 k 3 6 151

*9

42.5 i 2.9 615 & 3.5

+

+ +

14.1 f 3.5

38.9 & 5.5 202

29

139

9

2546 k 17 202 & 12

The experiments were performed using permeabilized cells (35 p g protein Experiment I. 30 pg protein Experiment S.D. from 4 separate determinations.

11). Values in the table are means \.r.261-7

Neuroblastoma cell adenylate cyclase: direct activation by adenosine and prostaglandins.

Jouriiul 01 Newocltrmmtry, 1976. Vol. 26, pp. 265-273. Pcrgamon Press. Printed i n Great Brltaln. NEUROBLASTOMA CELL ADENYLATE CYCLASE: DIRECT ACTI...
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