Naunyn-Schmiedeberg's Naunyn-Schmiedeberg's Arch. Pharmacol. 305, 233-240 (1978)

Archivesof

Pharmacology 9 by Springer-Verlag 1978

Phosphorylated Derivatives of Phloretin Inhibit Cyclic AMP Accumulation in Neuronal and Glial Tumor Cells in Culture R. Ortmann*, D. Nutto, and R. Jackisch Pharmakologisches Institut der Universit~it Freiburg, Hermann-Herder-Strasse 5, D-7800 Freiburg, Federal Republic of Germany

Summary. The potencies of polyphloretin phosphate,

di-4-phloretin phosphate, 4-phloretin phosphate and phloretin to inhibit the stimulation of cAMP accumulation by prostaglandins, isoproterenol and adenosine were studied in 2 clonal cell lines of CNS origin. The sequence of potency to inhibit PGE 1 effects was the same in neuroblastoma (N4TG3) and human astrocytoma cells (1321N1): di-4-phloretin phosphate > polyphloretin phosphate > phloretin > 4-phloretin phosphate. The inhibition of PGE 1 stimulated cAMP accumulation by the most prostaglandin-specific inhibitor di-4-phloretin phosphate was rapidly established after its addition, fully reversible after a 30 rain preincubation period and independent of the presence of calcium. Kinetic studies of the inhibition of PGE1 effects by di-4-phloretin-phosphate suggest a different type of inhibition in t321N1 and N4TG3 cells. Key words: Prostaglandin-antagonists - cAMP accu-

mulation - PGE~ -- Clonal cell lines - Phloretin.

Introduction

While a great number of substances is known to interfere with the synthesis of prostaglandins (Flower, 1974), rather few compounds have been described which inhibit specifically the actions of prostaglandins.

Besides the dibenzoxazepine derivative SC 19220, the PG analogue 7-oxa-13-prostynoic acid, morphine (Collier and Roy, 1974) and nonsteroid antiinflammatory drugs (Ortmann, 1976; Ortmann and Perkins, 1977), phosphorylated polymers of the dihydrochalcone phloretin have been shown to be antagonists of prostaglandins in various systems (Sanner, 1974). Since the original observation of antiprostaglandin activity of polyphloretin phosphate (Beitch and Eakins, 1969), the mechanism of its prostaglandin-antagonism has been studied mainly in smooth muscle or vascular preparations. It was found that antiprostaglandin specificity, the potency and kinetics of its inhibitory action vary greatly in different system. Sephadex fractionation of PPP and the use of dimers (DPP) and monomers (MPP) showed that there are significant differences in the inhibitory potencies of these phloretin derivatives (Burka and Eyre, 1974; Ishizawa and Miyazaki, 1976; Eakins et al., 1973; Sato et al., 1972). We studied the effect of PPP, DPP, MPP and phloretin on the agonist stimulated cAMP accumulation in human astrocytoma cell line 1321N1 and the murine neuroblastoma cell line N4TG3. The experiments were performed to define the most potent prostaglandin antagonstic derivative of phloretin, its prostaglandinspecificity and some aspects of its inhibitory mechanism.

Materials and Methods * Present address: Research Department, Pharmaceutical Division, Ciba-Geigy Ltd. Basle, Switzerland Send offprint requests to R. Jackisch at the above address

The following abbreviations are used in this report: polyphloretin phosphate: PPP; 4-phloretin phosphate: MPP; di-4-phloretin phosphate: DPP; 3-isobutyl-methylxanthine: IBMX; trichloroacetic acid: TCA; N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid: HEPES; Ethylenglycol-bis (~-amino ethylether) N,N'-tetraacetic acid: EGTA; Prostaglandin E~: PGEa (~25j) 2'-O-succinyl cyclic AMP tyrosine methyl ester: 125J-TME SCAMP.

Materials. PGE 1 was a gift from J. Pike, Upjohn Co., Kalamazoo,

Michigan, U.S.A. All phosphorylated derivatives of phloretin were from A.B. Leo, Helsingborg, Sweden. 3-isobutylmethyl-xanthinewas from Ega-Chemie, Steinheim, Federal Republic of Germany; all other substances were purchased from Sigma Chemical Co., Miinchen, Federal Republic of Germany. Cell Culture Conditions. The origin and growth conditions of the human astrocytoma cell line 1321N1 (Ponten and McIntyre, 1968; Clark et al., 1975) and the 6-thioguanine resistant neuroblastoma cell line N4TG3 (Amano et al., 1974; Schultz and Hamprecht, 1973) have

0028-1298/78/0305/0233/$01.60

234

Naunyn-Schmiedeberg's Arch. Pharmacol. 305 (1978)

Table 1. Effects of PGE l, isoproterenol and adenosine on intracellular levels of cAMP in N4TG3 and 1321N1 cells Agonist

None Isoproterenol PGE 1 Adenosine

~321 N1

N4TG3

Maximal response a (pmoles cAMP/rag prot/5 min)

EC5o b [~tM]

Maximal response a (pmoles cAMP/rag prot/5 min)

ECso b [gM]

20 2 200 1140 330 ~

-

24 24 1200 600

-

0.1 1 50 ~

0.03 20

a In the presence of l mMIBMX.F~rfurtherdetai~sseeMeth~ds.Maxima~resp~nsewasmeasuredat~aMag~nistc~ncentrati~nf~rPGE1 and isoproterenol and 3000 ~tM for adenosine b ECs0 were graphically determined from dose response curves c In the presence of 0.1 mM papaverine instead of IBMX

been described. 1321N1 cells were kindly provided by R.B.Clark, Worcester, Mass., U.S.A. N4TG3 cells by B. Hamprecht, MiJnchen, FRG. Culture dishes ("Lux", 35 ram) were seeded with 1 x 105 N4TG3 cells or 2 x 105 1321N1 cells in 2 ml Dulbecco's modified Eagle's minimal essential medium supplemented with 10 ~ fetal calf serum (BCK Biocult, Karlsruhe, FRG) and grown for 3 days. Protein was measured by the biuret method, cell numbers were counted with a model A Coulter counter. Experimental Incubation Conditions. For incubation the growth medium was aspirated, the cells washed twice with serum free growth medium and challenged for 5 min with the agonists in serum free growth medium at 37~ containing 1 mM IBMX or 0.1 mM papaverine (when adenosine was the agonist in 1321N 1 cells). When not otherwise stated, phloretin or its phosphorylated derivatives were added immediately before the agonist. Experimental incubations were stopped by aspirating the medium and adding 1 ml 5 ~o TCA to the culture dishes. The TCA extract was extracted with water saturated ether and the water phase used for cAMP determination without further purification. The phosphorylated phloretin derivatives were dissolved in water, phloretin in ethylenglycolmonoethylether, PGE~ in !0 ~ ethanol, isoproterenol and adenosine in 0.001 N HCI and diluted 100-fold into the incubation medium~ At the concentrations used none of the solvents used had a significant effect on cAMP levels. In both cell lines maximal responses to all 3 agonists varied considerably in different experiments. Half maximal concentrations of the agonists (ECs0) did, however, not change. Table I therefore depicts the results of representative experiments. Experiments with suspended N4TG3 cells: Cells were incubated for 1 rain with a calcium- and magnesium-free trypsin solution (0.005 ~ trypsin, pH 9.0), suspended in serum free growth medium and washed once in the same medium by low speed centrifugation. Incubations were started in serum free medium containing 1 mM IBMX by adding PGE 1 to the cell suspension stirred at 37~ Aliquots of 200 gl were withdrawn at the indicated times and pipetted immediately into 0.8 ml cold 6.25~ TCA. TCA extracts were analyzed for cAMP after centrifugation as described for attached cells. Basal cAMP levels in suspended N4TG3 cells varied from 10 30 pmoles/mg protein. cAMP-Radioimmunoassay (12sj) 2'O-succinyl cyclic AMP tyrosine methyl ester (125j-TME SCAMP) was prepared by the method of Hunter and Greenwood (1962) and purified according to Cailla and Delaage (t 972). Synthesis, purification and coupling of 2'-O-succinyl cAMP to human serum albumin and the immunization of rabbits were performed according to Steiner et al. (1972) with modifications suggested by Weinryb (1972). The conditions of the cAMP radioimmunoassay and erossreactions of the cAMP antiserum with other

adenine or guanine nucleotides have been described elsewhere (Ortmann, 1978). None of the prostaglandin-antagonists at the highest concentration used, interfered with the recovery of cAMP added to Ihe TCA extract after the ether extraction step. Each determination represents mean + S.D. of at least 4 cAMP determinations (2 dishes assayed in duplicates). Duplicate determinations varied less than +5Vo.

Results

Adenylate Cyclase System of the Cell Lines Used T h e effect o f v a r i o u s a g o n i s t s o n the c A M P c o n t e n t o f 1321N1 a n d N 4 T G 3 cells is s u m m a r i z e d in T a b l e 1. 1321N1 cells h a v e b e e n p r e v i o u s l y s h o w n to h a v e 3 d i f f e r e n t r e c e p t o r s m e d i a t i n g t h e effects o f p r o staglandins, catecholamines and adenosine on adenylate cyclase ( C l a r k et al., 1975; O r t m a n n a n d P e r k i n s , 1977). I n N 4 T G 3 cells c A M P a c c u m u l a t i o n is s t i m u lated o n l y b y P G E ~ a n d a d e n o s i n e , w h e r e a s c a t e c h o l a m i n e s h a v e little o r n o effect ( S c h u l t z a n d H a m p r e c h t , 1973). T a b l e 1 s h o w s t h a t P G E 1 has a m u c h h i g h e r a f f i n i t y to its r e c e p t o r ( E C s 0 = 0.03 g M ) in N 4 T G 3 t h a n in 1321N1 cells ( E C s 0 = 1 g M ) . I B M X acts as an a n t a g o n i s t o f a d e n o s i n e in 1321N1 b u t a p p a r e n t l y n o t i n N 4 T G 3 cells ( O r t m a n n , u n p u b l i s h e d o b s e r v a t i o n ) . T h e r e f o r e a d e n o s i n e e x p e r i m e n t s in 1321N1 cells w e r e d o n e in the p r e s e n c e o f 0.1 m M p a p a v e r i n e i n s t e a d o f I B M X . All s u b s e q u e n t e x p e r i m e n t s w e r e d o n e at h a l f maximally active agonist concentrations.

Comparison of Different Phloretin Derivatives Dose response curves of the inhibitory actions of PPP, DPP, MPP and phloretin on PGE1 stimulated cAMP a c c u m u l a t i o n in b o t h cell lines a r e s h o w n in Fig. 1. D P P was t h e m o s t p o t e n t i n h i b i t o r in b o t h celt lines. T h e d i f f e r e n c e in i n h i b i t o r y a c t i o n b e t w e e n t h e d i m e r D P P a n d the m o n o m e r M P P was s u r p r i s i n g l y great, its I C s 0 b e i n g a b o u t 1 0 0 - - 1 2 0 t i m e s h i g h e r t h a n the I C s o

R. Ortmann et al. : Phosphorylated Phloretin Derivatives Inhibit cAMP Accumulation

235

Table 2. Agonist specificity of phosphorylated phloretin derivatives and phloretin. IC50 values [pM], i.e. inhibitor concentrations which block 50 % of the accumulation of cAMP in the presence of the indicated agonists in 1321N1 and N4TG3 cells are given. IC50 were graphically determined from dose-effect curves. Numbers in parenthesis indicate number of experiments. Concentrations + S.D. are given when at least 3 experiments have been performed Inhibitor

1321N1 PGE1 [1 pM]

N4TG3 PGE1 [0.03 ~M]

1321N 1 [soproterenoI [0.1 pMI

1321 N 1 Adenosine [50 ~tM]

N4TG3 Adenosine [20 ~M]

PPP DPP MPP Phloretin

49 + 15 (4) 18 + 5(5) 2000 (2) 160 + 90 (4)

25 4 500 120

> 196 (3) 320 + 10(3) > 2400 (2) 400 + I00 (3)

196 (2) 400 (2) > 2400 (2) 130 • 60 (3)

> 196 (2) >160 a(3) > 900 (3) 380 + 150 (4)

+ 17 (3) + 1(4) + 100 (3) + 70 (5)

a Since N4TG3 cells were partially detached from their substrate at higher DPP concentrations, experiments with higher DPP concentrations than 160 gM were not done

100

'

'

'

" '

'

I

I

possessed the highest anti-prostaglandin specificity: In N4TG3 cells its ICso for adenosine as agonist was more than 40 times higher than the ICso of DPP in the presence of PGE 1. In 1321N1 cells adenosine and isoproterenol effects on cAMP content were inhibited by DPP at about 20 times higher concentrations than the effects elicited by PGE~. PPP exerted a prostaglandin-specific inhibition at rather high concentrations (100-200 pg/ml). In one experiment with 1321N1 cells the inhibitory action of a low molecular and a high molecular weight fraction of PPP was compared with PPP. On a weight basis no significant difference in potency of the 3 compounds was observed.

'

60

20 I

u g

100

I

I

~

I

I

~ N/, TG3

80 60 40 2O I

I

10 - 5

I

I

10 - l '

I

10-3

I

M

Inhibitor

Fig. l. Effects of PPP, DPP, MPP and phloretin on PGE 1 stimulated cAMP accumulation in N4TG3 and I321N1 and cells. Dose effect curves of increasing concentrations of PPP ( . *), DPP (A A), MPP (e ~ and phloretin (II --) in 1321N1 cells (1 ~tM PGE 0 and N4TG3 cells (0.03 FM PGE1) are shown

determined for DPP in the presence of PGE 1 in 1321N1 and N4TG3 cells respectively. The unphosphorylated dihydrochalcone phloretin had an inhibitory activity which was only slightly less than the inhibitory potency of ppp1. The effects of PPP, DPP, MPP and phloretin on PGE~-, isoproterenol- and adenosine-stimulated cAMP accumulation are compared in Table 2. MPP showed very little prostaglandin-specificity at high concentrations, while the effects of all agonists were inhibited by phloretin in about the same concentration range, its ICso varying between 120-400 pM. DPP 1 The exact molecular weight of PPP is not known. Calculations were based on a molecular weight of 4.600 (B. H6gberg, personal communication). Low and high molecular fractions of PPP prepared by Sephadex fractionation were supplied by Leo AG (Sweden)

Effect of DPP on Basal cAMP Levels DPP decreased dose dependently basal cAMP levels of N4TG3 cells, with a ICso of more than 470 gM. In a typical experiment the basal level of cAMP (24_ 1 pmoles/mg protein) was decreased 3 7 ~ by 470pM DPP, a concentration 150 times higher than the ICs0 determined for PGE 1. Release of cAMP Into the Incubation Medium The observed inhibition of intracellular cAMP accumulation by PPP, DPP or phloretin was not due to facilitation of the release of cAMP into the medium. cAMP excreted into the incubation medium varied between 2 - 8 % and 5 - 10 % of total cAMP in 1321N1 and N4TG3 cells, respectively. The amount of cAMP excreted into the medium in the presence or absence of PGE a was not increased by the addition of up to 158 pM DPP, 65 gM PPP and 360 pM phloretin. In the same experiments intracellular cAMP accumulation was inhibited up to 90 % by DPP, 70 % by PPP and 68 by phloretin. Onset and Reversal of DPP Inhibition The time course of intracellular cAMP accumulation of attached 1321N1 and N4TG3 cells in the presence and

236

Naunyn-Schmiedeberg's Arch. Pharmacol. 305 (1978) i

1000

i

i

i

i

Table 3. Reversibility of DPP-inhibition in 1321N1 cells. Results

are given in ~ of control (Y _+ S.D., n = 3) 1321N1 cells were preincubated for 30 rain at 37~ in incubation medium containing 1 mM IBMX in the presence (right column) of absence (left column) of the indicated concentrations of DPP. After 30 min all dishes were washed 2 times with 2 ml incubation medium and challenged with PGE t (I ~M) in the presence (left column) or absence (right column) of the indicated concentrations of DPP. Control dishes received no DPP during preincubation or incubation period. Control dishes contained 473+ 25pmoles cAMP/mg protein

800 600 .E

~oo

o

E

200 f

I

I

I

I

~1000

DPP {~tM]

800 o. 600 ~00 200 I

I

I

2

r

I

4

I

!

I

6

I

27 80 270

I

8 I0 Minutes

~ of control (Y + S.D.) No DPP during preincubation (DPP present during incubation)

Cells preincubated with DPP 30 min (no DPP during incubation)

60 • 5 40 _+ 1 22 + 2

102 + 7 105 _+ 8 79 +_ 5

Fig. 2. Time course of POE x stimulated cAMP accumulation in the

presence and absence of DPP. 1321N1 cells (top) were incubated with 1 ~tM PGEI in the absence ( A - - A ) and presence of 32 ~tM DPP (t .'). N4TG3 (bottom) cells were incubated with 0.03 p-M PGEx in the absence (A A) and presence (O-- @) of 16 laM DPP. DPP and PGE 1 were added simultaneously to the incubation medium

i

1

!

I

I

I

t

I

I

~J (J

=o 20 t--X U3

< 30 U

~. 5

least 2 min after addition o f 30 n M P G E 1 (not shown). Figure 3 depicts the effect o f 27 g M DPP, added 45 s after 30 n M P G E 1 to suspended N 4 T G 3 cells: c A M P accumulation stopped immediately after the addition ( 1 0 - 1 5 s) and reached a new steady state after 2 0 25 s. By addition o f 10 I~M PGEa a linear increase o f c A M P accumulation was again established within 10 s. Table 3 summarizes experiments with 1321N 1 cells, showing that the inhibitory effect o f D P P was easily reversed by washing the cells with m e d i u m after a 3 0 m i n preincubation period. Experiments with N 4 T G 3 cells (27 laM D P P ) gave comparable results and showed in addition that D P P inhibition did not increase during a 30 rain preincubation period.

O

E I

1

2

Minutes Fig. 3. Inhibition o f P G E 1 mediated cAMP accumulation by DPP in suspended N4TG3 cells. 3.8 x l0 p cells were suspended in 5 ml medium. 0.03 ~tM PGE 1 was added at the beginning of the experiment, DPP (16 IxM) was added after 45 s (arrow), PGE1 (10 laM)

after 110 s (arrow). Viability of the cells (trypanblue test) was between 75-95 ~ at the end of the experiments. Each point represents the average of duplicate determinations

absence o f D P P is shown in Fig. 2. The degree o f inhibition o f P G E 1 effects by D P P added immediately before the agonist did not change during a 10 min incubation period in both cell lines. These results suggest that the inhibitory action o f D P P is fully established at least 1 min after its addition. The levels o f intra- and extracellular c A M P in suspended N 4 T G 3 cells increased almost linearly for at

Kinetic Studies of DPP-Inhibition Dose response curves o f P G E 1 in the absence and presence o f D P P showed m a r k e d differences in the effect o f D P P for N 4 T G 3 (Fig. 4) and 1321N1 cells (Fig. 5). D P P acted partly like a competitive antagonist in b o t h cell lines by shifting the dose response curves to the right. However, a decrease in the maximal c A M P accumulation by D P P was observed in 1321N1 cells, where the maximal stimulation by 300 ~tM PGE1 was reduced by 50 ~ in the presence o f 79 laM DPP. This observation is n o t consistent with competitive inhibition. I n N 4 T G 3 cells no plateau o f the dose response curves was reached under our experimental conditions. However, a decrease in the maximal c A M P accumulation as seen in the a s t r o c y t o m a cell line c a n n o t be excluded f r o m o u r data for higher concentrations o f P G E 1 or the antagonist D P P iia N 4 T G 3 cells. The same concentration o f D P P (79 ~tM) which showed a m a r k e d inhibition o f

R. Ortmann et al. : Phosphorylated Phloretin Derivatives Inhibit cAMP Accumulation

I

I

I

I

237

I

100

i

i

i, i

i

100

80 8O

o= =~ 6 0

6o

y

E

40

,.e

40

20

I

I

I

I

1

I

10-B 10-7 10 -6 10-5 10-t' M 4 84

I

I

I

I

I

I

lO-e 10-7 10-6 10-5 10-~. 10--3

10-8 10-7 10-6 10-5 10-t. 10-3

M

[Isoproterenot]

s

Fig. 4. Dose response curves of PGE t stimulated cAMP accumulation in N4TG3 cells in the presence and absence of DPP. N4TG3 cells were incubated with the indicated concentrations ofPGE a in the absence (O ~) and presence of 7.9 gM (O O), 32 pM (B -') and 79 gM (y y) DPP. N = 3, + S.D Fig. 5. Dose response curves of PGE 1 and isoproterenol stimulated cAMP accumulation in 1321N1 cells in the presence and absence of DPP. 1321N1 cells were incubated with the indicated concentrations ofPGE t (left) and isoproterenol (right) in the absence (O------O) and presence of 23 gM ( I -') or 79 ~tM DPP (~~)

PGE 1 effects in 1321N1 cells (Fig. 5, left) did not alter the dose response curves generated with isoproterenol (Fig. 5, right).

Table 4. Inhibitory of DPP and structurally related compounds on PGE 1 stimulated cAMP accumulation in N4TG3 and 1321N1 cells. ICso values were graphically determined from dose effect curves of the inhibitors in the presence of the indicated concentrations of PGE 1. Results are the average of 2 experiments

Role of Calcium and of the pH of the Incubation Medium Calcium was not required for the inhibitory action of DPP of PPP in both cell lines: In N4TG3 cells the cAMP accumulation stimulated by 0.03 gM PGE t was inhibited by 4.7 gM DPP essentially to the same extent in Krebs-bicarbonate solution (Umbreit et al., 1964) containing 1.7 m M calcium (47 + 4 ~ of control: N = 3) as in the absence of calcium and the presence of 0.1 mM EGTA (42 + 1 ~ of control; N = 3). Similar results were obtained with 44 gM PPP as inhibitor (34 _+ 1 ~ of control in the presence, 31 + 3 ~o of control in the absence of calcium; N = 3). The pH dependency of DPP inhibition on cAMP accumulation of PGE 1 stimulated N4TG3 cells was studied in HEPES-(20 mM) - buffered medium: the inhibition of DPP (4.8 gM) was more pronounced at pH 6.0 (61 ~ inhibition) than at pH 8.2 (23 ~ inhibition). This may indicate that uncharged DPP is the active inhibitory species.

Effect of Structurally Related Compounds of DPP Table 4 depicts the effects of DPP and of some structurally related compounds on the PGE1 stimu-

Inhibitor

ICso [gM] N4TG3 (0.03~tM PGE 0

4-Diphloretinphosphate (Nr. 1235 Leo) 4-Dihydrochalconephosphate mono-3,5-dimethyl phenyl ester (Nr. 1262 Leo) 4-Phloretinphosphate mono-3,5-dimethyl phenyl ester (Nr. 1258 Leo) Diphenylphosphate (Nr. 1263 Leo)

1321N1 (1 ~M PGE 0

3

20

40

20

10

20

>> 300

>> 300

lation of cAMP accumulation in N4TG3 and 1321N1 cells. Again there were marked differences in the inhibitory potency of these compounds in the 2 cell lines: In N4TG3 cells DPP was the most potent antagonist and substitution of a phloretin molecule by a dimethylphenyl-group (Leo Nr. 1258) and by 4-

238 dihydrochalcone (Leo Nr. 1262) decreased its inhibitory potency. In 1321N1 cells the inhibition by all 3 compounds was not significantly different. Diphenylphosphate (Leo Nr. 1263) did not inhibit PGE~ stimulation in both cell lines at the highest concentration tested (300 ~tM).

Discussion

PPP has been shown to inhibit not only the effects of prostaglandins but also to interfere with their metabolism (Marrazzi and Matschinsky, 1972) and to inhibit various enzymes (Diczfalusy et al., 1953) including cAMP dependent protein kinase (Kuehl et al., 1971). To study the anti-prostaglandin activity of putative prostaglandin-antagonists like DPP or PPP it is therefore important to use an experimental model where other possible actions of the antagonists are not interfering with the determination of their prostaglandin-antagonistic action. Therefore the adenylate cyclase system of the two cell lines used in this study seems to be a suitable model for the characterization of prostaglandin-antagonists: 1. The generation of cAMP is the first measurable intracellular event after the interaction of prostaglandins with the regulatory site of adenylate cyclase on the outer membrane. Possible other effects of the prostaglandin antagonists therefore do not interfere with cAMP determination. 2. The antagonists did not alter the extrusion of intracellular cAMP into the incubation medium (see results). 3. All experiments were performed at maximally effective concentrations of the phosphodiesterase inhibitors IBMX or papaverine. The observed inhibitory effects are therefore probably not due to an enhanced degradation of cAMP. 4. The specificity of the antagonists for various agonists can be determined, because the adenylate cyclase systems of both cell lines are not only stimulated by prostaglandins but also by isoproterenol (1321N1 only) and adenosine. 5. Agonist induced changes in cAMP accumulation can be measured in intact cells without prior homogenization. It has been shown that breaking of ceils or tissues in many cases drastically alters the properties of the adenylate cyclase system (Suet al., 1976). DPP is the most potent and most PG-specific phosphorylated derivative of phloretin in both cell lines. This confirms the finding of Eakins et al. (1973) that phloretin derivatives with primary phosphoric acid ester groups like 4-phloretin phosphate (MPP) are significantly less potent than the dimers (DPP) which contain secondary phosphoric acid ester groups. The

Naunyn-Schmiedeberg's Arch. Pharmacol. 305 (1978)

inhibitory action of DPP is easily reversed by washing and fully established immediately after its addition to the incubation medium. The inhibition of PGE1 effects on adenylate cyclase by phloretin was found to be almost irreversible after a 30 min preincubation period (Ortmann, unpublished observation). Together with the kinetic data discussed below it is therefore unlikely that uptake of DPP into the cell is involved in its inhibitory action on cAMP accumulation. However, a very fast and reversible uptake mechanism for DPP cannot be excluded by our data. Kuehl (1973) suggested that polymers of phloretin might have to be dephosphorylated prior to their inhibitory action. We found that the onset of the inhibitory action of DPP is very rapid (Figs. 2, 3) and does not decrease during a 30 min preincubation period. Therefore cleavage of an ester bond or dephosphorylation of DPP is not involved in its inhibitory action (Both products of DPP hydrolysis - MPP and phloretin - are significantly less potent inhibitors than DPP). Another aspect of the mechanism of action of DPP is illustrated by the comparison of the dose response curves of PGE1 in the presence and absence of various DPP concentrations in N4TG3 cells (Fig. 4) and 1321N1 cells (Fig. 5). DPP seems to act competitively in neuroblastoma cells with - under our experimental conditions - n o detectable effect on the maximal stimulation of cAMP accumulation even at concentrations of DPP which are 20 times higher than the IC5o determined in the presence of PGE1. In 1321N1 cells DPP showed 2 effects: i) ~t shift of the dose response curve of PGE~ to the right and ii) a marked decrease of the apparent maximal rate of cAMP accumulation at higher concentrations. 79 gM DPP - a concentration which is 3 times higher than the ICso of DPP for PGE~ - decreased the maximal response of adenylate cyclase by 50 ~o- The same concentration of DPP did, however, not change the dose response curves of isoproterenol, showing that this inhibition by DPP is rather prostaglandin specific. A very similar pattern of inhibitory effects in 1321N1 cells has been described for meclofenamic acid and PPP (Ortmann and Perkins, 1977). We suggest that the competitive part of the DPP inhibition of PGEt effects represents the specific interaction with the prostaglandin receptor of adenylate cyclase, while the insurmountable part, seen mainly in the 1321N1 cells, reflects the binding of DPP to non-receptor binding sites of the membrane. Binding studies with labelled prostaglandins are necessary to fully elucidate the mechanism of inhibitory action of DPP. Carbachol was reported to inhibit PGE1, isoproterenol and adenosine stimulated cAMP accumulation in 1321N1 cells (Gross and Clark, 1977) and PGE 1 and adenosine effects in neuroblastoma cells (Matsuzawa

R. Ortmann et al. : Phosphorylated Phloretin Derivatives Inhibit cAMP Accumulation and Nierenberg, 1975). This effect was calcium dependent in 1321 N1 cells and correlated with an increase in c G M P in both cell lines. In contrast D P P inhibition o f P G E 1 stimulation of adenylate cyclase is not calcium dependent (see results) and not accompanied by a change of c G M P levels in 1321N1 cells (Ortmann, unpublished results). Together with the observation that the local anaesthetic lidocain (100 gM) does not inhibit the c A M P accumulation stimulated by isoproterenol (Gross and Clark, 1977) and does not influence the inhibitory effect of D P P on P G E 1 stimulation in 1321N1 cells (Ortmann, unpublished observation), it seems likely that ion fluxes across the membrane are not a crucial event during the stimulation of adenylate cyclase or its inhibition by DPP. This is supported by the finding of Skolnick and Daly (1975) that tetracain (50 gM) did not prevent the increase of c A M P levels in rat cerebral cortex elicited by methoxamine. Tetracain did however, prevent the accumulation of c A M P stimulated by depolarizing agents (Shimizu et al., 1973). The absence of calcium does not influence the stimulation o f c A M P accumulation by P G E 1 in both cell lines. F o r other cell lines decreased PGE1 effects on c A M P accumulation in the absence of calcium have been reported (Brand et al., 1977). In guinea pig cerebral cortex slices however, omission of calcium did increase the accumulation of c A M P elicited by various agonists (Schultz and Kleefeld, 1975). Omission of calcium from the incubation medium elicites therefore different effects on adenylate cyclase systems, representing an interesting property o f different regulatory mechanisms of cyclic nucleotide levels. Eakins et al. (1971) compared the inhibitory effects of PPP with other phosphorylated polymers of related compounds. They concluded that in the isolated jird colon the phloretin moiety was essential for the inhibitory action. Our results show (Table 4) that replacement of the 2 phloretin molecules of D P P by phenyt groups abolishes all inhibitory potency (Diphenyl phosphate, Nr. 1263 Leo). Substitution ofphloretin by structurally more related molecules (3.5 dimethylphenylester, dihydrochalcone) generated different results in the 2 cell lines: While there was no difference in potency of these differently substituted compounds in 1321N1 cells, D P P was the most potent antagonist of P G E x in N 4 T G 3 cells. Beside the different kinetic results (Figs. 4 and 5), this difference in the structureactivity-relationship of D P P analogues observed in the 2 cell lines suggests that phosphorylated derivatives of phloretin do not interact with the same receptor site in N 4 T G 3 and 1321N1 cells. Phloretin inhibits the stimulation of c A M P accumulation by all three agonists to a similar degree, being only 3 - 5 times less potent than PPP. An interesting

239

model for the inhibitory effects of phloretin on transport processes across biological membranes and lipid bilayers was proposed by Andersen et al. (1976). They suggested that uncharged phloretin is able to reduce an existing positive potential difference between the membrane interior and the adjacent aqueous phase due to its large dipole moment. A detailed study of the inhibitory effect of phloretin (Ortmann, in preparation) on the adenylate cyclase system described here, suggests that this type of inhibitory action of phloretin is caused by a different mechanism. So far it is uncertain by which mechanism the phophorylation of phloretin brings about such dramatic changes in affinity and prostagtandin specificity of phloretin, MPP and DPP.

Acknowledgement. This work was supported by the Deutsche Forschungsgemeinschaft(SFB 70). We thank Leo AG, Helsingborg, Sweden for the phosphorylated derivatives of phloretin and the Upjohn company for a sample of PGE~. References Amano, T., Hamprecht, B., Kemper, W. : High activity of choline acetyltransferase induced in neuroblastoma x glia hybrid cells. Exp. Cell Res. 85, 399-408 (1974) Andersen, O. S., Finkelstein, A., Katz, J., Cass, A.: Effect of phloretin on the permeability of thin lipid membranes. J. Gen. Physiol. 67, 749- 771 (1976) Beitch, B. R., Eakins, K. E. : The effects of prostaglandins on the intraocular pressure of the rabbit. Br. J. Pharmacol. 37, 158167 (1969) Brand, M., Traber, J., Buchen, C., Hamprecht, B. : 6th Meeting of the intern, society for neurochemistry, Kobenhagen, p. 438 (Abstract) 1977 Burka, J. F., Eyre, P. : Studies of prostaglandins and prostaglandin antagonists on bovine pulmonary vein in vitro. Prostaglandins 6, 333 - 343 (1974) Cailla, H., Delaage, M. : Succinylderivativesof adenosine-3'Y-cyclic monophosphate: Synthesis and purification. Anal. Biochem.48, 62- 72 (1972) Clark, R. B., Su, Y. F., Ortmann, R., Cubeddu, L. X., Johnson, G. L., Perkins, J. P. : Factors influencing the effect of hormones on the accumulation of cyclic AMP in cultured human astrocytoma cells. Metabolism 24, 343-358 (1975) Collier, H. O. J., Roy, A. C.: Morphine-like drugs inhibit the stimulation by E prostaglandins of cAMP formation by rat brain homogenate. Nature 248, 24- 27 (1974) Diczfalusy, E., Fern6, O., Fex, H., H6gberg, B., Linderot, T., Rosenberg, Th. : Synthetic high molecular weight enzyme inhibitors. I. Polymeric phosphates of phloretin and related compounds. Acta Chem. Scand. 7, 913-920 (1953) Eakins, K. E., Miller, J. D., Karim, S. M. M.: The nature of the prostaglandin blocking activity of polyphloretin phosphate. J. Pharmacol. Exp. Ther. 176, 441-447 (1971) Eakins, K. E., Fex. H., Fredholm, B., H6gberg, B., Veige, S. : On the prostaglandin inhibitory action of polyphloretin phosphate. Adv. Biosci. 9, 135-138 (1973) Flower, J. R.: Drugs which inhibit prostaglandin biosynthesis. Pharmacol. Rev. 26, 33-67 (1974) Gross, R. A, Clark, R. B.: Regulation of adenosine-3'5"monophosphate content in human astrocytoma cells by isoproterenol and carbachol. Mol. Pharmacol. 13, 242-250 (1977)

240 Hunter, W. M., Greenwood, F. C.: Preparation of Iodine-131labeled human growth hormone of high specific activity. Nature 194, 495-496 (1962) Ishizawa, M., Miyazaki, E. : Inhibitory actions of polyphloretin phosphate and related compounds on the response to prostaglandin in the smooth muscle of guinea pig stomach. Prostaglandins 11, 829-840 (1976) Kuehl, F. A. : The regulatory role of the prostaglandins on the cAMP system. Adv. Biosci. 9, 155-172 (1973) Kuehl, F. A., Humes, J. L., Mandel, L. R., Cirillo, V. J., Zanetti, M. E., Ham, E. A. : prostaglandin antagonists: studies on the mode of action of polyphloretin phosphate. Biochem. Biophys. Res. Commun. 44, 1464-1470 (1971) Marrazzi, M. A., Matschinsky, F. M.: Properties of 15 hydroxy prostaglandin dehydrogenase: structural requirements for substrate binding. Prostaglandins 1, 373-387 (1972) Matsuzawa, H., Nierenberg, M. : Receptor mediated shifts in cGMP and cAMP levels in neuroblastoma cells. Proc. Natl. Acad. Sci. U.S.A. 72, 3472-3476 (1975) Ortmann, R. : Non steroid antiinflammatory drugs inhibit the effects of prostaglandins on the adenylate cyclase of astrocytoma cells. Naunyn-Schmiedeberg's Arch. Pharmacol. 294, R 5 (Abstract) 1976 Ortmann, R. : Effect of PGI2 and stable endoperoxide analogues on cyclic nucleotide levels in clonal cell lines of CNS origin. FEBS Letters 90, 348- 352 (1978) Ortmann, R., Perkins, J. P.: Stimulation of cAMP formation by prostaglandins in human astrocytoma cells: Inhibition by nonsteroidal antiinflammatory agents. J. Biol. Chem. 252, 60186025 (1977) Pont6n, J., Mc Intyre, E. H. : Long term culture of normal and neoplastic human glia. Acta Path. Microbiol. Scand. 74, 4 6 5 486 (1968) Sanner, J. J. : Substances that inhibit the action of prostaglandins. Arch. Int. Med. 133, 133-146 (1974)

Naunyn-Schmiedeberg's Arch. Pharmacol. 305 (1978) Sato, S., Kowalski, K., Burke, G. : Effects of a prostaglandinantagonist, polyphloretin phosphate, on basal and stimulated thyroid function. Prostaglandins 1,345-363 (1972) Shimizu, H., Takenoshita, M., Huang, M., Daly, J. W.: Accumulation of adenosine-3'5'-monophosphate in brain slices: Interaction of local anesthetics and depolarizing agents. J. Neurochem. 20, 91-95 (1973) Schultz, J., Hamprecht, B.: Adenosine-3'5'-monophosphate in cultured neuroblastoma cells: effect of adenosine, phosphodiesterase inhibitors and benzazepines Naunyn-Schmiedeberg's Arch. Pharmacol. 278, 215-225 (1973) Schultz, J., Kleefeld, G.: Stimulation of adenosine-Y5'-monophosphate formation in guinea pig cerebral cortex slices in a calcium free medium. Naunyn-Schmiedeberg's Arch. Pharmacol. 287, 289~ 296 (1975) Skolnick, P., Daly, J. W. : Stimulation of adenosineY5'-monophosphate formation in rat cerebral cortex slices by methoxamine: Interaction with an alpha adrenergic receptor. J. Pharmacol. Exp. Ther. 193, 549-558 (1975) . ~ Steiner, A. L., Parker, C. W., Kipnis, D. M. : Radioimmunoassay for cyclic nucleotides. (I). Preparation of antibodies and iodinated cyclic nucleotides. J. Biol. Chem. 247, 1106 - 1113 (1972) Su, Y.-F., Johnson, G. L., Cubeddu, L. X., Leichtling, B. H., Ortmann, R., Perkins, J. P.: Regulation of adenosine-3'5'monophosphate content of human astrocytoma cells: Mechanism of agonist specific desensitization. J. Cycl. Nucl. Res. 2, 271-285 (1976) Umbreit, W. W., Borris, R. H., Stauffer, J. F.: Manometric techniques, 4th ed., p. 132. Minneapolis: Burgess Publ. Co. 1964 Weinryb, I.: Protein binding assays for cyclic AMP: Radioimmunoassay and cyclic AMP dependent protein kinase binding assay. In: Methods in cyclic nucleotide research (M. Chasin ed.), chapter 2. New York: Marcel Dekker, Inc. 1972

Received April 17/Accepted October I1, 1978