273

Biochimica et Biophysica Acta, 544 (1978) 2 7 3 - - 2 8 3 © Elsevier/North-Holland Biomedical Press

BBA 28718

MODULATION OF ADENYLATE CYCLASE ACTIVITY BY SULFATED GLYCOSAMINOGLYCANS II. EFFECTS OF MUCOPOLYSACCHARIDES AND DEXTRAN SULFATE ON THE ACTIVITY OF ADENYLATE CYCLASE DERIVED FROM VARIOUS TISSUES

A B R A H A M A M S T E R D A M a, A V I N O A M R E C H E S b, Y E H U D I T H A M I R a, Y A E L M I N T Z a and Y O R A M S A L O M O N a

a Department o f Hormone Research, The Weizmann Institute of Science, Rehovot and b Department of Neurology, Hadassah University Hospital, Jerusalem (Israel) (Received April 21st, 1978)

Summary Heparin was found to be the most potent inhibitor of rat ovarian luteinizing hormone-sensitive adenylate cyclase (I50 = 2 pg/ml) when compared to other naturally occurring glycosaminoglycans. This inhibition was also apparent when this enzyme was stimulated by follicle-stimulating hormone or prostaglandin E2. Heparin was also found to inhibit glucagon-sensitive rat hepatic adenylate cyclase, and the prostaglandin El-sensitive enzyme from rat ileum and human platelets. In contrast, heparin stimulated the dopamine sensitive adenylate cyclase from rat caudate nucleus. The sulfated polysugar dextran sulfate exerts similar effec£s on adenylate cyclase activity of the rat ovary and was shown to inhibit hormone binding to rat ovarian plasma membrane in a manner similar to that exerted by heparin. In contrast to heparin, dextran sulfate inhibited dopamine-sensitive adenylate cyclase from rat caudate nucleus.

Introduction Recent reports indicate that various sulfated glycosaminoglycans are widely distributed in tissues of vertebrates and invertebrates [1,2]. The pattern of the sulfated glycosaminoglycan component in transformed hepatoma cells was found to be strain-specific and different from that found in normal hepatoAbbreviations: hCG, human chorionic gonadotropin; FSH, follicle stimulating hormone; LH, luteinizing hormone; poly(Glu), poly(glutamic acid); TSH, thyroid stimulating hormone; EGTA, ethyleneglycol bis-

(~-aminoethylether)-N-Nt-tetraacetic acid.

274 cytes [3]. Moreover, sulfated glycosaminoglycans such as heparan sulfate have been implicated in important cell functions such as cell aggregation, contact inhibition and cell division [2,4,5]. We have previously characterized the inhibitory effects of heparin on the ovarian LH-sensitive adenylate cyclase [6,7]. Moreover, it was shown that heparin interferes with the binding of the gonadotrophic hormone to cell specific receptors. Ovarian cells also contain adenylate cyclase stimulated by FSH and by prostaglandin E2 [ 8]. Itwas therefore of interest to examine whether adenylate cyclase stimulated by these hormones would also be suppressed by heparin. In addition we wished to compare the action of heparin to that of other glycosaminoglycans on ovarian adenylate cyclase. This study was extended to other adenylate cyclase systems of those present in liver, brain, ileum mucosa and platelets. As will be demonstrated, heparin inhibited -- though to different extents -- the glucagon-sensitive adenylate cyclase from rat liver, the prostaglandin El-sensitive adenylate cyclase from human platelets, and that of rat ileum mucosa. In contrast, it was found that heparin stimulated the dopaminesensitive adenylate cyclase from rat caudate nucleus. The possibility that heparin and other glycosaminoglycans exert some of their biological effects by modulating adenylate cyclase activity in the respective tissues is discussed. Materials and Methods Materials Highly purified heparan sulfate and highly purified dermatan sulfate were kindly supplied by Dr. M.B. Mathews, University of Chicago, under a contract (No. 1-AM-5-2205) from the U.S. National Institutes of Health (N.I.H.) (NIAMD). Dopamine (3,4-dihydroxyphenethylamine) dextran sulfate (M~ approx. 500 000) and hyaluronic acid grade III-s and theophilline were from Sigma Chemical Corp. Chondroitin 4-sulfate and chondroitin 6-sulfate were super-special grade and obtained from Miles Laboratories Inc., Kankakee, Ill. Polyglutamic acid (poly(Glu); M, approx. 10 000} was a gift from Dr. N. Lotan, Weizmann Institute of Science. Prostaglandin E I was kindly supplied by the Upjohn Company, Kalamazoo, Mich., U.S.A. Crystalline porcine glucagon was obtained from Eli Lilly and Co. Rat FSH-I-3 was kindly made available by the NIAMD, N.I.H. All other materials were as described previously [6]. Animals Wistar derived rats were used as previously described [6]. Tissue preparations Tissue homogenates or subcellular fraction were prepared as described below: Rat ovarian plasma membranes were prepared as previously described [9]. Rat liver plasma membranes were prepared as described by Pohl et al. [10]. Rat caudate nucleus. Male rats weighing 80--100 g were killed by decapitation. Caudate nuclei were removed and homogenized using a modification of the method described by Clement-Cormier et al. [11] in 50 vols. (w/v) of 2 mM

275

Tris-maleate buffer, pH 7.5, containing 2 mM EGTA. Rat ileum mucosal cell homogenates. Segments of rat ileum were inverted on a glass pipette which was rotated mechanically [12]. Epithelial cells were obtained by gently applying the rotating pipette onto the wall of a test-tube containing cold 150 mM NaC1 (Elchanati, E., personal communication). The resulting cell mass was gently homogenized and centrifugated at 1000 × g for 20 min. The supernatant was collected. The pellet containing mucus and cell debris was resuspended and centrifuged as described above. The resulting supernatant was pooled with the first one, and centrifuged at 40 000 × g for 20 min. The resulting pellet was suspended in 10 mM Tris-maleate buffer, pH 7.5, containing 1 mM dithiothreitol. Small samples of the suspension were frozen in liquid nitrogen and thawed only once shortly before use. Human platelet homogenates. Lysates of human platelets were prepared by a modification of the method described by Jakobs, K.H. et al. [13]. Blood was drawn from healthy volunteers who had not taken any drug for at least one week prior to blood collection. Blood was taken with citrate/phosphate/dextrose solution as an anticoagulant. The platelet pellet was washed and resuspended with 150 mM NaC1/10 mM Tris-maleate, pH 7.5. The suspension in small samples was frozen in liquid nitrogen and thawed only once, shortly before use. Adenylate cyclase assays. Adenylate cyclase activity was determined by measuring the conversion of [~-32P]ATP to cyclic [32p/AMP. Incubation conditions for the various tissues tested are listed below; termination of the reaction and isolation of cyclic [32P/AMP were performed according to Salomon et al. [14]. Rat ovary. Incubation conditions were as described previously [6]. Rat liver. Incubation conditions were as described by Londos et al. [15]. Rat caudate nucleus. The standard assay mixture (final volume 50 ~l) contained 25 mM Tris-maleate, pH 7.5, 2 mM magnesium acetate, 0.25 mM [~3~P]ATP (2--4 • 106 cpm), 0.2 mM EGTA, I mM dithiothreitol, 10 mM theophylline, 5.0 mM creatine phosphate, 50 units/ml creatine phosphokinase and test substance as indicated. The reaction was initiated by the addition of 3--8 pg protein of the caudate nuclei homogenate. Incubation was at 30°C for 5--10 min. Rat ileum mucosa. The assay mixture was identical with that described for rat caudate nucleus (above), except that GTP (10 pM) was included. The amount of tissue homogenate was 20--25 ~g protein per assay tube and incubation time was 20 min. Human platelets. The assay mixture (final volume 50 ul) contained 25 mM Tris-maleate, pH 7.5, 5 mM magnesium acetate, 0.5 mM [~-32P]ATP (2--4. 106 cpm), 1 mM dithiothreitol, 10 mM theophylline, 50 pM GTP, 5.0 mM creatine phosphate, 50 units/ml creatine phosphokinase, and the test substance as indicated. The reaction was initiated by the addition of 5--10 #g protein of platelet membrane preparation. Incubation was at 30°C for 10 min. Results

The effect of heparin on ovarian adenylate cyclase stimulated by FSH or prostaglandin E2 In the previous reports [6,7] it was shown that heparin inhibits LH and hCG

276

TABLE

I

INHIBITION

OF HORMONE-STIMULATED

RAT

OVARIN

ADENYLATE

CYCLASE

BY HEPARIN

Adenylate e y c l a s e a c t i v i t y i n p u r i f i e d rat o v a r i a n p l a s m a m e m b r a n e s w a s d e t e r m i n e d in t h e a b s e n c e o r p r e s e n c e o f h e p a x i n at t h e c o n c e n t r a t i o n indicated. The amounts of membrane protein (/~g/assay) were 4 . 2 a n d 3 , r e s p e c t i v e l y . All o t h e r d e t a i l s w e r e as d e s c r i b e d i n M e t h o d s . Cyclic AMP formed (pmol/15

Exp. 1

Basal FSH (0.01

Exp. 2

pM)

Basal L H ( 0 . 1 /~M) Prostaglandin E 2 (20 /zg/ml)

rain per mg protein)

No heparin

Hepaxin *

543 3136

261 1166

472 1885 1055

165 119 270

* 8 ~ g / m l E x p t . 1; 1 0 ~ g / m l E x p t . 2.

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P L.A-rEi_E7 S

c

~.

7 ....

-T ......

I

CAUDATE

$1500

NUCLEUS

I

/

°2

-i2500

DOPAMINE IOO/~M

__._._.._~___A-F-i000 E o

-~ t . . . . . . . - T

--4M-----%

B ;2000 & o~

E i500

B E

o~

r'J

c

I000 Z 500 -

•

--o--------//-----o

~

E 5O0

100 >100 >100 4 >100

2 16 n.d. >100 >100 >100 6 >100

(21%) (28%) (22%) (24%)

(10%) (27%) (27%) (10%)

8 >100 >100 >100 >100 >100 22 >I00

(12%) (26%) (7%) (+5%) (+10%) (+6%)

n.d., not determined.

the most effective inhibitors among the polyanions tested. Heparan sulfate and dermatan sulfate exerted comparable inhibitory effects at a much higher (tenfold} concentration. The sulfated mucopolysaccharides chondroitin 4-sul-

% sol-

£ '~ 2 0 0 0 ] -

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7-;

1

j

25

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4

5

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