Effect of forskolin on alterations of vascular permeability induced with bradykinin, prostaglandin E1, adenosine, histamine and carrageenin in rats

Life Sciences, Vol. 33, pp. 65-73 Printed in the U.S.A.

EFFECT

OF

Pergamon Press

FORSKOLIN ON ALTERATIONS OF VASCULAR PERMEABILITY INDUCED BRADYKININ, PROSTAGLANDIN E_, ADENOSINE, HISTAMINE AND

WITH

CARRAGEEN~N IN RATS Kazuo Sugio and John W. Daly

National

Laboratory of Bioorganic Chemistry, Bldg. 4, Room 212, Institute of Arthritis, Diabetes, and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20205 U.S.A. (Received in final form April 20, 1983)

Sugary The effect of the diterpene forskolin on vascular permeability alone and in combination with bradykinin, prostaglandin E~, adenosine or I histamine has been investigated in rats. Vascular permeability in rat skin was measured using [1251]-labelled bovine serum albumin ([1251]BSA) as a tracer. In addition, the effect of forskolin on footpad edema induced by the injection of a mixture of 2% carrageenin was determined. Forskolin caused a marked potentiation of the increase in vascular permeability in rat skin elicited by the intradermal injection of histamine or bradykinin. However, forskolin caused a significant suppression of the prostaglandin El-induced vascular permeability response and at a low concentration suppressed the response to adenosine. Forskolin greatly potentiated the footpad edema induced with carrageenin in rats. Intravenous administration of the enzyme bromelain, which reduces plasma kininogen levels, inhibited the footpad edema induced with carrageenin or with a mixture of carrageenin and forskolin. Parenteral administration of a prostaglandin synthetase inhibitor, indomethacin, suppressed the footpad edema induced with earrageenin, but did not inhibit the footpad edema induced with a mixture of carrageenin and forskolin. An antihistamine, cyproheptadine, had no effect on carrageenininduced footpad edema either in the presence or absence of forskolin. These results suggest that both bradykinin and prostaglandins are essential for the development of carrageenin-induced footpad edema and that bradykinin plays an important role in the potentiative effect of forskolin on footpad edema induced with carrageenin in rats. Chemical mediators such as the prostaglandins, bradykinin and histamine appear to play important roles in the development of the rat carrageenininduced footpad edema, used widely as a model for acute inflammation (i). Such mediators have been divided into two classes, both of which increase plasma exudates (vascular permeability) in acute inflammation (2). The first class includes the prostaglandin E-group, while the second class includes bradykinin and histamine. The mechanism whereby prostaglandins increase vascular permeability is as yet unclear, but might involve activation of adenylate cyclase, since E-class prostaglandins do activate adenylate cyclase in many cell types (3,4). The second class of agents, including bradykinin and histamine, have been proposed to increase vascular permeability through formation of intracellular gaps between vascular endothelial cells (5,6). 0024-3205/83

$3.00 + .00

66

Forskolin

and Phlogistic

Agents

Vol.

33, No.

I, 1983

C i a s s one c o m p o u n d s , i n p a r t i c u l a r prostaglandins of the E-class, markedly potentiate the increase in vascular permeability eiicited by a g e n t s o f c l a s s two ( 2 ) . The d i t e r p e n e forskoiin has recentIy b e e n shown t o be a s p e c i f i c activator of adenylate cyciases in intact cells of virtualiy all tissues (7). The e f f e c t of this specific a g e n t on v a s c u l a r p e r m e a b i i i t y was a s s e s s e d . Fors k o l i n was f o u n d t o i n c r e a s e t h e v a s c u l a r p e r m e a b i i i t y responses induced with bradykinin and h i s t a m i n e , but to suppress the plasma exudation induced with prostaglandin E1 . F o r s k o l i n , therefore, provided a tool to re-examine the role of chemical mediators in the carrageenin-induced inflanmlation. Forskolint r e a t m e n t was f o u n d t o i n c r e a s e t h e i a t e s t a g e s o f c a r r a g e e n i n - i n d u c e d footpad edema. This effect o f f o r s k o I i n was b l o c k e d i f k i n i n o g e n l e v e l s w e r e r e d u c e d w i t h t h e enzyme b r o m e l a i n , b u t was s t i l l manifest if prostaglandin synthesis was i n h i b i t e d with indomethacin or if histamine-responses were b l o c k e d w i t h cyproheptadine. Materials

and Methods

Animals Male rats of S p r a g u e - D a w l e y throughout the experiments. All tap water ad libitum.

strain (body weight, i80-200 g) were used rats were maintained on laboratory food and

Materials

Carrageenin (lammda c a r r a g e e n i n , Sigma C o . , St. Louis, MO), [1251]labelled bovine serum aibumin ([125I]BSA, specific activity, 60 ~ C i / m m o l , New E n g l a n d N u c l e a r C o . , B o s t o n , HA), b r o m e l a i n (Sigma C o . , S t . L o u i s , MO), i n d o methacin (Sigma C o . , S t . L o u i s , NO), f o r s k o l i n ( C a l b i o c h e m - B e h r i n g C o . , La Jolla, CA), c y p r o h e p t a d i n e (Merck S h a r p Dohme, W e s t p o i n t , PA) w e r e u s e d . 7-Desacetyl forskolin, forsko]in 7-ethyl carbonate, i4,]5-dihydroforskolin and 1,9-dideoxyforskolin w e r e k i n d l y p r o v i d e d by Dr. J . Reden, Hoechst Pharmaceuticais L t d . , Bombay. Carrageenin-induced

footpad

edema

The f o o t p a d edema was i n d u c e d by t h e i n j e c t i o n o f 0 . 1 ml e a c h o f c a r r a g e e n i n ( r i g h t r e a r f o o t ) and a m i x t u r e o f c a r r a g e e n i n p l u s f o r s k o l i n (left rear foot) into the subpiantar space of the rat feet uniess otherwise noted. Forskolin was d i s s o l v e d in absolute ethanol a n d a d d e d t o 2 mi o f c a r r a g e e n i n solution so t h a t 0 . 1 ml o f f i n a l solution c o n t a i n e d 100 Ng f o r s k o l i n and 5% e t h a n o l a n d 2% c a r r a g e e n i n . F o o t p a d edema was m e a s u r e d u s i n g a v o l u m e m e t e r . The f o o t p a d edema was c a l c u l a t e d by m e a s u r i n g t h e d i f f e r e n c e s in the footpad volumes before injection and a p p r o p r i a t e times after injection of carrageenin. Induction

and measurement

of vascular

permeability

response

in rat skin

Vascular permeability r e s p o n s e s w e r e i n d u c e d on s h a v e d d o r s a l s k i n o f t h e r a t s by t h e i n t r a d e r m a l injection of phlogistic agents (8), Vascular permeability was m e a s u r e d u s i n g [ 1 2 5 I ] - l a b e l l e d bovine serum albumin ([125I]BSA) as a tracer. The [12SI]BSA was p u r i f i e d i n o r d e r t o remove r a d i o a c t i v e low m o l e c u l a r impurities (9,10). In brief, about 1 ml of commercial [1251]BSA in 0.9~ NaCI solution was loaded on a Sephadex G-IO0 column (20 x 300 mm) and eluted with 0.9% NaCI solution at a rate of 15-20 ml/hr. A fraction of the eluate corresponding to the elution volume of bovine serum albumin was collected. About I NCi of this purified [125I]BSA in 0.2 ml of 0.9% NaCI solution containing 1% Evans blue was injected into the tail vein of rats. Immediately after the injection of tracer, phlogistic agents (0.I ml) were injected intradermally on the back of the rat using ether anesthesia. Rats were then killed

Vol. 33, No. i, 1983

Forskolin and Phlogistic Agents

67

after 15 min by cutting the carotid arteries and the dorsal skin was peeled off and separated from adherent subcutaneous tissues. The blue area (20 mm diameter) of the rat skin at each site of injection with phlogistic agents was carefully cut as a sample. The radioactivity of [1251] BSA of each skin sample was measured in a gamma well-type scintillation counter (Beckman gamma 9000). Exudation of [1251]BSA was calculated by subtracting radioactivities of the control skin site injected with Tyrode's solution from that of the rat skin sites injected with phlogistic agents and was expressed in terms of percent of total radioactivity injected into each rat. Five separate injections were done on the back of each rat allowing control and dose response measurements to be done in a single animal. Drug treatment of animals Bromelain was dissolved in 0.9% NaCI solution and injected (3 or i0 mg/kg) into the tail vein of the rats i hr before the administration of a mixture of carrageenin and forskolin. Indomethacin or cyproheptadine were administered orally in sesame oil or water, respectively, 1 hr before the injection of a mixture of carrageenin and forskolin. The significance of the differences in the results between the treated and control groups was determined according to the Student t-test. Results Effect of forskolin on alterations of vascular responses induced with bradykinin, histamine, prostaglandin E 1 and adenosine in rat skin The effect of forskolin on plasma exudation induced with intradermal injection of bradykinin, prostaglandin El, histamine and adenosine in rat skin was investigated (Table I). Bradykinin (~ Ng), histamine (i ~g), prostaglandin EI(I Ng), and adenosine (I00 ~g) markedly increased plasma exudation (vascular permeability) compared with relatively small effects of forskolin (I0 Ng). However, increasing doses of forskolin from 0.1 to i0 Hg to a fixed dose of histamine (i Ng) or bradykinin (i Ng) greatly potentiated plasma exudation. The potentiation had not maximized even at i0 ~g of forskolin. In contrast, forskolin significantly inhibited the plasma exudation induced with a fixed dose of prostaglandin E (i Hg)The inhibition was maximal at the lowest tested concentration (0~i ~g) of forskolin. Forskolin at 0.i ~g markedly inhibited the plasma exudation elicited by a fixed dose of adenosine (i Ng)At i and i0 pg forskolin the inhibition appeared to reverse, perhaps due to the direct effects of forskolin on vascular permeability, which would obscure any inhibition of adenosine-effects. The potentiation of bradykinin-elicited increases in vascular permeability by forskolin analogs correlated with their ability to activate adenylate cyclase. Forskolin, forskolin 7-ethyl carbonate and 7-desacetylforskolin, all active with respect to stimulation of adenylate cyclase (ii), caused a marked potentiation of bradykinin-response (418% for forskolin, 287% for 7-ethyl carbonate, 286% for 7-desacetylforskolin, i0 Dg injected subdermally). 14,15-Dihydroforskolin and 1,9-dideoxyforskolin, which are much less active than forskolin and inactive, respectively as activators of adenylate cyclase (II), did not significantly potentiate the bradykininresponse. Similarly, only forskolin, forskolin 7-ethyl carbonate, and 7-desacetylforskolin significantly inhibited the prostaglandin-elicited increase in vascular permeability (81, 88 and 95% inhibition, 0.I Pg injected subdermally). Time course of the footpad edema induced with carrageenin or a mixture of carrageenin and forskolin A time course study was performed after injection of carrageenin or a mixture of carrageenin and forskolin. Measurement of the footpad volume was

68

Forskolin and Phlogistic Agents

Vol. 33, No. i, 1983

TABLE I Effect of Forskolin on the Increase in Vascular Permeability Elicited by Bradykinin, Histamine, Adenosine and Prostaglandin E 1 in Rat Skin

Plasma exudation Effect of Forskolin (% of [1251]BSA injected) (% change)

Phlogistic agents

Group i. Forskolin Forskolin Forskolin

(n=7) (0.i ~g) (1.0 ~g) (i0 ~g)

0.006 + 0.003 0.009 ¥ 0.004 0.050 $ 0.009

Group 2. (n=7) Bradykinin (I ~g) BK (I ~g) + FK (0.i pg) BK (I Ng) + FK (I.0 Ng) BE (l ~g) + FK (i0 ~g)

0.132 0.310 0.467 0.552

+ ¥ $ $

0.023 0.038 0.047 0.047

100 234 353 418

p<0.Ol p<0.01 p<0.01

Group 3. (n=7) Histamine (i Ng) Hist (i ~g) + FK (0.i Ng) Hist (i Ng) + FK (I.0 pg) Hist (I ~g) + FK (I0 ~g)

0.166 0.282 0.460 1.032

+ $ $ $

0.026 0.036 0.067 0.077

I00 169 277 622

p
Group 4. (n=7) Prostaglandin E1 PGE1 (1 pg) + F~ PGE~ (1 Ng) + FK PGE~ (1 Ng) + FK

0.110 0.021 0.035 0.028

+ $ $ $

0.023 0.009 0.009 0.008

100 19 32 25

p<0.O1 p<0.O1 p
Group 5. (n=7) Adenosine (1Ng) Ado ( 1 N g ) + FK ( 0 . 1 ~g) Ado (1 Ng) + FK ( 1 . 0 Ng) Ado (1 ~g) + FK (10 ~g)

0.048 0.013 0.053 0.089

+ $ $ $

0.013 0.009 0.018 0.012

100 27 110 185

p<0.05 N.S. p<0.05

Group 6. (n=6) A d e n o s i n e (100 Ado (100 Ng) + Ado (100 ~g) + Ado (100 ~g) +

0.270 0.171 0.232 0.304

+ + + $

0.068 0.050 0.061 0.055

100 63 85 111

N.S. N.S. N.S.

(1 Ng) (0.1Ng) ( 1 . 0 Ng) (10 ~g)

Ng) FK ( 0 . 1 N g ) FK ( 1 . 0 ~g) FK (10 ~g)

Forskolin (FK) and prostaglandin El (PGEI) were dissolved in ethanol and diluted with Tyrode's solution to 0~5% (v/~) ethanol. Adenosine (Ado), histamine (Hist) and bradykinin (BK) were dissolved in Tyrode's solution. These phlogistic agents were injected intradermally. Plasma exudation into skin patches were measured as described in Methods. Values are means + S.E.M. N.S. = not significant, p>O.l.

Vol. 33, No. i, 1983

Forskolin and Phlogistic Agents

0.9

oCarr.+

69

FK,lOOpg

0.'/'

o5

=

0.3,

! ~

o.i i Time after

~ Ihe

~ injection

~

~h,

of cerrageenin

FIG. 1 Time Course of the Footpad Edema Induced with Carrageenin or a Mixture of Carrageenin and Forskolin. The footpad edema was induced by the injection of each 0.i ml of 2% carrageenin (right foot) and a mixture of carrageenin and forskolin (left foot) into the subplantar space of the rat hind feet. The footpad edema was measured immediately before and I, 2, 3, 4, and 5 hrs after the injection of phlogistic agents by using a volume meter. The vertical lines represent means + S.E.M. (n = 5). TABLE II Dose-Response Relationship for the Effect of Forskolin on Rat Carrageenin-Induced Footpad Edema in Rats

Treatment

No. of

Increase in footpad edema (ml)

rats

mean + S.E.M.

% of control

Carrageenin (control)

7

0.52 + 0.051

I00

Carr + Forskolin (i Ng)

7

0.63 + 0.030

121 (N.S.)

Carr + Forskolin (i0 ~g)

7

0.76 + 0.039

146 (p<0.01)

Carr + Forskolin (i00 Ng)

7

0.98 + 0.051

188 (p
The footpad edema was measured 2 hr after injection of carrageenin or a mixture of carrageenin (Cart) and forskolin. N.S. = not significant.

70

Forskolin and Phlogistic Agents

Vol. 33, No. i, 1983

done immediately before a n d 1, 2 , 3 , 4 a n d 5 h r a f t e r forskolin and carrageenin injection. Forskolin (100 ~g) by itself did not induce the footpad edema (data not shown). Forskolin also did not have any effect on f o o t p a d edema induced with carrageenin within 1 hr after treatment, but a potentiative effect of forskolin on c a r r a g e e n i n - i n d u c e d e d e m a was a p p a r e n t at 2 hr and persisted for 5 hr (Fig. 1). F o o t p a d e d e m a was d e t e r m i n e d at 2 hr in all of the following experiments with carrageenin. Dose-response relationship for the effect of forskolin on the footpad edema induced with carrageenin Forskolin at i ~g had no significant effect on carrageenin-induced footpad edema, while 10 Ng and i00 pg increased edema by 46% and 88%, respectively (Table If). Effect of bromelain, indomethacin and ~proheptadirle pretreatment on footpad edema induced with carra$eenin or a mixture of carrageenin and forskolin in order to define possible mechanisms involved in the potentiative effect of forskolin on carrageenin-induced edema, rats were treated with either i) the enzyme bromelain which reduces plasma kininogens; ii) indomethacin which inhibits prostaglandin synthetase; or iii) cyproheptadine, which blocks histamine and serotonin responses. Suda et al. (12) have reported that bromelain at rats reduces plasma kininogen b y a b o u t 60% f o r 6 h r s ment. Doses of bromelain o f 3 a n d 10 m g / k g w e r e u s e d ments. Bromelain a t 10 m g / k g c a u s e d t h e s i g n i f i c a n t edema induced either with carrageenin alone or with a and forskolin (Table III).

t h e d o s e o f 10 m g / k g i n after bromelain treatin the present experisuppression of footpad mixture of carrageenin

It has been shown that indomethacin at the doses of 1-10 mg/kg reduces the level of prostaglandin E in exudates from carrageenin-induced inflammation sites (13). Doses of indomethacin o f 3 a n d 10 m g / k g w e r e u s e d i n t h e p r e s e n t experiments. The footpad edema induced with carrageenin was s i g n i f i c a n t l y inhibited by administration o f 10 m g / k g o f i n d o m e t h a c i n (Table III). However, this dose of indomethacin h a d no s i g n i f i c a n t effect on t h e footpad edema provoked with a mixture of carrageenin and forskolin. The anti-histamine and anti-serotonin drug cyproheptadine has been reported to completely inhibit the vascular permeability responses induced with histamine and serotonin in rat skin (14). Doses of cyproheptadine of 3 a n d 10 m g / k g w e r e u s e d i n t h e p r e s e n t experiments. Cyproheptadine did not inhibit the footpad edema induced with either carrageenin a l o n e o r t h e combination of carrageenin and forskolin (Table III). Discussion Carrageenin-induced footpad edema in rats is widely used as a model for acute inflammation. Non-steroidal anti-inflammatory drugs such as aspirin, phenylbutazone and indomethacin have extensively been studied for their antiinflammatory activities in such a model (15). However, the precise mechanism for the development of carrageenin-induced footpad edema in rats remains to be elucidated. DiRosa et al. (I) suggested that the process of carrageenininduced footpad edema has at least two phases. Histamine and serotonin appear to play important roles in the first phase of edema induced with carrageenin, since this phase of edema is inhibited by the injection of anti-histamine and anti-serotonin drugs (I). Prostaglandins and bradykinin appear to play important roles in the late phase of edema, since this phase of the edema can be reduced by lowering plasma kininogen level or blocking prostaglandin synthesis (16).

(i0 mg/kg)

Cyproheptadine

6

6

6

7

6

7

6

6

6

i00 109 (N.S.) 91 (N.S.)

0.69 + 0.015 0.58 + 0.018

34 (p<0.01)

0.18 + 0.029

0.64 + 0.042

77 (N.S.)

0.42 + 0.088

i00

28 (p<0.01)

0.16 + 0.062

0.55 + 0.057

81 (N.S.)

I00

0.71 + 0.060

(ml)

0.89 + 0.033

0.99 + 0.054

0.92 + 0.026

0.77 ~ 0.044

0.83 + 0.074

0.82 + 0.045

0.17 + 0.032

0.58 + 0.061

0.99 + 0.075

Cart + FK (I00 pg)

in footpad volume

0.58 + 0.046

% of control

Carrageenin

Increase

in Rats

98 (N.S.)

108 (N.S.)

i00

94 (N.S.)

i01 (N.S.)

100

17 (p<0.01)

59 (p<0.01)

i00

% of control

The footpad edema was induced by injection of carrageenin or a mixture of carrageenin (Cart) and forskolin 1 hr after administration of bromelain, indomethacin or cyproheptadine and measured 2 hr later. Values represent means ± S.E.M.

(3 mg/kg)

Cyproheptadine

(vehicle)

(i0 mg/kg)

Indomethacin

Control

(3 mg/kg)

Indomethacin

(vehicle)

(i0 mg/kg)

Bromelain

Control

(3 mg/kg)

(vehicle)

rats

No. of

E f f e c t o f B r o m e l a i n , I n d o m e t h a c i n and C y p r o h e p t a d i n e on Edema I n d u c e d w i t h C a r r a g e e n i n o r a M i x t u r e o f C a r r a g e e n i n a n d F o r s k o l i n

Bromelain

Control

Pretreatment

Footpad

TABLE I I I

OQ

o 0Q ~.

=-

m m

o

o

ko oo Lo

o

72

Forskolin and Phlogistic Agents

Vol. 33, No. 1, 1983

The role of cyclic AMP in development of carrageenin-induced footpad edema is unclear. Although many of the physiological effects of prostaglandins are mediated by activation of adenylate cyclase (3), whether or not a prostaglandin-sensitive adenylate cyclase is involved in inflammatory processes is unclear (see ref. 4). Cyclic AMP administered parenterally inhibits both carrageenin-induced edema and increases in vascular permeability after thermal injury, bradykinin or histamine (17). However, it remains unclear whether this is a direct effect of cyclic AMP. Forskolin, being a general activator of adenylate cyclase (7) appeared a potentially useful tool for the investigation of possible involvement of cyclic AMP-systems in inflammation. Forskolin was found to potentiate plasma exudation elicited by class two mediators such as histamine and bradykinin (Table I). Adenosine and prostaglandins, substances well known to activate adenylate cyclase (3,18) also potentiate plasma exudation elicited by bradykinin or histamine (2,19). Forskolin analogs which do not activate adenylate cyclase did not potentiate bradykinin-elicited increases in vascular permeability (see Results). Although the data indicate that forskolin potentiates bradykinin-responses through activation of adenylate cyclase, whether this is a direct effect on endothelial cells or an indirect effect related to increased venular flow is unknown. Forskolin is a potent vasodilator (20). A remarkable effect was found when forskolin was used in combination with prostaglandin E 1 . Forskol~n at very low concentrations greatly inhibited the response to prostaglandin E.. Forskolin at very low concentrations also I inhibited tile response to adenosine. It is unclear why forskolin should inhibit responses elicited by agents which supposedly act via the same mechanism as forskolin, namely through activation of adenylate cyclase. Possible mechanisms to explain this anomaly remain, at present, obscure. Forskolin analogs which do not activate adenylate cyclase did not inhibit prostaglandin El-elicited increases in vascular permeability (see Results). The selective and indeed opposite effect of forskolin on responses to class two mediators of inflammation such as bradykinin and histamine and on responses to class one mediators such as prostag]andin g I (Table I), provide a tool for the investigation of the role of such mediators in carrageenininduced edema. Forskolin was found to potentiate carrageenin-induced edema during the period of 2-5 hr after treatment (Fig. l). The first phase of carrageenin-induced footpad edema is thought to involve histamine and serotonin (i). Since forskolin potentiates histamine-induced plasma exudation (Table I), it is surprising that forskolin has no potentiative effect on footpad edema during the initial phase (Fig. l). Bradykinin and prostaglandins have been proposed to be involved in the later phase of footpad edema, i.e., the phase which forskolin potentiates. Since forskolin potentiates bradykinin-induced plasma exudation and inhibits prostaglandin-induced plasma exudation (Table I), it would appear likely that the forskolin-bradykinin interaction is of major importance to the augmentation of carrageenin-induced footpad edema by forskolin. The effects of forskolin were now investigated in rats pretreated with either i) bromelain to reduce plasma levels of bradykinin precursors, ii) indomethacin to block synthesis of prostaglandins, or iii) cyproheptadine to block histamine and serotonin receptors. The kininogendepleting agent, bromelain, when administered into the rat tail vein inhibited the footpad edema induced with either carrageenin or a mixture of carrageenin and forskolin (Table III). Furthermore, in bromelain-treated rats, forskolin no l o n g e r augmented carrageenin-induced edema. In contrast, while indomethacin greatly reduced footpad edema i n d u c e d w i t h c a r r a g e e n i n alone, i t h a d no effect on e d e m a i n d u c e d w i t h a m i x t u r e of carrageenin and forskolin (Table III). Cyproheptadine h a d no e f f e c t on t h e f o o t p a d e de ma i n d u c e d w i t h c a r r a geenin alone or with the combination of carrageenin and forskolin. The results suggest that both prostaglandins and bradykinin have essential roles

Vol. 33, No. i, 1983

Forskolin and Phlogistic Agents

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for the development of carrageenin-induced footpad edema, and that bradykinin is obligatory to the potentive effect of forskolin on footpad edema-induced with carrageenin in rats. The data with indomethacin suggest that bradykinin is relatively ineffective in supporting edema in the absence of prostaglandin or forskolin. REFERENCES i. 2. 3. 4. 5. 6. 7. 8. 9. i0. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.

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