Agents and Actions, vol. 23, 1/2 (1988)

0065-4299/88/020103-03 $1.50+ 0.20/0 9 1988 Birkhauser Verlag, Basel

Oxygen free radical scavengers (OFRS) prevent vasodepression induced by anaphylaxis, dextran or u.v.-irradiation in mice and rats H. Bekemeier, F. Hansel and Chr. Kuhn Dept of Pharmacology, Section of Pharmacy, Martin-Luther-University Halle-Wittenberg, GDR-4050 Halle, German Democratic Republic

Introduction

If legs of animals with inflammation (dextran, antigen or adjuvant-induced) are isolated and perfused, the efficacy ofco-perfused or i.a. injected vasoconstrictors (norepinephrine (NE), vasopressin, angiotensin II, PGF2a, ATP-Na2, or substance P) is found to be reduced, i.e., vasodepression develops [1]. Injection into animals (i.v., s.c. etc.) of high doses of histamine, PAFacether and PGI2 can also induce vasodepression. Inflammation-induced vasodepression can partly be prevented or restored by prophylactic administration or co-perfusion of antihistaminics, salicylic acid, acetylsalicylic acid, PAF-antagonists or glucocorticoids whereas other nonsteroidal antiinflammatory drugs (indomethacin, diclofenac, phenylbutazone) are rather inactive in this respect. Here, we report on the protection by OFRS administration against antigen-, dextranor ultra violet light (u.v.)-induced vasodepression.

Method Ill

Outbred strains of female Wistar rats (110-170 g; Falcke, Barby) or male AB-mice (25-30 g; Berger, Langburkersdort) were used. Dextran paw edema: Mice were injected intraplantarly with 0.01 ml 3% dextran solution (MW75000) into each hind paw. 20 min later hindlegs were prepared as described elsewhere [1].

A naphylactic paw edema: Mice received 0.1 mg HSA together with 2.109 germs of Bordetella pertuss. in 0.02 ml of an aqueous suspension and 14 days later provocation was induced by injecting 0.05 mg HSA in 0.01 ml of an aqueous solution in both hindlegs. 30 min later hindlegs were prepared [1]. U.v.-erythema: The back of rats was clipped to an area of about 40 cm 2 and then, after 500 IU hepafin i.p., rats were irradiated with a commercial sun lamp Q 64 (VEB Thelta, Zella-Mehlis, 800 W) without any filter for 10 min at a distance of about 50 cm. 5 min later a hindleg was prepared [1]. Results and discussion

When mice are intraplantarly injected with dextran, vasodepression develops. At the maximum of vasodepression, 20-60 min after injection, the maximum pressure amplitude (EArn) of NE was only 78+7% in comparison to 100% in normal mice (Table). When animals were pretreated with the new lipoxygenase inhibitor and antioxidant M 516 (1-[2,5-Dihydroxybenzyliden-]aminoadamantane), vasodepression was dose-dependently prevented, so that, at the dose of 200 mg/kg, EArn was practically 100%. In mice with anaphylactic paw edema, in which EArn ranged from 56-79%, M516 was less effective, since EArn= 100% could not be attained by pretreatment. At the dose of 100 mg/kg, EArnwas

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Agents and Actions, voL 23, 1/2 (1988)

Table 1

Maximum pressure amplitudes (EArn %; mean_+ s.d.) of norepinephrine in the isolated perfused hindlegs of mice or rats with dextran-, HSA- or u.v.-induced vasodepression without or with pretreatment by OFRS and related substances. EArn in healthy, untreated animals ~ 100%. U.v.-irradiation

Anaphylactic paw edema Pretreatment mg/kg p.o.

EArn

25 37 50 100

M516

7.5 25 50

Hydroquinone

56_+ 8 61_+ 6 69_+ 5' 91+ 6' 84_+ 9 b 78+ 8 85 + 11 87_+ 12 78-+ 12

Pretreatment mg/kg p.o. (8) (7) (8) (7) (7) (10) (8) (7) (6)

M516

Hydroquinone

EArn

15 50 100 200 2.5 7.5 15

30 Mannitol

125"

66+ 6 73_+ 5

Ascorbic acid

50 100

76-+ 10 86_+ 12 74+ 10

r

(7) (7)

Mannitol

(12) (7) (6)

lhiourea

200

88_+10

(8)

5 10 15

61_+ 14 72-+ 14 92-+ 8 b 96-+ 9 b

(8) (6) (8) (6)

BW 755 c

Ethanol

M 516

78+ 7

50 100 200

91_+ 8' 95 +--13 99_+ 9 b

30

(8) (30) (8) (10) (12)

70_+ 9 100-+ 7 ~

(9) (9)

(26) (8) (10) (8) (27) (9) (10) (8)

86_+ 7

(21)

25 50 100

BW 755 c

10 15 25

94+ 4 b 94-+ 4 b 98_+ 6 b

(14)

(8) (7) (7)

80_+ 8'

NaHSO3

0.5"* 1,0"* 1.6"*

(32) (8) (8) (8) (8) (8) (8) (8) (8)

75_+ 8 80_+ 16 95_+ 9 b 97-+ 12 b

72-+ 8 74-+ 10 100-+ 11 ~ 93-+ 8 ~ 79-+ 17 90_+ 13 92-+21" 111 _+24b

Dextran paw edema -

62.5* 125" 250*

81_+ 6 95_+ 9 b 97_+ 9 c 84_+ 9 77+ 7 80-+ 6 78-+ 6 101 _+ 11 c 96-+ 10'

(7) (7) (7)

* i,p.; ** ml/kg; ( ) number of animals; a, b, c means significantly different from control group of each substance group by p < 0.05, 0.01, and 0.001, respect. (t-test)

only 84%, and at the dose of 200 mg/kg, it was 83% (data not shown in the Table). Accordingly, the antioxidant hydroquinone as a partial structure of M 516, administered 30 min prior to perfusion, gave only little protection in this model. Moreover, at higher doses, hydroquinone itself induced vasodepression as was the case for M 516. Furthermore, the hydroxyl radical ('OH) scavenger mannitol, given 20 min prior to perfusion at the relatively high dose of 125 mg/kg i.p. also only weakly protected from vasodepression. Ascorbic acid was apparently without effect. This holds true whether it was administered 30 (Table), 15 or 60 (data not shown in the Table)

min prior to perfusion. Co-administration of 50, 100, and 200 mg/kg p.o. of both M 516 and ascorbic acid 30 min prior to perfusion (simultaneously with provocation) yielded EArn of 78_+ 8, 85--+ 11, and 94+ 12%, respectively, whereas in the anaphylactic animals the value was 84___ 11% (n--7, 8, 8, and 12). This means that the action of M 516 was not essentially influenced by ascorbic acid admixture. Finally, BW 755 c was one of the best substances in preventing vasodepression in the anaphylactic paw edema model (Table). In u.v.-irradiated rats, EArn lay between 70-86% (Table), i.e., vasodepression was only small. In this model, OFRS gave good protection. Man-

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Agents and Actions, vol. 23, 1/2 (1988)

nitol, at doses of 125 and 250 mg/kg i.p. immediately prior to irradiation, protected by 95 and 97%, respectively (Table). Similar results were obtained with thiourea, ethanol, sodium hydrogen sulphite, M516, hydroquinone and BW755c. M 516 and hydroquinone, at higher doses, again caused vasodepression themselves, possibly, by radical formation via semiquinone [2]. In order to gain insight into the underlying mechanism of protection from vasodepression, superoxide dismutase (SOD) and catalase were injected intravenously into animals immediately prior to u.v.-irradiation. SOD (Peroxinorm| gratefully acknowledged gift from Dr. Wirker, Grtinenthal GmbH), 3 mg/kg, did not prevent vasodepression, whereas catalase (Reanal, Budapest), 10 mg/ kg did. The respective data were 83+ 14 (n= 8) for SOD (u.v.-irradiated animals control value: 85+6 (n=8) and 101+_24 (n=10; p < 0.01) for catalase (u.v.-irradiated animals control value: 73+__17 (n= 10)). Simultaneous injection of both SOD and catalase gave protection against vasodepression as did catalase alone (data not shown). Heat-inactivated catalase was without effect. The explanation could be that in the models used, besides other mediators, OFR are formed by 02 reduction at the site of inflammation (see Fig.). OFR, then through the blood stream, enter the general circulation and damage membranes and blood vessel muscles, thereby also inducing hydroperoxy and alkoxy radical formation and vasodepression develops. Moreover, formation of OFR is apparently not only induced in the blood vessel system by hydroperoxy and alkoxy radical production but also by activation of xanthine oxidase and/or NADPH oxidase. Activation ofprostaglandin synthetase seems to be of less importance, since cyclooxygenase inhibitors, except salicylates, do not substantially prevent or restore inflammation-induced vasodepression. The suggestion that OFR are generated in the blood vessel system in inflammation-induced vasodepression is supported by the fact that vasodepression does not spontaneously improve after leg separation and that vasoreactivity can be fully restored in the isolated leg by OFRS co-perfusion. The OFR mainly formed in the models tested is probably 02 (Fig.) which, in view of the high SOD capacity of the blood (caeruloplasmin, Cucomplexes a.o.) is readily dismutated to H202.

~t

e.a~s

. 9.

I

/

x~Jteladasm/ c ~

F

3/ 2

~

RO.+OH" o

ROH+ R.

~

~ * R H / al~nm

~ -2 a00.

v

~OOH.*R.

,~,lr --~

~ )

curet ot-tocoplx,rol ?

Figure Formation of reactive oxygen species and some free oxygen radical scavengers.

Therefore, increasing SOD capacity by i.v. SOD injection did not influence vasodepression. Neither "O~-nor H202 are more than weakly tissue damaging [2]. However, H202 is the parent substance of the aggressive "OH and singlet oxygen, 102 (Fig.). Therefore, i.v. injection of catalase is, indeed, protective against vasodepression because H202 is destroyed by catalase and formation of "OH and/ or 102 should be reduced. Recently, Dowling et al. [3] described the detection of H202, "OH and 102 in an arthritis-like rat model, and we have found vasodepression in adjuvant arthritis [1]. The effectivity of OFRS against vasodepression (Table) is in good agreement with these findings and the mechanisms shown in the Figure. Since vasodepression is significantly less im.proved by OFRS in the anaphylactic model than m the dextran and u.v. models, other mediators than OFR seem to be involved to a higher degree in anaphylaxis.

References [1] H. Bekemeier and R. Hirschelmann, Determination of reactivity of resistance blood vessels in the isolated perfused legs of animals with inflammation as exemplified in adjuvant arthritis, Agents and Actions 17, 363-365 (1985). [2] W.H. Bannister and J.V. Bannister (eds.), Biological and clinical aspects of superoxide and superoxide dismutase, Elsevier North Holland, Inc., New York-Amsterdam-Oxford 1980. [3] E.J. Dowling, A. M. Symons and D. V. Parke, Free radical production at the site of an acute inflammatory reaction as measured by chemiluminescence, Agents and Actions 19, 203-207 (1986).