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Journal of Venomous Animals and Toxins

Print version ISSN 0104-7930On-line version ISSN 1678-4936

J. Venom. Anim. Toxins vol. 2 n. 2 Botucatu  1996

http://dx.doi.org/10.1590/S0104-79301996000200008 

Short communication

 

 

MEASUREMENT OF BRADYKININ, PROTEIN, Na+, Cl-, K+, AND Zn++ CONCENTRATIONS IN RAT URINE AFTER INTRAPERITONEAL INJECTION OF Tityus serrulatus VENOM.

 

 

L. A. F. FERREIRA , M. BERGAMASCO , L. ANGNES

1 Laboratory of Biochemistry and Biophysics, Butantan Institute, State of São Paulo, Brazil; 2 Department of Chemistry, University of São Paulo, State of São Paulo, Brazil.

 

 

ABSTRACT. Experiments were carried out in vivo by injecting Tityus serrulatus crude venom in rats followed by biological assays on the isolated guinea-pig ileum, to show the effects of scorpion venom on kallikrein-kinin system. Our results showed effects such as significant decrease of total kinin rate and a decrease of Zn , Na , Cl and K ions in rat urine 24 and 48 hours after the injection of Tityus serrulatus crude venom.
 KEY WORDS: Bradykinin, protein, ions,Tityus serrulatus.

 

 

INTRODUCTION

Kinins are vasoactive peptides released from high and low molecular weight kininogen and T-kininogen following cleavage by kallikrein, trypsin and/or cathepsin (13).

The kinins are present in biological fluids of mammals and are responsible for a variety of biological effects(13). It has been recently demonstrated that the kinins are involved in the induction and maintenance of chronic inflammation(4) and stimulate the release of tumor necrosis factor (TNF) and interleukin-1 (IL-1) from macrophages (14).

Renal and cardiovascular disturbances, and pulmonar edema may be observed in scorpion envenomation (1,3,5,6,7) caused by the involvement of kinin-kallikrein system (8,10).

Four kinins were studied in urine of man and other animals: bradykinin, kallidin, Met-Lys-bradykinin and des-Arg-bradykinin.

This paper shows the effects of Tityus serrulatus crude venom in rat urine 24 and 48 hours after venom injection.

The urine was collected from Wistar rats, (n=4). Tityus serrulatus dried venom was obtained at Butantan Institute , São Paulo, Brazil.

Adult male Wistar rats (80-100 g) and female guinea-pigs (160-180 g) were used. Bradykinin was purchased from Sigma Co. (St. Louis, MO U.S.A.). Other reagents were obtained from Merck Darmstadt, F.R.G.

Urine was collected from rats intraperitoneally injected with 1 mg of protein of Tityus serrulatus venom dissolved in 100µl of saline/kg/rat for 24 and 48 h (injected groups). The controls were injected with 100µl of saline solution for 24 and 48 h (control groups). Both groups were kept in metabolic cages (10 x 17 cm) and given water but no food during the experiment. In regard to the control groups, we collected 63 ml and 77 ml of urine after 24 h.

In regard to the injected group, we collected 52 ml and 56 ml of urine for 48 h. The urine was centrifuged for 15 min at 3015 g at 5ºC, using a Sorvall superspeed RC2-B automatic refrigerated centrifuge SS-34/55-1 rotor and then was assayed on the isolated guinea-pig ileum.

Protein was determined by the method of Lowry et al.(11), with bovine serum albumin as standard. The activity of the kinins was measured on the isolated ileum of female guinea-pigs (n=6) weighing 160-180 g. A 1-2 cm long ileum was suspended in a 5 ml muscle chamber with Tyrode solution containing atropine (1 mg/l). Bradykinin was used as standard. The isotonic concentrations/min were recorded using a kymograph and measured on a log dose-response curve.

Sodium and potassium were analyzed by flame photometry (digimed flame photometer, model 2004) using a mixture of air/butane. To minimize matrix interferences, the standard addition method was preferred. To determine the amount of chloride in the samples, potentiometric titrations were performed. A silver electrode and Ag/AgCl (KCl=3.0 M) were connected to a Metrohm Herisau pH-Meter model E 516. Preliminary experiments showed that the chloride contamination from the reference electrode did not affect the results in the time necessary to perform the titration. Zinc determinations were performed in an atomic absorption spectrophotometer Perkin-Elmer model 403.

Kinin in fresh urine is species-dependent, and bradykinin is the major kinin (6% from fresh rat urine, kallidin 3% and des-Arg-bradykinin 3%) (12).

Several authors have studied the structural, biological and functional properties of scorpion venoms and observed that the kinins were involved in some responses to scorpion envenomation (8,9,10).

In order to study the effects of T. serrulatus venom on rat urine, we collected urine for 24 and 48 hours, and the kinins and ions were measured by biological assay and photometric and atomic absorption methods, respectively.

The results showed a decrease of kinin concentrations of injected groups compared with that of the control groups. Decrease of kinins was 34.5% in the injected groups after 24 h and 56.2% in the injected groups after 48 h, showing a significative decrease in both groups (Table 1 and Table 2).

 

TABLE 1. The effects of Tityus serrulatus crude venom on rat urine after 24 hours. Results are reported as means ± SD.

 

* Rats injected with 1 mg/kg rat from T. serrulatus crude venom.
** p < 0.05, Student "t" test; ***ppm = mg/1.

 

 

TABLE 2. The effects of Tityus serrulatus crude venom on rat urine after 48 hours. Results are reported as means ± SD.

 

* Rats injected with 1 mg/kg rat from T. serrulatus crude venom.
** p < 0.02, Student "t" test; *** ppm = mg/1.

 

In addition, we observed a sensible decrease of the urine volume in the injected groups in spite of water ingestion. The volume of urine of injected groups after 24h compared with that of control groups was 17.5% after 24h and 27.3% after 48h. This observation agrees with the findings of Freire Maia and Campos(6) who showed that scorpion venom decreased the urine volume. Both groups showed concentrated, turbid and dark urine.

The ion concentrations of Zn , Na , Cl and K of urine of injected groups were measured and compared with those of the control groups. The urine of the injected groups showed an interesting decrease of the above mentioned ions: 17.28% after 48 h (data shown as percentage mean) Table 1 and Table 2. The ion concentrations found in the urine may be one of the limiting factors of kallikrein activity in the release of kinins. In our experiment, we observed a complexity between Zn and protein, which could explain the plasmatic and urinary kallikrein activity after injection of Tityus serrulatus crude venom in rats (unpublished data).

Earlier reports have demonstrated that the lack of NaCl supressed the bradykinin rate from plasma or tissues causing hypertension in rats, (2), since the decrease of renal sodium concentration seems to be the firts stage of hypertension.

The results presented here in are important and consistent with the view that T. serrulatus crude venom may possess some compounds that cause decrease of concentration of kinins in rat urine. This may be an important tool in the study of scorpion envenomation therapy.

 

ACKNOWLEDGEMENTS

We are grateful to Neusa de Lima for typing this manuscript. This work was supported by grants from Butantan Foundation.

 

REFERENCES

01 AMARAL CFS., BARBOSA AJA., LEITE VHR., TAFURI WL., REZENDE NA. Scorpion sting- induced pulmonary oedema: evidence of increased alveolocapillary membrane permeability. Toxicon, 1994, 32, 999-1003.

02 BENETOS A., BOUAZIZ H., SAFAR M. Salt sensitivity and bradykinin activity in rats. Agents Actions Suppl., 1978, 38, 228-34.

03 CUPO P., JURCA M., MARQUES MAM., OLIVEIRA JSM., HERING SE. Severe scorpion envenomation in Brazil. Clinical, laboratory and anatomopathological aspects. Rev. Inst. Med. Trop., São Paulo, 1994, 36, 67-76.

04 ERDOS EG. Kininases. In: ERDÖS EG. Ed. Bradykinin, kallidin and kallikrein. New York: Springer Verlag, 1979: 428-87.

05 FREIRE-MAIA L., ALMEIDA HO., CUNHA-MELLO JR., AZEVEDO AD., BARROSO J. Mechanism of the pulmonary oedema induced by intravenous injection of scorpion toxin in the rat. Agents Actions, 1978, 8, 113-8.

06 FREIRE-MAIA L., CAMPOS J.A. Response to the letter to the Editor by Gueron and Ovysyshcher on the treatment of the cardiovascular manisfestations of scorpion envenomation. Toxicon, 1987, 25, 125-30.

07 FREIRE-MAIA L., CAMPOS JA. Pathophysiology and treatment of scorpion poisoning. In: WORLD CONGRESS ON ANIMAL, PLANT AND MICROBIAL TOXINS, 9, Stillwater, 1994. Proceedings... Oxford: Pergamon Press, 1994.

08 FERREIRA LAF., ALVES EW., HENRIQUES OB. Peptide T, a novel bradykinin potentiator isolated from Tityus serrulatus scorpion venom. Toxicon, 1993, 31, 941-7.

09 FERREIRA LAF., BERGAMASCO M., HENRIQUES OB. Crude venom from the scorpion Tityus serrulatus inhibits angiotensin-converting enzyme activity in the rat. J. Nat. Toxins, 1993, 3, 145-53.

10 ISMAIL M., AMAL JY., FATTAM I., DABBES TT. Experimental treatment protocols for scorpion envenomation: A review of common therapies and an effect of kallikrein - kinin inhibitors. Toxicon, 1992, 30, 1257-79.

11 LOWRY OH., ROSENBROUGH NJ., FARR AL., RANDALL RJ. Protein measurement with the pholin phenol reagent. J. Biol. Chem., 1951, 193, 265-75.

12 MINDROIU T., SCICLI G., PERINI F., CARRETERO OA., SCICLI AG. Identification of a new kinin in human urine. J. Biol. Chem., 1986, 261, 7407-11.

13 PISANO JJ. Kinins in nature. In: ERDÖS EG. Ed. Bradykinin, kallidin and kallikrein. New York: Springer Verlag, 1979: 273-85.

14 TIFFANY CW., BURCH RM. Bradykinin stimulates tumor necrosis factor and interleukin-1 release from macrophages. FEBS Lett., 1989, 247, 189-92.

 

 

 CORRESPONDENCE TO:
L. A. F. FERREIRA - Laboratório de Bioquímica e Biofísica, Instituto Butantan, Av.Vital Brasil, 1500, CEP 05503-900, São Paulo, Brasil.

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