Pulmonary oedema produced by scorpion venom augments a phenyldiguanide-induced reflex response in anaesthetized rats

Abstract

1. The involvement of pulmonary oedema produced by scorpion venom in augmenting a phenyldiguanide (PDG)-induced reflex response was evaluated in urethane-anaesthetized rats. 2. PDG-induced bradycardiac, hypotensive and apnoeic responses, expressed as time-response area, exhibited similarities before or after venom treatment. Hence, the time-response area of bradycardia was taken as a reflex parameter. Pulmonary oedema was determined by physical evaporation and histological methods. 3. Exposure to Indian red scorpion (Buthus tamulus, BT; i.v.) venom for 30 min increased the pulmonary water content (P < 0.05; Student's t test) and augmented the PDG-induced bradycardiac reflex response by more than 2 times (P < 0.001). The increase of pulmonary water content was maximal with 100 microg kg-1 of venom and the augmentation was maximal with 10 microg kg-1. In a separate series of experiments, the venom (100 microg kg-1)-induced pulmonary oedema was confirmed by histological and physical methods. In this group also, the venom augmented the reflex to the same magnitude. 4. Pulmonary oedema (physical and histological) and augmentation of the bradycardiac reflex response after BT venom (100 microg kg-1; i.v.) were absent in animals pretreated with aprotinin, a kallikrein-kinin inhibitor (6000 KIU; i. v.). 5. Ondansetron (10 microg kg-1; i.v.), a 5-HT3 receptor antagonist, failed to block the venom-induced pulmonary oedema (physical and histological) but blocked the venom-induced augmentation of the reflex. 6. The results of this study indicate that the venom-induced augmentation of the PDG reflex is associated with pulmonary oedema involving kinins utilizing 5-HT3 receptors.

Figure 1. Time-response pattern of the PDG reflex

The data (means ±s.e.m.) show the time-response relationship of the PDG (10 μg kg⁻¹, i.v.) reflex on heart rate (HR), mean arterial pressure (MAP), and respiratory rate (RR) obtained from 5 different experiments. Note the parallel shift in the time-response curve before (○) and after i.v. injection of 100 μg kg⁻¹ of venom (•). There was tachypnoea after 30 s. In many places error bars are within the symbols. The responses after venom are significantly different from those before (P < 0.05, 2-way ANOVA).

Figure 2. Effect of different concentrations of BT venom on the PDG (10 μg kg⁻¹)-induced bradycardiac reflex response

The mean ±s.e.m. values were obtained from 5-8 different experiments for each point. The values after addition of 10 μg kg⁻¹ of venom are significantly different from the saline group (P < 0.001, Student's t test for unpaired observations). The dashed line indicates the time-matched response after saline (101 ± 1.20 % of initial; n = 8).

Figure 3. Effect of different concentrations of BT venom on the pulmonary water content

The pulmonary water content was determined by drying to constant weight. The mean ±s.e.m. values were from 5-8 different experiments for each point. The dashed line indicates the pulmonary water content from saline-treated animals (78.4 ± 0.71; n = 8). *P < 0.05 and **P < 0.001 as compared with saline control group (Student's t test for unpaired observations).

Figure 4. Aprotinin and ondansetron blocked the venom (100 μg kg⁻¹)-induced augmentation of the bradycardiac reflex response elicited by PDG

The mean ±s.e.m. values were obtained from 5-8 different experiments for each bar. The animals were pretreated with saline (n = 8), aprotinin (Apr; n = 6) or ondansetron (Ond; n = 6) for 15 min followed by venom (100 μg kg⁻¹). The reflex response after pretreatment with aprotinin (6000 KIU; before venom) was 101 ± 1.23 % of initial response whereas after pretreatment with ondansetron (10 μg kg⁻¹) the reflex response was absent but reappeared after exposure to venom for 30 min as shown (Ond + venom). * Significant difference from the saline only group (n = 8; P < 0.001, Student's t test for unpaired observations). Apr + venom, response to PDG (10 μg kg⁻¹) 30 min after venom in aprotinin-pretreated animal; Ond + venom, response to PDG 30 min after venom in ondansetron-treated animal.

Figure 5. Aprotinin blocked pulmonary oedema formation by BT venom

Pretreatment with aprotinin blocked the venom (100 μg kg⁻¹)-induced increase in pulmonary water content. The animals were pretreated with saline (n = 8), aprotinin (n = 6) or ondansetron (n = 6) for 15 min followed by venom (100 μg kg⁻¹). After 30 min exposure to venom, the reflex response was obtained before the lungs were excised for determination of water content by drying. The mean ±s.e.m. values were obtained from 5-8 different experiments for each bar. * Significant difference from saline only group (n = 8; P < 0.05; Student's t test for unpaired observations). Apr + venom, pulmonary water content 30 min after venom in aprotinin (6000 KIU)-pretreated animal; Ond + venom, pulmonary water content 30 min after venom in ondansetron (10 μg kg⁻¹)-treated animal.

Figure 6. Photomicrographs of lung tissue obtained from animals treated with saline and venom after pretreatment with saline, aprotinin or ondansetron

A, lung tissue from a saline-treated animal showing the normal alveolar pattern without oedema. B, lung tissue from an animal exposed to venom (100 μg kg⁻¹; i.v.) after pretreatment with saline, showing dilatation of air spaces, venocapillary congestion, haemorrhage, colloid material in the alveoli and leucocytic infiltration. C, lung tissue from a venom-treated animal pretreated with aprotinin showing the normally appearing alveoli. D, lung tissue from a venom-treated animal pretreated with ondansetron (10 μg kg⁻¹) showing greatly dilated air spaces, haemorrhage, necrosis and a severe degree of leucocytic infiltration in places. Oedema and dilatation of alveoli are more marked in D as compared with B. Similar types of observations were made in 4 additional experiments in each group. The scale bar in C indicates 200 μm and applies to all panels.