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OBJECTIVE To examine the efficiency of hemoperfusion in removing South American rattlesnake (Crotalus durissus terrificus) venom from rats compared with neutralization by antivenom. DESIGN An exploratory experimental investigation in rats involving the injection of snake venom with or without subsequent hemoperfusion or antivenom administration. SETTING Basic animal research laboratory in a private university. ANIMALS Normal, healthy male Wistar rats (0.29-0.40 kg, 3-6 months old) from a commercial breeder. INTERVENTIONS Four experimental groups of randomly allocated rats (n = 3/group) were studied: Group 1: rats were injected with a single dose of venom (5 mg/kg, IM, in the right thigh) with no other treatment; blood samples were collected minutes before death to determine leukocyte, platelet, and erythrocyte counts; Group 2 (Control): rats underwent hemoperfusion alone for 60 min using a hemoperfusion cartridge designed for protein adsorption (by granulated charcoal) and protein precipitation (by tannic acid); Group 3 (Venom + antivenom): rats were injected with venom (5 mg/kg, IM) and, 10 min later, were treated with antivenom at the venom:antivenom ratio recommended by the manufacturer; Group 4 (Venom + hemoperfusion): Rats were injected with venom (5 mg/kg, IM) and, 10 min later, were hemoperfused for 60 min. In groups 2-4, blood samples were collected for leukocyte, platelet, and erythrocyte counts 24 h after venom. MEASUREMENTS AND MAIN RESULTS Rats injected with venom alone (Group 1) developed signs of neurotoxicity and ataxia and died in 9.0 ± 0.43 h but showed no changes in leukocyte or erythrocyte counts. In contrast, there were no deaths in groups 2-4. The lack of deaths in Groups 3 and 4 indicated that antivenom and hemoperfusion, respectively, protected against the lethal effects of the venom. CONCLUSIONS Hemoperfusion with a double-action hemoperfusion cartridge capable of protein adsorption and precipitation protected rats against C. d. terrificus venom.

The metalloendopeptidase 24.15 (EP24.15) is ubiquitously present in the extracellular environment as a secreted protein. Outside the cell, this enzyme degrades several neuropeptides containing from 5 to 17 amino acids (e.g. gonadotropin releasing hormone, bradykinin, opioids and neurotensin). The constitutive secretion of EP24.15 from glioma C6 cells was demonstrated to be stimulated linearly by reduced concentrations of extracellular calcium. In the present report we demonstrate that extracellular calcium concentration has no effect on the total amount of the extracellular (cell associated + medium) enzyme. Indeed, immuno-cytochemical analyses by confocal and electron microscopy suggested that the absence of calcium favors the enzyme shedding from the plasma membrane into the medium. Two putative calcium-binding sites on EP24.15 (D93 and D159) were altered by site-directed mutagenesis to investigate their possible contribution to binding of the enzyme at the cell surface. These mutated recombinant proteins behave similarly to the wild-type enzyme regarding enzymatic activity, secondary structure, calcium sensitivity and immunoreactivity. However, immunocytochemical analyses by confocal microscopy consistently show a reduced ability of the D93A mutant to associate with the plasma membrane of glioma C6 cells when compared with the wild-type enzyme. These data and the model of the enzyme's structure as determined by X-ray diffraction suggest that D93 is located at the enzyme surface and is consistent with membrane association of EP24.15. Moreover, calcium was also observed to induce a major change in the EP24.15 cleavage site on distinctive fluorogenic substrates. These data suggest that calcium may be an important modulator of ep24.15 cell function.

Severe cases of human envenoming by caterpillars of the saturniid moth Lonomia obliqua in Brazil can result in renal damage, leading to renal failure, and intracerebral hemorrhaging. In this work, we used immunohistochemical staining with rabbit antiserum raised against L. obliqua venom to examine venom distribution in selected tissues of the brain (cerebellum and hippocampus), kidneys, and liver of male Wistar rats injected with a single dose of venom (200 microg/kg, i.v.) and sacrificed 6, 18, 24, and 72 hours later. The immunolabeling of GFAP was also examined to assess the venom effects on perivascular astrocytic end-feet in the microvasculature of the hippocampus and cerebellum. Venom was detected in the kidneys (6 and 18 hours) and in the liver (6 hours) but not in the brain at any of the time intervals examined. In contrast, immunolabeling for GFAP revealed astrogliosis in the cerebellum and enhanced expression of this protein in the glial processes of the cerebellum and hippocampus, with a maximum response from 24 hours onwards. The high immunoreactivity seen in the kidneys agreed with the renal damage and dysfunction reported for some patients. The lack of venom detection in the brain, despite the altered expression of GFAP in astrocytes, suggested either that the venom does not enter this organ or that its entrance is transient and fast. Alternatively, the circulating venom may induce the release of mediators that could serve as second messengers to provoke the late astrocytic reactivity and astrogliosis. It is possible that both of these mechanisms may contribute to the effects observed.

The effects of the Brazilian herbal medicine Catuama and each of its plant constituents (Paullinia cupana, Trichilia catigua, Zingiber officinalis and Ptychopetalum olacoides) were investigated on rabbit corpus cavernosum (RbCC) using a bioassay cascade. Catuama caused short-lived and dose-dependent relaxations (11% +/- 7%, 26% +/- 5% and 82% +/- 9%, at doses of 1, 3 and 10 mg, respectively). Neither the nitric oxide synthesis inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME; 10 microM) nor the soluble guanylate cyclase inhibitor ODQ (10 microM) significantly affected the Catuama-induced relaxations. Similarly, the selective ATP-dependent K(+) channel (K(ATP)) blocker glibenclamide (10 microM), the muscarinic receptor antagonist atropine (1 microM) and the voltage-dependent Na(+) channel blocker tetrodotoxin (1 microM) all failed to affect significantly the Catuama-induced relaxations. These results indicate that the relaxations induced by Catuama involve neither nitric oxide release nor K(ATP) channel activation. The extracts of P. cupana, Z. officinalis and P. olacoides caused short-lived and dose-dependent RbCC relaxations, whereas T. catigua evoked long-lasting relaxations which were occasionally preceded by a brief contractile effect. The extract of P. cupana was the most active in relaxing RbCC strips. The relaxations induced by all extracts were not significantly affected by L-NAME (10 microM). The infusion of ODQ (10 microM) had no significant effect on the P. cupana- and Z. officinalis-induced relaxations but reduced by >50% (p < 0.05) those evoked by P. olacoides and T. catigua. Incubations of RbCC with Catuama(10 mg/mL for 0.25 to 5 min) caused increases of cAMP levels (143% increase at 5 min of incubation). Incubations of RbCC with P. cupana extract (1 mg/mL) increased the cAMP levels by 200% whereas higher doses (10 and 100 mg/mL) caused smaller increases in the nucleotide levels (150% and 89%, respectively). The extracts of Z. officinalis and P. olacoides (same doses) caused smaller increases of the cAMP levels compared with the P. cupana extract, whereas T. catigua (1-100 mg) did not increase the levels of this nucleotide above the basal values. Our results show that of the four extracts assayed, P. cupana was the most effective, indicating that it is the main extract responsible for the relaxing effect of Catuama on rabbit cavernosal tissue.

Bothrops jararacussu venom and its major toxin, bothropstoxin-I (BthTX-I), possess myotoxic and neurotoxic activities. The ability of commercial equine antivenom to neutralize these activities was studied in mouse isolated phrenic nerve–diaphragm (PND) and extensor digitorum longus (EDL) preparations by indirect stimulation (0.1 HZ, 0.2 ms). The time required to produce 50% neuromuscular blockade in the PND and EDL preparations was, respectively, 70 ± 11.5 min and 58 ± 8 min for B. jararacussu venom (50 μg/mL), and 31 ± 6 min and 30 ± 3 min for BthTX-I (20 μg/mL). After a 120-min incubation, the creatine kinase (CK) concentrations in the EDL preparations were 3464 ± 346 U/L and 3422 ± 135 U/L following exposure to venom (50 μg/mL) and BthTX-I (20 μg/mL), respectively. Antivenom neutralized the neuromuscular blockade induced by the venom and toxin in PND preparations in a dose-dependent fashion, but only partially neutralized this effect in EDL. Antivenom also effectively prevented the venom- and toxin-induced release of CK from EDL. In contrast, histological analysis showed that the morphological damage caused by B. jararacussu venom and BthTX-I in the EDL was only partially prevented by the anti- venom. These results indicate that commercial equine antiserum fully protects against the neurotoxic action of B. jararacussu and BthTX-I in PND preparations, but only partially protects against the neurotoxic and myotoxic actions of the venom and its toxin in EDL preparations. Care must therefore be exercized in extrapolating results from different preparations even when similar pharmacological or physiological responses are involved. © 2000 John Wiley & Sons, Inc. Muscle Nerve 23: 1832–1839, 2000