Potent nonopioid antinociceptive activity of telocinobufagin in models of acute pain in mice.

INTRODUCTION: In recent decades, several researches have been conducted in search of new analgesics that do not present the side effects of opioids. In this context, animal venoms contain natural painkillers that have been used for the development of new analgesics. OBJECTIVE: The aims of this study were to evaluate the antinociceptive effects of telocinobufagin (TCB), a bufadienolide isolated from Rhinella jimi venom, in murine acute pain models, and to verify the participation of the opioid system in these effects. METHODS: TCB was purified from R. jimi venom by high-performance liquid chromatography, and its structure was confirmed by spectrometric techniques. TCB was administered intraperitoneally (i.p.) (0.062, 0.125, 0.25, 0.5, and 1 mg·kg-1) and orally (p.o.) (0.625, 1.125, 2.5, 5, and 10 mg·kg-1) in mice, which were then subjected to pain tests: acetic acid-induced writhing, formalin, tail-flick, and hot-plate. Involvement of the opioid system in TCB action was evaluated by naloxone i.p. injected (2.5 mg·kg-1) 20 minutes before TCB administration. In addition, the TCB action on the µ, δ, and κ opioid receptors was performed by radioligand binding assays. RESULTS: In all the tests used, TCB showed dose-dependent antinociceptive activity with more than 90% inhibition of the nociceptive responses at the doses of 1 mg·kg-1 (i.p.) and 10 mg·kg-1 (p.o.). Naloxone did not alter the effect of TCB. In addition, TCB did not act on the µ, δ, and κ opioid receptors. CONCLUSION: The results suggest that TCB may represent a novel potential nonopioid therapeutic analgesic for treatment of acute pains.

Effects of cardiotonic steroids on isolated perfused kidney and NHE3 activity in renal proximal tubules.

Cardiotonic steroids (CS) are known as modulators of sodium and water homeostasis. These compounds contribute to the excretion of sodium under overload conditions due to its natriuretic property related to the inhibition of the renal Na+/K+-ATPase (NKA) pump α1 isoform. NHE3, the main route for Na+ reabsorption in the proximal tubule, depends on the Na+ gradient generated by the NKA pump. In the present study we aimed to investigate the effects of marinobufagin (MBG) and telocinobufagin (TBG) on the renal function of isolated perfused rat kidney and on the inhibition of NKA activity. Furthermore, we investigated the mechanisms for the cardiotonic steroid-mediated natriuretic effect, by evaluating and comparing the effects of bufalin (BUF), ouabain (OUA), MBG and TBG on NHE3 activity in the renal proximal tubule in vivo. TBG significantly increased GFR, UF, natriuresis and kaliuresis in isolated perfused rat kidney, and inhibits the activity of NKA at a much higher rate than MBG. By stationary microperfusion technique, the perfusion with BUF, OUA, TBG or MBG promoted an inhibitory effect on NHE3 activity, whereas BUF was the most effective agent, and demonstrated a dose-dependent response, with maximal inhibition at 50nM. Furthermore, our data showed the role of NKA-Src kinase pathway in the inhibition of NHE3 by CS. Finally, a downstream step, MEK1/2-ERK1/2 was also investigated, and, similar to Src inhibition, the MEK1/2 inhibitor (U0126) suppressed the BUF effect. Our findings indicate the involvement of NKA-SRc-Kinase-Ras-Raf-ERK1/2 pathway in the downregulation of NHE3 by cardiotonic steroids in the renal proximal tubule, promoting a reduction of proximal sodium reabsorption and natriuresis.

A new structurally atypical bradykinin-potentiating peptide isolated from Crotalus durissus cascavella venom (South American rattlesnake).

Venom glands of some snakes synthesize bradykinin-potentiating peptides (BPP's) which increase bradykinin-induced hypotensive effect and decrease angiotensin I vasopressor effect by angiotensin-converting enzyme (ACE) inhibition. The present study shows a new BPP (BPP-Cdc) isolated from Crotalus durissus cascavella venom: Pro-Asn-Leu-Pro-Asn-Tyr-Leu-Gly-Ile-Pro-Pro. Although BPP-Cdc presents the classical sequence IPP in the C-terminus, it has a completely atypical N-terminal sequence, which shows very low homology with all other BPPs isolated to date. The pharmacological effects of BPP-Cdc were compared to BBP9a from Bothrops jararaca and captopril. BPP-Cdc (1 µM) significantly increased BK-induced contractions (BK; 1 µM) on the guinea pig ileum by 267.8% and decreased angiotensin I-induced contractions (AngI; 10 nM) by 62.4% and these effects were not significantly different from those of BPP9a (1 µM) or captopril (200 nM). Experiments with 4-week hypertensive 2K-1C rats show that the vasopressor effect of AngI (10 ng) was decreased by 50 µg BPP-Cdc (69.7%), and this result was similar to that obtained with 50 µg BPP9a (69.8%). However, the action duration of BPP-Cdc (60 min) was 2 times greater than that of BPP-9a (30 min). On the other hand, the hypotensive effect of BK (250 ng) was significantly increased by 176.6% after BPP-Cdc (50 µg) administration, value 2.5 times greater than that obtained with BPP9a administered at the same doses (71.4%). In addition, the duration of the action of BPP-Cdc (120 min) was also at least 4 times greater than that of BPP-9a (30 min). Taken together, these results suggest that BPP-Cdc presents more selective action on arterial blood system than BPP9a. Besides the inhibition of ACE, it may present other mechanisms of action yet to be elucidated.

Natriuretic effect of bufalin in isolated rat kidneys involves activation of the Na+-K+-ATPase-Src kinase pathway.

Bufadienolides are structurally related to the clinically relevant cardenolides (e.g., digoxin) and are now considered as endogenous steroid hormones. Binding of ouabain to Na(+)-K(+)-ATPase has been associated, in kidney cells, to the activation of the Src kinase pathway and Na(+)-K(+)-ATPase internalization. Nevertheless, whether the activation of this cascade also occurs with other cardiotonic steroids and leads to diuresis and natriuresis in the isolated intact kidney is still unknown. In the present work, we perfused rat kidneys for 120 min with bufalin (1, 3, or 10 µM) and measured its vascular and tubular effects. Thereafter, we probed the effect of 10 µM 3-(4-chlorophenyl)1-(1,1-dimethylethyl)-1H-pyrazolo[3,4-d]pyrimidin-4amine (PP2), a Src family kinase inhibitor, and 1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio] butadiene (UO126), a highly selective inhibitor of both MEK1 and MEK2, on bufalin-induced renal alterations. Bufalin at 3 and 10 µM profoundly increased several parameters of renal function in a time- and/or concentration-dependent fashion. At a concentration that produced similar inhibition of the rat kidney Na(+)-K(+)-ATPase, ouabain had a much smaller diuretic and natriuretic effect. Although bufalin fully inhibited the rat kidney Na(+)-K(+)-ATPase in vitro, its IC(50) (33 ± 1 µM) was threefold higher than the concentration used ex vivo and all its renal effects were blunted by PP2 and UO126. Furthermore, the phosphorylated (activated) ERK1/2 expression was increased after bufalin perfusion and this effect was totally prevented after PP2 pretreatment. The present study shows for the first time the direct diuretic, natriuretic, and kaliuretic effects of bufalin in isolated rat kidney and the relevance of Na(+)-K(+)-ATPase-mediated signal transduction.

Isolation and pharmacological effects of leptoxin, a novel proteic toxin from Leptodactylus pentadactylus skin secretion.

The in vivo and in vitro pharmacological effects of leptoxin, one of the most lethal protein toxins known at present date (LD(50) 0.5+/-0.03 microg/kg i.v., mice) isolated from Leptodactylus pentadactylus skin secretion, were studied. In rats, leptoxin (1.0 microg/kg, i.v.) induced cardiorespiratory collapse with abundant tracheal secretion followed by sudden death. The cardiovascular shock, pulmonary edema and mortality were not prevented by pretreating the animals with effective doses of pharmacological blockers, i.e., atropine with or without bilateral vagotomy, phentolamine, propranolol, hexamethonium, captopril, dexamethasone, indomethacin, L-NAME, promethazine, Ginkgolide BN-52021 or tezosentan. Pulmonary macroscopic examination revealed increased tracheobronchial secretion, hemorrhagic areas and edema. Microscopic examination showed intense vascular congestion, alveolar and septal interstitial hemorrhage and alveolar edema, without infiltrated inflammatory cells. Leptoxin increased pulmonary index (0.67+/-0.09 vs. 1.55+/-0.24; p<0.05) and the Evans blue concentration in the bronchoalveolar fluid (1.24+/-0.17 vs. 4.17+/-1.47 microg/microL; p<0.01) and in the lung parenchyma (40.73+/-3.27 vs. 65.33+/-4.51 microg/microL; p<0.03). Leptoxin increased the pulmonary perfusion pressure from 13.7+/-5.3 to 54.0+/-6.3 mmHg. It also induced a vasoconstrictor effect in the perfused mesenteric vascular bed that could be explained by a hyperreactivity to phenylephrine. Thus, the results suggest that leptoxin-induced death occurs by acute pulmonary edema due to increased microvascular pulmonary pressure evoked by direct vasoconstriction. Despite its strong toxicity, the role of leptoxin in L. pentadactylus skin remains unknown.

Leptoglycin: a new Glycine/Leucine-rich antimicrobial peptide isolated from the skin secretion of the South American frog Leptodactylus pentadactylus (Leptodactylidae).

Antimicrobial peptides are components of innate immunity that is the first-line defense against invading pathogens for a wide range of organisms. Here, we describe the isolation, biological characterization and amino acid sequencing of a novel neutral Glycine/Leucine-rich antimicrobial peptide from skin secretion of Leptodactylus pentadactylus named leptoglycin. The amino acid sequence of the peptide purified by RP-HPLC (C(18) column) was deduced by mass spectrometric de novo sequencing and confirmed by Edman degradation: GLLGGLLGPLLGGGGGGGGGLL. Leptoglycin was able to inhibit the growth of Gram-negative bacteria Pseudomonas aeruginosa, Escherichia coli and Citrobacter freundii with minimal inhibitory concentrations (MICs) of 8 microM, 50 microM, and 75 microM respectively, but it did not show antimicrobial activity against Gram-positive bacteria (Staphylococcus aureus, Micrococcus luteus and Enterococcus faecalis), yeasts (Candida albicans and Candida tropicalis) and dermatophytes fungi (Microsporum canis and Trichophyton rubrum). No hemolytic activity was observed at the 2-200 microM range concentration. The amino acid sequence of leptoglycin with high level of glycine (59.1%) and leucine (36.4%) containing an unusual central proline suggests the existence of a new class of Gly/Leu-rich antimicrobial peptides. Taken together, these results suggest that this natural antimicrobial peptide could be a tool to develop new antibiotics.

Antinociceptive activity of Calotropis procera latex in mice.

This work evaluated the antinociceptive effect of proteins from the Calotropis procera (Asclepiadaceae) latex using three different experimental models of nociception in mice. The latex protein fraction administered intraperitoneally in male mice at the doses of 12.5, 25 and 50 mg/kg showed the antinociceptive effect in a dose dependent manner compared to the respective controls in all assays. Inhibitions of the acetic acid-induced abdominal constrictions were observed at the doses of 12.5 (67.9%), 25 (85%) and 50 (99.5%) mg/kg compared to controls. Latex protein at the doses of 25 (39.8%; 42%) and 50 mg/kg (66.6%; 99.3%) reduced the nociception produced by formalin in the 1st and 2nd phases, respectively, and this effect was not reversed by pretreatment with naloxone (1 mg/kg). In the hot plate test, an increase of the reaction time was observed only at 60 min after the treatment with latex at the doses of 25 (79.5%) and 50 (76.9%) mg/kg, compared to controls and naloxone was ineffective to reverse the effect. It was concluded that the protein fraction derived from the whole latex of Calotropis procera possesses antinociceptive activity, which is independent of the opioid system.

Primary structure, behavioral and electroencephalographic effects of an epileptogenic peptide from the sea anemone Bunodosoma cangicum.

The primary structure of cangitoxin (CGX), a 4958 Da peptide from the sea anemone Bunodosoma cangicum, was determined: GVACRCDSDGPTVRGNSLSGTLWLTGGCPSGWHNCRGSGPFIGYCCKK. CGX contains all the 11 residues that are conserved and the 5 that are conservatively substituted within or between the type 1 and type 2 sequences of sea anemone peptides with specific action on voltage-sensitive sodium channels. Furthermore, it also has 6 identities (Asp9, Arg14, Asn16, Leu18, Trp33 and Lys48) and 1 homology (Arg36) in the 8 residues of the pharmacophore of the sea anemone ApB which are essential for interaction with mammalian sodium channels. The intrahippocampal injection of CGX induces several sequential behavioral alterations--episodes of akinesia alternating with facial automatisms and head tremor, salivation, rearing, jumping, barrel-rolling, wet dog shakes and forelimb clonic movements--and the electroencephalography analysis shows that they were followed by important seizure periods that gradually evolved to status epilepticus that lasted 8-12 h, similar to that observed in the acute phase of the pilocarpine model of epilepsy. These results suggest that CGX may be an important tool to develop a new experimental model of status epilepticus which may contribute to understanding the etiology of epilepsy and to test the effects of new antiepileptic drugs.