Krishnamurti M. Carvalho

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.

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.

Anticonvulsant action of Calotropis procera latex proteins.

Calotropis procera (Ait.) R.Br. is a laticiferous plant belonging to the Apocynaceae family. C. procera latex proteins were evaluated with respect to anticonvulsant and sedative activity in mouse models of pentylenetetrazol (PTZ)-, pilocarpine-, and strychnine-induced convulsions or turning behavior and pentobarbital-induced sleep. In the strychnine- and pilocarpine-induced seizure models, C. procera latex proteins caused no significant alterations in latencies to convulsions and death, as compared with controls. In the PTZ-induced seizure model, administration of C. procera latex proteins in high doses (50 or 100mg/kg) and diazepam caused significant increases in latencies to convulsions and death. C. procera latex proteins (50 or 100mg/kg) and 2mg/kg diazepam caused a decrease in sleep latency and an increase in sleep time compared with the control group and groups treated with 5 or 10mg/kg. Our results suggest that C. procera latex proteins have a central nervous system-depressant activity as reflected in their potentiation of pentobarbital-induced sleeping time and their anticonvulsant action in the PTZ-induced seizure model.

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 (BPPs) 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.

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.

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.

Central Nervous System Activity of Acute Administration of Latex Proteins from Calotropis procera in Mice

Relevant properties have been detected in protein fraction (LP) isolated from latex of Calotropis procera. In order to best characterize LP potentiality, this work describes some effects of LP on central nervous system (CNS). Three models were performed: elevated plus maze (EPM), open field (OF), and hole-board (HB) tests. LP was administered intraperitoneally to male mice. To assess the probable action mechanism, drugs acting on glutamatergic (ketamine, i.p.), gabaergic (diazepam and flumazenil, i.p.) and dopaminergic (haloperidol, i.p.) systems were used on OF. Decreases in the number of entries in the open and closed arms of EPM were observed at 100 mg/kg and this effect may be due to the reduction in the locomotor activity previously observed. A reduction in locomotor activity was observed in animals which received LP 50 and 100 mg/kg. There was a decrease in rearing at all doses; a reduction in grooming was seen only at 50 and 100 mg/kg. Ketamine reversed the effects of LP, and haloperidol reinforced the hypolocomotor effects of LP. In HB, there was a decrease in the number of head dips after LP 100 mg/kg; such effect may also be due to the reduction of the locomotor activity. The results suggest that LP from C. procera have depressant effects on the CNS, but do not present any anxiolytic effect. These effects may reflect a dopaminergic action. The active doses were considerably higher than those observed for anticancer, anti-inflammatory and anti-nociceptive activities described in the literature.