Harpalycin 2 inhibits the enzymatic and platelet aggregation activities of PrTX-III, a D49 phospholipase A2 from Bothrops pirajai venom.

BACKGROUND: Harpalycin 2 (HP-2) is an isoflavone isolated from the leaves of Harpalyce brasiliana Benth., a snakeroot found in northeast region of Brazil and used in folk medicine to treat snakebite. Its leaves are said to be anti-inflammatory. Secretory phospholipases A2 are important toxins found in snake venom and are structurally related to those found in inflammatory conditions in mammals, as in arthritis and atherosclerosis, and for this reason can be valuable tools for searching new anti-phospholipase A2 drugs. METHODS: HP-2 and piratoxin-III (PrTX-III) were purified through chromatographic techniques. The effect of HP-2 in the enzymatic activity of PrTX-III was carried out using 4-nitro-3-octanoyloxy-benzoic acid as the substrate. PrTX-III induced platelet aggregation was inhibited by HP-2 when compared to aristolochic acid and p-bromophenacyl bromide (p-BPB). In an attempt to elucidate how HP-2 interacts with PrTX-III, mass spectrometry, circular dichroism and intrinsic fluorescence analysis were performed. Docking scores of the ligands (HP-2, aristolochic acid and p-BPB) using PrTX-III as target were also calculated. RESULTS: HP-2 inhibited the enzymatic activity of PrTX-III (IC50 11.34 ± 0.28 µg/mL) although it did not form a stable chemical complex in the active site, since mass spectrometry measurements showed no difference between native (13,837.34 Da) and HP-2 treated PrTX-III (13,856.12 Da). A structural analysis of PrTX-III after treatment with HP-2 showed a decrease in dimerization and a slight protein unfolding. In the platelet aggregation assay, HP-2 previously incubated with PrTX-III inhibited the aggregation when compared with untreated protein. PrTX-III chemical treated with aristolochic acid and p-BPB, two standard PLA2 inhibitors, showed low inhibitory effects when compared with the HP-2 treatment. Docking scores corroborated these results, showing higher affinity of HP-2 for the PrTX-III target (PDB code: 1GMZ) than aristolochic acid and p-BPB. HP-2 previous incubated with the platelets inhibits the aggregation induced by untreated PrTX-III as well as arachidonic acid. CONCLUSION: HP-2 changes the structure of PrTX-III, inhibiting the enzymatic activity of this enzyme. In addition, PrTX-III platelet aggregant activity was inhibited by treatment with HP-2, p-BPB and aristolochic acid, and these results were corroborated by docking scores.

Renal and vascular effects of the natriuretic peptide isolated from Crotalus durissus cascavella venom.

Crotalus durissus cascavella is a snake that is usually found in the scrublands of northeast Brazil. The components of its venom may have effects on the vascular and renal systems. Recently, a new bradykinin inhibitory peptide has been identified in the venom of the Crotalinae family. The aim of the present study was to investigate the renal and vascular effects of the natriuretic peptide isolated from the venom of Crotalus durissus cascavella (NP2_Casca). The chromatographic profile showed the fractionation of substances identified as convulxin, gyroxin, crotoxin and crotamine, as well as fractions V and VI. The electrophoretic profile of fraction V consisted of several bands ranging from approximately 6kDa to 13kDa, while fraction VI showed only two main electrophoretic bands with molecular weights of approximately 6 and 14kDa. Reverse-phase chromatography showed that NP2_Casca corresponds to about 18% of fraction VI and that this fraction is the main natriuretic peptide. NP2_Casca was compared to other natriuretic peptides from other sources of snake venom. All amino acid sequences that were compared showed a consensus region of XGCFGX, XLDRIX and XSGLGCX. The group treated with NP2_Casca showed an increase in perfusion pressure, renal vascular resistance, urinary flow and glomerular filtration rate. The percent of total and proximal tubular transport of sodium was reduced significantly after administration of the peptide. The mean arterial pressure showed a dose-dependent decrease after infusion of NP2_Casca, and an increase in nitrite production. In the aortic ring assay, NP2_Casca caused a relaxant effect in endothelium-intact thoracic aortic rings precontracted with phenylephrine in the presence and absence of isatin. NP2_Casca failed to relax the aortic rings precontracted with an isosmotic potassium Krebs-Henseleit solution. In conclusion, the natriuretic peptide isolated from Crotalus durissus cascavella venom produced renal and vascular effects. NP2_Casca reduced total and proximal sodium tubular transport, leading to an increase in sodium excretion, thereby demonstrating a diuretic action. A hypotensive effect was displayed in an arterial pressure assay, with an increase in nitrite production, suggesting a possible vasoactive action.

Endothelial and metabolic disorders in adolescence: low birth weight is not an isolated risk factor.

BACKGROUND: Low birth weight (LBW) seems to be a determining factor for many cardiovascular diseases in adult life. OBJECTIVES: The objectives of this study were to investigate the occurrence of metabolic syndrome (MetS) and endothelial dysfunction in adolescents with LBW and compare them with subjects with normal birth weights (NBW). METHODS: This retrospective cohort study evaluated 172 adolescents (86 in each group) aged 10-20 years, who were born in a reference maternity hospital. The following criteria were adopted: International Diabetes Federation parameters for the diagnosis of MetS and flow-mediated brachial artery dilatation for endothelial dysfunction. Student's t-test or Mann-Whitney tests were used for continuous variables (depending on the normality of distribution evaluated by the Shapiro-Wilk test), and the Fisher exact test was used for categorical variables. The significance level was determined at 0.05. RESULTS: MetS was present in three cases of LBW and in four cases of NBW (p=0.70). The number of components of the syndrome was, on average, 0.6 and 0.5 in LBW and NBW, respectively (p=0.77). Endothelial dysfunction occurred in 25 (29.1%) cases of LBW and in 31 (36.1%) cases of NBW (p=0.33). CONCLUSION: LBW did not represent a risk factor in the occurrence of MetS or endothelial dysfunction in adolescence.

Isolation, homology modeling and renal effects of a C-type natriuretic peptide from the venom of the Brazilian yellow scorpion (Tityus serrulatus).

Mammalian natriuretic peptides (NPs) have been extensively investigated for use as therapeutic agents in the treatment of cardiovascular diseases. Here, we describe the isolation, sequencing and tridimensional homology modeling of the first C-type natriuretic peptide isolated from scorpion venom. In addition, its effects on the renal function of rats and on the mRNA expression of natriuretic peptide receptors in the kidneys are delineated. Fractionation of Tityus serrulatus venom using chromatographic techniques yielded a peptide with a molecular mass of 2190.64 Da, which exhibited the pattern of disulfide bridges that is characteristic of a C-type NP (TsNP, T. serrulatus Natriuretic Peptide). In the isolated perfused rat kidney assay, treatment with two concentrations of TsNP (0.03 and 0.1 µg/mL) increased the perfusion pressure, glomerular filtration rate and urinary flow. After 60 min of treatment at both concentrations, the percentages of sodium, potassium and chloride transport were decreased, and the urinary cGMP concentration was elevated. Natriuretic peptide receptor-A (NPR-A) mRNA expression was down regulated in the kidneys treated with both concentrations of TsNP, whereas NPR-B, NPR-C and CG-C mRNAs were up regulated at the 0.1 µg/mL concentration. In conclusion, this work describes the isolation and modeling of the first natriuretic peptide isolated from scorpion venom. In addition, examinations of the renal actions of TsNP indicate that its effects may be related to the activation of NPR-B, NPR-C and GC-C.

Vasoconstrictor effect of Africanized honeybee (Apis mellifera L.) venom on rat aorta.

BACKGROUND: Apis mellifera stings are a problem for public health worldwide, particularly in Latin America due to the aggressiveness of its Africanized honeybees. Massive poisoning by A. mellifera venom (AmV) affects mainly the cardiovascular system, and several works have described its actions on heart muscle. Nevertheless, no work on the pharmacological action mechanisms of the AmV in isolated aorta has been reported. Thus, the present work aimed to investigate the actions of AmV and its main fractions, phospholipase A2 (PLA2) and melittin, on isolated aorta rings and a probable action mechanism. RESULTS: AmV and the complex PLA2 + melittin (0.1-50 µg/mL) caused contraction in endothelium-containing aorta rings, but neither isolated PLA2 nor melittin were able to reproduce the effect. Endothelium removal did not change the maximum vasoconstrictor effect elicited by AmV. Ca2+-free medium, as well as treatment with phentolamine (5 µM), verapamil (10 µM), losartan (100 µM), and U-73122 (10 µM, a phospholipase C inhibitor), eliminated the AmV-induced contractile effects. CONCLUSIONS: In conclusion, AmV caused contractile effect in aorta rings probably through the involvement of voltage-operated calcium channels, AT1 and α-adrenergic receptors via the downstream activation of phospholipase C. The protein complex, PLA2 + melittin, was also able to induce vasoconstriction, whereas the isolated proteins were not.

Inhibitory effects of sertraline in rat isolated perfused kidneys and in isolated ring preparations of rat arteries.

OBJECTIVES: Sertraline is often prescribed to patients suffering with end stage renal disease, but its action on kidney has not been investigated. We aimed to investigate the pharmacological action of sertraline on rat kidney with emphasis on the underlying mechanisms involved in the vascular actions of the drug. METHODS: The effects of sertraline were evaluated in rat isolated perfused kidneys and on ring preparations of mesenteric or segmental rat renal artery. KEY FINDINGS: In kidneys, sertraline prevented the effects of phenylephrine on perfusion pressure, glomerular filtration rate, urinary flow and renal vascular resistance. In mesenteric rings sertraline inhibited phenylephrine-induced contractions with potency 30-times lower than verapamil. Sertraline reversed sustained contractions induced by phenylephrine or 60mm K(+) within a similar concentration range. In segmental isolated rings, sertraline also reversed contractions induced by phenylephrine or 60mm K(+) with the same concentration range, but with higher potency compared with mesenteric preparations. Under Ca(2+) -free conditions, sertraline did not change the intracellularly-mediated phasic contractions induced by phenylephrine or caffeine. Sertraline was ineffective against contractions induced by extracellular Ca(2+) restoration after thapsigargin treatment and Ca(2+) store depletion with phenylephrine. Conversely, sertraline decreased the contractions induced by Ca(2+) addition in tissues under high K(+) solution or phenylephrine plus verapamil. CONCLUSIONS: In rat isolated kidneys and in rat ring preparations of mesenteric or renal vessels, sertraline had antispasmodic effects that appeared to be caused by a direct action on vascular smooth muscle cells. Its actions were ineffective against Ca(2+) -releasing intracellular pathways, but appeared to interfere with sarcolemmal Ca(2+) influx with reduced permeability of both receptor- and voltage-gated Ca(2+) channels.

Hypotensive effects of the Crotalus durissus cascavella venom: involvement of NO.

Crotalus durissus cascavella is a snake native of northeastern Brazil. The aim of the study was to investigate the effects of C. d. cascavella venom on rat mean arterial pressure and vascular reactivity in the mesenteric vascular bed. The venom evoked a dose-dependent decrease in mean arterial pressure, cardiac and respiratory frequency with increased plasma nitrite levels. L-NAME (10 mg/kg) blunted both the hypotension and increased nitrite production observed after the venom administration. To investigate the effects of C. d. cascavella in resistance vessels, the vascular mesenteric bed was studied, and the results suggested that the hypotensive effect of the venom is not dependent on a direct vasodilatory activity. In conclusion, C. d. cascavella venom presented indirect hypotensive effects with the involvement of nitric oxide.

Purification and characterization of the biological effects of phospholipase A(2) from sea anemone Bunodosoma caissarum.

Sea anemones contain a variety of biologically active substances. Bunodosoma caissarum is a sea anemone from the Cnidaria phylum, found only in Brazilian coastal waters. The aim of the present work was to study the biological effects of PLA(2) isolated from the sea anemone B. caissarum on the isolated perfused kidney, the arteriolar mesenteric bed and on insulin secretion. Specimens of B. caissarum were collected from the São Vicente Channel on the southern coast of the State of São Paulo, Brazil. Reverse phase HPLC analysis of the crude extract of B. caissarum detected three PLA(2) proteins (named BcPLA(2)1, BcPLA(2)2 and BcPLA(2)3) found to be active in B. caissarum extracts. MALDI-TOF mass spectrometry of BcPLA(2)1 showed one main peak at 14.7 kDa. The N-terminal amino acid sequence of BcPLA(2)1 showed high amino acid sequence identity with PLA(2) group III protein isolated from the Mexican lizard (PA23 HELSU, HELSU, PA22 HELSU) and with the honey bee Apis mellifera (PLA(2) and 1POC_A). In addition, BcPLA(2)1 also showed significant overall homology to bee PLA(2). The enzymatic activity induced by native BcPLA(2)1 (20 microg/well) was reduced by chemical treatment with p-bromophenacyl bromide (p-BPB) and with morin. BcPLA(2)1 strongly induced insulin secretion in presence of high glucose concentration. In isolated kidney, the PLA(2) from B. caissarum increased the perfusion pressure, renal vascular resistance, urinary flow, glomerular filtration rate, and sodium, potassium and chloride levels of excretion. BcPLA(2)1, however, did not increase the perfusion pressure on the mesenteric vascular bed. In conclusion, PLA(2), a group III phospholipase isolated from the sea anemone B. caissarum, exerted effects on renal function and induced insulin secretion in conditions of high glucose concentration.

Vasoconstrictor effect of Africanized honeybee (Apis mellifera L.) venom on rat aorta

Background : Apis mellifera stings are a problem for public health worldwide, particularly in Latin America due to the aggressiveness of its Africanized honeybees. Massive poisoning by A. mellifera venom (AmV) affects mainly the cardiovascular system, and several works have described its actions on heart muscle. Nevertheless, no work on the pharmacological action mechanisms of the AmV in isolated aorta has been reported. Thus, the present work aimed to investigate the actions of AmV and its main fractions, phospholipase A2 (PLA2) and melittin, on isolated aorta rings and a probable action mechanism. Results : AmV and the complex PLA2 + melittin (0.1-50 g/mL) caused contraction in endothelium-containing aorta rings, but neither isolated PLA2 nor melittin were able to reproduce the effect. Endothelium removal did not change the maximum vasoconstrictor effect elicited by AmV. Ca2+-free medium, as well as treatment with phentolamine (5 M), verapamil (10 M), losartan (100 M), and U-73122 (10 M, a phospholipase C inhibitor), eliminated the AmV-induced contractile effects. Conclusions : In conclusion, AmV caused contractile effect in aorta rings probably through the involvement of voltage-operated calcium channels, AT1 and -adrenergic receptors via the downstream activation of phospholipase C. The protein complex, PLA2 + melittin, was also able to induce vasoconstriction, whereas the isolated proteins were not.

Vasoconstrictor effect of Africanized honeybee (Apis mellifera L. ) venom on rat aorta

Background : Apis mellifera stings are a problem for public health worldwide, particularly in Latin America due to the aggressiveness of its Africanized honeybees. Massive poisoning by A. mellifera venom (AmV) affects mainly the cardiovascular system, and several works have described its actions on heart muscle. Nevertheless, no work on the pharmacological action mechanisms of the AmV in isolated aorta has been reported. Thus, the present work aimed to investigate the actions of AmV and its main fractions, phospholipase A2 (PLA2) and melittin, on isolated aorta rings and a probable action mechanism. Results : AmV and the complex PLA2 + melittin (0.1-50 μg/mL) caused contraction in endothelium-containing aorta rings, but neither isolated PLA2 nor melittin were able to reproduce the effect. Endothelium removal did not change the maximum vasoconstrictor effect elicited by AmV. Ca2+-free medium, as well as treatment with phentolamine (5 μM), verapamil (10 μM), losartan (100 μM), and U-73122 (10 μM, a phospholipase C inhibitor), eliminated the AmV-induced contractile effects. Conclusions : In conclusion, AmV caused contractile effect in aorta rings probably through the involvement of voltage-operated calcium channels, AT1 and α-adrenergic receptors via the downstream activation of phospholipase C. The protein complex, PLA2 + melittin, was also able to induce vasoconstriction, whereas the isolated proteins were not.(AU)