Banisteriopsis caapi extract: Implications for Neuroinflammatory Pathways in Locus coeruleus Lesion Rodent Model.

ETHNOPHARMACOLOGY RELEVANCE: Ayahuasca is a beverage obtained from the decoctions of Banisteriopsis caapi (Spruce ex Griseb.) Morton and Psychotria viridis Ruiz & Pav., used throughout the Amazon as a medicinal beverage for healing and spiritual exploration. The Banisteriopsis caapi extract consists of harmine, harmaline, and tetrahydroharmine (THH); which inhibit the isoforms of monoamine oxidase A and B. In the central nervous system (CNS), it can increase the norepinephrine (NE) concentration, produced in the Locus coeruleus (LC), reducing inflammation that is associated with some neurological disease, such as Parkinson's disease and Alzheimer's disease AIM OF THE STUDY: evaluate the effects of treatment with B. caapi extract on the neuroinflammatory profile in animals with selective LC lesions MATERIAL AND METHODS: male Wistar rats with LC lesions induced by 6-hydroxydopamine were treated with B. caapi extract. Subsequently, behavioral tests were conducted, including the elevated plus maze, rotarod, and open field. Tyrosine hydroxylase positive (TH+) neurons and IBA-1 positive microglia were quantified from the LC inflammatory markers and free radical products were assessed RESULTS: Both 6-Hydroxydopamine hydrochloride and the Banisteriopsis caapi extract causes reduction of LC neurons, at the concentration and frequency used. The LC depletion and the treatment of B. caapi extract interfere with locomotion. B.caapi extract and the LC lesion increased the number and activation of inflammatory cells, such as microglia. B. caapi extract decreases IL-10 in the hippocampus and BDNF gene expression. CONCLUSION: This study suggests that B. caapi extract (at the concentration and frequency used) promotes noradrenergic neuron depletion and creates a proinflammatory environment in the CNS.

Memory reactivation mediates emotional valence updating of contextual memory in mice with protracted morphine withdrawal.

Mice subjected to morphine locomotor sensitization develop increased anxiety-behavior expression during protracted morphine withdrawal. This behavioral change is dependent on reexposure to the context of locomotor sensitization and reflects a state of conditioned anxiety. In this study, the effect of memory reconsolidation on the expression of conditioned anxiety in mice with protracted morphine withdrawal was examined. Five experimental protocols involving male C57BL/6 mice were used in which the animals were subjected to locomotor sensitization induced by morphine and reexposed to the context associated with the drug effect 28 days after locomotor sensitization and immediately after subjected to elevated plus maze. In experiment 1, mice were subjected or not to memory reactivation session and was observed that memory reactivation 27 days after sensitization reduced conditioned anxiety. In experiment 2, mice were subjected to memory reactivation, 24 h, 6 h or 1 h before contextual reexposure, and the effect of memory reactivation coincided with the temporal requirement for reconsolidation. In experiment 3, which involved exposure to a situation of acute stress immediately before memory reactivation, the mice demonstrated a return to increased conditioned anxiety. To confirm the influence of reconsolidation, in experiments 4 and 5, mice subjected to memory reactivation were treated with Nimodipine, diazepam or cyclohexamine, substances commonly used as pharmacological controls in reconsolidation experiments. Treatment with each substance separately inhibited the effect of reactivation in experiment 5 (presence of acute stressor) but not in experiment 4 (absence of acute stressor). These results suggest that, in our experimental model, reconsolidation is mediated through updating of the emotional valence of contextual memory associated with the administration of morphine.

Fmr1 exon 14 skipping in late embryonic development of the rat forebrain.

BACKGROUND: Fragile X syndrome, the major cause of inherited intellectual disability among men, is due to deficiency of the synaptic functional regulator FMR1 protein (FMRP), encoded by the FMRP translational regulator 1 (FMR1) gene. FMR1 alternative splicing produces distinct transcripts that may consequently impact FMRP functional roles. In transcripts without exon 14 the translational reading frame is shifted. For deepening current knowledge of the differential expression of Fmr1 exon 14 along the rat nervous system development, we conducted a descriptive study employing quantitative RT-PCR and BLAST of RNA-Seq datasets. RESULTS: We observed in the rat forebrain progressive decline of total Fmr1 mRNA from E11 to P112 albeit an elevation on P3; and exon-14 skipping in E17-E20 with downregulation of the resulting mRNA. We tested if the reduced detection of messages without exon 14 could be explained by nonsense-mediated mRNA decay (NMD) vulnerability, but knocking down UPF1, a major component of this pathway, did not increase their quantities. Conversely, it significantly decreased FMR1 mRNA having exon 13 joined with either exon 14 or exon 15 site A. CONCLUSIONS: The forebrain in the third embryonic week of the rat development is a period with significant skipping of Fmr1 exon 14. This alternative splicing event chronologically precedes a reduction of total Fmr1 mRNA, suggesting that it may be part of combinatorial mechanisms downregulating the gene's expression in the late embryonic period. The decay of FMR1 mRNA without exon 14 should be mediated by a pathway different from NMD. Finally, we provide evidence of FMR1 mRNA stabilization by UPF1, likely depending on FMRP.

Kinetic profile of N,N-dimethyltryptamine and ß-carbolines in saliva and serum after oral administration of ayahuasca in a religious context.

Ayahuasca is a beverage obtained from Banisteriopsis caapi plus Psychotria viridis. B. caapi contains the ß-carbolines harmine, harmaline, and tetrahydroharmine that are monoamine oxidase inhibitors and P. viridis contains N,N-dimethyltryptamine (DMT) that is responsible for the visionary effects of the beverage. Ayahuasca use is becoming a global phenomenon, and the recreational use of DMT and similar alkaloids has also increased in recent years; such uncontrolled use can lead to severe intoxications. In this investigation, liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to study the kinetics of alkaloids over a 24 h period in saliva and serum of 14 volunteers who consumed ayahuasca twice a month in a religious context. We compared the area under the curve (AUC), maximum concentration (Cmax ), time to reach Cmax (Tmax ), mean residence time (MRT), and half-life (t1/2 ), as well as the serum/saliva ratios of these parameters. DMT and ß-carboline concentrations (Cmax ) and AUC were higher in saliva than in serum and the MRT was 1.5-3.0 times higher in serum. A generalized estimation equations (GEEs) model suggested that serum concentrations could be predicted by saliva concentrations, despite large individual variability in the saliva and serum alkaloid concentrations. The possibility of using saliva as a biological matrix to detect DMT, ß-carbolines, and their derivatives is very interesting because it allows fast noninvasive sample collection and could be useful for detecting similar alkaloids used recreationally that have considerable potential for intoxication.

Inhaled Lavandula angustifolia essential oil enhances extinction learning and inhibits memory updating in mice submitted to the contextual fear conditioning.

ETHNOPHARMACOLOGICAL RELEVANCE: Lavender (Lavandula angustifolia) essential oil (EO) has a long history of use in emotional illness, including anxiety disorders. Cognitive mechanisms of learning and memory play a pivotal role in the etiology and maintenance of anxiety since exposure to cues related to aversive situations induces high arousal and anticipatory anxiety. Memory become labile after its reactivation and can be modulated by reconsolidation or extinction. Inhibition of memory reconsolidation or facilitation of memory extinction may be effective in preventing or minimizing the effect of contextual cues on anticipatory anxiety. AIM OF THE STUDY: We investigated the effect of Lavandula angustifolia EO in the memory updating of conditioned contextual fear. MATERIALS AND METHODS: Adult male C57Bl6 mice were submitted to fear conditioning. Two days after conditioning the mice underwent a reactivation session in a hybrid context and were then immediately exposed to vaporized water or essential oil at concentrations of 1%, 2.5% or 5% for 3 h. Two days later, the mice were tested in the original or an altered context and their freezing behavior was measured. In addition, mice were subjected to a fear memory recovery protocol followed by a reinstatement session. RESULTS: In the contextual fear test, 1% essential oil, but not 2.5% or 5%, reduced the freezing behavior response, whereas after a reinstatement session, exposure to 1% essential oil increased the freezing behavior response. CONCLUSIONS: These results suggest that Lavandula angustifolia essential oil enhances memory extinction and, consequently, inhibits memory updating.

Anxiety-like behavior in acute and protracted withdrawal after morphine-induced locomotor sensitization in C57BL/6 male mice: The role of context.

Contextual memory plays an important role in development and maintenance of drug addiction. However, little is known about of the role contextual memory in the emergence of a negative emotional state in the withdrawal period. Therefore, this study investigated anxiety-like behavior in acute and protracted morphine withdrawal of mice submitted to a locomotor sensitization protocol and the influence of contextual memory on this behavior. Male adult C57Bl6 mice were subjected to morphine locomotor sensitization and anxiety-like behavior was assessed by using the elevated plus maze test (EPM). To evaluate associative memory, the mice were re-exposed to the context of locomotor sensitization immediately before EPM. As expected, repeated morphine administrations promoted locomotor sensitization, seen as a gradual increase in the distance traveled during the acquisition phase. There was an increase in anxiety-like behavior upon acute withdrawal, as indicated by a decrease in open arms activity (OAA), but this effect dissipated over time. However, when the context was presented, mice in protracted withdrawal showed enhanced anxiety-like behavior, indicated by an increase in closed arms activity (CAA). This effect was context specific since re-exposure in an alternative context did not change the anxiety-like behavior. Treatment with diazepam counteracted the decrease in OAA in acute withdrawal and the increase in CAA induced by context re- exposure during protracted abstinence. Thus, repeated morphine administration induced a negative emotional state when the drug was discontinued. The context associated with drug exposure played a pivotal role in the appearance of anxiety-like behavior, even long after drug discontinuation. There were differences in the patterns of anxiety behaviors in acute (unconditioned anxiety-like behavior) and protracted (conditioned anxiety-like behavior) withdrawal since the former was characterized by a passive behavioral strategy and the latter by an active behavioral strategy.

Effect of Ritualistic Consumption of Ayahuasca on Hepatic Function in Chronic Users.

Ayahuasca is a beverage obtained from decoctions of the liana Banisteriopsis caapi plus the shrub Psychotria viridis. This beverage contains a combination of monoamine oxidase inhibitors (harmine, harmaline, and tetrahydroharmine) and N,N-dimethyltryptamine, the main substance responsible for its visionary effect. The ritualistic use of ayahuasca is becoming a global phenomenon. Most members of ayahuasca churches consume this beverage throughout their life, and many reports have discussed the therapeutic potential of this beverage. Ayahuasca is consumed orally, and the liver, as the major organ for the metabolism and detoxification of xenobiotics absorbed from the alimentary tract, may be susceptible to injury by compounds present in the ayahuasca decoction. In this study, we evaluated biochemical parameters related to hepatic damage in the serum of 22 volunteers who consumed ayahuasca twice a month or more for at least one year. There was no significant alteration in the following parameters: alanine aminotransferase, aspartate aminotransferase, bilirubin, creatinine, urea, lactate dehydrogenase, alkaline phosphatase, and gamma glutamyl transferase. These findings indicate that chronic ayahuasca consumption in a religious context apparently does not affect hepatic function.

Mutagenicity of Ayahuasca and Their Constituents to the Salmonella/Microsome Assay.

Ayahuasca is a beverage used in religious rituals of indigenous and nonindigenous groups, and its therapeutic potential has been investigated. Ayahuasca is obtained by decoction of the Banisteriopsis caapi that contains ß-carbolines (harmine, harmaline, and tetrahydroharmine) plus Psychotria viridis that contains N,N-dimethyltryptamine. Although plants used in folk medicine are recognized as safe, many of them have genotoxic potential. The Salmonella/microsome assay is usually the first line of the mutagenicity evaluation of products intended for therapeutic use. Our objective was to evaluate the mutagenicity of ayahuasca beverage and their constituents using the Salmonella/microsome assay with TA98 and TA100. We analyzed two ayahuasca samples, and also beverage samples prepared each individual plant P. viridis and B. caapi. Harmine and harmaline were also tested. All beverage samples were chemically characterized and both ayahuasca samples could be considered representative of the beverages consumed in religious rituals. Both ayahuasca samples were mutagenic for TA98 and TA100 with and without S9, with similar potencies. The beverage obtained from P. viridis was not mutagenic, and beverage obtained from B. caapi was mutagenic for TA98 with and without S9. Harmine was nonmutagenic and harmaline was mutagenic only for TA98 without S9. Harmaline fully explain the mutagenicity observed with TA98 without S9 of both ayahuasca samples and the B. caapi beverage samples. We conclude that the ayahuasca samples are mutagenic and this effect is partially explained by harmaline, one of the ß-carbolines present in the beverage. Other mutagenic compounds seem to be present and need to be further investigated. Environ. Mol. Mutagen. 60:269-276, 2019. © 2018 Wiley Periodicals, Inc.

Inhaled Lavandula angustifolia essential oil inhibits consolidation of contextual- but not tone-fear conditioning in rats.

Although the current treatment for anxiety is effective, it promotes a number of adverse reactions and medical interactions. Inhaled essential oils have a prominent action on the central nervous system, with minimal systemic effects, primarily because of reduced systemic bioavailability. The effects of drugs on the consolidation of fear conditioning reflects its clinical efficacy in preventing a vicious cycle of anticipatory anxiety leading to fearful cognition and anxiety symptoms. In this study, we investigated the effects of inhaled Lavandula angustifolia essential oil on the consolidation of aversive memories and its influence on c-Fos expression. Adult male Wistar rats were subjected to a fear conditioning protocol. Immediately after the training session, the rats were exposed to vaporized water or essential oil (1%, 2.5% and 5% solutions) for 4h. The next day, the rats underwent contextual- or tone-fear tests and 90min after the test they were euthanized and their brains processed for c-Fos immunohistochemistry. In the contextual-fear test, essential oil at 2.5% and 5% (but not 1%) reduced the freezing response and its respective c-Fos expression in the ventral hippocampus and amygdala. In the tone-fear test, essential oil did not reduce the freezing response during tone presentation. However, rats that inhaled essential oil at 2.5% and 5% (but not 1%) showed decreased freezing in the three minutes after tone presentation, as well as reduced c-Fos expression in the prefrontal cortex and amygdala. These results show that the inhalation of L. angustifolia essential oil inhibited the consolidation of contextual- but not tone-fear conditioning and had an anxiolytic effect in a conditioned animal model of anxiety.

The use of aromatase inhibitors in boys with short stature: what to know before prescribing?

Aromatase is a cytochrome P450 enzyme (CYP19A1 isoform) able to catalyze the conversion of androgens to estrogens. The aromatase gene mutations highlighted the action of estrogen as one of the main regulators of bone maturation and closure of bone plate. The use of aromatase inhibitors (AI) in boys with short stature has showed its capability to improve the predicted final height. Anastrozole (ANZ) and letrozole (LTZ) are nonsteroidal inhibitors able to bind reversibly to the heme group of cytochrome P450. In this review, we describe the pharmacokinetic profile of both drugs, discussing possible drug interactions between ANZ and LTZ with other drugs. AIs are triazolic compounds that can induce or suppress cytochrome P450 enzymes, interfering with metabolism of other compounds. Hydroxilation, N-dealkylation and glucoronidation are involved in the metabolism of AIs. Drug interactions can occur with azole antifungals, such as ketoconazole, by inhibiting CYP3A4 and by reducing the clearance of AIs. Antiepileptic drugs (lamotrigine, phenobarbital, and phenytoin) also inhibit aromatase. Concomitant use of phenobarbital or valproate has a synergistic effect on aromatase inhibition. Therefore, it is important to understand the pharmacokinetics of AIs, recognizing and avoiding possible drug interactions and offering a safer prescription profile of this class of aromatase inhibitors. Arch Endocrinol Metab. 2017;61(3):391-7.

The use of aromatase inhibitors in boys with short stature: what to know before prescribing?

ABSTRACT Aromatase is a cytochrome P450 enzyme (CYP19A1 isoform) able to catalyze the conversion of androgens to estrogens. The aromatase gene mutations highlighted the action of estrogen as one of the main regulators of bone maturation and closure of bone plate. The use of aromatase inhibitors (AI) in boys with short stature has showed its capability to improve the predicted final height. Anastrozole (ANZ) and letrozole (LTZ) are nonsteroidal inhibitors able to bind reversibly to the heme group of cytochrome P450. In this review, we describe the pharmacokinetic profile of both drugs, discussing possible drug interactions between ANZ and LTZ with other drugs. AIs are triazolic compounds that can induce or suppress cytochrome P450 enzymes, interfering with metabolism of other compounds. Hydroxilation, N-dealkylation and glucoronidation are involved in the metabolism of AIs. Drug interactions can occur with azole antifungals, such as ketoconazole, by inhibiting CYP3A4 and by reducing the clearance of AIs. Antiepileptic drugs (lamotrigine, phenobarbital, and phenytoin) also inhibit aromatase. Concomitant use of phenobarbital or valproate has a synergistic effect on aromatase inhibition. Therefore, it is important to understand the pharmacokinetics of AIs, recognizing and avoiding possible drug interactions and offering a safer prescription profile of this class of aromatase inhibitors. Arch Endocrinol Metab. 2017;61(3):391-7.

Pharmacological Characterization of the Edema Caused by Vitalius dubius (Theraphosidae, Mygalomorphae) Spider Venom in Rats.

Bites by tarantulas (Theraphosidae, Mygalomorphae) in humans can result in mild clinical manifestations such as local pain, erythema, and edema. Vitalius dubius is a medium-sized, nonaggressive theraphosid found in southeastern Brazil. In this work, we investigated the mediators involved in the plasma extravasation caused by V. dubius venom in rats. The venom caused dose-dependent (0.1-100 µg/site) edema in rat dorsal skin. This edema was significantly inhibited by ((S)1-{2-[3(3-4-dichlorophenyl)-1-(3-iso-propoxyphenylacetyl)piperidine-3-yl]ethyl}-4-phenyl-1-azoniabicyclo[2.2.2]octone, chloride) (SR140333, a neurokinin NK1 receptor antagonist), indomethacin [a nonselective cyclooxygenase (COX) inhibitor], cyproheptadine (a serotonin 5-hydroxytryptamine1/2 and histamine H1 receptor antagonist), and N(ω)-nitro-L-arginine methyl ester (a nitric oxide synthase inhibitor). In contrast, mepyramine (a histamine H1 receptor antagonist), D-Arg-[Hyp(3),Thi(5),D-Tic(7),Oic(8)-]-BK (JE 049, a bradykinin B2 receptor antagonist), and ((S)-N-methyl-N-[4-(4-acetylamino-4-phenylpiperidino)-2-(3,4-di-chlorophenyl)butyl]benzamide) (SR48968, a neurokinin NK2 receptor antagonist) had no effect on the venom-induced increase in vascular permeability. In rat hind paws, the venom-induced edema was attenuated by ketoprofen (a nonselective COX inhibitor) administered 15 minutes postvenom. Preincubation of venom with commercial antiarachnid antivenom attenuated the venom-induced edema. These results suggest that the enhanced vascular permeability evoked by V. dubius venom involves serotonin, COX products, neurokinin NK1 receptors, and nitric oxide formation. The attenuation of hind paw edema by ketoprofen suggests that COX inhibitors could be useful in treating the local inflammatory response to bites by these spiders.

Ritualistic Use of Ayahuasca versus Street Use of Similar Substances Seized by the Police: A Key Factor Involved in the Potential for Intoxications and Overdose?

The ritualistic use of ayahuasca is becoming a global phenomenon. This beverage contains a combination of monoamine oxidase inhibitors (harmine, harmaline, and tetrahydroharmine) and N,N-dimethyltryptamine, the main substance responsible for its visionary effect. The recreational use of similar alkaloids and N,N-dimethyltryptamine has increased in recent years, mainly because of their hallucinogenic effects. In the present study, the concentrations of psychoactive alkaloids in three powder samples seized by the São Paulo State Police and nine ayahuasca aqueous extracts were analyzed by HPLC-DAD in an attempt to distinguish between illicit drugs and the religious beverage. The alkaloids detected (µg/mL) in the ayahuasca aqueous extracts were N,N-dimethyltryptamine (402-2070.3), harmaline (27.5-181.3), harmine (294.5-2893.8), and tetrahydroharmine (849.5-2052.5), whereas, of the three powder samples, one contained only N,N-dimethyltryptamine (82% and 2% w/w, respectively) while the other contained only harmaline (16%, w/w) and harmine (12%, w/w). The ritualistic use of ayahuasca involves oral intake and the probability of overdose is minimized by serotonergic stimulation of vagal pathways, leading to vomiting and diarrhea. In contrast, the recreational use of N,N-dimethyltryptamine involves consumption mainly by smoking or inhalation, both of which markedly increase its bioavailability and the potential for intoxications.

Differential patterns of expression of neuropeptide Y throughout abstinence in outbred Swiss mice classified as susceptible or resistant to ethanol-induced locomotor sensitization.

Several studies have focused on the negative emotional state associated with drug abstinence. The peptide NPY plays an important role given its involvement in drug addiction, anxiety, and mood disorders. Interestingly, it is well established that outbred Swiss mice exhibit a prominent behavioral variability to ethanol-induced locomotor sensitization. Here, we investigated whether mice that were either susceptible or resistant to ethanol sensitization differed in their NPY expression during abstinence. The mice were treated daily with ethanol (2 g/kg, i.p.) or saline for 21 days. According to the locomotor activity after the last injection, the ethanol group was classified as sensitized (EtOH_High) or non-sensitized (EtOH_Low). To evaluate NPY expression, some of the mice were sacrificed at 18 h or 5 days of abstinence, and others were challenged at the 5th day of abstinence with ethanol (1.4 g/kg) and sacrificed after 1.5 h. At 5 days of abstinence, NPY expression increased in the orbital cortex, dorsomedial striatum, and dentate gyrus in the EtOH_High mice. These changes were counteracted by the ethanol challenge. In the EtOH_Low mice, NPY expression increased in the dentate gyrus only after 18 h of abstinence. Lastly, a decreased level of NPY was found in the prelimbic cortex of the EtOH_Low mice at 5 days of abstinence, and this was reversed by ethanol challenge. Therefore, behavioral variability in ethanol sensitization confers differential neurochemical features during the subsequent abstinence, including distinct patterns of NPY expression.

Histological and functional renal alterations caused by Bothrops alternatus snake venom: expression and activity of Na+/K+-ATPase.

BACKGROUND: Acute renal failure is a serious complication of human envenoming by Bothrops snakes. The ion pump Na+/K+-ATPase has an important role in renal tubule function, where it modulates sodium reabsorption and homeostasis of the extracellular compartment. Here, we investigated the morphological and functional renal alterations and changes in Na+/K+-ATPase expression and activity in rats injected with Bothrops alternatus snake venom. METHODS: Male Wistar rats were injected with venom (0.8 mg/kg, i.v.) and renal function was assessed 6, 24, 48 and 72 h and 7 days post-venom. The rats were then killed and renal Na+/K+-ATPase activity was assayed based on phosphate release from ATP; gene and protein expressions were assessed by real time PCR and immunofluorescence microscopy, respectively. RESULTS: Venom caused lobulation of the capillary tufts, dilation of Bowman's capsular space, F-actin disruption in Bowman's capsule and renal tubule brush border, and deposition of collagen around glomeruli and proximal tubules that persisted seven days after envenoming. Enhanced sodium and potassium excretion, reduced proximal sodium reabsorption, and proteinuria were observed 6 h post-venom, followed by a transient decrease in the glomerular filtration rate. Gene and protein expressions of the Na+/K+-ATPase α1 subunit were increased 6h post-venom, whereas Na+/K+-ATPase activity increased 6 h and 24 h post-venom. CONCLUSIONS: Bothrops alternatus venom caused marked morphological and functional renal alterations with enhanced Na+/K+-ATPase expression and activity in the early phase of renal damage. GENERAL SIGNIFICANCE: Enhanced Na+/K+-ATPase activity in the early hours after envenoming may attenuate the renal dysfunction associated with venom-induced damage.

Renal kinetics of Bothrops alternatus (Urutu) snake venom in rats.

The renal kinetics of Bothrops alternatus venom (0.8 mg/kg, i.v.) was studied in conscious male Wistar rats. Blood, urine and renal tissue samples were collected at various intervals after envenoming. Venom was quantified by ELISA in serum, renal tissue and urine. Urine volume was measured and the urine assayed for urobilinogen, glucose, bilirubin, ketones, urine specific gravity, occult blood, pH, protein, nitrite and leucocytes. Circulating venom showed biexponential kinetics, with no venom being detected after 7 days post-venom. Venom was detected in renal tissue 30 min post-venom but decreased progressively thereafter, in parallel with serum venom concentrations. Immunohistochemistry detected venom in glomeruli, proximal and distal tubules, and vascular and perivascular tissue. Venom was detected in urine 3, 6 and 24 h post-venom. Oliguria occurred 3 h to 7 days post-venom, urine acidification occurred 3-6 h post-venom, urine specific gravity increased in the first 3 h and proteinuria was also greatest in this period. Creatinine clearance decreased progressively until 24-48 h post-venom, then returned to normal. Glucose, ketones, leucocytes and occult blood were detected mainly during the first 6 h post-venom. These results indicate reversible alterations in renal function, with renal elimination of the venom.

Biological activities of a lectin from Bothrops jararacussu snake venom.

Snake venoms contain saccharide-binding lectins. In this work, we examined the biological activities of a lectin (BjcuL) purified from Bothrops jararacussu snake venom by chromatography on non-derivatized Sepharose 4B and Sephacryl S-200 HR. The protein, a homodimer with subunits of 14.5 kDa, gave a single immunoprecipitin line in immunoelectrophoresis and cross-reacted in ELISA with antivenoms raised against Bothrops spp. (lanceheads), Micrurus spp. (coral snakes), Crotalus durissus terrificus (South American rattlesnake), and arthropod (Loxosceles gaucho, Phoneutria nigriventer and Tityus serrulatus) venoms. BjcuL agglutinated human formaldehyde-fixed erythrocytes at > or = 100 ng/ml and was inhibited by lactose and EDTA (> or = 2 mM) and high concentrations (> 100 mM) of glucose and sucrose, but not by N-acetylglucosamine. BjcuL had no direct hemolytic activity and was devoid of esterase, PLA2 and proteolytic activities. The lectin (up to 200 microg/ml) did not aggregate human platelet-rich plasma (PRP) or washed platelets (WP), nor did it alter the aggregation induced by ADP in PRP or by thrombin in WP. When injected into mouse hind paws, BjcuL (10-100 microg/paw) caused edema and increased vascular permeability, with a maximum effect after 1h that persisted for up to 6 h (edema) or gradually decreased after the peak interval (vascular permeability). No hemorrhage was observed in BjcuL-injected paws. In anesthetized rats, B. jararacussu venom (200 microg/kg, i.v.) produced sustained hypotension (maximum decrease of approximately 60%) whereas a similar dose of BjcuL decreased the blood pressure by approximately 15%, with a rapid return to the resting level.

Peptidase activities in rats treated chronically with N(omega)-nitro-L-arginine methyl ester (L-NAME).

The chronic treatment of rats with N(omega)-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide (NO) biosynthesis, results in hypertension. This inhibition of NO production results in activation of the renin-angiotensin system, with increased activity of the carboxypeptidase angiotensin I-converting enzyme (ACE). Since chronic NO inhibition increases ACE activity, we hypothesized that this inhibition could also affect the activities of other peptidases involved in cardiovascular functions. To test this possibility, we examined the activities of aminopeptidase M (APM), dipeptidyl peptidase IV (DPP IV), metalloendopeptidase 24.15 (MEP 24.15) and neutral endopeptidase 24.11 (NEP 24.11) in rat brain, heart, kidney, liver, lung and thoracic aorta. Male Wistar rats were treated chronically with L-NAME (80mgkg(-1) per day) administered in the drinking water for 4 weeks and their organs then removed and processed for the determination of peptidase activities. Treatment with L-NAME did not significantly alter the activities of the four peptidases in brain, heart, kidney, liver and lung. In contrast, in aorta, the activity of APM was slightly but significantly reduced whereas those of DPP IV and MEP 24.15 were markedly enhanced; NEP 24.11 was not detected in this tissue. Immunoblotting for DPP IV and MEP 24.15 showed increased expression in aortic tissue. Neither L-NAME (1-100microM) nor the NO donors sodium nitroprusside and 3-morpholinosydnonimine (SIN-1; 1-100microM) had any consistent effect on the activity of recombinant MEP 24.15 or renal DPP IV. The importance of MEP 24.15 in peptide metabolism was confirmed in pentobartibal-anesthetized rats pretreated with the MEP 24.15 inhibitor N-[1-(R,S)-carboxy-3-phenylpropyl]-Ala-Aib-Tyr-p-aminobenzoate (JA2), which significantly potentiated the hypotensive response to bradykinin. The altered peptidase activities seen in aorta may contribute to modulating vascular responses in this model of hypertension.

Novel natural peptide substrates for endopeptidase 24.15, neurolysin, and angiotensin-converting enzyme.

Endopeptidase 24.15 (EC; ep24.15), neurolysin (EC; ep24.16), and angiotensin-converting enzyme (EC; ACE) are metallopeptidases involved in neuropeptide metabolism in vertebrates. Using catalytically inactive forms of ep24.15 and ep24.16, we have identified new peptide substrates for these enzymes. The enzymatic activity of ep24.15 and ep24.16 was inactivated by site-directed mutagenesis of amino acid residues within their conserved HEXXH motifs, without disturbing their secondary structure or peptide binding ability, as shown by circular dichroism and binding assays. Fifteen of the peptides isolated were sequenced by electrospray ionization tandem mass spectrometry and shared homology with fragments of intracellular proteins such as hemoglobin. Three of these peptides (PVNFKFLSH, VVYPWTQRY, and LVVYPWTQRY) were synthesized and shown to interact with ep24.15, ep24.16, and ACE, with K(i) values ranging from 1.86 to 27.76 microm. The hemoglobin alpha-chain fragment PVNFKFLSH, which we have named hemopressin, produced dose-dependent hypotension in anesthetized rats, starting at 0.001 microg/kg. The hypotensive effect of the peptide was potentiated by enalapril only at the lowest peptide dose. These results suggest a role for hemopressin as a vasoactive substance in vivo. The identification of these putative intracellular substrates for ep24.15 and ep24.16 is an important step toward the elucidation of the role of these enzymes within cells.