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The acrosome reaction of spermatozoa is a complex, calcium-dependent, regulated exocytosis. Fusion at multiple sites between the outer acrosomal membrane and the cell membrane causes the release of the acrosomal contents and the loss of the membranes surrounding the acrosome. However, very little is known about the molecules that mediate and regulate this unique fusion process. Here, we show that N-ethylmaleimide-sensitive factor (NSF), a protein essential for most fusion events, is present in the acrosome of several mammalian spermatozoa. Moreover, we demonstrate that calcium-dependent exocytosis of permeabilized sperm requires active NSF. Previously, we have shown that the addition of the active (GTP-bound) form of the small GTPase Rab3A triggers exocytosis in permeabilized spermatozoa. In the present report we show that Rab3A is necessary for calcium-dependent exocytosis. The activation of Rab3A protects NSF from N-ethylmaleimide inhibition and precludes the exchange of the endogenous protein with recombinant dominant negative mutants of NSF. Furthermore, Rab3A activation of acrosomal exocytosis requires active NSF. Our results suggest that, upon calcium stimulation, Rab3A switches to its active GTP-bound form, triggering the formation of a protein complex in which NSF is protected. This process is suggested to be an essential part of the molecular mechanism of membrane fusion leading to the release of the acrosomal contents.

Recent studies have shown that gallic acid and its alkylesters induce apoptosis in different cell lines. Since new compounds with biological activity and less cytotoxicity to normal cells are necessary for cancer therapy, the aim of this study was to evaluate the cytotoxic effect of 1-(3,4,5-trihydroxyphenyl)-dodecylbenzoate on human acute myeloid leukemia K562 cells and on human acute lymphoblastic leukemia Jurkat cells. The cell viability was determined by MTT method. The apoptosis induction was assessed by bromide and acridine orange staining and by Annexin V-FITC Apoptosis Detection kit. The cell cycle analysis was carried out by flow cytometry using propidium iodide. Cytometric analysis was also performed to evaluate the expression of the following proteins: AIF, p53, Bcl-2 and Bax. The mitochondrial potential was also assessed by flow cytometry using MitoView633 kit. The results showed that the compound significantly reduced the cell viability of K562 and Jurkat cells in a concentration and time dependent manner (IC50 of 30 μM). The compound induced cell cycle arrest in G0/G0 phase and significantly increased the proportion of cells in the sub-G0/G0 phase. Apoptosis was confirmed by the sight of morphological characteristics of apoptosis and by phosphatidylserine externalization (73.47±5.71% of cells expressing annexin). The results also showed that the compound promotes a modification in Bax:Bcl-2 ratio and increases p53 expression. Thus, it is possible to conclude that 1-(3,4,5-trihydroxyphenyl)-dodecylbenzoate induces apoptosis by inhibiting the antiapoptotic protein Bcl-2 and by increasing the release of AIF, Bax and p53. In addition, it blocks the cell cycle at G0/G0, stopping cell proliferation. So far, the results suggest that this compound may have a potential therapeutic effect against leukemia cells. [ABSTRACT FROM AUTHOR]

Background: Recent advancements in next-generation sequencing (NGS) technology have ushered in significant improvements in sequencing speed and data throughput, thereby enabling the simultaneous analysis of a greater number of samples within a single sequencing run. This technology has proven particularly valuable in the context of microbial community profiling, offering a powerful tool for characterizing the microbial composition at the species level within a given sample. This profiling process typically involves the sequencing of 16S ribosomal RNA (rRNA) gene fragments. By scaling up the analysis to accommodate a substantial number of samples, sometimes as many as 2,000, it becomes possible to achieve cost-efficiency and minimize the introduction of potential batch effects. Our study was designed with the primary objective of devising an approach capable of facilitating the comprehensive analysis of 1,711 samples sourced from diverse origins, including oropharyngeal swabs, mouth cavity swabs, dental swabs, and human fecal samples. This analysis was based on data obtained from 16S rRNA metagenomic sequencing conducted on the Illumina MiSeq and HiSeq sequencing platforms., Results: We have designed a custom set of 10-base pair indices specifically tailored for the preparation of libraries from amplicons derived from the V3-V4 region of the 16S rRNA gene. These indices are instrumental in the analysis of the microbial composition in clinical samples through sequencing on the Illumina MiSeq and HiSeq platforms. The utilization of our custom index set enables the consolidation of a significant number of libraries, enabling the efficient sequencing of these libraries in a single run., Conclusions: The unique array of 10-base pair indices that we have developed, in conjunction with our sequencing methodology, will prove highly valuable to laboratories engaged in sequencing on Illumina platforms or utilizing Illumina-compatible kits., (© 2024. The Author(s).)

Background and Aim: Prolonged stress causes deleterious effects on both the organism and its microbiota. In this study, we examined the effects of exposure to variable frequency ultrasound (US) on the gut microbiota-liver-brain axis of mice., Materials and Methods: This study was conducted on 20 mature clinically healthy sexually naive C57BL/6J male mice (42-45 days old). Group 1 (Normal) consisted of healthy intact mice (n = 10). Group 2 (Stress) consisted of mice subjected to US-induced stress (n = 10) for 20 days with alternating frequencies (20-45 kHz). Stool samples were collected on days 0, 10, and 20, and the corresponding DNA was later subjected to 16SrRNA sequencing. After mice were sacrificed on day 21, the leukocyte count, blood serum biochemical parameters, and liver and brain antioxidant status were measured. Behavioral testing was performed on days 17, 18, and 19., Results: Ultrasound lead to higher stress and anxiety levels; increase in creatinine by 8.29% and gamma-glutamyltransferase activity by 5 times, a decrease in alkaline phosphatase activity by 38.23%, increase of de Ritis coefficient by 21.34%; increased liver and brain superoxide dismutase level by 20.8% and 21.5%, respectively; the stress-related changes in the gut microbiota composition - Bacteroidaceae and Firmicutes ., Conclusion: Subjecting mice to 20 days of US-induced stress leads to systemic disorders due to oxidative stress and a decrease in the diversity of the gut microbiota., Competing Interests: The authors declare that they have no competing interests., (Copyright: © Chernukha, et al.)

Objective: To investigate the effects of diet on the gut microbiota and to assess the relationship of these factors with depression., Material and Methods: Microorganisms that predominate in depressed patients were identified and associations of the identified organisms with the patients' diet were performed. Fourteen depressed patients and 14 healthy volunteers with the same socio-demographic parameters were included in the study. The Hamilton Depression Scale, Generalized Anxiety Disorder Questionnaire, and the Center for Epidemiologic Studies Questionnaire were used., Results: Erysipelatoclostridium and Clostridium innocuum species were 11.3 and 14.4 times higher in depressed patients compared with healthy controls. Fusicatenibacter saccharivorans, Faecalibacterium prausnitzii and Roseburia faecis species , as well as members of the genus Roseburia were statistically significantly more abundant in the healthy volunteers group (6.5, 2.14, 8.75 and 5.2 times more frequently compared to patients). The presence of these microorganisms was correlated with dietary components., Conclusion: Our study revealed groups of microorganisms that differ in healthy volunteers and depressed patients. The association of these microorganisms with the diet was shown, which partially confirmed the influence of a «healthy diet» on the development of depressive disorders.

Probiotic bacteria are widely accepted as therapeutic agents against inflammatory bowel diseases for their immunostimulating effects. In the last decade, more evidence has emerged supporting the positive effects of probiotics on the course of neurodegenerative and psychiatric diseases. This brief review summarizes the data from clinical studies of probiotics possessing antidepressant properties and focuses on the potential genes and proteins underlying these mechanisms. Data from small-sample placebo-controlled pilot studies indicate that certain strains of bacteria can significantly reduce the symptoms of depression, especially in depressed patients. Despite the disparity between studies attempting to pinpoint the bacterial putative genes and proteins accounting for these mechanisms, they ultimately show that bacteria are a potential source of metabiotics-microbial metabolites or structural components. Since the constituents of cells-namely, secreted proteins, peptides and cell wall components-are most likely to be entangled in the gut-brain axis, they can serve as starting point in the search for probiotics with concrete properties.

Ethanol consumption during pregnancy may induce profound changes in fetal CNS development. We postulate that some of the effects of ethanol on striatal glutamatergic transmission and neurotrophin expression could be modulated by allopregnanolone, a neurosteroid modulator of GABAA receptor activity. We describe the acute pharmacological effect of allopregnanolone (65 μg/kg, s.c.) administered to juvenile male rats (day 21 of age) on the corticostriatal glutamatergic pathway, in both control and prenatally ethanol-exposed rats (two ip injections of 2.9 g/kg in 24% v/v saline solution on gestational day 8). Prenatal ethanol administration decreased the K(+)-induced release of glutamate regarding the control group. Interestingly, this effect was reverted by allopregnanolone. Regarding BDNF, allopregnanolone decreases the content of this neurotrophic factor in the striatum of control groups. However, both ethanol alone and ethanol plus allopregnanolone treated animals did not show any change regarding control values. We suggest that prenatal ethanol exposure may produce an alteration of GABAA receptors which blocks the GABA agonist-like effect of allopregnanolone on rapid glutamate release, thus disturbing normal neural transmission. Furthermore, the reciprocal interactions found between GABAergic neurosteroids and BDNF could underlie mechanisms operating during the neuronal plasticity of fetal development.

There is a growing amount of evidence for a neuroprotective role of progesterone and its neuroactive metabolite, allopregnanolone, in animal models of neurodegenerative diseases. By using a model of hemiparkinsonism in male rats, injection of the neurotoxic 6-OHDA in left striatum, we studied progesterone's effects on rotational behavior induced by amphetamine or apomorphine. Also, in order to find potential explanatory mechanisms, we studied expression and activity of nigrostriatal 3α-hydroxysteroid oxidoreductase, the enzyme that catalyzes progesterone to its active metabolite allopregnanolone. Coherently, we tested allopregnanolone for a possible neuromodulatory effect on rotational behavior. Also, since allopregnanolone is known as a GABAA modulator, we finally examined the action of GABAA antagonist bicuculline. We found that progesterone, in addition to an apparent neuroprotective effect, also increased ipsilateral expression and activity of 3α-hydroxysteroid oxidoreductase. It was interesting to note that ipsilateral administration of allopregnanolone reversed a clear sign of motor neurodegeneration, that is, contralateral rotational behavior. A possible GABAA involvement modulated by allopregnanolone was shown by the blocking effect of bicuculline. Our results suggest that early administration of progesterone possibly activates genomic mechanisms that promote neuroprotection subchronically. This, in turn, could be partially mediated by fast, nongenomic, actions of allopregnanolone acting as an acute modulator of GABAergic transmission.

In the present paper we show for the first time that pregnenolone sulfate (Preg-S) impairs rats' memory for novel object recognition when injected in lateral septum (1.2 microM). The effect of Preg-S is clearly related to the moment the reagent is administered: if administered shortly after the training phase, or prior to the test phase of the experiment, there is no amnesic effect. It is only amnesic when administered 30 min before training. Accordingly, Preg-S does not appear to affect the storage of new memories or their retrieval but rather the acquisition itself. Based on the described afferences and efferences of lateral septum, we suggest a possible stimulatory effect of Preg-S regarding glutamate receptors and/or an inhibitory effect of GABA receptors located in local interneurons or recurrent axon collaterals, both of which have been reported to exist in the aforementioned nucleus.

Our objective was to determine the effect of phosphodiesterase (PDE) inhibition on: 1) tyrosine phosphorylation of human spermatozoa at the tail level; and 2) sperm motion parameters and hyperactivated motility. The study was conducted with normozoospermic and asthenozoospermic samples incubated under in vitro capacitating conditions. The main outcome measures were computer-assisted sperm motion analysis and fluorescent immunodetection of phosphotyrosine-containing proteins. Pentoxifylline (PTX) was used as PDE inhibitor because of its wide use in the clinic. PTX-treatment significantly increased sperm velocity, hyperactivated motility and tyrosine-phosphorylation, both in normo and asthenozoospermic samples. Tyrosine-phosphorylation of tail proteins was highly conspicuous in both types of samples, showing no differential pattern after PTX-treatment. Normozoospermic samples treated with pentoxifylline showed an increase in the number of spermatozoa displaying hyperactivated movement and tyrosine-phosphorylation at the tail level. Preliminary data on asthenozoospermic samples exhibiting altered motion characteristics and defective phosphorylation of sperm-tail proteins showed that both defects can be concomitantly overcome by pentoxifylline treatment. Tyrosine-phosphorylation of sperm-tail proteins is underlying the enhancement of hyperactivated motility resulting from PDE inhibition by pentoxifylline.

The acrosome reaction is a unique type of regulated exocytosis. The single secretory granule of the sperm fuses at multiple points with the overlying plasma membrane. In the past few years we have characterized several aspects of this process using streptolysin O-permeabilized human spermatozoa. Here we show that Rab3A triggers acrosomal exocytosis in the virtual absence of calcium in the cytosolic compartment. Interestingly, exocytosis is blocked when calcium is depleted from intracellular stores. By using a membrane-permeant fluorescent calcium probe, we observed that the acrosome actually behaves as a calcium store. Depleting calcium from this compartment by using a light-sensitive chelator prevents secretion promoted by Rab3A. UV inactivation of the chelator restores exocytosis. Rab3A-triggered exocytosis is blocked by calcium pump and inositol 1,4,5-trisphosphate (IP(3))-sensitive calcium channel inhibitors. Calcium measurements inside and outside the acrosome showed that Rab3A promotes a calcium efflux from the granule. Interestingly, release of calcium through IP(3)-sensitive calcium channels was necessary even when exocytosis was initiated by increasing free calcium in the extraacrosomal compartment in both permeabilized and intact spermatozoa. Our results show that a calcium efflux from the acrosome through IP(3)-sensitive channels is necessary downstream Rab3A activation during the membrane fusion process leading to acrosomal exocytosis.

The present study describes the phytochemical analysis and analgesic activity of Curcuma zedoaria rhizomes grown in Brazil. The results showed that the hydroalcoholic extract, fractions, specially dichloromethane, and a pure compound, denoted as curcumenol (1), exhibited potent and dose-related analgesic activity when evaluated in several models of pain in mice, including writhing, formalin and capsaicin. Compound (1), which seems to be the main active principle from this plant, presented promising analgesic effects, being several times more potent than different reference drugs evaluated in the same experimental models. The calculated ID50 values (micromol/kg, i.p) were 22 and 12 when evaluated in writhing and capsaicin tests, respectively, and 29 micromol/kg in relation to the second phase of the formalin model. The lack of effect in the hot plate test suggests that (1) act by a mechanism which do not involves the participation of the opioid system. The phytochemical analysis indicated that the chemical composition of the plant grown in Brazil is similar to that grown in other countries. The results confirm and justify the popular use of this plant for the treatment of dolorous processes.

Acrosomal exocytosis is a calcium-dependent secretion event causing the release of the acrosomal contents and the loss of the membranes surrounding the acrosome. The synaptotagmins are a family of calcium-binding proteins that participate in the exocytosis of synaptic vesicles. The ubiquitous synaptotagmin VI isoform was found in human sperm cells by Western blot analysis. Immunocytochemistry at the optical and electron microscopy levels localized the protein to the outer acrosomal membrane. Calcium-triggered acrosomal exocytosis in permeabilized sperm cells was abrogated by a specific anti-synaptotagmin VI antibody, indicating that the protein is required for the process. Moreover, a recombinant fusion protein between glutathione S-transferase and the two calcium and phospholipid binding domains of synaptotagmin VI completely inhibited calcium-triggered exocytosis. Interestingly, phorbol ester-dependent in vitro phosphorylation of this recombinant protein abolished its inhibitory effect. We previously showed that, in permeabilized spermatozoa, addition of active Rab3A triggers acrosomal exocytosis at very low calcium concentration. Rab3A-promoted exocytosis was inhibited by the cytosolic domain of synaptotagmin VI and by the anti-synaptotagmin VI antibody, indicating that synaptotagmin is also necessary for Rab-mediated acrosomal content release. In conclusion, the results strongly indicate that synaptotagmin VI is a key component of the secretory machinery involved in acrosomal exocytosis., (Copyright 2001 Academic Press.)

Values of inhibition constants, Ki, and concentrations required for 50% inhibition, IC50, for a collection of structurally diverse competitive inhibitors of calf spleen purine nucleoside phosphorylase have been determined employing inosine as substrate. These values have been employed to create predictive quantitative structure-activity relationships (QSAR) which link structure to values of Ki and IC50. These QSAR models have substantial power to predict values and the associated uncertainties for Ki and IC50 for unknown, structurally diverse inhibitors of purine nucleoside phosphorylase.

This study investigated the vasorelaxant action of the sesquiterpene polygodial, isolated from the bark of Drymis winteri, on rat portal vein in vitro, contracted by various agonists. Polygodial (21-342 microM) preincubated 20 min before, produced graded antagonism of the contractile responses caused by bradykinin, endothelin-1, noradrenaline, the stable analogue of thromboxane A2 U46619, substance P, neurokinin B, and senktide (an NK3-selective agonist). Polygodial, at the same concentration, also produced graded inhibition of the contractile response induced by potassium chloride and by phorbol ester. At the median inhibitory concentration (IC50) level, polygodial was approximately 114- to 177-fold more active in inhibiting mediated contractions to senktide and phorbol ester. When assessed in the tonic contraction induced by endothelin-1 (0.5 nM) or by phorbol (3 microM), polygodial (0.1-100 microM) produced concentration-dependent relaxation, with maximal inhibition (E(max)) of 62 +/- 2% and 100%, respectively. Finally, polygodial (0.1-100 microM) inhibited the rhythmic spontaneous contractions of the rat portal vein (E(max) of 75 +/- 2%). Taken together, these results suggest that the vasorelaxant actions caused by polygodial in rat portal vein are, at least in part, associated with inhibition of calcium influx through voltage-sensitive channels and interaction with protein kinase C-dependent mechanisms. In addition, these data confirm and extend our previous suggestion that polygodial preferentially antagonizes tachykinin-mediated contraction, especially the NK3-mediated responses.

The present study was conducted in order to isolate and identify the phytochemical constituents responsible for analgesic effects shown by a methanolic extract obtained from Sebastiania schottiana roots. Conventional chromatographic procedures led to the isolation of moretenone, glutinol, beta-sitosterol and stigmasterol. The structural elucidation of these compounds was done on the basis of spectral data (IR, 1H- and 13C-NMR) and comparison with authentic samples. Either glutinol or moretenone exhibited marked analgesic action against acetic acid induced abdominal constrictions in mice by intraperitoneal route, indicating 16 to 26-fold higher efficacy than aspirin and paracetamol. When analyzed in a formal-in test, both compounds and standard drugs inhibited only the second phase (inflammatory pain). Our results suggest that the roots of S. schottiana contain analgesic compounds which justify, at least partially, the popular use of this plant for the treatment of urinary problems.

In the present work we have investigated the analgesic activity of various extracts prepared from Aleurites moluccana leaves by using the writhing test in mice. The results showed that the hydroalcoholic extract and the hexane fraction exhibit potent antinociceptive action. We have also isolated and identified some chemical constituents from the hexane fraction. In addition, the nature of substances present in ethyl acetate and butanol fractions were evaluated by TLC. Such fractions revealed the presence of phenolic compounds. Chromatographic procedures carried out with hexane fraction revealed the presence of n-hentriacontane, α-amyrin, β-amyrin, stigmasterol, β-sitosterol and campesterol, which were identified by spectroscopic data and HRGC or HRGC/MS techniques. The compounds considerably inhibited acetic acid-induced abdominal constrictions, being more efficacious than aspirin and paracetamol. In addition, preliminary HPLC analysis indicated, using a UV detector, one main constituent in alcoholic extract., (Copyright © 1998 Gustav Fischer Verlag. Published by Elsevier GmbH.. All rights reserved.)

Some phytoconstituents present in the R. Gardneriana Pl. Tr. leaves, a Brazilian medicinal plant, were isolated by column chromatography. Their preliminary analgesic effects were evaluated against formalin-induced pain in mice. The results demonstrated that four biflavonoids, identified as volkensiflavone, GB-2 a, fukugetin and fukugeside are the main active components of the ethyl acetate fraction. Such compounds exhibited significative analgesic activity in relation to the second phase (inflammatory pain) of the formalin test, suggesting that they are the compounds responsible for the pharmacological effects previously observed for the hydroalcoholic extract of this plant., (Copyright © 1997 Gustav Fischer Verlag. Published by Elsevier GmbH.. All rights reserved.)

The effect of four semi-purified compounds obtained from Mandevilla velutina crude extract by silica gel chromatography fractionization were analysed for their inhibitory effects on uterine contractions induced by bradykinin (BK), lysylbradykinin (L-BK), acetylcholine (ACh) and oxytocin, in vitro. None of the compounds tested affected uterine tone. Pre-incubation for 20 min with fraction 12 (20 to 80 micrograms ml-1), isolated from M. velutina produced a parallel and concentration-dependent displacement to the right of the concentration-response curves for BK and L-BK (1 to 1000 nM). Schild plots of these data were linear (correlation close to unity) and yielded a nominal pA2 value (as g ml-1) of 5.1 and 4.9, respectively, and the values of the slopes were not significantly different from unity. Fraction 11 (10 to 40 micrograms ml-1) also produced a parallel and concentration-dependent displacement to the right of the BK concentration-response curve. The Schild plot gave a mean pA2 value (g ml-1) of 5.4 and a slope not significantly different from unity. Fraction 12 did not influence the uterine contractile responses induced by ACh (0.1 to 100 microM) and oxytocin (0.01 to 30 miu ml-1) at concentrations less than 80 micrograms ml-1. Fraction 16 (20 to 80 micrograms ml-1) antagonized the action of BK only at concentrations greater than 40 micrograms ml-1, whereas fraction 5 (20 to 80 micrograms ml-1) was completely inactive against BK-induced responses. 5 As observed previously with the crude extract, the onset of the BK antagonistic action of these compounds was rapid and completely reversible following intermittent washing of the preparation for 30-60 min. 6 These results indicate the existence of at least two compounds in the crude extract of Mandevilla velutina that act as competitive and selective kinin antagonists on the isolated uterus of the rat.

A crude aqueous alcoholic extract of Mandevilla velutina (Apocynaceae) rhizomes produced a concentration-dependent displacement to the right of the concentration-response curve to bradykinin (Bk) in the isolated uterus of the rat. Schild analysis of the data revealed a linear relationship (r = 0.99) and yielded a pA2 value of 3.3 + 0.08 (- log g ml-1) but the slope differed significantly from unity. The anti-Bk action was of rapid onset and was reversible upon washout. Contractions induced by acetylcholine, oxytocin, angiotensin II and BaCl2 were unaffected by the extract. This represents the first report of a selective Bk antagonist of plant origin. The isolation of the active principle(s) may result in a useful pharmacological tool for elucidating the physiological and physiopathological roles of endogenous Bk.

1. Pure non-peptide compounds obtained in crystal form from the crude extract of the plant Mandevilla velutina (Apocynaceae) were analysed for their antagonistic effects on rat uterine and guinea-pig smooth muscle contractions induced by bradykinin (Bk), lisyl-bradykinin (L-Bk), acetylcholine (ACh), oxytocin and histamine, in vitro. 2. Pre-incubation of rat uterine muscle with compounds MV 8608, MV 8609, MV 8610, MV 8611 and MV 8612 (5 to 40 micrograms ml-1) caused parallel and concentration-dependent rightward displacements of the Bk concentration-response curves (1 to 1000 nM). Schild plots of these data were linear (correlation close to 1) and yielded nominal pA2 values (g ml-1) of 5.7, 5.6, 5.4, 5.7 and 5.3, respectively. Compounds MV 8608, MV 8611 and MV 8612 (5 to 20 micrograms ml-1) also caused concentration-dependent and parallel displacements to the right of the concentration-response curve to L-Bk (1 to 10,000 nM). The Schild plots were linear and furnished nominal pA2 values (g ml-1) of 5.4, 5.8 and 5.1, respectively. With the exception of the antagonist effect of compound MV 8606 against Bk-induced contraction, all compounds behaved as simple competitive kinin antagonists since the calculated slopes were not different from unity. 3. In the guinea-pig ileum, both MV 8608 and MV 8612 (5 to 20 micrograms ml-1), produced concentration-dependent rightward displacements of the concentration-response curve to Bk (0.1 to 1000 nM) when the experiments were performed in the presence but not in the absence of atropine (2.5 microM). However, in contrast to the result obtained in the rat uterus, compound MV 8608 also caused a significant reduction of the maximal response to Bk. The Schild plot for compound MV 8612 was linear (correlation close to unity) and furnished a nominal pA2 value (g ml-1) of 5.3 and a slope not different from unity. 4. In rat uterine muscle, compounds MV 8608 and MV 8612 at concentrations producing marked rightward displacements of the kinin concentration-response curves (10 and 20 micrograms ml-1), did not influence the uterine contractile response to oxytocin or ACh, indicating some selectivity towards kinin receptors. Similarly, compound MV 8612 (10 and 20 ygml 1) did not interfere with the sensitivities or the maximal responses to ACh and histamine in the guinea-pig ileum, whereas compound MV 8608 (10 and 20ug ml-1) caused a slight reduction of ACh- and histamine-induced maximal contractions, allied to decrease of the sensitivity to histamine at concentrations of 20pgml-1 or more. 5. These results extend our previous data, indicating the existence of several non-peptide compounds in the crude extract of Mandevilla velutina that act as simple, competitive, selective and reversible kinin receptor antagonists in the rat isolated uterus and guinea-pig ileum smooth muscle.

The effect of a crude aqueous/alcoholic extract of Mandevilla velutina on the contractile responses induced by bradykinin (Bk), [des-Arg9]-Bk and noradrenaline (NA) in rings of arterial and venous rabbit vessels was analysed. The contractile responses induced by Bk, [des-Arg9]-Bk differed between the rings of the aorta, jugular and mesenteric veins. Rings of the aorta and mesenteric vein were stimulated by both peptides, but were more sensitive to [des-Arg9]-Bk. The jugular vein was not only more sensitive to Bk but also did not respond to [des-Arg9]-Bk, even in the presence of a high concentration of this peptide. Pre-incubation for 20 min with the crude extract of mandevilla velutina rhizomes (1 mg ml-1) antagonized the contractions produced by both peptides. This blockade was surmountable by about a 10-30 fold increase in the concentration of both Bk and [des-Arg9]-Bk. In the aorta and mesenteric vein the NA-induced contractions were not affected by 1 mg ml-1 of the extract. Bk (1 to 100 nM) caused a concentration-related contractile response in rings of the rabbit jugular vein. Incubation for 20 min with the crude extract (0.25-1 mg ml-1) caused a concentration-dependent displacement to the right of the Bk concentration-response curves and depressed the maximal response. The onset of action of the crude extract was rapid and was reversible after intermittent washing of the preparations for 30-60 min. 6 These findings confirm and extend our previous work and indicate that the crude extract of Mandevilla velutina selectively antagonizes the action of Bk and related peptides on both B1- and B2-receptors present in rabbit vascular muscle.