Chemical constituents from Bakeridesia pickelii Monteiro (Malvaceae) and the relaxant activity of kaempferol-3-O-beta-D-(6"-E-p -coumaroyl) glucopyranoside on guinea-pig ileum

The phytochemical investigation of Bakeridesia pickelii Monteiro led to the isolation of seven compounds: beta-sitosterol, a mixture of sitosteryl-3-O-beta-D-glucopyranoside and stigmasteryl-3-O-beta-D-glucopyranoside, vanillic acid, p-coumaric acid, quercetin 3-O-beta-D-glucopyranoside (isoquercitrin) and kaempferol-3-O-beta-D-(6"-E-p -coumaroyl) glucopyranoside (tiliroside), which was isolated as the major component. Their structures were elucidated on the basis of spectroscopic data such as IR, ¹H and 13C NMR, including two-dimensional techniques. Tiliroside relaxed the guinea-pig ileum pre-contracted with KCl 40 mM (EC50 = 9.5 ± 1.0 x 10-5 M), acetylcholine 10-6 M (EC50 = 2.3 ± 0.9 x 10-5 M) or histamine 10-6 M (EC50 = 4.1 ± 1.0 x 10-5 M) in a concentration-dependent manner.

Effects of piperitenone oxide on the intestinal smooth muscle of the guinea pig

We investigated the effects of piperitenone oxide (PO), a major constituent of the essential oil of Mentha x villosa, on the guinea pig ileum. PO (30 to 740 µg/ml) relaxed basal tonus without significantly altering the resting membrane potential. In addition, PO relaxed preparations precontracted with either 60 mM K+ or 5 mM tetraethylammonium in a concentration-dependent manner. At concentrations from 0.1 to 10 µg/ml PO potentiated acetylcholine-induced contractions, while higher concentrations (>30 µg/ml) blocked this response. These higher PO concentrations also inhibited contractions induced by 60 mM K+. PO also blocked the components of acetylcholine contraction which are not sensitive to nifedipine or to solutions with nominal zero Ca2+ and EGTA. These results show that PO is a relaxant of intestinal smooth muscle and suggest that this activity may be mediated at least in part by an intracellular effect

The effect of L-arginine on guinea-pig and rabbit airway smooth muscle function in vitro

We have investigated the effects of L-arginine, D-arginine and L-lysine on airway smooth muscle responsiveness to spasmogens in vitro. Both L-arginine and D-arginine (100 mM) significantly reduced the contractile potency and maximal contractile response to histamine but not to methacholine or potassium chloride in guinea-pig epithelium-denuded isolated trachea. Similarly, the contractile response to histamine was significantly reduced by L-arginine (100 mM) in rabbit epithelium-denuded isolated bronchus. The amino acid L-lysine (100 mM) failed to significantly alter the contractile potency of histamine in guinea-pig isolated trachea (P>0.05). In guinea-pig isolated trachea precontracted with histamine, both L-arginine and D-arginine produced a concentration-dependent relaxation which was not significantly altered by epithelium removal or by the presence of the nitric oxide synthase inhibitor, NG-nitro L-arginine methyl ester (L-NAME; 50 µM). Thus, at very high concentrations, arginine exhibit a non-competitive antagonism of histamine-induced contraction of isolated airway preparations that was independent of the generation of nitric oxide and was not dependent on charge. These observations confirm previous studies of cutaneous permeability responses and of contractile responses of guinea-pig isolated ileal smooth muscle. Taken together, the data suggest that high concentrations of arginine can exert an anti-histamine effect.

Binding sites and actions of Tx1, a neurotoxin from the venom of the spider Phoneutria nigriventer, in guinea pig ileum

Tx1, a neurotoxin isolated from the venom of the South American spider Phoneutria nigriventer, produces tail elevation, behavioral excitation and spastic paralysis of the hind limbs after intracerebroventricular injection in mice. Since Tx1 contracts isolated guinea pig ileum, we have investigated the effect of this toxin on acetylcholine release, as well as its binding to myenteric plexus-longitudinal muscle membranes from the guinea pig ileum. [125I]-Tx1 binds specifically and with high affinity (Kd = 0.36 ± 0.02 nM) to a single, non-interacting (nH = 1.1), low capacity (Bmax 1.1 pmol/mg protein) binding site. In competition experiments using several compounds (including ion channel ligands), only PhTx2 and PhTx3 competed with [125I]-Tx1 for specific binding sites (K0.5 apparent = 7.50 x 10-4 g/l and 1.85 x 10-5 g/l, respectively). PhTx2 and PhTx3, fractions from P. nigriventer venom, contain toxins acting on sodium and calcium channels, respectively. However, the neurotoxin PhTx2-6, one of the isoforms found in the PhTx2 pool, did not affect [125I]-Tx1 binding. Tx1 reduced the [3H]-ACh release evoked by the PhTx2 pool by 33%, but did not affect basal or KCl-induced [3H]-ACh release. Based on these results, as well as on the homology of Tx1 with toxins acting on calcium channels (w-Aga IA and IB) and its competition with [125I]-w-Cono GVIA in the central nervous system, we suggest that the target site for Tx1 may be calcium channels.

Crotoxin, the major toxin from the rattlesnake Crotalus durissus terrificus, inhibits ³H-choline uptake in guinea pig ileum

We examined the effect of crotoxin, the neurotoxic complex from the venom of the South American rattlesnake Crotalus durissus terrificus, on the uptake of ³H-choline in minces of smooth muscle myenteric plexus from guinea pig ileum. In the concentration range used (0.03-1 µM) and up to 10 min of treatment, crotoxin decreased ³H-choline uptake by 50-75% compared to control. This inhibition was time dependent and did not seem to be associated with the disruption of the neuronal membrane, because at least for the first 20 min of tissue exposure to the toxin (up to 1 µM) the levels of lactate dehydrogenase (LDH) released into the supernatant were similar to those of controls. Higher concentrations of crotoxin or more extensive incubation times with this toxin resulted in elevation of LDH activity detected in the assay supernatant. The inhibitory effect of crotoxin on ³H-choline uptake seems to be associated with its phospholipase activity since the equimolar substitution of Sr2+ for Ca2+ in the incubation medium or the modification of the toxin with p-bromophenacyl bromide substantially decreased this effect. Our results show that crotoxin inhibits ³H-choline uptake with high affinity (EC25 = 10 ± 5 nM). We suggest that this inhibition could explain, at least in part, the blocking effect of crotoxin on neurotransmission.

The mechanism of gentisic acid-induced relaxation of the guinea pig isolated trachea: the role of potassium channels and vasoactive intestinal peptide receptors

We examined some of the mechanisms by which the aspirin metabolite and the naturally occurring metabolite gentisic acid induced relaxation of the guinea pig trachea in vitro. In preparations with or without epithelium and contracted by histamine, gentisic acid caused concentration-dependent and reproducible relaxation, with mean EC50 values of 18 µM and Emax of 100% (N = 10) or 20 µM and Emax of 92% (N = 10), respectively. The relaxation caused by gentisic acid was of slow onset in comparison to that caused by norepinephrine, theophylline or vasoactive intestinal peptide (VIP). The relative rank order of potency was: salbutamol 7.9 > VIP 7.0 > gentisic acid 4.7 > theophylline 3.7. Gentisic acid-induced relaxation was markedly reduced (24 ± 7.0, 43 ± 3.9 and 78 ± 5.6%) in preparations with elevated potassium concentration in the medium (20, 40 or 80 mM, respectively). Tetraethylammonium (100 µM), a nonselective blocker of the potassium channels, partially inhibited the relaxation response to gentisic acid, while 4-AP (10 µM), a blocker of the voltage potassium channel, inhibited gentisic acid-induced relaxation by 41 ± 12%. Glibenclamide (1 or 3 µM), at a concentration which markedly inhibited the relaxation induced by the opener of ATP-sensitive K+ channels, levcromakalim, had no effect on the relaxation induced by gentisic acid. Charybdotoxin (0.1 or 0.3 µM), a selective blocker of the large-conductance Ca2+-activated K+ channels, caused rightward shifts (6- and 7-fold) of the gentisic acid concentration-relaxation curve. L-N G-nitroarginine (100 µM), a NO synthase inhibitor, had no effect on the relaxant effect of gentisic acid, and caused a slight displacement to the right in the relaxant effect of the gentisic acid curve at 300 µM, while methylene blue (10 or 30 µM) or ODQ (1 µM), the inhibitors of soluble guanylate cyclase, all failed to affect gentisic acid-induced relaxation. D-P-Cl-Phe6,Leu17[VIP] (0.1 µM), a VIP receptor antagonist, significantly inhibited (37 ± 7%) relaxation induced by gentisic acid, whereas CGRP (8-37) (0.1 µM), a CGRP antagonist, only slightly enhanced the action of gentisic acid. Taken together, these results provide functional evidence for the direct activation of voltage and large-conductance Ca+2-activated K+ channels, or indirect modulation of potassium channels induced by VIP receptors and accounts for the predominant relaxation response caused by gentisic acid in the guinea pig trachea.

Short-term evaluation of non-absorbable microgranular hydroxyapatite infiltration in the guinea pig subepidermal abdominal region

Non-absorbable microgranular hydroxyapatite was infiltrated into the subepidermal abdominal region of guinea pigs in order to assess the possibility of using this material to correct deficiencies in orbital volume. Microgranular hydroxyapatite (2.0 ml) was subepidermally infiltrated into the abdominal region of 20 guinea pigs. The animals were divided into four experimental groups of 5 animals each, which were killed 7 (G1), 15 (G2), 30 (G3) and 60 (G4) days after infiltration. The area and the largest and smallest diameters of the nodules formed by infiltration were evaluated at the site of infiltration and histological examination was performed. The mean granuloma area was similar in all groups. Histopathological examination showed that the material remained isolated from surrounding tissues by a pseudocapsule that became denser throughout the experiment. A host reaction started with young fibroblastic tissue that evolved to dense tissue until cartilaginous tissue was formed in G4, progressively advancing towards the center of the granuloma from G1 to G4. Non-absorbable microgranular hydroxyapatite is an inert material that was well tolerated by the animals studied, with maintenance of the infiltrated volume, and may perhaps be useful to fill anophthalmic cavities.

Inotropic effects of extracts of Psidium guajava L. (guava) leaves on the guinea pig atrium

Many pharmacological effects have been ascribed to extracts of Psidium guajava L. (guava) leaves. However, in spite of its widespread use in Brazilian folk medicine and a reasonable number of scientific reports about it, we could not find any study dealing with its action on the mammalian myocardium. In the present study, by measuring isometric force, we observed that the crude extract of P. guajava (water-alcohol extract obtained by macerating dry leaves) depresses the guinea pig atrial contractility in a concentration-dependent fashion (N = 8 hearts, 15 trials). The compound with cardiac activity was concentrated by extraction in a Soxhlet apparatus using 17 M glacial acetic acid after removing the less polar fractions (hexane, chloroform, acetone, ethanol and methanol), suggesting that this compound is a highly polar substance. In the isolated guinea pig left atrium the acetic acid fraction (10-800 mg/l) of P. guajava 1) reversibly decreased myocardial force in a concentration-dependent fashion (EC50 = 0.07g/l, N = 5 hearts, 9 trials, P<0.05), 2) increased the atrial relaxation time measured at 20% of the force amplitude up to 35% (91 ± 15 to 123 ± 30 ms, N = 3 hearts, 6 trials, P<0.05), 3) abolished the positive staircase effect (Bowditch phenomenon) in a concentration-dependent fashion suggesting a decrease of the cellular inward calcium current (N = 4 hearts, 8 trials, P<0.05), and 4) its inotropic effect was abolished by cholinergic receptor blockade with 1.5 mM atropine sulfate, indicating a cholinergic involvement in the mechanism of action of the extract (N = 7 hearts, 15 trials, P<0.05). The acetic acid extract was 20 times more potent than crude extract (EC50 = 1.4 g/l). The results showed that extracts from P. guajava leaves depress myocardial inotropism.

Cholinergic-opioidergic interaction in the central amygdala induces antinociception in the guinea pig

Several studies have demonstrated the involvement of the central nucleus of the amygdala (CEA) in the modulation of defensive behavior and in antinociceptive regulation. In a previous study, we demonstrated the existence of a cholinergic-opioidergic interaction in the CEA, modulating the defensive response of tonic immobility in guinea pigs. In the present study, we investigated a similar interaction in the CEA, but now involved in the regulation of the nociceptive response. Microinjection of carbachol (2.7 nmol) and morphine (2.2 nmol) into the CEA promoted antinociception up to 45 min after microinjection in guinea pigs as determined by a decrease in the vocalization index in the vocalization test. This test consists of the application of a peripheral noxious stimulus (electric shock into the subcutaneous region of the thigh) that provokes the emission of a vocalization response by the animal. Furthermore, the present results demonstrated that the antinociceptive effect of carbachol (2.7 nmol; N = 10) was blocked by previous administration of atropine (0.7 nmol; N = 7) or naloxone (1.3 nmol; N = 7) into the same site. In addition, the decrease in the vocalization index induced by the microinjection of morphine (2.2 nmol; N = 9) into the CEA was prevented by pretreatment with naloxone (1.3 nmol; N = 11). All sites of injection were confirmed by histology. These results indicate the involvement of the cholinergic and opioidergic systems of the CEA in the modulation of antinociception in guinea pigs. In addition, the present study suggests that cholinergic transmission may activate the release of endorphins/enkephalins from interneurons of the CEA, resulting in antinociception.

Inotropic effect of Citrus sinensis (L.) Osbeck leaf extracts on the guinea pig atrium

The objective of the present investigation was to determine the contractile effect of crude and acetone leaf extracts of Citrus sinensis (L.) Osb. on mammalian myocardium. Crude leaf extracts have been used in folk medicine to treat neurological disorders. Some flavonoids isolated from this plant presented a positive inotropic effect on myocardium. This motivated us to test the extracts on the atria of guinea pigs of both sexes (300-500 g) and surprisingly we observed inotropic depression instead of an increase in force. The maximum effect of the crude extract was 79.4 ± 8.1% of the control force amplitude (N = 5 hearts, 10 trials, 27 ± 0.1ºC, stimulus: 2 Hz, 400 V, 0.5 ms). The EC50 for crude, ethanol, acetic, aqueous, and acetone extracts was 300, 300, 600, 1000, and 140 µg/ml, respectively, with a Hill constant of 1.8, 2.0, 2.5, 2.0, and 1.4, respectively. Blockade of cholinergic, beta-adrenergic, or opioid membrane receptors with 1.5 µM atropine sulfate, 1 µM propranolol, and 10 µM naloxone, respectively, did not change the effect of the crude extract. The acetone extract abolished the Bowditch positive staircase phenomenon (N = 5 hearts, 10 trials, 27 ± 0.1ºC), suggesting a possible reduction of the calcium inward current, and also promoted the so-called Woodworth phenomenon. The effect was concentration-dependent and indicated the existence of another inhibitory contractile mechanism such as the simultaneous activation of some of the membrane potassium channels reducing the myocardial action potential duration and further decreasing the cellular calcium entry.

Negative inotropic and chronotropic effects on the guinea pig atrium of extracts obtained from Averrhoa carambola L. leaves

It has been reported that star fruit can lead to a fatal outcome in uremic patients. The intoxication syndrome consists of hiccups, mental confusion, dizziness, and vomiting. On the other hand, folk medicine uses teas and infusions of carambola leaves to treat headache, vomiting, cough, insomnia, and diabetes. This motivated us to determine if Averrhoa carambola can act on the contractility and automaticity of the guinea pig heart. We measured the atrial isometric force in stimulated left atria and determined the chronotropic changes in spontaneously beating right atria. The carambola leaf extracts (1.5 mg/ml) abolished the contractile force in a concentration-dependent manner. Among the crude, methanolic, ethanolic, aqueous, and acetic extracts, the aqueous one was the most potent (EC50 = 520 ± 94 µg/ml; flavonoids and tannins are the main constituents; Na+ and K+ contents in 1.0 mg/ml of aqueous extract were 0.12 ± 0.016 and 1.19 ± 0.15 mM, respectively). The aqueous extract abolished the positive Bowditch staircase phenomenon and reduced the inotropic response to CaCl2 (0.17-8.22 mM), events that are dependent on the cellular Ca2+ inward current. The adrenergic, muscarinic or opioid membrane receptors do not seem to participate in the mechanism of action of the cardioactive substance(s). In spontaneously beating atria, the aqueous extract promoted a negative chronotropic effect that was antagonized by 0.1 µM isoproterenol bitartrate. With this agonist, the EC50 of the aqueous extract increased from 133 ± 58 to 650 ± 100 µg/ml. These data regarding the effect of A. carambola on guinea pig atrial contractility and automaticity indicate an L-type Ca2+ channel blockade.

The effect of Nystatin on the inner ear : an experimental guinea pig study

Objectifs: Le Nystatin est un antibiotique efficace pour le traitement d’otomycose. Bien que sa sécurité au niveau de l’oreille externe soit bien établie, son utilisation n’est pas recommandée lorsqu’il y a une perforation tympanique. L’objectif de cette étude est d’évaluer le potentiel ototoxique du Nystatin lorsque celui-ci est appliqué directement au niveau de l’oreille moyenne. Méthodes: Nous avons fait une étude expérimentale avec 18 cochons d’Indes de souche Hartley que nous avons divisés en deux groupes. En exposant l’oreille moyenne de chaque animal au Nystatin (groupe I) ou à la néomycine (groupe II) et chaque oreille controlatérale à une solution physiologique (NaCl), la fonction auditive a été évaluée avec un test de potentiels évoqués auditif du tronc cérébral avant et après les injections. Une étude par microscopie électronique a permis une comparaison histologique de l’état des cellules ciliées cochléaires entre les 2 groupes. Résultats: Les pertes auditives moyennes du groupe « Nystatin » étaient de 13.0 dB et comparables aux pertes moyennes observées dans les oreilles ayant été injectées avec du NaCl (4.0 dB dans le groupe I et 15.1 dB dans le groupe II). Le groupe de contrôle « néomycine » a subi une perte auditive moyenne de 39.3 dB, ce qui représente une différence cliniquement et statistiquement significative (p<0.001). L’étude histologique avec une microscopie à balayage électronique a démontré une conservation de l’architecture des cellules ciliées cochléaires dans les groupe Nystatin et NaCl. La néomycine a causé une destruction marquée de ces structures. Conclusions: Le Nystatin ne provoque pas d’atteinte auditive ni de destruction des cellules ciliées externes après injection directe dans l’oreille moyenne chez le cochon d’Inde.

Impact of neonatal total parenteral nutrition and early glucose-enriched diet on glucose metabolism and physical phenotypes in Guinea Pig

Les oxydants infusés avec la nutrition parentéral (NP) néonatale induisent une modification du métabolisme des lipides et du glucose, donnant lieu à l’âge adulte à un phénotype de carence énergétique (faible poids, baisse de l’activité physique). L’hypothèse qu’une diète précoce riche en glucose prévient ces symptômes plus tard dans la vie, fut évalué chez le cobaye par un ANOVA en plan factoriel complet à deux facteurs (p < 0:05) : NP du jour 3 à 7, suivit d’une nourriture régulière (chow) (NP+) vs. chow à partir du 3ième jour (NP-), combiné avec une eau de consommation enrichie en glucose (G+) ou non (G-) à partir de la 3ième semaine. Les paramètres suivant ont été mesurés à l’âge de 9 semaine: taux de croissance, activité physique, activité de phosphofructokinase-1 et glucokinase (GK), niveau hépatique de glucose-6-phosphate (G6P), glycogène, pyruvate et potentiel redox du glutathion, poids du foie, glycémie, tolérance au glucose, concentrations hépatiques et plasmatiques en triacylglycérides (TG) et cholestérol. Le groupe G+ (vs. G-) avait un taux de croissance plus bas, une activité de GK et une concentration en G6P plus élevée, et un potentiel redox plus bas (moins oxydé). Le niveau plasmatique de TG était moins élevé dans le groupe NP+ (vs. NP-). Les traitements n’eurent aucun effet sur les autres paramètres. Ces résultats suggèrent qu’indépendamment de la NP, une alimentation riche en glucose stimule la glycolyse et déplace l’état redox vers un statut plus réduit, mais ne surmonte pas les effets de la NP sur le phénotype physique de carence énergétique.

Mast cell tryptase and proteinase-activated receptor 2 induce hyperexcitability of guinea-pig submucosal neurons

Mast cells that are in close proximity to autonomic and enteric nerves release several mediators that cause neuronal hyperexcitability. This study examined whether mast cell tryptase evokes acute and long-term hyperexcitability in submucosal neurons from the guinea-pig ileum by activating proteinase-activated receptor 2 (PAR2) on these neurons. We detected the expression of PAR2 in the submucosal plexus using RT-PCR. Most submucosal neurons displayed PAR2 immunoreactivity, including those colocalizing VIP. Brief (minutes) application of selective PAR2 agonists, including trypsin, the activating peptide SL-NH2 and mast cell tryptase, evoked depolarizations of the submucosal neurons, as measured with intracellular recording techniques. The membrane potential returned to resting values following washout of agonists, but most neurons were hyperexcitable for the duration of recordings (> 30 min-hours) and exhibited an increased input resistance and amplitude of fast EPSPs. Trypsin, in the presence of soybean trypsin inhibitor, and the reverse sequence of the activating peptide (LR-NH2) had no effect on neuronal membrane potential or long-term excitability. Degranulation of mast cells in the presence of antagonists of established excitatory mast cell mediators (histamine, 5-HT, prostaglandins) also caused depolarization, and following washout of antigen, long-term excitation was observed. Mast cell degranulation resulted in the release of proteases, which desensitized neurons to other agonists of PAR2. Our results suggest that proteases from degranulated mast cells cleave PAR2 on submucosal neurons to cause acute and long-term hyperexcitability. This signalling pathway between immune cells and neurons is a previously unrecognized mechanism that could contribute to chronic alterations in visceral function.