Vasorelaxant effects of ellagitannins isolated from Cuphea carthagenensis.

Cuphea carthagenensis (Jacq.) J. F. Macbr. is a popular plant in Brazilian folk medicine owing to its hypotensive and central nervous system depressant effects. This study aimed to validate the hypotensive effect of the plant's aqueous extract (AE) in rats and examine the vascular actions of three hydrolyzable tannins, oenothein B, woodfordin C, and eucalbanin B, isolated from AE. Systolic blood pressure in unanesthetized rats was determined using the non-invasive tail-cuff method. Oral treatment of normotensive rats with 0.5 and 1.0 g/kg/day AE induced a dose-related hypotensive effect after 1 week. In rat aortic rings pre-contracted with noradrenaline, all ellagitannins (20 - 180 µM) induced a concentration-related vasorelaxation. This effect was blocked by either removing the endothelium or pre-incubating with NG-nitro-l-arginine methyl ester (10 µM), an inhibitor of nitric oxide (NO) synthase. In KCl-depolarized rat portal vein preparations, the investigated compounds did not affect significantly the maximal contractile responses and pD2 values of the concentration-response curves to CaCl2. Our results demonstrated the hypotensive effect of C. carthagenensis AE in unanesthetized rats. All isolated ellagitannins induced vasorelaxation in vitro via activating NO synthesis/NO release from endothelial cells, without altering the Ca2+ influx in vascular smooth muscle preparations. Considering the low oral bioavailability of ellagitannins, the determined in vitro actions of these compounds are unlikely to account for the hypotensive effect of AE in vivo. It remains to be determined the role of the bioactive ellagitannin-derived metabolites in the hypotensive effect observed after oral treatment of unanesthetized rats with the plant extract.

Bauhinia Protease Inhibitors Attenuate Gastric Ulcer by Blocking Neutrophil Enzymes.

Proteases play a pivotal role in many signaling pathways; inhibitors of well-established proteases have shown a substantial therapeutic success. This study aimed to examine the in vivo effects of 3 protease inhibitors isolated from Bauhinia species: i) Bauhinia mollis elastase inhibitor, which blocks human neutrophil elastase (Kiapp 2.8 nM) and cathepsin G (Kiapp 1.0 nM) activities; ii) Bauhinia mollis trypsin inhibitor, a trypsin inhibitor (Kiapp 5.0 nM); and iii) Bauhinia bauhinioides cruzipain inhibitor, which inhibits elastase (Kiapp 2.6 nM), cathepsin G (Kiapp 160.0 nM), and the cysteine proteases cathepsin L (Kiapp 0.2 nM). Bauhinia bauhinioides cruzipain inhibitor, Bauhinia mollis elastase inhibitor, and Bauhinia mollis trypsin inhibitor were isolated using acetone and ammonium sulfate fractionations, DEAE-Sephadex, trypsin-Sepharose, and Resource-Q chromatographies. Mice and rats were treated intraperitoneally with 1 dose of inhibitor; gastric mucosal lesions were induced by cold-restraint stress. Oral pretreatment of mice with Bauhinia mollis elastase inhibitor or Bauhinia mollis trypsin inhibitor (1 - 10 mg/kg) did not show anti-ulcer effect, while Bauhinia bauhinioides cruzipain inhibitor (0.1 - 1.0 mg/kg) produced a similar reduction of the index of mucosal damage at all effective doses (30 to 33% < control). In rats at doses lower than those used in mice, Bauhinia mollis elastase inhibitor and Bauhinia bauhinioides cruzipain inhibitor reduced the index of mucosal damage by 66% and 54% of controls, respectively. The results indicate a protective effect against gastric mucosal lesions associated with elastase inhibition but not inhibition of trypsin activities. Moreover, the lack of Bauhinia mollis elastase inhibitor efficacy observed in mice may possibly be related to the reported structural differences of elastase in mice and rats.

Effects of N-acetylcysteine on skeletal muscle structure and function in a mouse model of peripheral arterial insufficiency.

OBJECTIVE: Abnormalities in skeletal muscle structure and function are important contributors to exercise intolerance and functional decline in peripheral arterial disease. In this study, we tested the hypothesis that administration of N-acetylcysteine (NAC) would improve fatigue resistance and ameliorate the histopathological changes in skeletal muscle in a mouse model of peripheral arterial disease. We also anticipated that NAC treatment would lower the levels of biomarkers of oxidative damage in the ischemic muscle. METHODS: Male Balb/c mice were subjected to bilateral ligation of the femoral artery and, after 2 weeks of recovery, received daily intraperitoneal injections of either NAC (150 mg/kg) or saline for 15 days. At the end of the treatment, the extensor digitorium longus (EDL) and soleus muscles were excised for assessment of contractile function in vitro and histological analysis. Free malondialdehyde and protein carbonyl levels were measured in the gastrocnemius muscle. RESULTS: In the soleus muscle, force after 10 minutes of submaximal tetanic stimulation (60 Hz, 300 ms trains, 0.3 trains/s) was higher (P < .05) in NAC-treated animals (45% ± 3% of the initial value; n = 7) when compared with controls (30.3% ± 3%; n = 8). No differences were found in fatigue development between groups in the EDL muscle (ligated NAC, 35.7% ± 1.9%; ligated saline, 37.5% ± 1.1%). In addition, there was a tendency for lower levels of connective tissue deposition in the soleus of animals treated with NAC (n = 6) when compared with those that received only saline (n = 9) (ligated NAC, 16% ± 2% vs ligated saline, 24% ± 2%; P = .057). No differences were found in lipid peroxidation or protein carbonyl levels between ligated saline and ligated NAC groups. CONCLUSIONS: Taken together, these results indicate that treatment with NAC improves fatigue resistance in the soleus but not the EDL muscle in a model of peripheral arterial insufficiency. CLINICAL RELEVANCE: Despite the increasing burden of peripheral arterial disease (PAD) and its detrimental consequences on the quality of life of the patients, few pharmacological therapies have shown to evoke meaningful effects on functional performance in these individuals. N-acetylcysteine is approved for clinical use, has minimal side effects and most important, has shown to consistently improve exercise performance in animals and humans. In this study, we showed, for the first time, that treatment with this drug at a dose amenable for clinical application evoked marked effects on fatigue resistance in the soleus muscle in a mouse model of PAD. These encouraging findings set the stage for translational studies to determine the acute and long-term impact of this drug on walking capacity in patients with PAD.

Intestine of dystrophic mice presents enhanced contractile resistance to stretching despite morphological impairment.

Protein dystrophin is a component of the dystrophin-associated protein complex, which links the contractile machinery to the plasma membrane and to the extracellular matrix. Its absence leads to a condition known as Duchenne muscular dystrophy (DMD), a disease characterized by progressive skeletal muscle degeneration, motor disability, and early death. In mdx mice, the most common DMD animal model, loss of muscle cells is observed, but the overall disease alterations are less intense than in DMD patients. Alterations in gastrointestinal tissues from DMD patients and mdx mice are not yet completely understood. Thus, we investigated the possible relationships between morphological (light and electron microscopy) and contractile function (by recording the isometric contractile response) with alterations in Ca²âº handling in the ileum of mdx mice. We evidenced a 27% reduction in the ileal muscular layer thickness, a partial damage to the mucosal layer, and a partial damage to mitochondria of the intestinal myocytes. Functionally, the ileum from mdx presented an enhanced responsiveness during stretch, a mild impairment in both the electromechanical and pharmacomechanical signaling associated with altered calcium influx-induced contraction, with no alterations in the sarcoplasmic reticulum Ca²âº storage (maintenance of the caffeine and thapsigargin-induced contraction) compared with control animals. Thus, it is evidenced that the protein dystrophin plays an important role in the preservation of both the microstructure and ultrastructure of mice intestine, while exerting a minor but important role concerning the intestinal contractile responsiveness and calcium handling.

Unlocking the role of dorsal hippocampal α4ß2 nicotinic acetylcholine receptors in Ethanol-Induced conditioned place preference in mice.

Alcohol Use Disorder (AUD) presents a significant and challenging public health concern, marked by a dearth of effective pharmacological treatments. Understanding the neurobiological underpinnings of AUD is of paramount importance for the development of efficacious interventions. The process of addiction entails the acquisition of associative behaviors, prominently engaging the dorsal region of the hippocampus for encoding these associative memories. Nicotinic receptor systems have been implicated in mediating the rewarding effects of ethanol, as well as memory and learning processes. In our current investigation, we delved into the role of α4ß2 nicotinic acetylcholine receptors (nAChRs) within the dorsal hippocampus in the context of ethanol-induced conditioned place preference (CPP), a robust model for scrutinizing the rewarding properties and drug-associated behaviors. To establish CPP, ethanol (2 g/kg) was administered intraperitoneally during a 8-day conditioning phase. Fos immunohistochemistry was employed to assess the involvement of discrete subregions within the dorsal hippocampus in ethanol-induced CPP. Additionally, we probed the influence of α4ß2 nAChRs on CPP via microinjections of a selective nAChR antagonist, dihydro-ß-erythroidine (DHBE, at dosages of 6, 12, and 18 µg/0.5 µL per hemisphere) within the hippocampus. Our results unveiled that ethanol-induced CPP was associated with an increase Fos -positive cells in various subregions of the dorsal hippocampus, including CA1, CA2, CA3, and the dentate gyrus. Intrahippocampal administration of DHBE (at doses of 6 and 18 µg/0.50 µL per hemisphere) effectively blocked ethanol-induced CPP, while leaving locomotor activity unaffected. These findings underscore the critical involvement of the dorsal hippocampus and α4ß2 nAChRs in the acquisition of ethanol-associated learning and reward.

Loss of neuronal projections in the dystrophin-deficient mdx mouse is not progressive.

Lack of dystrophin is known to reduce several cerebral fiber systems. To investigate if the loss of fibers is progressive, we analyzed projections of the trigeminal sensory system to the red nucleus in 3, 6, and 12 month old dystrophin-deficient mdx mice. The retrograde tracer fluorogold was injected in the magnocellular part of the red nucleus, and the number of labeled neurons in the oral part of the spinal trigeminal nucleus (Sp5O) was counted. We found that the number of labeled Sp5O neurons was reduced by 50% in mdx mice compared to age-matched control mice. The number of labeled Sp5O neurons did not change significantly between 3 and 12 months neither in mdx nor in control mice. In addition, the number of labeled neurons in the interstitial system of the trigeminal nerve was reduced by 43% in mdx mice. We conclude that fiber loss did not continue beyond the age of 3 months. Our data suggest that lack of full-length dystrophin impairs neuronal migration or axonal outgrowth, or increases neuronal death during fetal or early life.

Altered release and uptake of gamma-aminobutyric acid in the cerebellum of dystrophin-deficient mice.

Dystrophin deficiency caused by mutations of the related gene leads to muscle wasting in Duchenne muscular dystrophy (DMD). Some patients with DMD also present with intellectual disability and various degrees of neurological disorders, which have been related to a decreased number of postsynaptic gamma-aminobutyric acid type A receptors (GABAARs) in the hippocampus (HPC) and cerebellum (CBL). The aim of this study was to examine the relevance of dystrophin in the presynaptic GABAergic function in brain regions in which this protein is normally abundant. [3H]-GABA release, induced by nicotinic receptor (nAChR) activation or K+ depolarization, and [3H]-GABA uptake were determined using synaptosomes extracted from the cortex (CTX), HPC, and CBL of littermate control and mdx mice. Superfusion of the synaptosomes with nicotine or high K+ solutions led to a concentration-dependent and Ca2+-dependent [3H]-GABA release in control and mdx synaptosomes. [3H]-GABA release induced by 10 µM nicotine in mdx CBL synaptosomes was 47% less than that in control mice. K+-induced [3H]-GABA release did not differ between control and mdx synaptosomes. α7-containing and ß2-containing nAChRs were involved in nicotine-induced [3H]-GABA release in control and mdx synaptosomes. Kinetic analysis of [3H]-GABA uptake in mdx CBL synaptosomes showed a reduced (50%) half-maximal uptake time (t1/2) and increased (44%) rate of [3H]-GABA uptake (Vmax) compared to controls. The apparent transporter affinity (Km) for GABA was not altered. Our findings show that dystrophin deficiency in mdx mice is associated with significant changes in the release and uptake of GABA in the CBL. These presynaptic alterations may be related to the reported decrease in postsynaptic GABAAR in the same brain region. The results indicate possible dysfunction of GABAergic synapses associated with dystrophin deficiency in the CBL, which may contribute to the cognitive and neurobehavioral disorders in mdx mice and patients with DMD.

Morphological changes in the trigemino-rubral pathway in dystrophic (mdx) mice.

The lack of dystrophin that causes Duchenne muscle disease affects not only the muscles but also the central nervous system. Dystrophin-deficient mdx mice present changes in several brain fiber systems. We compared the projections from the trigeminal sensory nuclear complex to the red nucleus in control and mdx mice using retrograde tracers. Injection of 200 nL 2% fluorogold into the red nucleus caused labeling in the mesencephalic trigeminal nucleus, the principal sensory nucleus and the oral, interpolar, and caudal subnuclei of the spinal trigeminal nucleus in both control and mdx mice. Injection of latex microbeads labeled with rhodamine and fluorescein gave results similar to those seen with fluorogold. The number of labeled neurons in the trigeminal sensory nuclear complex was significantly reduced in mdx mice. In the oral subnucleus of the spinal trigeminal nucleus this reduction was 50%. These results indicate that the trigemino-rubral pathway is reduced in dystrophin-deficient mice.

In vivo inhibition of gastric acid secretion by the aqueous extract of Scoparia dulcis L. in rodents.

The freeze-dried aqueous extract (AE) from the aerial parts of Scoparia dulcis was tested for its effects on experimental gastric hypersecretion and ulcer in rodents. Administration of AE to animals with 4h pylorus ligature potently reduced the gastric secretion with ED(50)s of 195 mg/kg (rats) and 306 mg/kg (mice). The AE also inhibited the histamine- or bethanechol-stimulated gastric secretion in pylorus-ligated mice with similar potency suggesting inhibition of the proton pump. Bio-guided purification of the AE yielded a flavonoid-rich fraction (BuF), with a specific activity 4-8 times higher than the AE in the pylorus ligature model. BuF also inhibited the hydrolysis of ATP by H(+),K(+)-ATPase with an IC(50) of 500 microg/ml, indicating that the inhibition of gastric acid secretion of Scoparia dulcis is related to the inhibition of the proton pump. Furthermore, the AE inhibited the establishment of acute gastric lesions induced in rats by indomethacin (ED(50)=313 mg/kg, p.o.) and ethanol (ED(50)=490 mg/kg, p.o.). No influence of the AE on gastrointestinal transit allowed discarding a possible CNS or a cholinergic interaction in the inhibition of gastric secretion by the AE. Collectively, the present data pharmacologically validates the popular use of Scoparia dulcis in gastric disturbances.

Inhibition of the gastric H+,K+ -ATPase by plectrinone A, a diterpenoid isolated from Plectranthus barbatus Andrews.

This work assessed the mechanism underlying the antisecretory gastric acid effect of Plectranthus barbatus Andrews (Lamiaceae) and active constituents. Popularly known as "false-boldo", this plant is used in Brazilian folk medicine to treat gastrointestinal and hepatic ailments. The plant aqueous extract (AE) and isolated compounds were assayed in vivo in pylorus-ligated mice, and in vitro on acid secretion measured as [(14)C]-aminopyrine ([(14)C]-AP) accumulation in rabbit gastric glands and gastric H(+),K(+)-ATPase preparations. Injected into the duodenal lumen, the AE of the plant leaves (0.5 and 1.0 g/kg) decreased the volume (62 and 76%) and total acidity (23 and 50%) of gastric acid secretion in pylorus-ligated mice. Bioguided purification of the AE yielded an active fraction (IC(50)=24 microg/ml) that inhibited acid secretion in rabbit gastric glands with a potency 10 to 18 times greater than that of the originating extract, on both the basal and stimulated acid secretion by histamine (His) (1 microM) or bethanechol (100 microM). At the same concentrations the gastric H(+),K(+)-ATPase activity was also inhibited. The active constituent was chemically identified as the abietanoid dienedione plectrinone A which reduced the H(+),K(+)-ATPase activity with IC(50)=171 microM. The results indicate that inhibition of the gastric proton pump by this diterpenoid may account for the antisecretory acid effect and reputed anti ulcer activity of Plectranthus barbatus.

Behavioral effects of a neurotoxic compound isolated from Clibadium surinamense L (Asteraceae).

Clibadium surinamense L, popularly known as cunambi, is a native plant from the Northern region of Brazil illegally used for predatory fishing. Previous results from our laboratory have demonstrated that the oral treatment of mice with the ethanolic extract (EE) of the leaves of the plant induced generalized tonic-clonic seizures followed by death within 30 min. The aims of the present paper were to characterize the convulsant effect of the hexanic extract (HE) of the stems and leaves of C. surinamense and, by bioguided purification, to identify the active principle and its mechanism of action. The leaves and stems were extracted with hexane (100 g/L) in Soxhlet for 36 h (yield of 2.4%), the solvent was evaporated and the powder dissolved in 1.5% saline/Tween 80. Male mice (30-35 g) treated with HE (22.5-360 mg/kg, p.o.) showed behavioral alterations consistent with CNS stimulation. The intensity and duration of the effect were proportional to the administered doses. The behavioral alterations, measured with a graded score of seizure severity, revealed that pretreatment with carbamazepine (30 mg/kg, i.p., 60 min) or phenytoin (50 mg/kg, i.p., 30 min) did not alter the HE convulsive effect. In contrast, phenobarbital (30 mg/kg, i.p., 60 min) or diazepam (2 mg/kg, i.p., 30 min) reduced the HE effect, increasing the ED(50) for clonic seizures from 64.4 to 89.8 mg/kg and 168.9 mg/kg, respectively. Purification of the HE in a silica gel column eluted with a hexane/ethyl acetate gradient yielded a single fraction with convulsant effect in which cunaniol acetate was identified by (1)H NMR as the main active compound. These results indicated that inhibition of GABAergic transmission by cunaniol acetate might be responsible for the convulsant effects of C. surinamense L in mice, but do not exclude a direct cunaniol action labilizing neuronal excitability.

Analysis of catalytic properties of tripeptidyl peptidase I (TTP-I), a serine carboxyl lysosomal protease, and its detection in tissue extracts using selective FRET peptide substrate.

Tripeptidyl peptidase I (TPP-I), also named ceroid lipofuscinosis 2 protease (CLN2p), is a serine carboxyl lysosomal protease involved in neurodegenerative diseases, and has both tripeptidyl amino- and endo- peptidase activities under different pH conditions. We developed fluorescence resonance energy transfer (FRET) peptides using tryptophan (W) as the fluorophore to study TPP-I hydrolytic properties based on previous detailed substrate specificity study (Tian Y. et al., J. Biol. Chem. 2006, 281:6559-72). Tripeptidyl amino peptidase activity is enhanced by the presence of amino acids in the prime side and the peptide NH2-RWFFIQ-EDDnp is so far the best substrate described for TPP-I. The hydrolytic parameters of this peptide and its analogues indicated that the S4 subsite of TPP-I is occluded and there is an electrostatic interaction of the positively charged substrate N-terminus amino group and a negative locus in the region of the enzyme active site. KCl activated TPP-I in contrast to the inhibition by Ca(2+) and NaCl. Solvent kinetic isotope effects (SKIEs) show the importance of the free N-terminus amino group of the substrates, whose absence results in a more complex solvent-dependent enzyme: substrate interaction and catalytic process. Like pure TPP-I, rat spleen and kidney homogenates cleaved NH2-RWFFIQ-EDDnp only at F-F bond and is not inhibited by pepstatin, E-64, EDTA or PMSF. The selectivity of NH2-RWFFIQ-EDDnp to TPP-I was also demonstrated by the 400 times higher k(cat)/K(M) compared to generally used substrate, NH2-AAF-MCA and by its resistance to hydrolysis by cathepsin D that is present in high levels in kidneys.

Mild dystrophic damage in the androgen-sensitive levator ani muscle of the mdx mouse.

The time course of muscular dystrophy on the androgen-sensitive levator ani muscle was compared to that of the diaphragm of dystrophic (mdx) mice aged 1-20 months. Muscle growth, isometric contractile properties and caffeine-induced contractures were determined to assess the hormone myotrophic effect, muscle strength and sarcoplasmic reticulum function, respectively, of both control and dystrophic muscles. Histological analysis of mdx muscles showed variable fiber size, centronucleated cells, infiltration of connective tissue, and necrosis which was less severe in the levator ani than in the diaphragm muscle. Tetanic tension per unit weight in the mdx levator ani was reduced (29%) after aging, while the contraction time remained unchanged. The tetanic tension of the mdx diaphragm muscle decreased with age from 3 to 20 months (20-64%), and the relaxation time was prolonged after aging (22%). Gonadectomy of young adult mdx mice caused atrophy of the levator ani muscle, accelerated muscle wasting, reduced the tetanic force (31%), but it did not affect the contraction time and caffeine responses. The results showed that testosterone does not prevent the progress of muscle disease in the mdx levator ani, but androgen withdrawal accelerates muscle wasting suggesting a normonal beneficial effect.

Kininogenase activity of Thalassophryne nattereri fish venom.

Accidents caused by the venomous fish Thalassophryne nattereri are characterized by edema, intense pain and necrosis at the site of the sting. This study assessed the nociceptive and edematogenic activities of T. nattereri venom after injection into the mouse hindpaw and determination of the paw licking duration and weight. Subplantar injections of the venom (0.1-6 microg) induced a dose-related increase of the paw licking time and paw swelling with maximal values at 3 microg (209.5 +/- 57.5 s and 135.0 +/- 6.8 mg, respectively). Pretreatment of mice with either indomethacin (10 mg/kg, i.p.), a cyclooxygenase inhibitor, dexamethasone (1 mg/kg, s.c.), a steroid anti-inflammatory agent, cyproheptadine (1 mg/kg, i.p.), antagonist of serotonin receptors or L-NAME (100 mg/kg, s.c.), inhibitor of nitric oxide syntase, did not affect the venom-induced nociceptive and edematogenic responses. Injection of the opioid analgesic fentanyl (0.1 mg/kg, s.c.) reduced the paw licking time induced by 1 microg venom by 84% of control, without affecting the paw swelling. Both nociceptive and edematogenic responses were reduced after treatment with a specific tissue kallikrein inhibitor (TKI, 100 mg/kg, i.p.) by 78% and 24% from control values, respectively. Administration of a specific plasma kallikrein inhibitor (PKSI(527,) 100 mg/kg, s.c.) did not affect the venom-induced nociceptive response, but it decreased the paw edema by 15% from control. After injection of the angiotensin-converting enzyme inhibitor captopril (100 mg/kg, i.p.) the venom-induced nociceptive end edematogenic responses were increased by two-fold. The role of kallikreins possibly present in the venom was further assessed by hydrolysis of human kininogen and kininogen-derived synthetic peptides, showing the release of kallidin (Lys-bradykinin). The hydrolysis was inhibited by metal chelating agents but not by serino-, aspartyl- or cysteino-proteinase inhibitors. The data suggest that a protease with tissue-kallikrein-like activity plays a major role in nociception and edema induced by T. nattereri venom and this should be considered to achieve efficient treatments for human accidents with this venom.

A comparative study of two clerodane diterpenes from Baccharis trimera (Less.) DC. on the influx and mobilization of intracellular calcium in rat cardiomyocytes.

Baccharis trimera (Less.) D.C. (Asteraceae) is a medicinal species native to South America and used in Brazilian folk medicine to treat gastrointestinal and liver diseases, kidney disorders and diabetes. The aqueous extract (AE) of the aerial parts of this species presented two mainly constituents: the ent-clerodane diterpene (Fig. 1) and the neo-clerodane diterpene (Fig. 2). The objective of this work was to study their activities on the blockade of Ca(2+)-induced contractions in KCL-depolarized rat portal vein preparations, and on the influx and mobilization of cytosolic calcium in rat cardiomyocytes by fluorescence measurements. The results showed that both the neo- and the ent-clerodane diterpenes reduced the maximal contractions induced by CaCl2, in KCl depolarized rat portal vein preparations, without modifying the EC50. The data on the concentration of cytosolic calcium ([Ca(2+)]c) showed that, while the neo-clerodane diterpene stimulates the mobilization of [Ca(2+)]c in rat cardiomyocytes, this effect was not observed with the ent-clerodane diterpene. On the other hand, the influx of calcium was not altered by the neo-clerodane diterpene, but was reduced in the presence of the ent-clerodane diterpene, indicating that this compound induces a blockade of the voltage-dependent calcium channels.

Quantitative changes of nicotinic receptors in the hippocampus of dystrophin-deficient mice.

Lack of dystrophin in Duchenne muscle dystrophy (DMD) and in the mutant mdx mouse results in progressive muscle degeneration, structural changes at the neuromuscular junction, and destabilization of the nicotinic acetylcholine receptors (nAChRs). One-third of DMD patients also present non-progressive cognitive impairments. Considering the role of the cholinergic system in cognitive functions, the number of nAChR binding sites and the mRNA levels of α4, ß2, and α7 subunits were determined in brain regions normally enriched in dystrophin (cortex, hippocampus and cerebellum) of mdx mice using specific ligands and reverse-transcription polymerase chain reaction assays, respectively. Membrane preparations of these brain regions were obtained from male control and mdx mice at 4 and 12 months of age. The number of [³H]-cytisine (α4ß2) and [¹²5I]-α-bungarotoxin ([¹²5I]-αBGT, α7) binding sites in the cortex and cerebellum was not altered with age or among age-matched control and mdx mice. A significant reduction in [³H]-cytisine (48%) and [¹²5I]-αBGT (37%) binding sites was detected in the hippocampus of mdx mice at 12 months of age. When compared with the age-matched control groups, the mdx mice did not have significantly altered [³H]-cytisine binding in the hippocampus, but [¹²5I]-αBGT binding in the same brain region was 52% higher at 4 months and 20% lower at 12 months. mRNA transcripts for the nAChR α4, ß2, and α7 subunits were not significantly altered in the same brain regions of all animal groups. These results suggest a potential alteration of the nicotinic cholinergic function in the hippocampus of dystrophin-deficient mice, which might contribute to the impairments in cognitive functions, such as learning and memory, that have been reported in the dystrophic murine model and DMD patients.

Effects of hecogenin and its possible mechanism of action on experimental models of gastric ulcer in mice.

This study investigates the gastroprotective effects of hecogenin, a steroid saponin isolated from Agave sisalana, on experimental models of gastric ulcer. Male Swiss mice were used in the models of ethanol- and indometacin-induced gastric ulcer. To clarify the hecogenin mechanism of action, the roles of nitric oxide (NO), sulfhydryls (GSH), K⁺(ATP) channels and prostaglandins were also investigated, and measurements of lipid peroxidation (TBARS assay) and nitrite levels in the stomach of hecogenin-treated and untreated animals were performed. Furthermore, the effects of hecogenin on myeloperoxidase (MPO) release from human neutrophils were assessed in vitro. Our results showed that hecogenin (3.1, 7.5, 15, 30, 60 and 90 mg/kg, p.o.) acutely administered, before ethanol or indomethacin, exhibited a potent gastroprotective effect. Although the pretreatments with L-NAME, an iNOS inhibitor, and capsazepine, a TRPV1 receptor agonist, were not able to reverse the hecogenin effect, this was reversed by glibenclamide, a K⁺(ATP) blocker, and indomethacin in the model of ethanol-induced gastric lesions. The hecogenin pretreatment normalized GSH levels and significantly reduced lipid peroxidation and nitrite levels in the stomach, as evaluated by the ethanol-induced gastric lesion model. The drug alone increased COX-2 expression and this effect was further enhanced in the presence of ethanol. It also decreased MPO release and significantly protected the gastric mucosa. In conclusion, we showed that hecogenin presents a significant gastroprotective effect that seems to be mediated by K⁺(ATP) channels opening and the COX-2/PG pathway. In addition, its antioxidant and anti-inflammatory properties may play a role in the gastroprotective drug effect.

Antisecretory actions of Baccharis trimera (Less.) DC aqueous extract and isolated compounds: analysis of underlying mechanisms.

ETHNOPHARMACOLOGICAL RELEVANCE: Baccharis trimera (Less.) DC. (Asteraceae) is a species native to South America used in Brazilian folk medicine to treat gastrointestinal and liver diseases, kidney disorders and diabetes. Previous studies from this laboratory confirmed the antacid and antiulcer activities of the plant aqueous extract (AE) in rat and mouse models. AIM OF THE STUDY: To investigate the mechanisms involved in the antacid action of AE and isolated compounds from Baccharis trimera. MATERIALS AND METHODS: AE was assayed in vivo in cold-restraint stress gastric ulcers and in pylorus-ligated mice. Nine fractions (F2-F10) previously isolated from AE were assayed in vitro on acid secretion measured as [(14)C]-aminopyrine ([(14)C]-AP) accumulation in rabbit gastric glands, and on gastric microsomal H(+), K(+)-ATPase preparations. Chlorogenic acids (F2, F3, F6, F7), flavonoids (F9), an ent-clerodane diterpene (F8) and a dilactonic neo-clerodane diterpene (F10) have been identified in these fractions. RESULTS: Intraduodenal injection of AE (1.0 and 2.0 g/kg) in 4h pylorus-ligated mice decreased the volume (20 and 50%) and total acidity (34 and 50%) of acid secretion compared to control values. Administered orally at the same doses AE protected against gastric mucosal lesions induced in mice by restraint at 4°C. Exposure of isolated rabbit gastric glands to fractions F8 (10-100 µM) and F9 (10-300 µg/ml) decreased the basal [(14)C]-AP uptake by 50 and 60% of control (Ratio=6.2±1.1), whereas the remaining fractions were inactive. In the presence of the secretagogues F2 and F4 (30-300 µg/ml) decreased the [(14)C]-AP uptake induced by histamine (His) with a 100-fold lower potency than that of ranitidine. F5 and F6 reduced the [(14)C]-AP uptake stimulated by carbachol (CCh), but they were 10 to 20-fold less potent than atropine. F8 (diterpene 2) and F9 (flavonoids) decreased both the His- and CCh-induced [(14)C]-AP uptake, whereas F10 (diterpene 1) was inactive against the [(14)C]-AP uptake stimulated by secretagogues. Diterpene 2 was the most active of all tested compounds being 7-fold less potent than ranitidine and equipotent to atropine in reducing acid secretion in vitro. This compound also reduced the gastric H(+), K(+)-ATPase activity by 20% of control, while the remaining fractions were inactive on the proton pump in vitro. CONCLUSIONS: The results indicate that Baccharis trimera presents constituents that inhibit gastric acid secretion by acting mainly on the cholinergic regulatory pathway. The plant extract also contains compounds that exert moderate inhibition of the histaminergic regulatory pathway of acid secretion and the gastric proton pump. Altogether these active constituents appear to provide effective inhibition of acid secretion in vivo, which may explain the reputed antiulcer activity of the plant extract.

Natural compounds endowed with cholinergic or anticholinergic activity. Enhancement of acetylcholine release by a quaternary derivative of L-hyoscyamine.

New compounds that target nicotinic receptors (nAChRs) have been sought to correct disorders affecting cholinergic transmission in central and peripheral synapses. A quaternary derivate of l-hyoscyamine, phenthonium (Phen), was shown by our group to enhance the spontaneous acetylcholine (ACh) release without altering the nerve-induced transmitter release at the neuromuscular junction. The effect was unrelated to membrane depolarization, and was not induced by an increase of calcium influx into the nerve terminal. Phen also presented a competitive antimuscarinic activity and blocked noncompetitively the neuromuscular transmission. In this work we re-examined the mechanisms underlying the facilitatory actions of Phen on [(3)H]-ACh release in isolated ganglia of the guinea pig ileal myenteric plexus. Exposure of the preparations to Phen (10-50 microM) increased the release of [(3)H]-ACh by 81 to 68% over the basal. The effect was not affected by the ganglionic nAChR antagonist hexamethonium (1 nM) at a concentration that inhibited the increase of [(3)H]-ACh release induced by the nicotinic agonist dimethylphenylpiperazinium (DMPP, 30 microM). Association of Phen (10 microM) with DMPP potentiated the facilitatory effect of Phen. [(3)H]-ACh release was not altered by the muscarinic antagonists atropine (1 nM) or pirenzepine (1 microM). However, both antagonists inhibited the release of [(3)H]-ACh induced by either the muscarinic M1 agonist McN-343 (10 microM) or Phen (20 microM). The facilitatory effect of Phen was not altered by CdCl(2) (50 mM), but it was potentiated in the presence of tetraethylammonium (40 mM). The results indicate that the facilitatory action of Phen appears to be mediated by an increase of the inwardly rectifying potassium channels conductance probably related to the compound antimuscarinic activity.

Allosteric interaction of the anticholinergic drug [N-(4-phenyl)-phenacyl-l-hyoscyamine] (Phenthonium) with nicotinic receptors of post-ganglionic sympathetic neurons of the rat vas deferens.

Phenthonium (Phen), a quaternary analog of hyoscyamine, is a blocker of muscarinic activity and an allosteric blocker of alpha(1)2betagammaepsilon nicotinic receptors. Specifically, Phenthonium increases the spontaneous release of acetylcholine at the motor endplate without depolarizing the muscle or inhibiting cholinesterase activity. This paper compares Phenthonium's effects on sympathetic transmission and on ganglionic nicotinic receptor activation. Neurotransmitter release and twitch of the rat vas deferens were induced either by electrical stimulation or by 1,1-dimethyl-4-phenylpiperazine (DMPP) activation of nicotinic receptors. Contractions independent of transmitter release were induced by noradrenaline and adenosine 5'-triphosphate (ATP). Phenthonium inhibited transmitter release and depressed twitch without changing the responsiveness to noradrenaline or ATP. Twitch depression did not occur after K(+)-channel blockade with 4-aminopyridine (4-AP) or charybdotoxin. DMPP had a similar effect, but high concentrations induced contraction of non-stimulated organs. Incubation of Phenthonium inhibited further DMPP twitch depression and non-competitively depressed the contractile responses elicited by DMPP. Furthermore, mecamylamine, but neither methyllycaconitine nor atropine, blocked the contraction elicited by DMPP. Phenthonium and DMPP are K(+)-channel openers that primarily inhibit sympathetic transmission. Contraction induced by DMPP was probably mediated by neuronal nicotinic receptor other than the alpha7 subtype. The blockade of DMPP contractile response was unrelated to Phenthonium's antimuscarinic or K(+)-channel opening activities. Since Phenthonium's quaternary chemical structure limits its membrane diffusion, the non-competitive inhibition of DMPP excitatory responses should be linked to allosteric interaction with neuronal nicotinic receptors that putatively qualify Phenthonium as a novel modulator of cholinergic synapses.