Convergent evidences from human and animal studies implicate angiotensin I-converting enzyme activity in cognitive performance in schizophrenia.

In schizophrenia (SCZ), higher angiotensin I-converting enzyme (ACE) levels have been reported in patient's blood and cerebrospinal fluid (CSF). Hereby, we propose to explore whether the ACE activity levels are associated to cognitive performance in SCZ. Seventy-two patients with SCZ or schizoaffective disorder diagnosis, and 69 healthy controls (HCs) underwent a cognitive battery with parallel collection of peripheral blood samples to measure ACE activity. Significant higher ACE activity levels were confirmed in the plasma of SCZ patients compared with HCs (Student's t=-5.216; P<0.001). ACE activity significantly correlated to Hopkins delayed recall measures (r=-0.247; P=0.004) and Hopkins total (r=-0.214; P=0.012). Subjects grouped as high ACE activity (above average) had worse performance compared with low ACE activity level group for Hopkins delayed recall measure, even after correction for clinical condition, age, gender and years of education (P=0.029). The adjusted R squared for this final model was 0.343. This result was evident only comparing extreme groups for ACE activity, when splitting the sample in three groups with similar number of subjects. To clarify this finding, we performed an evaluation of the cognitive performance of transgenic mice with three copies of ACE gene in novel object recognition (NOR) test, which showed that such animals presented impairment in NOR (P<0.05) compared with two copies of wild-type animals. The results observed in SCZ patients and animal model suggest both the association of ACE to cognitive deficits in SCZ. This finding may support the evaluation of novel treatment protocols and/or of innovative drugs for specific intervention of cognitive deficits in SCZ envisioning concomitant ACE activity and behavior evaluations.

Altered acetylcholine release in the hippocampus of dystrophin-deficient mice.

Mild cognitive impairments have been described in one-third of patients with Duchenne muscle dystrophy (DMD). DMD is characterized by progressive and irreversible muscle degeneration caused by mutations in the dystrophin gene and lack of the protein expression. Previously, we have reported altered concentrations of α7- and ß2-containing nicotinic acetylcholine receptors (nAChRs) in hippocampal membranes of dystrophic (mdx) mice. This suggests that alterations in the central cholinergic synapses are associated with dystrophin deficiency. In this study, we examined the release of acetylcholine (ACh) and the level of the vesicular ACh transporter (VAChT) using synaptosomes isolated from brain regions that normally have a high density of dystrophin (cortex, hippocampus and cerebellum), in control and mdx mice at 4 and 12months of age. ACh release evoked by nicotinic stimulation or K(+) depolarization was measured as the tritium outflow from superfused synaptosomes preloaded with [(3)H]-choline. The results showed that the evoked tritium release was Ca(2+)-dependent and mostly formed by [(3)H]-ACh. ß2-containing nAChRs were involved in agonist-evoked [(3)H]-ACh release in control and mdx preparations. In hippocampal synaptosomes from 12-month-old mdx mice, nAChR-evoked [(3)H]-ACh release increased by 57% compared to age-matched controls. Moreover, there was a 98% increase in [(3)H]-ACh release compared to 4-month-old mdx mice. [(3)H]-ACh release evoked by K(+) depolarization was not altered, while the VAChT protein level was decreased (19%) compared to that of age-matched controls. In cortical and cerebellar preparations, there was no difference in nAChR-evoked [(3)H]-ACh release and VAChT levels between mdx and age-matched control groups. Our previous findings and the presynaptic alterations observed in the hippocampi of 12-month-old mdx mice indicate possible dysfunction of nicotinic cholinergic synapses associated with dystrophin deficiency. These changes may contribute to the cognitive and behavioral abnormalities described in dystrophic mice and patients with DMD.

Inhibition of gastric acid secretion by a standardized aqueous extract of Cecropia glaziovii Sneth and underlying mechanism.

Cecropia glazioui Sneth (Cecropiaceae) is used in folk medicine in tropical and subtropical Latin America as cardiotonic, diuretic, hypotensive, anti-inflammatory and anti-asthmatic. The hypotensive/antihypertensive activity of the plant aqueous extract (AE) and isolated butanolic fraction (BuF) has been confirmed and putatively related to calcium channels blockade in vascular smooth musculature [Lapa, A.J., Lima-Landman, M.T.R., Cysneiros, R.M, Borges, A.C.R., Souccar, C., Barreta, I.P., Lima, T.C.M., 1999. The Brazilian folk medicine program to validate medicinal plants - a topic in new antihypertensive drug research. In: Hostettman, K., Gupta, M.P., Marston, A. (Eds.), Proceedings Volume, IOCD/CYTED Symposium, Panamá City, Panamá, 23-26 February 1997. Chemistry, Biological and Pharmacological Properties of Medicinal Plants from the Americas. Harwood Academic Publishers, Amsterdam, pp. 185-196; Lima-Landman, M.T., Borges, A.C., Cysneiros, R.M., De Lima, T.C., Souccar, C., Lapa, A.J., 2007. Antihypertensive effect of a standardized aqueous extract of Cecropia glaziovii Sneth in rats: an in vivo approach to the hypotensive mechanism. Phytomedicine 14, 314-320]. Bronchodilation and antidepressant-like activities of both AE and BuF have been also shown [Delarcina, S., Lima-Landman, M.T., Souccar, C., Cysneiros, R.M., Tanae, M.M., Lapa, A.J., 2007. Inhibition of histamine-induced bronchospasm in guinea pigs treated with Cecropia glaziovi Sneth and correlation with the in vitro activity in tracheal muscles. Phytomedicine 14, 328-332; Rocha, F.F., Lima-Landman, M.T., Souccar, C., Tanae, M.M., De Lima, T.C., Lapa, A.J., 2007. Antidepressant-like effect of Cecropia glazioui Sneth and its constituents -in vivo and in vitro characterization of the underlying mechanism. Phytomedicine 14, 396-402]. This study reports the antiulcer and antisecretory gastric acid activities of the plant AE, its BuF and isolated compounds with the possible mechanism involved. Both AE and BuF were assayed on gastric acid secretion of pylorus-ligated mice, on acute models of gastric mucosal lesions, and on rabbit gastric H(+), K(+)-ATPase preparations. Intraduodenal injection of AE or BuF (0.5-2.0g/kg, i.d) produced a dose-related decrease of the basal gastric acid secretion in 4-h pylorus-ligated mice. At 1.0g/kg, BuF decreased the volume (28%) and total acidity (33%) of the basal acid secretion, and reversed the histamine (2.5mg/kg, s.c.)- or bethanecol (1.0mg/kg, s.c.)-induced acid secretion to basal values, indicating inhibition of the gastric proton pump. Pretreatment of mice with the BuF (0.05-0.5g/kg, p.o.) protected against gastric mucosal lesions induced by 75% ethanol, indomethacin (30mg/kg, s.c.) or restraint at 4 degrees C. BuF also decreased the gastric H(+), K(+)-ATPase activity in vitro proportionately to the concentration (IC(50)=58.8microg/ml). The compounds isolated from BuF, consisting mainly of cathechins, procyanidins and flavonoids [Tanae, M.M., Lima-Landman, M.T.R., De Lima, T.C.M., Souccar, C., Lapa, A.J., 2007. Chemical standardization of the aqueous extract of Cecropia glaziovii Sneth endowed with antihypertensive, bronchodilator, antacid secretion and antidepressant-like activities. Phytomedicine 14, 309-313], inhibited the in vitro gastric H(+), K(+)-ATPase activity at equieffective concentrations to that of BuF. The results indicate that C. glazioui constituents inhibit the gastric proton pump; this effect may account for the effective antisecretory and antiulcer activities of the standardized plant extract.

Antidepressant-like effect of Cecropia glazioui Sneth and its constituents - in vivo and in vitro characterization of the underlying mechanism.

The present study aimed to characterize the antidepressant-like effect of a standardized aqueous extract (AE) of Cecropia glazioui Sneth and its purified fractions on in vivo (forced swimming test), ex vivo (hippocampal monoamines levels) and in vitro (serotonin, noradrenaline and dopamine uptake) tests, searching for the active principles and the underlying mechanisms of action. Treatment with AE, or with its butanolic fraction (BuF), the latter rich in catechins, procyanidins and flavonoids, reduced the immobility of rats in the forced swimming test indicating an antidepressant-like effect. Biochemical analysis of the hippocampal neurotransmitters in BuF-treated rats showed significant increase in monoamines levels. BuF and six of its purified constituents inhibited the uptake of [(3)H]-serotonin, [(3)H]-dopamine and [(3)H]-noradrenaline by synaptosomes of different brain regions. Catechin, catechin (4alpha-->8) ent-catechin (Procyanidin B3 isomer) and epicatechin (4beta-->8) epicatechin (Procyanidin B2) were the most active compounds. Comparatively, the uptake of [(3)H]-noradrenaline was the most affected. These results show that the antidepressant-like effect promoted by C. glazioui extract is most likely due to the blockade of the monoamines uptake in the CNS.

ACE activity during the hypotension produced by standardized aqueous extract of Cecropia glaziovii Sneth: a comparative study to captopril effects in rats.

To evaluate the effect of the standardized aqueous extract (AE) of Cecropia glaziovii Sneth on the plasma angiotensin I converting enzyme (ACE-EC 3.4.15.1) activity, rats were treated with a single dose of AE (1 g/kg, p.o.) or repeatedly (0.5 g/kg/bid, p.o.) for 60 days. Captopril (50 mg/kg, p.o.) was used as positive control on the same animals. The effects on the blood pressure were recorded directly from the femoral artery (single dose), or indirectly by the tail cuff method (repeated doses) in conscious rats. The plasma ACE activity was determined spectrofluorimetrically using Hypuril-Hystidine-Leucine as substrate. The arterial blood pressure, heart rate and plasma ACE activity were not significantly modified within 24 h after a single dose administration of AE. Comparatively, blood pressure in captopril treated rats was reduced by 7-16% and heart rate was increased by 10-20% from 30 min to 24 h after drug administration. ACE activity after captopril presented a dual response: an immediate inhibition peaking at 30 min and a slow reversal to 32% up-regulation after 24 h. To correlate the drug effects upon repeated administration of either compound, normotensive rats were separated in three groups: animals with high ACE (48.8+/-2.6 nmol/min/ml), intermediate ACE (39.4+/-1.4 nmol/min/ml) and low ACE (23.5+/-0.6 nmol/min/ml) activity, significantly different among them. Repeated treatment with AE reduced the mean systolic blood pressure (121.7+/-0.5 mm Hg) by 20 mm Hg after 14 days. The hypotension was reversed upon washout 60 days afterwards. Likely, repeated captopril administration decreased blood pressure by 20 mm Hg throughout treatment in all groups. After 30 days treatment with AE (0.5 g/kg/bid, p.o.) the plasma ACE activity was unchanged in any experimental group. After captopril (50 mg/kg/bid, p.o.) administration the plasma ACE activity was inhibited by 50% within 1 h treatment but it was up-regulated by 120% after 12 h in all groups. It is concluded that the hypotension produced by prolonged treatment with AE of C. glaziovii is unrelated to ACE inhibition.

Chemical standardization of the aqueous extract of Cecropia glaziovii Sneth endowed with antihypertensive, bronchodilator, antiacid secretion and antidepressant-like activities.

This study reports the extraction process and standardization of the aqueous extract (AE) of a Cecropia species aiming its pharmacological characterization as a phytomedicine to be used in primary health care. The plant was originally collected in its environment, and was thereafter specially cultivated for the present work. To standardize the plant AE, several 2.0% tea of the dried leaves were prepared under controlled conditions and freeze dried. The AE (20% yield) was partitioned with n-butanol yielding the butanolic fraction (BuF; 1% yield). The activity of AE on vital organ functions (cardiovascular, respiratory, gastrointestinal and central nervous system) was determined in vivo. The effects of AE were compared to those of BuF in the same models and the relative potency determined. BuF was further evaluated in representative in vitro models to assess possible mechanisms of action. Chemical constituents of BuF were isolated in preparative HPLC columns yielding 10 highly purified compounds chemically identified as catechins (2), procyanidins (4), flavonoids (2), mixed sugars (1) and chlorogenic acid. All the compounds were identified by chemical analytic instrumentation (13C-NMR, 1H-NMR, LC-MS). Their relative concentrations in AE were ca 12% catechins, 19% procyanidins and 19% flavonoids. The pharmacological activity of the standardized AE is reported in accompanying papers.

Antihypertensive effect of a standardized aqueous extract of Cecropia glaziovii Sneth in rats: an in vivo approach to the hypotensive mechanism.

Cecropia glaziovii Sneth is a common tree at the Southeastern Brazilian coast. As many other species of the genus, it shares the reputed folk use to treat heart failure, cough, asthma and bronchitis. The plant has been cultivated under controlled conditions and the 2% aqueous extract (AE) prepared with the dried leaves was standardized by its chemical contents on catechins, flavonoids and procyanidins. The present paper reports the antihypertensive activity of AE and of n-butanol fraction (BuF), an enriched semi-purified butanolic fraction used to isolate the main chemical constituents. Oral administration of AE and BuF induced hypotension in normotensive rats. The effect of AE (0.5 g/kg/bi, p.o.) was time and dose-dependent peaking at 2-3 weeks after daily administration. BuF was faster but not more active than AE. Both extracts decreased the hypertension of spontaneous hypertensive rats, the hypertension induced in rats by L-NAME treatment and that induced by constriction of one renal artery. The antihypertensive effect was maintained for as long as 60 days of treatment and was reversible upon drug washout at the same rate of its establishment. Acute i.v. administration of BuF to anesthetized rats induced a fast short-lasting hypotension and inhibited the pressor responses to noradrenaline, angiotensin I and angiotensin II by 40%. These results were indirect indications that the hypotension induced by AE is not related to ACE inhibition, increased NO synthesis, or specific blockade of alpha1 and AT1 receptors. It can be suggested that BuF interferes with the calcium handling mechanisms in smooth muscle cells and neurons. Intravenous injection of five out of nine compounds isolated from BuF produced immediate but short-lasting hypotension that does not correlate with the onset of the hypotension after oral treatment. This finding suggests that they may not be the compounds directly responsible for the delayed and sustained hypotension after per os administration of AE. The many compounds isolated from AE are under evaluation to determine its pharmacokinetics, mechanisms of action and interactions necessary to yield the plant effect. Although its mechanism is still unknown, AE seems to be an effective and safe antihypertensive phytomedicine.

Inhibition of histamine-induced bronchospasm in guinea pigs treated with Cecropia glaziovi Sneth and correlation with the in vitro activity in tracheal muscles.

A standardized aqueous extract (AE) and a purified fraction (BuF) of Cecropia glaziovi Sneth leaves were tested in unrestrained guinea pigs challenged with histamine. Changes of the respiratory pressure and rate were recorded in a whole body plethysmograph before and after treatment. The concentration of histamine necessary to produce bronchospasm was increased by five-fold following administration of AE (1.0 g/kg p.o.), and by two-fold after treatment with the semi-purified procyanidin/flavonoids enriched BuF (0.1 g/kg p.o.). Both effects were blocked by previous treatment with propranolol (10.0 mg/kg i.p.). In vitro incubation of BuF (0.1-1.0 mg/ml) decreased by 13-55% the maximal response of guinea pig tracheal muscle to histamine, without significant change of EC50. The results confirmed old reports on the useful pulmonary effects of Cecropia extracts. The bronchodilation observed in vivo seems to be related to beta-adrenergic activity observed in vitro only with high concentrations of the purified extract.

Pharmacological study of Stachytarpheta cayennensis Vahl in rodents.

Freeze-dried aqueous extracts (AEs, 0.1-1g/kg body wt., p.o.) obtained from entire or selected parts of Stachytarpheta cayennensis were tested for their effects on gastric secretion, gastric motility, inflammation and pain in rodents, with the purpose of validating the plant's ethnomedical uses. The AE-Total, AE-Flowers and AE-Leaves but not AE-Stems inhibited the gastric acid secretion in pylorus-ligated rats with varying potency. Purification of AEs yielded the semipurifed fractions EtFs rich in iridoids. All the EtFs with exception of EtF-Stems inhibited gastric acid secretion of pylorus ligated mice. While AE-Total stimulated the intestinal transit of mice by 43%, AE-Leaves delayed it by 38%. These effects on intestinal transit were not observed when the EtFs were tested. Only AE-Leaves and AE-Flowers altered the gastric emptying of semisolids, increasing it by 45% and 69%, respectively. These results indicate that the compounds related to inhibition of gastric acid secretion and gastrointestinal motility are different. The AE-Total reduced abdominal writhing induced by acetic acid potently (ED50 value = 700 mg/kg, p. o.) without altering the writhes induced by acetylcholine. Attempts to identify the mechanism of analgesia were unsuccessful since the AE-Total did not show analgesic effects when tested in different models of pain such as formalin and capsaicin or the tail-flick test. Pretreatment of animals with AE-Total did not show antiinflammatory activity in any of the acute (paw edema induced by carrageenin, dextran or histamine, pleurisy induced by carrageenin and capsaicin-induced mouse ear edema) or chronic (air pouch) models used. No toxic signs were observed after administration of the different extracts up to 2 g/kg body wt., p.o. Collectively, the results confirmed folk information indicating presence of analgesic, mild laxative and potent inhibition of gastric secretion activities in the aqueous extracts of S. cayennensis. The results do not, however confirm the folk use of the plant as an antiinflammatory medicine.

Solanum paniculatum L. (Jurubeba): potent inhibitor of gastric acid secretion in mice.

Solanum paniculatum L. is used commonly in Brazilian folk medicine for the treatment of liver and gastrointestinal disorders. The freeze-dried aqueous extracts (WEs) obtained from distinct parts of the plant (flowers, fruits, leaves, stems and roots) were tested to determine their antiulcer and antisecretory gastric acid activities using mice. The aqueous extracts of roots, stems and flowers inhibited gastric acid secretion in pylorus-ligated mice with ED50 values of 418, 777 and 820 mg/kg body wt. (i.d.), respectively. Extracts of leaves (0.5-2 g/kg body wt., i.d.) did not affect gastric secretion, whereas fruit extracts (0.5-2 g/kg body wt., i.d.) stimulated gastric acid secretion. The stimulatory effect of the fruit extract was inhibited by pretreatment with atropine (5 mg/kg body wt., i.m.) but not with ranitidine (80 mg/kg body wt., i.p.) suggesting that the fruit extract activates the muscarinic pathway of gastric acid secretion. In contrast, administration of the root extract into the duodenal lumen inhibited histamine- and bethanechol-induced gastric secretion in pylorus-ligated mice. In addition, the aqueous extract of roots (ED50 value, 1.2 g/kg body wt., p.o.) protected the animals against production of gastric lesions subsequent to the hypersecretion induced in mice by stress following cold restraint. This effect was not reproduced when the lesions were induced by blockade of prostaglandins synthesis via subcutaneous injection of indomethacin. Thus, antiulcer activity of the plant extracts appears to be related directly to a potent anti-secretory activity. No toxic signs were observed following administration of different extracts up to 2 g/kg body wt., p.o. Collectively, the results validate folk use of Solanum paniculatum L. plant to treat gastric disorders.

Inhibition of angiotesin-converting enzyme by quercetin alters the vascular response to brandykinin and angiotensin I.

Quercetin, one of the most widely distributed flavonoids in the plant kingdom, inhibits various enzymes. This study examined its inhibitory effect on the angiotensin-converting enzyme activity through the cardiovascular response to bradykinin and angiotensin I. Quercetin pre-treatment (88.7 micromol/kg p.o., 45 min; 14.7 micromol/kg i.v., 5 min) significantly potentiated the hypotensive effect of bradykinin (10 nmol/kg i.v.). This association was significantly attenuated by an antagonist of the B2 receptor. In addition, the hypertensive response to angiotensin I (0.1 nmol/kg i.v.) was significantly reduced by quercetin pretreatment using the same parameters as before. These results suggest an inhibitory effect of quercetin on the angiotensin-converting enzyme activity, similar to that of captopril. Quercetin was equally effective when given orally or intravenously.

Nicotinic receptor/ionic channel complex (AChR) in androgen-dependent skeletal muscle cultures.

The kinetic properties of the nicotinic receptor/ionic channel complex (AChR) were compared in cell cultures obtained from androgen-dependent skeletal muscles of the perineal complex (P) and from muscles less dependent upon sex hormones (the thigh musculature, T). Because the development of P is delayed compared to other skeletal muscles in the rat, cultures were performed taking into account the age of the donor (4- or 6-day-old rats), and the time interval the cells remained in culture (7 days and 15 days). The ionic channel conductance (gamma) and the mean channel open time (tau) were determined with the patch-clamp technique in the cell-attached configuration at room temperature. Cultures from P and T muscles were morphologically identical in size and shape, independent of the animals' age at plating or on the plating time. In all of them, the AChR was spread over the cell membrane. More than one AChR ionic channel conductance was observed in P and T cultures, and the prevalent value of gamma in either culture ranged from 30 pS to 35 pS. In P fibers from 4-day-old rats cultured for 7 days (P 4/7), the distribution of channel open times fitted a double exponential, while in T 4/7 they were fitted with a single exponential. In cultures from P and T muscles obtained from older rats (6 days old) and in those cells remaining in culture for a prolonged time (15 days), the channel open times also fitted a double exponential. Because P and T cultures lack trophic neuronal influences, the difference observed between the tau of P 4/7 and T 4/7 was thought to be the hormone requirement of P muscles to grow and differentiate. Likewise, the difference observed between T 4/7 and T 4/15 may indicate the need for neurotrophic influences to maintain higher tau values in older cultures. Since this requirement is not found in cultured fibers, tau would tend to assume slower values approaching those of P without hormone activation.

Diterpene from Baccharis trimera with a relaxant effect on rat vascular smooth muscle.

A bioassay monitored fractionation of a chloroform extract from the aerial parts of Baccharis trimera yielded a mixture that blocked the Ca2+-induced contractions of KCl- depolarized rat portal vein preparations. Pharmacological tests of two pure compounds isolated from the mixture revealed the dilactonic clerodane diterpene as the active compound.

Evaluation of the antiurolithiatic activity of the extract of Costus spiralis Roscoe in rats.

The antiurolithiatic activity of the water extract of Costus spiralis Roscoe was tested on formation of calculi on implants of calcium oxalate crystals or zinc disc in the urinary bladder of rats. The plant is a species from the family Zingiberaceae used in Brazilian folk medicine in urinary affections and for expelling urinary stones. Implantation of the foreign body in the urinary bladder of adult rats induced formation of urinary stones and hypertrophy of the smooth musculature. Oral treatment with the extract of Costus spiralis Roscoe (0.25 and 0.5 g/kg per day) after 4 weeks surgery reduced the growth of calculi, but it did not prevent hypertrophy of the organ smooth musculature. The contractile responses of isolated urinary bladder preparations to the muscarinic agonist bethanecol, in the presence and absence of the extract (0.3-3 mg/ml) or atropine (0.3-3 nM) did not differ among the experimental groups. The results indicate that the extract of Costus spiralis Roscoe is endowed with antiurolithiatic activity confirming thus folk information. The effect, however, was unrelated to increased diuresis or to a change of the muscarinic receptor affinity of the bladder smooth musculature to cholinergic ligands.

Natural probes for cholinergic sites: L-bebeerine actions on the neuromuscular transmission, the nicotinic receptor/ionic channel complex, and contraction of skeletal muscles.

The mechanisms underlying the muscle relaxant activity of 1-bebeerine (BB), a tertiary alkaloid isolated from the roots of Chondrodendron platyphyllum, were examined in mammalian and amphibian skeletal muscles. Injections of BB (0.05-1 g/kg, i.p.) in rats caused a dose-related flaccid paralysis and respiratory arrest at high doses. In isolated rat diaphragm and toad sartorius muscles, BB depressed the indirectly elicited muscle twitches (IC50: 228 microM and 5.4 microM, respectively, at 22 degrees C) and blocked the nerve-elicited muscle action potential. The neuromuscular blockade was not reversed by neostigmine (10 microM). High concentrations of BB (170 and 340 microM) caused muscle contracture unrelated to the junctional blockade, and intensified by increasing the bath temperature. Analysis of the contraction properties showed that BB (40 and 80 microM) increased the twitch/tetanus ratio (46% and 125%) and prolonged the relaxation time; the falling phase of the directly elicited action potential in toad sartorius muscle fibers was slower probably by a decreased potassium conductance. BB (0.1-340 microM) reduced the binding of [125l]alpha--bungarotoxin to the junctional ACh receptor of the rat diaphragm (IC50: 47.7 microM, at 37 degrees C. At low concentrations BB (1.5-15 microM) induced either opening or blockade of the ACh receptor-ionic channel. The results showed that BB blocked noncompetitively the neuromuscular transmission through a mechanism that affects the ACh recognition site and the ionic channel properties. The alkaloid also produced muscle contracture and changed the contractile properties through its extra-junctional action at the calcium handling by the sarcoplasmic reticulum or the contractile machinery.

Natural probes for cholinergic sites: L-bebeerine actions on the neuromuscular transmission, the nicotinic receptor/ionic channel complex, and contraction of skeletal muscles.

The mechanisms underlying the muscle relaxant activity of 1-bebeerine (BB), a tertiary alkaloid isolated from the roots of Chondrodendron platyphyllum, were examined in mammalian and amphibian skeletal muscles. Injections of BB (0.05-1 g/kg, i.p.) in rats caused a dose-related flaccid paralysis and respiratory arrest at high doses. In isolated rat diaphragm and toad sartorius muscles, BB depressed the indirectly elicited muscle twitches (IC50: 228 microM and 5.4 microM, respectively, at 22 degrees C) and blocked the nerve-elicited muscle action potential. The neuromuscular blockade was not reversed by neostigmine (10 microM). High concentrations of BB (170 and 340 microM) caused muscle contracture unrelated to the junctional blockade, and intensified by increasing the bath temperature. Analysis of the contraction properties showed that BB (40 and 80 microM) increased the twitch/tetanus ratio (46

and 125

) and prolonged the relaxation time; the falling phase of the directly elicited action potential in toad sartorius muscle fibers was slower probably by a decreased potassium conductance. BB (0.1-340 microM) reduced the binding of [125l]alpha--bungarotoxin to the junctional ACh receptor of the rat diaphragm (IC50: 47.7 microM, at 37 degrees C. At low concentrations BB (1.5-15 microM) induced either opening or blockade of the ACh receptor-ionic channel. The results showed that BB blocked noncompetitively the neuromuscular transmission through a mechanism that affects the ACh recognition site and the ionic channel properties. The alkaloid also produced muscle contracture and changed the contractile properties through its extra-junctional action at the calcium handling by the sarcoplasmic reticulum or the contractile machinery.

Mechanism of neuromuscular blockade induced by phenthonium, a quaternary derivative of (-)-hyoscyamine, in skeletal muscles.

1. The mechanisms underlying the postjunctional blockade induced by phenthonium [N-(4-phenyl) phenacyl 1-hyoscyamine] were investigated in mammalian and amphibian muscles. This muscarinic antagonist was previously shown to enhance specifically the spontaneous acetylcholine (ACh) release at concentrations that blocked neuromuscular transmission. 2. In both rat diaphragm and frog sartorius muscles, phenthonium (Phen, 1-100 microM) depressed the muscle twitches elicited by nerve stimulation (IC50: 23 microM and 5 microM, respectively), and blocked the nerve-evoked muscle action potential. The neuromuscular blockade was not reversed after incubation with neostigmine. 3. Equal concentrations of Phen decreased the rate of rise and prolonged the falling phase of the directly elicited action potential in frog sartorius muscle fibres, indicating that the drug also affects the sodium and potassium conductance. 4. Phen (50 and 100 microM) protected the ACh receptor against alpha-bungarotoxin (BUTX) blockade in the mouse diaphragm allowing recording of endplate potentials and action potentials after 5 h wash with physiological salt solution. 5. Phen (10-100 microM) produced a concentration- and voltage-dependent decrease of the endplate current (e.p.c.), and induced nonlinearity of the current-voltage relationship. At high concentrations Phen also shortened the decay time constant of e.p.c (tau(e.p.c.)) and reduced its voltage sensitivity. 6. At the same range of concentrations, Phen also reduced the initial rate of [125I]-BUTX binding to junctional ACh receptors of the rat diaphragm (apparent dissociation constant = 24 microM), the relationship between the degree of inhibition and antagonist concentration being that expected for a competitive mechanism. 7. It is concluded that Phen affects the electrical excitability of the muscle fibre membrane, and blocks neuromuscular transmission through a mechanism that affects the agonist binding to its recognition site and ionic channel conductance of the nicotinic ACh receptor.

Gender does not influence neuromuscular properties in dimorphic skeletal muscles of the toad.

The aim of this work was to study gender differences on the physiology of the dimorphic brachial musculature involved in the clasp reflex of the toad (Bufo marinus L.). The neuromuscular transmission, the sensitivity to acetylcholine (ACh) and the cholinesterase activity were compared on the forelimb sternoradialis muscles (SR) from male and female toads. The interosseous muscles of the first finger were used to compare the properties of the nicotinic receptor/ionic channel complex (AChR). All the muscles studied were dimorphic, i.e. significantly smaller in the female than in the male frog in otherwise similar size animals. The SR of either sex contracted to bath application of ACh with similar EC50. In physiological solution the frequency of the miniature end-plate potentials (mepps) was very low (0.1 s-1) and no gender difference was detected. The mepp amplitudes were 0.62 +/- 0.03 and 0.58 +/- 0.03 mV in SR from male and female toads, respectively. To increase exocytosis the muscles were incubated in hypertonic solution (158 mM NaCl). Under this condition mepp frequency was increased by five and seven times and mepp amplitude increased by 1.3 and 1.6 times in SR from male and female toads, respectively. The cholinesterase activity measured by the colorimetric method, did not differ in SR from male and female toads. In muscle fibers dissociated from the dimorphic interosseous muscles of male and female toads, the ionic channel conductance was 43 +/- 5.3 and 44 +/- 4.5 pS, respectively. The mean channel open time was voltage-dependent and not significantly different in preparations from both genders. These observations indicate that neither the ACh-nicotinic receptor interaction, nor the AChR complex kinetics and the nicotinic excitation-contraction coupling or the cholinesterase activity differ in dimorphic muscles from Bufo genders. No gender difference was detected in neuromuscular transmission of the studied muscle. Only a slight increase in mepp frequency and amplitude could be detected when the muscles were incubated in hypertonic solution.

Inhibition of gastric acid secretion by the aqueous extract and purified extracts of Stachytarpheta cayennensis.

Stachytarpheta cayennensis Schauer (Verbenaceae) is used in folk medicine to treat gastric and intestinal disturbances. The freeze-dried aqueous extract of the whole plant tested to rodents up to the dose of 2 g kg-1, p.o., did not produce signs of toxicity. The extract (0.5-2 g kg-1, p.o.) increased the intestinal motility and protected mice against ulcers induced by restraintin-cold, ethanol or indomethacin. Injected into the duodenal lumen the extract inhibited the basal acid secretion as well as that induced by histamine and bethanecol in pylorus-ligated mice. Partition of the aqueous extract in organic solvents yielded semipurified fractions whose antiacid activity guided further chemical purification. All the fractions were chromatographically characterized, the main substances in the active extract being flavonoids and amines; some substances were revealed only under UV light. The most purified active fraction obtained presented a specific activity 5-10 times higher than that detected in the original extract. Data from pharmacological studies indicate that the antiulcer activity of S. cayennensis is related to a specific inhibition of gastric acid secretion. Cholinergic and histaminergic stimulation of acid secretion were similarly reduced by the extracts suggesting inhibition of common steps in both pathways, possibly at the level of histamine release/H2 receptor interaction, or at the proton pump. Whatever the mechanisms involved, the present data confirm the plant effectiveness as antiacid/antiulcer and laxative.

Phenthonium induces a transient increase of acetylcholine efflux from motor nerve terminals.

Phenthonium (10-50 microM), a quaternary derivative of 1-hyoscyamine, increases the frequency of miniature end-plate potentials (2-5 fold) and blocks the nicotinic receptor-ionic channel in skeletal muscles. When tested on rat diaphragms previously incubated with [3H]choline, phenthonium (50 microM) increased the spontaneous release of radiolabelled acetylcholine (ACh) from 11.6 +/- 6.4 to 110.5 +/- 40.2 x 10(3) dpm/g within 15 min. The effect was transient, declining to 24.6 +/- 14.7 after 50 min. Subsequent electrical stimulation still in the presence of phenthonium increased the efflux to 164.7 +/- 45.3. The fractional release relative to the level before stimulation did not differ from controls. Phenthonium (20 microM) did not increase the spontaneous ACh release but doubled the efflux induced by nerve stimulation. The present results, compared to previous electrophysiological findings, indicate that quantal and nonquantal release are increased by phenthonium. They also show that the transient effect is not due to ACh depletion in nerve terminals.