Glória Queiroz

Ethnopharmacological notes about ancient uses of medicinal plants in Trás-os-Montes (northern of Portugal)

AIM OF THE STUDY In order to preserve the ancestral knowledge, an ethnopharmacological study has been carried out in two councils belonging to Trás-os-Montes region a small area located in the northern of Portugal. In that area, medicinal plants, most of the species wild, are still in use among farmers, shepherds and other people who live far from villages and built-up areas. MATERIALS AND METHODS Among the 46 people that were interviewed (mean age of 66 years old), 88 species belonging to 42 families of vascular plants were identified for treatment of various human ailments. An ethnopharmacological report is made consisting of species names, vernacular names, popular uses of the plants and their pharmacological properties. RESULTS AND CONCLUSION The most dominant family is Lamiaceae (18%) and the most frequently part of the plant used for the treatment of diseases are leaves (37.9%). The largest number of taxa is used to treat gastrointestinal disorders (73.9%).

Release of ATP from cultured rat astrocytes elicited by glutamate receptor activation

The release of ATP was studied in cultures of astrocytes derived from the brain hemispheres of newborn rats. There was a basal efflux of ATP, which was increased up to 19-fold by glutamate (300-1000 microM). N-methyl-D-aspartate (20-500 microM), alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA; 30-100 microM) and kainate (20 microM). The N-methyl-D-aspartate receptor-selective antagonist 2-amino-5-phosphonopentanoate (100 microM) blocked the effect of N-methyl-D-aspartate but not the effects of AMPA, kainate and glutamate. The AMPA receptor-selective antagonist 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(f)quinoxaline (30 microM) blocked the effect of AMPA and also of glutamate and N-methyl-D-aspartate, but not the effect of kainate. The kainate receptor-selective antagonist D-glutamyl-amino-methanesulfonate (30 microM) blocked the effect of kainate but not of glutamate. Glutamate (1000 microM) did not increase the release of lactate dehydrogenase from astrocytes. Excitatory amino acids are known to release adenyl compounds in the brain. The present results identify one adenyl compound thus released, namely ATP, and identify astrocytes as one source. The release is brought about by activation of any of the three ionotropic glutamate receptor types-N-methyl-D-aspartate, AMPA and kainate receptors. AMPA receptors seem to mediate at least a part of the effect of glutamate itself, but the involvement of other receptors cannot be ruled out. ATP and its degradation products, such as adenosine, once released, may exert acute as well as trophic effects on neurons and glial cells.

A study of the mechanism of the release of ATP from rat cortical astroglial cells evoked by activation of glutamate receptors.

Glutamate and the selective agonists at ionotropic glutamate receptors N-methyl-D-aspartate, alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) and kainate release ATP from superfused primary cultures of rat cortical astrocytes. The mechanism of this release was investigated. The release of ATP elicited by N-methyl-D-aspartate and kainate was abolished or greatly reduced in the absence of external calcium as well as in the presence of cadmium (1 mM) and nicardipine (10 microM). The release of ATP elicited by AMPA, in contrast, was not changed by these interventions. The calcium ionophore ionomycin (5 microM) released ATP in the presence but not in the absence of external calcium. No release was obtained with alpha-latrotoxin. Of several compounds tested as potential blockers of ATP transporters or channels only glibenclamide (100 microM) and diphenylamine-2-carboxylate (500 microM), which block the cystic fibrosis transmembrane conductance regulator, caused any change: both reduced the effect of AMPA without changing the effects of N-methyl-D-aspartate and (only glibenclamide tested) kainate. Lithium (1 mM) abolished the release of ATP evoked by glutamate and AMPA and significantly reduced the release evoked by N-methyl-D-aspartate and kainate. The three glutamate receptor agonists did not increase the release of lactate dehydrogenase. The results confirm the previous observation that activation of N-methyl-D-aspartate, AMPA and kainate receptors induces release of ATP from astrocytes in culture. Two different mechanisms seem to be involved. The N-methyl-D-aspartate- and kainate-induced release of ATP requires an influx of calcium, is not due to neuron-like exocytosis, is not mediated by cystic fibrosis transmembrane conductance regulator or a mechanism regulated by cystic fibrosis transmembrane conductance regulator, and is reduced (by an unknown mechanism) but not abolished by lithium. The AMPA-induced release does not require extracellular calcium, may be mediated by cystic fibrosis transmembrane conductance regulator or a mechanism regulated by cystic fibrosis transmembrane conductance regulator, and is abolished (by an unknown mechanism) by lithium. The ability of astrocytes to both release ATP and respond to ATP suggests that ATP may act as an autocrine or paracrine messenger between these glial cells.

Purinoceptor modulation of noradrenaline release in rat tail artery: tonic modulation mediated by inhibitory P2Y- and facilitatory A2A-purinoceptors

1 The effects of analogues of adenosine and ATP on noradrenaline release elicited by electrical stimulation (5 Hz, 2700 pulses) were studied in superfused preparations of rat tail artery. The effects of purinoceptor antagonists, of adenosine deaminase and of adenosine uptake blockade were also examined. Noradrenaline was measured by h.p.l.c. electrochemical detection. 2 The A1‐adenosine receptor agonist, N6‐cyclopentyladenosine (CPA; 0.1 − 100 nM) reduced, whereas the A2A‐receptor agonist 2‐p‐(2‐carboxyethyl)phenethylamino‐5′‐N‐ethylcarboxamidoadenosine (CGS 21680; 3 − 30 nM) increased evoked noradrenaline overflow. These effects were antagonized by the A1‐adenosine receptor antagonist, 8‐cyclopentyl‐1,3‐dipropylxanthine (DPCPX; 20 nM) and the A2‐adenosine receptor antagonist, 3,7‐dimethyl‐1‐propargylxanthine (DMPX; 100 nM), respectively. The P2Y‐purinoceptor agonist, 2‐methylthio‐ATP (1 − 100 μm) reduced noradrenaline overflow, an effect prevented by the P2‐purinoceptor antagonist, cibacron blue 3GA (100 μm) and suramin (100 μm). 3 Adenosine deaminase (2 u ml−1), DMPX (100 nM) and inhibition of adenosine uptake with S‐(p‐nitrobenzyl)‐6‐thioinosine (NBTI; 50 nM) decreased evoked noradrenaline overflow. DPCPX alone did not change noradrenaline overflow but prevented the inhibition caused by NBTI. The P2Y‐purinoceptor antagonist, cibacron blue 3GA (100 μm) increased evoked noradrenaline overflow as did suramin, a non‐selective P2‐antagonist. 4 It is concluded that, in rat tail artery, inhibitory (A1 and P2Y) and facilitatory (A2A) purinoceptors are present and modulate noradrenaline release evoked by electrical stimulation. Endogenous purines tonically modulate noradrenaline release through activation of inhibitory P2Y and facilitatory A2A purinoceptors, whereas a tonic activation of inhibitory A1 purinoceptors seems to be prevented by adenosine uptake.

Facilitatory and inhibitory modulation by endogenous adenosine of noradrenaline release in the epididymal portion of rat vas deferens.

SummaryThe present study aimed at determining the modulation by adenosine of the release of noradrenaline in the epididymal portion of the rat vas deferens. The tissues were treated with pargyline and perifused in the presence of desipramine and yohimbine. Up to four periods of electrical stimulation were applied (5 Hz, 9 min).The A1-adenosine receptor selective agonist R-N6-phenylisopropyladenosine (R-PIA; 100–900 nmol·l−1) reduced, whereas the A2A-receptor selective agonist 2-p-(2-carboxyethyl)phenethylamino-5′-N-ethylcarboxamidoadenosine (CGS21680; 3–30nmol·l−1) increased the electrically-evoked noradrenaline overflow in a concentration-dependent manner. The nonselective agonist 5′-N-ethy1carboxamidoadenosine (NECA; 30–300 nmol·l−1) reduced noradrenaline overflow, but the effect did not depend on the concentration. Adenosine deaminase at the concentration of 0.5 μ·ml−1 decreased but at that of 2.0 μ·ml−1 increased noradrenaline overflow. The inhibitors of adenosine uptake, S-(4-nitrobenzyl)-6-thioinosine (NBTI; 50 nmol·l−1) and dipyridamole (3 μmol·l−1), increased the electrically-evoked noradrenaline overflow. The A1-adenosine receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX; 20 nmol·l−1) caused an increase whereas the A2-adenosine receptor antagonist 3,7-dimethyl-1-(2-propynyl)xanthine (DMPX; 0.1 μmol·l−1) caused a decrease. NBTI (50 nmol·l−1), partially antagonized the effect of both DPCPX (20 nmol·l−1) and DMPX (0.1 μmol·l−1).It is concluded that, in the epididymal portion of the rat vas deferens, endogenous adenosine tonically modulates the release of noradrenaline evoked by electrical stimulation, through activation of both inhibitory (A1) and facilitatory (A2A) adenosine receptors.

Release inhibitory receptors activation favours the A2A‐adenosine receptor‐mediated facilitation of noradrenaline release in isolated rat tail artery

Interactions between A2A‐adenosine receptors and α2‐, A1‐ and P2‐ release‐inhibitory receptors, on the modulation of noradrenaline release were studied in isolated rat tail artery. Preparations were labelled with [3H]‐noradrenaline, superfused with desipramine‐containing medium, and stimulated electrically (100 pulses at 5 Hz or 20 pulses at 50 Hz). Blockade of α2‐autoreceptors with yohimbine (1 μM) increased tritium overflow elicited by 100 pulses at 5 Hz but not by 20 pulses at 50 Hz. The selective A2A‐receptor agonist 2‐p‐(2‐carboxyethyl)phenethylamino‐5′‐N‐ethylcarboxamidoadenosine (CGS 21680; 1 – 100 nM) enhanced tritium overflow elicited by 100 pulses at 5 Hz. Yohimbine prevented the effect of CGS 21680, which was restored by the A1‐receptor agonist N6‐cyclopentyladenosine (CPA; 100 nM) or by the P2‐receptor agonist 2‐methylthioadenosine triphosphate (2‐MeSATP; 80 μM). CGS 21680 (100 nM) failed to increase tritium overflow elicited by 20 pulses at 50 Hz. The α2‐adrenoceptor agonist 5‐bromo‐6‐(2‐imidazolin‐2‐ylamino)‐quinoxaline (UK 14304; 30 nM), the A1‐receptor agonist CPA (100 nM) or the P2‐receptor agonist 2‐MeSATP (80 μM) reduced tritium overflow. In the presence of these agonists CGS 21680 elicited a facilitation of tritium overflow. Blockade of potassium channels with tetraethylammonium (TEA; 5 mM) increased tritium overflow elicited by 100 pulses at 5 Hz to values similar to those obtained in the presence of yohimbine but did not prevent the effect of CGS 21680 (100 nM) on tritium overflow. It is concluded that, in isolated rat tail artery, the facilitation of noradrenaline release mediated by A2A‐adenosine receptors is favoured by activation of release inhibitory receptors.

Involvement of G-protein βγ subunits on the influence of inhibitory α2-autoreceptors on the angiotensin AT1-receptor modulation of noradrenaline release in the rat vas deferens

The influence of alpha2-autoreceptors on the facilitation of [3H]-noradrenaline release mediated by angiotensin II was studied in prostatic portions of rat vas deferens preincubated with [3H]-noradrenaline. Angiotensin II enhanced tritium overflow evoked by trains of 100 pulses at 8 Hz, an effect that was attenuated by the AT1-receptor antagonist losartan (0.3-1 microM), at concentrations suggesting the involvement of the AT1B subtype. The effect of angiotensin II was also attenuated by inhibition of phospholipase C (PLC) and protein kinase C (PKC) indicating that prejunctional AT1-receptors are coupled to the PLC-PKC pathway. Angiotensin II (0.3-100 nM) enhanced tritium overflow more markedly, up to 64%, under conditions that favor alpha2-autoinhibition, observed when stimulation consisted of 100 pulses at 8 Hz, than under poor alpha2-autoinhibition conditions, only up to 14%, observed when alpha2-adrenoceptors were blocked with yohimbine (1 microM) or when stimulation consisted of 20 pulses at 50 Hz. Activation of PKC with 12-myristate 13-acetate (PMA, 0.1-3 microM) also enhanced tritium overflow more markedly under strong alpha2-autoinhibition conditions. Inhibition of Gi/o-proteins with pertussis toxin (8 microg/ml) or blockade of Gbetagamma subunits with the anti-betagamma peptide MPS-Phos (30 microM) attenuated the effects of angiotensin II and PMA. The results indicate that activation of AT1-receptors coupled to the PLC-PKC pathway enhances noradrenaline release, an effect that is markedly favoured by an ongoing activation of alpha2-autoreceptors. Interaction between alpha2-adrenoceptors and AT1-receptors seems to involve the betagamma subunits released from the Gi/o-proteins coupled to alpha2-adrenoceptors and protein kinase C activated by AT1-receptors.

The effect of age on glucose uptake and GLUT1 and GLUT4 expression in rat skeletal muscle

During the life span, phenotypic and structural modifications on skeletal muscle contribute to a reduction on glucose uptake either in basal state or triggered by insulin, but the underlying mechanisms for this decline are not entirely identified. A reduction in the expression of skeletal muscle glucose transporters (GLUTs), glucose transporter type 1 (GLUT1) and glucose transporter type 4 (GLUT4), has been associated to such phenomena, but unlike the case of insulin, only few studies have addressed the effect of age on muscle‐contraction‐induced glucose uptake. The aim of the study was to investigate the influence of age on GLUT1 and GLUT4 expression in skeletal muscle and its relation to the glucose uptake induced by muscle contraction. For this purpose, soleus muscle from Wistar rats aged 4, 10, 22 and 42 weeks were isolated and electrically stimulated (30 min, 10 Hz, 20 V, 0.2 ms). After stimulation, glucose uptake and GLUT1 and GLUT4 expression and localisation were evaluated. Muscle contraction caused an increase in glucose uptake in all studied groups. In addition, the absolute rates of glucose uptake were negatively correlated with age. The expression of GLUT4 was lower in older animals, whereas no relation between age and GLUT1 expression was found. Immunohistochemistry confirmed the ontogenic effect on GLUT4 expression and suggested an age‐related modification on GLUT1 distribution within the muscle fibres; for instance, this protein seems to be present mainly out of the sarcoplasm. The present findings demonstrate that the ability of muscle contraction to increase glucose uptake is not influenced by age, whereas glucose uptake under basal conditions decreases with age. Copyright

Adenosine A2A receptor-mediated facilitation of noradrenaline release involves protein kinase C activation and attenuation of presynaptic inhibitory receptor-mediated effects in the rat vas deferens

In the epididymal portion of rat vas deferens, facilitation of noradrenaline release mediated by adenosine A2A receptors, but not that mediated by β2‐adrenoceptors or by direct activation of adenylyl cyclase, was attenuated by blockade of α2‐adrenoceptors and abolished by simultaneous blockade of α2‐adrenoceptors, adenosine A1 and P2Y receptors. The adenosine A2A receptor‐mediated facilitation was not changed by inhibitors of protein kinase A, protein kinase G or calmodulin kinase II but was prevented by inhibition of protein kinase C with chelerythrine or bisindolylmaleimide XI. Activation of protein kinase C with phorbol 12‐myristate 13‐acetate caused a facilitation of noradrenaline release that was abolished by bisindolylmaleimide XI and reduced by antagonists of α2‐adrenoceptors, adenosine A1 and P2Y receptors. Activation of adenosine A2A receptors attenuated the inhibition of noradrenaline release mediated by the presynaptic inhibitory receptors. This effect was mimicked by phorbol 12‐myristate 13‐acetate and prevented by bisindolylmaleimide XI. It is concluded that adenosine A2A receptors facilitate noradrenaline release by a mechanism that involves a protein kinase C‐mediated attenuation of effects mediated by presynaptic inhibitory receptors, namely α2‐adrenoceptors, adenosine A1 and P2Y receptors.

Presynaptic Adenosine and P2Y Receptors

Adenine-based purines, such as adenosine and ATP, are ubiquitous molecules that, in addition to their roles in metabolism, act as modulators of neurotransmitter release through activation of presynaptic P1 purinoceptors or adenosine receptors (activated by adenosine) and P2 receptors (activated by nucleotides). Of the latter, the P2Y receptors are G protein-coupled, whereas the P2X receptors are ligand-gated ion channels and not covered in this review.