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Cristina Giaroni,
Luca Canciani,
Elena Zanetti,
Daniela Giuliani, Rossana Pisani,
Rita Oldrini,
Elisabetta Moro,
Marco Trinchera,
Francesca Crema,
Sergio Lecchini,
Gianmario Frigo
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ABSTRACT: The existence of a close relation between presynaptic inhibitory alpha(2)-adrenoceptor and mu-opioid receptor pathways is well established. Such interplay may occur during chronic conditions that give rise to neuroadaptive changes involving both receptor systems. The aim of this study was to examine the effect of chronic treatment with the tricyclic antidepressant drug, desipramine, on alpha(2)-adrenoceptors and mu-opioid receptors in the guinea pig brain. Guinea pigs were treated with 10 mg/kg desipramine, injected i.p. for 21 days, every 24 h. The levels of expression of alpha(2)-adrenoceptors and mu-opioid receptors, the G protein receptor regulatory kinase, GRK2/3 and signal transduction inhibitory G proteins in synaptosomes of the guinea pig hippocampus and cortex were evaluated by immunoblotting. Quantitative analysis of alpha(2)-adrenoceptor and mu-opioid receptor mRNA levels has been carried out by competitive reverse transcriptase polymerase chain reaction. The expression levels of alpha(2)-adrenoceptors and mu-opioid receptors and the respective mRNAs were found unchanged in the cortex, after chronic desipramine treatment. In these experimental conditions alpha(2)-adrenoceptor and mu-opioid receptor levels decreased, while the relevant transcripts increased, in the hippocampus. GRK2/3 levels remained unchanged and increased, respectively, in the cortex and the hippocampus, after chronic exposure to desipramine. In the same experimental conditions, Galpha(i1), Galpha(i2), Galpha(o) and Galpha(z) levels remained unchanged, while Galpha(i3) levels decreased, in the cortex; whereas, Galpha(i1), Galpha(i2) and Galpha(i3) levels significantly increased, and Galpha(o) and Galpha(z) levels remained unchanged, in the hippocampus. On the whole, the present data suggest that alpha(2)-adrenoceptor and mu-opioid receptor expression and transcription are similarly influenced by chronic treatment with desipramine, in the guinea pig cortex and hippocampus. Furthermore, alterations in the levels of regulatory GRK2/3 and of inhibitory signal transduction G proteins, relevant to activation of both receptor pathways, have been documented. The distinct pattern of adaptations of the different protein studied in response to chronic desipramine treatment in both regions is discussed.
European Journal of Pharmacology 02/2008; 579(1-3):116-25. · 2.52 Impact Factor
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ABSTRACT: The existence of a functional interplay between alpha(2)-adrenoceptor and opioid receptor inhibitory pathways modulating neurotransmitter release has been demonstrated in the enteric nervous system by development of sensitivity changes to alpha(2)-adrenoceptor, mu- and kappa-opioid receptor agents on enteric cholinergic neurons after chronic sympathetic denervation. In the present study, to further examine this hypothesis we evaluated whether manipulation of alpha(2)-adrenoceptor pathways by chronic treatment with the antidepressant drug, desipramine (10 mg/kg i.p. daily, for 21 days), could entail changes in enteric mu- and kappa-opioid receptor pathways in the myenteric plexus of the guinea pig distal colon. In this region, subsensitivity to the inhibitory effect of both UK14,304 and U69,593, respectively alpha(2A)-adrenoceptor and kappa-opioid receptor agonist, on the peristaltic reflex developed after chronic desipramine treatment. On opposite, in these experimental conditions, supersensitivity developed to the inhibitory effect of [D-Ala, N-Me-Phe4-Gly-ol5]-enkephalin (DAMGO), mu-opioid receptor agonist, on propulsion velocity. Immunoreactive expression levels of alpha(2A)-adrenoceptors, mu- and kappa-opioid receptors significantly decreased in the myenteric plexus of the guinea pig colon after chronic desipramine treatment. In these experimental conditions, mRNA levels of alpha(2A)-adrenoceptors, mu- and kappa-opioid receptors significantly increased, excluding a direct involvement of transcription mechanisms in the regulation of receptor expression. Levels of G protein-coupled receptor kinase 2/3 and of inhibitory G(i/o) proteins were significantly reduced in the myenteric plexus after chronic treatment with desipramine. Such changes might represent possible molecular mechanisms involved in the development of subsensitivity to UK14,304 and U69,593 on the efficiency of peristalsis. Alternative molecular mechanisms, including a higher efficiency in the coupling between receptor activation and downstream intracellular effector systems, possibly independent from inhibitory G(i/o) proteins, may be accounted for the development of supersensitivity to DAMGO. Increased sensitivity to the mu-opioid agonist might compensate for the development of alpha(2A)-adrenoceptor and kappa-opioid receptor subsensitivity. On the whole, the present data further strengthen the concept that, manipulation of alpha(2)-adrenergic inhibitory receptor pathways in the enteric nervous system entails changes in opioid inhibitory receptor pathways, which might be involved in maintaining homeostasis as suggested for mu-opioid, but not for kappa-opioid receptors.
European Journal of Pharmacology 01/2007; 553(1-3):269-79. · 2.52 Impact Factor
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ABSTRACT: Up to now, baclofen (a GABA(B) receptor agonist) has been used for the treatment of severe spasticity unresponsive to oral antispasmodics. Although in humans it is usually administered at 2 mg/ml, the dosage to be used in the treatment of other diseases is unknown. For this reason, it is important to determine the safe maximum dosage and toxicity at the clinically used concentration. Primary cortical neurons represent a useful model to test the safety of baclofen. We performed a colorimetric assay (MTT test) as well as electron microscopy investigations, to determine neuronal survival after the treatment with baclofen at a concentration of 2 and 4 mg/ml. Our results demonstrated that, in our experimental model, neither concentration affected neuronal survival. Considering the above results, we can conclude that at the used concentrations, this drug is safe and its clinical use should be encouraged.
European Journal of Pharmacology 12/2006; 550(1-3):33-8. · 2.52 Impact Factor
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ABSTRACT: The existence of a functional interplay between α2-adrenoceptor and opioid receptor inhibitory pathways modulating neurotransmitter release has been demonstrated in the enteric nervous system by development of sensitivity changes to α2-adrenoceptor, μ- and κ-opioid receptor agents on enteric cholinergic neurons after chronic sympathetic denervation. In the present study, to further examine this hypothesis we evaluated whether manipulation of α2-adrenoceptor pathways by chronic treatment with the antidepressant drug, desipramine (10 mg/kg i.p. daily, for 21 days), could entail changes in enteric μ- and κ-opioid receptor pathways in the myenteric plexus of the guinea pig distal colon. In this region, subsensitivity to the inhibitory effect of both UK14,304 and U69,593, respectively α2A-adrenoceptor and κ-opioid receptor agonist, on the peristaltic reflex developed after chronic desipramine treatment. On opposite, in these experimental conditions, supersensitivity developed to the inhibitory effect of [D-Ala, N-Me-Phe4-Gly-ol5]-enkephalin (DAMGO), μ-opioid receptor agonist, on propulsion velocity. Immunoreactive expression levels of α2A-adrenoceptors, μ- and κ-opioid receptors significantly decreased in the myenteric plexus of the guinea pig colon after chronic desipramine treatment. In these experimental conditions, mRNA levels of α2A-adrenoceptors, μ- and κ-opioid receptors significantly increased, excluding a direct involvement of transcription mechanisms in the regulation of receptor expression. Levels of G protein-coupled receptor kinase 2/3 and of inhibitory Gi/o proteins were significantly reduced in the myenteric plexus after chronic treatment with desipramine. Such changes might represent possible molecular mechanisms involved in the development of subsensitivity to UK14,304 and U69,593 on the efficiency of peristalsis. Alternative molecular mechanisms, including a higher efficiency in the coupling between receptor activation and downstream intracellular effector systems, possibly independent from inhibitory Gi/o proteins, may be accounted for the development of supersensitivity to DAMGO. Increased sensitivity to the μ-opioid agonist might compensate for the development of α2A-adrenoceptor and κ-opioid receptor subsensitivity. On the whole, the present data further strengthen the concept that, manipulation of α2-adrenergic inhibitory receptor pathways in the enteric nervous system entails changes in opioid inhibitory receptor pathways, which might be involved in maintaining homeostasis as suggested for μ-opioid, but not for κ-opioid receptors.
European Journal of Pharmacology - EUR J PHARMACOL. 01/2006; 553(1):269-279.
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ABSTRACT: The relations between apparent affinity for substrates and operating rates have been investigated by two-electrode voltage clamp in the GABA transporter rGAT1 expressed in Xenopus oocytes. We have measured the transport current induced by the presence of GABA, as well as the charge equilibration rate in the absence of the neurotransmitter, in various experimental conditions known to affect the transporter characteristics. The apparent affinities for GABA and for Na(+) were also determined in the same conditions. Two pharmacological actions and three mutated isoforms have been examined. In all cases significant correlations were found between the charge equilibration rates and apparent affinities for both substrates. In particular in the transport process, the apparent affinity for GABA appears to be inversely related to the sum of the unidirectional charge equilibration rates (alpha+beta), while the Na(+) apparent affinity is directly related to their ratio (beta/alpha). Together these observations suggest a kinetic basis for GABA affinity with higher turnover rates resulting in lower affinity, and indicate that an efficient uptake requires a compromise between these two parameters.
The Journal of Physiology 01/2005; 562(Pt 2):333-45. · 4.72 Impact Factor
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ABSTRACT: The ability of the two highly homologous Na(+)/Cl(-)-dependent neutral amino acid transporters KAAT1 and CAATCH1, cloned from the midgut epithelium of the larva Manduca sexta, to transport different amino acids depends on the cotransported ion, on pH, and on the membrane voltage. Different organic substrates give rise to transport-associated currents with their own characteristics, which are notably distinct between the two proteins. Differences in amplitude, kinetics, and voltage dependence of the transport-associated currents have been observed, as well as different substrate selectivity patterns measured by radioactive amino acid uptake assays. These diversities represent useful tools to investigate the structural determinants involved in the substrate selectivity. To identify these regions, we built four chimeric proteins between the two transporters. These proteins, heterologously expressed in Xenopus laevis oocytes, were analyzed by two-electrode voltage clamp and uptake measurements. Initially, we exchanged the first three domains, obtaining the chimeras C3K9 and K3C9 (where numbers indicate the transmembrane domains and letters represent the original proteins), which showed electrophysiological and [(3)H]amino acid uptake characteristics resembling those of KAAT1 and CAATCH1, respectively. Subsequent substitution of the last four domains in C3K9 and K3C9 gave the proteins C3K5C4 and K3C5K4, which showed the same behavior as KAAT1 and CAATCH1 in electrophysiological and transport determinations. These results suggest that in KAAT1 and CAATCH1, only the central transmembrane domains (from 4 to 8) of the protein are responsible for substrate selectivity.
AJP Cell Physiology 10/2004; 287(3):C754-61. · 3.54 Impact Factor
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ABSTRACT: The existence of a functional interplay between α2-adrenoceptor and opioid receptor inhibitory pathways modulating neurotransmitter release has been demonstrated in the enteric nervous system by development of sensitivity changes to α2-adrenoceptor, μ- and κ-opioid receptor agents on enteric cholinergic neurons after chronic sympathetic denervation. In the present study, to further examine this hypothesis we evaluated whether manipulation of α2-adrenoceptor pathways by chronic treatment with the antidepressant drug, desipramine (10 mg/kg i.p. daily, for 21 days), could entail changes in enteric μ- and κ-opioid receptor pathways in the myenteric plexus of the guinea pig distal colon. In this region, subsensitivity to the inhibitory effect of both UK14,304 and U69,593, respectively α2A-adrenoceptor and κ-opioid receptor agonist, on the peristaltic reflex developed after chronic desipramine treatment. On opposite, in these experimental conditions, supersensitivity developed to the inhibitory effect of [D-Ala, N-Me-Phe4-Gly-ol5]-enkephalin (DAMGO), μ-opioid receptor agonist, on propulsion velocity. Immunoreactive expression levels of α2A-adrenoceptors, μ- and κ-opioid receptors significantly decreased in the myenteric plexus of the guinea pig colon after chronic desipramine treatment. In these experimental conditions, mRNA levels of α2A-adrenoceptors, μ- and κ-opioid receptors significantly increased, excluding a direct involvement of transcription mechanisms in the regulation of receptor expression. Levels of G protein-coupled receptor kinase 2/3 and of inhibitory Gi/o proteins were significantly reduced in the myenteric plexus after chronic treatment with desipramine. Such changes might represent possible molecular mechanisms involved in the development of subsensitivity to UK14,304 and U69,593 on the efficiency of peristalsis. Alternative molecular mechanisms, including a higher efficiency in the coupling between receptor activation and downstream intracellular effector systems, possibly independent from inhibitory Gi/o proteins, may be accounted for the development of supersensitivity to DAMGO. Increased sensitivity to the μ-opioid agonist might compensate for the development of α2A-adrenoceptor and κ-opioid receptor subsensitivity. On the whole, the present data further strengthen the concept that, manipulation of α2-adrenergic inhibitory receptor pathways in the enteric nervous system entails changes in opioid inhibitory receptor pathways, which might be involved in maintaining homeostasis as suggested for μ-opioid, but not for κ-opioid receptors.
European Journal of Pharmacology.
