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Agonist-induced beta-adrenoceptor internalization on intact human mononuclear leukocytes : effect of temperature of mononuclear leukocyte separation
No full textAgonist-induced beta-adrenergic receptor internalization on intact human mononuclear leukocytes: effect of temperature of mononuclear leukocyte separation.
No full textThe hydrophilic ligand 3H-CGP 12177 was used to measure beta-adrenergic receptors on intact human mononuclear leukocytes (MNLs). A single homogeneous class of receptor sites was found, with KD value of 0.71 +/- 0.04 nmol/L and Bmax of 3.0 +/- 0.4 fmol/10(6) cells (mean +/- SEM; n = 12). The receptor affinity (KD) and density (Bmax) were similar when measured on MNLs, purified lymphocytes, and a T-lymphocyte-enriched population from the same individual. Preincubation of intact MNLs with 1 mumol/L isoproterenol at 37 degrees C for 20 minutes reduced the number of surface receptors, measured by 3H-CGP 12177 binding at 4 degrees C for 20 hours, by approximately 70% (receptor internalization) without affecting KD. This effect was reversible, and surface receptors completely reappeared when binding was investigated at 37 degrees C for 40 minutes. Receptor internalization was similar when either isolated MNLs or whole blood was incubated with isoproterenol. Agonist-induced receptor internalization was stable during MNL isolation from whole blood at 4 degrees C but was partially or completely lost from MNLs prepared at 20 degrees C
Regulation of beta adrenergic receptors on rat mononuclear leukocytes by stress: receptor redistribution and down-regulation are altered with aging.
No full textIn vitro incubation of mononuclear leukocytes (MNL) with catecholamines desensitizes beta adrenergic receptors, meaning isoproterenol-stimulated cyclic AMP accumulation decreases. This desensitization is accompanied by two patterns of receptor changes: first, reduction of surface receptors (defined as binding of [3H]dihydroalprenolol inhibited by 1 microM CGP 12177 [4-3-tertiarybutylamino-2-hydroxypropoxy)-benzimidazole- 2-on-hydrochloride]) without any change in the total number of [3H] dihydroalprenolol binding sites inhibited by 1 microM propranolol (receptor redistribution); then reduction of the total number of receptors (receptor down-regulation). In the present study we investigated receptor redistribution and down-regulation under physiological conditions by raising endogenous catecholamines in the rat by stress. In young rats a single immobilization stress induced MNL beta adrenergic receptor redistribution: the number of surface receptors was reduced by about 50% but the total number remained the same. Receptor redistribution was prevented completely in rats pretreated with beta-blocking nadolol. Repeated stress down-regulated the MNL beta adrenergic receptors as shown by a reduction in the total number of sites. We also investigated the regulation of beta adrenergic receptors in three age-groups. After 60 min of immobilization stress the number of MNL surface receptors was reduced in young (4-month-old) rats but not in mature (12-month-old) or aged (26-month-old) rats. Using an alternative stress procedure, after single or repeated open-field sessions, we found receptor redistribution and down-regulation, respectively, in young rats. None of these adaptive receptor response was observed in 26-month-old rats
Effect of acute and chronic tramadol on [3H]-norepinephrine-uptake in rat cortical synaptosomes.
No full text1 Tramadol hydrochloride is a centrally acting opioid analgesic whose efficacy and potency is only five to ten times lower than that of morphine. Opioid, as well as non-opioid mechanisms, may participate in the analgesic activity of tramadol. 2 [3H]-NE uptake in isolated rat cortical synaptosomes was studied in the presence of tramadol, desipramine, methadone, and morphine. Desipramine and tramadol inhibited synaptosomal [3H]-NE uptake with apparent Kis of 7.3 +/- 0.66 and 1.4 +/- 0.0045 microM, respectively. Methadone was active at a 10-fold higher concentration (Ki: 87 +/- 5.6 microM). In contrast, morphine essentially failed to inhibit [3H]-5-HT uptake (Ki: 0.75 +/- 0.40 M). 3 Methadone, morphine, and tramadol were active in the hot plate test with ED50s of 6.2, 9.3, and 40 mg kg-1, respectively. 4 [3H]-NE uptake was examined in synaptosomes prepared from rats 30 min after receiving a single dose of morphine, methadone or tramadol. Only tramadol (31 mg kg-1, i.p.) decreased uptake of the transmitter, with an ED50 equal to that in the hot plate test. 5 Animals were chronically treated for 15 days with increasing doses of tramadol (20 to 125 mg kg-1, i.p.). Twenty-four hours after the last drug injection, a challenge dose of tramadol (40 mg kg-1, i.p.) was administered. Chronic tramadol was still able to reduce [3H]-NE uptake by 35%. 6 These results further support the hypothesis that [3H]-NE uptake inhibition may contribute to the antinociceptive effects of tramadol. The lack of tolerance in [3H]-NE uptake, together with the absence of behavioural alteration after chronic tramadol treatment proposes that tramadol holds potential over classical opioids in the treatment of pain disorders
Effect of acute and chronic tramadol on [3H]-5-HT-uptake in rat cortical synapyosomes
No full text1. Tramadol hydrochloride is a centrally acting opioid analgesic, the efficacy and potency of which is only five to ten times lower than that of morphine. Opioid, as well as non-opioid mechanisms, may participate in the analgesic activity of tramadol. 2. [3H]-5-hydroxytryptamine (5-HT) uptake in rat isolated cortical synaptosomes was studied in the presence of tramadol, desipramine, fluoxetine, methadone and morphine. Methadone and tramadol inhibited synaptosomal [3H]-5-HT uptake with apparent Kis of 0.27 +/- 0.04 and 0.76 +/- 0.04 microM, respectively. Morphine essentially failed to inhibit [3H]-5-HT uptake (Ki 0.50 +/- 0.30 M). 3. Methadone, morphine and tramadol were active in the hot plate test with ED50s of 3.5, 4.3 and 31 mg kg-1, respectively. At the highest tested dose (80 mg kg-1) tramadol produced only 77 +/- 5.3% of the maximal possible effect. 4. When [3H]-5-HT uptake was examined in synaptosomes prepared from rats 30 min after a single dose of morphine, methadone or tramadol, only tramadol (31 mg kg-1, s.c., equal to the ED50 in the hot plate test) and methadone (35 mg kg-1, s.c., equal to the ED90 in the hot plate test) decreased uptake. 5. Animals were chronically treated for 15 days with increasing doses of tramadol or methadone (5 to 40 mg kg-1 and 15 to 120 mg kg-1, s.c., respectively). Twenty-four hours after the last drug injection, a challenge dose of methadone (35 mg kg-1, s.c.) or tramadol (31 mg kg-1, s.c.) was administered. [3H]-5-HT uptake was not affected in synaptosomes prepared from rats chronically-treated with methadone, whereas chronic tramadol was still able to reduce this parameter by 42%. 6. Rats chronically-treated with methadone showed a significant increase in [3H]-5-HT uptake (190%) 72 h after drug withdrawal. In contrast, [3H]-5-HT uptake in rats chronically-treated with tramadol (110%) did not differ significantly from control animals. 7. These results further support the hypothesis that [3H]-5-HT uptake inhibition may contribute to the antinociceptive effects of tramadol. The lack of tolerance development of [3H]-5-HT uptake, together with the absence of behavioural alterations after chronic tramadol treatment, suggest that tramadol has an advantage over classical opioids in the treatment of pain disorders
Abecarnil enhances recovery from diazepam tolerance.
No full textTreatment with diazepam (25 mg/kg; p.o., twice-daily for 17 days) induced tolerance to the anticonvulsant effect of diazepam against bicuculline-induced convulsions in mice. Cross-tolerance was observed to the anticonvulsant action of clonazepam, imidazenil but not abecarnil. While substitution of clonazepam (12 mg/kg; p.o., twice-daily for 15 days) for diazepam did not affect tolerance to diazepam, substitution of imidazenil (17 mg/kg; p.o., twice-daily for 15 days) for diazepam significantly increased sensitivity to the anticonvulsant effect of diazepam, although tolerance was not abolished. Tolerance to diazepam progressively decreased either after suspension of diazepam administration or replacement treatment with abecarnil (20 mg/kg; p.o., twice-daily). Complete recovery of diazepam efficacy was detected after 8 and 15 days of administration of abecarnil and vehicle, respectively. Binding experiments using [H-3]-flumazenil showed that K-d values did not differ among treatment groups. A significant decrease in B-max (-42%) was observed in the cortex of diazepam-tolerant mice whether or not also treated with imidazenil and clonazepam. Conversely, chronically diazepam-treated mice, that further received abecarnil for either 8 or 15 days or vehicle for 15 days showed B-max values similar to those of vehicle-treated mice never exposed to diazepam. Results suggest that repeated abecarnil administration to diazepam-tolerant mice can facilitate re-adaptation of receptors to the diazepam-free state. It is proposed that replacement therapy with abecarnil after long-term treatment with conventional benzodiazepines (BDZs) may provide a novel approach for reducing tolerance to their anticonvulsant effects. (C) 1999 Elsevier Science Ltd. All rights reserved
In vivo and in vitro evidence of dopaminergic system down regulation induced by chronic L-DOPA.
No full textMelatonin protects primary cultures of cerebellar granule neurons from kainate but not from N-methyl -D-Aspartate excitotoxicity
No full textthe antiexcitotoxic efficacy of melatonin, a putative endogenous hydroxyl radical scavenger, was studied in primary cultures of rat cerebellar granule neurons. Excitotoxicity was induced in 7- to 9-day-old cultures by an exposure to glutamate (15 min in the absence of magnesium) or to glutamate receptor agonists, kainate (30 min), and N-methyl-D-aspartate (60 min in the absence of magnesium). Thereafter, cultures were returned to the culture-conditioned medium for 18 h at the end of which time viability was assessed by quantitative staining with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide. Cotreatment with melatonin (500 microM) protected the neurons completely from the toxicity of kainate (up to 1 mM) and shifted the ED50 for glutamate from 55 +/- 2.6 to 97 +/- 3.6 microM. Melatonin cotreatment was ineffective in protecting the neurons from N-methyl-D-aspartate toxicity. When melatonin was added to the cultures only before or after kainate treatment, there was no resultant protection from kainate toxicity. The neuroprotective effect of melatonin does not appear to be related to the direct action of melatonin on ionotropic glutamate receptors. That is, the kainate-stimulated inward currents measured by a patch-clamp technique in voltage clamped neurons and the kainate-stimulated increase in free cytosolic calcium measured at the single-cell level using digital imaging fluorescent microscopy with fura-2 were not affected by melatonin. Moreover, the binding of [3H]glutamate to rat cerebellar membranes was not competed off by melatonin. Further studies are needed to evaluate the pharmacologic relevance of the neuroprotective action of melatonin
In vitro and in vivo protective effects of melatonin against glutamate oxidative stress and neurotoxicity.
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