Biomedical subjects

T Rubino

Publications and source records attributed to T Rubino.

At least 19 recordsLinked to original sources

PubMed2004

Differential diurnal variations of anandamide and 2-arachidonoyl-glycerol levels in rat brain.

The endogenous ligands of cannabinoid receptors, also known as endocannabinoids, have been implicated in many physiological and pathological processes of the central nervous system. Here we show that the levels of the two major endocannabinoids, anandamide and 2-arachidonoyl-glycerol (2-AG), in four areas of the rat brain, change dramatically between the light and dark phases of the day. While anandamide levels in the nucleus accumbens, pre-frontal cortex, striatum and hippocampus were significantly higher in the dark phase, the opposite was observed with 2-AG, whose levels were significantly higher during the light phase in all four regions. We found that the activity of the fatty acid amide hydrolase, which catalyzes the metabolism of anandamide, was significantly lower during the dark phase, thus providing a possible explaination for the increase in anandamide levels. However, the activities of monoacylglycerol lipase and diacylglycerol lipase, two of the possible enzymes catalyzing the degradation and biosynthesis of 2-AG, respectively, changed significantly only in the striatum. These data suggest that the levels of the two major endocannabinoids might be under the control of endogenous factors known to undergo diurnal variations, and underscore the different roles, suggested by previous studies, of anandamide and 2-AG in neurophysiological processes.

Amidohydrolases↗

PubMed2003

Mu opioid receptor signaling in morphine sensitization.

We used a previously reported model of morphine sensitization that elicited a complex behavioral syndrome involving stereotyped and non stereotyped activity. To identify the mechanism of these long-lasting processes, we checked the density of mu opioid receptors, receptor-G-protein coupling and the cyclic AMP (cAMP) cascade. In morphine-sensitized animals mu opioid receptor autoradiography revealed a significant increase in the caudate putamen (30% versus controls), nucleus accumbens shell (16%), prefrontal and frontal cortex (26%), medial thalamus (43%), hypothalamus (200%) and central gray (89%). Concerning morphine's activation of G proteins in the brain, investigated in the guanylyl 5'-[gamma-(35)S]thio]triphosphate ([(35)S]GTPgammaS) binding assay, a significant increase in net [(35)S]GTPgammaS binding was seen in the caudate putamen (39%) and hypothalamus (27%). In the caudate putamen this was due to an increase in the amount of activated G proteins, and in the hypothalamus to a greater affinity of G proteins for guanosine triphosphate (GTP). The main second messenger system linked to the opioid receptor is the cAMP pathway. In the striatum basal cAMP levels were significantly elevated in sensitized animals (70% versus controls) and [D-Ala(2), N-Me-Phe(4), Gly(5)-ol]-enkephalin (DAMGO) significantly inhibited forskolin-stimulated cAMP production in control (30%) but not in sensitized rats. In the hypothalamus no significant changes were observed in basal cAMP levels and DAMGO inhibition. These cellular events induced by morphine pre-exposure could underlie the neuroadaptive processes involved in morphine sensitization.

Animals↗

PubMed2003

Cellular mechanisms of Delta 9-tetrahydrocannabinol behavioural sensitization.

We investigated the cellular events linked to the induction of cannabinoid behavioural sensitization. In sensitized rats, autoradiographic binding studies with [3H]CP-55,940 showed a significant increase in cannabinoid receptor binding, specifically in the cerebellum, with no changes in the other brain areas where basal CB1-receptor expression is observed. In vitro autoradiography of CP-55,940-stimulated [35S]GTP gamma S binding provided a picture of cannabinoid receptor-mediated G protein activation. Basal [35S]GTP gamma S binding was not affected, whereas sensitized rats showed a significant increase of net [35S]GTP gamma S binding in the caudate putamen and cerebellum. Autoradiographic studies suggested that only these two areas had altered receptor functionality. We therefore focused our intracellular investigations only there, first surveying the responsiveness of the cAMP system to cannabinoids. CP-55,940 was unable to inhibit forskolin-induced cAMP accumulation in the cerebellum of sensitized animals, but no difference was observed between groups in the caudate putamen. Finally, we surveyed the levels of CREB and AP-1 binding activity, in the same two areas and found no difference in sensitized rats. The intracellular picture in sensitized rats suggests that besides the cAMP cascade, other signalling pathways may participate in the development of cannabinoid sensitization.

Animals↗

PubMed2001

Cannabinoids inhibit nitric oxide production in bone marrow derived feline macrophages.

Feline immunodeficiency virus (FIV) infection causes a widespread natural immunodeficiency syndrome in cats that is considered a suitable animal model for studying human immunodeficiency virus (HIV) infection and pathogenesis. Short term cultures of bone marrow derived feline macrophages stimulated with recombinant feline interferon-gamma (r-IFN-gamma) and lipopolysaccharide (LPS) were shown to produce nitric oxide. Feline macrophages were shown to express cannabinoid receptors, and nitric oxide production decreased after in vitro exposure to synthetic cannabinoid CP-55940. Both cannabinoid receptors, CB1 and CB2, were involved in this process, since the inhibition was reversed by selective cannabinoid antagonists for both of these receptors.

Animals↗

PubMed2001

The psychoactive ingredient of marijuana induces behavioural sensitization.

Here we describe, for the first time, the occurrence of behavioural sensitization after chronic exposure to Delta9-tetrahydrocannabinol. Rats were treated twice a day, for five days, with increasing doses (5, 10, 20, 40, 40 mg/kg i.p.) of Delta9-tetrahydrocannabinol or its vehicle and after 20 days of withdrawal, animals were challenged with 5 mg/kg (i.p.) of the drug and their behaviour was assessed. Contrary to the motor inhibition induced in control rats, challenge with Delta9-tetrahydrocannabinol in pre-exposed animals elicited a complex behavioural syndrome mainly characterized by oral stereotyped items. Due to the relevance of behavioural sensitization in drug-seeking behaviour that persists long after discontinuation of drug use, our findings suggest that cannabinoids could trigger neurobiological alteration not dissimilar from those observed with more harmful abused drugs.

Animals↗

PubMed2000

Chronic delta-9-tetrahydrocannabinol treatment increases cAMP levels and cAMP-dependent protein kinase activity in some rat brain regions.

When Delta(9)-tetrahydrocannabinol (Delta(9)-THC,15 mg/kg) was injected intraperitoneally twice a day for 6 days, tolerance to its analgesic effect appeared to be complete. Chronic exposure to Delta(9)-THC caused a significant reduction in CB1 receptor binding in all brain areas that contain this receptor. Cannabinoid receptor density was markedly reduced in the cerebellum (52%), hippocampus (40%) and globus pallidum (47%) compared to 30% in the cortex and striatum. Chronic exposure enhanced the cAMP pathway, as shown by the significant increase of cAMP levels and PKA activity in the areas with receptor down-regulation (cerebellum, striatum and cortex). We propose that the increase in cAMP cascade is part of the biochemical basis of cannabinoid tolerance.

Animals↗

PubMed2000

Long-term treatment with SR141716A, the CB1 receptor antagonist, influences morphine withdrawal syndrome.

The role of the cannabinoid system in morphine withdrawal was examined through long-term CB1 receptor antagonist administration in morphine pellet implanted rats. SR141716A chronic treatment (5mg/kg i.p. twice a day for four days) did not influence the development of tolerance to the morphine analgesic effect but significantly reduced the intensity of naloxone-induced opiate withdrawal in tolerant rats: Specifically there was a significant reduction in the number of digging, teeth chattering and penile licking and the incidence of diarrhoea while other signs such as writhing, head dog shakes and rearing were unaffected. These results suggest that the pharmacological treatment with SR141716A could be of some interest in ameliorating opiate withdrawal syndrome.

Analysis of Variance↗

PubMed2000

In vivo characterization of the specific cannabinoid receptor antagonist, SR141716A: behavioral and cellular responses after acute and chronic treatments.

To characterize the behavioral and biochemical effects of the cannabinoid CB1 antagonist SR141716A, we injected the compound intraperitoneally (ip) at doses from 0.625 mg/kg to 5 mg/kg in rats. SR141716A per se induced a dose-dependent increase of some behavioral signs such as wet dog and head shakes, forepaw fluttering, grooming, and facial rubbing. When the highest dose of SR141716A (5 mg/kg ip) was injected once a day for four days, tolerance developed to most of the behavioral signs, although with different time courses, except for grooming behavior, which was still significantly different from controls after the fourth injection although reduced by 38% from the first. To characterize the biochemical mechanism underlying these effects, we designed a series of biochemical studies on specific cerebral areas from rats treated with the highest dose of SR141716A (5 mg/kg ip). Thirty minutes after SR141716A injection, cAMP accumulation in the cortex, striatum, hippocampus, mesencephalon, and cerebellum was the same as in controls, whereas protein kinase A (PKA) activity was significantly increased in the hippocampus (65%) and striatum (87%). To explain this difference, we performed a cAMP assay at an early time (10 min) and found a significant increase in the striatum and hippocampus, suggesting that the change in cAMP level is the earliest event in the G protein-coupled receptor transduction pathway ending in a pharmacological effect after 30 min. When the same assays were done in tolerant animals, no change was seen in either cAMP levels or PKA activity in the brain areas considered. To conclude, we found in vivo that SR141716A acts through activation of the cAMP cascade and our results represent an important point for developing potential therapeutic application for SR141716A.

Animals↗

PubMed2000

Changes in the cannabinoid receptor binding, G protein coupling, and cyclic AMP cascade in the CNS of rats tolerant to and dependent on the synthetic cannabinoid compound CP55,940.

Chronic exposure to CP55,940 produced a significant down-regulation of cannabinoid receptors in the striatum, cortex, hippocampus, and cerebellum of rat brain. At 24 h after SR141716-precipitated withdrawal, we observed a tendency to return to basal levels in the striatum and cortex, whereas the specific binding remained lower in the hippocampus and cerebellum. When we surveyed cannabinoid receptor-activated G proteins, in chronic CP55,940-treated rats the guanosine 5'-O:-(3-[(35)S]thiotriphosphate) ([(35)S]GTPgammaS) binding assay revealed a decrease of activated G proteins in the striatum, cortex, and hippocampus, whereas no significant changes were seen in the cerebellum. At 24 h after the SR141716-precipitated withdrawal, [(35)S]GTPgammaS binding increased compared with that of rats chronically exposed to CP55,940, attaining the control level except for cerebellum, where we observed a trend to overcome the control amounts. Concerning the cyclic AMP (cAMP) cascade, which represents the major intracellular signaling pathway activated by cannabinoid receptors, in the cerebral areas from rats chronically exposed to CP55,940 we found alteration in neither cAMP levels nor protein kinase A activity. In the brain regions taken from CP55, 940-withdrawn rats, we only observed a significant up-regulation in the cerebellum. Our findings suggest that receptor desensitization and down-regulation are strictly involved in the development of cannabinoid tolerance, whereas alterations in the cAMP cascade in the cerebellum could be relevant in the mediation of the motor component of cannabinoid abstinence.

Animals↗

PubMed2000

Loss of cannabinoid-stimulated guanosine 5'-O-(3-[(35)S]Thiotriphosphate) binding without receptor down-regulation in brain regions of anandamide-tolerant rats.

The endogenous cannabinoid anandamide has been reported to produce well-defined behavioral tolerance, but studies on the possible mechanisms underlying this process are few and often contradictory. The present study was designed to survey the cellular events involved in anandamide tolerance, in terms of the effects on receptor number, coupling with G proteins, and activation of the cyclic AMP (cAMP) cascade. Chronic treatment of rats with anandamide (20 mg/kg i.p. for 15 days) resulted in behavioral tolerance without any change in cannabinoid receptor binding in the brain regions studied (striatum, cortex, hippocampus, and cerebellum), suggesting that receptor down-regulation was not involved in the development of anandamide behavioral tolerance. In contrast, prolonged exposure to anandamide significantly reduced agonist-stimulated guanosine 5'-O:-(3-[(35)S]thiotriphosphate) binding in the same areas, with losses of >50%, suggesting that receptor desensitization may be part of the molecular mechanism underlying this tolerance. Finally, concerning the cAMP cascade-the most well-known intracellular signaling pathways activated by CB(1) receptors-in the brain regions from rats tolerant to anandamide, we found no alteration in cAMP levels or in protein kinase A activity. We propose that anandamide, unlike Delta(9)-tetrahydrocannabinol and other cannabinoids, does not alter the receptor system at multiple levels but that desensitization of the CB(1) receptor might account for behavioral tolerance to the drug.

Animals↗

PubMed1999

Presence of a reduced opioid response in interleukin-6 knock out mice.

Cytokines are known to influence neuronal functions. The purpose of this study was to investigate the putative role of the cytokine interleukin-6 (IL-6) in the pathways involved in opioid-mediated responses, by using IL-6-deficient mice. We reported that with a thermal stimulus IL-6-knock-out (IL-6KO) mice presented nociceptive thresholds similar to those measured in their controls. However, they showed a reduced analgesic response both to the restraint stress and to the administration of low doses of morphine. Hypothalamic levels of the opioid peptide beta-endorphin were significantly higher in IL-6KO mice than they were in their controls. The development of tolerance to the analgesic effect of morphine was more rapid in IL-6-deficient mice than in wild-type controls. Binding experiments showed that the number of opioid receptors in the midbrain, but not in the hypothalamus, decreased in IL-6KO mice. Autoradiographic binding analysis revealed that the density of mu receptors diminished while the delta-opioid receptors did not. These results suggest that IL-6 is necessary for a correct development of neuronal mechanisms involved in the response to both endogenous and exogenous opiates.

Analgesics, Opioid↗

PubMed1998

Immune function alterations in mice tolerant to delta9-tetrahydrocannabinol: functional and biochemical parameters.

We studied the effect of acute (1 h) or chronic exposure (7 and 14 days) to delta9-tetrahydrocannabinol (delta9-THC) on immune parameters in male Swiss mice. One hour after a dose of 10 mg/kg s.c., the splenocyte proliferative response to ConA and NK activity were not inhibited, but there was a significant decrease in the production of IL-2. After 7 days of treatment, when mice were tolerant to delta9-THC-induced analgesia, these functional parameters were strongly inhibited and there was a persistent reduction in IL-2 and IFNgamma. With 14 days exposure to the drug, splenocyte proliferation was significantly reduced only with 5 microg/ml ConA, and NK activity was still significantly depressed (about 37%). IL-2 had returned to the control value, whereas IFNgamma was still 40% down. Flow cytometry analysis of spleen cell composition indicated no changes after the acute and 7 day treatments, but at 14 days there was a 20% decrease in the number of T lymphocytes, mirrored by a 26% increase of B lymphocytes. In conclusion, in vivo exposure to psychoactive doses of delta9-THC has profound effects on immune function. This implies some important questions in relation to the liberalization of marijuana and its therapeutic uses.

Animals↗

PubMed1998

Short-term assessment of periradicular corticosteroid injections in lumbar radiculopathy associated with disc pathology.

We evaluated periradicular injection of corticosteroids performed by neuroradiologists under fluoroscopic guidance in the treatment of lumbosacral nerve root pain in 40 patients (average age 48 years) presenting with lumbosciatica or radiculopathy not responding to conservative treatment. Patients with root pain due to infectious, neoplastic or inflammatory diseases were excluded, as were patients who needed immediate surgery. Pain was evaluated using a visual analogue scale (VAS) 10 and 90 days after the injection. After 10 days a substantial decrease in root pain was observed, with a statistically significant decrease in mean VAS. This decrease was observed in 90% of patients, and it persisted after 90 days in 85%. Side effects were rare, mild and disappeared spontaneously. They were related to the drug injected (corticosteroids).

Adrenal Cortex Hormones↗

PubMed1998

Short-term evaluation of periradicular corticosteroid injections in the treatment of lumbar radiculopathy associated with disc disease.

OBJECTIVES: The purpose of this study was to evaluate the feasibility and efficacy of periradicular corticosteroid injections performed under fluoroscopic guidance in the treatment of pain originating in the lumbosacral nerve roots. METHOD: Forty patients presenting with lumbosciatica or femoral neuralgia unresponsive to conventional conservative treatment were admitted to the study. Patients with nerve root pain due to infectious, tumorous or inflammatory diseases were excluded, as were patients who needed immediate surgery. Pain was evaluated using a visual analog scale on the day of the injection and ten (D10) and ninety (D90) days later. RESULTS: All patients were evaluated as scheduled on D10 and D90. The mean visual analog scale score decreased from 53.3 mm at baseline to 30.1 mm on D10 and to 21.8 mm on D90. Ninety per cent of patients had a visual analog pain score decrease on D10 and 85% on D90. Reported side effects were two cases of mild spontaneously regressive acne and one case of radicular pain exacerbation of less than one day's duration. CONCLUSION: Periradicular injections of corticosteroids done under fluoroscopic guidance as an outpatient procedure was effective and safe in our study and may deserve to be used as part of the conservative management of lumbar nerve root pain before resorting to more invasive methods.

Acne Vulgaris↗

PubMed1998

Cannabinoid-precipitated withdrawal: a time-course study of the behavioral aspect and its correlation with cannabinoid receptors and G protein expression.

To characterize the time course of the behavioral and biochemical aspects of the cannabinoid withdrawal syndrome, we injected the cannabinoid antagonist SR141716A (5 mg/kg i.p.) in rats made tolerant to CP-55,940 (0.4 mg/kg i.p., twice daily for 6.5 days), 1, 24 and 96 h after the last CP-55,940 injection. Because the CB1 receptor and G protein alpha subunit are involved in cannabinoid tolerance, we observed their changes throughout the brain during the withdrawal syndrome by use of in situ hybridization. In vehicle-pretreated rats SR141716A per se induced abnormal behavior significantly different from the vehicle group: wet dog shakes, forepaw fluttering and scratching. These signs remained significantly elevated even after the second and third antagonist doses. SR141716A significantly modified the mRNA levels of G alpha s and G alpha i subunits in some brain areas without affecting CB1 receptor and G alpha o expression. These findings led us to conclude that SR141716A may have intrinsic activity. Concerning cannabinoid withdrawal, the first SR141716A injection in tolerant rats resulted in behavioral signs different from those observed with the antagonist alone; this moderate withdrawal syndrome was characterized by turning, chewing and digging. Additional SR141716A doses 24 and 96 h later did not induce a significant abstinence syndrome. In situ hybridization after the first SR141716A injection showed that CB1 receptor and G protein alpha subunits, whose levels were low in tolerance, recovered their basal level of expression. Thus, the general desensitization of the cannabinoid receptor and of the transduction system in tolerance are recovered in abstinent rats and might be part of the molecular mechanisms underlying cannabinoid dependence.

Animals↗

PubMed1997

Modulation of rat brain cannabinoid receptors after chronic morphine treatment.

Intraperitoneal injection of delta9-THC (7.5 mg/kg) in rats made tolerant to morphine by s.c. implantation of morphine pellets had a much greater analgesic effect than in placebo pellet plus delta9-THC treatment. To investigate whether this was due to some change in cannabinoid receptor levels and/or expression induced by chronic morphine, we designed this autoradiographic binding study coupled with in situ hybridization on sagittal sections of the treated rat brains. Binding showed a significant increase in CB1 receptor density (15%) specifically in the caudate-putamen, in parallel with a significant enhancement of CB1 mRNA in the same area (20%). We suggest that morphine chronic treatment leads to a functional modulation between the opioid and cannabinoid systems at least for analgesia in a specific area, in this case the striatum.

Analgesics↗

PubMed1997

Changes in rat spleen cannabinoid receptors after chronic CP-55,940: an autoradiographic study.

We examined whether cannabinoid receptor density changes in the rat spleen after in vivo chronic exposure to cannabinoids. Rats received daily injections of 0.4 mg/kg IP of the synthetic cannabinoid receptor ligand CP-55,940 for 11 days. One h after the last injection on day 11, the rats were killed and spleen coronal sections were processed for receptor binding autoradiography with 10 nM of [3H]CP-55,940 in the absence or presence of unlabeled CP-55,940 (10 microM). Densitometric analysis of the autoradiograms showed significant loss of [3H]CP-55,940 binding of about 42% in chronic cannabinoid-treated, tolerant rats. Our findings indicate that cannabinoid receptors basically present in immune spleen cells are down-regulated by chronic exposure to cannabinoids, suggesting a role in immune modulation and in the impairment of immune function.

Animals↗

PubMed1997

Chronic treatment with a synthetic cannabinoid CP-55,940 alters G-protein expression in the rat central nervous system.

Prolonged exposure of rats to the synthetic cannabinoid receptor ligand, CP-55,940 (0.4 mg/kg, i.p. for 11 days), induced tolerance to analgesia, to the reduction in spontaneous locomotor activity and the incidence of splayed hind limbs. One hour after the last injection on day 11, the rats were killed and in situ hybridization was used to investigate the effect of treatment on G-protein alpha-subunit expression throughout the brain. Chronic cannabinoid exposure markedly reduced G alpha(s), G alpha(i) and G alpha(o) mRNA levels. The message for the alpha(s)-subunit was decreased in all the brain areas containing the basal autoradiographic signal; the decrease ranging from 25% in the thalamus to 45% in the mesencephalon. Also the basal G alpha(i) expression was reduced in tolerant rats showing the greatest decrease in the forebrain (63%) in the cerebellum (58%) and in the mesencephalon (38%). The reduction in G alpha(o) expression (25%) was more localized, being present only in the rostral portion of the brain (cortex, striatum and olfactory area). The alterations in alpha-subunits gene expression were not followed by any change in the amount of proteins. Our results indicate that, besides the receptor modification, alteration to the G-protein expression could be a molecular event associated with the development of cannabinoid tolerance.

Analgesics↗