Salmonella Typhimurium exhibits fluoroquinolone resistance mediated by the accumulation of the antioxidant molecule H2S in a CysK-dependent manner.

OBJECTIVES: To evaluate the contribution of cysK and cysM to the fluoroquinolone (ofloxacin) antibiotic resistance in Salmonella Typhimurium, and their impact on H2S and cysteine production through targeted mutagenesis. METHODS: Salmonella Typhimurium 14028s and its cysK and cysM mutants were tested for their susceptibility to ofloxacin, as determined by a broth microdilution test (to determine the MIC) and survival curves. H2S levels were measured by the Pb(AC)2 method and cysteine levels were determined using 5,5-dithio-bis-2-nitrobenzoic acid. DNA damage induced by antibiotic treatment was determined by PFGE. Finally, expression of cysK and cysM genes under antibiotic treatment was determined by real-time reverse transcription PCR. RESULTS: As determined by MIC, the ΔcysK strain was more resistant to ofloxacin, a reactive oxygen species (ROS)-producing fluoroquinolone, than the WT and ΔcysM strains, which correlates with survival curves. Moreover, the ΔcysK strain exhibited higher H2S levels and lower cysteine levels than the WT strain. Finally, the ΔcysK strain exhibited lower DNA damage upon challenge with ofloxacin than the WT and ΔcysM strains. These results are in accordance with lower expression of cysK under ofloxacin treatment in the WT strain. CONCLUSIONS: This work demonstrated that cysteine metabolism in Salmonella Typhimurium modulated H2S levels, conferring resistance to second-generation fluoroquinolones.

Mean glandular dose in the mammary gland and dose of radiation in the thyroid gland and lens in women with and without breast implants during different modalities of mammography.

There have always been concerns about the secondary effects of diagnostic methods that use ionizing radiation. During mammography, the parameters to be concerned about are the mean glandular dose and the scatter dose. We evaluated the dose of radiation to the breast, thyroid gland, and lens in digital mammography in women with and without implants, in tomosynthesis in women with and without implants, and in contrast-enhanced mammography. MATERIALS AND METHODS: The study included 212 women with and without disease who were attended at the Centro Clínico de Estereotaxia, CECLINES, in Caracas, Venezuela, between June 2017 and August 2017; the women were classified into five groups according to the mammographic modality used to evaluate them and whether or not they had implants. The statistical analysis included descriptive statistics for the study population. We used the Mann-Whitney U to compare the mean glandular dose and dose in the thyroid gland and lens between groups. RESULTS: The mean glandular dose and the dose of radiation received in the thyroid and lens were within the acceptable range. In a few exceptions, the mean glandular dose per view was slightly higher than 3 mGy. The scatter dose to the thyroid gland and the lens during mammography has a very small contribution to the annual dose equivalent. CONCLUSION: The mean glandular dose and the scatter dose to the thyroid gland and lens delivered during tomosynthesis and 2D mammography in women with implants were higher than those delivered during other mammographic techniques in women without implants.

[Evaluation of the research performed in Spanish hospitals]. / Valoración de la investigación que se realiza en los hospitales españoles.

OBJECTIVES: No study that analyzes how the investigators who work in the Spanish hospitals perceived and evaluate the current research system has been performed. This work, carried out by the Scientific Forum of the Lilly Foundation, aims to improve the level of information on the research activity performed in the hospitals. SUBJECTS AND METHODS: By means of a «self-administered¼ interview made up of 34 items and aimed at physicians and other research professionals who work in the Spanish hospitals (272 surveyed), 3 questions were analyzed: a) general situation of biomedical research in Spain; b) administration of available resources: need for better resources, and c) evaluation and giving priority to biomedical research. RESULTS: The use of the data has shown strengths in the system such as the initiatives to promote research through contracts with FIS and post-residency (7.6/10 points); the beneficial effects of research and care quality (7.3/10); or support of the pharmaceutical industry through the sponsoring of clinical trials (6.9/10). However, it has also shown that there are some weaknesses in the organization of the centers, as for example, those referring to the differentiated allocation of the care cost of the research activity (5.1/10); to the coordination between them and the health care centers (2.8/10); to the integration and organization among care, teaching and research (3.6/10); and to decide the priorities (5.2/10) and evaluation (5.2/10) of the research activities. Furthermore, the value of the research as a fundamental activity of the hospitals is emphasized (4.7/10). CONCLUSIONS: The investigators surveyed consider that biomedical research in recent decades in the hospital setting has significantly improved and has had a positive effect in the number of publications. The subjects surveyed consider that research should have greater institutional support and recognition, and a more translational orientation, which would be translated into better quality of life of the citizens and registry of patents.

Mean glandular dose in the mammary gland and dose of radiation in the thyroid gland and lens in women with and without breast implants during different modalities of mammography. / Dosis glandular promedio en glándula mamaria y dosis de radiación en glándula tiroides y cristalino en mujeres con y sin implantes mamarios durante diferentes modalidades mamográficas.

There have always been concerns about the secondary effects of diagnostic methods that use ionizing radiation. During mammography, the parameters to be concerned about are the mean glandular dose and the scatter dose. We evaluated the dose of radiation to the breast, thyroid gland, and lens in digital mammography in women with and without implants, in tomosynthesis in women with and without implants, and in contrast-enhanced mammography. MATERIALS AND METHODS: The study included 212 women with and without disease who were attended at the Centro Clínico de Estereotaxia, CECLINES, in Caracas, Venezuela, between June 2017 and August 2017; the women were classified into five groups according to the mammographic modality used to evaluate them and whether or not they had implants. The statistical analysis included descriptive statistics for the study population. We used the Mann-Whitney U to compare the mean glandular dose and dose in the thyroid gland and lens between groups. RESULTS: The mean glandular dose and the dose of radiation received in the thyroid and lens were within the acceptable range. In a few exceptions, the mean glandular dose per view was slightly higher than 3mGy. The scatter dose to the thyroid gland and the lens during mammography has a very small contribution to the annual dose equivalent. CONCLUSION: The mean glandular dose and the scatter dose to the thyroid gland and lens delivered during tomosynthesis and 2D mammography in women with implants were higher than those delivered during other mammographic techniques in women without implants.

Evaluation of functionality of type II toxin-antitoxin systems of Clostridioides difficile R20291.

Clostridioides difficile is a nosocomial, Gram-positive, strictly anaerobic, spore-forming pathogen capable of colonizing and proliferating in the human intestine. In bacteria, it has been shown that the Toxin-Antitoxin systems mediate the cellular response to external stress by initiating processes such as biofilm formation and programmed cell death. This work aims to evaluate the functionality of four type II TA modules of Clostridioides difficile R20291. We performed bioinformatic analysis to search for putative TA systems using the TADB platform. Then we performed a heterologous expression assay to evaluate the functionality of these systems. Our results showed that the MazEF and RelBE systems were functional, suggesting that their corresponding toxins possess an endoribonuclease activity. In conclusion, MazEF and RelBE systems of C. difficile R20291 are functional in a heterologous expression system.

Inhibitory effect of biofilm-forming Lactobacillus kunkeei strains against virulent Pseudomonas aeruginosa in vitro and in honeycomb moth (Galleria mellonella) infection model.

Biofilms correspond to complex communities of microorganisms embedded in an extracellular polymeric matrix. Biofilm lifestyle predominates in Pseudomonas aeruginosa, an opportunistic Gram negative pathogen responsible for a wide spectrum of infections in humans, plants and animals. In this context, anti-biofilm can be considered a key strategy to control P. aeruginosa infections, thereby more research in the field is required. On the other hand, Lactobacillus species have been described as beneficial due to their anti-biofilm properties and their consequent effect against a wide spectrum of pathogens. In fact, biofilm-forming Lactobacilli seem to be more efficient than their planktonic counterpart to antagonise pathogenic bacteria. In this work, we demonstrated that Lactobacillus kunkeei, a novel Lactobacillus species isolated from honeybee guts, can form biofilms in vitro. In addition, the L. kunkeei biofilm can, in turn, inhibit the formation of P. aeruginosa biofilms. Finally, we found that L. kunkeei strains attenuate infection of P. aeruginosa in the Galleria mellonella model, presumably by affecting P. aeruginosa biofilm formation and/or their stability. Since L. kunkeei presents characteristics of a probiotic, this work provides evidence arguing that the use of this Lactobacillus species in both animals (including insects) and humans could contribute to impair P. aeruginosa biofilm formation.

Lose to win: marT pseudogenization in Salmonella enterica serovar Typhi contributed to the surV-dependent survival to H2O2, and inside human macrophage-like cells.

The difference in host range between Salmonella enterica serovar Typhimurium (S. Typhimurium) and Salmonella enterica serovar Typhi (S. Typhi) can be partially attributed to the gain of functions, to the loss of functions (i.e. pseudogenization), or to a combination of both processes. As previously reported, the loss of functions by pseudogenization may play a role in bacterial evolution, especially in host-restricted pathogens such as S. Typhi. The marT-fidL operon, located at the SPI-3, encodes the MarT transcriptional regulator and a hypothetical protein (i.e. FidL) with no significant similarities to known proteins, respectively. Even though predicted S. Typhimurium FidL exhibit 99.4% identity with S. Typhi FidL, marT has been annotated as a pseudogene in S. Typhi. In this work, we found that S. Typhi expressing S. Typhimurium marT-fidL exhibited an increased accumulation of reactive oxygen species (ROS), leading to a decreased survival in presence of H2O2. Moreover, we found that that the presence of a functional copy of S. Typhimurium marT-fidL in S. Typhi resulted in a repression of surV (STY4039), an ORF found in the S. Typhi SPI-3 but absent from S. Typhimurium SPI-3, that contribute to the resistance to H2O2 by decreasing the accumulation of ROS. Finally, we observed that the presence of S. Typhimurium marT-fidL in S. Typhi negatively affected the survival inside macrophage-like cells, but not in epithelial cells, after 24h post infection. Therefore, this work provides evidence arguing that marT pseudogenization in Salmonella Typhi contributed to the surV-dependent survival against H2O2, and inside human macrophage-like cells. This is a good example of how the loss of functions (marT pseudogenization) and the gain of functions (presence of surV) might contribute to phenotypic changes improving virulence.

Pharmacological and biochemical interactions between opioids and cannabinoids.

Opioids and cannabinoids are among the most widely consumed drugs of abuse in humans. A number of studies have shown that both types of drugs share several pharmacological properties, including hypothermia, sedation, hypotension, inhibition of both intestinal motility and locomotor activity and, in particular, antinociception. Moreover, phenomena of cross-tolerance or mutual potentiation of some of these pharmacological effects have been reported. In recent years, these phenomena have supported the possible existence of functional links in the mechanisms of action of both types of drugs. The present review addresses the recent advances in the study of pharmacological interactions between opioids and cannabinoids, focusing on two aspects: antinociception and drug addiction. The potential biochemical mechanisms involved in these pharmacological interactions are also discussed together with possible therapeutic implications of opioid-cannabinoid interactions.

Pseudogenization of sopA and sopE2 is functionally linked and contributes to virulence of Salmonella enterica serovar Typhi.

The difference in host range between Salmonella enterica serovar Typhimurium (S. Typhimurium) and S. enterica serovar Typhi (S. Typhi) can be partially attributed to pseudogenes. Pseudogenes are genomic segments homologous to functional genes that do not encode functional products due to the presence of genetic defects. S. Typhi lacks several protein effectors implicated in invasion or other important processes necessary for full virulence of S. Typhimurium. SopA and SopE2, effectors that have been lost by pseudogenization in S. Typhi, correspond to an ubiquitin ligase involved in cytokine production by infected cells, and to a guanine exchange factor necessary for invasion of epithelial cells, respectively. We hypothesized that sopA and/or sopE pseudogenization contributed to the virulence of S. Typhi. In this work, we found that S. Typhi expressing S. Typhimurium sopE2 exhibited a decreased invasion in different epithelial cell lines compared with S. Typhi WT. S. Typhimurium sopA completely abolished the hypo-invasive phenotype observed in S. Typhi expressing S. Typhimurium sopE2, suggesting that functional SopA and SopE2 participate concertedly in the invasion process. Finally, the expression of S. Typhimurium sopA and/or sopE2 in S. Typhi, determined changes in the secretion of IL-8 and IL-18 in infected epithelial cells.

Subchronic treatment with morphine inhibits the hypertension induced by isolation stress in the rat.

As preliminary evidence for the implication of opioids in the increase in blood pressure due to the stress of brief social deprivation, hypertension has been shown to be antagonized by acute administration of opiate receptor blockers. As a further evidence of involvement of opioids in the hypertensive response to this type of stress, cross-tolerance ought to be capable of being demonstrated in isolated animals, treated with an opiate. When rats were treated subchronically with morphine in the drinking water throughout the isolation period (1-15 days), readings of blood pressure did not show any variation, as compared to group-housed control rats. However, 7 days after withdrawal of morphine readings of arterial pressure in the isolated rats increased above the levels of the group-housed control animals. These findings support the idea that an endogenous opioid system is implicated in the induction of readings of high blood pressure due to the stress of social deprivation.

Association between hypoalgesia and hypertension in rats after short-term isolation.

By isolating young rats (90-100 g) a state of hypertension and tachycardia was induced after 7 days or a longer period of social deprivation. Clonidine, a drug used to treat hypertension in man, readily reversed the high blood pressure and heart rate in this experimental model of hypertension. In two different tests, an elevated nociceptive threshold was shown to be present in isolated animals as compared to group-housed rats. Naloxone was found to reverse this hypoalgesic state. The opiate antagonist also diminished the high blood pressure in the socially-deprived animals. Moreover, after 7 days of isolation, 24 hr of housing the rats in groups of five made the level of blood pressure and the sensitivity to pain return to control values. In this experimental model, in which hypertension was linked to stressful housing conditions, the data suggest that high blood pressure and hypoalgesia are closely associated.

Anandamide decreases naloxone-precipitated withdrawal signs in mice chronically treated with morphine.

The effect of anandamide, a putative endogenous ligand of the cannabinoid receptor, has been studied in a naloxone-precipitated morphine withdrawal syndrome in mice. Animals were chronically treated with increasing doses of morphine (from 8 to 45 mg/kg) over 5 days or implanted with morphine pellets (72 hr). Typical signs of withdrawal (jumping and body weight loss) were examined after naloxone administration (1 mg/kg). In these conditions, anandamide (5 mg/kg, i.v.) decreased both the number of jumps, measured over 30 min (81.2% +/- 3.15 and 92.2% +/- 3.5 decrease in chronically administered morphine and pellet implanted mice, respectively), and the body weight loss at 30 and 60 min (30 min: 2.6% +/- 0.4 vs 4.4% +/- 0.2 and 3.7% +/- 0.4 vs 5.3% +/- 0.4; 60 min: 3.2% +/- 0.5 vs 5.0% +/- 0.4 and 4.1% +/- 0.5 vs 6.0% +/- 0.5 in chronically treated morphine and pellet implanted mice respectively) after naloxone administration. This suggests, as shown in the case of delta 9-tetrahydrocannabinol, a modulation by anandamide of pathways involved in the expression of physical signs of opioid dependence and support its role as an endogenous cannabinoid agonist.

Repeated administration of delta9-tetrahydrocannabinol produces a differential time related responsiveness on proenkephalin, proopiomelanocortin and corticotropin releasing factor gene expression in the hypothalamus and pituitary gland of the rat.

The purpose of the present study was to explore the time related effects of repeated administration of delta9-tetrahydrocannabinol on opioid and corticotropin releasing factor gene expression in the hypothalamus and pituitary gland of the rat. By using in situ hybridization histochemistry, the effects of delta9-tetrahydrocannabinol (THC, 5 mg/kg per day; i.p.) were examined after 1, 3, 7 and 14 days of repeated administration on; (1) proenkephalin gene expression in the paraventricular (PVN) and ventromedial nuclei (VMN) of the hypothalamus, (2) proopiomelanocortin gene expression in the arcuate nucleus (ARC) of the hypothalamus and anterior (AL) and intermediate lobe (IL) of the pituitary gland, and (3) corticotropin releasing factor gene expression in the PVN. The results revealed that, in most of the hypothalamic and pituitary regions examined, repeated cannabinoid administration upregulates opioid and corticotropin releasing factor gene expression. However, the onset, the degree of magnitude of gene expression reached and the time related effects produced by repeated administration with delta9-tetrahydrocannabinol are dependent upon the brain and pituitary regions examined. Taken together, the results of the present study suggest that cannabinoids produce a time related differential responsiveness in opioid and corticotropin releasing factor gene expression, in areas of the hypothalamus and pituitary that may be related, at least in part, to a molecular integrative response to behavioral, endocrine and neurochemical alterations that occur in cannabinoid drug abuse.

Inhibition of opioid-degrading enzymes potentiates delta9-tetrahydrocannabinol-induced antinociception in mice.

Delta9-tetrahydrocannabinol (delta9-THC) elicits antinociception in rodents through the central CB1 cannabinoid receptor subtype. In addition. Delta9-THC stimulates the release of dynorphin-related peptides leading to kappa-opioid spinal antinociception. In this work we describe the effect of a mixture of thiorphan (a neutral endopeptidase EC3.4.24.11 inhibitor) and bestatin (an aminopeptidase inhibitor), administered i.c.v., on the antinociceptive effect of peripherally administered delta9-THC in mice. As in the case of morphine or DAMGO ([D-Ala2.N-Me-Phe4,Gly-ol]enkephalin), a mu-selective opioid receptor agonist, the mixture of enkephalin-degrading enzyme inhibitors also enhanced the antinociceptive effect of delta9-THC. This effect was blocked by the CB1 cannabinoid receptor antagonist, SR-141,716-A, as well as by naloxone. The kappa-opioid receptor antagonist nor-binaltorphimine, administered i.t., also antagonized the effect of this combination. Similar results were obtained with the mu-opioid receptor antagonist beta-funaltrexamine after i.c.v. administration. These results demonstrate the involvement of both mu-opioid supraspinal and kappa-opioid spinal receptors in the interaction of both opioid and cannabinoid systems regulating nociception in mice.

Synthesis and structure-activity relationships of a new model of arylpiperazines. 1. 2-[[4-(o-methoxyphenyl)piperazin-1-yl]methyl]-1, 3-dioxoperhydroimidazo[1,5-alpha]pyridine: a selective 5-HT1A receptor agonist.

A series of new bicyclohydantoin-arylpiperazines was prepared and evaluated for affinity at 5-HT1A, alpha 1, and D2 receptors. Most of the compounds showed very low affinity for D2 receptors, and most of them demonstrated moderate to high affinity for 5-HT1A and alpha 1 receptor binding sites. SAR observations indicated that the length of the alkyl chain between the arylpiperazine and the hydantoin moiety is of great importance for 5-HT1A/alpha 1 affinity and selectivity, n = 1 being the optimal value. Compound 1h, 2-[[4-(o-methoxyphenyl)piperazin-1-yl] methyl]-1,3-dioxoperhydroimidazo [1,5-alpha]pyridine, bound at 5-HT1A sites with nanomolar affinity (Ki = 31.7 nM) and high selectivity over alpha 1, D2, and 5-HT2A receptors (Ki > 1000, > 10 000, and > 1000 nM, respectively). Preliminary studies showed that this agent is probably functioning as a partial to full 5-HT1A agonist, and it displayed anxiolytic activity on the social interaction test in mice.

Synthesis and structure-activity relationships of a new model of arylpiperazines. 5. Study of the physicochemical influence of the pharmacophore on 5-HT(1a)/alpha(1)-adrenergic receptor affinity: synthesis of a new derivative with mixed 5-HT(1a)/d(2) antagonist properties.

In this paper we have designed and synthesized a test series of 32 amide arylpiperazine derivatives VI in order to gain insight into the physicochemical influence of the pharmacophores of 5-HT(1A) and alpha(1)-adrenergic receptors. The training set was designed applying a fractional factorial design using six physicochemical descriptors. The amide moiety is a bicyclohydantoin or a diketopiperazine (X = -(CH(2))(3)-, -(CH(2))(4)-; m = 0, 1), the spacer length is 3 or 4 methylene units, which are the optimum values for both receptors, and the aromatic substituent R occupies the ortho- or meta-position and has been selected from a database of 387 substituents using the EDISFAR program. The 5-HT(1A) and alpha(1)-adrenergic receptor binding affinities of synthesized compounds VI (1-32) have been determined. This data set has been used to derive classical quantitative structure-activity relationships (QSAR) and neural networks models for both receptors (following paper). A comparison of these models gives information for the design of the new ligand EF-7412 (46) (5-HT(1A): K(i) = 27 nM; alpha(1): K(i) > 1000 nM). This derivative displays affinity for the dopamine D(2) receptor (K(i) = 22 nM) and is selective versus all other receptors examined (5-HT(2A), 5-HT(3), 5-HT(4) and Bz; K(i) > 1000 nM). EF-7412 (46) acts as an antagonist in vivo in pre- and postsynaptic 5-HT(1A) receptor sites and as an antagonist in the dopamine D(2) receptor. Thus, EF-7412 (46) is a derivative with mixed 5-HT(1A)/D(2) antagonist properties and this derivative could be useful as a pharmacological tool.

Impairment of stress adaptive behaviours in rats by the CCKA receptor antagonist, devazepide.

1. Cholecystokinin (CCK) is released during stress both in limbic and hypothalamic areas suggesting that CCK could participate in modulating neuroendocrine as well as behavioural responses to stress. 2. In this study we have examined the effect of CCK receptor antagonists on the retention of the immobility response to a forced-swim stress in rats. In this test, rats are forced to swim during 15 min (conditioning period) and 24 h later, the duration of immobility is measured during a period of 5 min (re-test period). During the conditioning period rats display a period of vigorous activity, followed by progressive inactivity. During the re-test period rats remain 70-80% of the time in an immobile posture. 3. The CCKA receptor antagonist, devazepide (MK-329) but not the CCKB receptor antagonist, L-365,260, administered s.c. immediately before the conditioning period, decreased the duration of acquired immobility during the re-test period. The effect of devazepide was prevented by cholecystokinin octapeptide (CCK-8; 40 micrograms kg-1, s.c) as well as by the selective glucocorticosteroid GII receptor agonist, dexamethasone (30 micrograms kg-1, s.c.) 4. Neither corticosterone nor ACTH plasma levels measured both after the re-test period and after the conditioning period were modified by devazepide treatment. 5. The results suggest a role for CCK in the behavioural adaptation to stress and indicate a relationship between CCK systems and glucocorticoids in the neuronal mechanisms involved in the acquisition of adaptive behaviours to stress.

Inhibition of the hypothalamic-pituitary-adrenal axis in food-deprived rats by a CCK-A receptor antagonist.

The circadian activity of the hypothalamic-pituitary-adrenal (HPA) axis is regulated by caloric flow in rats. During the dark cycle, it has been shown that, in fasted rats, the time-course profile of plasma concentrations of adrenocorticotropin (ACTH) and corticosterone parallels the profile of food intake in ad libitum fed animals. Cholecystokinin (CCK) is involved in regulating food intake in rodents. CCK-8 reduces food intake by acting on CCK-A receptors subtype. This work aims at establishing an eventual relationship between the modulatory role of CCK on food intake and its effect on HPA axis activity during fasting. We studied the effect of CCK-A and CCK-B receptor antagonists on food intake during the first period of the dark cycle. Under these conditions we observed that the CCK-A receptor antagonist, SR-27897 (0.3 mg kg(-1)), but not the CCK-B receptor antagonist, L-365260 (1 mg kg(-1)), increases food-intake. In a second series of experiments we observed that the increase of both ACTH and corticosterone plasma level elicited by fasting, was prevented by SR-27897, but not by L-365260. These results indicate that CCK-A receptor blockade during fasting prevents the activation of the HPA axis.

Chronic administration of cannabinoids regulates proenkephalin mRNA levels in selected regions of the rat brain.

This study was designed to examine the interactions between the cannabinoid and enkephalinergic systems in the rat brain. To this aim, we have examined the effects of subchronic (5 days) administration (10 mg.kg-1.day-1; i.p.) of delta 9 -tetrahydrocannabinol (THC) or R-methanandamide (AM356) and chronic (18 days) administration with the synthetic cannabinoid receptor agonist CP-55,940 (1 mg.kg-1.day-1; i.p) on proenkephalin (PENK) mRNA levels in several brain regions of the rat. Twenty micrometer brain sections from striatum, nucleus accumbens, paraventricular nucleus, ventromedial nucleus, periaqueductal grey matter and mammillary nucleus were hybridized with an oligonucleotide probe complementary to PENK using in situ hybridization technique. Subchronic administration of THC or AM356 increased PENK mRNA levels in the ventromedial nucleus of the hypothalamus, (82%) and (39%), in the periaqueductal grey matter, (97%) and (49%), and mammillary nucleus, (43%) and (9%), respectively. In contrast, both drugs were without effect in the striatum and nucleus accumbens. On the other hand, chronic administration of CP-55,940 increased PENK mRNA levels in the striatum (44%), nucleus accumbens (25%), paraventricular (31%) and ventromedial nuclei of the hypothalamus (41%). These results revealed that chronic cannabinoid administration increases opioid gene expression in the rat central nervous system and suggest an interaction between the cannabinoid and enkephalinergic systems that may be part of a molecular integrative response to behavioral and neurochemical alterations that occur in cannabinoid drug abuse.

Time-dependent differences of repeated administration with Delta9-tetrahydrocannabinol in proenkephalin and cannabinoid receptor gene expression and G-protein activation by mu-opioid and CB1-cannabinoid receptors in the caudate-putamen.

The purpose of the present study was to examine the time-related effects of repeated administration of Delta9-tetrahydrocannabinol during 1, 3, 7 and 14 days on cannabinoid and mu-opioid receptor agonist-stimulated [35S]GTPgammaS binding, and CB1 cannabinoid receptor and proenkephalin gene expression in the caudate-putamen. Repeated administration with Delta9-tetrahydrocannabinol produced a time-related reduction in cannabinoid receptor synthesis and activation of signal transduction mechanisms in the caudate-putamen. Indeed, WIN-55,212-2-stimulated [35S]GTPgammaS binding decreased 24% on day 1 and then progressively decreased finding a 42% decrease on day 14. Similarly, CB1 cannabinoid receptor mRNA levels decreased (22%) on day 3, reaching 50% reduction on day 7. In contrast, a pronounced increase is detected in DAMGO-stimulated [35S]GTPgammaS binding and proenkephalin mRNA levels in the caudate-putamen. The highest degree of increase was reached on day 7 of the treatment (35% of proenkephalin mRNA levels and 62% of DAMGO-stimulated [35S]GTPgammaS binding) and then values slightly decreased on day 14. Taken together, the results of the present study indicate that, in the caudate-putamen, repeated administration with Delta9-tetrahydrocannabinol produces a time-related increase in proenkephalin gene expression and mu-opioid receptor activation of G-proteins, and a time-related decrease in CB1 cannabinoid receptor gene expression and reduction in CB1 cannabinoid receptor activation of G-proteins. These results also suggest a possible interaction between the cannabinoid and opioid systems in the caudate-putamen which may be potentially relevant in the understanding of the alterations of motor behavior that occur after prolonged exposure to cannabinoids.