Proteomic analysis of post-nuclear supernatant fraction and percoll-purified membranes prepared from brain cortex of rats exposed to increasing doses of morphine.
ABSTRACT: BACKGROUND: Proteomic analysis was performed in post-nuclear supernatant (PNS) and Percoll-purified membranes (PM) prepared from fore brain cortex of rats exposed to increasing doses of morphine (10-50 mg/kg) for 10 days. RESULTS: In PNS, the 10 up (?)- or down (?)-regulated proteins exhibiting the largest morphine-induced change were selected, excised manually from the gel and identified by MALDI-TOF MS/MS: 1-(gi|148747414, Guanine deaminase), ?2.5×; 2-(gi|17105370, Vacuolar-type proton ATP subunit B, brain isoform), ?2.6×; 3-(gi|1352384, Protein disulfide-isomerase A3), ?3.4×; 4-(gi|40254595, Dihydropyrimidinase-related protein 2), ?3.6×; 5-(gi|149054470, N-ethylmaleimide sensitive fusion protein, isoform CRAa), ?2.0×; 6-(gi|42476181, Malate dehydrogenase, mitochondrial precursor), ?1.4×; 7-(gi|62653546, Glyceraldehyde-3-phosphate dehydrogenase), ?1.6×; 8-(gi|202837, Aldolase A), ?1.3×; 9-(gi|31542401, Creatine kinase B-type), ?0.86×; 10-(gi|40538860, Aconitate hydratase, mitochondrial precursor), ?1.3×. The identified proteins were of cytoplasmic (1, 4, 5, 7, 9), cell membrane (2), endoplasmic reticulum (3) and mitochondrial (6, 8, 10) origin and 9 of them were significantly increased, 1.3-3.6×. The 4 out of 9 up-regulated proteins (4, 6, 7, 10) were described as functionally related to oxidative stress; the 2 proteins participate in genesis of apoptotic cell death.In PM, the 18 up (?)- or down (?)-regulated proteins were identified by LC-MS/MS and were of plasma membrane [Brain acid soluble protein, ?2.1×; trimeric G? subunit, ?2.0x], myelin membrane [MBP, ?2.5×], cytoplasmic [Internexin, ?5.2×; DPYL2, ?4.9×; Ubiquitin hydrolase, ?2.0×; 60S ribosomal protein, ?2.7×; KCRB, ?2.6×; Sirtuin-2, ?2.5×; Peroxiredoxin-2, ?2.2×; Septin-11, ?2.2×; TERA, ?2.1×; SYUA, ?2.0×; Coronin-1A, ?5.4×] and mitochondrial [Glutamate dehydrogenase 1, ?2.7×; SCOT1, ?2.2×; Prohibitin, ?2.2×; Aspartate aminotransferase, ?2.2×] origin. Surprisingly, the immunoblot analysis of the same PM resolved by 2D-ELFO indicated that the "active", morphine-induced pool of G? subunits represented just a minor fraction of the total signal of G? which was decreased 1.2x only. The dominant signal of G? was unchanged. CONCLUSION: Brain cortex of rats exposed to increasing doses of morphine is far from being adapted. Significant up-regulation of proteins functionally related to oxidative stress and apoptosis suggests a major change of energy metabolism resulting in the state of severe brain cell "discomfort" or even death.
Project description:Atrial fibrillation (AF) is the most common cardiac arrhythmia affecting approximately 2.2 million Americans. Because several studies have suggested that changes in mitochondrial function and morphology may contribute to AF, we developed a novel proteomic workflow focused on the identification of differentially expressed mitochondrial proteins in AF patients. Right human atrial tissue was collected from 20 patients, 10 with and 10 without AF, and the tissue was subjected to hydrostatic pressure cycling-based lysis followed by label-free mass spectrometric (MS) analysis of mitochondrial enriched isolates. Approximately 5% of the 700 proteins identified by MS analysis were differentially expressed between the AF and non-AF samples. We chose four differentially abundant proteins for further verification using reverse phase protein microarray analysis based on their known importance in energy production and regulatory association with atrial ion channels: four and a half LIM, destrin, heat shock protein 2, and chaperonin-containing TCP1. These initial study results provide evidence that a workflow to identify AF-related proteins that combines a powerful upfront tissue cell lysis with high resolution MS for discovery and protein array technology for verification may be an effective strategy for discovering candidate markers in highly fibrous tissue samples.
Project description:Recent warnings from Health Canada regarding codeine for children have led to increased use of nonsteroidal anti-inflammatory drugs and morphine for common injuries such as fractures. Our objective was to determine whether morphine administered orally has superior efficacy to ibuprofen in fracture-related pain.We used a parallel group, randomized, blinded superiority design. Children who presented to the emergency department with an uncomplicated extremity fracture were randomly assigned to receive either morphine (0.5 mg/kg orally) or ibuprofen (10 mg/kg) for 24 hours after discharge. Our primary outcome was the change in pain score using the Faces Pain Scale - Revised (FPS-R). Participants were asked to record pain scores immediately before and 30 minutes after receiving each dose.We analyzed data from 66 participants in the morphine group and 68 participants in the ibuprofen group. For both morphine and ibuprofen, we found a reduction in pain scores (mean pre-post difference ± standard deviation for dose 1: morphine 1.5 ± 1.2, ibuprofen 1.3 ± 1.0, between-group difference [?] 0.2 [95% confidence interval (CI) -0.2 to 0.6]; dose 2: morphine 1.3 ± 1.3, ibuprofen 1.3 ± 0.9, ? 0 [95% CI -0.4 to 0.4]; dose 3: morphine 1.3 ± 1.4, ibuprofen 1.4 ± 1.1, ? -0.1 [95% CI -0.7 to 0.4]; and dose 4: morphine 1.5 ± 1.4, ibuprofen 1.1 ± 1.2, ? 0.4 [95% CI -0.2 to 1.1]). We found no significant differences in the change in pain scores between morphine and ibuprofen between groups at any of the 4 time points (p = 0.6). Participants in the morphine group had significantly more adverse effects than those in the ibuprofen group (56.1% v. 30.9%, p < 0.01).We found no significant difference in analgesic efficacy between orally administered morphine and ibuprofen. However, morphine was associated with a significantly greater number of adverse effects. Our results suggest that ibuprofen remains safe and effective for outpatient pain management in children with uncomplicated fractures.ClinicalTrials.gov, no. NCT01690780.
Project description:BACKGROUND AND PURPOSE:Chronic administration of medication can significantly affect metabolic enzymes leading to physiological adaptations. Morphine metabolism in the liver has been extensively studied following acute morphine treatment, but such metabolic processes in the CNS are poorly characterized. Long-term morphine treatment is limited by the development of tolerance, resulting in a decrease of its analgesic effect. Whether or not morphine analgesic tolerance affects in vivo brain morphine metabolism and blood-brain barrier (BBB) permeability remains a major question. Here, we have attempted to characterize the in vivo metabolism and BBB permeability of morphine after long-term treatment, at both central and peripheral levels. EXPERIMENTAL APPROACH:Male C57BL/6 mice were injected with morphine or saline solution for eight consecutive days in order to induce morphine analgesic tolerance. On the ninth day, both groups received a final injection of morphine (85%) and d3-morphine (morphine bearing three 2 H; 15%, w/w). Mice were then killed and blood, urine, brain and liver samples were collected. LC-MS/MS was used to quantify morphine, its metabolite morphine-3-glucuronide (M3G) and their respective d3-labelled forms. KEY RESULTS:We found no significant differences in morphine CNS uptake and metabolism between control and tolerant mice. Interestingly, d3-morphine metabolism was decreased compared to morphine without any interference with our study. CONCLUSIONS AND IMPLICATIONS:Our data suggests that tolerance to the analgesic effects of morphine is not linked to increased glucuronidation to M3G or to altered global BBB permeability of morphine.
Project description:The effect of the i.c.v. administration of pertussis toxin (PTX) and antisense oligodeoxynucleotide directed against the alpha subunit of different Gi-proteins (anti-Gialpha1, anti-Gialpha2, anti-Gialpha3) on amnesia induced by morphine was evaluated in the mouse passive avoidance test. The administration of morphine (6 - 10 mg kg(-1) i.p.) immediately after the training session produced amnesia that was prevented by PTX (0.25 microg per mouse i.c.v.) administered 7 days before the passive avoidance test. Anti-Gialpha1 (6.25 microg per mouse i.c.v.) and anti-Gialpha3 (12.5 microg per mouse i.c.v.), administered 18 and 24 h before the training session, prevented the morphine amnesia. By contrast, pretreatment with anti-Gialpha2 (3.12 - 25 microg per mouse i.c.v.) never modified the impairment of memory processes induced by morphine. At the highest effective doses, none of the compounds used impaired motor coordination, as revealed by the rota rod test, nor modified spontaneous motility and inspection activity, as revealed by the hole board test. These results suggest the important role played by Gi1 and Gi3 protein subtypes in the transduction mechanism involved in the impairment of memory processes produced by morphine.
Project description:Recent studies suggest an association between particulate matter (PM) air pollution and gastrointestinal (GI) disease. In addition to direct deposition, PM can be indirectly deposited in oropharynx via mucociliary clearance and upon swallowing of saliva and mucus. Within the GI tract, PM may alter the GI epithelium and gut microbiome. Our goal was to determine the effect of PM on gut microbiota in a murine model of PM exposure via inhalation. C57BL/6 mice were exposed via inhalation to either concentrated ambient particles or filtered air for 8-h per day, 5-days a week, for a total of 3-weeks. At exposure's end, GI tract tissues and feces were harvested, and gut microbiota was analyzed. Alpha-diversity was modestly altered with increased richness in PM-exposed mice compared to air-exposed mice in some parts of the GI tract. Most importantly, PM-induced alterations in the microbiota were very apparent in beta-diversity comparisons throughout the GI tract and appeared to increase from the proximal to distal parts. Changes in some genera suggest that distinct bacteria may have the capacity to bloom with PM exposure. Exposure to PM alters the microbiota throughout the GI tract which maybe a potential mechanism that explains PM induced inflammation in the GI tract.
Project description:Sphingosine 1-phosphate (S1P) is a pleiotropic lipid mediator involved in the regulation of immune cell trafficking and vascular permeability acting mainly through G-protein-coupled S1P receptors (S1PRs). However, mechanism underlying how S1PRs are coupled with G-proteins remains unknown. Here we have uncovered that palmitoylation of a prototypical subtype S1P1R is prerequisite for subsequent inhibitory G-protein (Gi) coupling. We have identified DHHC5 as an enzyme for palmitoylation of S1P1R. Under basal conditions, S1P1R was functionally associated with DHHC5 in the plasma membranes (PM) and was fully palmitoylated, enabling Gi coupling. Upon stimulation, the receptor underwent internalisation leaving DHHC5 in PM, resulting in depalmitoylation of S1P1R. We also revealed that while physiological agonist S1P-induced endocytosed S1P1R readily recycled back to PM, pharmacological FTY720-P-induced endocytosed S1P1R-positive vesicles became associated with DHHC5 in the later phase, persistently transmitting Gi signals there. This indicates that FTY720-P switches off the S1P signal in PM, while switching on its signal continuously inside the cells. We propose that DHHC5-mediated palmitoylation of S1P1R determines Gi coupling and its signalling in a spatio/temporal manner.
Project description:1. The effects of morphine, nalorphine, acetazolamide, and 10% CO(2) on brain metabolite concentrations of 24h-starved rats were studied. 2. A single dose of morphine (20mg/kg body wt.) caused an increase in brain glucose concentration (42%) and decreased concentrations of lactate (24%), pyruvate (29%), citrate (20%), alpha-oxoglutarate (16%), malate (14%) and creatine phosphate (10%) after 30min. No changes were found in adenine nucleotide concentrations. 3. The same dose of morphine increased arterial CO(2) from 5.07 to 7.60 kN/m(2) (38 to 57 Torr), decreased the pH from 7.41 to 7.31 and decreased O(2) from 14.1 to 10.8kN/m(2) (106 to 81 Torr) at 30min. 4. Rats injected with morphine three times daily (20mg/kg body wt.) for 2 weeks had no changes in brain metabolite concentrations or in blood gases 30min after their last injection. 5. Nalorphine (an antagonist of morphine) caused essentially no changes in brain metabolite concentrations in normal rats. When nalorphine (20mg/kg) was administered to rats previously treated with morphine three times daily for 2 weeks, there was an increase in brain glucose (100%), lactate (23%), pyruvate (18%) and citrate (10%) concentrations. 6. Acetazolamide (an inhibitor of carbonic anhydrase) and 10% CO(2) increased the arterial CO(2) from 4.79 to 6.78kN/m(2) (36 to 51 Torr) and from 5.32 to 10.8kN/m(2) (40 to 81 Torr) respectively. 7. Both acetazolamide and 10% CO(2) caused changes in brain metabolite concentrations similar to those for acutely administered morphine. Thus 10% CO(2) caused increased brain glucose concentration (123%) and decreased brain lactate (46%), pyruvate (34%), citrate (26%), alpha-oxoglutarate (33%), malate (45%) and creatine phosphate (7%) concentrations. No changes in adenine nucleotide concentrations were found. 8. The results indicate that the effect of morphine on brain metabolite concentrations may be accounted for by the increased [CO(2)]. 9. These findings constitute a consistent pattern of metabolic changes after acute morphine administration, morphine addiction, and withdrawal from morphine addiction.
Project description:Latanoprostene bunod is a novel nitric oxide (NO)-donating prostaglandin F2? receptor agonist in clinical development for the reduction of intraocular pressure (IOP) in patients with open-angle glaucoma or ocular hypertension. We evaluated the effect of latanoprostene bunod 0.024% instilled once daily (QD) on lowering IOP over a 24-h period in healthy Japanese subjects following 14 days of treatment.This was a single-arm, single-center, open-label clinical study of 24 healthy Japanese male volunteers. A baseline IOP profile was established in both eyes in the sitting position at 8 PM, 10 PM, 12 AM, 2 AM, 4 AM, 8 AM, 10 AM, 12 PM, and 4 PM using a Goldmann applanation tonometer. Subjects subsequently instilled latanoprostene bunod 0.024% QD at 8 PM for 14 days in both eyes. The absolute and change from baseline in sitting IOP was assessed on day 14.The mean (SD) age of the subjects was 26.8 (6.3) years, and mean (SD) baseline IOP was 13.6 (1.3) mmHg in the study eye. Latanoprostene bunod 0.024% instilled QD for 14 days reduced IOP at all the evaluated time points (P < 0.001) with a mean (SD) 24-h reduction of 3.6 (0.8) mmHg or 27% from the baseline in the study eye. Peak and trough IOP lowering occurred at 8 AM and 8 PM (12 and 24 h following instillation) with a mean reduction of 4.2 (1.8) mmHg, or 30%, and 2.8 (2.2) mmHg, or 20%, respectively. Punctate keratitis and ocular hyperemia, both mild in severity, were the most common adverse events.Latanoprostene bunod ophthalmic solution 0.024%, dosed QD for 14 days, significantly lowered mean IOP in healthy Japanese subjects during the entire 24-h period. Studies of latanoprostene bunod in patients diagnosed with normal tension glaucoma are warranted.Clinicaltrials.gov identifier NCT01895985.Bausch & Lomb, Inc.
Project description:Deep brain stimulation (DBS) modulates the neuronal activity in specific brain circuits and has been recently considered as a promising intervention for refractory addiction. The insula cortex is the hub of interoception and is known to be involved in different aspects of substance use disorder. In the present study, we investigate the effects of continuous high frequency DBS in the anterior insula (AI) on drug-seeking behaviors and examined the molecular mechanisms of DBS action in morphine-addicted rats. Sprague-Dawley rats were trained to the morphine-conditioned place preference (CPP, day 1-8) followed by bilaterally implanted with DBS electrodes in the AI (Day 10) and recovery (Day 10-15). Continuous high-frequency (HF) -DBS (130 Hz, 150 ?A, 90 ?s) was applied during withdrawal (Day 16-30) or extinction sessions. CPP tests were conducted on days 16, 30, 40 during withdrawal session and several rats were used for proteomic analysis on day 30. Following the complete extinction, morphine-CPP was reinstated by a priming dose of morphine infusion (2 mg/kg). The open field and novel objective recognition tests were also performed to evaluate the DBS side effect on the locomotion and recognition memory. Continuous HF-DBS in the AI attenuated the expression of morphine-CPP post-withdrawal (Day 30), but morphine addictive behavior relapsed 10 days after the cessation of DBS (Day 40). Continuous HF-DBS reduced the period to full extinction of morphine-CPP and blocked morphine priming-induced recurrence of morphine addiction. HF-DBS in the AI had no obvious effect on the locomotor activity and novel objective recognition and did not cause anxiety-like behavior. In addition, our proteomic analysis identified eight morphine-regulated proteins in the AI and their expression levels were reversely changed by HF-DBS. Continuous HF-DBS in the bilateral anterior insula prevents the relapse of morphine place preference after withdrawal, facilitates its extinction, blocks the reinstatement induced by morphine priming and reverses the expression of morphine-regulated proteins. Our findings suggest that manipulation of insular activity by DBS could be a potential intervention to treat substance use disorder, although future research is warranted.
Project description:Chronic exposure to opioids induces adaptations in brain function that lead to the formation of the behavioral and physiological symptoms of drug dependence and addiction. Genome wide analysis of molecular effects of protracted morphine or saccharin intake in C57BL/6J mice over a period of 7 months provides an opportunity to observe the alterations in the brain that accompany long-term drug addiction. Overall design: Microarray experiment was designed to compare chronic and acute effects of protracted morphine or saccharin self-administration. Therefore we have analyzed 4 groups: animals chronically drinking saccharin with a saline or morphine injection, and mice chronically drinking morphine with saline or morphine injection. Animals were sacrificed 4 hours after receiving either a saline (10 ml/kg) or morphine (20 mg/kg, ip) injection.