Nociceptin/orphanin FQ (N/OFQ) was recently identified as the endogenous ligand for the opioid-receptor like (ORL1) receptor. Although the ORL1 receptor shows sequence homology with the opioid receptors, the nociceptin/ORL1 ligand–receptor system has very distinct pharmacological actions compared to the opioid receptor system. Recently, several small-molecule ORL1 receptor ligands were reported by pharmaceutical companies. Most of these ligands had close structural similarities with known neuroleptics and opiates. In this study, we screened several available neuroleptics and opiates for their binding affinity and functional activity at ORL1 and the opioid receptors. We also synthesized several analogs of known opiates with modified piperidine N-substituents in order to characterize the ORL1 receptor ligand binding pocket. Substitution with the large, lipophilic cyclooctylmethyl moiety increased ORL1 receptor affinity and decreased μ receptor affinity and efficacy in the fentanyl series of ligands but had a different effect in the oripavine class of opiate ligands. Our results indicate that opiates and neuroleptics may be good starting points for ORL1 receptor ligand design, and the selectivity may be modulated by appropriate structural modifications.
Opioid receptor; ORL1 receptor; Nociceptin/orphanin FQ; Structure activity; [35S]GTPγS binding
Obesity contributes to a multitude of serious health problems. Given the demonstrated role of the endogenous cannabinoid system in appetite regulation, the purpose of the present study was to evaluate structural analogs of two cannabinoids, rimonabant (cannabinoid CB1 receptor antagonist) and O-2050 (sulfonamide analog of Δ8-tetrahydrocannabinol), that showed appetite suppressant effects in previous studies. Structure–activity relationships of these two lead compounds were examined in several assays, including cannabinoid CB1 and CB2 receptor binding, food intake, and an in vivo test battery (locomotor activity, antinociception, ring immobility, and body temperature) in mice. Rimonabant and O-2050 reliably decreased feeding in mice; however, their analogs decreased feeding only at higher doses, even though some compounds had quite good cannabinoid CB1 binding affinity. Results of the in vivo test battery were inconsistent, with some of the compounds producing effects characteristic of cannabinoid agonists while other compounds were inactive or were antagonists against an active dose of Δ9-tetrahydrocannabinol. These results demonstrate that reduction of food intake is not a characteristic effect of pyrazole and sulfonamide cannabinoid analogs with favorable cannabinoid CB1 binding affinity, suggesting that development of these classes of cannabinoids for the treatment of obesity will require evaluation of their effects in a broad spectrum of pharmacological assays.
Antagonist; Cannabinoid; Feeding; Hemopressin; Pyrazole; Rimonabant; Sulfonamide; (Mouse)
The effect of chronic citalopram or escitalopram administration on 5-HT1A receptor function in the dorsal raphe nucleus was determined by measuring [35S]GTPγS binding stimulated by the 5-HT1A receptor agonist (R)-(+)-8-OH-DPAT (1nM-10μM). Although chronic administration of citalopram or escitalopram has been shown to desensitize somatodendritic 5-HT1A autoreceptors, we found that escitalopram treatment decreased the efficacy of 5-HT1A receptors to activate G-proteins, whereas citalopram treatment did not. The binding of [3H]8-OH-DPAT to the coupled, high affinity agonist state of the receptor was not altered by either treatment. Interestingly, escitalopram administration resulted in greater occupancy of serotonin transporter sites as measured by the inhibition of [3H]cyanoimipramine binding. As the binding and action of escitalopram is limited by the inactive enantiomer R-citalopram present in racemic citalopram, we propose that the regulation of 5-HT1A receptor function in the dorsal raphe nucleus at the level of receptor-G protein interaction may be a result of greater inhibition of the serotonin transporter by escitalopram.
quantitative autoradiography; serotonin-1A; citalopram; escitalopram; [35S]GTPγS binding; [3H]8-OH-DPAT
γ-Hydroxybutyrate (GHB) is a drug of abuse with multiple mechanisms of action. Consistent with its ability to modulate dopaminergic systems, GHB reportedly shares behavioral effects with neuroleptics and interacts with them in a synergistic manner. Here, we examined the ability of GHB and haloperidol to induce catalepsy and to affect operant responding. When given alone, both compounds induced catalepsy and decreased response rate. When given together, however, they produced these effects in an additive manner. This is further evidence that GHB has neuroleptic-like effects, but suggests that GHB interacts additively, not synergistically, with neuroleptics. The mechanisms involved in GHB- and haloperidol-induced catalepsy are different because the N-methyl-D-aspartate (NMDA) receptor antagonist, dizocilpine (MK-801), attenuated the cataleptic effects of haloperidol, but enhanced those of GHB. The latter finding suggests that other NMDA receptor antagonists (e.g., the drugs of abuse—phencyclidine and ketamine) may also interact synergistically with GHB.
GHB (γ-hydroxybutyrate); Haloperidol; Dizocilpine; Catalepsy; Synergism
Methamphetamine is currently one of the most widely abused drugs worldwide, with hyperthermia being a leading cause of death in methamphetamine overdose situations. Methamphetamine-induced hyperthermia involves a variety of cellular mechanisms, including increases in hypothalamic interleukin-1 beta (IL-1β) expression. Methamphetamine also interacts with sigma receptors and previous studies have shown that sigma receptor antagonists mitigate many of the behavioral and physiological effects of methamphetamine, including hyperthermia. The purpose of the current study was to determine if the attenuation of methamphetamine-induced hyperthermia by the sigma receptor antagonists, AZ66 and SN79, is associated with a concomitant attenuation of IL-1β mRNA expression, particularly in the hypothalamus. Methamphetamine produced doseand time-dependent increases in core body temperature and IL-1β mRNA expression in the hypothalamus, striatum, and cortex in male, Swiss Webster mice. Pretreatment with the sigma receptor antagonists, AZ66 and SN79, significantly attenuated methamphetamine-induced hyperthermia, but further potentiated IL-1β mRNA in the mouse hypothalamus when compared to animals treated with methamphetamine alone. These findings suggest sigma receptor antagonists attenuate methamphetamine-induced hyperthermia through a different mechanism from that involved in the modulation of hypothalamic IL-1β mRNA expression.
Hyperthermia; Hypothalamus; Interleukin-1β; Methamphetamine; Sigma Receptor
This review selectively explores some areas of pain research that, until recently, have been poorly understood. We have chosen four topics that relate to clinical pain and we discuss the underlying mechanisms and related pathophysiologies contributing to these pain states. A key issue in pain medicine involves crucial events and mediators that contribute to normal and abnormal pain signaling, but remain unseen without genetic, biomarker or imaging analysis. Here we consider how the altered genetic make-up of familial pains reveals the human importance of channels discovered by preclinical research, followed by the contribution of receptors as stimulus transducers in cold sensing and cold pain. Finally we review recent data on the neuro-immune interactions in chronic pain and the potential targets for treatment in cancer-induced bone pain.
Valproic acid (valproate), an anticonvulsant and a mood stabilizer, is a potent histone deacetylase inhibitor and a widely utilized pharmacological tool for neuroepigenetic research including DNA methylation. However, only nuclear but not mitochondrial DNA (mtDNA) has been investigated for the effects of valproate on the formation of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC). Using mouse 3T3-L1 cells, we investigated the effects of short (1 day) and prolonged (3 days) valproate treatment on global mtDNA 5mC content, global and mtDNA sequence-specific 5hmC content, mRNA levels for ten-eleven-translocation (TET) enzymes involved in 5hmC formation, and the mitochondrial content of TET proteins. Only 5hmC but not 5mC content in mtDNA was affected (decreased) by valproate, and only after the prolonged treatment. This action of valproate was mimicked by MS-275, a class I histone deacetylase inhibitor. The prolonged but not the short valproate treatment decreased the expression of Tet1 mRNA and reduced the mitochondrial content of the TET1 protein. Hence, a likely scenario for a valproate-induced 5hmC decrease in mtDNA may involve nuclear histone deacetylase inhibition (mitochondria do not contain histones) causing the initial increase of Tet1 transcription, which is followed by a delayed compensatory decrease of Tet1 expression and a reduced presence of TET1 protein in mitochondria. Further research is needed to elucidate the functional implications of epigenetic modifications of mtDNA. The observed effects of valproate on mitochondrial epigenetics may have implications for a better understanding of both therapeutic and unwanted effects of this drug and possibly other histone deacetylase inhibitors.
5-Methylcytosine (5mC); 5-Hydroxymethylcytosine (5hmC); Mitochondrial DNA (mtDNA); Valproic acid (valproate); Ten-eleven-translocation (TET); DNA methyltransferase 1 (DNMT1); Epigenetics
Selective inhibitors of sodium-glucose cotransporter 2 (SGLT2)-mediated reabsorption of glucose in the proximal tubule of the kidney are being developed for the treatment of diabetes. SGLT2 shares high degree of homology with SGLT3; however, very little is known about the expression and functional role of SGLT3 in the human kidney. Indeed, the SGLT2 inhibitors that are currently in clinical trials might affect the expression and/or the activity of SGLT3. Therefore, the present study examined the expression of SGLT3 mRNA and protein in human kidney and in a human proximal tubule HK-2 cell line. The results indicated that human SGLT3 (hSGLT3) message and protein are expressed both in vivo and in vitro. We also studied the activity of hSGLT3 protein following its over-expression in mammalian kidney-derived COS-7 cells and in HK-2 cells treated with the imino sugar deoxynojirimycin (DNJ), a potent agonist of hSGLT3. Over-expression of hSGLT3 in COS-7 cells increased intracellular sodium concentration by 3-fold without affecting glucose transport. Activation of hSGLT3 with DNJ (50 μM) increased sodium uptake in HK-2 cells by 5.5 fold and this effect could be completely blocked with SGLT inhibitor phlorizin (50 μM). These results suggest that SGLT3 is expressed in human proximal tubular cells where it serves as a novel sodium transporter. Up-regulation of the expression of SGLT3 in the proximal tubule in diabetic patients may contribute to the elevated sodium transport in this segment of the nephron that has been postulated to promote hyperfiltration and renal injury.
Human kidney; HK-2 cells; Proximal tubule; SGLT3; Sodium; Glucose
The dopamine transporter removes the neurotransmitter from the synapse, regulating dopamine availability. The transporter can be internalized and its function is blocked by cocaine and other ligands. Melittin inhibits dopamine transporter function and causes internalization of the recombinant transporter in stably transfected HEK-293 cells, but the specific pathways for internalization and disposition of the transporter are unknown. Here we report that melittin treatment increased both transporter internalization and colocalization with clathrin, effects that were blocked by pretreatment with cocaine. Density gradient centrifugation revealed that melittin treatment caused the dopamine transporter to associate with a density fraction containing the early endosome marker Rab 5A. Confocal microscopy revealed that melittin treatment also increased transporter colocalization with Rab 5A and decreased colocalization with the late endosome marker Rab 7 and the recycling endosome marker Rab 11. Following 60 min of melittin treatment, the transporter was trafficked back to the membrane. By comparison, phorbol ester treatment increased transporter colocalization with early endosome antigen 1 and Rab 7 in a time-dependent manner. Cocaine treatment alone does not affect transporter trafficking in these cells. Results indicate multiple dopamine transporter internalization and recycling pathways that depend on transporter-ligand interactions and post-translational modifications.
Dopamine transporter; Melittin; Trafficking; Protein kinase
Short application of the volatile anesthetic isoflurane at reperfusion after ischemia exerts strong protection of the heart against injury. Mild depolarization and acidification of the mitochondrial matrix are involved in the protective mechanisms of isoflurane, but the molecular basis for these changes is not clear. In this study, mitochondrial respiration, membrane potential, matrix pH, matrix swelling, ATP synthesis and -hydrolysis, and H2O2 release were assessed in isolated mitochondria. We hypothesized that isoflurane induces mitochondrial depolarization and matrix acidification through direct action on both complex I and ATP synthase. With complex I-linked substrates, isoflurane (0.5 mM) inhibited mitochondrial respiration by 28±10%, and slightly, but significantly depolarized membrane potential and decreased matrix pH. With complex II- and complex IV-linked substrates, respiration was not changed, but isoflurane still decreased matrix pH and depolarized mitochondrial membrane potential. Depolarization and matrix acidification were attenuated by inhibition of ATP synthase with oligomycin, but not by inhibition of mitochondrial ATP- and Ca2+-sensitive K+ channels or uncoupling proteins. Isoflurane did not induce matrix swelling and did not affect ATP synthesis and hydrolysis, but decreased H2O2 release in the presence of succinate in an oligomycin- and matrix pH-sensitive manner. Isoflurane modulated H+ flux through ATP synthase in an oligomycin-sensitive manner. Our results indicate that isoflurane-induced mitochondrial depolarization and acidification occur due to inhibition of the electron transport chain at the site of complex I and increased proton flux through ATP synthase. K+ channels and uncoupling proteins appear not to be involved in the direct effects of isoflurane on mitochondria.
isoflurane; mitochondria; mitochondrial potassium channels; uncoupling proteins; ATP-synthase
Chronic inflammation in lung diseases contributes to lung tissue destruction leading to the formation of chemotactic collagen fragments such as N-acetylated Proline–Glycine–Proline (N-ac-PGP). In this study, we investigated in more detail the mechanism of action of N-ac-PGP in neutrophilic inflammation. N-ac-PGP was chemotactic for human neutrophils via pertussis toxin sensitive G protein-coupled receptors in vitro and directly activated this cell type, which led to cytosolic calcium mobilization and release of CXCL8. Furthermore, using a selective CXCR2 antagonist confirmed that N-ac-PGP-induced neutrophil chemotaxis is mediated through CXCR2 activation. To determine whether N-ac-PGP was solely responsible for the migration and activation of human neutrophils in vitro and not the released CXCL8 upon stimulation with N-ac-PGP, an antibody directed against CXCL8 was used. Performing chemotaxis and calcium influx assays in the presence of this antibody did not alter the effects of N-ac-PGP whereas effects of CXCL8 were attenuated. These experiments indicate that N-ac-PGP, in addition to the direct induction of chemotaxis, also directly activates neutrophils to release CXCL8. In vivo, this may lead in the long term to a self-maintaining situation enhanced by both N-ac-PGP and CXCL8, leading to a further increase in neutrophil infiltration and chronic inflammation.
N-ac-PGP; Chemotaxis; Neutrophil; Chronic airway inflammation; CXCL8
Opiate abuse reportedly can exaggerate complications of human immunodeficiency virus type-1 (HIV-1) infection in the central nervous system (CNS), while opiate drugs are often indicated in the treatment of HIV-1-related neuropathic pain. Despite this quandary, few studies have assessed the relationship between the duration or extent of HIV-1 infection and the intrinsic neurobehavioral responsiveness to opioids. To address this problem, doxycycline (DOX)-inducible HIV-Tat1-86 transgenic mice were used as a model for HIV-1-associated neurocognitive disorders, which permitted the regulation of Tat exposure and duration. The effects of continuous Tat induction on the activity of morphine were examined at weekly intervals using standard behavioral assays for nociception and motor function. In the spinal cord, Tat mRNA levels did not increase until the second and third weeks following induction, which corresponded to a significant loss of morphine antinociception as assessed in the tail-flick test. Alternatively, in the striatum, sustained increases in Tat mRNA expression during the second week of induction coincided with significant decreases in rotarod performance and interactions with morphine. Importantly, the behavioral effects of morphine differed depending on the timing and location of Tat expression; with increases in Tat transcript levels in the spinal cord and striatum corresponding to significant alterations in morphine-dependent nociception and rotarod performance, respectively. Assuming Tat levels contribute to the clinical manifestations of HIV-1, the results suggest that regional differences in viral load and opioid phenotype might influence the nature and degree that opiate responsiveness is altered in HIV-1 infected individuals.
Neuro-acquired immunodeficiency syndrome (neuroAIDS); opioid drug abuse; nociception; spinal cord; striatum
Cholecystokinin (CCK) is one of the most abundant neuropeptides in the brain where it interacts with two G protein-coupled receptors (CCK1 and CCK2). Both types of CCK receptors are coupled to Gq/11 proteins resulting in increased function of phospholipase C (PLC) pathway. Whereas CCK has been suggested to increase neuronal excitability in the brain via activation of cationic channels, the types of cationic channels have not yet been identified. Here, we co-expressed CCK2 receptors and TRPC5 channels in human embryonic kidney (HEK) 293 cells and studied the effects of CCK on TRPC5 channels using patch-clamp techniques. Our results demonstrate that activation of CCK2 receptors robustly potentiates the function of TRPC5 channels. CCK-induced activation of TRPC5 channels requires the functions of G-proteins and PLC and depends on extracellular Ca2+. The activation of TRPC5 channels mediated by CCK2 receptors is independent of IP3 receptors and protein kinase C. CCK-induced opening of TRPC5 channels is not store-operated because application of thapsigargin to deplete intracellular Ca2+ stores failed to alter CCK-induced TRPC5 channel currents significantly. Bath application of CCK also significantly increased the open probability of TRPC5 single channel currents in cell-attached patches. Because CCK exerts extensive effects in the brain, our results may provide a novel mechanism to explain its roles in modulating neuronal excitability.
cholecystokinin; G protein; protein kinase; channel; calcium; TRP
Folic acid enhances endothelial function and improves outcome in primary prevention of cardiovascular disease. The exact intracellular signalling mechanisms involved remain elusive and were therefore the subject of this study. Particular focus was placed on folic acid-induced changes in posttranslational modifications of endothelial nitric oxide synthase (eNOS). Cultured endothelial cells were exposed to folic acid in the absence or presence of phosphatidylinositol-3' kinase/Akt (PI3K/Akt) inhibitors. The phosphorylation status of eNOS was determined via western blotting. The activities of eNOS and PI3K/Akt were evaluated. The interaction of eNOS with caveolin-1, Heat-Shock Protein 90 and calmodulin was studied using co-immunoprecipitation. Intracellular localisation of eNOS was investigated using sucrose gradient centrifugation and confocal microscopy. Folic acid promoted eNOS dephosphorylation at negative regulatory sites, and increased phosphorylation at positive regulatory sites. Modulation of phosphorylation status was concomitant with increased cGMP concentrations, and PI3K/Akt activity. Inhibition of PI3K/Akt revealed specific roles for this kinase pathway in folic acid-mediated eNOS phosphorylation. Regulatory protein and eNOS protein associations were altered in favour of a positive regulatory effect in the absence of bulk changes in intracellular eNOS localisation. Folic acid-mediated eNOS activation involves the modulation of eNOS phosphorylation status at multiple residues and positive changes in important protein–protein interactions. Such intracellular mechanisms may in part explain improvements in clinical vascular outcome following folic acid treatment.
Vascular biology; Endothelium; Folic acid; Nitric oxide synthase; Kinase; Phosphorylation
The effects of endomorphin-2 or endomorphin-1 microinjected into the centromedial amygdala on the thermally induced tail-flick response were studied in male CD rats. Microinjection of endomorphin-2 (8.7–35.0 nmol) given into the centromedial amygdala time- and dose-dependently decreased the tail-flick latencies. On the other hand, endomorphin-1 (8–32.6 nmol) given into the same site did not cause any change of the tail-flick latency. However, endomorphin-1 (32.6 nmol) or endomorphin-2 (35.0 nmol) given into the basolateral site of amygdala did not affect the tail-flick latency. Pretreatment with the antiserum against dynorphin A(1–17) (200 µg) significantly reversed the decrease of the tail-flick latency induced by endomorphin-2. The decrease of the tail-flick latency induced by endomorphin-2 was also blocked by the endomorphin-2 selective μ-opioid receptor antagonist 3-methoxynaltrexone (6.4 pmol) and by the Nmethyl-D-aspartate (NMDA) receptor antagonist MK-801 (30 nmol), but not by the κ-opioid receptor antagonist nor-binaltorphimine (6.6 nmol). It is concluded that endomorphin-2, but not endomorphin-1, given into the centromedial amygdala stimulates a 3-methoxynaltrexone-sensitive μ-opioid receptor subtype to induce the release of dynorphin A(1–17), which then acts on the NMDA receptor, but not κ-opioid receptor for producing hyperalgesia. This conclusion is further supported by the additional findings that dynorphin A(1–17) (2.3 nmol) given into the centromedial amygdala also caused the decrease of the tail-flick latency, which was similarly blocked by the NMDA receptor antagonist MK-801 (30 nmol), but not κ-opioid receptor antagonist nor-binaltorphimine (6.6 nmol).
opioid; hyperalgesia; amygdala; brain; rat
We have previously reported that selective blockade of brain dopamine D3 receptors by SB-277011A significantly attenuates cocaine self-administration and cocaine-induced reinstatement of drug-seeking behavior. In the present study, we investigated whether SB-277011A similarly inhibits methamphetamine self-administration and methamphetamine-induced reinstatement to drug-seeking behavior. Male Long–Evans rats were allowed to intravenously self-administer methamphetamine (0.05 mg/kg/infusion) under fixed-ratio 2 (FR2) or progressive-ratio (PR) reinforcement conditions, and some rats were tested for methamphetamine-induced reinstatement of drug-seeking behavior after extinction of self-administration. The effects of SB-277011A on each of these methamphetamine-supported behaviors were then tested. Acute intraperitoneal (i.p.) administration of SB-277011A failed to alter methamphetamine self-administration under FR2 reinforcement, but significantly lowered the break-point for methamphetamine self-administration under PR reinforcement. SB-277011A also significantly inhibited methamphetamine-triggered reinstatement of extinguished drug-seeking behavior. Overall, these data show that blockade of dopamine D3 receptors by SB-277011A attenuates the rewarding and incentive motivational effects of methamphetamine in rats, supporting the development of selective dopamine D3 antagonists for the treatment of methamphetamine addiction.
Methamphetamine; Dopamine D3 receptor; SB-277011A; Self-administration; Reward; Reinstatement
The study of the binding characteristics of σ ligands in vivo and in vitro requires radiolabeled probes with high affinity and selectivity. The radioligand presently used for in vitro studies of the σ1 receptor, [3H](+)-pentazocine, has significant limitations; it is difficult to synthesize, has limited chemical stability, and can be problematic to obtain. Evaluation of a series of novel 2(3H)-benzothiazolone compounds revealed SN56 to have subnanomolar and preferential affinity for the σ1 subtype, relative to σ2 and non-sigma, binding sites. The goal of this study was to characterize the binding of [3H]-SN56 to σ1 receptors isolated from rat brain. Standard in vitro binding techniques were utilized to 1) determine the specificity and affinity of binding to σ1 receptors, 2) confirm that [3H]-SN56 labels sites previously identified as σ1 by comparing binding to sites labeled by [3H](+)-pentazocine, and 3) characterize the kinetics of binding. The results indicate that [3H]-SN56 exhibits 1) specific, saturable, and reversible binding to the σ1 receptor, with Bmax = 340 ± 10 fmol/mg and Kd = 0.069 ± 0.0074 nM, 2) competitive displacement by classical sigma compounds, yielding σ1 Ki values consistent with those reported in the literature, and 3) binding kinetics compatible with a 90 min incubation, and filtration for separation of free and bound radioligand. The results of these studies suggest that [3H]-SN56 may serve as a viable alternative to [3H](+)-pentazocine in radioligand binding assays.
Radioligand Binding Assay; σ Receptor
Brain-derived neurotrophic factor (BDNF), one of the key brain neurotrophins, has been implicated in neuronal plasticity and memory. Recent studies document the importance of BDNF for normal long-term memory functions. However, there are few studies of the roles of BDNF in short term memory. Dopamine is likely to play important roles in BDNF gene expression in specific brain regions, including frontal cortical regions that are implicated in short term working memory processes that include spontaneous alternation. We have thus tested spatial working memory in dopamine transporter knockout (DAT KO) and wild-type mice. Spontaneous alternation in the Y-maze, an index of short-term spatial working memory in mice, was significantly decreased in DAT KO mice compared to wildtype mice. BDNF protein was significantly decreased in frontal cortex, though not in striatum or hippocampus, of the DAT KO mice. The data support the hypothesis that impaired spatial working memory in DAT KO mice may be related to decreased frontal cortical BDNF in these animals, and document apparent roles for BDNF in a short term memory process.
dopamine transporter knock out (DAT KO) mouse; Brain-derived neurotrophic factor (BDNF); Y-maze; frontal cortex
We have previously demonstrated that (+)-morphine and (−)-morphine pretreated spinally for 45 min stereoselectively attenuates the tail-flick inhibition produced by (−)-morphine given spinally in the mouse. The present study is then undertaken to determine if the same phenomenon observed in the mouse spinal cord can also take place in the ventral periaqueductal gray of the rat. Pretreatment with (+)-morphine for 45 min at 0.3 to 3.3 fmol dose-dependently attenuated the tail-flick inhibition produced by (−)-morphine (9 nmol) given into the ventral periaqueductal gray. Likewise, pretreatment with (−)-morphine for 45 min at a higher dose (3-900 pmol), which given alone did not affect the baseline tail-flick latency, also dose-dependently attenuated the tail-flick inhibition produced by (−)-morphine. Thus, (+)-morphine is approximately 270,000-fold more potent than (−)-morphine in attenuating the (−)-morphine-produced tail-flick inhibition. The attenuation of the (−)-morphine-produced tail-flick inhibition induced by (+)-morphine or (−)-morphine was dose-dependently reversed by (+)-naloxone (27.5 to 110 pmol) pretreatment for 50 min given into the ventral periaqueductal gray. Pretreatment with the sigma receptor antagonist BD1047 (N-[2-(3,4-Dichlorophenyl)ethyl]-N-methyl-2-(dimethylamino)ethylamine dihydrobromide) (11-45 nmol) for 45 min given into the ventral periaqueductal gray also reversed dose-dependently the attenuation of the (−)-morphine-produced tail-flick inhibition induced by (+)-morphine or (−)-morphine, indicating that the effects are mediated by the activation of the sigma receptors. Since (+)-morphine, (−)-morphine and (+)-naloxone do not have any affinity for the naloxone-inaccessible sigma receptors, we therefore propose that (+)-morphine and (−)-morphine attenuate the (−)-morphine-produced tail-flick inhibition via the activation of the naloxone-sensitive sigma receptor originally proposed by Tsao and Su (1997).
sigma receptors; analgesia; opioid; morphine; periaqueductal gray; rat
Endogenous renal dopamine is a major physiological regulator of renal ion transport; however its intracellular signaling pathways are not thoroughly understood. The present study examined the role of 20-hydroxyeicosatetraenoic acid (20-HETE), the major cytochrome P450 (CYP4A) metabolite of arachidonic acid formed in the renal cortex, on the natriuretic response to dopamine in Sprague Dawley rats. Infusion of dopamine (1.5 ug/kg/min, i.v.) increased urine flow (1.9 fold over basal), sodium excretion (UNaV, 2.7 fold), fractional sodium excretion (FENa, 3.3 fold) and proximal and distal delivery of sodium by 1.5 and 2-fold respectively. Administration of two inhibitors of the synthesis of 20-HETE, 1-aminobenzotriazole (ABT) and N-hydroxy-N’-(-4-butyl-2-methylphenyl)formamidine (HET0016) reduced the response to dopamine by 65%. Induction of the renal expression of CYP4A enzymes with clofibrate did not alter the response to dopamine. The natriuretic response to dopamine was lower in Dahl salt-sensitive rats in comparison to an SS.BN5 consomic strain in which transfer of chromosome 5 from Brown Norway to Dahl salt-sensitive rats upregulates the renal expression of CYP4A protein and the production of 20-HETE. Treatment with HET0016 blocked the renal effects of dopamine in SS.BN5 rats. We also examined the influence of 20-HETE in the natriuretic response to acute volume expansion that is in part mediated via the release of endogenous dopamine. The increase in urine flow, UNaV, FENa and distal FENa following volume expansion was markedly reduced in rats treated with ABT. These results suggest that 20-HETE plays at least a permissive role in the natriuretic response to dopamine.
dopamine; 20-HETE; CYP4A; sodium excretion
We have previously shown that β-endorphin plays a functional role in the rewarding effect of acute cocaine. Considering that β-endorphin has high affinity for the μ opioid receptor, we determined the role of this receptor in the rewarding action of acute cocaine. For comparison, we assessed the role of the μ opioid receptor in the rewarding effect of acute morphine. We also examined the effect of intracerebroventricular (i.c.v.) administration of β-funaltrexamine (β-FNA), an irreversible μ opioid receptor antagonist, on the rewarding action of acute cocaine as well as that of morphine. Using the conditioned place preference (CPP) paradigm as an animal model of reward, we first assessed the rewarding action of cocaine in mice lacking β-endorphin or the μ opioid receptor and their respective wild-type littermates/controls. Mice were tested for preconditioning place preference on day 1, conditioned once daily with saline/cocaine (30 mg/kg, i.p.) or cocaine/saline on days 2 and 3, and then tested for postconditioning place preference on day 4. We next studied the rewarding action of acute morphine in μ knockout mice and their wild-type controls. The CPP was induced by single alternate-day saline/morphine (10 mg/kg, s.c.) or morphine/saline conditioning. We finally determined the effect of β-FNA on CPP induced by cocaine or morphine in wild-type mice, in which mice were treated with saline or β-FNA (9ug/3 μl; i.c.v.) a day prior to the preconditioning test day. Our results revealed that morphine induced a robust CPP in wild-type mice but not in mice lacking the μ opioid receptor or in wild-type mice treated with β-FNA. In contrast, cocaine induced CPP in μ knockout mice as well as in wild-type mice treated with β-FNA. On the other hand, cocaine failed to induce CPP in mice lacking β-endorphin. These results illustrate that β-endorphin is essential for the rewarding action of acute cocaine, but the μ opioid receptor may not mediate the regulatory action of endogenous β-endorphin.
Cocaine; Morphine; β-FNA; Conditioned Place Preference (CPP); β-endorphin/μ opioid receptor system; Knockout mouse
HM30181, a potent and selective inhibitor of the adenosine triphosphate-binding cassette transporter P-glycoprotein (Pgp), was shown to enhance oral bioavailability and improve antitumour efficacy of paclitaxel in mouse tumour models. In search for a positron emission tomography (PET) radiotracer to visualise Pgp expression levels at the blood-brain barrier (BBB), we examined the ability of HM30181 to inhibit Pgp at the murine BBB. HM30181 was shown to be approximately equipotent with the reference Pgp inhibitor tariquidar in inhibiting rhodamine 123 efflux from CCRF-CEM T cells (IC50, tariquidar: 8.2±2.0 nM, HM30181: 13.1±2.3 nM). PET scans with the Pgp substrate (R)-[11C]verapamil in FVB wild-type mice pretreated i.v. with HM30181 (10 or 21 mg/kg) failed to show significant increases in (R)-[11C]verapamil brain uptake compared with vehicle treated animals. PET scans with [11C]HM30181 showed low and not significantly different brain uptake of [11C]HM30181 in wild-type, Mdr1a/b(−/−) and Bcrp1(−/−) mice and significantly, i.e. 4.7-fold (P<0.01), higher brain uptake, relative to wild-type animals, in Mdr1a/b(−/−)Bcrp1(−/−) mice. This was consistent with HM30181 being at microdoses a dual substrate of Pgp and breast cancer resistance protein (Bcrp). In vitro autoradiography on low (EMT6) and high (EMT6Ar1.0) Pgp expressing murine breast tumour sections showed 1.9 times higher binding of [11C]HM30181 in EMT6Ar1.0 tumours (P<0.001) which was displaceable with unlabelled tariquidar, elacridar or HM30181 (1 μM). Our data suggest that HM30181 is not able to inhibit Pgp at the murine BBB at clinically feasible doses and that [11C]HM30181 is not suitable as a PET tracer to visualise cerebral Pgp expression levels.
P-glycoprotein; blood-brain barrier; HM30181; tariquidar; positron emission tomography; microdosing
A study was made to determine if constitutively active adenosine receptors are present at mouse motor nerve endings. In preparations blocked by low Ca2+ / high Mg2+ solution, 8-cyclopentyl-1,3,dipropylxanthine (CPX, 10–100 nM), which has been reported to be both an A1 adenosine receptor antagonist and inverse agonist, produced a dose-dependent increase in the number of acetylcholine quanta released by a nerve impulse. Adenosine deaminase, which degrades ambient adenosine into its inactive congener, inosine, failed to alter the response to 100 nM CPX. 8-cyclopentyltheophylline (CPT, 3 μM), a competitive inhibitor at A1 adenosine receptors, prevented the increase in acetylcholine release produced by CPX. At normal levels of acetylcholine release, neither adenosine deaminase nor CPX affected acetylcholine release at low frequencies of nerve stimulation in (+)-tubocurarine blocked preparations. The results suggest that a proportion of the acetylcholine release process is controlled by constitutively active adenosine receptors at murine motor nerve endings, providing the first evidence for constitutive activity of G-protein-coupled receptors that modulate the function of mammalian nerve endings.
G-protein coupled receptors; constitutive activity; A1 adenosine receptor; neuromuscular junction; end-plate potential; neurotransmitter release
Drawbacks to opioid use include development of analgesic tolerance and persistent constipation. We previously reported that tolerance to morphine develops upon repeated exposure in the isolated ileum but not the isolated colon. The cellular mechanisms of antinociceptive tolerance vary among μ-opioid receptor agonists. In this study, we assess β-arrestin2 deletion on the development of tolerance to different opioids in ileum and colon circular muscle. Tolerance was determined by assessing the ability of repeated in-vitro opioid exposure to induce contraction of the circular muscle from C57BL/6 wild type (WT) and β-arrestin2 knockout (KO) mice. Repeated exposure every 30 min with in-between washes resulted in tolerance to all agonists in the ileum of both WT and KO mice. However, in the colon of WT mice, comparison of the contractions between the 4th exposure and 1st response was similar to DAMGO (100 ± 10%; N = 5) but reduced to fentanyl (62 ± 13%; N = 8) and etorphine (38 ± 4%; N = 7) indicative of tolerance to fentanyl and etorphine but not DAMGO. In contrast, all agonists produced tolerance in the colon of KO: DAMGO response at the 4th exposure decreased to 52 ± 10% (N = 5), fentanyl to 20 ± 5% (N = 6) and etorphine 33 ± 7% (N = 6). Differences in tolerance among opioid agonists in the colon suggest ligand bias. The deletion of β-arrestin2 in colon appears to be necessary for tolerance to DAMGO but not fentanyl or etorphine. β-arrestin2 potentially represents an important target for treating opioid-induced bowel dysfunction and warrants further exploration of its ligand bias.
μ-Opioid receptor; β-arrestin2; opioid; circular smooth muscle; tolerance
Prolonged morphine treatment increases pain sensitivity in many patients. Enhanced spinal Substance P release is one of the adaptive changes associated with sustained opioid exposure. In addition to pain transmitting second order neurons, spinal microglia and astrocytes also express functionally active Tachykinin NK1 (Substance P) receptors. In the present work we investigated the role of glial Tachykinin NK1 receptors in morphine withdrawal-mediated spinal microglia and astrocyte activation. Our data indicate that intrathecal co-administration (6 days, twice daily) of a selective Tachykinin NK1 receptor antagonist (N-acetyl-l-tryptophan 3,5-bis(trifluoromethyl)benzylester (L-732,138; 20 μg/injection) attenuates spinal microglia and astrocyte marker and pro-inflammatory mediator immunoreactivity as well as hyperalgesia in morphine-withdrawn rats. Furthermore, covalent linkage of the opioid agonist with a Tachykinin NK1 antagonist pharmacophor yielded a bivalent compound that did not augment spinal microglia or astrocyte marker or pro-inflammatory mediator immunoreactivity and did not cause paradoxical pain sensitization upon drug withdrawal. Thus, bivalent opioid/Tachykinin NK1 receptor antagonists may provide a novel paradigm for long-term pain management.
opioid-induced hyperalgesia; spinal glia; Tachykinin NK1 receptor; Tachykinin NK1 receptor antagonist