Extremely low-frequency electromagnetic field exposure during chronic morphine treatment strengthens downregulation of dopamine D2 receptors in rat dorsal hippocampus after morphine withdrawal

The aim of this study was to investigate the effect of extremely low-frequency electromagnetic field (ELF-EMF) exposure during morphine treatment on dopamine D2 receptor (D2R) density in the rat dorsal hippocampus following withdrawal. Rats were exposed t

Coupling between NMDA receptor and acid-sensing ion channel contributes to ischemic neuronal death

Acid-sensing ion channels (ASICs) composed of ASIC1a subunit exhibit a high Ca2+ permeability and play important roles in synaptic plasticity and acid-induced cell death. Here, we show that ischemia enhances ASIC currents through the phosphorylation at Ser478 and Ser479 of ASIC1a, leading to exacerbated ischemic cell death. The phosphorylation is catalyzed by Ca2+/calmodulin-dependent protein kinase II (CaMKII) activity, as a result of activation of NR2B-containing N-methyl-D-aspartate subtype of glutamate receptors (NMDARs) during ischemia. Furthermore, NR2B-specific antagonist, CaMKII inhibitor, or overexpression of mutated form of ASIC1a with Ser478 or Ser479 replaced by alanine (ASICla-S478A, ASIC1a-S479A) in cultured hippocampal neurons prevented ischemia-induced enhancement of ASIC currents, cytoplasmic Ca2+ elevation, as well as neuronal death. Thus, NMDAR-CaMKII cascade is functionally coupled to ASICs and contributes to acidotoxicity during ischemia. Specific blockade of NMDAR/CaMKII-ASIC coupling may reduce neuronal death after ischemia and other pathological conditions involving excessive glutamate release and acidosis.

POSTSYNAPTIC ALPHA-2 RECEPTOR STIMULATION IMPROVES MEMORY IN AGED MONKEYS - INDIRECT EFFECTS OF YOHIMBINE VERSUS DIRECT EFFECTS OF CLONIDINE

Very low doses (0.00001 mg/kg) of the alpha-2 adrenergic antagonist, yohimbine, improved working memory performance in a subset of aged monkeys. Improvement appeared to result from increased norepinephrine (NE) release onto postsynaptic alpha-2 adrenoceptors, as the response was blocked by the ''postsynaptic'' alpha-2 antagonist, SKF104078. Cognitive-enhancing effects of low dose yohimbine treatment may depend on aged animals retaining an intact, endogenous NE system. In contrast to yohimbine, the alpha-2 agonist, clonidine, has improved working memory in air aged animals examined. In the present study, clonidine's beneficial effects were also blocked by the postsynaptic antagonists SKF104078 and SKF104856, suggesting that clonidine acts by directly stimulating postsynaptic alpha-2 adrenoceptors. Beneficial doses of clonidine (0.01 mg/kg) and yohimbine (0.00001 mg/kg) were combined to see if they would produce additive effects on memory enhancement. This strategy was successful in young monkeys with intact NE systems but was not effective in the aged monkeys. These findings demonstrate that drugs that indirectly stimulate postsynaptic alpha-2 receptors by increasing NE release are not as reliable in aged monkeys as directly acting agonists that can replace NE at postsynaptic alpha-2 receptors.

Pharmacological and immunocytochemical investigation of the role of catecholamines on larval metamorphosis by beta-adrenergic-like receptor in the bivalve Meretrix meretrix

Catecholamines regulate several physiological processes in mollusks. Many pharmacological experiments have been conducted to determine the effects of adrenergic agonist and antagonist of catecholamine receptors on Meretrix meretrix metamorphosis. Results showed that adrenaline (AD) and noradrenaline (NA) had substantial effects (p < 0.05) on larval metamorphosis at concentrations ranging from 10 mu M to 100 mu M. 10 mu M beta-adrenergic receptor (AR) agonist isoproterenol showed the same inducement effect as that of NA and AD on metamorphosis, whereas the alpha-AR agonist phenylephrine had no significant effect at concentrations between 0.1 mu M and 100 mu M concentrations (p > 0.05). Furthermore, I mu M beta-AR antagonist propanolol, but not alpha-AR antagonist prazosin, depressed the larval metamorphosis induced by NA or AD. By immunocytochemistry, two cell bodies of beta-adrenergic-like receptor, C/A1, C/A2, were observed in the cerebral/apical ganglion of competent larvae. In addition, there were other immunoreactive dots near C/A1 and C/A2. The results of pharmacology and immunocytochemistry suggests that beta-adrenergic-like receptor located in the larval CNS, might play a considerable role in the larval metamorphosis of M meretrix by AD or NA. (c) 2006 Elsevier B.V. All rights reserved.

GABAA receptor chloride channels are involved in the neuroprotective role of GABA following oxygen and glucose deprivation in the rat cerebral cortex but not in the hippocampus.

Assays on "ex vivo" sections of rat hippocampus and rat cerebral cortex, subjected to oxygen and glucose deprivation (OGD) and a three-hour reperfusion-like (RL) recovery, were performed in the presence of either GABA or the GABA(A) receptor binding site antagonist, bicuculline. Lactate dehydrogenase (LDH) and propidium iodide were used to quantify cell mortality. We also measured, using real-time quantitative polymerase chain reaction (qPCR), the early transcriptional response of a number of genes of the glutamatergic and GABAergic systems. Specifically, glial pre- and post-synaptic glutamatergic transporters (namely GLAST1a, EAAC-1, GLT-1 and VGLUT1), three GABAA receptor subunits (α1, β2 and γ2), and the GABAergic presynaptic marker, glutamic acid decarboxylase (GAD65), were studied. Mortality assays revealed that GABAA receptor chloride channels play an important role in the neuroprotective effect of GABA in the cerebral cortex, but have a much smaller effect in the hippocampus. We also found that GABA reverses the OGD-dependent decrease in GABA(A) receptor transcript levels, as well as mRNA levels of the membrane and vesicular glutamate transporter genes. Based on the markers used, we conclude that OGD results in differential responses in the GABAergic presynaptic and postsynaptic systems.

Interazioni neurotrasmettitoriali nel meccanismo d’azione della cocaina: ruolo della serotonina e dell’adenosina

The putative 5-HT6 receptor agonist ST1936 has been shown to increase extracellular dopamine (DA) in the n.accumbens (NAc) Shell and in the medial prefrontal cortex (PFCX). These observations suggest that 5-HT6 receptors modulate DA transmission in mesolimbic and mesocortical terminal DA areas. To investigate the behavioral counterpart of this interaction I studied in rats the effect of 5-HT6 receptor blockade on cocaine stimulated overflow of DA in dialysates from the PFCX and from the NAc Shell and on cocaine i.v. selfadministration. Pretreatment with the 5-HT6 antagonist SB271046 reduced cocaine-induced increase of dialysate DA in the NAc Shell but not in the PFCX and impaired i.v. cocaine selfadministration. These suggest that 5-HT6 receptors play a role in cocaine reinforcement via their facilitatore interaction with DA projections to the NAc Shell. This 5-HT/DA interaction might provide the basis for a new pharmacotherapeutic strategy of cocaine addiction. Caffeine is one of the psychoactive substances most widely used as adulterant in illicit drugs, such as cocaine. Animal studies have demonstrated that caffeine is able to potentiate cocaine actions, although the enhancement of the cocaine reinforcing property by caffeine is less reported, and the results depend on the paradigms and experimental protocols used. In the present study I examined the ability of caffeine to enhance the motivational and rewarding properties of cocaine using the intravenous self-administration paradigm in rats. Additionally, the role of caffeine as a primer cue during extinction was evaluated. To this end, we assessed in naïve rats: 1) the ability of the combination of cocaine (0,125 mg/kg/infusion) and caffeine (0,0625 mg/kg/infusion) to maintain self-administration in fixed ratio (FR) and progressive ratio (PR) schedules of reinforcement compared with cocaine and caffeine alone; 2) the effect of caffeine in the maintenance of responding in the animals exposed to the combination of the drugs during cocaine extinction. Cocaine and the combination of cocaine and caffeine were self-administered on a FR and PR schedules of reinforcement, and the responding for the combination of the drugs was higher than cocaine alone. Caffeine was not reliably self-administered, but was able to maintain a drug-seeking behavior in rats previously exposed to cocaine plus caffeine. These findings suggest that the presence of caffeine enhances the reinforcing effects of cocaine and the motivational value of the drug. Our results highlight the role of active adulterants commonly used in illicit street drugs.

Targeting the EP1 receptor reduces Fas ligand expression and increases the antitumor immune response in an in vivo model of colon cancer

Despite studies demonstrating that inhibition of cyclooxygenase-2 (COX-2)-derived prostaglandin E2 (PGE2) has significant chemotherapeutic benefits in vitro and in vivo, inhibition of COX enzymes is associated with serious gastrointestinal and cardiovascular side effects, limiting the clinical utility of these drugs. PGE2 signals through four different receptors (EP1–EP4) and targeting individual receptor(s) may avoid these side effects, while retaining significant anticancer benefits. Here, we show that targeted inhibition of the EP1 receptor in the tumor cells and the tumor microenvironment resulted in the significant inhibition of tumor growth in vivo. Both dietary administration and direct injection of the EP1 receptor-specific antagonist, ONO-8713, effectively reduced the growth of established CT26 tumors in BALB/c mice, with suppression of the EP1 receptor in the tumor cells alone less effective in reducing tumor growth. This antitumor effect was associated with reduced Fas ligand expression and attenuated tumor-induced immune suppression. In particular, tumor infiltration by CD4+CD25+Foxp3+ regulatory T cells was decreased, whereas the cytotoxic activity of isolated splenocytes against CT26 cells was increased. F4/80+ macrophage infiltration was also decreased; however, there was no change in macrophage phenotype. These findings suggest that the EP1 receptor represents a potential target for the treatment of colon cancer.

Attenuation of inflammatory events in human intervertebral disc cells with a tumor necrosis factor antagonist.

STUDY DESIGN: The inflammatory responses of primary human intervertebral disc (IVD) cells to tumor necrosis factor α (TNF-α) and an antagonist were evaluated in vitro. OBJECTIVE: To investigate an ability for soluble TNF receptor type II (sTNFRII) to antagonize TNF-α-induced inflammatory events in primary human IVD cells in vitro. SUMMARY OF BACKGROUND DATA: TNF-α is a known mediator of inflammation and pain associated with radiculopathy and IVD degeneration. sTNFRs and their analogues are of interest for the clinical treatment of these IVD pathologies, although information on the effects of sTNFR on human IVD cells remains unknown. METHODS: IVD cells were isolated from surgical tissues procured from 15 patients and cultured with or without 1.4 nmol/L TNF-α (25 ng/mL). Treatment groups were coincubated with varying doses of sTNFRII (12.5-100 nmol/L). Nitric oxide (NO), prostaglandin E₂ (PGE₂), and interleukin-6 (IL6) levels in media were quantified to characterize the inflammatory phenotype of the IVD cells. RESULTS: Across all patients, TNF-α induced large, statistically significant increases in NO, PGE₂, and IL6 secretion from IVD cells compared with controls (60-, 112-, and 4-fold increases, respectively; P < 0.0001). Coincubation of TNF-α with nanomolar doses of sTNFRII significantly attenuated the secretion of NO and PGE₂ in a dose-dependent manner, whereas IL6 levels were unchanged. Mean IC₅₀ values for NO and PGE₂ were found to be 35.1 and 20.5 nmol/L, respectively. CONCLUSION: Nanomolar concentrations of sTNFRII were able to significantly attenuate the effects of TNF-α on primary human IVD cells in vitro. These results suggest this sTNFR to be a potent TNF antagonist with potential to attenuate inflammation in IVD pathology.

The dopamine metabolite 3-methoxytyramine is a neuromodulator.

Dopamine (3-hydroxytyramine) is a well-known catecholamine neurotransmitter involved in multiple physiological functions including movement control. Here we report that the major extracellular metabolite of dopamine, 3-methoxytyramine (3-MT), can induce behavioral effects in a dopamine-independent manner and these effects are partially mediated by the trace amine associated receptor 1 (TAAR1). Unbiased in vivo screening of putative trace amine receptor ligands for potential effects on the movement control revealed that 3-MT infused in the brain is able to induce a complex set of abnormal involuntary movements in mice acutely depleted of dopamine. In normal mice, the central administration of 3-MT caused a temporary mild hyperactivity with a concomitant set of abnormal movements. Furthermore, 3-MT induced significant ERK and CREB phosphorylation in the mouse striatum, signaling events generally related to PKA-mediated cAMP accumulation. In mice lacking TAAR1, both behavioral and signaling effects of 3-MT were partially attenuated, consistent with the ability of 3-MT to activate TAAR1 receptors and cause cAMP accumulation as well as ERK and CREB phosphorylation in cellular assays. Thus, 3-MT is not just an inactive metabolite of DA, but a novel neuromodulator that in certain situations may be involved in movement control. Further characterization of the physiological functions mediated by 3-MT may advance understanding of the pathophysiology and pharmacology of brain disorders involving abnormal dopaminergic transmission, such as Parkinson's disease, dyskinesia and schizophrenia.

Identification of beta-adrenergic receptors in human lymphocytes by (-) (3H) alprenolol binding.

Human lymphocytes are known to posessess a catecholamine-responsive adenylate cyclase which has typical beta-adrenergic specificity. To identify directly and to quantitate these beta-adenergic receptors in human lymphocytes, (-) [3H] alprenolol, a potent beta-adrenergic antagonist, was used to label binding sites in homogenates of human mononuclear leukocytes. Binding of (-) [3H] alprenolol to these sites demonstrated the kinetics, affinity, and stereospecificity expected of binding to adenylate cyclase-coupled beta-adrenergic receptors. Binding was rapid (t1/2 less than 30 s) and rapidly reversible (t1/2 less than 3 min) at 37 degrees C. Binding was a saturable process with 75 +/- 12 fmol (-) [3H] alprenolol bound/mg protein (mean +/- SEM) at saturation, corresponding to about 2,000 sites/cell. Half-maximal saturation occurred at 10 nM (-) [3H] alprenolol, which provides an estimate of the dissociation constant of (-) [3H] alprenolol for the beta-adrenergic receptor. The beta-adrenergic antagonist, (-) propranolol, potently competed for the binding sites, causing half-maximal inhibition of binding at 9 nM. beta-Adrenergic agonists also competed for the binding sites. The order of potency was (-) isoproterenol greater than (-) epinephrine greater than (-)-norepinephrine which agreed with the order of potency of these agents in stimulating leukocyte adenylate cyclase. Dissociation constants computed from binding experiments were virtually identical to those obtained from adenylate cyclase activation studies. Marked stereospecificity was observed for both binding and activation of adenylate cyclase. (-)Stereoisomers of beta-adrenergic agonists and antagonists were 9- to 300-fold more potent than their corresponding (+) stereoisomers. Structurally related compounds devoid of beta-adrenergic activity such as dopamine, dihydroxymandelic acid, normetanephrine, pyrocatechol, and phentolamine did not effectively compete for the binding sites. (-) [3H] alprenolol binding to human mononuclear leukocyte preparations was almost entirely accounted for by binding to small lymphocytes, the predominant cell type in the preparations. No binding was detectable to human erythrocytes. These results demonstrate the feasibility of using direct binding methods to study beta-adrenergic receptors in a human tissue. They also provide an experimental approach to the study of states of altered sensitivity to catecholamines at the receptor level in man.

Neuropathic pain activates the endogenous kappa opioid system in mouse spinal cord and induces opioid receptor tolerance.

Release of endogenous dynorphin opioids within the spinal cord after partial sciatic nerve ligation (pSNL) is known to contribute to the neuropathic pain processes. Using a phosphoselective antibody [kappa opioid receptor (KOR-P)] able to detect the serine 369 phosphorylated form of the KOR, we determined possible sites of dynorphin action within the spinal cord after pSNL. KOR-P immunoreactivity (IR) was markedly increased in the L4-L5 spinal dorsal horn of wild-type C57BL/6 mice (7-21 d) after lesion, but not in mice pretreated with the KOR antagonist nor-binaltorphimine (norBNI). In addition, knock-out mice lacking prodynorphin, KOR, or G-protein receptor kinase 3 (GRK3) did not show significant increases in KOR-P IR after pSNL. KOR-P IR was colocalized in both GABAergic neurons and GFAP-positive astrocytes in both ipsilateral and contralateral spinal dorsal horn. Consistent with sustained opioid release, KOR knock-out mice developed significantly increased tactile allodynia and thermal hyperalgesia in both the early (first week) and late (third week) interval after lesion. Similarly, mice pretreated with norBNI showed enhanced hyperalgesia and allodynia during the 3 weeks after pSNL. Because sustained activation of opioid receptors might induce tolerance, we measured the antinociceptive effect of the kappa agonist U50,488 using radiant heat applied to the ipsilateral hindpaw, and we found that agonist potency was significantly decreased 7 d after pSNL. In contrast, neither prodynorphin nor GRK3 knock-out mice showed U50,488 tolerance after pSNL. These findings suggest that pSNL induced a sustained release of endogenous prodynorphin-derived opioid peptides that activated an anti-nociceptive KOR system in mouse spinal cord. Thus, endogenous dynorphin had both pronociceptive and antinociceptive actions after nerve injury and induced GRK3-mediated opioid tolerance.

Direct evidence that Gi-coupled receptor stimulation of mitogen-activated protein kinase is mediated by G beta gamma activation of p21ras.

Stimulation of Gi-coupled receptors leads to the activation of mitogen-activated protein kinases (MAP kinases). In several cell types, this appears to be dependent on the activation of p21ras (Ras). Which G-protein subunit(s) (G alpha or the G beta gamma complex) primarily is responsible for triggering this signaling pathway, however, is unclear. We have demonstrated previously that the carboxyl terminus of the beta-adrenergic receptor kinase, containing its G beta gamma-binding domain, is a cellular G beta gamma antagonist capable of specifically distinguishing G alpha- and G beta gamma-mediated processes. Using this G beta gamma inhibitor, we studied Ras and MAP kinase activation through endogenous Gi-coupled receptors in Rat-1 fibroblasts and through receptors expressed by transiently transfected COS-7 cells. We report here that both Ras and MAP kinase activation in response to lysophosphatidic acid is markedly attenuated in Rat-1 cells stably transfected with a plasmid encoding this G beta gamma antagonist. Likewise in COS-7 cells transfected with plasmids encoding Gi-coupled receptors (alpha 2-adrenergic and M2 muscarinic), the activation of Ras and MAP kinase was significantly reduced in the presence of the coexpressed G beta gamma antagonist. Ras-MAP kinase activation mediated through a Gq-coupled receptor (alpha 1-adrenergic) or the tyrosine kinase epidermal growth factor receptor was unaltered by this G beta gamma antagonist. These results identify G beta gamma as the primary mediator of Ras activation and subsequent signaling via MAP kinase in response to stimulation of Gi-coupled receptors.

Structural basis of beta-adrenergic receptor subtype specificity studied with chimeric beta 1/beta 2-adrenergic receptors.

The beta 1- and beta 2-adrenergic receptors are two structurally related, but pharmacologically distinguishable, receptor subtypes, both of which activate adenylyl cyclase in a catecholamine-dependent manner through the guanine nucleotide-binding regulatory protein Gs. The receptors are approximately 50% identical in amino acid sequence and each is characterized by the presence of seven putative transmembrane domains. To elucidate the structural basis for the pharmacological distinctions between these two receptor subtypes, we constructed a series of chimeric beta 1/beta 2-adrenergic receptor genes and expressed them by injection of RNA into Xenopus laevis oocytes. The pharmacological properties of the expressed chimeric receptor proteins were assessed by radioligand binding and adenylyl cyclase assays utilizing subtype-selective agonists and antagonists. Our data indicate that transmembrane region IV is largely responsible for determining beta 1 vs. beta 2 properties with respect to agonist binding (relative affinities for epinephrine and norepinephrine). Transmembrane regions VI and VII play an important role in determining binding of beta 1 vs. beta 2 selective antagonists. However, a number of the other transmembrane regions also contribute, to a lesser extent, to the determination of beta-adrenergic receptor subtype specificity for agonists and antagonists. Thus, several of the membrane-spanning regions appear to be involved in the determination of receptor subtype specificity, presumably by formation of a ligand-binding pocket, with determinants for agonist and antagonist binding being distinguishable.

Beta-agonist- and prostaglandin E1-induced translocation of the beta-adrenergic receptor kinase: evidence that the kinase may act on multiple adenylate cyclase-coupled receptors.

beta-Adrenergic receptor kinase (beta-AR kinase) is a cytosolic enzyme that phosphorylates the beta-adrenergic receptor only when it is occupied by an agonist [Benovic, J. Strasser, R. H., Caron, M. G. & Lefkowitz, R. J. (1986) Proc. Natl. Acad. Sci. USA 83, 2797-2801.] It may be crucially involved in the processes that lead to homologous or agonist-specific desensitization of the receptor. Stimulation of DDT1MF-2 hamster smooth muscle cells or S49 mouse lymphoma cells with a beta-agonist leads to translocation of 80-90% of the beta-AR kinase activity from the cytosol to the plasma membrane. The translocation process is quite rapid, is concurrent with receptor phosphorylation, and precedes receptor desensitization and sequestration. It is also transient, since much of the activity returns to the cytosol as the receptors become sequestered. Stimulation of beta-AR kinase translocation is a receptor-mediated event, since the beta-antagonist propranolol blocks the effect of agonist. In the kin- mutant of the S49 cells (lacks cAMP-dependent protein kinase), prostaglandin E1, which provokes homologous desensitization of its own receptor, is at least as effective as isoproterenol in promoting beta-AR kinase translocation to the plasma membrane. However, in the DDT1MF-2 cells, which contain alpha 1-adrenergic receptors coupled to phosphatidylinositol turnover, the alpha 1-agonist phenylephrine is ineffective. These results suggest that the first step in homologous desensitization of the beta-adrenergic receptor may be an agonist-promoted translocation of beta-AR kinase from cytosol to plasma membrane and that beta-AR kinase may represent a more general adenylate cyclase-coupled receptor kinase that participates in regulating the function of many such receptors.

Beta-adrenergic receptor kinase: identification of a novel protein kinase that phosphorylates the agonist-occupied form of the receptor.

Agonist-promoted desensitization of adenylate cyclase is intimately associated with phosphorylation of the beta-adrenergic receptor in mammalian, avian, and amphibian cells. However, the nature of the protein kinase(s) involved in receptor phosphorylation remains largely unknown. We report here the identification and partial purification of a protein kinase capable of phosphorylating the agonist-occupied form of the purified beta-adrenergic receptor. The enzyme is prepared from a supernatant fraction from high-speed centrifugation of lysed kin- cells, a mutant of S49 lymphoma cells that lacks a functional cAMP-dependent protein kinase. The beta-agonist isoproterenol induces a 5- to 10-fold increase in receptor phosphorylation by this kinase, which is blocked by the antagonist alprenolol. Fractionation of the kin- supernatant on molecular-sieve HPLC and DEAE-Sephacel results in a 50- to 100-fold purified beta-adrenergic receptor kinase preparation that is largely devoid of other protein kinase activities. The kinase activity is insensitive to cAMP, cGMP, cAMP-dependent kinase inhibitor, Ca2+-calmodulin, Ca2+-phospholipid, and phorbol esters and does not phosphorylate general kinase substrates such as casein and histones. Phosphate appears to be incorporated solely into serine residues. The existence of this novel cAMP-independent kinase, which preferentially phosphorylates the agonist-occupied form of the beta-adrenergic receptor, suggests a mechanism that may explain the homologous or agonist-specific form of adenylate cyclase desensitization. It also suggests a general mechanism for regulation of receptor function in which only the agonist-occupied or "active" form of the receptor is a substrate for enzymes inducing covalent modification.