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H Akil

Publications and source records attributed to H Akil.

At least 91 records · Page 5Linked to original sources
PubMed1997

Opioid effects on mitogen-activated protein kinase signaling cascades.

BACKGROUND: The molecular mechanisms underlying both beneficial and undesirable opioid actions are poorly understood. Recently, the three currently known mammalian mitogen-activated protein kinase (MAPK) signaling cascades (extracellular signal-related kinase [ERK], stress-activated protein kinase, and p38 kinase) were shown to play important roles in transducing receptor-mediated signaling processes. METHODS: To determine whether any of these kinase cascades were activated by opioids, mu, delta, or kappa opioid receptors were transiently introduced into COS-7 cells together with MAPKs tagged to allow recognition by specific antibodies, and then exposed to opioids. Mitogen-activated protein kinase activation was determined by an in vitro MAPK activation assay. In addition, C6 glioma cells with either mu, delta, or kappa receptors stably introduced were exposed to opioids and MAPK activation determined by in vitro activation assay or antibody detection of activated forms. RESULTS: Transient experiments in COS cells revealed potent stimulation of ERK by mu and delta receptor activation, weak stimulation of stress-activated protein kinase by all receptor types, and no activation of p38. In stably transfected C6 glioma cells, only ERK activation was observed. Extracellular signal-related kinase induction was rapid, peaking 5 min after stimulation, and its activation was receptor-type specific. Mu and delta receptor stimulation activated ERK, but kappa stimulation did not. CONCLUSIONS: These results show that acute opioid signaling is not only inhibitory, but can strongly activate an important signaling cascade. Extracellular signal-related kinase activation may contribute to desirable responses to opioids, such as analgesia and sedation, and also to undesirable adaptive responses, such as tolerance, physical dependence, and possibly addiction. Further study of this system could provide greater insight into the molecular mechanisms underlying these clinical problems.

Animals↗
PubMed1997

Correlation between Changes in Stress-Induced Corticosterone Secretion and GR mRNA Levels.

The current study was conducted to determine the potential relationship between stress-induced corticosterone secretion and corticosteroid receptor mRNA levels after 5 days of intermittent stress. In particular, we were interested in the rate at which animals terminate a stress response, and how this termination may be altered by repeated stress. Adult male Sprague-Dawley rats were subjected to either 5 days of restraint stress or 5 days of an unpredictable stress paradigm. Restraint-stress induced corticosterone secretion was measured on Days 1 and 5 in both groups, and animals were killed on Day 6. Glucocorticoid receptor (GR), and mineralocorticoid (MR) mRNA levels were determined using in-situ hybridization techniques. Five days of restraint stress caused an habituation of the plasma corticosterone response to stress measured 60 and 90 min post-stress initiation; this pattern of corticosterone secretion was not observed in the animals subjected to unpredictable stress. Five days of either stress paradigm did not alter MR mRNA levels measured within the hippocampus or GR mRNA levels within the hippocampus or the medial parvocellular division of the paraventricular nucleus of the hypothalamus (mpPVN). However, an individual's GR mRNA levels measured within the CA1/2 region of the hippocampus and the mpPVN were significantly correlated with the degree of habituation of the corticosterone response to stress measured on Day 5. This suggests that an increase in the rate of termination of the stress response and levels of GR within the hippocampus and mpPVN may be functionally related.

Journal Article↗
PubMed1997

Opioid efficacy in a C6 glioma cell line stably expressing the delta opioid receptor.

A C6 glioma cell line stably transfected with the rat delta opioid receptor (C6delta) was used to characterize receptor binding and G protein activation by both peptide and nonpeptide delta opioid ligands. The ligand binding affinities for [3H]naltrindole and [3H]pCl-[D-Pen2,D-Pen5]enkephalin (DPDPE) were similar to those observed in monkey brain membranes. The nonpeptide agonists, BW373U86 and SNC80, as well as peptide agonist [D-Ser2, L-Leu5]enkephalyl-Thr maximally stimulated [35S]GTPgammaS binding by 640, 654 and 576%, respectively, over basal. The peptide agonists, DPDPE and deltorphin II, both stimulated [35S]GTPgammaS binding by 375%. Etorphine, diprenorphine, oxymorphindole and 7-spiroindanyloxymorphone were also partial agonists in this assay, although they were less efficacious than deltorphin II. Stimulation of [35S]GTPgammaS binding by agonists was blocked completely by pertussis toxin pretreatment. Both delta-1 and delta-2 selective antagonists 7-benzylidenenaltrexone and a benzofuran analog of naltrindole displayed high affinity for the cloned receptor (0.04 and 0.08 nM) and antagonized the stimulation of [35S]GTPgammaS binding by BW373U86 and DPDPE with similar potencies. Other evidence suggesting the lack of receptor subtypes includes the finding that stimulation of [35S]GTPgammaS binding by receptor subtype selective ligands DPDPE and deltorphin II was not additive. BW373U86, SNC80 and DPDPE maximally inhibited forskolin-stimulated adenylyl cyclase. These cells highly express a homogeneous population of delta opioid receptor that couple to inhibitory Go/Gi proteins. Ligand affinity for the delta opioid receptor correlates with ligand EC50 values for stimulation of [35S]GTPgammaS binding.

Adenylate Cyclase Toxin↗
PubMed1996

Moving from the orphanin FQ receptor to an opioid receptor using four point mutations.

It is unclear how receptor/ligand families that are evolutionarily closely related achieve functional separation. To address this question, we focus here on the newly discovered Orphanin FQ, a peptide homologous to the opioid peptide Dynorphin, and its receptor, the Orphanin FQ receptor, which is highly homologous to the opioid receptors. In spite of this high degree of homology in terms of both ligands and receptors, there is little direct cross-talk between the Orphanin FQ system and the endogenous opioid system. Thus, the opioid peptides show either relatively low affinity or no affinity toward the Orphanin FQ receptor; conversely, Orphanin FQ has no affinity toward any of the opioid receptors. We sought to investigate the molecular basis of such discrimination by attempting to reverse it and endowing the Orphanin FQ receptor with the ability to bind opioids. We report that by mutating as few as four amino acids, we can produce a receptor that recognizes pro-Dynorphin products with very high affinity and yet still binds Orphanin FQ as well as the wild-type receptor. This suggests that the Orphanin FQ receptor has developed features that specifically exclude the opioids and that these features are distinct from those required for the high affinity binding of its own endogenous ligand.

Analgesics↗
PubMed1996

Mapping the receptor domains critical for the binding selectivity of delta-opioid receptor ligands.

While a good deal has been learned about determinants of high affinity ligand/receptor interactions in G-protein-coupled receptors, less is known about mechanisms of ligand selectivity. The opioid receptors offer an excellent opportunity to study the mechanisms whereby structurally very similar receptors discriminate between different but structurally highly related ligands. In the current study, we use a series of chimeric constructs between the delta-opioid receptor and either the mu- or the kappa-opioid receptors to investigate the structural basis of binding selectivity of multiple classes of delta-opioid receptor selective ligands. Our results demonstrate that a region containing the sixth transmembrane domain (TM6) and the third extracellular loop (EL3) in the delta-opioid receptor is absolutely critical for delta-opioid receptor selectivity. The introduction of this region into the kappa-opioid receptor is sufficient to impart a delta profile for delta-opioid receptor selective alkaloids such as naltrindole and naltriben. In order to locate the amino acid residues that may be involved in ligand selectivity in TM6 and EL3 of the delta-opioid receptor, several mutations were introduced into that region. These mutations showed differential effects on peptide and alkaloid ligands. In addition, none of the individual mutations alone could account for the changes exhibited by the chimeric receptors. We conclude that the selectivity of most delta-opioid ligands is achieved through their interaction with many different residues in the TM6/EL3 region. Our results also support a view that the extracellular domains of peptide receptors may provide the basis of a sorting mechanism for ligand selectivity.

Binding, Competitive↗
PubMed1996

Regulation of glucocorticoid and mineralocorticoid receptor mRNAs in the hippocampus of the maternally deprived infant rat.

The hypothalamic-pituitary-adrenal (HPA) axis in the developing rat has a limited response to acute challenges between days 3 and 14 of life. Several hypotheses have been proposed to explain this quiescent state. Immaturity of brain, pituitary and adrenal elements or excessive feedback inhibition are common explanations. Recently, a series of studies by Levine and co-workers has shown that prolonged maternal deprivation (24 h) results in increased basal and stress induced corticosterone (CS) levels. An increased adrenal response to ACTH along with an enhanced and sustained ACTH response have been implicated in this phenomenon. A brain structure that appears to be important for normal HPA function is the hippocampus, a structure rich in corticosteroid receptors, which has been hypothesized to play a role in the basal tone of the HPA and in the magnitude and duration of stress responses. Thus, to study further the possible mechanisms leading to an enhanced and sustained ACTH response that is seen in maternally deprived pups, we used in situ hybridization to investigate hippocampal mineralocorticoid (MR) and glucocorticoid receptor (GR) gene expression in 12 groups of animals: six groups involved 24 h maternally deprived (DEP) and non-deprived (NDEP) rat pups at three ages (6-, 9-, and 12-days-old); the other six groups included pups similarly treated, but challenged with an exposure to a mild stressor (saline injection) and sacrificed 1 h thereafter. We found: (1) an age effect for almost every hippocampal subfield for both MR and GR mRNAs: MR increases with age, while GR decreases: (2) down-regulation of MR mRNA in CA1 region in the DEP animals; and (3) down-regulation of GR mRNA, also in CA1, in the saline-injected DEP and NDEP animals. Our results indicate that corticoid receptors in the developing CA1 hippocampal region appear to be sensitive to circulating CS. They also suggest that the relative ratio of GR and MR in the CA1 region may contribute to the enhanced and sustained CS response seen after a mild stressor in deprived animals.

Adrenocorticotropic Hormone↗
PubMed1996

The novel neuropeptide orphanin FQ fails to produce conditioned place preference or aversion.

An unbiased conditioned place preference procedure was used to determine whether the newly-identified neuropeptide orphanin FQ produced motivational effects after intracerebroventricular microinjections. Microinjections of orphanin FQ (0.1-100 nmol) failed to produce conditioned place preference or aversion, but a pronounced motor impairment was observed during conditioning sessions with the two highest doses. Thus, it appears that orphanin FQ lacks motivational effects when administered at behaviourally active doses.

Animals↗
PubMed1996

A chimeric analysis of the opioid receptor domains critical for the binding selectivity of mu opioid ligands.

The mu opioid receptor plays a key role in mediating the physiological, pharmacological, and behavioral effects of endogenous opioids and of opiate drugs such as morphine and heroin. This study examines the structural features critical to the selective binding of mu ligands to the mu receptor as opposed to the other two highly homologous opioid receptors, delta and kappa. We use a series of chimeric constructs between the mu and either the delta or the kappa receptors to investigate the structural bases of binding selectivity of multiple classes of mu-selective ligands. Our results demonstrate that a region comprising the sixth transmembrane domain and the third extracellular loop is critical for the mu/kappa discrimination by all mu-selective ligands. This region is also critical for mu/delta discrimination by the mu antagonists. However, mu agonists, particularly the peptides, exhibit more complex interactions, often relying on the N-terminal region surrounding the first extracellular loop for mu/delta discrimination. Thus, the same mu peptide ligand depends on different parts of the receptor to discriminate between mu and delta receptors on the one hand and mu and kappa on the other. In general, antagonists show the most consistent discrimination mechanisms regardless of construct, whereas agonists, particularly peptides, achieve selectivity by interacting with numerous domains of the receptors.

Animals↗
PubMed1996

Rats rapidly develop tolerance to the locomotor-inhibiting effects of the novel neuropeptide orphanin FQ.

We examined the effects of intracerebroventricular (i.c.v.) administration of orphanin FQ (OFQ) on locomotor activity in rats. The rats were habituated to locomotor-testing boxes and then injected i.c.v. with OFQ (0 - 10 nmoles). Acute injections of OFQ produced dose-orderly reductions in horizontal locomotion and rearing activity. This suppression of motor activity was characterized by a disruption of balance and muscle control. Within minutes of i.c.v. injection of the higher doses of OFQ, the rats exhibited flaccid muscle tone. They each lay in an atypical posture, pressing the abdomen against the floor, and splaying the hindlimbs. When these rats locomoted, their gate was unsteady. They wobbled from side to side, and frequently fell over. Repeated daily injections of OFQ resulted in a rapid development of tolerance to the OFQ-induced suppression of locomotion and rearing activity. Tolerance to the observed impairments of motor control were also apparent. In the rats that were repeatedly treated with the highest dose (10 nmol) of OFQ, tolerance to the motoric effects was still apparent after 7 days without OFQ treatment.

Analysis of Variance↗
PubMed1996

Psychomotor stimulant- and opiate-induced c-fos mRNA expression patterns in the rat forebrain: comparisons between acute drug treatment and a drug challenge in sensitized animals.

Amphetamine-, cocaine-, and morphine-induced c-fos expression patterns were examined following an injection protocol that has previously been shown to produce behavioral sensitization and enhanced dopamine release in the striatal complex. Drug-specific c-fos patterns were observed in both acute and sensitization injection paradigms. A sensitization pretreatment schedule did, however, alter the c-fos expression patterns induced by all the drugs in the caudate putamen, nucleus accumbens, and the cerebral cortex. In some striatal and cortical regions, there was an increase or recruitment of cells expressing c-fos whereas in others there was an apparent decrease or inhibition. The somatosensory cortex was one area where pretreatment with all three drugs increased c-fos expression. The results suggest that the neuronal networks that are modulated by systemic drug injections in the sensitized animal differ from those affected by the initial drug exposure; areas of overlap may indicate common "sensitization' circuits.

Animals↗
PubMed1996

Production and characterization of site-directed antibodies against dermorphin and dermorphin-related peptides.

To detect and purify endogenous dermorphin-like molecules in mammalian tissues, an immunological approach was developed. Site-directed antibodies against synthetic dermorphin and related dermorphin peptides were produced. The immunogenic forms of dermorphin were selected to obtain antibodies recognizing different epitopes overlapping the whole dermorphin molecule. One of them specifically recognized the crucial "opioid message" (the N-terminal part of the molecule), which is required for a ligand to exert its full opioid activity. The validity of our immunological approach was analyzed by studying the dermorphin-related peptide distribution in Phyllomedusa sauvagei skin. The finding that tetrapeptide Y-A-G-F-OH was present in Phyllomedusa sauvagei extracts suggested that either the Tyr3-Pro6 peptidic bond may be relatively unstable or endogenous proteolytic enzymes present in Phyllomedusa skin may inactivate this peptidic bond.

Analgesics, Opioid↗
PubMed1996

Immunohistochemical localization of the cloned kappa 1 receptor in the rat CNS and pituitary.

Several lines of evidence have demonstrated the presence of three opioid receptor types in the CNS and periphery. These receptors are referred to as mu, delta and kappa, and have been implicated in a wide variety of functions. The present study examines the localization of the kappa 1 receptor, a region of the receptor that has little homology with mu and delta receptors. Immunohistochemical studies in Zamboni-fixed rat tissue demonstrate immunoreactive perikarya and/or fibers in such regions as the deep layers of the parietal, temporal and occipital cortex, parasubiculum, central and medial amygdala, bed nucleus stria terminalis, nucleus accumbens, olfactory tubercle, endopiriform nucleus, claustrum, hypothalamic nuclei, median eminence, midline thalamic nuclei, zona incerta, central gray, caudal linear and dorsal raphe, substantia nigra, pars reticulata, ventral tegmental area, parabrachial nucleus, spinal trigeminal nucleus, nucleus of the solitary tract, spinal cord and the dorsal root ganglia. Specific kappa 1 receptor-like immunohistochemical staining is also observed in the pituitary, where immunoreactive perikarya and fibers are localized in the neural and intermediate lobes. Transfection and preabsorption controls suggest that the antibody is selective for the cloned kappa 1 receptor, and does not recognize mu or delta. This immunohistochemical localization corresponds well to previously described kappa 1 receptor mRNA and binding distributions and provides new insights into the cellular localization and pre- and postsynaptic organization of the kappa 1 receptor-like proteins in the rat brain and pituitary. The functional implications of these results are discussed in light of the kappa 1 receptors play in hormonal regulation, antinociception and reward.

Animals↗
PubMed1996

Interleukin-1 beta-mediated regulation of mu-opioid receptor mRNA in primary astrocyte-enriched cultures.

Opioids have been found to modulate the immune system by regulating the function of immunocompetent cells. Several studies suggest that the interaction between immune and opioid systems is not unidirectional, but rather reciprocal, in nature. In the CNS, one cellular target of immune system activation is the astrocytes. These glial cells have been shown to produce the opioid peptide, proenkephalin, to express the mu-, delta-, and kappa-opioid receptors, and to respond to the immune factor interleukin-1 beta (IL1 beta) with an increased proenkephalin synthesis. To characterize more completely the astrocytic opioid response to immune factor stimulation, we examined the effect of IL1 beta (1 ng/ml) on the mu-receptor mRNA expression in primary astrocyte-enriched cultures derived from rat (postnatal day 1-2) cortex, striatum, cerebellum, hippocampus, and hypothalamus. A 24-h treatment with IL1 beta produced a 70-80% increase in the mu-receptor mRNA expression in the striatal, cerebellar, and hippocampal cultures but had no effect on this expression in the cortical and hypothalamic cultures. This observation represents one of the few demonstrated increases in levels of the mu-receptor mRNA in vitro or in vivo, since the cloning of the receptor. The enhanced mu-receptor mRNA expression, together with the previous observation that IL1 beta stimulates proenkephalin synthesis in astrocytes, supports the IL1 beta-mediated regulation of an astroglial opioid peptide and receptor in vitro, a phenomenon that may be significant in the modulation of the gliotic response to neuronal damage. Therefore, the astroglial opioid "system" may be important in the IL1 beta-initiated, coordinated response to CNS infection, trauma, or injury.

Animals↗
PubMed1996

Differential pattern of c-fos mRNA in rat brain following central and systemic administration of interleukin-1-beta: implications for mechanism of action.

Interleukin-1-beta (IL-1 beta) is a potent activator of the hypothalamic-pituitary-adrenal (HPA) axis, resulting in the release of corticosteroids from the adrenal glands. This effect is evident after both central and peripheral administration, and controversy surrounds the mechanism(s) by which systemic administration of this peptide, which should not cross the blood-brain barrier, may activate the HPA axis. In the present study, IL-1 beta was administered systemically (5 micrograms/kg i.p.) or centrally (100 ng i.c.v.) to male rats. Both routes of administration of IL-1 beta resulted in significant and comparable activation of the HPA axis, as assessed by analysis of plasma conrticosterone. In addition, both routes of administration of IL-1 beta resulted in c-fos mRNA induction in specific regions, as determined by in situ hybridization. These included the meninges, cerebral vasculature, choroid plexus and circumventricular organs. Semiquantitative analysis revealed that both routes of administration resulted in significant and comparable induction of c-fos mRNA in the paraventricular nucleus of the hypothalamus, as compared with control animals. In contrast, in the nucleus tractus solitarius (NTS) and central nucleus of the amygdala (CeA), levels of c-fos mRNA were 3-4 times higher in animals treated intraperitoneally compared with intracerebroventricularly. A similar differential activation of c-fos mRNA was observed in the lateral divisions of the parabrachial nucleus (PBN) and bed nucleus of the stria terminalis (BNST). These data indicate that following systemic administration, IL-1 beta may activate specific brain areas through mechanisms distinct from those involved following central administration. The differential magnitude of the c-fos mRNA response in the NTS, PBN, CeA and BNST is consistent with vagal activation. Physiologically, these results suggest that IL-1 beta may have differential central effects depending on its source or point of entry to the brain.

Adrenal Glands↗
PubMed1996

Cloning and characterization of multiple opioid receptors.

Over the course of 1 to 2 years, the field has moved swiftly to investigate the functional and structural properties of the newly cloned opioid receptors. Achieving a better understanding of these macromolecules is likely to have profound implications for drug design aimed at the production of better analgesic drugs, for a more fundamental understanding of mechanisms of action of drugs of abuse, and for a more comprehensive knowledge base regarding the biology of opioid peptides in particular, and neuroactive peptides in general.

Animals↗
PubMed1996

Characterization of opioid agonist efficacy in a C6 glioma cell line expressing the mu opioid receptor.

In C6 glioma cells stably expressing a homogeneous population of the cloned rat mu opioid receptor, the binding affinities of opioid agonists and subsequent activation of G protein were examined. Opioid receptor number in membranes of these cells was high (10-30 pmol/mg protein [3H]diprenorphine binding sites). Opioids were found to bind to the receptor with high affinity [Tyr-D-Ala-Gly-(Me)Phe-Gly-ol (DAMGO) 0.23 nM; sufentanil 0.034 nM; morphine 0.16 nM]. Activation of G protein by opioid agonists was examined by measuring the stimulation of guanosine-5'-O-(3-[35S]thio)triphosphate ([35S]GTP gamma S) binding. Sufentanil increased [35S]GTP gamma S binding by 326% with an EC50 value of 2.39 nM. Agonist stimulation of [35S]GTP gamma S binding was stereoselective, naltrexone-reversible, and pertussis toxin-sensitive. The "intrinsic activity" of opioids at the mu receptor was reflected by the magnitude of agonist-mediated activation of G protein. The rank order of the stimulation of [35S]GTP gamma S binding was etonitazene = sufentanil = DAMGO = PLO17 = fentanyl > morphine > profadol > meperidine > butorphanol = nalbuphine = pentazocine > cyclazocine = nalorphine > levallorphan > naltrexone. High affinity binding of ligands to the mu opioid receptor was reduced by the addition of sodium and guanosine diphosphate at concentrations used in the [35S]GTP gamma S binding assay. Ligand affinity was reduced in a manner correlating with "intrinsic activity". DAMGO, 1229-fold, nalbuphine 35-fold, naltrexone, 3-fold. The results presented show that the stable expression of the rat mu opioid receptor in C6 cells provides an effective tool to examine opioid receptor signal transduction mechanisms and evaluate the activity of novel opioids at the mu receptor.

Animals↗
PubMed1996

Mutagenesis of residues adjacent to transmembrane prolines alters D1 dopamine receptor binding and signal transduction.

Proline is highly conserved in the presumed transmembrane alpha-helices of seven-transmembrane helix-containing, G protein coupled receptors. Unique properties of this imino acid have led to speculations of structural and perhaps dynamic importance for seven-transmembrane helix-containing receptor function. To avoid potentially deleterious consequences of proline-directed mutagenesis, substitutions were made in the X residue of X-Pro peptide bonds (where X is the residue on the amino-terminal side of proline), which may influence static geometries and potential agonist-induced conformational changes at the X-Pro peptide bond. In the fifth helix, Ile205 was substituted with either an alanine (I205A) or a tyrosine (I205Y). Similarly, in the sixth helix, Leu286 was substituted with either an alanine (L286A) or a tyrosine (L286Y). Mutant I205A demonstrated subtle changes in D1 pharmacology and signal transduction. The I205Y and L286Y mutations produced comparatively drastic impairments in both binding and signal transduction. Remarkably, the L286A mutation resulted in constitutive activity characterized by elevated basal signal transduction and increased agonist potencies. In addition, (R)-(+)-SCH23390, a classical antagonist at the wild-type D1 receptor, behaved as a partial agonist at L286A. This is the first report of a constitutively active receptor resulting from this point mutation and the first report of a constitutively active mutant dopamine receptor. These results are discussed in terms of binding pocket geometry and potential mechanisms of signal transduction.

Amino Acid Sequence↗