Norlaudanosoline and Nicotine Increase Endogenous Ganglionic Morphine Levels: Nicotine Addiction

Summary 1. Given the presence of morphine, its metabolites and precursors, e.g., norlaudanosoline, in mammalian and invertebrate tissues, it became important to determine if exposing normal excised ganglia to norlaudanosoline would result in increasing endogenous morphine levels.2. Mytilus edulis pedal ganglia contain 2.2 ± 0.41 ng/g wet weight morphine as determined by high pressure liquid chromatography coupled to electrochemical detection and radioimmunoassay.3. Incubation of M. edulis pedal ganglia with norlaudanosoline, a morphine precursor, resulted in a concentration- and time-dependent statistical increase in endogenous morphine levels (6.9 ± 1.24 ng/g).4. Injection of animals with nicotine also increased endogenous morphine levels in a manner that was antagonized by atropine, suggesting that nicotine addiction may be related to altering endogenous morphine levels in mammals.5. We surmise that norlaudanosoline is being converted to morphine, demonstrating that invertebrate neural tissue can synthesize morphine.     

Dopamine is necessary to endogenous morphine formation in mammalian brain in vivo

Morphine, the most used compound among narcotic analgesics, has been shown to be endogenously present in different mammalian/invertebrate normal tissues. In this study, we used mice that cannot make dopamine due to a genetic deletion of tyrosine hydroxylase specifically in dopaminergic neurons, to test the hypothesis that endogenous dopamine is necessary to endogenous morphine formation in vivo in mammalian brain. When dopamine was lacking in brain neurons, endogenous morphine was missing in brain mouse whereas it could be detected in brain from wild type rodent at a picogram range. Our data prove for the first time that endogenous dopamine is necessary to endogenous morphine formation in normal mammalian brain. Morphine synthesis appears to be originated from dopamine through L-tyrosine in normal brain tissue. Morphine synthesis is not considered to occur inside the same neuron in normal tissue; released dopamine might be transported into morphinergic neuron and further transformed into morphine. A physiological role for endogenous morphine is suggested considering that dopamine could modulate thermal threshold through endogenous morphine formation in vivo. Thus, dopamine and endogenous opiates/opioid peptides may be interconnected in the physiological processes; yet, endogenous morphine may represent a basic link of this chain.     

Effect of chronic morphine treatment on the biosynthesis of agmatine in rat brain and other tissues

Agmatine is an endogenous amine derived from the decarboxylation of arginine by arginine decarboxylase (ADC), and metabolized to putrescine by agmatinase. Exogenously administered agmatine has several biological actions including its ability to potentiate morphine analgesia and block symptoms of morphine tolerance/withdrawal in rats. To investigate the role of endogenous agmatine in this action, we sought to determine whether chronic exposure to morphine and induction of withdrawal modulate the synthesis of agmatine in rat brain and other tissues. Exposure of rats to morphine for three days significantly decreases the activity of ADC and the levels of agmatine in rat liver, kidney, brain, aorta and intestine with no changes in agmatinase activity. The precipitation of withdrawal syndrome by injecting naloxone further decreases ADC activity and agmatine levels in these tissues. We conclude that endogenous agmatine may play an important role in regulating morphine tolerance/dependence and withdrawal symptoms.     

Regulation of calcium channels in brain: implications for the clinical neurosciences

Calcium is a major second messenger in neurons and modulates many neuronal functions, including protein phosphorylation, phospholipid metabolism, cytoskeletal activity, and neurotransmitter release. These important events, which regulate neuronal activity, are directly dependent on the influx of extracellular calcium through voltage-sensitive calcium channels (VSCCs) in the neuronal membrane. Modulation of VSCC function represents an important strategy for regulating neuronal excitability. Although substantial evidence supports the ability of dihydropyridines to block VSCCs and contractility in cardiovascular tissue, their ability to block the majority of neuronal VSCCs remains controversial. Benzodiazepines, and other anticonvulsants, block depolarization-dependent 45Ca uptake through VSCCs in brain synaptosome preparations. In addition, benzodiazepines reduce voltage-gated calcium conductance as determined by voltage clamp studies of identified invertebrate neurons. Inhibition of VSCC activity may be an important mechanism by which these compounds produce their anticonvulsant and sedative effects. Intrasomal injection of calcium-calmodulin-dependent protein kinase modulates calcium conductance in invertebrate neurons, suggesting that protein phosphorylation may be an endogenous regulatory mechanism of VSCC activity. Developing novel pharmacological approaches to regulating VSCCs and understanding the endogenous regulatory mechanisms may lead to new therapeutic approaches to the treatment of neurological diseases.     

The Presence of Endogenous Morphine Signaling in Animals

Recent empirical findings have contributed valuable mechanistic information in support of a regulated de novo biosynthetic pathway for chemically authentic morphine and related morphinan alkaloids within animal cells. Importantly, we and others have established that endogenously expressed morphine represents a key regulatory molecule effecting local circuit autocrine/paracrine cellular signaling via a novel μ₃ opiate receptor coupled to constitutive nitric oxide production and release. The present report provides an integrated review of the biochemical, pharmacological, and molecular demonstration of μ₃ opiate receptors in historical linkage to the elucidation of mechanisms of endogenous morphine production by animal cells and organ systems. Ongoing research in this exciting area provides a rare window of opportunity to firmly establish essential biochemical linkages between dopamine, a morphine precursor, and animal biosynthetic pathways involved in morphine biosynthesis that have been conserved throughout evolution. Special issue article in honor of Dr. Ji-Sheng Han.     

Role for GDNF in biochemical and behavioral adaptations to drugs of abuse

The present study examined a role for GDNF in adaptations to drugs of abuse. Infusion of GDNF into the ventral tegmental area (VTA), a dopaminergic brain region important for addiction, blocks certain biochemical adaptations to chronic cocaine or morphine as well as the rewarding effects of cocaine. Conversely, responses to cocaine are enhanced in rats by intra-VTA infusion of an anti-GDNF antibody and in mice heterozygous for a null mutation in the GDNF gene. Chronic morphine or cocaine exposure decreases levels of phosphoRet, the protein kinase that mediates GDNF signaling, in the VTA. Together, these results suggest a feedback loop, whereby drugs of abuse decrease signaling through endogenous GDNF pathways in the VTA, which then increases the behavioral sensitivity to subsequent drug exposure.     

Morphine-induced receptor endocytosis in a novel knockin mouse reduces tolerance and dependence

Opioid drugs, such as morphine, are among the most effective analgesics available. However, their utility for the treatment of chronic pain is limited by side effects including tolerance and dependence. Morphine acts primarily through the mu-opioid receptor (MOP-R) , which is also a target of endogenous opioids. However, unlike endogenous ligands, morphine fails to promote substantial receptor endocytosis both in vitro, and in vivo. Receptor endocytosis serves at least two important functions in signal transduction. First, desensitization and endocytosis act as an "off" switch by uncoupling receptors from G protein. Second, endocytosis functions as an "on" switch, resensitizing receptors by recycling them to the plasma membrane. Thus, both the off and on function of the MOP-R are altered in response to morphine compared to endogenous ligands. To examine whether the low degree of endocytosis induced by morphine contributes to tolerance and dependence, we generated a knockin mouse that expresses a mutant MOP-R that undergoes morphine-induced endocytosis. Morphine remains an excellent antinociceptive agent in these mice. Importantly, these mice display substantially reduced antinociceptive tolerance and physical dependence. These data suggest that opioid drugs with a pharmacological profile similar to morphine but the ability to promote endocytosis could provide analgesia while having a reduced liability for promoting tolerance and dependence.     

Activation of post-synaptic dopamine D₁ receptors promotes the release of tissue plasminogen activator in the nucleus accumbens via PKA signaling

We have previously demonstrated that tissue plasminogen activator (tPA) plays an important role through the conversion of plasminogen to plasmin in the release of dopamine in the nucleus accumbens (NAc) evoked by depolarization or the systemic administration of drugs of abuse such as morphine and nicotine. In the present study, we examined the mechanisms by which drugs of abuse increase extracellular tPA activity in the NAc in vivo using in situ zymography. The dopamine D₁ receptor (D₁R) agonist SKF38393, but not D₂ receptor agonist quinpirole, significantly increased extracellular tPA activity in the NAc. The effect of SKF38393 was blocked by pre-treatment with the dopamine D₁R antagonist SCH23390. Microinjection of Rp-cAMPs, a protein kinase A inhibitor, into the NAc completely blocked the effect of SKF38393. Systemic administration of morphine and methamphetamine increased extracellular tPA activity in the NAc, and these effects were completely blocked by pre-treatment with SCH23390 and raclopride. The results suggest that activation of post-synaptic dopamine D₁Rs in the NAc leads to an increase in extracellular tPA activity via protein kinase A signaling. Furthermore, dopamine D₂ receptors are also involved in the release of tPA induced by morphine and methamphetamine.     

Polyamine deprivation alters formalin-induced hyperalgesia and decreases morphine efficacy

Although the exact functions of polyamines in the nervous system remain still unclear, they are thought to have a physiological role in intracellular signal processing and neurotransmission. Polyamine deprivation which consists in the reduction of both the endogenous and exogenous sources of polyamines is a promising treatment for cancer. In a previous study we have shown that this treatment provokes an analgesic effect in rats submitted to brief phasic nociceptive tests. The present study examined the effect of polyamine deprivation on pain-related behaviors and spinal c-fos expression evoked in the formalin test presumed to better reflect clinical pain, using morphine as analgesia control. Polyamine deprivation per se altered the characteristic pain-related behaviors, reducing the interphase depression of pain, without inducing changes in the spinal Fos staining. In addition this treatment prevented the antinociceptive effect of morphine both on behavioral responses and on spinal c-fos expression. In polyamine-deprived rats, despite morphine injection, nociceptive scores remained dramatically high during the intermediate and the late phases of the response and the number of Fos immunoreactive neurons remained largely higher in deeper layers than in morphine control rats. Altogether these data support a modulatory role of polyamines both on the neuronal circuitry mediating sensory information, and on mechanisms underlying morphine analgesia.     

Spatial autocorrelation patterns of stream invertebrates: exogenous and endogenous factors

Aim To investigate spatial autocorrelation of taxonomic stream invertebrate groups (richness and composition) at a large geographical scale and to analyse the importance of exogenous and endogenous factors. Location The Mediterranean Basin. Methods For exogenous factors, we used large‐scale factors related to climate, geology and river zonation; for endogenous factors, we used the dispersal mode of each taxonomic group. After describing and analysing spatial patterns of genus richness and genus composition of stream invertebrate groups in the Mediterranean Basin, we computed Moran’s I before and after accounting for the exogenous factors and related it to the endogenous factors. Results In relation to genus richness, most of the taxonomic groups did not show significant spatial autocorrelation, suggesting that no main large‐scale exogenous or endogenous factors were important and that local‐scale factors were probably controlling taxonomic richness. In contrast, for genus composition, all taxonomic groups except Odonata had significant spatial autocorrelation before accounting for the environment. After accounting for the environment, most taxonomic groups still had a significant spatial autocorrelation, but it decreased with their increasing dispersal ability (from Crustacea to Coleoptera). Thus, spatial taxonomic composition of groups with the strongest dispersal potential is mainly related to exogenous factors, whereas that of groups with weaker dispersal potential is related to a combination of exogenous and endogenous factors. Main conclusions Our results illustrate the importance of dispersal as an endogenous factor causing spatial autocorrelation and suggest that ignoring endogenous factors can lead to misunderstandings when explaining large‐scale community patterns.     

Agonist-specific regulation of delta-opioid receptor trafficking by G protein-coupled receptor kinase and beta-arrestin

Opioid receptors mediate multiple biological functions through their interaction with endogenous opioid peptides as well as opioid alkaloids including morphine and etorphine. Previously we have reported that the ability of distinct opioid agonists to differentially regulate mu-opioid receptor (mu OR) responsiveness is related to their ability to promote G protein-coupled receptor kinase (GRK)-dependent phosphorylation of the receptor (1). In the present study, we further examined the role of GRK and beta-arrestin in agonist-specific regulation of the delta-opioid receptor (delta OR). While both etorphine and morphine effectively activate the delta OR, only etorphine triggers robust delta OR phosphorylation followed by plasma membrane translocation of beta-arrestin and receptor internalization. In contrast, morphine is unable to either elicit delta OR phosphorylation or stimulate beta-arrestin translocation, correlating with its inability to cause delta OR internalization. Unlike for the mu OR, overexpression of GRK2 results in neither the enhancement of delta OR sequestration nor the rescue of delta OR-mediated beta-arrestin translocation. Therefore, our findings not only point to the existence of marked differences in the ability of different opioid agonists to promote delta OR phosphorylation by GRK and binding to beta-arrestin, but also demonstrate differences in the regulation of two opioid receptor subtypes. These observations may have important implications for our understanding of the distinct ability of various opioids in inducing opioid tolerance and addiction.     

The effect of an endogenous compound 1-methyl-1,2,3,4,-tetrahydroisoquinoline on morphine-induced analgesia, dependence and neurochemical changes in dopamine metabolism in rat brain structures.

1,2,3,4-Tetrahydroisoquinolines, among them the most interesting neuroprotective substance, an inhibitor of MAO, 1-methyl-1,2,3,4-tetrahydroisoquinoline (1MeTIQ), are endogenous compounds present in the central nervous system of mammals and humans. In this study, we investigated the effect of 1MeTIQ on morphine-induced analgesia, tolerance and abstinence syndrome as well as its effect on morphine-induced changes in dopamine metabolism in rat brain structures (nucleus accumbens, striatum, substantia nigra) using HPLC methodology. The experiments were carried out on male Wistar rats. Morphine analgesia was measured in the "hot-plate" test. To induce tolerance, morphine was given chronically (20 mg/kg i.p.) alone or following 1MeTIQ (50 mg/kg i.p.) injection. The development of dependence was assessed in the naloxone (2 mg/kg i.p.) precipitation test, after 10 days of morphine administration. The behavioral studies have shown that an endogenous compound, 1MeTIQ produced strong potentiation of morphine analgesia, prevented the development of morphine tolerance and inhibited expression of morphine abstinence syndrome in morphine-dependent rats. In neurochemical studies, we have demonstrated that 1MeTIQ antagonized morphine-induced changes in dopamine metabolism observed in rat brain structures. The main finding of this study was demonstration for the first time of an anti-abuse effect of an endogenous compound, 1MeTIQ, and its efficiency in counteracting morphine-induced addiction in the way useful from clinical point of view. The obtained results suggested a possibility of clinical application of 1MeTIQ in morphine addiction.     

Psychiatric research: psychoproteomics, degradomics and systems biology

While proteomics has excelled in several disciplines in biology (cancer, injury and aging), neuroscience and psychiatryproteomic studies are still in their infancy. Several proteomic studies have been conducted in different areas of psychiatric disorders, including drug abuse (morphine, alcohol and methamphetamine) and other psychiatric disorders (depression, schizophrenia and psychosis). However, the exact cellular and molecular mechanisms underlying these conditions have not been fully investigated. Thus, one of the primary objectives of this review is to discuss psychoproteomic application in the area of psychiatric disorders, with special focus on substance- and drug-abuse research. In addition, we illustrate the potential role of degradomic utility in the area of psychiatric research and its application in establishing and identifying biomarkers relevant to neurotoxicity as a consequence of drug abuse. Finally, we will discuss the emerging role of systems biology and its current use in the field of neuroscience and its integral role in establishing a comprehensive understanding of specific brain disorders and brain function in general.     

Psychiatric research: psychoproteomics,degradomics and systems biology

While proteomics has excelled in several disciplines in biology (cancer, injury and aging), neuroscience and psychiatryproteomic studies are still in their infancy. Several proteomic studies have been conducted in different areas of psychiatric disorders, including drug abuse (morphine, alcohol and methamphetamine) and other psychiatric disorders (depression, schizophrenia and psychosis). However, the exact cellular and molecular mechanisms underlying these conditions have not been fully investigated. Thus, one of the primary objectives of this review is to discuss psychoproteomic application in the area of psychiatric disorders, with special focus on substance- and drug-abuse research. In addition, we illustrate the potential role of degradomic utility in the area of psychiatric research and its application in establishing and identifying biomarkers relevant to neurotoxicity as a consequence of drug abuse. Finally, we will discuss the emerging role of systems biology and its current use in the field of neuroscience and its integral role in establishing a comprehensive understanding of specific brain disorders and brain function in general.     

Changes in morphine self-administration after exposure to ionizing radiation: Evidence for the involvement of endorphins

Recent findings have implicated endogenous opiates in radiation-induced behavioral change. The present experiment further investigated this hypothesis by observing alterations in morphine self-administration after irradiation. Under the presumption that the release of endogenous opiates would decrease the need for exogenously supplied morphine, we hypothesized that after radiation exposure morphine-experienced mice would self-administer less of the opiate. C57BL/6J mice had continuous access to two drinking flasks which contained either water or morphine in saccharine water. Irradiated mice drank significantly less morphine than did sham-irradiated controls. This decrease was naloxone-reversible and could not be entirely attributed to a generalized radiogenic hypodipsia or taste aversion. These results are consistent with the hypothesis that radiation-induced behavioral changes may be due, in part, to the fluctuations of endogenous opiates.     

Agonist-Specific Regulation of δ-Opioid Receptor Trafficking by G Protein-Coupled Receptor Kinase and β-Arrestin

Abstract

Opioid receptors mediate multiple biological functions through their interaction with endogenous opioid peptides as well as opioid alkaloids including morphine and etorphine. Previously we have reported that the ability of distinct opioid agonists to differentially regulate μ-opioid receptor (μOR) responsiveness is related to their ability to promote G protein-coupled receptor kinase (GRK)-dependent phosphorylation of the receptor (1). In the present study, we further examined the role of GRK and β-arrestin in agonist-specific regulation of the δ-opioid receptor (δOR). While both etorphine and morphine effectively activate the δOR, only etorphine triggers robust δOR phosphorylation followed by plasma membrane translocation of β-arrestin and receptor internalization. In contrast, morphine is unable to either elicit δOR phosphorylation or stimulate β-arrestin translocation, correlating with its inability to cause δOR internalization. Unlike for the μOR, overexpression of GRK2 results in neither the enhancement of δOR sequestration nor the rescue of δOR-mediated β-arrestin translocation. Therefore, our findings not only point to the existence of marked differences in the ability of different opioid agonists to promote δOR phosphorylation by GRK and binding to β-arrestin, but also demonstrate differences in the regulation of two opioid receptor subtypes. These observations may have important implications for our understanding of the distinct ability of various opioids in inducing opioid tolerance and addiction.     

Endogenous morphine modulates acute thermonociception in mice

The endogenous synthesis of morphine has been clearly demonstrated throughout the phylogenesis of the nervous system of mammals and lower animals. Endogenous morphine, serving as either a neurotransmitter or neurohormone, has been demonstrated in the nervous system of both vertebrates and invertebrates. As one of the effects of exogenous morphine is the modulation of pain perception, we investigated the effects that the depletion of endogenous morphine had on nociceptive transmission. The immunoneutralization of endogenous morphine from brain extracellular spaces was obtained through the intracerebroventricular administration of affinity purified anti-morphine IgG to mice, which then underwent the hot plate test. Endogenous morphine immunoneutralization decreased thermal response latency and attenuated the anti-nociceptive effect of the mu selective agonist DAMGO in hot plate test suggesting that endogenous morphine is involved in pain modulation.     

Effects of repeated morphine on locomotion, place preference and dopamine in heterozygous glial cell line-derived neurotrophic factor knockout mice

Glial cell line-derived neurotrophic factor (GDNF) has been shown to be involved in the maintenance of striatal dopaminergic neurons. Neurotrophic factors are crucial for the plasticity of central nervous system and may be involved in long-term responses to drug exposure. To study the effects of reduced GDNF on dopaminergic behaviour related to addiction, we compared the effects of morphine on locomotor activity, conditioned place preference (CPP) and extracellular accumbal dopamine in heterozygous GDNF knockout mice (GDNF+/-) with those in their wild-type (Wt) littermates. When morphine 30 mg/kg was administered daily for 4 days, tolerance developed towards its locomotor stimulatory action only in the GDNF+/- mice. A morphine 5 mg/kg challenge dose stimulated locomotor activity only in the GDNF+/- mice withdrawn for 96 h from repeated morphine treatment, whereas clear and similar sensitization of the locomotor response was seen after a 10 mg/kg challenge dose in mice of both genotypes. Morphine-induced CPP developed initially similarly in Wt and GDNF+/- mice, but it lasted longer in the Wt mice. The small challenge dose of morphine increased accumbal dopamine output slightly more in the GDNF+/- mice than in the Wt mice, but doubling the challenge dose caused a dose-dependent response only in the Wt mice. In addition, repeated morphine treatment counteracted the increase in the accumbal extracellular dopamine concentration we previously found in drug-naive GDNF+/- mice. Thus, reduced endogenous GDNF level alters the dopaminergic behavioural effects to repeatedly administered morphine, emphasizing the involvement of GDNF in the neuroplastic changes related to long-term effects of drugs of abuse.     

Cellular mechanisms governing synapse formation: lessons from identified neurons in culture

The accessibility of embryonic and adult neurons within invertebrate nervous systems has made them excellent subjects for neurobiological study. The ability to readily identify individual neurons, together with their great capacity for regeneration, has been especially beneficial to investigations of synapse formation and the specificity of neuronal connectivity. Many invertebrate neurons survive for long periods following isolation into primary cell culture. In addition, they readily extend new neuritic arbors and form electrical and chemical connections at sites of contact. Thus, cell culture approaches have allowed neuroscientists greater access to, and resolution of, events underlying neurite outgrowth and synaptogenesis. Studies of identified neuromuscular synapses ofHelisoma have determined a number of signaling mechanisms involved in transsynaptic communication at sites of neuron-target contact. At these sites, both anterograde and retrograde signals regulate the transformation of growth cones into functional presynaptic terminals. We have found that specific muscle targets induce both global and local changes in neurotransmitter secretion and intracellular calcium handling. Here we review recent studies of culturedHelisoma synapses and discuss the mechanisms thought to govern chemical synapse formation in these identified neurons and those of other invertebrate species.