Common intracellular actions of chronic morphine and cocaine in dopaminergic brain reward regions.
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Animals↗
Biomedical subjects
X Guitart
Publications and source records attributed to X Guitart.
Fischer and Lewis rat strains differ in basal levels of neurofilament proteins and their regulation by chronic morphine in the mesolimbic dopamine system.
We studied levels of neurofilament (NF) proteins in the ventral tegmental area (VTA), and other regions of the central nervous system, of two genetically inbred rat strains, Lewis (LEW) and Fischer (F344) rats. These strains represent genetically divergent populations of rats that have been used to study possible genetic factors involved in a variety of biological processes, including drug addiction: compared to F344 rats, LEW rats show a much higher preference for several classes of drugs of abuse. We found 30-50% lower levels of three NF proteins, NF-200 (NF-H), NF-160 (NF-M), and NF-68 (NF-L), in the VTA of LEW compared to F344 rats by use of immunolabeling and Coomassie blue staining. These strain differences were highly specific to this brain region, with no differences observed elsewhere in brain or spinal cord. Interestingly, chronic treatment of F344 rats with morphine decreased levels of these three NF proteins in the VTA, as found previously in outbred Sprague-Dawley rats (Beitner-Johnson, D., Guitart, X., and Nestler, E.J.:J. Neurosci., 12:2165-2176, 1992), whereas morphine had no effect on NF levels in the VTA of LEW rats. A similar strain difference was observed in chronic morphine regulation of tyrosine hydroxylase, with morphine increasing enzyme immunoreactivity in the VTA of F344 rats (as has been observed previously in Sprague-Dawley rats [Beitner-Johnson, D., and Nestler, E.J.:J. Neurochem., 57:344-347, 1991]), but not in LEW rats. In view of the observations that LEW and F344 rats show different levels of preference for several types of drugs of abuse, and of the evidence supporting a central role of the mesolimbic dopamine system in drug reward mechanisms, the results of the current study suggest the possibility that levels of NFs and tyrosine hydroxylase may mediate some aspects of drug reinforcement and contribute to individual genetic differences in vulnerability to drug addiction.
Animals↗
Chronic administration of lithium or other antidepressants increases levels of DARPP-32 in rat frontal cortex.
We studied the chronic actions of lithium on rat brain by investigating its effects on cyclic AMP-dependent protein phosphorylation by use of a back-phosphorylation procedure. We identified one heavily regulated phosphoprotein in frontal cortex as the 32-kDa dopamine- and cyclic AMP-regulated phosphoprotein (DARPP-32). Immunoblot experiments demonstrated that chronic lithium regulation of DARPP-32 back-phosphorylation is associated with equivalent increases in levels of DARPP-32 immunoreactivity. Lithium regulation of DARPP-32 immunoreactivity required chronic drug administration and was not observed in several other brain regions examined. Moreover, chronic administration of the antidepressant imipramine or tranylcypromine produced a similar increase in levels of DARPP-32 in frontal cortex, whereas other types of psychotropic drugs, including haloperidol, morphine, and cocaine, did not influence DARPP-32 levels. Increased levels of DARPP-32 could reflect a common functional effect on frontal cortex of long-term exposure to lithium and some other antidepressant medications, an effect possibly related to the clinical actions of these drugs.
Animals↗
Regulation of cyclic AMP response element-binding protein (CREB) phosphorylation by acute and chronic morphine in the rat locus coeruleus.
Previous studies have implicated adaptations in the cyclic AMP system in mechanisms of opiate tolerance, dependence, and withdrawal in the rat locus coeruleus. It has been speculated that such adaptations may occur at the level of gene expression. To understand better the mechanism by which opiates produce these intracellular adaptations, we studied morphine regulation of the state of phosphorylation of cyclic AMP response element-binding protein (CREB), a transcription factor that mediates some of the effects of the cyclic AMP system on gene expression. We show here, by use of a back phosphorylation and immunoprecipitation procedure, that acute morphine decreases the state of phosphorylation of CREB, an effect that becomes completely attenuated after chronic morphine administration. In contrast, acute precipitation of opiate withdrawal, via administration of an opiate receptor antagonist, increases the phosphorylation state of CREB. Such regulation of CREB phosphorylation could be part of the molecular pathway by which opiates produce changes in gene expression that lead to addiction.
Animals↗
Neurofilament proteins and the mesolimbic dopamine system: common regulation by chronic morphine and chronic cocaine in the rat ventral tegmental area.
The ventral tegmental area (VTA) and its dopaminergic projections appear to mediate some of the rewarding properties of opiates, cocaine, and other drugs of abuse. In a previous study, we demonstrated that chronic morphine and cocaine exert common actions on tyrosine hydroxylase, the rate-limiting enzyme in catecholamine biosynthesis, in this dopaminergic brain reward region (Beitner-Johnson and Nestler, 1991). In the present study, we investigated the effects of chronic morphine and cocaine on other phosphoproteins in the VTA by back phosphorylation and two-dimensional electrophoretic analysis. It was found that a number of phosphoproteins, in addition to tyrosine hydroxylase, were regulated similarly by the two drug treatments in this brain region. Several of these morphine- and cocaine-regulated phosphoproteins were identified as neurofilament (NF) proteins. Chronic, but not acute, administration of either morphine or cocaine was found to decrease levels of the three NF proteins, NF-200 (NF-H), NF-160 (NF-M), and NF-68 (NF-L), by between 15% and 50% in the VTA by back phosphorylation, immunolabeling, and Coomassie blue staining. Such regulation of NF proteins was selective, in that no detectable changes were observed in the levels of eight other major cytoskeletal or cytoskeletal-associated proteins analyzed. Furthermore, NF levels were not altered by chronic treatment with either imipramine or haloperidol, two psychotropic drugs without reinforcing properties, or by chronic stress. Morphine and cocaine regulation of NFs showed regional specificity, as NF levels were not altered in the substantia nigra, or other parts of the brain or spinal cord, by these drug treatments. NFs are thought to function as determinants of neuronal morphology and to be associated with axonal transport. Thus, decreased NF levels in the VTA in response to chronic morphine and chronic cocaine could lead to drug-induced alterations in the structural and functional properties of this brain region, which may represent, in turn, part of a common biochemical basis of morphine and cocaine addiction and craving.
Animals↗
Dopaminergic brain reward regions of Lewis and Fischer rats display different levels of tyrosine hydroxylase and other morphine- and cocaine-regulated phosphoproteins.
We studied cyclic AMP-dependent protein phosphorylation in the mesolimbic and nigrostriatal dopamine systems of two genetically inbred rat strains, Lewis (LEW) and Fischer (F344) rats. These strains represent genetically divergent populations of rats that have been used to study possible genetic factors involved in a variety of biological processes. We found striking differences in levels of tyrosine hydroxylase, and several other phosphoproteins, in the mesolimbic, but not the nigrostriatal, dopamine system between the two rat strains. Interestingly, in Sprague-Dawley rats, these same phosphoproteins are altered by chronic morphine and chronic cocaine specifically in the mesolimbic dopamine system, generally thought to be a brain reward pathway that mediates some of the reinforcing actions of many drugs of abuse. As LEW and F344 rats have been reported to show different levels of preference for several types of drugs of abuse, the results are consistent with the possibility that these phosphoproteins may mediate aspects of drug reinforcement and contribute to individual differences in vulnerability to drug addiction.
Animals↗
Ultrastructural changes induced by 12-O-tetradecanoylphorbol 13-acetate in pure cholinergic synaptosomes of Torpedo electric organ.
We have studied the morphological changes induced by the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) treatment on pure cholinergic synaptosomes from Torpedo electric organ. These changes were studied by both ultrathin sections and freeze-fracture techniques. We found that after a treatment with TPA, a redistribution of synaptic vesicles inside the nerve endings and exocytotic images could be observed. Also, TPA, under conditions that induced the acetylcholine release, did not change the density of intramembrane particles at the synaptosomal protoplasmic hemimembrane leaflet. Similar results were found when calcium was not present in the extrasynaptosomal medium, and our results suggest that acetylcholine release induced by phorbol ester is probably mediated by exocytosis of synaptic vesicles.
Acetylcholine↗
Identification of MARPP (14-20), morphine- and cyclic AMP-regulated phosphoproteins of 14-20 kDa, as myelin basic proteins: evidence for their acute and chronic regulation by morphine in rat brain.
MARPP(14-20), morphine- and cyclic AMP-regulated phosphoproteins of 14, 17-18, and 20 kDa, were identified originally by one-dimensional electrophoresis as a group of proteins whose state of phosphorylation was decreased by acute morphine and increased by chronic morphine in the rat locus coeruleus. We now show that MARPP(14-20) represent myelin basic proteins based on biochemical and immunochemical criteria. First, MARPP(14-20) were found to have isoelectric points of about 11 based on their migration on non-equilibrium pH gradient electrophoresis. Second, MARPP(14-20) were greatly enriched in myelin fractions of brain, and were not detectable, or present at very low levels, in other subcellular fractions of brain. Third, analysis of phosphorylated MARPP(14-20) by one- and two-dimensional gel electrophoresis demonstrated precise comigration with immunolabeled myelin basic proteins. In contrast, MARPP(14-20) were distinguished from histones, another group of low molecular weight, highly basic phosphoproteins, in these subcellular fractionation and immunochemical studies. Finally, we confirmed using two-dimensional electrophoresis, that changes observed previously by one-dimensional electrophoresis in the phosphorylation of MARPP(14-20) in response to acute and chronic morphine, and to acute forskolin, occur in myelin basic proteins. It was also found that changes in the state of phosphorylation of myelin basic proteins in response to chronic morphine occur without a change in the total amount of the proteins as determined by immunoblot analysis. The results demonstrate that the phosphorylation of myelin basic proteins is regulated by morphine in the nervous system, and raise the possibility that regulation of these proteins contributes to mechanisms underlying some of the acute and chronic actions of opiates in brain.
Animals↗
Opiates depress ACh and ATP release from cholinergic synaptosomes by blocking calcium uptake.
We have studied the effects of opiates on ATP and acetylcholine (ACh) release from cholinergic nerve terminals isolated from the electric organ of Torpedo marmorata. The release of ATP was inhibited by morphine and this action was reversed by naloxone. Morphine, [D-Met2-Pro5]enkephalinamide and [D-Ala2-Leu5]enkephalin also inhibited acetylcholine release. Naloxone prevented these inhibitory effects. The action of enkephalin on ACh release was less effective than that of morphine. The calcium uptake by nerve terminals of Torpedo electric organ was also inhibited by morphine, either under resting or depolarizing conditions, and this effect was reversed by naloxone. Using the quick freeze-fracture method, the structural changes induced by morphine in the presynaptic membrane were also studied. Morphine prevents the rearrangement of intramembrane particles (IMPs) at both freeze-fractured faces of the synaptosomal presynaptic membrane after depolarization. It is concluded that opiates depress the ATP and the ACh releases from cholinergic synaptosomes by inhibiting the calcium uptake by the nerve terminals and the rearrangement of the IMPs after potassium-induced depolarization. Furthermore, ACh release, but not ATP release, seems to be related with the rearrangement of IMPs in the presynaptic membrane.
Acetylcholine↗
Phorbol esters induce neurotransmitter release in cholinergic synaptosomes from Torpedo electric organ.
The effect of phorbol esters and so the involvement of Ca2+/phospholipid-dependent protein kinase (protein kinase C;PKC) in the release of acetylcholine (ACh) was studied using Torpedo electric organ synaptosomes. 12-O-Tetradecanoylphorbol 13-acetate (TPA), a known activator of PKC, induced neurotransmitter release in a concentration-dependent manner and increased the potassium-evoked release of ACh. The effect of TPA was shown to be independent of the extrasynaptosomal calcium concentration. TPA-induced ACh release was reversed by H-7, an inhibitor of PKC activity. This drug showed no effect on potassium-evoked ACh release. Botulinum toxin, a strong blocker of potassium-induced ACh release in that synaptosomal preparation, showed no inhibitory effect on the TPA-induced ACh release. Our results suggest that activation of PKC potentiates the release of an ACh pool that is not releasable by potassium depolarization, independently of the extracellular calcium concentration.
1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
Identification of MARPP-58, a morphine- and cyclic AMP-regulated phosphoprotein of 58 kDa, as tyrosine hydroxylase: evidence for regulation of its expression by chronic morphine in the rat locus coeruleus.
Previously, we have identified a number of morphine- and cyclic AMP-regulated phosphoproteins (MARPPs) in the rat locus coeruleus (LC) and other brain regions. We now show that one of these phosphoproteins, a 58 kDa protein designated MARPP-58, is tyrosine hydroxylase. First, MARPP-58 comigrates with immunolabeled, immunoprecipitated, and purified tyrosine hydroxylase on 1- and 2-dimensional electrophoresis. Second, MARPP-58, immunoprecipitated tyrosine hydroxylase, and purified tyrosine hydroxylase yield identical 1-dimensional phosphopeptide maps. Third, MARPP-58 exhibits a regional and subcellular distribution in brain consistent with tyrosine hydroxylase. Identification of MARPP-58 as tyrosine hydroxylase made it possible to determine whether increases in MARPP-58 phosphorylation induced by chronic morphine in the LC reported previously are associated with alterations in enzyme activity and expression in this brain region. We show that chronic treatment of rats with morphine increases levels of tyrosine hydroxylase activity, immunoreactivity, and mRNA in the LC. Induction of the enzyme by chronic morphine was blocked by concomitant treatment of rats with the opiate receptor antagonist naltrexone, indicating that morphine produces this effect through the activation of opiate receptors. Consistent with previous observations that the chronic morphine-induced change in MARPP-58 phosphorylation is specific to the LC, changes observed in enzyme activity, immunoreactivity, and mRNA were not observed in a number of other brain regions studied. The results indicate that chronic morphine regulates the expression of tyrosine hydroxylase specifically in the LC and suggest that such regulation reflects long-term adaptations of LC neurons to chronic morphine at the level of gene expression.
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Corticosterone differentially regulates the expression of Gs alpha and Gi alpha messenger RNA and protein in rat cerebral cortex.
The possibility that glucocorticoids regulate specific guanine nucleotide binding regulatory proteins (G proteins) was investigated in rat cerebral cortex. Corticosterone was administered to normal and bilaterally adrenalectomized rats, and hormone regulation of individual G-protein subunits was investigated in cerebral cortex in three ways: (i) immunoblot analysis of subunit protein, (ii) hybridization blot analysis of subunit mRNA, and (iii) ADP-ribosylation analysis of stimulatory G protein (Gs alpha) subunits. Chronic (7 days) corticosterone administration to normal rats increased levels of Gs alpha immunoreactivity, mRNA, and ADP-ribosylation but decreased levels of inhibitory G protein (Gi alpha) mRNA and tended to decrease levels of Gi alpha immunoreactivity. In contrast, levels of Go alpha and G beta immunoreactivity and mRNA were not influenced by corticosterone treatment. In adrenalectomized rats, corticosterone treatment produced a 25-50% increase in the levels of Gs alpha immunoreactivity, mRNA, and ADP-ribosylation, whereas the hormone produced a 20-35% decrease in the levels of Gi alpha immunoreactivity and mRNA. Adrenalectomy, without corticosterone replacement, produced the opposite effects on Gs alpha and Gi alpha compared to sham-operated controls, indicating that these G proteins are regulated by this class of steroid hormone under physiological conditions in vivo. The results indicate that specific G-protein subunits--namely, Gs alpha and Gi alpha--are under the coordinated control of glucocorticoids in rat brain and demonstrate that G proteins are physiological targets of glucocorticoids in vivo. Possible roles played by these G-protein responses in mediating the effects of glucocorticoids on brain function are discussed.
Adenosine Diphosphate Ribose↗
Identification of morphine- and cyclic AMP-regulated phosphoproteins (MARPPs) in the locus coeruleus and other regions of rat brain: regulation by acute and chronic morphine.
Regulation of cAMP-dependent protein phosphorylation by acute and chronic morphine was studied in the rat locus coeruleus (LC), a brain region that appears to play an important role in mediating morphine action in animals, including humans. Rats were treated chronically with morphine under conditions known to induce states of tolerance and dependence. Protein phosphorylation was then studied in extracts of LC and other brain regions with back-phosphorylation assays under different conditions and with 1- and 2-dimensional electrophoretic procedures. Evidence was obtained to suggest that chronic morphine increased the phosphorylation state of proteins of 165, 55, and 14-20 kDa and increased the total amount of proteins of 145, 71, 62, 58, and 51 kDa in the LC. Concomitant treatment of rats with naltrexone, an opiate receptor antagonist, blocked the ability of morphine to influence each of these proteins, indicating that morphine regulation of protein phosphorylation occurred through the specific activation of opiate receptors. Regulation of 165, 71, 62, 58, 55, and 51 kDa by chronic morphine was specific to the LC among the brain regions studied, whereas regulation of 145 and 14-20 kDa was also observed in the frontal cortex, neostriatum, and dorsal raphe. Most of the phosphoproteins whose phosphorylation state or total amount was increased by chronic morphine treatment in the LC in vivo were also shown to have their phosphorylation state decreased by acute morphine treatment in isolated LC nuclei ex vivo. In addition, the phosphorylation state of most of these morphine-regulated phosphoproteins was stimulated by forskolin or cAMP analogs in isolated LC and by cAMP in broken cell preparations of this brain region, supporting the view that these proteins are physiological substrates for cAMP-dependent protein kinase in the LC. Phosphoproteins regulated by morphine and cAMP were designated "MARPPs," morphine- and cAMP-regulated phosphoproteins, whereas those regulated by morphine but not by cAMP were designated "MRPPs." Taken together, the results of this study indicate that chronic morphine produces specific alterations in cAMP-dependent protein phosphorylation in the LC and raise the possibility that regulation of these specific phosphoproteins contributes to the development of tolerance and/or dependence in these neurons.
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Depolarization-stimulated protein phosphorylation in pure cholinergic nerve endings.
Cholinergic synaptosomes obtained from the electric organ of Torpedo marmorata have been used to study chemical stimulation-stimulated protein phosphorylation. Cholinergic synaptosomes were exposed to elevated K+0 concentrations or other chemical depolarizing agents such as gramicidin or secretagogues as the calcium ionophore A23187. During depolarization several synaptosomal proteins increase their state of phosphorylation. This phenomenon depends on the presence of Ca2+ in the external medium. These results suggest that stimulation of protein phosphorylation may be implicated in the acetylcholine release process and could represent a modulation mechanism in the neurotransmitter release machinery at this cholinergic synapse.
Animals↗
Botulinum neurotoxin inhibits depolarization-stimulated protein phosphorylation in pure cholinergic synaptosomes.
Botulinum neurotoxin, a strong blocker of acetylcholine release at peripheral cholinergic synapses, inhibits depolarization-stimulated protein phosphorylation in pure cholinergic synaptosomes isolated from the electric organ of Torpedo marmorata. Moreover, tetrodotoxin has the same effect on protein phosphorylation when cholinergic synaptosomes are depolarized by veratridine. Correlation between presynaptic protein phosphorylation and acetylcholine release is suggested by the fact that botulinum neurotoxin blocks specifically neurotransmitter release without affecting membrane depolarization and calcium fluxes in our synaptosomal preparation.
Animals↗