Desipramine Elicits the Expression of Opiate Receptors and Sulfogalactosylceramide Synthesis in Rat C6 Glioma Cells

In the course of our studies on lipidoses induced by amphiphilic drugs, we have investigated the ef- of desipramine, a tricyclic antidepressant, on glial cells in culture. We noted that the addition of desipramine to the culture medium of C6 glioma cells resulted in the modification of the lipid profile of the cell membranes. Of particular interest was the presence, in the desipramine-treated cells, of an additional lipid comigrating on thin layer chromatography with sulfogalactosylceramide (S-GalCer). Addition of radiolabelled sulfuric acid in the culture medium of the desipramine-treated cells resulted in the incorporation of [35S]sulfate in the newly synthesized lipid. Furthermore, this lipid was localized selectively by indirect immunofluorescence using a specific rabbit anti-S-GalCer antibody on the cell surface of desipramine-treated, but not control, C6 cells. Desipramine also increased the activity of 3'-phosphoadenosine-5'-phosphosulfate sulfotransferase (the enzyme responsible for the synthesis of S-GalCer). Since it has been suggested that S-GalCer may be involved in opiate receptors, we looked for opiate binding sites on C6 glioma cells after exposure to desipramine. We found that dihydromorphine was able to bind to the desipramine-treated C6 cell membrane. The binding of [3H]dihydromorphine (180 fmol/mg protein) was stereospecific and had a KD of 30-60 nM. Furthermore, morphine reduced both the basal and isoproterenol-stimulated cyclic AMP levels of the desipramine-treated C6 cells. This effect was blocked by naloxone. In these respects, the opiate binding sites induced after treatment of C6 glioma cells with desipramine fulfill the requirements of a true opiate receptor.     

Pattern of Protein Synthesis in Monkey Cells Infected by Simian Virus 40

After infection of several permanent monkey cell lines by simian virus 40 (SV40), four additional protein bands can be detected by simple sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole-cell extracts. These bands appear only after the onset of viral deoxyribonucleic acid (DNA) synthesis, and inhibitors of DNA synthesis prevent their appearance. Three of them correspond to three previously identified capsid components, VP1, VP2, and VP3. The fourth protein band, which does not correspond to a previously identified virion component, is induced by SV40 infection of CV-1 and BSC-1 cultures but not by infection of MA-134 cultures.     

Second messenger and protein phosphorylation mechanisms underlying opiate addiction: Studies in the rat locus coeruleus

We have studied the role of second messenger and protein phosphorylation pathways in mediating changes in neuronal function associated with opiate addiction in the rat locus coeruleus. We have found that chronic opiates increase levels of the G-protein subunits Gi and Go, adenylate cyclase, cyclic AMP-dependent protein kinase, and a number of phosphoproteins (including tyrosine hydroxylase) in this brain region. Electrophysiological data have provided direct support for the view that this up-regulation of the cyclic AMP system contributes to opiate tolerance, dependence, and withdrawal exhibited by these neurons. As the adaptations in G-proteins and the cyclic AMP system appear to occur at least in part at the level of gene expression, current efforts are aimed at identifying the mechanisms, at the molecular level, by which opiates regulate the expression of these intracellular messenger proteins in the locus coeruleus. These studies will lead to an improved understanding of the biochemical basis of opiate addiction.Special issue dedicated to Dr. Paul Greengard     

Effects of 5,5''-Dithiobis-(2-Nitrobenzoic Acid) on Opiate Binding to Both the Membrane-Bound Receptor and the Partially Purified Opiate Receptor

Pretreatment of partially purified opiate receptor from rat brains with 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) decreased opiate agonist binding more effectively than that of antagonist. This agent, at a concentration that inhibits only 3H-agonist binding, increases the IC50 values of agonists but not those of antagonists. We also observed similar effects of DTNB on opiate binding to the membrane-bound receptor that are in good agreement with the published data. Moreover, there was an excellent correlation between the IC50 values of the two different preparations. However, opiate binding to the partially purified receptor was about a thousandfold more sensitive to DTNB than binding to this membrane-bound receptor. Dithiothreitol, a sulfide bond reducing agent, reversed the effects of DTNB on the opiate binding.     

Basic research on heredity and alcohol: Implications for clinical application

Abstract

Alcoholism has been described as a behavioral condition comprised of symptoms of alcohol dependence and the psycho‐socio‐biologic consequences of chronic alcohol dependence. Progress in clarifying the role of genetic factors in explaining differences in onset of dependence upon alcohol, frequency of consequences of chronic alcohol use, and transmission of patterns of alcoholism within a family pedigree has been based upon use of diagnostic methods that reliably and validly separate alcohol dependence from alcohol abuse. Twin methods, which control for genotypic variation, and adoption studies, which control for differences in rearing, have provided significant support for a genetic vulnerability hypothesis for development of alcoholism and a genetic heterogeneity hypothesis for type of alcoholism. The author reviews data from basic and clinical investigation of two subtypes of alcoholism: one associated with antisocial personality, and one that is “familial” (family‐history‐positive alcoholism). Significant differences in onset and clinical course for these subtypes suggest that differential plans for matching treatment to the individual alcoholic may be warranted.     

Involvement of the hypothalamus in opiate-stimulated prolactin secretion

Administration of opiate agonists to rats is known to elevate plasma prolactin, an effect which is antagonised by the opiate antagonist naloxone. However, this appears not to be a result of a direct action at the pituitary gland. We report here that opiate agonists stimulate prolactin secretion from isolated adenohypophysial cells when they are coincubated with hypothalamic fragments. Both morphine and Met-enkephalin stimulated prolactin secretion by 1.84 fold and 1.50 fold respectively, and this was antagonised by naloxone. These findings support the hypothesis that one site of action of opioid compounds on pituitary hormone secretion is at the level of hypothalamus.     

Age-Dependent Striatal Excitotoxic Lesions Produced by the Endogenous Mitochondrial Inhibitor Malonate

Abstract: Cocultures of spinal cord and dorsal root ganglion cells contain relatively high concentrations of k -opiate receptors. We have previously shown that acute k -opiate agonist treatment reduces phosphorylation of synapsin I stimulated by depolarizing agents (such as 60 m M KCl). Here we show that prolonged opiate treatment increases the levels of synapsin I immunoreactivity in the cells. Several opiate agonists, such as U50488, ethylketocyclazocine, dynorphin, and [D-Ala²,D-Leu⁵]enkephalin, caused a 3.0–3.4-fold increase in the immunoreactive level of synapsin I. The effect of the k -agonist U50488 on the up-regulation of synapsin I was dose dependent and was blocked by the k -opiate antagonist norbinaltorphimine. The results suggest that continued activation of opiate receptors by chronic agonist treatment up-regulates the levels of synapsin I. This increase in synapsin I could contribute to the development of tolerance to opiates.     

Loss of Protein Kinase C-αβ in Brain of Heroin Addicts and Morphine-Dependent Rats

Abstract: The biochemical status of human brain protein kinase C (PKC)-αβ during opiate dependence was studied by means of immunoblotting techniques in postmortem brain of heroin addicts who had died by opiate overdose. In the frontal cortex, a marked decrease (53%, p < 0.05) in the immunoreactivity of PKC-αβ was found in heroin addicts compared with matched controls. The loss of PKC-αβ in the brain of human addicts paralleled that observed in the frontal cortex of rats after chronic treatment with morphine (10–100 mg/kg i.p. for 5 days) (PKC-αβ decreased by 34%, p < 0.05). Chronic treatment with naloxone (1 mg/kg i.p. every 12 h for 5 days) did not alter PKC-αβ immunoreactivity in the rat brain. However, in morphine-dependent rats, naloxone-precipitated withdrawal induced a rapid and strong behavioral reaction with a concomitant up-regulation of PKC-αβ immunoreactivity to control values. These results indicated that the decrease of brain PKC-αβ induced by heroin/morphine is a μ-opioid receptor-mediated effect. The chronic administration of opiates has been associated with a marked sensitization of the adenylyl cyclase/cyclic AMP system, although this phenomenon is not exclusive of the opioid system but the general cellular adaptation to chronic inhibition of adenylyl cyclase. In this context, chronic treatment of rats with other inhibitory agonists (e.g., clonidine, 1 mg/kg i.p. every 12 h for 14 days) acting through receptors (e.g., α₂-adrenoceptors) also coupled to adenylyl cyclase did not alter brain PKC-αβ immunoreactivity. Together these findings suggest that the brain PKC system might play a major role in opiate addiction.     

Regulation of G Protein-Coupled Receptor Kinase 2 in Brains of Opiate-Treated Rats and Human Opiate Addicts

Abstract: The effects of opiate drugs (heroin, morphine, and methadone) on the levels of G protein-coupled receptor kinase 2 (GRK2) were studied in rat and human brain frontal cortices. The density of brain GRK2 was measured by immunoblot assays in acute and chronic opiate-treated rats as well as in opiate-dependent rats after spontaneous or naloxone-precipitated withdrawal and in human opiate addicts who had died of an opiate overdose. In postmortem brains from human addicts, total GRK2 immunoreactivity was not changed significantly, but the level of the membrane-associated kinase was modestly but significantly increased (12%) compared with matched controls. In rats treated chronically with morphine or methadone modest increases of the enzyme levels (only significant after methadone) were observed. Acute treatments with morphine and methadone induced dose- and time-dependent increases (8–22%) in total GRK2 concentrations [higher increases were observed for the membrane-associated enzyme (46%)]. Spontaneous and naloxone-precipitated withdrawal after chronic morphine or methadone induced a marked up-regulation in the levels of total GRK2 in the rat frontal cortex (18–25%). These results suggest that GRK2 is involved in the short-term regulation of μ-opioid receptors in vivo and that the activity of this regulatory kinase in brain could have a relevant role in opiate tolerance, dependence, and withdrawal.     

Three autocrine feedback loops determine HIF1 alpha expression in chronic hypoxia

Hypoxia occurs in cancer, prolonged exercise, and long-term ischemia with durations of several hours or more, and the hypoxia-inducible factor 1 (HIF1) pathway response to these conditions differs from responses to transient hypoxia. We used computational modeling, validated by experiments, to gain a quantitative, temporal understanding of the mechanisms driving HIF1 response. To test the hypothesis that HIF1 alpha protein levels during chronic hypoxia are tightly regulated by a series of molecular feedbacks, we took into account protein synthesis and product inhibition, and analyzed HIF1 system changes in response to hypoxic exposures beyond 3 to 4 h. We show how three autocrine feedback loops together regulate HIF 1 alpha hydroxylation in different microenvironments. Results demonstrate that prolyl hydroxylase, succinate and HIF1 alpha feedback determine intracellular HIF1 alpha levels over the course of hours to days. The model provides quantitative insight critical for characterizing molecular mechanisms underlying a cell's response to long-term hypoxia.     

Chronic morphine alters the presynaptic protein profile: identification of novel molecular targets using proteomics and network analysis

Opiates produce significant and persistent changes in synaptic transmission; knowledge of the proteins involved in these changes may help to understand the molecular mechanisms underlying opiate dependence. Using an integrated quantitative proteomics and systems biology approach, we explored changes in the presynaptic protein profile following a paradigm of chronic morphine administration that leads to the development of dependence. For this, we isolated presynaptic fractions from the striata of rats treated with saline or escalating doses of morphine, and analyzed the proteins in these fractions using differential isotopic labeling. We identified 30 proteins that were significantly altered by morphine and integrated them into a protein-protein interaction (PPI) network representing potential morphine-regulated protein complexes. Graph theory-based analysis of this network revealed clusters of densely connected and functionally related morphine-regulated clusters of proteins. One of the clusters contained molecular chaperones thought to be involved in regulation of neurotransmission. Within this cluster, cysteine-string protein (CSP) and the heat shock protein Hsc70 were downregulated by morphine. Interestingly, Hsp90, a heat shock protein that normally interacts with CSP and Hsc70, was upregulated by morphine. Moreover, treatment with the selective Hsp90 inhibitor, geldanamycin, decreased the somatic signs of naloxone-precipitated morphine withdrawal, suggesting that Hsp90 upregulation at the presynapse plays a role in the expression of morphine dependence. Thus, integration of proteomics, network analysis, and behavioral studies has provided a greater understanding of morphine-induced alterations in synaptic composition, and identified a potential novel therapeutic target for opiate dependence.     

The development of opiate tolerance may dissociate from dependence

Experiments on opiate sensitive peripheral tissue preparations such as the mouse vas deferens and the guinea-pig ileum have demonstrated the ability to induce very high degrees of selective tolerance towards particular opiate agonists. Interestingly, the highly tolerant mouse vas deferens failed to display any sign of dependence as judged by the inability of naloxone to precipitate a withdrawal sign. In analogy, the present studies on the guinea-pig ileum revealed a striking dissociation in the degree of tolerance and dependence developed. Opiate receptor binding studies on both tissues point to distinct differences in the opiate-induced effector mechanisms. It is concluded that adaptational changes upon chronic opiate receptor activation may occur at multiple sites within the effector system of the opiate receptor.     

Translational control in early sea urchin embryogenesis: initiation factor eIF4F stimulates protein synthesis in lysates from unfertilized eggs of Strongylocentrotus purpuratus

We have used cell-free translation systems from unfertilized eggs and embryos of the sea urchin Strongylocentrotus purpuratus to analyze the mechanisms limiting protein synthesis in early embryogenesis. Unfertilized egg lysates supplemented with nuclease-treated reticulocyte lysate were stimulated 2-4-fold in incorporation of radioactive amino acid into protein. Thirty-minute zygote lysates supplemented in this way were not stimulated. These results suggested that a component limiting translation in the unfertilized egg lysate was provided by the nuclease-treated lysate and that this component was no longer limiting protein synthesis following fertilization. In view of these results, partially fractionated lysates and individual purified translational components from mammalian cells were tested for stimulation of the unfertilized egg lysate. A 1000000g supernatant devoid of ribosomal subunits also stimulated the unfertilized egg lysate. Thus, the stimulation was not due to the addition of active ribosomal subunits but to soluble elements in the reticulocyte lysate. Of the soluble components tested, only the cap-binding protein complex eIF4F caused a dramatic stimulation of the unfertilized egg lysate (2-3.5-fold). The 30-min zygote lysate was not stimulated by eIF4F or by any of the other components tested, supporting the hypothesis that a block in the translational machinery is removed at fertilization. A rabbit reticulocyte shift assay was used to analyze whether mRNA is limiting in early development. When unfertilized egg lysate was added to the shift assay, there was no shift in radioactivity from 43S to 80S complexes, indicating the unfertilized egg mRNA is not available for translation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cortical Components of Reaction-Time during Perceptual Decisions in Humans

The mechanisms of perceptual decision-making are frequently studied through measurements of reaction time (RT). Classical sequential-sampling models (SSMs) of decision-making posit RT as the sum of non-overlapping sensory, evidence accumulation, and motor delays. In contrast, recent empirical evidence hints at a continuous-flow paradigm in which multiple motor plans evolve concurrently with the accumulation of sensory evidence. Here we employ a trial-to-trial reliability-based component analysis of encephalographic data acquired during a random-dot motion task to directly image continuous flow in the human brain. We identify three topographically distinct neural sources whose dynamics exhibit contemporaneous ramping to time-of-response, with the rate and duration of ramping discriminating fast and slow responses. Only one of these sources, a parietal component, exhibits dependence on strength-of-evidence. The remaining two components possess topographies consistent with origins in the motor system, and their covariation with RT overlaps in time with the evidence accumulation process. After fitting the behavioral data to a popular SSM, we find that the model decision variable is more closely matched to the combined activity of the three components than to their individual activity. Our results emphasize the role of motor variability in shaping RT distributions on perceptual decision tasks, suggesting that physiologically plausible computational accounts of perceptual decision-making must model the concurrent nature of evidence accumulation and motor planning.     

Factors conducive to increased protein feeding by the blowfly Phormia regina

ABSTRACT. Each period of elevated protein consumption by Phormia regina was associated with some recognizable physiological event that requires protein synthesis (e.g. tanning, adult fat body development, vitellogenesis). Injecting cycloheximide to inhibit protein synthesis prior to the onset of such events delayed the events and also the development of the associated periods of protein hunger. Injecting cycloheximide after a known period of protein synthesis had occurred, however, did not postpone the normally associated protein hunger even while the inhibitor was fully active. The findings conform to a hypothesis that an unidentified deficit, created by protein synthesis, underlies the tendency by Phormia to increase its ingestion of proteinaceous materials. The effects which different hormones have on protein hunger are evidently indirect results of their control of different periods of protein synthesis.     

Relations between hippocampal network free oscillations and the theta activity

The relations between the component of the hippocampal network free oscillations, i.e., sharp potential waves (SPW) and the theta component of the hippocampal EEG are considered. It is commonly accepted that, in the course of changes from the state corresponding to a weak activation of the hippocampal network in the theta rhythm by the septum through the state corresponding to a medium activation to the state of a high activation, the inverse dependence between the magnitudes of these components in the EEG spectra takes place. However, our work shows that this dependence can be deranged in the rat EEG during quiet wakefulness that corresponds to the medium activation of the hippocampal network in theta rhythm. It was also shown that the RF stimulation can evoke abnormal signals with well-pronounced theta activity against the background of a considerable SPW component. This phenomenon demonstrates a possibility of a strong derangement of the dependence, which is, as a rule, observed under the natural conditions. The hypothesis concerning the organization and regulation of the investigated components of the hippocampal EEG is proposed.     

The effect of metabolic acidosis on the synthesis and turnover of rat renal phosphate-dependent glutaminase.

Regulation of the mitochondrial phosphate-dependent glutaminase activity is an essential component in the control of renal ammoniagenesis. Alterations in acid-base balance significantly affect the amount of the glutaminase that is present in rat kidney, but not in brain or small intestine. The relative rates of glutaminase synthesis were determined by comparing the amount of [35S]methionine incorporated into specific immunoprecipitates with that incorporated into total protein. In a normal animal, the rate of glutaminase synthesis constitutes 0.04% of the total protein synthesis. After 7 days of metabolic acidosis, the renal glutaminase activity is increased to a value that is 5-fold greater than normal. During onset of acidosis, the relative rate of synthesis increases more rapidly than the appearance of increased glutaminase activity. The increased rate of synthesis reaches a plateau within 5 days at a value that is 5.3-fold greater than normal. Recovery from chronic acidosis causes a rapid decrease in the relative rate of glutaminase synthesis, but a gradual decrease in glutaminase activity. The former returns to normal within 2 days, whereas the latter requires 11 days. The apparent half-time for glutaminase degradation was found to be 5.1 days and 4.7 days for normal and acidotic rats respectively. These results indicate that the increase in renal glutaminase activity associated with metabolic acidosis is due primarily to an increase in its rate of synthesis. From the decrease in activity that occurs upon recovery from acidosis, the true half-life for the glutaminase was estimated to be 3 days.