Protein phosphorylation in primary astrocyte cultures treated with and without dibutyryl cyclic AMP

Protein phosphorylation was investigated in primary rat astrocyte cultures treated with and without dibutyryl cyclic AMP. Astrocytes maintained in dibutyryl cyclic AMP for several weeks displayed increased phosphate incorporation in 5 protein bands (55, 52, 45, 43 and 28 kDa) while incorporation in one band (42 kDa) was decreased. Phosphate incorporation in several other protein bands was unchanged. Calcium-dependent phosphate incorporation was also altered by prior exposure of the cells to dibutyryl cyclic AMP: addition of calcium to broken cell preparations resulted in increased incorporation in 75, 53 and 52 kDa while decreased incorporation occurred in 100 kDa. These differences in protein phosphorylation may be related to the previously reported biochemical and morphological changes brought about by dibutyryl cyclic AMP and may provide insights into the mechanisms of reactive gliosis.     

Relationship between phosphorylation of blood platelet proteins and secretion of platelet granule constituents II. Effects of different inhibitors.

The increased phosphorylation of platelet polypeptides in the mol. wt. range 48,000 to 40,000 (P48-40) caused by exposure of intact platelets to collagen fibres was inhibited by concentrations of tetracaine, verapamil, indomethacin, N-ethylmaleimide, cytochalasin B, N⁶, 2′-0-dibutyryl cyclic AMP or prostaglandin E₁ that also inhibited the release of platelet 5-hydroxytryptamine by collagen. The smaller increase in phosphorylation of polypeptides in the mol. wt. range 25,000 to 19,000 (P25-19) induced by collagen was also shown to be inhibited by these drugs with the apparent exceptions of prostaglandin E₁ and cytochalasin B. In the former case this was because prostaglandin E₁ itself significantly increased phosphorylation of one or more polypeptides contained in P25-19. The results support previous evidence of a close functional relationship between the increased phosphorylation of specific platelet polypeptides and the secretion of platelet granule constituents and suggest a possible difference in the mechanisms responsible for the inhibitory actions of N⁶, 2′-0-dibutyryl cyclic AMP and prostaglandin E₁.     

Protein phosphorylation in isolated synaptic junctional structures: changes during development

Endogenous phosphorylation in subcellular fractions enriched in synaptic plasma membranes (SPM) and purified synaptic junction (SJ) has been examined in vitro at various stages of postnatal development. Protein kinase activity was measured using both endogenous and exogenous (histones) proteins as substrates. Protein phosphorylation that is regulated by cyclic AMP also was investigated. SPM and SJ fractions displayed large increments in kinase activities and cyclic AMP-stimulated protein phosphorylation between postnatal days 5 and 15. SJ fractions exhibited a dramatic age-dependent change in the cyclic AMP-stimulated phosphorylation of endogenous proteins of molecular weights 73,000 (1a), 68,000 (1b), 65,000 (1c) and 55,000. Experiments in which isolated SJs were phosphorylated with soluble cyclic AMP-dependent kinase showed that the phosphorylation of proteins 1a, 1b and 1c resulted from their de novo appearance in newborn SJ fractions and not from a maturation-dependent coupling of kinases and substrates that were already present in newborn SJ fractions. The levels of regulatory (R) subunits of cyclic AMP-dependent kinases in synaptic fractions were measured by [³H]cyclic AMP binding and photoaffinity labeling with [³²P]8-N₃-cyclic AMP and were observed to change little during postnatal development. These findings show that there is a strong correlation between the in situ appearance of synapses and the enzymatic maturation of endogenous, cyclic AMP-stimulated protein phosphorylation in SJ fractions isolated at different stages of development.     

Human brain protein phosphorylation in vitro: Cyclic AMP stimulation of electrophoretically-separated substrates

In vitro phosphorylation of electrophoretically-separated brain proteins was studied in human frontal cortex obtained 3-16 h post-mortem from 13 patients ages 3 days-82 years with extensive, mild or no neuropathological involvement. In 12 of the 13 cases, cyclic AMP increased incorporation of phosphate into acid-precipitable protein. Analysis of the autoradiographic profiles of separate proteins indicated that phosphorylation of the doublet of molecular weight 86-80,000 was stimulated by cyclic AMP in certain samples. This doublet corresponded to the cyclic AMP stimulated doublet from rat frontal cortex we have termed band D-1,2 (proteins Ia and Ib of Ueda and Greengard). Of special interest was the fact that, while co-migration was observed in the other phosphoprotein bands studied, band D-1,2 of humans consistently migrated slightly less than rat protein band D-1,2. This difference was not a function of post-mortem time, subcellular fraction or buffer used in the reaction phosphorylation assay. The use of post-mortem tissue was not a contributing factor as the retardation in band D-1,2 migration was still observed when post-mortem rat brain was used for comparison. In two human post-mortem samples, there was no measureable band D-1,2 phosphorylation even in the presence of cyclic AMP. This was the case in both homogenate and crude synaptosome/mitochondrial preparations. Band F-1 (mol. wt. = 47,000) was not observed in any of the human samples studied. This is consistent with prior studies in rat which show that band F-1 phosphorylation is not detected in post-mortem brain, Band F-2 (mol. wt. 41,000) recently identified as pyruvate dehydrogenase, was lightly phosphorylated under the reaction conditions used in this study.     

Phosphorylation of GluR1, ERK,and CREB during spontaneous withdrawal from chronic heroin self‐administration

Negative motivational symptoms are observed soon after withdrawal from chronic opiate administration, and are thought to mediate dependence. Examination of brain region‐specific signaling changes that accompany early withdrawal may shed light on neural mechanisms underlying negative reinforcement and dependence. Thus, we measured alterations in protein phosphorylation in multiple limbic brain regions in rats undergoing 24 h spontaneous or naltrexone‐precipitated withdrawal from chronic (6 h/day) i.v. heroin self‐administration. Region‐specific increases in cyclic AMP‐dependent GluR₁^S845 phosphorylation were found in the nucleus accumbens shell, basolateral amygdala, hippocampal CA1 and CA3 subregions, and premotor cortex from 12 to 24 h of spontaneous withdrawal, and there were no changes in prefrontal cortex, nucleus accumbens core or caudate‐putamen. Increased GluR₁^S845 phosphorylation was detected earlier (12 h withdrawal) in the central amygdala and ventral tegmental area. In contrast, prominent increases in extracellular signal‐regulated kinase phosphorylation were found in both prefrontal and premotor cortex, and CA1 and CA3 between 12 and 24 h withdrawal. Phosphorylation of striatal cyclic AMP response element binding protein increased in the caudate‐putamen but not in the nucleus accumbens. Naltrexone administration after 24 h withdrawal increased extracellular signal‐regulated kinase phosphorylation in the central amygdala, and nucleus accumbens core and shell. Thus, spontaneous withdrawal from heroin self‐administration produces region‐ and time‐dependent changes in cyclic AMP and extracellular signal‐regulated kinase activity that could contribute to the behavioral manifestation of opiate dependence. Synapse 63:224–235, 2009. © 2008 Wiley‐Liss, Inc.     

Ca and calmodulin-regulated endogenous tubulin kinase activity in presynaptic nerve terminal preparations

Synaptosomal tubulin was shown to be the major substrate for a Ca²⁺-calmodulin regulated protein kinase in synaptosome soluble fractions as determined by two-dimensional gel electrophoresis and peptide mapping. Ca²⁺ activated this endogenous tubulin kinase system in presynaptic nerve terminal preparations. The Ca²⁺-dependent activation of the tubulin kinase system was mediated by the Ca²⁺ binding protein, calmodulin. Trifluoperazine, a known inhibitor of calmodulin, significantly blocked the calmodulin-stimulated [³²P]phosphate incorporation into synaptic tubulin. This inhibition of endogenous tubulin phosphorylation could be reversed by addition of exogenous calmodulin to the reaction mixture. The concentrations of Ca²⁺ and calmodulin required to produce a half-maximal stimulation of the tubulin kinase were 0.8 μM and 0.3 μM respectively. Greater than 70% of soluble tubulin present in the nerve terminal was phosphorylated in less than 50 s by this kinase system. Evidence is presented indicating that the synaptic Ca²⁺-calmodulin tubulin kinase is a distinct enzyme system from the previously described cyclic AMP microtubule-associated kinase. The anticonvulsant phenytoin inhibited the Ca²⁺-calmodulin stimulated phosphorylation of tubulin, and α- and β-tubulin were identified as major components of previously designated synaptic phosphoprotein bands DPH-L and DPH-M. Existence of the kinase as a calmodulin-tubulin-kinase complex is suggested from kinetic studies. The Ca²⁺-calmodulin tubulin kinase is very labile and specialized isolation procedures were necessary to retain activity. The activation of the tubulin kinase by Ca²⁺ and calmodulin may play a role in the functional utilization of tubulin in the nerve terminal and may mediate some of the effects of Ca² on synaptic function.     

Increase of calmodulin-stimulated striatal particulate phosphorylation response in chronic haloperidol-treated rats

Calcium- and calmodulin-dependent protein kinase and phosphatase activities were studied in rat striatal particulate preparations. The effect of Ca²⁺ (0.1–0.5 mM) on phosphorylation was completely abolished in the preparation which had been washed 3 times with a buffer containing ethylene glycol bis-(β-aminoethyl ether)-N,N′-tetraacetic acid (EGTA, 1.2 mM). Ca²⁺-stimulated phosphorylation was restored in a dose-dependent manner after calmodulin (1 μg) was added to the assays. Ca²⁺ and calmodulin promoted the phosphate incorporation into two major striatal protein bands with estimated M_r at 52 and 40 kdaltons. The presence of phosphatase in the EGTA-pretreated preparations was negligible. Chronic treatment in rats with haloperidol (1 mg/kg, 20 days) produced a significant decrease in the Ca²⁺-independent phosphorylation but an increase in the extent of Ca²⁺ and calmodulin-dependent phosphorylation in the striatum. The chronic haloperidol treatment did not alter the striatal [¹²⁵I]calmodulin binding curve. In vitro, haloperidol (even at 10⁻⁴ M) had no effect on calmodulin-dependent phosphorylation. Haloperidol (10⁻⁴ M) did not reduce the number but decreased the rate of [¹²⁵I]calmodulin binding to the striatal particulates. These data suggest that the link between dopamine receptors and calmodulin-dependent enzyme is dissociated in vitro. On the other hand, the potential sensitivity of calmodulin-dependent protein kinase in the chronic haloperidol-treated rats correlated with the supersensitive dopamine receptor responses occurred in these rats. Therefore, calmodulin-dependent protein kinase may biochemically regulate dopamine receptor function in the striatum in vivo.     

Regulation of Chromogranin A and Chromogranin B (Secretogranin I) Synthesis in Bovine Cultured Chromaffin Cells

In the present study we investigated the regulation of Chromogranin A (CGA) and Chromogranin B (CGB) biosynthesis in bovine chromaffin cells maintained in primary culture. Cellular proteins were labelled with [³⁵S]methionine and the incorporated radioactivity was used as an index of the synthesis rate. The radioactivity incorporated into CGA was determined by immunoprecipitation, and that into CGB was quantified by a dot immunobinding assay using specific antibodies. Incubation of cells with carbamylcholine, nigh K⁺ or histamine, three potent stimulators of catecholamine secretion in chromaffin cells, increased the rate of CGA and CGB synthesis. On the other hand bradykinin, angiotensin II and prostaglandin E₂, which cause little secretion, also produced an increase in both CGA and CGB synthesis. These results suggest that in chromaffin cells, the biosynthesis of chromogranins is not closely linked to the secretory activity. Inhibition of protein kinase C by sphingosine or by long-term treatment with phorbol esters, completely abolished the synthesis of CGA and CGB induced by carbamylcholine, bradykinin and prostaglandin E² but decreased only partially the stimulating effect of histamine. Thus, protein kinase C may not be the sole effector involved in the secretagogue-induced modulation of Chromogranin synthesis. Forskolin, an activator of adenylate cyclase had no effect on CGA synthesis, but significantly enhanced the incorporation of radioactivity into CGB. The effect of forskolin was not modified by protein kinase C inhibitors and was additive to that induced by phorbol esters indicating that cyclic AMP did not stimulate CGB synthesis through a protein kinase C-dependent pathway. These observations suggest that the biosynthesis of CGA and CGB in chromaffin granules is independently regulated.     

Norepinephrine-mediated protein phosphorylation in astrocytes

Studies were conducted to determine if norepinephrine activates both protein kinase C and the cyclic AMP-dependent protein kinase in cultured rat astrocytes using phosphoproteins as markers. Norepinephrine was found to decrease 32P incorporation into an acidic 80,000 M(R) protein. A similar response was observed with isoproterenol and cyclic AMP analogs. In contrast, phorbol myristate acetate (PMA) increased 32P incorporation into this protein. Further studies looked at phosphorylation sites on glial fibrillary acidic protein and vimentin using two-dimensional tryptic phosphopeptide maps. The pattern of phosphorylation of these two proteins by norepinephrine resembles that of 8-bromo cyclic AMP and isoproterenol, and not that of PMA. Additionally, the effect of norepinephrine on the phosphorylation of GFAP and vimentin was blocked by alprenolol. One difference noted between norepinephrine and isoproterenol was the phosphorylation of an 18,000 M(R) protein. Norepinephrine increased, and isoproterenol decreased, 32P incorporation into this protein; however, the mechanism which mediates the norepinephrine effect remains to be determined. Overall, these studies indicate that the most prominent phosphorylation events mediated by norepinephrine are the consequence of the activation of cyclic AMP-dependent protein kinase.     

Altered responsiveness of adenosine 3′,5′-monophosphate-generating systems in brain slices from adult rats after neonatal treatment with 6-hydroxydopamine

Sprague-Dawley rat pups were administered 6-hydroxydopamine (100 mg/kg) subcutaneously on each of the first 4 days after birth. At 4–5 mo of age the rats and untreated littermates were killed, and the accumulation of [¹⁴C]cyclic AMP elicited by various agents in [¹⁴C]adenine-labeled brain slices was investigated. The accumulation of cyclic AMP elicited by norepinephrine was significantly greater in slices from neocortex and midbrain of 6-hydroxydopamine-treated rats than in corresponding slices from littermate controls. Accumulations in slices from medulla-pons and cerebellum were not significantly altered by the neonatal treatment with 6-hydroxydopamine. Thus, an enhanced response to norepinephrine pertains both in a region, the neocortex, where norepinephrine-levels were permanently reduced by neonatal treatment with 6-hydroxydopamine and in a region, the midbrain, where adult levels of norepinephrine were nearly doubled by the neonatal treatment with 6-hydroxydopamine. Enhanced responses of cyclic AMP-generating systems in neocortical slices from 6-hydroxydopamine-treated rats also pertained with isoproterenol, prostaglandin E₁ and an adenosine-norepinephrine combination, but not with adenosine alone. Depletion of norepinephrine throughout the brain by administration of either reserpine or intraventricular 6-hydroxydopamine to adult rats enhanced responses to norepinephrine in slices from neocortex, but not midbrain. Reserpine had no effect on responses in either neocortex or midbrain of rats treated neonatally with 6-hydroxydopamine.