Phosphorylation and nucleotidylation of proteins in Rhodocyclus gelatinosus

Abstract Cells of Rhodocyclus gelatinosus were radioactively labeled by addition of [³²P]orthophosphate, [¹⁴C]inosine or [¹⁴C]orotic acid during anaerobic growth on citrate in the light. Protein analysis by two-dimensional gel electrophoresis and autoradiography of the gels revealed the presence of several radioactively labeled protein species in this organism. The molecular mass and the isoelectric point of all these proteins were determined. Treatment of the ³²P-labeled protein fractions with acid and alkaline phosphatase clearly showed that at least 8 protein species were modified by phosphorylation. The experiments conducted with the ¹⁴C-labeled precursors of purines and pyrimidines indicated the presence of 4 protein species which were modified by a compound containing a purine and phosphate, and a single protein simultaneously being labeled with pyrimidine and phosphate.     

Pseudomonas testosteroni: new data about growth and steroid metabolism

Abstract Escherichia coli cells were labeled with [³²P]orthophosphate under anaerobic conditions using nitrate as electron acceptor. By 2-dimensional gel electrophoresis, 20 protein species were found to be radioactively labeled; their isoelectric points and molecular masses were determined. Treating the labeled extract with alkaline phosphatase revealed evidence for the presence of 9 proteins modified by phosphorylation. Treatment with snake venom phosphodiesterase also indicated that 2 protein species in E. coli were modified by phosphate-containing compounds which were bound to the protein by a phosphodiester bond.     

Neuronal Cyclin-Dependent Kinase-5 Phosphorylation Sites in Neurofilament Protein (NF-H) Are Dephosphorylated by Protein Phosphatase 2A

Abstract: Neurofilament (NF) protein [high molecular mass (NF-H)] is extensively phosphorylated in vivo. The phosphorylation occurs mainly in its characteristic KSP (Lys-Ser-Pro) repeat motifs. There are two major types of KSP motifs in the NF-H tail domain: KSPXKX and KSPXXX. Recent studies by two different laboratories have demonstrated the presence of a cdc2-like kinase [cyclin-dependent kinase-5 (cdk5)] in nervous tissue that selectively phosphorylates KSPXKX and XS/TXK motifs in NF-H and lysine-rich histone (H1). This article describes the identification of phosphatases dephosphorylating three different substrates: histone (H1), NF-H in a NF preparation, and a bacterially expressed C-terminal tail domain of NF-H, each containing KSPXKX repeats phosphorylated in vitro by cdk5. Among various phosphatases identified, protein phosphatase (PP) 2A from rabbit skeletal muscle appeared to be the most effective phosphatase in in vitro assays. Three phosphatase activity peaks—P1, P2, and P3—were partially purified from frozen rat spinal cord by ion exchange and size exclusion column chromatography and then characterized on the basis of biochemical, pharmacological, and immunochemical studies. One of the three peaks was identified as PP2A, whereas the others were mixtures of both PP2A and PP1. These three peaks could dephosphorylate cdk5-phosphorylated ³²P-histone (H1), ³²P-NF-H in the NF preparation, and ³²P-NF-H tail fusion protein. These studies suggest the involvement of PP2A or a PP2A-like activity in the regulation of the phosphorylation state of KSPXKX motifs in NF-H.     

Differential Inactivation of Postsynaptic Density-Associated and Soluble Ca2+/Calmodulin-Dependent Protein Kinase II by Protein Phosphatases 1 and 2A

Abstract: Autophosphorylation of Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) at Thr²⁸⁶ generates Ca²⁺-independent activity. As an initial step toward understanding CaMKII inactivation, protein phosphatase classes (PP1, PP2A, PP2B, or PP2C) responsible for dephosphorylation of Thr²⁸⁶ in rat forebrain subcellular fractions were identified using phosphatase inhibitors/activators, by fractionation using ion exchange chromatography and by immunoblotting. PP2A-like enzymes account for >70% of activity toward exogenous soluble Thr²⁸⁶-autophosphorylated CaMKII in crude cytosol, membrane, and cytoskeletal extracts; PP1 and PP2C account for the remaining activity. CaMKII is present in particulate fractions, specifically associated with postsynaptic densities (PSDs); each protein phosphatase is also present in isolated PSDs, but only PP1 is enriched during PSD isolation. When isolated PSDs dephosphorylated exogenous soluble Thr²⁸⁶-autophosphorylated CaMKII, PP2A again made the major contribution. However, CaMKII endogenous to PSDs (³²P autophosphorylated in the presence of Ca²⁺/calmodulin) was predominantly dephosphorylated by PP1. In addition, dephosphorylation of soluble and PSD-associated CaMKII in whole forebrain extracts was catalyzed predominantly by PP2A and PP1, respectively. Thus, soluble and PSD-associated forms of CaMKII appear to be dephosphorylated by distinct enzymes, suggesting that Ca²⁺-independent activity of CaMKII is differentially regulated by protein phosphatases in distinct subcellular compartments.     

Allosteric Interactions Among Agonists and Antagonists at 5-Hydroxytryptamine3 Receptors

Abstract: Cooperation in the action of agonists suggests that there are multiple binding sites on 5-hydroxytryptamine₃ (5-HT₃) receptors. The purpose of this study was to characterize these binding sites and their interactions on both native and cloned 5-HT₃ receptors. The affinities of competitive 5-HT₃ receptor antagonists were similar regardless of whether the receptors were labeled with [³H]RS-42358, [³H]granisetron, or 1-( m -[³H]chlorophenyl)biguanide ([³H]mCPG). By contrast, the affinities of the agonists 5-HT, mCPG, and phenylbiguanide were approximately 10-fold higher when the receptors were labeled with [³H]mCPG. The dissociation of [³H]mCPG, [³H]RS-42358, and [³H]RS-25259, but not [³H]granisetron, from both cloned and native 5-HT₃ receptors was markedly slower in the presence of 5-HT or 2-methyl-5-HT than in the presence of antagonists such as RS-42358. This suggests that the binding of these agonists to unoccupied sites on the receptor can increase the receptor's affinity for prebound ligands and thereby slow their dissociation. These data support previous indications of positive cooperation among multiple binding sites on both native and cloned 5-HT₃ receptors, and they extend this idea by demonstrating that agonists can modify the interaction of some, but not all, antagonists with the receptor.     

Regulation of membrane phospholipid catabolism in senescing carnation flowers

Evidence has been obtained for the involvement of μ M levels of Ca²⁺ in phospholipid catabolism during petal senescence by following the breakdown of [U-¹⁴C]-phosphatidylcholine by microsomal membranes from cut carnation ( Dianthus caryophyllus L. cv. White-sim) flowers. Phospholipid degradation was mediated by three membrane-associated lipases, viz. phospholipase D (EC 3.1.4.4), phosphatidic acid phosphatase (EC 3.1.3.4) and lipolytic acyl hydrolase. The activities of phospholipase D and phosphatidic acid phosphatase were stimulated by 30 and 100%, respectively, in the presence of 40 μ M free Ca²⁺, and the Ca²⁺-stimulation of phosphatidic acid phosphatase was calmodulin-dependent. When L-3-phosphatidyl-[2-³H]-inositol and L-3-phosphatidyl-[N-methyl-³H]-choline were used as substrates, inositol and choline accounted for 95 and 99%, respectively, of the water-soluble radiolabelled products. This suggests a predominance of phospholipase D activity over phospholipase C activity in these membranes.
Breakdown of membrane phospholipids in senescing carnations is known to be accelerated by treatment of young flowers with ethylene. To determine whether this involves a specific turnover of phosphatidylinositol as observed in animal systems in response to certain agonists, young flowers pre-labelled with ³²PO^3-₄ were treated with 10 ppm ethylene. All phospholipids incorporated the label, but no enhanced turnover of phosphatidylinositol was observed. Inositol 1,4,5-triphosphate did not release Ca²⁺ from preloaded microsomal vesicles at concentrations known to be effective in animal systems (i.e. < 5 μ M ) although release of Ca²⁺ was observed when a higher (20 μ M ) concentration was used.     

Differential regulation of the Cdk5-dependent phosphorylation sites of inhibitor-1 and DARPP-32 by depolarization

While cyclin-dependent kinase 5 (Cdk5) is of growing importance to neuronal signaling, its regulation remains relatively unexplored. Examination of the mechanism by which NMDA modulates the phosphorylation of protein phosphatase inhibitor-1 at Ser6 and Ser67 and dopamine- and cAMP-regulated phosphoprotein M _r 32 000 at Thr75 revealed that generalized depolarization, rather than specific activation of NMDA receptors, was sufficient to induce decreases in these Cdk5 sites. Although no evidence for the involvement of the Cdk5 cofactors p35 or p39, or for L- and T-type voltage-gated Ca²⁺ channels, was found, evaluation of the role of phosphatases and extracellular cations revealed differential regulation of the three sites. NMDA-induced decreases in the phosphorylation of Thr75 of dopamine- and cAMP-regulated phosphoprotein M _r 32 000 required protein phosphatase 1/2A activity and extracellular Ca²⁺. In contrast, the effects on Ser6 and Ser67 of inhibitor-1 were not cation specific; either Na⁺ or Ca²⁺ sufficed. Furthermore, while the decrease in phosphorylation of Ser6 was partially dependent on protein phosphatase 2B, that of Ser67 was independent of the major protein serine/threonine phosphatases, likely indicating the presence of a pathway by which NMDA inhibits Cdk5 activity. Thus, in the striatum the regulation of phosphorylation of Cdk5-dependent sites by NMDA occurs through multiple distinct pathways.     

Use of antibiotic resistance mutations to track strains of obligately anaerobic bacteria introduced into the rumen of sheep

Selective plating procedures were used to follow the fate of rifampicin-resistant mutant strains of the obligately anaerobic species Bacteroides multiacidus and Selenomonas ruminantium after their introduction at numbers around 10⁷/ml into the rumen of sheep. Bacteroides multiacidus strain F100 showed an initially rapid rate of loss (49%/h) but subsequently numbers declined more gradually approaching the limits of detection (10³/ml) after 100 h. Viable cell numbers also decreased in vitro upon addition of F100 cells to whole rumen contents, but remained stable upon addition to cell-free rumen fluid, suggesting protozoal predation. F100 cells were able to grow in vitro in whole rumen contents in the presence of an added utilizable substrate such as sorbitol, but addition of sorbitol to the rumen failed to enhance survival in vivo . In the case of S. ruminantium , introduced rif^R strains persisted in the rumen at levels around 10⁶ ml for at least 30 days.     

Rapid Communication: cis-Methyldioxolane Specifically Recognizes the m2 Muscarinic Receptor

Abstract: cis -Methyldioxolane (CD) is a muscarinic receptor agonist. [³H] CD has been used to label a subpopulation of muscarinic receptors described as exhibiting high agonist affinity. Pharmacological evidence suggests that the population of receptors labeled by [³H] CD consists of m2 and/or m4 subtypes; however, no studies have directly addressed the subtype selectivity of [³H] CD. The present study characterizes binding of this ligand to individual human receptor subtypes expressed in transfected Chinese hamster ovary cells. Results indicate that [³H] CD binds with high affinity only to Hm2 receptors but not to all Hm2 receptors. Twenty-eight percent of Hm2 receptors bound [³H] CD with a K _D of 3.5 ± 0.5 nM. Binding was eliminated in the presence of guanosine 5'- O -(3-thiotriphosphate), indicating that the Hm2 receptors labeled by [³H] CD are those that are associated with GDP-bound G protein. Binding of [³H] CD by only a subpopulation of Hm2 receptors is in agreement with data generated from studies of [³H] CD binding in mammalian brain. Because muscarinic receptors have been implicated to play a role in the pathogenesis of both Alzheimer's and Parkinson's disease, as well as the neurotoxicity of organophosphorus compounds, knowledge of the binding specificity of the muscarinic agonist [³H] CD should aid research in these areas.     

Probable Contribution of Protein Phosphorylation by Protein Kinase C to Spicule Formation in Sea Urchin Embryos

The formation of spicules and development of pluteus arms in sea urchin embryos were strongly blocked by H-7 (1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride) but were not affected by HA1004 ( N -(2-guanidinoethyl)-5-isoquinolinesulfonamide hydrochloride). Archenteron formation occurred normally in the presence of these compounds. Late gastrulae (28 hr after fertilization) were exposed to ³²Pi for 30 min at 20°C, and then dissociated and their primary mesenchyme cells with spicules, embryo-wall cells and archenteron cells were separated. Then, the radioactivities in the protein fractions of these separated cells were measured. Results showed that culture of embryos with H-7 strongly inhibited ³²p incorporation into proteins in primary mesenchyme cells but caused little inhibition of its incorporations in embryo-wall cells and archenteron cells. The effective concentrations of H-7 for inhibition of ³²p incorporation were within the range that blocked spicule formation and growth of pluteus arms in embryos. HA1004 only slightly inhibited ³²p incorporation into protein in mesenchyme cells, embryo-wall cells and archenteron cells of embryos exposed to ³²Pi. Protein kinase C activity was detectable only in isolated primary mesenchyme cells associated with spicule structures. These suggest that phosphorylation of proteins by protein kinase C contributes to the formation of spicule structures.     

Antibodies to a Segment of Tyrosine Hydroxylase Phosphorylated at Serine 40

Abstract: A synthetic peptide corresponding to residues 32–47 of rat tyrosine hydroxylase (TH) was phosphorylated by protein kinase A at Ser⁴⁰ and used to generate antibodies in rabbits. Reactivity of the anti-pTH_32–47 antibodies with phospho- and dephospho-Ser⁴⁰ forms of TH protein and peptide TH_32–47 was compared with reactivity of antibodies to nonphosphorylated peptide and to native TH protein. In antibody-capture ELISAs, anti-pTH_32–47 was more reactive with the phospho-TH than with the dephospho-TH forms. Conversely, antibodies against the nonphosphorylated peptide reacted preferentially with the dephospho-TH forms. In western blots, labeling of the ∼60-kDa TH band by anti-pTH_32–47 was readily detectable in lanes containing protein kinase A-phosphorylated native TH at 10–100 ng/lane. In blots of supernatants prepared from striatal synaptosomes, addition of a phosphatase inhibitor was necessary to discern labeling of the TH band with anti-pTH_32–47. Similarly, anti-pTH_32–47 failed to immunoprecipitate TH activity from supernatants prepared from untreated tissues, whereas prior treatment with either 8-bromoadenosine 3',5'-cyclic monophosphate or forskolin enabled removal of TH activity by anti-pTH_32–47. Lastly, in immunohistochemical studies, anti-pTH_32–47 selectively labeled catecholaminergic cells in tissue sections from perfusion-fixed rat brain.     

Regulation of l-DOPA Biosynthesis by Site-Specific Phosphorylation of Tyrosine Hydroxylase in AtT-20 Cells Expressing Wild-Type and Serine 40-Substituted Enzyme

Abstract: De novo l -DOPA biosynthesis was studied in stably transfected AtT-20 cells expressing wild-type- or [Leu⁴⁰]-recombinant tyrosine hydroxylase (rTH). Basal rates of DOPA accumulation were much higher by cells expressing rTH in which Leu was substituted for Ser⁴⁰ (S40L-rTH) than by those expressing wild-type rTH (WT-rTH). Treatment of WT-rTH cells with forskolin produced an increase in DOPA accumulation and a concomitant increase in WT-rTH phospho-Ser⁴⁰ content, whereas DOPA production by cells expressing S40L-rTH was entirely unaffected by forskolin. After forskolin treatment of ³²P_i-prelabeled cells, WT-rTH was phosphorylated at Ser⁸, Ser¹⁹, Ser³¹, and Ser⁴⁰, whereas ³²P incorporation into S40L-rTH was restricted to Ser⁸, Ser¹⁹, and Ser³¹. Relatively prolonged treatment of AtT-20 cells expressing WT-rTH with either a depolarizing agent (elevated potassium) or a phosphatase inhibitor (okadaic acid) increased DOPA production and increased the phosphorylation state of Ser⁴⁰; but, unlike forskolin, these treatments also increased DOPA production by cells expressing S40L-rTH. Thus, the present studies demonstrate that Ser⁴⁰ phosphorylation mediates forskolin-induced increases in DOPA biosynthesis directly but that mechanisms other than Ser⁴⁰ phosphorylation can mediate the increases in DOPA biosynthesis produced either by depolarization or by protein phosphatase inhibition.     

A Comparison of In Vitro- and In Vivo Phosphorylated Neurofilament Polypeptides

Abstract: Neurofilament polypeptides phosphorylated in vitro by incubation of neurofilament-enriched preparations from rat CNS with [γ-³²P]ATP were compared with the corresponding polypeptides labeled in vivo by injection of ³²P_i into the lateral ventricles of rats. Autoradiography of sodium dodecyl sulfate (SDS)-polyacrylamide gels revealed that the major phosphorylated species in both preparations were the three neurofilament subunits, which have molecular weights of 200K, 145K, and 68K. However, the relative levels of ³²P detected in the three in vitro -labeled subunits differed from the relative in vivo levels. The two larger neurofilament polypeptides displayed similar ³²P isoprotein distribution patterns on two-dimensional gels, whereas additional isoproteins were seen in the in vitro -labeled 68K species. Limited proteolysis in SDS-polyacrylamide gels revealed the presence of common phosphopeptides in the corresponding pairs of in vitro- and in vivo-labeled subunits, but the in vivo -labeled 145K and in vitro -labeled 200K polypeptides contained additional digestion products. Two-dimensional peptide mapping of the 68K polypeptide digested with a mixture of trypsin and chymotrypsin indicated that this component was phosphorylated at a single, identical site, both in vivo and in vitro. These results indicate that the protein kinase that copurifies with neurofilament preparations may be involved in their in vivo phosphorylation.     

Regulation of the heat-shock response depends on divalent metal ions in an hfIB mutant of Escherichia coli

HfIB, also called FtsH, is an essential Escherichia coli protein involved in the proteolysis of the heat-shock regulator σ³² and of the phage regulator λcll. The hfIB1 (Ts) allele (formerly called ftsH1 ) conferring temperature-sensitive growth at 42°C is suppressed by loss of the ferric-uptake repressor Fur and by anaerobic growth. We show here that suppression requires TonB-dependent Fe(III) transport in the hfIB1 (Ts) fur mutant during aerobic growth at 42°C and Feo-dependent Fe(II) transport during anaerobic growth at 42°C. Temperature-resistant growth of hfIB1 (Ts) strains is also observed at 42°C in the presence of a high concentration of Fe(II), Ni(II), Mn(II) or Co(II) salts, but not in the presence of Zn(II), Cd(II), Cu(II), Mg(II), Ca(II) or Cr(III) salts. However, neither Ni(II) nor a fur mutation permits growth in the complete absence of HfIB. The heat-shock response, evaluated by an htpG :: lacZ fusion, is overinduced in hfIB1 (Ts) strains at 42°C because of stabilization of σ³². Growth in the presence of Ni(II) or in the absence of the Fur repressor abolishes this overinduction in the hfIB1 (Ts) strain, and, in the hfIB1 (Ts) fur mutant, σ³² is no longer stabilized at 42°C. These results reinforce the recent observation that HfIB is a metalloprotease active against σ³² in vitro and suggest that it can associate functionally in vivo with Fe(II), Ni(II), Mn(II) and Co(II) ions.     

Heat shock regulation in the ftsH null mutant of Escherichia coli: dissection of stability and activity control mechanisms of σ32in vivo

The heat shock response of Escherichia coli is regulated by the cellular level and the activity of σ³², an alternative sigma factor for heat shock promoters. FtsH, a membrane-bound AAA-type metalloprotease, degrades σ³² and has a central role in the control of the σ³² level. The ftsH null mutant was isolated, and establishment of the Δ ftsH mutant allowed us to investigate control mechanisms of the stability and the activity of σ³² separately in vivo . Loss of the FtsH function caused marked stabilization and consequent accumulation of σ³² (≈20-fold of the wild type), leading to the impaired downregulation of the level of σ³². Surprisingly, however, Δ ftsH cells express heat shock proteins only two- to threefold higher than wild-type cells, and they also show almost normal heat shock response upon temperature upshift. These results indicate the presence of a control mechanism that downregulates the activity of σ³² when it is accumulated. Overproduction of DnaK/J reduces the activity of σ³² in Δ ftsH cells without any detectable changes in the level of σ³², indicating that the DnaK chaperone system is responsible for the activity control of σ³² in vivo . In addition, CbpA, an analogue of DnaJ, was demonstrated to have overlapping functions with DnaJ in both the activity and the stability control of σ³².     

Assimilation of methane and inorganic carbon by microbial communities mediating the anaerobic oxidation of methane

The anaerobic oxidation of methane (AOM) is a major sink for methane on Earth and is performed by consortia of methanotrophic archaea (ANME) and sulfate-reducing bacteria (SRB). Here we present a comparative study using in vitro stable isotope probing to examine methane and carbon dioxide assimilation into microbial biomass. Three sediment types comprising different methane-oxidizing communities (ANME-1 and -2 mixture from the Black Sea, ANME-2a from Hydrate Ridge and ANME-2c from the Gullfaks oil field) were incubated in replicate flow-through systems with methane-enriched anaerobic seawater medium for 5–6 months amended with either ¹³CH₄ or H¹³CO₃^-. In all three sediment types methane was anaerobically oxidized in a 1:1 stoichiometric ratio compared with sulfate reduction. Similar amounts of ¹³CH₄ or ¹³CO₂ were assimilated into characteristic archaeal lipids, indicating a direct assimilation of both carbon sources into ANME biomass. Specific bacterial fatty acids assigned to the partner SRB were almost exclusively labelled by ¹³CO₂, but only in the presence of methane as energy source and not during control incubations without methane. This indicates an autotrophic growth of the ANME-associated SRB and supports previous hypotheses of an electron shuttle between the consortium partners. Carbon assimilation efficiencies of the methanotrophic consortia were low, with only 0.25–1.3 mol% of the methane oxidized.     

Differential Labeling of Depot and Active Acetylcholine Pools in Nondepolarized and Potassium-Depolarized Rat Brain Synaptosomes

Abstract: To test the hypothesis that a pool of newly synthesized acetylcholine (ACh) turns over independently of preformed ACh, compartmentation and K⁺ -evoked release of ACh were examined in perfused synaptosomal beds intermittently stimulated by 50 m M K⁺. In resting synaptosomes, endogenous and labeled ACh was distributed between synaptic vesicles and the cytoplasm in a dynamic equilibrium ratio of 4:6. In the absence of new ACh synthesis, five sequential K⁺ -depolarizations caused a decremental release of preformed labeled ACh totaling 30% of the initial transmitter store. Further depolarization evoked little additional release, despite the fact that 60% of the labeled ACh remained in these preparations. Release of the preformed [¹⁴C]ACh was unaltered while new ACh was being synthesized from exogenous [³H]choline. Since the evoked release of [³H]ACh was maintained while that of [¹⁴C]ACh was decreasing, the [³H]ACh/[¹⁴C]ACh ratio in perfusate increased with each successive depolarization. This ratio was six to ten times higher than the corresponding ratio in vesicles or cytoplasm. These results indicate that the newly synthesized ACh did not equilibrate with either the depot vesicular or cytoplasmic ACh pools prior to release.     

Thymidine incorporation in Lake Cisó: Problems in estimating bacterial secondary production across oxic-anoxic interfaces

Abstract Heterotrophic bacterial activity was measured by means of the ³H-thymidine (³H-TdR) incorporation technique in Lake Cisó, a small holomictic lake with anoxic hypolimnion. We tested several methodological questions across the vertical profile: TdR concentration at which maximal incorporation is reached, linearity of incorporation and isotope dilution, during holomixis and stratification periods. The TdR concentration at which maximal incorporation is reached changed seasonally and vertically. During holomixis, maximal incorporation was not always reached at concentrations up to 40 nM. Uptake was always linear in short incubation times and decreased from epi- to hypolimnion. The isotope dilution technique indicated a degree of participation in DNA synthesis higher than 50%, although a linear relationship between the inverse of ³H-TdR incorporation and increasing ‘cold’ thymidine concentration was not always observed. Autoradiographic experiments showed a low percentage of bacteria taking up ³H-TdR in both aerobic and anaerobic samples. The percentage of total labeled bacteria seemed to be generally higher in the metalimnion (11% maximal value) than in the hypolimnion. Labeled Amoebobacter and Chromatium cells were detected in field samples. Amoebobacter cells photoassimilated TdR in culture. Therefore, our results show that ³H-TdR incorporation is not an appropriate technique to estimate bacterial secondary production in anaerobic systems and in oxic-anoxic interfaces.     

Effect of Protein Kinase C Activation on the Release of [3H]Acetylcholine in the Presence of Vesamicol

Abstract: The present work tested whether pharmacological activation of protein kinase C (PKC) influences the release of [³H]-acetylcholine ([³H]ACh) synthesized in the presence of vesamicol, an inhibitor of the vesicular acetylcholine transporter (VAChT). Newly synthesized [³H]ACh was released from hippocampal slices by field stimulation (15 Hz) in the absence of vesamicol, but as expected [³H]ACh synthesized during exposure to vesamicol was not released significantly by stimulation. Treatment of slices with the PKC activator phorbol myristate acetate (PMA) decreased the inhibitory effect of vesamicol on [³H]ACh release. The effect of PMA was dose-dependent, was sensitive to calphostin C, a PKC-selective inhibitor, and could not be mimicked by α-PMA, an inactive phorbol ester. PMA did not alter the release of [³H]ACh in the absence of vesamicol, suggesting that the site of PKC action could be related to the VAChT. In agreement with this observation, immunoprecipitation of VAChT from ³²P-labeled synaptosomes showed that phosphorylation occurs and that incorporation of ³²P in the VAChT protein increases in the presence of PMA. We suggest that PKC alters the output of [³H]ACh formed in the presence of vesamicol and also provide circumstantial evidence for a role of phosphorylation of VAChT in this process.     

Density of NMDA-Coupled and Uncoupled 1-[1-(2-[3H]Thienyl)cyclohexyl]piperidine Recognition Sites in the Brain and Spinal Cord: Differential Effects of NMDA Agonists and Antagonists

Abstract: Binding of 1-[1-(2-[³H]thienyl)cyclohexyl]piperidine ([³H]TCP) to mouse brain and spinal cord membranes was studied using compounds selective for the NMDA-coupled 1-(1-phenylcyclohexyl)piperidine (PCP) and/or σ recognition sites. In both tissues, [³H]TCP labeled two populations of binding sites. Density of the low-affinity sites was approximately the same in both tissues, but the population of the high-affinity [³H]TCP sites was three times bigger in the brain than in the spinal cord. Self- and cross-displacement studies showed that the high-affinity [³H]TCP binding sites could be identical with NMDA receptor-coupled PCP sites, whereas the low-affinity [³H]TCP sites may be associated with σ binding sites in both tissues. The NMDA-coupled PCP sites labeled in the presence of 6.25 n M [³H]TCP constituted a much higher percentage of the total binding in the brain (75%) than in the spinal cord (44%). Consistent with this, reintroduction of glycine and glutamate significantly increased, but DA antagonists significantly inhibited [³H]TCP binding in the brain but not in the spinal cord. Together, these data suggest that a large component of [³H]TCP-labeled binding sites in the spinal cord may be associated with σ but not the NMDA receptor-coupled PCP sites.