Proteasomes Associated with the Blm10 Activator Protein Antagonize Mitochondrial Fission through Degradation of the Fission Protein Dnm1

The conserved Blm10/PA200 activators bind to the proteasome core particle gate and facilitate turnover of peptides and unfolded proteins in vitro. We report here that Blm10 is required for the maintenance of functional mitochondria. BLM10 expression is induced 25-fold upon a switch from fermentation to oxidative metabolism. In the absence of BLM10, Saccharomyces cerevisiae cells exhibit a temperature-sensitive growth defect under oxidative growth conditions and produce colonies with dysfunctional mitochondria at high frequency. Loss of BLM10 leads to reduced respiratory capacity, increased mitochondrial oxidative damage, and reduced viability in the presence of oxidative stress or death stimuli. In the absence of BLM10, increased fragmentation of the mitochondrial network under oxidative stress is observed indicative of elevated activity of the mitochondrial fission machinery. The degradation of Dnm1, the main factor mediating mitochondrial fission, is impaired in the absence of BLM10 in vitro and in vivo. These data suggest that the mitochondrial functional and morphological changes observed are related to elevated Dnm1 levels. This hypothesis is supported by the finding that cells that constitutively overexpress DNM1 display the same mitochondrial defects as blm10Δ cells. The data are consistent with a model in which Blm10 proteasome-mediated turnover of Dnm1 is required for the maintenance of mitochondrial function and provides cytoprotection under conditions that induce increased mitochondrial damage and programmed cell death.     

Glutamate-Stimulated Phosphorylation of a Specific Protein in P2 Fractions of Rat Cerebral Cortex

Incubation of P2 fractions from rat cerebral cortex with 32Pi in the presence of L-glutamate caused an increased phosphorylation of a protein with apparent molecular weight of 43,000 (P43) as demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography. This glutamate-stimulated phosphorylation of P43 was already detectable 10 s after the addition of glutamate and was dependent on the concentrations of glutamate in the incubation medium. Other excitatory amino acids such as D-glutamate, L-aspartate, D,L-cysteic acid, L-cysteinesulfinic acid, and D,L-alpha-aminoadipic acid did not stimulate the phosphorylation of P43. In contrast, alpha-ketoglutarate and succinate stimulated the phosphorylation of this protein. Glutamate-stimulated phosphorylation of P43 seemed not to be mediated by either cAMP or cGMP and was inhibited by the presence of Ca2+ in the incubation medium. Experiments performed with metabolic inhibitors indicated that glutamate-stimulated protein phosphorylation is localized in mitochondria. This conclusion is supported by the occurrence of glutamate-stimulated phosphorylation of P43 in mitochondrial fractions from several peripheral tissues. The present results are consistent with the hypothesis that P43 is a component of the pyruvate dehydrogenase complex of mitochondria.     

Effect of Zinc on Calmodulin-Stimulated Protein Kinase II and Protein Phosphorylation in Rat Cerebral Cortex

The effect of increasing concentrations of Zn2+ (1 microM-5 mM) on protein phosphorylation was investigated in cytosol (S3) and crude synaptic plasma membrane (P2-M) fractions from rat cerebral cortex and purified calmodulin-stimulated protein kinase II (CMK II). Zn2+ was found to be a potent inhibitor of both protein kinase and protein phosphatase activities, with highly specific effects on CMK II. Only one phosphoprotein band (40 kDa in P2-M phosphorylated under basal conditions) was unaffected by addition of Zn2+. The vast majority of phosphoprotein bands in both basal and calcium/calmodulin-stimulated conditions showed a dose-dependent inhibition of phosphorylation, which varied with individual phosphoproteins. Two basal phosphoprotein bands (58 and 66 kDa in S3) showed a significant stimulation of phosphorylation at 100 microM Zn2+ with decreased stimulation at higher concentrations, which was absent by 5 mM Zn2+. A few Ca2+/calmodulin-stimulated phosphoproteins in P2-M and S3 showed biphasic behavior; inhibition at less than 100 microM Zn2+ and stimulation by millimolar concentrations of Zn2+ in the presence or absence of added Ca2+/calmodulin. The two major phosphoproteins in this group were identified as the alpha and beta subunits of CMK II. Using purified enzyme, Zn2+ was shown to have two direct effects on CMK II: an inhibition of Ca2+/calmodulin-stimulated autophosphorylation and substrate phosphorylation activity at low concentrations and the creation of a new Zn(2+)-stimulated, Ca2+/calmodulin-independent activity at concentrations of greater than 100 microM that produces a redistribution of activity biased toward autophosphorylation and an alpha subunit with an altered mobility on sodium dodecyl sulfate-containing gels.     

Accumulation and Elimination of Coliphage S-13 by the Hard Clam, Mercenaria mercenaria

Accumulation and elimination of viral particles by hard clams, Mercenaria mercenaria, were studied with the coliphage S-13 as a working model. Escherichia coli uptake and elimination were simultaneously monitored. Clams were exposed to low levels of S-13 (7 particles/ml) in running seawater for several days, achieving titers in tissues from 2 to more than 1,000 times the levels to which they had been exposed. Bacterial accumulation (previously established by other workers) was comparable. Upon exposure to virus-free running water, clams polluted to relatively low levels (100 plaque-forming units/ml) eliminated most of their bacterial contaminants in 24 to 48 hr. Viral contaminants, however, persisted for several days to weeks even under ideal conditions for clam activity, provided that the temperature remained below the inactivation threshold for the virus. Most of the accumulated virus appeared to be sequestered in the digestive gland. These sequestered particles are refractory to those mechanisms responsible for elimination of bacterial contaminants. This discrepancy points out the need for caution in evaluating the efficiency of shellfish depuration processes, especially if only a bacterial criterion is used as a monitoring system.     

Dopamine Oxidation Alters Mitochondrial Respiration and Induces Permeability Transition in Brain Mitochondria

Both reactive dopamine metabolites and mitochondrial dysfunction have been implicated in the neurodegeneration of Parkinson's disease. Dopamine metabolites, dopamine quinone and reactive oxygen species, can directly alter protein function by oxidative modifications, and several mitochondrial proteins may be targets of this oxidative damage. In this study, we examined, using isolated brain mitochondria, whether dopamine oxidation products alter mitochondrial function. We found that exposure to dopamine quinone caused a large increase in mitochondrial resting state 4 respiration. This effect was prevented by GSH but not superoxide dismutase and catalase. In contrast, exposure to dopamine and monoamine oxidase-generated hydrogen peroxide resulted in a decrease in active state 3 respiration. This inhibition was prevented by both pargyline and catalase. We also examined the effects of dopamine oxidation products on the opening of the mitochondrial permeability transition pore, which has been implicated in neuronal cell death. Dopamine oxidation to dopamine quinone caused a significant increase in swelling of brain and liver mitochondria. This was inhibited by both the pore inhibitor cyclosporin A and GSH, suggesting that swelling was due to pore opening and related to dopamine quinone formation. In contrast, dopamine and endogenous monoamine oxidase had no effect on mitochondrial swelling. These findings suggest that mitochondrial dysfunction induced by products of dopamine oxidation may be involved in neurodegenerative conditions such as Parkinson's disease and methamphetamine-induced neurotoxicity.     

Ergopeptine-Sensitive Calcium-Dependent Protein Phosphorylation System in the Brain

We studied a protein phosphorylation system that is regulated by the dopamine-mimetic ergot bromocriptine. Bromocriptine was found to inhibit selectively the endogenous phosphorylation of a threonine residue(s) in 50,000- and 60,000-dalton proteins in a synaptosome fraction. The bromocriptine-sensitive phosphorylation is stimulated by calcium and by calmodulin, and occurs predominantly in the brain. The inhibitory effect of bromocriptine was not mimicked by 3,4-dihydroxyphenylethylamine or by any of the neurotransmitters and related agents tested, but was mimicked, although less effectively, by other ergots that contain peptide moieties. In the hippocampus, the brain region with the highest content of the 50,000- and 60,000-dalton proteins, the ergopeptine-sensitive protein phosphorylation appears to be localized to interneurons or cell bodies whose axons synapse outside the hippocampus. The results raise the possibility that some of the bromocriptine- and ergopeptine-induced pharmacological effects in the CNS may be mediated by the inhibition of the calcium/calmodulin-dependent phosphorylation of these specific proteins.     

Altered Protein Tyrosine Phosphorylation in Alzheimer''s Disease

The activity of protein tyrosine kinase was determined in extracts from Alzheimer's disease brains and age- and postmortem time-matched control brains at autopsy using the synthetic peptide substrate poly(Glu4Tyr1). The specific activity of protein tyrosine kinases in the particulate fraction decreased roughly twofold (p less than 0.02) in Alzheimer's disease frontal cortex relative to unaffected control cortex. Cytosolic protein tyrosine kinase activity in Alzheimer's disease tissue was not significantly different from that in control tissue. In contrast to reduced particulate protein tyrosine kinase activity, analysis of Western blots of cytosolic and particulate fractions revealed increases in cytosolic antiphosphotyrosine immunoreactive polypeptides with molecular masses of 55 and 60 kDa. Quantitative immunohistochemistry and morphometry of frontal cortex sections with the antiphosphotyrosine antibody indicated increased antiphosphotyrosine staining in the neurons, although the number of antiphosphotyrosine-positive neurons per square millimeter decreased. Also, increased antiphosphotyrosine staining was observed in the hippocampal neurons. These results suggest that altered protein tyrosine kinases and protein tyrosine phosphorylation are involved in the pathology of Alzheimer's disease.     

Meclizine Inhibits Mitochondrial Respiration through Direct Targeting of Cytosolic Phosphoethanolamine Metabolism

We recently identified meclizine, an over-the-counter drug, as an inhibitor of mitochondrial respiration. Curiously, meclizine blunted respiration in intact cells but not in isolated mitochondria, suggesting an unorthodox mechanism. Using a metabolic profiling approach, we now show that treatment with meclizine leads to a sharp elevation of cellular phosphoethanolamine, an intermediate in the ethanolamine branch of the Kennedy pathway of phosphatidylethanolamine biosynthesis. Metabolic labeling and in vitro enzyme assays confirmed direct inhibition of the cytosolic enzyme CTP:phosphoethanolamine cytidylyltransferase (PCYT2). Inhibition of PCYT2 by meclizine led to rapid accumulation of its substrate, phosphoethanolamine, which is itself an inhibitor of mitochondrial respiration. Our work identifies the first pharmacologic inhibitor of the Kennedy pathway, demonstrates that its biosynthetic intermediate is an endogenous inhibitor of respiration, and provides key mechanistic insights that may facilitate repurposing meclizine for disorders of energy metabolism.     

Calmodulin-Dependent Protein Phosphorylation in Synaptic Junctions

Synaptic junctions (SJs) from rat forebrain were examined for Ca2+/calmodulin (CaM)-dependent kinase activity and compared to synaptic plasma membrane (SPM) and postsynaptic density (PSD) fractions. The kinase activity in synaptic fractions was examined for its capacity to phosphorylate endogenous proteins or exogenous synapsin I, in the presence or absence of Ca2+ plus CaM. When assayed for endogenous protein phosphorylation, SJs contained approximately 25-fold greater amounts of Ca2+/CAM-dependent kinase activity than SPMs, and fivefold more activity than PSDs. When kinase activities were measured by phosphorylation of exogenous synapsin I, SJs contained fourfold more activity than SPMs, and 10-fold more than PSDs. The phosphorylation of SJ proteins of 60- and 50-kilodalton (major PSD protein) polypeptides were greatly stimulated by Ca2+/CaM; levels of phosphorylation for these proteins were 23- and 17-fold greater than basal levels, respectively. Six additional proteins whose phosphorylation was stimulated 6-15-fold by Ca2+/CAM were identified in SJs. These proteins include synapsin I, and proteins of 240, 207, 170, 140, and 54 kilodaltons. The 54-kilodalton protein is a highly phosphorylated form of the major PSD protein and the 170-kilodalton component is a cell-surface glycoprotein of the postsynaptic membrane that binds concanavalin A. The CaM-dependent kinase in SJ fractions phosphorylated endogenous phosphoproteins at serine and/or threonine residues. Ca2+-dependent phosphorylation in SJ fractions was strictly dependent on exogenous CaM, even though SJs contained substantial amounts of endogenous CaM (15 micrograms CaM/mg SJ protein). Exogenous CaM, after being functionally incorporated into SJs, was rapidly removed by sequential washings. These observations suggest that the SJ-associated CaM involved in regulating Ca2+-dependent protein phosphorylation may be in dynamic equilibrium with the cytoplasm. These findings indicate that a brain CaM-dependent kinase(s) and substrate proteins are concentrated at SJs and that CaM-dependent protein phosphorylation may play an important role in mechanisms that underlie synaptic communication.     

Protein Phosphorylation in Astrocytes Mediated by Protein Kinase C: Comparison with Phosphorylation by Cyclic AMP-Dependent Protein Kinase

The protein kinase C activator, phorbol 12-myristate 13-acetate (PMA), has been found recently to transform cultured astrocytes from flat, polygonal cells into stellate-shaped, process-bearing cells. Studies were conducted to determine the effect of PMA on protein phosphorylation in astrocytes and to compare this pattern of phosphorylation with that elicited by dibutyryl cyclic AMP (dbcAMP), an activator of the cyclic AMP-dependent protein kinase which also affects astrocyte morphology. Exposure to PMA increased the amount of 32P incorporation into several phosphoproteins, including two cytosolic proteins with molecular weights of 30,000 (pI 5.5 and 5.7), an acidic 80,000 molecular weight protein (pI 4.5) present in both the cytosolic and membrane fractions, and two cytoskeletal proteins with molecular weights of 60,000 (pI 5.3) and 55,000 (pI 5.6), identified as vimentin and glial fibrillary acidic protein, respectively. Effects of PMA on protein phosphorylation were not observed in cells depleted of protein kinase C. In contrast to the effect observed with PMA, treatment with dbcAMP decreased the amount of 32P incorporation into the 80,000 protein. Like PMA, treatment with dbcAMP increased the 32P incorporation into the proteins with molecular weights of 60,000, 55,000 and 30,000, although the magnitude of this effect was different. The effect of dbcAMP on protein phosphorylation was still observed in cells depleted of protein kinase C. The results suggest that PMA, via the activation of protein kinase C, can alter the phosphorylation of a number of proteins in astrocytes, and some of these same phosphoproteins are also phosphorylated by the cyclic AMP-dependent mechanisms.     

Depolarisation-Dependent Protein Phosphorylation and Dephosphorylation in Rat Cortical Synaptosomes Is Modulated by Calcium

The effect of calcium on protein phosphorylation was investigated using intact synaptosomes isolated from rat cerebral cortex and prelabelled with 32Pi. For nondepolarised synaptosomes a group of calcium-sensitive phosphoproteins were maximally labelled in the presence of 0.1 mM calcium. The phosphorylation of these proteins was slightly decreased in the presence of strontium and absent in the presence of barium, consistent with the decreased ability of these cations to activate calcium-stimulated protein kinases. Addition of calcium alone to synaptosomes prelabelled in its absence increased phosphorylation of a number of proteins. On depolarisation in the presence of calcium certain of the calcium-sensitive phosphoproteins were further increased in labelling above nondepolarised levels. These increases were maximal and most sustained after prelabelling at 0.1 mM calcium. On prolonged depolarisation at this calcium concentration a slow decrease in labelling was observed for most phosphoproteins, whereas a greater rate and extent of decrease occurred at higher calcium concentrations. At 2.5 mM calcium a rapid and then a subsequent slow dephosphorylation was observed, indicating two distinct phases of dephosphorylation. Of all the phosphoproteins normally stimulated by depolarisation, only phosphoprotein 59 did not exhibit the rapid phase of dephosphorylation at high calcium concentrations. Replacing calcium with strontium markedly decreased the extent of change observed on depolarisation whereas barium decreased phosphorylation changes even further. Taken together these data suggest that an influx of calcium into synaptosomes initially activates protein phosphorylation, but as the levels of intrasynaptosomal calcium rise protein dephosphorylation predominates. Other phosphoproteins were dephosphorylated immediately on depolarisation in the presence of calcium. The fine control of protein phosphorylation levels exerted by calcium supports the idea that the synaptosomal phosphoproteins could play a role in modulating events such as neurotransmitter release in the nerve terminal.     

Depolarisation-Dependent Protein Phosphorylation in Rat Cortical Synaptosomes: Factors Determining the Magnitude of the Response

Abstract: The sequence of molecular events linking depolarisation-dependent calcium influx to the release of neurotransmitters from nerve terminals is unknown; however, calcium-stimulated protein phosphorylation may play a role. In this study the incorporation of phosphate into proteins was investigated using an intact postmitochondrial pellet isolated from rat cerebral cortex. The rate and relative incorporation of label into individual phosphoproteins depended on the prelabelling time and buffer concentrations of calcium and phosphate. After prelabelling for 45 min, depolarisation caused a >20% increase in the labelling of 10 phosphoproteins, and this initial increase was maximal with 41 mM K⁺ for 5 s, or 30 μ M veratridine for 15 s, in the presence of 1 mM calcium. Both agents also led to an initial dephosphorylation of four phosphoproteins. Depolarisation for 5 min led to a significant decrease in the labelling of all phosphoproteins. All of the depolarisation-stimulated changes in protein phosphorylation were calcium-dependent. The depolarisation conditions found to optimally alter the phosphorylation of synaptosomal proteins find many parallels in studies on calcium uptake and neurotransmitter release. However, the uniform responses of such a large number of phosphoproteins to the multitude of depolarisation conditions studied suggest that the changes could equally well relate to recovery events such as biosynthesis of neurotransmitters and regulation of intraterminal metabolic activity.     

Depolarizing Agents Regulate the Phosphorylation of Myelin Basic Protein in Rat Optic Nerves

The regulation of the state of phosphorylation of myelin basic protein has been studied in intact rat optic nerves incubated in vitro. For this purpose the endogenous state of phosphorylation was preserved and the "back-phosphorylation" technique was used to determine the amount of dephosphorylated protein present in extracts of the nerves. Our results indicate that when nerves were incubated in the presence of depolarizing agents, the state of phosphorylation of myelin basic protein was increased. This effect was calcium-dependent and was partly inhibited by chlorpromazine.     

Effect of Osmotic Shock on Protein Phosphorylation in Dunaliella salina Cells

Protein phosphorylation and dephosphorylation are considered as important regulatory mechanisms by which the activity of key enzymes and receptor molecules is altered within cells in response to a wide variety of external stimuli. Previous work is mainly on the purification and characteristics of protein kinase, but the role in stimulus-coupled responses in plants is not very clear. Experiments of in vitro protein phosphorylation demonstrated that in the extract of soluble protein of Dunaliella salina (Dunal) Teed. the activity of some protein kinases was, to some extent, dependent on the calcium concentration. The effects of calcium, verapamil, EGTA and A23187 on the in vivo protein phosphorylation also showed that calcium was important. In comparison, the autoradiograph of the in vivo phosphorylation was different from that of the in vitro phosphorylation. Addition of calcium or molybdate, an inhibitor of phosphatase, increased the extent of protein phesphorylation to a much higher level in hypoosmotic shocked samples (OSS), whereas as in hyperosmotic shocked samples(ESS) ,the extent of protein phosphorylation was lower than the control. In the absence of calcium or molybdate, the stimulation of protein phosphorylation by osmotic shock was hardly observed. The reason for this could be that the osmotic shock inhibited calcium uptake and/or activated protein phosphatase. The response of the intensely labelled 24 kD protein to osmotic shock was further studied. In the absence of calcium, the protein in OSS was more highly phesphorylated than the control and ESS. An increase of the eytosol calcium concentration stimulated phosphorylation of this protein in OSS, but had little effect on ESS. Such differences of calcium effects on protein phosphorylation indicated that the respective mechanisms of signal transduction mediated by protein phosphorylation may not be alike.     

Effects of Cyclic Nucleotides and Calcium/Calmodulin on Protein Phosphorylation in the CNS of Hirudo medicinalis

Protein phosphorylation plays an important role in the regulation of neural functions. We have studied the phosphorylation of proteins in homogenates of segmental ganglia of the leech Hirudo medicinalis. We describe a number of proteins whose phosphorylation is dependent on calcium/calmodulin or cyclic nucleotides. Most of the proteins whose phosphorylation is increased in the presence of calcium seem to be substrates for cyclic nucleotide-dependent protein kinases. Only two of the phosphoproteins described appear to be specific substrates for calcium/calmodulin protein kinase(s), and at least six phosphoproteins appear to be specific substrates for cyclic nucleotide-dependent kinase(s). The leech nervous system, with large and identifiable neurons, provides a good tool for studies of neural functions, such as learning. The results are discussed in the context of the role of protein phosphorylation on learning processes.     

New Aminoacyl-tRNA Synthetase-like Protein in Insecta with an Essential Mitochondrial Function

Aminoacyl-tRNA synthetases (ARS) are modular enzymes that aminoacylate transfer RNAs (tRNA) for their use by the ribosome during protein synthesis. ARS are essential and universal components of the genetic code that were almost completely established before the appearance of the last common ancestor of all living species. This long evolutionary history explains the growing number of functions being discovered for ARS, and for ARS homologues, beyond their canonical role in gene translation. Here we present a previously uncharacterized paralogue of seryl-tRNA synthetase named SLIMP (seryl-tRNA synthetase-like insect mitochondrial protein). SLIMP is the result of a duplication of a mitochondrial seryl-tRNA synthetase (SRS) gene that took place in early metazoans and was fixed in Insecta. Here we show that SLIMP is localized in the mitochondria, where it carries out an essential function that is unrelated to the aminoacylation of tRNA. The knockdown of SLIMP by RNA interference (RNAi) causes a decrease in respiration capacity and an increase in mitochondrial mass in the form of aberrant mitochondria.     

Protein Phosphorylation in Intact Superior Cervical Ganglion During Regeneration

The incorporation of radioactive phosphate into proteins of both normal and regenerating superior cervical ganglion nerve of the rat is reported. Incorporation studies carried out by in vitro and in vivo methods are compared. In the in vitro method, excised intact ganglia or their homogenates were incubated in the presence of inorganic phosphate or ATP, respectively, under various conditions. Proteins were analyzed by gel electrophoresis followed by autoradiography, in which quantitative but not qualitative differences between regenerating and control cases were apparent. In the in vivo procedure, inorganic phosphate was injected into the living animal 4 h before removal of ganglia. At least fivefold more proteins became labeled in vivo than in vitro, whereas no similarity in the pattern of labeling between the two methods was observed. For example, the most heavily labeled protein in the in vivo method, tentatively identified as microtubule-associated protein-2, was not detected on autoradiograms of proteins labeled by the in vitro method. In this latter method, an 85-kDa species and growth-associated protein-43 were always labeled, and the extent of their phosphorylation was enhanced by the additional presence of phosphatidylserine and Ca2+, a result indicating that these labeled species are substrates of protein kinase C. The in vitro conditions also led to the labeling of proteins identified as alpha- and beta-tubulin. Comparison of the methods suggests that removal of the ganglion interferes with the function of protein phosphorylation systems and that this effect involves elements of the cytoskeleton.     

Impulse Conduction Regulates Myelin Basic Protein Phosphorylation in Rat Optic Nerve

The influence of action potential conduction in myelinated axons on the state of phosphorylation of myelin basic protein was studied in rat optic nerve incubated in vitro. For this purpose we used a technique that permits continuous recording of the responses of nerves to electrical stimulation together with the "back-phosphorylation" assay. Our results indicate that action potential conduction, but not electrical stimulation, increased the state of phosphorylation of myelin basic protein. The increment in basic protein phosphorylation was related to the number of impulses conducted, up to a maximal change which occurred after 12 X 10(3) impulses. Also, the effect of action potential conduction was reversible, since the state of myelin basic protein phosphorylation returned to control levels within 5 min of stopping stimulation. These findings raise the interesting possibility that myelin basic protein phosphorylation plays a role in some dynamic function of myelin, perhaps related to ion transport or to the process of recovery of ionic gradients.     

Protein Phosphorylation in Bovine Adrenal Medullary Chromaffin Cells: Histamine-Stimulated Phosphorylation of Tyrosine Hydroxylase

Histamine can cause the release of catecholamines from bovine adrenal medullary chromaffin cells by a mechanism distinct from that of the depolarizing agents nicotine or high K+ buffer. It was the aim of this study to determine the protein phosphorylation responses to histamine in these cells and to compare them with those induced by depolarization. A number of proteins showed increases in phosphorylation in response to histamine especially when analyzed on two-dimensional polyacrylamide gel electrophoresis or by phosphopeptide mapping; one protein of 20,000 daltons was markedly dephosphorylated. Emphasis was given to the effects of histamine on tyrosine hydroxylase (TOH) phosphorylation, because this protein showed the most prominent changes on one-dimensional gels. Histamine acted via H1 receptors to increase TOH phosphorylation; the response was blocked by the H1 antagonist mepyramine and could be mimicked by the H1 agonist thiazolylethylamine, but not by the H2 agonist dimaprit. The H3 agonist (R) alpha-methylhistamine increased TOH phosphorylation at high concentrations, but the response was blocked entirely by mepyramine. Histamine rapidly increased the phosphorylation of TOH, with a maximum reached within 5 s and maintained for at least 30 min. This was in marked contrast to nicotine-stimulated protein phosphorylation of TOH, which was rapidly desensitized. The initial phosphorylation response to histamine was independent of extracellular Ca2+ for at least 3 min, but the sustained response required extracellular Ca2+. This was in contrast to the situation with both nicotine and high K+ buffer, which under the conditions used here caused a response which was dependent on extracellular Ca2+ at all times investigated. In the presence of histamine, the phosphopeptide profiles for TOH were essentially the same with or without Ca2+, suggesting that the same protein kinases were involved, but at longer times there was evidence of new phosphorylation sites. The mechanism or mechanisms whereby histamine modulates TOH phosphorylation are discussed with emphasis on the differences from depolarizing agents.