Microtubule-associated protein 2 phosphorylation is decreased in the human epileptic temporal lobe cortex.

Microtubule-associated protein 2 (MAP2) is an abundant component of the neuronal cytoskeleton whose function is related to the outgrowth and stability of neuronal processes, to synaptic plasticity and neuronal cell death. We have sought to study whether abnormal patterns of neuronal activity which are characteristic of epileptic patients are associated to alterations of MAP2 phosphorylation. An antibody (305) that selectively recognizes a phosphorylated epitope in a proline-rich region of the MAP2 molecule has been used to analyze neocortical biopsy samples from temporal lobe epileptic patients, whose electrocorticogram activity had been previously monitored. Immunoblot analysis showed that samples with greater spiking activity displayed significantly diminished MAP2 phosphorylation. Immunocytochemical analysis revealed the occurrence of discrete areas in the neocortex with highly decreased or no immunostaining for antibody 305, which showed a clear, although non-significant, tendency to appear more frequently in areas with greater spiking activity. To further support an association between epileptiform activity and MAP2 dephosphorylation an experimental model of epileptiform activity in cultures of rat hippocampal neurons was used. Neurons were cultured during 15 days in the presence of kynurenic acid, an antagonist of glutamate receptors. At this time, kynurenic acid was removed from the culture medium and neurons developed seizure-like activity. Using antibody 305, we found a decrease of MAP2 phosphorylation that was already visible after 15 min of kynurenic acid withdrawal. We therefore propose that MAP2 phosphorylation is decreased in the neocortex of epileptic patients and that this decrease is a likely consequence of seizure activity. Also, MAP2 dephosphorylation may lead to alterations of the neuronal cytoskeleton and eventually to neuronal damage and loss, which is typical of epileptic patients.

bFGF stimulates GAP-43 phosphorylation at ser41 and modifies its intracellular localization in cultured hippocampal neurons.

Cultured hippocampal neurons have been used to study GAP-43 phosphorylation and subcellular distribution. By immunofluorescence, GAP-43 was found associated with adherent membrane patches that remained attached to the substratum after in situ permeabilization with Nonidet-NP40. This association increases during neuronal development and is stabilized by the actin cytoskeleton. Basic fibroblast growth factor (bFGF) promotes GAP-43 translocation from the cytosol to adherent membrane patches and, at the same time, stimulates GAP-43 phosphorylation, mainly at the protein kinase C (PKC) site (Ser41). Inhibition of PKC prevented bFGF-stimulated GAP-43 phosphorylation and translocation, while activation by phorbol esters mimicked bFGF effects, suggesting that phosphorylation at Ser41 regulates GAP-43 subcellular localization. Using biochemical fractionation and phosphorylation analysis, it was found that Ser41 phosphorylation was highest in cytoskeleton-associated GAP-43 and lowest in membrane-associated GAP-43. It is proposed that GAP-43 is continuously cycling between intracellular compartments depending on its phosphorylation state and could be taking part in initial adhesive complexes assembled during growth cone advance.

Microscale purification of proteins exhibiting anomalous electrophoretic migration: application to the analysis of GAP-43 phosphorylation.

Quite often, in vivo analysis of posttranslational protein modifications is complicated by the lack of specific antibodies or unsatisfactory immunoprecipitation efficiency. Here, we present an alternative method to immunoprecipitation that takes advantage of the anomalous electrophoretic behavior exhibited by GAP-43. This method can be applied to other proteins which show similar characteristics, such as myristoylated alanine-rich C kinase, NAP-22, and Neurogranin, among others. All these proteins display relative mobility values that depend on the concentration of polyacrylamide used in the resolving gel. Cell extracts or tissue homogenates are first separated by SDS-PAGE in 15% polyacrylamide gels, and the bands containing GAP-43 are identified, excised from the gel, and rerun on a second SDS-PAGE in 7.5% polyacrylamide/6 M urea gels. To quickly identify the position of GAP-43 in the first gel, a small amount of fluorescein-labeled recombinant GAP-43 was added to the initial extracts. The method, applied to the analysis of GAP-43 phosphorylation in rat hippocampal slices, can be typically completed in less than 4 h. The excellent yields of purification obtained contributed to a greater accuracy and increased reliability of the radioactivity measurements. It also allowed further processing of the samples, including the analysis of the different phosphorylation sites by proteolytic digestion and peptide mapping.

Glutamate stimulates neurogranin phosphorylation in cultured rat hippocampal neurons.

Neurogranin is a calmodulin-binding and a protein kinase C substrate, that is expressed in telencephalic regions of the rat brain and has been associated with signal transduction and long-term potentiation (LTP). We here report that neurogranin is present in cultured hippocampal neurones, although in amounts lower than those present in the adult hippocampus, and that is also phosphorylated 'in vivo'. Glutamate receptor activation rapidly and significantly increases neurogranin phosphorylation, which achieves maximal phosphate labeling after ionotropic receptor stimulation (kainate and N-methyl-D-aspartate) and more moderate one after metabotropic receptor activation. It is proposed that neurogranin phosphorylation responds to changes in intracellular free Ca2+ and, also, that an increase in neurogranin phosphorylation contributes to enhance and extend calmodulin action, and therefore participate in post-synaptic signal transduction and LTP.

Neurogranin in the development of the rat telencephalon.

We have used a novel antibody to map the distribution of the protein kinase C substrate protein RC3/neurogranin during the development of the rat telencephalon. Neurogranin appearance in the rat brain is biphasic: it shows an early stage of anatomically restricted, low-intensity expression, and a juvenile stage of anatomically widespread, high-intensity expression. Most of the structures that express neurogranin during development conserve it in the adult stage. Neurogranin expression starts on embryonic day 18 in two different sites-the amygdalar primordium and in the piriform cortex-and is confined to these structures until the first postnatal day (P1). On P1, neurogranin expression increases dramatically in intensity, and appears in the olfactory cortex, isocortex, subiculum and hippocampus. In the striatum, expression starts on P1 and extends to the caudoputamen and parts of the globus pallidus and septum. Particularly complex patterns of labelling can be seen in the amygdala and cerebral cortex. Cortical layers showing early expression are the presumptive layers 4 and 5 in the somatosensory cortex, and layers 2 and 5 in the anterior cingulate and agranular insular cortices. Immunoreactivity is found mostly in cell bodies during the early and juvenile stages, but by the end of the first postnatal week it starts being more apparent in the neuropil. This phenomenon probably reflects the intracellular translocation of neurogranin to distal parts of the dendrites and dendritic spines. This process culminates by the end of the second postnatal week, when the adult pattern is reached. According to the timing and anatomy of its distribution, expression of neurogranin seems to be independently regulated in each telencephalic region by specific signalling mechanisms. It is proposed, on this basis, that neurogranin could be implicated in neuronal differentiation and synaptogenesis during telencephalic development.

Expression of protein kinase C isozymes in hippocampal neurones in culture.

Several protein kinase C (PKC) isozymes were analyzed by immunoblot and immunocytochemistry in cultures of hippocampal neurones at several stages of differentiation. Our findings reveal the existence of two distinct patterns of expression. Firstly, conventional PKC isozymes alpha, beta and gamma, that are expressed at very low levels during the initial stages and then increase continuously with time of culture. Secondly, novel PKC isozymes delta, epsilon and zeta, whose contents increase very early to reach a maximum after three days of culture and then progressively decline. Specific proteolysis for PKC isozymes beta and gamma was observed throughout the period studied. The developmental profile obtained for the different PKC isozymes is discussed in relation to the differentiation of hippocampal neurones in culture.

An increase in phosphorylation of microtubule-associated protein 2 accompanies dendrite extension during the differentiation of cultured hippocampal neurones.

Hippocampal neurones, from embryonic rats, were cultured for different times and the extension of dendrite-like processes was analysed morphologically and by immunofluorescence, using microtubule-associated protein 2 (MAP2) as a marker. Simultaneously, the changes in phosphorylation in MAP2 were analyzed and a correlation between dendrite sprouting and an increase in MAP2 phosphorylation was found. Phospho-MAP2 was cleaved by Staphylococcus aureus V8 protease limited proteolysis and its phosphopeptide pattern was compared to that obtained with two protein kinases (calcium/calmodulin-dependent kinase and protein kinase C) in vitro. An involvement of calcium/calmodulin-dependent protein kinase in the phosphorylation of MAP2, occurring simultaneously with dendrite extension during neuronal differentiation in vitro, is suggested.

Localization of differentially phosphorylated isoforms of microtubule-associated protein 1B in cultured rat hippocampal neurons.

The development and plasticity of axons and dendrites in mammalian neurons may depend on the presence and phosphorylation state of cytoskeletal proteins, including certain microtubule-associated proteins. One of these proteins, microtubule-associated protein 1B, is modified by different protein kinases, which give rise to two major types of phosphorylated isoforms. The distribution of these isoforms in cultured hippocampal neurons has been studied using antibodies to specific phosphorylation-sensitive epitopes. Mode I-phosphorylated MAP1B is largely restricted to developing axonal processes, particularly at their distal regions including their growth cones where no mode I-dephosphorylated MAP1B is present. Axonal maturation is accompanied by dephosphorylation of MAP1B at mode I sites. Thus, mode I-phosphorylated MAP1B may be a marker for axonal growth. In contrast, mode II-phosphorylated MAP1B is abundant in the axonal and somatodendritic compartments, and no increased dephosphorylation occurs during maturation. These results are compatible with a role for the mode I phosphorylation of MAP1B (which might be catalysed by proline-directed protein kinases) in supporting a rapid axonal-specific growth mechanism and a more general role for the mode II phosphorylation of MAP1B (which seems to be catalysed by casein kinase II) in controlling axonal and dendritic growth and remodeling.

Microtubule-associated protein 1B (MAP1B) is present in glial cells phosphorylated different than in neurones.

A panel of four anti-MAP1B antibodies have been used to study the presence and post-translational modification of MAP1B in primary cultures of glial cells. Two antibodies (150 and 125) recognize phosphorylated epitopes whereas the other two (531 and 842) recognize non-phosphorylated phosphorylatable epitopes on the MAP1B molecule. Immunofluorescence and Western blot analysis with antibodies 531 and 842 revealed the presence of small amounts of MAP1B-like immunoreactivity in type 1 astrocytes and a greater content in more differentiated glial cells found in long-term cultures. By immunofluorescence, these latter cells gave positive immunostaining with antibody 125, which recognizes a phosphorylated epitope phosphorylated by casein kinase II. Antibody 150, which reacts to a phosphorylated epitope on the MAP1B molecule, did not show any detectable immunoreactivity in glial cells cultures, either by immunofluorescence or Western blot. All four antibodies recognized hippocampal neurones in culture, with especially intense immunostaining in cell bodies and axons, and reacted strongly with protein present in hippocampal neurones extracts showing an electrophoretic mobility similar to that of brain MAP1B. In mixed optic nerve glial cell cultures, anti-galactocerebroside (GalC) positive cells gave also positive staining with antibodies 531 and 125. We propose that MAP1B is present in cultures of glial cells in moderate amounts and with a phosphorylation state different than in neurones. Thus, less differentiated glial cells, such as type 1 astrocytes, have a small amount of MAP1B, mainly in a non-phosphorylated form, which is spread diffusely in the cytoplasm and probably does not interact with microtubules. More differentiated glial cells, such as oligodendrocytes, show a greater content in MAP1B which, at least in part, is phosphorylated by a casein kinase II-like activity.

Rapid dephosphorylation of microtubule-associated protein 2 in the rat brain hippocampus after pentylenetetrazole-induced seizures.

We have studied the effect of Pentylenetetrazole (PTZ)-induced seizures on the state of phosphorylation of microtubule-associated protein 2 (MAP-2) from rat hippocampus. A method for the in vivo 32P-labeling of hippocampal proteins has been established, consisting of intracerebro-ventricular injection of 32PO4 of high specific activity. The results obtained indicate that PTZ induces a rapid and transient dephosphorylation of high-molecular-mass MAP-2, which is prevented when the N-methyl-D-aspartate receptor antagonist MK-801 is previously administered. Phosphopeptide mapping of 32P-labeled MAP-2 obtained from hippocampi of PTZ-treated rats reveals a pattern of phosphorylation distinct from that obtained from control saline-treated rats or MK-801 plus PTZ treated rats. We discuss the possible implications of N-methyl-D-aspartate-receptor activation and MAP-2 dephosphorylation on the plastic changes induced in rat brain hippocampus after induced epileptiform activity.

MAP2 phosphorylation parallels dendrite arborization in hippocampal neurones in culture.

Cultures of hippocampal neurones have been used to study a possible correlation between the pattern of dendritic growth and post-translational modification of Microtubule-Associated Protein 2 (MAP2). During the first three days in vitro, a small increase in the total amount of MAP2 is observed, whereas the level of phosphorylation increases exponentially, being particularly dramatic after three days in vitro. Analysis of dendrite morphology by MAP2 immunofluorescence, revealed a parallel exponential growth in dendrite arborization with maximum rates at the third day of culture. We propose a correlation between dendrite arborization and phosphorylation of MAP2, that could be mediated by the establishment of cell contacts.

Loracarbef (LY163892) versus amoxicillin/clavulanate in the treatment of acute purulent bacterial bronchitis.

In this single-blind study, 488 patients with acute bronchitis were randomly assigned to receive 400 mg of loracarbef twice daily or 500/125 mg of amoxicillin/clavulanate three times daily for seven days. Treatment efficacy was evaluated in 98 patients treated with loracarbef and in 99 treated with amoxicillin-clavulanate in whom pretreatment positive cultures of pathogens susceptible to both study drugs were found. Streptococcus pneumoniae, Haemophilus influenzae, Moraxella (Branhamella) catarrhalis, and Klebsiella pneumoniae were isolated in pure or mixed cultures in 64% of the evaluable patients; S pneumoniae was found in 26%. Among the evaluable patients, the rate of favorable clinical responses (cure and improvement) in the loracarbef group (96 of 98 patients; 98.0%) was similar to that in the amoxicillin/clavulanate group (96 of 99 patients; 97.0%); the favorable bacteriologic response rates were also similar (93.7% vs 92.9%, respectively). Eight patients in the loracarbef group and nine in the amoxicillin/clavulanate group discontinued treatment because of adverse events. The events were presumed to be drug related in five of the loracarbef group and in seven of the amoxicillin/clavulanate group. During therapy, diarrhea was the most frequently reported event in both groups. However, it occurred in only 8.2% of the loracarbef-treated patients compared with 22.5% of the amoxicillin/clavulanate patients (P less than 0.001). It is concluded that both loracarbef and amoxicillin/clavulanate are safe and effective in the treatment of acute purulent bacterial bronchitis.

Loracarbef (LY163892) versus amoxicillin/clavulanate in the treatment of acute bacterial exacerbations of chronic bronchitis.

In this single-blind study, 579 patients with chronic bronchitis were randomly assigned to receive 400 mg of loracarbef twice daily or 500/125 mg of amoxicillin/clavulanate thrice daily for seven days. Treatment efficacy was evaluated in 129 of the loracarbef-treated patients and 120 amoxicillin/clavulanate-treated patients in whom pretreatment positive cultures of pathogens susceptible to both antibiotics were isolated. Three organisms predominated in either pure or mixed cultures in 57.0% of the evaluable patients: Haemophilus influenzae, Streptococcus pneumoniae, or Moraxella (Branhamella) catarrhalis; H influenzae was isolated in 25.0% of the patients with single pathogens. Among the evaluable patients, favorable clinical responses (cure or improvement) were noted in 93.8% of the loracarbef-treated patients and in 95.0% of the amoxicillin/clavulanate-treated patients. A favorable bacteriologic response (pathogen eliminated or presumed eliminated) was found in 82.2% of loracarbef-treated patients and 90.0% of amoxicillin/clavulanate-treated patients. Six patients in the loracarbef group and 14 in the amoxicillin/clavulanate group discontinued treatment because of adverse events. The events were judged to be drug related in four loracarbef-treated patients and in 11 amoxicillin/clavulanate-treated patients. The incidence of diarrhea and other gastrointestinal symptoms was significantly more frequent in the amoxicillin/clavulanate group (13.5% and 5.6%) than in the loracarbef group (4.5% and 1.7%), while the incidence of severe headaches was significantly more frequent in the loracarbef than the amoxicillin/clavulanate group (7.2% vs 3.1%). It is concluded that loracarbef and amoxicillin/clavulanate are safe and effective in the treatment of acute bacterial exacerbations of chronic bronchitis.

Loracarbef (LY163892) versus amoxicillin/clavulanate in bronchopneumonia and lobar pneumonia.

In a single-blind study, 134 patients with bronchopneumonia or lobar pneumonia were randomly assigned to receive 400 mg of loracarbef twice daily or 500/125 mg of amoxicillin/clavulanate three times daily for 10 to 14 days. Treatment efficacy was evaluated in 38 patients treated with loracarbef and in 39 treated with amoxicillin/clavulanate in whom pre-treatment positive cultures of pathogens susceptibile to the study drugs were isolated. Streptococcus pneumoniae and Haemophilus influenzae were cultured as single pathogens in 40.3% of the evaluable patients. Among the evaluable patients, 100% of the loracarbef group and 92.3% of the amoxicillin/clavulanate group had a favorable clinical response (cure or improvement). Bacteriologic response was favorable and the pathogen was eliminated or presumed eliminated in 97.4% of the loracarbef-treated patients and in 92.3% of the amoxicillin/clavulanate-treated patients. Treatment was discontinued in one loracarbef-treated patient because Ludwig's angina, unrelated to the study drug, was diagnosed and in five amoxicillin/clavulanate-treated patients because of diarrhea (two patients), rash (two patients), and nausea and vomiting (one patient). Diarrhea, the most frequently cited adverse event, was reported by 6.1% of the loracarbef-treated patients and 11.8% of the amoxicillin/clavulanate-treated patients. Asthenia was reported by 0% and 8.8% of the loracarbef and amoxicillin/clavulanate patients, respectively. It is concluded that both loracarbef and amoxicillin/clavulanate are safe and effective in the treatment of acute bacterial pneumonia.

Na+ dependence of tyrosine transport across the synaptosomal membrane reflects changes in the morphology of synaptosomes.

The Na+ dependence of tyrosine uptake into rat brain synaptosomes and synaptosomal plasma membrane vesicles (SPMV) was examined in the present study. At low tyrosine concentrations, the isoosmotic substitution of Na+ by sucrose in the incubation medium led to an increase of tyrosine uptake in synaptosomes and to a decrease in SPMV. The removal of extracellular Ca2+ and Mg2+ and addition of isoosmotic sucrose completely prevented the augmented tyrosine uptake in Na+-free incubated synaptosomes. Morphological differences were found at the electron-microscopic level when synaptosomes were incubated in Na+-free and Na+-containing media. The internal volume measured for synaptosomes incubated in a Na+-free medium was almost half of that obtained in a Na+-containing medium, in good agreement with the observations made with the electron microscope. Also, the omission of Ca2+ and Mg2+ resulted in a specific swelling of only the synaptosomes incubated in Na+-free medium. When synaptosomes and SPMV were preloaded with several neutral amino acids, the tyrosine uptake rate was greatly increased, indicating fully operational exchange mechanisms for these amino acids. We propose that the enhancement of high-affinity synaptosomal tyrosine uptake observed in Na+-free medium is a consequence of a specific shrinkage of the synaptosomes and a parallel increase of the exchange rate with endogenous neutral amino acids.

Ontogeny of the novel tachykinins neurokinin A, neurokinin B and neuropeptide K in the rat central nervous system.

Neurokinin A, neurokinin B and neuropeptide K content has been measured in several regions of the rat central nervous system at different stages of postnatal development. For this, we have employed a combination of HPLC separation and radioimmunoassay detection using a neurokinin A antiserum which also recognizes neurokinin B and neuropeptide K. All 3 tachykinins were detectable during postnatal development in the various regions studied (hypothalamus, striatum, substantia nigra, cerebral cortex and spinal cord). Interestingly, a general increase in the tachykinin concentrations was observed during the second week of life. Some of these concentrations reached values on postnatal day 15 which far exceeded those observed in the adult. After day 15 most areas showed a slow decline in their tachykinin content until adult values were finally achieved. The developmental profiles obtained for these tachykinins are in good agreement with previous studies on the ontogeny of substance P and its receptors and support the view that tachykinins may play an important role in the organization and maturation of the developing central nervous system.

In vitro and in vivo release of neurokinin A-like immunoreactivity from rat substantia nigra.

In vivo and in vitro perfusion techniques have been used to study the release of neurokinin A-like immunoreactivity from the rat substantia nigra. Potassium depolarization and electrical field stimulation evoked calcium-dependent release from nigral slices. Potassium depolarization was also effective in vivo. Tetrodotoxin (1 microM) completely blocked electrically stimulated release but only diminished release in response to depolarizing potassium. Neurokinin A-like immunoreactivity release showed frequency dependence and a clear facilitation phenomenon between 5 and 25 Hz. High-performance liquid chromatography analysis of the immunoreactivity released in vitro revealed the presence of neurokinin A, neuropeptide K and neurokinin B, along with their sulphoxide forms. A marked depletion of neuropeptide K and neurokinin B content was observed when the tachykinin content of the nigral slices was examined before and after stimulation. However, the neurokinin A content of the slices was unchanged or even increased, suggesting an accelerated processing of neurokinin A precursors during the stimulation. The tachykinin peptides were degraded at different rates by substantia nigra homogenates; degradation was fastest for neuropeptide K and slowest for neurokinin A. The addition of a mixture of peptidases inhibitors (thiorphan, phosphoramidon, bestatin and captopril) substantially reduced the degradation of all three tachykinins, but did not completely block degradation. GABA-A receptor antagonists such as bicuculline and, particularly, picrotoxin potentiated the stimulated neurokinin A-like immunoreactivity release in vitro, but the GABA-agonist muscimol had no effect. Picrotoxin was even more potent in vivo. The results presented in this study demonstrate that neurokinin A, neuropeptide K and neurokinin B can be released by depolarizing stimuli from rat substantia nigra. Furthermore, the features exhibited by this release suggest that these peptides may have a neurotransmitter/neuromodulator role in the rat substantia nigra.

Evidence for release of calcitonin gene-related peptide and neurokinin A from sensory nerve endings in vivo.

Sensitive radioimmunoassays for calcitonin gene-related peptide and the tachykinin, neurokinin A, have been used to show that acute administration of the sensory neurotoxin capsaicin (10 mg/kg i.p.) to normal adult rats, causes a substantial release of calcitonin gene-related peptide immunoreactivity (15-fold increase) and neurokinin A immunoreactivity (4- to 5-fold increase) into the plasma. Neonatal administration of capsaicin (50 mg/kg s.c.) produced a long term deficit in the lumbar dorsal root ganglia content of calcitonin gene-related peptide (76% depletion), and neurokinin A immunoreactivity (86% depletion) in rats killed 6 weeks after administration. Acute capsaicin treatment of neonatally capsaicin-treated rats revealed that these animals still showed a capsaicin-evoked release of calcitonin gene-related peptide and neurokinin A immunoreactivity into the plasma. The increase in plasma content was, however, substantially less than that seen in normal (vehicle-treated) rats and was proportional to the initial basal plasma level of the respective peptides. Immunohistochemical staining using an anti-calcitonin gene-related peptide antiserum revealed that, despite the neonatal capsaicin treatment and loss of dorsal root ganglia content, the lumbar dorsal horn had a near normal pattern of calcitonin gene-related peptide immunoreactivity. This observation was supported by radioimmunoassays carried out on lumbar dorsal horn samples obtained from the same rats, which showed no significant decrease in calcitonin gene-related peptide immunoreactivity, whilst the dorsal horn content of neurokinin A was some 70% below control values.(ABSTRACT TRUNCATED AT 250 WORDS)

Opioid peptides inhibit noradrenergic transmission in the preoptic area to block LH secretion: evidence from neonatally androgenised rats.

A series of experiments are described which show (a) that the release of noradrenaline in the preoptic area of rats is regulated by a presynaptic opioid receptor and, (b) that changes in the opioid control of preoptic noradrenaline release are associated with the anovulatory condition found in adult female rats which have been exposed to androgen neonatally. The experiments support the hypothesis that opioid-noradrenergic interaction in the preoptic area is an important component in the neural mechanism regulating gonadotrophin secretion.