Modulation of muscarinic signaling in PC12 cells overexpressing neuronal Ca2+ sensor-1 protein.

It has been suggested that overexpression of neuronal Ca2+ sensor-1 (NCS-1) protein is implicated in the pathophysiology of neurodisorders such as schizophrenia, bipolar disturbance and X-linked mental retardation. The mechanism by which NCS-1 would be involved in the causes and/or consequences of these neurodisorders is still far from elucidation. Independent evidence has pointed NCS-1 as a key regulator of synaptic efficacy by altering the expression and activity of voltage-gated channels, inhibiting internalization of dopaminergic receptors, and altering phosphoinositide metabolism. In this study, we examined the possible participation of NCS-1 protein in signal transmission dependent on muscarinic receptor activation, using PC12 cells stably expressing NCS-1 (PC12-NCS-1). Carbachol (CCH; 300 microM) was able to evoke glutamate release more efficiently from PC12-NCS-1 (15.3+/-1.0nmol/mg of protein) than wild type cells (PC12-wt; 8.3+/-0.9nmol/mg of protein). This increase of glutamate release induced by CCH was independent on extracellular Ca2+ influx. Additionally, a larger increase of cytoplasmic levels of InsP3 (663.0+/-63.0 and 310.0+/-39.0% of fluorescence in A.U.) and [Ca2+]i (766.4+/-40.0 and 687.8+/-37.1nmol/L) was observed after CCH stimulus of PC12-NCS-1 compared with PC12-wt. Clearly distinction between intracellular Ca2+ dynamics was also observed in PC12-NCS-1 and PC12-wt. A larger increase followed by fast decay of [Ca2+]i was observed in PC12-NCS-1. A plateau with a delayed decay of [Ca2+]i was characteristic of PC12-wt [Ca2+]i response. Both enhancement of InsP3 production and glutamate release observed in PC12-NCS-1 were blocked by atropine (10 microM). Together, our data show that overexpression of NCS-1 in PC12 cells induces an enhancement of intracellular second messenger and transmitter release dependent on CCH response, suggesting that muscarinic signaling is "up-regulated" in this cell model.

Expression and localization of voltage dependent potassium channel Kv4.2 in epilepsy associated focal lesions.

An increasing number of observations suggest an important role for voltage-gated potassium (Kv) channels in epilepsy. We studied the cell-specific distribution of Kv4.2, phosphorylated (p) Kv4.2 and the Kv4.2 interacting protein NCS-1 using immunocytochemistry in different epilepsy-associated focal lesions. In hippocampal sclerosis (HS), Kv4.2 and pKv4.2 immunoreactivity (IR) was reduced in the neuropil in regions with prominent neuronal cell loss. In both HS and malformations of cortical development (MCD), intense labeling was found in neuronal somata, but not in dendrites. Strong NCS-1 IR was observed in neurons in all lesion types. Western blot analysis demonstrated an increase of total Kv4.2 in all lesions and activation of the ERK pathway in HS and ganglioglioma. These findings indicate that Kv4.2 is expressed in both neuronal and glial cells and its regulation may involve potassium channel interacting proteins, alterations in the subcellular localization of the channel, as well as phosphorylation-mediated posttranslational modifications.

Activity-dependent regulation of the subcellular localization of neuronal calcium sensor-1 in the avian cochlear nucleus.

Neurons in the avian cochlear nucleus, nucleus magnocellularis (NM), are highly sensitive to manipulations of afferent input, and removal of afferent activity through cochlear ablation results in the death of approximately 20-40% of ipsilateral NM neurons. The intracellular cascades that determine whether an individual NM neuron will die or survive are not fully understood. One early event observed in NM following deafferentation is a rapid rise in intracellular calcium concentration. In most cellular systems, the activity of calcium-binding proteins is believed to accommodate calcium influx. The calcium-binding protein, neuronal calcium sensor-1 (NCS-1), is an intracellular neuronal calcium sensor belonging to the EF-hand superfamily. NCS-1 has been implicated in calcium-dependent regulation of signaling cascades. To evaluate NCS-1 action in NM neurons, the localization of NCS-1 protein was examined. Double-label immunofluorescence experiments revealed that NCS-1 expression is evident in both the presynaptic nerve terminal and postsynaptic NM neuron. The postsynaptic expression of NCS-1 typically appears to be closely associated with the cell membrane. This close proximity of NCS-1 to the postsynaptic membrane could allow NCS-1 to function as a modulator of postsynaptic signaling events. Following deafferentation, NM neurons were more likely to show diffuse cytoplasmic NCS-1 labeling. This increase in the number of cells showing diffuse cytoplasmic labeling was observed 12 and 24 h following cochlea ablation, but was not observed 4 days following surgery. This activity-dependent regulation of NCS-1 subcellular localization suggests it may be associated with, or influenced by, processes important for the survival of NM neurons.

Over-expression of NCS-1 in AtT-20 cells affects ACTH secretion and storage.

The effect of over-expressing neuronal calcium sensor 1 (NCS-1) upon stimulated adrenocorticotrophin (ACTH) secretion was studied in AtT-20 cells. Stably-transfected AtT-20 cell lines over-expressing NCS-1 were obtained and compared to wild type AtT-20 cells. Corticotrophin releasing factor (CRF-41)-stimulated ACTH secretion from NCS-1 over-expressing cells was significantly reduced from that obtained in wild type AtT-20 cells. The effects of other stimulants of ACTH secretion from wild type AtT-20 cells were not attenuated in NCS-1 over-expressing cells. Calcium, guanosine 5'-O-(3'-thiotriphosphate) (GTP-gamma-S) and mastoparan stimulated ACTH secretion from permeabilised wild type AtT-20 and NCS-1 over-expressing AtT-20 cells with significantly greater ACTH secretion obtained in NCS-1 over-expressing cells. This study shows that in intact cells over-expression of NCS-1 reduces exocytotic ACTH release, while in permeabilised cells increases ACTH release. NCS-1 has multiple cellular targets and that directly and indirectly via these targets acts to increase the releasable ACTH pool while inhibiting CRF-41 stimulus-secretion coupling.

Interaction of neuronal calcium sensor-1 (NCS-1) with phosphatidylinositol 4-kinase beta stimulates lipid kinase activity and affects membrane trafficking in COS-7 cells.

Phosphatidylinositol 4-kinases (PI4K) catalyze the first step in the synthesis of phosphatidylinositol 4,5-bisphosphate, an important lipid regulator of several cellular functions. Here we show that the Ca(2+)-binding protein, neuronal calcium sensor-1 (NCS-1), can physically associate with the type III PI4Kbeta with functional consequences affecting the kinase. Recombinant PI4Kbeta, but not its glutathione S-transferase-fused form, showed enhanced PI kinase activity when incubated with recombinant NCS-1, but only if the latter was myristoylated. Similarly, in vitro translated NCS-1, but not its myristoylation-defective mutant, was found associated with recombinant- or in vitro translated PI4Kbeta in PI4Kbeta-immunoprecipitates. When expressed in COS-7 cells, PI4Kbeta and NCS-1 formed a complex that could be immunoprecipitated with antibodies against either proteins, and PI 4-kinase activity was present in anti-NCS-1 immunoprecipitates. Expressed NCS-1-YFP showed co-localization with endogenous PI4Kbeta primarily in the Golgi, but it was also present in the walls of numerous large perinuclear vesicles. Co-expression of a catalytically inactive PI4Kbeta inhibited the development of this vesicular phenotype. Transfection of PI4Kbeta and NCS-1 had no effect on basal PIP synthesis in permeabilized COS-7 cells, but it increased the wortmannin-sensitive [(32)P]phosphate incorporation into phosphatidylinositol 4-phosphate during Ca(2+)-induced phospholipase C activation. These results together indicate that NCS-1 is able to interact with PI4Kbeta also in mammalian cells and may play a role in the regulation of this enzyme in specific cellular compartments affecting vesicular trafficking.

Overexpression of rat neuronal calcium sensor-1 in rodent NG108-15 cells enhances synapse formation and transmission.

The role of rat neuronal calcium sensor-1 (NCS-1), a Ca2+-binding protein, in synapse formation and transmitter release was examined in mouse neuroblastoma x rat glioma hybrid NG108-15 cells in culture. Wild-type NG108-15 cells expressed rodent NCS-1. Endogenous NCS-1 was partially co-localized with the synaptic protein SNAP-25 at the plasma membrane in both cell bodies and processes, but not with the Golgi marker [beta]-COP, an individual coat subunit of the coatomer complex present on Golgi-derived vesicles. In NG108-15 cells co-cultured with rat myotubes, partial co-localization of SNAP-25 and NCS-1 was observed at the plasma membrane of neurites and growth cones, some of which had synaptic contacts to muscle cells. Transient co-transfection of the rat NCS-1 cDNA and green fluorescent protein (GFP) resulted in NCS-1 overexpression in about 30 % of the cells as determined by fluorescence microscopy. The rate of functional synapse formation with co-cultured rat myotubes increased 2-fold as determined by the presence of miniature endplate potentials (MEPPs) in NCS-1-overexpressing NG108-15 cells compared to non- and mock-transfected cells. The number of neurites per cell, branches per neurite and length of neurites was slightly less in cells that were either transiently transfected (GFP-NCS-1-fluorescence positive) or stably transformed with NCS-1 compared to GFP-NCS-1-negative, non-transfected or mock-transfected NG108-15 cells. The number of action potentials that elicited endplate potentials increased in NG108-15 cells stably transformed with rat NCS-1. The mean number of quanta per impulse (m) increased 5-fold. These results show that NCS-1 functions to facilitate synapse formation, probably because of the increased quantal content of evoked acetylcholine release.

Accumulation of synaptosomal-associated protein of 25 kDa (SNAP-25) and other proteins associated with the secretory pathway in GH4C1 cells upon treatment with estradiol, insulin, and epidermal growth factor.

Treatment of rat pituitary GH4C1 cells with estradiol, insulin, and epidermal growth factor induces secretory granule accumulation, PRL storage, and stabilization of ICA512, a membrane protein associated with secretory granules. In these investigations we found that the same treatment induced accumulation over 2-fold of other proteins in the secretory pathway, including synaptosomal-associated protein of 25 kDa (SNAP-25), synaptotagmin III, synaptobrevin, synaptophysin, and cyclophilin B, and did not affect accumulation of others, including synaptotagmin I, calnexin, and glucose-regulated protein 94. The induction of proteins was not a coordinate event, because epidermal growth factor alone maximally stimulated SNAP-25 accumulation, but not that of synaptotagmin III. Induction of SNAP-25 accumulation occurred without an increase in its synthesis, and induction of cyclophilin B occurred without an increase in its messenger RNA accumulation, suggesting that accumulation may be caused by stabilization of the proteins. SNAP-25 immunofluorescence was located in the cytoplasm and on the plasma membrane and sometimes was heavily concentrated in protrusions from the cell surface, especially in hormone-treated cells. Frequenin immunofluorescence was also sometimes concentrated in intense patches, but did not colocalize with SNAP-25. Growth hormone and prolactin immunofluorescence was not found in the protrusions and sometimes did not colocalize with each other when they were present in the same cell. Hormone treatment of GH4C1 cells therefore induces accumulation of specific proteins in all parts of the secretory pathway and causes morphological changes in addition to accumulating secretory granules.

Overexpression of frequenin, a modulator of phosphatidylinositol 4-kinase, inhibits biosynthetic delivery of an apical protein in polarized madin-darby canine kidney cells.

Polyphosphoinositides regulate numerous steps in membrane transport. The levels of individual phosphatidylinositols are controlled by specific lipid kinases, whose activities and localization are in turn regulated by a variety of effectors. Here we have examined the effect of overexpression of frequenin, a modulator of phosphatidylinositol 4-kinase activity, on biosynthetic and postendocytic traffic in polarized Madin-Darby canine kidney cells. Endogenous frequenin was identified in these cells by polymerase chain reaction, Western blotting, and indirect immunofluorescence. Adenoviral-mediated overexpression of frequenin had no effect on early Golgi transport of membrane proteins, as assessed by acquisition of resistance to endoglycosidase H. However, delivery of newly synthesized influenza hemagglutinin from the trans-Golgi network to the apical cell surface was severely inhibited in cells overexpressing frequenin, whereas basolateral delivery of the polymeric immunoglobulin receptor was unaffected. Overexpression of frequenin did not affect postendocytic trafficking steps including apical and basolateral recycling and basal-to-apical transcytosis. We conclude that frequenin, and by inference, phosphatidylinositol 4-kinase, plays an important and selective role in apical delivery in polarized cells.

Basolateral mechanisms of intracellular pH regulation in the colonic epithelial cell line HT29 clone 19A.

The intracellular pH (pHi) of the colonic tumour cell line HT29 cl.19A was studied by microspectrofluorometry using the pH-sensitive dye BCECF. Single cells within a confluent monolayer, grown in a polarized manner on permeable supports, were examined. An amiloride-sensitive Na+/H+ exchange and a stilbene-insensitive Cl-/HCO3- exchange mechanism have been identified in the basolateral membrane. Removal of Na+ from the basolateral solution caused a decrease of pHi by 0.50 +/- 0.09 unit (n = 4). Amiloride or Na(+)-free solution at the apical side had no effect on pHi. Cl- removal at the basolateral side led to an increase of pHi by 0.20 +/- 0.03 unit (n = 4) whereas apical removal had no influence on pHi. This effect was independent of Na+ and was insensitive to 0.2 mM 4,4'-diisothiocyanatodihydrostilbene-2,2'-disulphonic acid. A basolateral Cl-/HCO3- exchanger is the most likely explanation for this observation. The Na+/H+ exchange mechanism in the basolateral membrane is an acid extruder, whereas the Cl-/HCO3- exchanger is an acid loader. Both of these mechanisms are important for the maintenance of intracellular pH in HT29 cl.19A cells.

Chronic myelogenous leukemia-derived basophilic granulocytes express a functional active receptor for the anaphylatoxin C3a.

The receptor for the inflammatory peptide C3a has scarcely been examined on human cells. This work demonstrates that human tumor-derived basophilic granulocytes express C3a receptors, and presents parts of the hitherto unknown C3a-signal transduction. When incubated with IL-3, these cells specifically liberated histamine on C3a stimulation. Independent from IL-3, 240,000 +/- 100,000 receptors per cell with a Kd of 5.6 +/- 0.9 nM were determined. [Ca2+]i increased from 120 +/- 35 nM to 300 +/- 80 nM after a C3a challenge, as measured by digital imaging fluorescence microscopy, and rested at its basal level in the presence of C3a-desArg, the immediate catabolic product of C3a in vivo. This [Ca2+]i increase could be completely desensitized homologously by C3a as well as inhibited by up to 75% by pertussis toxin. Thus, tumor-derived basophils are suitable for cloning of the human C3a receptor.