UMLS:C0027819 - FACTA Search

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Query: UMLS:C0027819 (neuroblastoma)
27,800 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The relationship between the depletion of IP3-releasable intracellular Ca2+ stores and the activation of Ca(2+)-selective membrane current was determined during the stimulation of M1 muscarinic receptors in N1E-115 neuroblastoma cells. External Ca2+ is required for refilling Ca2+ stores and the voltage-independent, receptor-regulated Ca2+ current represents a significant Ca2+ source for refilling. The time course of Ca2+ store depletion was measured with fura-2 fluorescence imaging, and it was compared with the time course of Ca2+ current activation measured with nystatin patch voltage clamp. At the time of maximum current density (0.18 + .03 pA/pF; n = 48), the Ca2+ content of the IP3-releasable Ca2+ pool is reduced to 39 + 3% (n = 10) of its resting value. Calcium stores deplete rapidly, reaching a minimum Ca2+ content in 15-30 s. The activation of Ca2+ current is delayed by 10-15 s after the beginning of Ca2+ release and continues to gradually increase for nearly 60 s, long after Ca2+ release has peaked and subsided. The delay in the appearance of the current is consistent with the idea that the production and accumulation of a second messenger is the rate-limiting step in current activation. The time course of Ca2+ store depletion was also measured after adding thapsigargin to block intracellular Ca2+ ATPase. After 15 min in thapsigargin, IP3-releasable Ca2+ stores are depleted by > 90% and the Ca2+ current is maximal (0.19 + 0.05 pA/pF; n = 6). Intracellular loading with the Ca2+ buffer EGTA/AM (10 microM; 30 min) depletes IP3-releasable Ca2+ stores by between 25 and 50%, and it activates a voltage-independent inward current with properties similar to the current activated by agonist or thapsigargin. The current density after EGTA/AM loading (0.61 + 0.32 pA/pF; n = 4) is three times greater than the current density in response to agonist or thapsigargin. This could result from partial removal of Ca(2+)-dependent inactivation.
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PMID:The relationship between depletion of intracellular Ca2+ stores and activation of Ca2+ current by muscarinic receptors in neuroblastoma cells. 864

Following mobilization with the inositol 1,4,5-trisphosphate (IP3)-generating agonist bradykinin, Ca2+ stores in neuroblastoma x glioma hybrid, NG108-15 cells require extracellular Ca2+ to refill. The process by which this store refills with Ca2+ was characterized by recording bradykinin-induced intracellular free Ca2+ concentration transients as an index of the degree of refilling of the store. Cyclopiazonic acid, a microsomal Ca2+ ATPase inhibitor, reversibly depleted intracellular Ca2+ stores in these cells, but did not recruit detectable Ca2+ influx, suggesting that these cells lack substantial capacitative Ca2+ entry. The paucity of voltage-sensitive Ca2+ channels in undifferentiated NG108-15 cells, suggested that a channel analogous to that proposed to mediate capacitative Ca2+ entry in nonexcitable cells might assist refilling IP3-sensitive Ca2+ stores in these cells. The possibility that compounds shown previously to inhibit capacitative Ca2+ entry in nonexcitable cells might inhibit the refilling of the IP3-sensitive store in NG108-15 cells was explored. The IP3-sensitive store was depleted by exposure to bradykinin, allowed to refill briefly in the presence of the test compound and then challenged again with bradykinin to evaluate the degree of refilling of the store. The imidazole derivatives, econazole (10 microM), L-651582 (10 microM) and SKF 96365 (20 microM), all completely blocked the bradykinin-induced Ca2+ response. Calmodulin antagonists, W-7 (100 microM) and trifluoperazine (10 microM), were also effective, although at concentrations well above those required to inhibit calmodulin. Because of the high concentrations required to inhibit bradykinin responses, the possibility that these agents might have additional effects was explored. Compounds were tested in a paradigm in which the store was preloaded with Ca2+ before treatment. All of these agents depleted, at least partially, the preloaded store. Econazole was the least effective of the compounds tested for releasing stores, although it was comparable to the other compounds for inhibition of refilling. Although NG108-15 cells refill intracellular Ca2+ stores by a plasmalemmal Ca2+ leak, this leak shares a pharmacology similar to the capacitative Ca2+ entry pathway described for nonexcitable cells.
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PMID:Pharmacologic characterization of refilling inositol 1,4,5-trisphosphate-sensitive Ca2+ stores in NG108-15 cells. 875 Sep 56

1. Most neurotransmitters are inactivated by uptake into presynaptic nerve terminals and into glial cells. We recently provided evidence for uptake of amines in postsynaptic neurones. Uptake was evident at nanomolar concentrations of prazosin, but at concentrations of unlabelled prazosin greater than 10(-7) M, there was a further activation of uptake, manifested by a paradoxical increase in accumulation of the radioligand. We have now studied further characteristics of amine uptake in immortalised gonadotrophin-releasing hormone (GnRH) neurones. Control cells included SK-N-SH neuroblastoma cells (which possess presynaptic type amine transporters) and non-neuronal (COS-7) cells. 2. [3H]-prazosin bound to intact GnRH cells and was displaced by unlabelled prazosin in concentrations of 10(-9) to 10(-7) M. However, at higher concentrations of unlabelled prazosin, there was an increase in apparent [3H]-prazosin binding, as we had previously described. This paradoxical increase in accumulation of the radioligand was abolished by desipramine. 3. Desipramine had no effect on the association of prazosin with COS-7 cells. There was no paradoxical increase in accumulation of [3H]-prazosin in COS-7 cells, indicating that this effect requires the presence of a desipramine-blockable uptake process. 4. The increase in binding of the radioligand that was observed in the GnRH cells is not a general property of neuronal transporters; in SK-N-SH cells, there was no increase in accumulation of (-)-[3H]-noradrenaline in the presence of concentrations of unlabelled (-)-noradrenaline greater than 10(-7) M. 5. The uptake of prazosin and the increase in accumulation of [3H]-prazosin were abolished in the cold, indicating that this is an active, energy-requiring process. 6. Desipramine-sensitive uptake of prazosin was demonstrable in the GnRH cells in the absence of sodium. Further, the Na+/K(+)-ATPase inhibitor, vanadate, abolished noradrenaline uptake in SK-N-SH cells but had no effect on prazosin uptake in GnRH cells. Thus, the uptake of prazosin does not derive its energy from the sodium pump. 7. Prazosin uptake was inhibited by the V-ATPase inhibitor bafilomycin A1, the H+/Na+ ionophore, monensin and the organic base, chloroquine, indicating that uptake derives its energy from a proton pump. In contrast to other proton-dependent amine transporters, the uptake of prazosin was unaffected by reserpine. 8. Increasing extracellular pH did not increase the uptake of prazosin into GnRH cells, indicating that it is unlikely to be due to non-specific diffusion and concentration of a lysosomotropic drug into intracellular acidic particles. 9. The uptake of prazosin was unaffected by steroid hormones. 10. In COS-7 cells transfected with alpha 1-adrenoceptor cDNA, [3H]-prazosin was displaced by unlabelled prazosin without causing an increase in binding of the radioligand. This indicated that the increase in accumulation of the radioligand is unlikely to be due simply to some function of alpha 1-adrenoceptors. 11. Thus, peptidergic neurones possess an uptake process with properties that are distinguishable from known amine transporters.
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PMID:Functional properties of the uptake of amines in immortalised peptidergic neurones (transport-P). 882 51

Recent studies have demonstrated that opioid agonists affect the cytosolic Ca2+ concentration ([Ca2+]i) either by regulating plasma membrane Ca(2+)-channel activity or by mobilizing intracellular Ca2+ stores. The present report documents the [Ca2+]i increase induced by opioid agonists in a human neuroblastoma cell line, SK-N-BE, expressing delta-opioid receptors. In the presence, as well as in the absence, of extracellular Ca2+, opioid agonists enhanced significantly [Ca2+]i, whereas carbachol, known to mobilize specifically inositol 1,4,5-trisphosphate-sensitive intracellular Ca2+ stores, acted only in the presence of extracellular Ca2+. The opioid-induced increase in [Ca2+]i was not affected by treatments modifying the trimeric Gl, Go, and Gs protein transduction mechanisms or the activity of adenylyl cyclase. The Ca(2+)-ATPase pump-inhibiting sesquiterpene lactone, thapsigargin, did not modify the opioid-induced [Ca2+]i response, whereas it abolished the effects of carbachol. The Ryana speciosa alkaloid, ryanodine, at concentrations known to block endoplasmic reticulum ryanodine receptors, decreased significantly the response to opioids without affecting the effects of carbachol. Thus, our results suggest that, in SK-N-BE cells, delta-opioid receptors mobilize Ca2+ from intracellular ryanodine-sensitive stores and the mechanism involved is independent of Gl/Go Gs proteins and protein kinase A activation.
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PMID:The delta-opioid receptor regulates activity of ryanodine receptors in the human neuroblastoma cell line SK-N-BE. 893 79

Ca2+ entry following Ca2+ store depletion was examined in the human neuroblastoma cell line, SH-SY5Y, by measuring the concentration of intracellular free Ca2+ ([Ca2+]i) with fura-2. Application of the muscarinic agonist oxotremorine-M (oxo-M) caused an increase in [Ca2+]i. This consisted of a peak, mediated by release of Ca2+ from internal stores followed by a sustained plateau, mediated by Ca2+ entry across the plasma membrane. The Ca2+ entry resulted from depletion of intracellular Ca2+ stores This pathway was further characterized in the presence of thapsigargin, an inhibitor of the Ca2+ ATPase involved in replenishing IP3-sensitive stores. Stores were first depleted with oxo-M and thapsigargin in the absence of extracellular Ca2+. After washout of oxo-M, subsequent exposure to Ca2+ evoked reproducible increases in [Ca2+]i. Application of oxo-M plus Ca2+ had little effect on the increases in [Ca2+]i, indicating that in SH-SY5Y cells, agonist-dependent pathways contribute little to Ca2+ entry following store depletion. Mn2+, Sr2+ and Ba2+ were permeable through this pathway. Mn2+ and Ba2+ also showed slight permeability in the absence of store depletion. Ca2+ entry following store depletion was blocked by La3+ (IC50 = 75 nM) and by SKF 96365. La3+ blocked Mn2+ entry through the pathway activated by store depletion but did not affect basal Mn2+ permeability. These results indicate that SH-SY5Y neuroblastoma cells have an agonist-independent Ca2+ entry pathway activated by store depletion.
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PMID:Ca2+ entry following store depletion in SH-SY5Y neuroblastoma cells. 901 69

We investigated the influence of ion compositions on the membrane potential in LA-N-1 human neuroblastoma cells using bisoxonol as a potential-sensitive fluorescent dye. The ability of K+, ouabain, veratridine, and maitotoxin to induce membrane depolarization was evaluated. Increasing concentrations of K+ ions from 10 to 50 mM caused a dose-dependent increase of bisoxonol fluorescence, which was completely independent on Na+ and Ca2+. Ouabain (5 mM), an inhibitor of the Na+, K(+)-ATPase, failed to induce membrane depolarization. Veratridine (40 and 100 microM), a Na+ channel activator, only in the presence of 10 micrograms of Leiurus scorpion venom reduced the membrane potential. Maitotoxin (MTX) from 3 to 10 ng/mL depolarized LA-N-1 cells in a dose-dependent manner, and produced a rapid and sustained increase of intracellular free calcium monitored by means of fluorescent probe fura-2. The MTX-induced depolarization and the increase in cytosolic free calcium concentration were dependent on extracellular Ca2+ ions. On the other hand, Na+ ions also seem to be, although only partially, implicated in the MTX effects, since both the blockade of tetrodotoxin (TTX)-sensitive voltage-operated Na+ channels and the removal of Na+ ions were able to reduce the depolarization. In conclusion, our data indicate that the depolarizing action of MTX on LA-N-1 cells is Ca(2+)- and Na(+)-dependent, although the latter only partially, and that this effect is dependent on Ca2+ influx into the cells likely through a voltage-insensitive calcium-entry system.
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PMID:Membrane depolarization in LA-N-1 cells. The effect of maitotoxin is Ca(2+)- and Na(+)-dependent. 916 86

SecA is the peripheral subunit of the preprotein translocase of Escherichia coli. SecA consists of two independently folding domains, i.e., the N-domain bearing the high-affinity nucleotide binding site (NBS-I) and the C-domain that harbors the low-affinity NBS-II. ATP induces SecA insertion into the membrane during preprotein translocation. Domain-specific monoclonal antibodies (mAbs) were developed to analyze the functions of the SecA domains in preprotein translocation. The antigen binding sites of the obtained mAbs were confined to five epitopes. One of the mAbs, i.e., mAb 300-1K5, recognizes an epitope in the C-domain in a region that has been implicated in membrane insertion. This mAb, either as IgG or as Fab, completely inhibits in vitro proOmpA translocation and SecA translocation ATPase activity. It prevents SecA membrane insertion and, more strikingly, reverses membrane insertion and promotes the release of SecA from the membrane. Surface plasmon resonance measurements demonstrate that the mAb recognizes the ADP- and the AMP-PNP-bound state of SecA either free in solution or bound at the membrane at the SecYEG protein. It is concluded that the mAb actively reverses a conformation essential for membrane insertion of SecA. The other mAbs directed to various epitopes in the N-domain were found to be without effect, although all bind the native SecA. These results demonstrate that the C-domain plays an important role in the SecA membrane insertion, providing further evidence that this process is needed for preprotein translocation.
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PMID:Inhibition of preprotein translocation and reversion of the membrane inserted state of SecA by a carboxyl terminus binding mAb. 923 48

While emphasis has been placed upon those proteins which either mediate or respond to the rapid influx of calcium following depolarization, there has been little emphasis upon those proteins which aid in the reequilibration of the membrane potential. In an effort to identify presynaptic membrane proteins implicated in neurosecretion, monoclonal antibodies were screened against proteins which cosegregated with neuronal voltage-dependent calcium channels (VDCC) following immunoprecipitation. One monoclonal antibody (mAb 9A7) identified a 110-kDa protein. Micropeptide sequencing of (i) the mAb 9A7 immunoaffinity purified antigen and (ii) the 110-kDa protein present in the neuronal (N-type) VDCC preparation (McEnery et al., 1991, Proc. Natl. Acad. Sci. 88, 11095-11099) indicated identity with the alpha subunit(s) of the Na,K-ATPase. Further characterization by Western blotting, immunochemical localization, and immunoaffinity purification indicated that mAb 9A7 not only recognized the alpha3 isoform which is predominant in neuronal tissues but also identified the alpha1 and alpha2 isoforms. mAb 9A7 exhibited a wide cross-species reactivity and recognized human, rat, and mouse alpha subunit isoforms at an internal epitope. The pan-specificity of mAb 9A7 and the differential mobility of the alpha1 isoform relative to the alpha2 and alpha3 permitted parallel detection of multiple alpha isoforms. Western blot analysis of undifferentiated rat pheochromocytoma cell line (PC12) and human neuroblastoma (IMR32) cells indicated coexpression of the alpha1 and alpha3 isozymes. Upon differentiation of IMR32 cells by dibutrylyl-cAMP, a substantial increase in the alpha3 relative to the alpha1 isoform was observed. While the enrichment of total Na,K-ATPase may reflect the increased demand for ATP-dependent ion transport as IMR32 cells become more excitable, the specific increase in the alpha3 isoform suggests a unique role of this isoform during IMR32 cell differentiation.
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PMID:Parallel detection of Na,K-ATPase alpha subunit isoforms by pan-specific monoclonal mAb 9A7. 924 94

The contribution of an electrogenic proton pump to the membrane potential of neuroblastoma x glioma hybrid NG 108-15 cells was determined with whole-cell voltage and current recordings and cell volume measurements with the preparation bathed in symmetrical 140 mM KCl solutions. The effects of the K+ channel blockers tetraethylammonium and 4-aminopyridine and of the H+-ATPase inhibitor bafilomycin A1 on the membrane potential and input resistance revealed that the membrane potential is generated by an outward H+ pump current of 5-15 pA in equilibrium with an inward passive current. This conclusion is supported by both current- and voltage-clamp results obtained when the preparation was bathed in a Na+-containing external solution after K+ channel blockade with Cs+ in the pipette. Additional support was obtained by measurement of the volume of cells incubated in solutions containing 140 mM KCl. Tetraethylammonium induced a bafilomycin-sensitive increase in inward K+ current and an increase in cell volume of 76% which we believe to be a consequence of the K+ influx. Finally, comparison of membrane potentials obtained in experiments using Na+-containing external, and K+-containing pipette solutions and after K+ channel blockade with Cs+ in the pipette also showed that, under normal physiological conditions, the resting membrane potential is essentially determined by an electrogenic H+ pump.
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PMID:A proton pump contributes to neuroblastoma x glioma cell membrane potentials. 930 8

Thapsigargin, a specific inhibitor of the endoplasmic reticular Ca(2+)-ATPase, has been used previously to mobilize calcium release from intracellular calcium stores. We now show that thapsigargin (1-10 microM) induces apoptosis in a neuroblastoma cell line (SH-SY5Y) and in fetal rat cerebrocortical cultures. Cell death measured by lactate dehydrogenase release was observed 24-48 hours after treatment with thapsigargin. In both cases, DNA extracts from thapsigargin treated cells showed laddering, typical of endonuclease-mediated internucleosomal cleavages. The presence of DNA fragments was also confirmed by an ELISA designed for detecting nucleosomes in apoptotic cells. Cycloheximide reduced the extent of DNA fragmentation and injury in thapsigargin-treated cells. Dantrolene, an inhibitor of calcium release from intracellular stores partially abolished the effect of thapsigargin, suggesting that the initial Ca2+ rise may be the signalling event in this apoptotic cell death pathway. We propose that thapsigargin-induced cell death in cultured neuronal cells may be a useful system to study the molecular and genetic events involved in apoptosis.
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PMID:Thapsigargin induces apoptosis in SH-SY5Y neuroblastoma cells and cerebrocortical cultures. 931 98

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