Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 24
Filter
Add more filters










Publication year range
1.
Oncogene ; 36(20): 2846-2856, 2017 05 18.
Article in English | MEDLINE | ID: mdl-27991924

ABSTRACT

The growth of disseminated tumor cells into metastatic lesions depends on the establishment of a favorable microenvironment in the stroma of the target organs. Here we show that mice treated with anakinra, an antagonist of the interleukin (IL)-1ß receptor (IL-1R), or harboring a targeted deletion of IL-1R are significantly less prone to develop bone tumors when inoculated in the arterial circulation with human prostate cancer (PCa) cells expressing IL-1ß. Interestingly, human mesenchymal stem cells exposed in vitro to medium conditioned by IL-1ß-expressing cancer cells responded by upregulating S100A4, a marker of cancer-associated fibroblasts (CAFs), and this effect was blocked by anakinra. Analogously, the stroma adjacent to skeletal metastases generated in mice by IL-1ß-expressing cancer cells showed a dramatic increase in S100A4, COX-2 and the alteration of 30 tumor-related genes as measured by Nanostring analysis. These effects were not observed in the stroma associated with the rare and much smaller metastases generated by the same cells in IL-1R knockout animals, confirming that tumor-secreted IL-1ß generates skeletal CAFs and conditions the surrounding bone microenvironment. In skeletal lesions from patients with metastatic PCa, histological and molecular analyses revealed that IL-1ß is highly expressed in cancer cells in which the androgen receptor (AR) is not detected (AR-), whereas this cytokine is uniformly absent in the AR-positive (AR+) metastatic cells. The stroma conditioned by IL-1ß-expressing cancer cells served as a supportive niche also for coexisting IL-1ß-lacking cancer cells, which are otherwise unable to generate tumors after independently seeding the skeleton of mice. This niche is established very early following tumor seeding and hints to a role of IL-1ß in promoting early colonization of PCa at the skeletal level.


Subject(s)
Bone Neoplasms/metabolism , Bone Neoplasms/secondary , Cell Communication , Prostatic Neoplasms/pathology , Tumor Microenvironment , Animals , Biomarkers , Cell Line, Tumor , Cell Proliferation , Disease Models, Animal , Gene Expression , Heterografts , Humans , Interleukin-1beta/metabolism , Male , Mesenchymal Stem Cells/metabolism , Mice , Neoplasm Metastasis , Phenotype , Receptors, Androgen/genetics , Receptors, Androgen/metabolism , Signal Transduction , Stromal Cells/metabolism
2.
Oncogene ; 28(3): 412-21, 2009 Jan 22.
Article in English | MEDLINE | ID: mdl-18850002

ABSTRACT

Bone resorption by osteoclasts is thought to promote the proliferation of prostate cancer cells disseminated to the skeleton (Mundy, 2002). Using a mouse model of experimental metastasis, we found that although late-stage metastatic tumors were indeed surrounded by osteoclasts, these cells were spatially unrelated to the small foci of cancer cells in early-stage metastases. This is the first evidence that survival and growth of disseminated prostate cancer cells immediately after their extravasation may not depend on osteoclast involvement. Interestingly, prostate cancer cells expressing the alpha-receptor for platelet-derived growth factor (PDGFRalpha) progress during early-stages of skeletal dissemination, whereas cells expressing lower levels or lacking this receptor fail to survive after extravasation in the bone marrow. However, non-metastatic cells acquire bone-metastatic potential upon ectopic overexpression of PDGFRalpha. Finally, functional blockade of human PDGFRalpha on prostate cancer cells utilizing a novel humanized monoclonal antibody -- soon to undergo phase-II clinical trials -- significantly impairs the establishment of early skeletal metastases. In conclusion, our results strongly implicate PDGFRalpha in prostate cancer bone tropism through its promotion of survival and progression of early-metastatic foci, providing ground for therapeutic strategies aimed at preventing or containing the initial progression of skeletal metastases in patients affected by prostate adenocarcinoma.


Subject(s)
Antibodies, Monoclonal/therapeutic use , Bone Neoplasms/prevention & control , Bone Neoplasms/secondary , Prostatic Neoplasms/pathology , Receptor, Platelet-Derived Growth Factor alpha/antagonists & inhibitors , Adolescent , Adult , Animals , Blotting, Western , Bone Marrow/metabolism , Bone Marrow/pathology , Bone Neoplasms/metabolism , Bone and Bones/metabolism , Bone and Bones/pathology , Flow Cytometry , Humans , Immunoenzyme Techniques , Male , Mice , Mice, SCID , Middle Aged , Phosphorylation , Tumor Cells, Cultured , Young Adult
3.
Proc Natl Acad Sci U S A ; 97(14): 8075-80, 2000 Jul 05.
Article in English | MEDLINE | ID: mdl-10869418

ABSTRACT

Recent in vitro and in vivo studies have shown that the chemokine fractalkine is widely expressed in the brain and localized principally to neurons. Central nervous system expression of CX(3)CR1, the only known receptor for fractalkine, has been demonstrated exclusively on microglia and astrocytes. Thus, it has been proposed that fractalkine regulates cellular communication between neurons (that produce fractalkine) and microglia (that express its receptor). Here we show, for the first time, that hippocampal neurons also express CX(3)CR1. Receptor activation by soluble fractalkine induces activation of the protein kinase Akt, a major component of prosurvival signaling pathways, and nuclear translocation of NF-kappaB, a downstream effector of Akt. Fractalkine protects hippocampal neurons from the neurotoxicity induced by the HIV-1 envelope protein gp120(IIIB), an effect blocked by anti-CX(3)CR1 antibodies. Experiments with two different inhibitors of the phosphatidylinositol 3-kinase, a key enzyme in the activation of Akt, and with a phospholipid activator of Akt demonstrate that Akt activation is responsible for the neuroprotective effects of fractalkine. These data show that neuronal CX(3)CR1 receptors mediate the neurotrophic effects of fractalkine, suggesting that fractalkine and its receptor are involved in a complex network of both paracrine and autocrine interactions between neurons and glia.


Subject(s)
Chemokines, CX3C , Chemokines, CXC/metabolism , Hippocampus/cytology , I-kappa B Proteins , Membrane Proteins/metabolism , Neurons/physiology , Protein Serine-Threonine Kinases , Receptors, Cytokine/metabolism , Receptors, HIV/metabolism , Animals , Astrocytes/physiology , CX3C Chemokine Receptor 1 , Cell Survival , Chemokine CX3CL1 , Culture Techniques/methods , DNA-Binding Proteins/metabolism , NF-KappaB Inhibitor alpha , NF-kappa B/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Phosphatidylinositols/metabolism , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-akt , Rats , Receptors, Cytokine/genetics , Receptors, HIV/genetics , Recombinant Proteins/metabolism , Signal Transduction
4.
FASEB J ; 13(11): 1291-301, 1999 Aug.
Article in English | MEDLINE | ID: mdl-10428754

ABSTRACT

The effects of growth factors have been shown to depend on the position of a cell in the cell cycle. However, the physiological basis for this phenomenon remains unclear. Here we show that the majority of both CEINGE clone3 (cl3) and human embryonic kidney 293 cells, when arrested in a quiescent phase (G(0)), responded to platelet-derived growth factor BB (PDGF-BB) with non-oscillatory Ca(2+) signals. Furthermore, the same type of Ca(2+) response was also observed in CEINGE cl3 cells (and to a lesser extent in HEK 293 cells) blocked at the G(1)/S boundary. In contrast, CEINGE cl3 cells synchronized in early G(1) or released from G(1)/S arrest responded in an oscillatory fashion. This cell cycle-dependent modulation of Ca(2+) signaling was not observed on epidermal growth factor and G-protein-coupled receptor stimulation and was not due to differences in the expression of PDGF receptors (PDGFRs) during the cell cycle. We demonstrate that inhibition of sphingosine-kinase, which converts sphingosine to sphingosine-1-phosphate, caused G(0) as well as G(1)/S synchronized cells to restore the oscillatory Ca(2+) response to PDGF-BB. In addition, we show that the synthesis of sphingosine and sphingosine-1-phosphate is regulated by the cell cycle and may underlie the differences in Ca(2+) signaling after PDGFR stimulation.


Subject(s)
Calcium/physiology , Cell Cycle/physiology , Platelet-Derived Growth Factor/pharmacology , Signal Transduction/drug effects , Signal Transduction/physiology , Sphingolipids/metabolism , Cell Culture Techniques , Cell Cycle/drug effects , Humans , Phosphotransferases (Alcohol Group Acceptor)/physiology , Platelet-Derived Growth Factor/physiology
5.
Proc Natl Acad Sci U S A ; 95(24): 14500-5, 1998 Nov 24.
Article in English | MEDLINE | ID: mdl-9826729

ABSTRACT

The HIV-1 envelope protein gp120 induces apoptosis in hippocampal neurons. Because chemokine receptors act as cellular receptors for HIV-1, we examined rat hippocampal neurons for the presence of functional chemokine receptors. Fura-2-based Ca imaging showed that numerous chemokines, including SDF-1alpha, RANTES, and fractalkine, affect neuronal Ca signaling, suggesting that hippocampal neurons possess a wide variety of chemokine receptors. Chemokines also blocked the frequency of spontaneous glutamatergic excitatory postsynaptic currents recorded from these neurons and reduced voltage-dependent Ca currents in the same neurons. Reverse transcription-PCR demonstrated the expression of CCR1, CCR4, CCR5, CCR9/10, CXCR2, CXCR4, and CX3CR1, as well as the chemokine fractalkine in these neurons. Both fractalkine and macrophage-derived chemokine (MDC) produced a time-dependent activation of extracellular response kinases (ERK)-1/2, whereas no activation of c-JUN NH2-terminal protein kinase (JNK)/stress-activated protein kinase, or p38 was evident. Furthermore, these two chemokines, as well as SDF-1alpha, activated the Ca- and cAMP-dependent transcription factor CREB. Several chemokines were able also to block gp120-induced apoptosis of hippocampal neurons, both in the presence and absence of the glial feeder layer. These data suggest that chemokine receptors may directly mediate gp120 neurotoxicity.


Subject(s)
Chemokines/pharmacology , Gene Expression Regulation , HIV Envelope Protein gp120/toxicity , HIV-1 , Hippocampus/physiology , Neurons/physiology , Neurotoxins/toxicity , Receptors, Chemokine/genetics , Signal Transduction/physiology , Animals , Calcium/metabolism , Cell Death/drug effects , Cells, Cultured , DNA Primers , Embryo, Mammalian , Evoked Potentials/drug effects , Evoked Potentials/physiology , Gene Expression Regulation/drug effects , Hippocampus/cytology , Humans , Mice , Neurons/cytology , Neurons/drug effects , Rats , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction/drug effects , Synapses/drug effects , Synapses/physiology , Synaptic Transmission/drug effects , Synaptic Transmission/physiology
7.
J Biol Chem ; 272(7): 4351-8, 1997 Feb 14.
Article in English | MEDLINE | ID: mdl-9020156

ABSTRACT

Ca2+ signaling induced by platelet-derived growth factor (PDGF) was investigated in the oligodendroglial cell lines CG4 and CEINGE clone 3, using fura-2 microfluorimetry and video imaging. CEINGE cl3 cells, immortalized with polyoma middle T antigen, were found to uniformly express the polyoma middle T antigen protein as well as 2',3'-cyclic nucleotide 3'-phosphodiesterase, a specific marker for oligodendroglia. PDGF-BB induced both oscillatory and non-oscillatory Ca2+ responses in CEINGE cl3 cells as well as in CG4 cells, grown either as O-2A progenitors or differentiated oligodendrocytes. However, in CG4 cells the percentage of oscillatory Ca2+ responses was higher than that observed in CEINGE cl3 cells. In contrast, oscillatory Ca2+ responses were not observed in PC-12 cells transfected with beta-PDGF receptor (PDGFR) or in NIH 3T3 fibroblasts. CG4 cells expressed only the alpha-PDGFR, whereas CEINGE cl3 cells expressed both alpha and beta isoforms. When CEINGE cl3 cells were exposed to PDGF-AA, which binds only to the alpha-PDGFR, the percentage of oscillatory Ca2+ responses was higher than that observed after PDGF-BB stimulation. We previously reported that block of the enzyme sphingosine kinase, and a consequent increase in intracellular sphingosine levels in CEINGE cl3 cells caused an increase in the percentage of oscillatory Ca2+ responses induced by PDGF-BB. However, in CG4 cells block of sphingosine kinase did not increase the oscillatory Ca2+ response elicited by PDGF-BB, although the addition of exogenous sphingosine induced an oscillatory Ca2+ response in 77% of cells studied. We hypothesize that the alpha-PDGFR is less effective than the beta-PDGFR in stimulating the activity of sphingosine kinase. The results also suggest that alpha- and beta-PDGFRs may differently regulate sphingolipid metabolism.


Subject(s)
Calcium/metabolism , Oligodendroglia/metabolism , Platelet-Derived Growth Factor/pharmacology , Receptors, Platelet-Derived Growth Factor/metabolism , Signal Transduction , 3T3 Cells , Animals , Becaplermin , Blotting, Western , Cell Line , Cell Line, Transformed , Clone Cells , Fluorescent Antibody Technique , Mice , Neomycin/pharmacology , Oligodendroglia/cytology , PC12 Cells , Proto-Oncogene Proteins c-sis , Rats , Receptor, Platelet-Derived Growth Factor beta , Receptors, Platelet-Derived Growth Factor/genetics , Sphingosine/analogs & derivatives , Sphingosine/pharmacology
9.
J Biol Chem ; 271(1): 295-301, 1996 Jan 05.
Article in English | MEDLINE | ID: mdl-8550576

ABSTRACT

The roles of sphingosine and sphingosine 1-phosphate in Ca2+ signaling following platelet-derived growth factor (PDGF) receptor stimulation were investigated in the oligodendrocyte cell line CEINGE cl3, using single-cell fura-2 microfluorimetry and videoimaging. Two different Ca2+ responses were observed, which differed in their delays and kinetics. The first response, which occurred after a shorter delay, exhibited a single Ca2+ peak often followed by a plateau, while the second type of response was characterized by a longer delay and by Ca2+ spikes with different frequencies and amplitudes. The latter phenomenon was never observed after stimulation of G protein-coupled receptors for ATP, ET-1, and BK. The incubation with the inhibitor of sphingosine kinase, DL-threo-dihydrosphingosine, significantly increased the percentage of cells responding to PDGF-BB exposure with Ca2+ spikes (87 versus 47%), while it did not modify the Ca2+ response elicited by exposure to ATP, ET-1, or BK. Exposure to exogenous 10 microM sphingosine or 1 microM sphingosine 1-phosphate produced oscillatory and non-oscillatory Ca2+ responses, respectively, similar to those elicited by PDGF-BB. A second application of PDGF-BB, 30 min after the first, was normally ineffective in producing a Ca2+ response. However, if the second exposure was preceded by the inhibition of sphingosine 1-phosphate formation, an oscillatory Ca2+ response occurred in all cells. We conclude that intracellular levels of sphingosine and sphingosine 1-phosphate may differentially modulate Ca2+ signaling triggered by PDGF receptor stimulation in CEINGE cl3-transformed oligodendrocytes.


Subject(s)
Calcium/metabolism , Lysophospholipids , Oligodendroglia/metabolism , Platelet-Derived Growth Factor/physiology , Signal Transduction , Sphingosine/analogs & derivatives , Sphingosine/physiology , Becaplermin , Cell Line , Oligodendroglia/cytology , Proto-Oncogene Proteins c-sis
10.
J Neurosci ; 16(2): 519-30, 1996 Jan 15.
Article in English | MEDLINE | ID: mdl-8551336

ABSTRACT

We examined the properties of glutamate agonist-induced Ca2+ fluxes in cultured CG-4 and O-2A progenitor cells from rat cortex. Kainate-induced Ca2+ fluxes in these cells were found to be attributable to the activation of AMPA receptors. Thus, these fluxes were enhanced by cyclothiazide but not by concanavalin A and were blocked completely by GYKI-53655. We simultaneously examined kainate-induced Ca2+ entry and Na+ currents in these cells under voltage-clamp conditions. Both of these parameters were blocked by Joro spider toxin (JSTx) in undifferentiated cells. However, neither JSTx nor Argiotoxin 636 effectively blocked either parameter in cells differentiated into type II astrocytes. This change in toxin sensitivity occurred slowly over a period of several days. Similar results were obtained in Ca(2+)-imaging studies. When cells were differentiated into oligodendrocytes, they showed an intermediate sensitivity to block by JSTx as assessed using imaging and voltage-clamp studies. Analysis of the expression of AMPA-receptor subunits showed an increase in the concentration of glutamate receptor-2 (GluR2) in CG-4 cells as they differentiated into type II astrocytes and oligodendrocytes. These results demonstrate that the AMPA receptors in cells of the O-2A lineage flux appreciable amounts of Ca2+ but may contain variable amounts of edited GluR2 subunits.


Subject(s)
Calcium/metabolism , Neuroglia/drug effects , Neurotoxins/pharmacology , Receptors, AMPA/drug effects , Receptors, AMPA/physiology , Animals , Brain/drug effects , Cells, Cultured , Ion Channels/physiology , Kainic Acid/pharmacology , Permeability , Rats , Receptors, Glutamate/physiology , Time Factors
11.
Eur J Pharmacol ; 294(1): 11-5, 1995 Dec 27.
Article in English | MEDLINE | ID: mdl-8788411

ABSTRACT

2',4'-dimethylbenzamilamiloride (DMB), a somewhat selective inhibitor of the Na(+)-Ca2+ exchanger, in concentrations of 10, 30 and 100 microM did not produce any significant effect on baseline prolactin release from anterior pituitary cells in primary culture. When prolactin secretion was stimulated by the inhibitor of the Na(+)-K(+)-ATPase, ouabain, that activates the Na(+)-Ca2+ exchanger as a Ca(2+)-influx pathway, DMB was able to produce inhibition of prolactin secretion. 5-(N,N-hexamethylene) amiloride (HMA), another amiloride analog which specifically inhibits the Na(+)-H+ antiporter and has no inhibitory activity on the Na(+)-Ca2+ exchanger, at the concentrations of 0.1, 1 and 10 microM, did not affect basal prolactin release whereas it significantly reduced prolactin release stimulated by thyrotropin releasing hormone (TRH) (1 microM). These results suggest that the Na(+)-Ca2+ antiporter is involved in the process of prolactin release elicited by the inhibition of the Na(+)-K(+)-ATPase whereas the Na(+)-H+ antiporter is involved in the prolactin secretion elicited by TRH.


Subject(s)
Antiporters/physiology , Pituitary Gland, Anterior/metabolism , Prolactin/metabolism , Sodium-Hydrogen Exchangers/physiology , Amiloride/analogs & derivatives , Amiloride/pharmacology , Animals , Antiporters/antagonists & inhibitors , Cell Membrane/drug effects , Cell Membrane/metabolism , Cells, Cultured , Enzyme Inhibitors/pharmacology , Female , Ouabain/pharmacology , Pituitary Gland, Anterior/cytology , Pituitary Gland, Anterior/drug effects , Rats , Rats, Wistar , Sodium-Calcium Exchanger , Sodium-Hydrogen Exchangers/antagonists & inhibitors , Sodium-Potassium-Exchanging ATPase/antagonists & inhibitors , Thyrotropin-Releasing Hormone/antagonists & inhibitors , Thyrotropin-Releasing Hormone/pharmacology
12.
J Neurochem ; 63(5): 1900-7, 1994 Nov.
Article in English | MEDLINE | ID: mdl-7523602

ABSTRACT

LAN-1 is a human neuroblastoma cell line that, in the undifferentiated state, does not respond to membrane depolarization with an elevation of [Ca2+]i, monitored by fura-2 single-cell microfluorimetry. The exposure of LAN-1 cells to the differentiating agent retinoic acid induced the appearance of [Ca2+]i elevation elicited by 55 mM K+. Maitotoxin, a putative activator of voltage-sensitive Ca2+ channels, did not evoke an elevation of [Ca2+]i in undifferentiated LAN-1 cells, but produced a marked and sustained increase in [Ca2+]i when superfused in retinoic acid-treated cells. Both high K(+)- and maitotoxin-induced [Ca2+]i elevation in retinoic acid-differentiated LAN-1 cells was reversed by the lanthanide Gd3+, an inorganic Ca(2+)-entry blocker, and by the snail toxin omega-conotoxin GVIA, which interacts with the N subtype of voltage-sensitive Ca2+ channels. In contrast, both Bay K 8644 and nimodipine, dihydropyridines that selectively activate or block, respectively, the L-channel subtype, were completely ineffective. The tumor promoter phorbol 12-myristate 13-acetate (100 nM), a protein kinase C activator, inhibited the elevation of [Ca2+]i due to Ca2+ influx elicited by membrane depolarization. K(+)-induced [Ca2+]i elevation appeared 24 h after the addition of retinoic acid and reached the highest magnitude after 72 h. Furthermore, 8 days after the removal of the differentiating agent from the culture medium, the high K(+)-induced increase of [Ca2+]i was still present.(ABSTRACT TRUNCATED AT 250 WORDS)


Subject(s)
Calcium/metabolism , Marine Toxins/pharmacology , Membrane Potentials/physiology , Neuroblastoma/metabolism , Neuroblastoma/pathology , Oxocins , Tretinoin/pharmacology , 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester/pharmacology , Calcium/analysis , Cell Differentiation/drug effects , Cytophotometry , Humans , Membrane Potentials/drug effects , Neuroblastoma/chemistry , Nimodipine/pharmacology , Peptides/pharmacology , Potassium/pharmacology , Tetradecanoylphorbol Acetate/pharmacology , Time Factors , Tumor Cells, Cultured , omega-Conotoxin GVIA
13.
J Biol Chem ; 269(27): 18021-7, 1994 Jul 08.
Article in English | MEDLINE | ID: mdl-8027061

ABSTRACT

The effect of sequential stimulation of different inositol (1,4,5)-trisphosphate (IP3)-linked receptors on the functioning of intracellular Ca2+ stores was evaluated in single LAN-1 human neuroblastoma cells by means of fura-2 microfluorimetry. Homologous restimulation both in the absence and in the presence of extracellular Ca2+ with endothelin-1 (ET-1), Lys-bradykinin (BK), and ATP did not elicit an intracellular Ca2+ increase, whereas a [Ca2+]i elevation after carbachol (CCh) re-exposure was obtained only in the presence of extracellular Ca2+. Since thapsigargin and ionomycin, in the absence of extracellular Ca2+, were still able to release Ca2+ after ET-1, BK, and ATP but not after CCh, it can be argued that in the first case the stores were not completely depleted. This evidence was also confirmed by the fact that LAN-1 cells, sequentially exposed in different order to ET-1, BK, ATP, and upon extracellular Ca2+ removal, showed an increase of [Ca2+]i although progressively reduced in magnitude. By contrast, when CCh was perfused as the first agonist, it completely precluded any further Ca2+ mobilization by the other three agonists. In addition, the lack of potentiation of the Ca2+ response when BK and ET-1 were superfused together and the potentiation of Ca2+ response elicited by ET-1 after BK, when the plasma membrane Ca2+ efflux pathways were blocked by lanthanum during the first agonist exposure, indicated that LAN-1 cells can recycle cytoplasmic Ca2+ when exposed to ET-1, BK, ATP but not when exposed to CCh. This inhibitory effect of CCh (perfused for 90 s) on Ca2+ refilling was strictly dependent on the time of receptor occupancy since the exposure to CCh for a shorter period (15 s) produced the same effect on Ca2+ refilling when ET-1, BK, and ATP were perfused, as first agonist, for 90 s. Furthermore, the entity of Ca2+ refilling after 15 s of BK receptor occupancy was similar to that observed after 90 s. This seems to suggest that the receptors for ET-1, BK, and ATP maintain the transductional mechanisms in an activated state for a time shorter than the time of receptor occupancy. This was confirmed by the fact that IP3 levels during a 90-s BK exposure fell to prestimulated value within 30 s, whereas after CCh they reached a sustained plateau phase, after the peak.


Subject(s)
Calcium/metabolism , Inositol 1,4,5-Trisphosphate/metabolism , Receptors, Cell Surface/metabolism , Type C Phospholipases/metabolism , Cell Membrane/enzymology , Cytoplasm/metabolism , Enzyme Activation , Humans , Neuroblastoma , Receptors, Bradykinin/metabolism , Tumor Cells, Cultured
14.
Proc Natl Acad Sci U S A ; 91(6): 2036-40, 1994 Mar 15.
Article in English | MEDLINE | ID: mdl-8134346

ABSTRACT

In type I astrocytes from rat cerebral cortex, vasoactive intestinal peptide (VIP) at concentrations below 1 nM evoked an increase in intracellular calcium ion concentration. This response, however, was observed in only 18% of the astrocytes examined. alpha-Adrenergic stimulation with phenylephrine or norepinephrine also resulted in an intracellular calcium response in these cells and the threshold sensitivity of astrocytes to phenylephrine was vastly different from cell to cell. Treatment of these astrocytes with VIP (0.1 nM) together with phenylephrine at subthreshold concentrations produced large increases in intracellular Ca2+ concentration ([Ca2+]i) and oscillations. The continued occupation of the alpha-adrenergic receptor was required for sustained synergism. Both alpha-receptor stimulation and stimulation with the mixture of agonists induced the cellular calcium response by triggering release of calcium from cellular stores, since the response persisted in the absence of extracellular calcium. Furthermore, thapsigargin pretreatment, which depletes intracellular stores, abolished the agonist-induced [Ca2+]i response. VIP (0.1 nM) and phenylephrine were found to increase cellular levels of inositol phosphates; however, there was no apparent additivity in this response when the agonists were added together. These observations suggest a calcium-mediated second messenger system for the high-affinity VIP receptor in astrocytes and that alpha-adrenergic receptors act synergistically with the VIP receptor to augment an intracellular calcium signal. The synergism between diverse receptor types may constitute an important mode of cellular signaling in astroglia.


Subject(s)
Astrocytes/metabolism , Calcium/metabolism , Receptors, Adrenergic, alpha/metabolism , Vasoactive Intestinal Peptide/metabolism , Animals , Astrocytes/cytology , Cells, Cultured , Norepinephrine/pharmacology , Phenylephrine/pharmacology , Rats , Signal Transduction
15.
Neurosci Lett ; 159(1-2): 159-62, 1993 Sep 03.
Article in English | MEDLINE | ID: mdl-8264960

ABSTRACT

We have used the polyoma middle T oncogene to immortalize cells from rat embryo encephalon. Immunostaining experiments with monoclonal antibodies demonstrated that the cells of one of the obtained lines, named CEINGE CL3, are stained by anti-vimentin and anti-S100 antibodies, are not stained by anti-neurofilaments (NF) or anti-glial fibrillary acidic-protein (GFAP) antibodies. Only a subset of the CEINGE CL3 cells (20-30%) is stained by an anti-galactocerebroside antibody. Northern blot analysis demonstrated that these cells express low levels of proteolipid protein mRNA, whereas polymerase chain reaction (PCR) amplification failed to evidentiate the presence of both NF and GFAP mRNAs. Either retinoic acid or forskolin treatments or a combination of them are able to induce morphological changes that are accompanied by a complete growth arrest.


Subject(s)
Oligodendroglia/ultrastructure , Animals , Antibodies, Monoclonal , Antigens, Polyomavirus Transforming/pharmacology , Blotting, Northern , Brain/embryology , Cell Line , Colforsin/pharmacology , Female , Glial Fibrillary Acidic Protein/immunology , Glial Fibrillary Acidic Protein/metabolism , Neurofilament Proteins/immunology , Neurofilament Proteins/metabolism , Oligodendroglia/drug effects , Oncogenes , Phenotype , Pregnancy , Rats , Rats, Sprague-Dawley , Tretinoin/pharmacology
16.
J Biol Chem ; 267(30): 21928-35, 1992 Oct 25.
Article in English | MEDLINE | ID: mdl-1328249

ABSTRACT

Type IV collagen (Coll IV), a component of the extracellular matrix, stimulates motility in the A2058 human melanoma cell line, a response that is inhibited by pertussis toxin (PT). Fibronectin (FN)-induced chemotaxis in this cell line is not affected by PT. To understand the mechanism of cellular signaling, single cell intracellular Ca2+ responses to Coll IV and FN were studied using Fura-2 and digital imaging fluorescence microscopy. Coll IV, at a dose that stimulates motility (100 micrograms/ml, 185 nM), induces a significant rise in cytosolic free Ca2+ concentration ([Ca2+]i) within 100 s. This response is not inhibited by PT. Treatment of the cells with FN 30 micrograms/ml (70 nM), a dose that stimulates near-maximal chemotaxis, does not increase [Ca2+]i appreciably. Removal of extracellular Ca2+ fails to inhibit the Coll IV-stimulated rise in Ca2+ in all cells. Depletion of extracellular Ca2+ and pretreatment of cells with Ca2+ channel blockers only partially inhibits Coll IV-induced motility. Depletion of intracellular Ca2+ inhibits both chemotaxis and the Coll IV-induced increase in intracellular Ca2+. Coll IV does not stimulate membrane phosphoinositide hydrolysis. We conclude that Coll IV treatment induces an inositol 1,4,5-trisphosphate-independent release of intracellular Ca2+ stores which appears to play a necessary role in the chemotactic response of A2058 cells but is not mediated by a PT-sensitive G-protein. This response is not seen in cells exposed to FN, suggesting different intracellular signaling mechanisms for stimulated motility between these two extracellular matrix molecules.


Subject(s)
Calcium/metabolism , Chemotaxis , Collagen/physiology , Neoplasm Invasiveness , Calcium Channel Blockers/pharmacology , Chromatography, Thin Layer , Fibronectins/metabolism , Fura-2 , GTP-Binding Proteins/metabolism , Humans , Melanoma , Microscopy, Fluorescence , Phosphatidylinositols/metabolism , Tumor Cells, Cultured
17.
J Neurochem ; 59(1): 1-9, 1992 Jul.
Article in English | MEDLINE | ID: mdl-1319463

ABSTRACT

The LAN-1 clone, a cell line derived from a human neuroblastoma, possesses muscarinic receptors. The stimulation of these receptors with increasing concentrations of carbachol (CCh; 1-1,000 microM) caused a dose-dependent increase of the intracellular free Ca2+ concentration ([Ca2+]i). This increase was characterized by an early peak phase (10 s) and a late plateau phase. The removal of extracellular Ca2+ reduced the magnitude of the peak phase to approximately 70% but completely abolished the plateau phase. The muscarinic-activated Ca2+ channel was gadolinium (Gd3+) blockade and nimodipine and omega-conotoxin insensitive. In addition, membrane depolarization did not cause any increase in [Ca2+]i. The CCh-induced [Ca2+]i elevation was concentration-dependently inhibited by pirenzepine and 4-diphenylacetoxy-N-methylpiperidine methiodide, two rather selective antagonists of M1 and M3 muscarinic receptor subtypes, respectively, whereas methoctramine, an M2 antagonist, was ineffective. The coupling of M1 and M3 receptor activation with [Ca2+]i elevation does not seem to be mediated by a pertussis toxin-sensitive guanine nucleotide-binding protein or by the diacylglycerol-protein kinase C system. The mobilization of [Ca2+]i elicited by M1 and M3 muscarinic receptor stimulation seems to be dependent on an inositol trisphosphate-sensitive intracellular store. In addition, ryanodine did not prevent CCh-induced [Ca2+]i mobilization, and, finally, LAN-1 cells appear to lack caffeine-sensitive Ca2+ stores, because the methylxanthine was unable to elicit intracellular Ca2+ mobilization, under basal conditions, after a subthreshold concentration of CCh (0.3 microM), or after thapsigargin.


Subject(s)
Calcium/metabolism , Neuroblastoma/metabolism , Receptors, Muscarinic/metabolism , Calcium Channel Blockers/pharmacology , Calcium Channels/metabolism , Calcium Channels/physiology , Carbachol/pharmacology , Electrophysiology , Humans , Inositol 1,4,5-Trisphosphate/pharmacology , Intracellular Membranes/metabolism , Muscarinic Antagonists , Neuroblastoma/pathology , Osmolar Concentration , Pertussis Toxin , Protein Kinase C/metabolism , Tumor Cells, Cultured , Virulence Factors, Bordetella/pharmacology
18.
Funct Neurol ; 7(3): 239-42, 1992.
Article in English | MEDLINE | ID: mdl-1323509

ABSTRACT

Endothelin-1 (ET-1) produced a dose-dependent increase of intracellular Ca++ concentrations [Ca++]i characterized by an early peak phase and a delayed plateau in LAN-1 human neuroblastoma cells. The ET-1 receptor showed a rapid desensitization since a second pulse application of ET-1 did not elicit a further [Ca++]i increase. Furthermore thapsigargin, an endoplasmic reticulum Ca(++)-ATPase inhibitor, completely abolished the ET-1 induced intracellular Ca++ elevation.


Subject(s)
Calcium/metabolism , Endothelins/pharmacology , Intracellular Fluid/drug effects , Receptors, Cell Surface/drug effects , Tumor Cells, Cultured/drug effects , Cell Line , Dose-Response Relationship, Drug , Humans , Intracellular Fluid/physiology , Microscopy, Fluorescence , Neuroblastoma , Receptors, Cell Surface/physiology , Receptors, Endothelin
19.
Neurochem Int ; 20 Suppl: 95S-99S, 1992 Mar.
Article in English | MEDLINE | ID: mdl-1365465

ABSTRACT

2', 4'-Dimethylbenzamiloride (DMB), an inhibitor of Na(+)-Ca++ antiporter dose-dependently (10-100 microM) inhibited Na(+)-dependent 45Ca++ efflux from brain synaptosomes. This compound was also able to stimulate basal release of [3H]DA from superfused TIDA neurons. Another amiloride analogue, 5-N-methyl-N-guanidinocarbonylmethylamiloride (MGCMA, 100-300 microM), which lacks of inhibitory properties on the Na(+)-Ca++ antiporter, failed to modify basal [3H]DA release from TIDA neurons. In addition, when the antiporter operates as a Ca(++)-influx pathway, DMB dose-dependently inhibited Na(+)-dependent 45Ca++ uptake in brain synaptosomes, whereas it did not prevent K(+)-induced 45Ca++ uptake, which reflets the activation of voltage-operated Ca++ channels. Finally DMB inhibited ouabain-induced [3H]DA release, which depends on the activation of the Na(+)-Ca++ exchanger due to the inhibition of the Na+/K(+)-ATPase pump.


Subject(s)
Brain/metabolism , Carrier Proteins/metabolism , Dopamine/metabolism , Hypothalamus/metabolism , Nerve Endings/metabolism , Neurons/metabolism , Amiloride/analogs & derivatives , Amiloride/pharmacology , Animals , Calcium/metabolism , Carrier Proteins/antagonists & inhibitors , Choline/pharmacology , Female , Hypothalamus/cytology , Ouabain/pharmacology , Potassium/pharmacology , Rats , Rats, Wistar , Sodium-Calcium Exchanger , Synaptosomes/metabolism
20.
Brain Res ; 574(1-2): 33-41, 1992 Mar 06.
Article in English | MEDLINE | ID: mdl-1638405

ABSTRACT

Cytoplasmic calcium concentration was measured in neurosecretory nerve terminals (neurosecretosomes) isolated from rat neurohypophyses by fura-2 fluorescence measurements and digital video microscopy. Hormone release and cytoplasmic calcium concentration were measured during depolarizations induced by elevated extracellular potassium concentration. During prolonged depolarizations with 55 mM [K+]o, the cytoplasmic calcium concentration remained elevated as long as depolarization persisted, while secretion inactivated after the initial sharp rise. The amplitude and duration of the increase in [Ca2+]i was dependent on the degree of depolarization such that upon low levels of depolarizations (12.5 mM or 25 mM [K+]o), the calcium responses were smaller and relatively transient, and with higher levels of depolarization (55 mM [K+]o) the responses were sustained and were higher in amplitude. Responses to low levels of depolarization were less sensitive to the dihydropyridine calcium channel blocker, nimodipine, while the increase in [Ca2+]i induced by 55 mM [K+]o became transient, and was significantly smaller. These observations suggest that these peptidergic nerve terminals possess at least two different types of voltage-gated calcium channels. Removal of extracellular sodium resulted in a significant increase in [Ca2+]i and secretion in the absence of depolarizing stimulus, suggesting that sodium-calcium exchange mechanism is operative in these nerve terminals. Although the [Ca2+]i increase was of similar magnitude to the depolarization-induced changes, the resultant secretion was 10-fold lower, but the rate of inactivation of secretion, however, was comparable.


Subject(s)
Calcium/metabolism , Cytosol/metabolism , Nerve Endings/metabolism , Pituitary Gland, Posterior/metabolism , Animals , Calibration , Fura-2 , Male , Membrane Potentials/drug effects , Membrane Potentials/physiology , Nerve Endings/drug effects , Nimodipine/pharmacology , Pituitary Gland, Posterior/drug effects , Pituitary Gland, Posterior/innervation , Pituitary Hormones, Posterior/metabolism , Rats , Rats, Inbred Strains , Time Factors , Video Recording
SELECTION OF CITATIONS
SEARCH DETAIL
...