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1.
Retrovirology ; 18(1): 24, 2021 08 24.
Article in English | MEDLINE | ID: mdl-34429135

ABSTRACT

The HIV co-receptors, CCR5 and CXCR4, are necessary for HIV entry into target cells, interacting with the HIV envelope protein, gp120, to initiate several signaling cascades thought to be important to the entry process. Co-receptor signaling may also promote the development of neuroHIV by contributing to both persistent neuroinflammation and indirect neurotoxicity. But despite the critical importance of CXCR4 and CCR5 signaling to HIV pathogenesis, there is only one therapeutic (the CCR5 inhibitor Maraviroc) that targets these receptors. Moreover, our understanding of co-receptor signaling in the specific context of neuroHIV is relatively poor. Research into co-receptor signaling has largely stalled in the past decade, possibly owing to the complexity of the signaling cascades and functions mediated by these receptors. Examining the many signaling pathways triggered by co-receptor activation has been challenging due to the lack of specific molecular tools targeting many of the proteins involved in these pathways and the wide array of model systems used across these experiments. Studies examining the impact of co-receptor signaling on HIV neuropathogenesis often show activation of multiple overlapping pathways by similar stimuli, leading to contradictory data on the effects of co-receptor activation. To address this, we will broadly review HIV infection and neuropathogenesis, examine different co-receptor mediated signaling pathways and functions, then discuss the HIV mediated signaling and the differences between activation induced by HIV and cognate ligands. We will assess the specific effects of co-receptor activation on neuropathogenesis, focusing on neuroinflammation. We will also explore how the use of substances of abuse, which are highly prevalent in people living with HIV, can exacerbate the neuropathogenic effects of co-receptor signaling. Finally, we will discuss the current state of therapeutics targeting co-receptors, highlighting challenges the field has faced and areas in which research into co-receptor signaling would yield the most therapeutic benefit in the context of HIV infection. This discussion will provide a comprehensive overview of what is known and what remains to be explored in regard to co-receptor signaling and HIV infection, and will emphasize the potential value of HIV co-receptors as a target for future therapeutic development.


Subject(s)
HIV Infections/drug therapy , HIV-1/pathogenicity , Neuroinflammatory Diseases/virology , Receptors, CCR5/metabolism , Receptors, CXCR4/metabolism , Receptors, HIV/metabolism , Signal Transduction , Animals , CCR5 Receptor Antagonists/pharmacology , CCR5 Receptor Antagonists/therapeutic use , Clinical Trials as Topic , HIV Infections/complications , HIV-1/drug effects , Humans , Mice , Neuroinflammatory Diseases/immunology , Neuroinflammatory Diseases/physiopathology , Receptors, CCR5/immunology , Receptors, CXCR4/antagonists & inhibitors , Receptors, CXCR4/immunology , Receptors, HIV/immunology
2.
Cell Death Dis ; 1: e33, 2010 Apr 01.
Article in English | MEDLINE | ID: mdl-21364640

ABSTRACT

Homeostatic chemokines, such as CXCL12, can affect neuronal activity by the regulation of inhibitory and excitatory neurotransmission, but the mechanisms involved are still undefined. Our previous studies have shown that CXCL12 protects cortical neurons from excitotoxicity by promoting the function of the gene-repressor protein Rb, which is involved in the recruitment of chromatin modifiers (such as histone deacetylases (HDACs)) to gene promoters. In neurons, Rb controls activity-dependent genes essential to neuronal plasticity and survival, such as the N-methyl-D-aspartic acid (NMDA) receptor's subunit NR2B, the expression of which in the tetrameric ion channel largely affects calcium signaling by glutamate. In this study, we report that CXCL12 differentially modulates intracellular responses after stimulation of synaptic and extrasynaptic NMDA receptors, by a specific regulation of the NR2B gene that involves HDACs. Our results show that CXCL12 selectively inhibits NR2B expression in vitro and in vivo altering NMDA-induced calcium responses associated with neuronal death, while promoting prosurvival pathways that depend on stimulation of synaptic receptors. Along with previous studies, these findings underline the role of CXCL12/CXCR4 in the regulation of crucial components of glutamatergic transmission. These novel effects of CXCL12 may be involved in the physiological function of the chemokine in both developing and mature brains.


Subject(s)
Chemokine CXCL12/pharmacology , Histone Deacetylases/metabolism , Neurons/cytology , Neurons/enzymology , Protein Subunits/metabolism , Receptors, N-Methyl-D-Aspartate/metabolism , Signal Transduction/drug effects , Animals , Cell Nucleus/drug effects , Cell Nucleus/metabolism , Cell Survival/drug effects , Cells, Cultured , Cytoprotection/drug effects , Down-Regulation/drug effects , Histone Deacetylase Inhibitors/pharmacology , Humans , N-Methylaspartate/toxicity , Neurons/drug effects , Neurotoxins/toxicity , Rats , Receptors, CXCR4/metabolism , Receptors, Neurotransmitter/metabolism , Time Factors
3.
Cell Death Differ ; 15(10): 1663-72, 2008 Oct.
Article in English | MEDLINE | ID: mdl-18583990

ABSTRACT

Postmitotic neurons need to keep their cell cycle under control to survive and maintain a differentiated state. This study aims to test the hypothesis that the chemokine CXCL12 regulates neuronal survival and differentiation by promoting Rb function, as suggested by previous studies showing that CXCL12 protects neurons from apoptosis induced by Rb loss. To this end, the effect of CXCL12 on Rb expression and transcriptional activity and the role of Rb in CXCL12-induced neuronal survival were studied. CXCL12 increases Rb protein and RNA levels in rat cortical neurons. The chemokine also stimulates an exogenous Rb promoter expressed in these neurons and counteracts the inhibition of the Rb promoter induced by E2F1 overexpression. Furthermore CXCL12 stimulates Rb activity as a transcription repressor. The effects of CXCL12 are mediated by its specific receptor CXCR4, and do not require the presence of glia. Finally, shRNA studies show that Rb expression is crucial to the neuroprotective activity of CXCL12 as indicated by NMDA-neurotoxicity assays. These findings suggest that proper CXCR4 stimulation in the mature CNS can prevent impairment of the Rb-E2F pathway and support neuronal survival. This is important to maintain CNS integrity in physiological conditions and prevent neuronal injury and loss typical of many neurodegenerative and neuroinflammatory conditions.


Subject(s)
Cell Cycle/physiology , Cell Survival/physiology , Chemokine CXCL12/metabolism , Neurons/physiology , Retinoblastoma Protein/metabolism , Animals , Cell Differentiation/physiology , Cells, Cultured , Cerebral Cortex/physiology , Chemokine CXCL12/genetics , Gene Expression Regulation , Gene Silencing , N-Methylaspartate/metabolism , Neurons/cytology , Promoter Regions, Genetic , Rats , Receptors, CXCR4/genetics , Receptors, CXCR4/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Retinoblastoma Protein/genetics
4.
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
5.
Trends Neurosci ; 22(10): 471-9, 1999 Oct.
Article in English | MEDLINE | ID: mdl-10481195

ABSTRACT

Many HIV-1-positive individuals suffer from a variety of neurological problems known collectively as the HIV-1-related cognitive-motor complex. However, the molecular mechanisms that underlie HIV-1-induced neuropathology are unclear. They might include a combination of indirect effects, which result from the release of neurotoxins from activated astrocytes and microglia, and the direct effects of HIV-1-related proteins, such as gp120, on neurons. As the interaction of gp120 with immune cells has been shown to require the participation of chemokine receptors, this article explores the possibility that such receptors participate in the events underlying HIV-1-induced neuropathology. It is now clear that many types of cell in the brain possess chemokine receptors, including microglia, glia and neurons, and the interaction of gp120 with neuronal chemokine receptors initiates apoptotic death of neurons in vitro. Such effects might be modified by the actions of chemokines that act at these same receptors. However, the importance of this direct interaction with neurons in vivo and its relevance in the pathogenesis of AIDS-related dementia still needs to be established. Furthermore, the existence of chemokine receptors on neurons suggests that chemokines might regulate neuronal functions physiologically.


Subject(s)
AIDS Dementia Complex/immunology , Chemokines/immunology , HIV Envelope Protein gp120/immunology , AIDS Dementia Complex/virology , Brain Chemistry , Humans
7.
Neuropharmacology ; 37(10-11): 1431-43, 1998.
Article in English | MEDLINE | ID: mdl-9849678

ABSTRACT

We compared the toxin sensitivity, Ca2+ flux response and rectification properties of recombinant alpha-amino-3-hydroxy-5-methyl-4-isoazolepropionic acid (AMPA) receptors obtained by transfecting human embryonic kidney (HEK) 293 cells with different ratios of GluR1 and GluR2 cDNAs (10:1 to 1:10). Simultaneous measurements of kainate-activated Ca2+ fluxes and inward currents, using fura-2 microfluorimetry under voltage clamp conditions, suggested the existence of GluR2 containing channels which are permeable to Ca2+ and insensitive to Joro spider toxin (JSTx). Imaging experiments showed that JSTx inhibition of the Ca2+ response induced by kainate was reduced by increasing the relative amount of GluR2. However, even at GluR1/GluR2(R) ratios of 1:1 and 1:4, cells were still able to flux Ca2+ when stimulated by kainate. GluR2 similarly inhibited the ability of JSTx to reduce kainate-evoked inward currents in whole cell patch-clamp experiments. Variations in the rectification properties of the AMPA currents, induced by changes in the cDNA ratio, were not always correlated with the changes in toxin sensitivity and [Ca2+]i response. Thus, cells with almost linear I-V relationships were partially blocked by JSTx and still Ca2+ permeable. Our results indicate a dissociation between the toxin sensitivity and Ca2+ flux through GluR2 containing AMPA receptors and suggest that receptors with diverse Ca2+ permeabilities are generated by the expression of variable amounts of GluR2.


Subject(s)
Calcium/metabolism , Neurotoxins/pharmacology , Receptors, AMPA/drug effects , Spider Venoms/pharmacology , Animals , Cell Line , Kainic Acid/pharmacology , Patch-Clamp Techniques , Receptors, AMPA/metabolism , Recombinant Proteins
8.
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
9.
Neurochem Res ; 22(3): 257-65, 1997 Mar.
Article in English | MEDLINE | ID: mdl-9051659

ABSTRACT

Cerebellar granule cells (CGC) at different stages of maturation in vitro (1 or 6 DIV), were treated with beta 25-35 and acetyl-L-carnitine arginine amide (ST857) in presence of 25 mM KCl in the culture medium, and neuronal viability was assessed. Three days of treatment slightly modified the survival of 1 DIV-treated cells, which degenerate and die five days later beta-amyloid matching. Similarly, a significative neurotoxic effect was observed on 6 DIV treated-cells after 5 days of exposure to the peptide, while the death occurred within 8 days. ST857 coincubated with beta 25-35 was able to rescue neurons from beta 25-35-induced neurotoxicity. We also studied the changes in Ca2+ homeostasis following glutamate stimulation, in control and beta-amyloid treated single cells, either in presence or in absence of ST857. beta 25-35 did not affect basal [Ca2+]i, while modified glutamate-induced [Ca2+]i increase, causing a sustained plateau phase of [Ca2+]i, that persisted after the removal of the agonist. ST857 pretreatment completely reverted this effect suggesting that, in CGC chronically treated with beta 25-35, ST857 could protect the cells by neurotoxic insults of the peptide likely interfering with the cellular mechanisms involved in the control of Ca2+ homeostasis.


Subject(s)
Acetylcarnitine/analogs & derivatives , Amyloid beta-Peptides/toxicity , Arginine/analogs & derivatives , Cerebellum/drug effects , Nerve Growth Factors/pharmacology , Neurons/drug effects , Neurotoxins/toxicity , Peptide Fragments/toxicity , Acetylcarnitine/pharmacology , Amyloid beta-Peptides/antagonists & inhibitors , Animals , Arginine/pharmacology , Basal Metabolism , Cells, Cultured , Cerebellum/cytology , Drug Evaluation, Preclinical , Glutamic Acid/pharmacology , Homeostasis/drug effects , Nerve Degeneration/drug effects , Neurotoxins/antagonists & inhibitors , Peptide Fragments/antagonists & inhibitors , Rats , Rats, Wistar , Video Recording
10.
J Neurosci ; 16(13): 4080-8, 1996 Jul 01.
Article in English | MEDLINE | ID: mdl-8753870

ABSTRACT

We found that TGF-beta1, a cytokine that previously has been reported to have neuroprotective effects, was able to prevent the toxicity induced by the HIV-1 coat protein gp120 in hippocampal pyramidal neuron cultures. In the presence of glia, gp120 induced time- and dose-dependent cell death, which was more pronounced in mature (7-19 d in culture) than in young neurons (2-7 d in culture). Staining with nuclear dyes (propidium iodide and Hoechst 33342), in situ detection of DNA fragments, and DNA analysis on agarose gels indicated that apoptosis was mainly responsible for the death caused by the viral protein. However, after several days of treatment, death-displaying necrotic features also occurred. Neurotoxicity induced by gp120 was dependent on the activation of NMDA receptors and required the presence of glia as well as new protein synthesis. Thus, the effect of gp120 was abolished by the NMDA receptor antagonist APV and partially reduced by cycloheximide. Only modest neurotoxicity was observed in pure neuronal cultures deprived of the glia feeder layer. Fura-2-based videoimaging showed that treatment with gp120 enhanced the ability of NMDA to increase neuronal [Ca2+]i. The impairment of neuronal Ca2+ homeostasis was prevented completely by TGF-beta1. Therefore, it is likely that the neuroprotective action of the cytokine is attributable to its ability to stabilize neuronal [Ca2+]i.


Subject(s)
HIV Envelope Protein gp120/pharmacology , Hippocampus/drug effects , Neurons/drug effects , Neuroprotective Agents/pharmacology , Neurotoxins/pharmacology , Transforming Growth Factor beta/pharmacology , Animals , Apoptosis , Calcium/metabolism , Cell Survival/drug effects , Cells, Cultured , Hippocampus/cytology , Intracellular Membranes/metabolism , Necrosis , Nerve Tissue Proteins/biosynthesis , Neuroglia/physiology , Neurons/pathology , Neurons/physiology , Rats/embryology , Receptors, N-Methyl-D-Aspartate/physiology
11.
J Neurochem ; 66(5): 1995-2003, 1996 May.
Article in English | MEDLINE | ID: mdl-8780028

ABSTRACT

We studied the neurotoxic effects of beta 25-35 amyloid fragment (beta 25-35) on cerebellar granule cells and the intracellular mechanisms involved. Treatment for 3 days with peptide greatly reduced the survival of 1 day in vitro (DIV) cultures kept in 5 mM KCI but slightly modified the survival of 25 mM KCI-cultured cerebellar granule cells. We also studied the effect of glutamate on survival of undifferentiated cerebellar granules. We report no neurotoxic effect of glutamate on 3-DIV-treated cultures; whereas in beta 25-35-pretreated cells, a significant glutamate toxicity was observed. Treatment of 6-DIV cells with beta 25-35, performed with 25 mM KCI, induced a late but significant neurotoxic effect after 5 days of exposure, and death occurred within 8 days. Differentiated cerebellar granule cells were also sensitive to glutamate-related neurotoxicity, and this effect was enhanced by beta 25-35 pretreatment. To study the molecular mechanisms underlying the neurotoxic effects of beta 25-35, changes in calcium homeostasis after glutamate stimulation were evaluated in control and beta 25-35-treated cells. beta 25-35 did not affect basal [Ca2+]i but modified glutamate-induced [Ca2+]i increase, causing a sustained plateau phase that persisted even after the removal of the agonist. These results show that beta 25-35 induces neurotoxicity in cerebellar granule cells and that this effect is related to modifications in the control of calcium homeostasis.


Subject(s)
Amyloid beta-Peptides/pharmacology , Calcium/metabolism , Cerebellum/physiology , Homeostasis/drug effects , Neurotoxins/pharmacology , Peptide Fragments/pharmacology , Animals , Cell Survival/drug effects , Cells, Cultured , Cerebellum/cytology , Drug Synergism , Glutamic Acid/pharmacology , Neurons/drug effects , Neurons/physiology , Rats , Rats, Wistar
12.
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
13.
J Mol Endocrinol ; 14(3): 375-89, 1995 Jun.
Article in English | MEDLINE | ID: mdl-7669227

ABSTRACT

We used the PCR amplification technique in an attempt to characterize further the dopamine D2L receptor expressed in the prolactin-secreting pituitary MMQ cell clone, derived from the prolactin- and ACTH-secreting Buffalo rat 7315 alpha pituitary tumour. By semiquantitative PCR amplification we were unable to detect the mRNA encoding the D2S receptor isoform, which derives from the well-known process of alternative splicing, producing two D2 receptor subtypes (D2L and D2S) in such tissues as the anterior pituitary and the corpus striatum. Although the pharmacology of the D2 receptor has been established in many studies on both native receptors and transfected receptor isoforms, because of the lack of tissues naturally expressing only one receptor isoform, MMQ cells represent the first example of cells uniquely or prevalently expressing only the D2L receptor, conceivably coupled to its native transduction mechanisms. These considerations prompted us to evaluate the pharmacology and the second messenger systems known to be modulated by dopamine. Scatchard analysis of [3H]spiperone binding resulted in a linear plot, consistent with the existence of a single class of binding sites, with a Kd of 0.055 +/- 0.002 nM and a Bmax of 27 +/- 3.5 fmol/mg protein. Competition experiments confirmed the GTP-dependence and the order of potency for agonist and antagonist ligands consistent with binding to a D2 receptor. The inhibitory effects of dopamine on adenylyl cyclase activity, inositol phosphate production and intracellular free calcium concentrations, the latter presumably via the opening of K+ channels, and prolactin secretion, as well as the reversal of the effect by the D2-selective antagonist (-)sulpiride and pretreatment with pertussis toxin, are consistent with the known biological actions of dopamine at D2 receptors. Based on our observations, the MMQ cell line can be considered a useful tool for investigating ligand-receptor interactions to develop new selective dopaminergic D2L ligands for the therapy of dopamine-related disorders such as schizophrenia, depression, Parkinson's disease and drug addiction.


Subject(s)
Neoplasm Proteins/deficiency , Pituitary Neoplasms/chemistry , Prolactin/metabolism , Receptors, Dopamine D2/deficiency , Receptors, Dopamine D2/physiology , Signal Transduction/physiology , Adenylyl Cyclases/metabolism , Animals , Base Sequence , Calcium/physiology , Clone Cells/chemistry , Cloning, Molecular , Dopamine Agonists/pharmacology , Dopamine Antagonists/pharmacology , Guanosine Triphosphate/physiology , Hemolytic Plaque Technique , Inositol Phosphates/physiology , Ion Channel Gating , Membrane Potentials/drug effects , Molecular Sequence Data , Neoplasm Proteins/genetics , Pituitary Neoplasms/genetics , Pituitary Neoplasms/metabolism , Pituitary Neoplasms/pathology , Polymerase Chain Reaction , Potassium Channels/physiology , Radioligand Assay , Rats , Rats, Inbred BUF , Receptors, Dopamine D2/classification , Receptors, Dopamine D2/drug effects , Receptors, Dopamine D2/genetics , Second Messenger Systems , Tumor Cells, Cultured
14.
Biochem Biophys Res Commun ; 209(2): 630-8, 1995 Apr 17.
Article in English | MEDLINE | ID: mdl-7537494

ABSTRACT

In PC Cl3 rat thyroid cell line noradrenaline-induced Ca2+ response, mainly due to the activation of alpha 1B receptors, is characterized by a rapid peak phase, due to the Ca2+ mobilization from inositol trisphosphate-sensitive internal stores, followed by a sustained plateau, representing the capacitative calcium entry. The plateau phase elicited by noradrenaline returns to the basal value within 100 sec from the removal of agonist. The tyrosine kinases inhibitor genistein completely abolishes the plateau upon noradrenaline withdrawal. On the contrary, the tyrosine phosphatases blocker, vanadate, potentiates the plateau phase of calcium response to noradrenaline and prevents the gradual decrease of [Ca2+]i after removal of noradrenaline. The noradrenaline-induced Ca2+ influx, due to the activation of alpha 1A receptor-operated Ca2+ entry is not affected by vanadate. The treatment with noradrenaline induced the tyrosine phosphorylation of specific substrates in lysates derived from PC Cl3 cells, an effect inhibited by genistein pretreatment. These results show that a balance between tyrosine phosphorylation and dephosphorylation is required for the regulation of capacitative calcium entry following noradrenaline stimulation of alpha 1B receptor, whilst the influx of Ca2+ directly operated by alpha 1A receptor activation seems to be independent of the tyrosine phosphorylating pathway.


Subject(s)
Calcium/metabolism , Inositol 1,4,5-Trisphosphate/metabolism , Norepinephrine/pharmacology , Protein Tyrosine Phosphatases/metabolism , Protein-Tyrosine Kinases/metabolism , Receptors, Adrenergic, alpha/physiology , Thyroid Gland/metabolism , Animals , Cells, Cultured , In Vitro Techniques , Phosphoproteins/metabolism , Phosphotyrosine , Rats , Receptors, Adrenergic, alpha/classification , Signal Transduction , Tyrosine/analogs & derivatives , Tyrosine/metabolism , Vanadates/pharmacology
15.
J Neurosci ; 15(2): 1172-9, 1995 Feb.
Article in English | MEDLINE | ID: mdl-7532699

ABSTRACT

Cerebellar granule cells deprived of depolarizing concentration of extracellular potassium, [K+]o, undergo apoptosis. We here report that this apoptotic process is associated with an immediate and permanent decrease in the levels of free intracellular calcium, [Ca2+]i. Although forskolin and IGF-1 are both able to prevent apoptosis, only forskolin is able to counteract the instantaneous decrease of [Ca2+]i. However, the early effect of forskolin on [Ca2+]i is lost after longer incubation in low [K+]o. The calcium antagonist nifedipine is able to inhibit the survival effect of high [K+]o, while not affecting forskolin and IGF-1 promoted survival, as assessed by viability and genomic DNA analysis. Accordingly, the L-type calcium channels agonist Bay K8644 significantly enhanced the survival of low KCl treated neurons. To temporally characterize the signal transduction events and the essential transcriptional step in cerebellar granule cells apoptosis, we determined the time course of the rescue capacity of high [K+]o, forskolin, IGF-1, and actinomycin D. Addition of high KCl, forskolin, or IGF-1 6 hr after the initial KCl deprivation saves 50% of cells. Remarkably, 50% of neurons loss the potential to be rescued by actinomycin D after only 1 hr in low [K+]o. Finally, we show that the survival promoting activities of high [K+]o, forskolin, and IGF-1 do not require RNA synthesis. We conclude that [Ca2+]i is involved in the survival promoting activity exerted by high [K+]o but not in those of forskolin and IGF-1, and that all three agents, although rescuing neurons from apoptosis through distinct mechanisms of action, do not necessitate RNA transcription.


Subject(s)
Apoptosis/drug effects , Cerebellum/cytology , Colforsin/pharmacology , Insulin-Like Growth Factor I/pharmacology , Neurons/physiology , Potassium Chloride/pharmacology , 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester/pharmacology , Animals , Calcium/metabolism , Electrophysiology , Intracellular Membranes/metabolism , Neurons/drug effects , Nifedipine/pharmacology , Potassium/metabolism , RNA/biosynthesis , Rats , Rats, Wistar , Transcription, Genetic
16.
Endocrinology ; 134(1): 424-31, 1994 Jan.
Article in English | MEDLINE | ID: mdl-7903935

ABSTRACT

The aim of the present study was to characterize the adrenergic receptors mediating the effects of norepinephrine on PC C13 rat thyroid cells and identify the molecular mechanisms by which TSH regulates the noradrenergic response. We studied TSH regulation of norepinephrine-induced cytosolic calcium increase by means of the fluorescent probe fura-2. In PC C13 cells grown and maintained in a medium containing TSH (PC C13 6H), norepinephrine caused a higher increase in cytosolic calcium than in PC C13 starved from TSH 5 days before the experiments (PC C13 5H). In both group of cells the calcium response to norepinephrine was concentration dependent and reduced by the removal of extracellular calcium ions. Reintroduction of TSH in the culture medium of the PC C13 5H cells induced the recovery of the norepinephrine-stimulated intracellular calcium rise similarly to that in the native PC C13 6H. This effect was complete after a 48-h incubation period and was abolished by the simultaneous treatment of the cells with the protein synthesis inhibitor cycloheximide, suggesting that TSH may stimulate the synthesis of alpha 1-adrenergic receptors in PC C13 cells. Because in these cells we found that TSH increased cAMP levels as well as inositol phosphate production, we tested whether the activation of a protein kinase-A and/or protein kinase-C was involved in TSH regulation of the adrenergic response. We found that the treatment of PC C13 5H cells with forskolin restored the effect of norepinephrine on the calcium level, and that KT5720, an inhibitor of the protein kinase-A, was able to prevent the recovery of the noradrenergic response induced by the readdition of TSH to the culture medium of PC C13 5H. Conversely, treatment of PC C13 5H cells with the protein kinase-C activator phorbol 12-myristate 13-acetate was ineffective. Norepinephrine also stimulated inositol phosphate production in PC C13 6H and, to a lesser extent, in PC C13 5H, but it did not affect the cAMP levels in the two groups of cells. To characterize alpha 1-adrenergic receptor subtypes mediating the effects of norepinephrine in PC C13 cells, we used antagonists of alpha 1A and alpha 1B receptors (WB4101 and chlorethylclonidine respectively).(ABSTRACT TRUNCATED AT 400 WORDS)


Subject(s)
Calcium/metabolism , Cytosol/metabolism , Norepinephrine/pharmacology , Receptors, Adrenergic, alpha/physiology , Thyroid Gland/metabolism , Adrenergic alpha-Antagonists/pharmacology , Animals , Binding Sites , Calcium Channel Blockers/pharmacology , Cell Line , Cyclic AMP/metabolism , Cyclic AMP-Dependent Protein Kinases/metabolism , Inositol Phosphates/metabolism , Intracellular Membranes/metabolism , Rats , Thyroid Gland/cytology , Thyrotropin/pharmacology
17.
J Psychopharmacol ; 8(2): 109-17, 1994 Jan.
Article in English | MEDLINE | ID: mdl-22298538

ABSTRACT

The effect of aniracetam (10, 50, 100 mg/kg i.p. daily for 15 days) on both behavioural and biochemical parameters was investigated in the adult rat. Animals given aniracetam (50 mg/kg 1 h before the trial) showed a significant increase in the percentage of conditioned active avoidance responses and a reduction of latency times. Aniracetam significantly counteracted the scopolamine-induced memory failure at the passive avoidance (step down) test, while it did not modify the locomotion of the animals. In purified frontocortical and hippocampal synaptic membranes of rats treated with aniracetam (50 mg/kg i.p. daily for 15 days) a potentiation of basal, carbamylcholine-, dopamine- and norepinephrine-stimulated adenylyl cyclase activity was observed, while forskolin-stimulated enzyme activity was not modified. With regard to inositol phosphate production measured in fronto-cortical synaptoneurosomes, aniracetam potentiated the stimulation by angiotensin II, while the stimulation by carbamylcholine, not affected by 10 and 50 mg/kg aniracetam, was notably, although not significantly, decreased by 100 mg/kg aniracetam. Furthermore, in synaptosomes derived from hippocampus, aniracetam (50 mg/kg i.p. daily for 15 days) caused an increase of both basal and K(+)-stimulated intrasynaptosomal Ca(2+) concentration. In conclusion, a correlation between the improvement of behavioural performance and the modulation of transducing systems by aniracetam seems to take place in brain areas, such as frontal cortex and hippocampus, known to play a major role in the control of cognitive functions.

18.
J Mol Endocrinol ; 11(3): 325-34, 1993 Dec.
Article in English | MEDLINE | ID: mdl-8148040

ABSTRACT

We studied the effect of adenosine on prolactin secretion by the anterior pituitary, and the transduction mechanisms whereby the purine exerts its action. Adenosine inhibited prolactin release in basal and in vasoactive intestinal peptide (VIP)- or TRH-stimulated conditions. Pertussis toxin pretreatment reduced the inhibition of VIP-stimulated prolactin secretion which was induced by adenosine, while it completely abolished the effect of the purine on TRH-evoked prolactin release. In membrane preparations of anterior pituitary cells, adenosine reduced the adenylate cyclase activity stimulated by VIP. Such an inhibition was not blocked by pertussis toxin pretreatment. Furthermore, the purine reduced TRH-stimulated inositol phosphate production in cultured anterior pituitary cells, an effect that was reversed by pretreatment with pertussis toxin. In addition, the nucleoside did not significantly affect the TRH-induced rise in intracellular calcium. In conclusion, our data show that adenosine inhibits prolactin secretion, acting on purinergic receptors coupled to the adenylate cyclase enzyme and phospholipase C. The effect of the nucleoside on adenylate cyclase seems to be achieved either by the involvement of an adenosine receptor coupled to the catalytic subunit of the enzyme via a pertussis toxin-sensitive G protein, or by the activation of a site directly coupled to the catalytic subunit of the adenylate cyclase (the P site). Its effect on phospholipase C seems to be mediated by a purinergic receptor coupled to the intracellular effector via a pertussis toxin-sensitive G protein.


Subject(s)
Adenosine/pharmacology , Adenylate Cyclase Toxin , Pertussis Toxin , Pituitary Gland, Anterior/drug effects , Pituitary Gland, Anterior/metabolism , Prolactin/metabolism , Virulence Factors, Bordetella/pharmacology , Adenylyl Cyclases/metabolism , Animals , Calcium/metabolism , Female , GTP-Binding Proteins/metabolism , In Vitro Techniques , Inositol Phosphates/biosynthesis , Rats , Rats, Wistar , Receptors, Purinergic P1/drug effects , Receptors, Purinergic P1/physiology , Second Messenger Systems/drug effects , Second Messenger Systems/physiology , Thyrotropin-Releasing Hormone/pharmacology , Type C Phospholipases/metabolism , Vasoactive Intestinal Peptide/pharmacology
19.
Eur Neuropsychopharmacol ; 3(2): 95-101, 1993 Jun.
Article in English | MEDLINE | ID: mdl-8364354

ABSTRACT

The aim of the present study has been to evaluate the effect of acetyl-L-carnitine (ALCAR) on brain adenylate cyclase (AC) activity in adult and aged rats. In in vitro studies, ALCAR (1, 10 and 100 microM) did not exert any effect on frontal cortex basal AC activity. Acute and subchronic administrations of ALCAR were carried out in 4- and 25-month-old male Fisher rats and AC activity was determined in rat frontal cortex under both basal and stimulated conditions. The acute treatment of young rats with ALCAR (100 and 500 mg/kg s.c.) did not affect AC activity, whereas the subchronic administration of 250 mg/kg s.c. ALCAR enhanced the stimulation of AC by carbamylcholine (CCh), norepinephrine (NE) and dopamine (DA), without affecting the basal AC activity. Basal AC activity in old rats was lower than in young rats and was not modified by acute and subchronic ALCAR administration. Moreover, the response of the enzyme to CCh, NE and DA was potentiated by the subchronic administration of ALCAR. The concentration-response curve of CCh stimulation of AC activity in ALCAR-treated rats is shifted to the left in both young and aged rats. We conclude that ALCAR, subchronically administered, is able to enhance receptor-stimulated AC response in frontal cortex of both young and aged rats.


Subject(s)
Acetylcarnitine/pharmacology , Adenylyl Cyclases/metabolism , Aging/metabolism , Brain/enzymology , Animals , Brain/drug effects , Carbachol/pharmacology , Cerebral Cortex/drug effects , Cerebral Cortex/enzymology , Dopamine/pharmacology , Male , Norepinephrine/pharmacology , Rats , Rats, Inbred F344
20.
Life Sci ; 52(10): 891-9, 1993.
Article in English | MEDLINE | ID: mdl-8383262

ABSTRACT

The effect of thyrotropin and norepinephrine on cytosolic calcium levels were evaluated in normal (PC C13) and transformed (PC E1A, PC Py and PC E1APy) rat thyroid epithelial cell lines. A different pattern of response to both norepinephrine and thyrotropin was observed among the distinct cell lines. In PC C13 the cytosolic calcium rise induced by norepinephrine, characterized by an early transient spike followed by a second phase of sustained calcium levels, was greatly enhanced by thyrotropin. The effect of norepinephrine on calcium concentrations was less affected by thyrotropin in PC C13 transformed by the adenovirus E1A oncogene. Conversely, in Polyoma middle-T transformed PC C13 the increase in cytoplasmic calcium was still sensitive to thyrotropin. The most malignant PC E1APy were totally independent of thyrotropin.


Subject(s)
Adenoviridae/genetics , Adenovirus E1A Proteins/genetics , Antigens, Viral, Tumor/genetics , Calcium/metabolism , Cell Transformation, Viral/genetics , Genes, Viral/genetics , Norepinephrine/pharmacology , Polyomavirus/genetics , Signal Transduction/physiology , Thyroid Gland/metabolism , Thyrotropin/pharmacology , Amino Acid Sequence , Animals , Cell Line , Cell Transformation, Viral/drug effects , Cytosol/drug effects , Cytosol/metabolism , Epithelial Cells , Gene Expression/drug effects , Genes, Viral/drug effects , Homeostasis/drug effects , Homeostasis/physiology , Molecular Sequence Data , Oncogenes/genetics , Polyomavirus/drug effects , Rats , Signal Transduction/drug effects , Stimulation, Chemical , Thyroid Gland/cytology , Thyroid Gland/drug effects
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