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1.
J Cell Mol Med ; 28(9): e18274, 2024 05.
Article in English | MEDLINE | ID: mdl-38676362

ABSTRACT

TRP channels, are non-specific cationic channels that are involved in multiple physiological processes that include salivation, cellular secretions, memory extinction and consolidation, temperature, pain, store-operated calcium entry, thermosensation and functionality of the nervous system. Here we choose to look at the evidence that decisively shows how TRP channels modulate human neuron plasticity as it relates to the molecular neurobiology of sleep/circadian rhythm. There are numerous model organisms of sleep and circadian rhythm that are the results of the absence or genetic manipulation of the non-specific cationic TRP channels. Drosophila and mice that have had their TRP channels genetically ablated or manipulated show strong evidence of changes in sleep duration, sleep activity, circadian rhythm and response to temperature, noxious odours and pattern of activity during both sleep and wakefulness along with cardiovascular and respiratory function during sleep. Indeed the role of TRP channels in regulating sleep and circadian rhythm is very interesting considering the parallel roles of TRP channels in thermoregulation and thermal response with concomitant responses in growth and degradation of neurites, peripheral nerves and neuronal brain networks. TRP channels provide evidence of an ability to create, regulate and modify our sleep and circadian rhythm in a wide array of physiological and pathophysiological conditions. In the current review, we summarize previous results and novel recent advances in the understanding of calcium ion entry via TRP channels in different sleep and circadian rhythm conditions. We discuss the role of TRP channels in sleep and circadian disorders.


Subject(s)
Circadian Rhythm , Sleep , Transient Receptor Potential Channels , Circadian Rhythm/physiology , Circadian Rhythm/genetics , Animals , Humans , Sleep/physiology , Transient Receptor Potential Channels/metabolism , Transient Receptor Potential Channels/genetics
2.
Cell Signal ; 74: 109711, 2020 10.
Article in English | MEDLINE | ID: mdl-32702440

ABSTRACT

The CD34 protein is regarded as a marker of stem cells from multiple origins. Recently a mesenchymal progenitor CD34 positive cell identified from traumatized human skeletal muscle demonstrates differentiation capability into vascular endothelial cells, osteoblasts and adipocytes. Here they were treated with a small inhibitory RNA for CD34, which significantly reduced the cellular level of the CD34 protein. These treated cells had a reduced capacity to proliferate, and migrate. They were both unable to differentiation down multiple pathways and to undergo vascular endothelial differentiation as reflected by a lack of expression of VE cadherin, Tie 2 and CD31. Additionally the cells were unable to form tube-like structures in an endothelial tube assay. These treated cells were also unable to undergo osteogenesis, as revealed by lack of alizarin red and alkaline phosphatase staining and were unable to undergo adipogenesis as revealed by lack of oil red O staining. Finally, when CD34 was expressed in cells lacking this protein, the cells were able to undergo vascular endothelial differentiation as revealed by expression of Tie2, VE-cadherin and CD31. These data indicate that in cells derived from traumatized muscle the CD34 protein is required for enhanced proliferation, migration and differentiation down multiple pathways.


Subject(s)
Antigens, CD34/physiology , Mesenchymal Stem Cells , Muscle, Skeletal , Adipocytes/cytology , Adipogenesis , Cell Differentiation , Cell Movement , Cells, Cultured , Endothelial Cells/cytology , Humans , Mesenchymal Stem Cells/cytology , Mesenchymal Stem Cells/metabolism , Muscle Cells/cytology , Muscle, Skeletal/cytology , Muscle, Skeletal/metabolism , Osteoblasts/cytology , Osteogenesis
3.
Front Physiol ; 9: 1731, 2018.
Article in English | MEDLINE | ID: mdl-30559679

ABSTRACT

Angiogenesis is the growth of blood vessels mediated by proliferation, migration, and spatial organization of endothelial cells. This mechanism is regulated by a balance between stimulatory and inhibitory factors. Proangiogenic factors include a variety of VEGF family members, while thrombospondin and endostatin, among others, have been reported as suppressors of angiogenesis. Transient receptor potential (TRP) channels belong to a superfamily of cation-permeable channels that play a relevant role in a number of cellular functions mostly derived from their influence in intracellular Ca2+ homeostasis. Endothelial cells express a variety of TRP channels, including members of the TRPC, TRPV, TRPP, TRPA, and TRPM families, which play a relevant role in a number of functions, including endothelium-induced vasodilation, vascular permeability as well as sensing hemodynamic and chemical changes. Furthermore, TRP channels have been reported to play an important role in angiogenesis. This review summarizes the current knowledge and limitations concerning the involvement of particular TRP channels in growth factor-induced angiogenesis.

4.
Front Physiol ; 8: 392, 2017.
Article in English | MEDLINE | ID: mdl-28649203

ABSTRACT

According to the International Association for the Study of Pain (IASP) pain is characterized as an "unpleasant sensory and emotional experience associated with actual or potential tissue damage". The TRP super-family, compressing up to 28 isoforms in mammals, mediates a myriad of physiological and pathophysiological processes, pain among them. TRP channel might be constituted by similar or different TRP subunits, which will result in the formation of homomeric or heteromeric channels with distinct properties and functions. In this review we will discuss about the function of TRPs in pain, focusing on TRP channles that participate in the transduction of noxious sensation, especially TRPV1 and TRPA1, their expression in nociceptors and their sensitivity to a large number of physical and chemical stimuli.

5.
Cell Immunol ; 311: 63-70, 2017 01.
Article in English | MEDLINE | ID: mdl-27816167

ABSTRACT

B6.MRL/lpr mice, an autoimmune strain, have an accelerated injury time course, increased intensity of tissue damage, and increased CD4+ T cell infiltration in the mesenteric ischemia/reperfusion injury model. In this study, the mechanism by which CD4+ T cells were recruited into injured tissue was addressed. Fingolimod (FTY720) was utilized to assess the role of infiltrating CD4+ T cells. FTY720 treatment was more effective in attenuating injury in B6.MRL/lpr mice then in control mice. Reduced CD4+ cell infiltration and tissue injury correlated with decreased neutrophil infiltration and pro-inflammatory cytokine generation. Inhibiting downstream Sphingosine-1-phosphate (S1P) receptor signaling, specifically GαI mediated signaling, did not inhibit injury, suggesting differential utilization of the S1P receptors between control and MRL/lpr strains. Analysis of S1P receptor expression exposed a predominance of S1P2 in the B6.MRL/lpr strain. Reliance on alternate S1P receptors in the autoimmune strain will alter the progress of inflammation and tissue injury.


Subject(s)
Blood Vessels/drug effects , CD4-Positive T-Lymphocytes/immunology , Fingolimod Hydrochloride/therapeutic use , Immunosuppressive Agents/therapeutic use , Receptors, Lysosphingolipid/metabolism , Reperfusion Injury/drug therapy , Animals , Autoimmunity , Blood Vessels/pathology , Cell Movement , Cells, Cultured , Cytokines/metabolism , Disease Models, Animal , Disease Susceptibility , Gene Expression Regulation , Humans , Inflammation Mediators/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Inbred MRL lpr , Neutrophils/immunology , Receptors, Lysosphingolipid/genetics , Reperfusion Injury/immunology , Sphingosine-1-Phosphate Receptors
6.
J Biol Chem ; 291(13): 6982-8, 2016 Mar 25.
Article in English | MEDLINE | ID: mdl-26817842

ABSTRACT

The store-operated Ca(2+)entry-associated regulatory factor (SARAF) has recently been identified as a STIM1 regulatory protein that facilitates slow Ca(2+)-dependent inactivation of store-operated Ca(2+)entry (SOCE). Both the store-operated channels and the store-independent arachidonate-regulated Ca(2+)(ARC) channels are regulated by STIM1. In the present study, we show that, in addition to its location in the endoplasmic reticulum, SARAF is constitutively expressed in the plasma membrane, where it can interact with plasma membrane (PM)-resident ARC forming subunits in the neuroblastoma cell line SH-SY5Y. Using siRNA-based and overexpression approaches we report that SARAF negatively regulates store-independent Ca(2+)entry via the ARC channels. Arachidonic acid (AA) increases the association of PM-resident SARAF with Orai1. Finally, our results indicate that SARAF modulates the ability of AA to promote cell survival in neuroblastoma cells. In addition to revealing new insight into the biology of ARC channels in neuroblastoma cells, these findings provide evidence for an unprecedented location of SARAF in the plasma membrane.


Subject(s)
Arachidonic Acid/pharmacology , Cell Membrane/drug effects , Endoplasmic Reticulum/drug effects , Gene Expression Regulation , Membrane Proteins/metabolism , Neurons/drug effects , Arachidonic Acid/metabolism , Calcium/metabolism , Calcium Channels/genetics , Calcium Channels/metabolism , Cell Line, Tumor , Cell Membrane/metabolism , Cell Survival/drug effects , Endoplasmic Reticulum/metabolism , Humans , Intracellular Calcium-Sensing Proteins , Ion Transport , Membrane Proteins/antagonists & inhibitors , Membrane Proteins/genetics , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , Neurons/cytology , Neurons/metabolism , ORAI1 Protein , Protein Binding , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Signal Transduction , Stromal Interaction Molecule 1
7.
Int Rev Cell Mol Biol ; 317: 97-183, 2015.
Article in English | MEDLINE | ID: mdl-26008785

ABSTRACT

Regulators of G-protein-signaling (RGS) proteins are a category of intracellular proteins that have an inhibitory effect on the intracellular signaling produced by G-protein-coupled receptors (GPCRs). RGS along with RGS-like proteins switch on through direct contact G-alpha subunits providing a variety of intracellular functions through intracellular signaling. RGS proteins have a common RGS domain that binds to G alpha. RGS proteins accelerate GTPase and thus enhance guanosine triphosphate hydrolysis through the alpha subunit of heterotrimeric G proteins. As a result, they inactivate the G protein and quickly turn off GPCR signaling thus terminating the resulting downstream signals. Activity and subcellular localization of RGS proteins can be changed through covalent molecular changes to the enzyme, differential gene splicing, and processing of the protein. Other roles of RGS proteins have shown them to not be solely committed to being inhibitors but behave more as modulators and integrators of signaling. RGS proteins modulate the duration and kinetics of slow calcium oscillations and rapid phototransduction and ion signaling events. In other cases, RGS proteins integrate G proteins with signaling pathways linked to such diverse cellular responses as cell growth and differentiation, cell motility, and intracellular trafficking. Human and animal studies have revealed that RGS proteins play a vital role in physiology and can be ideal targets for diseases such as those related to addiction where receptor signaling seems continuously switched on.


Subject(s)
Calcium Signaling/physiology , Gene Expression Regulation , RGS Proteins/metabolism , Receptors, G-Protein-Coupled/antagonists & inhibitors , Receptors, G-Protein-Coupled/physiology , Signal Transduction , Animals , Humans , Protein Transport
8.
Biochim Biophys Acta ; 1853(5): 1145-53, 2015 May.
Article in English | MEDLINE | ID: mdl-25712868

ABSTRACT

STIM1 is a ubiquitous Ca2+ sensor of the intracellular, agonist-sensitive, Ca2+ stores that communicates the filling state of the Ca2+ compartments to plasma membrane store-operated Ca2+ (SOC) channels. STIM1 has been presented as a point of convergence between store-operated and voltage-operated Ca2+ influx, both inducing activation of SOC channels while suppressing Cav1.2 channels. Here we report that Homer proteins play a relevant role in the communication between STIM1 and Cav1.2 channels. HEK-293 cells transiently expressing Cav1.2 channel subunits α1, ß2 and α2δ-1 exhibited a significant Ca2+ entry upon treatment with a high concentration of KCl. In Cav1.2-expressing cells, treatment with thapsigargin (TG), to induce passive discharge of the intracellular Ca2+ stores, resulted in Ca2+ influx that was significantly greater than in cells not expressing Cav1.2 channels, a difference that was abolished by nifedipine and diltiazem. Treatment with TG induces co-immunoprecipitation of Homer1 with STIM1 and the Cav1.2 α1 subunit. Impairment of Homer function by introduction of the synthetic PPKKFR peptide into cells, which emulates the proline-rich sequences of the PPXXF motif, or using siRNA Homer1, reduced the association of STIM1 and the Cav1.2 α1 subunit. These findings indicate that Homer is important for the association between both proteins. Finally, treatment with siRNA Homer1 or the PPKKFR peptide enhanced the nifedipine-sensitive component of TG response in Cav1.2-expressing cells. Altogether, these findings provide evidence for a new role of Homer1 supporting the regulation of Cav1.2 channels by STIM1.


Subject(s)
Calcium Channels, L-Type/metabolism , Carrier Proteins/metabolism , Membrane Proteins/metabolism , Neoplasm Proteins/metabolism , Amino Acid Sequence , Animals , Calcium/metabolism , Diltiazem/pharmacology , HEK293 Cells , Homer Scaffolding Proteins , Humans , Immunoprecipitation , Intracellular Space/drug effects , Intracellular Space/metabolism , Molecular Sequence Data , Nifedipine/pharmacology , Peptides/chemistry , Peptides/pharmacology , Protein Binding/drug effects , Protein Subunits/metabolism , Rabbits , Rats , Stromal Interaction Molecule 1 , Thapsigargin/pharmacology
9.
PLoS One ; 9(12): e114318, 2014.
Article in English | MEDLINE | ID: mdl-25490403

ABSTRACT

Here we show that distinct subpopulations of cells exist within traumatic human extremity wounds, each having the ability to differentiate into multiple cells types in vitro. A crude cell suspension derived from traumatized muscle was positively sorted for CD29, CD31, CD34, CD56 or CD91. The cell suspension was also simultaneously negatively sorted for either CD45 or CD117 to exclude hematopoietic stem cells. These subpopulations varied in terms their total numbers and their abilities to grow, migrate, differentiate and secrete cytokines. While all five subpopulations demonstrated equal abilities to undergo osteogenesis, they were distinct in their ability to undergo adipogenesis and vascular endotheliogenesis. The most abundant subpopulations were CD29+ and CD34+, which overlapped significantly. The CD29+ and CD34+ cells had the greatest proliferative and migratory capacity while the CD56+ subpopulation produced the highest amounts of TGFß1 and TGFß2. When cultured under endothelial differentiation conditions the CD29+ and CD34+ cells expressed VE-cadherin, Tie2 and CD31, all markers of endothelial cells. These data indicate that while there are multiple cell types within traumatized muscle that have osteogenic differentiation capacity and may contribute to bone formation in post-traumatic heterotopic ossification (HO), the major contributory cell types are CD29+ and CD34+, which demonstrate endothelial progenitor cell characteristics.


Subject(s)
Extremities/injuries , Extremities/pathology , Lower Extremity/injuries , Stem Cells/pathology , Adipogenesis , Cell Differentiation , Cell Movement , Cell Proliferation , Endothelial Cells/pathology , Flow Cytometry , Humans , Lower Extremity/pathology , Ossification, Heterotopic/pathology , Ossification, Heterotopic/physiopathology , Osteogenesis , Stem Cells/metabolism
10.
Cell Signal ; 25(5): 1328-37, 2013 May.
Article in English | MEDLINE | ID: mdl-23395841

ABSTRACT

STIM1 acts as an endoplasmic reticulum Ca(2+) sensor that communicates the filling state of the intracellular stores to the store-operated channels. In addition, STIM1 is expressed in the plasma membrane, with the Ca(2+) binding EF-hand motif facing the extracellular medium; however, its role sensing extracellular Ca(2+) concentrations in store-operated Ca(2+) entry (SOCE), as well as the underlying mechanism remains unclear. Here we report that divalent cation entry stimulated by thapsigargin (TG) is attenuated by extracellular Ca(2+) in a concentration-dependent manner. Expression of the Ca(2+)-binding defective STIM1(D76A) mutant did not alter the surface expression of STIM1 but abolishes the regulation of divalent cation entry by extracellular Ca(2+). Orai1 and TRPC1 have been shown to play a major role in SOCE. Expression of the STIM1(D76A) mutant did not alter Orai1 phosphoserine content. TRPC1 silencing significantly attenuated TG-induced Mn(2+) entry. Expression of the STIM1(K684,685E) mutant impaired the association of plasma membrane STIM1 with TRPC1, as well as the regulation of TG-induced divalent cation entry by extracellular Ca(2+), which suggests that TRPC1 might be involved in the regulation of divalent cation entry by extracellular Ca(2+) mediated by plasma membrane-resident STIM1. Expression of the STIM1(D76A) or STIM1(K684,685E) mutants reduced store-operated divalent cation entry and resulted in loss of dependence on the extracellular Ca(2+) concentration, providing evidence for a functional role of plasma membrane-resident STIM1 in the regulation of store-operated divalent cation entry, which at least involves the EF-hand motif and the C-terminal polybasic lysine-rich domain.


Subject(s)
Calcium Channels/metabolism , Calcium/metabolism , Cations, Divalent/metabolism , Membrane Proteins/metabolism , Neoplasm Proteins/metabolism , Amino Acid Substitution , Calcium Channels/genetics , Cell Membrane/metabolism , Gene Expression/drug effects , HEK293 Cells , Humans , Ion Transport/drug effects , Membrane Proteins/genetics , Neoplasm Proteins/genetics , ORAI1 Protein , Phosphorylation , Polylysine/chemistry , Protein Structure, Tertiary , RNA Interference , RNA, Small Interfering/metabolism , Stromal Interaction Molecule 1 , TRPC Cation Channels/antagonists & inhibitors , TRPC Cation Channels/genetics , TRPC Cation Channels/metabolism , Thapsigargin/pharmacology
11.
J Nucl Med ; 52(12): 1956-63, 2011 Dec.
Article in English | MEDLINE | ID: mdl-22049461

ABSTRACT

UNLABELLED: Sensitive, specific, and noninvasive detection of angiogenesis would be helpful in discovering new strategies for the treatment of cardiovascular diseases. Recently, we reported the (64)Cu-labeled C-type atrial natriuretic factor (CANF) fragment for detecting the upregulation of natriuretic peptide clearance receptor (NPR-C) with PET on atherosclerosis-like lesions in an animal model. However, it is unknown whether NPR-C is present and overexpressed during angiogenesis. The goal of this study was to develop a novel CANF-integrated nanoprobe to prove the presence of NPR-C and offer sensitive detection with PET during development of angiogenesis in mouse hind limb. METHODS: We prepared a multifunctional, core-shell nanoparticle consisting of DOTA chelators attached to a poly(methyl methacrylate) core and CANF-targeting moieties attached to poly(ethylene glycol) chain ends in the shell of the nanoparticle. Labeling of this nanoparticle with (64)Cu yielded a high-specific-activity nanoprobe for PET imaging NPR-C receptor in a mouse model of hind limb ischemia-induced angiogenesis. Histology and immunohistochemistry were performed to assess angiogenesis development and NPR-C localization. RESULTS: (15)O-H(2)O imaging showed blood flow restoration in the previously ischemic hind limb, consistent with the development of angiogenesis. The targeted DOTA-CANF-comb nanoprobe showed optimized pharmacokinetics and biodistribution. PET imaging demonstrated significantly higher tracer accumulation for the targeted DOTA-CANF-comb nanoprobe than for either the CANF peptide tracer or the nontargeted control nanoprobe (P < 0.05, both). Immunohistochemistry confirmed NPR-C upregulation in the angiogenic lesion with colocalization in both endothelial and smooth muscle cells. PET and immunohistochemistry competitive receptor blocking verified the specificity of the targeted nanoprobe to NPR-C receptor. CONCLUSION: As evidence of its translational potential, this customized DOTA-CANF-comb nanoprobe demonstrated superiority over the CANF peptide alone for imaging NPR-C receptor in angiogenesis.


Subject(s)
Atrial Natriuretic Factor/metabolism , Nanoconjugates , Neovascularization, Pathologic/diagnostic imaging , Neovascularization, Pathologic/metabolism , Positron-Emission Tomography/methods , Animals , Atrial Natriuretic Factor/chemistry , Atrial Natriuretic Factor/pharmacokinetics , Binding, Competitive , Blood Circulation , Male , Mice , Multimodal Imaging , Neovascularization, Pathologic/pathology , Neovascularization, Pathologic/physiopathology , Oxygen Radioisotopes , Receptors, Atrial Natriuretic Factor/metabolism , Tomography, X-Ray Computed , Up-Regulation , Water
12.
J Biol Chem ; 286(14): 12257-70, 2011 Apr 08.
Article in English | MEDLINE | ID: mdl-21321120

ABSTRACT

Mammalian cells accumulate Ca2+ into agonist-sensitive acidic organelles, vesicles that possess a vacuolar proton-ATPase. Acidic Ca2+ stores include secretory granules and lysosome-related organelles. Current evidence clearly indicates that acidic Ca2+ stores participate in cell signaling and function, including the activation of store-operated Ca2+ entry in human platelets upon depletion of the acidic stores, although the mechanism underlying the activation of store-operated Ca2+ entry controlled by the acidic stores remains unclear. STIM1 has been presented as the endoplasmic reticulum Ca2+ sensor, but its role sensing intraluminal Ca2+ concentration in the acidic stores has not been investigated. Here we report that STIM1 and STIM2 are expressed in the lysosome-related organelles and dense granules in human platelets isolated by immunomagnetic sorting. Depletion of the acidic Ca2+ stores using the specific vacuolar proton-ATPase inhibitor, bafilomycin A1, enhanced the association between STIM1 and STIM2 as well as between these proteins and the plasma membrane channel Orai1. Depletion of the acidic Ca2+ stores also induces time-dependent co-immunoprecipitation of STIM1 with the TRPC proteins hTRPC1 and hTRPC6, as well as between Orai1 and both TRPC proteins. In addition, bafilomycin A1 enhanced the association between STIM2 and SERCA3. These findings demonstrate the location of STIM1 and STIM2 in the acidic Ca2+ stores and their association with Ca2+ channels and ATPases upon acidic stores discharge.


Subject(s)
Blood Platelets/metabolism , Cell Adhesion Molecules/metabolism , Membrane Proteins/metabolism , Neoplasm Proteins/metabolism , Blotting, Western , Calcium/metabolism , Calcium Channels , Cell Adhesion Molecules/genetics , Cells, Cultured , Electroporation , Fluorescent Antibody Technique , Humans , Immunoprecipitation , Macrolides/pharmacology , Membrane Proteins/genetics , Neoplasm Proteins/genetics , ORAI1 Protein , Protein Binding/drug effects , Sarcoplasmic Reticulum Calcium-Transporting ATPases/metabolism , Stromal Interaction Molecule 1 , Stromal Interaction Molecule 2
13.
Biochim Biophys Acta ; 1803(9): 1083-93, 2010 Sep.
Article in English | MEDLINE | ID: mdl-20600358

ABSTRACT

Different studies have reported that proteins involved in Ca(2+) entry are localized in discrete plasma membrane domains known as lipid rafts, which have been suggested to support store-operated Ca(2+) entry by facilitating STIM1 clustering in endoplasmic reticulum-plasma membrane junctions as well as the interaction of STIM1 with TRPC1. Here we report that treatment of HEK293 cells with thapsigargin (TG) results in the activation of Ca(2+) entry with two components, an early, La(3+)-sensitive, component and a late component that shows both La(3+)-sensitive and -insensitive constituents. Preincubation with methyl-beta-cyclodextrin (MbetaCD) prevented TG-induced activation of Ca(2+) entry but, in contrast, enhanced this process after its activation. Addition of MbetaCD after store depletion did not modify the La(3+)-sensitive store-operated divalent cation entry but increased La(3+)-insensitive non-capacitative Ca(2+) entry. Cell stimulation with TG results in a transient increase in Orai1 co-immunoprecipitation with STIM1, TRPC1 and TRPC6. TG-induced association of these proteins was significantly attenuated by preincubation for 30 min with MbetaCD, without altering surface expression of Orai1 or TRPCs. In contrast, the association of Orai1 with STIM1 or TRPC1 was unaffected when MbetaCD was added after store depletion with TG. Addition of MbetaCD to TG-treated cells promoted dissociation between Orai1 and TRPC6, as well as non-capacitative Ca(2+) entry. TRPC6 expression silencing indicates that MbetaCD-enhanced non-capacitative Ca(2+) entry was mediated by TRPC6. In conclusion, lipid raft domains are necessary for the activation but not the maintenance of SOCE probably due to the support of the formation of Ca(2+) signalling complexes involving Orai1, TRPCs and STIM1.


Subject(s)
Calcium Signaling , Calcium/metabolism , Membrane Microdomains/physiology , Calcium Channels/drug effects , Calcium Channels/metabolism , Calcium Channels/physiology , Calcium Signaling/drug effects , Calcium Signaling/physiology , Cations, Divalent/metabolism , Cells, Cultured , Enzyme Inhibitors/pharmacology , Humans , Membrane Microdomains/drug effects , Membrane Proteins/metabolism , Neoplasm Proteins/metabolism , ORAI1 Protein , Protein Binding , Stromal Interaction Molecule 1 , TRPC Cation Channels/metabolism , TRPC Cation Channels/physiology , TRPC6 Cation Channel , Thapsigargin/pharmacology , beta-Cyclodextrins/pharmacology
14.
Mol Cell Biol ; 30(14): 3519-30, 2010 Jul.
Article in English | MEDLINE | ID: mdl-20479129

ABSTRACT

In model organisms, resistance to inhibitors of cholinesterase 8 (Ric-8), a G protein alpha (G alpha) subunit guanine nucleotide exchange factor (GEF), functions to orient mitotic spindles during asymmetric cell divisions; however, whether Ric-8A has any role in mammalian cell division is unknown. We show here that Ric-8A and G alpha(i) function to orient the metaphase mitotic spindle of mammalian adherent cells. During mitosis, Ric-8A localized at the cell cortex, spindle poles, centromeres, central spindle, and midbody. Pertussis toxin proved to be a useful tool in these studies since it blocked the binding of Ric-8A to G alpha(i), thus preventing its GEF activity for G alpha(i). Linking Ric-8A signaling to mammalian cell division, treatment of cells with pertussis toxin, reduction of Ric-8A expression, or decreased G alpha(i) expression similarly affected metaphase cells. Each treatment impaired the localization of LGN (GSPM2), NuMA (microtubule binding nuclear mitotic apparatus protein), and dynein at the metaphase cell cortex and disturbed integrin-dependent mitotic spindle orientation. Live cell imaging of HeLa cells expressing green fluorescent protein-tubulin also revealed that reduced Ric-8A expression prolonged mitosis, caused occasional mitotic arrest, and decreased mitotic spindle movements. These data indicate that Ric-8A signaling leads to assembly of a cortical signaling complex that functions to orient the mitotic spindle.


Subject(s)
Antigens, Nuclear/metabolism , Dyneins/metabolism , GTP-Binding Protein alpha Subunits, Gi-Go/metabolism , Guanine Nucleotide Exchange Factors/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Nuclear Matrix-Associated Proteins/metabolism , Spindle Apparatus/metabolism , Animals , Base Sequence , Cell Cycle Proteins , Cell Division/physiology , Cell Line , Dogs , GTP-Binding Protein alpha Subunits, Gi-Go/antagonists & inhibitors , GTP-Binding Protein alpha Subunits, Gi-Go/genetics , Guanine Nucleotide Exchange Factors/antagonists & inhibitors , Guanine Nucleotide Exchange Factors/genetics , HeLa Cells , Humans , Interphase/physiology , Metaphase/physiology , Pertussis Toxin/pharmacology , Protein Binding/drug effects , RNA, Small Interfering/genetics , Signal Transduction , Spindle Apparatus/drug effects
15.
J Nucl Med ; 51(1): 85-91, 2010 Jan.
Article in English | MEDLINE | ID: mdl-20008978

ABSTRACT

UNLABELLED: Cardiovascular disease is the leading cause of death worldwide. PET has the potential to provide information on the biology and metabolism of atherosclerotic plaques. Natriuretic peptides (NPs) have potent antiproliferative and antimigratory effects on vascular smooth-muscle cells (VSMCs) and, in atherosclerosis, participate in vascular remodeling, in which the expression of NP clearance receptors (NPR-Cs) is upregulated both in endothelium and in VSMCs. METHODS: We investigated the potential of a C-type atrial natriuretic factor (C-ANF) to image developing plaque-like lesions in vivo. C-ANF was functionalized with 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) and labeled with (64)Cu for noninvasive PET in a hypercholesterolemic rabbit with atherosclerotic-like lesions induced by air desiccation of a femoral artery, followed by balloon overstretch of the developing neointima. Histopathology and immunohistochemistry were performed to assess plaque development and NPR-C localization. RESULTS: (64)Cu-DOTA-C-ANF uptake in the atherosclerotic region was visible on small-animal PET images, with the highest target-to-background ratio (3.59 +/- 0.94) observed after the air desiccation-induced injury. Immunohistochemistry and immunofluorescence staining showed NPR-C near the luminal surface of the plaque and in VSMCs. PET and immunohistochemistry competitive blocking studies confirmed receptor-mediated tracer uptake in the plaque. With blocking, PET tracer localization of atherosclerotic to control arteries was decreased from 1.42 +/- 0.02 to 1.06 +/- 0.06 (P < 0.001). CONCLUSION: We demonstrated that (64)Cu-DOTA-C-ANF is a promising candidate tracer for in vivo PET of NPR-Cs on atherosclerotic plaques.


Subject(s)
Atherosclerosis/diagnostic imaging , Atrial Natriuretic Factor , Copper Radioisotopes , Natriuretic Peptide, C-Type , Organometallic Compounds , Radiopharmaceuticals , Animals , Atherosclerosis/pathology , Cholesterol/blood , Immunohistochemistry , Isotope Labeling , Magnetic Resonance Imaging , Natriuretic Peptide, C-Type/pharmacokinetics , Positron-Emission Tomography , Rabbits , Radiopharmaceuticals/chemical synthesis , Radiopharmaceuticals/pharmacokinetics , Receptors, Atrial Natriuretic Factor/drug effects , Receptors, Atrial Natriuretic Factor/metabolism
16.
J Biol Chem ; 285(11): 8045-53, 2010 Mar 12.
Article in English | MEDLINE | ID: mdl-20022948

ABSTRACT

There is a body of evidence suggesting that Ca(2+) handling proteins assemble into signaling complexes required for a fine regulation of Ca(2+) signals, events that regulate a variety of critical cellular processes. Canonical transient receptor potential (TRPC) and Orai proteins have both been proposed to form Ca(2+)-permeable channels mediating Ca(2+) entry upon agonist stimulation. A number of studies have demonstrated that inositol 1,4,5-trisphosphate receptors (IP(3)Rs) interact with plasma membrane TRPC channels; however, at present there is no evidence supporting the interaction between Orai proteins and IP(3)Rs. Here we report that treatment with thapsigargin or cellular agonists results in association of Orai1 with types I and II IP(3)Rs. In addition, we have found that TRPC3, RACK1 (receptor for activated protein kinase C-1), and STIM1 (stromal interaction molecule 1) interact with Orai1 upon stimulation with agonists. TRPC3 expression silencing prevented both the interaction of Orai1 with TRPC3 and, more interestingly, the association of Orai1 with the type I IP(3)R, but not with the type II IP(3)R, thus suggesting that TRPC3 selectively mediates interaction between Orai1 and type I IP(3)R. In addition, TRPC3 expression silencing attenuated ATP- and CCh-stimulated interaction between RACK1 and the type I IP(3)R, as well as Ca(2+) release and entry. In conclusion, our results indicate that agonist stimulation results in the formation of an Orai1-STIM1-TRPC3-RACK1-type I IP(3)R complex, where TRPC3 plays a central role. This Ca(2+) signaling complex might be important for both agonist-induced Ca(2+) release and entry.


Subject(s)
Calcium Channels/metabolism , Calcium Signaling/physiology , GTP-Binding Proteins/metabolism , Inositol 1,4,5-Trisphosphate Receptors/metabolism , Neoplasm Proteins/metabolism , Receptors, Cell Surface/metabolism , TRPC Cation Channels/metabolism , Calcium/metabolism , Calcium/pharmacology , Calcium Channels/genetics , Calcium Signaling/drug effects , Chelating Agents/pharmacology , Egtazic Acid/analogs & derivatives , Egtazic Acid/pharmacology , HeLa Cells , Humans , Inositol 1,4,5-Trisphosphate Receptors/genetics , Kidney/cytology , Membrane Proteins/metabolism , ORAI1 Protein , RNA, Small Interfering , Receptors for Activated C Kinase , Stromal Interaction Molecule 1 , TRPC Cation Channels/genetics , Thapsigargin/pharmacology
17.
Int Rev Cell Mol Biol ; 268: 59-93, 2008.
Article in English | MEDLINE | ID: mdl-18703404

ABSTRACT

Four major natriuretic peptides have been isolated: atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), C-type natriuretic peptide (CNP), and Dendroaspis-type natriuretic peptide (DNP). Natriuretic peptides play an important role in the regulation of cardiovascular homeostasis maintaining blood pressure and extracellular fluid volume. The classical endocrine effects of natriuretic peptides to modulate fluid and electrolyte balance and vascular smooth muscle tone are complemented by autocrine and paracrine actions that include regulation of coronary blood flow and, therefore, myocardial perfusion; modulation of proliferative responses during myocardial and vascular remodeling; and cytoprotective anti-ischemic effects. The actions of natriuretic peptides are mediated by the specific binding of these peptides to three cell surface receptors: type A natriuretic peptide receptor (NPR-A), type B natriuretic peptide receptor (NPR-B), and type C natriuretic peptide receptor (NPR-C). NPR-A and NPR-B are guanylyl cyclase receptors that increase intracellular cGMP concentration and activate cGMP-dependent protein kinases. NPR-C has been presented as a clearance receptor and its activation also results in inhibition of adenylyl cyclase activity. The wide range of effects of natriuretic peptides might be the base for the development of new therapeutic strategies of great benefit in patients with cardiovascular problems including coronary artery disease or heart failure. This review summarizes current literature concerning natriuretic peptides, their receptors and their effects on fluid/electrolyte balance, and vascular and cardiac physiology and pathology, including primary hypertension and myocardial infarction. In addition, we will attempt to provide an update on important issues regarding natriuretic peptides in congestive heart failure.


Subject(s)
Blood Vessels/pathology , Blood Vessels/physiology , Natriuretic Peptides/physiology , Animals , Cell Proliferation , Heart/physiology , Humans , Hypertension/physiopathology , Models, Cardiovascular , Myocardium/pathology , Oxidative Stress , Receptors, Peptide/physiology , Signal Transduction
18.
Am J Physiol Cell Physiol ; 294(6): C1323-31, 2008 Jun.
Article in English | MEDLINE | ID: mdl-18400989

ABSTRACT

Ca+ release-activated Ca2+ (CRAC) channels are activated when free Ca2+ concentration in the intracellular stores is substantially reduced and mediate sustained Ca2+ entry. Recent studies have identified Orai1 as a CRAC channel subunit. Here we demonstrate that passive Ca2+ store depletion using the inhibitor of the sarcoendoplasmic reticulum Ca2+-ATPase, thapsigargin (TG), enhances the surface expression of Orai1, a process that depends on rises in cytosolic free Ca2+ concentration, as demonstrated in cells loaded with dimethyl BAPTA, an intracellular Ca2+ chelator that prevented TG-evoked cytosolic free Ca2+ concentration elevation. Similar results were observed with a low concentration of carbachol. Cleavage of the soluble N-ethylmaleimide-sensitive-factor attachment protein receptor, synaptosomal-assiciated protein-25 (SNAP-25), with botulinum neurotoxin A impaired TG-induced increase in the surface expression of Orai1. In addition, SNAP-25 cleaving by botulinum neurotoxin A reduces the maintenance but not the initial stages of store-operated Ca2+ entry. In aggregate, these findings demonstrate that store depletion enhances Orai1 plasma membrane expression in an exocytotic manner that involves SNAP-25, a process that contributes to store-dependent Ca2+ entry.


Subject(s)
Calcium Channels/metabolism , Calcium/metabolism , Cell Membrane/metabolism , Exocytosis , Sarcoplasmic Reticulum/metabolism , Botulinum Toxins, Type A/pharmacology , Carbachol/pharmacology , Cell Membrane/drug effects , Chelating Agents/pharmacology , Egtazic Acid/analogs & derivatives , Egtazic Acid/pharmacology , Enzyme Inhibitors/pharmacology , Exocytosis/drug effects , HeLa Cells , Humans , ORAI1 Protein , Protein Transport , Sarcoplasmic Reticulum/drug effects , Sarcoplasmic Reticulum/enzymology , Sarcoplasmic Reticulum Calcium-Transporting ATPases/antagonists & inhibitors , Sarcoplasmic Reticulum Calcium-Transporting ATPases/metabolism , Synaptosomal-Associated Protein 25/metabolism , Thapsigargin/pharmacology , Time Factors , Up-Regulation
19.
Thromb Haemost ; 98(2): 375-84, 2007 Aug.
Article in English | MEDLINE | ID: mdl-17721620

ABSTRACT

We have investigated the intracellular mechanisms involved in microtubular remodelling by thrombin and its possible involvement in platelet aggregation and secretion. Platelet stimulation with thrombin induces a time- and concentration-dependent regulation of the microtubular content, which was found to be maximally effective at the concentration 0.1 U/ml. Thrombin (0.1 U/ml) evoked an initial decrease in the microtubule content detectable at 5 seconds (sec) and reached a minimum 10 sec after stimulation. The microtubular content then increased, exceeding basal levels again approximately 30 sec after stimulation. Inhibition of tyrosine phosphatases using vanadate abolished thrombin-induced microtubular depolymerisation while inhibition of tyrosine kinases by methyl-2,5-dihydroxycinnamate prevented microtubule polymerisation. Thrombin activates the cytosolic Bruton's tyrosine kinase (Btk) and Src proteins. Inhibition of Btk or Src by LFM-A13 or PP1, respectively, abolished thrombin-induced microtubular polymerisation, while maintaining intact its ability to induce initial depolymerisation. Microtubular disruption by colchicine significantly reduced thrombin-induced platelet aggregation and ATP secretion. Similar results were observed after inhibition of microtubular disassembly by paclitaxel. These findings indicate that thrombin induces microtubular remodelling by modifying the balance between protein tyrosine phosphorylation and dephosphorylation. The former seems to be required for microtubular polymerisation, while tyrosine dephosphorylation is required for microtubular depolymerisation. Both, initial microtubular disassembly and subsequent polymerisation are required for thrombin-induced platelet aggregation and secretion in human platelets.


Subject(s)
Blood Platelets/cytology , Microtubules/ultrastructure , Thrombin/pharmacology , Tyrosine/metabolism , Blood Platelets/drug effects , Blood Platelets/metabolism , Cells, Cultured , Humans , Kinetics , Microtubules/drug effects , Phosphorylation , Platelet Aggregation/drug effects , Protein Tyrosine Phosphatases/metabolism , Protein Tyrosine Phosphatases/physiology
20.
Int Rev Cytol ; 256: 35-67, 2007.
Article in English | MEDLINE | ID: mdl-17241904

ABSTRACT

The transient receptor potential (TRP) family of ion channels is composed of more than 50 functionally versatile cation-permeant ion channels expressed in most mammalian cell types. Considerable research has been brought to bear on the members of this family, especially with regard to their possible role as store-operated calcium channels, although studies have provided evidence that TRP channels exhibit a number of regulatory and functional aspects. Endogenous and transiently expressed TRP channels can be activated by different mechanisms grouped into four main categories: receptor-operated activation, store depletion-mediated activation, ligand-induced activation, and direct activation. This article reviews the biochemical characteristics of the different members of the TRP family and summarizes their involvement in a number of physiological events ranging from sensory transduction to development, which might help in understanding the relationship between TRP channel dysfunction and the development of several diseases.


Subject(s)
Intracellular Signaling Peptides and Proteins/physiology , Transient Receptor Potential Channels/physiology , Animals , Calcium/metabolism , Calcium Signaling , Humans , Models, Biological , Second Messenger Systems , Transient Receptor Potential Channels/genetics
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