Papers by Alexander Stokes
Current Biology, Sep 1, 2020
Multimodal TRPA1 Channel Responses Highlights d TRPA1 in pFRG/RTN astrocytes selectively monitors... more Multimodal TRPA1 Channel Responses Highlights d TRPA1 in pFRG/RTN astrocytes selectively monitors moderate hypoxia d During hypoxia, TRPA1 acutely accumulates in the plasma membrane of pFRG/RTN astrocytes d TRPA1 is reversibly coupled with O 2-dependent internalization via PHD and NEDD4-1 d Hypoxia triggers astrocytic ATP release via TRPA1 modulating respiratory center
Current Biology, 2020
Multimodal TRPA1 Channel Responses Highlights d TRPA1 in pFRG/RTN astrocytes selectively monitors... more Multimodal TRPA1 Channel Responses Highlights d TRPA1 in pFRG/RTN astrocytes selectively monitors moderate hypoxia d During hypoxia, TRPA1 acutely accumulates in the plasma membrane of pFRG/RTN astrocytes d TRPA1 is reversibly coupled with O 2-dependent internalization via PHD and NEDD4-1 d Hypoxia triggers astrocytic ATP release via TRPA1 modulating respiratory center
Journal of immunobiology, 2017
Jo u rn a l o f Immun o b io lo gy
Cell Calcium, Sep 1, 2014
There is well-established variability in the numbers of lipid bodies (LB) in macrophages, eosinop... more There is well-established variability in the numbers of lipid bodies (LB) in macrophages, eosinophils, and neutrophils. Similarly to the steatosis observed in adipocytes and hepatocytes during hyperinsulinemia and nutrient overload, immune cell LB hyper-accumulate in response to bacterial and parasitic infection and inflammatory presentations. Recently we described that hyperinsulinemia, both in vitro and in vivo, drives steatosis and phenotypic changes in primary and transformed mast cells and basophils. LB reach high numbers in these steatotic cytosols, and here we propose that they could dramatically impact the transcytoplasmic signaling pathways. We compared calcium release and influx responses at the population and single cell level in normal and steatotic model mast cells. At the population level, all aspects of FcεRI-dependent calcium mobilization, as well as activation of calcium-dependent downstream signalling targets such as NFATC1 phosphorylation are suppressed. At the single cell level, we demonstrate that LB are both sources and sinks of calcium following FcεRI cross-linking. Unbiased analysis of the impact of the presence of LB on the rate of trans-cytoplasmic calcium signals suggest that LB enrichment accelerates calcium propagation, which may reflect a Bernoulli effect. LB abundance thus impacts this fundamental signalling pathway and its downstream targets.
Toxicology Letters, Aug 1, 2014
Carbon nanotubes (CNT) are environmental challenges to the respiratory and gastrointestinal mucos... more Carbon nanotubes (CNT) are environmental challenges to the respiratory and gastrointestinal mucosa, and to the dermal immune system. Mast cells (MC) are pro-inflammatory immunocytes that reside at these interfaces with the environment. Mast cells are sources of pro-inflammatory mediators (histamine, serotonin, matrix-active proteases, eicosanoids, prostanoids, cytokines and chemokines), which are released in a calcium-dependent manner following immunological challenge or physico-chemical stimulation. Since C-60 fullerenes, which share geometry with CNT, are suppressive of mast cell-driven inflammatory responses, we explored the effects of unmodified SWCNT aggregates on mast cell signaling pathways, phenotype and proinflammatory function. We noted SWCNT suppression of antigen-induced signalling pathways and pro-inflammatory degranulation responses. Mast cells recognize unmodified SWCNT by remodeling the plasma membrane, disaggregating the cortical actin cytoskeleton and relocalizing clathrin. Clathrin was also identified as a component of an affinity-purified 'interactome' isolated from MC using an SWCNT affinity matrix for mast cell lysates. Together these data are consistent with the ability of SWCNT to suppress mast cell pro-inflammatory function via a novel recognition mechanism.
Regulatory Peptides, Dec 1, 2011
C57BL/6-Kit W-sh/W-sh mice are generally regarded as a mast cell-deficient model, as they lack th... more C57BL/6-Kit W-sh/W-sh mice are generally regarded as a mast cell-deficient model, as they lack the necessary kit receptor for mast cell development. Further characterization of this strain, however, indicates that C57BL/6-Kit W-sh/W-sh mice also have a disruption in the Corin gene. Corin is a transmembrane serine protease critical for processing atrial natriuretic peptide (ANP) from pro-ANP through proteolytic cleavage. Pro-ANP is produced, stored and released by cardiac myocytes in response to atrial stretch and the stress generated by increased afterload such as increased ventricular pressure from aortic stenosis or myocardial infarction. ANP inhibits the effects of the renin-angiotensin system to preserve homeostasis under conditions of increased hemodynamic load, and changes in the level of its activating enzyme Corin have been observed during the progression to heart failure. Here, we investigate the effect of increased hemodynamic load on Corin-deficient C57BL/6-Kit W-sh/W-sh mice. Ten-week old male mice were subjected to transverse aortic constriction for 8 wks and were monitored for changes in cardiac structure and function by echocardiography. Hearts were collected 8 wks after surgery for molecular and histological analyses. Corin-deficient C57BL/6-Kit W-sh/W-sh mice developed rapidly progressive and substantial left ventricular dilation, hypertrophy, and markedly impaired cardiac function during the 8 wks after surgery, compared to wildtype mice. Concomitant with this we observed increased levels of ANP transcript, but a lack of prepro-ANP or pro-ANP protein in heart tissue extracted from Corindeficient mice. Surprisingly, fibrosis was not increased in Corin-deficient mice when compared to wildtype mice. These data indicate that Corin's involvement in ANP processing is a key element in the heart's response to increased hemodynamic load. Further, C57BL/6-Kit W-sh/W-sh strain is an effective model for investigating the involvement of Corin and, conversely, a less than optimal model for investigating mast cell, and immunological, functions in certain cardiovascular pathologies.
Channels, 2019
Cannabinoid compounds are potential analgesics. Users of medicinal Cannabis report efficacy for p... more Cannabinoid compounds are potential analgesics. Users of medicinal Cannabis report efficacy for pain control, clinical studies show that cannabis can be effective and opioid sparing in chronic pain, and some constituent cannabinoids have been shown to target nociceptive ion channels. Here, we explore and compare a suite of cannabinoids for their impact upon the physiology of TRPV1. The cannabinoids tested evoke differential responses in terms of kinetics of activation and inactivation. Cannabinoid activation of TRPV1 displays significant dependence on internal and external calcium levels. Cannabinoid activation of TRPV1 does not appear to induce the highly permeant, pore-dilated channel state seen with Capsaicin, even at high current amplitudes. Finally, we analyzed cannabinoid responses at nociceptive channels other than TRPV1 (TRPV2, TRPM8, and TRPA1), and report that cannabinoids differentially activate these channels. On the basis of response activation and kinetics, state-selectivity and receptor selectivity, it may be possible to rationally design approaches to pain using single or multiple cannabinoids.
Journal of Cellular Biochemistry, 2004
TRPV ion channels transduce a range of temperature stimuli. We proposed that analysis of the prot... more TRPV ion channels transduce a range of temperature stimuli. We proposed that analysis of the proteinprotein interactions made by TRPV2 might give insight into the key issues surrounding this channel. These issues include the potential functional significance of TRPV2 in non-sensory tissues, the molecules involved in transducing its activation signal(s) and the mechanism by which its trafficking to the cell surface is regulated. Here we describe the interaction of TRPV2 channel with the RGA gene product. RGA is a four-transmembrane domain, intracellularly localized protein. RGA associates with TRPV2 in a rat mast cell line that is a native context for both proteins. The interaction between TRPV2 and RGA is transient and occurs intracellularly. RGA does not accompany TRPV2 to the cell surface. Formation of the TRPV2/ RGA complex is dependent upon a cellular glycosylation event, suggesting that RGA may play a chaperone or targeting role for TRPV2 during the maturation of the ion channel protein. These data record a novel protein-protein interaction for TRPV2 and provide a foundation for future study of the potential regulatory contribution of RGA to TRPV2 function.
Journal of Immunology, May 15, 2003
Cannabinoid modulation of immune responses is a pathological consequence of marijuana abuse and a... more Cannabinoid modulation of immune responses is a pathological consequence of marijuana abuse and a potential outcome of therapeutic application of the drug. Moreover, endogenous cannabinoids are physiological immune regulators. In the present report, we describe alterations in gene transcription that occur after cannabinoid exposure in a mast cell line, RBL2H3. Cannabinoid exposure causes marked changes in the transcript levels for numerous genes, acting both independently of and in concert with immunoreceptor stimulation via Fc⑀RI. In two mast cell lines, we observed mRNA and protein expression corresponding to both CB1 and CB2 cannabinoid receptor isoforms, contrary to the prevailing view that CB1 is restricted to the CNS. We show that coexpression of the two isoforms is not functionally redundant in mast cells. Analysis of signaling pathways downstream of cannabinoid application reveals that activation of extracellular signal-regulated kinase, AKT, and a selected subset of AKT targets is accomplished by CB2 ligands and nonselective CB1/CB2 agonists in mast cells. CB1 inhibition does not affect AKT or extracellular signal-regulated kinase activation by cannabinoids, indicating that CB2 is the predominant regulatory receptor for these kinases in this cell context. CB1 receptors are, however, functional in these mast cells, since they can contribute to suppression of secretory responses.
Channels, Jan 2, 2015
Two-pore channels (TPC1, 2, and 3) are recently identified endolysosmal ion channels, but remain ... more Two-pore channels (TPC1, 2, and 3) are recently identified endolysosmal ion channels, but remain poorly characterized. In this study, we show for the first time a role for TPC1 in cytokinesis, the final step in cell division. HEK 293 T-REx cells inducibly overexpressing TPC1 demonstrated a lack of proliferation accompanied by multinucleation and an increase in G2/M cycling cells. Increased TPC1 was associated with a concomitant accumulation of active RhoGTP and a decrease in phosphorylated myosin light chain (MLC). Finally, we demonstrated a novel interaction between TPC1 and citron kinase (CIT). These results identify TPC1 as a central component of cytokinetic control, specifically during abscission, and introduce a means by which the endolysosomal system may play an active role in this process.
Journal of Cellular Biochemistry, 2005
The transient receptor potential, sub-family Vanilloid (TRPV) 2 cation channel is activated in re... more The transient receptor potential, sub-family Vanilloid (TRPV) 2 cation channel is activated in response to extreme temperature elevations in sensory neurons. However, TRPV2 is widely expressed in tissues with no sensory function, including cells of the immune system. Regulation of GRC, the murine homolog of TRPV2 has been studied in insulinoma cells and myocytes. GRC is activated in response to certain growth factors and neuropeptides, via a mechanism that involves regulated access of the channel to the plasma membrane. This is likely to be an important primary control mechanism for TRPV2 outside the CNS. Here, we report that a regulated trafficking step controls the access of TRPV2 to the cell surface in mast cells. In mast cells, elevations in cytosolic cAMP are sufficient to drive plasma membrane localization of TRPV2. We have previously proposed that the recombinase gene activator protein (RGA), a fourtransmembrane domain, intracellular protein, associates with TRPV2 during the biosynthesis and early trafficking of the channel. We use a polyclonal antibody to RGA to confirm the formation of a physiological complex between RGA and TRPV2. Finally, we show that over-expression of the RGA protein potentiates the basal surface localization of TRPV2. We propose that trafficking and activation mechanisms intersect for TRPV2, and that cAMP mobilizing stimuli may regulate TRPV2 localization in non-sensory cells. RGA participates in the control of TRPV2 surface levels, and coexpression of RGA may be a key component of experimental systems that seek to study TRPV2 physiology.
Cellular Signalling, Oct 1, 2006
Certain TRP cation channels confer the ability to sense environmental stimuli (heat, cold, pressu... more Certain TRP cation channels confer the ability to sense environmental stimuli (heat, cold, pressure, osmolarity) across physiological and pathophysiological ranges. TRPA1 is a TRP-related channel that responds to cold temperatures, and pungent compounds that include the coldmimetic icilin and cannabinoids. The initial report of TRPA1 as a transformation-associated gene product in lung epithelia is at odds with subsequent descriptions of a tissue distribution for TRPA1 that is restricted to sensory neurons. Here, we report that the human TRPA1 protein is widely expressed outside the CNS, and is indeed dys-regulated during oncogenic transformation. We describe that TRPA1 associates with the tumor-suppressor protein CYLD. TRPA1 is a novel substrate for the de-ubiquitinating activity of CYLD, and this de-ubiquitination has the net effect of increasing the cellular pool of TRPA1 proteins. Oncogenic mutations in the CYLD gene may therefore be predicted to alter cellular levels of TRPA1.
eLife, May 24, 2016
Transcriptome and genome data from twenty stony coral species and a selection of reference bilate... more Transcriptome and genome data from twenty stony coral species and a selection of reference bilaterians were studied to elucidate coral evolutionary history. We identified genes that encode the proteins responsible for the precipitation and aggregation of the aragonite skeleton on which the organisms live, and revealed a network of environmental sensors that coordinate responses of the host animals to temperature, light, and pH. Furthermore, we describe a variety of stress-related pathways, including apoptotic pathways that allow the host animals to detoxify reactive oxygen and nitrogen species that are generated by their intracellular photosynthetic symbionts, and determine the fate of corals under environmental stress. Some of these genes arose through horizontal gene transfer and comprise at least 0.2% of the animal gene inventory. Our analysis elucidates the evolutionary strategies that have allowed symbiotic corals to adapt and thrive for hundreds of millions of years.
PubMed, 2014
Loss of plasma membrane asymmetry is a hallmark of apoptosis, but lipid bilayer asymmetry and los... more Loss of plasma membrane asymmetry is a hallmark of apoptosis, but lipid bilayer asymmetry and loss of asymmetry can contribute to numerous cellular functions and responses that are independent of programmed cell death. Exofacial exposure of phosphatidylserine occurs in lymphocytes and mast cells after antigenic stimulation and in the absence of apoptosis, suggesting that there is a functional requirement for phosphatidylserine exposure in immunocytes. In this review we examine current ideas as to the nature of this functional role in mast cell activation. Mechanistically, there is controversy as to the candidate proteins responsible for phosphatidylserine translocation from the internal to external leaflet, and here we review the candidacies of mast cell PLSCR1 and TMEM16F. Finally we examine the potential relationship between functionally important mast cell membrane perturbations and phosphatidylserine exposure during activation.
Adipocyte, Jan 2, 2019
Mast cell lipid bodies are key to initiation, maintenance and resolution of inflammatory response... more Mast cell lipid bodies are key to initiation, maintenance and resolution of inflammatory responses in tissue. Mast cell lines, primary bone marrow-derived mast cells and peripheral blood basophils present a 'steatotic' phenotype in response to chronic insulin exposure, where cells become loaded with lipid bodies. Here we show this state is associated with reduced histamine release, but increased capacity to release bioactive lipids. We describe the overall lipid phenotype of mast cells in this insulin-induced steatotic state and the consequences for critical cellular lipid classes involved in stages of inflammation. We show significant insulin-induced shifts in specific lipid classes, especially arachidonic acid derivatives, MUFA and PUFA, the EPA/DHA ratio, and in cardiolipins, especially those conjugated to certain DHA and EPAs. Functionally, insulin exposure markedly alters the FcεRI-induced release of Series 4 leukotriene LTC4, Series 2 prostaglandin PGD2, Resolvin-D1, Resolvin-D2 and Resolvin-1, reflecting the expanded precursor pools and impact on both the pro-inflammation and pro-resolution bioactive lipids that are released during mast cell activation. Chronic hyperinsulinemia is a feature of obesity and progression to Type 2 Diabetes, these data suggest that mast cell release of key lipid mediators is altered in patients with metabolic syndrome.
Phytotherapy Research, Jan 30, 2015
Kava is a soporific, anxiolytic and relaxant in widespread ritual and recreational use throughout... more Kava is a soporific, anxiolytic and relaxant in widespread ritual and recreational use throughout the Pacific. Traditional uses of kava by indigenous Pacific Island peoples reflect a complex pharmacopeia, centered on GABA-ergic effects of the well-characterized kavalactones. However, peripheral effects of kava suggest active components other than the CNS-targeted kavalactones. We have previously shown that immunocytes exhibit calcium mobilization in response to traditionally-prepared kava extracts, and that the kavalactones do not induce these calcium responses. Here, we characterize the complex calcium-mobilizing activity of traditionallyprepared and partially HPLC-purified kava extracts, noting induction of both calcium entry and store release pathways. Kava components activate intracellular store depletion of thapsigarginsensitive and-insensitive stores that are coupled to the calcium release activated (CRAC) current, and cause calcium entry through non-store-operated pathways. Together with the pepper-like potency reported by kava users, these studies lead us to hypothesize that kava extracts contain one or more ligands for the transient receptor potential (TRP) family of ion channels. Indeed, TRP-like conductances are observed in kava-treated cells under patch clamp. Thus TRP-mediated cellular effects may be responsible for some of the reported pharmacology of kava.
Journal of Ethnopharmacology, Mar 1, 2021
Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on ... more Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre-including this research content-immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active.
PLOS Neglected Tropical Diseases, Dec 20, 2016
Zika virus (ZIKV) is an emerging mosquito-borne pathogen. ZIKV infection is linked to the develop... more Zika virus (ZIKV) is an emerging mosquito-borne pathogen. ZIKV infection is linked to the development of severe fetal abnormalities that include spontaneous abortion, stillbirth, hydranencephaly, and microcephaly. ZIKV outbreaks have been recorded in the United States. We recently demonstrated the first congenital ZIKV infection in the United States. In this study, we investigated archived blood samples from six mothers who gave birth to babies with microcephaly and 12 mothers who gave birth to healthy babies in Hawaii between 2009 and 2012. We tested maternal blood for the presence of ZIKV IgM and IgG antibodies using commercially available human ZIKV IgM and IgG ELISA kits. Blood from one mother who delivered babies with microcephaly tested positive for ZIKV IgM antibody (16.6%) and blood from three mothers tested positive for ZIKV IgG antibody (50%). ZIKV showed a trend toward significance with microcephaly. ZIKV IgG antibody positive mothers were more likely to deliver babies with microcephaly than mothers who were negative for ZIKV IgG antibodies (Odds ratio [OR] = 11.0, 95% confidence interval [CI] = 0.8-147.9, p = 0.083). Similarly, ZIKV IgM antibody positive mothers were also more likely to deliver babies with microcephaly than mothers who were negative for ZIKV IgM antibody (OR = 6.8, 95% CI = 0.2-195.1). These data provide further evidence of a link between ZIKV infection and microcephaly and suggests presence of ZIKV positive cases and associated microcephaly in the United States as early as 2009.
Phytotherapy Research, Apr 4, 2012
Kava ('Awa) is a traditional water-based beverage in Pacific island communities, prepared from th... more Kava ('Awa) is a traditional water-based beverage in Pacific island communities, prepared from the ground root and stems of Piper methysticum. Kava use is associated with an ichthyotic dermatitis and delayed type hypersensitivity reactions. In the current study we collated preparative methodologies from cultural practitioners and recreational kava users in various Pacific communities. We standardized culturally-informed aqueous extraction methods and prepared extracts that were subjected to basic physicochemical analysis. Mast cells exposed to these extracts displayed robust intracellular free calcium responses, and concomitant release of proinflammatory mediators. In contrast, mast cells were refractory to single or combinatorial stimulation with kavalactones including methysticin, dihydromethysticin and kavain. Moreover, we reproduced a traditional modification of the kava preparation methodology, pre-mixing with the mucilage of Hibiscus taliaceus, and observed its potentiating effect on the activity of aqueous extracts in mast cells. Taken together, these data indicate that water extractable active ingredients may play a role in the physiological and pathophysiological effects of kava, and suggests that mast cell activation may be a mechanistic component of kava-related skin inflammations.
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Papers by Alexander Stokes