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Activation and Regulation of TRPV1: Studies in Recombinant Human Neuroblastoma TRPV1-SHSY5Y Cells
Stockholm University, Faculty of Science, Department of Neurochemistry. (Dr Anna Forsby)
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

TRPV1 is a transmembrane non-selective cation channel with preference for Ca2+. The receptor is primarily localised on dorsal root ganglion neurons and is activated by numerous endogenous and exogenous potentially irritating ligands eliciting pain. The TRPV1 expression and activity are regulated by several neurotrophic agents and inflammatory mediators via activation of phosphorylation cascades.

In this thesis a stably TRPV1-expressing cell clone of the human neuroblastoma cell line SHSY5Y was established with the purpose to study regulation of TRPV1 through a straightforward and reproducible approach.

In paper I it is reported that the neurotrophic factors insulin, and IGF-1 up-regulate TRPV1 in the TRPV1-SHSY5Y cells. Additionally, the involved signalling pathways are suggested. This is of interest because both TRPV1 activity and expression is altered in diabetic patients with painful neuropathies, and so is insulin and IGF-1 signalling.

Results from paper II show that the neuronally differentiating morphogen retinoic acid (RA) increases TRPV1 protein levels and TRPV1-mediated Ca2+ influxes. Furthermore, the basal Ca2+ level is increased after RA treatment in TRPV1-SHSY5Y cells but not in native SHSY5Y cells. The TRPV1-SHSY5Y cells also develop into a more mature neuronal phenotype than the native SHSY5Y cells after six days of RA-induced differentiation. Hence, TRPV1 might be involved in neurogenesis.

In paper III-IV it is concluded that the TRPV1-SHSY5Y cells can be used in a semi-high throughput screening (HTS)-mode to adress TRPV1-mediated Ca2+ influxes. In this assay it is shown that anionic linear aliphatic surfactants might be potent ligands of TRPV1. As a concluding remark, the TRPV1–SHSY5Y cells can be utilised to assess activation and regulation of TRPV1 in an easy-to-use and robust model system.

Place, publisher, year, edition, pages
Stockholm: Department of Neurochemistry, Stockholm University , 2009. , 91 p.
Keyword [en]
nociception, neurotrophic factors, eye irritation, stable transfection, Ca2+
National Category
Neurosciences
Research subject
Neurochemistry and Molecular Neurobiology
Identifiers
URN: urn:nbn:se:su:diva-30182ISBN: 978-91-7155-954-8 (print)OAI: oai:DiVA.org:su-30182DiVA: diva2:241963
Public defence
2009-11-13, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 13:00 (English)
Opponent
Supervisors
Note
At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: In press. Paper 4: In press.Available from: 2009-10-22 Created: 2009-10-06 Last updated: 2015-03-09Bibliographically approved
List of papers
1. Insulin and Insulin-Like Growth FactorType-I Up-Regulate the Vanilloid Receptor-1(TRPV1) in Stably TRPV1-ExpressingSH-SY5Y Neuroblastoma Cells
Open this publication in new window or tab >>Insulin and Insulin-Like Growth FactorType-I Up-Regulate the Vanilloid Receptor-1(TRPV1) in Stably TRPV1-ExpressingSH-SY5Y Neuroblastoma Cells
2007 (English)In: Journal of Neuroscience Research, ISSN 0360-4012, E-ISSN 1097-4547, Vol. 85, no 7, 1413-1419 p.Article in journal (Refereed) Published
Abstract [en]

The capsaicin receptor, transient receptor potential, vanilloid type 1 (TRPV1), is a Ca2+ permeable ion channel activated by noxious stimuli eliciting pain. Several reports have shown modulation of TRPV1 activity and expression by neuronal growth factors. Here, we study the long-term effects on TRPV1 expression mediated by insulin like growth factor type-I (IGF-I) and insulin in a stably TRPV1-expressing SH-SY5Y neuroblastoma cell line. We show that after 72 h of 10 nM IGF-I or insulin exposure, the TRPV1 protein level was up-regulated 2.5 and 2-fold, respectively. By blocking phosphatidylinositol-3-kinase (PI(3)K) or mitogen-activated protein kinase (MAPK) signaling we concluded that the increase in total TRPV1 protein content induced by IGF-I was controlled by PI(3)K signaling whereas insulin seemed to regulate TRPV1 protein expression via both PI(3)K and MAPK pathways. Inhibiting protein kinase C (PKC) blocked the effects of both IGF-I and insulin. Furthermore, the concentrations causing 50 % Ca2+ increase (EC50) after insulin and IGF-I-treatments were significantly lowered compared to untreated cells. We conclude that IGF-I and insulin enhance TRPV1 protein expression and activity, and impaired pain sensation might result from distorted TRPV1 regulation in the peripheral nervous system.

National Category
Neurosciences
Identifiers
urn:nbn:se:su:diva-30176 (URN)10.1002/jnr.21255 (DOI)000246929900005 ()
Available from: 2009-10-06 Created: 2009-10-06 Last updated: 2017-12-13Bibliographically approved
2. Surfactant-Induced TRPV1 Activity—A Novel Mechanismfor Eye Irritation?
Open this publication in new window or tab >>Surfactant-Induced TRPV1 Activity—A Novel Mechanismfor Eye Irritation?
2007 (English)In: Toxicological Sciences, ISSN 1096-6080, E-ISSN 1096-0929, Vol. 99, no 1, 174-180 p.Article in journal (Refereed) Published
Abstract [en]

The pain receptor transient receptor potential vanilloid type 1 (TRPV1) has been reported as one of the key components in the pain pathway.  Activation of the receptor causes a Ca2+ influx with secondary effects leading to neurogenic inflammation. Here we report specific activation of TRPV1 by detergent-containing hygiene products measured as intracellular Ca2+ influxes in stably TRPV1-expressing neuroblastoma SH-SY5Y cells. Children products marketed as “painless” (containing lower concentration of detergents), and conditioners (without detergents) did not induce specific TRPV1 activation. Furthermore, low concentrations of the detergent sodium lauryl sulfate (SLS) dose-dependently induced Ca2+ influxes that could be addressed to TRPV1. These results reveal a novel mechanistic pathway for surfactant-induced nociception, which may be an important endpoint in in vitro test batteries as alternatives to Draize’s rabbit eye test for classification of eye irritating products.

Identifiers
urn:nbn:se:su:diva-30178 (URN)10.1093/toxsci/kfm164 (DOI)000249148300018 ()
Available from: 2009-10-06 Created: 2009-10-06 Last updated: 2017-12-13Bibliographically approved
3. TRPV1 expression and activity during retinoic acid-induced neuronal differentiation
Open this publication in new window or tab >>TRPV1 expression and activity during retinoic acid-induced neuronal differentiation
2009 (English)In: Neurochemistry International, ISSN 0197-0186, E-ISSN 1872-9754, Vol. 55, no 8, 768-774 p.Article in journal (Refereed) Published
Abstract [en]

The transient receptor potential vanilloid subtype 1 (TRPV1) is a Ca2+-permeable channel primarily expressed in dorsal root ganglion neurons. Besides its function in thermogenic nociception and neurogenic inflammation, TRPV1 is involved in cell migration, cytoskeleton reorganisation and in neuronal guidance.  To explore the TRPV1 level and activity during conditions for neuronal maturation, TRPV1-expressing SHSY5Y neuroblastoma cells were differentiated into a neuronal phenotype using all-trans-retinoic acid (RA). We show that RA highly up-regulated the total and cell surface TRPV1 protein expression but the TRPV1 mRNA level was unaffected. The up-regulated receptors were localised to the cell bodies and the developed neurites. Furthermore, RA increased both the basal intracellular free Ca2+ concentration by 30 % as well as the relative capsaicin-induced Ca2+ influx. The results show that TRPV1 protein expression increases during RA-induced differentiation in vitro, which generates an altered intracellular Ca2+ homeostasis.

National Category
Biological Sciences Chemical Sciences
Identifiers
urn:nbn:se:su:diva-30177 (URN)10.1016/j.neuint.2009.07.011 (DOI)000271690000008 ()
Available from: 2009-10-06 Created: 2009-10-06 Last updated: 2017-12-13Bibliographically approved
4. Anionic linear aliphatic surfactants activate TRPV1: a possible endpoint for estimation of detergent induced eye nociception?
Open this publication in new window or tab >>Anionic linear aliphatic surfactants activate TRPV1: a possible endpoint for estimation of detergent induced eye nociception?
Show others...
2009 (English)In: Toxicology in Vitro, ISSN 0887-2333, E-ISSN 1879-3177, Vol. 23, no 8, 1472-1476 p.Article in journal (Refereed) Published
Abstract [en]

The transient receptor potential vanilloid type 1 (TRPV1) has been reported as one of the key components in the pain pathway. Activation of the receptor causes a Ca2+ influx in sensory C-fibres with secondary effects leading to neurogenic inflammation in the surrounding tissue. We have earlier reported specific activation of TRPV1 by surfactant-containing hygiene products. We have continued this project by investigating activation of the TRPV1 by shampoo and soap ingredients in low concentrations measured as intracellular Ca2+ influxes in stably TRPV1-expressing neuroblastoma SH-SY5Y cells. As a TRPV1 specific control, the TRPV1 antagonist capsazepine was used. The response was quantified as the product induced Ca2+ influx during 2 min in relation to the maximum response induced by the TRPV1 agonist capsaicin. The results show that anionic alkyl linear surfactant ingredients such as sodium lauryl sulphate, sodium laureth sulphate, ammonium lauryl sulphate, sodium C12-15 pareth sulphate and N-lauroylsarcosine concentration-dependently induced Ca2+ influx that could be addressed to TRPV1. The cationic surfactants benzalkonium chloride and cetylpyridinium chloride induced a Ca2+ influx that was not TRPV1 mediated as well as the zwitterionic surfactant cocamidopropyl betaine, the non-linear anionic surfactant sodium deoxycholate and the non-ionic surfactant Triton-X. These results reveal a new mechanistic pathway for surfactant-induced nociception.

Identifiers
urn:nbn:se:su:diva-32269 (URN)10.1016/j.tiv.2009.06.013 (DOI)000272276600006 ()19540328 (PubMedID)
Available from: 2009-12-07 Created: 2009-12-07 Last updated: 2017-12-12Bibliographically approved

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