Investigation of the transient receptor potential vanilloid 1 (TRPV1) ion channel

Winter Zoltán
Investigation of the transient receptor potential vanilloid 1 (TRPV1) ion channel.
Doktori értekezés, Szegedi Tudományegyetem.
(2013)

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Absztrakt (kivonat) idegen nyelven

The aims of this research were to determine sensory modalities that may be lost after the RTX treatment of newborn or adult mice, to dissect potential side-effect(s) of molecular neurosurgery, to gather information about the structure and function of the channel by investigating the effects of M2+ on the TRPV1 and by collecting the literature data on the functionally important point mutations of the channel for prospective in silico modeling. The findings of the research work can be summarized as follows: 1, • AITC had no effect on TRPV1-expressing cells. 2, • Mice treated either with one dose of RTX two days post-natal, or one dose of RTX at adult age, or daily with a dose of RTX for three days at adult age completely lost their sensitivity to vanilloids and to AITC. • Immunohistological staining and Western blotting experiments confirmed TRPV1 and TRPA1 loss in the DRG and TG of mice treated with one dose of RTX as neonates, or one dose of RTX at adult age, or daily with a dose of RTX for three days at adult age. • These data indicate that TRPA1 expression is coincident with TRPV1 expression in the vast majority of nociceptive neurons that normally confer pain signal to endogenous substances produced in pain and inflammation. 3, • Mice treated with RTX two days post-natal and tested at the age of two months interestingly showed no change in temperature sensitivity either to heat or to cold. Due to neuronal plasticity, mice treated at neonatal age can most likely compensate for the imbalance caused by that drop-out of C-type afferents neurons. • Mice treated with one dose of RTX at adult age become less sensitive to heat and, surprisingly, more sensitive to cold. Two hypotheses can be put forward to explain this finding: 1, The NGF-mediated overexpression of TRPM8 caused by TRPV1+ neuron loss. 2, The imbalance caused by the drop-out of C-type afferent neurons may cause alterations in the representation of sensory stimuli signaling heat and cold at the levels of the CNS. • Mice treated daily with a dose of RTX for three days at adult age lost their heat sensitivity to a higher extent as compared with mice subjected to acute treatment, because the heat threshold increase depends on the efficacy of the RTX treatment. The heat insensitivity of the animal becomes more pronounced if RTX ablates TRPV1+ nociceptors almost completely. Chronic, repeated RTX treatment gave the best results, while neonatal treatments were less effective. • Immunohistological staining and Western blotting experiments indicated that TRPM8+ cells remained intact in the DRG and TG of mice treated with one dose of RTX as neonates, or one dose of RTX at adult age, or daily with a dose of RTX for three days at adult age. • On the basis of these data, two markedly distinct populations of cold-responsive neurons can be distinguished: 1, TRPM8+ neurons that lack TRPV1 and TRPA1 expression and are resistant to RTX treatment, and 2, TRPA1+ neurons that express TRPV1 as well and can be deleted with either AITC or RTX. 4, • Neither Mg2+, Mn2+ nor La3+ significantly changed the function of TRPV1. • Zn2+ proved to be a weak and only partial inhibitor of the CAPS-induced 45Ca2+ uptake. • Co2+, Cd2+, Ni2+ and Cu2+ effectively blocked the vanilloid-induced Ca2+ entry into TRPV1/HaCaT cells, with the following sequence of potency: Co2+ > Cd2+ > Ni2+ > Cu2+. • Co2+ reduced both the heat and CAPS-induced 45Ca2+ influx to a similar extent as did RuRed, AMI or R4W2. • Increasing concentrations of Co2+ decreased only the Emax of Ca2+ entry in a vanilloid-induced 45Ca2+-uptake assay; the affinity of CAPS for TRPV1 did not change. Co2+ inhibition patterns indicated channel-blocking kinetics. • Increasing cold Ca2+ concentration, in a vanilloid-induced 45Ca2+-uptake assay, decreased the inhibitory effect of Co2+, showing that the effect of Co2+ on Ca2+ entry mainly depends on the competition for entry sites. • Increasing CAPS concentration in a vanilloid-induced 45Ca2+-uptake assay, caused a shift in the IC50 of Co2+, increasing agonist concentration enhancing the blocking ability of Co2+, suggesting that the increased efficiency of inhibition correlates with the different open-state conformations of the TRPV1 channel. • We presume that the efficacy of the TRPV1 blocking effect caused by M2+ is determined by the combined impact of the ionic radii of the M2+ and the stability constants of the complexes formed between M2+ and amino acids. Ca2+ is likely to have the ideal ionic radius and stability constant in its reactions with amino acids in order to be effectively passed along the carbonyl groups of the peptide backbone in the ion selectivity filter and the pore loop. The stronger the M2+-amino acid complex and the smaller the ionic radius is, the more probable it is that M2+ will block the Ca2+ influx through the TRPV1 channel. • (NH4)nSx histochemistry experiments on rTRPV1/HaCaT and rTRPV1/3T3 cell lines and sensory neuron cultures prepared from DRGs of rat embryos revealed that Co2+ not only competes with Ca2+, but also enters into the cytosol of TRPV1+ cells, through the TRPV1 channel. • (NH4)nSx histochemistry experiments following extracellular KCl exposure of rTRPV1/3T3 cells ruled out the role of VGCCs in Co2+ accumulation. • Co2+ decreased the frequency of vanilloid-evoked defending movements in tests of eye wiping. 5, • An overview of the currently available results of site-directed mutagenesis studies on TRPV1 receptor was created and published [185] containing summarized information on 112 unique mutated sites along the TRPV1. The mutations influence the effects or binding of different agonists, antagonists, and channel blockers, alter the responsiveness to heat, acid, and voltage dependence, affect the channel pore characteristics, and influence the regulation of the receptor function by phosphorylation, glycosylation, CaM, PIP2, ATP and lipid binding.

Mű típusa: Disszertáció (Doktori értekezés)
Doktori iskola: Gyógyszertudományok Doktori Iskola
Tudományterület / tudományág: orvostudományok > gyógyszerészeti tudományok
Magyar cím: Tranziens receptor potenciál vanilloid 1 (TRPV1) ioncsatorna vizsgálata
Idegen nyelvű cím: Investigation of the transient receptor potential vanilloid 1 (TRPV1) ion channel
Témavezető(k):
Témavezető neveBeosztás, tudományos fokozat, intézményEmail
Prof. Dombi GyörgyTanszékvezető egyetemi tanár, Ph.D. C.Sc., SZTE GYTK, Gyógyszeranalitiai Intézetg.dombi@pharm.u-szeged.hu
Dr. Szakonyi GerdaEgyetemi adjunktus, Ph.D., Pharm.D., SZTE GYTK, Gyógyszeranalitiai Intézetgerda.szakonyi@pharm.u-szeged.hu
Dr. Oláh ZoltánTudományos főmunkatárs, Ph.D., SZTE GYTK, Gyógyszeranalitiai Intézetoz@acheuron.com
EPrint azonosító (ID): 1885
Publikációban használt név : Winter Zoltán
A mû MTMT azonosítója: 2852024
doi: 10.14232/phd.1885
A feltöltés ideje: 2013. júl. 23. 21:41
Utolsó módosítás: 2017. jún. 13. 14:44
Egyebek (raktári szám): B 5607
URI: http://doktori.bibl.u-szeged.hu/id/eprint/1885
Védés állapota: védett

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