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The mechanisms of Ca2+ signaling in human lens epithelial cells upon mechanical stimulation
ID Spasovska, Elena (Author), ID Andjelić, Sofija (Mentor) More about this mentor... This link opens in a new window, ID Gosak, Marko (Comentor)

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Abstract
Calcium (Ca2+) is a ubiquitous second messenger that controls diverse cellular functions. Human lens epithelial cells (LECs) are not exception in this respect, because Ca2+ signaling plays a major role in lens homeostasis and its disturbances are also related to cataract formation. The major goal of the present work is analysis, identification and comparison of intercellular Ca2+ signaling in human LECs upon local mechanical stimulation and local mechanical stimulation after applied antagonist’s apyrase (ATP diphosphohydrolase) and carbenoxolone (CBX). The purpose of added antagonists was to determine and better understand paracrine and gap junctional mechanisms in the intercellular aspect of LECs. In our experiments we used fresh postoperative lens capsules (LCs) obtained during cataract surgery and ex vivo explants. We mechanically stimulated each anterior LECs sample with a glass micropipette, which induced an increase in intracellular Ca2+ concentration ([Ca(2+)]i). Changes in [Ca2+]i in the cytosol were monitored using the fluorescent dye Fura-2. We observed the propagation on the Ca2+ signal in form of a wave that propagated radially from the stimulation point. Basically, amplitude of Ca2+ transients was found to decrease with increasing distance from the stimulation point. The Ca2+ signaling analysis in postoperative aLCs with added CBX has shown that the Ca2+ wave propagation is almost completely diminished since only cells nearby the simulation point get activated. In contrast, the application of apyrase showed little effect on the intercellular Ca2+ signaling in fresh postoperative LCs. Furthermore, we additionally studied Ca2+ signaling in ex vivo explant LC culture. It turned out that apyrase has more blocking effect on the spread of Ca2+ signal in ex vivo culture while CBX showed more efficiency in postoperative lenses, which suggests that these pharmacological agents could be a promising tool in regenerative medicine, repair, and lens regeneration.

Language:English
Keywords:lens epithelial cells, calcium signaling, intercellular communication, cataract, eye lens, cultures of lens epithelial cells, mechanical stimulation, apyrase, carbenoxolone, gap junction, paracrine mechanism
Work type:Master's thesis/paper
Organization:BF - Biotechnical Faculty
Year:2020
PID:20.500.12556/RUL-117796 This link opens in a new window
COBISS.SI-ID:26682883 This link opens in a new window
Publication date in RUL:26.07.2020
Views:1371
Downloads:172
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Secondary language

Language:Slovenian
Title:Mehanizmi signalizacije Ca2+ v človeških lečnih epitelnih celicah po mehanski stimulaciji
Abstract:
Kalcij (Ca2+) je vseprisotni sekundarni sporočevalec, ki nadzoruje različne celične funkcije. V tem pogledu epitelijske celice človeške leče (ECL) niso izjema, saj ima Ca2+ signalizacija pomembno vlogo pri homeostazi leče, motnje signalizacije Ca2+ pa povezujejo tudi s tvorbo sive mrene. Glavni cilj našega dela je bila identifikacija, analiza in primerjava medcelične signalizacije Ca2+ v človeških ECL, ob lokalni mehanski stimulaciji, brez in z dodatno apliciranima agonistoma apirazo (ATP difosfohidrolaza) in karbenoksolonom (CBX). Namen dodanih agonistov je bil določanje in boljše razumevanje parakrinega mehanizma in presledkovnih stikov pri komunikaciji med ECL. V našem poskusu smo uporabili sveže postoperativne lečne ovojnice (LO), pridobljene med operacijo katarakte, in primarne lečne kulture. ECL smo mehansko stimulirali s stekleno mikropipeto, kar je povzročilo povečanje znotrajcelične koncentracije Ca2+ ([Ca2+]i). Spremembe [Ca2+]i v citosolu smo spremljali s pomočjo fluorescentnega barvila Fura-2 in ugotovili, da se Ca2+ signal širi v obliki vala, radialno od točke stimulacije skozi celice v vidnem polju, ter da se amplitude [Ca2+]i zmanjšujejo s povečevanjem oddaljenosti od točke stimulacije. Analiza signalizacije Ca2+ v postoperativnih LO z dodanim CBX je pokazala, da je širjenje Ca2+ valov skoraj popolnoma onemogočeno, saj se aktivirajo samo celice v bližini stimulacijske točke. Po drugi strani pa apiraza ni imela znatnega vpliva na medcelično signalizacijo Ca2+ pri svežih postoperativnih LO. Poleg tega smo dodatno preučevali signalizacijo Ca2+ v primarni lečni kulturi. V tem primeru se je izkazalo, da apiraza bolj zablokira širjenje Ca2+ signala med celicami lečne kulture, medtem ko CBX v primerjavi s postoperativno lečo tukaj učinkuje manj. Ti rezultati nakazujejo, da bi lahko bila preizkušana farmakološka agonista učinkovito orodje pri regenerativni medicini, popravilu in regeneraciji leč.

Keywords:lečne epitelijske celice, kalcijeva signalizacija, medcelična komunikacija, katarakta, očesna leča, kulture epitelijskih celic leče, mehanska stimulacija, apiraza, karbenoksolon, presledkovni stiki, parakrini mehanizem

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