Fiziološko prilagajanje občutljivosti aparata za zlivanje sekretornih mešičkov na kalcijeve ione celic beta trebušne slinavke

doktorska disertacija

Maša Skelin (Author), Marjan Rupnik (Mentor), Ivan Krajnc (Thesis defence commission member)

Abstract

Raziskovali smo vlogo proteinskih kinaz v procesu uravnavane eksocitoze v celicah beta trebušne slinavke. Za stimulacijo od Ca2+ odvisne sekrecije smo uporabili vlak depolarizacijskih pulzov ali počasno fotolizo, s katerima smo kontrolirali znotrajcelično aktivnost Ca2+ ionov. S pomočjo meritev kapacitivnosti celične membrane smo zasledovali eksocitozo, to je zlivanje sekretornih mešičkov s plazemsko membrano. Pri kontroli je zadostna sprememba Ca2+ sprožila vsaj dve fazi eksocitoze. Hitrost spremembe kapacitivnosti je v odvisnosti od [Ca2+]i razkrila saturacijsko kinetiko z visoko kooperativnostjo, pri čemer je bila polovica maksimalne hitrosti dosežena pri 2,9 ± 0,2 µM kalcija. Nato smo z dodajanjem cAMP ugotavljali vlogo PKA v procesu eksocitoze. Naši rezultati kažejo, da cAMP stimulira eksocitozo pri značilno nižji [Ca2+]i v primerjavi s kontrolo. Enake rezultate smo dobili, ko smo s 6-Phe-cAMP direktno stimulirali PKA, medtem ko aktivacija Epac2, ki je prav tako ena izmed poti delovanja cAMP, ni imela posebnega vpliva na spremembo občutljivosti sekretornega aparata. V nadaljevanju smo preučili še vlogo PKC in Cdk5 v procesu izločanja inzulina. Aktivacija PKC je znižala občutljivost prve faze eksocitoze v primerjavi z inhibicijo PKC, medtem ko je inhibicija Cdk5 zmanjšala hitrost zlivanja sekretornih mešičkov s plazemsko membrano, pri čemer je ostala občutljivost na [Ca2+]i nespremenjena. Ker literatura navaja, da PKC in Cdk5 fosforilirata vrsto proteinov, vključenih v mehanizem sekrecije, smo testirali vlogo Munc18-1, ki je po našem mnenju ena najbolj verjetnih fosforilacijskih tarč PKC in Cdk5. Povišana ekspresija PKC fosforilacijske mutante Munc18-1 je značilno znižala amplitudo prve faze eksocitoze in zvišala njeno občutljivost na [Ca2+]i. Amp1 je bila značilno nižja tudi pri povišani ekspresiji Cdk5 fosforilacijske mutante Munc18-1, vendar je bila njena občutljivost na [Ca2+]i tokrat značilno nižja. Prav tako je bila značilno nižja tudi hitrost zlivanja mešičkov s plazemsko membrano. Naši rezultati tako potrjujejo hipotezo, da cAMP v celicah beta spremeni občutljivost sekretornega aparata na Ca2+ ione z aktivacijo PKA. Tudi PKC in Cdk5 sta z delovanjem preko proteina Munc18-1 pomembna regulatorja eksocitoze, pri čemer je PKC pomembna za preprečevanje zlivanja mešičkov s plazemsko membrano pri nezadostni [Ca2+]i, Cdk5 pa sodeluje v pripravi mešičkov na zlitje.

Keywords

beta celice;eksocitoza;proteinske kinaze;občutljivost na kalcij;izločanje inzulina;

Data

Language:	Slovenian
Year of publishing:	2011
Source:	[Maribor
Typology:	2.08 - Doctoral Dissertation
Organization:	UM MF - Faculty of Medicine
Publisher:	M. Skelin]
UDC:	57
COBISS:	255650048
Views:	2103
Downloads:	225
Average score:	0 (0 votes)
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Other data

Secondary language:	English
Secondary title:	The physiological modulation of the sensitivity of the exocytotic vesicle fusion machinery to calcium in pancreatic beta cells
Secondary abstract:	In this study we characterize the role of protein kinases in insulin secretion. A train of depolarizing pulses or slow photo-release of caged Ca2+ were stimuli for the exocytotic activity. In controls, due to exocytosis after increased [Ca2+]i, the Cm change had typically two phases. We observed that the Ca2+-dependency of the rate of the first Cm change follows saturation kinetics with high cooperativity and half-maximal rate at 2.9 ± 0.2 µM. Next we tested the role of PKA in insulin secretion by using cAMP. Our results demonstrated that cAMP triggers Ca2+ -dependent exocytosis at significantly lower [Ca2+]i comparing to the control. The same results were obtained from 6-Phe-cAMP treated cells which directly activated PKA. On the other hand activation of Epac2, which is also one of the mechanisms through which cAMP is working, did not change the Ca2+ sensitivity. Next we tested the role of PKC and Cdk5 in the insulin secretion. PKA activation decreased the Ca2+ sensitivity of the exocytotic burst comparing to the PKA inhibition, while inhibition of Cdk5 changed only the rate of exocytosis whith no effect on the Ca2+ sensitivity. Since previous studies reported that PKC and Cdk5 phosphorylate various proteins involved in secretory machinery, we tested the role of Munc18-1 which is in our opinion one of the most suitable phosphorylation targets for PKC and Cdk5. Overexpression of Munc18-1 PKC phosphorylation mutant significantly decreased the amplitude while the Ca2+ sensitivity of the first phase of Cm change was significantly increased. The amp1 was again signifficantly decreased in cells with overexpressed Cdk5 phosphorylation mutant of Munc18-1. The Ca2+ sensitivity and the rate of the first phase of Cm change were also decreased. At the end we can confirm the hypothesis that cAMP changes the sensitivity of the secretory machinery to Ca2+ in pancreatic beta cells by acting through PKA. Furthermore, PKC and Cdk5 are through phosphorylation of Munc18-1 important regulators of exocytosis. While PKC is important for preventing the fusion of secretory granules with plasma membrane at insufficient [Ca2+]i, Cdk5 is involved in steps before fusion.
Secondary keywords:	beta cells;exocytosis;protein kinases;calcium sensitivity;insulin secretion;
URN:	URN:SI:UM:
Type (COBISS):	Dissertation
Thesis comment:	Univ. Maribor, Medicinska fak.
Pages:	VII, 118 f.
Keywords (UDC):	mathematics;natural sciences;naravoslovne vede;matematika;biological sciences in general;biologija;
ID:	12900