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1.
Calcium dependencies of regulated exocytosis in different endocrine cells
Jurij Dolenšek, Maša Skelin, Marjan Rupnik, 2011, review article

Abstract: Exocytotic machinery in neuronal and endocrine tissues is sensitive to changesin intracellular Ca2+ concentration. Endocrine cell models, that are most frequently used to study the mechanisms of regulated exocytosis, are pancreatic beta cells, adrenal chromaffin cells and pituitary cells. To reliably study the Ca2+ sensitivity in endocrine cells, accurate and fast determination of Ca2+ dependence in each tested cell is required. With slow photo-release it is possible to induce ramp-like increase in intracellular Ca2+ concentration ([Ca2+]i) that leads to a robust exocytotic activity. Slow increases in the [Ca2+]i revealed exocytotic phases with different Ca2+ sensitivities that have been largely masked in step-like flash photo-release experiments. Strikingly, in the cells of the three described model endocrine tissues (beta, chromaffin and melanotroph cells), distinct Ca2+ sensitivity ćclassesć of secretory vesicles have been observed: a highly Ca2+-sensitive, amedium Ca2+-sensitive and a low Ca2+- sensitive kinetic phase of secretory vesicle exocytosis. We discuss that a physiological modulation of a cellular activity, e.g. by activating cAMP/PKA transduction pathway, can switch the secretory vesicles between Ca2+ sensitivity classes. This significantly alterslate steps in the secretory release of hormones even without utilizationof an additional Ca2+ sensor protein.
Keywords: Calcium sensitivity, Exocytosis, Insulin-secreting cells, Chromaffin cells, Melanotrophs
Published: 05.06.2012; Views: 764; Downloads: 14
URL Full text (0,00 KB)

2.
FIZIOLOŠKO PRILAGAJANJE OBČUTLJIVOSTI APARATA ZA ZLIVANJE SEKRETORNIH MEŠIČKOV NA KALCIJEVE IONE CELIC BETA TREBUŠNE SLINAVKE
Maša Skelin, 2011, dissertation

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
Published: 19.03.2013; Views: 1372; Downloads: 112
.pdf Full text (13,49 MB)

3.
Cell physiology in tissue slices
Maša Skelin, Andraž Stožer, Jurij Dolenšek, Marjan Rupnik, 2013, review article

Abstract: Uporaba izoliranih endokrinih celic je omogočila razvoj številnih metod za oceno osnovne celične arhitekture in opredelitev pomembnega dela molekularnih mehanizmov, ki sodelujejo pri celičnih procesih, kot sta vzdražnost celic in izločanje hormonov v procesu eksocitoze po porastu znotrajcelične koncentracije kalcijevih ionov. Ta prizadevanja so vodila v oblikovanje dogovornih modelov, ki razlagajo aktivacijo in splošen način delovanja določenega celičnega tipa. Z uporabo svežih tkivnih rezin lahko presežemo dosedanje raziskave in razkrijemo celo vrsto porajajočih se lastnosti, ki jih dogovorni modeli ne morejo zlahka predvideti niti razložiti. V tem članku predstavljamo izbor naših najpomembnejših eksperimentalnih ugotovitev na celicah beta v tkivnih rezinah trebušne slinavke.
Keywords: celica beta, Langerhansov otoček, tkivna rezina trebušne slinavke miši, elektrofiziologija, konfokalna fluorescenčna mikroskopija, kalcijevo barvilo, teorija grafov
Published: 10.07.2015; Views: 395; Downloads: 23
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4.
Breeding of laboratory mice for biomedical research
Maša Skelin, Marjan Rupnik, Marko Volk, 2010, original scientific article

Keywords: mouse breeding, husbandry, animal welfare, identification
Published: 10.07.2015; Views: 273; Downloads: 4
URL Full text (0,00 KB)

5.
Intracellular serotonin modulates insulin secretion from pancreatic ß-cells by protein serotonylation
Nils Paulmann, Maik Grohmann, Jörg-Peter Voigt, Bettina Bert, Jakob Vowinckel, Michael Bader, Maša Skelin, Marko Jevšek, Heidrun Fink, Marjan Rupnik, Diego Walther, 2009, original scientific article

Abstract: While serotonin (5-HT) co-localization with insulin in granules of pancreatic ß-cells was demonstrated more than three decades ago, its physiological role in the etiology of diabetes is stili unclear. We combined biochemical and electrophysiological analyses of mice selectively deficient in peripheral tryptophan hydroxylase (Tph1-/-) and 5-HT to show that intracellular 5-HT regulates insulin secretion. We found that these mice are diabetic and have an impaired insulin secretion due to the lack of 5-HT in the pancreas. The pharmacological restoration of peripheral 5-HT levels rescued the impaired insulin secretion in vivo. These findings were further evidenced by patch clamp experiments with isolated Tph1-/- ß-cells, which clearly showed that the secretory defect is downstream of Ca2+ -signaling and can be rescued by direct intracellular application of 5-HT via the clamp pipette. In elucidating the underlying mechanism further, we demonstrate the covalent coupling of 5-HT by transglutaminases during insulin exocytosis to two key players in insulin secretion, the small GTPases Rab3a and Rab27a. This renders them constitutively active in a receptor-independent signaling mechanism we have recently termed serotonylation. Concordantly, an inhibition of such activating serotonylation in ß-cells abates insulin secretion. We also observed inactivation of serotonylated Rab3a by enhanced proteasomal degradation, which is in line with the inactivation of other serotonylated GTPases. Our results demonstrate that 5-HT regulates insulin secretion by serotonylation of GTPases within pancreatic ß-cells and suggest that intracellular 5-HT functions in various microenvironments via this mechanism in concert with the known receptor-mediated signaling.
Keywords: insulin secretion, serotonin, insulin, glucose, diabetes mellitus, guanosine triphosphatase, exocytosis, pancreas
Published: 16.06.2017; Views: 230; Downloads: 18
.pdf Full text (774,75 KB)

6.
Membrane potential and calcium dynamics in beta cells from mouse pancreas tissue slices
Marjan Rupnik, Andraž Stožer, Jurij Dolenšek, Borut Žalik, Maša Skelin, Marko Gosak, Denis Špelič, 2015, review article

Abstract: Beta cells in the pancreatic islets of Langerhans are precise biological sensors for glucose and play a central role in balancing the organism between catabolic and anabolic needs. A hallmark of the beta cell response to glucose are oscillatory changes of membrane potential that are tightly coupled with oscillatory changes in intracellular calcium concentration which, in turn, elicit oscillations of insulin secretion. Both membrane potential and calcium changes spread from one beta cell to the other in a wave-like manner. In order to assess the properties of the abovementioned responses to physiological and pathological stimuli, the main challenge remains how to effectively measure membrane potential and calcium changes at the same time with high spatial and temporal resolution, and also in as many cells as possible. To date, the most wide-spread approach has employed the electrophysiological patch-clamp method to monitor membrane potential changes. Inherently, this technique has many advantages, such as a direct contact with the cell and a high temporal resolution. However, it allows one to assess information from a single cell only. In some instances, this technique has been used in conjunction with CCD camera-based imaging, offering the opportunity to simultaneously monitor membrane potential and calcium changes, but not in the same cells and not with a reliable cellular or subcellular spatial resolution. Recently, a novel family of highly-sensitive membrane potential reporter dyes in combination with high temporal and spatial confocal calcium imaging allows for simultaneously detecting membrane potential and calcium changes in many cells at a time. Since the signals yielded from both types of reporter dyes are inherently noisy, we have developed complex methods of data denoising that permit for visualization and pixel-wise analysis of signals. Combining the experimental approach of high-resolution imaging with the advanced analysis of noisy data enables novel physiological insights and reassessment of current concepts in unprecedented detail.
Keywords: calcium sensors, membrane potential sensors, calcium imaging, membrane potential imaging, beta cell, pancreas, denoising, patch-clamp
Published: 22.06.2017; Views: 187; Downloads: 22
.pdf Full text (4,17 MB)

7.
Rab3a is critical for trapping alpha-MSH granules in the high Ca2+-affinity pool by preventing constitutive exocytosis
Simon Sedej, Maša Skelin, Oliver Schlüter, Marjan Rupnik, 2013, original scientific article

Abstract: Rab3a is a small GTPase of the Rab3 subfamily that acts during late stages of Ca2+-regulated exocytosis. Previous functional analysis in pituitary melanotrophs described Rab3a as a positive regulator of Ca2+-dependent exocytosis. However, the precise role of the Rab3a isoform on the kinetics and intracellular [Ca2+] sensitivity of regulated exocytosis, which may affect the availability of two major peptide hormones, -melanocyte stimulating hormone (-MSH) and -endorphin in plasma, remain elusive. We employed Rab3a knock-out mice (Rab3a KO) to explore the secretory phenotype in melanotrophs from fresh pituitary tissue slices. High resolution capacitance measurements showed that Rab3a KO melanotrophs possessed impaired Ca2+-triggered secretory activity as compared to wild-type cells. The hampered secretion was associated with the absence of cAMP-guanine exchange factor II/ Epac2-dependent secretory component. This component has been attributed to high Ca2+-sensitive release-ready vesicles as determined by slow photo-release of caged Ca2+. Radioimmunoassay revealed that -MSH, but not -endorphin, was elevated in the plasma of Rab3a KO mice, indicating increased constitutive exocytosis of -MSH. Increased constitutive secretion of -MSH from incubated tissue slices was associated with reduced -MSH cellular content in Rab3a-deficient pituitary cells. Viral re-expression of the Rab3a protein in vitro rescued the secretory phenotype of melanotrophs from Rab3a KO mice. In conclusion, we suggest that Rab3a deficiency promotes constitutive secretion and underlies selective impairment of Ca2+-dependent release of -MSH.
Keywords: melanotrophs, exocytosis
Published: 19.06.2017; Views: 177; Downloads: 20
.pdf Full text (1,90 MB)

8.
The relationship between membrane potential and calcium dynamics in glucose-stimulated beta cell syncytium in acute mouse pancreas tissue slices
Jurij Dolenšek, Andraž Stožer, Maša Skelin, Evan Miller, Marjan Rupnik, 2013, original scientific article

Abstract: Oscillatory electrical activity is regarded as a hallmark of the pancreatic beta cell glucose-dependent excitability pattern. Electrophysiologically recorded membrane potential oscillations in beta cells are associated with in-phase oscillatory cytosolic calcium activity ([Ca2+]i) measured with fluorescent probes. Recent high spatial and temporal resolution confocal imaging revealed that glucose stimulation of beta cells in intact islets within acute tissue slices produces a [Ca2+]i change with initial transient phase followed by a plateau phase with highly synchronized [Ca2+]i oscillations. Here, we aimed to correlate the plateau [Ca2+]i oscillations with the oscillations of membrane potential using patch-clamp and for the first time high resolution voltage-sensitive dye based confocal imaging. Our results demonstrated that the glucose-evoked membrane potential oscillations spread over the islet in a wave-like manner, their durations and wave velocities being comparable to the ones for [Ca2+]i oscillations and waves. High temporal resolution simultaneous records of membrane potential and [Ca2+]i confirmed tight but nevertheless limited coupling of the two processes, with membrane depolarization preceding the [Ca2+]i increase. The potassium channel blocker tetraethylammonium increased the velocity at which oscillations advanced over the islet by several-fold while, at the same time, emphasized differences in kinetics of the membrane potential and the [Ca2+]i. The combination of both imaging techniques provides a powerful tool that will help us attain deeper knowledge of the beta cell network.
Keywords: glucose, pancreas, mice
Published: 19.06.2017; Views: 202; Downloads: 27
.pdf Full text (2,57 MB)

9.
SNAP-25b-deficiency increases insulin secretion and changes spatiotemporal profile of $Ca^{2+}$ oscillations in $\beta$ cell networks
Teresa Daraio, Lidija Križančić Bombek, Marko Gosak, Ismael Valladolid-Acebes, Maša Skelin, Essam Refai, Per-Olof Berggren, Kerstin Brismar, Marjan Rupnik, Christina Bark, 2017, original scientific article

Abstract: SNAP-25 is a protein of the core SNARE complex mediating stimulus-dependent release of insulin from pancreatic $\beta$ cells. The protein exists as two alternatively spliced isoforms, SNAP-25a and SNAP-25b, differing in 9 out of 206 amino acids, yet their specific roles in pancreatic $\beta$ cells remain unclear. We explored the effect of SNAP-25b-deficiency on glucose-stimulated insulin release in islets and found increased secretion both in vivo and in vitro. However, slow photo-release of caged $Ca^{2+}$ in $\beta$ cells within pancreatic slices showed no significant differences in $Ca^{2+}$-sensitivity, amplitude or rate of exocytosis between SNAP-25b-deficient and wild-type littermates. Therefore, we next investigated if $Ca^{2+}$ handling was affected in glucose-stimulated [beta] cells using intracellular $Ca^{2+}$-imaging and found premature activation and delayed termination of [$Ca^{2+}$] i elevations. These findings were accompanied by less synchronized $Ca^{2+}$-oscillations and hence more segregated functional $\beta$ cell networks in SNAP-25b-deficient mice. Islet gross morphology and architecture were maintained in mutant mice, although sex specific compensatory changes were observed. Thus, our study proposes that SNAP-25b in pancreatic [beta] cells, except for participating in the core SNARE complex, is necessary for accurate regulation of $Ca^{2+}$-dynamics.
Keywords: insulin secretion, pre-diabetes
Published: 23.08.2017; Views: 166; Downloads: 25
.pdf Full text (3,80 MB)

10.
Vpliv aerobne vadbe na vzdržljivost otrok
Katja Simončič, 2018, undergraduate thesis

Abstract: Diplomsko delo z naslovom Vpliv aerobne vadbe na vzdržljivost otrok je razdeljeno na teoretični in empirični del. V teoretičnem delu smo pojasnili izhodišča o vplivu aerobne vadbe na vzdržljivost otrok. Skozi poglavja smo predstavili pojem vzdržljivosti, merjenje vzdržljivosti, vrste aerobne vadbe in načine za izboljšanje aerobne vzdržljivosti pri predšolskih otrocih. V empiričnem delu smo predstavili namen in cilje naše raziskave. Predstavili smo potek eksperimenta in dobljene rezultate oziroma ugotovitve, do katerih smo prišli na podlagi izvajanja osemnajstih vadbenih enot v časovnem obdobju dveh mesecev. Naš namen je bil ugotoviti vpliv aerobne vadbe na vzdržljivost otrok. Vpliv smo proučevali tako, da smo poleg eksperimentalne skupine v raziskavo vključili še kontrolno skupino, s katero programa redne aerobne vadbe nismo izvajali. V kvantitativni raziskavi so sodelovali otroci stari od 4 let in pol do 6 let iz vrtca Cerkvenjak (eksperimentalna skupina) in otroci vrtca Vitomarci (kontrolna skupina). Meritve smo opravili s testom 20-metrskega stopnjevalnega teka pred začetkom izvajanja in po končanem izvajanju programa aerobne vadbe; pri eksperimentalni skupini smo opravili še vmesno meritev. Zbrane podatke smo kvantitativno obdelali z računalniškim programom SPSS na nivoju deskriptivne statistike. Rezultate smo predstavili v preglednicah, iz katerih smo lahko razbrali, da aerobna vadba pozitivno vpliva na vzdržljivost otrok. Vzdržljivost se ob redni aerobni vadbi namreč povečuje, saj so otroci eksperimentalne skupine dosegli velik napredek od začetnega do končnega stanja izvajanja vadbe v nasprotju s kontrolno skupino, s katero nismo izvajali programa redne aerobne vadbe. Na podlagi naše raziskave smo ugotovili, da ima redna aerobna vadba dober vpliv na otroke, saj povečuje njihovo vzdržljivost.
Keywords: aerobna vadba, vpliv vadbe, vzdržljivost, aerobna vzdržljivost, predšolski otroci
Published: 25.09.2018; Views: 5; Downloads: 4
.pdf Full text (1,06 MB)

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