Marjan Slak Rupnik | Medical University of Vienna (original) (raw)
Papers by Marjan Slak Rupnik
PLoS ONE, 2013
Oscillatory electrical activity is regarded as a hallmark of the pancreatic beta cell glucose-dep... more 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 ([Ca 2+ ] 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 [Ca 2+ ] i change with initial transient phase followed by a plateau phase with highly synchronized [Ca 2+ ] i oscillations. Here, we aimed to correlate the plateau [Ca 2+ ] 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 [Ca 2+ ] i oscillations and waves. High temporal resolution simultaneous records of membrane potential and [Ca 2+ ] i confirmed tight but nevertheless limited coupling of the two processes, with membrane depolarization preceding the [Ca 2+ ] 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 [Ca 2+ ] i . The combination of both imaging techniques provides a powerful tool that will help us attain deeper knowledge of the beta cell network. Citation: Dolenšek J, Stožer A, Skelin Klemen M, Miller EW, Slak Rupnik M (2013) The Relationship between Membrane Potential and Calcium Dynamics in Glucose-Stimulated Beta Cell Syncytium in Acute Mouse Pancreas Tissue Slices. PLoS ONE 8(12): e82374.
PLoS ONE, 2013
Rab3a is a small GTPase of the Rab3 subfamily that acts during late stages of Ca 2+ -regulated ex... more Rab3a is a small GTPase of the Rab3 subfamily that acts during late stages of Ca 2+ -regulated exocytosis. Previous functional analysis in pituitary melanotrophs described Rab3a as a positive regulator of Ca 2+ -dependent exocytosis. However, the precise role of the Rab3a isoform on the kinetics and intracellular [Ca 2+ ] sensitivity of regulated exocytosis, which may affect the availability of two major peptide hormones, α-melanocyte stimulating hormone (α-MSH) and β-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 Ca 2+ -triggered secretory activity as compared to wild-type cells. The hampered secretion was associated with the absence of cAMP-guanine exchange factor II/ Epac2dependent secretory component. This component has been attributed to high Ca 2+ -sensitive release-ready vesicles as determined by slow photo-release of caged Ca 2+ . Radioimmunoassay revealed that α-MSH, but not β-endorphin, was elevated in the plasma of Rab3a KO mice, indicating increased constitutive exocytosis of α-MSH. Increased constitutive secretion of α-MSH from incubated tissue slices was associated with reduced α-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 Ca 2+ -dependent release of α-MSH. Citation: Sedej S, Klemen MS, Schlüter OM, Rupnik MS (2013) Rab3a Is Critical for Trapping Alpha-MSH Granules in the High Ca 2+ -Affinity Pool by Preventing Constitutive Exocytosis. PLoS ONE 8(10): e78883.
Neuromethods, 2013
A number of approaches have been developed during the last decades to assess exocytosis in neuron... more A number of approaches have been developed during the last decades to assess exocytosis in neuronal, endocrine, and other secretory cells. A detailed description of a selection of the methods that have been adapted to study regulated exocytosis in endocrine tissue slices is provided in this chapter. Such adaptation of the methods has been associated with a plethora of open issues that required innovative solutions, but innovation has been and shall be in the future plentifully rewarded by discoveries of novel, often emergent principles of cells' operation within an intact tissue. The use of tissue slices has enabled us to reduce negative effects of cell culturing. It helped us circumvent serious experimental challenges associated with the small size and enhanced sensitivity of embryonic endocrine cells from wild-type animals and those lacking important functional proteins and thus associated with perinatal lethality. More recent methods to study exocytosis specifically in the intact tissues include polar tracers and lipophilic dyes that can be used to directly visualize exocytosis with multiphoton excitation microscopy. A hallmark of the microscopic approach is that it offers positional information and the exocytic process can simultaneously be studied in a large number of cells. Optical approaches alone and in combination with electrophysiology hold promise for future excitement about the mechanism of regulated exocytosis.
Journal of Biological Chemistry, 2011
Fast neurotransmission and slower hormone release share the same core fusion machinery consisting... more Fast neurotransmission and slower hormone release share the same core fusion machinery consisting of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins. In evoked neurotransmission, interactions between SNAREs and the Munc18-1 protein, a member of the Sec1/Munc18 (SM) protein family, are essential for exocytosis, whereas other SM proteins are dispensable. To address if the exclusivity of Munc18-1 demonstrated in neuroexocytosis also applied to fast insulin secretion, we characterized the presence and function of Munc18-1 and its closest homologue Munc18-2 in β-cell stimulus-secretion coupling. We show that pancreatic β-cells express both Munc18-1 and Munc18-2. The two Munc18 homologues exhibit different subcellular localization, and only Munc18-1 redistributes in response to glucose stimulation. However, both Munc18-1 and Munc18-2 augment glucose-stimulated hormone release. Ramp-like photorelease of caged Ca(2+) and high resolution whole-cell patch clamp recordings show that Munc18-1 and Munc18-2 overexpression shift the Ca(2+) sensitivity of the fastest phase of insulin exocytosis differently. In addition, we reveal that Ca(2+) sensitivity of exocytosis in β-cells depends on the phosphorylation status of the Munc18 proteins. Even though Munc18-1 emerges as the key SM-protein determining the Ca(2+) threshold for triggering secretory activity in a stimulated β-cell, Munc18-2 has the ability to increase Ca(2+) sensitivity and thus mediates the release of fusion-competent granules requiring a lower cytoplasmic-free Ca(2+) concentration, [Ca(2+)](i)(.) Hence, Munc18-1 and Munc18-2 display distinct subcellular compartmentalization and can coordinate the insulin exocytotic process differently as a consequence of the actual [Ca(2+)](i).
Isolated endocrine cells have enabled the development of numerous methods to assess basic cellula... more Isolated endocrine cells have enabled the development of numerous methods to assess basic cellular architecture and define a significant part of the molecular machinery involved in cellular processes, such as excitability or the exocytotic release of hormones following cytosolic calcium increase. These efforts have generated consensus models explaining the activation or general operation of a particular cell type. The use of fresh tissue slices can go even further and uncover a series of emergent properties never explained or predicted by consensus models. In this paper, we present a selection of our most important experimental findings in the beta cells of pancreatic tissue slices.
PloS one, 2015
Regeneration of skeletal muscle after injury is limited by scar formation, slow healing time and ... more Regeneration of skeletal muscle after injury is limited by scar formation, slow healing time and a high recurrence rate. A therapy based on platelet-rich plasma (PRP) has become a promising lead for tendon and ligament injuries in recent years, however concerns have been raised that PRP-derived TGF-β could contribute to fibrotic remodelling in skeletal muscle after injury. Due to the lack of scientific grounds for a PRP -based muscle regeneration therapy, we have designed a study using human myogenic progenitors and evaluated the potential of PRP alone and in combination with decorin (a TGF-β inhibitor), to alter myoblast proliferation, metabolic activity, cytokine profile and expression of myogenic regulatory factors (MRFs). Advanced imaging multicolor single-cell analysis enabled us to create a valuable picture on the ratio of quiescent, activated and terminally committed myoblasts in treated versus control cell populations. Finally high-resolution confocal microscopy validated th...
Neuroreport, Jan 19, 1995
We investigated the role of ADP-ribosylation factor (ARF) in regulated exocytosis in patch-clampe... more We investigated the role of ADP-ribosylation factor (ARF) in regulated exocytosis in patch-clamped rat melanotrophs. Addition of brefeldin A (BFA) to inhibit activation of endogenous ARF protein was found to attenuate regulated secretory activity monitored as changes in membrane capacitance (Cm). A synthetic peptide to amino acids 46-61 of ARF (P-14) was also found to inhibit Ca(2+)-induced secretory activity in these cells. This inhibition was not apparent with a scrambled amino acid sequence of ARF-P14 peptide. This paper provides the first patch-clamp study to suggest that the small GTP-binding protein ARF is required to trigger release of secretory granules from rat pituitary melanotrophs.
Nature medicine, Jan 16, 2015
In the nervous system, NMDA receptors (NMDARs) participate in neurotransmission and modulate the ... more In the nervous system, NMDA receptors (NMDARs) participate in neurotransmission and modulate the viability of neurons. In contrast, little is known about the role of NMDARs in pancreatic islets and the insulin-secreting beta cells whose functional impairment contributes to diabetes mellitus. Here we found that inhibition of NMDARs in mouse and human islets enhanced their glucose-stimulated insulin secretion (GSIS) and survival of islet cells. Further, NMDAR inhibition prolonged the amount of time that glucose-stimulated beta cells spent in a depolarized state with high cytosolic Ca(2+) concentrations. We also noticed that, in vivo, the NMDAR antagonist dextromethorphan (DXM) enhanced glucose tolerance in mice, and that in vitro dextrorphan, the main metabolite of DXM, amplified the stimulatory effect of exendin-4 on GSIS. In a mouse model of type 2 diabetes mellitus (T2DM), long-term treatment with DXM improved islet insulin content, islet cell mass and blood glucose control. Furthe...
Clostridium difficile toxin B (TcdB) is a single-stranded protein consisting of a C-terminal doma... more Clostridium difficile toxin B (TcdB) is a single-stranded protein consisting of a C-terminal domain responsible for binding to the host cell membrane, a middle part involved in internalization, and the N-terminal catalytic (toxic) part. This study shows that TcdB is processed by a single proteolytic step which cleaves TcdB 10463 between Leu 543 and Gly 544 and the naturally occurring variant TcdB 8864 between Leu 544 and Gly 545 . The cleavage occurs at neutral pH and is catalysed by a pepstatin-sensitive protease localized in the cytoplasm and on the cytoplasmic face of intracellular membranes. The smaller N-terminal cleavage products [63 121 Da (TcdB 10463 ) and 62 761 Da (TcdB 8864 )] harbour the cytotoxic and glucosyltransferase activities of the toxins. When microinjected into cultured Chinese hamster lung fibroblasts, the N-terminal cleavage fragment shows full cytotoxic activity shortly after injection whereas the holotoxin initially exhibits a very low activity which, however, increases with time. Twenty minutes after the start of internalization of TcdB, the larger cleavage products [206 609 Da (TcdB 10463 ) and 206 245 Da (TcdB 8864 )] are found exclusively in a membrane fraction, whereas the N-terminal cleavage products appear mainly in the cytosol and associated with the membrane. This is in line with a proposed model according to which the longer, C-terminal, part of these toxins forms a channel allowing for the translocation of the toxic N-terminal part, which is subsequently cleaved off at the cytoplasmic face of an intracellular compartment, most likely endosomes.
Alpha-neurexins constitute a family of neuronal cell surface molecules that are essential for eff... more Alpha-neurexins constitute a family of neuronal cell surface molecules that are essential for efficient neurotransmission, because mice lacking two or all three alpha-neurexin genes show a severe reduction of synaptic release. Although analyses of alpha-neurexin knock-outs and transgenic rescue animals suggested an involvement of voltage-dependent Ca2+ channels, it remained unclear whether alpha-neurexins have a general role in Ca2+-dependent exocytosis and how they may affect Ca2+ channels. Here we show by membrane capacitance measurements from melanotrophs in acute pituitary gland slices that release from endocrine cells is diminished by >50% in adult alpha-neurexin double knock-out and newborn triple knock-out mice. There is a reduction of the cell volume in mutant melanotrophs; however, no ultrastructural changes in size or intracellular distribution of the secretory granules were observed. Recordings of Ca2+ currents from melanotrophs, transfected human embryonic kidney cells, and brainstem neurons reveal that alpha-neurexins do not affect the activation or inactivation properties of Ca2+ channels directly but may be responsible for coupling them to release-ready vesicles and metabotropic receptors. Our data support a general and essential role for alpha-neurexins in Ca2+-triggered exocytosis that is similarly important for secretion from neurons and endocrine cells.
PLoS Biology, 2009
While serotonin (5-HT) co-localization with insulin in granules of pancreatic b-cells was demonst... more While serotonin (5-HT) co-localization with insulin in granules of pancreatic b-cells was demonstrated more than three decades ago, its physiological role in the etiology of diabetes is still unclear. We combined biochemical and electrophysiological analyses of mice selectively deficient in peripheral tryptophan hydroxylase (Tph12/2) 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 Tph12/2 b-cells, which clearly showed that the secretory defect is downstream of Ca 2+ -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 b-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 b-cells and suggest that intracellular 5-HT functions in various microenvironments via this mechanism in concert with the known receptor-mediated signaling.
Scientific Reports, 2015
Collective beta cell activity in islets of Langerhans is critical for the supply of insulin withi... more Collective beta cell activity in islets of Langerhans is critical for the supply of insulin within an organism. Even though individual beta cells are intrinsically heterogeneous, the presence of intercellular coupling mechanisms ensures coordinated activity and a well-regulated exocytosis of insulin. In order to get a detailed insight into the functional organization of the syncytium, we applied advanced analytical tools from the realm of complex network theory to uncover the functional connectivity pattern among cells composing the intact islet. The procedure is based on the determination of correlations between long temporal traces obtained from confocal functional multicellular calcium imaging of beta cells stimulated in a stepwise manner with a range of physiological glucose concentrations. Our results revealed that the extracted connectivity networks are sparse for low glucose concentrations, whereas for higher stimulatory levels they become more densely connected. Most importantly, for all ranges of glucose concentration beta cells within the islets form locally clustered functional sub-compartments, thereby indicating that their collective activity profiles exhibit a modular nature. Moreover, we show that the observed non-linear functional relationship between different network metrics and glucose concentration represents a well-balanced setup that parallels physiological insulin release.
The Journal of Physiology, 2005
Clions are known regulators of Ca 2+ -dependent secretory activity in many endocrine cells. The s... more Clions are known regulators of Ca 2+ -dependent secretory activity in many endocrine cells. The suggested mechanisms of Claction involve the modulation of GTP-binding proteins, voltage-activated calcium channels or maturation of secretory vesicles. We examined the role of cytosolic Cl -([Cl -] i ) and Clcurrents in the regulation of secretory activity in mouse melanotrophs from fresh pituitary tissue slices by using the whole-cell patch-clamp. We confirmed that elevated [Cl -] i augments Ca 2--dependent exocytosis and showed that Clacts on secretory vesicle maturation. The latter process was abolished by a V-type H --ATPase blocker (bafilomycin), intracellular 4,4 -diisothiocyanatostilbene-2,2 -disulphonic acid (DIDS), a Clchannel blocker, and tolbutamide, a sulphonylurea implicated in secretory vesicle maturation. In a small subset of cells, block of plasmalemmal Clcurrent by DIDS reversibly enhanced endocytosis. The direct activation of G-proteins by GTP-γ-S, a non-hydrolysable GTP analogue, did not restore the impaired secretion observed in low [Cl -] i conditions. The amplitude of voltage-activated calcium currents was unaffected by the [Cl -] i . Furthermore, two Cl --permeable channels, calcium-activated Clchannels and GABA A receptors, appeared as major regulators of intracellular Clhomeostasis. In conclusion, the predominant underlying mechanism of Claction is mediated by intracellular Clfluxes during vesicle maturation, rather than activation of G-proteins or modulation of voltage-activated Ca 2+ channels.
The Journal of Physiology, 2004
We have prepared fresh pituitary gland slices from adult and, for the first time, from newborn mi... more We have prepared fresh pituitary gland slices from adult and, for the first time, from newborn mice to assess modulation of secretory activity via voltage-activated Ca(2+) channels (VACCs). Currents through VACCs and membrane capacitance have been measured with the whole-cell patch-clamp technique. Melanotrophs in newborns were significantly larger than in adults. In both newborn and adult melanotrophs activation of VACCs triggered exocytosis. All pharmacologically isolated VACC types contributed equally to the secretory activity. However, the relative proportion of VACCs differed between newborns and adults. In newborn cells L-type channels dominated and, in addition, an exclusive expression of a toxin-resistant R-type-like current was found. The expression of L-type VACCs was up-regulated by the increased oestrogen levels observed in females, and was even more emphasized in the cells of pregnant females and oestrogen-treated adult male mice. We suggest a general mechanism modulating endocrine secretion in the presence of oestrogen and particularly higher sensitivity to treatments with L-type channel blockers during high oestrogen physiological states.
The International Journal of Developmental Biology, 2004
We present a new strategy for the differentiation of embryonic stem (ES) cells into insulin-produ... more We present a new strategy for the differentiation of embryonic stem (ES) cells into insulin-producing cells via a multi-step process without selection and induction of nestin-positive cells. During ES cell differentiation, transcript levels of genes characteristic of early and mature beta cells including Pdx1, Pax4, insulin and islet amyloid pancreatic peptide are up regulated. Islet-like clusters are characterized by expression of C-peptide, insulin and partially cytokeratin 19 as well as by ion channel activity similar to that found in embryonic beta cells. Cells of islet-like clusters show glucose-dependent insulin release at terminal stage. At an intermediate stage, nestin is partially co-expressed with C-peptide and cytokeratin 19, whereas islet-like clusters at the terminal stage are nestin-negative. We conclude that expression of nestin and cytokeratin 19 is a normal property of ES cells preceding differentiation into C-peptide/insulin-producing cells without any selection for nestin-positive phenotypes.
Proceedings of the National Academy of Sciences, 2000
Although many proteins essential for regulated neurotransmitter and peptide hormone secretion hav... more Although many proteins essential for regulated neurotransmitter and peptide hormone secretion have been identified, little is understood about their precise roles at specific stages of the multistep pathway of exocytosis. To study the function of CAPS (Ca 2؉ -dependent activator protein for secretion), a protein required for Ca 2؉ -dependent exocytosis of dense-core vesicles, secretory responses in single rat melanotrophs were monitored by patch-clamp membrane capacitance measurements. Flash photolysis of caged Ca 2؉ elicited biphasic capacitance increases consisting of rapid and slow components with distinct Ca 2؉ dependencies. A threshold of Ϸ10 M Ca 2؉ was required to trigger the slow component, while the rapid capacitance increase was recorded already at a intracellular Ca 2؉ activity < 10 M. Both kinetic membrane capacitance components were abolished by botulinum neurotoxin B or E treatment, suggesting involvement of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)-dependent vesicle fusion. The rapid but not the slow component was inhibited by CAPS antibody. These results were further clarified by immunocytochemical studies that revealed that CAPS was present on only a subset of dense-core vesicles. Overall, the results indicate that dense-core vesicle exocytosis in melanotrophs occurs by two parallel pathways. The faster pathway exhibits high sensitivity to Ca 2؉ and requires the presence of CAPS, which appears to act at a late stage in the secretory pathway.
PLoS ONE, 2014
Gold nanoparticles (GNPs) are claimed as outstanding biomedical tools for cancer diagnostics and ... more Gold nanoparticles (GNPs) are claimed as outstanding biomedical tools for cancer diagnostics and photo-thermal therapy, but without enough evidence on their potentially adverse immunological effects. Using a model of human dendritic cells (DCs), we showed that 10 nm-and 50 nm-sized GNPs (GNP 10 and GNP 50 , respectively) were internalized predominantly via dynamin-dependent mechanisms, and they both impaired LPS-induced maturation and allostimulatory capacity of DCs, although the effect of GNP 10 was more prominent. However, GNP 10 inhibited LPS-induced production of IL-12p70 by DCs, and potentiated their Th2 polarization capacity, while GNP 50 promoted Th17 polarization. Such effects of GNP 10 correlated with a stronger inhibition of LPS-induced changes in Ca 2+ oscillations, their higher number per DC, and more frequent extraendosomal localization, as judged by live-cell imaging, proton, and electron microscopy, respectively. Even when released from heat-killed necrotic HEp-2 cells, GNP 10 inhibited the necrotic tumor cell-induced maturation and functions of DCs, potentiated their Th2/Th17 polarization capacity, and thus, impaired the DCs' capacity to induce T cell-mediated anti-tumor cytotoxicity in vitro. Therefore, GNP 10 could potentially induce more adverse DC-mediated immunological effects, compared to GNP 50 .
Planta, 1994
Enhanced elongation of coleoptile cells has been proposed to be related to a rise in secretory ac... more Enhanced elongation of coleoptile cells has been proposed to be related to a rise in secretory activity. Therefore, to obtain a direct measurement of exocytotic events in maize (Zea mays L.) coleoptile protoplasts we used the patch-clamp method to record changes in membrane capacitance (Cm) as a parameter proportional to fluctuations of the membrane surface area. The secretory activity of protoplasts was correlated with the cytosolic free Ca 2 + concentration ([Ca 2 +]cyt): dialyzing protoplasts with 1 laM [Ca2+]cy t caused a steady rise in Cm of 3.3_ pF-s 1. In contrast, dialysis with a solution containing <20 nM Ca 2+ produced a small and persistent decrease in Cm. This demonstrates that secretory activity in coleoptile cells can be controlled by factors which modulate [Ca2+]cy t.
Pfl�gers Archiv European Journal of Physiology, 2003
The tissue-slice technique has enabled major insights into neural and neuroendocrine physiology. ... more The tissue-slice technique has enabled major insights into neural and neuroendocrine physiology. Our aim was to adapt this technique to study the function of the endocrine pancreas. The preparation combines an in situ approach, as in gland perfusion, with a resolution characteristic of electrophysiological studies on single cells. The membrane potential in b-cells in the slices recorded using the whole-cell patch-clamp was close to the calculated reversal potential for K + . With sufficient ATP in the recording pipette the b-cells depolarized rapidly on exposure to an increased glucose concentration or stimulation with tolbutamide. The cells preserved bursting and spiking capacity for tens of minutes despite the whole-cell dialysis. In addition, the voltage clamp was used to monitor the changes in the membrane capacitance and to allow correlation of the electrical activity and the cytosolic calcium changes. The pancreatic tissue slice preparation is a novel method for studying the function of the b-and other pancreatic endocrine and exocrine cells under near-physiological conditions.
Pfl�gers Archiv European Journal of Physiology, 1995
... 3A, B Ca2+-dependent secretory response is attenuated by guanosine 5'-O-(2-thiodipho... more ... 3A, B Ca2+-dependent secretory response is attenuated by guanosine 5'-O-(2-thiodiphosphate),GDP[fi-S]. A Secretory responses recorded at 1 gM [Ca2+]i and 4 mM (top panel) and 154 mM (lower panel) [C1-]i in control cells and in the presence of GDP[fl-S] (500 gM). ...
PLoS ONE, 2013
Oscillatory electrical activity is regarded as a hallmark of the pancreatic beta cell glucose-dep... more 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 ([Ca 2+ ] 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 [Ca 2+ ] i change with initial transient phase followed by a plateau phase with highly synchronized [Ca 2+ ] i oscillations. Here, we aimed to correlate the plateau [Ca 2+ ] 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 [Ca 2+ ] i oscillations and waves. High temporal resolution simultaneous records of membrane potential and [Ca 2+ ] i confirmed tight but nevertheless limited coupling of the two processes, with membrane depolarization preceding the [Ca 2+ ] 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 [Ca 2+ ] i . The combination of both imaging techniques provides a powerful tool that will help us attain deeper knowledge of the beta cell network. Citation: Dolenšek J, Stožer A, Skelin Klemen M, Miller EW, Slak Rupnik M (2013) The Relationship between Membrane Potential and Calcium Dynamics in Glucose-Stimulated Beta Cell Syncytium in Acute Mouse Pancreas Tissue Slices. PLoS ONE 8(12): e82374.
PLoS ONE, 2013
Rab3a is a small GTPase of the Rab3 subfamily that acts during late stages of Ca 2+ -regulated ex... more Rab3a is a small GTPase of the Rab3 subfamily that acts during late stages of Ca 2+ -regulated exocytosis. Previous functional analysis in pituitary melanotrophs described Rab3a as a positive regulator of Ca 2+ -dependent exocytosis. However, the precise role of the Rab3a isoform on the kinetics and intracellular [Ca 2+ ] sensitivity of regulated exocytosis, which may affect the availability of two major peptide hormones, α-melanocyte stimulating hormone (α-MSH) and β-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 Ca 2+ -triggered secretory activity as compared to wild-type cells. The hampered secretion was associated with the absence of cAMP-guanine exchange factor II/ Epac2dependent secretory component. This component has been attributed to high Ca 2+ -sensitive release-ready vesicles as determined by slow photo-release of caged Ca 2+ . Radioimmunoassay revealed that α-MSH, but not β-endorphin, was elevated in the plasma of Rab3a KO mice, indicating increased constitutive exocytosis of α-MSH. Increased constitutive secretion of α-MSH from incubated tissue slices was associated with reduced α-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 Ca 2+ -dependent release of α-MSH. Citation: Sedej S, Klemen MS, Schlüter OM, Rupnik MS (2013) Rab3a Is Critical for Trapping Alpha-MSH Granules in the High Ca 2+ -Affinity Pool by Preventing Constitutive Exocytosis. PLoS ONE 8(10): e78883.
Neuromethods, 2013
A number of approaches have been developed during the last decades to assess exocytosis in neuron... more A number of approaches have been developed during the last decades to assess exocytosis in neuronal, endocrine, and other secretory cells. A detailed description of a selection of the methods that have been adapted to study regulated exocytosis in endocrine tissue slices is provided in this chapter. Such adaptation of the methods has been associated with a plethora of open issues that required innovative solutions, but innovation has been and shall be in the future plentifully rewarded by discoveries of novel, often emergent principles of cells' operation within an intact tissue. The use of tissue slices has enabled us to reduce negative effects of cell culturing. It helped us circumvent serious experimental challenges associated with the small size and enhanced sensitivity of embryonic endocrine cells from wild-type animals and those lacking important functional proteins and thus associated with perinatal lethality. More recent methods to study exocytosis specifically in the intact tissues include polar tracers and lipophilic dyes that can be used to directly visualize exocytosis with multiphoton excitation microscopy. A hallmark of the microscopic approach is that it offers positional information and the exocytic process can simultaneously be studied in a large number of cells. Optical approaches alone and in combination with electrophysiology hold promise for future excitement about the mechanism of regulated exocytosis.
Journal of Biological Chemistry, 2011
Fast neurotransmission and slower hormone release share the same core fusion machinery consisting... more Fast neurotransmission and slower hormone release share the same core fusion machinery consisting of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins. In evoked neurotransmission, interactions between SNAREs and the Munc18-1 protein, a member of the Sec1/Munc18 (SM) protein family, are essential for exocytosis, whereas other SM proteins are dispensable. To address if the exclusivity of Munc18-1 demonstrated in neuroexocytosis also applied to fast insulin secretion, we characterized the presence and function of Munc18-1 and its closest homologue Munc18-2 in β-cell stimulus-secretion coupling. We show that pancreatic β-cells express both Munc18-1 and Munc18-2. The two Munc18 homologues exhibit different subcellular localization, and only Munc18-1 redistributes in response to glucose stimulation. However, both Munc18-1 and Munc18-2 augment glucose-stimulated hormone release. Ramp-like photorelease of caged Ca(2+) and high resolution whole-cell patch clamp recordings show that Munc18-1 and Munc18-2 overexpression shift the Ca(2+) sensitivity of the fastest phase of insulin exocytosis differently. In addition, we reveal that Ca(2+) sensitivity of exocytosis in β-cells depends on the phosphorylation status of the Munc18 proteins. Even though Munc18-1 emerges as the key SM-protein determining the Ca(2+) threshold for triggering secretory activity in a stimulated β-cell, Munc18-2 has the ability to increase Ca(2+) sensitivity and thus mediates the release of fusion-competent granules requiring a lower cytoplasmic-free Ca(2+) concentration, [Ca(2+)](i)(.) Hence, Munc18-1 and Munc18-2 display distinct subcellular compartmentalization and can coordinate the insulin exocytotic process differently as a consequence of the actual [Ca(2+)](i).
Isolated endocrine cells have enabled the development of numerous methods to assess basic cellula... more Isolated endocrine cells have enabled the development of numerous methods to assess basic cellular architecture and define a significant part of the molecular machinery involved in cellular processes, such as excitability or the exocytotic release of hormones following cytosolic calcium increase. These efforts have generated consensus models explaining the activation or general operation of a particular cell type. The use of fresh tissue slices can go even further and uncover a series of emergent properties never explained or predicted by consensus models. In this paper, we present a selection of our most important experimental findings in the beta cells of pancreatic tissue slices.
PloS one, 2015
Regeneration of skeletal muscle after injury is limited by scar formation, slow healing time and ... more Regeneration of skeletal muscle after injury is limited by scar formation, slow healing time and a high recurrence rate. A therapy based on platelet-rich plasma (PRP) has become a promising lead for tendon and ligament injuries in recent years, however concerns have been raised that PRP-derived TGF-β could contribute to fibrotic remodelling in skeletal muscle after injury. Due to the lack of scientific grounds for a PRP -based muscle regeneration therapy, we have designed a study using human myogenic progenitors and evaluated the potential of PRP alone and in combination with decorin (a TGF-β inhibitor), to alter myoblast proliferation, metabolic activity, cytokine profile and expression of myogenic regulatory factors (MRFs). Advanced imaging multicolor single-cell analysis enabled us to create a valuable picture on the ratio of quiescent, activated and terminally committed myoblasts in treated versus control cell populations. Finally high-resolution confocal microscopy validated th...
Neuroreport, Jan 19, 1995
We investigated the role of ADP-ribosylation factor (ARF) in regulated exocytosis in patch-clampe... more We investigated the role of ADP-ribosylation factor (ARF) in regulated exocytosis in patch-clamped rat melanotrophs. Addition of brefeldin A (BFA) to inhibit activation of endogenous ARF protein was found to attenuate regulated secretory activity monitored as changes in membrane capacitance (Cm). A synthetic peptide to amino acids 46-61 of ARF (P-14) was also found to inhibit Ca(2+)-induced secretory activity in these cells. This inhibition was not apparent with a scrambled amino acid sequence of ARF-P14 peptide. This paper provides the first patch-clamp study to suggest that the small GTP-binding protein ARF is required to trigger release of secretory granules from rat pituitary melanotrophs.
Nature medicine, Jan 16, 2015
In the nervous system, NMDA receptors (NMDARs) participate in neurotransmission and modulate the ... more In the nervous system, NMDA receptors (NMDARs) participate in neurotransmission and modulate the viability of neurons. In contrast, little is known about the role of NMDARs in pancreatic islets and the insulin-secreting beta cells whose functional impairment contributes to diabetes mellitus. Here we found that inhibition of NMDARs in mouse and human islets enhanced their glucose-stimulated insulin secretion (GSIS) and survival of islet cells. Further, NMDAR inhibition prolonged the amount of time that glucose-stimulated beta cells spent in a depolarized state with high cytosolic Ca(2+) concentrations. We also noticed that, in vivo, the NMDAR antagonist dextromethorphan (DXM) enhanced glucose tolerance in mice, and that in vitro dextrorphan, the main metabolite of DXM, amplified the stimulatory effect of exendin-4 on GSIS. In a mouse model of type 2 diabetes mellitus (T2DM), long-term treatment with DXM improved islet insulin content, islet cell mass and blood glucose control. Furthe...
Clostridium difficile toxin B (TcdB) is a single-stranded protein consisting of a C-terminal doma... more Clostridium difficile toxin B (TcdB) is a single-stranded protein consisting of a C-terminal domain responsible for binding to the host cell membrane, a middle part involved in internalization, and the N-terminal catalytic (toxic) part. This study shows that TcdB is processed by a single proteolytic step which cleaves TcdB 10463 between Leu 543 and Gly 544 and the naturally occurring variant TcdB 8864 between Leu 544 and Gly 545 . The cleavage occurs at neutral pH and is catalysed by a pepstatin-sensitive protease localized in the cytoplasm and on the cytoplasmic face of intracellular membranes. The smaller N-terminal cleavage products [63 121 Da (TcdB 10463 ) and 62 761 Da (TcdB 8864 )] harbour the cytotoxic and glucosyltransferase activities of the toxins. When microinjected into cultured Chinese hamster lung fibroblasts, the N-terminal cleavage fragment shows full cytotoxic activity shortly after injection whereas the holotoxin initially exhibits a very low activity which, however, increases with time. Twenty minutes after the start of internalization of TcdB, the larger cleavage products [206 609 Da (TcdB 10463 ) and 206 245 Da (TcdB 8864 )] are found exclusively in a membrane fraction, whereas the N-terminal cleavage products appear mainly in the cytosol and associated with the membrane. This is in line with a proposed model according to which the longer, C-terminal, part of these toxins forms a channel allowing for the translocation of the toxic N-terminal part, which is subsequently cleaved off at the cytoplasmic face of an intracellular compartment, most likely endosomes.
Alpha-neurexins constitute a family of neuronal cell surface molecules that are essential for eff... more Alpha-neurexins constitute a family of neuronal cell surface molecules that are essential for efficient neurotransmission, because mice lacking two or all three alpha-neurexin genes show a severe reduction of synaptic release. Although analyses of alpha-neurexin knock-outs and transgenic rescue animals suggested an involvement of voltage-dependent Ca2+ channels, it remained unclear whether alpha-neurexins have a general role in Ca2+-dependent exocytosis and how they may affect Ca2+ channels. Here we show by membrane capacitance measurements from melanotrophs in acute pituitary gland slices that release from endocrine cells is diminished by &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt;50% in adult alpha-neurexin double knock-out and newborn triple knock-out mice. There is a reduction of the cell volume in mutant melanotrophs; however, no ultrastructural changes in size or intracellular distribution of the secretory granules were observed. Recordings of Ca2+ currents from melanotrophs, transfected human embryonic kidney cells, and brainstem neurons reveal that alpha-neurexins do not affect the activation or inactivation properties of Ca2+ channels directly but may be responsible for coupling them to release-ready vesicles and metabotropic receptors. Our data support a general and essential role for alpha-neurexins in Ca2+-triggered exocytosis that is similarly important for secretion from neurons and endocrine cells.
PLoS Biology, 2009
While serotonin (5-HT) co-localization with insulin in granules of pancreatic b-cells was demonst... more While serotonin (5-HT) co-localization with insulin in granules of pancreatic b-cells was demonstrated more than three decades ago, its physiological role in the etiology of diabetes is still unclear. We combined biochemical and electrophysiological analyses of mice selectively deficient in peripheral tryptophan hydroxylase (Tph12/2) 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 Tph12/2 b-cells, which clearly showed that the secretory defect is downstream of Ca 2+ -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 b-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 b-cells and suggest that intracellular 5-HT functions in various microenvironments via this mechanism in concert with the known receptor-mediated signaling.
Scientific Reports, 2015
Collective beta cell activity in islets of Langerhans is critical for the supply of insulin withi... more Collective beta cell activity in islets of Langerhans is critical for the supply of insulin within an organism. Even though individual beta cells are intrinsically heterogeneous, the presence of intercellular coupling mechanisms ensures coordinated activity and a well-regulated exocytosis of insulin. In order to get a detailed insight into the functional organization of the syncytium, we applied advanced analytical tools from the realm of complex network theory to uncover the functional connectivity pattern among cells composing the intact islet. The procedure is based on the determination of correlations between long temporal traces obtained from confocal functional multicellular calcium imaging of beta cells stimulated in a stepwise manner with a range of physiological glucose concentrations. Our results revealed that the extracted connectivity networks are sparse for low glucose concentrations, whereas for higher stimulatory levels they become more densely connected. Most importantly, for all ranges of glucose concentration beta cells within the islets form locally clustered functional sub-compartments, thereby indicating that their collective activity profiles exhibit a modular nature. Moreover, we show that the observed non-linear functional relationship between different network metrics and glucose concentration represents a well-balanced setup that parallels physiological insulin release.
The Journal of Physiology, 2005
Clions are known regulators of Ca 2+ -dependent secretory activity in many endocrine cells. The s... more Clions are known regulators of Ca 2+ -dependent secretory activity in many endocrine cells. The suggested mechanisms of Claction involve the modulation of GTP-binding proteins, voltage-activated calcium channels or maturation of secretory vesicles. We examined the role of cytosolic Cl -([Cl -] i ) and Clcurrents in the regulation of secretory activity in mouse melanotrophs from fresh pituitary tissue slices by using the whole-cell patch-clamp. We confirmed that elevated [Cl -] i augments Ca 2--dependent exocytosis and showed that Clacts on secretory vesicle maturation. The latter process was abolished by a V-type H --ATPase blocker (bafilomycin), intracellular 4,4 -diisothiocyanatostilbene-2,2 -disulphonic acid (DIDS), a Clchannel blocker, and tolbutamide, a sulphonylurea implicated in secretory vesicle maturation. In a small subset of cells, block of plasmalemmal Clcurrent by DIDS reversibly enhanced endocytosis. The direct activation of G-proteins by GTP-γ-S, a non-hydrolysable GTP analogue, did not restore the impaired secretion observed in low [Cl -] i conditions. The amplitude of voltage-activated calcium currents was unaffected by the [Cl -] i . Furthermore, two Cl --permeable channels, calcium-activated Clchannels and GABA A receptors, appeared as major regulators of intracellular Clhomeostasis. In conclusion, the predominant underlying mechanism of Claction is mediated by intracellular Clfluxes during vesicle maturation, rather than activation of G-proteins or modulation of voltage-activated Ca 2+ channels.
The Journal of Physiology, 2004
We have prepared fresh pituitary gland slices from adult and, for the first time, from newborn mi... more We have prepared fresh pituitary gland slices from adult and, for the first time, from newborn mice to assess modulation of secretory activity via voltage-activated Ca(2+) channels (VACCs). Currents through VACCs and membrane capacitance have been measured with the whole-cell patch-clamp technique. Melanotrophs in newborns were significantly larger than in adults. In both newborn and adult melanotrophs activation of VACCs triggered exocytosis. All pharmacologically isolated VACC types contributed equally to the secretory activity. However, the relative proportion of VACCs differed between newborns and adults. In newborn cells L-type channels dominated and, in addition, an exclusive expression of a toxin-resistant R-type-like current was found. The expression of L-type VACCs was up-regulated by the increased oestrogen levels observed in females, and was even more emphasized in the cells of pregnant females and oestrogen-treated adult male mice. We suggest a general mechanism modulating endocrine secretion in the presence of oestrogen and particularly higher sensitivity to treatments with L-type channel blockers during high oestrogen physiological states.
The International Journal of Developmental Biology, 2004
We present a new strategy for the differentiation of embryonic stem (ES) cells into insulin-produ... more We present a new strategy for the differentiation of embryonic stem (ES) cells into insulin-producing cells via a multi-step process without selection and induction of nestin-positive cells. During ES cell differentiation, transcript levels of genes characteristic of early and mature beta cells including Pdx1, Pax4, insulin and islet amyloid pancreatic peptide are up regulated. Islet-like clusters are characterized by expression of C-peptide, insulin and partially cytokeratin 19 as well as by ion channel activity similar to that found in embryonic beta cells. Cells of islet-like clusters show glucose-dependent insulin release at terminal stage. At an intermediate stage, nestin is partially co-expressed with C-peptide and cytokeratin 19, whereas islet-like clusters at the terminal stage are nestin-negative. We conclude that expression of nestin and cytokeratin 19 is a normal property of ES cells preceding differentiation into C-peptide/insulin-producing cells without any selection for nestin-positive phenotypes.
Proceedings of the National Academy of Sciences, 2000
Although many proteins essential for regulated neurotransmitter and peptide hormone secretion hav... more Although many proteins essential for regulated neurotransmitter and peptide hormone secretion have been identified, little is understood about their precise roles at specific stages of the multistep pathway of exocytosis. To study the function of CAPS (Ca 2؉ -dependent activator protein for secretion), a protein required for Ca 2؉ -dependent exocytosis of dense-core vesicles, secretory responses in single rat melanotrophs were monitored by patch-clamp membrane capacitance measurements. Flash photolysis of caged Ca 2؉ elicited biphasic capacitance increases consisting of rapid and slow components with distinct Ca 2؉ dependencies. A threshold of Ϸ10 M Ca 2؉ was required to trigger the slow component, while the rapid capacitance increase was recorded already at a intracellular Ca 2؉ activity < 10 M. Both kinetic membrane capacitance components were abolished by botulinum neurotoxin B or E treatment, suggesting involvement of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)-dependent vesicle fusion. The rapid but not the slow component was inhibited by CAPS antibody. These results were further clarified by immunocytochemical studies that revealed that CAPS was present on only a subset of dense-core vesicles. Overall, the results indicate that dense-core vesicle exocytosis in melanotrophs occurs by two parallel pathways. The faster pathway exhibits high sensitivity to Ca 2؉ and requires the presence of CAPS, which appears to act at a late stage in the secretory pathway.
PLoS ONE, 2014
Gold nanoparticles (GNPs) are claimed as outstanding biomedical tools for cancer diagnostics and ... more Gold nanoparticles (GNPs) are claimed as outstanding biomedical tools for cancer diagnostics and photo-thermal therapy, but without enough evidence on their potentially adverse immunological effects. Using a model of human dendritic cells (DCs), we showed that 10 nm-and 50 nm-sized GNPs (GNP 10 and GNP 50 , respectively) were internalized predominantly via dynamin-dependent mechanisms, and they both impaired LPS-induced maturation and allostimulatory capacity of DCs, although the effect of GNP 10 was more prominent. However, GNP 10 inhibited LPS-induced production of IL-12p70 by DCs, and potentiated their Th2 polarization capacity, while GNP 50 promoted Th17 polarization. Such effects of GNP 10 correlated with a stronger inhibition of LPS-induced changes in Ca 2+ oscillations, their higher number per DC, and more frequent extraendosomal localization, as judged by live-cell imaging, proton, and electron microscopy, respectively. Even when released from heat-killed necrotic HEp-2 cells, GNP 10 inhibited the necrotic tumor cell-induced maturation and functions of DCs, potentiated their Th2/Th17 polarization capacity, and thus, impaired the DCs' capacity to induce T cell-mediated anti-tumor cytotoxicity in vitro. Therefore, GNP 10 could potentially induce more adverse DC-mediated immunological effects, compared to GNP 50 .
Planta, 1994
Enhanced elongation of coleoptile cells has been proposed to be related to a rise in secretory ac... more Enhanced elongation of coleoptile cells has been proposed to be related to a rise in secretory activity. Therefore, to obtain a direct measurement of exocytotic events in maize (Zea mays L.) coleoptile protoplasts we used the patch-clamp method to record changes in membrane capacitance (Cm) as a parameter proportional to fluctuations of the membrane surface area. The secretory activity of protoplasts was correlated with the cytosolic free Ca 2 + concentration ([Ca 2 +]cyt): dialyzing protoplasts with 1 laM [Ca2+]cy t caused a steady rise in Cm of 3.3_ pF-s 1. In contrast, dialysis with a solution containing <20 nM Ca 2+ produced a small and persistent decrease in Cm. This demonstrates that secretory activity in coleoptile cells can be controlled by factors which modulate [Ca2+]cy t.
Pfl�gers Archiv European Journal of Physiology, 2003
The tissue-slice technique has enabled major insights into neural and neuroendocrine physiology. ... more The tissue-slice technique has enabled major insights into neural and neuroendocrine physiology. Our aim was to adapt this technique to study the function of the endocrine pancreas. The preparation combines an in situ approach, as in gland perfusion, with a resolution characteristic of electrophysiological studies on single cells. The membrane potential in b-cells in the slices recorded using the whole-cell patch-clamp was close to the calculated reversal potential for K + . With sufficient ATP in the recording pipette the b-cells depolarized rapidly on exposure to an increased glucose concentration or stimulation with tolbutamide. The cells preserved bursting and spiking capacity for tens of minutes despite the whole-cell dialysis. In addition, the voltage clamp was used to monitor the changes in the membrane capacitance and to allow correlation of the electrical activity and the cytosolic calcium changes. The pancreatic tissue slice preparation is a novel method for studying the function of the b-and other pancreatic endocrine and exocrine cells under near-physiological conditions.
Pfl�gers Archiv European Journal of Physiology, 1995
... 3A, B Ca2+-dependent secretory response is attenuated by guanosine 5'-O-(2-thiodipho... more ... 3A, B Ca2+-dependent secretory response is attenuated by guanosine 5'-O-(2-thiodiphosphate),GDP[fi-S]. A Secretory responses recorded at 1 gM [Ca2+]i and 4 mM (top panel) and 154 mM (lower panel) [C1-]i in control cells and in the presence of GDP[fl-S] (500 gM). ...