Biomedical subjects

Marjan Rupnik

Publications and source records attributed to Marjan Rupnik.

16 recordsLinked to original sources

PubMed2006

Important contribution of alpha-neurexins to Ca2+-triggered exocytosis of secretory granules.

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.

Animals↗

PubMed2006

Glutamate stimulation increases hormone release in rat melanotrophs.

In melanotrophs, neuroendocrine cells from the intermediate lobe of the rat pituitary gland, glutamate causes a rise in intracellular [Ca2+] suggesting the presence of ionotropic NMDA and non-NMDA AMPA/K receptors. However, the Ca(2+)-dependent release of the major peptide hormone, alpha-melanocyte stimulating hormone (alpha-MSH), in response to glutamate stimulation has not been studied yet in this cell model. Significant spontaneous secretion of the peptide, which results in hormone deposits on the perimeter of the cells, has been confirmed by using confocal microscopy. Co-staining with a membrane area marker FM 1-43, which co-localized with the immunocytochemically marked hormone deposits, showed that fusion-competent sites on the plasma membrane coincided with secretion-competent sites. Stimulation of the cells with glutamate and high K+ saline induced a significant increase in the plasma membrane area covered with alpha-MSH deposits compared to control cells incubated with glutamate and CNQX, a glutamate channel blocker. The optical approach to monitor the secretory activity of a single neuroendocrine cell revealed that glutamate stimulates the release of alpha-MSH at distinct exocytotic membrane domains only.

6-Cyano-7-nitroquinoxaline-2,3-dione↗

PubMed2005

Block of delayed-rectifier potassium channels by reduced haloperidol and related compounds in mouse cortical neurons.

Haloperidol is known as an antagonist of dopamine D2 receptors. However, it also blocks a variety of ion channels at concentrations above the therapeutic range. Reduced haloperidol (R-haloperidol), one of the main metabolites of haloperidol, has been reported to accumulate in certain tissues, particularly in brain cortex, and it may produce the pharmacological effects associated with haloperidol treatment. In this study, we assessed the effect of R-haloperidol and other related compounds on native delayed-rectifier potassium channels (K(DR)) in mouse cortical neurons by using the whole-cell patch-clamp technique. Although R-haloperidol has much lower affinity to D2 receptors than haloperidol, the IC50 of R-haloperidol to block K(DR) currents was 4.4 microM, similar to its parent compound. The binding site of R-haloperidol is on the cytoplasmic side of the channel because its quaternary derivative preferentially inhibited the currents from intracellular side. 4-Chlorophenyl-4-hydroxypiperidine (4C4HP) is the active fragment of haloperidol because other compounds containing this moiety, including L-741,626 (3-[4-(4-chlorophenyl)-4-hydroxypiperidin-L-yl]-methyl-1H-indole) and loperamide, also blocked K(DR) channels. The potency of the 4C4HP fragment positively correlated with the hydrophobicity index (clogP) of the compounds tested. We conclude that R-haloperidol is a K(DR) channel blocker, although it does not interfere with the normal channel function at a clinically relevant concentration.

Animals↗

PubMed2005

cAMP increases Ca2+-dependent exocytosis through both PKA and Epac2 in mouse melanotrophs from pituitary tissue slices.

Cyclic AMP regulates Ca(2+)-dependent exocytosis through a classical protein kinase A (PKA)-dependent and an alternative cAMP-guanine nucleotide exchange factor (GEF)/Epac-dependent pathway in many secretory cells. Although increased cAMP is believed to double secretory output in isolated pituitary cells, the direct target(s) for cAMP action and a detailed and high-time resolved analysis of the effect of intracellular cAMP levels on the secretory activity in melanotrophs are still lacking. We investigated the effect of 200 microM cAMP on the kinetics of secretory vesicle depletion in mouse melanotrophs from fresh pituitary tissue slices. The whole-cell patch-clamp technique was used to depolarize melanotrophs and increase the cytosolic Ca(2+) concentration ([Ca(2+)](i)). Exogenous cAMP elicited an about twofold increase in cumulative membrane capacitance change and approximately 34% increase of high-voltage activated Ca(2+) channel amplitude. cAMP-dependent mechanisms did not affect [Ca(2+)](i), since the application of forskolin failed to change [Ca(2+)](i) in melanotrophs, a phenomenon readily observed in anterior lobe. Depolarization-induced secretion resulted in two distinct kinetic components: a linear and a threshold component, both stimulated by cAMP. The linear component (ATP-independent) probably represented the exocytosis of the release-ready vesicles, whereas the threshold component was assigned to the exocytosis of secretory vesicles that required ATP-dependent reaction(s) and > 800 nM [Ca(2+)](i). The linear component was modulated by 8-pCPT-2Me-cAMP (Epac agonist), while either H-89 (PKA inhibitor) or Rp-cAMPS (the competitive antagonist of cAMP binding to PKA) completely prevented the action of cAMP on the threshold component. In line with this, 6-Phe-cAMP, (PKA agonist), increased the threshold component. From our study, we suggest that the stimulation of cAMP production by application of oestrogen, as found in pregnant mice, increases the efficacy of the hormonal output through both PKA and cAMP-GEFII/Epac2-dependent mechanisms.

Animals↗

PubMed2005

Cytosolic Cl- ions in the regulation of secretory and endocytotic activity in melanotrophs from mouse pituitary tissue slices.

Cl- ions are known regulators of Ca2+ -dependent secretory activity in many endocrine cells. The suggested mechanisms of Cl- action 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 Cl- currents 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 Ca2- -dependent exocytosis and showed that Cl- acts 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 Cl- channel blocker, and tolbutamide, a sulphonylurea implicated in secretory vesicle maturation. In a small subset of cells, block of plasmalemmal Cl- current by DIDS reversibly enhanced endocytosis. The direct activation of G-proteins by GTP-gamma-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 Cl- channels and GABAA receptors, appeared as major regulators of intracellular Cl- homeostasis. In conclusion, the predominant underlying mechanism of Cl- action is mediated by intracellular Cl- fluxes during vesicle maturation, rather than activation of G-proteins or modulation of voltage-activated Ca2+channels.

Algorithms↗

PubMed2005

Characterization of the cleavage site and function of resulting cleavage fragments after limited proteolysis of Clostridium difficile toxin B (TcdB) by host cells.

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.

Amino Acid Sequence↗

PubMed2005

Endocytosis-dominated membrane area decrease requires Rab5 protein in rat melanotrophs.

Eukaryotic cells internalize extracellular macromolecules by endocytosis and it was shown that Rab5 protein is required for this process. While it is clear that endocytosis consists of vesicle fission from the plasma membrane, the role of Rab5 protein in the plasma membrane surface area changes is still unclear. Here we studied whether Rab5 is required for membrane surface area changes in rat melanotrophs-cells deriving from the pituitary pars intermedia. The presence of this protein in melanotrophs was probed by immunocytochemistry and its putative role in membrane area dynamics was monitored electrophysiologically with membrane capacitance measurements as this parameter directly reflects changes in membrane surface area. We found that Rab5 protein exists in melanotrophs. At [Ca(2+)](i) < 3 microM, endocytosis-dominated membrane capacitance decrease was found to be blocked by microinjection of specific Rab5 antibody. At high [Ca(2+)](i), Rab5 antibody did not affect the steady-state increase in membrane capacitance, while it elevated the rate of membrane capacitance increase, which is consistent with an inhibition of endocytosis.

Animals↗

PubMed2004

Ammodytoxin, a neurotoxic secreted phospholipase A(2), can act in the cytosol of the nerve cell.

Recent identification of intracellular proteins that bind ammodytoxin (calmodulin, 14-3-3 proteins, and R25) suggests that this snake venom presynaptically active phospholipase A(2) acts intracellularly. As these ammodytoxin acceptors are cytosolic and mitochondrial proteins, the toxin should be able to enter the cytosol of a target cell and remain stable there to interact with them. Using laser scanning confocal microscopy we show here that Alexa-labelled ammodytoxin entered the cytoplasm of the rat hippocampal neuron and subsequently also its nucleus. The transport of proteins into the nucleus proceeds via the cytosol of a cell, therefore, ammodytoxin passed the cytosol of the neuron on its way to the nucleus. Although it is not yet clear how ammodytoxin is translocated into the cytosol of the neuron, our results demonstrate that its stability in the cytosol is not in question, providing the evidence that the toxin can act in this cellular compartment.

Animals↗

PubMed2004

Inhibition of ATP-sensitive potassium channels by haloperidol.

Chronic haloperidol treatment has been associated with an increased incidence of glucose intolerance and type-II diabetes mellitus. We studied the effects of haloperidol on native ATP-sensitive potassium (K(ATP)) channels in mouse pancreatic beta cells and on cloned Kir6.2/SUR1 channels expressed in HEK293 cells. The inhibitory effect of haloperidol on the K(ATP) channel was not mediated via the D2 receptor signaling pathway, as both D2 agonists and antagonists blocked the channel. K(ATP) currents were studied using the patch-clamp technique in whole-cell and outside-out patch configurations. Addition of haloperidol to the extracellular solution inhibited the K(ATP) conductance immediately, in a reversible and voltage-independent manner. Haloperidol did not block the channel when applied intracellularly in whole-cell recordings. Haloperidol blocked cloned Kir6.2/SUR1 and Kir6.2DeltaC36 K(ATP) channels expressed in HEK cells. This suggests that the drug interacts with the Kir6.2 subunit of the channel. The IC(50) for inhibition of the K(ATP) current by haloperidol was 1.6 microM in 2 mM extracellular K(+) concentration ([K(+)](o)) and increased to 23.9 microM in 150 mM [K(+)](o). The Hill coefficient was close to unity, suggesting that the binding of a single molecule of haloperidol is sufficient to close the channel. Haloperidol block of K(ATP) channels may contribute to the side effects of this drug when used therapeutically.

Adenosine Triphosphate↗

PubMed2004

Enlargeosome, an exocytic vesicle resistant to nonionic detergents, undergoes endocytosis via a nonacidic route.

Enlargeosomes, a new type of widely expressed cytoplasmic vesicles, undergo tetanus toxin-insensitive exocytosis in response to cytosolic Ca(2+) concentration ([Ca(2+)](i)) rises. Cell biology of enlargeosomes is still largely unknown. By combining immunocytochemistry (marker desmoyokin-Ahnak, d/A) to capacitance electrophysiology in the enlargeosome-rich, neurosecretion-defective clone PC12-27, we show that 1) the two responses, cell surface enlargement and d/A surface appearance, occur with similar kinetics and in the same low micromolar [Ca(2+)](i) range, no matter whether induced by photolysis of the caged Ca(2+) compound o-nitrophenyl EGTA or by the Ca(2+) ionophore ionomycin. Thus, enlargeosomes seem to account, at least in large part, for the exocytic processes triggered by the two stimulations. 2. The enlargeosome membranes are resistant to nonionic detergents but distinct from other resistant membranes, rich in caveolin, Thy1, and/or flotillin1. 3. Cell cholesterol depletion, which affects many membrane fusions, neither disrupts enlargeosomes nor affects their regulated exocytosis. 4. The postexocytic cell surface decline is [Ca(2+)](i) dependent. 5. Exocytized d/A-rich membranes are endocytized and trafficked along an intracellular pathway by nonacidic organelles, distinct from classical endosomes and lysosomes. Our data define specific aspects of enlargeosomes and suggest their participation, in addition to cell differentiation and repair, for which evidence already exists, to other physiological and pathological processes.

Animals↗

PubMed2004

In vivo and in vitro development of mouse pancreatic beta-cells in organotypic slices.

Taking tissue slices of the embryonic and newborn pancreas is a novel approach for the study of the perinatal development of this gland. The aim of this study was to describe the morphology and physiology of in vivo and in vitro developing beta-cells. In addition, we wanted to lay a foundation for the functional analysis of other pancreatic cells, either alone or as part of an integrative pancreatic physiology approach. We used cytochemistry and light microscopy to detect specific markers and the whole-cell patch-clamp to assess the function of single beta-cells. The insulin signal in the embryonic beta-cells was condensed to a subcellular compartment and redistributed throughout the cytosol during the first 2 days after birth. The hormone distribution correlated well with the development of membrane excitability and hormone release competence in beta-cells. Endocrine cells survived in the organotypic tissue culture and maintained their physiological properties for weeks. We conclude that our preparation fulfills the criteria for a method of choice to characterize the function of developing pancreas in wild-type and genetically modified mice that die at birth. We suggest organotypic culture for in vitro studies of the development and regeneration of beta-cells.

Animals↗

PubMed2004

Voltage-activated Ca(2+) channels and their role in the endocrine function of the pituitary gland in newborn and adult mice.

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.

Aging↗

PubMed2004

Properties of Ca(2+)-dependent exocytosis in cultured astrocytes.

Astrocytes, a subtype of glial cells, have numerous characteristics that were previously considered exclusive for neurons. One of these characteristics is a cytosolic [Ca2+] oscillation that controls the release of the chemical transmitter glutamate and atrial natriuretic peptide. These chemical messengers appear to be released from astrocytes via Ca(2+)-dependent exocytosis. In the present study, patch-clamp membrane capacitance measurements were used to monitor changes in the membrane area of a single astrocyte, while the photolysis of caged calcium compounds by a UV flash was used to elicit steps in [Ca2+]i to determine the exocytotic properties of astrocytes. Experiments show that astrocytes exhibit Ca(2+)-dependent increases in membrane capacitance, with an apparent Kd value of approximately 20 microM [Ca2+]i. The delay between the flash delivery and the peak rate in membrane capacitance increase is in the range of tens to hundreds of milliseconds. The pretreatment of astrocytes by the tetanus neurotoxin, which specifically cleaves the neuronal/neuroendocrine type of SNARE protein synaptobrevin, abolished flash-induced membrane capacitance increases, suggesting that Ca(2+)-dependent membrane capacitance changes involve tetanus neurotoxin-sensitive SNARE-mediated vesicular exocytosis. Immunocytochemical experiments show distinct populations of vesicles containing glutamate and atrial natriuretic peptide in astrocytes. We conclude that the recorded Ca(2+)-dependent changes in membrane capacitance represent regulated exocytosis from multiple types of vesicles, about 100 times slower than the exocytotic response in neurons.

Animals↗

PubMed2004

Embryonic stem cells differentiate into insulin-producing cells without selection of nestin-expressing cells.

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.

Animals↗

PubMed2003

A novel approach to in situ characterization of pancreatic beta-cells.

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 beta-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 beta-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 beta- and other pancreatic endocrine and exocrine cells under near-physiological conditions.

Adenosine Triphosphate↗

PubMed2002

Modeling excess retrieval in rat melanotroph membrane capacitance records.

We have used the patch-clamp technique to monitor changes in membrane capacitance (C(m)) elicited by fast and spatially homogeneous rises in cytosolic calcium concentration ([Ca(2+)](i)) using flash photolysis of NP-EGTA. Average peak [Ca(2+)](i) amplitudes of 20-25 microM triggered three different types of responses in C(m): (i) In 42% of cells, a rise in [Ca(2+)](i) activated a monotonic increase in C(m) followed by a slow decline to resting values; (ii) In 30% of cells, the rise in C(m) was clearly characterized by two dynamic components, consisting of a rapid and a slow exo-endocytosis cycle; (iii) In 28% of cells, after the initial rapid rise in C(m), endocytosis exhibited excess retrieval that was characterized by a decline in C(m) below resting C(m). The aim of this work is to develop a unified mathematical model with a minimum number of parameters that would describe all the observed types of responses. Three models were considered: Model A, a model with a single component of exo-endocytosis cycle; model B, a model consisting of a sum of two independent dynamic components; and model C, a model in which, in addition to the two dynamic components as in model B, excess retrieval due to a lipid flow through the reversal closing of the fusion pore during the rapid component of exo-endocytosis cycle was considered. The results show that the latter model describes all the types of responses in C(m) recorded in rat melanotrophs. The association of excess retrieval exclusively with the rapid, but not the slow, exocytosis indicates that some fusing vesicles mediate a lipidic flux during the reversal closing of the fusion pore, whereas those entering the slow phase of exocytosis may fuse with the plasma membrane completely and are retrieved by other endocytic machinery, independent of the lipid flow that might have occurred as the fusion pore opened permanently.

Animals↗