Marjan Rupnik

Negative impact of endocrine-disrupting compounds on human reproductive health

There is increasing concern about chemical pollutants that are able to mimic hormones, the so-called endocrine-disrupting compounds (EDCs), because of their structural similarity to endogenous hormones, their ability to interact with hormone transport proteins or because of their potential to disrupt hormone metabolic pathways. Thus, the effects of endogenous hormones can be mimicked or, in some cases, completely blocked. A substantial number of environmental pollutants, such as polychlorinated biphenyls, dioxins, polycyclic aromatic hydrocarbons, phthalates, bisphenol A, pesticides, alkylphenols and heavy metals (arsenic, cadmium, lead, mercury), have been shown to disrupt endocrine function. These compounds can cause reproductive problems by decreasing sperm count and quality, increasing the number of testicular germ cells and causing male breast cancer, cryptorchidism, hypospadias, miscarriages, endometriosis, impaired fertility, irregularities of the menstrual cycle, and infertility. Although EDCs may be released into the environment in different ways, the main sources is industrial waste water. The present paper critically reviews the current knowledge of the impact of EDCs on reproductive disorders in humans.

A novel approach to in situ characterization of pancreatic β-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 β-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 β-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 β- and other pancreatic endocrine and exocrine cells under near-physiological conditions.

Donor Islet Endothelial Cells in Pancreatic Islet Revascularization

OBJECTIVE Freshly isolated pancreatic islets contain, in contrast to cultured islets, intraislet endothelial cells (ECs), which can contribute to the formation of functional blood vessels after transplantation. We have characterized how donor islet endothelial cells (DIECs) may contribute to the revascularization rate, vascular density, and endocrine graft function after transplantation of freshly isolated and cultured islets. RESEARCH DESIGN AND METHODS Freshly isolated and cultured islets were transplanted under the kidney capsule and into the anterior chamber of the eye. Intravital laser scanning microscopy was used to monitor the revascularization process and DIECs in intact grafts. The grafts’ metabolic function was examined by reversal of diabetes, and the ultrastructural morphology by transmission electron microscopy. RESULTS DIECs significantly contributed to the vasculature of fresh islet grafts, assessed up to 5 months after transplantation, but were hardly detected in cultured islet grafts. Early participation of DIECs in the revascularization process correlated with a higher revascularization rate of freshly isolated islets compared with cultured islets. However, after complete revascularization, the vascular density was similar in the two groups, and host ECs gained morphological features resembling the endogenous islet vasculature. Surprisingly, grafts originating from cultured islets reversed diabetes more rapidly than those originating from fresh islets. CONCLUSIONS In summary, DIECs contributed to the revascularization of fresh, but not cultured, islets by participating in early processes of vessel formation and persisting in the vasculature over long periods of time. However, the DIECs did not increase the vascular density or improve the endocrine function of the grafts.

The regulated exocytosis of enlargeosomes is mediated by a SNARE machinery that includes VAMP4

The mechanisms governing the fast, regulated exocytosis of enlargeosomes have been unknown, except for the participation of annexin-2 in a pre-fusion step. We investigated whether any SNAREs are involved. In PC12-27 cells, which are enlargeosome-rich, the expressed SNAREs exhibited various distributions (trans-Golgi network, scattered puncta, plasma membrane); however, only VAMP4 was colocalized in discrete puncta with the enlargeosome marker desmoyokin. The exocytosis of the organelle, revealed by capacitance increases and by surface appearance of desmoyokin, was largely inhibited by microinjection of anti-VAMP4, anti-syntaxin-6 and anti-SNAP23 antibodies, by incubation with botulinum toxin E, and by transfection of VAMP4 and syntaxin-6 siRNAs. Microinjection of the antibodies anti-VAMP7, anti-VAMP8 and anti-syntaxin-4, and transfection with the VAMP8 siRNA were ineffective. Inhibition of enlargeosome exocytosis by VAMP4 siRNA also occurred in a cell type that was competent for neurosecretion, SH-SY5Y. Moreover, in cells expressing a VAMP4-GFP construct, enlargeosome exocytosis and surface appearance of fluorescence occurred concomitantly, and many ensuing surface patches were co-labelled by GFP and desmoyokin. VAMP4, an R-SNARE that has never been shown to participate in regulated exocytoses, therefore appears to be harboured in the membrane of enlargeosomes and to be a member of the machinery mediating their regulated exocytosis. Syntaxin-6 and SNAP23 appear also to be needed for the process to occur; however, the mechanism of their participation, whether direct or indirect, remains undefined.

Cytosolic chloride ions stimulate Ca2+-induced exocytosis in melanotrophs

We used the whole‐cell patch‐clamp technique to study the secretory activity of single cells by monitoring changes in membrane capacitance [Neher, E. and Marty, A. (1982) Proc. Natl. Acad. Sci. USA 79, 523–535] in anterior pituitary cells. Unexpectedly we have observed that increasing intracellular chloride ions stimulate Ca2+‐induced exocytosis in a dose‐dopendent fashion (K d = 12 mM). These results demonstrate a role of cytosolic chloride ions in the regulation of exocytotic secretion in anterior pituitary cells. It is suggested that chloride channels, in addition to playing a part in regulating membrane electrical activity [Korn, S.J., Bolden, A. and Horn, A. (1991) J. Physiol. 439, 423–437; Penner, R., Matthews, G. and Horn, A. (1988) Nature 334, 499–504] and cytosolic pH [Kaila, K. and Voipio, J. (1987) Nature 330, 163–165], are also involved in the modulation of cytosolic chloride concentration and thus in the control of exocytosis.

Raising the cytosolic Ca2+ concentration increases the membrane capacitance of maize coleoptile protoplasts: Evidence for Ca2+-stimulated exocytosis

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 Ca2+ concentration ([Ca2+]cyt): dialyzing protoplasts with 1 μM [Ca2+]cyt caused a steady rise in Cm of 3.3 ± pF·s−1. In contrast, dialysis with a solution containing <20 nM Ca2+ produced a small and persistent decrease in Cm. This demonstrates that secretory activity in coleoptile cells can be controlled by factors which modulate [Ca2+]cyt.

Cathophoresis paint insulated carbon fibre ultramicro disc electrode and its application to in vivo amperometric monitoring of quantal secretion from single rat melanotrophs

Abstract This paper reports the preparation and characterization of carbon fibre ultramicro disc electrodes (CUMDEs) by employing cathodic electrophoresis paint to electrically and chemically insulate a carbon fibre protruding from a pulled glass capillary housing. A thin and uniform paint coating was deposited electrochemically onto a carbon fibre of 7xa0μm in diameter, and then heat cured. The film thickness and its resistivity can be controlled by varying parameters such as the cathodic deposition voltage, the curing temperature, etc. Then, the coated carbon fibre was cut to expose a disc-shaped electrode surface with an overall tip diameter of about 12xa0μm. The electrodes were examined by optical and scanning electron microscopy and their electrochemical behaviour and properties were characterized using cyclic voltammetry, amperometry and electrochemical impedance spectroscopy. Cyclic voltammograms of hexacyanoferrate(III) in aqueous and of ferrocene in acetonitrile media displayed low-noise, low-background sigmoidal responses with virtually no current hysteresis. The high performance of the proposed cathophoresis paint insulated CUMDEs was verified successfully by carrying out in vivo amperometric monitoring of the secretory activity of stimulated single rat melanotrophs. The extremely low current noise (1–2xa0pA rms) and high temporal resolution ensure the suitability of the proposed electrode for studying peptide hormone release at a single cell level.

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

Abstract.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 β-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 β-cells. The insulin signal in the embryonic β-cells was condensed to a subcellular compartment and redistributed throughout the cytosol during the first 2xa0days after birth. The hormone distribution correlated well with the development of membrane excitability and hormone release competence in β-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 β-cells.

Using pancreas tissue slices for in situ studies of islet of Langerhans and acinar cell biology

Studies on the cellular function of the pancreas are typically performed in vitro on its isolated functional units, the endocrine islets of Langerhans and the exocrine acini. However, these approaches are hampered by preparation-induced changes of cell physiology and the lack of an intact surrounding. We present here a detailed protocol for the preparation of pancreas tissue slices. This procedure is less damaging to the tissue and faster than alternative approaches, and it enables the in situ study of pancreatic endocrine and exocrine cell physiology in a conserved environment. Pancreas tissue slices facilitate the investigation of cellular mechanisms underlying the function, pathology and interaction of the endocrine and exocrine components of the pancreas. We provide examples for several experimental applications of pancreas tissue slices to study various aspects of pancreas cell biology. Furthermore, we describe the preparation of human and porcine pancreas tissue slices for the validation and translation of research findings obtained in the mouse model. Preparation of pancreas tissue slices according to the protocol described here takes less than 45 min from tissue preparation to receipt of the first slices.

Exocytosis of Insulin : In Vivo Maturation of Mouse Endocrine Pancreas

The aim of this study was to define when an insulin‐positive cell becomes functional in vivo and starts to exocytose insulin in a regulated nutrient‐dependent manner. Insulin‐positive cells appear in embryonic life (midgestation) and complete their maturation, presumably around birth. In order to work with embryonic and newborn endocrine pancreas, we used organotypic slices. The mouse embryonic pancreas slices show high basal insulin release that is not further elevated by high glucose levels. Despite the presence of functional voltage‐activated ion channels, the cells are not electrically active in the presence of secretagogues. At birth, the high basal insulin release drops and, after postnatal day 2, the insulin‐positive cells show both adult‐like bursting electrical activity and hormone release induced by high glucose levels. These properties allowed us to define them as beta cells. Despite the apparent stability of the transcription factor profile reported in insulin‐positive cells during late‐embryonic life, functional beta cells appear only 2 days after birth.