Zuzana Bacova

Resveratrol Inhibits Electrical Activity and Insulin Release from Insulinoma Cells by Block of Voltage-Gated Ca2+ Channels and Swelling-Dependent Cl- Currents

The phytostilbene resveratrol (RV) improves the metabolic state in animal models by increasing the insulin responsiveness of tissues and there is evidence that RV affects insulin secretion from native β-cells and insulinoma cells. In whole cell patch clamp experiments on clonal rat INS-1E cells we used high extracellular glucose (20 mM), extracellular hypotonicity (30%) or tolbutamide (100 μM) to elicit membrane depolarizations and electrical activity. Application of RV (50 μM) repolarized the cells, terminated electrical activity and prevented the hypotonicity-induced depolarization. These effects were fully reversible and intermittent application of RV restored tolbutamide-induced electrical activity after desensitization. Glucose-induced depolarization was counteracted by RV in presence of iberiotoxin (50 nM), showing that the RV effect does not depend on BKCa channel activation. RV dose-dependently inhibited KATP currents, L- and T-type Ca2+ currents and swelling-dependent Cl- currents evoked by either hypotonicity or high extracellular glucose - ion conductances crucially involved in regulating the electrical activity of insulin secreting cells. We further show that RV blunts glucose-induced, but not basal insulin release. Our results indicate that RV counteracts/prevents stimulus-induced cell membrane depolarization and electrical activity by blocking voltage-gated Ca2+- and swelling-dependent Cl- currents despite the inhibition of KATP currents.

Are Molecules Involved in Neuritogenesis and Axon Guidance Related to Autism Pathogenesis

Abstract Autism spectrum disorder is a heterogeneous disease, and numerous alterations of gene expression come into play to attempt to explain potential molecular and pathophysiological causes. Abnormalities of brain development and connectivity associated with alterations in cytoskeletal rearrangement, neuritogenesis and elongation of axons and dendrites might represent or contribute to the structural basis of autism pathology. Slit/Robo signaling regulates cytoskeletal remodeling related to axonal and dendritic branching. Components of its signaling pathway (ABL and Cdc42) are suspected to be molecular bases of alterations of normal development. The present review describes the most important mechanisms underlying neuritogenesis, axon pathfinding and the role of GTPases in neurite outgrowth, with special emphasis on alterations associated with autism spectrum disorders. On the basis of analysis of publicly available microarray data, potential biomarkers of autism are discussed.

Intracerebroventricular oxytocin administration in rats enhances object recognition and increases expression of neurotrophins, microtubule-associated protein 2, and synapsin I

Brain oxytocin regulates a variety of social and affiliative behaviors and affects also learning and memory. However, mechanisms of its action at the level of neuronal circuits are not fully understood. The present study tests the hypothesis that molecular factors required for memory formation and synaptic plasticity, including brain‐derived neurotrophic factor, neural growth factor, nestin, microtubule‐associated protein 2 (MAP2), and synapsin I, are enhanced by central administration of oxytocin. We also investigated whether oxytocin enhances object recognition and acts as anxiolytic agent. Therefore, male Wistar rats were infused continuously with oxytocin (20 ng/µl) via an osmotic minipump into the lateral cerebral ventricle for 7 days; controls were infused with vehicle. The object recognition test, open field test, and elevated plus maze test were performed on the sixth, seventh, and eighth days from starting the infusion. No significant effects of oxytocin on anxious‐like behavior were observed. The object recognition test showed that oxytocin‐treated rats significantly preferred unknown objects. Oxytocin treatment significantly increased gene expression and protein levels of neurotrophins, MAP2, and synapsin I in the hippocampus. No changes were observed in nestin expression. Our results provide the first direct evidence implicating oxytocin as a regulator of brain plasticity at the level of changes of neuronal growth factors, cytoskeletal proteins, and behavior. The data support assumption that oxytocin is important for short‐term hippocampus‐dependent memory.

Different Signaling Pathways Involved in Glucose- and Cell Swelling-Induced Insulin Secretion by Rat Pancreatic Islets in Vitro

Background: The objective was to compare signal transduction pathways exploited by glucose and cell swelling in stimulating insulin secretion. Methods: Isolated rat (Wistar) pancreatic islets were stimulated in vitro by 20 mmol/l glucose or 30% hypotonic medium (202 mOsm/kg) in various experimental conditions. Results: Glucose did not stimulate insulin release in calcium free medium. Cell swelling-induced insulin release in calcium free medium, even in the presence of the membrane permeable calcium chelator BAPTA/AM (10 µmol/l). Protein kinase C (PKC) inhibitor bisindolylmaleimide VIII (1 µmol/l) abolished the stimulation of insulin secretion by glucose but did not affect the swelling-induced insulin release. PKC activator phorbol 12-13-dibutyrate (1 µmol/l) stimulated insulin secretion in medium containing Ca2+ and did not potentiate insulin secretion stimulated by hypotonic extracellular fluid. Dilution of the medium (10-30%) had an additive effect on the glucose-induced insulin secretion. Noradrenaline (1 µmol/l) abolished glucose-induced insulin secretion but did not inhibit hypotonic stimulation either in presence or absence of Ca2+. Conclusion: Glucose- and swelling-induce insulin secretion through separate signal transduction pathways. Hyposmotic stimulation is independent from both the extracellular and intracellular Ca2+, does not involve PKC activation, and could not be inhibited by noradrenaline. These data indicate a novel signaling pathway for stimulation of insulin secretion exploited by cell swelling.

Cell Swelling-Induced Peptide Hormone Secretion

Cell volume changes induced in various ways (anisosmotic environment, hormones, oxidative stress, substrate uptake) are an integral part of a signal transduction network regulating cell function. 1,2,3 Cell swelling has received increasing attention as a stimulus for a variety of intracellular phenomena. 4 One of the most remarkable effects of cell swelling is its powerful effect in inducing exocytosis of material in intracellular secretory vesicles. Secretion of essentially all so-packaged hormones 5-24 including those from hypothalamus (thyrotropin-releasing hormone, TRH; gonadotropin-releasing hormone, GnRH), pituitary (LH, FSH, ACTH, MSH, TSH, prolactin, beta endorphin), pancreas (insulin, somatostatin, glucagon), heart (atrial natriuretic hormone) and kidney (renin) are stimulated in a concentration-related manner by medium hyposmolarity or isosmolar medium containing permeant molecules such as ethanol or urea (reviewed in Ref. 21). Cell swelling-induced exocytosis is not restricted to endocrine cells and hormones; medium hyposmolarity also induces secretion of exocrine pancreatic enzymes 5 and myeloperoxidase from human polymorphonuclear leukocytes. 25

Thyrotropin-releasing hormone in rat heart: effect of swelling, angiotensin II and renin gene

Aim:  This study was performed to examine thyrotropin‐releasing hormone (TRH) secretion and regulation in rat heart.

Cell Swelling-Induced Signaling for Insulin Secretion Bypasses Steps Involving G Proteins and PLA2 and is N-ethylmaleimide Insensitive

Background: This study was undertaken to examine putative mechanisms of calcium independent signal transduction pathway of cell swelling-induced insulin secretion. Methods: The role of phospholipase A2, G proteins, and soluble N-ethylmaleimide-sensitive-factor attachment protein receptor (SNARE) in insulin secretion induced by 30% hypotonic medium was studied using isolated rat pancreatic islets. Results: In contrast to glucose stimulation, osmotically induced insulin secretion from pancreatic islets was not inhibited by 10 µmol/l bromoenol lactone, an iPLA2 (Ca2+ independent phospholipase) inhibitor. Similarly, preincubation of islets for 20 hours with 25 µg/ml mycophenolic acid to inhibit GTP synthesis fully abolished glucose-induced insulin secretion but was without effect on hypotonicity stimulated insulin release. Glucose-induced insulin secretion was prevented by preincubation with 20 nmol/l tetanus toxin (TeTx), a metalloprotease inactivating soluble SNARE. Cell swelling-induced insulin secretion was inhibited by TeTx in the presence of calcium ions but not in calcium depleted medium. The presence of N-ethylmaleimide (NEM, 5 mmol/l, another inhibitor of SNARE proteins) in the medium resulted in high basal insulin secretion and lacking response to glucose stimulation. In contrast, high basal insulin secretion from NEM treated islets further increased after hypotonic stimulation. Conclusion: G proteins and iPLA2 – putative mediators of Ca2+ independent signaling pathway participate in glucose but not in hypotonicity-induced insulin secretion. Hypotonicity-induced insulin secretion is sensitive to clostridial neurotoxin TeTx but is resistant to NEM.

The Role of Hypothalamic Neuropeptides in Neurogenesis and Neuritogenesis

The hypothalamus is a source of neural progenitor cells which give rise to different populations of specialized and differentiated cells during brain development. Newly formed neurons in the hypothalamus can synthesize and release various neuropeptides. Although term neuropeptide recently undergoes redefinition, small-size hypothalamic neuropeptides remain major signaling molecules mediating short- and long-term effects on brain development. They represent important factors in neurite growth and formation of neural circuits. There is evidence suggesting that the newly generated hypothalamic neurons may be involved in regulation of metabolism, energy balance, body weight, and social behavior as well. Here we review recent data on the role of hypothalamic neuropeptides in adult neurogenesis and neuritogenesis with special emphasis on the development of food intake and social behavior related brain circuits.

Oxytocin Receptor Ligands Induce Changes in Cytoskeleton in Neuroblastoma Cells

Aim of the present study was to evaluate effects of ligands of oxytocin receptors on gene expression of neurofilament proteins (nestin and microtubule-associated protein 2 (MAP2)) associated with neuronal differentiation and growth factors (brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF)) related to neuronal growth. Fluorescent staining of F-actin was used to observe morphology of cells. Co-treatment with oxytocin and oxytocin receptor antagonist—atosiban—resulted in significant increase of MAP2 gene expression in SK-N-SH cells. There was no effect of oxytocin on gene expression of growth factors BDNF and NGF. Surprisingly, oxytocin with atosiban significantly increased mRNA levels for both BDNF and NGF. Gene expression of vasopressin receptor (V1aR) significantly decreased in response to vasopressin. Atosiban decreased mRNA levels for oxytocin receptor (OXTR) and V1aR. Oxytocin significantly decreased OXTR and nestin mRNA levels and increased mRNA levels for BDNF and NGF in U-87 MG cells. The densest recruitment of F-actin filaments was observed in apical parts of filopodia in SK-N-SH cells incubated in oxytocin presence. Present data demonstrate complex role of ligands of oxytocin receptors in regulation of gene expression of intermediate filaments and thus, oxytocin might be considered as a growth factor in neuronal type of cells.

Cell swelling-induced insulin secretion from INS-1E cells is inhibited by extracellular Ca2+ and is tetanus toxin resistant.

Cell swelling-induced insulin secretion represents an alternative pathway of stimulation of insulin secretion. INS-1E rat tumor beta cells do not release insulin in response to cell swelling in presence of Ca2+ despite a good response to glucose challenge and appropriate increase in cell volume. Surprisingly, perifusion with Ca2+-depleted medium showed distinct secretory response of INS-1E cells to hypotonicity. Objective of this study was further characterization of the role of Ca2+ in secretory process in INS-1 and INS-1E cell lines. Ca2+ depleted hypotonic medium with 10 µM BAPTA/AM (intracellular chelator) induced insulin secretion from both types of cells. We demonstrated expression of L-type Ca2+ channel Cav1.2 and non-L-type Ca2+ channels Cav2.1 (P/Q-type), Cav2.2 (N-type), and Cav3.1 (T-type) in both cell lines. Inhibition of L type channel with nifedipine and/or P/Q type with ω-agatoxin IVA enabled distinct response to hypotonic medium also in INS-1E cells. Tetanus toxin (TeTx) in medium containing Ca2+ and a group of calcium channel blockers inhibited hypotonicity-induced insulin secretion from INS-1 cells but not from INS-1E cells. Conclusion: Hypotonicity-induced insulin secretion from INS-1E cells is inhibited by extracellular Ca2+, does not require intracellular Ca2+ and is TeTx resistant.