Mojca Kržan

Fusion-related release of glutamate from astrocytes

Although cell culture studies have implicated the presence of vesicle proteins in mediating the release of glutamate from astrocytes, definitive proof requires the identification of the glutamate release mechanism and the localization of this mechanism in astrocytes at synaptic locales. In cultured murine astrocytes we show an array of vesicle proteins, including SNARE proteins, and vesicular glutamate transporters that are required to fill vesicles with glutamate. Using immunocytochemistry and single-cell multiplex reverse transcription-PCR we demonstrate the presence of these proteins and their transcripts within astrocytes freshly isolated from the hippocampus. Moreover, immunoelectron microscopy demonstrates the presence of VGLUT1 in processes of astrocytes of the hippocampus. To determine whether calcium-dependent glutamate release is mediated by exocytosis, we expressed the SNARE motif of synaptobrevin II to prevent the formation of SNARE complexes, which reduces glutamate release from astrocytes. To further determine whether vesicular exocytosis mediates calcium-dependent glutamate release from astrocytes, we performed whole cell capacitance measurements from individual astrocytes and demonstrate an increase in whole cell capacitance, coincident with glutamate release. Together, these data allow us to conclude that astrocytes in situ express vesicle proteins necessary for filling vesicles with the chemical transmitter glutamate and that astrocytes release glutamate through a vesicle- or fusion-related mechanism.

Properties of Ca2+-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 Ca2+‐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 Ca2+‐dependent increases in membrane capacitance, with an apparent Kd value of ∼20 μM [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 Ca2+‐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 Ca2+‐dependent changes in membrane capacitance represent regulated exocytosis from multiple types of vesicles, about 100 times slower than the exocytotic response in neurons.

Number of microvesicles in peripheral blood and ability of plasma to induce adhesion between phospholipid membranes in 19 patients with gastrointestinal diseases

It was recently shown that the plasma protein-mediated attractive interaction between phospholipid membranes could in the budding process cause adhesion of the bud to the mother membrane [J. Urbanija, N. Tomsic, M. Lokar, A. Ambrozic, S. Cucnik, M. Kanduser, B. Rozman, A. Iglic, V. Kralj-Iglic, Coalescence of phospholipid membranes as a possible origin of anticoagulant effect of serum proteins, Chem. Phys. Lipids 150 (2007) 49-57]. Since in the in vivo conditions the budding of cell membranes leads to the release of microvesicles into the circulation, a hypothesis was put forward that the ability of plasma to cause adhesion between membranes supresses the microvesiculation process. In the present work, this hypothesis was tested in a population of 19 patients with gastrointestinal diseases. The number of microvesicles in peripheral blood of patients was determined by flow cytometry while the ability of plasma to cause adhesion between membranes was determined by adding patients plasma to the suspension of giant phospholipid vesicles created by electroformation method, and measuring the average effective angle of contact between the adhered vesicles. Statistically significant negative correlations between the number of microvesicles and the average effective angle of contact (Pearson coefficient -0.50, p=0.031) and between the number of microvesicles per number of platelets and the average effective angle of contact (Pearson coefficient -0.64, p=0.003) were found, which is in favor of the above hypothesis. Patients with gastrointestinal cancer had larger number of microvesicles (difference 140%, statistical significance 0.033) and smaller average effective angle of contact (difference 20%, statistical significance 0.013) compared to patients with other gastrointestinal diseases.

Prevention of microvesiculation by adhesion of buds to the mother cell membrane--a possible anticoagulant effect of healthy donor plasma.

Microvesicles (MVs) found in peripheral blood are derived from the budding of cell membranes and are associated with a higher risk of thrombosis. Recently, a hypothesis has been suggested that certain plasma proteins could suppress microvesiculation by mediating adhesion of the buds to the mother cell membrane. In a pilot study, we have tested this hypothesis by considering the relation between the amount of MVs in peripheral blood and the ability of plasma to induce adhesion between giant phospholipid vesicles (GPVs). MVs were isolated from human plasma and counted by flow cytometry. The adhesion between GPVs was measured by assessing the average angle of contact between the adhered vesicles. It was found that greater ability of plasma to induce adhesion relates to smaller concentration of MVs in plasma. The ratio between the concentration of MVs and the concentration of platelets proved the most efficient parameter to predict the propensity of the membrane to shed vesicles. Our results indicate that a stronger attractive interaction between GPVs mediated by plasma is associated with a smaller amount of MVs per platelets. Plasma that mediates stronger attractive interaction between GPVs might potentially be associated with a smaller risk of thrombosis.

The Quantum Nature of Drug-Receptor Interactions: Deuteration Changes Binding Affinities for Histamine Receptor Ligands

In this article we report a combined experimental and computational study concerning the effects of deuteration on the binding of histamine and two other histaminergic agonists to 3H-tiotidine-labeled histamine H2 receptor in neonatal rat astrocytes. Binding affinities were measured by displacing radiolabeled tiotidine from H2 receptor binding sites present on cultured neonatal rat astrocytes. Quantum-chemical calculations were performed by employing the empirical quantization of nuclear motion within a cluster model of the receptor binding site extracted from the homology model of the entire H2 receptor. Structure of H2 receptor built by homology modelling is attached in the supporting information (S1 Table) Experiments clearly demonstrate that deuteration affects the binding by increasing the affinity for histamine and reducing it for 2-methylhistamine, while basically leaving it unchanged for 4-methylhistamine. Ab initio quantum-chemical calculations on the cluster system extracted from the homology H2 model along with the implicit quantization of the acidic N–H and O–H bonds demonstrate that these changes in the binding can be rationalized by the altered strength of the hydrogen bonding upon deuteration known as the Ubbelohde effect. Our computational analysis also reveals a new mechanism of histamine binding, which underlines an important role of Tyr250 residue. The present work is, to our best knowledge, the first study of nuclear quantum effects on ligand receptor binding. The ligand H/D substitution is relevant for therapy in the context of perdeuterated and thus more stable drugs that are expected to enter therapeutic practice in the near future. Moreover, presented approach may contribute towards understanding receptor activation, while a distant goal remains in silico discrimination between agonists and antagonists based on the receptor structure.

Suppression of membrane microvesiculation--a possible anticoagulant and anti-tumor progression effect of heparin.

Heparins (unfractionated and low molecular weight (LMWH) heparins) primarily used as anticoagulants, were found to be effective also in slowing down the development of some types of cancer. On the other hand, the number of microvesicles in the peripheral blood originating from the budding of cell membranes (mostly platelets) is increased in hypercoagulabile states as well as in cancer, indicating a possible common underlying mechanism. It was hypothesized that by mediating an attractive interaction between phospholipid membranes heparin suppresses microvesiculation and thereby acts as an anticoagulant and anti-tumor agent. In this work, the effect of LMWH nadroparin on phospholipid membranes was tested in vitro in a system of giant phospholipid vesicles (GPVs) created by electroformation and observed under the phase contrast microscope. Plasma of different blood donors containing different concentrations of nadroparin was added to the suspension of GPVs to induce adhesion between GPVs. The attractive interaction between membranes was assessed by measuring the average effective angle of contact between the adhered GPVs. It was found in healthy donors, in a donor with gastrointestinal cancer and in a donor with rheumatoid arthritis that adding therapeutic doses of nadroparin to the plasma samples enhanced adhesion of phospholipid membranes in a dose and time-dependent manner while nadroparin alone had no effect within the therapeutic concentration range. The results are in favor of the hypothesis that suppression of microvesiculation underlies both, the anticoagulant and the anti-tumor progression effect of heparin.

Histamine and astrocyte function

Astrocytes support the brain through numerous functional interactions in health and disease. The recent advances in our knowledge of astrocyte involvement in various neurological disorders raised up several questions about their role and functioning in the central nervous system. From the evidence discussed in this review, we show that histamine importantly influences the main astrocytic activities such as ion homeostasis, energy metabolism, neurotransmitter clearance, neurotrophic activity and immune response. These processes are mediated through at least three histamine receptor subtypes, H1, H2 and H3, expressed on the astrocyte surface. Thus, we recognize histamine as an important player in the modulation of astrocytic functions that deserves further considerations in exploring involvement of astrocytes in neurological disorders.

Histamine (re)uptake by astrocytes: an experimental and computational study

Astrocytes participate in the clearance of neurotransmitters by their uptake and subsequent enzymatic degradation. Histamine as a polar and/or protonated molecule must use a carrier to be transported across the cell membrane, although a specific histamine transporter has not been elucidated, yet. In this work we upgraded the kinetic studies of histamine uptake into neonatal rat cultured type 1 astrocytes with quantum chemical calculations of histamine pKa values in conjunction with Langevin dipoles solvation model as the first step toward microscopic simulation of transport. Our results indicate that astrocytes transport histamine by at least two carrier mediated processes, a concentration gradient dependent passive and a sodium-dependent and ATP-driven active transport. We also demonstrated that histamine protonation states depend on the polarity of the environment. In conclusion we suggest that histamine, a polar molecule at physiological pH uses at least two different mechanisms for its uptake into astrocytes –an electrodiffusion and Na+-dependent and ouabain sensitive active process. We emphasize relevance of knowledge of histamines protonation states at the rate limiting step of its transport for microscopic simulation that will be possible when structure of histamine transporter is known.

Molecular and kinetic characterization of histamine transport into adult rat cultured astrocytes.

Astrocytes have a key role in the clearance and inactivation of histamine in the adult central nervous system, but transporters which mediate histamine uptake into astrocytes have not been fully characterized. We therefore investigated the kinetic and molecular characteristics of histamine uptake into cultured adult rat astrocytes. [(3)H]-histamine was taken up by astrocytes in a temperature-, time- and concentration-dependent manner and was inhibited up to 60-70% by 1mM ouabain or by substitution of NaCl with choline chloride. Specific [(3)H]-histamine uptake, determined as the difference between transport at 37 and 4°C, displayed saturation kinetics with the apparent Michaelis-Menten constant (K(m)) of 141 and 101μM and the apparent maximal uptake rate (V(max)) of 22.5 and 17.8pmol/min/mg protein, as estimated from the Woolf and the Eadie-Hofstee plots, respectively. Since our data suggested the presence of a carrier-operated histamine uptake system, we assessed the possible involvement of the organic cation transporters (OCT) 1, 2 and 3, which have been previously described to play a role in histamine transport in the central nervous system. Low level mRNA expression of all OCT isoforms was detected, but in contrast to rat brain cortex homogenate, where OCT3 was the most prominently expressed OCT isoform, OCT2 mRNA was the predominant OCT species in cultured astrocytes. However, OCT inhibitors corticosterone and decynium 22 (D22) had no effect or only modestly reduced [(3)H]-histamine uptake. Thus, our data indicate that adult rat astrocytes possess an efficient high-capacity, low-affinity carrier-operated histamine uptake system, which does not seem to involve OCTs.

Kinetic and pharmacological properties of [3H]-histamine transport into cultured type 1 astrocytes from neonatal rats

Abstract.Objective and design:Astrocytes actively participate in the inactivation of neurotransmitters. In this work we elucidated the contribution of astrocytes in clearance of histamine, a process which has not yet been fully clarified.Methods:The characteristics of [3H]-histamine uptake were determined in cultured neonatal rat type 1 astrocytes and histamine-N-methyl-transferase expression was determined using RT-PCR.Results:These cells transport [3H]-histamine in a time- and concentration-dependent manner. The histamine clearance by astrocytes was described by a mathematical model including two processes: electrodiffusion and active transport. A further analysis of kinetic parameters of a carrier-operated transport revealed a single transport system with Michaelis constant (Km) of 3.5 ± 0.8 μM and a maximal uptake rate (Vmax) of 7.9 ± 0.3 pmol/mg protein/min. From drugs tested amitriptyline, desipramine, mepyramine and cimetidine significantly decreased [3H]-histamine uptake. Taken-up histamine could be metabolically degraded in cultured astrocytes, since they express mRNA for enzyme histamine-N-methyltransferase.Conclusions:Astrocytes participate in the clearance of extracellular histamine by electrodiffusion and active transport by a yet not identified carrier. Taken up histamine can be converted to tele-methylhistamine within astrocytes thus indicating the involvement of astrocytes not only in clearance but also in the inactivation of histamine.