Penetration of substances into tumor tissue: A methodological study with microelectrodes and cellular spheroids

Summary A new method was tested for studies of penetration of substances into tumorlike tissue. The penetration of the ions K⁺, Cl⁻, and Ca²⁺ through several layers of tumor cells was demonstrated by using double barrelled, ion sensitive microelectrodes with extra thin tip diameters. Spheroids consisting of human glioma, U-118 MG, and human thyroid cancer, HTh-7, cells were used as models of tumor tissue. A microelectrode was inserted into the center of a spheroid. Thereafter, the concentration of the test substance was increased in the surrounding medium. The change in concentration inside the spheroid was recorded and the penetration pattern evaluated. All three types of tested ions penetrated easily through the spheroids. The K⁺ ions penetrated most efficiently, and the Ca²⁺ ions showed the slowest penetration. The Ca²⁺ ions penetrated somewhat more slowly in the U-118 MG spheroids (which had rather small extracellular spaces) than in the HTh-7 spheroids (which had larger extracellular spaces). Ion sensitive electrodes, which are easily available, were used in this study only to demonstrate the principle. We hope that the method described can be used for penetration studies of various substances. For example, all substances that can be detected by enzyme microelectrodes could be studied. The main advantage of the method is that the complete penetration pattern can be studied as a function of time in individual spheroids. Previously described methods require histological procedures for each analyzed penetration time. The work has been supported financially by the Max-Planck-Gesellschaft, Munich, the Swedish Cancer Society, and the Swedish National Defence Research Institute.     

Homogeneous penetration but heterogeneous binding of antibodies to carcinoembryonic antigen in human colon carcinoma HT-29 spheroids

Summary The monoclonal antibodies 38S1, directed against the carcinoembryonic antigen (CEA), were tested for penetration and binding in human colon carcinoma HT-29 spheroids. Penetration was studied with a method which has not previously been used in immunological investigations. The method, which allows unbound substances to be visualized, is based on freeze drying, vapour fixation, dry sectioning and dry autoradiography. The antibodies penetrated easily and all parts of the HT-29 spheroids seemed to be reached within 15 min. The penetration was even faster than in control glioma U-118MG spheroids that did not express CEA. Binding of the 38S1 antibodies was demonstrated after processing with conventional histology and autoradiography. The binding in the HT-29 spheroids was, after a 1-h incubation period, extremely heterogeneous and occurred mainly in the peripheral parts. More cells were binding the antibodies after 8-h and 32-h incubations and these cells were arranged in peripheral clusters. No binding at all was seen in the CEA-negative glioma spheroids. The distribution of CEA antigens in monolayers and in frozen sections of spheroids of HT-29 cells was analysed with immunohistochemical staining using polyclonal CEA antibodies. The CEA antigens were heterogeneously distributed in both spheroids and monolayers and were as heterogeneous as the binding of the monoclonal antibodies in the living spheroids. Thus, the heterogeneous binding in the living spheroids was not due to penetration barriers, but instead to the heterogeneity in the CEA antigen expression.     

Influence of ionizing radiation on oxygen profiles in different types of multicellular spheroids

Human glioma (U-118 MG and U-138 MG), human colorectal adenocarcinoma (HT-29), human thyroid carcinoma (HTh 7), and hamster embryonic lung (V79-379A) spheroids were irradiated with either single doses of 16 or 40 Gy or fractionated doses of eight times 5 Gy. Oxygen profiles in the spheroids were measured with microelectrodes at different times following irradiation, and these profiles were then compared with the oxygen profiles measured in parallel cultured nonirradiated spheroids. No significant radiation-induced changes in the oxygen profiles were seen in any of the spheroids within the first few days after irradiation. The glioma spheroids did not show any significant increase in oxygen tension even after longer times; however, they were growth inhibited, and the number of S-phase cells was strongly suppressed. Increases in oxygen tension did occur in the HT-29 and V79-379A spheroids but only appeared more than a week after irradiation, when degeneration had started. Histological changes and decrease in diameter were seen in the spheroids that started to degenerate about 5 days after irradiation. Thus radiation doses in the therapeutic range did not, for the spheroids studied, produce rapid increases in the oxygen tension. When a change occurred, it appeared rather late and was probably a consequence of cell degeneration.     

Effects of the 5-lipoxygenase inhibitors AA-863 and U-60,257 on human glioma cell lines

The effects of two specific 5-lipoxygenase inhibitors AA-863 and U-60,257 (piriprost) on the growth of two human glioma cell lines, U-343 MGa and U-251 MG were investigated. Both monolayer cultured cells and spheroids were studied. The results of the monolayer studies showed potent and dose dependent inhibitory effects on the proliferation of glioma cells (IC50/one week treatment/of AA-863: 9.0 microM, IC50 of U-60,257: 40.0 microM). The experiments made on the tumor spheroids suggested an inhibitory effect on proliferation and volume growth already at lower doses (AA-863: 0.4-2.0 microM, U-60,257: 1.0-5.0 microM), a dose range where effects were not found in monolayers. At higher doses (AA-863: 10.0-30.0 microM, U-60,257: 30.0-90.0 microM) the experiments with spheroids failed to demonstrate a further inhibitory effect on spheroid volume, probably attributed to phenomena such as swelling of cells, dissociation of spheroid structure and development of necrosis. The clearly dose dependent inhibitory effect on the proliferation of human glioma cells in monolayer culture and the inhibitory effects on spheroid growth with these specific inhibitors indicate a role for lipoxygenase products in the growth of gliomas.     

Glucose increases extracellular [Ca2+] in rat insulinoma (INS-1E) pseudoislets as measured with Ca2+-sensitive microelectrodes

Secretory granules of pancreatic β-cells contain high concentrations of Ca2+ ions that are co-released with insulin in the extracellular milieu upon activation of exocytosis. As a consequence, an increase in the extracellular Ca2+ concentration ([Ca2+]ext) in the microenvironment immediately surrounding β-cells should be expected following the exocytotic event. Using Ca2+-selective microelectrodes we show here that both high glucose and non-nutrient insulinotropic agents elicit a reversible increase of [Ca2+]ext within rat insulinoma (INS-1E) β-cells pseudoislets. The glucose-induced increases in [Ca2+]ext are blocked by pretreatment with different Ca2+ channel blockers. Physiological agonists acting as positive or negative modulators of the insulin secretion and drugs known to intersect the secretory machinery at different levels also induce [Ca2+]ext changes as predicted on the basis of their described action on insulin secretion. Finally, the glucose-induced [Ca2+]ext increase is strongly inhibited after disruption of the actin web, indicating that the dynamic [Ca2+]ext changes recorded in INS-1E pseudoislets by Ca2+-selective microelectrodes occur mainly as a consequence of exocytosis of Ca2+-rich granules. In conclusion, our data directly demonstrate that the extracellular spaces surrounding β-cells constitute a restricted domain where Ca2+ is co-released during insulin exocytosis, creating the basis for an autocrine/paracrine cell-to-cell communication system via extracellular Ca2+ sensors.     

Method for the determination of oxygen consumption rates and diffusion coefficients in multicellular spheroids

A method has been developed for the quantitative evaluation of oxygen tension (PO2) distributions in multicellular spheroids measured with O2-sensitive microelectrodes. The experimental data showed that multicellular tumor spheroids in stirred growth media were characterized by a diffusion-depleted zone surrounding the spheroids. This zone was elicited by an unstirred layer of medium next to the spheroid leading to a continuous decrease in the PO2 values from the bulk medium towards the spheroid surface. Theoretical considerations demonstrate that the volume-related O2 consumption rate, Q, in the spheroids can be assessed by measuring the PO2 gradient in the diffusion-depleted zone outside the spheroids. Accordingly, Krogh's diffusion constant, KS, in the spheroids can be determined through measuring the PO2 gradient within the spheroids. The results obtained suggest that multicellular spheroids represent useful in vitro tumor models for the experimental and theoretical analysis of the interrelationship among O2 supply to tumor cells, O2 metabolism in tumors tissue, and the responsiveness of cancer cells to treatment.     

受体酪氨酸激酶Axl对胶质母细胞瘤细胞增殖、凋亡和侵袭的影响

摘要 目的：研究受体酪氨酸激酶Axl在胶质母细胞瘤组织和细胞系U-118MG细胞中的表达情况及其对U-118MG细胞增殖、凋亡、侵袭的影响。方法：收集2015年3月至2018年5月在本院进行手术切除并经病理分型证实的胶质母细胞瘤组织标本（n=30）,另取脑外伤手术中因作内减压而切除的正常脑组织作为对照（n=28）。采用荧光实时定量 (qRT-PCR)检测正常脑组织和胶质母细胞瘤肿瘤组织中Axl mRNA表达水平;采用Western blot检测人小神经胶质HM细胞、U-118MG细胞以及Axl-shRNA转染后U-118MG细胞中Axl蛋白表达水平;采用CCK-8检测Axl-shRNA转染后U-118MG细胞增殖能力;采用流式细胞术检测Axl-shRNA转染后U-118MG细胞凋亡水平;采用Transwell小室实验检测Axl-shRNA转染后U-118MG细胞的侵袭能力。结果：在胶质母细胞瘤组织中Axl mRNA表达水平显著高于正常脑组织（P<0.05）;U-118MG细胞Axl蛋白表达水平显著高于人小神经胶质细胞系HM细胞,差异有统计学意义（P<0.05）;转染Axl-shRNA后,U-118MG细胞中Axl蛋白表达水平显著降低（P<0.05）。与U-118MG细胞和转染control-shRNA细胞相比, 转染Axl-shRNA的U-118MG细胞增殖能力降低（P<0.05）,凋亡水平升高（P<0.05）,侵袭能力降低（P<0.05）。结论：在胶质母细胞瘤组织和U-118MG细胞中,Axl表达水平显著增高,并且Axl表达水平与U-118MG细胞增殖、凋亡及侵袭密切关联。     

Increased nanoparticle penetration in collagenase-treated multicellular spheroids

The extracellular matrix of solid tumors presents a transport barrier that restricts nanoparticle penetration, thereby limiting the efficacy of nano-sized delivery vehicles for cancer imaging and therapy. In this study, the effect of nanoparticle size and collagenase treatment on penetration of carboxylated polystyrene nanoparticles was systematically assessed in a multicellular spheroid model. Penetration of the nanoparticles into the spheroid core was limited to particles smaller than 100 nm. Collagenase treatment of spheroids resulted in significantly increased penetration of nanoparticles up to 100 nm with only a minor increase in particle penetration observed for particles larger than 100 nm. Collagenase was immobilized onto the surface of nanoparticles for site-specific degradation of ECM proteins. Collagenase-coated, 100 nm nanoparticles demonstrated a 4-fold increase in the number of particles delivered to the spheroid core compared with control nanoparticles. Thus, nanoparticle delivery to solid tumors may be substantially improved by the incorporation of ECM-modulating enzymes in the delivery formulation.     

Penetration of substances into tumor tissue—A methodological study on cellular spheroids

Summary The penetration of [³H]thymidine, [³H]d-leucine, [¹²⁵I]albumin, and the drugs [³H]5-fluorouracil and [³H]vinblastine into human glioma spheroids (in vitro tumor models) was studied by a method based on rapid freezing, freeze drying, vapor fixation, wax embedding, dry sectioning, and contact autoradiography. No significant disturbances in the distribution of water soluble substances were observed. Thymidine andd-leucine penetrated the whole spheroids relatively fast, whereas albumin showed reduced penetration. the concentration of albumin was highest at the periphery of the spheroids, but only smaller amounts were detected in the deeper regions. A significant difference between the penetration patterns of the drugs studied was also observed. Fluorouracil penetrated rather freely, but the penetration of vinblastine was limited. The work was supported financially by Lennanders Foundation, OE and Edla Johanssons Foundation, and the Swedish Cancer Society.     

Single potassium channels of human glioma cells.

Single potassium channels in the membrane of human malignant glioma cells U-118MG were studied using the technique of patch clamp in cell-attached and inside-out configurations. Three types of potassium channels were found which differed from each other under conditions close to physiological in their conductance and gating characteristics. The lowest-conductance channel (20 pS near the reversal potential) showed a mild outward rectification up to 45 pS at positive voltages and spontaneous modes of high and low activity. At extreme values of potentials its activity was generally low. The intermediate conductance channel had an S-shaped I-V curve, giving a conductance of 63 pS at reversal, and a low and voltage independent opening probability. The high-conductance (215 pS) channel was found to be activated by both membrane potential and Ca2+ ions and blocked by internal sodium at high voltages. The current-voltage curves of all three channel types displayed saturation.     

Sustaining olfaction at low salinities: evidence for a paracellular route of ion movement from the hemolymph to the sensillar lymph in the olfactory sensilla of the blue crab Callinectes sapidus

Evidence reported previously suggests that in low-salinity conditions the integrity of the olfactory dendrites of the blue crab is sustained by a diffusion-generated ionic microenvironment within the aesthetascs. Diffusion of ions from the hemolymph to the sensillar lymph is proposed to maintain this microenvironment. In this study, using lanthanum as an electron-dense marker of extracellular fluid space, we find morphological evidence for paracellular continuity between the hemolymph and the sensillar lymph. Lanthanum penetrates extracellular fluid spaces within the aesthetascs when antennules are either perfused or bathed externally with solutions containing lanthanum nitrate. This was found in both freshwater- and seawater-acclimated animals. Evidence for ion diffusion from the aesthetascs was obtained using self-referencing, ion-selective microelectrodes. Both Ca2+ and K+ exhibit outwardly directed flux gradients associated with the aesthetasc tuft in low-salinity conditions. These findings are consistent with the concept that ion diffusion from the hemolymph to the sensillar lymph generates an ionic/osmotic microenvironment within the aesthetascs at low salinities.     

Intracellular mechanisms involved in leukotriene C4-stimulated adhesion of U-937 cells.

Leukotrienes C4 and D4 (LTC4 and LTD4) stimulated, 5- to 6-fold, the adhesion of the monoblastoid cell line U-937 to plastic. Half-maximal effects were observed around 1 nM. Leukotrienes E4 and B4 (LTE4 and LTB4) were less effective. The adhesive response to LTC4 was inhibited by pertussis toxin and was completely dependent on the presence of extracellular Ca2+. The LTC4-stimulated increases in inositol-phosphates and in intracellular Ca(2+)-concentration were insensitive to pertussis toxin. Activation of leukocyte adhesion is a novel action of cysteinyl-leukotrienes and the present study suggests that control of U-937-cell adhesion by LTC4 involves two pathways; one pertussis toxin insensitive pathway regulating intracellular Ca2+ in a manner partly dependent on extracellular Ca2+ and one pertussis toxin sensitive pathway not concerned with Cai(2+)-regulation.     

Effects of the PAF-analog and -antagonist CV-6209 on cultured human glioma cell lines

Cell lines of human glioma (U-343 MGa and U-251 MG) and human glia (U-533 CG) origin were cultured as monolayers and exposed to CV-6209, an alkyl-phospholipid analog and antagonist of platelet activating factor. This drug had very potent antiproliferative effects on the studied human glioma cell lines; IC50 was 0.9 microM after 48 h treatment and 0.2 microM after 2 weeks treatment. At these doses no growth inhibitory effect was noted on the normal glia cells. The effects on the glioma cells were reversible in the dose intervals, where cell proliferation, 3H-thymidine and 14C-methionine uptakes were greatly inhibited. The simultaneous administration of platelet activating factor [(R)PAF] did not influence the antiproliferative effects of CV-6209 on the cells cultured as monolayers. The structurally similar analog CV-3988 also had antiproliferative effects, although at 10 times higher concentration than CV-6209. Two other, structurally unrelated, PAF-antagonists (WEB-2086 and TCV-309) gave effects only at very high concentrations. The U-343 MGa cell line was also exposed to CV-6209 when growing as multicellular spheroids. The studies on the spheroid cultures also demonstrated good antitumoral effects with decreases of both the volume growth and the thymidine uptake. The simultaneous administration of (R)PAF reversed the inhibitory effect of CV-6209 on thymidine incorporation. This study demonstrates a strong antitumoral effect at low concentrations of CV-6209. The antiproliferative effects were probably primarily related to the ether-lipid structure and not to the PAF-antagonistic properties. The good antitumoral effect of CV-6209 on both monolayer and spheroid cultures and the possible PAF-antagonistic properties are discussed.     

Issues involved in the transmission of chemical signals through the brain extracellular space

Two classes of substances exist within the extracellular space: energetic and informational. Examples of the former are glucose, dissolved oxygen and CO2 while the latter include excitatory amino acids, cathecholamines and opiates. The simple ions Na+ and Cl- are generally associated with energetic processes while extracellular K+ and Ca2+ tend to be informational in function. Local release of an informational substance brings about a concentration gradient that causes the substance to be dispersed in the extracellular space by diffusion. This process is modified relative to a free aqueous medium by the constraints of volume fraction, tortuosity and uptake. Volume fraction is defined simply as the fraction of a brain region that is extracellular. If a given quantity of substance is released into a region with a reduced volume fraction then the substance will reach a higher concentration than it would in a free medium. Tortuosity is related to the increase in the path length of the random walk of a diffusing particle due to the necessity to navigate around cellular obstructions. Tortuosity manifests itself as a decrease in the diffusion coefficient. Uptake represents the movement of a substance from the extracellular space to the intracellular. Since initially a concentration gradient exists in this direction and all membranes have some permeability some concentration-dependent uptake always occurs. In addition there exist specific carrier-mediated uptake processes for some substances such as amino acids or catecholamines. In some regions the dispersal process can be dominated by uptake rather than diffusion. While volume fraction, tortuosity and uptake have all been demonstrated by a technique based on the use of radiolabels and other methods, these classical techniques have limited spatial and temporal resolution. The advent of methods based on micro-injection of substances by iontophoresis or pressure and subsequent detection with ion-selective microelectrodes (ISMs) or voltammetric microsensors (VMs) has opened a new window onto the dynamic local behavior of the extracellular space. In the last decade our laboratory and others have studied the migration of the test substances tetramethylammonium, tetraethylammonium, AsF6- and alpha naphthalene sulfonate, the endogenous ions K+ and Ca2+, the epileptogenic agent penicillin and the neurotransmitter dopamine. These studies have been carried out on the cerebellum and some other regions in a variety of species that include rat, turtle, skate and an intervertebrate, the cuttlefish.(ABSTRACT TRUNCATED AT 400 WORDS)     

C1q induces chemotaxis and K+ conductance activation coupled to increased cytosolic Ca2+ in mouse fibroblasts

Cultured mouse fibroblasts (L cells) respond to whole C with a slow hyperpolarization. Among the C components tested, C1q was found to be most effective. In contrast, the cell did not respond to C1, in which the collagen-like region of the C1q molecule is masked. The C1q-induced hyperpolarizing response was inhibited by collagen or C1q-specific antisera. Human diploid skin fibroblasts (Flow 1,000 cells) also exhibited similar membrane potential changes in response to whole C or C1q. After repeated applications of C1q, the cell membrane became unresponsive (desensitized). The treatment of L cells with pronase E inhibited the C1q-induced response, whereas the response to ATP, which is known to interact to its own receptor, was still preserved. The reversal potential of C responses was close to the K+ equilibrium potential. The hyperpolarizing response was inhibited by a blocker of Ca2+-activated K+ channels in fibroblasts (quinine), by deprivation of extracellular Ca2+ or by a Ca2+ channel blocker (nifedipine). By means of Ca2+-selective microelectrodes, the cytosolic free Ca2+ concentration was found to increase from 126 to 206 nM upon stimulation of L cells with C1q. Using an agarose-well method, L cells were observed to migrate predominantly toward C1q or whole C. It is concluded that the fibroblasts have the C1q receptor sensitive to pronase E and that activation of C1q receptors gives rise to Ca2+ influx, triggering an increase in the cytosolic free Ca2+ ions, which in turn induces a hyperpolarizing response as a result of the stimulation of Ca2+-activated K+ channels and initiates chemotaxis to C1q.     

Predicting the growth of glioblastoma multiforme spheroids using a multiphase porous media model

Tumor spheroids constitute an effective in vitro tool to investigate the avascular stage of tumor growth. These three-dimensional cell aggregates reproduce the nutrient and proliferation gradients found in the early stages of cancer and can be grown with a strict control of their environmental conditions. In the last years, new experimental techniques have been developed to determine the effect of mechanical stress on the growth of tumor spheroids. These studies report a reduction in cell proliferation as a function of increasingly applied stress on the surface of the spheroids. This work presents a specialization for tumor spheroid growth of a previous more general multiphase model. The equations of the model are derived in the framework of porous media theory, and constitutive relations for the mass transfer terms and the stress are formulated on the basis of experimental observations. A set of experiments is performed, investigating the growth of U-87MG spheroids both freely growing in the culture medium and subjected to an external mechanical pressure induced by a Dextran solution. The growth curves of the model are compared to the experimental data, with good agreement for both the experimental settings. A new mathematical law regulating the inhibitory effect of mechanical compression on cancer cell proliferation is presented at the end of the paper. This new law is validated against experimental data and provides better results compared to other expressions in the literature.     

Potassium dependence for sperm-egg fusion in mice

In this study, we examined the potassium requirements for sperm-egg fusion in mouse. Zona-free mouse eggs prepared by the method described by Boldt and Wolf were inseminated with capacitated sperm in culture media containing 0-6 mM extracellular K+, and scored for penetration. Penetration of zona-free eggs was dependent on extracellular K+, with no penetration observed under K(+)-free conditions. Media transfer experiments indicated that the lack of penetration observed was due to effects on fusion, and not on postpenetration events such as sperm head decondensation. To analyze whether the K+ effect was attributable to an effect on the sperm (i.e., occurrence of acrosome reactions), sperm were treated with the Ca2+ ionophore A23187 before insemination. Less than 5% of zona-free eggs were penetrated with ionophore-treated sperm under K(+)-free conditions, suggesting that K+ is required for fusion per se. Addition of ionophore to insemination cultures similarly did not overcome the block to fusion observed under K(+)-free conditions. The potassium channel blockers 4-aminopyridine (0.1-5 mM) and tetraethyl ammonium chloride (5-50 mM) had no inhibitory effect on fusion. These data indicate that extracellular K+ is required for sperm-egg fusion and that this requirement may not involve membrane K+ channels.     

Comparison of NO production level and proliferation of MCF-7 breast tumor cells under conditions of microenvironment modification

Connection between the level of NO generation and its cytotoxic influence was investigated on two models of tumor cells (breast adenocarcinoma MCF-7) culture: monolayer and spheroids. Conditions of microenvironment were modified by Ca2+ channels blockers: EGTA, nitrendipine and Cl-lanthan. It was discovered in the work that deviation in concentration of extracellular Ca2+ under the influence of EGTA leads to reducing of the level of extracellular NO to 50% (P < or = 0.05) of control and increasing of the number of live cells by 10-40% (P < or = 0.05). Nitrendipin has the properties selective Ca2+ channels blocker that reduced the level of NO by 35-70% (P < or = 0.05), depending on the substances concentration and time of incubation. At that time proliferation potential of cells increased by 10-35% (P < or = 0.05). NO production in MCF-7 cells under the influence of Cl-lanthan in concentration of 0.01 and 0.1 mM is reduced by 50-60%, as compared with the control. But it had no influence on the number of live cells. On 2-D and 3-D models of tumor growth it was demonstrated that extracellular NO production in monolayer of MCF-7 was 3.2 pmol for 10(6) cells and for spheroids it was 8.5 pmol.     

Distributions of oxygen,nutrient, and metabolic waste concentrations in multicellular spheroids and their dependence on spheroid parameters

The distribution of oxygen, nutrients and metabolic wastes in multicellular tumor spheroids and its dependence on the parameters characterizing the spheroid (i.e., spheroid geometry, diffusivity, and consumption/ production rates of biological substances) have been investigated by a theoretical analysis: 1. Parameter dependence is qualitatively demonstrated and visualized. 2. Reduction of the number of variables by specific coordinate transformations made it possible to generate nomograms from which concentration distributions for any choice of parameter values may easily be obtained. In particular, these nomograms may also be used for estimating concentration profiles of metabolic waste products, e.g. of lactate, which are expected to accumulate in the tumor spheroids. 3. An additional set of nomograms is given which is more convenient for determining time courses of these concentrations during spheroid growth. 4. A quantitative sensitivity analysis of parameter dependencies is performed to identify those parameters upon which a concentration of interest depends most critically in a given experimental situation. Offprint requests to: W Mueller-Klieser     

The antidepressant mirtazapine-induced cytosolic Ca2+ elevation and cytotoxicity in human osteosarcoma cells

The effect of the antidepressant mirtazapine on cytosolic free Ca2+ concentration ([Ca2+]i) and viability has not been explored in any cell type. This study examined whether mirtazapine alters Ca2+ levels and causes cell death in osteoblast-like cells using MG63 human osteosarcoma cells as a model. [Ca2+]i and cell viability were measured using the fluorescent dyes fura-2 and WST-1, respectively. Mirtazapine at concentrations above 250 microM increased [Ca2+]i in a concentration-dependent manner. The Ca2+ signal was reduced by 60% by removing extracellular Ca2+. The mirtazapine-induced Ca2+ influx was sensitive to blockade of nifedipine and verapamil. In Ca(2+)-free medium, after pretreatment with 1.5 mM mirtazapine, 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor), 2 microM CCCP (a mitochondrial uncoupler), and 1 microM ionomycin failed to release more stored Ca2+; conversely, pretreatment with thapsigargin, CCCP and ionomycin abolished mirtazapine-induced Ca2+ release. Inhibition of phospholipase C with 2 microM U73122 did not change mirtazapine-induced [Ca2+]i, increase. Seal of Ca2+ movement across the plasma membrane with 50 microM extracellular La3+ enhanced 1 microM thapsigargin-induced [Ca2+]i increase, suggesting that Ca2+ efflux played a role in lowering thapsigargin-induced [Ca2+]i increase; however, the same La3+ treatment did not alter mirtazapine-induced [Ca2+]i increase. At concentrations of 500 microM and 1000 microM, mirtazapine killed 30% and 60% cells, respectively. The cytotoxicity was not reversed by chelating cytosolic Ca2+ with BAPTA. Collectively, in MG63 cells, mirtazapine induced a [Ca2+]i increase by causing Ca2+ release from stores and Ca2+ influx from extracellular space. Furthermore, mirtazapine caused cytotoxicity at higher concentrations in a Ca(2+)-dissociated manner.