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

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.     

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.     

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.     

The ionic hemolymph composition of the terrestrial isopod Porcellio scaber Latr. during molt

We analyzed the ionic composition of the hemolymph of Porcellio scaber in four different stages of the molt cycle using capillary electrophoresis and calcium selective mini- and microelectrodes. The main ions in the hemolymph were K⁺, Ca²⁺, Na⁺, Mg⁺, and Cl⁻. The values for total calcium obtained by means of capillary electrophoresis and calcium selective minielectrodes did not differ significantly from each other. In situ measurements of the free Ca²⁺ concentration ([Ca²⁺]) by means of calcium-selective microelectrodes indicated that Ca²⁺ is not bound in the hemolymph. During molt the [Ca²⁺] is significantly larger than during intermolt. The [Ca²⁺] increased by 13%, 19% and 18% during premolt, intramolt, and postmolt, respectively. The concentration of the other cations and of Cl⁻ decreased significantly between premolt and intramolt. Thus, the rise of the [Ca²⁺] in the hemolymph is not due to a general increase in all ions, but rather to the resorption of cuticular calcium. Furthermore, the results suggest that K⁺, Na⁺, Mg⁺, and Cl⁻are extruded from the hemolymph during and/or after posterior ecdysis. Accepted: 5 August 1997     

Analysis of calcium signaling in live human Tongue cell 3D-Cultures upon tastant perfusion

Bridging the gap between two-dimensional cell cultures and complex in vivo tissues, three-dimensional cell culture models are of increasing interest in the fields of cell biology and pharmacology. However, present challenges hamper live cell imaging of three-dimensional cell cultures. These include (i) the stabilization of these structures under perfusion conditions, (ii) the recording of many z-planes at high spatio-temporal resolution, (iii) and the data analysis that ranges in complexity from whole specimens to single cells. Here, we addressed these issues for the time-lapse analysis of Ca²⁺ signaling in spheroids composed of human tongue-derived HTC-8 cells upon perfusion of gustatory substances. Live cell imaging setups for confocal and light sheet microscopy were developed that allow simple and robust spheroid stabilization and high-resolution microscopy with perfusion. Visualization of spheroids made of HTC-8 cells expressing the G-GECO fluorescent Ca²⁺ sensor revealed Ca²⁺ transients that showed similar kinetics but different amplitudes upon perfusion of bitter compounds Salicine and Saccharin. Dose-dependent responses to Saccharin required extracellular Ca²⁺. From the border towards the center of spheroids, compound-induced Ca²⁺ signals were progressively delayed and decreased in amplitude. Stimulation with ATP led to strong Ca²⁺ transients that were faster than those evoked by the bitter compounds and blockade of purinergic receptors with Suramin abutted the response to Saccharin, suggesting that ATP mediates a positive autocrine and paracrine feedback. Imaging of ATP-induced Ca²⁺ transients with light sheet microscopy allowed acquisition over a z-depth of 100 μm without losing spatial and temporal resolution. In summary, the presented approaches permit the study of fast cellular signaling in three-dimensional cultures upon compound perfusion.     

Galactocerebroside mediated transmembrane Ca2+ signalling in U-87 MG cells: Possible involvement of phosphoinositides

Antibody to galactocerebroside (anti- GalC) has been shown to evoke a Ca²⁺ response in cultured glioma U- 87 MG cells. The rise in [Ca²⁺]_i was due to release of Ca²⁺ from the intracellular stores and influx through the plasma membrane. The rise in [Ca²⁺]_i was markedly inhibited by neomycin sulphate and phorbol dibutyrate suggesting the involvement of phosphoinositides in Ca²⁺ mobilization. The Ca²⁺ response induced by anti- GalC was rapidly desensitized and repeated addition of anti- GalC did not elevate the [Ca²⁺]_i. Heterologous desensitization was observed with bradykinin and adenosine triphosphate. The intracellular Ca²⁺ store mobilized by anti- GalC appears to be the IPin3 sensitive pool of endoplasmic reticulum. The influx of Ca²⁺ is mediated by a channel. The Ca²⁺ influx was also prevented by pretreatment of cells with neomycin sulphate or phorbol dibutyrate. We propose that galactocerebroside may be associated with phospholipase C or other proteins linked to the phosphoinositide pathway of transmembrane signalling and anti- GalC activates the breakdown of phosphoinositides and thus mobilizes Ca²⁺ in U-87 MG cells.     

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.     

Effect of external K+, Ca2+, and Ba2+ on membrane potential and ionic conductance in rat astrocytes

Summary 1. The purpose of this study was (a) to identify if astrocytes show a similar non-Nernstian depolarization in low K⁺ or low Ca²⁺ solutions as previously found in human glial and glioma cells, and (b) to analyze the influence of the K⁺ conductance on the membrane potential of astrocytes.2. The membrane potential (E_m) and the ionic conductance were studied with whole-cell patch-clamp technique in neonatal rat astrocytes (5–9 days in culture) and in human glioma cells (U-251MG).3. In 3.0 mM K⁺, E_m was –75 ± 1.0 mV (mean ± SEM,n=39) in rat astrocytes and –79 ± 0.7 mV (n=5) in U-251MG cells. In both cell types E_m changed linearly to the logarithm of [K⁺]₀ between 3.0 and 160 mM K⁺. K⁺ free medium caused astrocytes to hyperpolarize to –93 ± 2.7 mV (n=21) and U-251MG cells to depolarize to –27 ± 2.1 mV (n=3).4. The I-E curve did not show inward rectification in astrocytes at this developmental stage. The slope conductance (g) exhibited only a small decrease (–19%) in K⁺ free solution and no significant change in 160 mM K⁺.5. Ba²⁺ (1.0 mM) depolarized astrocytes to –45 ± 2.9 mV (n=11), decreasing the slope conductance (g) by 42.4 ± 8.3% (n=11). Ca²⁺ free solution depolarized astrocytes to –53 ± 3.4 mV (n=12) and resulted in a positive shift of the I-E curve, increasing g by 15.3 ± 8.2% (n=8).6. Calculations indicated that a block of K⁺ channels explains the depolarizing effect of Ba²⁺. The effects of K⁺ free or Ca²⁺ free solutions on E_m can be explained by a transformation of K⁺ channels to non-specific leakage channels. That astrocytes show a different reaction to low K⁺ than glioma cells can be related to the lack of inwardly rectifying K⁺ channels in astrocytes at this developmental stage.     

Ca2+-dependent mechanism of membrane insertion and destabilization by the SARS-CoV-2 fusion peptide

Cell penetration after recognition of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus by the ACE2 receptor and the fusion of its viral envelope membrane with cellular membranes are the early steps of infectivity. A region of the Spike protein of the virus, identified as the “fusion peptide” (FP), is liberated at its N-terminal site by a specific cleavage occurring in concert with the interaction of the receptor-binding domain of the Spike. Studies have shown that penetration is enhanced by the required binding of Ca²⁺ ions to the FPs of coronaviruses, but the mechanisms of membrane insertion and destabilization remain unclear. We have predicted the preferred positions of Ca²⁺ binding to the SARS-CoV-2-FP, the role of Ca²⁺ ions in mediating peptide-membrane interactions, the preferred mode of insertion of the Ca²⁺-bound SARS-CoV-2-FP, and consequent effects on the lipid bilayer from extensive atomistic molecular dynamics simulations and trajectory analyses. In a systematic sampling of the interactions of the Ca²⁺-bound peptide models with lipid membranes, SARS-CoV-2-FP penetrated the bilayer and disrupted its organization only in two modes involving different structural domains. In one, the hydrophobic residues F833/I834 from the middle region of the peptide are inserted. In the other, more prevalent mode, the penetration involves residues L822/F823 from the LLF motif, which is conserved in CoV-2-like viruses, and is achieved by the binding of Ca²⁺ ions to the D830/D839 and E819/D820 residue pairs. FP penetration is shown to modify the molecular organization in specific areas of the bilayer, and the extent of membrane binding of the SARS-CoV-2 FP is significantly reduced in the absence of Ca²⁺ ions. These findings provide novel mechanistic insights regarding the role of Ca²⁺ in mediating SARS-CoV-2 fusion and provide a detailed structural platform to aid the ongoing efforts in rational design of compounds to inhibit SARS-CoV-2 cell entry.     

Osmolality and electrolyte concentration of hemolymph and the problem of ion and volume regulation of cells in higher insects

• 1.1. The study was carried out on 22 species of insects from 5 orders. The osmolality of their hemolymph varied from 319 to 421 mOsm/kg H₂O, concentration of Na⁺ 4.6 to 118 mM/l, K⁺ 6.3 to 73mM/l, Ca²⁺ 3.6 to 12.9 mM/l, Mg²⁺ 2.3 to 76 mM/l. The most abundant cation in the hemolymph of insects from higher orders is either K⁺ or Mg²⁺.
• 2.2. In the muscles of lower and higher insects K⁺ is usually within 80–120 mM/kg wet wt.
• 3.3. Most Ca²⁺ and Mg²⁺ in hemolymph is bound with protein and low molecular anions, concentration of free Ca²⁺ is 0.9-2.1mM/l Mg²⁺ 3.7–8.0 mM/l.
• 4.4. It is concluded that, in insects, potassium hemolymph, cell volume regulation and accumulation of ions in the cell, are ensured by an increased osmolality of hemolymph due to a high percentage contribution of low molecular organic substances which are retained in the hemolymph due to the absence of filtration apparatus in the Malpighian tubules.

     

The role of ion fluxes in Nod factor signalling in Medicago sativa

Using ion-selective microelectrodes, the basis of Nod factor-induced changes in the plasma membrane potential was analysed by measuring the extracellular free concentrations of Ca²⁺, K⁺, H⁺ and Cl^– in the root hair zone of alfalfa. After addition of the Rhizobium meliloti Nod factor NodRm-IV(C16:2,S) at a concentration of 0.1 μM, a decrease in [Ca²⁺] was observed first, which was followed after a few seconds by an increase of [Cl^–], by an alkalinization, and then by a delayed increase of [K⁺], all of which were transient changes. Simultaneously with the appearance of Cl^– ions in the root hair zone, a decrease in cytosolic [Cl^–] was measured. It was concluded that the depolarization was caused by temporary short-circuiting of the proton pump through the rapid release of Cl^– ions along their steep electrochemical gradient. Since under resting conditions the driving force for K⁺ ions was inwardly directed, their release was delayed until their driving force was inverted. This indicates that K⁺ serves as a charge balance that eventually stops depolarization and initiates repolarization. Since the decrease in [Ca²⁺] was observed seconds before the increase in [Cl^–] and the depolarization, it is argued that Ca²⁺ entering into the cell does not cause the depolarization directly, but might initiate it by triggering the activation of an anion channel that then releases the chloride ions. The observations that the Ca²⁺ ionophore A23187 mimicks the Nod factor response, and that the Ca²⁺ channel antagonist nifedipine inhibits this response, support the idea that Ca²⁺ plays a primary role in the transduction of the Nod signal in alfalfa.     

Rapid calcium exchange for protons and potassium in cell walls of Chara

Net fluxes of Ca²⁺, H⁺ and K⁺ were measured from intact Chara australis cells and from isolated cell walls, using ion-selective microelectrodes. In both systems, a stimulation in Ca²⁺ efflux (up to 100 nmol m^?2 s^?1, from an influx of ～40 nmol m^?2 s^?1) was detected as the H⁺ or K⁺ concentration was progressively increased in the bathing solution (pH 7.0 to 4.6 or K⁺ 0.2 to 10mol m^?3, respectively). A Ca²⁺ influx of similar size occurred following the reverse changes. These fluxes decayed exponentially with a time constant of about 10 min. The threshold pH for Ca²⁺ efflux (pH 5.2) is similar to a reported pH threshold for acid-induced wall extensibility in a closely related characean species. Application of NH₄⁺ to intact cells caused prolonged H⁺ efflux and also transient Ca²⁺ efflux. We attribute all these net Ca²⁺ fluxes to exchange in the wall with H⁺ or K⁺. A theoretical treatment of the cell wall ion exchanges, using the ‘weak acid Donnan Manning’ (WADM) model, is given and it agrees well with the data. The role of Ca²⁺ in the cell wall and the effect of Ca²⁺ exchanges on the measured fluxes of other ions, including bathing medium acidification by H⁺ efflux, are discussed.     

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。     

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.     

Overcoming the Problem of Non-Ideal Liquid Ion Exchanger Selectivity in Microelectrode Ion Flux Measurements

Ion-selective microelectrodes are a powerful tool in studies on various aspects of cell membrane biology in both animal and plant tissues. Further application of this technique is, however, limited to a large extent by the problem of non-ideal selectivity of the liquid ion exchanger used in the preparation of microelectrodes for ion flux measurements. Because of this problem, which is persistent in many commercial liquid ion exchangers, the microelectrode does not discriminate between the ion of interest and other interfering ions (for example, Mg²⁺ and Ca²⁺; Na⁺ and K⁺), thereby leading to inaccurate concentration readings and, consequently, inaccurate flux calculations. In this work we show that the existing analytical procedure to overcome this problem, using the inverted Nicolsky-Eisenman equation, is inadequate, and suggest an alternative analytical procedure that can be applied directly to the data obtained with commercially available liquid ion exchangers. We show that this alternative procedure allows accurate measurement of ionic concentrations with non-ideal ion-selective microelectrodes in the presence of interfering ions, and illustrate the method by direct experiment using Ca²⁺ and Mg²⁺ as a “case study”. Several more examples are given, further illustrating practical applications of the method for study of plant responses to salinity, osmotic and reactive oxygen species stresses.     

High Concentrations of Tricyclic Antidepressants Increase Intracellular Ca2+ in Cultured Neural Cells

We examined the effect of tricyclic antidepressants on intracellular Ca²⁺ signalling in cultured cells of neuronal and glial origin. High concentrations of amitriptyline and desipramine increased the intracellular Ca²⁺ in PC-12 and U-87 MG cells. In PC-12 cells amitriptyline induced a biphasic rise in intracellular Ca²⁺. A rapid and transient increase due to release of Ca²⁺ from intracellular pools was followed by sustained elevation of [Ca²⁺]_i due to influx from the extracellular medium. Desipramine evoked the Ca²⁺ release from intracellular pools but the influx of Ca²⁺ was not elicited. In U-87 MG cells both the drugs induced Ca²⁺ release from intracellular pools, however amitriptyline also induced a transient influx of Ca²⁺. To delineate the mechanisms involved in mobilization of Ca²⁺ by the drugs pharmacological agents that inhibit IP₃ formation in cells and Ca²⁺ channel blockers were used and changes in [Ca²⁺]_i and membrane potential were monitored. The results show that both the drugs release Ca²⁺ from IP₃ sensitive pools by activation of phospholipase C and amitriptyline in addition activates a non specific cation channel in the plasma membrane of cells. Paradoxically at relatively lower concentrations (< 50 M) amitriptyline and desipramine inhibited the Ca²⁺ signal induced by adenosine triphosphate in both the cell types. Our data demonstrate that tricyclic antidepressants at different doses may have inhibitory or stimulatory effects on cellular Ca²⁺ signalling.     

Cation (K+, Mg2+, Ca2+) exchange in Pb2+ accumulation by Saccharomyces cerevisiae

The relationship between Pb²⁺ accumulation and cation (K⁺, Mg²⁺, Ca²⁺) release in Saccharomyces cerevisiae was extensively investigated. As Pb²⁺ accumulation proceeded, the release of cellular metal ions such as K⁺, Mg²⁺ and Ca²⁺ was concomitantly released within 24 h, thereafter Pb²⁺ penetrated into the inner cellular parts and consequently plasmolysis of the cell was observed by TEM analysis. Pb²⁺ accumulation process in S. cerevisiae after 24 h was metabolism-independent because of the absence of cell viability. As the cell storage time was prolonged, the released amount of K⁺ was markedly increased, while the amount of accumulated Pb²⁺ was nearly constant regardless of cell storage time and the time required to reach an equilibrium state was shortened. The autoclaved cells had less Pb²⁺ accumulation capacity than the untreated cells, and the amounts of released K⁺ and Mg²⁺ were very low due to the denaturation of cell surface and cell membrane.     

Gangliosides inhibit PDGF-induced signal transduction events in U-1242 MG human glioma cells

In this study we investigated the responses of intracellular calcium ([Ca²⁺]_i) and protein kinase C (PKC) to PDGF in U-1242 MG cells. PDGF-BB stimulated [³H]PDBu binding approximately 2–3 fold. This response was inhibited by preincubating the cells with an inhibitor of phospholipase C (PLC), U73122, suggesting that PLC mediates the induction of PKC translocation by PDGF. PDGF also increased the concentration of [Ca²⁺]_i that was attenuated in a calcium-free medium. This indicates that PDGF-induced elevation of [Ca²⁺]_i is mainly due to influx of extracellular calcium. PDGF-stimulated translocation of PKC was inhibited by the intracellular calcium buffer BAPTA/AM. All gangliosides studied except GM3 inhibited these responses with similar efficacy. Collectively, these results indicate that the signal transduction pathway initiated by PDGF leading to PKC translocation in U-1242 MG cells is intact, and this pathway is inhibited by several gangliosides.Special issue dedicated to Dr. Leon S. Wolfe.     

Oscillations of cytoplasmic concentrations of Ca2+ and K+ in fused L cells

Summary Using Ca²⁺- and K⁺-selective microelectrodes, the cytosolic free Ca²⁺ and K⁺ concentrations were measured in mouse fibroblastic L cells. When the extracellular Ca²⁺ concentration exceeded several micromoles, spontaneous oscillations of the intracellular free Ca²⁺ concentration were observed in the submicromolar ranges. During the Ca²⁺ oscillations, the membrane potential was found to oscillate concomitantly. The peak of cyclic increases in the free Ca²⁺ level coincided in time with the peak of periodic hyperpolarizations. Both oscillations were abolished by reducing the extracellular Ca²⁺ concentration down to 10^–7 m or by applying a Ca²⁺ channel blocker, nifedipine (50 m). In the presence of 0.5mm quinine, an inhibitor of Ca²⁺-activated K⁺ channel, sizable Ca²⁺ oscillations still persisted, while the potential oscillations were markedly suppressed. Oscillations of the intracellular K⁺ concentration between about 145 and 140mm were often associated with the potential oscillations. The minimum phase of the K⁺ concentration was always 5 to 6 sec behind the peak hyperpolarization. Thus, it is concluded that the oscillation of membrane potential results from oscillatory increases in the intracellular Ca²⁺ level, which, in turn, periodically stimulate Ca²⁺-activated K⁺ channels.