Gap junctions in human synovial cells and tissue.

Our objective was to establish the existence of intercellular communication through gap junctions in synovial lining cells and in primary and passaged cultures of human synovial cells. Communication between cells was assessed using the nystatin perforated-patch method, fluorescent dye transfer, immunochemistry, transmission electron microscopy, and immunoblotting. Functional gap junctions were observed in primary and passaged cultures and were based on measurements of the transient current response to a step voltage. The average resistance between cells in small aggregates was 300 +/- 150 MOmega. Gap junctions were also observed between synovial lining cells in tissue explants; the size of the cell network in synovial tissue was estimated to be greater than 40 cells. Intercellular communication between cultured cells and between synovial lining cells was confirmed by dye injection. Punctate fluorescent regions were seen along intercellular contacts between cultured cells and in synovial membranes in cells and tissue immunostained for connexin43. The presence of the protein was verified in immunoblots. Regular 2-nm intermembrane gap separations characteristic of gap junctions were seen in transmission electron micrographs of synovial biopsies. The results showed that formation of gap-junction channels capable of mediating ionic and molecular communication was a regular feature of synovial cells, both in tissue and in cultured cells. The gap junctions contained connexin43 protein and perhaps other proteins. The physiological purpose of gap junctions in synovial cells is unknown, but it is reasonable to anticipate that intercellular communication serves some presently unrecognized function.

Resting potential of excitable neuroblastoma cells in weak magnetic fields.

The mechanism by which static and low-frequency magnetic fields are transduced into biological signals responsible for reported effects on brain electrical activity is not yet ascertained. To test the hypothesis that fields can cause a subthreshold change in the resting membrane potential of excitable cells, we measured changes in transmembrane current under voltage clamp produced in SH-SY5Y neuroblastoma cells, using the patch-clamp method in the whole-cell configuration. In separate experiments, cells were exposed to static fields of 1, 5, and 75 G, to time-varying fields of 1 and 5 G, and to combined static and time-varying fields tuned for resonance of Na+, K+, Ca2+, or H+. To increase sensitivity, measurements were made on cells connected by gap junctions. For each cell, the effect of the field was evaluated on the basis of 100 trials consisting of a 5-s exposure immediately followed by a 5-s control period. In each experiment, the field had no discernible effect on the transmembrane current in the vicinity of zero current (- 50 mV voltage clamp). The sensitivity of the measuring system was such that we would have detected a current corresponding to a change in membrane potential as small as 38 microV. Consequently, if sensitivity of mammalian cells to magnetic fields is mediated by subthreshold changes in membrane potential, as in sensory transduction of sound, light, and other stimuli, then the ion channels responsible for the putative changes are probably present only in specialized sensory neurons or neuroepithelial cells. A change in transmembrane potential in response to magnetic fields is not a general property of excitable cells in culture.

Reconstitution of the mitochondrial ATP-dependent potassium channel into bilayer lipid membrane.

Electrical properties and regulation of the mitochondrial ATP-dependent potassium channel were studied. The channel protein was solubilized from the mitochondrial membrane using an ethanol/water mixture. Reconstituted into a bilayer lipid membrane BLM), the protein formed a slightly voltage-dependent channel with a conductance of 10 pS in 100 mM KCl. Often, several channels worked simultaneously (clusters) when many channels were incorporated into the BLM. The elementary channel and the clusters were both highly potassium selective. At concentrations of 1 to 10 microM, ATP favors channel opening, while channels become closed at 1-3 mM ATP. GDP (0.5 mM) reactivated the ATP-closed channels without affecting the untreated channels. The sulfhydryl-reducing agent ditiothreitol increased the open probability at concentrations of 1 to 3 mM, but damaged the selectivity of the channel.

Intracellular signaling mechanisms of interleukin-1beta in synovial fibroblasts.

The possibility that membrane depolarization of synovial fibroblasts caused by interleukin-1beta (IL-1beta) was mediated by protein kinase C (PKC) and Ca2+ influx was studied using inhibitor and activator analysis. The effect of IL-1beta was blocked by bisindolylmaleimide I, an inhibitor of PKC, and by the Ca2+ channel blockers nifedipine and verapamil. In other experiments, PKC was activated using phorbol 12-myristate 13-acetate, and Ca2+ influx was increased by means of a Ca2+ ionophore. Simultaneous application of phorbol ester and Ca2+ ionophore in the absence of IL-1beta mimicked the depolarization caused by IL-1beta. The results were consistent with the hypothesis that, under the conditions studied, activation of PKC and Ca2+ influx are necessary and sufficient processes in the transduction of IL-1beta by synovial cells leading to membrane depolarization. The essential role of protein phosphorylation and Ca2+ influx in the early electrophysiological response of synovial fibroblasts to IL-1beta was therefore established. The role of IL-1beta-induced depolarization in regulating protein expression by the cells remains to be determined, but the results reported here, taken together with observations that protein phosphorylation and Ca2+ influx also mediate the effect of IL-1beta on protease production (1, 2), suggest that electrophysiological changes are actually part of the pathway for expression of proteases in response to IL-1beta.

Interleukin-1 beta switches electrophysiological states of synovial fibroblasts.

The role of electro-physiological events in signal transduction of interleukin-1 beta (IL-1 beta) was investigated in rabbit synovial fibroblasts using the perforated-patch method. Aggregated synovial fibroblasts using the perforated-patch method. Aggregated synovial fibroblasts occurred in two different electrophysiological states having membrane potentials (Vm) of -63 +/- 4 (n = 71) and -27 +/- 10 mV (n = 55) (high and low Vm, respectively). IL-1 beta affected the cells with high Vm; it switched the state of the cell from high to low Vm. This effect was strongly dependent on the external potential applied to the cell membrane. Low Vm (-30 mV) alone without IL-1 beta did not switch the state of the cells. Thus a synergistic effect involving the cytokine and cell Vm in switching the electrophysiological state of the cell was shown, indicating that electrophysiological changes are involved in signal transduction. Gap junctions between aggregated cells were necessary for the cells to have a high Vm and to respond to IL-1 beta. Gap junction resistance between adjacent cells was estimated as 300 +/- 100 M omega. Our findings suggest that the electrophysiological behavior of synovial fibroblasts is tightly connected to a signaling or intracellular mediator system that is triggered by IL-1 beta.

ATP-dependent potassium channel from rat liver mitochondria: inhibitory analysis, channel clusterization.

An inhibitor of potassium mitochondrial channels quinidine (0.1 mM) closed the ATP-sensitive potassium channels isolated from mitochondria and reconstituted into a bilayer lipid membrane. Inhibitors of cytoplasm membrane K-channels (ouabain, tetraethyl ammonium, and cesium ions) produced no effect on the ATP-sensitive mitochondria K-channels; 0.5-2 microM glybenclamide did not affect the reconstituted channels, either. The multiplicity of jumps of conductivity at small concentration of the protein and the maintenance of ion selectivity at switching various levels of conductivity lead us to assume that the large channels are clusters of elementary channels. The average frequency of channel-cluster switchovers between various levels of conductivity is much higher than at the elementary channel.

[Reaction of the brain receptor system to the effect of low intensity microwaves]. / Reaktsii retseptornykh sistem mozga na deistvie mikrovoln nizkoi intensivnosti.

We have found that two important brain synaptic receptor systems are very sensitive to the influence of low intensity microwaves. Both the excitatory glutamatergic and the inhibitory GABAergic are affected by microwave exposure. Power densities higher 50 microW/cm2 caused changes in the binding properties of these receptors to agonists. Low frequence modulation of microwaves was essential for these effects. Statistically significant effects on mean value of binding were observed at 16 Hz modulation. Exposure to microwave radiation changes the binding of agonists to glutamate and GABA receptors in different ways. An increase was observed for glutamate and a decrease for GABA. We suppose that the effect of microwaves is related to the stress reactions of the organism.

Ionic channels in Langmuir-Blodgett films imaged by a scanning tunneling microscope.

The molecular structure of channels formed by gramicidin A in a lipid membrane was imaged by a scanning tunneling microscope operating in air. The mono- and bimolecular films of lipid with gramicidin A were deposited onto a highly oriented pyrolitic graphite substrate by the Langmuir-Blodgett technique. It has been shown that under high concentration gramicidin A molecules can form in lipid films a quasi-regular, densely packed structure. Single gramicidin A molecules were imaged for the first time as well. The cavity of 0.4 +/- 0.05 nm in halfwidth was found on the scanning tunneling microscopy image of the gramicidin A molecule. The results of direct observation obtained by means of scanning tunneling microscope are in good agreement with the known molecular model of gramicidin A. It was shown that gramicidin A molecules can exist in a lipid monolayer as individual molecules or combined into clusters. The results demonstrate that scanning tunneling microscope can be used for high spatial resolution study of ionic channel structure.

[Action of microwaves with different modulation frequencies and exposure times on GABA receptor concentration in the cerebral cortex of rats]. / Deistvie mikrovoln s raznoi chastotoi moduliatsii i vremenem ékspozitsii na kontsentratsiiu retseptorov GAMK v kore mozga krys.

The effect of 800 mHz microwaves of 0, 3, 5, 7, 16, and 30 Hz modulation on GABA receptor concentration in rat brain cortex has been investigated. Irradiation of the whole body at a modulation frequency of 16 Hz readily decreases the GABA receptor concentration. Irradiation at other modulation frequencies is ineffective. Irradiation of the whole body modulated at 16 Hz with various exposure times (5, 15 and 60 min) has revealed the highest effect at 5 min, while at a longer exposure the effect decreases.

[The effect of ionizing radiation on the binding of muscimol by synaptic membranes in the rat brain]. / Deistvie ioniziruiushchei radiatsii na sviazyvanie mustsimola sinapticheskimi membranami mozga krysy.

A study was made of the effect of gamma-radiation on binding of muscimol, a GABA agonist, by synaptic membranes of rat brain cortex. Exposure to 2 Gy radiation was shown to reduce [3H]-muscimol binding to membranes.

[Antibody interaction with the amphotericin channel]. / Vzaimodeistvie antitel s amfoteritsinovym kanalom.

The monoclonal antibodies against asymmetric channel formed in the lipid bilayer of polyene antibiotic amphotericin B and cholesterol after addition of the antibiotic to the compartment from the cis side of the membrane were obtained. The effect of the antibodies on ion conductance of the channel depends on the distribution of cholesterol in the membrane. When cholesterol was present on both sides of the lipid bilayer, three antibody molecules bound to the channel from the trans side of the membrane, thus markedly increasing the lifetime of the open state of the channel. When cholesterol was present in the cis monolayer only, the antibodies, added to the trans compartment of the cell, reduced the membrane conduction.

Antibodies affect the ionic conductance of channels formed by amphotericin B in a lipid bilayer.

For the first time poly- and monoclonal antibodies (class IgM) against the polyene antibiotic amphotericin B were obtained affecting the properties of a channel formed by the antibiotic and cholesterol in a lipid bilayer when amphotericin B was added to the solution at one (cis) side of the membrane. In the case of the symmetric distribution of cholesterol in the lipid bilayer, three molecules of monoclonal antibodies bind firmly to the channel at the trans-side of the membrane, thus strongly increasing the mean lifetime of the channel in the open state, and not changing practically the ion conductance of its open state. The antibodies did not alter the properties of these channels when added at the cis-side of the membrane as well as of the channels formed in the lipid bilayer when amphotericin B was added at both membrane sides. The antibodies obtained did not affect the conductance of channels in which amphotericin B and cholesterol were replaced with their analogs levorin and 5 alpha-androstan-3 beta-one, which points to a high specificity of the immunoglobulins isolated. When cholesterol was present only in the cis-monolayer of the lipid bilayer and was absent in the trans-monolayer, the same monoclonal antibodies when added at the trans-side of the membrane blocked the conductance of the channel formed by adding the antibiotic to the solution at the cis-side of the bilayer. The obtained evidence is of interest in elucidating the general features of interaction of antibodies with the ionic channels of cellular and model membranes.

Blocking of the beta-latrotoxin channels in bimolecular lipid membranes by antibodies.

Asymmetric ion channels are formed in a bimolecular lipid membrane by beta-latrotoxin (LT) introduced to one (cis) side of the membrane. LT-specific antibodies added to the opposite (trans) side of the membrane block the current through the LT channels when a negative potential is applied to the cis side, no blockade is observed at positive potentials. LT-specific antibodies do not block the channel current when added to the cis compartment after removal of LT. LT-unspecific immunoglobulins have no influence on LT channel conductance.

Structure of planar membrane formed from liposomes.

Lipid vesicles with incorporated ion channels from polyene antibiotic amphotericin B were used to investigate structures of planar membranes formed by Shindler's techniques. A planar membrane assembled on the aperture in a lavsan film from two layers generated at the air-aqueous liposome suspension interface is not a simple bilayer but a bimolecular membrane containing numerous partly fused liposomes. A complete fusion of liposomal membranes with the planar bilayer is an unlikely event during membrane formation. A planar bimolecular lipid membrane without incorporated liposomes can be made by a method consisting of three stages: formation of a lipid layer on the air-water interface of a suspension containing liposomes, transfer of this layer along the surface of the solution into a chamber containing a solution without liposomes where a lipid monomolecular layer forms gradually (within about 20 min) at the air-water interface, assembling of the planar bilayer membrane from this monolayer. The knowledge of the planar membrane structure may be useful in experiments on incorporation of membrane proteins into a planar lipid bilayer.

[Action of a UHF field on GABA-ergic and acetylcholinergic systems in synaptic transmission]. / Deistvie SVCh polia na GAMKergicheskie i atsetilkholinergicheskie sistemy sinapticheskoi peredachi.

It was shown that exposure of rats to microwaves (800 MHz, 16 Hz modulation, 1-3 mW/cm2) decreased muscimol binding with synaptic membranes and reduced acetycholinesterase activity in the rat brain.

[Effect of ultra-high frequency electromagnetic waves on a postsynaptic membrane model]. / Deistvie sverkhvysokochastotnykh élektromagnitnykh voln na model' postsinapticheskoi membrany.

It was shown that high-frequency electromagnetic field increases the conductivity of ionic channels formed by the synaptic membrane fragments, which bind glutamate, in a bilayer lipid membrane. Heating of the whole electrolyte in a cell, under the effect of the electromagnetic field applied, was minor to be responsible for the effects observed.

[Electric conductivity of lipid membranes in the presence of proteolytic enzymes and their substrates]. / Elektroprovodnost' lipidnykh membran v prisutstvii proteoliticheskikh fermentov i ikh substratov

It is shown that there are spasmodic changes of electroconductivity in bimolecular membranes in the presence of alpha-chimotripsin and denatured ovalbumin or glycyl-d,L-beta-phenylalanine. These conductivity changes are not due to the interaction ob proteolysis products with lipid bilayer but result from the interaction of alpha-chimo trypsin with the substrate or competing inhibitor. Probability of the state with a specific conductivity depends on the membrane voltage. A modified membrane has a low ionic selectivity.