Jerzy W. Mozrzymas

Calcium and Fos Involvement in Brain‐Derived Ca2+‐Binding Protein (S100)‐Dependent Apoptosis in Rat Phaeochromocytoma Cells

Brain‐derived calcium‐binding protein S100 induces apoptosis in a significant fraction of rat phaeochromocytoma (PC12) cells. We used single cell techniques (patch clamp, videomicroscopy and immunocytochemistry) to clarify some of the specific aspects of S100‐induced apoptosis, the modality(ies) of early intracellular Ca2+ concentration increase and the expression of some classes of genes (c‐fos, c‐jun, bax, bcl‐x, p‐15, p‐21) known to be implicated in apoptosis of different cells. The results show that S100: (1) causes an increase of [Ca2+]i due to an increased conductance of L‐type Ca2+ channels; (2) induces a sustained increase of the Fos levels which is evident since the first time point tested (3 h) and remains elevated until to the last time point (72 h). All these data suggest that S100‐derived apoptosis in PC12 cells may be the consequence of a system involving an increase in L‐type Ca2+ channel conductance with consequent [Ca2+]i increase which up‐regulates, directly or indirectly, the expression of Fos.

Membrane stretch activates a potassium channel in pig articular chondrocytes.

Activity of stretch-activated potassium channels has been recorded in articular chondrocytes using patch-clamp technique. Pressure dependence is described by a sigmoidal function with a half-maximum effect at -20.5 mbar. Selectivity for potassium is demonstrated by agreement between the reversal potential measured at different [K+]o and the prediction of Nernst equation and by block of these channels by caesium.

A large-conductance voltage-dependent potassium channel in cultured pig articular chondrocytes

Abstractu2002The patch-clamp techniques were used to study voltage-dependent potassium channels in cultured pig articular chondrocytes. A predominant single-channel conductance of 125 pS was found. These channels were reversibly blocked by tetraethylammonium. In cell-attached patches, transient increases in the channel activity were observed, and defined as a switching between low and high activity modes (LAM and HAM). Open-time distributions could be described with two kinetics components (in LAM and HAM) having similar time constants (fast τ1 and slow τ2). In HAM, the area of the slow component was larger. The mean burst length was significantly longer in HAM than in LAM. In both modes, the burst-length distributions were fitted with a sum of three exponentials. In LAM and HAM, the time constants τ1 and τ2 were indistinguishable from those of the open-time distributions. The slowest time constant, τ3, was strongly voltage dependent, and was significantly longer in HAM than in LAM. In both LAM and HAM, the ensemble currents were characterised by a rapid rising phase followed by fast and profound inactivation. The activation kinetics were similar, but the inactivation was faster in HAM. In the outside-out configuration no evidence for mode switching was found. The kinetics of the rising phase of the ensemble currents were also similar to those observed using the cell-attached configuration, but the channels did not inactivate. In the whole-cell configuration, the mode switching was not present. The inactivation time constant showed a large scattering, and was much slower than that measured in the cell-attached patch mode. These currents were blocked by tetraethylammonium and 4-aminopyridine. Our results indicate that intracellular factors are involved in controlling the mode switching and the kinetics of the inactivation of potassium channels in pig articular chondrocytes.

ATP activates junctional and extrajunctional acetylcholine receptor channels in isolated adult rat muscle fibres

Single-channel recordings in the cell-attached configuration were made on freshly dissociated and cultured rat muscle fibers. We demonstrate that ATP (50-100 microM) elicits single channel currents in freshly isolated fibres. These ATP-activated channels have a slope conductance similar to that determined for ACh-activated channels but exhibit a shorter open time. We also show that ATP activates extrajunctional AChR-channels in the membrane of long-term cultured muscle fibres. Here the ATP activated channel has a slope conductance and open time characteristics similar to those of the ACh-activated channel.

Propofol blocks voltage-gated potassium channels in human T lymphocytes

The effect of propofol (PR) on voltage-gated potassium channels (KV) in human T lymphocytes (TL) was studied using the patch-clamp technique in the whole-cell configuration. PR was found to reversibly block the KV channels in a dose-dependent manner with a half-blocking concentration of approximately 40 microM. The decrease in the peak current caused by PR was voltage-independent. The activation time constant of the whole-cell potassium currents remained unaffected upon PR treatment, whereas both the rate and extent of the inactivation process were increased, indicating the open channel block mechanism. The PR half-blocking concentration was of the same order of magnitude as PR blood concentrations employed in anesthesia. Taking into account the extensive use of PR and the important role of KV channels in human TL, these results suggest a need for investigations into the effect of PR on TL cell-function regulation.

An electrophysiological study of the effects of myasthenia gravis sera and complement on rat isolated muscle fibres

The effect of human myasthenia gravis (MG) sera and complement on isolated adult rat muscle fibres was investigated. Heat-inactivated MG sera reduced the frequency of single acetylcholine receptor (AChR)-channel activity. One of the MG sera tested had a stronger effect on the extrajunctional type of AChRs than on the junctional type. The simultaneous addition of normal human serum (NHS), as source of complement, and MG serum to freshly dissociated muscle fibres caused contraction restricted to the endplate area and progressive depolarization of the muscle membrane, followed by contracture. An MG antibody-dependent complement-mediated damage of the muscle fibres is suggested.

Forskolin reduces the activity of the rat muscle embryonic type acetylcholine receptor channel

The action of forskolin (FSK), a stimulator of cAMP-dependent protein kinase (PKA), on nicotinic acetylcholine receptor-(nAChR-) channels was studied on cultured rat muscle fibres. The channel activity was estimated by determining Np, with N, being the number of channels and p, the single channel open probability. In order to elucidate the possible role of PKA in the modulation of nAChRs, FSK (10-50 microM) was added to the bath or to the pipette filling solution in the cell-attached configuration. The first protocol used was to test for indirectly-mediated cytosolic effects, the other, for any direct effects of the drug on nAChR-channels. Using both experimental protocols, no effects on the duration of single-channel openings or conductance were observed, while channel activity was significantly reduced. In particular, FSK 10 microM caused a reduction of Np only when applied to the non-patch membrane. FSK at higher concentrations, produced a more marked decrease of Np when present in the recording pipette. The present work provides evidence that the channel activity of muscle embryonic-type nAChRs can be influenced by a direct action of FSK, and is also significantly reduced by an indirectly-mediated cytosolic mechanism triggered by FSK.

Patch-clamp study on T-lymphocyte potassium conductance in patients with chronic renal failure.

Using the patch-clamp technique, we studied the differences in whole-cell potassium conductance (g(K)+) in T lymphocytes (TL) from three groups of patients suffering from renal failure: not dialyzed patients, dialyzed patients, and dialyzed patients treated with human recombinant erythropoietin (rHuEPO). The differences in g(K+) values in the group of not dialyzed patients in comparison with controls was not significant (p > 0.05). In the group of dialyzed patients, after roughly 6 years of the hemodialysis therapy, the g(K+) value was significantly higher than in controls. In dialyzed patients treated with rHuEPO, g(K+) value was significantly lower in comparison with control. Moreover, in dialyzed patients treated with rHuEPO, the time duration of dialysis therapy did not significantly affect the TL whole-cell conductance. We conclude that the g(K+) is changed in TL in renal failure patients and that the time duration of hemodialysis therapy as well as the use of rHuEPO affect the g(K+) value. Possible mechanisms underlying the observed changes in g(K+) values, as well as medical implications of obtained results are discussed.