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A Feltz

Publications and source records attributed to A Feltz.

At least 37 records · Page 2Linked to original sources
PubMed1995

Inhibition by cyclothiazide of neuronal nicotinic responses in bovine chromaffin cells.

1. The desensitizing acetylcholine (ACh) response of bovine chromaffin cells maintained in culture was examined using rapid agonist applications (of 2 s duration) which imposed nominal drug concentrations within 50 ms. This study was aimed, firstly, at identifying which of the alpha 3, alpha 4 and alpha 7 subunits known to be present in these cells is predominant in the ACh-evoked response and secondly, on the effects on these neuronal nicotinic ACh receptors (AChR) of cyclothiazide (CT), an agent acting as a modulator of a gating desensitization site on other ligand-gated channels. 2. Locally applied 100 microM ACh evoked peak currents (IACh) of -1.5 +/- 0.1 nA (n = 83) at a holding potential of -60 mV. The ACh dose-response curve yielded an estimated EC50 of 60 microM. This current was not sustained but desensitized during the application period; it displayed strong inward rectification, but desensitized equally whether the evoked current was inward or outward going. The latter observation excludes alpha 4 as a major contributor to the recorded current. Because the response was almost insensitive to a 1 microM alpha-bungarotoxin pretreatment (IACh = -1.2 +/- 0.1 nA; n = 6), and because 1, 1-dimethyl-4-phenylpiperazinium (DMPP) works as a potent agonist (peak current = -1.9 nA, n = 2 for 100 microM DMPP), the alpha 7 subunit is also a minor contributor to the response. Taken together, these observations suggest a dominant alpha 3 type of response. 3. Triple exponential fits were used to describe the characteristics of the ACh-evoked currents; one component to fit the rising phase, with 2 components to describe the decay phase. The decay times were 100 ms and 4 s for the fast and slow components respectively. The rate of the slow decay component increased systematically with recording time, approximately doubling from its initial value within 20-40 min. Furthermore there was a gradual rundown of the response, seen first as a loss of the late component of the current, measured at 2 s, with the peak current amplitude decreasing later in the recording.4. CT, when coapplied with ACh, produced a dose-dependent inhibition of the ACh-evoked peak current. The effect showed little voltage-dependency with 100 microM CT producing 46 +/- 5% (s.d.; n = 3)and 47 +/- 8% (s.d.; n = 7) inhibition at -100 and -60 mV respectively. At + 60 mV, inhibition was estimated to be 26 +/- 7% (s.d.; n = 3).5. After pre-exposure of the cells to CT by bath application, 10 and 30 microM CT produced poorly reversible 20 +/- 9% (n = 7) and 42 +/- 5% (n = 4) inhibitions of the peak current respectively. There were no discernible effects on the fitted decay constants at any CT concentration tested, although an increased inhibitory effect of CT was observed at higher concentrations (100 microM) on the amplitude of the late component measured at 2 s.6. Similar effects were observed in conditions chosen to isolate the alpha 3 type of receptor: namely when using DMPP as an agonist, or after a-bungarotoxin pretreatment.7. The 2,3-benzodiazepine, GYKI 53655, is known to antagonize the action of CT on AMPA receptors.Coapplication of 50 microM GYKI 53655 with ACh (100 microM) produced a 29 +/- 4% inhibition of the peak ACh-evoked current and 44 +/- 6% inhibition of its amplitude at 2 s (n = 4). This response was fully reversible. Brief applications of both CT (100 microM) and GYKI 53655 (50 microM) with ACh via the microperfusion system produced a fully reversible inhibition that was not significantly different from the values obtained with either CT or GYKI 53655 alone, with 37 +/- 6% inhibition of peak and 61 +/- 9%inhibition of the amplitude at 2 s (n = 3).8. The results obtained suggest that the CT effect is to impede recovery from a slow desensitization,with a more pronounced effect with longer CT applications. Globally, CT favours the 'rundown state' of the neuronal nicotinic AChR.

Acetylcholine↗
PubMed1995

Calcium-dependent inactivation of synaptic NMDA receptors in hippocampal neurons.

1. We examined whether synaptically activated N-methyl-D-aspartate (NMDA) receptors are regulated by intracellular calcium in cultured hippocampal neurons by comparing excitatory postsynaptic currents (EPSCs) to the previously described calcium-dependent regulation of whole cell NMDA currents. Standard whole cell recording and fast application methods were used. 2. Low-frequency (0.2 Hz) stimulation of EPSCs in the presence of 2-amino-5-phosphonovalerate (AP5) evoked a constant amplitude alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor-mediated EPSC. On removal of AP5 in Ca(2+)-containing solutions, the amplitude of the slow NMDA receptor-mediated EPSC decreased by approximately 50% during the next 10 stimuli. The decrease in the EPSC was dependent on the extracellular calcium concentration and stimulus frequency, consistent with Ca(2+)-dependent desensitization/inactivation of postsynaptic NMDA receptors. A whole cell prepulse of NMDA (10 microM, 10 s) in Ca(2+)-containing solutions inhibited the slow EPSC to a similar degree. A series of slow EPSCs also produced Ca(2+)-dependent inactivation of whole cell NMDA currents evoked in low calcium solutions. 3. These results demonstrate that synaptic NMDA receptors are inactivated by intracellular calcium and that calcium entry through synaptically activated NMDA receptors is sufficient to provide feedback inhibition of the slow EPSC.

Animals↗
PubMed1995

Synaptic NMDA receptor channels have a low open probability.

Realistic estimates of channel-gating parameters of synaptic receptors are essential to an understanding of synaptic transmission and modulation. However, the gating of N-methyl-D-aspartate (NMDA) channels appears to differ, depending on recording conditions; thus, it remains unclear what measurements are most relevant to synaptic receptors. To further explore this discrepancy, we examined the open probability (Po) of NMDA channels in whole-cell and outside-out patch recording from cultured hippocampal neurons. Currents were evoked by rapid application of saturating concentrations of NMDA in the presence or absence of the "irreversible" open channel blocker, MK-801 ((+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine) . The reduction of the peak amplitude and the acceleration of the decay of the current in MK-801 were used to derive Po by fitting the current traces to a multistate kinetic model. The Po in whole cell was low (0.04), similar to that previously measured for synaptically activated NMDA channels. In contrast, ensemble average currents from outside-out patches were much more rapidly blocked in the presence of MK-801, indicative of a significantly higher Po. The Po also gradually increased with the duration of recording in both whole-cell and outside-out configurations, suggesting that channel gating is sensitive to mechanical alterations of the patch or that washout of cytoplasmic factors leads to an increase in channel open probability.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Maintained L-type Ca2+ channel activity in excised patches of PTX-treated granule cells of the cerebellum.

Activity of high-threshold voltage activated neuronal Ca2+ channels, including dihydropyridine-sensitive (L-type) channels, rapidly disappears during cell dialysis in whole-cell recording conditions or after excision of a patch. To date, this phenomenom has been mainly related to phosphatase or protease activity. On the other hand, it has been suggested that Ca2+ channels may be regulated by G-proteins. Therefore, disruption of this regulatory pathway may also be involved directly or indirectly in the rundown process. Here, we show that treatment of cultured cerebellar granule cells with pertussis toxin (PTX) increases to 70% the probability for excising patches that display L-type Ca2+ channels activity in the inside-out recording configuration. Quantitative study indicates that, except a half decrease in the open probability, most features of the channel activity are retained after patch excision with minor modifications. The characteristics of the channel activity did not change with time during at least the first 9 min of the inside-out configuration. In addition, comparison of unitary currents recorded in the cell-attached, configuration on treated and nontreated cells demonstrates that the PTX treatment slows the activation kinetics of the current and increases the duration of channel openings evoked at -20 mV but not at 0 mV depolarizing potential. These data suggest that L-type Ca2+ channel activity are under a tonic regulation of a PTX-sensitive mechanism, which is implied in the run-down process.

Animals↗
PubMed1995

Facilitatory coupling between a glutamate metabotropic receptor and dihydropyridine-sensitive calcium channels in cultured cerebellar granule cells.

The effect of metabotropic glutamate receptor activation on Ca dihydropyridine (DHP)-sensitive channels recorded in the presence of 1 microM Bay K 8644 was examined on cultured cerebellar granule cells using the patch-clamp technique in the cell-attached configuration. Bath-applied agonist (trans-ACPD, 1S,3R-, and 1R,3S-ACPD isomers, and glutamate or quisqualate in the presence of CPP and CNQX) evoked an increase in Ca channel activity with a variable latency of 8.9 +/- 8.6 sec in 40% of the recorded cells. Neither L-CCG1, L-AP3, L-AP4, nor AMPA or NMDA activated Ca channels. Two dihydropyridine-sensitive channels present in this cell type were activated by trans-ACPD: the classical 24 pS L-type channel and a smaller-conductance 7 pS channel. The effect was shown to be mediated by neither intracellular Ca2+ nor a pertussis toxin (PTX)-sensitive G protein. Interestingly treatment with BAPTA-AM increased the number of responding patches and the activity was more sustained throughout the drug application. After overnight PTX treatment, activation of the Ca channels persisted even after washout of the agonist. These results indicate that mGluR1/mGluR5 probably mediate the facilitation of dihydropyridine-sensitive Ca channels.

Animals↗
PubMed1994

Forskolin blocks the transient K current of rat cerebellar granule neurons.

The action of forskolin (FSK) on voltage-activated K currents was investigated in cerebellar granule cells. FSK reversibly inhibited both A-type (IA) and non-inactivating K currents. IA blockade was more precisely characterized. This effect of FSK was shown to be voltage- and concentration-dependent with an IC50 of 19 microM at +50 mV. 1,9-Dideoxyforskolin (1,9-ddxFSK), a derivative of FSK that does not activate adenylate cyclase, specifically blocked IA, while cAMP-increasing agents had no direct effect on the K currents. The possibility that the non-cAMP mediated effect of FSK occurs through a channel-blocking mechanism and its eventual implications for neuronal excitability are discussed.

Animals↗
PubMed1994

Characteristics of calcium channels responsible for voltage-activated calcium entry in rat cerebellar granule cells.

The properties and characteristics of calcium channel openings in cerebellar granule cells were analysed by the cell-attached patch-clamp technique. At depolarized potentials, with 110 mM Ba2+ as the divalent charge carrier, 36% of the patches displayed activity that consisted of elementary events whose amplitude ranged from -0.3 to -1.75 pA at 0 mV, giving rise to a high threshold current. In this population of events at least four different types of channel openings were identified by their distinct biophysical and pharmacological properties. Two types of channel openings, with conductances around 24 and 7 pS, had similar characteristics in that both opened following two modes of gating characterized by brief (approximately 2 ms) and longer openings (approximately 8 ms) and both were sensitive to dihydropyridines. A further type of channel opening, with a conductance around 11 pS gated mainly with brief openings (approximately 1 ms), was shown to be insensitive to dihydropyridines but was undetectable in recordings from the cells that had been treated with omega-conotoxin. The last type of event was revealed after treatment of the cell with nicardipine or nifedipine and omega-conotoxin. The corresponding channel had a conductance of 19 pS and opened in one dominant mode characterized by brief openings (approximately 1 ms). The data obtained on single-channel activity of cerebellar granule cells are compared with the properties of the total current recorded in whole-cell conditions.

Animals↗
PubMed1994

Evolution of the Ca2+ current during dialysis of isolated bovine chromaffin cells: effect of internal calcium.

We have examined the internal Ca(2+)-dependence of the long-term evolution of whole cell high voltage activated Ca current in chromaffin cells. The evolution of the peak Ca current was characterized by 2 distinct phases: after an initial facilitation, there followed a rundown, which represented a reduction by 70% within some 10 min. The rundown process was shown not to depend on Ca2+ entry nor on membrane depolarization. It resulted from cell dialysis with a saline solution and, once initiated, it proceeded at a rate of 0.28 min-1 at 4 different Ca2+ concentrations (pCa 5-9). The facilitation is also initiated by cell dialysis but this process developed faster at higher internal Ca2+ concentrations. Thus, globally, high-voltage activated Ca2+ current runs down faster when using a recording pipette solution with a higher internal Ca2+ concentration (pCa 5 or 6). Some leupeptin-sensitive proteases may be involved in the initiation of facilitation and rundown processes.

Adrenal Medulla↗
PubMed1992

Voltage-dependent calcium entry in confluent bovine capillary endothelial cells.

Confluent bovine capillary endothelial cells display, when examined for voltage-dependent calcium entries using cell-attached channel recordings, two types of Ca2+ channels (4 and 23.5 pS in 110 mM Ba2+) both sensitive to the dihydropyridine Ca agonist BAY K 8644. In contrast to isolated cells, confluent cells display no T-type, low threshold activity, and Ca currents were typically only elicited at very depolarized potentials. In these cells, voltage-dependent calcium entries will only be made operative by substances able to shift their activation towards the resting potential.

Adrenal Medulla↗
PubMed1992

Voltage-gated Ca entry in isolated bovine capillary endothelial cells: evidence of a new type of BAY K 8644-sensitive channel.

Isolated bovine capillary endothelial cells have been examined for voltage-dependent Ca entry. All cells displayed a low threshold activity, with the main characteristics of a T-type transient current, when examined using whole-cell recording for activation and inactivation and cell-attached conditions or inside-out patches for the elementary conductance (8 pS). 25% of the cells displayed an additional sustained current in 5 mM CaCl2 above -40 mV, which was enhanced by application of BAY K 8644, but almost insensitive to superfusion with nicardipine. Two types of channels (2.8 and 21 pS, in 110 mM BaCl2) were shown to have a BAY K 8644 sensitivity. The large conductance channels were L-type channels. The smaller events were elicited at more hyperpolarized potentials (by some 30 mV). Their mean open time was 16 ms in control conditions. In presence of BAY K 8644, additional long open times were observed (up to 100 ms as compared to 7.8 ms for the time constants of the slow mode of the L-type channel). We refer to these channels as SB channels: of small conductance and sensitive to BAY K 8644. In the presence of nicardipine, SB channels are not noticeably modified, in contrast to the L-type openings which are abolished. Also, SB open times are close to control values when nicardipine is added after a BAY K 8644 application. We suggest that, at physiological concentrations of divalent ions, an SB-type activity is elicited above -40 mV which generates the low threshold sustained current.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1992

Inositol phosphate regulation of voltage-dependent calcium channels in cerebellar granule neurons.

The effects of intracellularly applied inositol phosphates on voltage-dependent calcium channel currents were assessed in rat cerebellar neurons using the whole-cell recording configuration of the patch-clamp technique. Intraneuronal perfusion of 10 microM inositol 1,4,5-trisphosphate (IP3) increased the amplitude of currents elicited by depolarization from a holding potential of -40 mV. IP3 did not modify current activation, but shifted the steady-state inactivation curve toward more positive values. The dose-response curve indicated an EC50 of 0.5 microM for IP3. Inositol 1,3,4,5-tetrakisphosphate (IP4), but not inositol 4,5,-bisphosphate, mimicked the effect of IP3. The effect of IP3 persisted in the presence of 100 micrograms/ml heparin and did not depend on intracellular calcium mobilization, as similar responses were not produced by 10 mM caffeine or by intrapipette calcium buffering at pCa 6 instead of pCa 7.7. Preincubation with omega-conotoxin led to a 55% inhibition of barium current; however, inhibition was reversed by IP3, which reestablished the control current amplitude. These results imply that IP3 and IP4 can elicit calcium entry by modifying both the gating characteristics and the pharmacological properties of voltage-dependent calcium channels.

Animals↗
PubMed1991

Inactivation characteristics reveal two calcium currents in adult bovine chromaffin cells.

1. Two calcium currents were identified by differences in their inactivation characteristics in adult chromaffin cells maintained in short-term primary culture (3-5 days). Calcium currents were recorded by means of the whole-cell configuration using an intracellular medium highly buffered for pH and pCa. 2. Calcium current evoked from a holding potential of -90 mV inactivated along two components: an initial transient with a time constant of 250 ms followed by a plateau. 3. Steady-state inactivation followed two processes which developed at two distinct membrane potentials. One process was half-inactivated at low voltages around -55 mV and affected mainly the initial transient component. The other process, which affected mainly the sustained component of the calcium current, was half-inactivated at voltages around -10 mV. The proportions of these two processes varied greatly from cell to cell. 4. The dihydropyridine antagonists (nicardipine and nifedipine applied at 10(-5) M) and the phenylalkylamine D600 (5 x 10(-6) M) shifted the half-inactivation value towards -55 mV, indicating the suppression of the sustained component. The snail toxin, omega-conotoxin, had the opposite effect; it shifted the half-activation value towards -10 mV. 5. The calcium channel agonist Bay K 8644 (10(-5) M) either had no effect or induced only a slight increase of the response, as did its (-)-enantiomer (10(-6) M). To interpret the present results, we suggest that the L-component was maximally activated in our recording conditions. 6. In chromaffin cells, the calcium current recorded in whole-cell conditions is composed of two components with properties close to those of N- and L-type currents described in sympathetic neurons.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1991

Two types of calcium channels are expressed in adult bovine chromaffin cells.

1. Calcium channel activity was recorded in chromaffin cells in the cell-attached condition, using 110 mM-Ba2+ as the permeant ion. 2. One type of calcium channel had a conductance of 16 pS, was completely inactivated at a holding potential of -20 mV and was insensitive to dihydropyridine agonists and antagonists. These characteristics correspond to a calcium channel of the N-type. 3. A second type of calcium channel was active at holding potentials of -30 mV and above, had a channel conductance of 31 pS, and was sensitive to the dihydropyridine agonist, Bay K 8644. The channel opened along two dominant modes with characteristic time constants of 0.5 and 5 ms. The main effect of Bay K 8644 was to increase the probability of both short and long openings with no change in their relative proportions (6 to 1 respectively). These characteristics correspond to a calcium channel of the L-type. 4. omega-Conotoxin affected the activity of both N- and L-type channels. It drastically reduced the number of N-type channel openings and produced complex changes in L-type channel activity. Long openings were less frequent and the conductance during short openings was slightly smaller than that measured in the presence of Bay K 8644. 5. The discussion focuses on modulation of L-type channel activity. Openings of L-type channels are rarely recorded in the cell-attached configuration under control conditions. Addition of Bay K 8644 is needed to reveal the presence of L-type channels. By contrast, L-type currents recorded in the whole-cell configuration are always observed and are insensitive to Bay K 8644. These results indicate that L-type channels are normally inoperable in chromaffin cells.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1990

Lipopolyamine-mediated transfection allows gene expression studies in primary neuronal cells.

A simple and efficient transfection technique based on lipopolyamine-coated DNA that can be used for gene transfer in cerebellar granular neurons is described. Gene transfer is achieved by exposure of cells to a DNA/lipid complex obtained by simple mixing of lipopolyamine and plasmid DNA. This procedure may represent a general tool of physiological investigations in primary cells. We show that the promoters of the introduced chimera genes are regulated by their respective trans-acting factors and may be modulated via membrane receptors and second messengers. This procedure has no noticeable toxic effects, nor does it seem to interfere with complex physiological behavior like neuronal differentiation.

Amino Acid Sequence↗
PubMed1989

Voltage-dependent transient calcium currents in freshly dissociated capillary endothelial cells.

Dissociated capillary endothelial cells display a voltage-dependent Ca current activating around the resting potential. The initial transient component of the current corresponds to a Ca channel of the T type. Some cells also display a plateau component corresponding to a distinct dihydropyridine-sensitive Ca channel. Depolarization induced by high external K+ elicits an increase in cytoplasmic Ca concentration. Confluent cells have been found to express the same Ca permeabilities.

Adrenal Medulla↗
PubMed1989

Decay kinetics of calcium currents in rat sensory neurones: analysis at two internal free calcium concentrations.

The patch-clamp technique in whole-cell configuration was used to investigate the kinetics of decay of calcium currents in rat sensory neurones. Whole-cell recording permitted control of the internal medium, particularly of the internal free calcium concentration, which was maintained at either 10(-9) M or 10(-6) M using a high concentration of Ca buffer. The inactivation decay of the total Ca current elicited above -10 mV was found to be faster at pCa 6 than at pCa 9. The total current contained three exponential components which were tentatively identified as the three types of Ca currents (IcaT, IcaN and IcaS). Kinetic analyses indicated that the control of the inactivation process by internal Ca results from an effect on both high-threshold Ca currents, IcaN and IcaS. The inactivation kinetics reported in the literature presents a large variability depending on the cell type. We propose that this variability may result from differences in the capacity of those cells to control their internal Ca.

Animals↗
PubMed1989

Voltage-activated calcium channels in rat Purkinje cells maintained in culture.

Cell-attached patch recordings were used to study calcium channels on the dendritic membrane of rat cerebellar Purkinje cells maintained in culture. Experiments were performed with isotonic BaCl2 (110 mM) in the pipette and isotonic potassium gluconate in the bath to zero the cell membrane potential. Two distinct types of voltage-activated calcium channels were identified. The first one had a small conductance (9 pS), was activated at a low threshold (congruent to -50 mV) and could be inactivated by holding the membrane potential at -30 mV. This channel had the same characteristics as the T channel described in other neuronal preparations. The second type of Ca channel activated at a high threshold (-30 or +10 mV depending on whether BAY K 8644 was added or not to the pipette solution) and was still activatable even when the membrane was held at -40 mV. In the presence of BAY K 8644 this channel had a conductance of 21 pS with long openings. All these characteristics are similar to those of the S (L) Ca channel described in many preparations. The present study is in agreement with our previous experiments on Purkinje dendrites, where we identified low and high threshold Ca currents using the whole-cell configuration. Up to now, no channel corresponding to the N current has been observed but we cannot exclude its presence.

Animals↗