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  • 1
    Electronic Resource
    Electronic Resource
    Human adrenal chromaffin cell calcium channels: drastic current facilitation in cell clusters, but not in isolated cells (1998)
    Springer
    Pflügers Archiv 436 (1998), S. 696-704 
    Details
    ISSN: 1432-2013
    Keywords: Key words Calcium channels ; Human ; Modulation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract  Human adrenal medullary chromaffin cells were prepared and cultured from a cystic tumoral adrenal gland whose medullary tissue was unaffected. Adrenaline-containing and noradrenaline-containing cells were identified using a confocal fluorescence microscope and antibodies against dopamine beta-hydroxylase (DBH) and phenylethanolamine N-methyltransferase (PNMT). Current/voltage (I/V) curves performed with the voltage-clamped cells bathed in 10 mM Ba2+ (holding potential, V h=–80 mV) revealed the presence of only high-threshold voltage-dependent Ca2+ channels; T-type Ca2+ channels were not seen. By using supramaximal concentrations of selective Ca2+ channel blockers, the whole-cell I Ba could be fractionated into various subcomponents. Thus, I Ba had a 25% fraction sensitive to 1 µM nifedipine (L-type channels), 21% sensitive to 1 µM ω-conotoxin GVIA (N-type channels), and 60% sensitive to 2 µM ω-agatoxin IVA (P/Q-type channels). The activation of I Ba was considerably slowed down, and the peak current was inhibited upon superfusion with 10 µM ATP. The slow activation and peak current blockade were reversed by strong depolarizing pre-pulses to +100 mV (facilitation). A drastic facilitation of I Ba was also observed in voltage-clamped human chromaffin cell surrounded by other unclamped cells; in contrast, in voltage-clamped cells not immersed in a cell cluster, facilitation was scarce. So, facilitation of Ca2+ channels in a voltage-clamped cell seems to depend upon the exocytotic activity of neighbouring unclamped cells, which is markedly increased by Ba2+. It is concluded that human adrenal chromaffin cells mostly express P/Q-types of voltage-dependent Ca2+ channels (60%). L-Type channels and N-type channels are also expressed, but to a considerably minor extent (around 20% each). This dominance of P/Q-type channels in human chromaffin cells clearly contrasts with the relative proportion of each channel type expressed by chromaffin cells of five other animal species studied previously, where the P/Q-type channels accounted for 5–50%. The results also provide strong support for the hypothesis that Ca2+ channels of human chromaffin cells are regulated in an autocrine/paracrine fashion by materials co-secreted with the catecholamines, i.e. ATP and opiates.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s004240050691
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  • 2
    Electronic Resource
    Electronic Resource
    Re-evaluation of the P/Q Ca2+ channel components of Ba2+ currents in bovine chromaffin cells superfused with solutions containing low and high Ba2+ concentrations (1996)
    Springer
    Pflügers Archiv 432 (1996), S. 1030-1038 
    Details
    ISSN: 1432-2013
    Keywords: Key words Calcium channels ; Chromaffin cells ; ω-Agatoxin IVA ; ω-Conotoxin ; GVIA ; ω-Conotoxin MVIIC ; Furnidipine
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract  This study was undertaken to reassess the set of voltage-dependent Ca2+ channel subtypes expressed by bovine adrenal chromaffin cells maintained in primary cultures. Previous views on the pharmacology of such channels had to be revised in the light of the novel data which arose from the use in this study of low and high micromolar concentrations of ω-agatoxin IVA, and low (2 mM) and high (10 mM) concentrations of the charge carrier Ba2+. Whole-cell Ba2+ currents (IBa) through Ca2+ channels were elicited in voltage-clamped chromaffin cells, with a holding potential of –80 mV and depolarising pulses to 0 mV. Mean peak I Ba was 425 pA in 2 mM Ba2+ (59 cells) and 787 pA in 10 mM Ba2+ (42 cells). In 2 mM Ba2+, ω-conotoxin MVIIC (3 μM) inhibited I Ba by 79%; in 10 mM Ba2+, the blockade developed much more slowly and reached only 44%. A low concentration of ω-agatoxin IVA (20 nM) inhibited I Ba by 9%; 2 μM inhibited I Ba by 60%. This blockade was similar in low and high Ba2+ concentrations. After giving furnidipine (3 μM) and ω-conotoxin GVIA (1 μM), 2 μM ω-agatoxin IVA inhibited the remaining current (about 40–45%); this blockade was independent of the Ba2+ concentration. The current could be fully blocked by the cocktail furnidipine/ω-conotoxin GVIA/high ω-agatoxin IVA, both in low and high Ba2+ concentrations. The large Q-type channel component of I Ba is blocked by micromolar concentrations of ω-agatoxin IVA and ω-conotoxin MVIIC. While solutions with a high Ba2+ concentration strongly delayed the development of blockade by ω-conotoxin MVIIC, the blockade by high concentrations of ω-agatoxin IVA was equally effective in solutions with a low or a high Ba2+ concentration. Hence, the use of appropriate Ba2+ and toxin concentrations in this study reveals that P-type Ca2+ channels are poorly expressed in bovine chromaffin cells; in contrast, a robust component of the current depends on Q-type Ca2+ channels. An R-type residual current is not present in these cells.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s004240050231
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  • 3
    Electronic Resource
    Electronic Resource
    Analogies and differences between ω-conotoxins MVIIC and MVIID: binding sites and functions in bovine chromaffin cells (1997)
    Springer
    Pflügers Archiv 435 (1997), S. 55-64 
    Details
    ISSN: 1432-2013
    Keywords: Key words Calcium channels ; Q channels ; Chromaffin cells ; ω-Conotoxin MVIIC ; ω-Conotoxin MVIID ; 45Ca2+ uptake ; Catecholamine release
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract  The characteristics of the binding sites for the Conus magus toxins ω-conotoxin MVIIC and ω-conotoxin MVIID, as well as their effects on K+-evoked 45Ca2+ entry and whole-cell Ba2+ currents (I Ba), and K+-evoked catecholamine secretion have been studied in bovine adrenal chromaffin cells. Binding of [125I] ω-conotoxin GVIA to bovine adrenal medullary membranes was displaced by ω-conotoxins GVIA, MVIIC and MVIID with IC50 values of around 0.1, 4 and 100 nM, respectively. The reverse was true for the binding of [125I] ω-conotoxin MVIIC, which was displaced by ω-conotoxins MVIIC, MVIID and GVIA with IC50 values of around 30, 80 and 1.200 nM, respectively. The sites recognized by ω-conotoxins MVIIC and MVIID in bovine brain exhibited higher affinities (IC50 values of around 1 nM). Both ω-conotoxin MVIIC and MVIID blocked I Ba by 70–80%; the higher the [Ba2+]o of the extracellular solution the lower the blockade induced by ω-conotoxin MVIIC. This was not the case for ω-conotoxin MVIID; high Ba2+ (10 mM) slowed down the development of blockade but the maximum blockade achieved was similar to that obtained in 2 mM Ba2+. A further difference between the two toxins concerns their reversibility; washout of ω-conotoxin MVIIC did not reverse the blockade of I Ba while in the case of ω-conotoxin MVIID a partial, quick recovery of current was produced. This component was irreversibly blocked by ω-conotoxin GVIA, suggesting that it is associated with N-type Ca2+ channels. Blockade of K+-evoked 45Ca2+ entry produced results which paralleled those obtained by measuring I Ba. Thus, 1 μM of each of ω-conotoxin GVIA and MVIIA inhibited Ca2+ uptake by 25%, while 1 μM of each of ω-conotoxin MVIIC and MVIID caused a 70% blockade. K+-evoked catecholamine secretory responses were not reduced by ω-conotoxin GVIA (1 μM). In contrast, at 1 μM both ω-conotoxin MVIIC and MVIID reduced the exocytotic response by 70%. These data strengthen the previously established conclusion that Q-type Ca2+ channels that contribute to the regulation of secretion and are sensitive to ω-conotoxins MVIIC and MVIID are present in bovine chromaffin cells. These channels, however, seem to possess binding sites for ω-conotoxins MVIIC and MVIID whose characteristics differ considerably from those described to occur in the brain; they might represent a subset of Q-type Ca2+ channels or an entirely new subtype of voltage-dependent high-threshold Ca2+ channel.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s004240050483
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  • 4
    Electronic Resource
    Electronic Resource
    Autocrine/paracrine modulation of calcium channels in bovine chromaffin cells (1998)
    Springer
    Pflügers Archiv 437 (1998), S. 104-113 
    Details
    ISSN: 1432-2013
    Keywords: Key words Calcium channels ; Modulation ; Presynaptic receptors
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract  Applying 10-s pulses of 10 mM Ba2+ to resting or K+-depolarized (70 mM) bovine adrenal chromaffin cells superfused with a nominal 0Ca2+ solution produced a large catecholamine secretory peak. In contrast, pulses of 10 mM Sr2+ or Ca2+ did not induce secretion from polarized resting cells, and induced smaller and narrower secretory peaks from depolarized cells; the areas of the secretory peaks from depolarized cells were 1.87, 3.06 and 27.4 nA s, respectively, for Ca2+, Sr2+ and Ba2+. Ca2+ channel currents in isolated cells or in cells surrounded by other unpatched cells (cell cluster) were studied with either the continuous-flow or the flow-stop method. When applied to an isolated cell, flow-stop reduced the amplitude of I Ca by 19%, I Sr by 31%, and I Ba by 53%, compared with the current amplitude measured under continuous-flow conditions. This decrease in current amplitude was accompanied by a pronounced slowing down of current activation and could be largely relieved by applying strong depolarizing prepulses (facilitation). Under continuous-flow conditions, 10 µM exogenous ATP reduced (about 50%) I Ca, I Sr and I Ba similarly. On the other hand, the use of Na+ as a charge carrier through Ca2+ channels, or intracellular dialysis with 1 mM BAPTA prevented the modulation of current by flow-stop. In cell clusters, activating secretion from unpatched cells, by either 10 mM Ba2+, 100 µM acetylcholine or 70 mM K+, caused a pronounced slowing down of current activation, as well as a decrease of its magnitude in the voltage-clamped cell immersed in the cluster. Such modulation of isolated cells was not observed. These data are compatible with the idea that the secretory activity of adrenal medullary chromaffin cells ”in situ” controls the activity of their Ca2+ channels through autocrine/paracrine mechanisms.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s004240050754
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    Paper (German National Licenses)
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