Currents In Bovine Chromaffin Cells Research Articles

Aluminum alters excitability by inhibiting calcium, sodium, and potassium currents in bovine chromaffin cells.

Aluminum (Al3+ ) has long been related to neurotoxicity and neurological diseases. This study aims to describe the specific actions of this metal on cellular excitability and neurotransmitter release in primary culture of bovine chromaffin cells. Using voltage-clamp and current-clamp recordings with the whole-cell configuration of the patch clamp technique, online measurement of catecholamine release, and measurements of [Ca2+ ]c with Fluo-4-AM, we have observed that Al3+ reduced intracellular calcium concentrations around 25% and decreased catecholamine secretion in a dose-dependent manner, with an IC50 of 89.1μM. Al3+ blocked calcium currents in a time- and concentration-dependent manner with an IC50 of 560 μM. This blockade was irreversible since it did not recover after washout. Moreover, Al3+ produced a bigger blockade on N-, P-, and Q-type calcium channels subtypes (69.5%) than on L-type channels subtypes (50.5%). Sodium currents were also inhibited by Al3+ in a time- and concentration-dependent manner, 24.3% blockade at the closest concentration to the IC50 (399 μM). This inhibition was reversible. Voltage-dependent potassium currents were low affected by Al3+ . Nonetheless, calcium/voltage-dependent potassium currents were inhibited in a concentration-dependent manner, with an IC50 of 447 μM. This inhibition was related to the depression of calcium influx through voltage-dependent calcium channels subtypes coupled to BK channels. In summary, the blockade of these ionic conductance altered cellular excitability that reduced the action potentials firing and so, the neurotransmitter release and the synaptic transmission. These findings prove that aluminum has neurotoxic properties because it alters neuronal excitability by inhibiting the sodium currents responsible for the generation and propagation of impulse nerve, the potassium current responsible for the termination of action potentials, and the calcium current responsible for the neurotransmitters release.

Dual Antidepressant Duloxetine Blocks Nicotinic Receptor Currents, Calcium Signals and Exocytosis in Chromaffin Cells Stimulated with Acetylcholine.

The inhibition of nicotinic acetylcholine receptors (nAChRs) has been proposed as a potential strategy to develop new antidepressant drugs. This is based on the observation that antidepressants that selectively block noradrenaline (NA) or serotonin (5-HT) reuptake also inhibit nAChRs. Dual antidepressants blocking both NA and 5-HT reuptake were proposed to shorten the delay in exerting their clinical effects; whether duloxetine, a prototype of dual antidepressants, also blocks nAChRs is unknown. Here we explored this question in bovine chromaffin cells (BCCs) that express native α3, α5, and α7 nAChRs and in cell lines expressing human α7, α3β4, or α4β2 nAChRs. We have found that duloxetine fully blocked the acetylcholine (ACh)-elicited nicotinic currents in BCCs with an IC50 of 0.86 µM. Such blockade seemed to be noncompetitive, voltage dependent, and partially use dependent. The ACh-elicited membrane depolarization, the elevation of cytosolic calcium ([Ca2+]c), and catecholamine release in BCCs were also blocked by duloxetine. This blockade developed slowly, and the recovery of secretion was also slow and gradual. Duloxetine did not affect Na+ or Ca2+ channel currents neither the high-K+-elicited [Ca2+]c transients and secretion. Of interest was that in cell lines expressing human α7, α3β4, and α4β2 nAChRs, duloxetine blocked nicotinic currents with IC50 values of 0.1, 0.56, and 0.85 µM, respectively. Thus, in blocking α7 receptors, which are abundantly expressed in the brain, duloxetine exhibited approximately 10-fold to 100- fold higher potency with respect to reported IC50 values for various antidepressant drugs. This may contribute to the antidepressant effect of duloxetine.

Enhancement of inward Ca 2+ currents in bovine chromaffin cells by green tea polyphenol extracts

Green tea contains four major polyphenol compounds: they are (−)-epigallocatechin-3-gallate (EGCG), (−)-epigallocatechin (EGC), (−)-epicatechin-3-gallate (ECG), and (−)-epicatechin (EC). Although all four polyphenol compounds are known to affect tumor suppression, little is known about whether they alter membrane properties. In this study, we examined the effects of ECG and EGCG on ionic currents and secretion. Membrane capacitance changes were used to monitor secretion in bovine chromaffin cells. ECG had the ability to reversibly enhance the inward Ca 2+ current by 21%, and inhibited the peak sodium current by 34%. EGCG had no effect on Ca 2+ current even though it differs from ECG by just a hydroxyl group. The EC 50 of ECG in enhancing Ca 2+ current was 7.6 μM. The maximum enhancement of Ca 2+ current was observed at 0 mV and the maximum current was shifted ∼10 mV in the hyperpolarizing direction. When cells were stimulated by trains of depolarizations, the exocytosis elicited was enhanced by ECG treatment and the largest enhancement of secretion was observed in later stimulations. EGCG, although it had no significant effect on Ca 2+ current, enhanced exocytosis and slowed endocytosis. These results suggest that green tea polyphenol compounds modulate stimulus-secretion coupling in bovine chromaffin cells.

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

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 omega-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 IBa was 425 pA in 2 mM Ba2+ (59 cells) and 787 pA in 10 mM Ba2+ (42 cells). In 2 mM Ba2+, omega-conotoxin MVIIC (3 microM) inhibited IBa by 79%; in 10 mM Ba2+, the blockade developed much more slowly and reached only 44%. A low concentration of omega-agatoxin IVA (20 nM) inhibited IBa by 9%; 2 microM inhibited IBa by 60%. This blockade was similar in low and high Ba2+ concentrations. After giving furnidipine (3 microM) and omega-conotoxin GVIA (1 microM), 2 microM omega-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/omega-conotoxin GVIA/high omega-agatoxin IVA, both in low and high Ba2+ concentrations. The large Q-type channel component of IBa is blocked by micromolar concentrations of omega-agatoxin IVA and omega-conotoxin MVIIC. While solutions with a high Ba2+ concentration strongly delayed the development of blockade by omega-conotoxin MVIIC, the blockade by high concentrations of omega-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.

A new cysteine framework in sodium channel blocking conotoxins.

Two novel sodium channel blocking peptides from the venom of the molluscivorous snail Conus pennaceus, muPnIVA and muPnIVB, are described. Elucidation of their amino acid sequences was complicated by a previously undescribed anomalous product of reduction and pyridylethylation, which occurs on N-terminal cysteine residues and gives a PTH derivative eluting at the same position as PTH-Trp in reverse-phase chromatography. The amino acid sequences of the toxins were determined by a combination of Edman degradation and mass-spectrometric techniques as CCKYGWTCLLGCSPCGC (PnIVA) and CCKYGWTCWLGCSPCGC (PnIVB). These toxins block sodium channels in molluscan neurons, but have no effect on sodium currents in bovine chromaffin cells or in rat brain synaptosomes. Although there is only one amino acid difference in the two sequences, PnIVB is approximately 6 times more potent than PnIVA in blockade of the sodium current in Lymnaea neurons. The PnIV sequences reveal a new cysteine residue framework for conotoxins (CC-----C---C--C-C). Strikingly, the only charged residue in PnIVA/B is Lys3. Iodination reaction experiments on the adjacent Tyr4 suggest that this region of the peptide must be solvent exposed and essential for activity. These structurally novel mu-conotoxins target a sodium channel subtype with low affinity for tetrodotoxin and therefore provide new probes for functional studies on sodium channels.

ω-Conotoxin GVIA blocks nicotine-induced catecholamine secretion by blocking the nicotinic receptor-activated inward currents in bovine chromaffin cells

We have studied the contribution of N-type voltage-dependent Ca 2+ channels to both norepinephrine and epinephrine secretion from bovine chromaffin cells induced by high K + or nicotine using ω-conotoxin GVIA, a selective blocker of N-type voltage-dependent Ca 2+ channels. We found that high K + (75 mM) induced catecholamine secretion was not affected by exposure of bovine chromaffin cells to ω-conotoxin GVIA (1 μM). However, nicotine-induced both norepinephrine and epinephrine secretion were similarly blocked (about 25%) by ω-conotoxin GVIA (1 μM). This effect could be explained by a potent (about 80%) and reversible blockade of the inward current induced by nicotine receptor activation in bovine chromaffin cells. The results indicate that besides the blockade of N-type voltage-dependent channels, ω-conotoxin GVIA is a potent and reversible blocker of the nicotinic receptor-induced currents in chromaffin cells.

Pb 2+ activates potassium currents in bovine adrenal chromaffin cells

Inorganic lead (Pb 2+) is a potent environmental toxin which adversely affects several aspects of neuronal and secretory cell function. In this report, we provide evidence that at subnanomolar concentrations, Pb 2+ activates the outward K + currents in bovine adrenal chromaffin cells. Whole-cell patch clamp combined with intracellular perfusion was employed to monitor outward K + currents in bovine chromaffin cells before and after intracellular application of EGTA-Pb buffers. Intracellular Pb 2+ ≥ 10 −10 M enhanced the K +-currents in a concentration dependent manner, with apparent K 0.5 ≊ 5 × 10 −10 M . Extracellular application of 40 nM Charybdotoxin (ChTX) blocked the Pb 2+-dependent component of outward currents, suggesting that Pb 2+ activates the large conductance Ca 2+-dependent K + channels.

G-protein activation mediates prepulse facilitation of Ca2+ channel currents in bovine chromaffin cells.

The effects of G-protein activation were investigated on tonic, large depolarization-induced Ca2+ channel facilitation in cultured bovine adrenal chromaffin cells. Under whole-cell voltage clamp, activation of G proteins by intracellular dialysis with 200 microM GTP-gamma S did not significantly affect prepulse facilitation or whole-cell Ba2+ current (IBa) density. In contrast, inactivation of G proteins by intracellular GDP-beta S or pertussis toxin (PTX) pretreatment completely abolished or markedly attenuated facilitation of IBa, respectively. GDP-beta S dialysis resulted in nearly a threefold increase in peak IBa density, whereas PTX pretreatment resulted in a 50% increase. Our results indicate that under control recording conditions (200 microM intracellular GTP), G proteins are tonically activated and suppress high-voltage-activated (HVA) Ca2+ channels in a voltage-dependent and voltage-independent manner. Local superfusion of chromaffin cells with normal bath solution produced a rapid and reversible increase (approximately 50%) in IBa amplitudes that also abolished prepulse facilitation. Together, these results demonstrate that tonic facilitation of HVA Ca2+ channels in bovine chromaffin cells involves the voltage-dependent relief of a G-protein-mediated suppression, imposed by chromaffin cell secretory products that feedback and activate G-protein-coupled autoreceptors.

A patch clamp study of the nicotinic acetylcholine receptor of bovine adrenomedullary chromaffin cells in culture.

1. Acetylcholine-induced currents recorded from bovine adrenal medullary chromaffin cells maintained in culture were studied during pressure or ionophoretic applications of ACh, using the 'whole-cell' and 'outside-out' configurations of the patch clamp technique. In standard salines, ACh evoked whole-cell currents of -38 pA to -1 nA at -60 mV, which had a reversal potential (EACh) of -7.1 +/- 0.6 mV. The ACh current-voltage relationship was characteristically linear at negative holding potentials and biphasic at positive holding potentials, displaying a region of almost zero slope conductance between 0 and +40 mV followed by a region of positive slope conductance at more positive potentials. 2. Relative permeation to cations was examined. Substitution of external Na+ by sucrose resulted in a -42 mV shift of EACh for a 10-fold reduction in [Na+]o. Using isotonic substitutions, the permeability ratios (relative to Na+) for monovalent cations were determined to be 1.32 +/- 0.02 for Cs+ (n = 11), 1.03 +/- 0.02 for Li+ (n = 8) and 0.18 +/- 0.02 for Tris+ (n = 7). Elevated external Ca2+ salines were found to shift EACh to more positive potentials, especially in the presence of low external Na+. 3. The nicotinic agonists nicotine, tetramethylammonium and lobeline evoked inward currents in bovine chromaffin cells. In contrast, decamethonium and the muscarinic agonist, methacholine, had no effect. 4. The nicotinic antagonists mecamylamine, trimetaphan, (+)-tubocurarine and hexamethonium caused dose-dependent reductions in the amplitude of ACh-evoked inward currents. The estimated IC50's were 0.25, 0.33, 0.63 and 2.2 microM respectively, for cells voltage clamped at -60 mV. High concentrations (> 2 microM) of the muscarinic antagonist, atropine, also produced a dose-dependent reduction in the amplitude of ACh-induced currents. 5. Inhibition by trimetaphan was voltage independent. With the other drugs the antagonism was voltage sensitive, increasing with membrane hyperpolarization. The voltage sensitivity was most marked for hexamethonium. Neither hexamethonium nor mecamylamine were found to depress ACh-evoked outward currents at concentrations which severely depressed inward currents. In addition to its antagonist actions, (+)-tubocurarine activated unitary currents in these cells and on isolated membrane patches. 6. The results indicate that nicotinic ion channels of bovine chromaffin cells have a similar ionic selectivity to monovalent cations, but that Ca2+ ions permeate the channels to a greater degree than at the motor endplate. The ACh current-voltage relationship resembles that described for other types of 'neuronal' nicotinic receptors.(ABSTRACT TRUNCATED AT 400 WORDS)

Modulation of secretion by dopamine involves decreases in calcium and nicotinic currents in bovine chromaffin cells.

1. Catecholamine secretion from cultured bovine adrenal chromaffin cells was decreased in a dose-dependent manner by the D2 dopamine agonists apomorphine and LY 17 1555. 2. 45Ca2+ uptake was similarly inhibited and whole-cell Ca2+ currents were reduced by apomorphine. 3. These inhibitory effects of D2 agonists depended on the secretagogue used, being much more pronounced for nicotine-evoked responses compared to high K+ stimulation, indicating another possible site of action of apomorphine up-stream of Ca2+ entry. 4. Inhibition by apomorphine of nicotine-evoked responses could not be explained by competitive antagonism against nicotine or DMPP (1,1-dimethyl-4-phenyl-piperazinium iodide). 5. Apomorphine caused reductions of inward whole-cell nicotinic current evoked by ACh and nicotine. 6. Inhibition of nicotine-evoked secretion and 22Na+ influx by apomorphine were not affected by tetrodotoxin, and voltage-dependent, whole-cell Na+ currents were unaltered by apomorphine. 7. No evidence was obtained for increases in K+ conductance by apomorphine. 8. Action potentials recorded in whole-cell current clamp were blocked by apomorphine when they were triggered by nicotinic depolarization but not when they were elicited by direct electrical stimulation. 9. Inclusion of GDP-beta-S in the pipette internal solution did not affect apomorphine-dependent inhibition of nicotinic-evoked responses, while the decrease in whole-cell Ca2+ current induced by apomorphine was completely inhibited in the presence of GDP-beta-S. 10. Increases in cyclic AMP caused by cholera toxin and forskolin did not change the apomorphine-dependent inhibitory effects on nicotine-evoked secretion, indicating that changes in cyclic AMP levels caused by dopamine receptor stimulation are probably not involved.