Secretion In Bovine Chromaffin Cells Research Articles

Alleviation of oxidative stress by potent and selective thioredoxin-mimetic peptides

One of the major enzymatic cell defenses providing protection from oxidative injury is the TrxR–Trx system. It consists of NADPH and thioredoxin reductase (TrxR), which maintain thioredoxin (Trx) in a reduced state. Perturbing the TrxR–Trx system with the selective TrxR inhibitor auranofin (AuF; 2,3,4,6-tetra-O-acetyl-1-thio-β-d-glucopyranosato-S-(triethylphosphine) gold) induces oxidative stress by keeping Trx in its oxidized state. We have prepared a family of tri- and tetra-oligopeptides derived from the canonical CxxC motif of the Trx active site and a modified CxC motif. These Trx-mimetic compounds are N- and C-terminal-blocked peptides that consist of two cysteine residues that flank the two-amino-acid CxxC motif (CB4 and CB6) or the single-amino-acid CxC motif (CB3). Catecholamine (CA) secretion in bovine chromaffin cells, which is a highly redox sensitive process, is abolished by AuF. The Trx-mimetic peptides effectively restore CA secretion, as monitored by amperometry in single cells. They also prevent the AuF-induced phosphorylation of p38 mitogen-activated protein kinase (MAPK) and c-Jun NH2-terminal kinase. In PC12 cells, the alleviation of AuF-induced ERK1/2-MAPK phosphorylation by Trx-like peptides parallels their effect of restoring CA secretion. CB3, CB4, and CB6 act intracellularly and are significantly more potent than the traditional antioxidants NAC, GSH, DTT, AD4 (NAC-amide), and ascorbic acid. Taken together, the CxxC and CxC peptides represent a new family of potent and selective redox compounds that could serve as potential candidates for prevention and treatment of oxidative-stress-related disorders.

The Involvement of Ser1898 of the Human L-Type Calcium Channel in Evoked Secretion

A PKA consensus phosphorylation site S1928 at the α 11.2 subunit of the rabbit cardiac L-type channel, CaV1.2, is involved in the regulation of CaV1.2 kinetics and affects catecholamine secretion. This mutation does not alter basal CaV1.2 current properties or regulation of CaV1.2 current by PKA and the beta-adrenergic receptor, but abolishes CaV1.2 phosphorylation by PKA. Here, we test the contribution of the corresponding PKA phosphorylation site of the human α 11.2 subunit S1898, to the regulation of catecholamine secretion in bovine chromaffin cells. Chromaffin cells were infected with a Semliki-Forest viral vector containing either the human wt or a mutated S1898A α 11.2 subunit. Both subunits harbor a T1036Y mutation conferring nifedipine insensitivity. Secretion evoked by depolarization in the presence of nifedipine was monitored by amperometry. Depolarization-triggered secretion in cells infected with either the wt α 11.2 or α 11.2/S1898A mutated subunit was elevated to a similar extent by forskolin. Forskolin, known to directly activate adenylyl-cyclase, increased the rate of secretion in a manner that is largely independent of the presence of S1898. Our results are consistent with the involvement of additional PKA regulatory site(s) at the C-tail of α 11.2, the pore forming subunit of CaV1.2.

Ciguatoxin-induced catecholamine secretion in bovine chromaffin cells: Mechanism of action and reversible inhibition by brevenal

Ciguatoxin (P-CTX-1B) from the dinoflagellate Gambierdiscus toxicus, belongs to the family of polyether neurotoxins responsible for the neurological poisoning disorder ciguatera. Although it is the most widespread marine-borne disease affecting humans, there is no current FDA-approved treatment available except for symptomatic therapies. In this paper, we report that P-CTX-1B promotes catecholamine secretion from bovine chromaffin cells, an effect that is insensitive to concomitant activation of capacitative Ca 2+ entry. Moreover, we confirm that brevenal, a polyether from the dinoflagellate Karenia brevis, blocks P-CTX-1B-induced catecholamine secretion. This effect is partially reversible. Our results therefore raise the prospect of finding functional antagonists for P-CTX-1B that could be useful for the treatment of ciguatera.

Mitochondrial calcium sequestration and protein kinase C cooperate in the regulation of cortical F‐actin disassembly and secretion in bovine chromaffin cells

Mitochondria play an important role in the homeostasis of intracellular Ca(2+) and regulate its availability for exocytosis. Inhibitors of mitochondria Ca(2+) uptake such as protonophore CCCP potentiate the secretory response to a depolarizing pulse of K(+). Exposure of cells to agents that directly (cytochalasin D, latrunculin B) or indirectly (PMA) disrupt cortical F-actin networks also potentiate the secretory response to high K(+). The effects of cytochalasin D and CCCP on secretion were additive whereas those of PMA and CCCP were not; this suggests different mechanisms for cytochalasin D and CCCP and a similar mechanism for PMA and CCCP. Mitochondria were the site of action of CCCP, because the potentiation of secretion by CCCP was observed even after depletion of Ca(2+) from the endoplasmic reticulum. CCCP induced a small increase in the cytosolic Ca(2+) concentration ([Ca(2+)](c)) that was not modified by the protein kinase C (PKC) inhibitor chelerythrine. Both CCCP and PMA induced cortical F-actin disassembly, an effect abolished by chelerythrine. In addition, rotenone and oligomycin A, two other mitochondrial inhibitors, also evoked cortical F-actin disassembly and potentiated secretion; again, these effects were blocked by chelerythrine. CCCP also enhanced the phosphorylation of PKC and myristoylated alanine-rich C kinase substance (MARCKS), and these were also inhibited by chelerythrine. The results suggest that the rapid sequestration of Ca(2+) by mitochondria would protect the cell from an enhanced PKC activation and cortical F-actin disassembly, thereby limiting the magnitude of the secretory response.

Differential participation of actin- and tubulin-based vesicle transport systems during secretion in bovine chromaffin cells.

The role of cytoskeletal elements in vesicle transport occurring during exocytosis was examined in adrenal medullary bovine chromaffin cells maintained in culture. Amperometric determination of depolarization-dependent catecholamine release from individual intact cells treated with actin or myosin inhibitors showed alterations in the fast and slow phases of secretion when compared with untreated cells. In contrast, microtubule disassemblers or stabilizers have a moderate effect on secretion, only affecting the release of slow secretory components. In experiments using confocal dynamic microscopy we have observed the drastic effect of actin and myosin inhibitors in abolishing vesicle movement throughout the cytoplasm, and the inhibition of granule mobility in deep perinuclear regions caused by the microtubule stabilizers. Following loss of mobility, vesicles were associated with filaments of F-actin or microtubules. In addition, the mobility of cortical vesicles was affected by actin-myosin inhibitors but not by microtubule inhibitors. The study of cortical cytoskeleton in living cells showed vesicles associated with dense tubular F-actin structures, with microtubules appearing as low density networks. These findings suggest that the distribution and density of both cytoskeletal elements in the cortical region may influence the recruitment of vesicle pools during secretion.

Molecular mechanisms of glutamate release by bovine chromaffin cells in primary culture

Previous work indicated that glutamate could be involved in the regulation of catecholamine secretion in bovine chromaffin cells. Thus, the question arises on the source of this putative regulatory glutamate. In this work we have examined the possibility that glutamate could be released from chromaffin cells. Data from this study indicate that chromaffin cells are able to release glutamate when they are stimulated by different depolarising agents such as 60 mM KCl, 1 mM 4-aminopyridine or 50 μM veratridine. The amount of glutamate released by these compounds was 0.32 nmol/10 6 cells (9.24% of cellular glutamate content), 0.275 (7.86%) and 0.158 (4.52%) for KCl, 4-AP and veratridine stimulation, respectively. All these catecholamine-secretagogues induced glutamate secretion by two mechanisms: 1) a Ca 2+-dependent, probably exocytotic, mechanism and 2) a Ca 2+-independent mechanism mediated by reversion of the electrogenic glutamate transporter. Analysis of Ca 2+-dependent and independent releases for different compounds carried out by several experimental approaches, indicate that Ca 2+-dependent release was the predominant mechanism for release induced by 4-aminopyridine (84% of total release) and high KCl (63%) whilst Ca 2+-independent release was predominant for veratridine (67%). The Ca 2+-dependent glutamate release evoked by depolarisation of chromaffin cells with high KCl and 4-AP could be split into both a fast and a slow kinetic component, which might correspond to the release of docked and mobilised chromaffin granules, respectively. On the other hand, depolarisation of cells with veratridine result in glutamate release with only the fast kinetic component. In the case of 60 mM KCl-evoked glutamate release, the fast component exhibited a decay time of <1 s and accounted for 0.63 nmol glu/6×10 6 cells (70% of total exocytotic release), whereas the slow component, which exhibited a decay time of 231 s, accounted for the release of 0.27 nmol glu/6×10 6 cells (30% of total exocytotic release). By contrast in the case of 4-aminopyridine the fast component of exocytosis only represents a 19% of total secretion and the slow a 81% with a decay time of 94 s. These data are very similar to those found in neurones and support the possible intracellular origin of glutamate having a role in the regulation of catecholamine secretion from chromaffin cells. In support of this, we have found that glutamate secretion could be evoked by stimulation of the nicotinic cholinergic receptors.

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.

Naloxone inhibits nicotine-induced receptor current and catecholamine secretion in bovine chromaffin cells

Nicotine-induced catecholamine (CA) secretion and inward ionic currents were inhibited by the opioid antagonist naloxone in cultured bovine chromaffin cells. Naloxone inhibited nicotine-induced CA secretion, as detected by an on-line real-time electrochemical technique, in a dose-dependent manner (IC 50=29 μM). In voltage-clamped chromaffin cells, nicotine (10 μM) evoked an average peak inward current of −146 pA that was inhibited by low concentrations of naloxone (42% at 0.1 μM). The antagonist also inhibited total charge influx associated with nicotinic receptor activation (53% at 0.1 μM). This provides strong evidence that naloxone modulation of nicotine-induced CA secretion does not involve opioid receptors but results from the direct interaction with the nicotinic receptor itself, which might also be the case for other related opioid compounds.

Effect of Glutamate Receptor Agonists on Catecholamine Secretion in Bovine Chromaffin Cells

In this study, the effects of glutamate and glutamate receptor agonists in cultured chromaffin cells from bovine adrenal medulla were investigated. It was found that glutamate increases basal catecholamine (CA) secretion in a dose-dependent manner. This effect is mimicked by specific agonists of the four known glutamate receptors N-methyl-D-aspartate (NMDA), quisqualate/(RS)-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), kainate (KA), and trans-(+)-1-amino-1,3-cyclopentane dicarboxylic acid (t-ACPD), which increased both basal and nicotine-evoked CA secretion. The NMDA receptor antagonist 2-amino-5-phosphonopentanoic acid, 6-cyano-7-nitroquinoxaline-2,3-dione, an antagonist of KA and AMPA receptors, and L-(+)-2-amino-3-phosphonopropionic acid, an antagonist of the t-ACPD receptor, inhibited the stimulatory effect of related glutamate agonists. Hexamethonium, an antagonist of the nicotinic receptor, failed to influence glutamate agonists except for a 15% inhibition of KA. The increase in CA secretion produced by a 100 µM concentration of glutamate agonists was about 20–60% of that obtained with 10 µM of nicotine, an agonist of the physiological stimulatory cholinergic receptor. The increase in CA secretion produced by glutamate was accompanied by both an increase in bisoxonol fluorescence, suggesting membrane depolarization, and by an increase in intracellular Ca²⁺ concentrations. Results obtained with image analysis on single cells indicated that the percentage of cells which respond to the stimulation of 50 µM of glutamate is 42%. From these results, we conclude that glutamate, through its four known glutamate receptors, can increase both basal and nicotine-evoked CA secretion in chromaffin cells by a process which involves membrane depolarization and an increase in intracellular calcium levels.

Importance of the Rab3a-GTP binding domain for the intracellular stability and function of Rabphilin3a in secretion.

We had previously demonstrated that Rab3a-GTP inhibits and the Rab3a-binding protein Rabphilin3a enhances secretion in bovine chromaffin cells. In this study, we investigated the role of Rab3a-GTP binding in the intracellular expression and the function of Rabphilin3a in regulated exocytosis in bovine chromaffin cells. Using transient transfections, we found that a minimal domain, Rp(51-190), that inhibits secretion coincides with a minimal domain that effectively binds Rab3a-GTP and allows intracellular stability of the construct. This domain includes a cysteine-rich, Zn2+-binding domain whose integrity is also required for Rab3a-GTP binding and the ability to inhibit secretion. A Rabphilin3a mutant, containing both C2 domains but defective in Rab3a-GTP, and wild-type Rabphilin3a both localized to chromaffin granules and stimulated secretion similarly despite lessened intracellular expression of the mutant protein. The data are consistent with a sequence of events in which a Rab3a-GTP x Rabphilin3a complex forms on the secretory granule as a precursor in a pathway that enhances secretion. The complex dissociates (perhaps because of GTP hydrolysis) to permit the enhancement of secretion by Rabphilin3a.

Effects of caffeine on Ca 2+ fluxes and secretion in bovine chromaffin cells

The effects of caffeine on Ca 2+ fluxes and catecholamine secretion in bovine adrenal chromaffin cells were examined. Caffeine inhibited secretion, 45Ca 2+ uptake and cytosolic Ca 2+ concentration ([Ca 2+] i) rise induced by the nicotinic receptor agonist 1,1-dimethyl-4-phenylpiperazinium (DMPP) and the Na + channel activator veratridine. The inhibitory effect of caffeine on high K +-induced secretion was smaller than that on DMPP- and veratridine-induced responses. Caffeine only slightly inhibited high K +-induced 45Ca 2+ uptake and did not affect [Ca 2+] i rise. Caffeine also inhibited muscarinic receptor-mediated inositol phosphate generation. Our results suggest that the inhibitory effects of caffeine on bovine chromaffin cells mainly occur at both muscarinic and nicotinic receptors as well as at the voltage-dependent Na + channels and to a smaller extent at site(s) distal to Ca 2+ entry. The effects of caffeine on nicotinic receptors but not on muscarinic receptors can be explained by its ability to raise intracellular cAMP.

ATP stimulated catecholamine secretion: Response in perfused adrenal glands and a subpopulation of cultured chromaffin cells

Extracellular ATP is shown to induce catecholamine secretion in bovine chromaffin cells. Our data indicate that cells in culture gradually increase their response to ATP, and we have separated freshly isolated cells on a density gradient and found that the lighter cells develop a much stronger response to ATP than do the heavier cells. To see if the ATP sensitivity is physiological, we have perfused intact adrenal glands. ATP induces a greater secretory response from glands than does acetylcholine without causing preferential secretion of norepinephrine or epinephrine. These data show that the response to ATP found in cultured cells is not an artifact of cell culture, and that ATP co-released with catecholamines from the storage vesciles may have a significant physiological role.

Q- and L-type Ca 2+ channels dominate the control of secretion in bovine chromaffin cells

Potassium-stimulated catecholamine release from superfused bovine adrenal chromaffin cells (70 mM K + in the presence of 2 mM Ca 2+ for 10 s, applied at 5-min intervals) was inhibited by the dihydropyridine furnidipine (3 μM) by 50%. ω-Conotoxin MVIIC (CTx-MVIIC, 3 μM) also reduced the secretory response by about half. Combined CTx-MVIIC plus furnidipine blocked 100% catecholamine release. 45Ca 2+ uptake and cytosolic Ca 2+ concentrations ([Ca 2+] i) in K +-depolarized cells were partially blocked by furnidipine or CTx-MVIIC, and completely inhibited by both agents. The whole cell current through Ca 2+ channels carried by Ba 2+ ( I Ba) was partially blocked by CTx-MVIIC. Although ω-conotoxin GVIA (CTx-GVIA, 1 μM) and ω-agatoxin IVA (Aga-IVA, 0.2 μM) partially inhibited 45Ca 2+ entry, I Ba and the increase in [Ca 2+] i, the combination of both toxins did not affect the K +-evoked secretory response. The results are compatible with the presence in bovine chromaffin cells of a Q-like Ca 2+ channel which has a prominent role in controlling exocytosis. They also suggest that Q- and L-type Ca 2+ channels, but not N- or P-types are localized near exocytotic active sites in the plasmalemma.

Calyculin A blocks bovine chromaffin cell calcium channels independently of phosphatase inhibition

The specific phosphatase inhibitor, Calyculin-A (CL-A), decreases high-K stimulated catecholamine secretion in bovine chromaffin cells. This effect can be split into two components: one needs long exposures to the drug to be elicited, and is sensitive to the protein kinase-inhibitor K252a; the other is observed after short incubations of CL-A, and is insensitive to K252a. Here we report that the latter component is due to an external block, by CL-A, of chromaffin cell calcium channels in a voltage-dependent, reversible and phosphorylation-independent manner.

Neuropeptide Y inhibits catecholamine secretion from single bovine chromaffin cells by a decrease in intracellular cAMP

In cultured bovine chromaffin cells, activation ofnicotinic acetylcholine receptors initiates a cationic inward current and a subsequent secretion of catecholamines. It has been shown that neuropeptide Y (NPY) and its C-terminal fiagment inhibit the nicotine-induced current.’ In the present study, fast cyclic voltammetry was used to determine nicotine-induced release of endogenous catecholamines from single chromaffin cells. A small carbon fibre electrode was placed about 5 pm apart from a cell, and the current resulting from the catecholamine oxidation was measured. Pressure application of nicotine (10 pM) for 10 s evoked a catecholamine secretion corresponding to 1.2 f 0.1 pM noradrenaline (n = 78). Repeated nicotine-applications at 3 min intervals produced stable secretion-responses. The secretion was abolished in the absence of Ca” andreduced by about 90% in the presence of hexamethonium (100 pM). NPY(18-36) (0.2-20 PM), when applied for 30 s, caused a marked oxidation signal by itself which disappeared within a 10 min washout period. However, the nicotine-induced secretion was decreased for a fUrther 15 min. 8-Bromo-CAMP (100 pM) increased the catecholamine secretion by about 80%. 8-Bromo-CAMP also abolished the effect of NPY( 18-36) (2 PM). Pressure application of K+ (50 mM; 10 s) induced a catecholamine secretion corresponding to 1.6 f 0.3 pM noradrenaline (n = 16). The K+-induced secretion was not affected by NPY( 18-36) (20 pM). In conclusion, NPY reduces nicotine induced catecholamine secretion in bovine chromaffin cells by decreasing the intracellular CAMP concentration.

Transient transfection studies of secretion in bovine chromaffin cells and PC12 cells. Generation of kainate-sensitive chromaffin cells

We have developed a transient transfection method to measure protein secretion from non-dividing, primary bovine chromaffin cells and from the continuous cell line, PC12. A plasmid coding human growth hormone (GH) was expressed in sufficient amounts in bovine chromaffin and PC12 cells to allow precise measurements of secretion from the small fraction (less than 1%) of transfected cells in a dish. GH was secreted in a similar proportion to endogenous catecholamine upon nicotinic stimulation, depolarization with elevated K+, and upon permeabilization with digitonin and subsequent stimulation with micromolar Ca2+. GH in homogenates from GH-transfected chromaffin cells cosedimented with catecholamine on discontinuous sucrose gradients. The data indicate that transiently expressed human GH in chromaffin and PC12 cells is localized predominantly in secretory vesicles in the regulated secretory pathway. With transient transfection there is a high probability of coexpression in the same cell of two plasmids which are cotransfected. Coexpression of a plasmid for GH and a plasmid for the non-N-methyl-D-aspartate glutamate receptor, GluR1, created chromaffin cells in which Ca(2+)-dependent GH secretion could be stimulated by the glutamatergic agonist kainate. The ability to coexpress a plasmid of interest with a plasmid for GH will allow the investigation of the role of other cloned proteins in the regulated secretory pathway in differentiated, non-dividing cells.

Multiple calcium-dependent processes related to secretion in bovine chromaffin cells

We have used the caged calcium compound DM-nitrophen to investigate the kinetics of calcium-dependent secretion in bovine chromaffin cells. Perfusion with partially calcium-loaded nitrophen often caused a loading transient-slow secretion for up to 1 min due to displacement of Ca 2+ by cytoplasmic Mg 2+. Flash photolysis elicited 100 μM [Ca 2+] i steps that evoked intense secretion, lasting a few seconds. In cells experiencing a loading transient, [Ca 2+] i steps evoked an especially fast secretion. A persistent, slow secretion often followed these fast phases. Distinct kinetic components may reflect secretion from pools that are differentially capable of release. Both secretion and movement of vesicles between pools appear to be [Ca 2+] i sensitive. Later [Ca 2+] i steps sometimes evoked a rapid capacitance decrease, indicating a fast, [Ca 2+] i-dependent phase of endocytosis.

Mixed nicotinic and muscarinic features of cholinergic receptor coupled to secretion in bovine chromaffin cells.

Acetylcholine evokes release from cultured bovine chromaffin cells by a mechanism that is believed to be classically nicotinic. However, we found that the full muscarinic agonist oxotremorine-M (Oxo-M) induced a robust catecholamine (CA) secretion. By contrast, muscarine, pilocarpine, bethanechol, and McN-A-343 did not elicit any secretory response. Desensitization of the response to nicotine by Oxo-M and desensitization of the response to Oxo-M by nicotine suggest that both nicotine and Oxo-M were acting at the same receptor. Additional experiments supporting this conclusion show that nicotine-induced secretion and Oxo-M-induced secretion were similarly blocked by various muscarinic and nicotinic antagonists. Moreover, secretion induced by nicotine and Oxo-M were Ca2+ dependent, and both agonists induced 45Ca2+ uptake. Equilibrium binding studies showed that [3H]Oxo-M bound to chromaffin cell membranes with a Kd value of 3.08 x 10(-8) M and a Hill coefficient of 1.00, suggesting one binding site for this ligand. Nicotine inhibited Oxo-M binding in a noncompetitive manner, suggesting that both ligands bind at two different sites on the same receptor. We propose that the receptor on bovine chromaffin cells that is coupled to secretion represents an unusual cholinergic receptor that has both nicotinic and muscarinic features.