Bovine Adrenal Chromaffin Cells Research Articles

Autocrine activation of P2X7 receptors mediates catecholamine secretion in chromaffin cells.

ATP is highly accumulated in secretory vesicles and secreted upon exocytosis from neurons and endocrine cells. In adrenal chromaffin granules, intraluminal ATP reaches concentrations over 100 mM. However, how these large amounts of ATP contribute to exocytosis has not been investigated. Exocytotic events in bovine and mouse adrenal chromaffin cells were measured with single cell amperometry. Cytosolic Ca2+ measurements were carried out in Fluo-4 loaded cells. Submembrane Ca2+ was examined in PC12 cells transfected with a membrane-tethered Ca2+ indicator Lck-GCaMP3. ATP release was measured using the luciferin/luciferase assay. Knockdown of P2X7 receptors was induced with short interfering RNA (siRNA). Direct Ca2+ influx through this receptor was measured using a P2X7 receptor-GCamp6 construct. ATP induced exocytosis in chromaffin cells, whereas the ectonucleotidase apyrase reduced the release events induced by the nicotinic agonist dimethylphenylpiperazinium (DMPP), high KCl, or ionomycin. The purinergic agonist BzATP also promoted a secretory response that was dependent on extracellular Ca2+. A740003, a P2X7 receptor antagonist, abolished secretory responses of these secretagogues. Exocytosis was also diminished in chromaffin cells when P2X7 receptors were silenced using siRNAs and in cells of P2X7 receptor knockout mice. In PC12 cells, DMPP induced ATP release, triggering Ca2+ influx through P2X7 receptors. Furthermore, BzATP, DMPP, and KCl allowed the formation of submembrane Ca2+ microdomains inhibited by A740003. Autocrine activation of P2X7 receptors constitutes a crucial feedback system that amplifies the secretion of catecholamines in chromaffin cells by favouring submembrane Ca2+ microdomains.

High signal-to-noise imaging of spontaneous and 5 ns electric pulse-evoked Ca2+ signals in GCaMP6f-expressing adrenal chromaffin cells isolated from transgenic mice.

In studies exploring the potential for nanosecond duration electric pulses to serve as a novel modality for neuromodulation, we found that a 5 ns pulse triggers an immediate rise in [Ca2+]i in isolated bovine adrenal chromaffin cells. To facilitate ongoing efforts to understand underlying mechanisms and to work toward carrying out investigations in cells in situ, we describe the suitability and advantages of using isolated murine adrenal chromaffin cells expressing, in a Cre-dependent manner, the genetically-encoded Ca2+indicator GCaMP6f. Initial experiments confirmed that Ca2+ responses evoked by a 5 ns pulse were similar between fluorescent Ca2+ indicator-loaded murine and bovine chromaffin cells, thereby establishing that 5 ns-elicited excitation of chromaffin cells occurs reproducibly across species. In GCaMP6f-expressing murine chromaffin cells, spontaneous Ca2+ activity as well as nicotinic receptor agonist- and 5 ns evoked-Ca2+ responses consistently displayed similar kinetic characteristics as those in dye-loaded cells but with two-twentyfold greater amplitudes and without photobleaching. The high signal-to-noise ratio of evoked Ca2+ responses as well as spontaneous Ca2+ activity was observed in cells derived from Sox10-Cre, conditional GCaMP6f mice or TH-Cre, conditional GCaMP6f mice, although the number of cells expressing GCaMP6f at sufficiently high levels for achieving high signal-to-noise ratios was greater in Sox10-Cre mice. As in bovine cells, Ca2+ responses elicited in murine GCaMP6f-expressing cells by a 5 ns pulse were mediated by the activation of voltage-gated Ca2+ channels but not tetrodotoxin-sensitive voltage-gated Na+ channels. We conclude that genetically targeting GCaMP6f expression to murine chromaffin cells represents a sensitive and valuable approach to investigate spontaneous, receptor agonist- and nanosecond electric pulse-induced Ca2+ responses in vitro. This approach will also facilitate future studies investigating the effects of ultrashort electric pulses on cells in ex vivo slices of adrenal gland, which will lay the foundation for using nanosecond electric pulses to stimulate neurosecretion in vivo.

A single high-intensity nanosecond electric pulse stimulates NALCN and TRPC4/5 channels leading to Na+ accumulation in adrenal chromaffin cells as detected by live cell Na+ fluorescence imaging.

We recently reported that a single 5 ns high intensity electric pulse (NEP) elicits an inwardly rectifying current carried mainly by Na+ in bovine adrenal chromaffin cells, with ∼75% of the current being carried by both TRPC4/5 channels and the Na+ leak channel (NALCN) (Yang et al. Arch Biochem Biophys 723: 109252, 2022). In this study we used Na+ fluorescence imaging to further investigate the involvement of these channels in mediating Na+ influx triggered by NEPs. Chromaffin cells were loaded with the Na+ fluorescent indicator ING-2/AM (2 μM; Ex/Em: 525/545 nm; Kd = 20 mM). Similar to patch clamp experiments, a single NEP (5 MV/m) consistently evoked Na+ accumulation in all cells, which was characterized by a gradual rise in fluorescence that reached a plateau by ∼4 min. Also consistent with the inward current monitored by patch clamp, a combination of the TRPC4/5 channel inhibitor M084 (40 µM) and the NALCN blocker CP-96345 (50 µM) blocked the increase in fluorescence by ∼69%. Surprisingly, Na+ influx was significantly reduced in the absence of extracellular Ca2+ or by blocking L-type voltage-gated Ca2+ channels with nitrendipine, nifedipine or verapamil. The NEP-evoked Na+ influx pathway did not involve nicotinic agonist receptor stimulation nor voltage-gated Na+ channels. Overall, these results suggest that a 5-ns pulse triggers a Na+ conductance through NALCN and TRPC4/5 channels, serving as a novel way to modify neural cell excitability. Future experiments will identify the mechanism by which the NEP activates Na+ influx through these channels and whether these channels can also be activated by conventional electric stimulation (i.e., milli- and micro-second electric pulses).

Ultrashort nanosecond electric pulses activate a conductance in bovine adrenal chromaffin cells that involves cation entry through TRPC and NALCN channels

In whole-cell voltage clamped bovine adrenal chromaffin cells maintained at a holding potential of −70 mV, a single 5 ns, 5 MV/m pulse elicited an inward current carried mainly by Na+ that displayed inward rectification and a reversal potential near −3 mV, a voltage consistent with a non-selective cation current. The broad-spectrum inhibitors of transient receptor potential (TRP) channels, La3+ (10 μM), Gd3+ (10 μM), SKF-96365 (50 μM) and 2-aminoethoxydiphenyl borane (2-APB; 100 μM), inhibited the current similarly by ∼72%, ∼83%, ∼68% and ∼76%, respectively. Depleting membrane cholesterol with methyl-β-cyclodextrin (MβCD; 1–6 mg/ml) or inhibiting phosphatidylinositol 4,5-bisphosphate (PIP2) synthesis with wortmannin (20 and 40 μM) produced a similar level of inhibition on the NEP-induced conductance as the broad spectrum TRP channel inhibitors. Moreover, no additive inhibitory effect was detected by combining MβCD (3 mg/ml), wortmannin (20 μM) and La3+ (10 μM), suggesting that each agent targeted different levels of the same pathway to exert a full effect. RT-PCR experiments revealed robust expression at the mRNA level of TRPC4, TRPC5 and TRPM7 channels for which specific blockers were available. Whereas the TRPM7 blocker FTY720 had no effect, the TRPC4/5 channel inhibitor M084 (20 μM) blocked the conductance by ∼50%, indicating that TRPC4 and/or TRPC5 channel(s) may be partially involved in mediating the NEP-induced current. CP-96345 (20 μM), a specific blocker of the sodium leak current channel (NALCN), also reduced the NEP-induced current. The inhibition was ∼30% and additive to that caused by the TRPC4/5 blocker M084. RT-PCR experiments confirmed the expression of this channel at the mRNA level. Taken as a whole, these data provide evidence that a large fraction of the current evoked by a 5 ns pulse in adrenal chromaffin cells may be carried by both TRPC4/5 channels and the NALCN channel. Understanding the biophysical properties of the NEP-elicited conductance in a neural-type cell will be extremely valuable for the future development of NEP stimulation approaches for neuromodulation.

Confocal Microscopy to Measure Three Modes of Fusion Pore Dynamics in Adrenal Chromaffin Cells.

Dynamic fusion pore opening and closure mediate exocytosis and endocytosis and determine their kinetics. Here, it is demonstrated in detail how confocal microscopy was used in combination with patch-clamp recording to detect three fusion modes in primary culture bovine adrenal chromaffin cells. The three fusion modes include 1) close-fusion (also called kiss-and-run), involving fusion pore opening and closure, 2) stay-fusion, involving fusion pore opening and maintaining the opened pore, and 3) shrink-fusion, involving shrinkage of the fusion-generated Ω-shape profile until it merges completely at the plasma membrane. To detect these fusion modes, the plasma membrane was labeled by overexpressing mNeonGreen attached with the PH domain of phospholipase C δ (PH-mNG), which binds to phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) at the cytosol-facing leaflet of the plasma membrane; vesicles were loaded with the fluorescent false neurotransmitter FFN511 to detect vesicular content release; and Atto 655 was included in the bath solution to detect fusion pore closure. These three fluorescent probes were imaged simultaneously at ~20-90 ms per frame in live chromaffin cells to detect fusion pore opening, content release, fusion pore closure, and fusing vesicle size changes. The analysis method is described to distinguish three fusion modes from these fluorescence measurements. The method described here can, in principle, apply to many secretory cells beyond chromaffin cells.

High intensity nanosecond electric pulses trigger a Na+ conductance carried by TRPC and NALCN channels in bovine adrenal chromaffin cells

Our group previously showed that a single 5 ns, 5 MV/m pulse (NEP) elicits an inward membrane current near the resting potential of bovine chromaffin cells that is partially carried by Na+. We hypothesize that the NEP-induced current depolarizes the cell, leading to activation of voltage-gated Ca2+ channels (VGCCs). Recently we found that the NEP-induced membrane current displayed inward rectification, a reversal potential near −3 mV, and sensitivity to block by non-selective inhibitors of Transient Receptor Potential (TRP) channels.

Permissive Modulation of Sphingosine-1-Phosphate-Enhanced Intracellular Calcium on BKCa Channel of Chromaffin Cells.

Sphingosine-1-phosphate (S1P), is a signaling sphingolipid which acts as a bioactive lipid mediator. We assessed whether S1P had multiplex effects in regulating the large-conductance Ca2+-activated K+ channel (BKCa) in catecholamine-secreting chromaffin cells. Using multiple patch-clamp modes, Ca2+ imaging, and computational modeling, we evaluated the effects of S1P on the Ca2+-activated K+ currents (IK(Ca)) in bovine adrenal chromaffin cells and in a pheochromocytoma cell line (PC12). In outside-out patches, the open probability of BKCa channel was reduced with a mean-closed time increment, but without a conductance change in response to a low-concentration S1P (1 µM). The intracellular Ca2+ concentration (Cai) was elevated in response to a high-dose (10 µM) but not low-dose of S1P. The single-channel activity of BKCa was also enhanced by S1P (10 µM) in the cell-attached recording of chromaffin cells. In the whole-cell voltage-clamp, a low-dose S1P (1 µM) suppressed IK(Ca), whereas a high-dose S1P (10 µM) produced a biphasic response in the amplitude of IK(Ca), i.e., an initial decrease followed by a sustained increase. The S1P-induced IK(Ca) enhancement was abolished by BAPTA. Current-clamp studies showed that S1P (1 µM) increased the action potential (AP) firing. Simulation data revealed that the decreased BKCa conductance leads to increased AP firings in a modeling chromaffin cell. Over a similar dosage range, S1P (1 µM) inhibited IK(Ca) and the permissive role of S1P on the BKCa activity was also effectively observed in the PC12 cell system. The S1P-mediated IK(Ca) stimulation may result from the elevated Cai, whereas the inhibition of BKCa activity by S1P appears to be direct. By the differentiated tailoring BKCa channel function, S1P can modulate stimulus-secretion coupling in chromaffin cells.

Exposure System Dependent Calcium Influx in Chromaffin Cells by Nanosecond Electric Pulse- Field Reflections at Dielectric Interface

Nanosecond electric pulses have been successfully used as a novel electrical stimulus for evoking a dependent catecholamine release from the bovine adrenal chromaffin cells. It was observed that the duration of calcium influx varies depending on the exposure system. For example, cells exposed to 5 ns and 5MV/m pulses by a gold strip exposure chamber elicited calcium responses similar to the physiological stimulus, nicotinic cholinergic receptor activation. On the other hand, recent exposure systems utilizing tungsten rod electrodes evoke an undesired calcium response that is relatively long-lived even by exposure of the same pulse.

Gadolinium Differentially Affects Membrane Permeabilization and Ca2+ Influx Induced by Nanosecond Electric Pulses in Bovine Adrenal Chromaffin Cells

Our studies show that exposing isolated bovine adrenal chromaffin cells to a single 5 ns, 5 MV/m pulse (NEP) causes activation of voltage-gated Ca2+ channels (VGCCs) that in turn leads to Ca2+ influx and catecholamine release. The trigger for VGCC activation is hypothesized to be membrane depolarization caused by an instantaneous, NEP-elicited inward membrane current that is carried in part by Na+. Recent studies characterizing the properties of the NEP-elicited inward current by whole-cell patch clamp electrophysiology have shown that a large component of this unknown conductance displays properties that are consistent with those of ion channel proteins, in particular, TRP channels.

Paradoxical effects on voltage-gated Na+conductance in adrenal chromaffin cells by twin vs single high intensity nanosecond electric pulses.

We previously reported that a single 5 ns high intensity electric pulse (NEP) caused an E-field-dependent decrease in peak inward voltage-gated Na+ current (INa) in isolated bovine adrenal chromaffin cells. This study explored the effects of a pair of 5 ns pulses on INa recorded in the same cell type, and how varying the E-field amplitude and interval between the pulses altered its response. Regardless of the E-field strength (5 to 10 MV/m), twin NEPs having interpulse intervals ≥ than 5 s caused the inhibition of TTX-sensitive INa to approximately double relative to that produced by a single pulse. However, reducing the interval from 1 s to 10 ms between twin NEPs at E-fields of 5 and 8 MV/m but not 10 MV/m decreased the magnitude of the additive inhibitory effect by the second pulse in a pair on INa. The enhanced inhibitory effects of twin vs single NEPs on INa were not due to a shift in the voltage-dependence of steady-state activation and inactivation but were associated with a reduction in maximal Na+ conductance. Paradoxically, reducing the interval between twin NEPs at 5 or 8 MV/m but not 10 MV/m led to a progressive interval-dependent recovery of INa, which after 9 min exceeded the level of INa reached following the application of a single NEP. Disrupting lipid rafts by depleting membrane cholesterol with methyl-β-cyclodextrin enhanced the inhibitory effects of twin NEPs on INa and ablated the progressive recovery of this current at short twin pulse intervals, suggesting a complete dissociation of the inhibitory effects of twin NEPs on this current from their ability to stimulate its recovery. Our results suggest that in contrast to a single NEP, twin NEPs may influence membrane lipid rafts in a manner that enhances the trafficking of newly synthesized and/or recycling of endocytosed voltage-gated Na+ channels, thereby pointing to novel means to regulate ion channels in excitable cells.

Ca2+ influx and clearance at hyperpolarized membrane potentials modulate spontaneous and stimulated exocytosis in neuroendocrine cells

Neuroendocrine adrenal chromaffin cells release neurohormones catecholamines in response to Ca2+ entry via voltage-gated Ca2+ channels (VGCCs). Adrenal chromaffin cells also express non-voltage-gated channels, which may conduct Ca2+ at negative membrane potentials, whose role in regulation of exocytosis is poorly understood. We explored how modulation of Ca2+ influx at negative membrane potentials affects basal cytosolic Ca2+ concentration ([Ca2+]i) and exocytosis in metabolically intact voltage-clamped bovine adrenal chromaffin cells. We found that in these cells, Ca2+ entry at negative membrane potentials is balanced by Ca2+ extrusion by the Na+/Ca2+ exchanger and that this balance can be altered by membrane hyperpolarization or stimulation with an inflammatory hormone bradykinin. Membrane hyperpolarization or application of bradykinin augmented Ca2+-carrying current at negative membrane potentials, elevated basal [Ca2+]i, and facilitated synchronous exocytosis evoked by the small amounts of Ca2+ injected into the cell via VGCCs (up to 20 pC). Exocytotic responses evoked by the injections of the larger amounts of Ca2+ via VGCCs (> 20 pC) were suppressed by preceding hyperpolarization. In the absence of Ca2+ entry via VGCCs and Ca2+ extrusion via the Na+/Ca2+ exchanger, membrane hyperpolarization induced a significant elevation in [Ca2+]i and asynchronous exocytosis. Our results indicate that physiological interferences, such as membrane hyperpolarization and/or activation of non-voltage-gated Ca2+ channels, modulate basal [Ca2+]i and, consequently, segregation of exocytotic vesicles and their readiness to be released spontaneously and in response to Ca2+ entry via VGCCs. These mechanisms may play role in homeostatic plasticity of neuronal and endocrine cells.

Integrating Optical and Electrochemical Approaches to Assess the Actions of Dynamin at the Fusion Pore.

Of the techniques currently available to monitor dense core granule exocytosis in adrenal chromaffin cells, two have proven particularly useful: carbon-fiber amperometry and total internal reflection fluorescence (TIRF) microscopy. Amperometry enables the detection of oxidizable catecholamines escaping a fusion pore with millisecond time resolution. TIRF microscopy, and its variant polarized-TIRF (pTIRF) microscopy, provides information on the characteristics of fusion pores at temporally later stages. Used in conjunction, amperometry and TIRF microscopy allow an investigator to follow the fate of a fusion pore from its formation to expansion orreclosure. The properties of fusion pores, including theirstructure and dynamics, have been shown by multiple groups to be modified by the dynamin GTPase (Dyn1). In this chapter, we describe how amperometry and TIRF microscopy enableinsights into dynamin-dependent effects on exocytosis in primary cultures of bovine adrenal chromaffin cells.

The Ca2+ channel subunit CaV β2a-subunit down-regulates voltage-activated ion current densities by disrupting actin-dependent traffic in chromaffin cells.

β-Subunits of the Ca2+ channel have been conventionally regarded as auxiliary subunits that regulate the expression and activity of the pore-forming α1 subunit. However, they comprise protein-protein interaction domains, such as a SRC homology 3 domain (SH3) domain, which make them potential signaling molecules. Here we evaluated the role of the β2a subunit of the Ca2+ channels (CaV β2a) and its SH3 domain (β2a-SH3) in late stages of channel trafficking in bovine adrenal chromaffin cells. Cultured bovine adrenal chromaffin cells were injected with CaV β2a or β2a-SH3 under different conditions, in order to acutely interfere with endogenous associations of these proteins. As assayed by whole-cell patch clamp recordings, Ca2+ currents were reduced by CaV β2a in the presence of exogenous α1-interaction domain. β2a-SH3, but not its dimerization-deficient mutant, also reduced Ca2+ currents. Na+ currents were also diminished following β2a-SH3 injection. Furthermore, β2a-SH3 was still able to reduce Ca2+ currents when dynamin-2 function was disrupted, but not when SNARE-dependent exocytosis or actin polymerization was inhibited. Together with the additional finding that both CaV β2a and β2a-SH3 diminished the incorporation of new actin monomers to cortical actin filaments, β2a-SH3 emerges as a signaling module that might down-regulate forward trafficking of ion channels by modulating actin dynamics.

Nuclear Factor-kappaB Gates Nav1.7 Channels in DRG Neurons via Protein-Protein Interaction.

SummaryIt is well known that nuclear factor-kappaB (NF-κB) regulates neuronal structures and functions by nuclear transcription. Here, we showed that phospho-p65 (p-p65), an active form of NF-κB subunit, reversibly interacted with Nav1.7 channels in the membrane of dorsal root ganglion (DRG) neurons of rats. The interaction increased Nav1.7 currents by slowing inactivation of Nav1.7 channels and facilitating their recovery from inactivation, which may increase the resting state of the channels ready for activation. In cultured DRG neurons TNF-α upregulated the membrane p-p65 and enhanced Nav1.7 currents within 5 min but did not affect nuclear NF-κB within 40 min. This non-transcriptional effect on Nav1.7 may underlie a rapid regulation of the sensibility of the somatosensory system. Both NF-κB and Nav1.7 channels are critically implicated in many physiological functions and diseases. Our finding may shed new light on the investigation into the underlying mechanisms.

Biophysical Properties of the Electropermeabilization-Induced Membrane Conductance in Patch Clamped Adrenal Chromaffin Cells

Our group recently showed that a single 5 ns high intensity electric pulse (NEP) elicited membrane permeabilization in whole-cell voltage clamped bovine adrenal chromaffin cells, inducing an instantaneous inward conductance at the holding potential of −70 mV. When Na+ was completely replaced in the bath solution with n-methyl-d-glucamine, the NEP-induced current was significantly reduced suggesting that the current was partially carried by Na+. Later, a slow voltage ramp protocol from −100 mV to 80 mV was used to inactivate Na+ channel, whereas K+ and Ca2+ channels were blocked by cesium, tetraethylammonium and nitrendipine. With this ramp protocol, the fact that the NEP-induced conductance showed a consistent reversal potential around −10 mV revealed the properties of the current that resembled protein ion channel-like activity. We further exposed cells to SKF-96365 and La3+, both compounds considered to be able to alter the activity of TRP channels. The results showed 33% and 59 % inhibition effect on the NEP-induced current. Therefore, one possibility of the NEP-induced conductance is a TRP-like channel that is activated via some sort of elctromechnical coupling triggered as a consequence of the NEP. It is known that many TRP channels are localized in caveolae, a subset of membrane lipid rafts involved in compartmentalized signal transduction. Methy-β-cyclodextrin, as cholesterol depletion drug which disrupts caveolae integrity, produced a strong inhibitory effect on the NEP-induced current suggesting the hypothesis that caveolae disruption altered TRP trafficking and/or compartmentalization leading to a diminished ability of NEP to activate these channels, although this observation cannot rule out the possibility that membrane cholesterol depletion altered the biophysical properties of the membrane in a way that would prevent or limit the formation of lipid-containing electronanopores.

Hydrogen sulphide facilitates exocytosis by regulating the handling of intracellular calcium by chromaffin cells.

Gasotransmitter hydrogen sulphide (H2S) has emerged as a regulator of multiple physiological and pathophysiological processes throughout. Here, we have investigated the effects of NaHS (fast donor of H2S) and GYY4137 (GYY, slow donor of H2S) on the exocytotic release of catecholamines from fast-perifused bovine adrenal chromaffin cells (BCCs) challenged with sequential intermittent pulses of a K+-depolarizing solution. Both donors caused a concentration-dependent facilitation of secretion. This was not due to an augmentation of Ca2+ entry through voltage-activated Ca2+ channels (VACCs) because, in fact, NaHS and GYY caused a mild inhibition of whole-cell Ca2+ currents. Rather, the facilitation of exocytosis seemed to be associated to an augmented basal [Ca2+]c and the K+-elicited [Ca2+]c transients; such effects of H2S donors are aborted by cyclopiazonic acid (CPA), that causes endoplasmic reticulum (ER) Ca2+ depletion through sarcoendoplasmic reticulum Ca2+ ATPase inhibition and by protonophore carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), that impedes the ability of mitochondria to sequester cytosolic Ca2+ during cell depolarization. Inasmuch as CPA and FCCP reversed the facilitation of secretion triggered by K+ in the presence of NaHS and GYY, is seems that such facilitation is tightly coupled to Ca2+ handling by the ER and mitochondria. On the basis of these results, we propose that H2S regulates catecholamine secretory responses triggered by K+ in BCCs by (i) mobilisation of ER Ca2+ and (ii) interference with mitochondrial Ca2+ circulation. In so doing, the clearance of the [Ca2+]c transient will be delayed and the Ca2+-dependent trafficking of secretory vesicles will be enhanced to overfill the secretory machinery with new vesicles to enhance exocytosis.

Phosphopeptidomics Reveals Differential Phosphorylation States and Novel SxE Phosphosite Motifs of Neuropeptides in Dense Core Secretory Vesicles.

Neuropeptides are vital for cell-cell communication and function in the regulation of the nervous and endocrine systems. They are generated by post-translational modification (PTM) steps resulting in small active peptides generated from prohormone precursors. Phosphorylation is a significant PTM for the bioactivity of neuropeptides. From the known diversity of distinct neuropeptide functions, it is hypothesized that the extent of phosphorylation varies among different neuropeptides. To assess this hypothesis, neuropeptide-containing dense core secretory vesicles from bovine adrenal medullary chromaffin cells were subjected to global phosphopeptidomics analyses by liquid chromatography (LC)-mass spectrometry (MS/MS). Phosphopeptides were identified directly by LC-MS/MS and indirectly by phosphatase treatment followed by LC-MS/MS. The data identified numerous phosphorylated peptides derived from neuropeptide precursors such as chromogranins, secretogranins, proenkephalin and pro-NPY. Phosphosite occupancies were observed at high and low levels among identified peptides and many of the high occupancy phosphopeptides represent prohormone-derived peptides with currently unknown bioactivities. Peptide sequence analyses demonstrated SxE as the most prevalent phosphorylation site motif, corresponding to phosphorylation sites of the Fam20C protein kinase known to be present in the secretory pathway. The range of high to low phosphosite occupancies for neuropeptides demonstrates cellular regulation of neuropeptide phosphorylation. Graphical Abstract ᅟ.

Slow fusion pore expansion creates a unique reaction chamber for co-packaged cargo.

A lumenal secretory granule protein, tissue plasminogen activator (tPA), greatly slows fusion pore dilation and thereby slows its own discharge. We investigated another outcome of the long-lived narrow fusion pore: the creation of a nanoscale chemical reaction chamber for granule contents in which the pH is suddenly neutralized upon fusion. Bovine adrenal chromaffin cells endogenously express both tPA and its primary protein inhibitor, plasminogen activator inhibitor 1 (PAI). We found by immunocytochemistry that tPA and PAI are co-packaged in the same secretory granule. It is known that PAI irreversibly and covalently inactivates tPA at neutral pH. We demonstrate with zymography that the acidic granule lumen protects tPA from inactivation by PAI. Immunocytochemistry, total internal reflection fluorescence (TIRF) microscopy, and polarized TIRF microscopy demonstrated that co-packaged PAI and tPA remain together in granules for many seconds in the nanoscale reaction chamber, more than enough time to inhibit tPA and create a new secreted protein species.

Chromaffin cells as a model to evaluate mechanisms of cell death and neuroprotective compounds.

In this review, we show how chromaffin cells have contributed to evaluate neuroprotective compounds with diverse mechanisms of action. Chromaffin cells are considered paraneurons, as they share many common features with neurons: (i) they synthesize, store, and release neurotransmitters upon stimulation and (ii) they express voltage-dependent calcium, sodium, and potassium channels, in addition to a wide variety of receptors. All these characteristics, together with the fact that primary cultures from bovine adrenal glands or chromaffin cells from the tumor pheochromocytoma cell line PC12 are easy to culture, make them an ideal model to study neurotoxic mechanisms and neuroprotective drugs. In the first part of this review, we will analyze the different cytotoxicity models related to calcium dyshomeostasis and neurodegenerative disorders like Alzheimer's or Parkinson's. Along the second part of the review, we describe how different classes of drugs have been evaluated in chromaffin cells to determine their neuroprotective profile in different neurodegenerative-related models.

The F-Actin Binding Protein Cortactin Regulates the Dynamics of the Exocytotic Fusion Pore through its SH3 Domain.

Upon cell stimulation, the network of cortical actin filaments is rearranged to facilitate the neurosecretory process. This actin rearrangement includes both disruption of the preexisting actin network and de novo actin polymerization. However, the mechanism by which a Ca2+ signal elicits the formation of new actin filaments remains uncertain. Cortactin, an actin-binding protein that promotes actin polymerization in synergy with the nucleation promoting factor N-WASP, could play a key role in this mechanism. We addressed this hypothesis by analyzing de novo actin polymerization and exocytosis in bovine adrenal chromaffin cells expressing different cortactin or N-WASP domains, or cortactin mutants that fail to interact with proline-rich domain (PRD)-containing proteins, including N-WASP, or to be phosphorylated by Ca2+-dependent kinases, such as ERK1/2 and Src. Our results show that the activation of nicotinic receptors in chromaffin cells promotes cortactin translocation to the cell cortex, where it colocalizes with actin filaments. We further found that, in association with PRD-containing proteins, cortactin contributes to the Ca2+-dependent formation of F-actin, and regulates fusion pore dynamics and the number of exocytotic events induced by activation of nicotinic receptors. However, whereas the actions of cortactin on the fusion pore dynamics seems to depend on the availability of monomeric actin and its phosphorylation by ERK1/2 and Src kinases, cortactin regulates the extent of exocytosis by a mechanism independent of actin polymerization. Together our findings point out a role for cortactin as a critical modulator of actin filament formation and exocytosis in neuroendocrine cells.