Exocytosis In Bovine Chromaffin Cells Research Articles

Modulation of Ca2+ Influx at Hyperpolarized Membrane Potentials Alters Depolarization-Triggered Exocytosis in Bovine Chromaffin Cells

Perturbations in Ca2+ entry via neuronal transient receptor potential (TRP) and ORAI family proteins have been shown to alter synaptic signaling and architecture, and suggested to play a role in a spectrum of neuropathological conditions. Specific mechanisms by which of TRP/ORAI channels modulate synaptic transmission are not completely understood. Here we employed a combination of imaging and electrophysiological techniques to monitor changes in intracellular Ca2+ concentration ([Ca2+]i), membrane currents at hyperpolarized membrane potentials, voltage-gated Ca2+ currents, and changes in membrane capacitance to investigate effects of modulation of Ca2+ entry at hyperpolarized membrane potentials on exocytosis in bovine chromaffin cells. A small inwardly directed membrane current (I hold), whose amplitude was dependent on the concentration of extracellular Ca2+ and Na+, was present at resting membrane potential. Augmentation of I hold either by hyperpolarization (from −50 mV to −90 mV) or stimulation with bradykinin (1 μM), a catecholamine secretagogue, elevated [Ca2+]i at hyperpolarized potentials and facilitated exocytotic responses triggered by short depolarizations (<500 ms in duration). Exocytotic responses triggered by long depolarizations (>500 ms in duration) were smaller at −90 mV holding potential compared with those at −50 mV holding potential. Reversal of Na+/Ca2+ exchanger by removal of Na+ from bath solution did not cause depolarization-independent exocytosis at −50 mV holding potential. Membrane hyperpolarization from −50 mV to −90 mV in Na+ -free bath solution evoked depolarization-independent exocytosis with the maximal release rate of about 6 fF/s. Our findings provide first direct evidence that 1) Ca2+ influx via native Ca2+ channels operating at negative voltages is functionally coupled with Na+/Ca2+ exchanger and that 2) perturbation in Ca2+ influx an negative voltages with physiologically relevant stimuli significantly modulate exocytotic responses to depolarization-triggered Ca2+ influx in neuroendocrine cells.

Novel synthetic sulfoglycolipid IG20 facilitates exocytosis in chromaffin cells through the regulation of sodium channels.

In search of druggable synthetic lipids that function as potential modulators of synaptic transmission and plasticity, we synthesized sulfoglycolipid IG20, which stimulates neuritic outgrowth. Here, we have explored its effects on ion channels and exocytosis in bovine chromaffin cells. IG20 augmented the rate of basal catecholamine release. Such effect did not depend on Ca(2+) mobilization from intracellular stores; rather, IG20-elicited secretion entirely dependent on Ca(2+) entry through L-subtype voltage-activated Ca(2+) channels. Those channels were recruited by cell depolarization mediated by IG20 likely through its ability to enhance the recruitment of Na(+) channels at more hyperpolarizing potentials. Confocal imaging with fluorescent derivative IG20-NBD revealed its rapid incorporation and confinement into the plasmalemma, supporting the idea that IG20 effects are exerted through a plasmalemmal-delimited mechanism. Thus, synthetic IG20 seems to mimic several physiological effects of endogenous lipids such as regulation of ion channels, Ca(2+) signaling, and exocytosis. Therefore, sulfoglycolipid IG20 may become a pharmacological tool for investigating the role of the lipid environment on neuronal excitability, ion channels, neurotransmitter release, synaptic efficacy, and neuronal plasticity. It may also inspire the synthesis of druggable sulfoglycolipids aimed at increasing synaptic plasticity and efficacy in neurodegenerative diseases and traumatic brain-spinal cord injury. The novel synthetic sulfoglycolipid IG20 mimics several physiological effects of endogenous lipids such as regulation of ion channels, Ca(2+) signaling, and exocytosis. This profile may eventually drive enhanced synaptic plasticity and efficacy.

The Role of Doc2B in Depolarization-Evoked and G Protein-Coupled Receptor Modulated Exocytosis in Mouse Chromaffin Cells

Previously we demonstrated that activation of Gq protein-coupled H1 histamine receptors potentiates stimulus-coupled exocytosis in bovine chromaffin cells, despite inhibiting Ca2+ influx through voltage-gated calcium channels (VGCCs). This potentiation is restricted to the immediately releasable pool (IRP) of vesicles coupled to VGCCs, and requires the priming protein Munc13-1. Doc2B is a calcium-binding protein, known to interact with Munc13 to promote exocytosis; whether it plays a role in agonist-regulated exocytosis is unknown. To address this question, we isolated chromaffin cells from Doc2B (+/+), (+/-) and (-/-) mice and examined the effects of PLC-coupled GPCR activation on depolarization-evoked exocytosis.We found that under control stimulus conditions, depolarization-evoked exocytosis, measured as changes in membrane capacitance, was not significantly different between Doc2B (+/+), (+/-) and (-/-) cells; similarly no differences were found between the sizes of the releasable vesicle pools (IRP, RRP, SRP). Equimolar replacement of extracellular Ca2+ by Ba2+ also revealed no significant differences in the synchronous and asynchronous phases of Ba2+-evoked exocytosis in Doc2B (+/+), (+/-) or (-/-) cells. Finally, although more than 80% of cells, independent of their genotype, responded to histamine with a reduction of Ca2+ influx, potentiation of exocytosis was surprisingly modest (2-fold) and, even in Doc2 (+/+) cells rarely observed (5/17 cells, (+/-): 5/24 cells, (-/-): 2/18). This is in stark contrast to bovine cells, where 80% of the cells show a 5-fold potentiation of exocytosis. Importantly, Doc2B (+/+), (+/-) and (-/-) cells responded to the DAG analog PMA with potentiation. Taken together, the results suggest that there are profound species differences in the coupling between GqPCRs and the exocytotic machinery in chromaffin cells and that Doc2B is not required for agonist-dependent potentiation of exocytosis in mice.

Calcium entry through slow-inactivating L-type calcium channels preferentially triggers endocytosis rather than exocytosis in bovine chromaffin cells

Calcium (Ca(2+))-dependent endocytosis has been linked to preferential Ca(2+) entry through the L-type (α(1D), Ca(V)1.3) of voltage-dependent Ca(2+) channels (VDCCs). Considering that the Ca(2+)-dependent exocytotic release of neurotransmitters is mostly triggered by Ca(2+) entry through N-(α(1B), Ca(V)2.2) or PQ-VDCCs (α(1A), Ca(V)2.1) and that exocytosis and endocytosis are coupled, the supposition that the different channel subtypes are specialized to control different cell functions is attractive. Here we have explored this hypothesis in primary cultures of bovine adrenal chromaffin cells where PQ channels account for 50% of Ca(2+) current (I(Ca)), 30% for N channels, and 20% for L channels. We used patch-clamp and fluorescence techniques to measure the exo-endocytotic responses triggered by long depolarizing stimuli, in 1, 2, or 10 mM concentrations of extracellular Ca(2+) ([Ca(2+)](e)). Exo-endocytotic responses were little affected by ω-conotoxin GVIA (N channel blocker), whereas ω-agatoxin IVA (PQ channel blocker) caused 80% blockade of exocytosis as well as endocytosis. In contrast, nifedipine (L channel blocker) only caused 20% inhibition of exocytosis but as much as 90% inhibition of endocytosis. Conversely, FPL67146 (an activator of L VDCCs) notably augmented endocytosis. Photoreleased caged Ca(2+) caused substantially smaller endocytotic responses compared with those produced by K(+) depolarization. Using fluorescence antibodies, no colocalization between L, N, or PQ channels with clathrin was found; a 20-30% colocalization was found between dynamin and all three channel antibodies. This is incompatible with the view that L channels are coupled to the endocytotic machine. Data rather support a mechanism implying the different inactivation rates of L (slow-inactivating) and N/PQ channels (fast-inactivating). Thus a slow but more sustained Ca(2+) entry through L channels could be a requirement to trigger endocytosis efficiently, at least in bovine chromaffin cells.

Ca2+ binding protein-1 inhibits Ca2+ currents and exocytosis in bovine chromaffin cells

Calcium binding protein-1 (CaBP1) is a calmodulin like protein shown to modulate Ca2+ channel activities. Here, we explored the functions of long and short spliced CaBP1 variants (L- and S-CaBP1) in modulating stimulus-secretion coupling in primary cultured bovine chromaffin cells. L- and S-CaBP1 were cloned from rat brain and fused with yellow fluorescent protein at the C-terminal. When expressed in chromaffin cells, wild-type L- and S-CaBP1s could be found in the cytosol, plasma membrane and a perinuclear region; in contrast, the myristoylation-deficient mutants were not found in the membrane. More than 20 and 70% of Na+ and Ca2+ currents, respectively, were inhibited by wild-type isoforms but not myristoylation-deficient mutants. The [Ca2+]( i ) response evoked by high K+ buffer and the exocytosis elicited by membrane depolarizations were inhibited only by wild-type isoforms. Neuronal Ca2+ sensor-1 and CaBP5, both are calmodulin-like proteins, did not affect N(+, Ca2+ currents, and exocytosis. When expressed in cultured cortical neurons, the [Ca2+]( i ) responses elicited by high-K+ depolarization were inhibited by CaBP1 isoforms. In HEK293T cells cotransfected with N-type Ca2+ channel and L-CaBP1, the current was reduced and activation curve was shifted positively. These results demonstrate the importance of CaBP1s in modulating the stimulus-secretion coupling in excitable cells.

Lysophospholipids regulate excitability and exocytosis in cultured bovine chromaffin cells

Bioactive lysophospholipids (LPLs) are released by blood cells and can modulate many cellular activities such as angiogenesis and cell survival. In this study, the effects of sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) on excitability and exocytosis in bovine chromaffin cells were investigated using the whole-cell configuration of the patch-clamp. Voltage-gated Ca(2+) current was inhibited by S1P and LPA pre-treatment in a concentration-dependent manner with IC(50)s of 0.46 and 0.79 mumol/L, respectively. Inhibition was mostly reversible upon washout and prevented by suramin, an inhibitor of G-protein signaling. Na(+) current was inhibited by S1P, but not by LPA. However, recovery of Na(+) channels from inactivation was slowed by both LPLs. The outward K(+) current was also significantly reduced by both LPLs. Chromaffin cells fired repetitive action potentials in response to minimal injections of depolarizing current. Repetitive activity was dramatically reduced by LPLs. Consistent with the reduction in Ca(2+) current, exocytosis elicited by a train of depolarizations and the ensuing endocytosis were both inhibited by LPL pre-treatments. These data demonstrate the interaction between immune and endocrine systems mediated by the inhibitory effects of LPLs on the excitability of adrenal chromaffin cells.

Potentiation of Exocytosis by Phospholipase C-Coupled G-Protein-Coupled Receptors Requires the Priming Protein Munc13-1

The vesicle priming protein Munc13-1 is regulated by diacylglycerol (DAG) and is therefore hypothesized to play a role in the control of neurotransmitter release by phospholipase C (PLC)-coupled receptors. We combined voltage-clamp recordings of voltage-gated Ca2+ channels (VGCCs) and high-resolution capacitance measurements to investigate the mechanism of receptor-mediated modulation of exocytosis in bovine chromaffin cells. Activation of endogenous H1 G(q)-protein-coupled receptors (G(q)PCRs) by histamine potentiated stimulus-coupled secretion despite concurrently inhibiting Ca2+ influx through VGCCs. Histamine increased the size of the readily releasable pool of vesicles and in particular a subpool of fusion-competent vesicles localized in close proximity to VGCCs. Pharmacological characterization showed that potentiation of exocytosis depended on the activation of PLC but not protein kinase C. Overexpression of wild-type Munc13-1 by adenoviral infection had no effect on histamine-induced potentiation per se, whereas DAG-insensitive Munc13-1(H567K) completely abolished it. This is the first endogenous mammalian G(q)PCR signaling pathway identified that engages Munc13-1 to increase stimulus-coupled secretion by recruiting vesicles to the immediately releasable pool. G(q)PCRs are therefore able to control exocytosis at the level of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex formation to produce rapid, short-term potentiation of the secretory output of neurons and endocrine cells.

Lysophospholipids elevate [Ca 2+] i and trigger exocytosis in bovine chromaffin cells

Sphingosine 1-phosphate (S1P) and lysophosphatidic acid (LPA) are responsible for many physiological functions, including angiogenesis, neuronal survival, and immunity. However, little is known about their effects in modulating the stimulus-secretion coupling in bovine chromaffin cells. The result of PCR showed that at least two receptors (S1P 3 and LPA 1) were expressed in bovine chromaffin cells. The elevation of [Ca 2+] i by S1P was fast and sustaining; but the elevation by LPA was slow and transient. The EC 50 for S1P and LPA in elevating the [Ca 2+] i were 0.55 ± 0.01 and 0.54 ± 0.40 μM, respectively. This elevation could be totally blocked by thapsigargin, 2-APB, and U73122. Pertussis toxin pretreatment inhibited about half of the elevation in [Ca 2+] i suggesting the involvement of G i and other G-proteins. Repetitive [Ca 2+] i elevations elicited by S1P, but not LPA, were inhibited by ryanodine. S1P was more effective than LPA in triggering exocytosis as measured by the changes in membrane capacitance. The whole-cell Ca 2+ current was inhibited by both lysophospholipids but Na + current was inhibited by S1P only. These results suggest the differential effects of LPA and S1P in releasing Ca 2+ from the intracellular Ca 2+ stores and modulating the stimulus-secretion coupling in bovine chromaffin cells.

A Comparison Between Acetylcholine-Like Action Potentials and Square Depolarizing Pulses in Triggering Calcium Entry and Exocytosis in Bovine Chromaffin Cells

Depending on experimental conditions, cell model, and pattern and type of depolarizing stimuli, the relationship between calcium entry ([Ca2+]c) and the release of neurotransmitters and hormones varies from exponential (power of 3-4) to near linear (power of 1.5) or linear function. Here, we present a study using the more physiological stimulation pattern based on acetylcholine (ACh)-like action potentials, in voltage-clamped bovine chromaffin cells, with the perforated-patch configuration of the patch-clamp technique and 2 mM extracellular calcium. Trains of ACh-like action potentials or square depolarizing pulses of increasing length were applied, and calcium currents (ICa), total calcium entry (QCa), and exocytosis (DeltaCm) measured.

Enhancement of Asynchronous and Train-Evoked Exocytosis in Bovine Adrenal Chromaffin Cells Infected With a Replication Deficient Adenovirus

Bovine adrenal chromaffin cells share many characteristics with neurons and are often used as a simple model system to study ion channels and neurotransmitter release. We infected bovine adrenal chromaffin cells with a replication deficient adenovirus that induces expression of the common reporters beta-galactosidase and Green Fluorescent Protein via a bicistronic sequence. In perforated-patch recordings performed 48-h postinfection, peak calcium currents were reduced 32%, primarily due to loss of omega-conotoxin-GVIA-sensitive current. In contrast, sodium currents were increased 17%. Exocytosis, detected as an increase in membrane capacitance immediately after a single step depolarization, was reduced in proportion to the decrease in calcium influx. However, capacitance continued to increase for seconds after the depolarization. The amplitude of this poststimulus drift, or asynchronous exocytosis, was approximately three times that which occurred in a small fraction of control cells. Exocytosis evoked by repetitive stimulation with a train of brief depolarizations was increased 50%. Intracellular calcium levels measured during and after stimulation were lower, not higher, in adenovirus-infected cells. Electroporated cells showed reduced calcium currents but no enhancement of exocytosis. Cells infected with UV-irradiated virus showed reduced calcium currents and enhancement of exocytosis, but the changes were smaller than those caused by intact virus. Our results are consistent with the idea that adenovirus capsid and adenoviral DNA contribute to a Ca2+ influx- and [Ca2+]i-independent enhancement of exocytosis in bovine chromaffin cells.

Temperature and PMA affect different phases of exocytosis in bovine chromaffin cells.

Amperometry was used to study secretory kinetics of single bovine chromaffin cells stimulated by transient depolarizations at different temperatures. The initial rate of release was moderately enhanced when the temperature was raised from 18 to 22 and 37 degrees C. Secretion increased drastically at a later period, 5-10 s after the initiation of stimulus. Interestingly, incubation of the cells with phorbol 12-myristate 13-acetate (PMA) clearly enhanced fast secretory components. In addition, the rate of secretion of the slower component recruited by prolonged depolarizations (t > 30 s) was unaffected at the range of temperatures normally used in secretory experiments (22-37 degrees C). A 'counting events' analysis of secretion, which avoids the influence of event charge changes, showed specific increases in a population of vesicles fusing between 7 and 12 s over the same range of temperatures, and a marked increase in vesicles fusing during the initial phase (1-5 s), of PMA-treated cell secretion. An analysis of temperature influence on transient components released by high sucrose, the secretion elicited by cell permeabilization with digitonin, and studies of the individual characteristics of amperometric events, allow us to conclude that an increase in the size of a secondary-released vesicle population is the main factor contributing to temperature-dependent enhancement of secretion, in clear contrast to the enhancement of fast releasable pools caused by phorbol esters.

Purinergic modulation of Ca2+ channels and exocytosis in bovine chromaffin cells

AbstractIt is now firmly established that adenosine 5′‐triphosphate (ATP) can act as a fast excitatory neurotransmitter by activation of postsynaptic purinergic receptors. Moreover, ATP also serves as a neuromodulator and inhibits neurotransmitter release by acting on presynaptic purinoceptors of the P2Y type. The mechanisms underlying inhibition of neurotransmission by P2Y purinoceptors may involve either inhibition of membrane excitability, particularly Ca2+entry through voltage‐activated Ca2+ channels, or direct action on the secretory apparatus. Bovine chromaffin cells from the adrenal medulla are a good model system to address this problem since: 1) they store and release ATP along with catecholamines by Ca2+ regulated exocytosis; 2) ATP binds to P2Y purinergic receptors to inhibit Ca2+ channels; and 3) they are amenable to combined capacitance measurement and voltage clamp recordings for the purpose of directly examining the relationship between voltage‐activated Ca2+ entry and exocytosis. This paper reviews our recently published results on the specific Ca2+ channel types that are inhibited by ATP and their relative contribution to the expected ATP regulation of catecholamine release from bovine chromaffin cells. While no direct effect of ATP on the secretory machinery could be evidenced, ATP regulation of exocytosis is voltage‐dependent as a reflection of the voltage dependence of ATP effects on Ca2+ channels. These data lend support to the idea of an ATP‐mediated autocrine regulation of secretion from chromaffin cells, though feed‐back regulation of the rate of release can be assumed to be a complex function of the occupancy of the purinoceptors and the electrical and secretory activity of the cell. Drug Dev. Res. 52:89–94, 2001. © 2001 Wiley‐Liss, Inc.

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.

Trimeric G Proteins Control Regulated Exocytosis in Bovine Chromaffin Cells: Sequential Involvement of Go Associated With Secretory Granules and Gi3 Bound to the Plasma Membrane

Regulated secretion requires both calcium and MgATP. Studies in diverse secretory systems indicate that ATP is required to prime the exocytotic apparatus whereas Ca2+ triggers the final ATP-independent fusion event. In this paper, we examine the possible role of trimeric G proteins in these two steps of exocytosis in chromaffin cells. We show that in the presence of low concentrations of Mg2+, mastoparan selectively stimulates G proteins associated with purified chromaffin granule membranes. Under similar conditions in permeabilized chromaffin cells, mastoparan inhibits ATP-dependent secretion but is unable to trigger ATP-independent release. This inhibitory effect of mastoparan on secretion was specifically reversed by anti-Galphao antibodies and a synthetic peptide corresponding to the carboxyl terminus of Galphao. In contrast, mastoparan required millimolar Mg2+ for the activation of plasma membrane-bound G proteins and stimulation of ATP-independent secretion in permeabilized chromaffin cells. The latter effect was completely inhibited by anti-Galphai3. By confocal immunofluorescence and immunoreplica analysis, we provide evidence that in chromaffin cells Go is preferentially associated with secretory granules, while Gi3 is essentially present on the plasma membrane. Our findings suggest that these two trimeric G proteins act in series in the exocytotic pathway in chromaffin cells: a secretory granule-associated Go protein controls the ATP-dependent priming reaction, whereas a plasma membrane-bound Gi3 protein is involved in the late calcium-dependent fusion step, which does not require ATP.

Role of heterotrimeric G proteins in membrane traffic.

Role of heterotrimeric G proteins in membrane traffic.