L-type calcium channels are preferentially coupled to endocytosis in bovine chromaffin cells

Exocytosis and endocytosis are Ca(2+)-dependent processes. The contribution of high-voltage activated Ca(2+) channels subtypes to exocytosis has been thoroughly studied in chromaffin cells. However, similar reports concerning endocytosis are unavailable. Thus, we studied here the effects of blockers of L (nifedipine), N (omega-conotoxin GVIA) and P/Q (omega-agatoxin IVA) Ca(2+) channel on Ca(2+) currents (I(Ca)), Ca(2+) entry (Q(Ca)), as well as on the changes in membrane capacitance (C(m)) in perforated-patch voltage-clamped bovine adrenal chromaffin cells. Using 500-ms pulses to 0 or +10 mV, given from a holding potential of -80 mV and 2 mM Ca(2+) we found that omega-conotoxin GVIA affected little the exo-endocytotic responses while omega-agatoxin IVA markedly blocked those responses. However, nifedipine blocked little exocytosis but almost completely inhibited endocytosis. We conclude that L-type Ca(2+) channels seem to be selectively coupled to endocytosis.     

Triggered exocytosis and endocytosis have different requirements for calcium and nucleotides in permeabilized bovine chromaffin cells

The intracellular requirements for membrane recapture in permeabilized chromaffin cells were compared to the requirements for exocytosis from the same cells.In permeabilized bovine chromaffin cells, calcium-driven exocytosis also triggers, with a short delay, uptake of extracellular horseradish peroxidase (HRP). This internalized HRP remains compartmentalized within the cell and migrates to a low density band on a Percoll gradient which is distinct from the heavier chromaffin granules.The amount of horseradish peroxidase internalized is similar in intact and leaky cells and is approximately equivalent to the volumes secreted. Endocytosis in both preparations is blocked by botulinum toxin, operates in a collapsed membrane potential, and is inhibited by low temperature. In permeabilized cells, exocytosis and coupled endocytosis are activated by the same concentrations of Ca²⁺ and MgATP. Although secretion requires Ca²⁺ and MgATP, once exocytosis has occurred the subsequent endocytosis can proceed in the virtual absence of Ca²⁺ or MgATP, and is largely unaffected by a variety of nucleotide triphosphates (including nonhydrolyzable analogues), and cyclic nucleotides.These data suggest that endocytosis can proceed, once exocytosis has been triggered, under conditions that are quite different from those necessary to support exocytosis, and that the specific requirements for Ca²⁺ and MgATP in secretion are for the exocytotic limb of the secretory cycle rather than for the associated endocytotic pathway.We are grateful to Mr. John Gibbs for excellent technical assistance, and to the Medical Research Council (UK) for financial support.     

Multiple calcium pathways induce the expression of SNAP-25 protein in chromaffin cells

Incubation of bovine adrenal chromaffin cells in high K+ (38 mM) during 24-48 h enhanced 2.5 to five times the expression of SNAP-25 protein and mRNA, respectively. This increase was reduced 86% by furnidipine (an L-type Ca2+ channel blocker) but was unaffected by either omega-conotoxin GVIA (an N-type Ca2+ channel blocker) or -agatoxin IVA (a P/Q-type Ca2+ channel blocker). Combined blockade of N and P/Q channels with omega-conotoxin MVIIC did, however, block by 76% the protein expression. The inhibitory effects of fumidipine were partially reversed when the external Ca2+ concentration was raised from 1.6 to 5 mM. These findings, together with the fact that nicotinic receptor activation or Ca2+ release from internal stores also enhanced SNAP-25 protein expression, suggest that an increment of cytosolic Ca2+ concentration ([Ca2+]), rather than its source or Ca2+ entry pathway, is the critical signal to induce the protein expression. The greater coupling between L-type Ca2+ channels and protein expression might be due to two facts: (a) L channels contributed 50% to the global [Ca2+]i rise induced by 38 mM K+ in indo-1-loaded chromaffin cells and (b) L channels undergo less inactivation than N or P/Q channels on sustained stimulation of these cells.     

Presence of dynamin--syntaxin complexes associated with secretory granules in adrenal chromaffin cells

Dynamin proteins have been implicated in many aspects of endocytosis, including clathrin-mediated endocytosis, internalization of caveolae, synaptic vesicle recycling, and, more recently, vesicular trafficking to and from the Golgi complex. To provide further insight into the function(s) of dynamin in neuroendocrine cells, we have examined its intracellular distribution in cultured chromaffin cells by subcellular fractionation, immunoreplica analysis, and confocal immunofluorescence. We found that dynamin, presumably the dynamin-2 isoform, is associated specifically with the membrane of purified secretory chromaffin granules. Oligomerization state analysis by sucrose density velocity gradients indicated that the granule-associated dynamin is in a monomeric form. Immunoprecipitation experiments coupled to double-labeling immunofluorescence cytochemistry revealed that the granular dynamin is associated with a syntaxin component that is not involved in the granule-bound SNARE complex. The possibility that dynamin participates in the coupling of the exocytotic and endocytotic reaction through the building of a granular membrane subset of proteins is discussed.     

Ouabain enhances exocytosis through the regulation of calcium handling by the endoplasmic reticulum of chromaffin cells

The augmentation of neurotransmitter and hormone release produced by ouabain inhibition of plasmalemmal Na⁺/K⁺-ATPase (NKA) is well established. However, the mechanism underlying this action is still controversial. Here we have shown that in bovine adrenal chromaffin cells ouabain diminished the mobility of chromaffin vesicles, an indication of greater number of docked vesicles at subplasmalemmal exocytotic sites. On the other hand, ouabain augmented the number of vesicles undergoing exocytosis in response to a K⁺ pulse, rather than the quantal size of single vesicles. Furthermore, ouabain produced a tiny and slow Ca²⁺ release from the endoplasmic reticulum (ER) and gradually augmented the transient elevations of the cytosolic Ca²⁺ concentrations ([Ca²⁺]_c) triggered by K⁺ pulses. These effects were paralleled by gradual increments of the transient catecholamine release responses triggered by sequential K⁺ pulses applied to chromaffin cell populations treated with ouabain. Both, the increases of K⁺-elicited [Ca²⁺]_c and secretion in ouabain-treated cells were blocked by thapsigargin (THAPSI), 2-aminoethoxydiphenyl borate (2-APB) and caffeine. These results are compatible with the view that ouabain may enhance the ER Ca²⁺ load and facilitate the Ca²⁺-induced-Ca²⁺ release (CICR) component of the [Ca²⁺]_c signal generated during K⁺ depolarisation. This could explain the potentiating effects of ouabain on exocytosis.     

Amine weak bases disrupt vesicular storage and promote exocytosis in chromaffin cells

The vesicular contents in bovine chromaffin cells are maintained at high levels owing to the strong association of its contents, which is promoted by the low vesicular pH. The association is among the catecholamines, Ca2+, ATP, and vesicular proteins. It was found that transient application of a weak base, methylamine (30 mM), amphetamine (10 microM), or tyramine (10 microM), induced exocytotic release. Exposure to these agents was also found to increase both cytosolic catecholamine and intracellular Ca2+ concentration, as measured by amperometry and fura-2 fluorescence. Amphetamine, the most potent amine with respect to evoking exocytosis, was found to be effective even in buffer without external Ca2+; however, the occurrence of spikes was suppressed when BAPTA-acetoxymethyl ester was used to complex intracellular Ca2+. Amphetamine-induced spikes in Ca2+-free medium were not suppressed by thapsigargin or ruthenium red, inhibitors of the sarco(endo)plasmic reticulum Ca2+-ATPase and mitochondrial Ca2+ stores. Atomic absorption measurements of amphetamine- and methylamine-treated vesicles reveal that intravesicular Ca2+ stores are decreased after a 15-min incubation. Taken together, these data indicate that amphetamine and methylamine can disrupt vesicular stores to a sufficient degree that Ca2+ can escape and trigger exocytosis.     

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 M GTP-S did not significantly affect prepulse facilitation or whole-cell Ba²⁺ current (I _Ba) density. In contrast, inactivation of G proteins by intracellular GDP-S or pertussis toxin (PTX) pretreatment completely abolished or markedly attenuated facilitation of I _Ba, respectively. GDP-S dialysis resulted in nearly a threefold increase in peak I _Ba density, whereas PTX pretreatment resulted in a 50% increase. Our results indicate that under control recording conditions (200 m 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 (50%) in I _Ba 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.This work was supported by a National Science Foundation grant (DCB-8812562), American Heart Association-Ohio Affiliate grant (SW-91-18), and an American Parkinson's Disease Association grant. C.A.D. was supported by a predoctoral National Research Service Award (National Institutes of Health training grant HL07571-08). The authors thank Kluener's Packing Co. for their generous supply of adrenal glands.     

Phospholipase D in calcium-regulated exocytosis: Lessons from chromaffin cells

Membrane fusion remains one of the less well-understood processes in cell biology. A variety of mechanisms have been proposed to explain how the generation of fusogenic lipids at sites of exocytosis facilitates secretion in mammalian cells. Over the last decade, chromaffin cells have served as an important cellular model to demonstrate a key role for phospholipase D1 (PLD1) generated phosphatidic acid in regulated exocytosis. The current model proposes that phosphatidic acid plays a biophysical role, generating a negative curvature and thus promoting fusion of secretory vesicles with the plasma membrane. Moreover, multiple signaling pathways converging on PLD1 regulation have been unraveled in chromaffin cells, suggesting a complex level of regulation dependant on the physiological context.     

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.     

Tight coupling of the t-SNARE and calcium channel microdomains in adrenomedullary slices and not in cultured chromaffin cells

Regulated exocytosis involves calcium-dependent fusion of secretory vesicles with the plasma membrane with three SNARE proteins playing a central role: the vesicular synaptobrevin and the plasma membrane syntaxin1 and SNAP-25. Cultured bovine chromaffin cells possess defined plasma membrane microdomains that are specifically enriched in both syntaxin1 and SNAP-25. We now show that in both isolated cells and adrenal medulla slices these target SNARE (t-SNARE) patches quantitatively coincide with single vesicle secretory spots as detected by exposure of the intravesicular dopamine beta-hydroxylase onto the plasmalemma. During exocytosis, neither area nor density of the syntaxin1/SNAP-25 microdomains changes on the plasma membrane of both preparations confirming that preexisting clusters act as the sites for vesicle fusion. Our analysis reveals a high level of colocalization of L, N and P/Q type calcium channel clusters with SNAREs in adrenal slices; this close association is altered in individual cultured cells. Therefore, microdomains carrying syntaxin1/SNAP-25 and different types of calcium channels act as the sites for physiological granule fusion in "in situ" chromaffin cells. In the case of isolated cells, it is the t-SNAREs microdomains rather than calcium channels that define the sites of exocytosis.     

Rundown of secretion after depletion of intracellular calcium stores in bovine adrenal chromaffin cells

In this study, the relationship between intracellular calcium stores and depolarization-evoked stimulation was examined in bovine chromaffin cells, using changes in membrane capacitance to monitor both exocytosis and endocytosis. Cells were voltage-clamped using the perforated whole-cell patch configuration to minimize alterations in intracellular constituents. Control cells exhibited reproducible secretory responses each time the cell was stimulated. However, the same stimulation protocol elicited progressively smaller secretory responses in cells where their intracellular calcium store was emptied by thapsigargin. Transient elevation of the intracellular calcium concentration with a brief histamine treatment enhanced subsequent secretory responses in control but not in thapsigargin-treated cells. A series of depolarizations to -20 mV, which allowed small amounts of Ca(2+) influx but which by itself did not trigger catecholamine secretion, enhanced subsequent exocytosis in both control and thapsigargin-treated cells. Caffeine-pretreated cells exhibited a rundown in the secretory response that was similar to that produced by thapsigargin. These results suggest that brief elevations of [Ca(2+)](i) could enhance subsequent secretory responses. In addition, the data suggest that intracellular calcium stores are vital for the maintenance of exocytosis during repetitive stimulation.     

Differential control of tyrosine hydroxylase activation and catecholamine secretion by voltage-operated Ca2+ channels in bovine chromaffin cells

Contributions of L-, N-, and P/Q-type voltage-operated Ca2+ channels to two responses of bovine adrenal chromaffin cells have been studied using the nonreceptor stimulus K+ depolarization. Tyrosine hydroxylase activity and catecholamine secretion were both increased by K+ over a similar concentration range and in a Ca(2+)-dependent manner. At a submaximal concentration of 20 mM K+, tyrosine hydroxylase activation was reduced by nitrendipine but unaffected individually by (+/-)-Bay K 8644, omega-conotoxin GVIA, omega-agatoxin IVA, and omega-conotoxin MVIIC. It was fully blocked by combined inhibition of L-, N-, and P/Q-type channels. With a maximal concentration of 50 mM K+, tyrosine hydroxylase activation was unaffected by nitrendipine as well as by each of the other drugs on its own; however, it was reduced by 71 % by combined inhibition of L-, N-, and P/Q-type channels. In contrast, catecholamine secretion with both 20 and 50 mM K+ was enhanced by (+/-)-Bay K 8644, partially inhibited by nitrendipine and omega-conotoxin MVIIC, and completely blocked by a combination of antagonists for L-, N-, and P/Q-type channels. The results show that Ca2+ entry through voltage-operated Ca2+ channels can differentially regulate distinct chromaffin cell responses and that this is an intrinsic property of the mechanisms by which Ca2+ entry activates these responses. It is not dependent on the parallel activation of other signaling events by receptors.     

Mobilizing store Ca(2+) in the presence of La(3+) evokes exocytosis in bovine chromaffin cells

The effect on exocytosis of La(3+), a known inhibitor of plasma membrane Ca(2+)-ATPases and Na(+)/Ca(2+) exchangers, was studied using cultured bovine adrenal chromaffin cells. At high concentrations (0.3-3 mM), La(3+) substantially increased histamine-induced catecholamine secretion. This action was mimicked by other lanthanide ions (Nd(3+), Eu(3+), Gd(3+), and Tb(3+)), but not several divalent cations. In the presence of La(3+), the secretory response to histamine became independent of extracellular Ca(2+). La(3+) enhanced secretion evoked by other agents that mobilize intracellular Ca(2+) stores (angiotensin II, bradykinin, caffeine, and thapsigargin), but not that due to passive depolarization with 20 mM K(+). La(3+) still enhanced histamine-induced secretion in the presence of the nonselective inhibitors of Ca(2+)-permeant channels SKF96365 and Cd(2+), but the enhancement was abolished by prior depletion of intracellular Ca(2+) stores with thapsigargin. La(3+) inhibited (45)Ca(2+) efflux from preloaded chromaffin cells in the presence or absence of Na(+). It also enhanced and prolonged the rise in cytosolic [Ca(2+)] measured with fura-2 during mobilization of intracellular Ca(2+) stores with histamine in Ca(2+)-free buffer. The results suggest that the efficacy of intracellular Ca(2+) stores in evoking exocytosis is enhanced dramatically by inhibiting Ca(2+) efflux from the cell.     

Identification of a key domain in annexin and 14-3-3 proteins that stimulate calcium-dependent exocytosis in permeabilized adrenal chromaffin cells

Calcium-dependent secretion in digitonin-permeabilized adrenal chromaffin cells is stimulated by exogenous annexin II and 14-3-3 proteins. These proteins share a conserved domain that has been suggested to be involved in specific protein-protein interactions. We examined whether this domain was involved in secretion by using a synthetic peptide (P16) of sequence KGDYQKALLYLCGGDD corresponding to the C-terminus of annexin II. P16, but not truncated peptides, prevented the stimulation of secretion by 14-3-3 proteins and produced a partial inhibition of control secretion. These data suggest that the shared annexin/14-3-3 domain is important in the mechanisms controlling Ca²⁺-dependent secretion and may play a key role in protein-protein interactions during exocytosis.     

Comparison of apex and bottom secretion efficiency at chromaffin cells as measured by amperometry

In chromaffin cells, the exocytosis of neuromediators involves the fusion between a secretory vesicle and the cell membrane. Many techniques based on electrophysiology, electrochemistry and fluorescence microscopy allow the study of such a complex process at active zones of single immobilized cells. These techniques can provide an effective analysis either at the apex, either at the base of the cell adhering onto a substrate. For instance, patch-clamp (electrophysiology) and amperometry (electrochemistry) deal with detection at the exposed top of the cell, whereas evanescent field microscopy concerns mainly its bottom, i.e., the zone on which the cell rests onto the surface. However, in chromaffin cells, comparison between the two sets of methods remains to be established and whether apex fusion events are comparable or not to those observed at the base of the cell is an open question. In this work, we compare both active zones upon using the same measurement method, viz., by performing electrochemical detection at these both poles (top and bottom) of bovine chromaffin cells. This is performed upon using carbon fiber microelectrodes (apical analysis) and planar ITO transparent (basal analysis) electrodes, respectively. Our results indicate that the processes monitored at each pole differ though the same technique is used.     

Acetylcholine-induced calcium signalling in adrenaline- and noradrenaline-containing adrenal chromaffin cells

Adrenal chromaffin cells secrete catecholamines in response to cholinergic receptor activation by acetylcholine (ACh). Characteristics of Ca(2+) transients induced by activation of nicotinic (nAChRs) and muscarinic (mAChRs) receptors were analyzed using Fura-2 fluorescent measurements on rat chromaffin cells. We first found two populations of chromaffin cells, which differently responded on AChR stimulation. In the first group (n-cells), consecutive ACh applications evoked persistent Ca(2+) transients, whereas desensitizing transients were observed in the other group (m-cells). The AChR agonists and antagonists precisely imitated or abolished the ACh action on n- and m-type cells, respectively. Cytochemical staining showed that n-cells contained adrenaline, whereas m-cells-noradrenaline. Thus, for the first time we found that nAChRs and mAChRs are differentially expressed in adrenergic and noradrenergic chromaffin cells, respectively. Our data suppose that chromaffin cells can be differentially regulated by incoming ACh signals and in such way release different substances-adrenaline and noradrenaline.     

The chromaffin granule proton pump and calcium-dependent exocytosis in bovine adrenal medullary cells

Summary Calcium-dependent exocytosis in leaky bovine adrenal medullary cells has a requirement for Mg-ATP. One possibility is that exocytosis depends in some way on the operation of the ATP-dependent proton pump that serves to maintain the core of the secretory vesicles both acid and at a positive potential with respect to the cytosol. This possibility has been tested in leaky cells by monitoring exocytosis under conditions where the secretory vesicle pH and potential gradients are measuredin situ. The results show rather clearly that exocytosis can persist, with unchanged Ca-activation kinetics, in the virtual absence both of a difference in pH between the cytosol and secretory vesicle core and also of a difference in potential across the vesicle membrane. The results do not, however, exclude a small modulating effect of vesicle pH or potential on exocytosis and shed no light on whether or not the plasma membrane potential, which is maintained close to zero in these experiments, influences exocytosis.     

A two-step model for acetylcholine control of exocytosis via nicotinic receptors

The view that Ca²⁺ entry through voltage-dependent Ca²⁺ channels (VDCC) and through nicotinic receptors for acetylcholine (nAChRs) causes equal catecholamine release responses in chromaffin cells, was reinvestigated here using new protocols. We have made two-step experiments consisting in an ACh prepulse followed by a depolarizing pulse (DP). In voltage-clamped bovine chromaffin cells an ACh prepulse caused a slow-rate release but augmented 4.5-fold the much faster exocytotic response triggered by a subsequent depolarizing pulse (measured with capacitance and amperometry). If the ACh prepulse was given with mecamylamine or in low external Ca²⁺, the secretion increase disappeared. This suggests a two-step model for the effects of ACh: (1) meager Ca²⁺ entry through nAChRs mostly serves to keep loaded with vesicles the secretory machine; and (2) in this manner, the cell is prepared to respond with an explosive secretion of catecholamine upon depolarization and fast high Ca²⁺ entry through VDCC.     

Quantitative investigations of amperometric spike feet suggest different controlling factors of the fusion pore in exocytosis at chromaffin cells

Around 30% of exocytosis events recorded by amperometry at carbon fiber microelectrodes exhibit a pre-spike feature (PSF) termed a “foot”. This wave is associated with the release of the neurotransmitters via a transitory fusion pore, whilst the large, main exocytotic spike is due to complete release. The amperometric data reported herein were obtained using bovine chromaffin cells stimulated with either potassium or barium ions, two commonly-employed elicitors of exocytosis. Identical trends are observed with both activators: (i) they induce the same ratio (close to 30%) of events with a foot in the population of amperometric spikes, and (ii) spikes with a foot can be divided into two primary categories, depending on the temporal variation of the current wave (viz. as a ramp, or a ramp followed by a plateau). Correlations between the characteristics of the whole current spike, and of its observed foot, have been sought; such analyses demonstrate that the maximum current of both foot and spike signals are highly correlated, but, in contrast, the integrated charges of both are poorly correlated. Moreover, the temporal duration of the PSF is fully uncorrelated with any parameter pertaining to the main current spike. On the basis of these reproducible observations, it is hypothesized that the characteristics (dimensions and topology, at least) of each secretory vesicle determine the probability of formation of the fusion pore and its maximum size, whilst molecular factors of the cell membrane control its duration, and, consequently, the amount delivered prior to the massive exocytosis of catecholamines observed as a spike in amperometry.     

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.