Qualitative analysis of membrane currents in glial cells from normal and gliotic tissue in situ: down-regulation of Na+ current and lack of P2 purinergic responses

A secretion from cultured bovine chromaffin cells was stimulated to examine the pattern of exocytotic fusion on the plasma membrane. Confocal microscopy revealed that dopamine-beta-hydroxylase immunofluorescence in intact cells stimulated for 20s with the nicotinic agonist 1,1-dimethyl-4-phenylpiperazinium was almost entirely punctate and evenly distributed on the cell surface. The basis for the fine, punctate appearance of dopamine-beta-hydroxylase was investigated. Dopamine-beta-hydroxylase presentation on the surface of permeabilized cells stimulated with 1-30 microM Ca2+ was punctate and similar to that on the plasma membrane of intact cells. The fluorescence intensities of both surface dopamine-beta-hydroxylase sites and internal chromaffin granules were estimated by computerized digital image analysis. The surface area of punctate surface dopamine-beta-hydroxylase (0.218 +/- 0.013 microm2, mean +/- S.E.M.) is similar to the surface area of a 0.28 microm diameter chromaffin granule (0.25 microm2). The average fluorescence intensity integrated over the area of the surface spots was 25-30% of the average chromaffin granule intensity, a fraction that is similar to the published values of 40-50% of the dopamine-beta-hydroxylase in the chromaffin granule being membrane bound. The surface density of the spots is consistent with the number of granules undergoing exocytosis. The spots do not tend to be clumped. The key conclusions from this work are that each individual punctate site of dopamine-beta-hydroxylase represents the fusion of a single chromaffin granule and that the distribution of dopamine-beta-hydroxylase spots over the cell surface is extensive and random, suggesting that each individual granule associates with its own release site.     

Fluorescence spectroscopic studies on interactions between liver annexin VI and nucleotides--a possible role for a tryptophan residue

Annexin VI is a 68-kDa calcium-, phospholipid-, and cytoskeletal-element-binding protein, which has been implicated in various processes, including calcium release and sequestration in calcifying cartilage, in a receptor-mediated endocytosis in human fibroblasts, and in secretion from chromaffin granules. In these processes it was found that, in addition to Ca2+ and annexin, the presence of ATP is also a prerequisite. In the present report we show that annexin VI binds ATP and the binding of nucleotide to protein is accompanied by quenching of an intrinsic fluorescence of annexin VI, which was found to be specific for 2'-(or 3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate, GTP and ATP, and dependent on the annexin conformation. The nucleotide-binding site within an annexin VI molecule is likely to be close to the tryptophan-containing domain of annexin VI. We propose that ATP plays the role of a physiological ligand for annexin VI, and its binding to annexin VI may represent an alternative cellular mechanism for the regulation of annexin-membrane interactions coupled to overall energy transitions in the cell.     

Transport via the regulated secretory pathway in semi-intact PC12 cells: role of intra-cisternal calcium and pH in the transport and sorting of secretogranin II

To gain insight into the mechanisms governing protein sorting, we have developed a system that reconstitutes both the formation of immature secretory granules and their fusion with the plasma membrane. Semi-intact PC12 cells were incubated with ATP and cytosol for 15 min to allow immature granules to form, and then in a buffer containing 30 microM [Ca2+]free to induce exocytosis. Transport via the regulated pathway, as assayed by the release of secretogranin II (SgII) labeled in the TGN, was inhibited by depletion of ATP, or by the inclusion of 100 microM GTP gamma S, 50 microM AlF3-5 or 5 micrograms/ml BFA. When added after immature granules had formed, GTP gamma S stimulated rather than inhibited exocytosis. Thus, exocytosis of immature granules in this system resembles the characteristics of fully matured granules. Transport of SgII via the regulated pathway occurred at a fourfold higher efficiency than glycosaminoglycan chains, indicating that SgII is sorted to some extent upon exit from the TGN. Addition of A23187 to release Ca2+ from the TGN had no significant effect on sorting of SgII into immature granules. In contrast, depletion of lumenal calcium inhibited the endoproteolytic cleavage of POMC and proinsulin. These results establish the importance of intra-cisternal Ca2+ in prohormone processing, but raise the question whether lumenal calcium is required for proper sorting of SgII into immature granules. Disruption of organelle pH gradients with an ionophore or a weak base resulted in the inhibition of transport via both the constitutive and the regulated pathways.     

Structure of the human sensorimotor system. I: Morphology and cytoarchitecture of the central sulcus

Chromaffin cells grafted to the brain of animals with experimental parkinsonism and patients with Parkinson's disease can restore nigrostriatal functions. Mechanisms underlying these beneficial effects are unknown, but may include growth factors rather than the minute amounts of dopamine (DA) liberated from chromaffin cells. We now report that protein from chromaffin granules, which release their contents by exocytosis, promotes survival and uptake of 3H-DA of mesencephalic DAergic neurons in vitro and protect against N-methylpyridinium ion toxicity. This neurotrophic effect is accompanied by cell proliferation and mediated by astroglial cells induced in these cultures. Inhibition of cell proliferation and concomitant astrogliosis by 5-fluorodeoxyuridine and alpha-aminoadipic acid abolishes the trophic effect. Two highly specific inhibitors of the epidermal growth factor receptor (EGFR) signal transduction pathway, 4,5-dianilinophthalimide (10 microM) and tyrphostin B56 (10 microM), selectively block the neurotrophic capacity of chromaffin granule protein. As expected, they also block the mitogenic effects of EGF and TGF-alpha. However, these two mitogens do not mimic the pronounced mitogenic and trophic actions of chromaffin granule protein. Culture medium conditioned by mesencephalic cells pretreated with chromaffin granule protein promotes survival of DAergic neurons without increasing numbers of astroglial cells. The effective molecule is unlikely to be glial cell line-derived neurotrophic factor, whose mRNA is not detectable in cultures treated with chromaffin granule protein. We conclude that chromaffin granules contain a putatively novel growth factor, which signals through the EGFR and may be responsible for the known protective and restorative actions of chromaffin cell grafts to the lesioned nigrostriatal system.     

Calcium-dependent neutrophil secretion: characterization and regulation by annexins

To gain direct access to the secretory machinery and study the regulation, mechanisms, and effectors of Ca2+-dependent neutrophil secretion, we developed an efficient and reproducible method of plasma membrane permeabilization using streptolysin O. We confirmed previous studies that permeabilized neutrophils secrete in response to calcium alone, but we also found that the Ca2+ dose-response is biphasic. Secretion is detectable at <1.0 microM Ca2+ and reaches a plateau between 1.0 and 60 to 80 microM. When stimulated with >80 microM Ca2+, secretion is two- to threefold greater than at lower [Ca2+], suggesting that two distinct mechanisms of Ca2+-dependent secretion that differ in their affinity for Ca2+ exist in neutrophils. Although permeabilization allows 100% leak of lactate dehydrogenase, maximum secretion from permeabilized cells is 80% that of f-met-leu-phe-stimulated intact cells, indicating that the essential components of the Ca2+-dependent secretory apparatus are predominantly, if not entirely, membrane bound. Permeabilization causes leakage of 100% of annexins V and VI, but 41% of annexin I and 12% of annexin III are retained. Immunofluorescence microscopy revealed that retained annexins I and III are associated with granule membranes. Addition of soluble annexins I and III to permeabilized cells increased Ca2+-induced secretion up to 15% and 90%, respectively, implying that both annexins participate in this secretory pathway. While annexin V is not required for secretion, it inhibits the low Ca2+-affinity mechanism of secretion.     

Survey of antimicrobial resistance in bacterial isolates from diseased cattle in France

Adrenaline and noradrenaline are released from adrenal medullary chromaffin cells by regulated exocytosis from stored secretory granules. Many aspects of the mechanisms by which exocytosis is activated in chromaffin cells are now understood in detail and these cells have provided an important model for the study of neuroendocrine secretion in general. Exocytosis is triggered by Ca2+ influx which activates a multistep process involving at least two Ca(2+)-binding proteins with distinct Ca2+ affinities. Several cytosolic and membrane proteins have been implicated by functional studies as components of the exocytotic machinery. The likely roles of these proteins in exocytosis are discussed in this review and the questions that remain for the understanding of the molecular basis of catecholamine release are highlighted.     

Inhibition of nicotinic receptor-mediated responses in bovine chromaffin cells by diltiazem

1. The effects of diltiazem on various functional parameters were studied in bovine cultured adrenal chromaffin cells stimulated with the nicotinic receptor agonist dimethylphenylpiperazinium (DMPP) or with depolarizing Krebs-HEPES solutions containing high K+ concentrations. 2. The release of [3H]-noradrenaline induced by DMPP (100 microM for 5 min) was gradually and fully inhibited by increasing concentrations of diltiazem (IC50 = 1.3 microM). In contrast, the highest concentration of diltiazem used (10 microM) inhibited the response to high K+ (59 mM for 5 min) by only 25%. 3. 45Ca2+ uptake into cells stimulated with DMPP (100 microM for 1 min) was also blocked by diltiazem in a concentration-dependent manner (IC50 = 0.4 microM). Again, diltiazem blocked the K(+)-evoked 45Ca2+ uptake (70 mM K+ for 1 min) only by 20%. In contrast, the N-P-Q-type Ca2+ channel blocker omega-conotoxin MVIIC depressed the K+ signal by 70%. In the presence of this toxin, diltiazem exhibited an additional small inhibitory effect, indicating that the compound was acting on L-type Ca2+ channels. 4. Whole-cell Ba2+ currents through Ca2+ channels in voltage-clamped chromaffin cells were inhibited by 3-10 microM diltiazem by 20-25%. The inhibition was readily reversed upon washout of the drug. 5. The whole-cell currents elicited by 100 microM DMPP (IDMPP) were inhibited in a concentration-dependent and reversible manner by diltiazem. Maximal effects were found at 10 microM, which reduced the peak IDMPP by 70%. The area of each curve represented by total current (QDMPP) was reduced more than the peak current. At 10 microM, the inhibition amounted to 80%; the IC50 for QDMPP inhibition was 0.73 microM, a figure close to the IC50 for 45Ca2+ uptake (0.4 microM) and [3H]-noradrenaline release (1.3 microM). The blocking effects of diltiazem developed very quickly and did not exhibit use-dependence; thus the drug blocked the channel in its closed state. The blocking effects of 1 microM diltiazem on IDMPP were similar at different holding potentials (inhibition by around 30% at -100, -80 or -50 mV). Diltiazem did not affect the current flow through voltage-dependent Na+ channels. 6. These data are compatible with the idea that diltiazem has little effect on Ca2+ entry through voltage-dependent Ca2+ channels in bovine chromaffin cells. Neither, does diltiazem affect INa. Rather, diltiazem acts directly on the neuronal nicotinic receptor ion channel and blocks ion fluxes, cell depolarization and the subsequent Ca2+ entry and catecholamine release. This novel effect of diltiazem might have clinical relevance since it might reduce the sympathoadrenal drive to the heart and blood vessels, thus contributing to the well established antihypertensive and cardioprotective effects of the drug.     

Isoproterenol evokes extracellular Ca2+ spikes due to secretory events in salivary gland cells

Secretory cells should in principle export substantial amounts of calcium via exocytosis since Ca2+ is sequestered in secretory granules. Based on a new technique for measurements of the extracellular calcium concentration in the vicinity of the cell membrane and on the droplet technique, we have monitored the rate of calcium extrusion from salivary gland acinar cells. Isoproterenol (ISP), a beta-adrenergic agonist and powerful secretogogue, evoked no change in the cytosolic free Ca2+ concentration ([Ca2+]i) but induced vigorous extracellular Ca2+ concentration ([Ca2+]i) spiking. The absence of [Ca2+]i elevation and the pulsatile nature of the changes in [Ca2+]i indicate that these spikes are most likely due to calcium release from secretory granules. The cholinergic agonist acetylcholine (ACh), which induces moderate secretion, evoked a marked rise in [Ca2+]i and a smooth rise in [Ca2+]i, most likely induced by plasma membrane calcium pumps, on which shortlasting [Ca2+]i spikes were superimposed. The rate of ISP-induced calcium efflux was very substantial. The calculated calcium loss during the first 100 s of supramaximal stimulation corresponded to a reduction of the total cellular calcium concentration of approximately 0.4 mM. We conclude that in salivary glands, calcium release via exocytosis is one of the main mechanisms extruding calcium from cells to the extracellular milieu.     

Thiopental and methohexital depress Ca2+ entry into and glutamate release from cultured neurons

BACKGROUND: Although barbiturates activate alpha-aminobutyric acid type A receptors as part of their hypnotic effect, these drugs also inhibit voltage-gated calcium channels. The authors determined if barbiturates could decrease neuronal intracellular Ca2+ transients and the resulting glutamate release. METHODS: Neonatal rat cerebellar granule neurons were isolated and cultured on coverslips and studied at 37 degrees C. Spectrofluorometric assays were used during identical conditions to monitor intracellular Ca2+ with the Ca2+ -sensitive fluorophore fura-2 and glutamate release by a glutamate dehydrogenase-coupled assay, which produced the reduced form of nicotinamide-adenine dinucleotide phosphate in proportion to the amount of glutamate released. Neurons were depolarized by a rapid increase in external [K+] from 5 to 55 mM. Control responses were compared with those in the presence of 10, 30, and 100 microM thiopental; 3, 10, and 30 microM methohexital; decreased external [Ca2+]; or voltage-gated calcium channel blockers. RESULTS: Thiopental and methohexital depressed the intracellular Ca2+ transient peak and plateau in a dose-dependent manner, as did decreased Ca2+. The intermediate dose of either drug caused approximately 50% decrease in peak intracellular Ca2+ and 60% decrease in glutamate release. In the presence of specific L- and/or N-type voltage-gated calcium channel blockade by nicardipine or omega-conotoxin-GVIA, respectively, 30 microM thiopental further decreased the intracellular Ca2+ transient. Thiopental caused a dose-dependent decrease in glutamate release, which was proportional to the decreased peak intracellular Ca2+. CONCLUSIONS: Thiopental and methohexital depress the depolarization-induced increase in intracellular Ca2+ and the accompanying glutamate release, actions which can contribute to the anesthetic and neuronal protective effects of these drugs.     

Mastoparan stimulates exocytosis at a Ca(2+)-independent late site in stimulus-secretion coupling. Studies with the RINm5F beta-cell line

Mastoparan, a tetradecapeptide from wasp venom, stimulated exocytosis in a concentration-dependent manner, which was enhanced by pertussis toxin pre-treatment, in the insulin secreting beta-cell line RINm5F. Mastoparan (3-20 microM) also elevated cytosolic free calcium concentration ([Ca2+]i), a rise that was not attenuated by nitrendipine. Divalent cation-free Krebs-Ringer bicarbonate (KRB) medium with 0.1 mM EGTA nullified the mastoparan-induced increase in [Ca2+]i, suggesting that the peptide increased Ca2+ influx but not through the L-type voltage-dependent Ca2+ channel. Depletion of the intracellular Ca2+ pool did not affect the mastoparan-induced elevation of [Ca2+]i. Remarkably, in divalent cation-free KRB medium with 0.1 mM EGTA and 2 microM thapsigargin in which mastoparan reduced [Ca2+]i, the mastoparan-stimulated insulin release was similar to that in normal Ca(2+)-containing KRB medium. Inhibitors of protein kinase C, such as bisindolylmaleimide, staurosporine, and 1-O-hexadecyl-2-O-methyl-rac-glycerol did not suppress the mastoparan-stimulated insulin release. Mastoparan at 10-20 microM did not increase cellular cAMP levels, nor did mastoparan at 5-10 microM affect [3H]arachidonic acid release. In conclusion, although mastoparan increased [Ca2+]i, this increase was not involved in the stimulation of insulin release. Rather, the data suggest that mastoparan directly stimulates exocytosis in a Ca(2+)-independent manner. As GTP-binding proteins (G proteins) are thought to be involved in the process of exocytosis and as mastoparan is known to exert at least some of its effects by activation of G proteins, an action of mastoparan to activate the putative stimulatory Ge (exocytosis) protein is likely.     

Hydroperoxide-induced increases in intracellular calcium due to annexin VI translocation and inactivation of plasma membrane Ca2+-ATPase

Oxidative stress can cause changes in intracellular free calcium concentration ([Ca2+]i) that resemble those occurring under normal cell signaling. In the alveolar macrophage, hydroperoxide-induced elevation of [Ca2+]i modulates the respiratory burst and other important physiologic functions. The source of Ca2+ released by hydroperoxide is intracellular but separate from the endoplasmic reticulum pool released by receptor-mediated stimuli (Hoyal, C. R., Gozal, E., Zhou, H., Foldenauer, K., and Forman, H. J. (1996) Arch. Biochem. Biophys. 326, 166-171). Previous studies in other cells have suggested that mitochondria are a potential source of oxidant-induced [Ca2+]i elevation. In this study we have identified another potential source of hydroperoxide-releasable intracellular calcium, that bound to annexin VI on the inner surface of the plasma membrane. Translocation of annexin VI from the membrane during exposure to t-butyl hydroperoxide matched elevation of [Ca2+]i as a function of time and t-butyl hydroperoxide concentration. The translocation was possibly due to a combination of ATP depletion and oxidative modification of membrane lipids and proteins. A sustained increase in [Ca2+]i occurring > 50 pmol/10(6) cells (50 microM under these conditions) appeared to be a consequence of membrane Ca2+-ATPase dysfunction. These results suggest that exposure to oxidative stress results in early alterations to the plasma membrane and concomitant release of Ca2+ into the cytosol. In addition it suggests a mechanism for participation of annexin VI translocation that may underlie the alterations in macrophage function by oxidative stress.     

Macrolides and drug interactions: review of the literature]

Annexin I is a member of the annexin family of calcium-dependent membrane binding proteins. The core domain of these proteins is similar in all annexins but the N-terminal domain is specific for each member. This domain is thought to regulate annexin function through phosphorylation. In annexin I, Ser-27 is one of the amino acids that can be phosphorylated by protein kinase C. Phosphorylations are thought to regulate some annexin I functions by increasing calcium requirement. Two mutants were prepared in this study: S27E and S27A proteins. The first mimics phosphorylation while the second prevents phosphorylation at residue 27. Wild-type annexin I and S27A mutant protein showed the same calcium dependence for phospholipid vesicles aggregation, while S27E mutant protein showed a higher calcium requirement and a low maximal extent of aggregation. By contrast, liposome binding and self-association required identical calcium concentrations for the wild-type and the two mutant proteins. To examine whether the regulation observed is due to modification of the N-terminal structure, we investigated conformational changes by using two approaches. Firstly we analysed proteinase sensibility. Limited proteolysis of the N-terminal tail showed similar patterns for the three proteins. Using drastic conditions of proteolysis, we observed strong resistance of the core domain to digestion in the presence of calcium. Secondly, since Ser-27 is located on the N-terminal domain that contains a tryptophan located at position 12, the fluorescence of this residue was analysed during Ca2+-binding of wild-type annexin I and S27E mutant protein. The results demonstrated that Ca2+ induces a slight change in the Trp environment of wild-type annexin I, corresponding to a burying of the residue. No changes in fluorescence features were observed with S27E mutant protein. The results obtained show that phosphorylation of the N-terminal tail plays a regulatory role in phospholipid vesicle aggregation, which is based on a mechanism distinct from protein self-association. This phosphorylation induces a conformational change in the tail probably related to aggregation property.     

Novel antiallergic agents. Part I: Synthesis and pharmacology of pyrimidine amide derivatives

Cytolytic T cells use two mechanisms to kill virally infected cells, tumor cells, or other potentially autoreactive T cells in short-term in vitro assays. The perforin/granule exocytosis mechanism uses preformed cytolytic granules that are delivered to the target cell to induce apoptosis and eventual lysis. FasL/Fas (CD95 ligand/CD95)-mediated cytolysis requires de novo protein synthesis of FasL by the CTL and the presence of the death receptor Fas on the target cell to induce apoptosis. Using a CD8(+) CTL clone that kills via both the perforin/granule exocytosis and FasL/Fas mechanisms, and a clone that kills via the FasL/Fas mechanism only, we have examined the requirement of intra- and extracellular Ca2+ in TCR-triggered cytolytic effector function. These two clones, a panel of Ca2+ antagonists, and agonists were used to determine that a large biphasic increase in intracellular calcium concentration, characterized by release of Ca2+ from intracellular stores followed by a sustained influx of extracellular Ca2+, is required for perforin/granule exocytosis. Only the sustained influx of extracellular Ca2+ is required for FasL induction and killing. Thapsigargin, at low concentrations, induces this small but sustained increase in [Ca2+]i and selectively induces FasL/Fas-mediated cytolysis but not granule exocytosis. These results further define the role of Ca2+ in perforin and FasL/Fas killing and demonstrate that differential Ca2+ signaling can modulate T cell effector functions.     

Pre- and post-synaptic muscarinic receptors in thin slices of rat adrenal gland

The effects of activation of muscarinic receptors on chromaffin cells and splanchnic nerve terminals were studied in a rat adrenal slice preparation. In chromaffin cells, muscarine induced a transient hyperpolarization followed by a depolarization associated with cell spiking. The hyperpolarization was blocked by charybdotoxin (1 microM) and tetraethylammonium chloride (TEA, 1 mM), but was not affected by 200 microM Cd2+ or removal of external Ca2+, consistent with activation of BK channels. This would follow internal Ca2+ mobilization, as shown by Ca2+ imaging with fura-2 on isolated chromaffin cells in culture. Under voltage-clamp, outward BK currents were insensitive to MT3 toxin, a specific muscarinic m4 receptor antagonist. In contrast, muscarine-induced depolarization was due to a m4 receptor-mediated inward current blocked by MT3 toxin. This current was permeable to cations and was associated with Ca2+ entry and subsequently, Ca2+-induced Ca2+ release. Finally, both muscarine (25 microM) and oxotremorine (10 microM) decreased the amplitude and frequency of KCI-evoked excitatory postsynaptic currents, without affecting quantal size, consistent with a presynaptic inhibitory effect. Taken together, our data suggest that activation of m4 and probably m3 muscarinic receptors results in a strong, long-lasting excitation of chromaffin cells, as well as an uncoupling of synaptic inputs onto these cells.     

The novel insulinotropic mechanism of pimobendan: direct enhancement of the exocytotic process of insulin secretory granules by increased Ca2+ sensitivity in beta-cells

Pimobendan is a new class of inotropic drug that augments Ca2+ sensitivity and inhibits phosphodiesterase (PDE) activity in cardiomyocytes. To examine the insulinotropic effect of pimobendan in pancreatic beta-cells, which have an intracellular signaling mechanism similar to that of cardiomyocytes, we measured insulin release from rat isolated islets of Langerhans. Pimobendan augmented glucose-induced insulin release in a dose-dependent manner, but did not increase cAMP content in pancreatic islets, indicating that the PDE inhibitory effects may not be important in beta-cells. This agent increased the intracellular Ca2+ concentration ([Ca2+]i) in the presence of 30 mM K+, 16.7 mM glucose, and 200 microM diazoxide, but failed to enhance the 30 mM K+-evoked [Ca2+]i rise in the presence of 3.3 mM glucose. Insulin release evoked by 30 mM K+ in 3.3 mM glucose was augmented. Then, the direct effects of pimobendan on the Ca2+-sensitive exocytotic apparatus were examined using electrically permeabilized islets in which [Ca2+]i can be manipulated. Pimobendan (50 microM) significantly augmented insulin release at 0.32 microM Ca2+, and a lower threshold for Ca2+-induced insulin release was apparent in pimobendan-treated islets. Moreover, 1 microM KN93 (Ca2+/calmodulin-dependent protein kinase II inhibitor) significantly suppressed this augmentation. Pimobendan, therefore, enhances insulin release by directly sensitizing the intracellular Ca2+-sensitive exocytotic mechanism distal to the [Ca2+]i rise. In addition, Ca2+/calmodulin-dependent protein kinase II activation may at least in part be involved in this Ca2+ sensitization for exocytosis of insulin secretory granules.     

Isolation of chromaffin cell thapsigargin-sensitive Ca2+ store in light microsomes from bovine adrenal medulla

1. A subcellular fractionation procedure for bovine adrenal glands was designed with the aim to study the biochemical properties of Ca2+ stores in chromaffin cells. 2. The thapsigargin-sensitive compartment of Ca2+ stores was found to be highly enriched in a light microsomal fraction (LMF) on a 15-30% linear sucrose gradient, and was found to be essentially devoid of contamination by plasma, mitochondrial or secretory granule membranes. 3. A Ca(2+)-pumping ATPase was identified in this LMF as a 97 kDa protein forming an acid-stable, Ca(2+)-dependent, thapsigargin-sensitive phosphorylated intermediate upon incubation with [gamma-32P]ATP, suggesting this protein to represent a SERCA-3 isoform of Ca2+ ATPases. 4. A major 162 kDa protein, previously demonstrated in the isolated chromaffin cells, was enriched in the LMF, distributing on sucrose gradients in parallel with the thapsigargin-sensitive Ca2+ uptake. 5. LMF appears to represent a part of the thapsigargin-sensitive Ca2+ store of chromaffin cells, and should be useful for further studies of the store properties at the subcellular and molecular level.     

Mechanisms determining the time course of secretion in neuroendocrine cells

Transmitter release from chromaffin cells differs from that in synapses in that it persists for a longer time after Ca2+ entry has stopped. This prolonged secretion is not due to a delay between vesicle fusion and transmitter release, nor to slow detection of released substance: step increases in capacitance due to single vesicle fusion precede the release detected by amperometry by only a few milliseconds. The persistence of secretion after a depolarization is reduced by addition of mobile calcium buffer. This suggests that most of the delay is due to diffusion of Ca2+ between channels and release sites, implying that Ca2+ channels and secretory vesicles are not colocalized in chromaffin cells, in contrast to presynaptic active zones.     

Regulation of the Ca2+ sensitivity of exocytosis by Rab3a

Ca2+ ions trigger the release of hormones and neurotransmitters and contribute to making the secretory vesicles competent for fusion. Here, we present evidence for the involvement of the GTP-binding protein Rab3a in the sensitivity of the exocytotic process to internal [Ca2+]. The secretory activity of bovine adrenal chromaffin cells was elicited by Ca2+ dialysis through a patch-clamp pipette and assayed by monitoring changes in cell membrane capacitance. Microinjection of antisense oligonucleotides directed to rab3a mRNA increased the secretory activity observed at low (0.2-4 microM) [Ca2+], but did not change the maximal activity observed at 10 microM free [Ca2+]. Moreover, after a train of depolarizing stimuli, the secretory activity of antisense-injected cells dialyzed with 10 microM [Ca2+] was increased significantly compared with that of control cells. This result suggests that the activity of either Rab3a or its partners might change upon stimulation. We conclude that Rab3a, together with its partners, participates in the Ca2+ dependence of exocytosis and that its activity is modulated further in a stimulus-dependent manner. These findings should provide some clues to elucidate the role of Rab3a in synaptic plasticity.     

Roles for protein kinase C and mitogen-activated protein kinase in nicotine-induced secretion from bovine adrenal chromaffin cells

Both the Ca2+/phospholipid-dependent protein kinases (protein kinases C, PKCs) and mitogen-activated protein kinases (MAPKs) have been implicated as participants in the secretory response of bovine adrenomedullary chromaffin cells. To investigate a possible role for these kinases in exocytosis and the relationship of these kinases to one another, intact chromaffin cells were treated with agents that inhibited each of the kinases and analyzed for catecholamine release and MAPK/extracellular signal-regulated kinase (ERK) kinase (MEK)/MAPK activation after stimulation with secretagogues of differential efficacy. Of the three secretagogues tested, inactivation of PKCs by long-term phorbol 12-myristate 13-acetate (PMA) treatment or incubation with GF109203X had the greatest inhibitory effect on nicotine-induced catecholamine release and MEK/MAPK activation, a moderate effect on KCl-induced events, and little, if any, effect on Ca2+ ionophore-elicited exocytosis and MEK/MAPK activation. These results indicate that PKC plays a significant role in events induced by the optimal secretagogue nicotine and a lesser role in exocytosis elicited by the suboptimal secretagogues KCl and Ca2+ ionophore. Treatment of cells with the MEK-activation inhibitor PD098059 completely inhibited MEK/MAPK activation (IC50 1-5 microM) and partially inhibited catecholamine release induced by all secretagogues. However, PD098059 was more effective at inhibiting exocytosis induced by suboptimal secretagogues (IC50 approximately 10 microM) than that induced by nicotine (IC50 approximately 30 microM). These results suggest a more prominent role for MEK/MAPK in basic secretory events activated by suboptimal secretagogues than in those activated by the optimal secretagogue nicotine. However, PD098059 also partially blocked secretion potentiated by short-term PMA treatment, suggesting that PKC can function in part by signaling through MEK/MAPK to enhance secretion. Taken together, these results provide evidence for the preferential involvement of MEK/MAPK in basic secretory events activated by the suboptimal secretagogues KCl and Ca2+ ionophore and the participation of both PKC and MEK/MAPK in optimal, secretion induced by nicotine.     

KSAYMRFamide (PF3/AF8) is present in the free-living nematode, Caenorhabditis elegans

The effect of cAMP on a ryanodine-sensitive Ca2+ release from microsomal vesicles of rat parotid acinar cells was studied. After a steady state of ATP-dependent 45Ca2+ uptake into the vesicles, cAMP was added to the medium with thapsigargin (TG) to block a reuptake of 45Ca2+. The addition of cAMP (1.0 mM) with TG released about 10% of the 45Ca2+ that had been taken up. The cAMP-induced 45Ca2+ release was strongly inhibited by pretreatment of the vesicles with 500 microMM ryanodine. Preincubation with cAMP (1 mM) abolished ryanodine (10 microM)-induced 45Ca2+ release. The presence of a specific inhibitor of cAMP-dependent protein kinase (PKA) H-89 (10 microMM) inhibited the cAMP-induced 45Ca2+ release. These results indicate that in rat parotid acinar cells, cAMP can activate a ryanodine-sensitive Ca2+ release mechanism in the endoplasmic reticulum and that this activation is via a PKA-dependent process.