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Granule-plasma membrane docking and fusion can only occur when proteins that enable these reactions are present at the granule-plasma membrane contact. Thus, the mobility of granule membrane proteins may influence docking and membrane fusion. We measured the mobility of vesicle associated membrane protein 2 (VAMP2), synaptotagmin 1 (Syt1), and synaptotagmin 7 (Syt7) in chromaffin granule membranes in living chromaffin cells. We used a method that is not limited by standard optical resolution. A bright flash of strongly decaying evanescent field produced by total internal reflection was used to photobleach GFP-labeled proteins in the granule membrane. Fluorescence recovery occurs as unbleached protein in the granule membrane distal from the glass interface diffuses into the more bleached proximal regions, enabling the measurement of diffusion coefficients. We found that VAMP2-EGFP and Syt7-EGFP are mobile with a diffusion coefficient of ∼3 × 10 -10 cm 2 /s. Syt1-EGFP mobility was below the detection limit. Utilizing these diffusion parameters, we estimated the time required for these proteins to arrive at docking and nascent fusion sites to be many tens of milliseconds. Our analyses raise the possibility that the diffusion characteristics of VAMP2 and Syt proteins could be a factor that influences the rate of exocytosis.

Large dense-core vesicles (LDCVs) contain a variety of neurotransmitters, proteins, and hormones such as biogenic amines and peptides, together with microRNAs (miRNAs). Isolation of LDCVs is essential for functional studies including vesicle fusion, vesicle acidification, monoamine transport, and the miRNAs stored in LDCVs. Although several methods were reported for purifying LDCVs, the final fractions are significantly contaminated by other organelles, compromising biochemical characterization. Here we isolated LDCVs (chromaffin granules) with high yield and purity from bovine adrenal medulla. The fractionation protocol combines differential and continuous sucrose gradient centrifugation, allowing for reducing major contaminants such as mitochondria. Purified LDCVs show robust acidification by the endogenous V-ATPase and undergo SNARE-mediated fusion with artificial membranes. Interestingly, LDCVs contain specific miRNAs such as miR-375 and miR-375 is stabilized by protein complex against RNase A. This protocol can be useful in research on the biological functions of LDCVs.

FTY-720 (Fingolimod) was one of the first compounds authorized for the treatment of multiple sclerosis. Among its other activities, this sphingosine analogue enhances exocytosis in neuroendocrine chromaffin cells, altering the quantal release of catecholamines. Surprisingly, the size of chromaffin granules is reduced within few minutes of treatment, a process that is paralleled by the homotypic fusion of granules and their heterotypic fusion with mitochondria, as witnessed by dynamic confocal and TIRF microscopy. Electron microscopy studies support these observations, revealing the fusion of several vesicles with individual mitochondria to form large, round mixed organelles. This cross-fusion is SNARE-dependent, being partially prevented by the expression of an inactive form of SNAP-25. Fused mitochondria exhibit an altered redox potential, which dramatically enhances cell death. Therefore, the cross-fusion of intracellular organelles appears to be a new mechanism to be borne in mind when considering the effect of FTY-720 on the survival of neuroendocrine cells.

Soluble N-ethylmaleimide-sensitive factor activating protein receptor (SNARE) proteins are the main catalysts for membrane fusion in the secretory pathway of eukaryotic cells. In vitro, SNAREs are sufficient to mediate effective fusion of both native and artificial membranes. Here we have established, to our knowledge, a new platform for monitoring SNARE-mediated docking and fusion between giant unilamellar vesicles (GUVs) and smaller liposomes or purified secretory granules with high temporal and spatial resolution. Analysis of fusion is restricted to the free-standing part of the GUV-membrane exhibiting low curvature and a lack of surface contact, thus avoiding adhesion-mediated interference with the fusion reaction as in fusion with supported bilayers or surface-immobilized small vesicles. Our results show that liposomes and chromaffin granules fuse with GUVs containing activated SNAREs with only few milliseconds delay between docking and fusion. We conclude that after initial contact in trans, SNAREs alone can complete fusion at a rate close to fast neuronal exocytosis., (Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

The Ca(2+) sensor synaptotagmin-1 is thought to trigger membrane fusion by binding to acidic membrane lipids and SNARE proteins. Previous work has shown that binding is mediated by electrostatic interactions that are sensitive to the ionic environment. However, the influence of divalent or polyvalent ions, at physiological concentrations, on synaptotagmin's binding to membranes or SNAREs has not been explored. Here we show that binding of rat synaptotagmin-1 to membranes containing phosphatidylinositol 4,5-bisphosphate (PIP2) is regulated by charge shielding caused by the presence of divalent cations. Surprisingly, polyvalent ions such as ATP and Mg(2+) completely abrogate synaptotagmin-1 binding to SNAREs regardless of the presence of Ca(2+). Altogether, our data indicate that at physiological ion concentrations Ca(2+)-dependent synaptotagmin-1 binding is confined to PIP2-containing membrane patches in the plasma membrane, suggesting that membrane interaction of synaptotagmin-1 rather than SNARE binding triggers exocytosis of vesicles.

ComplexinII (CpxII) and SynaptotagminI (SytI) have been implicated in regulating the function of SNARE proteins in exocytosis, but their precise mode of action and potential interplay have remained unknown. In this paper, we show that CpxII increases Ca(2+)-triggered vesicle exocytosis and accelerates its secretory rates, providing two independent, but synergistic, functions to enhance synchronous secretion. Specifically, we demonstrate that the C-terminal domain of CpxII increases the pool of primed vesicles by hindering premature exocytosis at submicromolar Ca(2+) concentrations, whereas the N-terminal domain shortens the secretory delay and accelerates the kinetics of Ca(2+)-triggered exocytosis by increasing the Ca(2+) affinity of synchronous secretion. With its C terminus, CpxII attenuates fluctuations of the early fusion pore and slows its expansion but is functionally antagonized by SytI, enabling rapid transmitter discharge from single vesicles. Thus, our results illustrate how key features of CpxII, SytI, and their interplay transform the constitutively active SNARE-mediated fusion mechanism into a highly synchronized, Ca(2+)-triggered release apparatus.

Clathrin-mediated endocytosis is the major pathway for recycling of granule membrane components after strong stimulation and high exocytotic rates. It resembles "classical" receptor-mediated endocytosis but has a trigger that is unique to secretion, the sudden appearance of the secretory granule membrane in the plasma membrane. The spatial localization, the relationship to individual fusion events, the nature of the cargo, and the timing and nature of the nucleation events are unknown. Furthermore, a size mismatch between chromaffin granules (∼300-nm diameter) and typical clathrin-coated vesicles (∼90 nm) makes it unlikely that clathrin-mediated endocytosis internalizes as a unit the entire fused granule membrane. We have used a combination of total internal reflection fluorescence microscopy of transiently expressed proteins and time-resolved quantitative confocal imaging of endogenous proteins along with a fluid-phase marker to address these issues. We demonstrate that the fused granule membrane remains a distinct entity and serves as a nucleation site for clathrin- and dynamin-mediated endocytosis that internalizes granule membrane components in small increments.

Exocytosis of neurosecretory vesicles is mediated by the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins syntaxin-1, synaptobrevin and SNAP-25, with synaptotagmin functioning as the major Ca(2+) sensor for triggering membrane fusion. Here we show that bovine chromaffin granules readily fuse with large unilamellar liposomes in a SNARE-dependent manner. Fusion is enhanced by Ca(2+), but only when the target liposomes contain phosphatidylinositol-4,5-bisphosphate and when polyphosphate anions, such as nucleotides or pyrophosphate, are present. Ca(2+)-dependent enhancement is mediated by endogenous synaptotagmin-1. Polyphosphates operate by an electrostatic mechanism that reverses an inactivating cis association of synaptotagmin-1 with its own membrane without affecting trans binding. Hence, the balancing of trans- and cis-membrane interactions of synaptotagmin-1 could be a crucial element in the pathway of Ca(2+)-dependent exocytosis.

We have studied how the F-actin cytoskeleton is involved in establishing the heterogeneous intracellular Ca(2+) levels ([Ca(2+)](i)) and in the organization of the exocytotic machinery in cultured bovine chromaffin cells. Simultaneous confocal visualization of [Ca(2+)](i) and transmitted light studies of the cytoskeleton showed that, following cell stimulation, the maximal signal from the Ca(2+)-sensitive fluorescent dye Fluo-3 was in the empty cytosolic spaces left by cytoskeletal cages. This was mostly due to the accumulation of the dye in spaces devoid of cytoskeletal components, as shown by the use of alternative Ca(2+)-insensitive fluorescent cytosolic markers. In addition to affecting the distribution of such compounds in the cytosol, the cytoskeleton influenced the location of L- and P-Q-type Ca(2+) channel clusters, which were associated with the borders of cytoskeletal cages in resting and stimulated cells. Indeed, syntaxin-1 and synaptotagmin-1, which are components of the secretory machinery, were present in the same location. Furthermore, granule exocytosis took place at these sites, indicating that the organization of the F-actin cytoskeletal cortex shapes the preferential sites for secretion by associating the secretory machinery with preferential sites for Ca(2+) entry. The influence of this cortical organization on the propagation of [Ca(2+)](i) can be modelled, illustrating how it serves to define rapid exocytosis.

Chromogranin A (CgA) is a hydrophilic acidic one-chain peptide containing 439 amino acids, preceded by NH2-terminal 18-amino-acid signal peptide; the complete pre-chromogranin A molecule thus encompasses 457 amino acids. It is a member of the chromogranin family that comprises several proteins. The CgA gene is a single-copy gene localized in the locus 14q32. Chromogranin A is produced by endocrine and neuroendocrine cells. The largest amount of CgA occurs in chromaffin granules of adrenal medulla and in the dense-core vesicles of sympathetic nerves. Its biological functions have not been completely elucidated, but it is known that it acts as a precursor of many biologically active peptides generated by cleavage at specific sites. It is the major soluble protein co-stored and co-released along with resident catecholamines and polypeptide hormones or cell-specific neurotransmitters. Because of its widespread distribution in neuroendocrine tissue, it can be used both as immunohistochemical marker and serum marker of neuroendocrine tumours. CgA has been used as a rather reliable tumour marker because its level is significantly increased in neuroendocrine tumours and changes of its level reflect the tumour response to therapy or tumour recurrence.

Voltage-gated L-type calcium channels (LTCCs) are expressed in adrenal chromaffin cells. Besides shaping the action potential (AP), LTCCs are involved in the excitation-secretion coupling controlling catecholamine release and in Ca (2+) -dependent vesicle retrieval. Of the two LTCCs expressed in chromaffin cells (CaV1.2 and CaV1.3), CaV1.3 possesses the prerequisites for pacemaking spontaneously firing cells: low-threshold, steep voltage-dependence of activation and slow inactivation. By using CaV1 .3 (-/-) KO mice and the AP-clamp it has been possible to resolve the time course of CaV1.3 pacemaker currents, which is similar to that regulating substantia nigra dopaminergic neurons. In mouse chromaffin cells CaV1.3 is coupled to fast-inactivating BK channels within membrane nanodomains and controls AP repolarization. The ability to carry subthreshold Ca (2+) currents and activate BK channels confers to CaV1.3 the unique feature of driving Ca (2+) loading during long interspike intervals and, possibly, to control the Ca (2+) -dependent exocytosis and endocytosis processes that regulate catecholamine secretion and vesicle recycling.

Although synaptophysin is one of the most abundant integral proteins of synaptic vesicle membranes, its contribution to neurotransmitter release remains unclear. One possibility is that through its association with dynamin it controls the fine tuning of transmitter release. To test this hypothesis, we took advantage of amperometric measurements of quantal catecholamine release from chromaffin cells. First, we showed that synaptophysin and dynamin interact in chromaffin granule-rich fractions and that this interaction relies on the C terminal of synaptophysin. Experimental maneuvers that are predicted to disrupt the association between these two proteins, such as injection of antibodies against dynamin or synaptophysin, or peptides homologous to the C terminal of synaptophysin, increased the quantal size and duration of amperometric spikes. In contrast, the amperometric current that precedes the spike remained unchanged, indicating that synaptophysin/dynamin association does not regulate the initial fusion pore, but it appears to target a later step of exocytosis to control the amount of catecholamines released during a single vesicle fusion event.

Processes underlying the formation of dense core secretory granules (DCGs) of neuroendocrine cells are poorly understood. Here, we present evidence that DCG biogenesis is dependent on the secretory protein secretogranin (Sg) II, a member of the granin family of pro-hormone cargo of DCGs in neuroendocrine cells. Depletion of SgII expression in PC12 cells leads to a decrease in both the number and size of DCGs and impairs DCG trafficking of other regulated hormones. Expression of SgII fusion proteins in a secretory-deficient PC12 variant rescues a regulated secretory pathway. SgII-containing dense core vesicles share morphological and physical properties with bona fide DCGs, are competent for regulated exocytosis, and maintain an acidic luminal pH through the V-type H(+)-translocating ATPase. The granulogenic activity of SgII requires a pH gradient along this secretory pathway. We conclude that SgII is a critical factor for the regulation of DCG biogenesis in neuroendocrine cells, mediating the formation of functional DCGs via its pH-dependent aggregation at the trans-Golgi network.

Changes in cellular cholesterol can affect exocytosis, but the influence of cholesterol in fusion pore kinetics is unclear. Using carbon fiber amperometry, we monitored quantal catecholamine release from rat chromaffin cells. To bypass any possible effect of cholesterol perturbation on ion channels or the colocalization of voltage-gated Ca(2+) channels with sites of exocytosis, exocytosis was stimulated via uniform elevation of cytosolic [Ca(2+)] (with whole-cell dialysis of a Ca(2+)-buffered solution). Under this condition, alterations of cellular cholesterol affected neither the mean number of amperometric events triggered per cell nor their quantal size and the kinetics of their main spike (which reflects the rapid release during and after rapid fusion pore dilation). In contrast, the reduction of cellular cholesterol shortened the "prespike foot" signals (which reflect the leakage of catecholamine via a semi-stable fusion pore) and reduced the proportion of "stand-alone foot" signals (which reflect the release via a flickering fusion pore that may close before it dilates significantly), whereas an oversupply of cholesterol had opposite effects. Acute extraction of cholesterol from the cytosol (via whole-cell dialysis of a cholesterol extractor) also shortened the prespike foot signals and reduced the proportion of stand-alone foot signals, but acute extracellular application of cholesterol extractor or "soluble" cholesterol had no effect. Our data raise the possibility that cholesterol molecules, particularly those in the cytoplasmic leaflet, helps to constrain the narrow waistline of a semi-stable fusion pore while it is flickering or before it starts to dilate rapidly.

The roles of nonmuscle myosin II and cortical actin filaments in chromaffin granule exocytosis were studied by confocal fluorescence microscopy, amperometry, and cell-attached capacitance measurements. Fluorescence imaging indicated decreased mobility of granules near the plasma membrane following inhibition of myosin II function with blebbistatin. Slower fusion pore expansion rates and longer fusion pore lifetimes were observed after inhibition of actin polymerization using cytochalasin D. Amperometric recordings revealed increased amperometric spike half-widths without change in quantal size after either myosin II inhibition or actin disruption. These results suggest that actin and myosin II facilitate release from individual chromaffin granules by accelerating dissociation of catecholamines from the intragranular matrix possibly through generation of mechanical forces.

Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) synthesis is required for calcium-dependent exocytosis in neurosecretory cells. We developed a PtdIns(4,5)P2 bead pulldown strategy combined with subcellular fractionation to identify endogenous chromaffin granule proteins that interact with PtdIns(4,5)P2. We identified two synaptotagmin isoforms, synaptotagmins 1 and 7; spectrin; alpha-adaptin; and synaptotagmin-like protein 4 (granuphilin) by mass spectrometry and Western blotting. The interaction between synaptotagmin 7 and PtdIns(4,5)P2 and its functional relevance was investigated. The 45-kDa isoform of synaptotagmin 7 was found to be highly expressed in adrenal chromaffin cells compared with PC12 cells and to mainly localize to secretory granules by subcellular fractionation, immunoisolation, and immunocytochemistry. We demonstrated that synaptotagmin 7 binds PtdIns(4,5)P2 via the C2B domain in the absence of calcium and via both the C2A and C2B domains in the presence of calcium. We mutated the polylysine stretch in synaptotagmin 7 C2B and demonstrated that this mutant domain lacks the calcium-independent PtdIns(4,5)P2 binding. Synaptotagmin 7 C2B domain inhibited catecholamine release from digitonin-permeabilized chromaffin cells, and this inhibition was abrogated with the C2B polylysine mutant. These data indicate that synaptotagmin 7 C2B-effector interactions, which occur via the polylysine stretch, including calcium-independent PtdIns(4,5)P2 binding, are important for chromaffin granule exocytosis.

Using carbon fiber amperometry, we exploited the natural variation in quantal size (Q) among individual granules in rat chromaffin cells to examine the influence of Q on quantal release kinetics. Although it is generally accepted that granules with larger Q have slower kinetics of release, we found that this trend was applicable only to granules with Q(1/3) < 0.6 pC(1/3). Granules with larger Q adapted specific mechanisms to maintain a rapid kinetic of release. The semistable fusion pores in the large-Q granules persisted for a longer duration and could reach a bigger size before the onset of very rapid dilation to allow a longer and larger foot signal. Most importantly, a large proportion of large-Q granules maintained a relatively short half-width in the main spike. This suggests that the most rapid phase of fusion pore dilation in many large-Q granules may be faster than that in small-Q granules. Moreover, cAMP selectively advanced the onset of the rapid dilation of the fusion pore in the large- but not the small-Q granules. Thus, our finding raises the possibility that fusion pore and/or granule matrix in small- and large-Q granules may have different molecular structures.

We report for the first time that morphine-6-glucuronide, a highly analgesic morphine-derived molecule, is present in adrenal chromaffin granules and secreted from chromaffin cells upon stimulation. We also demonstrate that phosphatidylethanolamine-binding protein (alternatively named Raf-1 kinase inhibitor protein or RKIP) acts as an endogenous morphine-6-glucuronide-binding protein. An UDP-glucuronosyltransferase 2B-like enzyme, described to transform morphine into morphine-6-glucuronide, has been immunodetected in the chromaffin granule matrix, and morphine-6-glucuronide de novo synthesis has been characterized, demonstrating the possible involvement of intragranular UDP-glucuronosyltransferase 2B-like enzyme in morphine-6-glucuronide metabolism. Once secreted into the circulation, morphine-6-glucuronide may mediate several systemic actions (e.g. on immune cells) based on its affinity for mu-opioid receptors. These activities could be facilitated by phosphatidylethanolamine-binding protein (PEBP), acting as a molecular shield and preventing morphine-6-glucuronide from rapid clearance. Taken together, our data represent an important observation on the role of morphine-6-glucuronide as a new endocrine factor.

Chromaffin granules are involved in catecholamine synthesis and traffic in the adrenal glands. The transporting membrane proteins of chromaffin granules play an important role in the ion homeostasis of these organelles. In this study, we characterized components of the electrogenic (86)Rb(+) flux observed in isolated chromaffin granules. In order to study single channel activity, chromaffin granules from the bovine adrenal medulla were incorporated into planar lipid bilayers. Four types of cationic channel were found, each with a different conductance. The unitary conductances of the potassium channels are 360 +/- 10 pS, 220 +/- 8 pS, 152 +/- 8 pS and 13 +/- 3 pS in a gradient of 450/150 mM KCl, pH 7.0. A multiconductance potassium channel with a conductivity of 110 +/- 8 pS and 31 +/- 4 pS was also found. With the exception of the 13 pS conductance channel, all are activated by depolarizing voltages. One type of chloride channel was also found. It has a unitary conductance of about 250 pS in a gradient of 500/150 mM KCl, pH 7.0.

Most nerve cells communicate with each other through synaptic transmission at chemical synapses. The regulated exocytosis of neurotransmitters, hormones, and peptides occurs at specialized membrane areas through Ca2+-triggered fusion of secretory vesicles with the plasma membrane . Prior to fusion, vesicles are docked at the plasma membrane and must then be rendered fusion-competent through a process called priming. The molecular mechanism underlying this priming process is most likely the formation of the SNARE complex consisting of Syntaxin 1, SNAP-25, and Synaptobrevin 2. Members of the Munc13 protein family consisting of Munc13-1, -2, -3, and -4 were found to be absolutely required for this priming process . In the present study, we identified the minimal Munc13-1 domain that is responsible for its priming activity. Using Munc13-1 deletion constructs in an electrophysiological gain-of-function assay of chromaffin-granule secretion, we show that priming activity is mediated by the C-terminal residues 1100-1735 of Munc13-1, which contains both Munc13-homology domains and the C-terminal C2 domain. Priming by Munc13-1 appears to require its interaction with Syntaxin 1 because point mutants that do not bind Syntaxin 1 do not prime chromaffin granules.

The biogenesis of dense-core secretory granules (DCGs), organelles responsible for the storage and secretion of neurotransmitters and neuropeptides in chromaffin cells, is poorly understood. Chromogranin A (CgA), which binds catecholamines for storage in the lumen of chromaffin granules, has been shown to be involved in DCG biogenesis in neuroendocrine PC12 cells. Here, we report that downregulation of CgA expression in vivo by expressing antisense RNA against CgA in transgenic mice led to a significant reduction in DCG formation in adrenal chromaffin cells. The number of DCGs formed in CgA antisense transgenic mice was directly correlated with the amount of CgA present in adrenal medulla. In addition, DCGs showed an increase in size, with enlargement in the volume around the dense core, a phenomenon that occurs to maintain constant "free" catecholamine concentration in the lumen of these granules. The extent of DCG swelling was inversely correlated with the number of DCGs formed, as well as the amount of CgA present in the adrenal glands of CgA antisense transgenic mice. These data indicate an essential role of CgA in regulating chromaffin DCG biogenesis and catecholamine storage in vivo.

The effects of the antihypertensive agent hydralazine (1 to 100 nmol/L) on the exocytotic process of single adrenal chromaffin cells have been studied using amperometry. Hydralazine does not reduce the frequency of exocytotic spikes but rapidly slows the rate of catecholamine release from individual exocytotic events by reducing the quantal size of catecholamine exocytosis. Confocal and standard epifluorescence microscopy studies show that hydralazine rapidly accumulates within secretory vesicles. The blockade of the vesicular H+ pump with bafilomycin A1 inhibits hydralazine uptake. Experiments with permeabilized cells show that hydralazine displaces catecholamines from secretory vesicles. The drug also displaces vesicular Ca2+, as shown by fura-2 microfluorimetry. These data suggest that hydralazine acts, at least partially, by interfering with the storage of catecholamines. These effects of hydralazine occurred within seconds, and at the tissue concentrations presumably reached in antihypertensive therapy; these concentrations are a thousand times lower than those described for relaxing vascular tissues in vitro. We proposed that these novel effects could explain many of the therapeutic and side effects of this drug that are likely exerted in sympathetic nerve terminals.

Tyrosine hydroxylase (TH) demonstrates by two-dimensional electrophoresis a microheterogeneity both as a soluble recombinant human TH (hTH1) and as a membrane-bound bovine TH (bTHmem). Part of the heterogeneity is likely due to deamidation of labile asparagine residues. Wild-type (wt)-hTH1 was found to be a substrate for the ubiquitin (Ub) conjugating enzyme system in a reconstituted in vitro system. When wt-hTH1 was expressed in a coupled transcription-translation TnT(R)-T7 reticulolysate system 35S-labelled polypeptides of the expected molecular mass of native enzyme as well as both higher and lower molecular mass forms were observed. The amount of high-molecular-mass forms increased by time and was enhanced in the presence of Ub and clasto-lactacystin beta-lactone. In pulse-chase experiments the amount of full-length hTH1 decreased by first-order kinetics with a half-time of 7.4 h and 2.1 h in the absence and presence of an ATP-regenerating system, respectively. The ATP-dependent degradation was inhibited by clasto-lactacystin beta-lactone. Our findings support the conclusion that hTH1 is ubiquitinated and at least partially degraded by the proteasomes in the reticulocyte lysate system. Finally, it is shown that the integral TH of the bovine adrenal chromaffin granule membrane (bTHmem) is ubiquitinated, most likely monoubiquitinated. Additional Ub-conjugates of this membrane, detected by Western blot analysis, have not yet been identified.

In the present study the influx of 86Rb+, a K+ analogue, was studied in mitochondria, microsomes and chromaffin granules prepared from adrenal gland medulla. The most active electrogenic 86Rb+ transport was found in the membrane fraction identified as chromaffin granules by marker enzyme estimation. The transport was found to be sensitive to ATP, ATP gamma S, ADP and to the triazine dyes, but not to AMP and cAMP. The inhibition induced by ATP was observed in the absence of externally added Mg2+, suggesting that a free nucleotide, rather than the ATP-Mg complex, was required for inhibition. Furthermore, the 86Rb+ influx was found to be inhibited by Mg2+ alone, but not by Ca2+ and antidiabetic sulfonylureas. The 86Rb+ influx was not stimulated by potassium channel openers. In conclusion, our results indicate that an electrogenic, ATP-sensitive potassium transport system operates in the chromaffin granule membrane.

Chromaffin granule transmitters such as chromogranin A and catecholamines have been used in the diagnosis of pheochromocytoma, but the diagnostic and prognostic value of chromogranin A have not been explored in malignant pheochromocytoma. We evaluated these transmitters in patients with pheochromocytoma (n=27), both benign (n=13) and malignant (n=14). Patients with benign pheochromocytoma were studied before and after surgical excision (n=6), whereas patients with malignant pheochromocytoma were evaluated before and after combination chemotherapy with regular cycles of cyclophosphamide/dacarbazine/vincristine (nonrandomized trial in n=9). During treatment, patient responses to chemotherapy were divided according to anatomic and clinical criteria: responders (n=5) versus nonresponders (n=4). Plasma chromogranin A rose progressively (P<0.0001) from control subjects (48.0+/-3.0 ng/mL) to benign pheochromocytoma (188+/-40.5 ng/mL) to malignant pheochromocytoma (2932+/-960 ng/mL). Parallel changes were seen for plasma norepinephrine (P<0.0001), though plasma epinephrine was actually lower in malignant than benign pheochromocytoma (P=0.0182). In bivariate analyses, chromogranin A, norepinephrine, and epinephrine discriminated between pheochromocytoma and control subjects (all P<0.0001), whereas in a multivariate analyses, norepinephrine was the best discriminator (P:=0.011). Chromogranin A was significantly different in benign versus malignant pheochromocytoma on both bivariate (P=0.0003) and multivariate (P:=0.011) analyses. After excision of benign pheochromocytoma, chromogranin A (P=0.028), norepinephrine (P=0.047), and epinephrine (P=0.037) all fell to values near normal. During chemotherapy of malignant pheochromocytoma (n=9), plasma chromogranin A (P=0.047) and norepinephrine (P=0.02) fell but not epinephrine. In 5 responders to chemotherapy, there were significant declines in chromogranin A (P=0.03) and norepinephrine (P=0.03) but not epinephrine; in 4 nonresponders, none of the transmitters changed. Plasma chromogranin A varied longitudinally with tumor response and relapse. We conclude that plasma chromogranin A is an effective tool in the diagnosis of pheochromocytoma, and markedly elevated chromogranin A may point to malignant pheochromocytoma. During chemotherapy of malignant pheochromocytoma, chromogranin A can be used to gauge tumor response and relapse.

The effects of nitric oxide (NO) on the late phase of exocytosis have been studied, by amperometry, on Ba(2+)-stimulated chromaffin cells. Acute incubation with NO or NO donors (sodium nitroprusside, spermine-NO, S-nitrosoglutathione) produced a drastic slowdown of the granule emptying. Conversely, cell treatment with N(omega)-nitro-l-arginine methyl ester (a NO synthase inhibitor) or with NO scavengers (methylene blue, 2-(4-carboxyphenyl)-4,4,5, 5-tetramethyl-imidazoline-1-oxyl-3-oxide potassium) accelerated the extrusion of catecholamines from chromaffin granules, suggesting the presence of a NO modulatory tone. The incubation with phosphodiesterase inhibitors (3-isobutyl-1-methylxanthine or zaprinast) or with the cell-permeant cGMP analog 8-bromo-cGMP, mimicked the effects of NO, suggesting the involvement of the guanylate cyclase cascade. NO effects were not related to changes in intracellular Ba(2+). NO did not modify the duration of feet. Effects were evident even on pre-fusioned granules, observed under hypertonic conditions, suggesting that the fusion pore is not the target for NO, which probably acts by modifying the affinity of catecholamines for the intragranular matrix. NO could modify the synaptic transmitter efficacy through a novel mechanism, which involves the regulation of the emptying of secretory vesicles.

Serpins represent a diverse class of endogenous protease inhibitors that regulate important biological functions. In consideration of the importance of regulated proteolysis within secretory vesicles for the production of peptide hormones and neurotransmitters, this study revealed the molecular identity of a novel serpin, endopin 1, that is localized to neurosecretory vesicles of neuropeptide-containing chromaffin cells (chromaffin granules). Endopin 1 of 68-70 kDa was present within isolated chromaffin granules. Stimulated cosecretion of endopin 1 with chromaffin granule components, [Met]enkephalin and a cysteine protease known as "prohormone thiol protease," demonstrated localization of endopin 1 to functional secretory vesicles. Punctate, discrete immunofluorescence cellular localization of endopin 1 in chromaffin cells was consistent with its secretory vesicle localization. Endopin 1 contains a unique reactive site loop with Arg as the predicted P1 residue, suggesting inhibition of basic residue-cleaving proteases; indeed, trypsin was potently inhibited (K(i(app)) of 5 nM), and plasmin was moderately inhibited. Although endopin 1 possesses homology with alpha(1)-antichymotrypsin, chymotrypsin was not inhibited. Moreover, endopin 1 inhibited the chromaffin granule prohormone thiol protease (involved in proenkephalin processing). These results suggest a role for the novel serpin, endopin 1, in regulating basic residue-cleaving proteases within neurosecretory vesicles of chromaffin cells.

The target-SNARE syntaxin 1A is an essential component of the core machinery required for regulated exocytosis (where SNARE is the soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptor). Syntaxin 1A interacts with a variety of other proteins, two of which, N-ethylmaleimide-sensitive fusion protein (NSF) and alpha-soluble NSF attachment protein (alpha-SNAP) have been suggested to impart a conformational rearrangement on this protein during a reaction referred to as priming. We have studied the effect of the primed state on the binding properties of syntaxin 1A and we have confirmed that primed syntaxin 1A no longer associated with alpha-SNAP or its cognate vesicle-SNARE, vesicle-associated membrane protein (VAMP). Under such conditions, however, it retained the ability to bind to nSec-1. It has been demonstrated that nSec-1, a regulatory protein also involved in neuronal exocytosis, binds syntaxin 1A with high affinity in vitro, although evidence for this physical interaction occurring in vivo has proven elusive. We analysed the subcellular distribution of these two proteins in fractions from bovine adrenal medulla and detected syntaxin 1A and nSec-1 in both plasma membrane and chromaffin-granule fractions. Using a cross-linking approach with chromaffin-granule membranes we detected a putative dimeric complex composed of approx. 54% total granule membrane nSec-1 and approx. 30% total syntaxin 1A. The results of this study therefore suggest the possibility of nSec-1 interactions with primed syntaxin 1A and demonstrate a potentially significant interaction of syntaxin 1A and nSec-1 on the membranes of chromaffin granules.

Unlabelled: Because nothing is known about whether metaiodobenzylguanidine (MIBG) has tyramine-like actions, the sympathomimetic effects of MIBG were determined in the isolated rabbit heart and compared with those of tyramine., Methods: Spontaneously beating rabbit hearts were perfused with Tyrode's solution (Langendorff technique; 37 degrees C; 26 mL/min), and the heart rate as well as the norepinephrine and dopamine overflow into the perfusate was measured before and after doses of MIBG or tyramine (0.03-10 micromol) given as bolus injections (100 microL) into the aortic cannula. Km and Vmax values for the neuronal uptake (uptake1) of 125I-MIBG and 14C-tyramine were obtained in human neuroblastoma (SK-N-SH) cells. The Ki of MIBG for inhibition of the 3H-catecholamine uptake mediated by the vesicular monoamine transporter was determined in membrane vesicles obtained from bovine chromaffin granules and compared with the previously reported Ki value for tyramine determined under identical experimental conditions., Results: By producing increases in heart rate and norepinephrine overflow, both compounds had dose-dependent sympathomimetic effects in the rabbit heart. MIBG was much less effective than tyramine in increasing heart rate (maximum effect 59 versus 156 beats/min) and norepinephrine overflow (maximum effect 35 versus 218 pmol/g). Tyramine also caused increases in dopamine overflow, whereas MIBG was a poor dopamine releaser. At a dose of 10 micromol, the increase in heart rate lasted more than 60 min after MIBG and about 20 min after tyramine injection. Accordingly, the norepinephrine overflow caused by 10 micromol MIBG and tyramine declined with half-lives of 57.8 and 2.2 min, respectively. The effects of both drugs were drastically reduced in hearts exposed to 2 micromol/L desipramine. The kinetic parameters characterizing the saturation of neuronal uptake by 125I-MIBG and 14C-tyramine were similar for the two compounds: Km values of MIBG and tyramine were 1.6 and 1.7 micromol/L, respectively, and Vmax values of MIBG and tyramine were 43 and 37 pmol/mg protein/min, respectively. However, in inhibiting the vesicular 3H-catecholamine uptake, MIBG was eight times less potent than tyramine., Conclusion: MIBG is much less effective than tyramine as an indirect sympathomimetic agent. This is probably a result of its relatively low affinity for the vesicular monoamine transporter and explains the relatively poor ability of the drug to mobilize norepinephrine stored in synaptic vesicles. The long duration of MIBG action results primarily from the drug not being metabolized by monoamine oxidase. The sympathomimetic effects of MIBG described here are not likely to come into play in patients given diagnostic or common therapeutic doses of radioiodinated MIBG.

NSF and alpha-SNAP have been shown to be required for SNARE complex disassembly and exocytosis. However, the exact requirement for NSF and alpha-SNAP in vesicular traffic through the secretory pathway remains controversial. We performed a study on the kinetics of exocytosis from bovine chromaffin cells using high time resolution capacitance measurement and electrochemical amperometry, combined with flash photolysis of caged Ca2+ as a fast stimulus. alpha-SNAP, a C-terminal mutant of alpha-SNAP, and NEM were assayed for their effects on secretion kinetics. Two kinetically distinct components of catecholamine release can be observed upon fast step-like elevation of [Ca2+]i. One is the exocytotic burst, thought to represent the readily releasable pool of vesicles. Following the exocytotic burst, secretion proceeds slowly at maintained high [Ca2+]i, which may represent vesicle maturation/recruitment, i.e. some priming steps after docking. alpha-SNAP increased the amplitude of both the exocytotic burst and the slow component but did not change their kinetics, which we examined with millisecond time resolution. In addition, NEM only partially inhibited the slow component without altering the exocytotic burst, fusion kinetics and the rate of endocytosis. These results suggest a role for alpha-SNAP/NSF in priming granules for release at an early step, but not modifying the fusion of readily releasable granules.

The vacuolar proton pump of clathrin-coated vesicles is composed of two general sectors, a cytosolic, ATP hydrolytic domain (V1) and an intramembranous proton channel, V0. V1 is comprised of 8-9 subunits including polypeptides of 50 and 57 kDa, termed SFD (Sub Fifty-eight-kDa Doublet). Although SFD is essential to the activation of ATPase and proton pumping activities catalyzed by holoenzyme, its constituent polypeptides have not been separated to determine their respective roles in ATPase functions. Recent molecular characterization of these subunits revealed that they are isoforms that arise through an alternative splicing mechanism (Zhou, Z., Peng, S.-B., Crider, B.P., Slaughter, C., Xie, X.S., and Stone, D.K. (1998) J. Biol. Chem. 273, 5878-5884). To determine the functional characteristics of the 57-kDa (SFDalpha)1 and 50-kDa (SFDbeta) isoforms, we expressed these proteins in Escherichia coli. We determined that purified recombinant proteins, rSFDalpha and rSFDbeta, when reassembled with SFD-depleted holoenzyme, are functionally interchangeable in restoration of ATPase and proton pumping activities. In addition, we determined that the V-pump of chromaffin granules has only the SFDalpha isoform in its native state and that rSFDalpha and rSFDbeta are equally effective in restoring ATPase and proton pumping activities to SFD-depleted enzyme. Finally, we found that SFDalpha and SFDbeta structurally interact not only with V1, but also withV0, indicating that these activator subunits may play both structural and functional roles in coupling ATP hydrolysis to proton flow.

Rab3a, a small GTP-binding protein, is believed to mediate Ca2+-dependent exocytosis. Consistent with such a role was the previously reported specific association of Rab3a with synaptic vesicles in neurons and secretory granules in adrenal chromaffin cells. Secretory vesicles are believed to be the final point of Rab3a membrane association, as it was shown by several groups that Rab3a dissociates from the secretory vesicle membrane during stimulated exocytosis. In chromaffin cells, Rab3a is not exclusively localized on secretory granules since a fraction is present on a previously unidentified subcellular compartment equilibrating at light sucrose density. This 'light' membraneous structure could be the starting point for reassociation of Rab3a with membranes involved in granule formation, or it could be a structure unrelated to granules. The present study used several subcellular fractionation techniques and immunomicroscopy to unravel the nature of the 'light' Rab3a-containing structures from bovine chromaffin cells in primary culture. After stimulation, amounts of both Rab3a-d and the granule marker dopamine-beta-hydroxylase (DbetaH) increase transiently in sucrose gradient fractions enriched in endosomal markers. A diaminobenzidine-induced density shift of endosomes alters the distribution of DbetaH and Rab3a-d. At the ultrastructural level, subplasmalemmal pleiomorphic organelles were detected by Rab3a-d-immunogold labelling. Taken together our data provide for the first time evidence that internalised secretory granule membranes go through an endosomal stage where Rab3a is present, resembling the neuronal synaptic vesicle cycle. This indicates that the endosome is an important trafficking route in the biogenesis/recycling of secretory vesicles in chromaffin cells, in which Rab3a could have an as yet unknown regulatory function, and could point to the existence of alternative recycling pathways for the chromaffin granule membrane.

Carboxypeptidase E (CPE) is a prohormone-processing enzyme and peripheral membrane protein of endocrine/neuroendocrine secretory granules. CPE has been shown to bind to an amino-terminal peptide of pro-opiomelanocortin (N-POMC) at pH 5.5 and hypothesized to be critically involved in the targeting of hormones such as POMC to the regulated secretory pathway [Cool, D. R., Normant, E., Shen, F., Chen, H. C., Pannell, L., Zhang, Y., and Loh, Y. P. (1997) Cell 88, 73-83]. To further explore the possibility that CPE serves to mediate the association of content proteins with the membrane during granule biogenesis, the binding of CPE to granule content proteins was investigated using an in vitro aggregation assay in which the selective precipitation of granule content proteins is induced by titration of the pH to <6.0. CPE was observed to co-aggregate efficiently with pituitary and chromaffin granule content proteins at concentrations well below those that promote its self-aggregation. In addition, CPE co-precipitated at pH 5.8 with purified prolactin and with insulin, which homophillically self-aggregate yet are structurally distinct from N-POMC. N-POMC when added to the assays did not inhibit the aggregation of CPE with prolactin or insulin, indicating that these interactions do not involve a binding site for N-POMC. The data show that CPE interacts at acidic pH with a variety of different content proteins, resembling in this regard other granule membrane proteins. The results support the idea that co-aggregation of abundant membrane proteins with content proteins is an important general mechanism for the sorting and retention of secretory granule proteins during granule maturation.

Proenkephalin-A (PEA) and its derived peptides (PEAP) have been described in neural, neuroendocrine tissues and immune cells. The processing of PEA has been extensively studied in the adrenal medulla chromaffin cell showing that maturation starts with the removal of the carboxyl-terminal PEAP209-239. In 1995, our laboratory has shown that antibacterial activity is present within the intragranular chromaffin granule matrix and in the extracellular medium following exocytosis. More recently, we have identified an intragranular peptide, named enkelytin, corresponding to the bisphosphorylated PEAP209-237, that inhibits the growth of Micrococcus luteus (Goumon, Y., Strub, J. M., Moniatte, M., Nullans, G., Poteur, L., Hubert, P., Van Dorsselaer, A., Aunis, D., and Metz-Boutigue, M. H. (1996) Eur. J. Biochem. 235, 516-525). As a continuation of this previous study, in order to characterize the biological function of antibacterial PEAP, we have here examined whether this COOH-terminal fragment is released from stimulated chromaffin cells and whether it could be detected in wound fluids and in polymorphonuclear secretions following cell stimulation. The antibacterial spectrum shows that enkelytin is active against several Gram-positive bacteria including Staphylococcus aureus, but it is unable to inhibit the Gram-negative bacteria growth. In order to relate the antibacterial activity of enkelytin with structural features, various synthetic enkelytin-derived peptides were tested. We also propose a computer model of synthetic PEAP209-237 deduced from 1H NMR analysis, in order to relate the antibacterial activity of enkelytin with the three-dimensional structure. Finally, we report the high phylogenetic conservation of the COOH-terminal PEAP, which implies some important biological function and we discuss the putative importance of enkelytin in the defensive processes.

In mast cells and granulocytes, exocytosis starts with the formation of a fusion pore. It has been suggested that neurotransmitters may be released through such a narrow pore without full fusion. However, owing to the small size of the secretory vesicles containing neurotransmitter, the properties of the fusion pore formed during Ca2+-dependent exocytosis and its role in transmitter release are still unknown. Here we investigate exocytosis of individual chromaffin granules by using cell-attached capacitance measurements combined with electrochemical detection of catecholamines, achieved by inserting a carbon-fibre electrode into the patch pipette. This allows the simultaneous determination of the opening of individual fusion pores and of the kinetics of catecholamine release from the same vesicle. We found that the fusion-pore diameter stays at <3 nm for a variable period, which can last for several seconds, before it expands. Transmitter is released much faster through this pore than in mast cells, generating a 'foot' signals which precedes the amperometric spike. Occasionally, the narrow pore forms only transiently and does not expand, allowing complete transmitter release without full fusion of the vesicle with the plasma membrane.

The annexins are characterized by their ability to bind phospholipid membranes in a Ca2+-dependent manner. Sequence variability between the N-terminal domains of the family members may contribute to the specific cellular function of each annexin. To identify proteins that interact with the N-terminal domain of synexin (annexin VII), a fusion protein was constructed composed of glutathione S-transferase fused to amino acids 1-145 of human synexin. Affinity chromatography using this construct identified sorcin as a Ca2+-dependent synexin-binding protein. Overlay assays confirmed the interaction. The glutathione S-transferase construct associates with recombinant sorcin over the range of pCa2+ = 4.7-3.1 with no binding observed at pCa2+ = 5.4. Overlay assays using deletion constructs of the synexin N-terminal domain mapped the sorcin binding site to the N-terminal 31 amino acids of the synexin protein. Additionally, synexin forms a complex with sorcin and recruits this protein to chromaffin granule membranes in a Ca2+-dependent manner. Sorcin is able to inhibit synexin-mediated chromaffin granule aggregation in a manner saturable with increasing sorcin concentrations, but does not influence the Ca2+ sensitivity of synexin-mediated granule aggregation. Therefore, the interaction between sorcin and synexin may serve to regulate the functions of these proteins on membrane surfaces in a Ca2+-dependent manner.

alpha-Latrotoxin is a potent stimulator of neurosecretion. Its action requires extracellular binding to high affinity presynaptic receptors. Neurexin I alpha was previously described as a high affinity alpha-latrotoxin receptor that binds the toxin only in the presence of calcium ions. Therefore, the interaction of alpha-latrotoxin with neurexin I alpha cannot explain how alpha-latrotoxin stimulates neurotransmitter release in the absence of calcium. We describe molecular cloning and functional expression of the calcium-independent receptor of alpha-latrotoxin (CIRL), which is a second high affinity alpha-latrotoxin receptor that may be the major mediator of alpha-latrotoxin's effects. CIRL appears to be a novel orphan G-protein-coupled receptor, a member of the secretin receptor family. In contrast with other known serpentine receptors, CIRL has two subunits of the 120 and 85 kDa that are the result of endogenous proteolytic cleavage of a precursor polypeptide. CIRL is found in brain where it is enriched in the striatum and cortex. Expression of CIRL in chromaffin cells increases the sensitivity of the cells to the effects of alpha-latrotoxin, demonstrating that this protein is functional in coupling to secretion. Syntaxin, a component of the fusion complex, copurifies with CIRL on an alpha-latrotoxin affinity column and forms stable complexes with this receptor in vitro. Interaction of CIRL with a specific presynaptic neurotoxin and with a component of the docking-fusion machinery suggests its role in regulation of neurosecretion.

Bovine adrenal medullary chromogranin A, the major soluble component of chromaffin granules, is a phosphorylated glycoprotein. In the present work, phosphorylation and glycosylation sites were determined using mild proteolysis, peptide separation, microsequencing, and mass analysis by electrospray and matrix-assisted laser desorption ionization time-of-flight techniques. Seven post-translational modification sites were detected. Two O-linked glycosylation sites, each consisting of the trisaccharide NeuAcalpha2-3Galbeta1-3GalNAcalpha1, were located in the middle part of the protein, on Ser186 and on Thr231. The former residue is present in the antibacterial peptide named chromacin. Four phosphorylation sites were located on serine residues at positions Ser81 in the N-terminal region of the protein and Ser307, Ser372, and Ser376 in the C-terminal end. One additional phosphorylation site was found on the tyrosine residue at position Tyr173, the N-terminal amino acid of chromacin. With the exception of the phosphorylation on Tyr173, all of the other post-translational modifications are located on highly conserved chromogranin A regions, implying some biological importance.

The speed at which secretory vesicles fuse with the plasma membrane is a key parameter for neuronal and endocrine functions. We determined the precise time courses for fusion of small clear and large dense-core vesicles in PC12 and chromaffin cells by simultaneously measuring both plasma membrane areas and release of vesicular contents. We found that instantaneous increases in cytosolic Ca2+ concentration evoked vesicle fusion, but with time constants that varied over four orders of magnitude among different types of vesicles and cells. This indicates that the molecular machinery for the final Ca2+-dependent fusion steps of exocytosis is highly variable and is as critical as Ca2+ signalling processes in determining the speed and amount of secretion of neurotransmitters and hormones. Our results suggest a new possibility that the molecules responsible for the final fusion reaction that leads to vesicle fusion are key determinants for neuronal plasticity and hormonal disorders.

Effects of indomethacin on catecholamine secretion evoked by receptor agonists, muscarine, bradykinin or histamine, in rat adrenal chromaffin cells were studied. Indomethacin at 200 microM increased a sustained component of secretion during stimulation with muscarine, bradykinin and histamine by a factor of 2.3, 2.1 and 2.9, respectively, whereas it did not significantly alter basal, high-K(+)- and nicotine-evoked secretions. Although indomethacin at above 400 microM dose-dependently increased basal secretion, the amount of secretion induced by indomethacin alone was much smaller than that in muscarine-evoked secretion as compared at the same concentration of indomethacin applied. Bradykinin-evoked secretion and its potentiation by indomethacin were not inhibited by 20 microM nifedipine but were suppressed by 0.5 mM Ni2+. The cyclooxygenase inhibitor, ibuprofen (200 microM) did not mimic the effect of indomethacin; prostaglandin E2 (20 microM) and arachidonic acid (100 microM) did not significantly alter either bradykinin-evoked secretion itself or its potentiation by indomethacin. Bradykinin increased the intracellular free Ca2+ concentration, [Ca2+]i, in cells loaded with indo-1, and this response was enhanced in the presence of indomethacin. These results suggest that indomethacin may promote Ca2+ entry to potentiate agonist-evoked catecholamine secretions through a novel action that is not directly related to the inhibition of cyclooxygenase activity with indomethacin.

The ADP-ribosylation factor (ARF) GTP-binding proteins are believed to function as regulators of vesicular budding and fusion along the secretory pathway. To investigate the role of ARF in regulated exocytosis, we have examined its intracellular distribution in cultured chromaffin cells by subcellular fractionation and immunoreplica analysis. We found that ARF6 is specifically associated with the membrane of purified secretory chromaffin granules. Chemical cross-linking and immunoprecipitation experiments suggested that ARF6 may be part of a complex with betagamma subunits of trimeric G proteins. Stimulation of intact chromaffin cells or direct elevation of cytosolic calcium in permeabilized cells triggered the rapid dissociation of ARF6 from secretory granules. This effect could be inhibited by AlF4- which selectively activates trimeric G proteins. Furthermore, a synthetic myristoylated peptide corresponding to the N-terminal domain of ARF6 strongly inhibited calcium-evoked secretion in streptolysin-O-permeabilized chromaffin cells. The possibility that ARF6 plays a role in the effector pathway by which trimeric G proteins control exocytosis in chromaffin cells is discussed.

Tissue-type plasminogen activator (t-PA) is a serine protease that plays a central role in the regulation of intravascular thrombolysis. The acute release of t-PA in vivo is induced by a variety of stimuli including exercise, trauma, and neural stimulation. These types of stimuli also result in sympathoadrenal activation and exocytotic release of amines and proteins from catecholamine storage vesicles of the adrenal medulla and sympathetic neurons. Therefore, we tested the hypothesis that t-PA is packaged in and released directly from catecholamine storage vesicles, using several chromaffin cell sources including the rat pheochromocytoma PC-12 chromaffin cell line, primary cultures of bovine adrenal chromaffin cells, and human pheochromocytoma. t-PA was expressed in chromaffin cells as detected by Northern blotting, immunoprecipitation of [35S]Met-labeled t-PA, and specific t-PA enzyme-linked immunosorbent assay of cell homogenates. In addition, chromaffin cell t-PA was enzymatically active by fibrin zymography. To explore the subcellular localization of the expressed t-PA, PC-12 cells were labeled with [3H]norepinephrine, homogenized, and subjected to sucrose density fractionation. [3H]Norepinephrine and t-PA antigen were co-localized to the same subcellular fraction with a major peak at 1.4 M sucrose, consistent with the buoyant density of catecholamine storage vesicles. In addition, catecholamine storage vesicle lysates isolated from human pheochromocytoma tumors were enriched approximately 30-fold in t-PA antigen, compared with tumor homogenate. Furthermore, exposure of PC-12 cells or primary bovine adrenal chromaffin cells to chromaffin cell secretagogues (60 microM nicotine, 55 mM KCl, or 2 mM BaCl2) resulted in co-release of t-PA in parallel with catecholamines. These data demonstrate that t-PA is expressed in chromaffin cells, is sorted into the regulated pathway of secretion, and is co-released with catecholamines by chromaffin cell stimulation. Catecholamine storage vesicles may be an important reservoir and sympathoadrenal activation an important physiologic mechanism for the rapid release of t-PA. In addition, expression of t-PA by chromaffin cells suggests a role for this protease in the proteolytic processing of chromaffin cell proteins.

Exocytotic membrane fusion and secretion are promoted by the concerted action of GTP and Ca2+, although the precise site(s) of action in the process are not presently known. However, the calcium-dependent membrane fusion reaction driven by synexin (annexin VII) is an in vitro model for this process, which we have now found to be further activated by GTP. The mechanism of fusion activation depends on the unique ability of synexin to bind and hydrolyze GTP in a calcium-dependent manner, both in vitro and in vivo in streptolysin O-permeabilized chromaffin cells. The required [Ca2+] for GTP binding by synexin is in the range of 50-200 microM, which is known to occur at exocytotic sites in chromaffin cells, neurons, and other cell types. Previous immunolocalization studies place synexin at exocytotic sites in chromaffin cells, and we conclude that synexin is an atypical G protein that may be responsible for both detecting and mediating the Ca2+/GTP signal for exocytotic membrane fusion.

SNAP-25 is located on the plasma membrane and essential for exocytosis of neurotransmitters. It was suggested that SNAP-25 and syntaxin 1 via the interaction with VAMP-2 located on synaptic vesicles mediate the docking of the vesicles with the plasma membrane. In the present study, by means of biochemical and morphological analyses, we showed that SNAP-25 is present on chromaffin granules as well as on the plasma membrane. Reconstitution and immunoprecipitation analyses revealed that SNAP-25 on chromaffin granules has essentially the same properties as does SNAP-25 on the plasma membrane.

The occurrence of high concentrations of ATP in certain cell organelles prompted us to study the self-association of ATP via the concentration dependence of the 1H-NMR chemical shifts for H2, H8 and H1' in D2O at pD 8.4 (25 degrees C) in the range 0.0025-0.4 M in the presence and absence of poly(alpha, L-lysine), where [Lys units] was 0.4 M. The experiment in the presence of poly(Lys) was repeated at pD 12.1. At pD 8.4, the poly(amino acid) is protonated, i.e. poly(H.Lys)n+, whereas at pD 12.1 only approximately 10% of the epsilon-amino groups are positively charged. The results in all three systems are consistent with the isodesmic model of indefinite non-cooperative stacking. The stacking tendency follows the series: ATP4- (K = 1.3 M-1; pD 8.4) < ATP4-/poly(H.Lys)n+ (K = 11.5 M-1; pD 8.4) > ATP4-/90% poly(Lys)/10% poly(H.Lys)n+ (K = 3.1 M-1; pD 12.1). It is evident that poly(H.Lys)n+ assists the association of ATP by a factor of approximately 10, and it is suggested that, via its positively charged epsilon-ammonium groups, poly(H.Lys)n+ acts as a matrix by aligning ATP4- ions via ionic interactions with the negatively charged phosphate residues. The intragranular concentrations of various constituents of several storage or secretory cell organelles, as reported in the literature, are tabulated. The chromaffin granules of the adrenal medulla and the dense granules of blood platelets contain particularly high concentrations of nucleotides ([ATP] is approximately 0.14 M in the chromaffin granules and 0.5 M in the dense granules of rabbit blood platelets) and amines, such as epinephrine or 5-hydroxytryptamine. These granules, and probably also the storage vesicles of certain neurons (which seem to have a similar composition), appear, if the total concentrations of the various solutes are considered, to be osmotically unstable, which means that the intragranular solutes must be associated. This aggregation is discussed, especially with regard to the nucleotides.

In this work, we have examined the subcellular distribution of cysteine string proteins (Csps) in bovine adrenal medullary chromaffin cells. Csps did not leak from digitonin-permeabilized chromaffin cells, suggesting that there is no cytosolic pool of the protein in these cells. Subcellular fractionation studies confirmed that there was essentially no Csp immunoreactivity in the cytosolic fraction. However, immunoreactivity was detected in the membrane fractions of these cells. Csp immunoreactivity codistributed with dopamine beta-hydroxylase, a granule marker protein, in sucrose gradient-separated granule fractions. Immunofluorescence studies showed that all chromaffin cells in culture were stained with a punctate appearance consistent with a granular localization. These results were confirmed by immunogold labeling, which demonstrated specific labeling of chromaffin granule membranes. In addition to its presence on synaptic vesicles, cysteine string protein is therefore a bona fide chromaffin granule membrane protein.

Adenosine 5'-tetraphosphate (Ap4) is a natural constituent of chromaffin granules with concentration values of 2.2 +/- 0.1 nmol/mg of protein and a ratio 245 +/- 40 times lower with respect to ATP (n = 4). The granular transport of epsilon-ATP resulted in a time- and concentration-dependent production of epsilon-adenosine tetraphosphate (epsilon-Ap4) at the intragranular level. The epsilon-Ap4 formation followed a hyperbolic saturation kinetic at low epsilon-ATP concentrations with K(m) value of 0.4 microM epsilon-ATP intragranular (1.15 pmol/mg of granular protein). Intragranular concentrations of epsilon-ATP higher than 500 pmol/mg of protein (approximately to 175 microM intragranular) resulted in a non-saturable production of epsilon-Ap4.

ATP is an excitatory neurotransmitter that is stored and cosecreted with catecholamines from cells of the adrenal medulla. While the transport of catecholamines into chromaffin granule ghosts has been extensively characterized, there is little information on the mechanism of ATP transport into these structures. Here we show that ATP transport is driven by the electrical component of the electrochemical proton gradient created by the chromaffin granule membrane H+-ATPase, and that the accumulated nucleotide is released from the vesicles by inhibition of the H+-ATPase. GTP and UTP are also substrates for this transporter, distinguishing it from the mitochondrial ADP/ATP exchanger. Accumulation of ADP and ATP (rather than exchange with intravesicular ATP) is demonstrated by high pressure liquid chromatography measurements. The anion transport inhibitor 4,4-diisothiocyanatostilbene-2,2-disulfonic acid (Ki = 27 microM) inhibits ATP transport, while atractyloside, the inhibitor of the mitochondrial ATP/ADP exchanger, is a very poor inhibitor. Finally, we have demonstrated a synergy between the accumulation of ATP and that of serotonin (i.e. more of each solute accumulates when the two are accumulated together), supporting the view that there is an interaction between serotonin and ATP that reduces their effective concentration within the ghosts.

The two major proteins of the secretory vesicles of neuroendocrine cells, chromogranin A (CGA) and chromogranin B (CGB), have been shown to undergo pH- and Ca2+-dependent conformational changes and aggregation and have been suggested to play essential roles during secretory vesicle biogenesis in the trans-Golgi network. CGA has been shown to exist primarily in a tetrameric state at pH 5.5 and primarily in a dimeric state at pH 7.5, and CGB has been shown to exist in a monomeric state at both pH 5.5 and pH 7.5. Using purified CGA and CGB, it recently has been shown that CGA interacts with CGB at pH 5.5 (Yoo, S. H.(1996) J. Biol. Chem. 271, 1558-1565). In expanding this investigation, we have studied the temperature dependence of the pH-dependent interaction of CGA and CGB by analytical ultracentrifugation and found that two molecules of CGA bound to two molecules of CGB at pH 5.5 with DeltaG0 values of -43.6 kcal/mol in the absence of Ca2+ at 37 degrees C and -40.3 kcal/mol in the presence of 0.1 mM Ca2+. However, one molecule of CGA bound to one molecule of CGB at pH 7.5 with DeltaG0 values of -13.6 kcal/mol in the absence of Ca2+ at 37 degrees C. The magnitude of DeltaG0 values increased with increasing temperatures at both pH values. However, the values for enthalpy and entropy changes decreased with increasing temperatures in both pH levels, suggesting formation of more ordered structures. In the absence of Ca2+ at pH 5. 5, the heterotetramerization reaction at 37 degrees C was entropically driven, whereas in the presence of Ca2+ (0.1 mM) the heterotetramerization was virtually an enthalpic reaction. On the other hand, the heterodimer formation in the absence of Ca2+ at pH 7. 5 showed large negative enthalpy and entropy changes at 37 degrees C, indicating an enthalpic interaction compensated by entropic changes. In view of the interaction of tetrameric CGA with tetrameric inositol 1,4,5-trisphosphate (IP3) receptor and the existence of heterotetrameric IP3 receptor in the cell, the heterotetramer formation by CGA and CGB not only raises the possibility of interaction between the heterotetrameric chromogranin and heterotetrameric IP3 receptor but also appears to reflect their important roles in the cell.

The vesicular monoamine transporter, which catalyses a H+/ monoamine antiport in monoaminergic vesicle membrane, is a very hydrophobic intrinsic membrane protein. After solubilization, this protein was found to have a high tendency to aggregate, as shown by SDS/PAGE, especially when samples were boiled in the classical Laemmli buffer before electrophoresis. This behavior was analysed in some detail. The aggregation was promoted by high temperatures, organic solvents and acidic pH, suggesting that it resulted from the unfolding of structure remaining in SDS. The aggregates were very stable and could be dissociated only by suspension in anhydrous trifluoroacetic acid. This SDS-resistant aggregation behaviour was shared by very few intrinsic proteins of the chromaffin granule membrane. Consequently, a purification procedure was based on this property. A detergent extract of chromaffin granule membranes enriched in monoamine transporter was heated and the aggregates were isolated by size-exclusion HPLC in SDS. The aggregates, containing the transporter, were dissociated in the presence of trifluoroacetic acid and analysed on the same HPLC column. This strategy might be of general interest for the purification of membrane proteins that exhibit SDS-resistant aggregation.