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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.

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.

This study examines chromaffin cell expression of the synaptosomal-associated protein SNAP-25 in the adrenal medulla by immunoblotting, immunocytochemistry and PCR. Both mRNAs coding for the SNAP-25 isoforms a and b were detected and SNAP-25 was found to be present in all chromaffin cells in adult rat adrenal gland sections. It was essentially restricted to a zone close to the cytoplasmic face of the plasma membrane in the majority of cells, but located extensively throughout the cytoplasm in a chromaffin cell sub-population, identified by double immunofluorescence labelling to have a noradrenergic phenotype. This differential SNAP-25 expression may reflect different stages in the phenotypic development of the sympathoadrenal lineage and be related to an additional functional role in noradrenergic chromaffin cells not associated with secretion.

The annexin (Anx) gene family comprises a set of calcium-dependent membrane binding proteins, which have been implicated in a wide variety of cellular processes including membrane fusion and calcium channel activity. We report here that cAMP activates Ca(2+)-dependent aggregation of both phosphatidylserine (PS) liposomes and bovine chromaffin granules driven by [des 1-12]annexin I (lipocortin I, Anx1). The mechanism of cAMP action involves an increase in AnxI-dependent cooperativity on the rate of such a reaction without affecting the corresponding k1/2 values. Cyclic AMP causes the values of the Hill coefficient (nH) for AnxI to change from 3 to 6 in both PS liposomes and chromaffin granules. By contrast, ATP inhibits the rate of aggregation activity without affecting the cooperativity or the extent of aggregation process. We were also able to photolabel Anx1 specifically with an 8-azido analogue of cAMP by a calcium-independent process. Such a process is saturable, yielding a Kd = 0.8 microM by Scatchard analysis. Specific displacement occurs in the presence of cAMP and ATP. Finally, we found that cAMP alters the conductance of calcium channels formed by AnxI in planar lipid bilayers. We interpret these data to indicate that AnxI binds both calcium and cAMP independently, and that both actions have functional consequences. This is the first report of a nucleotide binding function for a member of the annexin gene family.

Catecholamines are accumulated in vesicles by a proton gradient-dependent transport, which has mostly been studied in bovine chromaffin granules. The full sequence of a cDNA encoding a vesicular transporter from bovine chromaffin cells, bVMAT2, was recently reported. We now present an analysis of bVMAT2, expressed in transfected COS cells. Comparing the binding of a labelled ligand, [3H]TBZOH, and the rate of uptake, we find a much lower molecular turnover number than in chromaffin granules, probably indicating that a majority of expressed transporters are correctly folded and possess the ligand binding site but cannot actively transport monoamines because they are located in compartments which do not possess a proton gradient. The substrate specificity of uptake and its pharmacological sensitivity to various inhibitors closely resemble those previously observed in chromaffin granules. These results suggest that VMAT2 is the major transporter in bovine adrenal glands, and raise the question of the significance of the second related transporter, VMAT1, which is also expressed in this tissue.

Using oligonucleotide primers derived from the vesicular monoamine transporters sequences, a cDNA predicted to encode the bovine chromaffin granule amine transporter has been cloned (b-VMAT2). Surprisingly, its structure is more similar to the rat brain transporter (VMAT2), than to the rat adrenal counterpart (VMAT1). Unlike rat VMAT1, bovine VMAT2 appears to be expressed both in the adrenal medulla and the brain, as judged by Northern analysis. After modification/deletion of the seven amino acids at the N-terminus of the protein it was expressed in a functional form. The order of affinity of the bovine VMAT2 transporter to substrates is: serotonin > dopamine = norepinephrine > epinephrine. Also, the recombinant bovine adrenal transporter is highly sensitive to tetrabenazine, in sharp contrast to the rat adrenal transporter. The findings indicate, therefore, a clear species variation in which structure and function of the bovine adrenal transporter resemble the rat brain protein, while its tissue distribution is distinct from both types of rat proteins. In addition, the predicted protein sequence is identical to the experimentally determined N-terminus sequence of the purified vesicular amine transporter [Stern-Bach et al. (1992) Proc. Natl. Acad. Sci. USA 89, 9730-9733].

Secretory granules of the adrenal medulla have recently been shown to be able to sequester and release Ca2+, in addition to their previously established role as carriers of secretory products. In order to study the ability of these or any other secretory granules to participate in intracellular calcium homeostasis, it is imperative that they should be free of other contaminating Ca2+ sequestering organelles, and that the Ca2+ uptake and release mechanisms of those granules should remain intact throughout any chosen purification procedure. We report here that chromaffin granules which were purified by the isopycnic gradient medium Percoll, or even incubated with it, showed an attenuated ability to sequester Ca2+.

Alamethicin causes a concentration-dependent increase of [Ca2+]i in suspensions of bovine adrenal chromaffin cells loaded with fura-2. The basal levels of Cai2+ (234 +/- 37 nM; n = 4) increased to a maximum of 2347 +/- 791 nM (n = 3) with 100 micrograms/ml alamethicin. In the presence of 1 mM Cae2+ the increase reached a plateau within about 2-5 s. This increase was due to Ca2+ entry into chromaffin cells, since in the absence of Cae2+ alamethicin did not modify [Ca2+]i. This contrasts with ionomycin (1 microM) which produced a Cai2+ transient even in the absence of Cae2+. Mn2+ ions also entered chromaffin cells in the presence of alamethicin, as measured by the quenching of fura-2 fluorescence following excitation at 360 nm. Resting chromaffin cells had a measurable permeability to Mn2+ which was drastically increased by cell depolarization by K+ (50 mM) addition. This suggests that Mn2+ is able to permeate voltage-dependent Ca2+ channels. Ni2+ uptake into either resting or K(+)-stimulated chromaffin cells was undetectable, but addition of alamethicin induced rapid uptake of this cation. The alamethicin-induced entry of Ni2+ was decreased by 50 mM K+. Overall, the results are compatible with the formation by alamethicin of ion channels in chromaffin cell plasma membranes.

We have examined the interaction of chromaffin granules from bovine adrenal medulla with microtubules. Chromaffin granules were mixed with microtubules made of phosphocellulose-purified tubulin, and pelleted through a 1.6 M sucrose cushion at 12,000 x g for 10 min. Both components (granules and microtubules) were pelleted when added together but not separately. This result indicates that granules form a heavy complex with the microtubules. Such a complex was visualized by an electron microscopy of the granule/microtubule mixture. Treatment of the granules with trypsin abolished their ability to interact with the microtubules. The binding of the granules to the microtubules; (i) was not sensitive to ATP; and (ii) was completely inhibited by the cleavage of C-terminal peptides of alpha- and beta-subunits of tubulin with subtilisin. These relationships suggest that the granule binding is mediated by one of the structural microtubule-associated proteins rather than by microtubule-dependent translocators. For identification of protein(s) mediating the binding, the granules were solubilized with Triton X-100, soluble proteins were mixed with the microtubules, and microtubules with bound proteins were pelleted through a glycerol cushion. At least one granule protein interacting with the microtubules was found in the pellet. This protein was identified as MAP2 according to its electrophoretic mobility and reactivity with a MAP2 antibody. Affinity chromatography of solubilized proteins on a column containing taxol-stabilized microtubules also revealed MAP2 as a protein of chromaffin granules interacting with the microtubules.

Di(1,N6-ethenoadenosine)5',5'''-P1,P4-tetraphosphate, epsilon-(Ap4A), a fluorescent analog of Ap4A has been synthesized by reaction of 2-chloroacetaldehyde with Ap4A. At neutral pH this Ap4A analog presents characteristics maxima at 265 and 274 nm, shoulders at ca 260 and 310 nm and moderate fluorescence (lambda exc 307 nm, lambda em 410 nm). Enzymatic hydrolysis of the phosphate backbone produced a slight hyperchromic effect but a notorious increase of the fluorescence emission. Cytosolic extracts from adrenochromaffin tissue as well as cultured chromaffin cells were able to split epsilon(Ap4A) and catabolize the resulting epsilon-nucleotide moieties up to epsilon-Ado.

Tetanus toxin, a potent neurotoxin which blocks neurotransmitter release in the CNS, also inhibits Ca2+-induced catecholamine release from digitonin-permeabilized, but not from intact bovine chromaffin cells. In searching for intracellular targets for the toxin we studied the binding of affinity-purified tetanus toxin to bovine adrenal chromaffin granules. Tetanus toxin bound in a neuraminidase-sensitive fashion to intact granules and to isolated granule membranes, as assayed biochemically and visualized by electron microscopic techniques. The binding characteristics of the toxin to chromaffin granule membranes are very similar to the binding of tetanus toxin to brain synaptosomal membranes. We suggest that the toxin-binding site is a glycoconjugate of the G1b type (a polysialoganglioside or a glycoprotein-proteoglycan) which is localized on the cytoplasmic face of the granule membrane and might directly be involved in exocytotic membrane fusion.

Using rhodamine-phalloidin staining it was found that actin filaments are concentrated in the cortex of resting chromaffin cells. Cortical actin filaments were disassembled 15 s after stimulation by nicotine and had reassembled 30 s later. Actin filament disassembly following nicotinic stimulation was also detected using the DNase I inhibition assay. Disassembly was independent of external calcium, insensitive to trifluoperazine and was not elicited by high K+, muscarinic agonists or phorbol ester. Disassembly of cortical actin filaments may allow access of secretory granules to exocytotic sites and act in conjunction with a rise in intracellular free calcium to bring about the full secretory response due to nicotinic agonists.

Cytochrome b-561 in chromaffin granules interacts with antimycin and its alpha-peak shifts 1 nm towards red. When chromaffin granules were treated with Triton X-100 antimycin no effect was observed. Cytochrome b-561 is located in the plasma membrane isolated from the chromaffin cells. The plasma membrane b-561 does not seem to interact with antimycin. A number of NADH or NADPH (acceptor) oxidoreductase activity has been observed in isolated plasma membrane providing clues to the origin of plasma membrane dehydrogenase. The possible role of cytochrome b-561 in secretory granules other than its accredited energy conserving electron transport property is projected.

Binding of actin to chromaffin granules was confirmed and shown to be salt dependent and eliminated by prior trypsin treatment of the granules. However, purified granules bind less actin than do crude granules. A mitochondria-enriched fraction was found to bind substantially more actin per mg protein than did the secretory vesicle fraction. Binding of actin by the secretory vesicles therefore is not a good indication that actin plays an active role in exocytosis.

Oriented plasma membrane fragments from chromaffin cells, isolated on polylysine-coated polyacrylamide beads, bind synexin in a calcium dependent manner. Synexin binding was also detected on beads coated with chromaffin granule membranes, but not to beads coated with erythrocyte membranes or to uncoated beads. Synexin binding to plasma membrane coated beads showed a specific requirement for calcium (K1 2 = 200 microM) and was insensitive to other divalent cations such as magnesium, strontium and barium. Synexin binding to either plasma membrane or granule membrane coated beads was saturable, was partially reversible by EGTA and was directly observed by SDS-polyacrylamide gel electrophoresis.

Specific antisera were raised against synthetic peptide fragments of bovine chromogranin A. The soluble proteins of bovine chromaffin granules were subjected to two-dimensional immunoblotting with these antisera. The endogenous breakdown products of chromogranin A gave distinct patterns of immunostaining which enabled us to correlate these peptides with defined regions of the chromogranin A molecule. The results establish that within chromaffin granules degradation of chromogranin A by the endogenous proteases can start either at the C- or the N-terminal site.

The matrix of the chromaffin granule contains a family of acidic proteins, collectively known as the chromogranins. It has been suggested that this family results from protease action on the major component, chromogranin A. Evidence for this has now been obtained from in vitro translation of adrenal medullary messenger RNA and immunoprecipitation of translation products using an antiserum directed against chromogranin A, but which also recognises other chromogranins.

In a range of [Ca2+] similar to cytosolic transient, a drastic reduction from about 20 dyn/cm to almost zero was observed in the membrane elastic modulus of bovine chromaffin granules, isolated in a solution containing 0.3 M sucrose and 5 mM Hepes at pH 7.0, and measured by combination of osmotic swelling and dynamic light-scattering (DLS) methods. This result suggests that the granule membrane becomes extremely flexible as a prelude to exocytosis.

The average size of chromaffin granules isolated from bovine adrenal medullae was analyzed by a quasi-elastic laser light scattering method. The granule diameter increased by a factor of 1.3 by addition of Mg-ATP in the medium. The ATP effect was completely suppressed in the presence of an anion transport blocker (SITS), and partly depressed by a proton transport blocker (DCCD).

The glucose transporter was identified and characterized by cytochalasin B binding in subcellular membrane fractions of chromaffin tissue. The binding was saturable with Kd of about 0.3 microM for each subcellular fraction. The Bmax capacity was 12-16 pmol/mg protein for enriched plasma membrane fractions, 6.3 pmol/mg protein for microsomal membrane preparations and 5.4 pmol/mg protein for chromaffin granule membranes. Irreversible photoaffinity labelling of the glucose-protectable binding sites with [3H]cytochalasin B followed by solubilization and polyacrylamide gel electrophoresis from enriched plasma membrane preparations demonstrated the presence of three molecular species: 97 +/- 10, 51.5 +/- 6 and 30 +/- 4 kDa. The chromaffin granule membranes showed only a molecular species of 80 +/- 10 kDa.

The activity of the catecholamine transporter of chromaffin granules and the binding to these vesicles of reserpine, a transporter inhibitor, decrease during ghost preparation. In contrast, the number of binding sites of dihydrotetrabenazine, another transporter ligand, is constant. Dihydrotetrabenazine thus binds to an inactive transporter whereas reserpine binds only to active vesicles. Inactivation occurs during lysis of the granules, possibly because of an incomplete resealing. The turnover number of the transporter, determined by dividing the uptake activity by the density of dihydrotetrabenazine binding sites, has a maximal value (140 molecules/min) in intact granules. The reserpine to dihydrotetrabenazine binding ratio (10-25%) is an estimate of the proportion of correctly resealed vesicles.

The luminescent oxidation of luciferin has been used to monitor acetylcholine-induced ATP release from cultured bovine chromaffin cells. Acetylcholine (1-100 microM) evoked ATP release of up to 30% of the total cellular ATP. This secretion required external free calcium and could also be elicited by K+-induced membrane depolarization. The size of the cytosolic ATP compartment was estimated as 5% of the ATP in the cell by solubilising the cell membrane using digitonin (20 microM) or by application to the cells of brief pulses (2 microseconds) of high electric field (2000 V/cm). Blockers of the voltage-gated Ca2+ channel effectively blocked K+-induced ATP release, while the acetylcholine antagonists d-tubocurarine and beta-bungarotoxin inhibited the acetylcholine-induced release of ATP. These data support the concept that ATP is released together with the catecholamines by exocytosis of chromaffin granule contents.

Bovine parathyroid chromogranin A inhibits the cleavage of Z-Ala-Lys-Arg-AMC by either trypsin or IRCM-serine protease 1 (IRCM-SP1), a putative novel processing enzyme originally isolated from porcine pituitary anterior and neurointermediate lobes. On larger substrates, chromogranin A is a reversible competitive inhibitor of the cleavage at pairs of basic amino acids by IRCM-SP1. The substrates tested included pituitary ACTH and adrenal medulla pro-enkephalin-derived peptides such as the 8.6 kDa synenkephalin-containing precursor and peptide B. Chromogranin A is itself selectively processed by IRCM-SP1, and ACTH was shown to compete for such cleavage. These data suggest that chromogranins as a class of acidic proteins could participate in the tissue-specific processing of pro-hormones.

GTP-binding proteins have been proposed to be involved in some secretory processes. Bordetella pertussis toxin is known to catalyze ADP-ribosylation of several GTP-binding proteins. In this paper, the subcellular localization of B. pertussis toxin substrates has been explored in chromaffin cells of bovine adrenal medulla. With appropriate gel electrophoresis conditions, three ADP-ribosylated substrates of 39, 40 and 41 kDa were detectable in both plasma and granule membranes. The more intense labelling occurred on the 40 kDa component, while the 41 kDa species exhibited electrophoretic mobility similar to that of Gi alpha. Significant immunoreactivity with anti-Go alpha antibodies was detected at the level of the 39 kDa faster component. The association of G-proteins with granule and plasma membranes suggests the involvement of these proteins in the exocytotic process or in its regulation.

In our previous work, the existence of the precursor and mature forms of atrial natriuretic factor (ANF) within the bovine chromaffin granules has been reported. To confirm the endogenous character of these peptides, we demonstrate that nicotinic activation and depolarization by KCl increase their co-secretion from cultured chromaffin cells. The increase of intracellular levels of these atrial peptides by phorbol ester is potentiated by addition of forskolin. The release of ANF and their de novo synthesis within the cultured chromaffin cells emphasize the usefulness of this model in the study of the mechanisms of release and storage of these peptides in the neuronal tissues.

The proteinaceous components of the secretory vesicle contents isolated from bovine adrenal medulla bind Ca2+ (number of binding sites, 152 +/- 52 nmol Ca2+ per mg protein; dissociation constant, 54 +/- 8 microM (n = 5)). SDS-polyacrylamide gel electrophoresis and 45Ca2+ binding of the proteins following their separation and blotting on nitrocellulose revealed that Ca2+ binds to chromogranins. Moreover, it was shown that the chromogranins, like other known Ca2+-binding proteins, can be specifically stained with a cationic carbocyanine dye. The Ca2+-binding function of the chromogranins described here, in conjunction with recent findings concerning Ca2+ transport across chromaffin vesicle membranes and the widespread distribution findings concerning Ca2+ transport across chromaffin vesicle membranes and the widespread distribution of chromogranins in many different endocrine and nerve cells, points to the general importance of these proteins in the metabolism of Ca2+.

Catecholamine release from chromaffin cells in response to carbamylcholine and high K+ is transient. Monitoring intracellular free calcium ([Ca2+]i) using quin2 demonstrated a transient rise in [Ca2+]i in response to carbamylcholine. The termination of secretion due to carbamylcholine is probably a consequence of the return of [Ca2+]i to resting levels as the nicotinic receptors desensitise. Depolarisation with 55 mM K+ led to a long-lasting rise in [Ca2+]i which persisted after the secretory response had terminated. The maintained rise in [Ca2+]i appeared to be due to continued opening of verapamil-sensitive Ca2+ channels. These results suggest that inactivation of voltage-dependent Ca2+ channels does not account for the transient nature of the secretory response in chromaffin cells.

Bovine adrenomedullary chromaffin granules can be separated into two subpopulations by differential centrifugation. The subpopulation which sediments at the interface of two sucrose layers, 1.6 and 1.8 M respectively, is found to be enriched about 10-times in prohormone processing activity, as measured by in vitro degradation of synthetic peptide substrates. The enhanced proteolytic activity is not due to lysosomal contaminations which are very low and only slightly increased in the more active fraction. The low density of the enriched subpopulation suggests that we are dealing with immature granules. The physiological implications of this finding are discussed. Furthermore, the enriched fraction can be used as the starting material for the isolation of proenkephalin processing enzymes.