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Annexin 2 is a member of the annexin family which has been implicated in calcium-regulated exocytosis. This contention is largely based on Ca(2+)-dependent binding of the protein to anionic phospholipids. However, annexin 2 was shown to be associated with chromaffin granules in the presence of EGTA. A fraction of this bound annexin 2 was released by methyl-beta-cyclodextrin, a reagent which depletes cholesterol from membranes. Restoration of the cholesterol content of chromaffin granule membranes with cholesterol/methyl-beta-cyclodextrin complexes restored the Ca(2+)-independent binding of annexin 2. The binding of both, monomeric and tetrameric forms of annexin 2 was also tested on liposomes of different composition. In the absence of Ca(2+), annexin 2, especially in its tetrameric form, bound to liposomes containing phosphatidylserine, and the addition of cholesterol to these liposomes increased the binding. Consistent with this observation, liposomes containing phosphatidylserine and cholesterol were aggregated by the tetrameric form of annexin 2 at submicromolar Ca(2+) concentrations. These results indicate that the lipid composition of membranes, and especially their cholesterol content, is important in the control of the subcellular localization of annexin 2 in resting cells, at low Ca(2+) concentration. Annexin 2 might be associated with membrane domains enriched in phosphatidylserine and cholesterol.

We have studied the biosynthesis of chromaffin granules by labelling primary cultures of bovine chromaffin cells with either [35S]methionine or various precursors for lipids. After labelling the cells were subjected to subcellular fractionation including density gradient centrifugation. After [35S]methionine significant label (mainly represented by labelled chromogranin A) was found in the soluble proteins of chromaffin granules, whereas the membranes were relatively little labelled. However incorporation into membrane bound dopamine beta-hydroxylase and cytochrome b-561 could be demonstrated. Neither of the used lipid precursors ([3H]glycerol, [3H]choline, [3H]palmitic acid or [3H]arachidonic acid) was incorporated to any significant extent into the soluble components of chromaffin granules. Thus there is no evidence that this secretory compartment contains any lipids or acylated proteins. Incorporation of lipid precursors into the membranes of chromaffin granules was apparently low. After short chases labelled lysolecithin was not present in these organelles. However with prolonged chase times labelled lysolecithin, apparently appeared in chromaffin granules irrespective of whether the cells were stimulated or not. We can conclude that the reusable membranes of chromaffin granules have a very low lipid turnover. Lysolecithin is not transferred into these organelles during biosynthesis but is formed in them during their long life span. This formation of lysolecithin is independent of stimulation of these cells and therefore unlikely to be involved in exocytosis.

Contrary to the notion that the soluble intravesicular matrix proteins of the secretory vesicles of adrenal medullary chromaffin cells freely float in the vesicle, several vesicle matrix proteins of the secretory vesicles, including chromogranins A and B, bound to the vesicle membrane at intravesicular pH (5.5) and were freed from it when the pH was raised to a near physiological pH (7.5). Estimation of the fraction of vesicle matrix proteins that might remain bound to the vesicle membrane in the vesicle suggested that the majority (> 50-80%) of chromogranins A and B, as well as several other proteins, will stay bound to the membrane in the vesicle. Comparison of the amino-acid sequences of chromogranins A and B revealed two highly conserved regions, i.e., one near the N-terminus and the other being the C-terminal region. Since it has been demonstrated with chromogranin A that the conserved near N-terminal region of chromogranin A exhibited the pH-dependent membrane-binding activity (Yoo, S. H. (1993) Biophys. J., 64, A195), the same region in chromogranin B (residues 17-36) was tested using a synthetic chromogranin B peptide, and found to exhibit the pH-dependent membrane-binding activity. The pH-dependent binding of the matrix proteins at pH 5.5 and the automatic untethering at a physiological pH accord well with the rapid release and circulation of the vesicular contents in the bloodstream.

Tetanus toxin (about 1 nM) inhibits 70% of the nicotine-evoked release of catecholamines from intact adrenal medullary chromaffin cells after 20 h of incubation and 30% of the K(+)-evoked release. Inhibition of Ca(2+)-evoked release from detergent-permeabilized cells requires higher concentrations of toxin (about 1 microM) toxin, but is maximal after 12 min. Preincubation of the intact cells with ganglioside GT1 in the absence of toxin also inhibits evoked secretion. 125I-labelled toxin bound specifically to these cells; the binding capacity was greater at pH 6 (about 1 pmol toxin/mg cell protein) than at pH 7.4 (about 0.25 pmol). In both cases there were at least two binding components: one of high affinity (Kd about 1 nM) accounting for about 20% of total binding and one of lower affinity (Kd 10-20 nM). Preincubation of the cells with ganglioside increased the binding capacity, but did not affect the Kd of the lower affinity component. Similar observations could be made when binding was measured immunocytochemically. Extraction of gangliosides from chromaffin cells and overlay experiments with radiolabelled toxin showed that, as well as GM3, the major ganglioside component of chromaffin cell membranes, a ganglioside having the chromatographic mobility of GT1 was a major ligand for toxin.

Secretory processes via exocytosis in rat peritoneal mast cells were visualized by two complementary fluorescence techniques; one staining pre-exocytotic granules with a basic probe and the other staining post-exocytotic granules with acidic probes. Granules within mast cells were selectively stained with acridine orange and emitted orange yellow fluorescence. Upon stimulation with compound 48/80, release of acridine orange from granules was observed both in population and single cell measurements. This release was seen in some localized area of mast cells. Opening of pores between plasma membranes and granule membranes was monitored using acidic fluorescence probes such as 6-carboxyfluorescein or lucifer yellow CH. Not only granules located at peripheral region, but also granules near the core region participated in exocytosis. The existence of junctions between these granules was suggested. TMA-DPH, a lipophilic membrane probe, which was localized at plasma membrane before stimulation, diffused into granule membranes after stimulation. This shows that after stimulation, some constituents of plasma and granule membranes were mixed. Even after extensive degranulation, mast cells extruded acidic probes, indicating the plasma membranes still play a role of barrier. Activation of lateral motion of granules preceding to exocytosis was not observed. It was concluded that the visualization of secretory processes by fluorescence and image processing techniques will be useful for the study of molecular mechanisms underlying exocytosis.

Bovine chromaffin granules were treated with the ionophores nigericin or A23187 in sucrose solutions with the pH varying from 4.7 to 7.0. Nigericin and A23187 induced osmotic lysis of the granules in sucrose solutions at pH values below 5.8, but not at physiological pH. This effect is explained by a progressive protonation of the acidic chromogranins induced by the ionophore-promoted exchange of internal potassium- and calcium ions for external protons. The results support the view that the interactions between catecholamines and ATP with chromogranins play a significant role in osmotic pressure reduction of the granule interior.

The chromaffin granule membrane binding and aggregating properties of three annexins, synexin, p32 and p67, have been studied and compared. Each protein was activated to bind and aggregate membranes with a biphasic Ca2+ dependence, with one phase titrating between pCa 5.0-3.5 and the second at higher levels of calcium (pCa less than 3.5). cis-Unsaturated free fatty acids lowered these Ca2+ requirements by approximately one log unit. Barium and strontium were able to partially substitute for calcium, with the order of sensitivity Ca2+ greater than Sr2+ greater than Ba2+. The proteins appeared to bind to distinct but overlapping populations of receptor sites, and did so in a manner displaying positive cooperativity at the higher Ca2+ levels. The maximal efficacy of the proteins as membrane aggregators differed with synexin being 1-2-fold more efficacious than p32, which in turn was 7-fold more efficacious than p67. In combination, p67 was an effective inhibitor of granule aggregation induced by synexin or p32, while p32 was able to both promote and inhibit synexin-induced granule aggregation in a manner which varied with synexin concentration. The complexity of these annexin-membrane interactions may be a reflection of the multidomain structure of the annexins and may have implications for the differential functions of these proteins in cells.

The binding of the fluorescent probe bis-ANS to chromogranin A, the major protein of adrenal chromaffin vesicles, caused a marked enhancement and blue shift in the fluorescence emission spectrum. The emission maximum shifted from 515 nm to 480 nm and the yield increased approx. 75-fold upon addition of 10 microM chromogranin A to 1 microM bis-ANS. Adenine nucleotides had clear effects on the bis-ANS fluorescence signal, while other nucleotides such as GTP, UTP and CTP had no discernible effect. Specifically, ATP caused a decrease in the fluorescence, whereas ADP and AMP caused a fluorescence increase. These results indicate adenine nucleotide binding to chromogranin A. Substitution of ATP with epsilon-ATP, an ATP derivative with a modification on the six-membered ring of the adenine base, failed to reduce the fluorescence intensity. Therefore, it was concluded that adenine bases play an important role in the chromogranin A-adenine nucleotide interaction.

We have studied the effects of GABA on cytosolic free Ca2+ concentration ([Ca2+]i) as a means of investigating the role of GABA in adrenal catecholamine (CA) secretion. It was demonstrated that GABA caused an elevation of [Ca2+]i via the GABAA receptor in a concentration-dependent manner, which was well correlated with an increase of 45Ca uptake, an increase of CA release and a depolarization of chromaffin cells assessed with bis-oxonol fluorescence. Since the GABA-induced rise of [Ca2+]i was absolutely dependent on the presence of extracellular Ca2+ and partly sensitive to nifedipine, at least one entry route for Ca2+ facilitated by GABA via a voltage-sensitive Ca2+ channel was suggested. When extracellular Cl- was lowered, GABA-induced CA release, depolarization, and rise of [Ca2+]i were all markedly enhanced. It is possible that GABA plays a modulatory role in the regulation of adrenal CA secretion as a facilitatory modulator.

The prohormone chromogranin A is the major soluble component of secretory granules in chromaffin cells of adrenal medulla and in many other different endocrine cell types. The proteolytic processing of chromogranin A was studied in cultured bovine chromaffin cells using [35S]methionine to label proteins and a specific antibody to immunoprecipitate the native protein and its breakdown products. In resting cells, it was found that the degradation of chromogranin A is a slow process, since no degradation was observed after a 40 h incubation with radiolabelled methionine. Stimulation of cells with a single pulse or with successive pulses of nicotine did not significantly enhance the degree of proteolytic processing of chromogranin A. As it has recently been shown (Simon, J.P., Bader, M.F. and Aunis, D. Biochem. J. (1989) 260, 915-922) that protein kinase C may be involved in the regulation of chromogranin A synthesis, the possibility that prohormone processing may also be controlled by protein kinase C was examined using the activator of protein kinase C, 12-O-tetradecanoylphorbol 13-acetate (TPA). However, incubation of cells with TPA did not significantly modify chromogranin A processing, indicating that biosynthesis and proteolytic processing of chromogranin A are two distinctly regulated mechanisms. Glucocorticoids are known to exert regulatory control of chromaffin cell metabolism; however, incubation of cells with dexamethasone did not alter slow chromogranin A processing. Stimulation of labelled cells rapidly released newly synthesized chromogranin A into external medium. In addition, released chromogranin A was found to be actively processed into its 60 kDa and 43 kDa breakdown products. This extracellular proteolytic degradation mechanism may be of importance with regard to the function of chromogranin A as a prohormone.

The role of the transmembrane potential (delta phi), the proton concentration gradient (delta pH) and the proton electrochemical gradient (delta gamma H+) in monoamine uptake by bovine chromaffin granules or ghosts was investigated. In presence of ATP the permeant anion SCN- collapsed the delta phi (inside positive) and inhibited monoamine uptake by granules or well buffered ghosts. With lightly buffered ghosts, SCN- induced an acidification which resulted in a low inhibition of uptake. Cation efflux as well as anion influx affected the delta phi, and a transient valinomycin-mediated K+ efflux induced a lag in the uptake. The delta pH-driven noradrenalin uptake was also sensitive to delta phi, since superimposing a positive or a negative delta phi to the delta pH, respectively, increased or decreased the rate of noradrenalin accumulation. A delta pH was required for this increase of uptake rate, adrenalin accumulation. A delta pH was required for this increase of uptake rate, which was proportional to the delta phi. The pH-dependence of the ATP-induced monoamine uptake by granules pointed to the delta gamma H+ as the driving force. In contrast with the rate of uptake, which was not dependent on the anions present, the extent of amine incorporation was decreased when the internal anionic buffer concentration was decreased and, at a low internal buffer concentration, when ATP anion transport was blocked.

Proteins of the chromaffin granule membrane were covalently labeled in situ with sulfhydryl-specific fluorophores. Using MIANS (maleimide iodoaminonaphthyl sulfonate) as the donor and fluorescein mercury acetate or fluorescein-5-maleimide as the acceptor. Förster fluorescence resonance energy transfer (FRET) could be employed to measure the degree of inter-membrane and intra-membrane protein-protein contact upon Ca2+-induced aggregation of the membranes. The four major findings were: (1) Raising the Ca2+ concentration to approx. 500 microM causes the proteins to aggregate in the plane of the membrane. This is demonstrated by Ca2+-induced increases in the fluorescence resonance energy transfer in double labeled membranes. This effect is not protein-concentration dependent and occurs at calcium concentrations too low for granule aggregation, implying intra-membrane protein clustering or patching. To our knowledge this is the first direct demonstration of the fluid mosaic nature of subcellular organelles. (2) If two sets of granules are labeled separately, Ca2+-induced aggregation brings at least 74% of the labeled proteins into close transmembrane proximity. This effect is also observed at 10-100-fold slower rates in the absence of calcium and can be greatly reduced by depleting the granule membrane of labeled peripheral proteins. It is enhanced if the granules are aggregated by Ca2+ or K+. We conclude that (some) peripheral proteins can transfer from one membrane surface to another. (3) Aggregation of separately labeled sets of membranes by Ca2+ also produces transmembrane energy transfer since: (a) the Km for Ca2+-induced quantum transfer is in the same range as the Km for aggregation; (b) the reaction is protein-concentration dependent; (c) reversal of aggregation also (partially) reverses donor quenching. (4) A kinetic analysis of the transmembrane effect shows it to be 5-10-fold slower than aggregation itself, supporting earlier suggestions (Haynes, D.H., Kolber, M. and Morris, S.J., (1979) J. Theor. Biol. 81, 713-743) that lipid and protein rearrangements are secondary to granule membrane aggregation.

Ghosts derived from bovine chromaffin granules have a 32Pi-ATP exchange activity which is associated with the H+ pump of that membrane. This activity was low when compared to bacteria, chloroplasts or submitochondrial particles, but had similar properties (Km for ATP and Pi, ATP/Mg2+ ratio, pH profile, inhibition by dicyclohexylcarbodiimide and tributyltin) to the ATPase from above membranes. The 32Pi-ATP exchange activity was solubilized by cholate/octylglucoside mixtures. The soluble extract was lipid depleted by ammonium sulfate fractionation and partially purified by sucrose gradient centrifugation. The purified preparation was reconstituted with phospholipids by freeze-thawing. The reconstituted vesicles had a 32Pi-ATP exchange sensitive to dicyclohexylcarbodiimide and trybutyltin and an ATPase with a sensitivity to the inhibitors which varied with the reconstitution conditions. The alpha- and beta-subunits of F1-ATPase were major components of the preparation.

Carbamylcholine-stimulated catecholamine release from adrenal chromaffin cells was completely inhibited by pretreatment of the cells for 10 min with 1 microM calmidazolium. Catecholamine release due to 55 mM K+ and ionophore A23187 was also inhibited by calmidazolium but less effectively than release due to carbamylcholine. Inhibition of release appeared to be due to an effect of calmidazolium on a step distal to Ca2+ entry, since the carbamylcholine-stimulated rise in the concentration of intracellular free calcium, monitored using quin-2, was unaffected by calmidazolium. The possibility was considered that calmidazolium inhibited secretion through an effect on protein kinase C rather than calmodulin. However, the phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA), had no demonstrable effect on catecholamine release, arguing against a significant role for protein kinase C in secretion from adrenal chromaffin cells. These results give further support to the notion that calmodulin plays a role in the secretory process in chromaffin cells.

Membranes of chromaffin granules isolated from bovine adrenal medulla are shown to bind dihydrocytochalasin B with high affinity. These membranes also bound [3H]actin in a time- and Mg2+-dependent manner and electron microscopy showed the presence of membrane-attached actin filaments following addition of exogenous actin. Binding of [3H]actin was partially inhibited by cytochalasin B. Electron microscopic analysis of heavy meromyosin-decorated, membrane-attached filaments showed terminally (end-on) attached filaments with both possible polarities (i.e., filaments with arrowheads pointing both towards and away from the membranes). Treatment of samples with cytochalasin B preferentially inhibited growth of filaments with their 'barbed' ends pointing away from membranes. These results are discussed with respect to the role of actin in secretory granule function and the mechanism of cytochalasin action.

We showed earlier that insulin stimulated sugar transport in adrenal chromaffin cells (Bigornia, L. and Bihler, I. Biochim. Biophys. Acta 885, 335-344). Transport regulation and its Ca2+ -dependence was further investigated in isolated bovine adrenal chromaffin cells, serving as a model of a homogeneous neuronal cell population. Uptake of the nonmetabolizable glucose analogue, 3-O-methyl-D-glucose was stimulated by hyperosmolar medium, and this effect was abolished in the absence of external Ca2+, or depressed in the presence of La3+ or the slow Ca2+ channel blocker methoxyverapamil. Basal transport was also stimulated by factors (acetylcholine, carbamylcholine, low-Na+ medium), which cause Ca2+ -dependent catecholamine release, and these effects were abolished in Ca2+ -free medium. In addition insulin, acetylcholine, hyperosmolar and low-Na+ medium significantly increased 45Ca uptake. Thus, glucose transport in adrenal chromaffin cells was stimulated by insulin and hyperosmolarity in a Ca2+ -dependent manner, as in muscle. Sensitivity to secretory stimuli, a regulatory feature perhaps characteristic of this cell type, was also demonstrated. In contrast to muscle, sugar transport was not affected by Na+ -pump inhibition, metabolic inhibitors or the Na+ ionophore monensin, suggesting that Ca2+ influx by Na+/Ca2+ exchange does not play a significant role in the activation of sugar transport in chromaffin cells.

Carbon-13 NMR spectra of the reconcentrated chromaffin granule lysate have been obtained at 50 MHz and 62.9 MHz. The spectrum contains a number of assignable resonances in addition to those of the main soluble components (catecholamines, adenine nucleotides and chromogranin). Guanine and uridine nucleotides are present at levels of 0.13 and 0.08 mol/mol adenine nucleotides, respectively. Concentrations of cytidine nucleotides and NAD+ are below the detection limit (0.02 mol/mol adenine nucleotides). An unidentified low molecular weight species, thought to be an adenine-containing oligonucleotide, is also present. Ascorbic acid was observed at a concentration of 0.14 mol/mol adenine nucleotides, but both dopamine and dehydroascorbic acid were below the detection limit. Protein resonances agree well with the reported amino acid composition of chromogranin A, with the exception of tryptophan and glutamine which have not previously been measured. The concentrations of these residues are estimated to be 12 +/- 3 and 39 +/- 5 residues per 77 000 dalton unit of chromogranin A. Substantial intensity due to unsaturated fatty acid side-chains in solubilized lipid is seen in the olefinic carbon region and in the methylene region, suggesting the presence of lipoprotein. Unassigned carbohydrate resonances are also present, but are largely obscured by sucrose in the isolation medium.

Adenosine transporters in freshly isolated and cultured chromaffin cells were quantified by the [3H]dipyridamole binding technique, showing a maximal bound capacity of 0.4 +/- 0.05 pmol/10(6) cells (240,000 +/- 20,000 transporters by cell). Scatchard analysis showed a similar affinity for [3H]dipyridamole in isolated cells and subcellular fractions (Kd = 5 +/- 0.6 nM). For enriched plasma membrane preparations and chromaffin granule membranes, the maximal binding capacities were also very similar, 2.3 +/- 0.3 and 1.8 +/- 0.4 pmol/mg protein, respectively. When [3H]nitrobenzylthioinosine was employed as a radioligand, the maximal bound capacity in cultured chromaffin cells was 0.053 +/- 0.004 pmol/10(6) cells (32,000 +/- 3000 transporters per cell) with a high affinity constant (Kd = 0.25 +/- 0.03 nM); similar values were obtained in all subcellular fractions (Kd = 0.1 +/- 0.01). Also, plasma and chromaffin granule membranes showed similar maximal binding values (0.4 +/- 0.06 pmol/mg protein). Photoincorporation studies with [3H]nitrobenzylthioinosine into plasma membrane polypeptides showed the presence of three molecular species of 115 +/- 10; 58 +/- 6 and 42 +/- 5 kDa. Chromaffin granule membranes showed only the 105 +/- 9 and 51 +/- 4 molecular species.

The structure and function of chromaffin granules of human pheochromocytoma was extensively investigated in a highly purified granule fraction obtained from a single specimen of human pheochromocytoma tissue. Pheochromocytoma chromaffin granules were analyzed for catecholamine, ATP, enkephalin, phospholipid, cytochrome and ion content. Using a variety of techniques it was found that the membrane of these granules is highly impermeable to Na+, K+, and H+, and that the intragranular pH was maintained at 5.1 irrespective of suspending media. The presence of MgATP induces a transmembrane potential (delta psi) across the membrane of these granules which is positive inside and which corresponds to 90 mV. Both delta pH and delta psi are coupled to biogenic amine accumulation into the granules in a process which is reserpine sensitive. These properties are compared with those of chromaffin granules isolated from normal human tissue or from other animal species and are discussed in terms of possible explanation at a biochemical or subcellular level of the clinical manifestation of the pheochromocytoma.

We have investigated the interaction between isolated membrane vesicles from chromaffin granules and large unilamellar phospholipid vesicles (liposomes). Mixing of membrane lipids has been monitored continuously, utilizing the fluorescence resonance energy transfer assay described by Struck et al. ((1982) Biochemistry 20, 4093-4099). To demonstrate coalescence of the internal vesicle volumes the transfer of colloidal gold from the liposomes to the interior of the granule membrane vesicles has been examined. Efficient fusion of the liposomes with the granule membranes was observed. Significant fusion occurred in the absence of Ca2+, although the extent of interaction was enhanced in its presence. The sensitivity of the interaction to pretreatment of the granule membranes with trypsin showed the fusion reaction to be a protein-mediated process.

The thermostatics of cotransport are reviewed. A static-head equilibrium state across a cotransport system, without leaks, is thought to occur when the electrochemical potential of the driven solute, B prevents net flow of the driving solute, A. For a symport this gives the relationship (formula: see text) Where n is the stoichiometric coefficient, namely the number of moles of A transported per mole of B. (2) If either a symporter with a 2:1 stoichiometric coefficient and a 1:1 symporter, or alternatively, a 1:1 symporter and a 1:1 antiporter are placed in a series membrane array, then the predicted static-head equilibrium across the entire array conflicts with the zeroth law of thermodynamics. (3) There are two major reasons for this failure of cotransport theory; these are: (A) the thermostatic relationships derived shown in Point 1 are based on the assumption that the cotransport process takes place within a closed system. However, the membrane and the external reservoirs are open to the cotransported ligands. It follows that A and B in the external reservoirs can vary independently of the changes within the cotransport process. As no chemical reaction between A and B occurs in the external solutions, reactions within the membrane phase do not affect the equilibrium between the transported ligands in the open reservoirs. (B) It is assumed that the law of mass action can be applied to the cotransport chemical reactions within the membrane phase, without any allowance for the fact that these reactions occur within a 'small thermodynamic system'. Any proper analysis of the chemical potential of the transported intermediate must consider the effects of lower order ligand-carrier forms, which coexist and compete for space with the higher order cotransported forms on the binding matrix. If account is taken of this necessity, then a simple extension of the work of Hill and Kedem (1966) J. Theor. Biol. 10, 399-441 shows that: (a) the static-head equilibrium state cannot exist; (b) the stoichiometry of cotransport, whether symport, or antiport, does not affect the static-head distribution of cotransported ligands; (c) the hypothetical net charge of the transported ligand-carrier complex does not affect static-head equilibrium; (d) the only equilibrium state where there is zero net flow of both driving and driven transported ligand is at true equilibrium when the ligands are uniformly distributed across the membrane. (4) It is deduced that cotransport is not entirely an affinity-driven, but is partially an entropy-driven process.(ABSTRACT TRUNCATED AT 400 WORDS)

1. The accessibility of phospholipids in the membrane of the adrenomedullary storage vesicles (chromaffin granules) has been studied. 2. The reaction of 2,4,6-trinitrobenzenesulphonic acid with both intact granules and their ghosts, results in the labelling of 70% of the phosphatidylethanolamine. 3. The action of phospholipase A2 (from bee venom), phospholipase C (from Bacillus cereus) and sphingomyelinase C (from Staphylococcus aureus) on granules and their ghosts was followed as a function of time. No significant difference was observed between the intact granules and their ghosts. 4. In the intact granules the various treatments led to varying amounts of lysis although again no evidence was obtained that such lysis in any way increased the amount of accessible phospholipid. 5. Highly purified granule preparations were also compared with the so-called "large granule" fraction and no significant differences were detected. 6. Approx. 67% of phosphatidylethanolamine + phosphatidic acid, 50% of phosphatidylserine + phosphatidylinositol, 65% of phosphatidylcholine and 20% of sphingomyelin is accessible to enzymatic degradation. In total, approx. 50% of all the phospholipids reacted. 7. It is also shown that, unlike in enzymatic treatment, all the phosphatidylcholine can be exchanged in the presence of a phospholipid exchange protein (prepared from beef liver). 8. It is concluded that transmembrane movement of phosphatidylcholine is slow in isolated membranes of chromaffin granules. The presence of the exchange protein, however, in conjunction with membrane proteins and specific phospholipid arrangements may catalyse this transmembrane movement.

The subcellular distribution of ascorbate and catecholamines has been studied in homogenates of bovine adrenal medulla and cortex. 1. The recovery of the vitamin was found to be 4.10 +/- 0.22 and 9.57 +/- 1.37 mumol/g wet weight for the medulla and cortex, respectively. A major fraction (34.4%) of the vitamin was recovered in the particulate fraction of the medulla as compared to about 8% in the corresponding fraction of the cortex. In comparison, 78.9% of the catecholamines were found in the particulate fraction of the medulla. 2. Analytical differential centrifugation of medulla homogenates revealed a sedimentation profile of ascorbate which was identical to that obtained for noradrenalin and adrenalin. The co-sedimentation of these compounds indicates that ascorbate is an essential component of the heavy as well as the light population of chromaffin granules. The stoichiometry of catecholamines to ascorbate was approx. 25:1 in both subpopulations. 3. Based on an estimated volume fraction of approximately 13% for the chromaffin granules, as determined morphometrically (Kryvi, H., Flatmark, T. and Terland, O. (1979) Eur. J. Cell Biol. 20, 76-82), a concentration gradient (chromaffin granules:cytosol) of approx. 4 was estimated for ascorbate in the cells of adrenal medulla. 4. No ascorbate 2-sulfate was detected in any of the subcellular fractions isolated, and the content of dehydroascorbate in isolated chromaffin granules was less than 1% of the total ascorbate value.

The possible role of metalloendoproteinase in stimulus-secretion coupling in adrenal chromaffin cells was examined using the metalloendoproteinase inhibitors 1,10-phenanthroline and carbobenzoxy-Gly-Phe-NH2. Catecholamine release elicited by nicotine or by depolarisation with 55 mM K+ was almost completely abolished by 0.5 mM 1,10-phenanthroline. Carbobenzoxy-Gly-Phe-NH2 (2.5 mM) inhibited catecholamine release in response to nicotine but enhanced that due to 55 mM K+. The rise in intracellular free calcium, [Ca2+]i, in response to either nicotine or 55 mM was inhibited by about 50% by both inhibitors. One site of action of metalloendoproteinase inhibitors may, therefore, be at the level of the regulation of [Ca2+]i. Catecholamine release and the rise in [Ca2+]i elicited by the calcium ionophore ionomycin were not reduced by the inhibitors. These results show that metalloendoproteinase inhibitors have complex effects on chromaffin cells including effects on the regulation of [Ca2+]i but do not inhibit calcium-activated exocytosis itself.

A method is described for the preparation of (1) the heavy population of bovine adrenal chromaffin granules (SH (average sedimentation coefficient) = 12 400 S in 0.25 M sucrose) essentially free from contamination with mitochondria and other organelles, and (2) a subpopulation of this heavy population which is highly enriched in noradrenalin (greater than or approximately 95% of the total catecholamine is noradrenalin). The method is based on isopycnic gradient centrifugation using a self-generating gradient of polyvinylpyrrolidone-coated colloidal silica particles (Percoll) in 0.5 M sucrose medium. The isolated population of noradrenalin granules appeared highly electron dense in transmission electron microscopy and revealed a rather narrow size distribution. The specific content of amine and adenine nucleotides (with reference to total granule protein) was markedly higher than for the total population of heavy chromaffin granules. The molar ratio of amines to adenine nucleotides was, however, lower in the noradrenalin granules, i.e. 4.8 vs. 11.9.

We have previously shown that plasma membranes from adrenal medulla possess specific high-affinity binding sites for prostaglandins (PGs) E1 and E2. We have now investigated the binding of PGE2 to intact bovine adrenal chromaffin cells and the effects of prostaglandins on the release of catecholamines from these cells. Adrenal chromaffin cells specifically bound PGE2 with a dissociation constant of 2 nM and a concentration of about 40,000 binding sites per cell. Low concentrations of PGE2 inhibited the nicotine-stimulated release of catecholamines from these cells. The effect of PGE2 was biphasic, the maximal inhibitory effect being observed at a concentration of between 1 and 10 nM. Higher concentrations (1 microM) of PGE2 had minimal inhibitory effects on nicotine-evoked noradrenaline release, but instead had a direct stimulatory effect in the absence of cholinergic agonists. Although the stimulatory effects of high concentrations of PGE2 were reproducibly observed in all cell preparations, only about one-half of the cultures tested responded to the inhibitory effects of this prostaglandin. It is possible that PGE2 plays a modulatory role in the regulation of catecholamine secretion from the adrenal medulla.

The temperature-dependent release of core constituents from isolated chromaffin granules in isotonic sucrose has been a controversial and puzzling phenomenon that has been interpreted either as selective catecholamine efflux from different catecholamine pools or as temperature-dependent lysis. We have analysed the kinetics, temperature dependence and physical basis of this process. Our results demonstrate that, upon increasing the ambient temperature, chromaffin granules show a shift in their osmotic fragility to higher osmolarities, which is linearly dependent on temperature and leads to measurable lysis in 0.26 M buffered sucrose at temperatures above 12 degrees C. It is possible to demonstrate both protein and dopamine beta-hydroxylase release when lysis as a function of temperature is measured in 0.26 M buffered sucrose. Real time measurements of the lysis kinetics were recorded on cassettes and analysed by a computer program for exponential decay kinetics. It is shown that the temperature-dependent lysis proceeds in two separate phases, the fast one of which is associated with temperature-dependent shift in the osmotic fragility curve. It has no characteristics of any exponential decay kinetics. The slow phase, when followed over several hours, leads to complete lysis of the granules in a sigmoidal time course at 30 degrees C. We conclude from the absence of exponentiality that there is no basis on which to assume the existence of different catecholamine pools. The fast phase of temperature-dependent lysis can be best explained as a simple temperature-dependent increase of the granule core solution's osmotic pressure, while the slow phase is probably caused by sucrose permeation into the granules. On the basis of these results, we warn against any efflux experiments measuring the temperature-dependent transmitter release from secretory vesicles with highly concentrated core solutions.

The dye, oxonol-V (bis(3-phenyl-5-oxoisoxazol-4-yl)pentamethine oxonol), can be used to estimate the transmembrane potential of chromaffin granules. The potentials result either from a resting-state Donnan equilibrium (inside negative at pH 6.6) or from an ATP-driven proton pump. The fluorescence and absorption changes generated by ATP addition depended on the pH of the medium and the dye-to-vesicle ratio. Energization resulted in an increase in the number of oxonol-V binding sites, the new binding sites having the same dissociation constant. The rate of dye association was higher with resting than with energized chromaffin granules. The absorption change was associated with a red shift whereas the fluorescence change involved a quenching due to the increase in dye concentration on the membrane. The absorption and fluorescence changes varied linearly with the transmembrane potential difference when the interior potential was positive relative to the medium.