Diadenosine Tetraphosphate Is Co-Released with ATP and Catecholamines from Bovine Adrenal Medulla

Secretion of adenosine(5')tetraphospho(5')adenosine (Ap4A) and ATP from perfused bovine adrenal glands stimulated with acetylcholine or elevated potassium levels was measured and compared with that of catecholamines. We have found a close correlation between the release of Ap4A and catecholamines elicited with all the secretagogues used in the presence of either Ca2+ or Ba2+, suggesting co-release of both constituents from the chromaffin granules. By contrast, ATP secretion, as measured with luciferase, showed a significantly different time course regardless of the secretagogue used. ATP secretion consistently decreased after 1-2 min of stimulation at a time when Ap4A and catecholamine secretions were still increasing. Measures of degradation of injected [3H]ATP to the gland during stimulation showed little difference in the level of uptake or decomposition of ATP throughout the pulse. However, a reexamination of ATP secretion by monitoring its products of degradation (AMP, adenosine, and inosine) by HPLC techniques showed that Ap4A, ATP, and catecholamines are indeed secreted in parallel from the perfused adrenal gland.     

Fluorescence properties of catecholamines in isolated storage organelles of adrenal medulla

THe quantum yield, the life time and the degree of polarization of the fluorescence of intact chromaffin granules isolated from bovine adrenal medulla were compared to those of catecholamines solutions and catecholamine/ATP mixtures. Rising concentrations of catecholamines in aqueous solutions exhibited increasing quenching and decreasing life times indicating that the quenching was collision induced. Similar effects occurred in mixtures of catecholamines with ATP and Ca²⁺ showing that the nucleotide did not remarkedly hinder the mobility of the catechol group. In suspensions of whole granules stron quenching and shortening of life time was observed compared with solutions of disrupted granules. Fluorescence yield and life time were decreased by about the same factor suggesting that storage of the amines was not correlated with a major immobilization of the catechol group. The degree of polarization of intact granules was higher than that of solutions of catecholamines alone, but similar to catecholamine/ATP mixtures with concentrations corresponding to those found in the granules. This indicates an interaction of catecholamines with ATP in the granules. The results are in agreement with a storage model for catecholamines in the chromaffin granules of adrenal medulla in which catecholamines are bound to ATP, but in a non-rigid way.     

Specificity and properties of the nucleotide carrier in chromaffin granules from bovine adrenal medulla.

1. The influence of various substances on the uptake of [3H]ATP and [14C]-noradrenaline into isolated bovine chromaffin granules was investigated. The carrier-mediated [3H]ATP uptake is specifically inhibited by SO42-, PO43- and phosphoenolpyruvate. Compounds with carboxylic acid or sulphonic acid groups had no significant inhibitory effects on either uptake. 2. 35SO42-, 32PO43- and phosphoenol[14C]pyruvate are taken up into chromaffin granules by a temperature-dependent process that is inhibited by atractyloside, uncouplers of oxidative phosphorylation and lipid-permeant anions. The apparent Km of 35SO42- uptake is 0.4 mM. 3. These results indicate that the nucleotide carrier in chromaffin granules has a broad specificity, transporting compounds with two strong negative charges. 4. Amino acid probes influence the uptake of ATP and catecholamines differently. Pyridoxal phosphate inhibits both uptake processes, 4,4'-di-isothiocyanostilbene-2,2'-disulphonic acid preferentially blocks ATP uptake, whereas phenylglyoxal blocks only ATP transport. It is suggested that the nucleotide carrier possesses arginine residues in a functionally important position. 5. The significance of these results obtained on isolated granules for the function of chromaffin granules within the cell is discussed.     

Carbachol-Induced Cosecretion of Immunoreactive Atrial Natriuretic Peptides with Catecholamines from Cultured Bovine Adrenal Medullary Cells

The distribution and secretion of atrial natriuretic peptides (ANPs) were investigated in bovine adrenal medulla. (1) Cultured bovine adrenal medullary cells (2 x 10(6)/dish) contained 100.4 +/- 6.0 fmol of immunoreactive ANP (IR-ANP) and 207.3 +/- 6.6 nmol of catecholamines as epinephrine plus norepinephrine. (2) Stimulation of nicotinic but not muscarinic acetylcholine receptors caused a cosecretion of IR-ANP and catecholamines corresponding to the ratio of IR-ANP to catecholamines in cultured bovine adrenal medullary cells. (3) Carbachol-stimulated secretion of IR-ANP was dependent on the presence of extracellular Ca2+. (4) Chromaffin granules isolated from bovine adrenal medulla contained large amounts of IR-ANP and catecholamines, in the same ratio as did cultured adrenal medullary cells. (5) Reverse-phase HPLC analysis showed that both stored and secreted IR-ANP consisted of two components, which eluted at the position of ANP(99-126) or ANP(1-126). These results indicate that ANPs are stored as ANP(99-126) and ANP(1-126) in chromaffin granules, and are cosecreted in parallel with catecholamines in a Ca2+-dependent manner by the stimulation of nicotinic acetylcholine receptors.     

Differential regulation of multiple populations of granules in rat adrenal chromaffin cells by culture duration and cyclic AMP

We employed carbon fiber amperometry to measure the amount of catecholamine released from individual granules (i.e. the quantal size, Q) of rat chromaffin cells. The distribution of Q1/3 of amperometric events could be reasonably described by the summation of at least three Gaussians, suggesting that rat chromaffin cells contained at least three distinct populations of granules, with a small, medium or large modal Q. After 3 days of culture, the mean cellular Q reduced by approximately 14%, which did not arise from a uniform percentage decrease in the Q of every granule. Instead, the rundown involved a > 11% decrease in the proportional release from large Q granules and a > 19% decrease in the modal Q-value of small Q granules. In contrast, when cells were cultured with dibutyryl-cAMP (dBcAMP) for 3 days, their mean cellular Q increased by approximately 38% (relative to time-matched controls). This increase in Q was not associated with any shift in the proportional release from the three populations of granules. Instead, cAMP increased the average amount of catecholamines released from all three populations of granules. Our data raise the possibility that distinct populations of granules in rat chromaffin cells can be regulated either differentially or uniformly.     

Plasma Membrane and Chromaffin Granule Characteristics in Digitonin-Treated Chromaffin Cells

Digitonin permeabilizes the plasma membranes of bovine chromaffin cells to Ca2+, ATP, and proteins and allows micromolar Ca2+ in the medium to stimulate directly catecholamine secretion. In the present study the effects of digitonin (20 microM) on the plasma membrane and on intracellular chromaffin granules were further characterized. Cells with surface membrane labeled with [3H]galactosyl moieties retained label during incubation with digitonin. The inability of digitonin-treated cells to shrink in hyperosmotic solutions of various compositions indicated that tetrasaccharides and smaller molecules freely entered the cells. ATP stimulated [3H]norepinephrine uptake into digitonin-treated chromaffin cells fivefold. The stimulated [3H]norepinephrine uptake was inhibited by 1 microM reserpine, 30 microM NH4+, or 1 microM carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). The data indicate that [3H]norepinephrine was taken up into the intracellular storage granules by the ATP-induced H+ electrochemical gradient across the granule membrane. Reduction of the medium osmolality from 310 mOs to 100 mOs was required to release approximately 50% of the catecholamine from chromaffin granules with digitonin-treated chromaffin cells which indicates a similar osmotic stability to that in intact cells. Chromaffin granules in vitro lost catecholamine when the digitonin concentration was 3 microM or greater. Catecholamine released into the medium by micromolar Ca2+ from digitonin-treated chromaffin cells that had subsequently been washed free of digitonin could not be pelleted in the centrifuge and was not accompanied by release of membrane-bound dopamine-beta-hydroxylase. The studies demonstrate that 20 microM of digitonin caused profound changes in the chromaffin cell plasma membrane permeability but had little effect on intracellular chromaffin granule stability and function. It is likely that the intracellular chromaffin granules were not directly exposed to significant concentrations of digitonin. Furthermore, the data indicate that during catecholamine release induced by micromolar Ca2+, the granule membrane was retained by the cells and that catecholamine release did not result from release of intact granules into the extracellular medium.     

The chromaffin granule — Plasma membrane interaction as a model for exocytosis: Quantitative release of the soluble granular content

Bovine adrenal medullary plasma membranes induce the release of soluble components from chromaffin granules in a Ca²⁺ dependent manner. This interaction can be modulated by changing the temperature. Correlation of the concentrations of four released soluble markers (catecholamines, soluble protein, ATP and dopamine-β-hydroxylase) in samples incubated at different temperatures revealed that those markers were liberated simultaneously. Their ratio did not differ significantly from the ratio measured in chromaffin granule lysates. These results suggest the release of the total granular content upon incubation with plasma membranes. Further analysis of the free catecholamines showed a preferential release of adrenalin.     

Release of aldosterone and catecholamines from the interrenal gland of Triturus carnifex in response to adrenocorticotropic hormone (ACTH) administration

The influence of adrenocorticotropic hormone (ACTH) on the interrenal gland of Triturus carnifex was investigated by in vivo administration of synthetic ACTH. The effects were evaluated by examination of the ultrastructural morphological and morphometrical features of the tissues as well as the circulating serum levels of aldosterone, noradrenaline (NA), and adrenaline (A). In June and November, ACTH administration increased aldosterone release (from 281.50 +/- 1.60 pg/ml in carrier-injected newts to 597.02 +/- 3.35 pg/ml in June; from 187.45 +/- 1.34 pg/ml in carrier-injected animals to 651.00 +/- 3.61 pg/ml in November). The steroidogenic cells showed clear signs of stimulation, together with a reduction of lipid content in June and an increase of lipid content in November. Moreover, ACTH administration decreased the mean total number of secretory vesicles in the chromaffin cells in June (from 7.73 +/- 0.60 granules/microm2 in carrier-injected animals to 5.91 +/- 0.40 granules/microm2) and November (from 7.78 +/- 0.75 granules/microm2 in carrier-injected newts to 4.87 +/- 0.40 granules/microm2). In June, however, when T. carnifex chromaffin cells contain almost exclusively NA granules (NA: 7.42 +/- 0.86 granules/microm2; A: 0.32 +/- 0.13 granules/microm2), ACTH decreased NA content (5.52 +/- 0.32 granules/microm2) increasing NA release (from 639.82 +/- 3.30 pg/ml in carrier-injected to 880.55 +/- 4.52 pg/ml). In November, when both catecholamines, NA (3.92 +/- 0.34 granules/microm2) and A (3.84 +/- 0.33 granules/microm2), are present in the chromaffin cells, ACTH administration reduced A content (1.02 +/- 0.20 granules/microm2), enhancing adrenaline secretion (from 681.30 +/- 3.62 pg/ml in carrier-injected newts to 1,335.73 +/- 9.03 pg/ml). The results of this study indicate that ACTH influences the steroidogenic tissue, eliciting aldosterone release. The effects on the chromaffin tissue, increase of NA or A secretion, according to the period of chromaffin cell functional cycle, may be direct and/or mediated through the increase of aldosterone release. Finally, the lack of an increase of A content in the chromaffin cells, or A serum level, following ACTH administration in June might suggest an independence of PNMT enzyme on corticosteroids.     

An osmotic mechanism for exocytosis from dissociated chromaffin cells

Dissociated chromaffin cells from bovine adrenal medulla were stimulated to secrete epinephrine and dopamine beta-hydroxylase with a variety of secretagogues in a study designed to test the hypothesis that the chemiosmotic lysis reaction of isolated chromaffin granules might in some way be related to the mechanism of release during exocytosis. Increasing the osmotic strength of the incubation medium with either NaCl or sucrose led to suppression of secretion of epinephrine from the cells regardless of whether secretion was induced with veratridine or acetylcholine. Suppression of secretion was approximately exponential with respect to osmotic strength. Epinephrine secretion occurred only if the medium contained a permeant anion such as chloride, and secretion induced by veratridine was suppressed when Na isethionate replaced NaCl in the medium. In an extensive study with different monovalent anions veratridine supported epinephrine secretion according to the following activity series: Br-, I-, NO3- greater than methylsulfate, SCN- greater than Cl greater than acetate much greater than isethionate. A similar series, except for the potency of NO3-, was observed with A23187 as agonist. In general, the anion series for granule lysis was analogous. However, there was a poor quantitative correlation between the anion dependence of chemiosmotic granule lysis and the anion dependence of cell secretion. Anion transport inhibitors such as probenecid and pyridoxal phosphate also inhibited secretion while the stilbene disulfonates were inactive. The ineffectiveness of the stilbene disulfonates further distinguished chemiosmotic granule lysis from cell secretion. Secretion of catecholamines, induced by veratridine or nicotine, a cholinergic agonist, was suppressed when NaCl in the medium was replaced by isosmotic sucrose and unexpectedly low levels of dopamine beta-hydroxylase were observed in some cases. In sum, these properties of secreting chromaffin cells resembled some properties of isolated chromaffin granules incubated in ATP and Cl-, but were different in a number of instances. We, therefore, have interpreted our data to indicate that while some mechanistic relationships may indeed exist between the release event in exocytosis from chromaffin cells and the chemiosmotic lysis reaction characteristic of isolated chromaffin granules, an understanding of the energetics of exocytosis awaits the discovery of reasons for the quantitative differences between the two systems.     

Effects of noradrenaline administration on the interrenal gland of the newt, Triturus carnifex: evidence of intra-adrenal paracrine interactions

The existence of paracrine control of steroidogenic activity by adrenochromaffin cells in Triturus carnifex was investigated by in vivo noradrenaline (NA) administration. The effects were evaluated by examination of the ultrastructural morphological and morphometrical features of the tissues as well as the serum levels of aldosterone, NA, and adrenaline (A). In March and July, NA administration increased aldosterone release (from 187.23 +/- 2.93 pg/ml to 878.31 +/- 6.13 pg/ml in March; from 314.60 +/- 1.34 pg/ml to 622.51 +/- 2.65 pg/ml in July) from steroidogenic cells. The cells showed clear signs of stimulation, as evidenced by a strong reduction of lipid content. Moreover, NA administration decreased the mean total number of secretory vesicles in the chromaffin cells in March (from 7.24 +/- 0.18 granules/micro2 to 5.57 +/- 1.88 granules/micro2) and July (from 7.74 +/- 0.74 granules/micro2 to 6.04 +/- 1.13 granules/micro2). In March, however, when T. carnifex chromaffin cells contain both catecholamines, NA (3.88 +/- 0.13 granules/micro2) and A (3.36 +/- 0.05 granules/micro2) in almost equal quantities, NA administration reduced A content (1.29 +/- 1.04 granules/micro2) in the chromaffin cells, enhancing adrenaline secretion (from 681.27 +/- 1.83 pg/ml to 1527.02 +/- 2.11 pg/ml). In July, when the chromaffin cells contain almost exclusively NA granules (NA: 7.42 +/- 0.86 granules/micro2; A: 0.32 +/- 0.13 granules/micro2), NA administration reduced the number of NA granules (5.45 +/- 1.10 granules/micro2), thereby increasing noradrenaline release from the chromaffin cells (from 640.19 +/- 1.65 pg/ml to 1217.0 +/- 1.14 pg/ml). The results of this study indicate that NA influences the steroidogenic cells, eliciting aldosterone release. Noradrenalin effects on the chromaffin cells, increase of NA or A secretion, according to the period of chromaffin cell functional cycle, may be direct and/or mediated through the steroidogenic cells. The existence of intra-adrenal paracrine interactions in T. carnifex is discussed.     

Effects of adrenaline administration on the interrenal gland of the newt, Triturus carnifex: evidence of intraadrenal paracrine interactions

The existence of paracrine control of steroidogenic activity by adrenochromaffin cells in Triturus carnifex was investigated by in vivo adrenaline (A) administration. The effects were evaluated by examination of the ultrastructural morphological and morphometrical features of the tissues as well as the serum levels of aldosterone, noradrenaline (NA), and adrenaline. In March and July, adrenaline administration reduced aldosterone release (from 187.23 +/- 2.93 pg/ml to 32.28 +/- 1.85 pg/ml in March; from 314.60 +/- 1.34 pg/ml to 87.51 +/- 2.57 pg/ml in July) from steroidogenic cells. The cells showed clear signs of lowered activity: they appeared full of lipid, forming large droplets. Moreover, adrenaline administration decreased the mean total number of secretory granules in the chromaffin cells in July (from 7.74 +/- 0.74 granules/microm(2) to 5.14 +/- 1.55 granules/microm(2)). In this period T. carnifex chromaffin cells contain almost exclusively NA granules (NA: 7.42 +/- 0.86 granules/microm(2); A: 0.32 +/- 0.13 granules/microm(2)). Adrenaline administration reduced noradrenaline content (4.36 +/- 1.40 granules/microm(2)) in the chromaffin cells, enhancing noradrenaline secretion (from 640.19 +/- 1.65 pg/ml to 1030.16 +/- 3.03 pg/ml). In March, adrenaline administration did not affect the mean total number of secretory vesicles (from 7.24 +/- 0.18 granules/microm(2) to 7.25 +/- 1.97 granules/microm(2)). In this period the chromaffin cells contain both catecholamines, noradrenaline (3.88 +/- 0.13 granules/microm(2)), and adrenaline (3.36 +/- 0.05 granules/microm(2)), in almost equal quantities; adrenaline administration reduced adrenaline content (1.74 +/- 0.84 granules/microm(2)), increasing adrenaline release (from 681.27 +/- 1.83 pg/ml to 951.77 +/- 4.11 pg/ml). The results of this study indicate that adrenaline influences the steroidogenic cells, inhibiting aldosterone release. Adrenaline effects on the chromaffin cells (increase of noradrenaline or adrenaline secretion) vary according to the period of chromaffin cell functional cycle. The existence of intraadrenal paracrine interactions in T. carnifex is discussed.     

Growth characteristics of postnatal rat adrenal medulla in culture

Explants and enzyme-dispersed cells of adrenal medulla from 10-12 day old rats were studied in culture for up to 3 weeks. Adrenomedullary chromaffin cells, nerve cells and satellite cells were clearly discernible. The nerve cells were few in number and did not show catecholaminespecific fluorescence. Chromaffin cells stored catecholamines, as judged by the Falck and Hillarp method, in varying amounts decreasing with age of the cultures and the distance from the explants. Exocytosis profiles observed with the electron microscope suggested that cultured chromaffin cells also released catecholamines. Moreover, the cells formed processes and frequently migrated into the outgrowth. After 6 days in culture, the great majority of chromaffin cells stored noradrenaline as revealed by electron microscopy with few adrenaline-storing cells being visible. Granular vesicles (approximately 80-240 nm in diameter) with cores of different electron densities were occasionally present in the same cell suggesting the occurrence of mixtures of primary and secondary amines. Apart from "chromaffin" granules, small clear and dense-cored vesicles (approximately 40-60 nm) were found both in the somata and cell processes. Chromaffin cells and their processes were often closely apposed and occasionally formed specialized attachment zones. As a whole, chromaffin cells in culture resembled small granule-containing cells in sympathetic ganglia. 0.5 mM dbcAMP prevented dedifferentiation of chromaffin cells as judged by the lack of processes, the size and amount of "chromaffin" granules and the high number of adrenaline-storing cells present after 6 days in culture. NGF caused a striking increase in the number of axons growing out from explants.     

Bovine Chromaffin Cells Release a Transforming Growth Factor-β-Like Molecule Contained Within Chromaffin Granules

Abstract: Bovine chromaffin cells contain within their storage vesicles and release upon cholinergic stimulation a complex mixture of proteins and peptides. We present data suggesting that one of these proteins resembles transforming growth factor (TGF)-β in terms of its biological activity. The assay used to assess the activity of TGF-β is based on cells transfected with a plasminogen activator inhibitor-1 promoter-luciferase construct. The assay is highly specific in detecting TGF-β1, -β2, and -β3 but does not detect several cytokines and growth factors, such as fibroblast growth factor-2, transforming growth factor-α, platelet-derived growth factor-AB, insulin-like growth factor-I, or neurotrophin-3 or -4. Moreover, we show that this assay does not detect a wide range of TGF-β superfamily members (activin A, bone morphogenetic protein-2, -4, -6, and -7, growth/differentiation factor-5, and glial cell line-derived neurotrophic factor). Chromaffin granules contain ∼1 ng of TGF-β/10 mg of protein. The biological activity elicited by the chromaffin granule component can be neutralized by using an antibody against TGF-β1/β2/β3. TGF-β is releasable from cultured chromaffin cells stimulated with the cholinergic agonist carbachol (10⁻⁵ M ). These data suggest that TGF-β is stored in chromaffin granules and can be released by exocytosis.     

The ionogenic nature of the secretory-granule membrane. Electrokinetic properties of isolated chromaffin granules

1. Chromaffin granules isolated from the bovine adrenal medulla possess an electrophoretic mobility of -1.12mum.s(-1).cm.V(-1), corresponding to a surface zeta potential of -14.4mV and surface charge density of 1.38x10(-6)C.cm(-2). 2. The mobility of chromaffin granules is pH-dependent, indicating an amphoteric surface with an isoelectric point at pH3.0 and acidic groups with a pK(a) of 3.11. 3. Addition of bi- and ter-valent cations decreased the mobility of chromaffin granules in a dose-dependent fashion with a relative potency of La(3+)>Mn(2+)>Ca(2+) >Sr(2+)>Mg(2+)>Ba(2+). 4. Treatment with neuraminidase decreased the mobility of erythrocytes by 84%, whereas chromaffin-granule mobility was decreased by only 14%. This correlates well with the small complement of neuraminic acid present in the granule membrane. 5. The nature, origin and significance of the anionic surface charge of the chromaffin granule is discussed. It is concluded that the net negative charge at the surface of shear derives chiefly from a single type of chemical group, namely -CO(2) (-), contributed by the alpha-carboxyl group of constituent proteins, the phospholipid phosphatidylserine and, to a lesser extent, the sialic acid component of glycoproteins.     

Composition of the aqueous phase of chromaffin granules.

Nuclear magnetic resonance spectroscopy has been used to determine the composition of the aqueous phase of bovine chromaffin granules. Relative concentrations of catecholamines (epinephrine plus norepinephrine), ATP and chromogranins have been measured from integrated intensities in the proton spectra using computer simulation techniques. Most or all of the catecholamines (97 +/- 8%) are present in the aqueous phase and contribute to the high resolution spectrum. The catecholamine:ATP molar ratio (4.41 +/- 0.45) determined by NMR is close to the value (4.45) derived from biochemical assay indicating that most or all of the ATP is present with catecholamine in the aqueous phase. Catecholamine:protein ratios show that approximately 45% of the soluble protein freed by lysis is not NMR visible. Intensity from this fraction does not appear under highly denaturing conditions (8 M urea) but reappears after hydrolysis. This behavior is similar to that of recently isolated soluble lipoprotein complexes. Variations in the NMR spectra associated with (1) different preparative procedures; (2) different suspension media, and (3) increasing osmolality are described. The fact that high concentrations of epinephrine and ATP (approximately 700 mM total) are dissolved in the aqueous phase implies that solution phase interactions at least partially ionic in nature are responsible for the low internal osmolality of chromaffin granules in vivo. Ordered phases containing a substantial fraction of the total catecholamine in an osmotically inactive form are not present.     

Molecular mobilities and the lowered osmolality of the chromaffin granule aqueous phase

Carbon-13 spin-lattice relaxation times, T₁, have been measured in whole adrenal medullary tissue slices, in suspensions of isolated chromaffin granules, in the reconcentrated chromaffin granule lysate, and in various model solutions containing catecholamines, ATP, chromogranins and Ca²⁺. Reorientational correlation times have been calculated at 10°C using T₁ data and nuclear Overhauser enhancemments for protonated carbons on both catecholamines and nucleotides. Correlation times in all media are relatively short and characteristic of highly fluid aqueous phases. Adrenalin and ATP exhibit substantial differences in correlation times in all media, however, the ratio γ_R(ATP):γ_R(catecholamine) ranging from 2.4 in simple 3:1 adrenalin-ATP solutions to 4 in intact chromaffin granules. This difference, as well as the relatively high absolute reorientational mobilities of both components, confirms the importance of labile ionic interactions between ATP and catecholamines, but rules out the presence of high concentrations of base-stacked structures. Participation of the chromogranins in ternary complexes with catecholamines and ATP appears to be of minor importance. Ionic interactions to the protein are not reflected in either ¹³C T₁ values or chemical shifts of arginine or glutamate sidechain resonances, or in the ¹³C chemical shifts of ATP or catecholamines. Very labile protein-ATP binding appears to be reflected in the correlation time measurements, however, which show selective immobilization of ATP relative to catecholamine in the presence of soluble protein. Osmotic measurements indicate that solutions containing adrenaline, ATP and Ca²⁺ are highly nonideal, but probably not sufficiently so to account fully for the osmotic stabilization of the chromaffin granule aqueous phase. Even in the absence of specific intermolecular complexation, the chromogranins, through their polyelectrolyte properties, exert a significant influence on the intragranular osmolality. The osmotic lowering due to polyion-counterion interactions has been estimated semiquantitatively using a theory developed by Oosawa.     

The participation of annexin II (calpactin I) in calcium-evoked exocytosis requires protein kinase C

Permeabilized adrenal chromaffin cells secrete catecholamines by exocytosis in response to micromolar calcium concentrations. Recently, we have demonstrated that chromaffin cells permeabilized with digitonin progressively lose their capacity to secrete due to the release of certain cytosolic proteins essential for exocytosis (Sarafian T., D. Aunis, and M. F. Bader. 1987. J. Biol. Chem. 34:16671-16676). Here we show that one of the released proteins is calpactin I, a calcium-dependent phospholipid-binding protein known to promote in vitro aggregation of chromaffin granules at physiological micromolar calcium levels. The addition of calpactin I into digitonin- or streptolysin-O-permeabilized chromaffin cells with reduced secretory capacity as a result of the leakage of cytosolic proteins partially restores the calcium-dependent secretory activity. This effect is specific of calpactin I since other annexins (p32, p37, p67) do not stimulate secretion at similar or higher concentrations. Calpactin I requires the presence of Mg-ATP, suggesting that a phosphorylating step may regulate the activity of calpactin. Calpactin is unable to restore the secretory activity in cells which have completely lost their cytosolic protein kinase C or in cells having their protein kinase C inhibited by sphingosine or downregulated by long-term incubation with TPA. In contrast, calpactin I prephosphorylated in vitro by purified protein kinase C is able to reconstitute secretion in cells depleted of their protein kinase C activity. This stimulatory effect is also observed with thiophosphorylated calpactin I which is resistant to cellular phosphatases or with phosphorylated calpactin I introduced into cells in the presence of microcystin, a phosphatase inhibitor. These results suggest that calpactin I is involved in the exocytotic machinery by a mechanism which requires phosphorylation by protein kinase C.     

Antibacterial Peptides Are Present in Chromaffin Cell Secretory Granules

1. Antibacterial activity has recently been associated with the soluble matrix of bovine chromaffin granules. Furthermore, this activity was detected in the contents secreted from cultured chromaffin cells following stimulation.2. The agents responsible for the inhibition of Gram+ and Gram– bacteria growth are granular peptides acting in the micromolar range or below. In secretory granules, these peptides are generated from cleavage of chromogranins and proenkephalin A and are released together with catecholamines into the circulation.3. Secretolytin and enkelytin are the best characterized; these two peptides share sequence homology and similar antibacterial activity with insect cecropins and intestinal diazepam-binding inhibitor. For some of the peptides derived from chromogranin A, posttranslational modifications were essential since antibacterial activity was expressed only when peptides were phosphorylated and/or glycosylated.4. The significance of this activity is not yet understood. It may be reminiscent of some primitive defense mechanism or may serve as a first barrier to bacteria infection during stress, as these peptides are secreted along with catecholamines.     

Regulation of catecholamine release in human adrenal chromaffin cells by β-adrenoceptors

The adrenal gland plays a fundamental role in the response to a variety of stress situations. After a stress condition, adrenal medullary chromaffin cells release, by exocytosis, high quantities of catecholamine (epinephrine, EP; norepinephrine, NE), especially EP. Once in the blood stream, catecholamines reach different target organs, and induce their biological actions through the activation of different adrenoceptors. Adrenal gland cells may also be activated by catecholamines, through hormonal, paracrine and/or autocrine system. The presence of functional adrenoceptors on human adrenal medulla and their involvement on catecholamines secretion was not previously evaluated. In the present study we investigated the role of β(1)-, β(2)- and β(3)-adrenoceptors on catecholamine release from human adrenal chromaffin cells in culture. We observed that the β-adrenoceptor agonist (isoproterenol) and β(2)-adrenoceptor agonist (salbutamol) stimulated catecholamine (NE and EP) release from human adrenal chromaffin cells. Furthermore, the β(2)-adrenoceptor antagonist (ICI 118,551; 100 nM) and β(3)-adrenoceptor antagonist (SR 59230A; 100 nM) inhibited the catecholamine release stimulated by isoproterenol and nicotine in chromaffin cells. The β(1)-adrenoceptor antagonist (atenolol; 100 nM) did not change the isoproterenol- neither the nicotine-evoked catecholamine release from human adrenal chromaffin cells. Moreover, our results show that the protein kinase A (PKA), protein kinase C (PKC), mitogen-activated protein kinase (MAPK) and phospholipase C (PLC) are intracellular mechanisms involved in the catecholamine release evoked by salbutamol. In conclusion, our data suggest that the activation of β(2)- and β(3)-adrenoceptors modulate the basal and evoked catecholamine release, NE and EP, via an autocrine positive feedback loop in human adrenal chromaffin cells.