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At least 19 recordsLinked to original sources
PubMed1985

Fe2+-induced lysis and lipid peroxidation of chromaffin granules.

Chromaffin granules, the catecholaminergic storage granules from adrenal chromaffin cells, lysed in 10(-9)-10(-7) M Fe2+. Lysis was accompanied by the production of malondialdehyde which results from lipid peroxidation. Both chromaffin granule lysis and malondialdehyde production were inhibited by the free radical trapping agent butylated hydroxytoluene but not by catalase and/or superoxide dismutase. The results suggest that lysis resulted from a direct transfer of electrons from Fe2+ to a component of the chromaffin granule membrane without the participation of either superoxide or hydrogen peroxide and may have resulted from lipid peroxidation. In some experiments, ascorbate alone induced chromaffin granule lysis which was inhibited by EDTA, EGTA, or deferoxamine. The lysis was probably caused by trace amounts of reducible polyvalent cation. Lysis sometimes occurred when Ca2+ was added with EGTA (10 microM free Ca2+ concentration) and was consistently observed together with malondialdehyde production in the presence of Ca2+, EGTA, and 10 microM Fe2+ (total concentration). The apparent Ca2+ dependency for chromaffin granule lysis and malondialdehyde production was probably caused by a trace reducible polyvalent ion displaced by Ca2+ from EGTA and not by a Ca2+-dependent reaction involving the chromaffin granule.

Animals↗
PubMed1983

Evidence for a divalent cation dependent catecholamine storage complex in chromaffin granules.

Chromaffin granules, the secretory vesicles of the adrenal medulla, are stable in isotonic sucrose solutions at room temperature; however, when low concentrations of the ionophore A23187 are added, rapid lysis ensues which is dependent on the presence of a divalent cation chelator and is prevented by the addition of either Ca2+ or Mg2+. As little as 10 microM Ca2+ totally inhibit lysis of chromaffin granules by A23187, while 28 mM KCl have no effect. Lysis by A23187 at 4.7 microM is almost 100% in the presence of EDTA in isotonic sucrose in 1 h and can be suppressed by raising the osmotic strength of the medium with half maximal inhibition at 0.57 M sucrose, demonstrating that A23187 causes osmotic lysis of chromaffin granules as a consequence of the withdrawal of divalent cations from the core solution. Our results strongly suggest that divalent cations are involved in the formation of a ionic complex in the core solution which lowers its effective osmotic pressure.

Animals↗
PubMed1983

A possible high molecular weight precursor to vasoactive intestinal polypeptide sequestered into pheochromocytoma chromaffin granules.

Chromaffin granules, the catecholamine storage granules of pheochromocytoma were isolated from five human pheochromocytoma tumors. Vasoactive intestinal polypeptide (VIP) immunoreactivity was detected in all chromaffin granule preparations, paralleling the synthetic VIP antibody binding curve over a range of serial dilutions. In addition, gel filtration revealed an immunoreactive peptide peak coeluting with VIP. However, high molecular weight immunoreactive material was also detected on the column. This high molecular weight material was further characterized by sodium dodecyl sulfate gel electrophoresis, followed by electroblotting onto nitrocellulose paper and detection by anti-VIP antibodies with a secondary antibody conjugated to horseradish peroxidase. A 70 000 dalton immunoreactive band was identified, in which reactivity with anti-VIP antibody was inhibited by VIP; this band did not cross react with non-related antibodies. This 70 000 dalton protein may be an intermediate molecule in the biosynthesis and processing of VIP.

Adrenal Gland Neoplasms↗
PubMed1994

Diadenosine 5',5"-P1,P4-tetraphosphate (Ap4A), ATP and catecholamine content in bovine adrenal medulla, chromaffin granules and chromaffin cells.

The level of diadenosine 5',5"-P1-P4-tetraphosphate (diadenosine tetraphosphate or Ap4A), catecholamines, ATP and other nucleotides has been investigated in perchloric acid extracts of bovine adrenal medulla, chromaffin granules and cultured chromaffin cells. As a control, the amount of Ap4A and ATP has also been measured in human blood platelets. The following values (nmol/mg protein) were found in adrenal medulla: Ap4A, 0.019 +/- 0.004; ATP, 109 +/- 11; ADP, 23.8 +/- 5.8; AMP, 11.3 +/- 1.5; p4A, 0.18 +/- 0.08; catecholamines, 460 +/- 57. The level of Ap4A, catecholamines and ATP (nmol/mg protein) found in chromaffin granules and in chromaffin cells were, respectively: (0.15 +/- 0.07; 2175 +/- 99; 531 +/- 66) and (0.22 +/- 0.14; 1143 +/- 277; 222 +/- 53). In all the cases investigated, the ratio catecholamines/ATP and catecholamines/Ap4A were around 5 and in the order of 10(3), respectively. The amount of Ap4A found here, in bovine adrenal medulla, chromaffin granules and chromaffin cells, is two orders of magnitude lower than previously reported.

Adenosine Triphosphate↗
PubMed1992

Immunolocalization of synexin (annexin VII) in adrenal chromaffin granules and chromaffin cells: evidence for a dynamic role in the secretory process.

Synexin (annexin VII) is a Ca(2+)- and phospholipid-binding protein which has been proposed to play a role in Ca(2+)-dependent membrane fusion processes. Using a monoclonal antibody against synexin, Mab 10E7, and immunogold, we carried out a semiquantitative localization study of synexin in bovine adrenal medullary chromaffin granules, and in resting and nicotine-stimulated adrenal chromaffin cells. Isolated chromaffin granules contained very little synexin, whereas chromaffin granules aggregated with synexin (24 micrograms/mg) and Ca2+ (1 mM) clearly showed synexin-associated immunogold particles in the vicinity of the granule membrane (1.88 gold particles per granule profile). In isolated, cultured adrenal chromaffin cells, synexin was present in the nucleus (5.5 particles/microns 2) and in the cytosol (5.3 particles/microns 2), but mainly around the granule membrane in the granular cell area (11.7 particles/microns 2). During the active phase of cholinergically stimulated catecholamine secretion, the amount of synexin label was reduced by 33% in the nucleus, by 23% in the cytosol, and by 51% in the granule area. The plasma membrane contained a small amount of synexin, which did not significantly change upon stimulation of the cells. We conclude that synexin is involved in the secretory process in chromaffin cells.

Adrenal Medulla↗
PubMed2006

The heterogeneity of ion channels in chromaffin granule membranes.

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

Animals↗
PubMed1989

[Peptidylglycine alpha-amidating monooxygenase from bovine heart atrium secretory granules and adrenal chromaffin granules].

About 40-60% of the peptidylglycine alpha-amidating amonooxygenase activity in the lysates of secretory granules from bovine atria and adrenal medulla isolated and lyzed in the presence of pepstatin, phenylmethylsulfonyl gluoride, N-ethylmaleimide and catalase, was found to be in the soluble form. The remaining part bound to the membrane fraction was extracted with Triton X-100. The procedure of purification of the soluble form of peptidylglycine alpha-amidating monooxygenase from both atrial and chromaffin granules in electrophoretically homogeneous enzyme preparations was developed. The enzyme is made up of a single subunit with a molecular mass of 68 kDa and contains one copper atom per molecule. The EPR spectra of peptidylglycine alpha-amidating amonooxygenase and dopamine beta-monooxygenase were found to be practically identical, thus indicating that the copper environment in the both enzymes is the same. Both peptidylglycine alpha-amidating monooxygenase and dopamine beta-monooxygenase are inhibited by the neurocuprein apoform, an extremely acidic protein isolated from brain and secretory granules of different endocrine tissues.

Adrenal Medulla↗
PubMed1982

Osmotic pressures of solutions of ATP and catecholamines relating to storage in chromaffin granules.

The chromaffin granule, which is the catecholamine storage organelle of the adrenal medulla, contains at least 0.73 M ions, yet it is isotonic with 0.3 osM solutions. One hypothesis which accounts for this disparity is formation of a complex between major constituents of the granule: the catecholamines, the proteins, and the ATP. In this paper we show by vapor pressure osmometry, which affords a direct measure of colligative properties, that ATP-catecholamine mixtures form highly nonideal solutions. At 37 degrees C, solutions containing 0.6 M epinephrine and 0.15 M ATP show an effective osmotic pressure of only 0.25 osM. The existence of polymeric complexes is implied by the fact that the increase of osmotic pressure with increasing concentrations of ATP and catecholamine falls off substantially at concentrations approaching those in the chromaffin granules. Neither inorganic ions nor calcium chelators cause regain of ideal colligative behavior. Osmotic measurements on model compounds suggest that the primary interaction is between the phosphate and amino groups. There is also evidence that the effects are not wholly due to the formation of discrete complexes; factors of nonideal solution behavior also play a role in lowering the osmotic pressure. These observations show that the stability of the chromaffin granule in situ can be accounted for, perhaps entirely, by spontaneous interactions among nucleotides and catecholamines.

Adenosine Triphosphate↗
PubMed1995

Bovine chromaffin cells release a transforming growth factor-beta-like molecule contained within chromaffin granules.

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)-beta in terms of its biological activity. The assay used to assess the activity of TGF-beta is based on cells transfected with a plasminogen activator inhibitor-1 promoter-luciferase construct. The assay is highly specific in detecting TGF-beta 1, -beta 2, and -beta 3 but does not detect several cytokines and growth factors, such as fibroblast growth factor-2, transforming growth factor-alpha, platelet-derived growth factor-AB, insulin-like growth factor-1, or neurotrophin-3 or -4. Moreover, we show that this assay does not detect a wide range of TGF-beta 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 approximately 1 ng of TGF-beta/10 mg of protein. The biological activity elicited by the chromaffin granule component can be neutralized by using an antibody against TGF-beta 1/beta 2/beta 3. TGF-beta is releasable from cultured chromaffin cells stimulated with the cholinergic agonist carbachol (10(-5) M). These data suggest that TGF-beta is stored in chromaffin granules and can be released by exocytosis.

Animals↗
PubMed1996

Characterization of ATP transport into chromaffin granule ghosts. Synergy of ATP and serotonin accumulation in chromaffin granule ghosts.

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

Adenosine Triphosphate↗
PubMed1972

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.

Adrenal Medulla↗
PubMed1995

Long term stimulation changes the vesicular monoamine transporter content of chromaffin granules.

Bovine chromaffin cells cultured for 5 days in the presence of depolarizing concentrations of K+ ions show a decreased number of secretory (chromaffin) granules per cell. These cells were still capable of exocytosis. Their contents in catecholamine and chromogranin A, components of the granule matrix, and cytochrome b561, a major protein of the granule membrane, were decreased to 35, 30, and 50% of control cells, respectively. However, in the same cells, the number of [3H]dihydrotetrabenazine binding sites, a specific ligand of the vesicular monoamine transporter, was increased to 180% of controls. In situ uptake of noradrenaline in permeabilized cells indicated that [3H]dihydrotetrabenazine binding sites were associated with a functional vesicular monoamine transporter. When analyzed by isopycnic centrifugation, these sites cosedimented with catecholamine, chromogranin A, and cytochrome b561, in a peak with a density lighter than that from controls. The composition of this peak suggests that it contains incompletely matured secretory granules, with a 3-5-fold increase in the vesicular monoamine transporter content of this membrane. This increase might indicate that an adaptative process occurs which allows a faster filling of the granules in continuously secreting cells.

Animals↗
PubMed1982

ATP-activated exchange of catecholamines by isolated intact chromaffin granules.

Isolated chromaffin granules were found to accumulate exogenous (R)-[3H]-norepinephrine at 37 C in the presence of Mg2+-ATP by a process involving one-to-one exchange for endogenous epinephrine. By contrast, omission of Mg2+-ATP from the incubation medium resulted in a substantial unidirectional efflux of the endogenous epinephrine independent of external catecholamine concentrations. However, the effects of ATP on the spontaneous efflux and the exchange process were found to occur with different kinetic parameters and differential sensitivity to a stilbene disulfonate inhibitor of the granule ATPase, 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid. These data lead us to conclude that ATP catalyzes bidirectional catecholamine flux across the granule membrane (not net accumulation) by an ATPase-dependent mechanism. In addition, ATP has the ability to suppress unidirectional efflux of catecholamines from granules by a mechanism independent of the ATPase or the exchange mechanism. The data also allow us to distinguish among three possible models for how these processes might be occurring.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗
PubMed1981

The structure and organization of dopamine-beta-hydroxylase in the chromaffin granule membrane.

Chromaffin granules have been purified from bovine adrenal medullae. The granule membranes have been cross-linked with the disulphide-bridged bifunctional imido ester, dimethyl-3,3'-dithiobissuccinimidylpropionate hydrochloride. Analysis of the cross-linked proteins by electrophoresis on agarose/acrylamide gels revealed components of M(r) 300 000 and 150 000. Further analysis of samples by electrophoresis in a second dimension containing a reducing agent revealed the monomeric species from which the cross-linked polypeptides were formed. The major component in the second dimension exhibited a molecular weight of approx. 80 000 and could be identified with dopamine-beta-hydroxylase (3,4-dihydroxyphenylethylamine ascorbate:oxygen oxidoreductase (beta-hydroxylating), EC 1.14.17.1). It is proposed that dopamine-beta-hydroxylase in the intact granule membrane is arranged as a tetramer consisting of two disulphide-bridged dimers of the 80 000 subunit in close apposition. This structural arrangement of the membrane-bound form of dopamine-beta-hydroxylase is identical with that previously proposed for the soluble, intra-granular form of the enzyme.

Adrenal Medulla↗
PubMed1983

Processing of enkephalin-containing peptides in isolated bovine adrenal chromaffin granules.

Intact chromaffin granules isolated from bovine adrenal medulla were incubated at 37 degrees C for up to 22 hr. Processing of enkephalin-containing (EC) peptides in the granules was followed by the change in their size distribution as shown by chromatography on Sephadex G-75 columns. A gradual shift toward lower molecular weight EC peptides was observed during the incubation, indicating processing of the higher molecular weight to lower molecular weight EC peptides. The total amount of [Met]-enkephalin, free and in peptide linkage, remained constant indicating that little or no nonspecific degradation occurred during the experiment. HPLC resolution of the fraction containing the low molecular weight EC peptides showed that free enkephalins as well as [Met]enkephalin-Arg6-Phe7 and [Met]enkephalin-Arg6-Gly7-Leu8 accumulated while [Met]enkephalin-Arg6 and [Met]enkephalin-Lys6 disappeared. All the above data indicate the presence of an atypical trypsin activity and the presence of a carboxypeptidase B-like activity within the granules. From the rates of accumulation of the low molecular weight EC peptides and the disappearance of the higher molecular weight EC peptides, a processing rate of 65-70 pmol/g tissue per hr was estimated, which calculates to a lifetime of 6-8 days for EC peptides in the granules. Under steady-state conditions this rate of processing appears to be too low to produce significant amounts of free enkephalins from larger EC peptides. This is well in accord with previous observations that relatively small amounts of free enkephalins are found in bovine adrenal medulla.

Animals↗
PubMed1989

A sodium/proton antiporter in chromaffin-granule membranes.

Chromaffin granules, the secretory vesicles of the adrenal medulla, have a Na+/H+ exchange activity in their membranes which brings their proton gradient into equilibrium with a Na+ gradient. This explains why Na+ is mildly inhibitory to amine transport (which is driven by the H+ gradient) The activity can be demonstrated by using accumulation of 22Na+ in response to a pH gradient that is either imposed by diluting membrane 'ghosts' into alkaline media, or generated by ATP hydrolysis. It can also be monitored indirectly by fluorescence measurements in which the pH inside 'ghost' is monitored by quenching of a fluorescent weak base. This method has been used to monitor Na+ entry into acid-loaded 'ghosts' of H+ entry into methylamine accumulation. The exchanger appears to be reversible and non-electrogenic, with a stoichiometry of 1:1. Using an indirect assay we measured an apparent Km for Na+ of 4.7 mM, and a Ki for amiloride, a competitive inhibitor, of 0.26 mM. Direct assays using 22Na+ suggested a higher Km. Ethylisopropylamiloride was not inhibitory.

Adenosine Triphosphate↗