Hydroxytryptamine transport by the bovine chromaffin-granule membrane.
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ABSTRACT:
SUBMITTER: Phillips JH
PROVIDER: S-EPMC1183946 | biostudies-other | 1978 Mar
REPOSITORIES: biostudies-other
Similar Datasets
Project description:Bovine chromaffin granules were lysed and their membranes resealed to give osmotically sensitive 'ghosts'. These swell in the presence of salts and MgATP. It is shown that this is due to proton entry accompanied by anions. The rate of swelling depends on the anion present, but swelling is not limited to media containing permeant anions. It is quite marked in solutions of sulphates, phosphates and acetates. It is not uncoupler-sensitive, suggesting that at least one component of swelling is due to coupled proton and anion entry (non-electrogenic proton translocation). Direct measurements of transmembrane pH and potential gradients generated in the presence of MgATP shows that these are rapidly established in sucrose media, and are rather little affected by the presence of salts. They contribute roughly equally to the total protonmotive force. The potential gradient is establihsed very rapidly, but the pH gradient is generated over several minutes. The gradients are not completely dissipated by uncoupler, and it is shown that, in media containing sulphate but no permeant anion, sulphate can be taken up by the 'ghosts'. There thus appear to be two mechanisms of proton translocation across the membrane, both dependent on ATP hydrolysis: an electrogenic transfer of protons, and proton movement linked to an anion transporter of broad specificity.
| S-EPMC1183945 | biostudies-other
Project description:Bovine chromaffin-granule ghosts accumulate 45Ca2+ in a temperature- and osmotic-shock-sensitive process; the uptake is saturable, with Km 38 microM and Vmax. 28 nmol/min per mg at 37 degrees C. Entry occurs by exchange with Ca2+ bound to the inner surface of the membrane. It is inhibited non-competitively by Na+, La3+ and Ruthenium Red (Ki 10.7 mM, 7 microM and 2 microM respectively), and competitively by Mg2+ (ki 0.9 mM). Uptake was not stimulated by ATP. Na+ induces Ca2+ efflux; Ca2+ can re-enter the ghosts by a process of Ca2+/Na+ exchange. La3+ inhibits Ca2+ efflux during Ca2+-exchange, and Ca2+ efflux induced by Na+, suggesting that Ca2+ uptake and efflux, and Ca2+/Na+ exchange, are catalysed by the same protein. Na+ enters ghosts during CA2+ efflux, but the kinetics of its entry are not exactly similar to the kinetics of Ca2+ efflux. Initially 1-2 Na+ enter per Ca2+ lost, but at equilibrium 3-4 Na+ have replaced each Ca2+. There is no evidence that either Ca2+ uptake or efflux by Ca2+/Na+ exchange is electrogenic, suggesting that the stoichiometry of exchange is Ca2+/2Na+. This exchange reaction may have a role in depleting cytoplasmic Ca2+ after depolarization-induced Ca2+ entry through the adrenal medulla plasma membrane; there is some evidence that there may be an additional entry mechanism for Na+ across the granule membrane.
| S-EPMC1163507 | biostudies-other
Project description:1. Highly purified resealed chromaffin-granule ;ghosts' were assayed for ATPase and ATP-driven H(+)-translocation and 5-hydroxytryptamine-uptake activities, and for 5-hydroxytryptamine uptake driven by an imposed transmembrane H(+)-gradient. The effects of several inhibitors on these activities were studied. 2. Dicyclohexylcarbodi-imide inhibits all of these activities, but not in parallel; at low concentrations it decreases the permeability of the membrane to protons. 3. 4-Chloro-7-nitrobenzofuran (Nbf-Cl) and silicotungstate inhibit ATP-dependent activities, without effect on 5-hydroxytryptamine uptake driven by an imposed H(+)-gradient. 4. Tributyltin chloride inhibits all of the activities. 5. Treatment of the ;ghosts' with low concentrations of urea inhibits 5-hydroxytryptamine uptake and ATP-dependent generation of a transmembrane H(+)-gradient, without inhibiting ATPase activity. 6. Nbf-Cl and silicotungstate are without effect on the rate of leakage of 5-hydroxytryptamine from preloaded ;ghosts', whereas dicyclohexylcarbodi-imide and tributyltin chloride accelerate the rate of leakage. 7. Treatment of the membranes with (14)C-labelled Nbf-Cl labels several proteins; membranes treated with dicyclohexyl[(14)C]carbodi-imide are labelled predominantly in a protein of low molecular weight, which may be analogous to the mitochondrial H(+)-conducting proteolipid. 8. It is concluded that Nbf-Cl and silicotungstate inhibit the H(+)-translocating ATPase of the granule membrane; that dicyclohexylcarbodi-imide inhibits the ATPase, and inhibits 5-hydroxytryptamine accumulation by accelerating leakage of the amine; and that the effects of tributyltin chloride are due to inhibition of the ATPase, and collapse of the transmembrane H(+)-gradient through OH(-)-anion exchange.
| S-EPMC1162091 | biostudies-other
Project description:Bovine adrenomedullary cells in culture have been used to study the role of myosin in vesicle transport during exocytosis. Amperometric determination of calcium-dependent catecholamine release from individual digitonin-permeabilized cells treated with 3 microM wortmannin or 20 mM 2,3-butanedione monoxime (BDM) and stimulated by continuous as well as repetitive calcium pulses showed alteration of slow phases of secretion when compared with control untreated cells. The specificity of these drugs for myosin inhibition was further supported by the use of peptide-18, a potent peptide affecting myosin light-chain kinase activity. These results were supported also by studying the impact of these myosin inhibitors on chromaffin granule mobility using direct visualization by dynamic confocal microscopy. Wortmannin and BDM affect drastically vesicle transport throughout the cell cytoplasm, including the region beneath the plasma membrane. Immunocytochemical studies demonstrate the presence of myosin types II and V in the cell periphery. The capability of antibodies to myosin II in abrogating the secretory response from populations of digitonin-permeabilized cells compared with the modest effect caused by anti-myosin V suggests that myosin II plays a fundamental role in the active transport of vesicles occurring in the sub-plasmalemmal area during chromaffin cell secretory activity.
| S-EPMC1223018 | biostudies-literature
Project description:The bovine homologue of p65, a calmodulin-binding protein located in the membranes of synaptic vesicles and endocrine secretory granules, has been studied by the use of monoclonal antibodies directed against this antigen and against dopamine beta-mono-oxygenase. The protein (apparent molecular mass 67 kDa; pI = 5.5-6.2) is partially degraded by treatment with neuraminidase or endoglycosidase F. Trypsin treatment of intact adrenal chromaffin granules or of granule membranes releases a soluble 39 kDa fragment of p65 which corresponds to the whole of its cytoplasmic domain. This domain contains both the epitope for the monoclonal antibody cgm67 and the calmodulin-binding site. The 20 amino acids at the N-terminus of this fragment are identical to part of the rat p65 sequence.
| S-EPMC1151502 | biostudies-other
Project description:'Ghosts' of bovine chromaffin granules, in which the complex mixture of proteins and solutes normally found in the granule matrix is replaced by buffered sucrose are osmotically sensitive. They shrink when the osmotic pressure of the suspension medium is increased, and swell if solute entry is facilitated by the addition of ionophores. Swelling in the presence of ionophores has been used to investigate the passive ion permeability of these membranes. They have a very low permeability to K+ ions (of the order of 10(-10) cm/s); their permeability to protons, Na+ and choline ions is too low to be detected by these methods. Their passive permeability to anions decreases in the order: CNS- greater than I- greater than CCl3CO2- greater than Br- greater than Cl- greater than SO4(2)- greater than CH3CO2-, HCO3-, F-, PO4(3)- the permeability to hiocyanate being of the order of 10(-7) cm/s. Coupled proton and anion entry is extremely slow, except for weak acids. Fluoride, unexpectedly, also appears to enter rapidly when proton/K+ exchange is facilitated by nigericin. In the presence of K+ salts, nigericin, like valinomycin, induces lysis of intact granules, an effect that is not dependent on the presence of a permeant anion, but is dependent on the pH gradient across the membrane.
| S-EPMC1183763 | biostudies-other
Project description:Phosphorylation of bovine chromaffin granules by ATP leads to the formation of diphosphoinositide in the granule membrane. Both phosphatidylinositol kinase and its substrate are components of this membrane, and triphosphoinositide is not formed under the conditions of the assay. The reaction is Mg(2+)-dependent and is stimulated by Mn(2+) and F(-) ions. The initial reaction is rapid, with a broad pH profile and a ;transition' temperature for its activation energy at 27 degrees C. The apparent K(m) for ATP is 5mum. ATP, N-ethylmaleimide, Cu(2+) ions and NaIO(4) are inhibitory. The phospholipids of chromaffin-granule membranes have been analysed: 6.8% of the lipid P is found in phosphatidylinositol, and only 2-3% in phosphatidylserine. Comparison of the rate of phosphorylation of intact and lysed granules suggests that the sites for phosphorylation are on the outer (cytoplasmic) surface of the granules, and diphosphoinositide may therefore make an important contribution to the charge of the chromaffin granule in vivo.
| S-EPMC1165992 | biostudies-other
Project description:Resealed chromaffin-granule ;ghosts' were used to study the steady-state kinetics of catecholamine transport. The pump has a high affinity for (-)-noradrenaline, (-)-adrenaline, tyramine and 5-hydroxytryptamine (serotonin), but a lower affinity for (+)-noradrenaline. The measured rates of incorporation do not conform to Michaelis-Menten kinetics, but affinity constants for the former substrates are in the range 8-18mum. Reserpine is a potent inhibitor. Incorporation as a function of ATP concentration also fails to show simple kinetics; the affinity constant for ATP is deduced to be about 3mm at 1mm-MgCl(2). Adenylyl (betagamma-methylene)diphosphonate is a competitive inhibitor at low concentrations, but inhibits more strongly at high concentrations. The pump has a transition temperature at 29 degrees C and does not seem to be identical with the Mg(2+)-stimulated adenosine triphosphatase of chromaffin granules.
| S-EPMC1168499 | biostudies-other
Project description:Catecholamines are accumulated by bovine chromaffin-granule "ghosts" in the presence of MgATP at 25 degrees C. With low concentrations of catecholamine, ratios of internal to external amine concentration of up to 20 000 were obtained. These values fit well with a transport model in which amine accumulation is both electrogenic and dependent on a pH gradient across the membrane.
| S-EPMC1162331 | biostudies-other
Project description:Neuroendocrine chromaffin cells represent an excellent model to study the molecular mechanisms associated with the exo-endocytotic cycle of neurotransmitter release. In this study, EGFP-Lifeact and confocal microscopy has been used to analyze the re-organization of the cortical F-actin cytoskeleton associated to organelle transport during secretion with unprecedented detail. In these cells secretory events accumulate in temperature-sensitive and myosin II-dependent F-actin expansions and retractions affecting specific regions of the sub-membrane space. Interestingly, not only vesicles but also mitochondria are transported toward the plasmalemma during these expansions. Simultaneously, we found F-actin cytoskeletal retraction withdraws vesicles from the sub-plasmalemmal space, forming novel empty internal spaces into which organelles can be transported. In addition to these well-coordinated, F-actin-myosin II dependent processes that drive the transport of the majority of vesicles, fast transport of chromaffin vesicles was observed, albeit less frequently, which used F-actin comet tails nucleated from the granular membrane. Thus, upon cell stimulation F-actin structures use diverse mechanisms to transport organelles to and from the membrane during the exo-endocytotic cycle taking place in specific areas of cell periphery.
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