scholarly journals Hydroxytryptamine transport by the bovine chromaffin-granule membrane

1978 ◽  
Vol 170 (3) ◽  
pp. 673-679 ◽  
Author(s):  
J H Phillips
Keyword(s):  
Low Ph ◽  
Ph Gradient ◽  
Chromaffin Granule ◽  
Concentration Gradients ◽  
Granule Membrane ◽  
Chromaffin Granule Ghosts

5-Hydroxytryptamine is accumulated by resealed chromaffin-granule ‘ghosts’ if a pH gradient (acid inside) is imposed across their membranes by preincubating them at low pH. This uptake, like that driven by MgATP, is reserpine- and uncoupler-sensitive. This strongly suggests that catecholamines are taken up by intact granules in response to a pH gradient. In line with this, it is shown that 5-hydroxytryptamine decreases the pH gradient generated in the presence of MgATP, an effect that is inhibited by reserpine; nigericin, which discharges the pH gradient in the presence of K+, inhibits uptake. Permeant anions, however, also inhibit uptake. It is suggested that this may be because they permit equilibration of amine cations directly across the membrane, down concentration gradients.

scholarly journals Stoichiometry of catecholamine/proton exchange across the chromaffin-granule membrane

1980 ◽  
Vol 192 (1) ◽  
pp. 273-278 ◽  
Author(s):  
J H Phillips ◽  
D K Apps
Keyword(s):  
Transport Model ◽  
Proton Exchange ◽  
Ph Gradient ◽  
Chromaffin Granule ◽  
Amine Concentration ◽  
Low Concentrations ◽  
Granule Membrane ◽  
Chromaffin Granule Ghosts

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.

scholarly journals Reduction of membrane-bound dopamine β-hydroxylase from the cytoplasmic surface of the chromaffin-granule membrane

1982 ◽  
Vol 202 (3) ◽  
pp. 759-770 ◽  
Author(s):  
M Grouselle ◽  
J H Phillips
Keyword(s):  
Accumulation Rate ◽  
Chromaffin Granule ◽  
Cytoplasmic Surface ◽  
Dopamine Beta Hydroxylase ◽  
Matrix Surface ◽  
The Matrix ◽  
Granule Membrane ◽  
Membrane Bound ◽  
Chromaffin Granule Ghosts ◽  
Low Ionic Strength

Resealed bovine chromaffin-granule ‘ghosts’ were used for assaying the membrane-bound form of dopamine beta-hydroxylase. Hydroxylation of the substrate tyramine is dependent on its accumulation within the ‘ghosts’, where the active site of the enzyme is located. Free tyramine in the medium is at a low concentration, low ionic strength and a relatively high pH (7.0), so that even in the presence of a reducing agent (co-reductant) the unaccumulated amine is hydroxylated at a negligible rate. ‘Ghosts’ contain an endogenous co-reductant, which is shown to be catecholamine remaining in the membrane itself after granule lysis. Catecholamine that is free in solution in the medium or in the interior of the ‘ghosts’ is not effective as co-reductant, nor is ascorbate, in contrast with the situation with soluble dopamine beta-hydroxylase. Ferrocyanide is an active co-reductant, however, giving a hydroxylation rate approximately equal to the tyramine accumulation rate: it does not enter the ‘ghosts’, nor does the enzyme appear to utilize ferrocyanide sealed inside the ‘ghosts’. A mechanism must therefore exist for transferring electrons across the membrane from the cytoplasmic surface to the matrix surface. NADH is not an electron donor for the enzyme, nor is cytochrome b-561 involved.

scholarly journals Inhibition of adenosine triphosphatase, 5-hydroxytryptamine transport and proton-translocation activities of resealed chromaffin-granule ‘ghosts’

1980 ◽  
Vol 190 (2) ◽  
pp. 273-282 ◽  
Author(s):  
David K. Apps ◽  
James G. Pryde ◽  
Raul Sutton ◽  
John H. Phillips
Keyword(s):  
Molecular Weight ◽  
Atpase Activity ◽  
Adenosine Triphosphatase ◽  
Proton Translocation ◽  
Low Molecular Weight ◽  
Chromaffin Granule ◽  
Low Concentrations ◽  
Tributyltin Chloride ◽  
Granule Membrane ◽  
Chromaffin Granule Ghosts

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 14C-labelled Nbf-Cl labels several proteins; membranes treated with dicyclohexyl[14C]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.

scholarly journals Transport of Ca2+ and Na+ across the chromaffin-granule membrane

1981 ◽  
Vol 200 (1) ◽  
pp. 99-107 ◽  
Author(s):  
J H Phillips
Keyword(s):  
Plasma Membrane ◽  
Exchange Reaction ◽  
Osmotic Shock ◽  
Ruthenium Red ◽  
Chromaffin Granule ◽  
Inner Surface ◽  
Granule Membrane ◽  
Chromaffin Granule Ghosts ◽  
Kinetics Of ◽  
Entry Mechanism

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.

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