scholarly journals Ion permeability of isolated chromaffin granules.

1976 ◽  
Vol 68 (6) ◽  
pp. 601-631 ◽  
Author(s):  
R G Johnson ◽  
A Scarpa
Keyword(s):  
Time Course ◽  
Choline Chloride ◽  
Catecholamine Release ◽  
Ionophore A23187 ◽  
Chromaffin Granules ◽  
Ion Permeability ◽  
Chromaffin Granule ◽  
Internal Ph ◽  
Apparent Concentration ◽  
Granule Membrane

The passive ion permeability, regulation of volume, and internal pH of isolated bovine chromaffin granules were studied by radiochemical, potentiometric, gravimetric, and spectrophotometric techniques. Chromaffin granules behave as perfect osmometers between 340 and 1,000 mosM in choline chloride, NaCl, and KCl as measured by changes in absorbance at 430 nm or from intragranular water measurements using 3H2O and [14C]polydextran. By suspending chromaffin granules in iso-osmotic media of various metal ions and selectively increasing the permeability to either the cation or the anion by intrinsically permeable ions or specific ionophores, it was possible to determine by turbidity and potentiometric measurements the permeability to the counterion. These measurements indicate that the chromaffin granule is impermeable to the cations tested (Na+, K+, and H+). Limited H+ permeability across the chromaffin granule membrane was also shown by means of the time course of pH re-equilibration after pulsed pH changes in the surrounding media. The measurement of [14C]methylamine distribution indicates that a significant deltapH exists across the membrane, inside acidic, which at an external value of 6.85 has a value of 1.16. The deltapH is relatively insensitive to changes in the composition of the external media and can be enhanced or collapsed by the addition of ionophores and uncouplers. Measurement at various values of external pH indicates an internal pH of 5.5. Use of the ionophore A23187 indicates that Ca++ and Mg++ can be accumulated against an apparent concentration gradient with calcium uptake exceeding 50 nmol/mg of protein at saturation. These measurements also show that Ca++ and Mg++ are impermeable. Measurement of catecholamine release under conditions where intravesicular calcium accumulation is maximal indicates that catecholamine release does not occur. The physiological significance of the high impermeability to ions and the existence of a large deltapH are discussed in terms of regulation of uptake, storage, and release of catecholamines in chromaffin granules.

scholarly journals Passive ion permeability of the chromaffin-granule membrane

1977 ◽  
Vol 168 (2) ◽  
pp. 289-297 ◽  
Author(s):  
J H Phillips
Keyword(s):  
Osmotic Pressure ◽  
Complex Mixture ◽  
Low Permeability ◽  
Chromaffin Granules ◽  
Ion Permeability ◽  
Chromaffin Granule ◽  
Passive Permeability ◽  
Granule Membrane ◽  
Granule Matrix ◽  
Permeant Anion

‘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.

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

1972 ◽  
Vol 130 (3) ◽  
pp. 825-832 ◽  
Author(s):  
E. K. Matthews ◽  
R. J. Evans ◽  
P. M. Dean
Keyword(s):  
Surface Charge ◽  
Negative Charge ◽  
Single Type ◽  
Chromaffin Granules ◽  
Chromaffin Granule ◽  
Granule Membrane ◽  
Dose Dependent Fashion ◽  
Ph Dependent ◽  
Dose Dependent ◽  
Anionic Surface

1. Chromaffin granules isolated from the bovine adrenal medulla possess an electrophoretic mobility of -1.12μm·s-1·cm·V-1, corresponding to a surface ζ potential of -14.4mV and surface charge density of 1.38×10-6C·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 pKa 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 La3+»Mn2+>Ca2+ >Sr2+>Mg2+>Ba2+. 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 -CO2-, contributed by the α-carboxyl group of constituent proteins, the phospholipid phosphatidylserine and, to a lesser extent, the sialic acid component of glycoproteins.

scholarly journals Bovine chromaffin granule membranes undergo Ca(2+)-regulated exocytosis in frog oocytes.

1992 ◽  
Vol 116 (2) ◽  
pp. 359-365 ◽  
Author(s):  
D Scheuner ◽  
C D Logsdon ◽  
R W Holz
Keyword(s):  
Chromaffin Cells ◽  
Secretory Vesicle ◽  
Xenopus Laevis Oocytes ◽  
Chromaffin Granules ◽  
Chromaffin Granule ◽  
Vesicle Membrane ◽  
Sequence Of Events ◽  
Dopamine Beta Hydroxylase ◽  
Granule Membrane ◽  
Adrenal Chromaffin

We have devised a new method that permits the investigation of exogenous secretory vesicle function using frog oocytes and bovine chromaffin granules, the secretory vesicles from adrenal chromaffin cells. Highly purified chromaffin granule membranes were injected into Xenopus laevis oocytes. Exocytosis was detected by the appearance of dopamine-beta-hydroxylase of the chromaffin granule membrane in the oocyte plasma membrane. The appearance of dopamine-beta-hydroxylase on the oocyte surface was strongly Ca(2+)-dependent and was stimulated by coinjection of the chromaffin granule membranes with InsP3 or Ca2+/EGTA buffer (18 microM free Ca2+) or by incubation of the injected oocytes in medium containing the Ca2+ ionophore ionomycin. Similar experiments were performed with a subcellular fraction from cultured chromaffin cells enriched with [3H]norepinephrine-containing chromaffin granules. Because the release of [3H]norepinephrine was strongly correlated with the appearance of dopamine-beta-hydroxylase on the oocyte surface, it is likely that intact chromaffin granules and chromaffin granule membranes undergo exocytosis in the oocyte. Thus, the secretory vesicle membrane without normal vesicle contents is competent to undergo the sequence of events leading to exocytosis. Furthermore, the interchangeability of mammalian and amphibian components suggests substantial biochemical conservation of the regulated exocytotic pathway during the evolutionary progression from amphibians to mammals.

scholarly journals Phosphatidylinositol kinase. A component of the chromaffin-granule membrane

1973 ◽  
Vol 136 (3) ◽  
pp. 579-587 ◽  
Author(s):  
John H. Phillips
Keyword(s):  
Activation Energy ◽  
Initial Reaction ◽  
Chromaffin Granules ◽  
Chromaffin Granule ◽  
Ph Profile ◽  
Phosphatidylinositol Kinase ◽  
Cytoplasmic Surface ◽  
Granule Membrane ◽  
Kinase A

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 Mg2+-dependent and is stimulated by Mn2+and F−ions. The initial reaction is rapid, with a broad pH profile and a ‘transition’ temperature for its activation energy at 27°C. The apparent Km for ATP is 5μm. ATP, N-ethylmaleimide, Cu2+ions and NaIO4 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.

The membrane of the chromaffin granule

1971 ◽  
Vol 261 (839) ◽  
pp. 293-303 ◽  
Keyword(s):  
Membrane Fusion ◽  
Adrenal Medulla ◽  
Single Unit ◽  
Morphological Characteristics ◽  
Chromaffin Granules ◽  
Unit Membrane ◽  
Chromaffin Granule ◽  
Granule Membrane ◽  
Microsomal Membranes ◽  
Biochemical Analyses

Chromaffin granules of the adrenal medulla are surrounded by a single unit membrane. So far no special morphological characteristics of these membranes have been described. However, biochemical analyses have revealed the special properties of these membranes. The lipids are characterized by a high content of lysolecithin. It has been suggested that this specifically localized phospholipid is essential for the secretion of catecholamines, which involves membrane fusion. The proteins of the granule membrane have also been investigated. Two major components appear to be specific for chromaffin granules of several species. Three enzymes, namely an Mg 2+ -activated ATPase, dopamine β-hydroxylase and cytochrome b-559, are also known to be present in the granule membranes. The membranes of these organelles have no common structural backbone with microsomal membranes.

scholarly journals Spectrophotometric measurements of transmembrane potential and pH gradients in chromaffin granules.

1980 ◽  
Vol 75 (2) ◽  
pp. 109-140 ◽  
Author(s):  
G Salama ◽  
R G Johnson ◽  
A Scarpa
Keyword(s):  
Time Course ◽  
Electrical Potential ◽  
Accurate Method ◽  
Rapid Quenching ◽  
Chromaffin Granules ◽  
Experimental Conditions ◽  
Ph Gradients ◽  
Nernst Potential ◽  
Carbonyl Cyanide ◽  
Granule Membrane

The electrical potential (delta psi) and proton gradient (alpha pH) across the membranes of isolated bovine chromaffin granules and ghosts were simultaneously and quantitatively measured by using the membrane-permeable dyes 3,3'dipropyl-2,2'thiadicarbocyanine (diS-C3-(5)) to measure delta psi and 9-aminoacridine or atebrin to measure delta pH. Increases or decreases in the delta psi across the granular membrane could be monitored by fluorescence or transmittance changes of diS-C3-(5). Calibration of the delta psi was achieved by utilization of the endogenous K+ and H+ gradients, and valinomycin or carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), respectively, with the optical response of diS-C3-(5) varying linearly with the Nernst potential for H+ and K+ over the range -60 to +90 mV. The addition of chromaffin granules to a medium including 9-aminoacridine or atebrin resulted in a rapid quenching of the dye fluorescence, which could be reversed by agents known to cause collapse of pH gradients. From the magnitude of the quenching and the intragranular water space, it was possible to calculate the magnitude of the alpha pH across the chromaffin granule membrane. The time-course of the potential-dependent transmittance response of diS-C3-(5) and the delta pH-dependent fluorescence of the acridine dyes were studied simultaneously and quantitatively by using intact and ghost granules under a wide variety of experimental conditions. These results suggest that membrane-permeable dyes provide an accurate method for the kinetic measurement of delta pH and delta psi in an amine containing subcellular organelle.

The adrenal paraneurone: tubulin organization

1984 ◽  
Vol 62 (5) ◽  
pp. 502-511 ◽  
Author(s):  
M. F. Bader ◽  
F. Bernier-Valentin ◽  
B. Rousset ◽  
D. Aunis
Keyword(s):  
Cell Body ◽  
Intracellular Transport ◽  
Cultured Cells ◽  
Specific Binding ◽  
Secretory Granules ◽  
Immunofluorescent Staining ◽  
Chromaffin Granules ◽  
Two Dimensional Gel Electrophoresis ◽  
Granule Membrane

When chromaffin cells from the bovine adrenal medulla are maintained in culture, they develop neuritelike processes which end with growth-cone-like structures. Chromaffin granules were found to migrate from the cell body to the neurite endings. Thus, the intracellular transport of secretory granules, existing in vivo, seems to occur in an exaggerated way in the cultured cells. These cells offer an excellent model for studying the mechanism of transport, particularly the role of microtubules. By immunofluorescent staining, we observed that tubulin antibodies decorate a complex network visible along the neurites. Colchicine treatment induced the disappearance of this network followed by a return of granules in the cell body and a retraction of neurites. To test the presence of tubulin in the chromaffin granule membrane, we used two-dimensional gel electrophoresis and a radioimmunoassay. Our results indicate that tubulin is not a significant component of chromaffin granules. However, binding experiments show that granule membranes are able to bind tubulin through high affinity binding sites. These results show that microtubules appear involved in neurite formation and probably in granule transport. Tubulin is not an integral constituent of the granule membrane, but is present as a result of a reversible specific binding. This insertion of tubulin into the membrane might represent a step in the association between microtubules and secretory granules.

scholarly journals Calcium metabolism in rat hepatocytes

1978 ◽  
Vol 170 (3) ◽  
pp. 615-625 ◽  
Author(s):  
S Foden ◽  
P J Randle
Keyword(s):  
Cyclic Amp ◽  
Time Course ◽  
Rat Hepatocytes ◽  
Ionophore A23187 ◽  
Total Calcium ◽  
Free Medium ◽  
Adrenergic Agonists ◽  
Calcium Efflux ◽  
Similar Time ◽  
Carbonyl Cyanide

1. The total calcium concentration in rat hepatocytes was 7.9 microgram-atoms/g dry wt.; 77% of this was mitochondrial. Approx. 20% of cell calcium exchanged with 45Ca within 2 min. Thereafter incorporation proceeded at a low rate to reach 28% of total calcium after 60 min. Incorporation into mitochondria showed a similar time course and accounted for 20% of mitochondrial total calcium after 60 min. 2. The alpha-adrenergic agonists phenylephrine and adrenaline + propranolol stimulated incorporation of 45Ca into hepatocytes. Phenylephrine was shown to increase total calcium in hepatocytes. Phenylephrine inhibited efflux fo 45Ca from hepatocytes perifused with calcium-free medium. 3. Glucagon, dibutryl cyclic AMP and beta-adrenergic agonists adrenaline and 3-isobutyl-1-methyl-xanthine stimulated calcium efflux from hepatocytes perifused with calcium-free medium. The effect of glucagon was blocked by insulin. Insulin itself had no effect on calcium efflux and it did not affect the response to dibutyryl cyclic AMP. 4. Incorporation of 45Ca into mitochondria in hepatocytes was stimulated by phenylephrine and inhibited by glucagon and by carbonyl cyanide p-trifluoromethoxyphenylhydrazone. The effect of glucagon was blocked by insulin. 5. Ionophore A23187 stimulated hepatocyte uptake of 45Ca, uptake of 45Ca into mitochondria in hepatocytes and efflux of 45Ca into a calcium-free medium.

Sign in / Sign up

Export Citation Format

Share Document