Adenosine triphosphate-evoked catecholamine release in chromatin granules. Osmotic lysis as a consequence of proton translocation

Casey, Robert P.; Njus, David; Radda, George K.; Sehr, Peter

doi:10.1042/bj1580583

Cited by 112 publications

(43 citation statements)

References 15 publications

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“…As observed In many laboratones further purificatlOn of secretory veslcles on sucrose density gradients gives nse to great fragility when Incubated at 37°C. Even for secretory vesicles Isolated only by differential centnfugatlOn leakage dunng Incubation in 300 mM sucrose media was found to be much hlgher [27] than leakage from veslcles punfied further on Percoll gradients (Fig.2). Secretory veslcles further punfied on sucrose-metnzamlde gradients Incubated at 37°C In 300 mM sucrose release about 50% of their content wlthin 5 min, followed by a slow further release over the next hour [28] _ By contrast secretory vesicles recovered from Percoll gradlents release only 7.5% of total adrenalin after 5 mm of InCUbatlOn at 37°C, with a gradual release of further content withIn 1 h (FIg.2).…”

Section: Discussionmentioning

confidence: 99%

Latent acetylcholinesterase in secretory vesicles isolated from adrenal medulla

Gratzl

Krieger-Brauer

Ekerdt

1981

Biochimica et Biophysica Acta (BBA) - Biomembranes

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A new procedure is described for the preparation of highly purified and stable secretory vesicles from adrenal medulla. Two forms of acetylcholinesterase, a membrane bound form as weil as a soluble form, were found within these vesicles. The secretory vesicles, isolated by differential centrifugation, were further purified on a continuous isotonic PercoU™ gradient. In this way, secretory vesicles were separated from mitochondrial, mierosomal and cell membrane contamination. The secretory vesicles recovered from the gradient contained an average of 2.26 ~mol adrenalin/mg protein. On incubation for 30 min at 37°C in media differing in ionic strength, pH, Mg 2 + and Ca 2 + concentration, the vesicles released less than 20% of total adrenalin. Acetylcholinesterase could hardly be detected in the secretory vesicle fraction when assayed in isotonic media. However, in hypotonie media «400 mosmol/kg) or in Triton X-loO (0.2% final concentration) acetylcholinesterase activity was markedly higher. During hypotonie treatment or when secretory vesicles were specifically lyzed with 2 mM Mg 2 + and 2 mM A TP, adrenalin as weil as part of acetylcholinesterase was released from the vesicular conte nt. On polyacrylamide gel electrophoresis this soluble enzyme exhibited the same electrophoretie mobility as the enzyme released into the perfusate from adrenal glands upon stimulation. In addition to the soluble enzyme a membrane bound form of acetylcholinesterase exists within secretory vesicles, which sediments with the secretory vesicle membranes and exhibits a different electrophoretic mobility compared to the soluble enzyme. It is concluded, that the soluble enzyme found within isolated secretory vesicles is secreted via exocytosis, whilst the membrane-bound form is transported to the cell membrane during this process, contributing to the biogenesis of the cell membrane.

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Section: Discussionmentioning

confidence: 99%

Latent acetylcholinesterase in secretory vesicles isolated from adrenal medulla

Gratzl

Krieger-Brauer

Ekerdt

1981

Biochimica et Biophysica Acta (BBA) - Biomembranes

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“…The integrity of granules during incubations was followed by light scattering measurements at 540 nm [29,30] using a Varian Techtron 635 double-beam spectrophotometer equiped with thermostated cuvettes. In preliminary experiments the extent of lysis was also evaluated by determination of soluble protein released into the incubation mixture.…”

Section: Incorporation Of Exogenous [14c]lysophosphatidylcholine In Imentioning

confidence: 99%

Localization of lysophosphatidylcholine in bovine chromaffin granules

Filgueiras

Besselaar

Bosch

1979

Biochimica et Biophysica Acta (BBA) - Biomembranes

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SummaryOne of the unique features of the chromaffin granule membrane is the presence of about 17 mol% lysophosphatidylcholine. Lysophosphatidylcholine isolated from the granules could be degraded by approx. 94% by lysophospholipase. This result is consistent with chemical analyses data showing that about 9% of this lysophospholipid is l'-alkenyl glycerophosphocholine.The localization of the acylglycerophosphocholine in the chromaffin granule membrane was studied by using pure bovine liver lysophospholipases. In intact granules only about 10% of the total lysophosphatidylcholine was directly available for enzymic hydrolysis. In contrast, when granule membranes (ghosts) were treated with lysophospholipases approx. 60% of the lysophosphatidylcholine was deacylated. These values did not increase after pre-treatment of intact granules or ghosts with trypsin. Added 1-[1-14C]palmitoyl-sn-glycero-3-phosphocholine did not mix with the endogenous lysophosphatidylcholine pool(s) and remained completely accessible to added lysophospholipases.

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“…There is compelling evidence that the ATPase of the chromaffin vesicle membrane is a H+ pump that can increase the intravesicular H+ concentration (6,7,14). One It has been suggested that catecholamine concentrates within the chromaffin vesicle by the same mechanism as methylamine (4,8).…”

Section: Methodsmentioning

confidence: 99%

“…Membrane 6-fold in this preparation. Because there is strong evidence that the membrane-bound ATPase is a H+ pump, the effects on the H+ concentration gradient were investigated.…”

Section: Methodsmentioning

confidence: 99%

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Evidence that catecholamine transport into chromaffin vesicles is coupled to vesicle membrane potential

Holz

1978

Proc. Natl. Acad. Sci. U.S.A.

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The effects of ATP, Mg2+, and various agents on pH gradient, membrane potential, and catecholamine transport across membranes of intact bovine chromaffin vesicles were investigated. Methylamine and thiocyanate (SCN-) distributions across the vesicle membrane were used to estimate the H+ concentration gradient and membrane potential, respectively. The H+ concentration ratio (intravesicular:medium) equals 16 when the medium pH is 6.9 and is unaltered by ATP and Mg2+. In the absence of ATP and Mg2+, the steady-state intravesicular S14CN-concentration is lower than the medium concentration. ATP and Mge+ cause an increased influx and a decreased efflux of SCN-that results in SCN-being concentrated in the vesicles 6-to 8-fold over the medium. The findings are consistent with an ATP,Mg2+-induced potential of approximately 50 mV (intravesicular side positive). Carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), a H+ translocater, and N-ethylmaleimide (NEM), a sulfhydryl reagent, decrease the SCN-ratio and, thus, the membrane potential in the presence of ATP and Mge+. The have no effect on the H+ concentration gradient. The rate ofcatecholamine uptake into vesicles is increased 4-to 6-fold by ATP and Me+. The ATP,Mge+-stimulated uptake is inhibited by FCCP and NEM over the same concentration ranges that reduce the SCNdistribution (membrane potential). FCCP increases and NEM decreases vesicular membrane ATPase activity. Thus, catecholamine uptake is correlated to an inside-positive membrane potential, and not to ATPase activity. If catecholamine uptake is coupled to membrane potential, then a charged species must be involved in the transport mechanism. Reserpine and rotenone inhibit catecholamine influx but have no effect on the H+ electrochemical gradient; they probably act at a step before coupling to the membrane potential (or the H+ electrochemical gradient). Atractyloside, an inhibitor of nucleotide transport, has no effects on catecholamine transport or the H+ electrochemical gradient. Chromaffin vesicles are the catecholaminergic storage vesicles in adrenal medullary cells. They are intimately involved in both secretion (via exocytosis) and intracellular synthesis of catecholamines. Catecholamine transport into vesicles is important for both these processes. Because the in vitro rate of uptake of catecholamines into chromaffin vesicles is increased severalfold by ATP and magnesium (1), it had been thought that the Mg2+-dependent ATPase associated with the vesicle membrane is a catecholamine transport enzyme. Although partial correlations have been obtained between ATPase activity and catecholamine transport (2, 3), lack of a simple quantitative correlation suggests that the ATPase may be indirectly rather than directly involved.Elements of a chemiosmotic system involving the H+ electrochemical gradient are present in chromaffin vesicles (Fig.

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Adenosine triphosphate-evoked catecholamine release in chromatin granules. Osmotic lysis as a consequence of proton translocation

Cited by 112 publications

References 15 publications

Latent acetylcholinesterase in secretory vesicles isolated from adrenal medulla

Latent acetylcholinesterase in secretory vesicles isolated from adrenal medulla

Localization of lysophosphatidylcholine in bovine chromaffin granules

Evidence that catecholamine transport into chromaffin vesicles is coupled to vesicle membrane potential

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