Electron Transfer across Posterior Pituitary Neurosecretory Vesic]Le Membranes*

Secretory vesicles from the neurohypophysis have a transmembrane electron carrier very similar to that found in adrenal medullary Chromaffin granules. Two different tests show that ascorbic acid contained in the vmicles will reduce an external electron acceptor. First, reduction of cytochrome c or ferricyanide in the medium by a neurosecretory vesicle suspension can be followed spectrophotometrically. Second, the membrane potential (inside positive) generated by electron transfer can be monitored wing the membrane poten- tial-sensitive optical probe Oxonol VI. As in chromaffin granules, this electron transfer is probably media- ted by cytochrome basl. It may function to regenerate internal ascorbic acid and to provide reducing equivalents needed by the intravesicular amidating enzyme.


Electron Transfer across Posterior Pituitary Neurosecretory Vesic]Le Membranes*
(Received for publication, August 1, 1984) Secretory vesicles from the neurohypophysis have a transmembrane electron carrier very similar to that found in adrenal medullary Chromaffin granules. Two different tests show that ascorbic acid contained in the vmicles will reduce an external electron acceptor. First, reduction of cytochrome c or ferricyanide in the medium by a neurosecretory vesicle suspension can be followed spectrophotometrically. Second, the membrane potential (inside positive) generated by electron transfer can be monitored wing the membrane potential-sensitive optical probe Oxonol VI. As in chromaffin granules, this electron transfer is probably mediated by cytochrome basl. It may function to regenerate internal ascorbic acid and to provide reducing equivalents needed by the intravesicular amidating enzyme.
The neurosecretory vesicles in the nerve endings of the posterior pituitary are responsible for the storage and secretion mainly of vasopressin and oxytocin. In recent years, it has become apparent that these vesicles are metabolicaliy quite active. Like many secretory vesicles, the vesicle membrane possesses an inwardly directed H*translocating ATPase (1,2). The membranes also contain a c~ochrome bS1 spectro~hotometrically and i~munolo~cally similar to the one found in adrenal medullary chromaf~n vesicles (3). In addition, the neurosecretory vesicles contain an amidating enzyme which converts the COOHterminus of vasopressin and oxytocin into an amide group during processing of the prohormone (4-6), a modification which is required for the biolo~cal activity of the peptides (8). The amidating enzyme contains copper and uses ascorbic acid as an electron donor (4-7).
Some insight into the functions of the ATPase and cytochrome bssl may be obtained by comparing the neurosecretory vesicle with the chromaf~n vesicle, the catechola~ne storage vesicle of the adrenal medulla. C h~m a f~n vesicles also have a copper~con~in~ng enzyme, dopamine ~-h y d r o x y l~e , which uses ascorbic acid as an electron donor (9, 10). Eipper et d. (5,6) have stressed the parallels between the smidating enzyme found in the pituitary and dopamine @-hydroxylase found in the adrenal. (see also Ref. 3). In the chromaffin vesicle, cytochrome bat is thought to be involved in transfer-* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "ndvertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Supported by an Established Investigatorship from the American
Heart A s s~i a t~o n and by Grant GM-30500 from the Nationai Institutes of Health. ring electrons across the membranes from the cytosol to the vesicle interior to regenerate the internal ascorbic acid needed by dopamine &hydroxylase (11)(12)(13). While the H+-transloeating ATPase in chromaf~n granules ~n c t i o n s in the transport of catecho~amines, it also is thought to power the transport of electrons into the vesicles via c~ochrome b561.
Therefore, by analogy with chromaffin vesicles, neurosecretory vesictes may also require cytochrome bssx as an electron carrier for regenerating intravesicular ascorbic acid needed, in this case, by the amidation enzyme. One of the functions of the ~~-t r a n s l~a t i n~ ATPase may be to drive electrons into the vesicles via c~o c h r o~e bml. For these reasons, we have looked €or a transmembrane electron transfer system in the neurohypophyseal secretory vesicles. The approach we have taken is similar to that taken by Njus et aZ. (12) in studying electron transfer across the chromaffin vesicle membrane, Using intravesicular ascorbic acid as an endogenous electron donor, we have added external electron acceptors (ferricytochrome c and ferricyanide) and looked for electron transfer from the internal ascorbate to the external acceptor.

P r e~~~i o n
of N e u~s e e r e t o~ Vesicles--Bovine posterior pituitaries were obtained from a local siaugh~rhouse and brought to the laboratory on ice. The posterior lobes were trimmed clean of membranes and intermediate iobe tissue, minced, and homogenized in 0.25 M sucrose, 10 mM Hepes,' pH 7.0 (sucrose medium). Neurosecretory vesicles were isolated as described by Russell f14) using the metrizamide gradient procedure. Metrizamide in the purified neurosecretory vesicle fraction was removed by diluting the vesicfes in sucrose medium and centrifuging at l~, O O O X g for 45 min. The pellet was resuspended in a small volume of sucrose medium. The preparation was kept below 4 "C throughout the procedure.
at 24 "C. Absorbance measurements were made at 625 nm referenced to 587 nm.
Assay of Ascorbic Acid-HPLC analysis of ascorbic acid was performed exactly as described previously (15, 16). Ascorbic acid was separated using a 5-pm radial compression CIS column and quantitated using an ultraviolet detector set at 254 nm (Waters Associates). The mobile phase was 0.2% dicyclohexylamine phosphate run at 3.0 ml/min and 1500 p.s.i. Ascorbic acid eluted at 5.6 min. methine oxonol) was obtained from Molecular Probes, Junction City,

RESULTS
Reduction of External Electron Acceptor-Cytochrome c is slowly reduced by suspensions of intact neurosecretory vesicles as indicated by the increase in absorbance a t 550 nm (Fig. 1). The rate of reduction depends upon the composition of the medium: in 140 mM potassium gluconate, between 1.1 and 1.6 neq of cytochrome c are reduced per min per mg of vesicle protein. In 130 mM KzS04, the rate is only about 0.5 neq/min/mg. In these experiments, KCN was added to prevent reduced cytochrome c from being reoxidized by cytochrome oxidase which might be present in mitochondrial membranes contaminating the neurosecretory vesicle preparation. Ascorbate oxidase was also added 5 min prior to the measurement to ensure that cytochrome c was not being reduced by ascorbic acid external to the vesicles. That cytochrome c is being reduced indirectly by an intravesicular electron donor is shown by the difference in reduction caused by intact and lysed vesicles. The rate of cytochrome c reduc- tion does not depend on the pH of the medium when the vesicles are intact (Fig. 2C). When the vesicles are lysed with Triton X-100, however, the rate of reduction is markedly slowed at pH 5.5 (Fig. 2 0 ) . This is expected if ascorbic acid is the electron donor because the reduction potential of the electron couple ascorbate:semidehydroascorbate is shifted toward higher values at more acidic pH (18,19).
To show that the vesicles contain ascorbic acid and that cytochrome c is being reduced by this intravesicular ascorbic acid, we examined cytochrome c reduction by lysed vesicles (Fig. 2A). Whereas ascorbate oxidase does not affect reduction of cytochrome c by intact vesicles (Fig. l), it abolishes over 80% of the reducing capacity of vesicles lysed by treatment with 0.2% Triton X-100. This suggests that ascorbic acid sequestered inside the vesicles is responsible for most and perhaps all of the cytochrome c reduction.
Ferricyanide is also reduced by neurosecretory vesicles (Fig.  3) but at a much faster rate (about 100 neq/min/mg of protein). Moreover, the rate of ferricyanide reduction in sul-  fate medium is comparable to the rate in gluconate (data not shown). Because neurosecretory vesicles reduce ferricyanide fairly quickly, the total extent of ferricyanide reduction can be measured. Intact vesicles reduce about 150 nmol of ferri-cyanide/mg of vesicle protein. Vesicles lysed by treatment with 0.2% Triton X-100 reduce a comparable amount of ferricyanide (Fig. 4). Moreover, ascorbate oxidase destroys most of the ferricyanide-reducin~ capacity of the lysed but not of the intact vesicles, indicating that the reduction is attributable to intravesicular ascorbic acid. Ascorbic Acid Content of Neurosecretory Vesicles-The ascorbic acid content of the neurosecretory vesicles was estimated by four different methods: 1) the rate at which lysed vesicles reduce cytochrome c, 2) the capacity of lysed vesicles to reduce ferricyanide, 3) the capacity of intact vesicles to reduce DCIP, and 4) direct measurement of extracted ascorbic acid by high-perfo~ance liquid chromatography. We compared the rates of cytochrome c reduction caused by lysed neurosecretory vesicles and by known amounts of ascorbic acid (Fig. 2, A and 3). Since the rate of cytochrome c reduction varies with ascorbate concentration (data not shown), the rate can be used to estimate the amount of ascorbate present in the vesicle lysate. In Fig. 2  nmol/mg as shown by the slope in Fig.  4. These values correspond to ascorbate contents of 75 and 58 nmol/mg, respectively. The extent of DCIP reduction was measured by adding neurosecretory vesicles (364 pg of protein) to 25 nmol of DCIP and measuring the decrease in absorbance at 600 nm. Intact vesicles reduce 55 nmol of DCIP/mg of vesicle protein, suggesting that the vesicles contain an equivalent amount of ascorbate. In four preparations of neurosecretory vesicles, the ascorbic acid content was 19.4 +. 6.0 nmol/mg of protein (average +: S.D.) as assayed by high-performance liquid chromatography. The different techniques summarized in Table I all demonstrate the presence of ascorbic acid within the neurosecretory vesicles. The value obtained by HPLC is considerably lower than those estimated by cytochrome c, ferricyanide, or DCIP reduction. This discrepancy may be caused by incomplete extraction of ascorbic acid or by losses occurring during the extraction and assay procedure. In any case, the ascorbate content is between 20 and 70 nmol/mg of protein. Using the value of 3.02 pl/mg of protein for the intravesicular volume (1, 2), this translates into an internal ascorbic acid concentration between 7 and 20 mM.
Membrane Potential Associated with Electron Transfer-Reduction of external electron acceptor by internal ascorbic

Reducing capacity of neurosecretory vesicles
Ascorbate content was measured by HPLC, and DCIP reduction was measured spectrophotometrically as described in the text. Since DCIP reduction and ascorbic acid oxidation are two-electron reactions, reducing capacity was taken as twice the measured values. The extent of ferricyanide reduction was measured from the slope in Fig.  4. Reducing capacity was deduced from the rate of cytochrome c reduction by neurosecretory vesicle lysate (Fig. 2) as described under "Results." acid must involve electron transfer across the vesicle membrane. This charge transfer should depend on the membrane potential. By fueling the inwardly directed H+-translocating ATPase, MgATP creates a membrane potential (inside positive) which should oppose the flow of electrons from internal ascorbate to external ferricyanide under our experimental conditions. In accordance with this, MgATP slows the rate of ferricyanide reduction in intact vesicles (Fig. 5). The protonophore 53-13, which carries H+ across the membrane, should dissipate the membrane potential and accelerate the efflux of electrons. This, too, is observed (Fig. 5).

Method of measu~ment
Electron transfer from internal ascorbic acid to external ferricyanide should itself create a membrane potential. This can be observed using the voltage-sensitive optical probe Oxonol VI (Fig. 6A). When ferricyanide is added to a suspension of neurosecretory vesicles, a membrane potential (inside positive) is generated across the neurosecretory vesicle membrane. This response is indeed a membrane potential since it is abolished by the protonophore S-13. It requires an external electron acceptor since ferrocyanide will not substitute for ferricyanide. It also acquires an internal electron donor since it is not observed in vesicles depleted of ascorbic acid by pretreatment with 2,6-dichloroindop~enol (data not shown). Cytochrome c does not elicit a detectable membrane potential probably because it supports a much slower rate of electron transfer. The magnitude and duration of the electron transferdependent membrane potential increase with the amount of ferricyanide added up to a concentration of about 20 p~ (Fig.  7). This potential, however, is still considerably smaller than that generated by the H+-t~anslocating ATPase (Fig. 6B). DISCUSSION It is apparent that cytochrome c and ferricyanide become reduced when added to a suspension of neurosecretory vesi- Dependence of membrane potential on ferricyanide concentration. Vesicles (404 pg of protein) were suspended in 3 ml of 140 mM potassium gluconate, 1 mM EDTA, 10 mM Pipesflris, 5 p~ Oxonol VI, pH 6.5. Absorbance changes were initiated by adding the following quantitites of ferricyanide: 200 nmol (a), 60 nmol (b), 20 nmol (e), 6 nmol ($1, and 2 nmol (e). S-13 (8 nmol) was added at the time indicated by the arrow.
cles. The evidence that the reducing agent is internal ascorbic acid is as follows. First, cytochrome c and ferricyanide are reduced by intact vesicles treated with ascorbate oxidase and by the vesicle lysate in the absence of the enzyme, but not by the vesicle lysate treated with ascorbate oxidase (Figs. 2 and  4). Second, the amount of ferricyanide reduced by both intact and lysed neurosecretory vesicles correlates roughly with the ascorbic acid content of the vesicles (Fig. 3 and Table I). Third, the rate of cytochrome c reduction by lysed vesicles is consistent with the ascorbate content of the vesicles (Table   I), and the pH dependence of that rate in the vesicle lysate is consistent with the pH dependence of the midpoint reduction potential of ascorbic acid (Fig. 2). Fourth, intact vesicles oxidized with DCIP will not reduce cytochrome c, and the amount of DCIP required to completely oxidize the neurosecretory vesicle suspension correlates with the ascorbic acid content of the vesicles (Table I). Finally, when neurosecretory vesicle contents are subjected to HPLC with electrochemical detection, only one peak of reducing material is seen, and this corresponds to ascorbic acid. We conclude, therefore, that we are observing electron transfer from intravesicular ascorbic acid to the external acceptors, ferricyanide and cytochrome c.
This is the first report that ascorbic acid is present at high concentrations within neurosecretory vesicles. The amount of ascorbate in the vesicles estimated from the reducing capacity (cytochrome c, ferricyanide, and DCIP reduction) is between 45 and 60 nmol/mg of vesicle protein ( Table I). Quantitation of ascorbic acid using an HPLC method yields a value of 19.4 nmol/mg of vesicle protein, which is significantly lower than that estimated by the other techniques. This is probably caused by incomplete extraction of ascorbic acid from the vesicles or by oxidation occurring during the extraction procedure. The possibility that the vesicles contain a significant concentration of another reducing agent is unlikely because more than 80% of the reducing power of the vesicle lysate was abolished by treatment with ascorbate oxidase, and only one peak (ascorbic acid) was seen when the vesicle lysate was subjected to HPLC with electrochemical detection. However, the presence of an intravesicular reducing system that is responsible for cycling ascorbic acid cannot be discounted. Ascorbic acid is not taken up by neurosecretory vesicles.' Thus, the question remains as to whether ascorbic acid is packaged in nascent vesicles or is accumulated in a permeable form (perhaps dehydroascorbic acid) and later reduced within the vesicle by electron transfer.
Several factors affect the rate of electron transfer across the neurosecretory vesicle membrane. First, the composition of the medium seems to have an important effect on the rate of cytochrome c reduction. This is probably attributable to anion binding by cytochrome c (20), since the medium affects the rate, whether the electrons are contributed by intact or lysed neurosecretory vesicles or by pure ascorbic acid. The membrane potential also influences the rate of electron transfer (Fig. 5). Because electron transfer from internal ascorbate to external cytochrome c will generate a membrane potential (inside positive), this membrane potential will inhibit electron transfer. Dissipating the membrane potential by adding the uncoupler S-13 relaxes this constraint on electron transfer and accelerates the rate. MgATP increases the membrane potential (inside positive) and hence slows electron transfer.
Since the membrane potential affects the rate of electron transfer, electron efflux must be electrogenic and should itself create a membrane potential (inside positive). The membrane potential probe Oxonol VI confirms this expectation (Fig. 6). Adding the electron acceptor ferricyanide elicits a membrane potential in neurosecretory vesicles. The electron donor ferrocyanide does not. That the absorbance change reflects an increase in internal positive membrane potential is indicated by the fact that the change is abolished by the uncoupler S- 13. Finally, that this response requires an internal electron donor is indicated by the fact that it cannot be elicited from DCIP-treated vesicles.
We have postulated that cytochrome b56l is responsible for the electron transfer activity. The reasons for this are several. First, this cytochrome in chromaffin vesicles has a midpoint potential of +lo0 to +140 mV (21,22). This is the appropriate potential for an electron carrier mediating electron flow between ascorbic acid (+110 mV at the intravesicular pH of 5.5) * M. Levine and J. T. Russell, unpublished observations. and cytochrome c (+240 mV). Second, both chromaffin vesicles and neurosecretory vesicles have cytochrome b561 (3), and both vesicles catalyze electron transfer. Finally, although the rates of electron transfer in the two membranes have been measured under different conditions, the rates correlate with the relative amounts of cytochrome in the membranes. Chromaffin vesicle membranes contain 7 nmol of cytochrome bssl/mg of membrane protein, while neurosecretory vesicle membranes contain about 1 nmol/mg (3). Chromaffin vesicle ghosts containing 30 mM ascorbic acic reduce 19 g~ cytochrome c at a rate of about 20 nmol/min/mg of membrane p r~t e i n .~ Neurosecretory vesicles catalyze electron transfer at about one-seventh this rate (Fig. 1) given that only 40% of the vesicle protein is in the membrane.
Neurosecretory vesicles offer some advantages over chromaffin vesicles as a system in which to investigate electron transfer. The catecholamines, stored at massive concentrations in chromaffin vesicles, complicate studies of electron transfer. It is particularly difficult to observe ferricyanide reduction by chromaffin vesicles because catecholamines leaking out of the vesicles reduce ferricyanide directly. Studies of electron transfer in neurosecretory vesicles, however, are free of complications caused by this additional reductant.
The role of the electron transfer system in vivo may be to provide reducing equivalents for the intravesicular amidating enzyme. This enzyme uses ascorbic acid as an electron donor, so electrons must be imported from a cytosolic reductant to regenerate the intravesicular ascorbate. This inward electron transfer will be promoted by the membrane potential (inside positive) established by the H+-translocating ATPase. Moreover, because the midpoint reduction potential of ascorbic acid is pH-dependent, ascorbic acid will be more stable at the lower pH prevailing in the vesicle interior so that it will, in fact, take electrons from cytosolic ascorbic acid. Since many biologically important peptide hormones are carboxyl-terminally amidated, this system may also be found in other peptidergic vesicles. Indeed, cytochrome b561 has been identified in pituitary intermediate lobe secretory vesicles as well as in some regions of the brain (3). Thus, this system appears to be common to both peptidergic and aminergic vesicles and perhaps should be sought in other types of vesicles as well.