The effect of ascorbic acid on the conversion of do- pamine to norepinephrine was investigated in isolated chromaffin granules from bovine adrenal medulla. As-.
THEJOURNALOF BIOLOGICAL CHEMISTRY
Vol. 260, No. 29, Issue of December 15, pp. 15598-15603,1985 Printed in U.S.A.
Ascorbic AcidRegulation of Norepinephrine Biosynthesis in Isolated Chromaffin Granules fromBovine Adrenal Medulla” (Received for publication, April 12, 1985)
Mark Levine, Kyoji Morita, Eliahu Heldman, and Harvey B. Pollard From theLaboraton, of Cell Biology and Genetics, National Instituteof Arthritis, Diabetes, and Digestiue and Kidney Diseases, National InstitutesLf Health, Bethesda, Maryland 20892
The effect of ascorbic acid on the conversion of dopamine to norepinephrine was investigated in isolated chromaffin granules from bovine adrenal medulla. Ascorbic acid was shown to double the rate of [3H]norepinephrine formation from [3H]dopamine, despite no demonstrable accumulation of ascorbic acid into chromaffin granules. The enhancement of norepinephrine biosynthesis by ascorbic acid was dependent on the external concentrations of dopamine and ascorbate. The apparent K, of the dopamine &hydroxylation system for external dopamine was approximately 20 p~ in the presence or absence of ascorbic acid. However, the apparentmaximum velocity of norepinephrine formation was nearlydoubled in thepresence of ascorbic acid. By contrast, the apparent K , and V,,, of dopamine uptake into chromaffin granules were not affected by ascorbic acid. Norepinephrine formation was increased by ascorbic acid when the concentration of ascorbate was 200 pM or higher; a concentration of 2 mM appeared to induce the maximal effect under the experimental conditions used here. The effect of ascorbic acid on conversion of dopamine to norepinephrine required Mg-ATP-dependent dopamine uptakeinto chromaffin granules. In contrast to ascorbic acid, other reducing agents such as NADH, glutathione, and homocysteine were unable to enhance norepinephrine biosynthesis. These data suggest that ascorbic acid provides reducing equivalents forhydroxylation of dopamine despite the lack of ascorbate accumulation into chromaffin granules. These findings imply the functional existence of an electron carrier system in the chromaffin granule which transfers electrons from external ascorbic acid for subsequent intragranular norepinephrine biosynthesis.
exclusively within chromaffin granules (4-6), most newly transported ascorbic acid remains in thecytosol of chromaffin cells (7-9).l In addition, ascorbic acid under several conditions is unable to enter isolated chromaffin granules to provide reducing equivalents to dopamine 6-monooxygenase (10,11). Ascorbic acid, however, does enhance norepinephrine biosynthesis in chromaffin cells (11)and inpermeabilized chromaffin cells? Thus, a mechanism is likely to exist for dopamine /3-monooxygenase to receive electrons from ascorbic acid. One attractive hypothesis for such a mechanism is that ascorbic acid provides reducing equivalents to intragranular dopamine P-monooxygenase via a transmembrane electron carrier system (12). This proposal has been investigated indirectly for dopamine P-monooxygenase using chromaffin granule ghosts (13) and phospholipid vesicles (14). An analogous system which might also require transmembrane transfer of electrons is theascorbate-dependent amidating enzyme found inposterior pituitary vesicles (15). However, the concept of ascorbate contributing reducing equivalents to the interior of the granule by means of an electron transfer process has remained experimentally problematic. For example, the demonstrated direction of electron transfer inchromaffin granule ghosts and pituitary vesicles is opposite to thatnecessary for biosynthesis of norepinephrine (13) and vasopressin (15). Furthermore, it has yetto be shown that a biosynthetic product results from electron transfer in chromaffin granules or pituitary granules. In fact,recent experiments using resealed chromaffin granule ghosts seemed to question explicitly whether ascorbic acid could enhance internal dopamine 0-monooxygenase activity at all (16). These experiments thus appeared to generate doubtabout the role of ascorbic acid as a cofactor for dopamine P-monooxygenase and any interaction of ascorbate with the putative electron transfer system. It therefore seemed important to address the functional relationship between ascorbic acid and electron transfer by Norepinephrine is synthesized from dopamine by the action investigating norepinephrine biosynthesis directly. Inthe of the enzyme dopamine @-monooxygenase(3,4-dihydroxy- present paper we have studied the effect of ascorbic acid on phenethylamine,ascorbate:oxygenoxidoreductase; EC dopamine uptake and subsequent norepinephrine formation 1.14.17.1) (1-3). Dopamine P-monooxygenase requires an in isolated chromaffin granules. We report here that ascorbic electron donor for reduction of associated copper, and ascorbic acid enhancesnorepinephrine biosynthesis in chromaffin acid has been described as the optimalreducing agent in vitro granules in a manner which is very similar to that found in (1-3). However, in chromaffin cells, the mechanism of elec- chromaffin cells, without accumulation of ascorbate into chrotron transfer to dopamine P-monooxygenase from ascorbate maffin granules. These data provide evidence that ascorbate appears to be more complex than thesimple reduction of the action on dopamine P-monooxygenase in vivo may be mediaisolated enzyme. One problem is that dopamine P-monooxy- ted by transfer of electrons across the chromaffin granule genase and newly transported ascorbic acid are localized to membrane. different sites. While dopamine 6-monooxygenase is found
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Morita, K., Levine, M., Heldman, E., and Pollard, H. B. (1985) J. Biol. Chem., in press. Morita, K., Levine, M., and Pollard, H. B. (1985) J. Neurochem., in press.
15598
Biosynthesis MATERIALS AND METHODS
Norepinephrine
15599 nanomoles of norepinephrine synthesized as a function of
nanomoles of dopamine transported. As shown inFig. lC, the Norepinephrine Biosynthesis in Chromaffin Granules-Chromaffin granules were prepared from bovine adrenal medulla by differential ratio of nanomoles norepinephrine formed to nanomoles of centrifugation (17), with modifications as described (18).The density dopamine transported was 0.36 in the presence of ascorbic of the final granule suspensionwas adjusted to A = 5 by dilution with acid and 0.18 in its absence. Thus, over a wide range of 0.3 M sucrose. intragranular dopamine concentrations there was more new Dopamine uptake and norepinephrine biosynthesis were investi- norepinephrine synthesized in thepresence of ascorbate than gated using the isolated chromaffin granule preparation. The stand- in theabsence of ascorbate. ard incubation mixture in a final volume of 3.5 ml contained 0.3 M Ascorbic Acid Accumulation into Chromafin Granules--It sucrose, 50 mM HEPES' (pH 6.9), 33 pg/ml catalase, MgS04 (0.255.0 mM), ATP (0.25-5.0 mM), ascorbic acid (0.2-5.0 mM), and [3H]was conceivable that ascorbic acid could havepromoted condopamine (10-100 PM). The reaction was started by the addition of version of dopamine to norepinephrine bygaining direct 0.25 ml of the granule suspension to the incubation mixture. After access to the granule interior and thus to dopamine 8-monincubating at 37 "C for various times the reaction was terminated by ooxygenase. However, ascorbic acid is not transported into transferring each tube to an ice-water bath and adding 2 ml of ice- chromaffin granules in the presence or absence of 1 mM Mgcold 0.3 M sucrose. The granules were centrifuged immediately at 40,000 X g for 20 min at 4 "C,and washed with 0.3 M sucrose. The ATP (IO,ll),despite coincident dopamine uptake (11).Since granule pellets were lysed by addition of 1ml of 0.4 N perchloric acid the experiments in Fig. 1 were performed in the presence of and freezing and thawing. The lysate was centrifuged at 40,000 X g 2.5 mM Mg-ATP (see Fig. 4), we investigated whether changes for 20 min, and the supernatantfraction was saved for determination in Mg-ATP concentration would induce ascorbate accumulaof catecholamine biosynthesis and dopamine uptake. tion. We found that ascorbic acid was not accumulated in Norepinephrinesynthesis and dopamine uptake were analyzed using a cation exchange resin according to the method of Itoh et al. chromaffin granules incubated with up to 5 mM Mg-ATP for (19) as described (11) or by high pressure liquid chromatography (20). 60 min (Table I). While the actual amount of ascorbic acid Dopamine uptake was calculated to be the sum of measured dopamine dropped slightly during the incubations, the ratio of ascorbic transport andnorepinephrine biosynthesis. acid to epinephrine remained constant. These data demonAscorbicAcid Accumulation in Isolated Chromaffin Granulesstrated that under these conditions there was no substantial Ascorbic acid accumulation in the isolated granule preparation was change in ascorbate content of granules. Thus, the increase determined using the incubation mixturesand techniques as described above. However, after centrifugation of the granules, granule lysis in norepinephrine biosynthesis found in Fig. 1B could not be was accomplished by freeze thawing of the pellets in waterwith 5 mM accounted for by increased internal ascorbate content of the thiourea. Ascorbic acid accumulation was measured by high pressure chromaffin granules incubated in ascorbic acid. The accelerliquid chromatography as described (8). ation of norepinephrine biosynthesis therefore appeared to be Chemicals and Other A~says-3,4-[ring-2,5,6-~H]Dopamine (46.7 mediated by external ascorbic acid. Ci/mmol) was purchased from New England Nuclear. Other chemiNorepinephrine Biosynthesis: Variation of Dopamine, Ascals were of commercially available reagent grade. of external ascorbic Protein determinations were according to themethod of Lowry et corbic Acid, and Mg-ATP-The effect acid on norepinephrine biosynthesis in isolated chromaffin al. (21). Each experimental result presentedthe mean -+ S.D. granules was further investigated by changing different components of the incubation medium. We first studied norepinephrine biosynthesis when the concentration of dopamine Norepinephrine Biosynthesis and Dopamine Uptakein Iso- was varied, as shown in Fig. 2A. At dopamine concentrations lated Chromaffin Granules-IsolatedChromaffin granules of 20 p~ and above, norepinephrine biosynthesis was inwere incubated with 50 p~ radiolabeled dopamine with or creased in the presence of 2 mM ascorbic acid.We calculated without 2 mM ascorbic acid. This concentration of ascorbic the apparent K,,, of the dopamine hydroxylation system for acid was chosen to approximate the intracellular concentra- external dopamine to be 20.6 p~ in the presence of ascorbic tion of ascorbate in chromaffin cells that had been supple- acid and 18.6 p~ without ascorbic acid. These values are mented for 3 h with ascorbic acid (7-9). As shown in Fig. lA, essentially identical. By contrast, the apparent V,, of norchromaffin granules readily transported dopamine regardless epinephrine formation was nearly doubled in thepresence of of the presence or absence of ascorbate. The rateof dopamine ascorbic acid. Thus, ascorbic acid appeared to increase the uptake intochromaffin granules was 0.64 nmol/mg of protein/ apparent V,,, of dopamine hydroxylation without affecting min in the absence of ascorbic acid and 0.66 nmol/mg of the apparentK , of dopamine hydroxylation. protein/min in thepresence of ascorbic acid. Dopamine uptake was also measured under varying concenBy contrast with the independence of dopamine transport trations of dopamine in the presence or absence of 2 mM on ascorbate, norepinephrine biosynthesis was enhanced by ascorbic acid. As shown in Fig. lB, chromaffin granules in- ascorbic acid. As shown in Fig. 2B, dopamine uptake was cubated with ascorbic acid synthesized norepinephrine at 0.24 similar whether or not ascorbic acid was present. The apparnmollmg of protein/min, compared to the rateof 0.10 nmol/ ent X, of catecholamine transport for dopaminewas 67.7 p~ mgof protein/min in granules incubated without ascorbic in thepresence of ascorbic acidand 64.0 p~ without ascorbic of, dopamine transport was also acid. A lag period of approximately 20 min occurred before acid. The apparent V, the enhancement became apparent, and thelinear increase in unaffected by ascorbic acid. Thus, in contrastto norepinephnorepinephrine synthesis was evident by 30 min. No norepi- rine formation from dopamine, the presence of ascorbic acid ,f dopamine uptake. nephrine was converted to epinephrine at all time points did not affect the apparent K,,, or Vo for dopaminein thepresence or under these conditions as measured by high performance In addition, the apparentK,,, absence of ascorbic acid was in close agreement with the liquid chromatography. The increase in norepinephrine biosynthesis in thepresence apparent K,,, for norepinephrine (22) and for isoproterenol of ascorbic acid could not be explained by an increase in (23). The conversion of dopamine to norepinephrine was measdopamine transported into these granules, as seen from Fig. 1A. To express this more clearly, we compared the values of ured using different concentrations of ascorbic acid with 50 p~ dopamine (Fig. 3). Ascorbic acid at all concentrations 'The abbreviation used is: HEPES, 4-(2-hydroxyethyl)-l-pipera- increased norepinephrine formation as a saturable function. zineethanesulfonic acid. We wereable to calculate the apparentK, of norepinephrine RESULTS
15600
Norepinephrine Biosynthesis
A
B @ ASC
20
I
2
0
4 0 6 0 TIME Irnin)
8
0
I
0 12 24 36 48 DOPAMINE UPTAKE (nmolesimg protein)
FIG. 1. A, dopamine uptake in the presence or absence of ascorbic acid is shown. Isolated chromaffin granules were incubated with 50 p~ [3H]dopamineand 2.5 mM Mg-ATP in thepresence (0)or absence (0)of 2 mM ascorbic acid at 37 “C for varying times. Dopamine uptake represents the sum of newly transported dopamine plus newly synthesized norepinephrine and was determined as described in the text. Each point represents the mean f S.D. of three determinations. B, norepinephrine biosynthesis in the presence or absence of ascorbic acid is shown. Norepineprhine biosynthesis was determined in the same chromaffin granules as inA. Isolated chromaffin granules were incubated with 50 p~ [3H]dopamine and 2.5 mM Mg-ATP for different times in thepresence (0)or absence (0) of ascorbic acid. [3H]Norepinephrine synthesis was measured as described under “Materials and Methods.” Each point represents the mean f S.D. of three determinations. C, norepinephrine formation as a function of dopamine uptake in the presence and absence of ascorbic acid is shown. Norepinephrine biosynthesis from B was displayed as a function of dopamine uptake from A in thepresence (0)or absence (0)of ascorbic acid. n = 3. TABLE I Ascorbic acid content in Chromaffin granule preparationsas a function of Mg-ATP concentration Chromaffiri granules were incubated for 60 min with 2.0 mM ascorbic acid and varying concentrations of Mg-ATP. The data is corrected for trapping at 0 time of external ascorbic acid; trapped ascorbic acid was found to represent 59.6 & 2.0%of total ascorbic acid measured. Ascorbic acid and epinephrine were determined as described in the text. n = 3.
of Mg-ATP dopamine uptake was not affected by 2 mM ascorbic acid (data not shown). These data suggest for the experimental conditions used here that Mg-ATP-dependent dopamine uptake into chromaffin granules is necessary for external ascorbic acid to stimulate intragranular norepinephrine biosynthesis. Specificity of Ascorbic Acid for Increasing Norepinephrine Biosynthesis-The specificity of ascorbic acid as a source of reducing equivalents for norepinephrine formationin the Ascorbic Acid Content Mg-ATP . chromaffin granule preparation was examined. Chromaffin rnM nmoles/mg protein prnoles/nmole epiniphrine granules were incubated with physiologic and nonphysiologic reducing agents, and norepinephrine biosynthesis and dopa0 Minutes 60 Minutes 0 Minutes 60 Minutes mine uptake were determined. As seen in Table 11, norepinephrine biosynthesis in the presence of 2 mM ascorbic acid 0 17.320.4 15.0k2.6 36.4r1.1 32.1-+11.1 was 22.86 k 1.10 nmol of norepinephrine/mg of protein/h. By .o 16.9k0.4 16.5k1.5 38.8k1.3 37.0k2.7 1 contrast, norepinephrine biosynthesis in the absence of as15.0k0.8 15.0k0.4 36.3k2.5 33.7r2.1 2.5 corbic acid was 11.02 +- 0.46 nmol of norepinephrine/mg of protein/h. None of the other reducing agents tested,including 15.4k0.8 14.5k1.1 36.9k1.3 29.8k4.8 5.0 NADH, glutathione, homocysteine, dithiothreitol, and thiourea, were effective in increasing norepinephrine formation. formation for external ascorbic acid to be 240.8 p~ by using Dithiothreitol inhibited dopamine 0-monooxygenase activity the rate of norepinephrine formation and theexternal ascor- in chromaffin granules, possibly due to chelation of copper bic acid concentration, since ascorbic acid did not penetrate (25). the chromaffin granule membrane. As shown in Fig. 3, we Corresponding dopamine uptake for these experiments is also found that ascorbic acid at each concentration did not also shown inTable 11. As seen inthesedata, dopamine affect dopamine uptake.Therefore, the ascorbate-induced uptake was relatively constant for each reducing agent. With increases in norepinephrine biosynthesis could not be attrib- lower concentrations (0.5 and 1 mM) of the agents in Table uted toparallel increases in dopamine uptake. 11, the same results were obtained for norepinephrine formaIt remained possible that ascorbic acid stimulated extra- tion anddopamine uptake (data notshown). All of these data granular hydroxylation of dopamine. To test thispossibility, suggest that ascorbic acid specifically increased conversion of chromaffin granules were incubated with 50 FM dopamine dopamine to norepinephrine. There was no inhibition of and 2 mM ascorbic acid with varying concentrations of Mg- dopamine uptake induced by the other reducing agents, which ATP (Fig. 4). Mg-ATP was required for ascorbic acid to could otherwise explain the specificity of ascorbic acid in increase norepinephrine formation. In the absence ofMgincreasing norepinephrine biosynthesis. ATP, ascorbic acid did not increase conversion of dopamine Two other pharmacological agents were tested as controls to norepinephrine (Fig. 4). As expected (24), without Mg- in their ability to influence norepinephrine biosynthesis in ATP, dopamine uptake was reduced 10-fold; in the presence the isolated granule system. N-Ethylmaleimide inhibits the
15601
Norepinephrine Biosynthesis
FIG. 2. Effect norepinephrine pamine uptake centrations of
of ascorbic acid on
formation and dowith varying condopamine. Isolated
chromaffin granules were incubated with different concentrations of [3H]dopamine and 2.5 mM Mg-ATP in the presence (0)or absence (0)of 2 mM ascorbic acidat 37 "Cfor 60 min. Norepinephrine formation (A)and dopamine uptake ( B ) were determined as described in thetext. n = 3.
L 40
20
60
80
100
20
DOPAMINE (@MI
40 60 DOPAMINE (pM)
80
loo
160
100
90
I
I
I
0
1
2
ASCORBIC ACID, mM
FIG. 3. Norepinephrine formation and dopamine uptake in the presence of varying concentrations of ascorbic acid. Isolated chromaffin granules were incubated with 50 I.LM[3H]dopamine, 2.5 mM Mg-ATP, and varying concentrations of ascorbic acid at 37 "C for 60 min. Norepinephrine formation (0)and dopamine uptake (0) were determined as described in the text and displayed as per cent of control. n = 3.
ATPase and subsequent catecholamine transport (25, 26). Thus, in thepresence of N-ethylmaleimide new norepinephrine biosynthesis would not be expected to occur. As predicted, N-ethylmaleimide inhibited dopamine uptake to approximately 5% of control, and norepinephrine biosynthesis was similarly inhibited (Table 11).We also incubated chromaffin granules with diethyldithiocarbamate, an inhibitor of dopamine P-monooxygenase (25). We expected and observed that dopamine uptake would occur but dopamine P-monooxygenase activity would be inhibited. Indeed, norepinephrine biosynthesis was less than 20% of control. The isolated chromaffin granule system thus responded as predicted to inhibition of both dopamine transport and dopamine P-monooxygenase activity and appears to be a satisfactory system for studying norepinephrine biosynthesis.
0
1
2
3
4
5
Mg ATP (mM) FIG. 4. Norepinephrine formation as function of Mg-ATP concentration. Isolated chromaffin granules were incubated with 50 I.LM[3H]dopamine in the presence or absence of 2 mM ascorbic acid with different concentrations of Mg-ATP at 37 "C for 60 min. The difference in the amount of norepinephrine formed in the presence and absence of ascorbic acid was determined as described in thetext. n = 3. DISCUSSION
In this paper we demonstrate that ascorbic acid increases norepinephrine biosynthesis from dopaminein isolated chromaffin granules without changing dopamine uptake, and confirm that ascorbic acid is not transported into chromaffin granules. In contrast to the isolated enzyme (1-3), the effect of external ascorbic acid on the activation of intragranular dopamine P-hydroxylase is necessarilyindirect, and does not apparently involve other components of the cell cytosolsince none are present. It appears likely that thechromaffin granule model system used in these experiments reflects ascorbate action within chromaffin cells. In cultured chromaffin cells incubated in ascorbic acid for several hours, the ascorbate intracellular concentration is approximately 2 mM (7-9). Most of this
15602
Biosynthesis
Norepinephrine
TABLEI1 Effect of various reducing agents on norepinephrine formation and dopamine uptake Isolated chromaffin granules were incubated with 50 PM [3H] dopamine, 2.5 mM Mg-ATP, and thereducing agents as noted for 60 min at 37 "C. Norepinephrine formation and dopamine uptake were determined as described in the text. The concentrations of reducing agents and N-ethylmalemide were 2 mM, and diethyldithiocarbamate was 1 mM. n = 3. Dopamine
Agent nmolimg proteinlhour
Control Ascotbc Acid NADH Glutathione Homocysteine Thiourea Dithhhreitol
11.02t.46 22,86*1.10 12.58+.36 10.04t.26 57.01 10.51t.36 14.04t.41 1.26*.08
% Control nmollmg proteinlhour
1W
57.09tl.M w ne1 .H
237
114 91 95 127 11
% Control
41.2823.95 e2.74 56.55 2 2.02 55.22*.33
116
103 95
83
1W 33 97
66.14t.72
~""""~"""""
Diethyldilhio -
carbamate Nethylmaleimide
1.79e.10 58.09 l.n+.n4.30
16 16
t8.38 2.85
104 8
newly accumulatedascorbicacid is in the cytosol (7-9); Chromaffin cells incubated with ascorbic acid under these conditions have double the rate of new norepinephrine synthesis as compared to controls (11). Similarly, digitonintreated chromaffin cells can be incubated with 2 mM ascorbic acid, without apparent ascorbate entry into chromaffin granules.' These cells also have approximately twice the rate of new norepinephrine synthesis compared to controls? In this paper, wenow demonstrate that chromaffin granules incubated in 2 mM ascorbicacidhavetwice the rate ofnew norepinephrine synthesis compared to controls without ascorbate. Thus, the chromaffin granule system alone appears to explain in a quantitative fashion the datafrom chromaffin cells and digitonin-treated chromaffin cells. These data suggest that the action of ascorbic acid on the isolated granule system accurately reflects the action of ascorbic acidin chromaffin cell preparations. Furthermore, all of these data support the concept that ascorbic acidenhances norepinephrine formation in the adrenal gland in vivo. An initial analysis of our data, however, would appear to diverge from previousconceptions about norepinephrine biosynthesis in chromaffin granules. Since the K, of dopamine @-monooxygenase for ascorbate in vitro is 0.6-1.0 mM (1,27), and theconcentration of internal granular ascorbate has been estimated to be 22 mM (28), it might be considered surprising that external ascorbic acid can increase norepinephrine formation at all, with the apparent K, of norepinephrine formation for ascorbic acid being240 KM. Specifically, since the concentration of intragranular ascorbate wouldbewell in excess of the K , of dopamine @-monooxygenasein vitro for ascorbate (28), external ascorbate might not be expected to regulate norepinephrine biosynthesis. To resolve these apparent discrepancies, it is important to apply our current understanding of norepinephrine biosynthesis in chromaffin cells. We now know that ascorbate can enhance norepinephrine formation in chromaffincells by enhancing dopamine ,f3-monooxygenase activity (11). However, ascorbic acidcannot enter isolated chromaffin granules (10, II), and appears to enter particulate components of chromaffin cells sparingly and over many hours (7-9).' We therefore expected that ascorbic acid would regulate norepinephrine biosynthesis in isolated chromaffin granules, but without entering into the granules. Ourexpectations seem to be correct, as shown in the experiments here. Thus, these
experiments confirm that cytosolic ascorbate regulates dopamine @-monooxygenaseactivity in cultured chromaffin cells. Corollary implications are that dopamine 8-monooxygenase in isolated chromaffin granules may not truly have excess ascorbic acid availableand regeneration of intragranular ascorbate is necessary, that theenzyme is unableto fully utilize intragranular ascorbic acid unlessextragranular ascorbate is also present, or that the in vivo and in vitro K, values of dopamine P-monooxygenase for ascorbate are not comparable. In this regard, the previous estimate of the intragranular ascorbate concentration of 22 mM (28) may be too high (11, 29). Our own estimate is a maximum of approximately 9 mM ascorbic acid ( l l ) , which is in agreement with earlier observations (30). In addition, this value of 9 mM maybe an overestimate (31) because it is also inclusive of ascorbate in mitochondria,lysosomes, and other particulate structures. Therefore, contrary to initial expectations, it is quite possible that intragranular oxidation and depletion of intragranular ascorbic acid could require external ascorbate to regenerate the reduced cofactor. Just as the intragranular ascorbate concentration may be too high, it is also possible that the K, of isolated dopamine P-monooxygenase forascorbate may be lowerthan theK, of dopamine P-monooxygenase within granules. In particular, an endogenous inhibitor of dopamine 0-monooxygenase has been characterized whichcompetitively inhibits ascorbate stimulation of dopamine B-monooxygenase activity in vitro. Such an inhibitor couldincrease the K,,, of dopamine Pmonooxygenaseforascorbicacid (32). There alsomaybe other soluble or membrane bound components of chromaffin granules which could likewise affect the K, of the intragranular enzymefor ascorbate when compared to the isolated enzyme. In addition, it is important to emphasize that the data herein reflect the apparent K, of intragranular norepinephrine formation mediated by external ascorbate. This apparent K,,, may be a different entity than theK, of isolated dopamine /3-monooxygenase for ascorbate. Indeed, in contrast to the isolated enzyme, our data demonstrate that external ascorbate increases norepinephrine formation without itself havingdirect access to theintragranular enzyme, and that only intragranular dopamine undergoes enhanced @-hydroxylationin the presence of exogenous ascorbate. Therefore, at least one intermediate must be involved in transferring electrons from external ascorbate across the chromaffin granule membrane. One likely candidate for involvement in electron transfer is cytochrome bsl. Cytochrome bS1 can transport electrons from insideto outside of artificial phospholipidvesicleswith intravesicular ascorbate as the electron donor(14). This protein is also the presumed insideto-outside electron carrier in preparations using chromaffin granule ghosts loaded with ascorbate (13) and posterior pituitary vesicles containing endogenous ascorbate (15). It is noteworthy that theapparent K, of norepinephrineformation for external ascorbate, 240 p ~ is,similar to theK, of reduction of cytochrome b561 by ascorbate, 340 @A (33). One interpretation of our data, therefore, is that cytochrome b561 might mediate transfer of electrons from external ascorbic acid to internal electron acceptors in chromaffin granules. It may also be tempting to assume that, once transfer of reducing equivalents from ascorbic acid occurs, subsequent norepinephrine formation simply followsthe behavior of isolated dopamine 6-monooxygenaseand ascorbic acid.It might be expected, then, that intragranular ascorbic acid reduces dopamineP-monooxygenase in chromaffin granules. Intragranular ascorbic acid would then be oxidized and could be regenerated by the transfer of reducing equivalents across the
Norepinephrine Biosynthesis granule membrane, perhaps mediated by cytochrome (13). However, our data arealso compatible with alternative explanations. It is possible, for example, that extragranular ascorbic acid or cytochrome bM1reduces other intermediates which are membrane-bound or soluble intragranular components (29). Furthermore, the actual reduction of dopamine P-monooxygenase within chromaffin granules maybe dependent on intragranular ascorbate directly, indirectly, or not all (29). Our current experiments cannot distinguish between these alternatives, nor do the available data permit us to firmly identify the complete pathway of electron transfer to dopamine @-monooxygenase. However,it appears that the different possibilities can now be explored by rigorously comparing the biosynthetic capability of the intact chromaffin granule system to chromaffin granule ghosts, artificial vesicles, and chromaffin cells. REFERENCES ~~
~
1. Levin, E. Y., Levenberg, B., and Kaufman, S. (1960) J. Biol. Chem. 235,2080-2086 2. Levin, E. Y., and Kaufman, S. (1961) J. Biol. Chem. 236,20432049 3. Friedman, S.,and Kaufman, S. (1965) J. Biol. Chem. 240,47634773 4. Kirshner, N. (1957) J. Bwl Chem. 226,821-825 5. Hortnagl, H., Winkler, H., and Lochs, H. (1972) Bwchem. J. 129,187-195 6. Laduron, P. (1975) FEBS Lett. 52,132-134 7. Levine, M., and Pollard, H. B. (1983) FEBS Lett. 158,134-138 8. Levine, M., Asher, A., Pollard, H., and Zinder, 0.(1983) J. Bwl. Chem. 258,13111-13115 9. Diliberto, E. J., Jr., Heckman, G. D., and Daniels, A. J. (1983) J. Bwl. Chem. 258,12886-12894 10. Tirrell, J., and Westhead, E. (1979) Neuroscience 4,181-186
15603
11. Levine, M., Morita, K., and Pollard, H. B. (1985) J. Biol. Chem. 260,12942-12947 12. Wakefield, L. M., Cass, A. E., and Radda, G. K. (1982) Fed. Proc. 41,893 13. Njus, D., Knoth, J., Cook, C., and Kelley, P. M. (1983) J. Biol. Chem. 258,27-31 14. Srivastava, M., Duong, L. T., and Fleming, P. J. (1984) J. Biol. Chem. 259,8072-8074 15. Russell, J. T., Levine, M., and Njus, D. (1985) J, Biol.Chem. 260,226-231 16. Grouselle, M., and Phillips, J. H. (1982) Biochem. J. 202, 759770 17. Taugner, G. (1972) Biochem. J. 130,969-973 18. Pollard, H. B., Zinder, O., Hoffman, P. G., and Nikodejevic, 0. (1976) J. BioL Chem. 251,4544-4550 19. Itoh, T., Matsuoka, M., Nakazima, K., Tagawa, K., and Imaizumi, R. (1962) Jpn. J. Pharmacol. 1 2 , 130-136 20. Kelner. K.. Levine. R. A.. Morita. K.. and Pollard. H. B. (1985) Neuioch;?m. Int. 7, 373-378 21. Lowry, 0.H.,Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Bwl. Chem. 193,265-275 22. Ramu, A., Pazoles, C. J., Creutz, C. E., and Pollard, H. B. (1981) J. Biol. Chem. 256,1229-1234 23. Ramu, A., Levine, M., and Pollard, H. B. (1983) Proc. Natl. Acad. Sci. U.S. A. 80,2107-2111 24. Kirshner. N. (1962) J. Biol. Chem. 237.2311-2317 25. Nagatsu,'T. (i973)'Biochemistry of Catecholamines Chap. I, Uni.
I
,
I
versity Park Press, Baltimore 26. Phillips, J. H. (1974) Biochem. J. 144,311-318 27. Miras-Portugal, M.-T., Aunis, D., and Mandel, P. (1975) Biochimie 57,669-675 28. Ingebretsen, 0.C., Terland, O., and Flatmark, T.(1980) Biochim. Biophys. Acta 628,182-186 29. Levine, M., and Morita, K. (1985) Vitam. Horm. 4 2 , 1-64 30. Terland, O., and Flatmark, T. (1975) FEBS Lett. 59,52-56 31. Hagan, P. (1954) Biochem. J. 56,44-46 32. Duch, D., and Kirshner, N. (1971) Biochim. Biophys. Acta 236, 628-638 33. Flatmark, T., and Terland, 0.(1971) Bwchim.Biophys.Acta 253,487-491
