Ascorbic Acid and Mg-ATP Co-regulate Dopamine &Monooxygenase ...

Vol. 263,No. 36,Issue of December 25, pp. 19353-19362,1988 Printed in U.S.A.

THEJOURNAL OF BIOLOGICAL CHEMISTRY

Ascorbic Acid and Mg-ATP Co-regulate Dopamine &Monooxygenase Activity in Intact Chromaffin Granules* (Received for publication, May 20, 1988)

Mark Levine$, William Hartzells, and Avner Bdolahll From the Laboratory of Cell Biology and Genetics and DigestiveDiseases Brunch, National Instituteof Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland20892

Ascorbic acid and Mg-ATP were found to regulate norepinephrine biosynthesis in intact secretory vesicles synergistically and specifically, using the model system of isolated bovine chromaffin granules. Dopamine uptake into chromaffin granules was shown to be unrelated to the presence of Mg-ATP and ascorbic acid at externaldopamine concentrations of 7.5 and 10 mM. Under these conditions of dopamine uptake, norepinephrine biosynthesis was enhanced 5-6-fold by MgATP and ascorbic acid compared to control experiments with dopamine only. Furthermore, norepinephrine formation wasenhanced approximately %fold by ascorbic acid and Mg-ATP together compared to norepinephrineformationingranules incubated with either substance alone. The action of Mg-ATP and ascorbic acid together was synergistic and independent of dopamine content of chromaffin granules as well as of dopamine uptake. The apparent K , of norepinephrine formation for external ascorbic acid was 376 PM and for externalMg-ATP was 132PM, consistent with the largeramounts of cytosolic ascorbic acid and ATP that areavailable to chromaffin granules. Other physiologic reducing agents were not able to increase norepinephrine biosynthesis in the presence or absence of Mg-ATP. In addition, maximum enhancement of norepinephrine biosynthesis occurred only with the nucleotide ATP and the cation magnesium. The mechanism of the effect of ascorbic acid and Mg-ATP on norepinephrine biosynthesis was investigated and appeared to be independent of a positive membrane potential. The effect was also not mediated by direct action of ADP, ATP, or magnesium on the activity of soluble or particulate dopamine 8-monooxygenase. These data indicate that Mg-ATP and ascorbic acid specifically and synergistically eo-regulate dopamine #?-monooxygenaseactivity in intact chromaffin granules, independent of substrate uptake. Although the mechanism is not known, the data areconsistent with the possibility that the chromaffin granule ATPase mediates these effects.

Ascorbic acid is essential for maximal biosynthesis of the * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisernent” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ To whom correspondence should be addressed Bldg. 8, Rm. 419, NIH, Bethesda, MD 20892. § Supported in part by The Foundation for Nutritional Advancement and The Nature’s Plus Foundation. Present address: Medical College of Virginia, Richmond, VA 23290. ll Present address: Dept. of Zoology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel.

catecholamine norepinephrine in chromaffin tissue (1).The requirement for ascorbic acid isshared by the isolated enzyme dopamine P-monooxygenase (2, 3) and by the enzyme in situ in cultured chromaffin cells and in intact chromaffin granules (1, 4), thesecretory vesicles of the adrenal medulla. However, in situ the requirement for ascorbic acid is more complex as compared to the isolated enzyme. In chromaffin tissue, dopamine P-monooxygenaseappears to be localized to the inner aspect of chromaffin granules (5, 6). Although ascorbic acid is found in chromaffin granules, ascorbic acid does not enter isolated chromaffin granules (7, 8). Instead, reducing equivalents may be transferred from external ascorbic acid across the granule membrane for subsequent norepinephrine biosynthesis (4,9-13). At present, however, our understanding is incomplete of the specific steps that occur between electron transfer from external ascorbic acid and synthesisof norepinephrine within chromaffin granules (11, 14, 15). Electron transfer from external ascorbic acid appears to conserve intragranular ascorbic acid, which might otherwise be consumed as a function of dopamine P-monooxygenase activity (16). Cytochrome is a likely candidate as the membrane protein which mediates electron transfer from external ascorbic acid (10, 13, 17-19). In chromaffin granule ghosts, electron transfer via cytochrome bssl has been suggested to be coupled to membrane potential generated by Mg-ATP (9, 11, 13). By contrast, it is unknown what the role of Mg-ATP maybe in the intact secretory vesicles. It is possible that Mg-ATP may also maintain apositive membrane potential to increase electron transfer, similar to the proposals in chromaffin granule ghosts (9, 11, 13, 20). Another possibility is that Mg-ATP or Mg-ADP might directly regulate the enzyme dopamine P-monooxygenase in granules, as Mg-ADP has been shown to do for the isolated enzyme (21-23). Mg-ATP has also been proposed to be necessary for replenishing intragranular protonsconsumed by turnover of dopamine P-monooxygenase (11). Alternatively, Mg-ATP may simply drive uptake of the substrate dopamine into chromaffin granules for subsequent norepinephrine biosynthesis (4, 15, 24), but have no other role in norepinephrine formation. Due to these different possibilities, information about Mg-ATP requirements for dopamine 8monooxygenase activity in intact chromaffin granules could provide new insight into theentire mechanism of norepinephrine formation mediated by ascorbic acid (9, 11, 14). We have therefore directly tested the roles of Mg-ATP and external ascorbic acid in norepinephrine formation in chromaffin granules. The dataindicate that external ascorbic acid and Mg-ATP are required together for maximal norepinephrine biosynthesis, independent of dopamine uptake. The effects of external ascorbic acid and Mg-ATP on norepinephrine formation are specific and synergistic, but may be unrelated

19353

19354

Ascorbic Acid, Mg-ATP, and Norepinephrine Biosynthesis

to maintenance of membrane potential or direct nucleotide regulation of dopamine @-monooxygenase.

acid were purchased from Sigma. Other reagents were of the highest grade available commercially. RESULTS

MATERIALS ANDMETHODS

Preparation of Chromaffin Granules, Granule Membranes, and Granule Lysates-Chromaffin granules were prepared from bovine adrenal medulla by differential centrifugation in 0.3 M sucrose (25), with modifications as described (26). The density of the granule suspension was adjusted to A540= 5 nm using 0.3 M sucrose, Chromaffin granule lysates were prepared by subjecting chromaffin granules to two cycles of freezing on dry ice and thawing at 25 "C. Immediately after the second thaw, the lysate was placed on ice until used. Unresealed chromaffin granule membranes were prepared by centrifugation of the chromaffin granule lysate at 9500 X g for 30 min. The precipitate was washed once, resuspended in 20 mM acetate, pH 5.5,and placed on ice until used. Norepinephrine Biosynthesis Using Intact Granules, Granule Membranes, and Granule Lysates-Dopamine uptake and norepinephrine biosynthesis were studied in the isolated chromaffin granule preparation as described (4),with minor modifications. The incubation mixture had a final volume of 3.5 ml and contained 0.3 M sucrose, 55 mM HEPES,' pH 6.8, pg/ml 33 catalase, 0-2.5 mM MgS04, 0-2.5 mM ATP, 0-2 mM ascorbic acid, and 0.1-10.0 mM [3H]dopamine. The reaction was started by adding 0.25 ml of the granule preparation, containing approximately 0.4 mg of protein, to the incubation mixture. The reactions were incubated for varying times at 37 "C and terminated by placing the tubes on ice and adding 2 ml of ice-cold sucrose. The tubes were immediately centrifuged at 30,000 X g for 20 min; the resulting pellets were washed twicewith 40 mM HEPES, pH 6.8, in 0.3 M sucrose. The granule pellets werelysed with 0.4 M perchloric acid in 10%acetic acid and frozen at -70 "C for subsequent HPLC analysis. Chromaffin granule integrity was assessed by quantitation of total norepinephrine, epinephrine, anddopamine in every sample pellet by high pressure liquid chromatography with coulometric electrochemical detection. No lysis of chromaffin granules occurred, as indicated by constant catecholamine content for all conditions at all time points in each experiment. The membrane and lysate preparations were also tested for their ability to synthesize norepinephrine. The reaction mixtures of 0.5 ml contained 50 mM sodium acetate, pH 5.5, 5 mM fumarate, 50 pg/ml catalase (500units/ml), 0 or 2 mM MgSO,, 0 or 2 mM MgS04-ADP, 0 or 2 mM MgSO4-ATP, 0 or 2 mM Na-ADP, 0 or 2 mM Na-ATP, 0 or 5 mM ascorbic acid, and 50 p1 of lysate or membranes (2-8pg of protein/tube except as indicated). The reactions were initiated by addition of dopamine without radiolabel, final concentration 5 mM. After 30 min incubation at 37 "C, the reaction was stopped by adding 0.1 ml 2 M perchloric acid in 50% acetic acid to obtain a final concentration of 0.4 M perchloric acid in 10% acetic acid. The samples were frozen at -70 "C for subsequent HPLC analysis. A~says-[~H]Norepinephrine biosynthesis and [3H]dopamine uptake were measured by combining the techniques of high pressure liquid chromatography, coulometric electrochemical detection, and scintillation spectrometry. The mobile phase contained 50 mM trichloroacetic acid, 50 mM sodium phosphate (monobasic), and 0.02% sodium dodecyl sulfate, in 10:90 acetonitrile/water (v:v), adjusted to pH 3.5 with NaOH and used as described previously (1).Unlabeled dopamine, norepinephrine, and epinephrine were also measured by high pressure liquid chromatography with electrochemical detection using the same mobile phase (1). For analysis, samples were thawed, vortexed, and centrifuged for 5 min at 12,000 X g in atabletop Mkrofuge (Beckman); the supernatant was injected without further modification. Protein determinations were according to theBradford method as described for application in adrenal medulla (27). Each experimental result is presented as the mean S.D. for at least three datapoints. Chemi~als-3,4-[ring-2,5,6-~H]Dopamine (40-50 Ci/mmol) was purchased from Du Pont-New England Nuclear and used within 4 weeks of shipment. Dopamine hydrochloride (28,35,45)and ascorbic

*

Dopamine Uptake into Chromaffin Granules-To investigate whether Mg-ATP with and without ascorbic acid enhanced norepinephrine formation in chromaffin granules, it was first necessary to exclude an effect of Mg-ATP and ascorbic acid on dopamine uptake. Dopamine uptake is MgATP-dependent when the external dopamine concentration is in the micromolar range (24). However, we expected that as externaldopamine concentration were increased, dopamine uptake would occur by diffusion, independent of Mg-ATP (24, 28). We therefore incubated chromaffin granules with varying concentrations of [3H]dopamine, in the presence of ascorbic acid alone, Mg-ATP alone, in the presence of both ascorbic acid and Mg-ATP, and without ascorbic acid and Mg-ATP. The concentration of ascorbic acid was 2 mM, and of MgATP was 2.5 mM; both of these concentrations are estimated to be found in the cytosol of chromaffin cells (29-33). As shown in Fig. 1, [3H]Dopamine uptake was independent of Mg-ATP and ascorbic acid at concentrations of 7.5 mM external dopamine or higher. [3H]Dopamine uptake under all four conditions was nearly identical at 10 mM external dopamine. Ascorbic acid alone, as expected, did not enhance I3H] dopamine uptake. These data indicate that in the presence of 10 mM external dopamine, dopamine uptake was similar in the presence and absence of Mg-ATP and ascorbic acid. To measure the time course of dopamine uptake, chromaffin granules were incubated with 10 mM [3H]dopamine for varying times in the presence and absence of Mg-ATP andascorbic acid (Fig. 2 4 ) . These data indicatethat dopamine uptake was similar under all conditions and was linear from 15 to 75 min. Norepinephrine Biosynthesis in ChromaffinGranulesSince dopamine uptake using 10 mM external dopamine was similar with and without Mg-ATP and ascorbic acid for at least 60 min, we used 10 mM external dopamine to determine norepinephrine biosynthesis. [3H]N~repinephrinebiosynthesis was measured as a function of time in the presence of both ascorbic acid and Mg-ATP, ascorbic acid alone, MgATP alone, and neither substance (control). As shown in Fig.

External Dopamine (mM)

FIG. 1. Total [SH]dopamineuptake in the presence and absence of ascorbic acid and Mg-ATP as a function of external dopamine concentration. Isolated chromaffin granules were incubated for 60 min with 1-10 mM external dopamine with radiolabeled tracer. Reaction mixtures contained 2 mM ascorbic acid and 2.5 mM The abbreviations used are: HEPES, 4-(2-hydroxyethyl)-l-piper- Mg-ATP (O),2.5 mM Mg-ATP (A), 2 mM ascorbic acid (V),or buffer azineethanesulfonic acid; FCCP, carbonyl cyanidep-trifluoromethox- alone as control (0).Total [3H]dopamineuptake is the sum of [3H] yphenylhydrazone; HPLC, high pressure liquid chromatography; dopamine content and [3H]norepinephrine biosynthesis as determined by HPLC and scintillation spectrometry. ApH, proton concentration gradient across chromaffin granules.


19355

A

Minutes

C

D

m

t

.a 125

.

_"-- 4"" 25

0

15

30

45

80 Total ['HIDoparnine Uptake lnmol)irng protein

FIG. 2. A , total 13H]dopamineuptake in the presence and absence of ascorbic acid and Mg-ATP as a function of time. Chromaffin granules were incubated with 10 mM external dopamine with radiolabeled tracer for varying amounts of time. Reaction mixtures contained 2 mM ascorbic acid and 2.5 mM Mg-ATP (O),2.5 mM Mg-ATP (A), 2 mM ascorbic acid (V), or buffer alone (0).Total [3H]dopamineuptake is the sum of [3H]dopaminecontent and [3H]norepinephrine biosynthesis as determined by HPLC and scintillation spectrometry. B , ['Hlnorepinephrine biosynthesis in the presence and absence of ascorbic acid and Mg-ATP as a function of time. Chromaffin granules were incubated with 10 mM external dopamine with radiolabeled tracer for varying amounts of time. Reaction mixtures contained 2 mM ascorbic acid and 2.5 mM Mg-ATP (O),2.5 mM Mg-ATP (A), 2 mM ascorbic acid (V), or buffer alone (0).[3H]Norepinephrine biosynthesis was measured by HPLC and scintillation spectrometry. Chromaffin granules are the same as in Fig. 2 A . C, synergistic effect of ascorbic acid and Mg-ATP on [3H] norepinephrine biosynthesis. Chromaffin granules were incubated with 10 mM external dopamine and radiolabeled tracer for varying times. [3H]Norepinephrinebiosynthesis was determined in the presence of 1 mM ascorbic acid and 1 mM Mg-ATP (O),1 mM Mg-ATP (A), or 1 mM ascorbic acid (VI. [3H]Norepinephrine biosynthesis in the presence of buffer alone was subtracted from each time point. Predicted [3H]norepinephrinebiosynthesis as the sum of [3H]norepinephrinebiosynthesis with ascorbic acid alone (V) plus Mg-ATP alone (A) is indicated by (0). D, 3H percent conversion to [3H]norepinephrinefrom [3H]dopamine,as a function of total ['Hldopamine uptake. Chromaffin granules were incubated with 10 mM external dopamine with radiolabeled tracer for varying amounts of time. Percent conversion or biosynthesis of [3H]norepinephrine from the substrate total [3H]dopamine is expressed as a function of total [3H]dopamine uptake. Reaction mixtures contained 2 mM ascorbic acid and 2.5 mM Mg-ATP (O),2.5 mM Mg-ATP (A), 2 mM ascorbic acid (V), or buffer alone (0).Calculations are based on the data in Fig. 2, A and B .

2B, [3H]norepinephrine biosynthesis in the presence of ascorbicacid andMg-ATP was enhanced 4-&foldmore than control and approximately %fold more than in the presence of either ascorbic acid or Mg-ATP alone. Norepinephrine biosynthesis was linear over time. It was also found that dopamine P-monooxygenase activity in chromaffin granules was saturated with respect to external dopamine at concentrations of 7.5-10 mM dopamine, with no granule lysis (data not shown). The results in Fig. 2B raised the question of whether the effects of ascorbic acid and Mg-ATP were additive or synergistic. As shown in Fig. 2C, [3H]norepinephrine biosynthesis in the presence of both ascorbic acid and Mg-ATP was always more than the sum of predicted biosynthesis with ascorbic acid alone plus Mg-ATP alone. These data indicate that ascorbic acid and Mg-ATP together had a synergistic effect on norepinephrine biosynthesis in intactchromaffin granules. The synergy may be physiologicallyrelevent. To test whether the enhancement in norepinephrine biosynthesis could be related to changes in dopamine uptake, we

calculated the percent conversion to norepinephrine from the substrate dopamine, as a function of dopamine uptake (Fig. 2 0 ) . These data demonstrate that theconversion of dopamine to norepinephrine was increased by ascorbic acid and MgATP together, compared with either substance alone or with control. Furthermore, the increased conversion was independent of total dopamine uptake and occurred across the entire range of dopamine uptake. These data indicate that increased norepinephrine biosynthesis induced by ascorbic acid and MgATP together cannot be accounted for simply by increased dopamine uptake. Since regulation of norepinephrine biosynthesis by both ascorbic acid andMg-ATP occurred in intact chromaffin granules, we predicted that the effect would be present at a variety of external dopamine concentrations. Therefore, chromaffin granules were incubated over a range of concentrations of [3H]dopamine, and [3H]norepinephrine biosynthesis was measured. In the presence of Mg-ATP, with or without ascorbic acid, chromaffin granules were incubated with 0.1-12.5 mM external dopamine; concentrations of external dopamine

Ascorbic Acid, Mg-A

19356

TP,Norepinephrine and Biosynthesis

of 15 mM or higher induced granule lysis (data not shown). acid and Mg-ATP was also apparent from Fig. 2 0 . Thus, Without Mg-ATP, in the presence or absence of ascorbic acid, these data suggest that enhancement of norepinephrine bioexternal dopamine concentration was 0.1-30 mM external synthesis by ascorbic acid and Mg-ATP may occur in chrodopamine, with no granule lysis. Since there are differences maffin granules evenwhen dopamine uptake is Mg-ATPin dopamine uptake in the presence and absence of Mg-ATP, dependent and that theenhancement of biosynthesis may be especially at lower dopamine concentrations (see Fig. l),we independent of the requirement of Mg-ATP for dopamine expressed t3H]norepinephrine biosynthesis as a function of transport. 3H total dopamine uptake. As shown in Fig. 3, these data We determined whether the effects of ascorbic acid and demonstrate that [3H]norepinephrine biosynthesis was in- Mg-ATP on norepinephrine biosynthesis could be accounted creased by Mg-ATP and ascorbic acid together, regardless of for by pre-existing intragranular dopamine. Chromaffin gran[3H]dopamine uptake. The effect of Mg-ATP and ascorbic ules were incubated with [3H]dopaminefor various times and acid waspresent across a wide range of t3H]doparnineuptake, intragranular dopamine content was measured as a function without granule lysis. Thus, enhancement of norepinephrine of [3H]dopamineuptake. As shown in Fig. 4,dopamine conformation by ascorbic acid and Mg-ATP was independent of tent of chromaffin granules was almost entirely accounted for the amount of intragranular dopamine, at a variety of external by t3H]dopamine. Therefore, the amount of pre-existing dodopamine concentrations. pamine in chromaffin granules was much too small to influNorepinephrine biosynthesis asa function of dopamine ence the effect of Mg-ATP and ascorbic acid on ['Hlnorepiuptake in Fig. 3 included the range of catecholamine uptake nephrine biosynthesis. which occurs by Mg-ATP-dependenttransport, approxiDopamine P-Monooxygenme Activity: Concentration Demately less than 300 nmol of catecholamine uptake/mg pro- pendence on Mg-ATP and Ascorbic Acid-The dependence of tein (28). In this range, if external dopamine requires Mg- norepinephrine formation on Mg-ATP was determined using ATP for uptake, the additional effect of Mg-ATP on norepi- chromaffin granules incubated with 10 mM dopamine, so that nephrine biosynthesis independent of dopamine transport dopamine uptake was independent of Mg-ATP. Chromaffin cannot be measured directly (4,15). However, dopamine up- granules were incubated with varying concentrations of Mgtake similar to that generated by active transport will occur ATP in thepresence and absence of 1mM ascorbic acid (Fig. in the absence of Mg-ATP if the external dopamine concen- 5 A ) . In the presence of ascorbic acid, Mg-ATP enhanced tration is sufficient for diffusion to occur. By comparison of norepinephrine formation at concentrations of 25 pM and these different conditions for dopamine uptake, it is possible above. As expected, enhanced norepinephrine biosynthesis to determine the effects of Mg-ATP on norepinephrine bio- could not be explained by changes in dopamine uptake, since synthesis as a function of dopamine uptake, even if external dopamine uptake was nearly identical in the presence and dopamine requires Mg-ATP for uptake under some condi- absence of ascorbic acid and at all concentrationsof Mg-ATP tions. As seen in Fig. 3, at amounts of dopamine uptake less (Fig. 5 B ) . The apparent K, of norepinephrine formation for than 300 nmol/mg protein, the combined effect of ascorbic Mg-ATP was 132 p ~which , is similar to the K, of catecholacid and Mg-ATP was still present.A similar effect of ascorbic amine transport for Mg-ATP (28) and to the K, of granule membrane ATPase activity for Mg-ATP (34). Determination of the apparent K,,, of norepinephrine formation for external ascorbic acid was performed in the same manner. Chromaffin granules were incubated with 10 mM

0

100200300400500600700800900 Total L3H1 Dopamine Uptake (nmol)/mg protein/hour

FIG. 3. ['HINorepinephrine biosynthesis as a function of total [3H]dopamine uptake in the presence and absence of PHI Dopamine Uptake (nmol)/mg Protein ascorbic acid and Mg-ATP. Chromaffin granules were incubated FIG. 4. Dopamine content as a function of ['Hldopamine for 60 min with varying concentrations of external dopamine with radiolabeled tracer. Reaction mixtures contained 2 mM ascorbic acid uptake. Chromaffin granules were incubated with 10 mM external and 2.5 mM Mg-ATP (O),2.5 mM Mg-ATP (A), 2 mM ascorbic acid dopamine with radiolabeled tracer from 0 to 75 min. Reaction mix(V), or buffer alone (0).In reaction mixtures with Mg-ATP, with or tures contained 2 mM ascorbic acid and 2.5 mM Mg-ATP (@), 2.5 mM without ascorbic acid, external dopamine concentrations were 0.1- Mg-ATP (A), 2 mM ascorbic acid (V), or buffer alone (0).[3H] 12.5 mM. In reaction mixtures without Mg-ATP, with or without Dopamine uptake was determined using HPLCand scintillation ascorbic acid, external dopamine concentrations were 0.1-30 mM. spectrometry, and dopamine content was measured by HPLC with Total ['Hldopamine uptake is the sum of [3H]dopaminecontent and electrochemical detection, as described under "Materials and Methods." ['Hlnorepinephrine biosynthesis.


19357

B

a L

A

f

a001

T

MQ-ATP

Mg-ATP

(m)

FIG. 5. A , [3H]norepinephrine biosynthesis as a function of external Mg-ATP concentration. Chromaffin granules were incubated for 60 min with 0.025-1.0 mM Mg-ATP and 10 mM dopamine with radiolabeled tracer, in the presence (0)and absence (0)of ascorbic acid. B , total [3H]dopamine uptake as a function of external MgATP concentration. Total [3H]dopamine uptake was determined in chromaffin granules from Fig. 5A, in the of ascorbic acid. Total [3H]dopamine uptake is the sum of [3H]dopamine content presence ( 0 )and absence (0) and [3H]norepinephrinebiosynthesis as determined by HPLC and scintillation spectrometry.

TABLEI Effect of different reducing agents on[3H]norepinephrine biosynthesis Chromaffin granules were incubated with 2.0 m M of the reductants indicated and 10 mM external dopamine with radiolabeled tracer, in the presence and absence of2.5 mM Mg-ATP. [3H]Norepinephrine biosynthesis and total[3H]dopamineuptake were measured a t 60 min by HPLC with scintillation spectrometry. Reducing Agent Acid, Ascorbic

Mg-ATP

Acid Ascorbic Homocysteine. Mg-ATP Homocysteine

-p L 25

x 2

0

50

200 Ascorbic Acid (pM)

loo

500

L loo0

FIG. 6. ['HINorepinephrine biosynthesis as a function of

external ascorbic acid concentration. Chromaffin granules were

Glutathione, Mg-ATP Glutathione NADPH, Mg-ATP NADPH

I'M1 Norepinephrine Biosynthesis lnmollmg proteinlhr)

Total I'M1 Dopamine Uptake lnmollmg protelnlhrl

171.60f11.83

450.46i23.92

58.91f 1.04

382.25f 13.89

62.17f7.06 33.93*1.80

553.18f46.75 379.95k 17.74

57.52f4.34 32.44f2.68

467.24i27.40 353.59f17.32

71.47 f 1.31 40.22 f 3.55

519.87f7.41 381.88f 10.76

incubated for 60 min with 0.025-1.0 mM ascorbic acid and 10 mM dopamine with radiolabeled tracer, in the presence (0)and absence (0) of Mg-ATP.

Thiourea, Mg-ATP

Mg-ATP (no reducing agent)

74.49f 2.28

464.62k17.30

dopamine and varying concentrations of ascorbic acid in the presence and absence of 1 mM Mg-ATP. Norepinephrine formation was increased by concentrations of external ascorbic acid at 50 p~ or above (Fig. 6). These findings could not be accounted for by differences in dopamine uptake at each ascorbic acid concentration in the presence and absence of 1 mM Mg-ATP (data not shown). The apparent K, of norepinephrine formation for external ascorbic acid under these . apparentK , is similar to theK,,, conditions was 376 p ~This of cytochrome b661for ascorbic acid (18) and to the apparent K, of norepinephrine formation for external ascorbic acid when dopamine uptake is dependent on Mg-ATP (4).

Control Ino additions)

40.44f5.13

484.71+X44

Specificity of Norepinephrine Formation for Ascorbic Acid and Mg-ATP-We tested the requirement for ascorbic acid

as thereducing substance in thepresence and absence of MgATP. As shown in TableI, ascorbic acid and Mg-ATPinduced maximal norepinephrine biosynthesis which was independent

Thiourea

117.12f2.67 72.14k3.93

500.99f29.51 405.09*11.38

of dopamine uptake. NADPH, homocysteine, and glutathione had minimal effects on norepinephrine biosynthesis in the presence and absence of Mg-ATP. The inability of these reducing agents to increase norepinephrine biosynthesis could not be explained by changes in dopamine uptake. Dopamine uptake without Mg-ATP or ascorbic acid (control) was similar to dopamine uptake with Mg-ATP and each of the reducing agents. In the presence of Mg-ATP, thiourea produced some enhancement of norepinephrine formation (Table I), which is consistent with earlier observations (4). However, thiourea had no effect on increasing norepinephrine formation in lysed chromaffin granules (data not shown). Therefore, thiourea may only be able to donate extragranularreducing equivalents

19358

Ascorbic Mg-ATP, Acid,

and Norepinephrine Biosynthesis

indirectly to dopamineP-monooxygenase. These dataprovide hancement of norepinephrine formation was absent. Again, additional evidence that an electron transfer system for nor- these observations are consistent with the ability of different epinephrine formation exists in situ and that the likely exter- cations to support ATPase-mediatedprocesses in chromaffin nal electron donoris ascorbic acid. granules (24, 26). The specificity of Mg-ATP for enhancing norepinephrine Possible Mechanisms for Mg-ATP and Ascorbic Acid-deformation was alsoinvestigated.Chromaffin granules were pendent Norepinephrine Formation-There are severalhyincubated with radiolabeleddopamine andvarious nucleotides potheses to account for the effect of Mg-ATP on norepinephand cations, in the presence and absence of ascorbicacid rine formation in the presence of ascorbic acid.One attractive (Table 11). Maximalnorepinephrinebiosynthesis occurred explanation is that a positivemembrane potentialis necessary with Mg-ATP and ascorbic acid. By contrast, Mg-ADP and for maximal synthesis of norepinephrine and that Mg-ATP Mg-AMP were much less effective in increasing norepineph- maintains this requisite membrane potential in intact chrorine biosynthesis. Furthermore, ascorbic acid and the nonevidence using maffingranules (9, 11). Thereisindirect hydrolyzable ATP analog methylene ATP did not increase chromaffin granule ghosts, but not intact granules, to support norepinephrine formation,implying that ATP must hydrobe this possibility. For example, chromaffin granule ghosts inlyzed to affect norepinephrine formation. Of the other nucle- cubated without permeant anions have an increased amount otides tested, Mg-GTP with ascorbic acid had some ability to of reduced cytochrome b561in the presence of Mg-ATP, and enhance norepinephrine biosynthesis. However, the maximal the increase is uncoupler-sensitive (11).These data suggest activity occurredonly with Mg-ATP andascorbic acid. All of that maintenance of a positive membrane potential by Mgthesedataareinagreementwiththeability of different ATP could drive cytochrome bSG1reduction and subsequent nucleotides to stimulate catecholamine transport(24) and to norepinephrine formation. This possibility was investigated generate a transmembrane potential (26), both of which are inintactchromaffingranules using the proton ionophore indices of ATPase activity. Finally, the cation magnesium FCCP. The activityof FCCP as a proton ionophore was first wasalso specifically requiredfor maximalnorepinephrine tested by measuring its effects on Mg-ATP dependent dopabiosynthesis, along with ATP andascorbic acid. After replace- mine uptake in thepresence of 1 mM ascorbic acid. FCCP at ment of magnesium by sodium, zinc, or no cation, the enconcentrations of 1-50 p~ inhibited Mg-ATP-dependent dopamine uptake almostcompletely (Fig. 7A).These dataimply TABLEI1 that FCCP at all concentrations studied effectively decreases Effect of various nucleotides and cationson norepinephrine the chromaffin granule membrane potential despite the presbiosynthesis i n intact chromaffin granules ence of Mg-ATP (35, 45). We then measured the effect of Chromaffin granules were incubated with 10 mM dopamine, radi- FCCP on norepinephrine biosynthesis. Chromaffin granules olabeled tracer, and 0.5 mM of the indicated nucleotides and/or were incubatedwith 10 mM [3H]dopamine, 1 mM ascorbic cations for 60 min, in the presence or absence of 1 mM ascorbic acid. All cations were used as sulfate salts. [3H]Norepinephrine biosyn- acid, and 1-50 p~ FCCP. As shown in Fig. 7B, norepinephrine thesis and [3H]dopamine content were determined by HPLC with biosynthesis was unaffected at every concentration of FCCP. scintillation spectrometry. Furthermore, the amountsof norepinephrine biosynthesis a t 30 minwithandwithoutFCCPin Fig. 7B were nearly Dopamine Norepinephrine identical quantitatively to norepinephrine biosynthesis found Content Biosynthesis Condition nmollmg protelnlhour nmoUmg protoidhour in all the other experiments in this paper. Thus, these data Mg ATP + Ascorbate 181.1Oi2.83 311.43f19.88 suggest that the effect of Mg-ATP and ascorbic acidon Mg ATP - Ascorbate 52.77i3.70 356.56i29.15 norepinephrine biosynthesismay be independentof the action Mg ADP + Ascorbate 98.72f2.84 279.15i12.16 of Mg-ATP to maintaina positive membrane potential. 315.88i 56.84 27.91 f6.43 Mg ADP - Ascorbate It is also possible that the effect of Mg-ATP and ascorbic 256.78i4.46 60.94f1.19 Mg AMP + Ascorbate acid onnorepinephrinebiosynthesiscan be explained by 346.42f16.96 28.62f1.77 Mg AMP - Ascorbate direct action of Mg-ADP on dopamineP-monooxygenase. In Mg Mathylane ATP 30.93 i 5.70 350.31 f38.76 chromaffingranuleghostpreparations, Mg-ADP has been +Ascorbate reported to enhance theVmaxof octopamine formation, as an aloneAscorbate 63.63i1.88 264.74i 15.49 index of dopamine @-monooxygenaseactivity (21,22). For the 367.Mi7.21 24.62i4.76 Mg UTP + Ascorbate isolated enzyme, Mg-ADP decreases the K,,, of dopamine p347.64i 13.14 30.69i1.67 Mg UTP - Ascorbate monooxygenase for ascorbic acid and tyramine without afMg CTP + Ascorbate 101.98f3.72 257.41 i2.43 fecting the Vmaxof the enzyme (23). Since Mg-ADPmay enhance enzyme activity by modifying 228.86i5.78 Mg GTP + Ascorbate 125.74i11.88 294.25i21.25 Mg GTP - Asorbate 37.10i1.43 V,, or K,, we tested theeffects of Mg-ADP and Mg-ATPon norepinephrine formation. In intact chromaffin granules, the Mg GDP + Ascorbate 64.12i3.33 204.33i32.13 concentration of ATP is approximately 20-25%of that of 379.98i29.05 31.18i2.18 Mg +Ascorbate catecholamines (36). Becauseintragranular concentrationsof 355.42i 19.36 32.83i2.16 Mg -Ascorbate ATP may approach 0.16 M (37), we used dilute granule lysate 381.11i18.41 29.14i.a Ns ATP + Ascorbate preparations with catecholamine concentrationsof less than 376.23f 32.47 31.88i3.85 Na ATP - Ascorbate 20 p~ and a calculated ATP concentrationof less than 5 pM. 213.53f25.91 62.90i 4.94 Zn ATP + Ascorbate 363.91 i 19.71 m.93i4.55 Zn ATP- Ascorbate In addition, we used unresealed granule membrane preparations with a catecholamine concentration of less than 1 pM. ATP (no added R2.11i8.00 315.12i21.74 divalent cation1 As shown in Table 111, ascorbic acid without magnesium or +Ascorbate nucleotides increased norepinephrine formation.By contrast, =.mi 13.07 29.74i1.04 ATP (no added divalet cation) ascorbic acid and 2 mM Mg-ATP or Mg-ADP increased the -Ascorbate rate of norepinephrine synthesis only slightly above the rate 27.64il.W 380.04i1.96 Control (no added induced by ascorbic acid alone;this slight enhancement could divsknt cation, be accounted for by the addition of magnesium sulfate and no nucleotide. no ascorbate not of the nucleotides. Since catecholamine transport or dif-

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Ascorbic Acid,Mg-ATP, and Norepinephrine Biosynthesis A

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