Enhancement of norepinephrine biosynthesis by ascorbic acid in cultured bovine chromaffin cells

Ascorbic acid donates electrons to dopamine beta-monooxygenase during the hydroxylation of dopamine to norepinephrine in vitro. However, the possible role of ascorbic acid in norepinephrine biosynthesis in vivo has not been defined. We therefore investigated the effect of newly accumulated ascorbic acid on catecholamine biosynthesis in cultured bovine adrenal chromaffin cells. Cells supplemented for 3 h with ascorbic acid accumulated 9-fold more ascorbic acid than found in control cells. Under these conditions, the cells loaded with ascorbate were found to double the rate of norepinephrine biosynthesis from [14C]tyrosine compared to control. By contrast, the amounts present of [14C] 3,4-dihydroxyphenylalanine and [14C]dopamine synthesized from [14C]tyrosine were unaffected by the preloading of ascorbic acid. Ascorbate preloaded cells incubated with [3H]dopamine also showed a similar increase in the rate of norepinephrine formation, without any change in dopamine transport into the cells. Thus, these data were consistent with ascorbate action at the dopamine beta-monooxygenase step. In order to determine if ascorbate could interact directly with dopamine beta-monooxygenase localized within chromaffin granules, we studied whether isolated chromaffin granules could accumulate ascorbic acid. Ascorbic acid was not transported into chromaffin granules by an uptake or exchange process, despite coincident [3H]dopamine uptake which was Mg-ATP dependent. These data indicate that ascorbic acid does augment norepinephrine biosynthesis in intact chromaffin cells, but by a mechanism that might enhance the rate of dopamine hydroxylation indirectly.     

Ascorbic acid within chromaffin granules. In situ kinetics of norepinephrine biosynthesis

Ascorbic acid requirements for norepinephrine biosynthesis were investigated in intact bovine chromaffin granules using the physiologic substrate dopamine and a novel coulometric electrochemical detection high pressure liquid chromatography system for ascorbic acid. 10 mM external dopamine, 1 mM Mg-ATP, and 1 mM ascorbic acid produced maximal norepinephrine biosynthesis without granule lysis. When external ascorbic acid was omitted, intragranular ascorbic acid was consumed in a 1:1 ratio with respect to norepinephrine biosynthesis. The initial concentration of intragranular ascorbic acid was 10.5 mM, which was depleted in stepwise fashion to 15 lower concentrations over the range of 9.2-0.2 mM. Chromaffin granules containing these varying concentrations of intragranular ascorbic acid were then incubated with 1 mM exogenous ascorbic acid, and norepinephrine biosynthesis from dopamine was determined. The apparent Km of norepinephrine biosynthesis for intragranular ascorbic acid was 0.57 mM by Eadie-Hofstee analysis and 0.68 mM by Lineweaver-Burk analysis. These data indicate that intragranular ascorbic acid is available and required for norepinephrine biosynthesis, that ascorbic acid is a true co-substrate for dopamine beta-monooxygenase, and that intragranular ascorbic acid is maintained by extragranular ascorbic acid. Continued norepinephrine biosynthesis in granules is dependent on both intragranular and extragranular concentrations of the vitamin. Furthermore, in situ kinetics of dopamine beta-monooxygenase for ascorbic acid may be most accurately determined using intact granules and the true physiologic substrate.     

Ascorbic acid regulation of norepinephrine biosynthesis in isolated chromaffin granules from bovine adrenal medulla

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 Km of the dopamine beta-hydroxylation system for external dopamine was approximately 20 microM in the presence or absence of ascorbic acid. However, the apparent maximum velocity of norepinephrine formation was nearly doubled in the presence of ascorbic acid. By contrast, the apparent Km and Vmax 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 microM 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 uptake into 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 for hydroxylation 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.     

Role of Mg-ATP in Norepinephrine Biosynthesis in Intact Chromaffin Granules

Abstract: Dopamine β-mdriooxygenase converts dopamine to norepinephrine in intact chromaffin granules using intragranular ascorbic acid as a cosubstrate. Mg-ATP with external ascorbic acid is required for maximal norepinephrine biosynthesis. Mechanisms to explain these requirements were investigated specifically using intact granules. The effect of Mg-ATP was independent of membrane potential (ΔΨ) because norepinephrine biosynthesis was unchanged whether ΔΨ was positive or collapsed. Furthermore, the effect of Mg-ATP was independent of absolute intragranular and extragranular pH as well as the pH difference across the chromaffin granule membrane (ΔpH). Nevertheless, norepinephrine biosynthesis was inhibited by N -ethylmaleimide, 4-chloro-7-nitrobenzofurazane, and N , N -dicyclohexylcarbodiimide, specific inhibitors of the secretory vesicle ATPase that may directly affect proton pumping. Biosynthesis occurred normally with other ATPase inhibitors that do not inhibit the ATPase in secretory vesicles. The data indicate that the effect of Mg-ATP with ascorbic acid is mediated by the granule membrane ATPase but independent of maintaining ΔΨ and ApH. An explanation of these findings is that Mg-ATP, via the granule ATPase, may change the rate at which protons or dopamine are made available to dopamine β-monooxygenase.     

Stimulatory Effect of Ascorbic Acid on Norepinephrine Biosynthesis in Digitonin-Permeabilized Adrenal Medullary Chromaffin Cells

The regulatory role of ascorbic acid in norepinephrine biosynthesis was studied using digitonin-permeabilized chromaffin cells. When permeabilized chromaffin cells were incubated with [3H]3,4-dihydroxyphenylethylamine ([3H]dopamine) in calcium-free medium, the amounts of radioactive dopamine and norepinephrine measured in the cell fraction were increased as a function of incubation time and dopamine concentration. Both the accumulation of dopamine and the formation of norepinephrine were shown to require the presence of Mg-ATP in the medium. These results indicate that the permeabilization of chromaffin cells by digitonin treatment does not disrupt the functions of chromaffin granules, including dopamine uptake, norepinephrine formation, and storage of these amines. Using this permeabilized cell system, the effect of ascorbic acid on the rates of dopamine uptake and hydroxylation was investigated. The formation of norepinephrine was stimulated by ascorbic acid at concentrations of 0.5-2 mM in the presence of Mg-ATP. By contrast, dopamine uptake was not affected by the presence or absence of ascorbic acid in the medium. These findings provide evidence that ascorbic acid may stimulate the conversion of dopamine to norepinephrine by increasing dopamine beta monooxygenase activity rather than by increasing the substrate supply of dopamine. These observations also suggest that the rate of norepinephrine biosynthesis in adrenal medullary cells may be regulated by the concentration of ascorbic acid within the cell cytoplasm.     

Ascorbic acid specifically enhances dopamine beta-monooxygenase activity in resting and stimulated chromaffin cells

Ascorbic acid enhancement of norepinephrine formation from tyrosine in cultured bovine chromaffin cells was characterized in detail as a model system for determining ascorbate requirements. In resting cells, ascorbic acid increased dopamine beta-monooxygenase activity without changing tyrosine 3-monooxygenase activity. [14C]Norepinephrine specific activity was increased by ascorbic acid, while [14C]dopamine specific activity was unchanged. Dopamine content, dopamine biosynthesis, tyrosine content, and tyrosine uptake were also unaffected by ascorbic acid. Furthermore, increased norepinephrine formation could not be attributed to changes in norepinephrine catabolism. Enhancement of dopamine beta-monooxygenase activity was specific for ascorbic acid, since other reducing agents with higher redox potentials were unable to increase norepinephrine formation. The specific effect of ascorbic acid on enhancement of norepinephrine formation was also observed in chromaffin cells stimulated to secrete with carbachol, acetylcholine, veratridine, and potassium chloride. In stimulated cells with and without ascorbate, there were no differences in dopamine content, tyrosine uptake, dopamine specific activity, and norepinephrine catabolism. These data indicate that, under a wide variety of conditions, only one catecholamine biosynthetic enzyme activity, dopamine beta-monooxygenase, is specifically stimulated by ascorbic acid alone in cultured chromaffin cells. This model system exemplifies a new approach for determining ascorbic acid requirements in cells and animals.     

Semidehydroascorbic acid as an intermediate in norepinephrine biosynthesis in chromaffin granules.

We investigated whether semidehydroascorbic acid was an intermediate in norepinephrine synthesis in chromaffin granules and in electron transfer across the chromaffin granule membrane. Semidehydroascorbic acid was measured in intact granules by electron spin resonance. In the presence of intragranular but not extragranular ascorbic acid, semidehydroascorbic acid was formed within granules in direct relationship to dopamine beta-monooxygenase activity. However, semidehydroascorbic acid was not generated when granules were incubated with epinephrine instead of the substrate dopamine, with dopamine beta-monooxygenase inhibitors, without oxygen, and when intragranular ascorbic acid was depleted. Experiments using the impermeant paramagnetic broadening agents [K3 [Cr(C2O4)3].3H2O] and Ni(en)3(NO3)2 provided further evidence that semidehydroascorbic acid was generated only within granules. We also investigated semidehydroascorbic acid formation in the presence of intragranular and extragranular ascorbic acid. Under these conditions, semidehydroascorbic acid was formed on both sides of the granule membrane, and formation was coupled to dopamine beta-monooxygenase activity. These data indicate that dopamine beta-monooxygenase is reduced by single electron transfer from intragranular ascorbic acid, that transmembrane electron transfer occurs by single electron transfer, and that transmembrane electron transfer is directly coupled to formation of intragranular semidehydroascorbic acid via dopamine beta-monooxygenase activity.     

Evidence for an ascorbate shuttle for the transfer of reducing equivalents across chromaffin granule membranes

Adrenal chromaffin granules must shuttle reducing equivalents from the cytosol inward to reduce ascorbic acid oxidized during norepinephrine biosynthesis by intragranular dopamine-beta-hydroxylase. A transmembrane electron shuttle between the external (cytosolic) and intragranular ascorbate pools was demonstrated in vitro in intact bovine chromaffin granules undergoing tyramine- or dopamine-stimulated dopamine-beta-hydroxylase turnover. Incubation of intact chromaffin granules with tyramine results in a time-dependent decrease in reduced intragranular ascorbate and production of octopamine. The rate of ascorbate oxidation is a function of the extragranular concentrations of tyramine over the range 50 microM to 2 mM and is 95% inhibited by addition of the dopamine-beta-hydroxylase inhibitor disulfiram. The stoichiometry of octopamine synthesized/ascorbate oxidized closely approximates unity. The presence of extragranular dopamine also induces oxidation of intragranular ascorbate which is inhibited by blocking dopamine transport with reserpine. On the other hand, incubation with octopamine, which is also transported by the granules, causes no net decrease in reduced intragranular ascorbate. The presence of 400 microM extragranular ascorbate abolishes the observed tyramine-induced intragranular ascorbate oxidation. The addition of ascorbate extragranularly 30 min after addition of tyramine reverses the oxidation of intragranular ascorbate. The measurement of [14C]ascorbate distribution ratios in granule pellets and supernatants indicates that there is no transmembrane transport of ascorbate. Extravesicular NADH had no significant effect on matrix ascorbate levels during beta-hydroxylation. These data provide new in vitro evidence that chromaffin granules shuttle reducing equivalents inwardly from an extra- to an intravesicular ascorbate pool and that cytosolic ascorbate is the source of the intragranular reducing equivalents required during norepinephrine biosynthesis.     

Demonstration of the ascorbate dependence of membrane-bound dopamine beta-monooxygenase in adrenal chromaffin granule ghosts

Chromaffin granule ghosts from bovine adrenal medullae have been used to examine the ability of membrane-bound dopamine beta-monooxygenase to interact directly with intravesicular ascorbate and to investigate vectorial electron transfer from external ascorbate across the ghost membrane. Ghosts prepared by a modification of published procedures were shown to be fully active in both dopamine uptake and norepinephrine production. Dopamine uptake is dependent on the presence of a magnesium and ATP ionic complex, is abolished by reserpine, and reaches a steady-state level in the presence of dopamine beta-monooxygenase, ascorbate, catalase, and fumarate. Omission of ascorbate either inside or outside the ghosts greatly enhances dopamine accumulation, which reaches levels of approximately 30 nmol/mg under these conditions. Correspondingly, in the presence of all components, norepinephrine production reached approximately 100 nmol/mg in 30 min of incubation. Norepinephrine production was strictly magnesium-ATP-dependent, inhibited by either reserpine or dopamine beta-monooxygenase inactivation, and was markedly reduced when ascorbate was omitted from either inside or outside the ghosts. In the presence of limiting amounts of internal ascorbate, rapid norepinephrine production occurred which corresponded to the amount of initial ascorbate present, followed by a much slower endogenous norepinephrine production observable after complete depletion of internal ascorbate. The endogenous rate of norepinephrine production likely represents epinephrine-supported dopamine beta-monooxygenase turnover. Taken together, the data demonstrate that facile norepinephrine production by membrane-bound dopamine beta-monooxygenase occurs only when internal ascorbate is present, terminates upon depletion of internal ascorbate, and can only be sustained at a significant rate when reducing equivalents from external ascorbate are available.     

Plasma Membrane and Chromaffin Granule Characteristics in Digitonin-Treated Chromaffin Cells

Digitonin permeabilizes the plasma membranes of bovine chromaffin cells to Ca2+, ATP, and proteins and allows micromolar Ca2+ in the medium to stimulate directly catecholamine secretion. In the present study the effects of digitonin (20 microM) on the plasma membrane and on intracellular chromaffin granules were further characterized. Cells with surface membrane labeled with [3H]galactosyl moieties retained label during incubation with digitonin. The inability of digitonin-treated cells to shrink in hyperosmotic solutions of various compositions indicated that tetrasaccharides and smaller molecules freely entered the cells. ATP stimulated [3H]norepinephrine uptake into digitonin-treated chromaffin cells fivefold. The stimulated [3H]norepinephrine uptake was inhibited by 1 microM reserpine, 30 microM NH4+, or 1 microM carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). The data indicate that [3H]norepinephrine was taken up into the intracellular storage granules by the ATP-induced H+ electrochemical gradient across the granule membrane. Reduction of the medium osmolality from 310 mOs to 100 mOs was required to release approximately 50% of the catecholamine from chromaffin granules with digitonin-treated chromaffin cells which indicates a similar osmotic stability to that in intact cells. Chromaffin granules in vitro lost catecholamine when the digitonin concentration was 3 microM or greater. Catecholamine released into the medium by micromolar Ca2+ from digitonin-treated chromaffin cells that had subsequently been washed free of digitonin could not be pelleted in the centrifuge and was not accompanied by release of membrane-bound dopamine-beta-hydroxylase. The studies demonstrate that 20 microM of digitonin caused profound changes in the chromaffin cell plasma membrane permeability but had little effect on intracellular chromaffin granule stability and function. It is likely that the intracellular chromaffin granules were not directly exposed to significant concentrations of digitonin. Furthermore, the data indicate that during catecholamine release induced by micromolar Ca2+, the granule membrane was retained by the cells and that catecholamine release did not result from release of intact granules into the extracellular medium.     

Effects of dopamine beta-monooxygenase substrate analogs on ascorbate levels and norepinephrine synthesis in adrenal chromaffin granule ghosts

Chromaffin granule ghosts from bovine adrenal medullae have been used to investigate the effects of prototypic dopamine beta-monooxygenase substrate analogs of two distinct classes on intravesicular reduced ascorbic acid (AscH2) levels and on norepinephrine synthesis. Phenyl-2-aminoethyl sulfide (PAES), a sulfur-containing substrate, was shown to concentrate within ghosts, a process that was time and ATP dependent, but reserpine insensitive. Dopamine beta-monooxygenase oxygenation of PAES resulted in accumulation of the oxygenation product, PAESO, without affecting intravesicular levels of AscH2. Similarly, incubations of ghosts with phenyl-2-aminoethyl selenide (PAESe) also resulted in rapid, time- and ATP-dependent, but reserpine-insensitive uptake. However, oxygenation of PAESe by dopamine beta-monooxygenase within ghosts was found to cause a marked decrease in intravesicular AscH2, without buildup of the oxygenated product, phenyl 2-aminoethyl selenoxide. These results illustrate two basic differences between the consequences of PAES and PAESe turnover: while PAES accumulation proceeds concomitant with PAESO production and without AscH2 depletion, PAESe accumulation proceeds with a marked lowering of internal AscH2 but without observable product formation. Both PAES and PAESe were capable of competing with dopamine, the physiological substrate, for enzymatic oxygenation and/or vesicular uptake, and were capable of significantly reducing norepinephrine synthesis. In experiments where ghosts were preincubated with either PAES or PAESe with delayed addition of dopamine, it was clear that neither compound nor their oxygenated products interfered with electron transport via cytochrome b561. These results are consistent with the hypothesis that the physiological activity observed with both PAES and PAESe may be related to their ability to gain entrance to adrenergic neurons and decrease norepinephrine synthesis within neurotransmitter storage vesicles.     

Ascorbic acid regeneration in chromaffin granules. In situ kinetics.

We have investigated in intact chromaffin secretory vesicles the kinetics, specificity, and mechanism of intragranular ascorbic acid regeneration by extragranular ascorbic acid. The apparent Km of internal ascorbic acid regeneration for external ascorbic acid was 280 microM by Lineweaver-Burk analysis and 287 microM by Eadie-Hofstee analysis. Intragranular ascorbic acid regeneration was specifically mediated by extragranular ascorbic acid or its isomer isoascorbic acid; the reducing agents glutathione, thiourea, homocysteine, NADH, and NADPH did not support regeneration. The structural analog D-glucose did not inhibit regeneration by external ascorbic acid, suggesting specificity at the membrane site of electron transfer. The driving force for regeneration of intragranular ascorbic acid was independent of membrane potential, absolute intragranular and extragranular pH, and ATPase activity, but might be coupled to the pH difference across the chromaffin granule membrane. Since the apparent Km of regeneration was approximately 10-fold below the cytosolic concentration of ascorbic acid, the reaction may proceed at Vmax in situ.     

Electron transfer in chromaffin-vesicle ghosts containing peroxidase.

In chromaffin vesicles, the enzyme dopamine beta-monooxygenase converts dopamine to norepinephrine. It is believed that reducing equivalents for this reaction are supplied by intravesicular ascorbic acid and that the ascorbate is regenerated by importing electrons from the cytosol with cytochrome b-561 functioning as the transmembrane electron carrier. If this is true, then the ascorbate-regenerating system should be capable of providing reducing equivalents to any ascorbate-requiring enzyme, not just dopamine beta-monooxygenase. This may be tested using chromaffin-vesicle ghosts in which an exogenous enzyme, horseradish peroxidase, has been trapped. If ascorbate and peroxidase are trapped together within chromaffin-vesicle ghosts, cytochrome b-561 in the vesicle membrane is found in the reduced form. Subsequent addition of H2O2 causes the cytochrome to become partially oxidized. H2O2 does not cause this oxidation if either peroxidase or ascorbate are absent. This argues that the cytochrome is oxidized by semidehydroascorbate, the oxidation product of ascorbate, rather than by H2O2 or peroxidase directly. The semidehydroascorbate must be internal because the ascorbate from which it is formed is sequestered and inaccessible to external ascorbate oxidase. This shows that cytochrome b-561 can transfer electrons to semidehydroascorbate within the vesicles and that the semidehydroascorbate may be generated by any enzyme, not just dopamine beta-monooxygenase.     

Activation of dopamine beta-monooxygenase by external and internal electron donors in resealed chromaffin granule ghosts

Membrane ghosts derived from chromaffin vesicles of bovine adrenal medullas have been used to examine the mechanism of reduction of dopamine beta-monooxygenase in its compartmentalized state. The rate of the dopamine beta-monooxygenase-catalyzed conversion of dopamine to norepinephrine is greatly stimulated by the presence of ATP, reflecting substrate hydroxylation on the ghost interior subsequent to the active transport of dopamine. We demonstrate a 2-3-fold increase in the turnover rate for ghosts resealed with 0.2-2 mM potassium ferrocyanide, conditions leading to a slight decrease in the rate of dopamine transport. These data provide the first evidence that an intravesicular pool of reductant can activate dopamine beta-monooxygenase, as required by models in which vesicular ascorbate behaves as enzyme reductant. Although there is sufficient catecholamine (endogenous plus substrate) to keep internal ferrocyanide reduced in these experiments, an additional 2-3-fold increase in turnover occurs in the presence of 0.2-2 mM ascorbate on the ghost exterior. The magnitude of this activation is found to be constant at all concentrations of internal ferrocyanide (both below and above saturation), implying that reductants on opposite sides of the membrane behave independently. Replacement of ascorbate by potassium ferrocyanide as external reductant leads to almost identical results, and we are able to rule out an inward transport of dehydroascorbate as the source of activation by external ascorbate. We conclude that external reductants are capable of reducing membrane-bound dopamine beta-monooxygenase from the exterior face of the vesicle, either by direct reduction or through a membrane-bound mediator. It appears that two viable modes for reduction of dopamine beta-monooxygenase may exist in vivo, involving the reduction of membrane-bound enzyme by cytosolic ascorbate as well as the reduction of soluble enzyme by the pool of intravesicular ascorbate present in chromaffin vesicles.     

Relation between the membrane and soluble forms of dopamine beta-monooxygenase

The quantitative ratio of membrane-bound and soluble forms of dopamine beta-monooxygenase from chromaffin granules obtained under different experimental conditions was determined. The amount of the membrane-bound form of dopamine beta-monooxygenase made up to no less than 60% of the total enzyme pool, when the granules were obtained and lyzed in the presence of pepstatin, phenylmethylsulfonyl fluoride, N-ethylmaleimide and catalase. In the absence of protectors practically all the enzyme can be obtained in the soluble form without detergent treatment. The effects of some ionic and nonionic detergents on the enzymatic activity of both forms of dopamine beta-monooxygenase were studied. No inhibition of dopamine beta-monooxygenase by 2% octyl glucoside or 1% Triton X-100 was observed. A comparative analysis of specific activities, subunit compositions, antigenic and physico-chemical properties of membrane-bound and soluble forms of dopamine beta-monooxygenase was carried out.     

Continuous spectrophotometric assays for dopamine beta-monooxygenase based on two novel electron donors: N,N-dimethyl-1,4-phenylenediamine and 2-aminoascorbic acid.

Based on the novel chromophoric electron donors, N,N-dimethyl-1,4-phenylenediamine (DMPD) and 2-amino-2-deoxy-L-ascorbic acid (2-aminoascorbic acid), two sensitive, convenient, and continuous spectrophotometric assays for dopamine beta-monooxygenase (EC 1.14.17.1) are described. Both, DMPD and 2-aminoascorbic acid are kinetically and stoichiometrically well-behaved electron donors for dopamine beta-monooxygenase with kinetic parameters comparable to the most efficient physiological electron donor, ascorbic acid. During dopamine beta-monooxygenase turnover, DMPD is converted to its chromophoric cation radical which is stable under the standard assay conditions. The rate of the enzyme-dependent formation of DMPD cation radical under standard assay conditions could easily be followed at 515 nm with high accuracy and reproducibility. Similarly, dopamine beta-monooxygenase-mediated oxidation of 2-aminoascorbic acid results in the formation of the known, stable chromophoric product, 2,2'-nitrilodi-2(2')-deoxy-L-ascorbic acid (red pigment), which has a very strong absorption maximum at 385 nm. Both the above assays are superior to the existing assays in their convenience, reproducibility, and sensitivity for routine kinetic analysis of dopamine beta-monooxygenase and may be adopted as a simple color test for the enzyme. We propose that the above assays could also be adopted to design continuous and sensitive spectrophotometric assays for ascorbate oxidase, peptidyl alpha-amidating monooxygenase, and the chromaffin granule electron transport protein, cytochrome b561, due to their remarkable similarity to dopamine beta-monooxygenase in the chemistry of catalysis with regard to the electron donor.     

Cell type-specific storage of dopamine beta-monooxygenase

Expression of dopamine beta-monooxygenase (DBM), the enzyme that converts dopamine into norepinephrine, is limited to adrenal chromaffin cells and a small population of neurons. We studied DBM trafficking to regulated granules by stably expressing rat DBM in AtT-20 corticotrope tumor cells, which contain regulated granules, and in Chinese hamster ovary (CHO) cells, which lack regulated granules. The behavior of exogenous DBM in both cell lines was compared with endogenous DBM in adrenal chromaffin cells. CHO cells secreted active DBM, indicating that production of active enzyme does not require features unique to neuroendocrine cells. Pulse-chase experiments indicated that early steps in DBM maturation followed a similar time course in AtT-20, CHO, and adrenal chromaffin cells. Use of a conformation-sensitive DBM antiserum indicated that acquisition of a folded structure occurred with a similar time course in all three cell types. Cell type-specific differences in DBM trafficking became apparent only when storage in granules was examined. As expected, DBM was stored in secretory granules in chromaffin cells; CHO cells failed to store DBM. Despite the fact that AtT-20 cells have regulated granules, exogenous DBM was not stored in these granules. Thus storage of DBM in secretory granules requires cell type specific factors.     

Role of ascorbic acid in dopamine beta-hydroxylation. The endogenous enzyme cofactor and putative electron donor for cofactor regeneration

The role(s) of ascorbic acid in dopamine beta-hydroxylation was studied in primary cultures of bovine adrenomedullary chromaffin cells and in isolated bovine adrenomedullary chromaffin vesicles. Dopamine beta-hydroxylase activity was assessed by measuring the rate of conversion of tyramine to octopamine. The ascorbic acid content of chromaffin cells declined with time in culture and the dopamine beta-hydroxylase activity of ascorbate-depleted cells was low. Ascorbate additions to ascorbate-depleted cells increased both the intracellular ascorbate concentrations and the rates of dopamine beta-hydroxylation. Ascorbate uptake into the cells was rapid; however, the onset of enhanced octopamine synthesis by added ascorbate was delayed by several hours and closely followed the time course for accumulation of the newly taken up ascorbate into the chromaffin vesicle. The amount of octopamine synthesized by the chromaffin cells exceeded the intracellular ascorbate content and ascorbate levels were maintained during dopamine beta-hydroxylation in the absence of external ascorbate. This suggests an efficient recycling of ascorbate. In contrast to intact cells, ascorbic acid was depleted during octopamine synthesis in isolated chromaffin vesicles. The molar ratio of octopamine formed to ascorbate depleted was close to unity. Thus, the recycling of intravesicular ascorbate depends on an extravesicular factor(s). The depletion of intravesicular ascorbate during dopamine beta-hydroxylation was prevented by the addition of nonpermeant extravesicular electron donors such as ascorbate or glucoascorbate. This suggests that intravesicular ascorbate is maintained in the reduced state by electron transport across the vesicle membrane. These results are compatible with the hypothesis that both intra- and extravesicular ascorbate participate in the regulation of dopamine beta-hydroxylase. Intravesicular ascorbate is the cofactor for the enzyme. Cytosolic ascorbate is most likely the electron donor for the vesicle-membrane electron transport system which maintains the intravesicular cofactor concentration.     

Nature of rate-limiting steps in a compartmentalized enzyme system. Quantitation of dopamine transport and hydroxylation rates in resealed chromaffin granule ghosts

Using isolated chromaffin granule ghosts from bovine adrenal medullae, we have studied the kinetics of dopamine beta-monooxygenase (D beta M) activity as it is linked to dopamine transport. Measurements of the initial rates of transport and of transport-linked norepinephrine formation suggested that enzyme activity may be partially rate-limiting in the coupled carrier/enzyme system. This was confirmed by (i) measurements of initial rates of norepinephrine formation using deuterated substrate, which gave isotope effects greater than 2.0, and (ii) kinetic measurements using ghosts pulsed with varying concentrations of labeled dopamine, which indicated substantial substrate accumulation in the vesicle interior as a function of time. Initial rates of product formation, when combined with approximations of internal substrate concentrations, allowed estimates of Kcat and Km for intravesicular D beta M. Activation by external reductant was apparent in both initial rate parameters and the measurements of transients. Under conditions of optimal D beta M activity, the enzyme rate parameters (kcat = 0.31 nmol/s.mg and Km = 2 mM) indicated partial rate limitation compared to dopamine transport (kcat = 0.38 nmol/s.mg and Km = 32 microM). Compartmental analysis of the time curves, performed using numerical nonlinear least squares methods, gave least squares estimates of rate constants for a simple carrier mechanism and kcat values for D beta M which were consistent with estimates from initial rates.     

Specificity and properties of the nucleotide carrier in chromaffin granules from bovine adrenal medulla.

1. The influence of various substances on the uptake of [3H]ATP and [14C]-noradrenaline into isolated bovine chromaffin granules was investigated. The carrier-mediated [3H]ATP uptake is specifically inhibited by SO42-, PO43- and phosphoenolpyruvate. Compounds with carboxylic acid or sulphonic acid groups had no significant inhibitory effects on either uptake. 2. 35SO42-, 32PO43- and phosphoenol[14C]pyruvate are taken up into chromaffin granules by a temperature-dependent process that is inhibited by atractyloside, uncouplers of oxidative phosphorylation and lipid-permeant anions. The apparent Km of 35SO42- uptake is 0.4 mM. 3. These results indicate that the nucleotide carrier in chromaffin granules has a broad specificity, transporting compounds with two strong negative charges. 4. Amino acid probes influence the uptake of ATP and catecholamines differently. Pyridoxal phosphate inhibits both uptake processes, 4,4'-di-isothiocyanostilbene-2,2'-disulphonic acid preferentially blocks ATP uptake, whereas phenylglyoxal blocks only ATP transport. It is suggested that the nucleotide carrier possesses arginine residues in a functionally important position. 5. The significance of these results obtained on isolated granules for the function of chromaffin granules within the cell is discussed.