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.     

Activity of tyrosine hydroxylase (EC 1.14.16.2) from chromaffin and nervous tissue of the Atlantic cod, Gadus morhua

1. The activity of tyrosine hydroxylase (TH; EC 1.14.16.2) was estimated in vitro in crude tissue homogenates from the posterior cardinal veins (chromaffin tissue) and the coeliac ganglion (adrenergic neurons) of the Atlantic cod, Gadus morhua. 2. TH from the chromaffin tissue showed its highest activity at pH = 6.0 and a temperature of 30-35 degrees C, and was stimulated by low concentrations of catalase (20 micrograms/ml). 3. Estimations of the absolute TH activity in vitro showed values of 110 +/- 30 nmol/g X hr for the chromaffin tissue and 1120 +/- 280 nmol/g X hr for the coeliac ganglion.     

On the uptake of exogenous catecholamines by adrenal chromaffin cells and nerve endings

Summary Light-microscopic autoradiography has revealed characteristic labelling patterns in adrenal medullary cells following the intravenous administration of different catecholamines. The uptake patterns for [³H] dopa, [³H] dopamine, [³H] noradrenaline and [³H] adrenaline have been compared. In all cases A cells were more active than NA cells and cells situated in the zone nearest the cortex demonstrated a markedly higher rate of uptake than central cells. It was concluded that adjacent chromaffin cells with very similar morphology may differ as much as 50 fold in their capacities to incorporate exogenous amines. The adrenergic nature of the innervation of the vessels of the adrenal cortex and capsule in the mouse was confirmed.     

In Vitro Synthesis of Dopamine and Noradrenaline in the Isolated Rat Pineal Gland: Day-Night Variations and Effects of Electrical Stimulation

Isolated rat pineal glands were incubated in vitro in a medium containing [14C]dopamine or [14C]tyrosine, and the tissue contents of 14C-labelled and total dopamine and noradrenaline were determined by HPLC followed by electrochemical detection and scintillation spectrometry. During incubation with [14C]dopamine, the labelled amine accumulated in pineal glands and was partially converted into [14C]noradrenaline. Nomifensine, a neuronal amine uptake blocker, largely inhibited the accumulation of [14C]dopamine and the formation of [14C]noradrenaline. These experiments demonstrated dopamine beta-hydroxylase activity in the sympathetic nerves of the pineal gland. During incubation with [14C]tyrosine, formation of [14C]dopamine and [14C]noradrenaline was observed in the pineal tissue, indicating that noradrenaline can also be synthesized from dopamine, endogenously formed in the gland. Electrical stimulation of the stalk region of the pineal gland during incubation with [14C]dopamine enhanced the accumulation of [14C]dopamine and synthesis of [14C]noradrenaline. Electrical stimulation also enhanced the formation of [14C]dopamine during incubation with [14C]tyrosine. Compared to that at midday, the tissue content of endogenous noradrenaline at midnight was enhanced by 50% and that of dopamine by 450%. The in vitro accumulation of [14C]dopamine, as well as the synthesis of [14C]dopamine and [14C]noradrenaline, was also increased at midnight. In conclusion, sympathetic nerves in the rat pineal gland contain tyrosine hydroxylase and dopamine beta-hydroxylase, the two enzymes required for the synthesis of noradrenaline.(ABSTRACT TRUNCATED AT 250 WORDS)     

Catecholamines in sow graafian follicles at proestrus and at diestrus

Endogenous dopamine, noradrenaline, and adrenaline were detected in the sow graafian follicular wall and in the follicular fluid. Noradrenaline represented the highest level and adrenaline the lowest. Dopamine and noradrenaline concentrations found in the follicular fluid were lower at early proestrus than at mid-diestrus, whereas adrenaline levels in the fluid did not differ at either stage of the estrous cycle. The sow follicular wall contained less dopamine, noradrenaline and adrenaline at early proestrus than at mid-diestrus. Concomitantly, a decrement of [3H]-dopamine and [3H]-noradrenaline uptake, and of dopamine-beta-hydroxylase activity was detected at early proestrus compared to levels detected at mid-diestrus. The findings in sow graafian follicles show the existence of relationships between hormonal status, dopamine, noradrenaline and adrenaline endogenous levels and uptake, and dopamine-beta-hydroxylase activity. Possible links between estradiol, follicle-stimulating hormone (FSH), and luteinizing hormone (LH) levels during the pig estrous cycle and ovarian catecholamines are discussed, as is a plausible involvement of these neurotransmitters in the contractile activity of the theca layer and the processes of follicular rupture and ovulation.     

Accumulation of intra-arterially administered [3H]adrenaline and [3H]noradrenaline in various tissues of the Atlantic cod, Gadus morhua

The accumulation of intra-arterially administered radiolabelled adrenaline and noradrenaline was studied in various tissues of the Atlantic cod, Gadus morhua. The largest uptake was seen in the posterior cardinal vein (chromaffin tissue), head kidney, kidney, heart and gill filaments. All these tissues, except the heart, also accumulated noradrenaline to a greater extent than adrenaline. The heart, spleen, gas gland and muscularis mucosae of the swimbladder instead favoured adrenaline accumulation. Small amounts of the injected label (both adrenaline and noradrenaline) were also recovered in the intestine, liver and hypothalamus. The lowest detectable amine accumulation was seen in the rest of the brain and in the skeletal muscle. It is suggested that innervation density, blood flow to the tissue and the concentration of circulating and endogenously stored amine, as well as the affinity of the amine for the degrading enzymes and a possible stereospecificity of the uptake mechanisms, determine the rate and preference of accumulation between the amines.     

Lack of Selectivity Between the Uptake of [3H] Adrenaline and [3H]Noradrenaline into Rat Hypothalamic Slices

The uptake of [3H]adrenaline and [3H]noradrenaline into rat hypothalamic slices was compared for determination of whether adrenaline uptake was independent of uptake into noradrenergic neurones. Kinetic analysis revealed a similar high-affinity uptake process for both adrenaline and noradrenaline, with Km and Vmax values within similar ranges. These uptakes were inhibited by desipramine and maprotiline in a dose-dependent manner, but the selective dopamine and 5-hydroxytryptamine uptake inhibitors benztropine and fluoxetine, respectively, were without effect. Competition for uptake sites by unlabelled adrenaline with [3H]adrenaline and [3H]-noradrenaline and by unlabelled noradrenaline with [3H]-adrenaline and [3H]noradrenaline was very similar. Lesioning of the major adrenaline-containing cell group (C1 cell group) decreased the hypothalamic adrenaline concentration but had no effect on hypothalamic [3H]adrenaline or [3H]noradrenaline uptake. The results suggest that exogenous adrenaline is largely taken up by high-affinity sites on noradrenergic nerve terminals.     

Simultaneous radioenzymatic determination of plasma and tissue adrenaline,noradrenaline and dopamine within the femtomole range

A radiometric-enzymatic assay for measuring simultaneously femtomole quantities of adrenaline, noradrenaline and dopamine has been developed. The three catecholamines are first converted to their O-methylated analogues by catechol-O-methyltransferase in the presence of S-adenosyl-methionine-³H and thereafter extracted following addition of sodium tetraphenylborate. This extraction, together with an improved quick chromatographic separation and the oxidation of the adrenaline and noradrenaline derivatives to vanillin, yields an extremely high sensitivity and specificity of the method.The present assay allows the determination of adrenaline, noradrenaline and dopamine in tissue samples with a protein content of 100 μg or less and in plasma volumes of 20 – 100 μl. The amine content of 40 – 50 samples can be determined in two days by one person.Due to the high sensitivity achieved, this method promises to be a valid alternative to the gas chromatography-mass spectrometry technique.     

Intra-adrenal interactions in fish: catecholamine stimulated cortisol release in sea bass (Dicentrarchus labrax L.)

The effect of the catecholamines, adrenaline and noradrenaline, on sea bass (Dicentrarchus labrax) and sea bream (Sparus auratus) interrenal cortisol production was studied in vitro using a dynamic superfusion system technique. Increasing concentrations of catecholamines (10(-6), 10(-8) and 10(-10) M) stimulated cortisol production in a dose-dependent manner, in sea bass only. The increase in cortisol production stimulated by adrenaline (10(-6) M) and noradrenaline (10(-6) M) was inhibited by sotalol (2 x 10(-5) M), but not by prazosin suggesting that catecholamines stimulate cortisol release through the beta-receptor subtype. To evaluate catecholamine-induced signal transduction in head kidney cells, measurements of cAMP production and [H3]myo-inositol incorporation were determined in head kidney cell suspensions. Adrenaline and noradrenaline (10(-6) M) increased cAMP production, but had no effect on total inositol phosphate accumulation. These results indicate that catecholamines released from the chromaffin cells within the interrenal tissue may act as a paracrine factor to stimulate interrenal steroidogenesis in the sea bass.     

Resonance Raman studies on the blue-green-colored bovine adrenal tyrosine 3-monooxygenase (tyrosine hydroxylase). Evidence that the feedback inhibitors adrenaline and noradrenaline are coordinated to iron

Tyrosine 3-monooxygenase (tyrosine hydroxylase) is a non-heme iron, tetrahydropterin-dependent enzyme which catalyzes the rate-limiting step in the biosynthesis of catecholamines. The highly purified bovine adrenal enzyme contains an unusual blue-green chromophore with lambda max at around 700 nm (epsilon = 1.3 (mM subunit enzyme)-1 cm-1). On excitation at 605.2 nm, resonance-enhanced Raman vibrations are observed at 454, 494, 527, 604, 635, 835, 1130, 1271, 1320, 1426, and 1476 cm-1. The excitation profiles of the modes of 1276 and 1476 cm-1 (from 488 to 620 nm) follow the contour of the 700 nm absorption band. The vibrations observed strongly indicate the presence of a bidentate catecholamine-Fe(III) complex in the enzyme as isolated which gives rise to the characteristic charge-transfer transitions. This is further supported by the release of 0.11 +/- 0.04 mol of noradrenaline and 0.25 +/- 0.06 mol of adrenaline per mol of enzyme subunit on denaturation of the enzyme. The energies of the catecholate to Fe(III) charge-transfer transitions indicate a mixture of histidines and carboxylate(s) coordinated to the iron center in tyrosine hydroxylase. At neutral pH, the enzymatic activity was inhibited more than 50% by 10 microM dopamine, noradrenaline, and adrenaline. The high affinity of the catecholamines to the nonphosphorylated form of tyrosine hydroxylase may have significance in vivo since catecholamines are potent feedback inhibitors of the enzyme.     

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.     

Dopamine-selective response in membrane potential by homooxacalix[3]arene triether host incorporated in PVC liquid membrane.

Selectivities of membrane potential changes for catecholamines and inorganic cations were investigated with lipophilic derivatives of calix[6]arene and related hosts incorporated in poly(vinyl chloride) (PVC) matrix liquid membranes. Homooxacalix[3]arene triether displayed an excellent selectivity for dopamine against other catecholamines (adrenaline, noradrenaline) and also against inorganic cations (K+, Na+).     

Effects of Reserpine and Tetrabenazine on Catecholamine and ATP Storage in Cultured Bovine Adrenal Medullary Chromaffin Cells

The in vivo storage relationship between catecholamines and ATP in chromaffin vesicles of cultured bovine adrenal medulla cells was investigated using drugs that block vesicular catecholamine uptake. Three-day treatments with reserpine and tetrabenazine causing 85-90% depletion of catecholamines resulted in 41-46% reductions in cellular ATP content. Subcellular fractionation of reserpine-treated cells indicated that the ATP is lost from the chromaffin vesicle pool. This was confirmed in experiments using metabolic inhibitors to differentiate the vesicular and extravesicular ATP pools. The vesicular ATP loss was not proportional to that of catecholamines, resulting in a reduction by 50% in the chromaffin vesicle mole ratio of catecholamines to ATP after 48 h of treatment. In metabolic labeling studies, it was found that reserpine treatment reduced the incorporation of [3H]adenosine into vesicular ATP selectively, but it reduced the incorporation of 32Pi into both the vesicular and extravesicular pools. The reduction of the [3H]adenosine incorporation was not due to diminished vesicular nucleotide uptake resulting from low catecholamine levels, because when the catecholamines were depleted by tetrabenazine pretreatment followed by removal of the drug before labeling, no reduction in [3H]adenosine incorporation was observed. When present during the labeling, tetrabenazine was found to be a reversible inhibitor of plasma membrane adenosine uptake. The observed loss of adenine nucleotides from catecholamine-depleted chromaffin vesicles in vivo provides evidence that interactions between ATP and catecholamines are important in the vesicular storage of high concentration of these compounds.     

Biochemical characterization of various populations of isolated bovine adrenal chromaffin cells

Various populations of bovine adrenal chromaffin cells were isolated first by successive digestions with collagenase (original cell preparation) followed by sedimentation through a stepwise bovine serum albumin gradient (cell layers I, II and III). At the fine structural level, the ratios between the number of adrenaline-cells and noradrenaline-cells were 1.9 in the original cell preparation and 0.9, 2.0 and 4.6 in cell layers I, II and III, respectively. The catecholamine content of each cell population was also measured by spectrofluorometry. The original cell preparation contained 20.1 and 12.2 nmol per 10⁶ cells of adrenaline and noradrenaline, respectively. Each cell layer had similar total amount of catecholamines (from 38.3 to 40 nmol per 10⁶ cells) but their adrenaline/noradrenaline content ratios varied from 0.6 in cell layer 1 to 1.3 and 3.3 in cell layers II and III, respectively. Incubation of the cells in the presence of acetylcholine (50 μM) induced a release of catecholamines which was proportional to the cell content of each amine. However, the percentage of total cell content released was much higher in cell layer I (20%) than in cell layers II (8%) and III (5%). Finally, each cell population was also analyzed for its ability to respond to a muscarinic stimulation of cyclic GMP level and to bind [³H]etorphine, a highly potent opiate agonist. Acetylcholine induced 3.15-, 2.15- and 4.21-fold increases in the levels of cyclic GMP in the original cell preparation, cell layers II and III, respectively, but not in cell layer I. Conversely, the high affinity opiate binding site for [³H]etorphine was almost exclusively confined to cell layer III (B_max of 28.4 fmol per 10⁶ cells as compared with 2.8–7.5 fmol in the other cell preparations). These results indicate that bovine adrenal chromaffin cells can be separated according to their content in adrenaline and noradrenaline and their response to nicotinic, muscarinic and opiate stimuli.     

Neuropeptide Y co-exists and co-operates with noradrenaline in perivascular nerve fibers

Neuropeptide Y (NPY)-immunoreactive nerve fibers were numerous around arteries and few around veins. NPY probably co-exists with noradrenaline in such fibers since chemical or surgical sympathectomy eliminated both NPY and noradrenaline from perivascular nerve fibers and since double staining demonstrated dopamine-beta-hydroxylase, the enzyme that catalyzes the conversion of dopamine to noradrenaline, and NPY in the same perivascular nerve fibers. Studies on isolated blood vessels indicated that NPY is not a particularly potent contractile agent in vitro. NPY greatly enhanced the adrenergically mediate contractile response to electrical stimulation and to application of adrenaline, noradrenaline or histamine, as studied in the isolated rabbit gastro-epiploic and femoral arteries. The potentiating effect of NPY on the response to electrical stimulation is probably not presynaptic since NPY affected neither the spontaneous nor the electrically evoked release of [3H]noradrenaline from perivascular sympathetic nerve fibers.     

Histological, histochemical and ultrastructural studies on the interrenal and chromaffin cells of the fathead minnow, Pimephales promelas Rafmesque

Light and electron microscopic examination of fathead minnow head kidneys revealed that the interrenal and chromaffin cells were intermingled and always closely associated with the cardinal veins and their tributaries. Histochemical tests for lipids in the interrenal cells were positive, and two types of chromaffin cells were indicated by chromaffin reactions. Interrenal cells contained abundant smooth endoplasmic reticulum and mitochondria with tubulo-vesicular cristae, characteristic of steroid-producing cells. Only one interrenal cell type was found. Two types of chromaffin cells were present with differences in cytoplasmic density and in types of granules. In light cells, adrenaline granules were most common, and in dark cells noradrenaline granules predominated.     

The effects of repeated physical stress or fasting on catccholamine storage and release in the rainbow trout, Oncorhynchus mykiss

Rainbow trout, Oncorhynchus mykiss, were subjected to either physical stress (twice daily chasing to exhaustion for 5 days) or a period of 2 months of fasting. Following these treatments, the levels of catecholamines, adrenaline and noradrenaline, stored within the kidney and posterior cardinal vein (PCV) were determined. The ability of the catecholamine-storing chromaffin cells to release catecholamines in response to cholinergic stimulation was measured using an in situ saline-perfused PCV preparation. In the physically stressed fish, the concentration (μg catecholamine g^?1 tissue) of noradrenaline within the anterior and middle thirds of the kidney increased; the concentration of adrenaline was unchanged in all tissues. The content (μg) of noradrenaline or adrenaline, within the various tissues, was similar in both groups of fish with the exception of a higher noradrenaline content in the middle third of the kidney in the physically stressed fish. The total catecholamine content (μg catecholamine) of these tissues (kidney+PCV) was unaffected by physical stress. With the exception of a lower concentration of adrenaline in the middle third of the kidney, the concentrations of catecholamines were unaffected by fasting. There was a trend towards a greater content (μg) of noradrenaline within all of the tissue regions of the fed fish, however, a significant difference was only observed in the anterior third of the kidney. The content of adrenaline in the fed fish was greater in all regions of the kidney as well as the middle third of the PCV. The total catecholamine content (kidney + PCV) was lower in the fasted fish owing to significantly lower PCV and kidney masses. Prolonged physical stress caused a decrease in the responsiveness of the chromaffin cells to the cholinoceptor agonist carbachol (10^?8 to 10^?4mol). The ED₅₀ (the dose of carbachol required to elicit a half maximal response) for catecholamine release was 0·96 ± 10^?6mol carbachol in the physically stressed fish and 0·84 ± 10^?7 in the control fish. Fasting did not alter the pattern of catecholamine release. The ED₅₀ values were 0·96 ± 10^?7 and 1·24 ± 10^?7 mol for fasted and fed fish, respectively. Thus, a physical stress affected both catecholamine storage and release whereas fasting affected only storage and not the release process.     

Effects of adrenaline administration on the interrenal gland of the newt, Triturus carnifex: evidence of intraadrenal paracrine interactions

The existence of paracrine control of steroidogenic activity by adrenochromaffin cells in Triturus carnifex was investigated by in vivo adrenaline (A) administration. The effects were evaluated by examination of the ultrastructural morphological and morphometrical features of the tissues as well as the serum levels of aldosterone, noradrenaline (NA), and adrenaline. In March and July, adrenaline administration reduced aldosterone release (from 187.23 +/- 2.93 pg/ml to 32.28 +/- 1.85 pg/ml in March; from 314.60 +/- 1.34 pg/ml to 87.51 +/- 2.57 pg/ml in July) from steroidogenic cells. The cells showed clear signs of lowered activity: they appeared full of lipid, forming large droplets. Moreover, adrenaline administration decreased the mean total number of secretory granules in the chromaffin cells in July (from 7.74 +/- 0.74 granules/microm(2) to 5.14 +/- 1.55 granules/microm(2)). In this period T. carnifex chromaffin cells contain almost exclusively NA granules (NA: 7.42 +/- 0.86 granules/microm(2); A: 0.32 +/- 0.13 granules/microm(2)). Adrenaline administration reduced noradrenaline content (4.36 +/- 1.40 granules/microm(2)) in the chromaffin cells, enhancing noradrenaline secretion (from 640.19 +/- 1.65 pg/ml to 1030.16 +/- 3.03 pg/ml). In March, adrenaline administration did not affect the mean total number of secretory vesicles (from 7.24 +/- 0.18 granules/microm(2) to 7.25 +/- 1.97 granules/microm(2)). In this period the chromaffin cells contain both catecholamines, noradrenaline (3.88 +/- 0.13 granules/microm(2)), and adrenaline (3.36 +/- 0.05 granules/microm(2)), in almost equal quantities; adrenaline administration reduced adrenaline content (1.74 +/- 0.84 granules/microm(2)), increasing adrenaline release (from 681.27 +/- 1.83 pg/ml to 951.77 +/- 4.11 pg/ml). The results of this study indicate that adrenaline influences the steroidogenic cells, inhibiting aldosterone release. Adrenaline effects on the chromaffin cells (increase of noradrenaline or adrenaline secretion) vary according to the period of chromaffin cell functional cycle. The existence of intraadrenal paracrine interactions in T. carnifex is discussed.     

Uptake and metabolism of catecholamines in the awake dog

The contributions of uptake, metabolism and excretion to the removal of circulating dopamine (D) have been studied in comparison to those of adrenaline (A) and noradrenaline (NA). In two experiments radioactive catecholamines were infused during 80 min in an awake dog. In the first experiment [14C]D and D, L-[3H]A was used and in the second experiment these catecholamines were infused together with D, L-[14C]NA. Renal excretion of 14C-radioactivity was almost equal in both experiments, as was the case with the accumulation of 14C-components in plasma, demonstrating that the uptake of D was comparable to that of NA. The removal of [14C]D, [14C]NA and [3H]A, by uptake was 50, 50 and 13.5% respectively after 1 h. The conversion by metabolism was 46, 46 and 81%. Renal excretion was 3.5, 2 and 0.5%. Thus only 0.5, 2 and 5% was left in the extracellular fluid (ECF). In a report on similar experiments in anaesthetized dogs much higher levels of unchanged NA in plasma were measured. Probably this is due to anaesthesia inhibiting uptake. In the pulmonary circulation 14C-radioactivity was extracted at a constant rate during infusion which can mainly be attributed to extraneuronal uptake of [14C]D and to neuronal uptake of [14C]NA. Besides extraneuronal uptake of [3H]A in the lung expiration of [3H]water may contribute to the pulmonary extraction.     

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.