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.     

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.     

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.     

Activity of membranous dopamine beta-monooxygenase within chromaffin granule ghosts. Interaction with ascorbate

The role of intra- and extravesicular ascorbate has been investigated in dopamine beta-monooxygenase (D beta M) turnover using adrenal medulla chromaffin granule ghosts. Resealing of vesicle ghosts with high levels of intravesicular ascorbate leads to viable vesicles, as evidenced from the high rates of the ATP-dependent accumulation of tyramine, Vmax = 14 +/- 1 nmol/min.mg and Km = 20 +/- 6 microM. However, the D beta M-catalyzed conversion of tyramine to octopamine occurs slowly, Vmax = 0.50 +/- 0.13 nmol/min.mg and Km = 29 +/- 18 mM. When ascorbate is present instead in the external buffer, the D beta M rate increases 3.6-fold for a final Vmax = 1.8 +/- 0.2 and Km = 1.2 +/- 0.3 mM. This relatively high rate of enzyme turnover is retained in ghosts resealed with a large excess of ascorbate oxidase, ruling out contamination by intravesicular ascorbate as the source of enzyme activity. The synergistic effect of intravesicular ascorbate was examined under conditions of 2 mM external ascorbate, showing that the enzymatic rate increases 2.7-fold, from 1.2 (0 internal ascorbate) to 3.2 +/- 0.4 nmol/min.mg (saturating internal ascorbate). This result confirms that high levels of internal ascorbate are not damaging to intravesicular D beta M. These studies demonstrate very clearly that external ascorbate is the preferred reductant for the membranous form of D beta M in chromaffin granule ghosts.     

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 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.     

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.     

Reserpic acid as an inhibitor of norepinephrine transport into chromaffin vesicle ghosts

Reserpic acid, a derivative of the antihypertensive drug reserpine, inhibits catecholamine transport into adrenal medullary chromaffin vesicles. Since it does not affect the membrane potential generated by the H+-translocating adenosine triphosphatase but inhibits ATP-dependent norepinephrine uptake with a Ki of about 10 microM, reserpic acid must block the H+/monoamine translocator. Because reserpic acid is much more polar than reserpine, it does not permeate the chromaffin vesicle membrane, nor is it transported into chromaffin vesicle ghosts in the presence of Mg2+-ATP. Although it inhibits norepinephrine transport when added externally, reserpic acid does not inhibit when trapped inside chromaffin vesicle ghosts. Therefore, reserpic acid must bind to the external face of the monoamine translocator and should be a good probe of the translocator's structural asymmetry.     

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.     

Ascorbic acid and Mg-ATP co-regulate dopamine beta-monooxygenase activity in intact chromaffin granules

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 external dopamine concentrations of 7.5 and 10 mM. Under these conditions of dopamine uptake, norepinephrine biosynthesis was enhanced 5-6-fold by Mg-ATP and ascorbic acid compared to control experiments with dopamine only. Furthermore, norepinephrine formation was enhanced approximately 3-fold by ascorbic acid and Mg-ATP together compared to norepinephrine formation in granules 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 Km of norepinephrine formation for external ascorbic acid was 376 microM and for external Mg-ATP was 132 microM, consistent with the larger amounts of cytosolic ascorbic acid and ATP that are available 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 beta-monooxygenase. These data indicate that Mg-ATP and ascorbic acid specifically and synergistically co-regulate dopamine beta-monooxygenase activity in intact chromaffin granules, independent of substrate uptake. Although the mechanism is not known, the data are consistent with the possibility that the chromaffin granule ATPase mediates these effects.     

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.     

Localization of the phlorizin site on Na, K-ATPase in red cell membranes.

Phlorizin at 2 X 10(-4) M inhibited Na+ and Rb+-activated ATPase activities in human red cell membranes by 43%. It inhibited the 86Rb uptake activity of erythrocytes by only 15%. 86Rb uptake into resealed ghosts was inhibited strongly when phlorizin and ATP were preloaded in the ghosts before resealing. Na,K-ATPase activity in the resealed ghosts was also inhibited in the presence of phlorizin inside but not outside the ghosts. These findings suggested that the phlorizin site is located inside the cell.     

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.     

Mechanism of the insect enzyme, tyramine beta-monooxygenase, reveals differences from the mammalian enzyme, dopamine beta-monooxygenase

Tyramine beta-monooxygenase (TbetaM) catalyzes the synthesis of the neurotransmitter, octopamine, in insects. Kinetic and isotope effect studies have been carried out to determine the kinetic mechanism of TbetaM for comparison with the homologous mammalian enzymes, dopamine beta-monooxygenase and peptidylglycine alpha-hydroxylating monooxygenase. A new and distinctive feature of TbetaM is very strong substrate inhibition that is dependent on the level of the co-substrate, O(2), and reductant as well as substrate deuteration. This has led to a model in which tyramine can bind to either the Cu(I) or Cu(II) forms of TbetaM, with substrate inhibition ameliorated at very high ascorbate levels. The rate of ascorbate reduction of the E-Cu(II) form of TbetaM is also reduced at high tyramine, leading us to propose the existence of a binding site for ascorbate to this class of enzymes. These findings may be relevant to the control of octopamine production in insect cells.     

Amine transport into chromaffin ghosts. Kinetic measurements of net uptake of biologically and pharmacologically relevant amines using an on-line amperometric technique

The kinetic parameters for net transport of dopamine, epinephrine, norepinephrine, 5-hydroxytryptamine, S alpha-methyldopamine, R alpha-methyldopamine, and 1R,2S alpha-methylnorepinephrine into highly purified bovine chromaffin ghosts were determined using an on-line amperometric technique. Chromaffin ghosts devoid of endogenous amines were formed from lysis of chromaffin granules under hypotonic conditions, extensive washing of the scattered membranes, followed by resuspension in iso-osmotic media and overnight dialysis. When chromaffin ghosts formed so as to generate and maintain a large delta pH were suspended in 185 mM KCl, 10 mM Hepes at pH 7.0, 37 degrees C, the addition of MgATP resulted in rapid acidification of the intravesicular space, which was maintained at pH 6.0 (+/- 0.1) for over 30 min. Kinetic net amine transport was subsequently measured with a glassy carbon electrode. The initial rates of uptake were found to follow Michaelis-Menten kinetics. Computer based statistical analysis of the data using distribution-free procedures yielded Km (and V) values as follows: in microM (nmol X mg protein-1 X min-1) dopamine, 16.2 (14.0); R-norepinephrine, 32.5 (12.9); R-epinephrine, 35.1 (15.2); 5-hydroxytryptamine, 4.7 (5.1); S alpha-methyldopamine, 17.7 (11.2); R alpha-methyldopamine, 44.2 (9.9); 1R,2S alpha-methylnorepinephrine, 76.5 (12.5). The physiologic and pharmacologic implications of these kinetic parameters are discussed.     

Characterization of alternate reductant binding and electron transfer in the dopamine beta-monooxygenase reaction

The steady-state limiting kinetic parameters Vmax, V/KDA, and V/KO2, together with deuterium isotope effects on these parameters, have been determined for the dopamine beta-monooxygenase (D beta M) reaction in the presence of structurally distinct reductants. The results show the one-electron reductant ferrocyanide to be nearly as kinetically competent as the presumed in vivo reductant ascorbate. Further, a reductant system of ferricyanide plus substrate dopamine yields steady-state kinetic parameters and isotope effects very similar to those measured solely in the presence of ferrocyanide, indicating a role for catecholamine in the rapid recycling of oxidized ferrocyanide. Use of substrate dopamine as the sole reductant is found to lead to a highly unusual kinetic independence of oxygen concentration, as well as significantly reduced values of Vmax and V/KDA, and we conclude that dopamine reduces enzymic copper in a rate-limiting step that is 40-fold slower than with ascorbate. The near-identical kinetic parameters measured in the presence of either ascorbate or ferrocyanide, together with markedly reduced rates with dopamine, are interpreted in terms of a binding site for reductant that is physically distinct from the substrate binding site. This view is supported by molecular modeling, which reveals ascorbate and ferrocyanide to possess an unexpected similarity in potential sites for interaction with enzymic residues. With regard to electron flux, identical values of V/KO2 have been measured with [2,2-2H2]dopamine as substrate both in the presence and in the absence of added ascorbate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dopamine and norepinephrine in the brains of hepatectomized rats

After complete hepatectomy in rats, there was a profound depletion of norepinephrine throughout the brain, whereas dopamine concentrations remained within the normal range. This suggests that in liver failure there is inhibition of norepinephrine synthesis at one of two processes subsequent to the formation of dopamine--that is, either the uptake of dopamine by vesicles or the hydroxylation of dopamine may be inhibited. However, other factors could contribute to the depletion of norepinephrine, including degeneration of its specific storage sites, displacement by false neurotransmitters, or an increased rate of catabolism. The present experiments do not allow a selection among these possibilities.     

The biosynthesis of ascorbate protects isolated rat hepatocytes from cumene hydroperoxide-mediated oxidative stress

Most animals synthesize ascorbate. It is an essential enzymatic cofactor for the synthesis of a variety of biological molecules and also a powerful antioxidant. There is, however, little direct evidence supporting an antioxidant role for endogenously produced ascorbate. Recently, we demonstrated that incubation of rat hepatocytes with 1-bromoheptane or phorone simultaneously depleted glutathione (GSH) and triggered rapid ascorbate synthesis. The present study investigates the hypothesis that endogenous ascorbate synthesis can confer protection against oxidative stress. Rat and guinea pig hepatocytes were depleted of GSH with 1-bromoheptane and subsequently treated with the oxidative stressor cumene hydroperoxide (CHP) in the presence or absence of the ascorbate synthesis inhibitor sorbinil. In rat hepatocytes, ascorbate content increased linearly (from 15.1 to 35.8 nmol/10(6) cells) over a 105-min incubation. Prior depletion of GSH increased CHP-induced cellular reactive oxygen species (ROS) production, lipid peroxidation, and cell death in rat and guinea pig hepatocytes. Inhibiting ascorbate synthesis, however, further elevated ROS production (2-fold), lipid peroxidation (1.5-fold), and cell death (2-fold) in rat hepatocytes only. This is the first time that endogenous ascorbate synthesis has been shown to decrease cellular susceptibility to oxidative stress. Protection by endogenously produced ascorbate may therefore need to be addressed when extrapolating data to humans from experiments using rodents capable of synthesizing ascorbate.     

Counterflow of L-glutamate in plasma membrane vesicles and reconstituted preparations from rat brain

Membrane vesicles from rat brain exhibit sodium-dependent uptake of L-[3H]glutamate in the absence of any transmembrane ion gradients. The substrate specificity of the process is identical with (Na+ + K+)-coupled L-glutamate accumulation. Although these vesicles are prepared after osmotic shock and are washed repeatedly, they contain about 1.5 nmol/mg of protein endogenous L-glutamate, apparently located inside the vesicles. The affinity of the process (Km approximately 1 microM) is similar to that of (Na+ + K+)-dependent accumulation by the L-glutamate transporter. Membrane vesicles have been disrupted by the detergent cholate, and the solubilized proteins have been subsequently reconstituted into liposomes. The reconstituted proteoliposomes also exhibit the above uptake--with the same characteristics--provided they contain entrapped cold L-glutamate. Counterflow is optimal when sodium is present on both sides of the membrane, but partial activity is still observed when sodium is present either on the inside or on the outside. Increasing the L-glutamate concentration above the Km results in counterflow completely independent of cis sodium. The initial rate of counterflow is 100-200-fold lower than that of net trans potassium dependent flux. The rate of net flux in the presence of trans sodium or lithium is about 10-fold lower than when choline or Tris are used instead. However, the rate of counterflow (no internal potassium present) was not stimulated by replacing internal sodium or lithium by internal choline. Therefore, optimal functioning of the transporter requires internal potassium while internal sodium and lithium are inhibitory.(ABSTRACT TRUNCATED AT 250 WORDS)