Evidence that dioxygen and substrate activation are tightly coupled in dopamine beta-monooxygenase. Implications for the reactive oxygen species

Oxygen activation occurs at a wide variety of enzyme active sites. Mechanisms previously proposed for the copper monooxygenase, dopamine beta-monooxygenase (DbetaM), involve the accumulation of an activated oxygen intermediate with the properties of a copper-peroxo or copper-oxo species before substrate activation. These are reminiscent of the mechanism of cytochrome P-450, where a heme iron stabilizes the activated O2 species. Herein, we report two experimental probes of the activated oxygen species in DbetaM. First, we have synthesized the substrate analog, beta,beta-difluorophenethylamine, and examined its capacity to induce reoxidation of the prereduced copper sites of DbetaM upon mixing with O2 under rapid freeze-quench conditions. This experiment fails to give rise to an EPR-detectable copper species, in contrast to a substrate with a C-H active bond. This indicates either that the reoxidation of the enzyme-bound copper sites in the presence of O2 is tightly linked to C-H activation or that a diamagnetic species Cu(II)-O2* has been formed. In the context of the open and fully solvent-accessible active site for the homologous peptidylglycine-alpha-hydroxylating monooxygenase and by analogy to cytochrome P-450, the accumulation of a reduced and activated oxygen species in DbetaM before C-H cleavage would be expected to give some uncoupling of oxygen and substrate consumption. We have, therefore, examined the degree to which O2 and substrate consumption are coupled in DbetaM using both end point and initial rate experimental protocols. With substrates that differ by more than three orders of magnitude in rate, we fail to detect any uncoupling of O2 uptake from product formation. We conclude that there is no accumulation of an activated form of O2 before C-H abstraction in the DbetaM and peptidylglycine-alpha-hydroxylating monooxygenase class of copper monooxygenases, presenting a mechanism in which a diamagnetic Cu(II)-superoxo complex, formed initially at very low levels, abstracts a hydrogen atom from substrate to generate Cu(II)-hydroperoxo and substrate-free radical as intermediates. Subsequent participation of the second copper site per subunit completes the reaction cycle, generating hydroxylated product and water.     

Dopamine Transporter Loss in 6-OHDA Parkinson’s Model Is Unmet by Parallel Reduction in Dopamine Uptake

The dopamine transporter (DAT) regulates synaptic dopamine (DA) in striatum and modulation of DAT can affect locomotor activity. Thus, in Parkinson’s disease (PD), DAT loss could affect DA clearance and locomotor activity. The locomotor benefits of L-DOPA may be mediated by transport through monoamine transporters and conversion to DA. However, its impact upon DA reuptake is unknown and may modulate synaptic DA. Using the unilateral 6-OHDA rat PD model, we examined [³H]DA uptake dynamics in relation to striatal DAT and tyrosine hydroxylase (TH) protein loss compared with contralateral intact striatum. Despite >70% striatal DAT loss, DA uptake decreased only ∼25% and increased as DAT loss approached 99%. As other monoamine transporters can transport DA, we determined if norepinephrine (NE) and serotonin (5-HT) differentially modulated DA uptake in lesioned striatum. Unlabeled DA, NE, and 5-HT were used, at a concentration that differentially inhibited DA uptake in intact striatum, to compete against [³H]DA uptake. In 6-OHDA lesioned striatum, DA was less effective, whereas NE was more effective, at inhibiting [³H]DA uptake. Furthermore, norepinephrine transporter (NET) protein levels increased and desipramine was ∼two-fold more effective at inhibiting NE uptake. Serotonin inhibited [³H]DA uptake, but without significant difference between lesioned and contralateral striatum. L-DOPA inhibited [³H]DA uptake two-fold more in lesioned striatum and inhibited NE uptake ∼five-fold more than DA uptake in naïve striatum. Consequently, DA uptake may be mediated by NET when DAT loss is at PD levels. Increased inhibition of DA uptake by L-DOPA and its preferential inhibition of NE over DA uptake, indicates that NET-mediated DA uptake may be modulated by L-DOPA when DAT loss exceeds 70%. These results indicate a novel mechanism for DA uptake during PD progression and provide new insight into how L-DOPA affects DA uptake, revealing possible mechanisms of its therapeutic and side effect potential.     

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.     

Kinetic analysis of a model of the sarcoplasmic reticulum Ca-ATPase, with variable stoichiometry, which enhances the amount and the rate of Ca transport

The sarcoplasmic reticulum (SR) Ca-dependent adenosinetriphosphatase (Ca-ATPase) actively transports Ca2+ from the myoplasm to the SR lumen. Under optimal conditions a 2:1 stoichiometry of Ca transport/ATP hydrolysis has been observed, but lower stoichiometries have been reported under several circumstances. A lower stoichiometry under conditions of high Ca2+ load, although thermodynamically less efficient, could in theory increase the rate and the maximal amount of Ca uptake. We analysed, by computing simulation, the transient kinetics of a model of the SR Ca-ATPase with variable stoichiometry. The model is based on current experimental reports and includes the most relevant properties of the system. The results show an acceleration in the rate of Ca uptake, an increase in the net Ca transport, and an increase in the rate of [Ca2+] reduction in the medium, which might be physiologically useful to increase the rate of Ca pumping at high Ca load of the sarcoplasmic reticulum.     

Pharmacological characterization of dopamine transport in cultured rat astrocytes.

The effects of GBR-12909 (selective DA uptake inhibitor), zimelidine (selective 5-HT uptake inhibitor) and nisoxetine (selective NE uptake inhibitor) on the uptake of 30 nM [3H]DA into cultured rat astrocytes were examined. [3H]DA uptake was inhibited by approximately 50% by GBR-12909 or zimelidine in a concentration-dependent manner (100 nM to approximately 10 microM). Furthermore, the inhibition curves of GBR-12909 were biphasic, and uptake was completely inhibited by a high concentration of GBR-12909 (100 microM). [3H]DA uptake was also inhibited by approximately 50% by nisoxetine in a concentration-dependent manner (0.1 to approximately 100 nM), and nisoxetine was more potent than GBR-12909 or zimelidine. The inhibitory potencies were in the order nisoxetine > GBR-12909 > zimelidine. The uptake of [3H]DA under Na+-free conditions was approximately 50% of that under normal conditions. Thus, DA was taken up by both Na+-dependent and Na+-independent mechanisms. Nisoxetine (100 nM), zimelidine (100 microM) and GBR-12909 (10 microM) inhibited [3H]DA uptake into astrocytes only in the presence of Na+. On the other hand, this uptake was completely inhibited by a high concentration of GBR-12909 (100 microM) in the absence of Na+. The present data suggest that the Na+-dependent uptake of [3H]DA in cultured rat astrocytes may occur in the NE uptake system. Furthermore, astrocytes express the extraneuronal monoamine transporter (uptake2), which is an Na+-independent system, and this transporter is involved in the inactivation of centrally released DA.     

Non-michaelian behavior of 5 alpha-reductase in human prostate

An in-depth analysis of the kinetics of 5 alpha-reductase in human prostatic tissue gave findings inconsistent with the claim that the enzyme is michaelian. In both hyperplastic and malignant tissue, the time-course of the conversion of testosterone (T) into dihydrotestosterone (DHT) was non-linear under conditions ensuring less than 15% conversion of substrate and cofactor. An initial rapid phase of conversion was followed by a long steady-state phase. This time-dependent change in conversion rate was not due to enzyme denaturation, fast inhibition by substrate or product effects. It resulted from a true slow transient kinetic process induced in the reactive enzyme by the substrates. Under our experimental conditions at pH 5.5, 5 alpha-reductase appeared to undergo a conformational change from an initially highly reactive form to a less reactive form. Since this "hysteretic" behavior was correlated with apparently negative cooperativity in enzyme kinetics, we postulate that, as previously described for other key metabolic enzymes, regulation of 5 alpha-reductase activity in the prostate depends on the molecular flexibility of the enzyme and on changes in the cooperativity of different enzyme forms over time. This original non-michaelian behavior may explain the conflicting kinetics reported so far in the literature for this enzyme. The clinical implications of 5 alpha-reductase hysteresis and its involvement in the damping of DHT production within the prostate are discussed.     

Contribution of Sulfate Conjugation, Deamination, and O-Methylation to Metabolism of Dopamine and Norepinephrine in Human Brain

Abstract: The kinetic constants were determined for dopamine (DA) and norepinephrine (NE) metabolism by phenolsulfotransferase (PST), type A and B monoamine oxidase (MAO), and membrane-bound and soluble catechol- O - methyltransferase (COMT) in frontal lobe preparations of human brain. PST and membrane-bound COMT were found to have the lowest K _m, values for both catecholamines. By means of the appropriate rate equations and the calculated kinetic constants for each enzyme, the activity of each enzymatic pathway was determined at varying concentrations of DA and NE. Results indicate that deamination by MAO is the principal pathway for the enzymatic inactivation of DA whereas NE is largely metabolized by MAO type A and membrane-bound COMT under the in vitro assay conditions used. At concentrations less than 100 μ M , soluble COMT'contributes less than 5% to the total catabolism of either catecholamine. PST can contribute up to 15% of the total DA metabolism and 7% of NE metabolism.     

Releases of Norepinephrine and Dopamine in Ventriculocisternal Perfusions in Hepatectomized and Laparotomized Rats

Abstract: The completely hepatectomized rat has frequently been used as a model to study changes in the economy of norepinephrine (NE) and dopamine (DA) in hepatic coma. Hypothermia characteristically develops in hepatectomized rats and also occurs in patients in hepatic coma and is associated with improved survival in both. The aims of the present study were to measure both release and uptake of NE and release of DA in brain in warm (37°C) and cool (30–32°C) rats at 3–5 h after laparotomy or hepatectomy. Ventriculocisternal perfusions of the brain were performed on rats under basal conditions and during releases evoked by 40 m M K⁺. Basal releases of NE and DA and evoked release of DA were greater in the warm hepatectomized rats than in all other groups. In some studies, 10⁻⁵ M amitriptyline was added to the perfusates to assess whether neuronal uptake was changed after hepatectomy. Uptake of released NE was equally robust in cool hepatectomized as in cool laparotomized rats but could not be measured in warm hepatectomized rats because of amitriptyline toxicity in these rats. Decreases in NE and increases in DA content were found in most areas of the brain after perfusion. Increased releases of NE and DA may contribute to the pathogenesis of hepatic encephalopathy.     

Role of ATP and beta-gamma-iminoadenosinetriphosphate in the stimulation of epinephrine and protein release from isolated adrenal secretory vesicles.

ATP-stimulated release of epinephrine and protein from isolated chromaffin granules of the bovine adrenal medulla has been characterized with respect to pH optimum, substrate requirements, and temperature. Chromaffin granules incubated at 37 °C under optimal conditions released virtually all of their epinephrine and soluble protein within 10 min. ATP-stimulated epinephrine release was optimal at pH 5.8–6.2 and was five times greater than at pH 7.0–7.4. Magnesium and chloride were absolutely required for this process. Magnesium stimulated release at concentrations up to 1.0 mm; however, it was moderately inhibitory at higher concentrations. The dependence of release on Cl^? exhibited positive cooperativity and was nonsaturable. At 90 mm Cl^? and 1.0 mm Mg²⁺, ATP-stimulated epinephrine release followed Michaelis-Menten kinetics with a $\text{K}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of 0.2 mm. The release of soluble chromaffin granule proteins closely paralleled epinephrine release under all conditions tested, while membrane components were not released. Analogs of ATP substituted at the β-γ position with methylene or imino groups were also capable of stimulating release from granules. These ATP analogs had reduced affinity and lower activity than ATP itself. ATP analogs substituted at the α-β position were essentially inactive but were potent inhibitors of ATP-stimulated release. We conclude that the regulation of release from granules by ATP is rapid and specific and may not depend on hydrolysis of ATP at the β-γ position. This ATP-dependent reaction may be involved in the cellular process of exocytosis.     

Free, glucuronide, and sulfate catecholamines in the rat: effect of hypoxia

The formation and excretion of conjugated catecholamines (CA) was studied in conscious rats after sympathetic stimulation by hypoxia (5.5-6% O2, 4 h). Hypoxia induced a rapid and intense increase of free epinephrine (E, X 12) and norepinephrine (NE, X 6) but only a limited enhancement of free dopamine (DA, X 2). Sulfate conjugates of E and NE had kinetics similar to the free forms, while glucuronides were only moderately and lately altered. In contrast to free and sulfated DA, DA glucuronide, the major plasma conjugate, was decreased (-25%). This result suggests that DA glucuronide, unlike other CA conjugates, is not related to detoxication but might supply a CA precursor. Urinary conjugates badly reflected plasma conjugates. In normoxic controls, CA conjugates prevailed in the plasma, whereas the free amines prevailed in the urine. Hypoxia increased mainly the excretion of E and NE glucuronide but not of the free amines. Urinary DA, free or conjugated, was decreased (-25%), a result in keeping with plasma DA glucuronide only. The poor relations between plasma and urine catecholamines pinpoint the importance of the kidney in CA handling.     

Characterization of a novel long-chain n-alkane-degrading strain, Dietzia sp. E1

The newly isolated strain E1, identified as a Dietzia sp., proved to have an excellent ability to degrade n-C12 to n-C38 alkane components of crude oil. The preferred substrate was the very long-chain alkane n-eicosane at an optimal temperature of 37 degrees C and an optimal pH of 8 under aerobic conditions. The growth and substrate uptake kinetics were monitored during the n-alkane fermentation process, and Dietzia sp. E1 cells were found to possess three distinct levels of cell-surface hydrophobicity. Gas chromatographic/mass spectrometric analysis revealed that intracellular substrate mineralization occurred through the conversion of n-alkane to the corresponding n-alkanal. The monoterminal oxidation pathway was presumably initiated by AlkB and CYP153 terminal alkane hydroxylases, both of their partial coding sequences were successfully detected in the genome of strain E1, a novel member of the Dietzia genus.     

A short-term diabetes induced changes of catecholamines and p38-MAPK in discrete areas of rat brain

Chronic diabetes is associated with the alteration of second messengers and CNS disorders. We have recently identified that protein kinases (CaMKII and PKC-alpha) and brain neurotransmitters are altered during diabetes as well as in hyperglycemic and acidotic conditions. In this study, we investigated the effects of acute diabetes on the levels of dopamine (DA), norepinephrine (NE), epinephrine (E) and p38-Mitogen-Activated Protein Kinase (p38-MAPK) in striatum (ST), hippocampus (HC), hypothalamus (HT), midbrain (MB), pons medulla (PM), cerebellum (CB) and cerebral cortex (CCX). Alloxan (45 mg/kg) diabetic untreated rats that showed hyperglycemia (>260 mg%), revealed significant increases of DA level in ST (1.5 fold), HC (2.2 fold) and PM (2.0 fold) and the E level also found to be increased significantly in HT (2.4 fold), whereas the NE level was decreased in CB (0.5 fold), after 7 days of diabetes. Immunoblotting study of p38-MAPK expression under identical conditions showed significant alterations in ST, HC, HT and PM (p<0.05) correlated with the changes of catecholamines (DA and E). On the other hand, the above changes were reversed in insulin-treated diabetic rats maintained under normal glucose level (80 -110 mg %). These results suggest that p38-MAPK may regulate the rate of either the synthesis or release of DA and E in corresponding brain areas, but not NE, under these conditions.     

Single doses of catecholamines in the rat: catecholaminotropic, but not hyperglycemic

1. As in two "lower" vertebrates, the lamprey and the eel, single intravascular injections of physiological doses (2.5 micrograms/kg) of epinephrine (E) into the rat immediately increased levels of plasma dopamine (DA) and norepinephrine (NE). 2. Single doses of DA (5 micrograms/kg) enhanced circulating NE and E, while NE (5 micrograms/kg) had no clear impact on the plasma levels of the other two catecholamines (CAs). 3. These data are at variance with findings in the eel, where all three CAs are mutually stimulatory; and in the lamprey, where only E stimulates release of the other two CAs. 4. It appears that E-stimulated CA release is widespread or ubiquitous among vertebrates, and that complex interactions between circulating CAs must be considered under experimental, physiological, and clinical conditions. 5. None of the injections had a significant hyperglycemic effect.     

Effects of dibutyryl cyclic AMP on tyrosine uptake and metabolism in neuroblastoma cultures

A series of thin-layer Chromatographic (TLC) systems were employed to study the effects of dibutyryl cyclic AMP (db-cAMP) on the metabolism of ³H-tyrosine in neuroblastoma cultures. The neuroblastoma monolayer cultures incubated with radiolabelled tyrosine synthesized di-hydroxyphenylalanine (DOPA), dopamine (DA), and norepinephrine (NE), in confirmation of previous reports identifying these compounds in neuroblastoma cultures. In addition, we found evidence suggesting the presence of metabolites of DA and NE, that is, homovanillic acid (HVA) and 3-methoxy-4-hydroxyphenylglycol (MHPG) together with 3-methoxy-4-hydroxymandelic acid (VMA). When these cultures were grown in the presence of db-cAMP for 3 days, tyrosine uptake was increased with a proportional increase in tyrosine hydroxylation. This effect persisted in the absence of db-cAMP, but it was not apparent with only 90 min exposure to db-cAMP. Suspension cultures showed the same baseline level of tyrosine uptake as did monolayer cultures, but the uptake in suspension cultures failed to increase with db-cAMP treatment. It is suggested that the db-cAMP induced differentiation of the neuroblastoma cells in monolayer cultures was associated with induction of a tyrosine uptake system.     

Catecholaminotropic effects of catecholamines in a teleost fish,Anguilla rostrata

Summary Injections of physiological and supraphysiological doses of epinephrine (E) into cardiaccannulated eels cause a dose-related increase of plasma dopamine (DA) and norepineprine (NE) within 3 min. Likewise, both exogenous DA and NE increase the plasma titers of the respective other two catecholamines (CAs). The baseline titers of NE and E are closely correlated. Lack of a correlation of the baseline titers of NE and E with that of DA appears to be due to a faster disappearance rate of DA from the circulation. E is strongly hyperglycemic, and the weaker glycemic action of NE may be mediated via E release. The effects of E seem to depend on a spurt-like increase rather than its titer per se. The ability of the eel to cope with very fast, excessive increases of plasma CAs raises the question of the underlying mechanisms.Abbreviations CA(s) catecholamine(s) - DA dopamine - NE norepinephrine - E epinephrine     

Regulation of pyridoxal-5′-phosphate level by biogenic amines in mouse brain

We investigated the relationship between the concentration of pyridoxal-5′-phosphate (PLP) and biogenic amine in mouse brain. The production of PLP from pyridoxal (PL) by pyridoxal kinase (PLK) was inhibited by the addition of dopamine (DA), norepinephrine (NE) and 5-hydroxytryptamine (5-HT), but not by that of epinephrine and N-acetyl-serotonin. DA and NE were combined with PLP by a non-enzymatic reaction, whereas 5-HT was bound only slightly with PLP. The conjugated product of PLP with DA was also detected by HPLC analysis when PLK activity was assayed using PL as a substrate in the presence of DA. In an in vivo investigation, the depletion of DA and 5-HT in mouse brain after an intraperitoneal injection of 5 mg/kg reserpine, led to slight elevation of the PLP level to 120% of the control level. By contrast, the increase in DA in the brain caused by intraperitoneal administration of 150 mg/kg L-DOPA caused the PLP concentration to decrease to 70% of the control level. However, no change in PLK activity in the brain was observed when the mice were treated with either reserpine or L-DOPA. These results suggested that the level of PLP in mouse brain was partly regulated by the concentration of biogenic amines, such as DA, NE and 5-HT, without apparent induction of PLK.     

Chronic Blockade of Nitric Oxide Synthesis Elevates Plasma Levels of Catecholamines and Their Metabolites at Rest and During Stress in Rats

Formation of nitric oxide, an endothelium-derived relaxing factor, can be inhibited by administration of N-nitro-L-arginine methylesther (L-NAME). In the present study, the activity of the sympathoadrenal system in rats with blood pressure (BP) elevation induced by L-NAME was investigated. L-NAME was administered in a dose of 50 mg/kg, i.p. every 12 h for 4 days. Blood samples were collected via chronically inserted arterial catheters in conscious, freely moving rats at rest and during immobilization stress. Plasma epinephrine (EPI), norepinephrine (NE), and dopamine (DA), as well as catecholamine metabolites dihydroxyphenylglycol (DHPG) and dihydroxyphenylacetic acid (DOPAC) were measured by HPLC method. In L-NAME treated animals, which showed a significant increase in BP, plasma EPI levels were markedly elevated both before and during stress. Plasma NE levels were not significantly increased, however, DHPG levels, which indicate NE turnover and reuptake, were highly elevated. Plasma DA levels were not changed after L-NAME administration but DA metabolite DOPAC showed a significant elevation both under basal conditions and during stress. Thus, the present results indicate that the prolonged blockade of nitric oxide synthesis that causes arterial hypertension is associated with an activation of the sympathoadrenal system.     

Substrate channeling and enzyme complexes for biotechnological applications

Substrate channeling is a process of transferring the product of one enzyme to an adjacent cascade enzyme or cell without complete mixing with the bulk phase. Such phenomena can occur in vivo, in vitro, or ex vivo. Enzyme–enzyme or enzyme–cell complexes may be static or transient. In addition to enhanced reaction rates through substrate channeling in complexes, numerous potential benefits of such complexes are protection of unstable substrates, circumvention of unfavorable equilibrium and kinetics imposed, forestallment of substrate competition among different pathways, regulation of metabolic fluxes, mitigation of toxic metabolite inhibition, and so on. Here we review numerous examples of natural and synthetic complexes featuring substrate channeling. Constructing synthetic in vivo, in vitro or ex vivo complexes for substrate channeling would have great biotechnological potentials in metabolic engineering, multi-enzyme-mediated biocatalysis, and cell-free synthetic pathway biotransformation (SyPaB).     

Effects of exogenous catecholamines on plasma catecholamines and glucose in the sea lamprey,Petromyzon marinus

Summary The effects of exogenous dopamine (DA), norepinephrine (NE) and epinephrine (E) on endogenous catecholamine (CA) titers and glycemia were studied with a highly specific and sensitive radioenzymatic assay (REA) in cardiac-cannulated, prespawning sea lampreys. Neither DA nor NE had a specific effect on the endogenous titers of the other two CAs, or on glycemia. In contrast, E caused a strong increase of both DA and NE at three different doses, one of which must have been in the physiological ranges. This increase may be due to direct stimulation of E on the NE and DA cells. E also caused hyperglycemia 45 min after the injection; however, this effect occurred only with unphysiologically high doses. An estimation of the disappearance rate of exogenous CAs revealed a mammalian-like speed, ranging from 3–5.5 min.Abbreviations CA catecholamines - DA dopamine - E epinephrine - NE norepinephrine - REA radioenzymatic assay     

Free dopamine in dog plasma: lack of relationship with sympathoadrenal activity

To investigate the relationship between dopamine (DA) released into the bloodstream and sympathoadrenal activity, levels of free DA, norepinephrine (NE), and epinephrine (E) in plasma were recorded in four dogs subjected to three tests: treadmill exercise at two work levels [55 and 75% maximal O2 uptake; 15 min], normobaric hypoxia (12% O2; 1 h), combined exercise and hypoxia. Normoxic exercise induced slight nonsignificant decreases in the arterial partial pressure of O2 (PaO2), increases in NE [median values and ranges during submaximal work vs. rest: 1086 (457-1,637) vs. 360 (221-646) pg/ml; P less than 0.01] and E [277 (151-461) vs. 166 (95-257) pg/ml; P less than 0.05], but it failed to alter the DA level. Hypoxia elicited large decreases in PaO2 [hypoxia vs. normoxia: 42.8 (40.3-50.0) vs. 97.6 (83.2-117.6) Torr; P less than 0.01], increases in DA [230 (105-352) vs. 150 (85-229) pg/ml; P less than 0.01] and NE [383 (219-1,165) vs. 358 (210-784) pg/ml; P less than 0.05], but it failed to alter the E level. Combined exercise and hypoxia further increased NE levels but did not alter the DA response to hypoxia alone. The data indicate that free DA in plasma may vary independently of the sympathoadrenal activity.