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PubMed1985

Splenic pooling of granulocytes.

The intrasplenic kinetics of granulocytes, isolated in plasma and labelled in plasma with 111In-tropolonate, have been studied in normal subjects, patients with negative studies for inflammatory disease and patients with positive studies, with the aim of identifying the nature of splenic activity seen after 111In-labelled granulocyte administration. Up to 40 min after injection, 111In activity was visible only in major blood vessels, liver and spleen, with slight, abnormal activity visible in most of those with positive scans. The time courses of uptake of hepatic and splenic activity were different, with liver activity rapidly reaching a plateau and splenic activity increasing mono-exponentially to a plateau achieved between 20 and 40 min. The clear difference between the shapes of the hepatic and splenic uptake curves and the magnitude of the splenic uptake rate constant indicated that splenic activity represented reversible uptake. The application of deconvolution analysis to the blood and splenic time-activity curves generated a splenic retention (or washout) curve consistent with dynamic exchange of granulocytes between blood and spleen. The slope of this curve indicated an intrasplenic granulocyte transit time of 9.3 (+/- SE 0.6) min. Taking splenic activity to be reversible, comparison of the 111In signal from the spleen 40 min after injection of 111In-labelled granulocytes with that given from the spleen after the injection of 111In-labelled erythrocytes (relative to their respective blood levels) indicated that intrasplenic granulocyte transit time was 14.4 (+/- SE 1.1) times that of erythrocytes. Based on actual erythrocyte time, this corresponds to a granulocyte transit time of 8.6-11.5 min, in close agreement with the estimate based on deconvolution analysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Colitis↗
PubMed2004

Time-dependent probability of quantum tunneling in terms of the quasisemiclassical method.

In view of the rapid progress in experiments of the tunneling dynamics in the time domain, we develop a quasisemiclassical method that is aimed at a study of the proton-transfer dynamics in a large system such as tropolone and its interesting derivatives, to which not only full quantum mechanics, but even a standard semiclassical theory is never easy to apply. In our very tractable method for multidimensional systems, the tunneling paths are generated in terms of the generalized classical mechanics, but the quantum phases arising from the action integral, the Maslov index, and the semicalssical amplitude factor as well in the semiclassical kernels are entirely neglected. This approach is called the quasisemiclassical method. One of the technical issues involved in the general semiclassical scheme is how to locate points from which a tunneling path emanates. Hence the studies of such tunneling points and the quasisemiclassical method should be examined collectively. We test several ways of determining the tunneling point, including those already proposed in the literature and a newly proposed one. It is shown numerically that the quasisemiclassical method with an appropriate choice of tunneling points reproduces the full quantum mechanical tunneling probability reasonably well. This case study indicates that the present conventional approach is promising to the study of large systems. The role of tunneling points in the initial process of tunneling is also discussed.

Journal Article↗
PubMed1974

Promotion of fluorescence upon binding of colchicine to tubulin.

Colchicine, which does not fluoresce in aqueous media and organic solvents, exhibits marked fluorescence on combination with brain tubulin, with a corrected excitation maximum at 362 nm, an emission maximum at 435 nm, and a quantum yield of about 0.03. From fluorescence measurements it was found that rat brain tubulin binds 0.83 moles of colchicine per dimer (molecular weight 110,000) with an association constant of 3.2 muM(-1) at pH 7.0 and 37 degrees . These results are in excellent agreement with those obtained with the binding of [(3)H]-colchicine. The enthalpy of binding is 10 kcal/mole, with an entropy change of 62 entropy units. The fluorescence can be ascribed to the tropolone moiety. However, the A ring of colchicine is also involved in binding. Denaturing agents abolish fluorescence, whereas podophyllotoxin, another antimitotic agent, decreases fluorescence competitively. Fluorescence is a convenient method for determining the binding of colchicine to tubulin that does not require the separation of free colchicine from bound colchicine and yields values for physical and biochemical parameters that are in excellent agreement with those obtained from the binding of [(3)H]colchicine.

Animals↗
PubMed1986

B ring regulation of colchicine binding kinetics and fluorescence.

Several properties of the colchicine-tubulin interaction such as association rate, reversibility, and the promotion of drug fluorescence have been related to the B ring of colchicine. The B ring itself retards the binding rate, and substitution at C-7 leads to further binding rate decreases that appear to be related to both substituent bulk and the presence of a N-acyl group. Thus, the decreasing order of binding rates is 2-methoxy-5-(2',3',4'-trimethoxyphenyl)tropone greater than deacetamidocolchicine greater than deacetylcolchicine greater than or equal to colcemid greater than colchicine greater than N-benzoyldeacetylcolchicine, etc. The apparent irreversibility of the binding seems more closely related to the presence of an N-acyl group rather than the bulk of the substituent at C-7. Substitution at C-7 also affects the tropolone fluorophore. Thus, amines (deacetylcholchicine, colcemid, or N-methylcolcemid) fluoresce poorly in the presence of tubulin, whereas substitution of the amino group with an acyl group enhances fluorescence. The presence of an N-acyl group at C-7 is essential for enhanced fluorescence. We conclude that, in addition to A- and the C-ring portion of the molecule, the B ring of colchicine is a third determinant recognized by the binding site on tubulin.

Animals↗
PubMed1996

Identification of cysteine 354 of beta-tubulin as part of the binding site for the A ring of colchicine.

The colchicine analog 3-chloroacetyl-3-demthylthio-colchicine (3CTC) is a competitive inhibitor of colchicine binding to tubulin, binds to tubulin at 37 degrees C, but not at 0 degree C, and covalently reacts with beta-tubulin at 37 degree C, but not at 0 degree C, in a reaction inhibited by colchicine site drugs. The approximate intramolecular distance between the oxygen at position C-3 in 3CTC and the chlorine atom of the 3-chloroacetyl group is 3 A. using decylagarose chromatography, we purified beta-tubulin that had reacted with 3-(chloromethyl-[14C] Carbonyl)-3- demethylthiocolchicine ([14C]3CTC). This beta-tubulin that had reacted with 3-(chloromethyl-[14C]carbonyl)- 3-demethythiocolchicine ([14C]3CTC). This beta-tubulin was digested with formic acid, cyanogen bromide, endoproteinase Glu-C, or endoproteinase Lys-C, and the radio-labeled peptide(s) were isolated. The sequences of these peptides indicated that as much as 90% of the covalent reaction between the [14C]3CTC and beta-tubulin occurred at cysteine 354. This finding indicates that the C-3 oxygen atom of colchicinoids is within 3 A of the sulfur atom of the Cys-354 residue, suggests that the colchicine A ring lies between Cys-354 and Cys-239, based on the known 9 A distance between these residues, and may indicate that the tropolone C ring lies between the peptide region containing Cys-239 and the amino-terminal beta-tubulin sequence, based on the labeling pattern observed following direct photoactivation of tubulin-bound colchicine.

Amino Acid Sequence↗
PubMed2003

Butyltin compounds in retail mollusc products.

Butyltin was found in 62 of 74 mollusc products purchased from major supermarket outlets in seven cities across Canada. Tribuyltin was detected in 41 samples, with levels up to 233 ng g(-1). Dibutyltin and monobutyltin levels ranged up to 88 and 53 ng g(-1), respectively. Products originating from East and South East Asia generally contained the highest levels. Gastropods contained lower levels than other molluscs. Enzymatically hydrolysed samples were extracted with 0.05% tropolone in hexane-diethyl ether (1 : 1). Ethyl derivatives were made by Grignard reaction and analysed by gas chromatography coupled to an atomic emission detector. Gas chromatograph-mass spectrometry confirmed the presence of butyltins in the mollusc samples.

Animals↗
PubMedDate not supplied

Survey of organotin compounds in blended wines.

An extraction method for butyltin, cyclohexyltin, octyltin and phenyltin compounds in wines was developed using 0.05% tropolone in 75% diethyl ether/pentane. Recoveries averaged 96.8% (range 78-121%). Methyl derivatives made by Grignard reaction were quantitated by gas chromatography (GC)-atomic absorption spectrometry (AAS). Twenty-nine of the 90 samples (32.2%) tested contained at least one of the analytes. Mono- and diocyltin were found in one sample only, at levels of 2.41 and 0.12 ng/ml respectively. Dibutyltin levels ranged from < 0.08 to 1.44 ng/ml. Monobutyl and tributyltin were found less frequently and at lower levels than dibutyltin. GC/mass spectrometric analysis confirmed the presence of butyl- and octyltins. No other organotin compounds were detected by either GC-AAS or GC-MS.

Gas Chromatography-Mass Spectrometry↗
PubMed1988

Migration patterns of dendritic cells in the mouse. Traffic from the blood, and T cell-dependent and -independent entry to lymphoid tissues.

Dendritic cells (DC) are critical accessory cells for primary immune responses and they may be important stimulators of transplantation reactions, but little is known of their traffic into the tissues. We have studied the migration of purified splenic DC and T lymphocytes, labeled with 111Indium-tropolone, in syngeneic and allogeneic mice. First we demonstrate that DC can migrate from the blood into some lymphoid and nonlymphoid tissues. Immediately after intravenous administration, radio-labeled DC were sequestered in the lungs, but they actively migrated into the liver and spleen and reached equilibrium levels between 3 and 24 h after transfer. At least half of the radiolabel accumulated in the liver, but the spleen was the principal site of DC localization in terms of specific activity (radiolabel per weight of tissue). DC were unable to enter Peyer's patches, or mesenteric and other peripheral lymph nodes from the bloodstream. This was also true in splenectomized recipients, where the otherwise spleen-seeking DC were quantitatively diverted to the liver. In contrast, T cells homed readily to the spleen and lymph nodes of normal mice and increased numbers were present in these tissues in splenectomized mice. Thus, unlike T cells, DC cannot recirculate from blood to lymph via the nodes. We then show that migration of DC from the blood into the spleen is dependent on the presence of T cells: DC did not enter the spleens of nude mice, but when they were reconstituted with T cells the numbers entering the spleen resembled those in euthymic mice. In nude mice, as in splenectomized recipients, the DC that would normally enter the spleen were quantitatively diverted to the liver. These findings suggest that there is a spleen-liver equilibrium for DC, that may be akin to that existing between spleen and lymph node for T cells. Finally, we followed the traffic of radiolabeled DC via the afferent lymphatics after subcutaneous footpad inoculation. DC accumulated in the popliteal nodes but did not migrate further to the inguinal nodes. There was no difference between euthymic and nude mice, showing that unlike traffic to the spleen, this route probably does not require T cells. These migration patterns were not affected by major histocompatibility barriers, and were only seen with viable, but not glutaraldehyde-fixed, DC.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMedDate not supplied

Application of gas chromatographic determination of organotin compounds to basic research on the metabolism of triphenyltin chloride in rats.

This paper describes the application of gas chromatographic determination of organotin compounds to basic research on the metabolism of triphenyltin chloride in rats. Phenyltin compounds were extracted (as chloride) from rat-liver homogenates and subcellular fractions with hydrochloric acid and n-hexane-benzene (3:2, containing 0.05% tropolone). These organotin compounds were pentylated with a Grignard reagent prior to capillary gas chromatography. The absolute detection limits were in the range of 3.6 to 4.4 pg as tin. The absolute recoveries of phenyltin compounds added to homogenized whole liver and each subcellular fraction sample ranged from 73.9 to 97.4%. The intracellular distribution of triphenyltin and its metabolites in the liver was observed for 72 h after a single oral dose of triphenyltin chloride was given to rats. The tri-, di-, and monophenyltin compounds in the fractions reached maximal amounts about 6, 48, and 72 h after administration, respectively. Also, the concentrations of monophenyltin compounds in the liver were typically lower than to those of di- and triphenyltin compounds. The highest contents of di- and triphenyltin compounds per protein were found in the microsomal fractions.

Animals↗
PubMed1990

Large-scale purification of bovine brain lactate dehydrogenase by affinity chromatography on immobilized colchicine.

Lactate dehydrogenase (LDH) [EC 1.1.1.27] in a crude extract (40-80% ammonium sulfate fraction) of bovine brain was adsorbed on an immobilized colchicine column and specifically eluted by addition of 1 mM NADH. The purity and subunit composition of the pooled LDH were estimated by two-dimensional gel electrophoresis. With an increase of NaCl concentration from 0 to 2.0 M, ligand saturation of LDH on immobilized colchicine increased from 6.8 to 14%, whereas that on immobilized Cibacron blue F3GA decreased from 2.1 to 0%. In the presence of high NaCl concentration, immobilized colchicine enabled both large- and small-scale purification of LDH by affinity chromatography and resulted in a yield of 117 mg from 1 kg of bovine brain in the presence of 2.5 M NaCl or higher recoveries of 54-96% from various tissues of one rat in the presence of 1.0 M NaCl. These results indicate that immobilized colchicine is an excellent adsorbent for the isolation and purification of LDH by affinity chromatography and has a high LDH-adsorbing capacity dependent upon a high NaCl concentration. Kinetic studies revealed that colchicine apparently competed with cofactor NAD for the active site of LDH and the Ki values of colchicine decreased with an increase of NaCl concentration. The chemical specificity of the colchicine-binding site of LDH was studied by the use of colchicine analogues and it is concluded that both the tropolone moiety (C-ring) and the amido bond in a side chain of colchicine structure are essential to the colchicine-LDH interaction.

Animals↗
PubMed1996

Widespread hemodynamic depression and focal platelet accumulation after fluid percussion brain injury: a double-label autoradiographic study in rats.

Cerebrovascular damage leading to subsequent reductions in local cerebral blood flow (lCBF) may represent an important secondary injury mechanism following traumatic brain injury (TBI). We determined whether patterns of 111-indium-labeled platelet accumulation were spatially related to alterations in lCBF determined autoradiographically 30 min after TBI. Sprague-Dawley rats (n = 8), anesthetized with halothane and maintained on a 70:30 (vol/vol) mixture of nitrous oxide/oxygen and 0.5% halothane, underwent parasagittal fluid percussion brain injury (1.7-2.2 atm). 111-Indium-tropolone-labeled platelets were injected 30 min prior to TBI while [14C]-iodoantipyrine was infused 30 min after trauma. Sham-operated animals (n = 7) underwent similar surgical procedures but were not injured. In autoradiographic images of the indium-labeled platelets, focal sites of platelet accumulation within the traumatized hemisphere were restricted to the pial surface (five of eight rats), the external capsule underlying the lateral parietal cortex (five of eight rats), and within cerebrospinal fluid (CSF) compartments (six of eight rats). In contrast, mild-to-moderate reductions in lCBF, not restricted to sites of platelet accumulation, were seen throughout the traumatized hemisphere. Flow reductions were most severe in coronal sections underlying the impact site. For example, within the lateral parietal cortex and hippocampus, lCBF was significantly reduced [p <0.01; analysis of variance (ANOVA)] from 1.71 +/- 0.34 (mean +/- SD) and 0.78 +/- 0.12 ml/g/min, respectively, versus 0.72 +/- 0.17 and 0.41 +/- 0.06 ml/g/min within the traumatized hemisphere. Significant flow reductions were also seen in remote cortical and subcortical areas, including the right frontal cortex and striatum. These results indicate that focal platelet accumulation and widespread hemodynamic depression are both early consequences of TBI. Therapeutic strategies directed at these early microvascular consequences of TBI may be neuroprotective by attenuating secondary ischemic processes.

Animals↗
PubMed1998

Posttraumatic cerebral ischemia after fluid percussion brain injury: an autoradiographic and histopathological study in rats.

OBJECTIVES: Mild-to-moderate reductions in local cerebral blood flow (ICBF) have been reported to occur in rats after moderate (1.7-2.2 atm) fluid percussion brain injury. The purpose of this study was to determine whether evidence for severe ischemia (i.e., mean ICBF < 0.25 ml/g/min) could be demonstrated after severe brain injury. In addition, patterns of indium-labeled platelet accumulation and histopathological outcome were correlated with the hemodynamic alterations. METHODS: Sprague-Dawley rats (n = 23), anesthetized with halothane and maintained on a 70:30 mixture of nitrous oxide:oxygen and 0.5% halothane, underwent normothermic (37 degrees C) parasagittal fluid percussion brain injury (2.4-2.6 atm). Indium-111-tropolone-labeled platelets were injected 30 minutes before traumatic brain injury (TBI), while 14C-iodoantipyrine was infused 30 minutes after trauma for ICBF determination. Sham-operated animals (n = 8) underwent similar surgical procedures but were not injured. For histopathological analysis, traumatized rats (n = 5) were perfusion-fixed 3 days after TBI. RESULTS: In autoradiographic images of indium-labeled platelets, abnormal platelet accumulation that was most pronounced overlying the pial surface was commonly associated with severe reductions in ICBF within underlying cortical regions 30 minutes after TBI. For example, within the lateral parietal cortex, ICBF was significantly reduced from 1.67 +/- 0.11 ml/g per minute (mean +/- standard error of the mean) in sham-operated animals to 0.23 +/- 0.03 ml/g per minute within the traumatized group. In addition to focal severe ischemia, moderate reductions in ICBF were detected throughout the traumatized hemisphere, including the frontal and occipital cortices, hippocampus, thalamus, and striatum. Mild decreases in ICBF were also observed throughout the contralateral cerebral cortex. At 3 days after severe TBI, histopathology demonstrated intracerebral and subarachnoid hemorrhage associated with cerebral contusion and selective neuronal necrosis. CONCLUSION: These data indicate that multiple cerebrovascular abnormalities, including subarachnoid hemorrhage, focal platelet accumulation, and severe ischemia, are important early events in the pathogenesis of cortical contusion formation after TBI. Injury severity is expected to be a critical factor in determining what therapeutic strategies are attempted in the clinical setting.

Animals↗
PubMed1988

Osteomyelitis.

The use of 111In-labelled granulocyte scintigraphy is recognized as a reliable method for detecting osteomyelitis and has similar sensitivity and significantly increased specificity compared to bone scintigraphy and 67Ga studies. Recent published work using pure granulocytes labelled with 111In tropolonate to detect osteomyelitis resulted in sensitivity of 100% and specificity of 92%. 99Tcm as an alternative granulocyte label offers advantages of convenience, lower radiation dose and higher image resolution. We have scanned 20 patients with suspected osteomyelitis using autologous granulocytes labelled with 99Tcm hexamethylpropyleneamineoxime (HMPAO), 12 of whom had prosthetic joints. The scan results were correlated with clinical, radiographic, microbiological and histological findings. Sensitivity was 100% and specificity was 93% which compares favourably with results obtained using 111In-labelled granulocytes. We believe that labelled granulocyte scintigraphy is a useful investigation in the diagnosis of osteomyelitis and that 99Tcm HMPAO appears to be at least as useful as 111In as the labelling agent.

Granulocytes↗
PubMed1994

Utility of 111In-labelled leukocytes in patients completing high-dose interleukin-2 therapy: a pilot study.

Interleukin-2 (IL-2) therapy has resulted in modest response rates in patients with renal carcinoma and melanoma, but few reproducible pre- or post-treatment parameters have been associated with response, and tumour localization with lymphokine-activated killer cells has only occasionally been demonstrated. Eight patients (seven with renal carcinoma and one with melanoma) treated on a protocol with chronic indomethacin and ranitidine with three courses of continuous infusion of IL-2 had peripheral blood leukocytes withdrawn 12-36 h after completion of IL-2 therapy, labelled with 111In tropolone and reinjected. Images were taken at 4 and 20 h after reinjection. Three of these patients achieved objective responses to therapy, but none demonstrated uptake of radioisotope-labelled leukocytes in known tumour-bearing areas. Two nonresponding patients (one renal carcinoma, one melanoma) demonstrated uptake in all known tumour areas; one further nonresponding patient demonstrated uptake in the region of a femoral metastasis, but not in other bulky areas of disease. No correlation between scan uptake, and leukocyte subsets could be demonstrated. Although occasional patients demonstrate tumoural accumulations of 111In-labelled leukocytes after completion of therapy with IL-2, this does not appear to be associated with response.

Carcinoma, Renal Cell↗
PubMed2001

Studies on white blood cell labelling: (99)Tc(m)-HMPAO preferentially labels granulocytes.

Mixed leucocytes have been labelled in small volumes of plasma using small aliquots of hexamethylpropylene amine oxime (HMPAO) from vials of Ceretec, which were fractionated after reconstitution and stored frozen under nitrogen. Labelling efficiency was found to be independent of HMPAO concentration >8.8 microg.ml-1 and independent of cell concentration at >8x10(7) ml-1. Results are presented from 145 patients referred for white cell scintigraphy. Labelling efficiency was 78.9+/-9.8%. The labelled mixed leucocytes were separated on a 3-step density gradient of Percoll/plasma which showed that (99)Tc(m)-HMPAO gave a granulocyte/lymphocyte specific activity ratio of 1.67; the ratio for (111)In-tropolone or oxine was 1.0, with no preference for granulocytes. It was noted that the labelling efficiency was reduced when there was a high proportion of lymphocytes present, and also that in one patient with non-Hodgkin's lymphoma the lymphocytes labelled as equally well as the granulocytes. The stability of the label was studied by fractionating the mixed cells after in vitro incubation and after 1 h in vivo; very little loss of label occurred.

Granulocytes↗
PubMed1994

Thiopurine methyltransferase activity in a French population: h.p.l.c. assay conditions and effects of drugs and inhibitors.

1. Thiopurine methyltransferase (TPMT) is a cytosolic enzyme involved in the catabolism of thiopurine drugs, which are used to treat cancer patients and organ transplant recipients. Because TPMT activity is polymorphic and under genetic control, large interindividual variations in the immunosuppressive activity and toxicity of these drugs may, at least in part, be inherited. 2. We have developed a specific h.p.l.c. method for measuring 6-methyl mercaptopurine formed from 6-mercaptopurine (6-MP) in red blood cell lysates during the TPMT assay procedure. In blinded assays of 55 samples from adult blood donors, the results of the h.p.l.c. method correlated with those of the radiochemical reference method (r = 0.83, P < 0.001). 3. Using this h.p.l.c. assay, we tested the effect of known inhibitors of TPMT activity (syringic acid, p-anisic acid and tropolone) in vitro and showed that they were highly inhibitory. We also found that drugs often administered concomitantly with 6-MP (prednisone, prednisolone, 6-methylprednisolone, cyclophosphamide, methotrexate, and trimethoprim-sulphamethoxazole) had little or no effect on TPMT activity in vitro. 4. In a group of 300 French individuals, TMPT activity was highly variable, ranging from 4.7 to 35.3 nmol h-1 ml-1 of packed red blood cells (nmol h-1 ml-1 PRBC) with a mean value of 19.3 +/- 4.9. TMPT activity was not influenced by sex. 5. This sensitive and reproducible h.p.l.c. assay for TPMT activity in red blood cells may prove useful for prospective clinical studies designed to optimise dosage regimens of thiopurine drugs (detection limit for 6-methyl mercaptopurine is 5 ng ml-1, intra- and inter-assay variations are 6.8 and 8.2%, respectively).

Adult↗
PubMed1982

Estimation of the turnover of 3-methoxytyramine in the rat striatum by HPLC with electrochemical detection: implications for the sequence in the cerebral metabolism of dopamine.

A highly sensitive method for the determination of 3-methoxytyramine (3-MT) in nervous tissue is described. The method is based on a rapidly performed isolation of 3-MT on small columns of Sephadex G 10, followed by reverse-phase high-performance liquid chromatography in conjunction with a rotating disk electrochemical detector. The detection limit of the assay (0.5-1 pmol/tissue sample) is about 10% of control value for microwave-killed rats. 3-MT as well as dopamine could be quantified in the same chromatographic run. Inhibition of catechol-O-methyl transferase with tropolone resulted in an exponential decline of 3-MT. From this exponential decline a turnover rate for 3-MT of 1.9 nmol/gh/h was calculated. In the same group of rats the turnover rate of homovanillic acid was 9.1 nmol/g/h. From these data it is concluded that in the rat striatum about 80% of homovanillic acid is formed from 3,4-dihydroxyphenylacetic acid and 20% from 3-MT.

Animals↗
PubMed1984

Monitoring the stimulated release of dopamine with in vivo voltammetry. II: Clearance of released dopamine from extracellular fluid.

Microvoltammetric electrodes implanted in the caudate nucleus of the anesthetized rat have been used to monitor dopamine released following electrical stimulation of the medial forebrain bundle. These electrodes are fabricated from unmodified carbon fibers and have been used with normal pulse voltammetry. Dopamine appears in the vicinity of the electrode when the stimulation is initiated, and disappears almost immediately when the stimulation is terminated. The data suggest that the effective diffusion distance is less than 100 micron. Postmortem analysis using liquid chromatography with electrochemical detection shows that dopamine released in this manner is metabolized to 3,4-dihydroxyphenylacetic acid (DOPAC); however, neither substance is observed electrochemically in the extracellular fluid within seconds after the stimulation. In addition, inhibitors of neuronal uptake of dopamine, amphetamine (1.8 or 15 mg X kg-1) or benztropine (25 mg X kg-1), or of dopamine metabolism, pargyline (150 mg X kg-1) or tropolone (100 mg X kg-1), do not significantly affect the rate at which dopamine disappears from extracellular fluid, although they can affect the amount released. These results suggest that dopamine cannot freely diffuse in the extracellular fluid because an extraneuronal uptake mechanism exists that clears dopamine from extracellular fluid into an extraneuronal pool where metabolism to 3,4-dihydroxyphenylacetic acid occurs. Dopamine can be observed during electrical stimulation of the ascending fibers because neuronal and extraneuronal uptake systems are unable to remove dopamine on these short time scales.

3,4-Dihydroxyphenylacetic Acid↗