Architectonic distribution of the serotonin transporter within the orbitofrontal cortex of the vervet monkey.

To elucidate the organization of the serotoninergic innervation within the orbitofrontal cortex (OFC), serotonin transporter (SERT) density was quantified by autoradiography using [(3)H]cyanoimipramine binding. In six adult vervet monkeys, 15 architectonic areas were delineated according to cytoarchitectonic (Nissl), myeloarchitectonic (Gallyas) and chemoarchitectonic (acetylcholinesterase) criteria to assess SERT distribution at two levels of organization: cortical area and cortical type. For cortical type, the 15 areas were evenly divided into three different categories primarily based upon the degree of granularization of layer IV: agranular, dysgranular, and granular. Within agranular and dysgranular, but not granular cortical types, SERT density was area-specific and progressively decreased in a medial to lateral gradient. Across cortical types, SERT density decreased in a caudal to rostral gradient: agranular>dysgranular>granular. A similar caudal to rostral gradient was seen when serotonin content was measured (using high performance liquid chromatography) in areas representative of each cortical type. Collectively, these results suggest that the serotoninergic innervation is organized according to both cortical type and area, and is thus structured to differentially modulate information processing within the OFC.

L-6-[18F]fluoro-dopa metabolism in monkeys and humans: biochemical parameters for the formulation of tracer kinetic models with positron emission tomography.

Characterization of peripheral and cerebral L-3,4-dihydroxy-6-[18F]fluorophenylalanine (FDOPA) metabolism in humans and monkeys has shown FDOPA to be an analogue of L-DOPA for the study of the dopaminergic system with positron emission tomography (PET). In human studies with carbidopa pretreatment, L-3,4-dihydroxy-6-[18F]fluoro-3-O-methylphenylalanine (3-OMFD) was the only FDOPA metabolite detected in plasma. FDOPA administration in monkeys resulted in selective accumulation of FDOPA metabolites in central dopaminergic regions, whereas 3-OMFD of peripheral origin was uniformly distributed among putamen, caudate, frontal cortex, and cerebellum. At 60 min, 3-OMFD and 6-[18F]fluorodopamine (FDA) each represented approximately 35% of the total activity, the remainder being FDOPA and FDA metabolites. These data on monkey and human FDOPA metabolism provide the basis for the configuration of an FDOPA tracer kinetic model with PET.

Kinetics and modeling of L-6-[18F]fluoro-dopa in human positron emission tomographic studies.

Kinetics of L-3,4-dihydroxy-6-[18F]fluorophenylalanine (FDOPA) in striatum and cerebellum were measured in 10 normal human subjects with positron emission tomography (PET) from 0 to 120 min after an intravenous bolus injection of the tracer. The time course of the arterial plasma concentrations of the tracer and its metabolites was also assayed biochemically. FDOPA compartmental models that are based on biochemical information were investigated for their consistency with the measured striatal and cerebellar tissue kinetics. A modeling approach was also developed for separating plasma FDOPA and metabolite time-activity curves from the measured total 18F time-activity curve in plasma. Results showed that a model consisting of three separate compartments for tissue FDOPA, tissue 6-[18F]fluorodopamine (FDA) and its metabolites, and tissue L-3,4-dihydroxy-6-[18F]fluoro-3-O-methylphenylalanine (3-OMFD) could describe adequately the striatal kinetics in humans. Based on this model, the FDOPA transport constant across the blood-brain barrier (BBB) (K1), the FDOPA decarboxylation rate constant (k3), and the turn-over rate constant of FDA and its metabolites (k4) could be estimated by model fitting to the tissue kinetics and were found for the normal subjects to be 0.031 +/- 0.006 ml/min/g (mean +/- SD), 0.041 +/- 0.015/min, and 0.004 +/- 0.002/min, respectively. About 50% of the FDOPA that crossed the BBB from plasma to striatum was decarboxylated. The decarboxylation constant with respect to plasma FDOPA (K3) was 0.015 +/- 0.003 ml/min/g. The BBB transport corresponded to a permeability-surface area product of 0.032 ml/min/g for FDOPA. For 3-OMFD, the BBB transport was 1.7 times faster. The effects of tissue heterogeneity on the FDOPA kinetics and on the estimated model parameters were also investigated. The usefulness and implications of these findings for interpretation of PET FDOPA studies are discussed.

Effects of large neutral amino acid concentrations on 6-[F-18]Fluoro-L-DOPA kinetics.

6-[F-18]Fluoro-L-3,4-dihydroxyphenylalanine (FDOPA) has been used to measure the central dopaminergic function in many species, including humans and monkeys. For transport across the blood brain barrier (BBB), FDOPA competes with plasma large neutral amino acids (LNAA). In this article we evaluate the effects of normal physiological LNAA concentration variation on BBB transport (K1) and the FDOPA uptake measurement, Ki. We also investigate a method for reducing the dependency of FDOPA quantitation on LNAA. Adult vervet monkeys (Cercopithecus aethiops sabaeus, n = 19) were fasted overnight before FDOPA positron emission tomography scans. Blood samples were drawn for LNAA determination, metabolite analysis, and compartmental modeling. The estimated K1 and Ki were both negatively correlated with LNAA concentrations (r2 = 0.51 and 0.62, respectively). Using an adjustment to K1 and Ki based on these correlations, the LNAA dependency was reduced (SD of the data for K1 was reduced by 33%, for Ki by 40%). Experiments with amino acid loading on an additional six animals indicate that BBB transport can be described using Michaelis-Menten kinetics. Results show a clear dependence of FDOPA uptake on plasma LNAA concentrations, which can be removed to increase the precision of FDOPA quantitation.

Radiofluorinated L-m-tyrosines: new in-vivo probes for central dopamine biochemistry.

In this work, we introduce 6-[18F]fluoro-L-m-tyrosine (6-FMT) and compare its in-vivo kinetic and bio-chemical behaviors in monkeys and rodents with those of 4-FMT and 6-[18F]fluoro-L-3, 4-dihydroxyphenylalanine (DOPA) (FDOPA). These radiofluorinated m-tyrosine presynaptic dopaminergic probes, resistant to peripheral 3-O-methylation, offer a nonpharmacological alternative to the use of catechol-O-methyltransferase inhibitors. Like FDOPA, 4-FMT and 6-FMT are analogs that essentially follow the L-DOPA pathway of central metabolism. After i.v. administration in nonhuman primates and rodents, these new radiofluorinated m-tyrosine analogs accumulate selectively in striatal structures and allow for the detection of additional innervation sites (e.g., brain stem) rich in aromatic amino acid decarboxylase. Bio-chemical analyses in rodents and monkeys revealed the specificity of their central and peripheral metabolism. Molecular and enzymatic mechanisms involved in their retention in central brain structures are consistent with involvement of dopaminergic neurons. The high signal-to-noise ratios observed make these radiofluorinated m-tyrosine analogs outstanding candidates for probing the integrity of central dopaminergic mechanisms in humans.

Social impulsivity inversely associated with CSF 5-HIAA and fluoxetine exposure in vervet monkeys.

Animal and human research suggests that the central serotonin system is involved in the inhibition of impulsive behavior. Two studies were designed to assess this relationship in male vervet monkeys (Cercopithecus aethiops sabaeus) using a standardized test of impulsivity in a social context: the Intruder Challenge. In the first study, an index of impulsivity in response to an unfamiliar adult male intruder (including latency to approach and aggressive and assertive interactions) was inversely correlated with levels of the serotonin metabolite, 5-hydroxyindoleacetic acid (5-HIAA) in cisternal cerebrospinal fluid (r = -0.33, p <.01, n = 138). The approach, but not aggressive, component of the Impulsivity Index was the primary contributor to this relationship (partial r = -0.27, p <.01). The second experiment compared responses to the Intruder Challenge after 9 weeks of daily treatment with fluoxetine (2 mg/kg, i.m.) or vehicle. Fluoxetine-treated subjects (n = 6) had significantly lower Impulsivity Index scores than controls (n = 12). The results from these two investigations provide evidence for serotonergic influences on social impulsivity.

Methods for improving quantitation of putamen uptake constant of FDOPA in PET studies.

To estimate the striatal uptake constant of [18F]-L-6-fluorodopa (FDOPA) in humans, we studied two methods that can account for the image resolution and the background level of FDOPA. These two methods utilize information obtained from multiple ROIs varying in size around the putamen and from profiles crossing the middle of the putamen. The estimation of the uptake constant was based on a model of one-dimensional activity variation. Simulated data were used to evaluate the adequacy of these two methods. Parametric images of FDOPA uptake constants were generated using Patlak analysis for five studies in normals and were then analyzed with the two methods. Results from the simulated data indicated a good agreement between the estimated values and the true simulated values. Results from studies in normals show stable estimates of the FDOPA uptake constant that are not affected by image resolution. The two methods were not sensitive to the misplacement of ROIs and profiles.

The effects of carbidopa administration on 6-[18F]fluoro-L-dopa kinetics in positron emission tomography.

Carbidopa (L-alpha-hydrazino-alpha-methyl-b-(3,4-dihydroxyphenyl) propionic acid is a known inhibitor of aromatic amino acid decarboxylase. In both humans and monkeys, we studied the effects of carbidopa on plasma and brain kinetics of 6-[18F]fluoro-L-DOPA (FDOPA), an analog of L-DOPA used for PET studies of the central dopaminergic system. Pretreatment with carbidopa resulted in increases in the plasma levels of FDOPA and 3-O-methyl-6-[18F]fluoro-L-DOPA (3-OMFD). Total striatal and cerebellar activities measured with PET were also increased. Furthermore, increases observed in the specific striatal activity (striatum minus cerebellum total activity) were correlated with increases in the plasma FDOPA curve. Carbidopa pretreatment did not affect the influx rate constant (K) for FDOPA from plasma to striatum in humans as determined by Patlak graphical analysis. Thus, an increase in measured striatal tomographic activity was secondary to the increase in plasma FDOPA levels rather than as a result of changes in the FDOPA influx rate constant.

Comparative in vivo metabolism of 6-[18F]fluoro-L-dopa and [3H]L-dopa in rats.

In vivo double-label experiments in rats were designed to correlate the peripheral and cerebral metabolism of 6-[18F]fluoro-L-DOPA [( 18F]FDOPA) with that of [3H]L-DOPA. Authentic samples of the major [18F]FDOPA metabolites were synthesized to identify the 18F-labeled metabolites. After carbidopa pretreatment and intravenous administration of the compound, the products of peripheral metabolism in plasma were analyzed at times from 3 to 60 min. In the periphery, amine conjugates were detected but they accounted for less than 15% of the total radioactivity; the major metabolites were 3-O-methyl-6-[18F]fluoro-L-DOPA and 3-O-methyl-[3H]L-DOPA. The rate and extent of 3-O-methylation of [18F]FDOPA exceeded that of [3H]L-DOPA. Both 3-O-methylated products entered the striatum and cerebellum where they contributed significant but uniform activity. Analysis of cerebral metabolism in these structures indicated a linear accumulation of total radioactivity: a striatum/cerebellum ratio of 2 was observed by 60 min. 6-[18F]Fluorodopamine (35%) and [3H]dopamine (55%) were the major metabolites formed in the striatum: however, the methylated [18F]FDOPA and [3H]DOPA products of predominantly peripheral origin represented 55% (18F) and 35% (3H) of the total radioactivity respectively. Other [3H]dopamine metabolites and their 18F-labeled analogs represented less than 10-15% at all times analyzed. The cerebellum radioactivity was composed only of [18F]FDOPA, [3H]DOPA and their 3-O-methylated products. These data will serve as the basis for the development of kinetic models of [18F]FDOPA metabolism that can be applied to the evaluation of central dopamine biochemistry with positron emission tomography in humans.

Evaluation of a stereotactic frame for repositioning of the rat brain in serial positron emission tomography imaging studies.

For serial imaging studies of the rat brain with positron emission tomography (PET), reproducible positioning of the head can facilitate spatial alignment of images and quantitative analysis. To achieve this aim, we constructed a plastic head frame and tested the positioning reproducibility on a high-resolution small-animal PET scanner, microPET. Two sets of ear bars, with tapers of either 18 degrees (sharp) or 45 degrees (blunt), were evaluated for their relative precision in securing the animal to the frame. For sequential positioning of an animal, average distances from the mean position of 0.51 mm (SD 0.41 mm) and 0.91 mm (SD 0.48 mm) were measured with the sharp and blunt ear bars, respectively. These results show that a rat brain can be reproducibly positioned using the frame, with a variation of position less than the spatial resolution of modern animal PET scanners. Brain regions of interest defined on one scan and copied across subsequent scans of a frame-repositioned animal resulted in an average coefficient of variation of 5.4% (SD 2.7%) using the sharp ear bars and 6.8% (SD 2.5%) using the blunt ear bars. This methodology has the potential to improve quantitative assessment for serial PET studies.

Ethological and 6-[18F]fluoro-L-DOPA-PET profiles of long-term vulnerability to chronic amphetamine.

A chronic 10-day amphetamine (Amp) protocol was used to induce significant long-term decrements of the striatal [18F]fluoro-L-DOPA influx rate constant (FDOPA Ki) in the vervet monkey. Longitudinal FDOPA-positron emission tomography (PET) assessment in Amp-treated subjects subsequently revealed a gradual recovery of striatal dopamine function: FDOPA Ki values were decreased by approximately 70% at 1 month, approximately 45% at 6 months, approximately 20% at 12 months and were similar to pre-Amp values at 24 months. Motoric and social behavioral measures were obtained on all subjects within a species-typical group setting. Behavioral observations were conducted during both basal and stressor-challenge conditions, the latter being created by placing a potential intruder-animal in an individual cage adjacent to the subject's group enclosure. During basal conditions, post-Amp stereotypies were present at 2 weeks and locomotor behaviors were increased throughout 1 month; both alterations occurred while FDOPA Ki values were significantly decreased. Social behaviors were also significantly affected; affiliative behavior was decreased up to 6 months while aggressive behavior was increased for 12 months. However, a different pattern of behavioral changes emerged under stressor-challenge conditions. Motoric and social changes were of greater magnitude and persisted longer than in basal settings while aggressive behavior remained elevated at 24 months. These results indicate that chronic Amp-induced decreases in FDOPA Ki values and behavioral alterations are reversible. Changes in striatal dopamine function as indexed with FDOPA-PET are not correlated with post-Amp alterations in behaviors and moreover, expression of those behaviors is context-dependent.

Recovery from methamphetamine induced long-term nigrostriatal dopaminergic deficits without substantia nigra cell loss.

After administration of methamphetamine (METH) (2x2 mg/kg, 6 h apart) to vervet monkeys, long term but reversible dopaminergic deficits were observed in both in vivo and post-mortem studies. Longitudinal studies using positron emission tomography (PET) with the dopamine transporter (DAT)-binding ligand, [11C]WIN 35,428 (WIN), were used to show decreases in striatal WIN binding of 80% at 1 week and only 10% at 1.5 years. A post-mortem characterization of other METH subjects at 1 month showed extensive decreases in immunoreactivity (IR) profiles of tyrosine hydroxylase (TH), DAT and vesicular monoamine transporter-2 (VMAT) in the striatum, medial forebrain bundle and the ventral midbrain dopamine (VMD) cell region. These IR deficits were not associated with a loss of VMD cell number when assessed at 1.5 years by stereological methods. Further, at 1.5 years, IR profiles of METH subjects throughout the nigrostriatal dopamine system appeared similar to controls although some regional deficits persisted. Collectively, the magnitude and extent of the dopaminergic deficits, and the subsequent recovery were not suggestive of extensive axonal degeneration followed by regeneration. Alternatively, this apparent reversibility of the METH-induced neuroadaptations may be related primarily to long-term decreases in expression of VMD-related proteins that recover over time.

6-[18F]fluoro-L-dopa metabolism in MPTP-treated monkeys: assessment of tracer methodologies for positron emission tomography.

6-[18F]Fluoro-L-DOPA (FDOPA) is an L-DOPA analog that is used to assess the functional integrity of central dopaminergic systems in vivo with positron emission tomography (PET). FDOPA metabolites from putamen of normal and MPTP-treated monkeys were characterized to correlate FDOPA metabolism changes with those of the endogenous dopamine system. In MPTP-lesioned putamen, 6-[18F]fluorodopamine and dopamine levels were less than 2% those of controls. Increases in endogenous dopamine metabolism were reflected by similar increases in 6-[18F]fluorodopamine metabolites. These results suggest that changes in the central dopamine system biochemistry can be monitored in vivo with FDOPA and PET.

Recovery of striatal dopamine function after acute amphetamine- and methamphetamine-induced neurotoxicity in the vervet monkey.

In six vervet monkeys, presynaptic striatal dopamine function was assessed longitudinally by [18F]fluoro-L-DOPA (FDOPA)-positron emission tomography (PET) after administration (2 x 2 mg/kg, i.m., 4 h apart) of either amphetamine (Amp), n = 3, or methamphetamine (MeAmp), n = 3. At 1-2 weeks postdrug, both Amp and MeAmp exposure effected similar decreases (60-70%) in the FDOPA influx rate constant (FDOPA Ki), an index of striatal dopamine synthesis capacity. Subsequent studies in these subjects showed that FDOPA Ki values were decreased by 45-67% at 3-6 weeks, by 25% at 10-12 weeks and by 16% in one Amp-treated subject at 32 weeks. Biochemical analysis showed that striatal dopamine concentrations were decreased by 75% at 3-4 weeks and by 55% at 10-12 weeks. These results indicate that in vervet monkey striatum, an acute Amp or MeAmp drug dosage produces extensive striatal dopamine system neurotoxicity. However, these effects were reversible; observed time-dependent recovery in both FDOPA Ki and dopamine concentrations indicates that neurochemical plasticity remains active in the adult primate striatum. At 3-4 and 10-12 weeks postdrug, the concurrent characterization of the striatal FDOPA Ki and dopamine concentrations for individual subjects showed that Ki decreases between 24 and 67% corresponded to dopamine depletions of 55-85%. These relatively larger postdrug decrements in steady-state striatal dopamine concentrations suggest that compensatory increases in dopamine synthesis capacity develop in the partially lesioned striatum. In contrast to the dopamine depletion in striatum, substantia nigra concentrations remained unchanged from referent values at both 3-4 and 10-12 weeks postdrug. Thus, the integrity of the substantia nigra could not be inferred from decreases in the striatal FDOPA Ki parameter. This disparity between striatum and substantia nigra reactivity to systemic administration of amphetamines suggests that each has unique dopamine system regulatory mechanisms.

Dizocilpine and reduced body temperature do not prevent methamphetamine-induced neurotoxicity in the vervet monkey: [11C]WIN 35,428 - positron emission tomography studies.

[11C]WIN 35,428 (WIN), a cocaine analog that binds to the dopamine transporter (DAT), and positron emission tomography (PET) were used to evaluate the potential neuroprotective effects of dizocilpine (MK-801) on methamphetamine (MeAmp) induced neurotoxicity in the striatal dopamine system of the vervet monkey. MK-801 (1 mg/kg, i.m.) was administered 30 min prior to a neurotoxic MeAmp dosage for this species (2 x 2 mg/kg, 4 h apart); control subjects received MeAmp. MK-801 treated subjects were anesthetized by the drug for 6-8 h; throughout that period, a 2-3 degrees C decrease in body temperature was measured. At 1-2 weeks post-MeAmp, decreases of approximately 75% in striatal WIN binding were observed for both MK-801/MeAmp and MeAmp subjects. Thus, in this non-human primate species, the combination of MK-801 pretreatment and reduced body temperature did not provide protection from the MeAmp-induced loss of DAT. Further, the absence of an elevated body temperature during the acute MeAmp exposure period indicated that hyperthermia, per se, was not a necessary concomitant of the MeAmp neurotoxicity profile as has been previously demonstrated in rodents. These results provide evidence that different regulatory factors maintain the integrity of the rodent and primate striatal dopamine systems.

[(18)F]p-MPPF: aA radiolabeled antagonist for the study of 5-HT(1A) receptors with PET.

This paper summarizes the present status of the researches conducted with [(18)F]4-(2'-methoxyphenyl)-1-[2'-[N-(2"-pyridinyl)-p-fluorobenzamido ]ethyl]-piperazine known as [(18)F]p-MPPF, a new 5-HT(1A) antagonist for the study of the serotonergic neurotransmission with positron emission tomography (PET). This includes chemistry, radiochemistry, animal data (rats, cats, and monkeys) with autoradiography and PET, human data with PET, toxicity, and metabolism.

Radiosurgery performed with the aid of a 3-mm collimator in the subthalamic nucleus and substantia nigra of the vervet monkey.

OBJECT: Radiosurgery for functional neurosurgery performed using a linear accelerator (LINAC) has not been extensively characterized in preclinical studies. In the present study, the properties of a newly designed 3-mm-diameter collimator were evaluated in a dedicated LINAC, which produced lesions in the basal ganglia of vervet monkeys. Lesion formation was determined in vivo in three animals by examining magnetic resonance (MR) images to show the dose-delivery precision of targeting and the geometry and extent of the lesions. Postmortem immunohistochemical studies were conducted to determine the extent of lesion-induced radiobiological effects. METHODS: In three male vervet monkeys, the subthalamic nucleus (STN; one animal) and the pars compacta of the lateral substantia nigra (SN; two animals) were targeted by a Novalis Shaped Beam Surgery System that included a 3-mm collimator and delivered a maximum dose of 150 Gy. Magnetic resonance images obtained 4, 5, and 9 months posttreatment were reviewed, and the animals were killed so that immunohistological characterizations could be made. CONCLUSIONS: The generation of precise radiosurgical lesions by a 3-mm collimator was validated in studies that targeted the basal ganglia of the vervet monkey. The extent of the lesions created in all animals remained restricted in diameter (< 3 mm) throughout the duration of the studies, as assessed by reviewing MR images. Histological studies showed that the lesions were contained within the STN and SN target areas and that there were persistent increases in glial fibrillary acidic protein immunoreactivity. Increases in immunoreactivity for tyrosine hydroxylase, the serotonin transporter, and the GluR1 subunit of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate glutamate receptor in penumbral regions of the lesion were suggestive of compensatory neuronal adaptations. This radiosurgical approach may be of particular interest for the induction of lesions of the STN and SN in studies of experimental parkinsonism, as well as for the development of potential radiosurgical treatments for Parkinson disease.

The effects of carbidopa on the metabolism of 6-[18F]fluoro-L-dopa in rats, monkeys and humans.

The effects of carbidopa on the peripheral metabolism of 6-[18F]fluoro-L-DOPA (FDOPA) were characterized in the rat, monkey and human along with its effects on cerebral FDOPA metabolism in the rat. After carbidopa pretreatment, FDOPA plasma metabolite profiles in all three species revealed extensive metabolism of FDOPA to 3-0-methyl-6-[18F]-fluoro-L-DOPA (3-OMFD). In humans, there were significant increases in FDOPA plasma levels for 30 min and in 3-OMFD levels for 120 min after FDOPA administration. 6-[18F]Fluorodopamine sulfate (FDA-sulfate) and [18F]fluoro-homovanillic acid (FHVA) levels were decreased, while at all times, free 6-[18F]-fluorodopamine (FDA) and 6-[18F]-3-4 dihydroxy-phenylacetic acid (FDOPAC) were not detected. In rat brain, the FDOPA metabolite profile at 30 min was significantly altered by carbidopa pretreatment; increases were noted for striatum FDA (700%) and 3-OMFD (230%), and for cerebellum FDOPA (370%) and 3-OMFD (300%). Thus, carbidopa pretreatment increased FDOPA plasma levels for a given FDOPA dose and essentially restricted peripheral FDOPA metabolism to 3-OMFD formation. The increase in FDOPA bioavailability to the brain resulted in greater selective FDA accumulation in striatum. As such, carbidopa pretreatment for FDOPA-positron emission tomography studies will significantly increase the amount of radioactivity that can be attributable to FDA in cerebral regions of interest.

Modelling approach for separating blood time-activity curves in positron emission tomographic studies.

A modelling approach is developed to generate the full time course of an injected radiotracer and its labelled metabolites in plasma/blood, based on measurements of the total radioactivities in withdrawn plasma/blood samples. A compartmental model is used to describe the conversion of an injected tracer to its metabolites in the body. The model equation is formulated with the total radioactivity concentration curve as the input function. The utility and characteristics of the approach in quantitative positron emission tomographic (PET) studies are shown with two examples. In the first example, using the tracer 6-[18F]fluoro-L-dopa (FDOPA), the approach is shown to derive the full time course of plasma FDOPA and its metabolites. In the second example of dynamic 15O oxygen PET, the approach is used to solve a deconvolution problem to give separated time-activity curves of 15O oxygen and 15O water in blood. The modelling approach improves the separation of blood/plasma time-activity curves and leads to better quantitative interpretation of PET results.

Choline and acetylcholine metabolism in PC12 secretory cells.

PC12, a clonal line of rat pheochromocytoma, synthesizes, stores, and secretes dopamine and acetylcholine. The cells take up choline by a saturable process and rapidly convert the accumulated choline to acetylcholine. This choline transport has a Km of 12 microM, is Na+ and energy independent, and is relatively insensitive to hemicholinium-3 (IC50 approximately 50 microM). Different ionic conditions can modulate the choline transport. Uptake was increased by pretreatment with 55 mM K+ whereas it was decreased in the presence of 55 mM K+. Choline uptake had similar characteristics in PC12 cells that had been induced to extend neurites by treatment with nerve growth factor. In undifferentiated PC12 cells, storage of newly synthesized acetylcholine was found in bound and free compartments as evidenced from subcellular fractionation. The free pool had a faster turnover rate. Most of the newly synthesized acetylcholine was rapidly degraded in the absence of a cholinesterase inhibitor while continuous incubation with labeled choline resulted in a slow incorporation of newly labeled acetylcholine into a bound pool. The accumulation of acetylcholine in the bound pool, but not acetylcholine synthesis, was inhibited by each of several agents that are known to interfere with the generation or maintenance of proton electrochemical gradients. The newly synthesized acetylcholine could be released from PC12 cells by incubation of the cells with 55 mM K+. These properties indicate that PC12 cells are a good system for studying acetylcholine metabolism by secretory cells.