High Concentrations In Brain Research Articles

Calcineurin as a multifunctional regulator.

Long recognized as an important regulatory mechanism in biosignal processes, modulation of the phosphorylation state of proteins has emerged as the most important mechanism for understanding signal transduction. In contrast to the multitude of protein kinases and the clear signal transduction pathways, relatively few protein phosphatases are known and their regulation is unclear. Among them, calcineurin, a calcium/calmodulin-dependent phosphatase (PP2B), is the best enzyme to unveil the phosphatase function, because it was shown to be the direct target for immunosuppressants CsA and FK506, which are powerful tools for understanding this function in diseases as well as in several tissues and organs. Although calcineurin has been found in the highest concentrations in brain, it has also been detected in many other mammalian tissues. Well characterized in T cell activation by analysing the transcription factor NFAT, the function of calcineurin, however, was less well understood in other tissues and organs. Since the mid-1990s, several novel functions of this phosphatase have been reported, revealing that it plays important roles as a multifunctional regulator under the direct regulation of calcium signaling.

High-affinity sodium-vitamin C co-transporters (SVCT) expression in embryonic mouse neurons.

The sodium-vitamin C co-transporters SVCT1 and SVCT2 transport the reduced form of vitamin C, ascorbic acid. High expression of the SVCT2 has been demonstrated in adult neurons and choroid plexus cells by in situ hybridization. Additionally, embryonic mesencephalic dopaminergic neurons express the SVCT2 transporter. However, there have not been molecular and kinetic analyses addressing the expression of SVCTs in cortical embryonic neurons. In this work, we confirmed the expression of a SVCT2-like transporter in different regions of the fetal mouse brain and in primary cultures of neurons by RT-PCR. Kinetic analysis of the ascorbic acid uptake demonstrated the presence of two affinity constants, 103 microM and 8 microM. A K(m) of 103 microM corresponds to a similar affinity constant reported for SVCT2, while the K(m) of 8 microM might suggest the expression of a very high affinity transporter for ascorbic acid. Our uptake analyses also suggest that neurons take up dehydroascorbic acid, the oxidized form of vitamin C, through the glucose transporters. We consider that the early expression of SVCTs transporters in neurons is important in the uptake of vitamin C, an essential molecule for the fetal brain physiology. Vitamin C that is found at high concentration in fetal brain may function in preventing oxidative free radical damage, because antioxidant radical enzymes mature only late in the developing brain.

Molecular mechanisms of thiamine utilization.

Thiamine is required for all tissues and is found in high concentrations in skeletal muscle, heart, liver, kidneys and brain. A state of severe depletion is seen in patients on a strict thiamine-deficient diet in 18 days, but the most common cause of thiamine deficiency in affluent countries is alcoholism. Thiamine diphosphate is the active form of thiamine, and it serves as a cofactor for several enzymes involved primarily in carbohydrate catabolism. The enzymes are important in the biosynthesis of a number of cell constituents, including neurotransmitters, and for the production of reducing equivalents used in oxidant stress defenses and in biosyntheses and for synthesis of pentoses used as nucleic acid precursors. Because of the latter fact, thiamine utilization is increased in tumor cells. Thiamine uptake by the small intestines and by cells within various organs is mediated by a saturable, high affinity transport system. Alcohol affects thiamine uptake and other aspects of thiamine utilization, and these effects may contribute to the prevalence of thiamine deficiency in alcoholics. The major manifestations of thiamine deficiency in humans involve the cardiovascular (wet beriberi) and nervous (dry beriberi, or neuropathy and/or Wernicke-Korsakoff syndrome) systems. A number of inborn errors of metabolism have been described in which clinical improvements can be documented following administration of pharmacological doses of thiamine, such as thiamine-responsive megaloblastic anemia. Substantial efforts are being made to understand the genetic and biochemical determinants of inter-individual differences in susceptibility to development of thiamine deficiency-related disorders and of the differential vulnerabilities of tissues and cell types to thiamine deficiency.

L-Glutamate as a Central Neurotransmitter: Looking Back

The high concentration in brain of unbound l-glutamic acid (in its anionic form, l-glutamate) fuelled considerable speculation as to its role in central nervous function more than 50 years ago. Claims in the 1940s that it could improve cognitive acuity in patients with mental impairment were particularly intriguing, though later refuted. In the early 1950s Hayashi [(1954) Keio J. Med. 3, 183-192] found that l-glutamate could cause convulsions and proposed that it might be a central synaptic transmitter. Soon thereafter, Curtis and colleagues [Curtis, Phillis and Watkins (1959) Nature (London) 183, 611] showed that l-glutamate depolarized and excited central neurons, as expected for an excitatory transmitter; however, various aspects of the action of l-glutamate seemed to argue strongly against a transmitter function. This negative view prevailed for some 20 years, before compelling evidence for such a role was adduced. Over the last two decades, extensive research has revealed a host of glutamate receptor subtypes, subserving several different functions in excitatory synaptic transmission. This paper gives a very brief and personal overview of the development of the field over the last 50 years from a mainly pharmacological standpoint.

L-Glutamate as a Central Neurotransmitter: Looking Back

The high concentration in brain of unbound l-glutamic acid (in its anionic form, l-glutamate) fuelled considerable speculation as to its role in central nervous function more than 50 years ago. Claims in the 1940s that it could improve cognitive acuity in patients with mental impairment were particularly intriguing, though later refuted. In the early 1950s Hayashi [(1954) Keio J. Med. 3, 183–192] found that l-glutamate could cause convulsions and proposed that it might be a central synaptic transmitter. Soon thereafter, Curtis and colleagues [Curtis, Phillis and Watkins (1959) Nature (London) 183, 611] showed that l-glutamate depolarized and excited central neurons, as expected for an excitatory transmitter; however, various aspects of the action of l-glutamate seemed to argue strongly against a transmitter function. This negative view prevailed for some 20 years, before compelling evidence for such a role was adduced. Over the last two decades, extensive research has revealed a host of glutamate receptor subtypes, subserving several different functions in excitatory synaptic transmission. This paper gives a very brief and personal overview of the development of the field over the last 50 years from a mainly pharmacological standpoint.

Pharmacokinetics and organ distribution of intravenous and oral methylene blue.

To determine the pharmacokinetics and organ distribution of i.v. and oral methylene blue, which is used to prevent ifosfamide-induced encephalopathy in oncology. The concentration of methylene blue in whole blood was measured using high-performance liquid chromatography in seven volunteers after i.v. and oral administration of 100 mg methylene blue with and without mesna. The distribution of methylene blue in different tissues was measured in rats after intraduodenal and i.v. application. The time course of methylene blue in whole blood after i.v. administration showed a multiphasic time course with an estimated terminal half-life of 5.25 h. Following oral administration, the area under the concentration-time curve was much lower (9 nmol/min/ml vs 137 nmol/min/ml). Co-administration of mesna, which could influence distribution by ion-pairing, did not alter the pharmacokinetics. The urinary excretion of methylene blue and its leucoform was only moderately higher after i.v. administration (18% vs 28% dose). Intraduodenal administration to rats resulted in higher concentrations in intestinal wall and liver but lower concentrations in whole blood and brain than i.v. methylene blue. Differences in organ distribution of methylene blue are mainly responsible for the different pharmacokinetics after oral and i.v. administration. If methylene blue acts in the liver, where ifosfamide is primarily activated to reactive and potentially toxic metabolites, oral and i.v. methylene blue are likely to be equally effective. However, if the site of action is the central nervous system, i.v. methylene blue which results in much higher concentrations in brain seems preferable.

Intrahippocampal spermidine administration improves inhibitory avoidance performance in rats.

Polyamines are polycations present at high concentrations in the mammalian brain. We investigated the effect of an intrahippocampal infusion of spermidine, a polyamine agonist, immediately post-training on the inhibitory avoidance learning paradigm in rats. Bilateral intrahippocampal microinjection of spermidine (0.02-20 nmol) caused an increase in test step-down latencies at low concentrations. Administration of arcaine (0.002-0.2 nmol), an antagonist of the N-methyl-D-aspartate (NMDA) receptor polyamine binding site, did not modify the test step-down latencies. In contrast, co-administration of arcaine and spermidine completely reversed the facilitatory effect of spermidine on the test step-down latencies. These results provide evidence that polyamines may be involved in learning and memory modulation.

Zinc and ascorbic acid coordinately promote lipid peroxidation in brain membranes.

Zn2+ is present at high concentrations in mammalian brain, and is released in chelatable form after excitation of certain glutamatergic neurons. Recent observations suggest that it may play an important role in excitotoxic-induced neural injury. Ascorbic acid has been widely studied as a stimulator or an inhibitor of lipid-peroxide formation, depending on concentration, and lipid peroxidation has been postulated to be involved in both acute and chronic neurogenerative diseases. We find that ascorbic acid and Zn2+, at concentrations that are achieved in the brain after prolonged synaptic depolarization, coordinately promote lipid-peroxide formation and cause dysfunction of membrane-bound proteins. This effect is unique to Zn2+, and other divalent cations do not share a similar synergism with ascorbate. We propose that the Zn2+-ascorbate interaction may be an overlooked mechanism of lipid-peroxide formation in brain injury.

The effect of treatment regimen on the development of tolerance to the sedative and anxiolytic effects of diazepam

Chronic treatment with benzodiazepines results in tolerance to their sedative and anxiolytic effects and there is considerable evidence that different mechanisms underlie the development of tolerance to different behavioural effects. The purpose of the present experiment was to compare the behavioural effects of chronic treatment with diazepam (15 mg/kg per day) given as daily subcutaneous injections or by osmotic minipump. Both regimens resulted in continual receptor occupancy, but the daily injections also provided a period of higher brain concentrations. Rats were tested in the holeboard, which provides measures of exploration and locomotor activity, and in the elevated plus-maze and social interaction tests of anxiety. For those in the subcutaneous injection group the tests were 2 h after injection, when brain concentrations were highest. Despite a higher brain concentration in the injected group, both groups showed tolerance to diazepam's sedative effects, after 7 days of treatment. In contrast, in the elevated plus-maze, there was tolerance to the anxiolytic effects in the pump group after 14 days, but a persisting anxiolytic effect in the injected group at 14 and 28 days. Whilst higher brain concentrations could explain this result in the plus-maze, they cannot account for the pattern observed in the social interaction test, where the injection group showed a significant anxiogenic effect at 28 days. Whereas the mechanism underlying tolerance to the sedative effects of diazepam was insensitive to the different treatment regimens, the results suggest that different adaptive mechanisms were triggered in the two tests of anxiety with a differential sensitivity to the treatment regimen. The adaptive mechanism predominating in the social interaction test was favoured by the injection regimen which produced intermittent peak concentrations. This mechanism seems to be an oppositional one, leading to an anxiogenic response, which was manifest despite high brain concentrations of diazepam at the time of testing.

Modulation of Calmodulin and Protein Kinase C Activities by Pencillium Mycotoxins

Calmodulin (CaM), a calcium-binding protein, is found in high concentrations in mammalian brain where it plays a pivotal role in a large number of cellular functions. Protein kinase C (PKC), a multifunctional cytosolic enzyme, in the presence of both Ca2+ and phospholipids, transduce extracellular signals into intracellu-lar events. Both CaM and PKC are partially involved in maintaining Ca2+ homeostasis in the cell. Any fluctuations in the intracel-lular Ca2+ can modulate cellular functions and may contribute to neuronal dysfunction. Hence, the present investigation was initiated to study the effects of some selected penicillium (naturally occurring tremorgenic) mycotoxins like secalonic acid, citreoviridin, and verruculogen on CaM activity, active conformation of CaM and PKC activity. Stimulation of CaM-deflcient bovine brain 3′-5′ phosphodieste rase (PDE) indicated CaM activity. The modification of CaM active conformation was studied by the binding of fluorescent probe N-phenyl-1-napthylamine (NPN) to CaM. Alterations in the fluorescence of dansyl-CaM was used to study the effect of these compounds on complex formation between CaM and PDE. Rat brain cytosolic PKC was studied using 32P-ATP as a measure of altered protein phosphorylation. The concentrations of mycotoxins used were in the range of 10 to 50 μM. All three mycotoxins inhibited CaM-stimulated PDE activity in a concentration-dependent manner. Citreoviridin and secalonic acid inhibited NPN fluorescence and Ca2+-dependent complex formation of dansyl-CaM and PDE. The IC50 values for NPN fluorescence of citreoviridin and secalonic acid were 13 μM and 19 μM respectively. However, verruculogen showed little effect on NPN fluorescence and the Ca2+-dependent complex formation of dansyl-CaM and PDE. These mycotoxins also inhibited PKC activity in a concentration-dependent manner with IC50 values of 19.8, 25.7, and 38.4 μM for secalonic acid, citreoviridin, and verruculogen, respectively. The results of our study suggest that these mycotoxins at very low concentrations are interacting with CaM and PKC. Such an effect could lead to impairment of neurotransmission and result in neurotoxicity.

Lactational Exposure and Neonatal Kinetics of Methylmercury and Inorganic Mercury in Mice

The concentration of mercury in milk and the distribution pattern in the sucking pup was followed over time after administration of a single iv injection of 0.5 mg/kg body wt of203Hg-labeled methylmercuric chloride or mercuric chloride to lactating mice on Day 10 of lactation. Mercury concentrations in milk of the dams and in whole body, blood, plasma, GI-tract, liver, kidneys, and brain of the offspring were followed up to 11 days after dosing (until lactational Day 21). Following the inorganic mercury dose to the dams, most of the mercury in milk was delivered to the pups during the first 24 h, but the maximum mercury concentration in plasma and tissues of pups was not reached until 7 days after dosing, indicating a prolonged absorption of inorganic mercury in the sucking pup. Pups of dams given methylmercury were exposed to a much lower and constant mercury concentration in milk. The estimated accumulated mercury dose via milk per pup of dams given methylmercury was less than half of that estimated after the inorganic mercury dose. When the accumulated dose via milk from methylmercury-exposed dams was compared to the amount of mercury in pup's carcass (whole body minus GI-tract including content), it was revealed that almost all mercury delivered via milk was absorbed, and that the suckling pups had a very low elimination of mercury until lactational Day 17. Lactational exposure following a maternal methylmercury or inorganic mercury dose resulted in almost similar mercury concentrations in liver, kidneys, and plasma of the suckling, but higher concentrations in brain (as most 14 times) and also twice as high mercury body burden in the methylmercury group. Thus, differences in kinetics indicate that lactational exposure of methylmercury is a greater hazard for the breast-fed infant than inorganic mercury.

Significant transport of doxorubicin into the brain with polysorbate 80-coated nanoparticles.

To investigate the possibility of delivering of anticancer drugs into the brain using colloidal carriers (nanoparticles). Rats obtained 5 mg/kg of doxorubicin by i.v. injection in form of 4 preparations: 1. a simple solution in saline, 2. a simple solution in polysorbate 80 1% in saline, 3. bound to poly(butyl cyanoacrylate) nanoparticles, and 4. bound to poly(butyl cyanoacrylate) nanoparticles overcoated with 1% polysorbate 80 (Tween 80). After sacrifice of the animals after 10 min, 1, 2, 4, 6, and 8 hours, the doxorubicin concentrations in plasma, liver, spleen, lungs, kidneys, heart and brain were determined after extraction by HPLC. No significant difference in the body distribution was observed between the two solution formulations. The two nanoparticle formulations very significantly decreased the heart concentrations. High brain concentrations of doxorubicin (>6 microg/g) were achieved with the nanoparticles overcoated with polysorbate 80 between 2 and 4 hours. The brain concentrations observed with the other three preparations were always below the detection limit (< 0.1 microg/g). The present study demonstrates that the brain concentration of systemically administered doxorubicin can be enhanced over 60-fold by binding to biodegradable poly(butyl cyanoacrylate) nanoparticles, overcoated with the nonionic surfactant polysorbate 80. It is highly probable that coated particles reached the brain intact and released the drug after endocytosis by the brain blood vessel endothelial cells.

Ibogaine acts at the nicotinic acetylcholine receptor to inhibit catecholamine release

In an effort to determine mechanisms of action of the putative anti-addictive agent ibogaine, we have measured its effects on catecholamine release in a model neuronal system, cultured bovine chromaffin cells. Various modes of stimulating catecholamine release were used including nicotinic ACh receptor activation, membrane depolarization with elevated K + and Na + channel activation with veratridine. In addition, because ibogaine has been reported to interact with kappa opioid receptors, we tested whether kappa receptor antagonists could reverse ibogaine's effects on catecholamine release. Ibogaine, at low concentration (<10 μM) was found to selectively inhibit nicotinic receptor-mediated catecholamine release, while having no significant effect on release evoked by either veratridine or membrane depolarization with elevated K +. The inhibitory actions of ibogaine and the kappa agonists were not reversed by preincubation with the opioid antagonists nor-binaltorphimine or naltrexone, suggesting that these inhibitory effects are not mediated by the kappa opioid receptor. The effects of low dose (10 μM) ibogaine were rapidly reversible, while the inhibitory effects of higher ibogaine doses persisted for at least 19 h following ibogaine washout. The results provide evidence for a mechanism of action ibogaine at the nicotinic ACh receptor. The results are consistent with a model in which the initial high transient brain concentrations (100 μM) of ibogaine act at multiple cellular sites and then have a selective action at the nicotinic ACh receptor cation channel following its metabolism to lower brain concentrations. The present findings are relevant to potential anti-addictive actions of ibogaine and to the development of drugs to combat nicotine addiction.

Neuronal defects and delayed wound healing in mice lacking fibroblast growth factor 2.

Basic fibroblast growth factor (FGF2) is a wide-spectrum mitogenic, angiogenic, and neurotrophic factor that is expressed at low levels in many tissues and cell types and reaches high concentrations in brain and pituitary. FGF2 has been implicated in a multitude of physiological and pathological processes, including limb development, angiogenesis, wound healing, and tumor growth, but its physiological role is still unclear. To determine the function of FGF2 in vivo, we have generated FGF2 knockout mice, lacking all three FGF2 isoforms, by homologous recombination in embryonic stem cells. FGF2(-/-) mice are viable, fertile and phenotypically indistinguishable from FGF2(+/+) littermates by gross examination. However, abnormalities in the cytoarchitecture of the neocortex, most pronounced in the frontal motor-sensory area, can be detected by histological and immunohistochemical methods. A significant reduction in neuronal density is observed in most layers of the motor cortex in the FGF2(-/-) mice, with layer V being the most affected. Cell density is normal in other regions of the brain such as the striatum and the hippocampus. In addition, the healing of excisional skin wounds is delayed in mice lacking FGF2. These results indicate that FGF2, although not essential for embryonic development, plays a specific role in cortical neurogenesis and skin wound healing in mice, which, in spite of the apparent redundancy of FGF signaling, cannot be carried out by other FGF family members.

Two faces of cholecystokinin: anxiety and schizophrenia.

It has been suggested that cholecystokinin (CCK), a gut-brain peptide found in high concentrations in the mammalian brain, might be implicated in the neurobiology of anxiety and panic disorder. The administration of CCK tetrapeptide induced panic attacks analogous to spontaneous ones in patients suffering from panic disorder and to a lesser degree in healthy volunteers. In animal models of anxiety, the pretreatment with CCK agonists and antagonists produced, respectively, anxiogenic- and anxiolytic-like action on the exploratory paradigms. On the other hand, CCK could also play a role in the pathophysiology of schizophrenia. The administration of CCK agonists (caerulein, CCK-8s) to rodents results in behavioural effects analogous to those of antipsychotic drugs. However, CCK agonists lack any activity in rodent behavioural models to reveal antipsychotic drugs. A significant reduction of CCK concentration and CCK receptors has been shown in cortical and limbic structures of patients suffering from schizophrenia. Nevertheless, administration of CCK agonists to these patients does not effect their symptoms. Two major conclusions should be drawn: first, CCK is involved in the neurobiology of anxiety; second, changes in the CCK system in schizophrenia could be linked to a cortical neurodegeneration related to this disease.

Vitamin C and nervous tissue. In vivo and in vitro aspects.

Ascorbic acid is present at high concentrations in the brain (Adlard et al., 1973). Besides its well-known role as a cofactor in catecholamine biosynthesis (Kaufman and Friedman, 1965), ascorbic acid has been shown to exert a variety of functions in the nervous system. The following is a brief overview concerning the roles of ascorbic acid in the nervous system. Readers may also refer to recent excellent reviews by Grunewald (1993) and Rebec and Pierce (1994).

Effects of TRH and atropine on induction and duration of anesthesia with propofol in rats.

The effects of IV TRH pretreatment on induction of anesthesia with propofol or pentobarbital were investigated in rats. The effects of IV TRH, administered after induction, on duration of propofol anesthesia and the interaction with atropine were also studied. The doses of propofol or pentobarbital were not influenced by TRH. TRH reduced duration of anesthesia after propofol, with higher brain concentrations of propofol at recovery. Atropine did not block this effect, but given alone prolonged duration of anesthesia. It is concluded that TRH shortens the duration of propofol anesthesia, probably due to a pharmacodynamic effect and not to a pharmacokinetic interaction.

Response of cerebral blood vessels to an endogenous inhibitor of nitric oxide synthase.

We examined effects of NG,NG-dimethyl-L-arginine (asymmetric dimethylarginine, ADMA), an endogenous inhibitor of nitric oxide (NO) synthase, on cerebral vascular responses using cranial windows in anesthetized rats and rabbits. Under control conditions in rats, topical application of 10 and 100 microM ADMA constricted the basilar artery by 9 +/- 2 and 19 +/- 1% (SE; P < 0.05, n = 8), respectively, from a baseline diameter of 213 +/- 19 microns. ADMA (10 and 100 microM) produced marked inhibition of vasodilation in response to acetylcholine without inhibiting vasodilatation in response to nitroprusside. ADMA (1-100 microM) inhibited activity of brain NO synthase (measured as the conversion of L-[14C]arginine to L-[14C]citrulline). In cerebrum and cerebellum, 50% inhibition of activity of NO synthase was produced by 2.3 +/- 0.4 and 1.8 +/- 0.1 microM ADMA, respectively. In rabbits, treatment with ADMA (300 microM) decreased baseline diameter of cerebral arterioles (control diameter = 93 +/- 10 microns) by 11 +/- 2% (P < 0.05, n = 10). In response to 1 microM acetylcholine, cerebral arterioles dilated by 36 +/- 6 and 13 +/- 4% (P < 0.05 vs. control) in the absence and presence of ADMA, respectively. Effects of ADMA were prevented by L-arginine. Thus ADMA inhibits activity of brain NO synthase and relaxation of cerebral blood vessels in response to acetylcholine. Because ADMA is produced in relatively high concentrations in brain, it may be an important endogenous modulator of cerebral vascular tone under resting conditions and in response to vasoactive stimuli.

CCKB receptors mediate CCK‐8S‐induced activation of dorsal hippocampus CA3 pyramidal neurons: An in vivo electrophysiological study in the rat

The sulphated octapeptide C-terminal fragment of cholecystokinin (CCK-8S) is present in high concentration in the mammalian brain, where it acts via two types of receptor denoted CCKA and CCKB. In the dorsal hippocampus, CCK-8S exerts a potent excitatory effect on pyramidal neurons. The present electrophysiological study was undertaken to determine which CCK receptor type mediates this neuronal activation. Using in vivo extracellular unitary recordings of CA3 pyramidal hippocampal neurons, we compared the effect of SNF-8702, a potent selective CCKB receptor agonist, to that of CCK-8S, and assessed the effects of selective CCKA and CCKB antagonists. CCK-8S and SNF-8702, microiontophoretically applied on the same neurons produced a similar degree and pattern of activation. Both CCK-8S- and SNF-8702-induced activations were suppressed by the microiontophoretic application of the CCKB antagonist CI-988, but not by that of the CCKA antagonist SR 27897. CCK-8S-induced activation was not significantly modified by the intravenous administration of the CCKA antagonists devazepide and SR 27897. However, it was reduced by the CCKB antagonist PD 135158, administered intravenously or intracerebroventricularly, and by the intravenous administration of the CCKB antagonist L-365,260. The intravenous administration of PD 135158 also reduced SNF-8702-induced activations. These results indicate that CCKB receptors mediate CCK-8S-induced activation of rat CA3 pyramidal neurons.

Cellubrevin and synaptobrevins: similar subcellular localization and biochemical properties in PC12 cells.

There is strong evidence to indicate that proteins of the synaptobrevin family play a key role in exocytosis. Synaptobrevin 1 and 2 are expressed at high concentration in brain where they are localized on synaptic vesicles. Cellubrevin, a very similar protein, has a widespread tissue distribution and in fibroblasts is localized on endosome-derived, transferin receptor-positive vesicles. Since brain cellubrevin is not detectable in synaptic vesicles, we investigated whether cellubrevin and the synaptobrevins are differentially targeted when co-expressed in the same cell. We report that in the nervous system cellubrevin is expressed at significant levels only by glia and vascular cells. However, cellubrevin is coexpressed with the two synaptobrevins in PC12 cells, a neuroendocrine cell line which contains synaptic vesicle-like microvesicles. In PC12 cells, cellubrevin has a distribution very similar to that of synaptobrevin 1 and 2. The three proteins are targeted to neurites which exclude the transferrin receptor and are enriched in synaptic-like microvesicles and dense-core granules. They are recovered in the synaptic-like microvesicle peak of glycerol velocity gradients, have a similar distribution in isopycnic fractionation and are coprecipitated by anti-synaptobrevin 2 immunobeads. Finally, cellubrevin, like the synaptobrevins, interact with the neuronal t-SNAREs syntaxin 1 and SNAP-25. These results suggest that cellubrevin and the synaptobrevins have similar function and do not play a specialized role in constitutive and regulated exocytosis, respectively.