On the Role of Extracellular ATP in the Induction of Long-Term Potentiation in the Hippocampus

Abstract: The involvement of a purinergic system in the mechanisms of ATP- and electrically induced long-term potentiation (LTP) has been investigated in mouse hippocampal slices. Extracellular ATP (500 n M ) and its slowly hydrolyzable analogue adenosine 5'- O -(3-thiotriphosphate) (ATP-γ-S; 2.5 µ M ) amplified permanently the magnitude of the population spike. This effect was antagonized by adenylimidodiphosphate (AMPPNP), a non-hydrolyzable analogue of ATP. AMPPNP, other ATP analogues [2-methylthioadenosine triphosphate (2-MeSATP) and α,β-methyleneadenosine 5'-triphosphate (α,β-methyleneATP)], or a purinergic receptor antagonist (Cibacron Blue 3G) tested in the concentration range of 3–40 µ M did not exert agonistic activity similar to that of ATP or ATP-γ-S, suggesting that ATP hydrolysis is required to exert this effect. All the tested nonhydrolyzable analogues reduced or prevented the establishment of stable, nondecremental LTP without blocking the short-lasting increase in the magnitude of the population spike immediately after electrical stimulation (short-term potentiation). These results indicate that ATP released by high-frequency stimulation contributes to the maintenance of stable LTP. The underlying mechanism operating in this process may involve a new type of ATP receptors or hydrolysis by ecto-ATPase. However, the findings that ATP-γ-S is less potent than ATP and that other ATP analogues known to act as agonists of purinergic receptors did not induce LTP but rather inhibited its maintenance are more consistent with the possibility that ecto-protein kinase, using extracellular ATP as a cosubstrate, plays a role in mechanisms underlying synaptic plasticity.     

Extracellular ATP Stimulates Norepinephrine Uptake in PC12 Cells

This study examined the effects of extracellular ATP on norepinephrine (NE) uptake, using PC12 cells as a model of noradrenergic neurons. Previous experiments with synaptosomes led to the hypothesis that extracellular ATP can regulate NE uptake via an ecto-protein kinase. In the present study, we examined the high-affinity uptake of NE (referred to as uptake 1) in PC12 cells in the presence of varying concentrations of extracellular ATP. In the presence of Ca2+, low concentrations of ATP (0.1 microM) increased uptake 1 by approximately 36%. This increase could be mimicked by adenosine-5'-O-(3-thiotriphosphate) tetralithium salt (ATP gamma S), an analogue of ATP which can be utilized by protein kinases, and not by 5'-adenylylimidodiphosphate tetralithium salt, a nonhydrolyzable analogue of ATP, GTP, ADP, and adenosine also had no effect on uptake 1. Preincubation of the cells with NE and ATP gamma S, followed by washing and assaying NE uptake 30 min later, resulted in a persistent increase in uptake 1. Similar pretreatment with ATP did not show this increase; however, simultaneous pretreatment with ATP and ATP gamma S blocked the activation produced by ATP gamma S alone. Kinetic analysis showed that ATP gamma S pretreatment produces an increase in the Vmax of uptake 1 without altering the apparent Km for NE. These results support the hypothesis that extracellular ATP can regulate NE uptake via an ecto-protein kinase.     

Oligodendroglial Signal Transduction Systems Are Regulated by Neuronal Contact

Abstract: Previous reports indicate that oligodendrocytes express signaling systems activated by classical neurotransmitters. Several signaling systems linked to mobilization of intracellular calcium have been demonstrated, and some of these are developmentally lost in vitro and in vivo. The experiments described here use oligodendrocyte-neuron cocultures to examine the effects of neuronal contact on the expression of these signaling pathways. Neonatal rat cerebral oligodendrocytes in contact with dorsal root ganglia (DRG) neurites responded to bath application of histamine, ATP, carbachol, glutamate, or bradykinin with increases in intracellular Ca²⁺ concentration. Similar results were obtained in coculture with superior cervical ganglia neurons. Preventing neuronal contact by transection of DRG neurites significantly reduced the percentage of oligodendrocytes responsive to each ligand, with the exception of bradykinin responsiveness, which was unaffected. Oligodendroglia isolated from adult rat spinal cord were also examined for responsiveness to these neuroligands. Few isolated adult oligodendroglia were responsive to these ligands, and coculture with DRG neurons failed to restore responsiveness. Neuroligand responsiveness was not induced in oligodendrocytes maintained 8 days in purified culture before establishment of cocultures. A significant reduction in the number of neuroligand-responsive oligodendroglia was noted for histamine, carbachol, glutamate, and ATP after including tetrodotoxin for the final 6 days of coculture. These results suggest that both neuronal contact and neuronal activity contribute to the maintenance of functional neurotransmitter-activated signaling pathways coupled to mobilization of intracellular calcium in oligodendrocytes.     

Intracellular signal transduction mechanisms of rat epididymal spermatozoa and their relationship to motility and metabolism

The role of intracellular signal transduction mechanisms in regulating the motility and metabolism of rat spermatozoa in undiluted caudal epididymal fluid (CEF) was examined. Samples of CEF containing immotile spermatozoa were exposed to drugs and other agents that either stimulate signal transduction pathways or mimic the action of their second messengers. Under these conditions, sperm motility in 25–30 nl of CEF was stimulated by calcium ions (Ca²⁺), N,2′ -O-dibutyryl-guanosine 3′:5′ -cyclic monophosphate (dibutryl cGMP), cyclic adenosine 3′:5′-monophosphate (cAMP), N⁶,2′-O-dibutyryladenosine 3′:5′ -cyclic monophosphate (dibutyryl cAMP), 8-bromoadenosine 3′:5′ -cyclic monophosphate (8-bromo cAMP), caffeine, theophylline and bicarbonate ions (HCO₃^?). Other agents such as magnesium ions (Mg²⁺), veratridine, phospholipase C (PLC), ionophore A23187, 1,2-dioctenoyl-sn-glycerol (DAG), phorbol 12-myristate 13-acetate, phospholipase A₂ (PLA₂), arachidonic acid, and melittin did not significantly influence motility. In the presence of radiolabelled energy substrates, untreated (immotile) spermatozoa in samples of CEF utilised D-[U-¹⁴C]glucose and [1-¹⁴C]acetate as exogenous energy sources for oxidative metabolism. No detectable ¹⁴C-lactate was produced, and none of the drugs altered the rate of glycolytic or oxidative metabolism. The findings suggest that the motility of rat caudal epididymal spermatozoa is regulated by Ca²⁺ and the guanylate cyclase and adenylate cyclase pathways, but not through the PLC and PLA₂ pathways. Also, their metabolism of exogenous substrate was uncoupled from the induction of motility, and their oxidative capacity exceeded the rate of flux of glucose-carbon through the glycolytic pathway. © 1994 Wiley-Liss, Inc.     

Oligodendroglial Signal Transduction Systems Are Developmentally Regulated

Abstract: Studies from several laboratories indicate that oligodendroglia exhibit signal transduction systems that can be activated by classical neurotransmitters. Previous studies from this laboratory indicate that oligodendroglia express neuroligand receptors linked to the regulation of Ca²⁺_i. Experiments presented in this article were designed to determine if developmental processes that influence the ability of oligodendroglia to respond to neuroligands with an increase in Ca²⁺_i proceed either in vitro or in vivo. Findings support the view that developmental processes markedly affected the sensitivity of these cells to both purinergic and cholinergic receptor agonists, whereas their responsiveness to either histamine or bradykinin appeared relatively stable over time. Approximately 90 and 75% of oligodendroglia responded to ATP or carbachol, respectively, after 4 days in vitro, whereas <10% of these cells responded to either of these neuroligands after 8 days in vitro. The decrease in the percentage of oligodendroglia responding to ATP, but not carbachol, could be prevented by including dibutyryl cyclic AMP in the culture medium during the final 4 days in vitro. However, once the loss in responsiveness to ATP had occurred, it could not be reversed by exposure to dibutyryl cyclic AMP. Developmental changes in the ATP sensitivity of oligodendroglia occurred in cells expressing galactocerebroside and myelin basic protein. The neuroligand sensitivity of oligodendroglia isolated from either neonatal, 2-, 3-, or 5-week-old spinal cord was examined to determine if developmental changes in oligodendroglial Ca²⁺ regulation occurred in vivo. The results of these experiments indicate that the percentage of oligodendroglia responding to either ATP or carbachol markedly decreased as a function of the age of the animal used to prepare the cultures; this was not the case for the stimulation of Ca²⁺_i by histamine. The decreased sensitivity of oligodendroglia isolated from older animals could not be reversed through the addition of dibutyryl cyclic AMP. Overall, the results of these experiments indicate that developmental processes selectively influence the sensitivity of oligodendroglia to specific neuroligands and suggest that oligodendroglial processes unrelated to myelin formation may be regulated by neuroligands in vivo.     

ATP binding is required for physiological activation of retinal guanylate cyclase

ATP bound to retinal guanylate cyclase (retGC)/membranes prior to the assay (pre-binding effect) and during the assay (direct effect) further enhances retGC activity stimulated by GC-activating proteins (GCAPs). Here we investigate differences between these two effects. We found that the pre-binding effect, but not the direct effect, was absent in membranes pre-washed with Mg(2+)-free hypotonic buffers, that the pre-binding effect, but not the direct effect, was strictly limited to GCAP-stimulated retGC activity, and that these two effects were independent and additive rather than being synergistic. Pre-incubation with amiloride enhanced GCAP2-activated retGC activity in a manner similar to that by ATP pre-binding; however, amiloride did not directly stimulate the retGC activity. These results indicate that these two effects are mechanistically different. Levels of retGC activation by these effects and conditions required for these effects indicate that only the mechanism involving ATP pre-binding is physiologically relevant to retGC activation.     

Studies of Signal Transduction of Salicylic Acid in Cucumber Cells

For the first time, the signal transduction pathway of salicylic acid (SA) was investigated by using 3H-labelling, thin-layer chromatography and anion exchange column chromatography. It was found that SA stimulated the activity of membrane bound phospholipase C (PLC), accelerated the bm&down of phosphatidylinositol-4-monophosphate (PIP) and phosphatidylinositol-4,5-bisphos- phate (PIP2) and increased the levels of inositol-1,4-bisphosphate (IP2), inositol-1, 4,5-trisphos- phate (IP3) and diacylglycerol (DAG). These indicated that signal transduction of SA was probably accomplished through the mediation of phosphatidylinositide signal transduction system in cucumber ( Cucumis sativa L. ).     

Metabolism of Extracellular Adenine Nucleotides by Cultured Rat Brain Astrocytes

Intact astrocytes cultured from newborn rat cerebral cortex rapidly converted extracellular ATP to ADP. The ATPase responsible was apparently not saturated, even at 750 microM ATP. In contrast, the conversion of ADP to AMP was slow, and the reaction was limiting for the subsequent dephosphorylation process. Adenosine formation was the only fate for AMP. The reaction was catalyzed by 5'-nucleotidase with an apparent Km of 55 microM for AMP and appeared to be inhibited by high concentrations of ATP and ADP. Astrocytes were able to take up adenosine with an apparent Km value of 45 microM. Uptake was inhibited by dipyridamole but not by anti-5'-nucleotidase IgG. The results support the proposal that astrocytes play a role in modulating synaptic events involving ATP and adenosine.     

牛脑皮层G_i的纯化及其与AC的偶联

用1％胆酸钠和20％饱和度的硫酸铵抽提牛脑皮层细胞膜得到含G蛋白和腺苷酸环化酶（AC）的制剂,通过Sepharose6B柱将两者分开,再将含G蛋白的级分用庚胺-Sepharose4B疏水柱、羟基磷灰石柱将其它亚型的G蛋白（主要是Gs和Go）从抑制型G蛋白（Gi）中除去,获得纯化的高活力的Gi,其GTP结合活力为17．6nmol／mg,比细胞膜Gi活力提高50倍;并具有较高的产率,从1g膜蛋白中可获得0．66mg的Gi,同时可获得无G蛋白污染的AC和少量的Gs蛋白．SDS-PAGE显示分子量为41000和36000的两条蛋白带,证实是Gi的α基和β亚基．进一步用重建脂酶体的方法检测Gi对AC的抑制作用,结果显示Gi对AC活力的抑制达40％左右,表明CAMP信息跨膜转导通路中Gi与AC之间具有较好偶联功能．     

Extracellular Calcium Sensing by Glial Cells: Low Extracellular Calcium Induces Intracellular Calcium Release and Intercellular Signaling

Abstract: Glial cells in primary mixed cultures or purified astrocyte cultures from mouse cortex respond to reduced extracellular calcium concentration ([Ca²⁺]_e) with increases in intracellular calcium concentration ([Ca²⁺]_i) that include single-cell Ca²⁺ oscillations and propagated intercellular Ca²⁺ waves. The rate and pattern of propagation of low [Ca²⁺]_e-induced intercellular Ca²⁺ waves are altered by rapid perfusion of the extracellular medium, suggesting the involvement of an extracellular messenger in Ca²⁺ wave propagation. The low [Ca²⁺]_e-induced Ca²⁺ response is abolished by thapsigargin and by the phospholipase antagonist U73122. The low [Ca²⁺]_e-induced response is also blocked by replacement of extracellular Ca²⁺ with Ba²⁺, Zn²⁺, or Ni²⁺, and by 100 µM La³⁺. Glial cells in lowered [Ca²⁺]_e(0.1–0.5 mM) show an increased [Ca²⁺]_i response to bath application of ATP, whereas glial cells in increased [Ca²⁺]_e (10–15 mM) show a decreased [Ca²⁺]_i response to ATP. These results show that glial cells possess a mechanism for coupling between [Ca²⁺]_e and the release of Ca²⁺ from intracellular stores. This mechanism may be involved in glial responses to the extracellular environment and may be important in pathological conditions associated with low extracellular Ca²⁺ such as seizures or ischemia.     

Modulation by Monoamines of Somatostatin-Sensitive Adenylate Cyclase on Neuronal and Glial Cells from the Mouse Brain in Primary Cultures

Primary cultures of mouse embryonic neuronal or glial cells from the cerebral cortex, striatum, and mesencephalon were used to identify and determine the cellular localization of somatostatin receptors coupled to an adenylate cyclase. Somatostatin inhibited basal adenylate cyclase activity on neuronal but not on glial crude membranes in the three structures examined. The somatostatin-inhibitory effect on neuronal crude membranes was still observed in the presence of (-)-isoproterenol, 3,4-dihydroxyphenylethylamine (dopamine, DA), or 5-hydroxytryptamine (5-HT, serotonin) used at a concentration (10(-5) M) inducing maximal adenylate cyclase activation. In addition, in most cases biogenic amines modified the pattern of the somatostatin-inhibitory effect, triggering either an increase in the peptide apparent affinity for its receptors or an increase in the maximal reduction of adenylate cyclase activity or both. However, 5-HT did not modify the somatostatin-inhibitory response on striatal and cortical neuronal crude membranes. The changes in somatostatin-inhibitory responses were interpreted as a colocalization of the amine and the peptide receptors on subtypes of neuronal cell populations. Finally, somatostatin was shown to inhibit adenylate cyclase activity following its activation by (-)-isoproterenol on glial crude membranes of the striatum and the mesencephalon but not on those of the cerebral cortex.     

Regulation of Norepinephrine Uptake by Adenine Nucleotides and Divalent Cations: Role for Extracellular Protein Phosphorylation

This study examined the hypothesis that ATP, released together with norepinephrine (NE) from brain noradrenergic nerve terminals, may serve as a cosubstrate for an extracellular protein phosphorylation system that regulates the reuptake of the transmitter, NE. The possible regulation of high-affinity uptake (uptake 1) of [3H]NE by divalent cations and ATP, both of which are involved in protein phosphorylation, was examined in rat cerebral cortical synaptosomes. A marked inhibition of uptake 1 by 5'-adenylylimidodiphosphate [App(NH)p], a nonhydrolyzable, competitive antagonist of ATP, was observed. A similar inhibition of uptake was observed when Ca2+ and Mg2+ were both omitted from the incubation medium. App(NH)p distinguished the actions of Ca2+ from those of Mg2+: Ca2+-stimulated uptake 1 was blocked by App(NH)p; Mg2+-stimulated uptake was not. In parallel experiments, the patterns of protein phosphorylation in crude and purified preparations of synaptosomes were examined under conditions similar to those used in uptake assays. A striking correlation was found between the inhibition of uptake 1, by either App(NH)p or Ca-omission, and inhibition of the phosphorylation of one specific, 39,000-dalton, Ca2+-dependent, protein component in synaptosomes. This 39K protein was distinct from the alpha subunit of pyruvate dehydrogenase, a mitochondrial protein of similar electrophoretic mobility. These findings are consistent with the possibility that an ectokinase on synaptosomes utilizes extracellular ATP and Ca2+ in phosphorylating a protein(s) associated with the regulation of NE uptake.     

Synergistic Activation of DNA Synthesis in Astrocytes by Fibroblast Growth Factors and Extracellular ATP

Abstract: The effects of extracellular ATP and polypeptide growth factors on DNA synthesis in primary cultures of rat astrocytes have been examined. It was found that ATP acts synergistically with either acidic or basic fibroblast growth factor to stimulate DNA synthesis. The specificity of this effect was demonstrated by the inability of ATP to potentiate DNA synthesis induced by platelet-derived growth factor or epidermal growth factor. ATP appears to act via P₂ purinergic receptors, because (a) it was more effective than adenosine and (b) the synergistic effect was observed with the hydrolysis-resistant P₂ agonists, ADPβS and ATPγS. The evidence suggests that extracellular ATP may be an important factor in regulating the extent of gliosis and, as such, may be involved in mechanisms of neural injury and repair.     

Adenylate kinase is a source of ATP for tumor mitochondrial hexokinase

It has proposed that hexokinase bound to mitochondria occupies a preferred site to wich ATP from oxidative phosphorylation is channeled directly (Bessman, S. (1966) Am. J. Medicine 40, 740–749). We have investigated this problem in isolated Zajdela hepatoma mitochondria. Addition of ADP to well-coupled mitochondria in the presence of an oxidizable substrate initiates the synthesis of glucose 6-phosphate via bound hexokinase. This reaction is only partially inhibited by oligomycin, carboxyatractyloside, carbonyl cyanide m-chlorophenylhydrazone (CCCP) ot any combination of these, suggesting a source of ATP in addition to oxidative phosphorylation. This source appears to be adenylate kinase, since Ado₂P₅, an inhibitor of the enzyme, suppresses hexokinase activity by about 50% when added alone or suppresses activity completely when added together with any of the inhibitors of oxidative phosphorylation. Ado₂P₅ does not uncouple oxidative phosphorylation nor does it inhibit ADP transport (state 3 respiration) or hexokinase. The relative amount of ATP contributed by adenylate kinase is dependent upon the ADP concentration. At low ADP concentraions, glucose phosphorylation is supported by oxidative phosphorylation, but as the adenine nucleotide translocator becomes saturated the ATP contributed by adenylate kinase increases due to the higher apparent K_m of the enzyme. Under conditions of our standard experiment ([ADP] = 0.5 mM), adenylate kinase provides about 50% of the ATP used by hexokinase in well-coupled mitochondria. In spite of this, externally added ATP supported higher rates of hexokinase activity than ADP. Our findings demonstrate that oxidative phosphorylation is not a specific or preferential source of ATP for hexokinase bound to hepatoma mitochondria. The apparent lack of a channeling mechanism for ATP to hexokinase in these mitochondria is discussed.     

Inhibition of a Low Km GTPase Activity in Rat Striatum by Calmodulin

In rat striatum, the activation of adenylate cyclase by the endogenous Ca2+-binding protein, calmodulin, is additive with that of GTP but is not additive with that of the nonhydrolyzable GTP analog, guanosine-5'-(beta, gamma-imido)triphosphate (GppNHp). One possible mechanism for this difference could be an effect of calmodulin on GTPase activity which has been demonstrated to "turn-off" adenylate cyclase activity. We examined the effects of Ca2+ and calmodulin on GTPase activity in EGTA-washed rat striatal particulate fractions depleted of Ca2+ and calmodulin. Calmodulin inhibited GTP hydrolysis at concentrations of 10(-9)-10(-6) M but had no effect on the hydrolysis of 10(-5) and 10(-6) M GTP, suggesting that calmodulin inhibited a low Km GTPase activity. The inhibition of GTPase activity by calmodulin was Ca2+-dependent and was maximal at 0.12 microM free Ca2+. Maximal inhibition by calmodulin was 40% in the presence of 10(-7) M GTP. The IC50 for calmodulin was 100 nM. In five tissues tested, calmodulin inhibited GTP hydrolysis only in those tissues where it could also activate adenylate cyclase. Calmodulin could affect the activation of adenylate cyclase by GTP in the presence of 3,4-dihydroxyphenylethylamine (DA, dopamine). Calmodulin decreased by nearly 10-fold the concentration of GTP required to provide maximal stimulation of adenylate cyclase activity by DA in the striatal membranes. The characteristics of the effect of calmodulin on GTPase activity with respect to Ca2+ and calmodulin dependence and tissue specificity parallel those of the activation of adenylate cyclase by calmodulin, suggesting that the two activities are closely related.(ABSTRACT TRUNCATED AT 250 WORDS)     

Extracellular ATP through P2 receptors activates AMP-activated protein kinase and suppresses superoxide generation in cultured mouse podocytes

Podocytes are an important constituent of the glomerular filtration barrier. The function of these glomerular cells is affected by extracellular nucleotides through P2 receptors. The activation of P2 receptors may lead to the activation of NAD(P)H oxidase, the key enzyme in oxidative stress, with the intracellular pathways leading to intracellular ATP depletion associated with an increase in the intracellular AMP:ATP ratio. This deregulation of the energy balance activates AMP-activated protein kinase (AMPK) to restore energy homeostasis. We investigated whether P2 receptor activation influences NAD(P)H oxidase-dependent rate of superoxide anion (O₂^•−) generation and AMPK activity in cultured mouse podocytes. The rate of O₂^•− generation was measured by chemiluminescence and changes in AMPK activity were determined by immunoblotting against AMPKα-Thr¹⁷²-P. The addition of 100 μM ATP induced a rapid and transient decrease in rate of O₂^•− generation and increased AMPK phosphorylation with maximal effects in the first minute (2.44 ± 0.09 versus 1.62 ± 0.06 nmol/mg protein/min, P < 0.05 and 0.64 ± 0.04 versus 0.97 ± 0.07, P < 0.05, respectively). Both parameters returned to control levels at 10 min. Suramin (300 μM, P2 receptor antagonist) and compound C (100 μM, AMPK inhibitor) completely, and STO-609 (25 μM, CaMKK-β inhibitor) partially, prevented ATP action in rate of O₂^•− generation and AMPK phosphorylation. Various ATP analogues (10 μM) mimicked the effects of ATP on rate of O₂^•− generation and AMPK phosphorylation. The data indicate that extracellular ATP, acting through P2 receptors upstream of CaMKK-β, modulates podocyte function through simultaneous effects on AMPK and NAD(P)H oxidase activities. This mechanism may play a role in restoring energy homeostasis after oxidative stress.     

Modulation of Phosphorylation of Neuronal Cytoskeletal Proteins by Neuronal Depolarization

1. The neuronal cytoskeletal protein tau and the carboxy tails of cytoskeletal proteins neurofilament-M (NF-M) and neurofilament-H (NF-H) are phosphorylated on serine residues by the cyclin-dependent kinase cdk-5.2. In aggregating neuronal–glial cultures we show that veratridine-mediated cation influx causes dephosphorylation of tau, NF-M and NF-H. Dephosphorylation was blocked specifically by cyclosporine A but not by okadiac acid at concentrations up to 200 nM.3. These results suggest that veratridine-triggered cation influx causes activation of PP-2B (calcineurin) leading to dephosphorylation of these cytoskeletal proteins.     

Extracellular Intermediates of Glucose Metabolism: Fluxes of Endogenous Lactate and Alanine Through Extracellular Pools in Embryonic Sympathetic Ganglia

The flux rates of lactate and alanine in and out of the cells of an intact tissue, which cannot be measured directly because some of the released materials are reabsorbed, were determined by computer analysis of uptakes and outputs by the whole tissue in the presence of various concentrations of these substances. The outputs of labeled lactate and alanine from [U-14C]glucose and the uptakes of [U-14C]lactate and [U-14C]alanine were measured on intact sympathetic ganglia excised from 15-day-old chicken embryos. The volume and time constant of the extracellular space were measured using labeled lactate, alanine, and sucrose. Models, which mathematically described the cellular uptakes and outputs as functions of the extracellular concentrations, were used to predict the exchanges that would be observed on the whole tissue, and their parameters were adjusted for best fit to the actual observations. The fitted models were then used to calculate the fluxes in and out of the cells and the concentrations in the extracellular space. The following results were obtained: (1) Cellular uptakes of lactate and alanine were both well described by familiar Michaelis-Menten kinetics. (2) The cellular output of [14C]-lactate from [14C]glucose declined with increase in the extracellular lactate concentration, whereas the cellular output of [14C]alanine from [14C]glucose rose with the extracellular alanine concentration. (3) Half-saturation values for cellular uptake, determined from the fitted equations, were 0.45 mM for lactate and 1.17 mM for alanine, both several-fold lower than less relevant estimates for the whole tissue made directly from the uptake observations. (4) As much as 45% of the carbon in the glucose consumed was released into the extracellular space as lactate and alanine, but much of this was reabsorbed. Implications for brain metabolism are discussed.