The uptake and metabolism of L-glutamate by tissue slices of the cestode Hymenolepis diminuta

The in vitro uptake of L-[3H]glutamate by tissue slices of the cestode Hymenolepis diminuta, denuded of tegument, was investigated. Two sodium concentration-dependent mechanisms, one of high affinity (Kt 1.8 X 10(-5) M; Vmax 4.76 pMoles/min/mg wet weight) and another of low affinity (Kt 2.2 X 10(-4) M; Vmax 50.7 pMoles/min/mg wet weight), were identified, in addition to a sodium insensitive component. Exchange of preloaded [3H]glutamate did not occur in tissue slices incubated in dilute unlabelled glutamate. Acidic amino acids, imipramine and fluoxetine were effective inhibitors of high and low affinity uptake, while glutamate receptor ligands, neurotransmitters and some antihelminthics generally were not. The concentrations present in, and the metabolism of glutamate by, tissue slices was examined by HPLC. The significance of the three modes of glutamate uptake and their possible role in the physiology of H. diminuta are discussed.     

Evolvement of systems of serotonin uptake in rat hypothalamus during its development]

Kinetics of 3H serotonin accumulation into slices from hypothalamus have been compared in adult, puppy and foetus rat. In 15 days-old, as in adult rat, there are two components of 5 HT accumulation corresponding to the low and high affinity transport systems. For this latter, Km and Vmax values are much higher in adult than in 15 days old rat (in adult, Km=1,3 X 10(-7) and Vmax=0,33 X 10(-10); in 15 days old rat, Km=0,5 X 10(-7) and Vmax=0,125 X 10(-10)). On the opposite, in the 7 days old rat and in the 21 days old foetus, it is only possible to arbitrarely define one uptake system corresponding to the following apparent values: in the 7 days old rat, Km= 5 X 10(-7) and Vmax=2 X 10(-10), in the foetus, Km=0,2 X 10(-7) and Vmax=0,15 X 10(-10). These results showed important developmental differences in affinity of 3H serotonin to hypothalamus. The low and high affinity uptake systems existing in adult are only individualized in the 15 days old little rat.     

The influx of tryptamine into snail (Helix pomatia) ganglia: comparison with 5-hydroxytryptamine.

Isolated ganglia possess the ability to concentrate tryptamine from an external medium by a process which is temperature sensitive and independent of sodium and other cations. Kinetic analysis of the accumulation process showed the influx of tryptamine to be a single mechanism with Km and Vmax values of 1.4 X 10(-4)M and 5 X 10(-8) mole/g/min. The influx of tryptamine is an unspecific process and is insensitive to a number of metabolic inhibitors and various analogues. The process of tryptamine influx is thus similar in principle to the low affinity uptake mechanism for 5-HT (see Osborne et al., 1975). The present data, which include some experiments on the release of 5-HT and tryptamine, are discussed from the point of view of a functional role for 5-HT and tryptamine in the snail CNS.     

Active Uptake of Substance P Carboxy-Terminal Heptapeptide (5–11) into Rat Brain and Rabbit Spinal Cord Slices

We previously reported that nerve terminals and glial cells lack an active uptake system capable of terminating transmitter action of substance P (SP). In the present study, we demonstrated the existence of an active uptake system for SP carboxy-terminal heptapeptide, (5-11)SP. When the slices from either rat brain or rabbit spinal cord were incubated with [3H](5-11)SP, the uptake of (5-11)SP into slices was observed. The uptake system has the properties of an active transport mechanism: it is dependent on temperature and sensitive to hypoosmotic treatment and is inhibited by ouabain and dinitrophenol (DNP). In the brain, (5-11)SP was accumulated by means of a high-affinity and a low-affinity uptake system. The Km and the Vmax values for the high-affinity system were 4.20 x 10(-8) M and 7.59 fmol/10 mg wet weight/min, respectively, whereas these values for the low-affinity system were 1.00 x 10(-6) M and 100 fmol/10 mg wet weight/min, respectively. In the spinal cord, there was only one uptake system, with a Km value of 2.16 x 10(-7) M and Vmax value of 26.2 fmol/10 mg wet weight/min. These results suggest that when SP is released from nerve terminals, it is hydrolysed into (5-11)SP before or after acting as a neurotransmitter, which is in turn accumulated into nerve terminals. Therefore, the uptake system may represent a possible mechanism for the inactivation of SP.     

Uptake of GABA by neuronal and nonneuronal cells in dispersed cell cultures of postnatal rat cerebellum.

A study was made of the time course and kinetics of [3H]GABA uptake by dispersed cell cultures of postnatal rat cerebellum with and without neuronal cells. The properties of GABA neurons were calculated from the biochemical difference between the two types of cultures. It was found that for any given concentration of [3H]GABA, or any time up to 20 min, GABA neurons in cultures 21 days in vitro had an average velocity of uptake several orders of magnitude greater than that of nonneuronal cells. In addition, the apparent Kmvalues for GABA neurons for high and low affinity uptake were 0.33 X 10(-6) M and 41.8 X 10(-4) M, respectively. For nonneuronal cells, the apparent Km for high affinity uptake was 0.29 X 10(-6) M. The apparent Vmax values for GABA neurons for high and low affinity uptake were 28.7 X 10(-6) mol/g DNA/min and 151.5 mmol/g DNA/min, respectively. For nonneuronal cells, the apparent Vmax for high affinity uptake was 0.06 X 10(-6) mol/g DNA/min. No low affinity uptake system for nonneuronal cells could be detected after correcting the data for binding and diffusion. By substituting the apparent kinetic constants in the Michaelis-Menten equation, it was determined that for GABA concentrations of 5 X 10(-9) M to 1 mM or higher over 99% of the GABA should be accumulated by GABA neurons, given equal access of all cells to the label. In addition, high affinity uptake of [3H]GABA by GABA neurons was completely blocked by treatment with 0.2 mM ouabain, whereas that by noneuronal cells was only slightly decreased. Most (75-85%) of the [3H]GABA (4.4 X 10(-6) M) uptake by both GABA neurons and nonneuronal cells was sodium and temperature dependent.     

Uptake and release of [3H]-serotonin in photophores of the midshipman fish, Porichthys notatus

A kinetic analysis of [3H]-5-HT uptake in the photocytes of the photophores of Porichthys notatus revealed a high affinity (Km: 1.71 X 10(-7] and low affinity component (Km: 1.10 X 10(-5) M). The high affinity uptake was sodium- and potassium-dependent but largely insensitive to temperatures between 0 and 20 C. Ouabain (5 X 10(-3) M) and dinitrophenol (10(-3) M) reduced uptake significantly. DMI, imipramine and fluoxetine, in that order of potency, greatly inhibited [3H]-5-HT uptake. Noradrenaline and adrenaline reduced uptake in a non-competitive manner, while dopamine, tryptophan, 5-hydroxytryptophan and Cypridina luciferin had little or not effect on uptake. Non-facilitated luminescent responses to electrical stimulation were accompanied by release of [3H]-5-HT accumulated in the photocytes. Facilitatory luminescence excitation consistently failed to induce the release of [3H]-5-HT. Electrical and adrenaline (10(-5) M) stimulation of photophores after [3H]-5-HT release has occurred, failed to elicit any additional luminescent response. The photophores were responsive to KCN (10(-3) M) under these conditions. The results indicate that a specific carrier-mediated transport system is responsible for photocytic [3H]-5-HT uptake, and that release of photocytic [3H]-5-HT is stringently regulated and followed by inhibition of luminescence excitability.     

Effects of ascorbic acid on the uptake of serotonin in differentiated neuroblastoma cells

Ascorbic acid causes concentration-dependent and time-dependent effects on [³H]-serotonin (³H-5HT) uptake into differentiated neuroblastoma N-2a cells. Preincubation of cells with ascorbic acid inhibits both passive diffusion and active transport of ³H-5HT (0.1 μM). The kinetic characteristics of the active uptake process change with ascorbic acid treatment, resulting in an increase in the Km from 0.27 μM to 3.0 μM and in the Vmax from 453 to 2369 fmol/min/10⁶ cells. This inhibitory effect of ascorbic acid appears to be due to its reducing properties.     

ATP-dependent calcium transport in rat parotid basolateral membrane vesicles. Modulation by agents which elevate cyclic AMP

ATP-dependent Ca2+ transport was studied in basolateral membrane vesicles prepared from rat parotid gland slices incubated without or with agents which increase cyclic AMP. Isoproterenol (10(-5) M), forskolin (2 X 10(-6) M) and 8-bromocyclic AMP (2 X 10(-3) M) all increased ATP-dependent 45Ca2+ uptake 1.5- to 3-fold. The effect of isoproterenol was concentration-dependent and blocked by the beta-adrenergic antagonist propranolol. Enhanced uptake did not appear an artifact of vesicle preparation as apparent vesicle sidedness, 45Ca2+ efflux rates, specific activity of marker enzymes and equilibrium Ca2+ content were identical in vesicle preparations from control and 8-bromocyclic AMP-treated slices. Kinetic studies showed the ATP-dependent Ca2+ transport system in vesicles from 8-bromocyclic AMP-treated slices displayed a approximately 50% increase in Vmax and in Km Ca2+, compared to controls. The data suggest that physiological secretory stimuli to rat parotid acinar cells, which involve cyclic AMP, result in a readjustment of the basolateral membrane ATP-dependent Ca2+ pump.     

Effects of cadmium treatment in vitro on the uptake and release of [3H]serotonin by human blood platelets

Effects of cadmium treatment on human platelets were studied with respect to uptake and release of 5-[3H]hydroxytryptamine (5-HT). The uptake of 5-[3H]HT in the presence of varying concentrations of CdCl2 (0.001-10 mM) was inhibited significantly with respect to control platelets and the inhibition was maximum at 1 mM CdCl2 concentration. From studies on the kinetics of 5-[3H]HT uptake a higher Km and significantly lower Vmax for CdCl2-treated platelets were observed. CdCl2 stimulated spontaneous release but inhibited thrombin-induced release of 5-[3H]HT. Spontaneous release of 5-[3H]HT induced by CdCl2 was not significantly altered in the presence of externally available CaCl2 (1 mM).     

Transport and metabolism of vitamin B6 in Salmonella typhimurium LT2.

Salmonella typhimurium LT2 concentrates radioactivity intracellularly from [3H]pyridoxal or [3H]pyridoxine up to 25 times the external concentration. After 1 min of uptake intracellular radioactivity is found as phosphorylated vitamin B6. The process is sensitive to temperature and is maximally active at pH 8.1, but under the conditions tested it is insensitive to monovalent cations or metabolic inhibitors, and does not require an exogenous energy source. The Km values for uptake of pyridoxine and pyridoxal are 2.0 x 10(-7) M and 1.2 x 10(-7) M, respectively; [3H]pyridoxamine is not transported. Evidence is presented for an uptake mechanism involving facilitated diffusion followed by trapping by pyridoxal kinase. S. typhimurium also appears to lack a periplasmic binding protein for vitamin B6.     

Basic and neutral amino acid transport in Aspergillus nidulans.

Arginine and methionine transport by Aspergillus nidulans mycelium was investigated. A single uptake system is responsible for the transport of arginine, lysine and ornithine. Transport is energy-dependent and specific for these basic amino acids. The Km value for arginine is 1 X 10(-5) M, and Vmax is 2-8 nmol/mg dry wt/min; Km for lysine is 8 X 10(-6) M; Kt for lysine as inhibitor of arginine uptake is 12 muM, and Ki for ornithine is mM. On minimal medium, methionine is transported with a Km of 0-I mM and Vmax about I nmol/mg dry wt/min; transport is inhibited by azide. Neutral amnio acids such as serine, phenylalanine and leucine are probably transported by the same system, as indicated by their inhibition of methionine uptake and the existence of a mutant specifically impaired in their transport. The recessive mutant nap3, unable to transport neutral amino acids, was isolated as resistant to selenomethionine and p-fluorophenylanine. This mutant has unchanged transport of methionine by general and specific sulphur-regulated permeases.     

Physiology of lysine permeases in Saccharomycopsis lipolytica.

Two active lysine transport systems were detected in Saccharomycopsis lipolytica. No excretion of lysine out of the cells could be obtained, even by chasing with L-lysine or by poisoning with sodium azide. The kinetic properties of one of the permeases, the high-affinity lysine permease, were studied in detail. Its Km was 1.91 +/- 0.23 X 10(-5) M. It proved highly specific, the only potent competitive inhibitors being (i) arginine and its analogs L-canavanine and L-ornithine, and (ii) the lysine analogs L-5 aminoethylcysteine and L-4,5-transdehydrolysine. It is suggested that the high-affinity lysine permease is common to L-lysine, L-ornithine, and L-arginine. The other amino acids tested behaved as noncompetitive inhibitors. The variation of uptake during a growth cycle was studied on ammonia-rich, ammonia-poor, and ammonia-free media. In each case, the uptake exhibited a peak in the early exponential growth phase. No new permease activity was detected during the lag phase or the stationary phase. Ammonia ions competitively inhibited the uptake and also decreased the Vmax value.     

5 Alpha-reductase and 3 alpha-hydroxysteroid dehydrogenase activities in isolated canine prostatic epithelial cells

Kinetic parameters of two enzymes, 5 alpha-reductase and 3 alpha-hydroxysteroid dehydrogenase, have been calculated for freshly isolated canine prostatic epithelial cells separated into secretory and non-secretory cells on the basis of their density in Percoll gradients. For 5 alpha-reductase, a Km value of 3 X 10(-6) M was obtained in both epithelial cell types, and similar Vmax values of 1.7 X 10(-12) and 1.9 X 10(-12) moles of dihydrotestosterone formed/min/10(6) cells (P greater than 0.50) were calculated in secretory and non-secretory cells, respectively. The Km of 3 alpha-hydroxysteroid dehydrogenase (dihydrotestosterone----3 alpha-androstanediol) varied between 2.2 and 2.8 X 10(-6) M (P greater than 0.50) with respective Vmax's of 9 and 24 X 10(-12) moles of 3 alpha-androstanediol formed/min/10(6) cells (P less than 0.005) in secretory and non-secretory cells. For the reverse reaction, that is the transformation of 3 alpha-androstanediol to dihydrotestosterone, the Km's obtained were 0.4 and 0.5 X 10(-6) M (P greater than 0.50) with Vmax's of 14 and 19 X 10(-12) moles of dihydrotestosterone formed/min/10(6) cells (P less than 0.50) in secretory and non-secretory cells, respectively. Vmax values for 3 alpha-hydroxysteroid dehydrogenase are 10-fold higher than the ones for 5 alpha-reductase. Moreover, Km values for the reaction 3 alpha-androstanediol----DHT are 5-fold lower than those calculated for the reverse reaction and for 5 alpha-reductase. This could explain why the major metabolic pathway in the canine prostate gland is the conversion of 3 alpha-androstanediol to dihydrotestosterone.     

Uptake of methylamine and methanol by Pseudomonas sp. strain AM1.

The uptake of methylamine and of methanol by the facultative methylotroph Pseudomonas sp. strain AM1 was investigated. It was found that this organism possesses two uptake systems for methylamine. One of these operates when methylamine is the sole source of carbon, nitrogen, and energy. It has a Km of 1.33 X 10(-4) M and a Vmax of 67 nmol/min per mg of cells (dry weight). The other system, found when methylamine is the sole nitrogen source only, has a Km of 1.2 X 10(-5) M and a Vmax of 8.9 nmol/min per mg of cells (dry weight). Both uptake systems were severely inhibited by azide, cyanide, carbonyl cyanide-m-chlorophenyl hydrazone, and N-ethylmaleimide, but only the high-affinity system was inhibited by ammonium ions with a Ki of 7.7 mM. Both systems were susceptible to osmotic shock treatment, competitively inhibited by ethylamine, and unaffected by most amino acids. Methanol uptake showed a Km of 4.8 microM and a Vmax of 60.6 nmol/min per mg of cells (dry weight) and was not inhibited by osmotic shock treatment. Azide, cyanide, and N-ethylmaleimide curtailed uptake, but carbonyl cyanide-m-chlorophenyl hydrazone merely reduced the rate of uptake. A methanol dehydrogenase mutant, M15A, was unable to take up methanol. It is proposed that methanol diffuses into the cell where it is rapidly oxidized by methanol dehydrogenase.     

Characterization of calcium transport by basal plasma membranes from human placental syncytiotrophoblast.

We have studied the mechanisms involved in calcium (Ca2+) transport through the basal plasma membranes (BPM) of the syncytiotrophoblast cells from full-term human placenta. These purified membranes were enriched 25-fold in Na+/K(+)-adenosine triphosphate (ATPase), 37-fold in [3H] dihydroalprenolol binding sites, and fivefold in alkaline phosphatase activity compared with the placenta homogenates. In the absence of ATP and Mg2+, a basal Ca2+ uptake was observed, which followed Michaelis-Menten kinetics, with a Km Ca2+ of 0.18 +/- 0.05 microM and Vmax of 0.93 +/- 0.11 nmol/mg/min. The addition of Mg2+ to the incubation medium significantly decreased this uptake in a concentration-dependent manner, with a maximal inhibition at 3 mM Mg2+ and above. The Lineweaver-Burk plots of Ca2+ uptake in the absence and in the presence of 1 mM Mg2+ suggest a noncompetitive type of inhibition. Preloading the BPM vesicles with 5 mM Mg2+ had no significant effect on Ca2+ uptake, eliminating the hypothesis of a Ca2+/Mg2+ exchange mechanism. This ATP-independent Ca2+ uptake was not sensitive to 10(-6) M nitrendipine nor to 10(-4) M verapamil. An ATP-dependent Ca2+ transport was also detected in these BPM, whose Km Ca2+ was 0.09 +/- 0.02 microM and Vmax 3.4 +/- 0.2 nmoles/mg/3 min. This Ca2+ transport requires Mg2+, the optimal concentration of Mg2+ being approximately 1 mM. Preincubation of the membrane with 10(-6) M calmodulin strongly enhanced the initial ATP-dependent Ca2+ uptake. Finally, no Na+/Ca2+ exchange process could be demonstrated.     

Detection and characterization of p-coumaric acid hydroxylase in mung bean, Vigna mungo, seedlings

A new p-coumaric acid (4-hydroxycinnamic acid) hydroxylase was detected in mung bean seedlings treated with tentoxin, a fungal toxin, in which polyphenol oxidase that hydroxylates a wide variety of monophenols in vitro was completely eliminated. The enzyme required molecular oxygen and showed a pH optimum of 5.0. The enzyme acted only on p-coumaric acid (Km, 3.0 X 10(-5) M), while its specificity for the electron donor was rather broad. The Km value for NADPH (1.5 X 10(-4) M) was much lower than that for L-ascorbic acid (1.0 X 10(-2) M), although the Vmax value was almost the same with both electron donors. The enzyme was potently inhibited by beta-mercaptoethanol (Ki, 3.5 X 10(-6) M) and diethyldithiocarbamate (Ki, 2.3 X 10(-4) M), but was insensitive to p-chloromercuribenzoate. The enzyme was localized in the cell organelles which sedimented between mitochondria and endplasmic reticulum on sucrose density gradient centrifugation. The enzyme activity in the seedling was changed in response to induction by light in a manner suggesting its involvement in biosynthesis of phenolic compounds in mung bean seedlings.     

Effects of valinomycin on hexose transport and cellular ATP pools in mouse fibroblasts

The K+ ionophore valinomycin at concentrations of 1 X 10(-8) M and over, stimulated 2-deoxy-D-glucose (2DG) and 3-O-methylglucose (3OMG) uptake in Swiss 3T3 fibroblasts. The rate-limiting step of 2DG uptake was transport rather than phosphorylation, in the control or valinomycin-treated cells. Kinetic analysis showed that valinomycin increased the Vmax for 2DG uptake without change of the Km. The valinomycin-stimulated 2DG uptake was insensitive to 10 micrograms/ml cycloheximide, and extracellular K+ concentrations between 0.1 and 50 mM. On the other hand, valinomycin at the concentration of 1 X 10(-8) M and over, induced a rapid decrease in cellular ATP content, followed by stimulation of 2DG uptake and recovery of the ATP content. A similar relationship between the reduction of cellular ATP content and the subsequent stimulation of 2DG uptake was observed when the cells were treated not only with 2,4-dinitrophenol and iodoacetic acid, but also with other monovalent cation ionophores or inhibitors of oxidative phosphorylation. These results suggest that valinomycin may posttranslationally stimulate hexose transport by increasing the number of functional carriers of hexose or changing their mobility, and the rapid decrease in cellular ATP pools by valinomycin may be a trigger of the stimulation of the hexose transport in Swiss 3T3 fibroblasts.     

Tricyclohexyltin hydroxide effects on cationic and substrate activation kinetics of beta-adrenergic-stimulated cardiac Ca2+-ATPase

Previous studies from this laboratory have indicated that tricyclohexyltin hydroxide (Plictran) is a potent inhibitor of both basal- and isoproterenol-stimulated cardiac sarcoplasmic reticulum (SR) Ca2+-ATPase, with an estimated IC-50 of 2.5 X 10(-8) M. The present studies were initiated to evaluate the mechanism of inhibition of Ca2+-ATPase by Plictran. Data on substrate and cationic activation kinetics of Ca2+-ATPase indicated alteration of Vmax and Km by Plictran (1 and 5 X 10(-8) M), suggesting a mixed type of inhibition. The beta-adrenergic agonist isoproterenol increased Vmax of both ATP- and Ca2+-dependent enzyme activities. However, the Km of enzyme was decreased only for Ca2+. Plictran inhibited isoproterenol-stimulated Ca2+-ATPase activity by altering both Vmax and Km of ATP as well as Ca2+-dependent enzyme activities, suggesting that after binding to a single independent site, Plictran inhibits enzyme catalysis by decreasing the affinity of enzyme for ATP as well as for Ca2+. Preincubation of enzyme with 15 microM cAMP or the addition of 2mM ATP to the reaction mixture resulted in slight activation of Plictran-inhibited enzyme. Pretreatment of SR with 5 X 10(-7) M propranolol and 5 X 10(-8) M Plictran resulted in inhibition of basal activity in addition to the loss of stimulated activity. Preincubation of heart SR preparation with 5 X 10(-5) M coenzyme A in combination with 5 X 10(-8) M Plictran partly restored the beta-adrenergic stimulation. These results suggest that some critical sites common to both basal- and beta-adrenergic-stimulated Ca2+-ATPase are sensitive to binding by Plictran, and the resultant conformational change may lead to inhibition of beta-adrenergic stimulation.     

Uptake of biogenic amines by glial cells in culture I. A neuronal-like transport system for serotonin

Rat C6 astrocytoma cells take up serotonin (5HT) via a high affinity carrier mediated system with Km of 1 micromolar, and a second component of lower affinity. This high affinity 5HT transport system is rapid, concentrative, and highly sodium and temperature dependent. Chlorimipramine and Lilly 110140 preferentially block the glial 5HT but not NE uptake. This preferential inhibition has previously been shown for synaptosomes and brain slices. Norepinerphrine (NE) and to a lesser extent dopamine (DA) block the glial 5HT uptake, suggesting a partial overlap between the catecholamine and indoleamine glial carrier systems. 5-Hydroxy but not 6-hydroxy dopamine inhibits the high affinity 5HT transport in glia. A variety of ring hydroxylated indoleamine analogs block this glial 5HT transport; of the compounds tested, 5, 7 dihydroxytryptamine is the least effective inhibitor. Phenylethylamine (PEA) and its 0-methylated derivatives block synaptosomal and glial 5HT transport equally well. These observations suggest that cultured C6 cells used as models of glia possess a 5HT transport system which kinetically and pharmacologically resembles a neuronal 5HT transport system.     

Thyrotropin-releasing hormone (TRH) and its analog (DN-1417): interaction with pentobarbital in choline uptake and acetylcholine synthesis of rat brain slices

TRH or its analog DN-1417 (gamma-butyrolactone-gamma-carbonyl-L-histidyl-L-proliamide) given 15 min after intravenous (i.v.) administration of pentobarbital (30 mg/kg) markedly shortened the pentobarbital-induced sleeping time in rats. This effect was almost completely abolished by intracerebroventricular pretreatment with atropine methylbromide (20 micrograms/rat), thereby suggesting the involvement of cholinergic mechanism. The action mechanism was investigated using rat brain slices. TRH (10(-6)-10(-4)M) or DN-1417 (10(-7)-10(-5)M) caused significant increases in the uptake of [3H]-choline into striatal slices. TRH(10(-4)M) or DN-1417(10(-5)M) also stimulated the conversion of [3H]-choline to [3H]-acetylcholine in striatal slices. A 30% reduction of acetylcholine synthesis from [3H]-choline in hippocampal slices and a 40% reduction of [3H]-choline uptake in slices of cerebral cortex, hippocampus and hypothalamus were observed in rats pretreated with pentobarbital (60 mg/kg, i.v.). TRH or DN-1417 (20 mg/kg, i.v.) given 15 min after the administration of pentobarbital markedly reversed both of the pentobarbital effects. Direct application of pentobarbital (5 X 10(-4)M) to slices in vitro also caused a 20-40% reduction of [3H]-choline uptake of cerebral cortex, hippocampus and diencephalon. A concomitant application of TRH(10(-4)M) or DN-1417(10(-5)M) and pentobarbital abolished the pentobarbital effect. These results provide neurochemical evidence that the antagonistic effects of TRH and DN-1417 on pentobarbital-induced narcosis are closely related to alterations in the rat brain choline uptake and acetylcholine synthesis, which are considered to be measures of the activity of cholinergic neurons.