Metabolism of [U-13C5]Glutamine in Cultured Astrocytes Studied by NMR Spectroscopy: First Evidence of Astrocytic Pyruvate Recycling

Abstract: Metabolism of [U-¹³C₅]glutamine was studied in primary cultures of cerebral cortical astrocytes in the presence or absence of extracellular glutamate. Perchloric acid extracts of the cells as well as redissolved lyophilized media were subjected to nuclear magnetic resonance and mass spectrometry to identify ¹³C-labeled metabolites. Label from glutamine was found in glutamate and to a lesser extent in lactate and alanine. In the presence of unlabeled glutamate, label was also observed in aspartate. It could be clearly demonstrated that some [U-¹³C₅]glutamine is metabolized through the tricarboxylic acid cycle, although to a much smaller extent than previously shown for [U-¹³C₅]glutamate. Lactate formation from tricarboxylic acid cycle intermediates has previously been demonstrated. It has, however, not been demonstrated that pyruvate, formed from glutamate or glutamine, may reenter the tricarboxylic acid cycle after conversion to acetyl-CoA. The present work demonstrates that this pathway is active, because [4,5-¹³C₂]glutamate was observed in astrocytes incubated with [U-¹³C₅]glutamine in the additional presence of unlabeled glutamate. Furthermore, using mass spectrometry, mono-labeled alanine, glutamate, and glutamine were detected. This isotopomer could be derived via the action of pyruvate carboxylase using ¹³CO₂ produced within the mitochondria or from labeled intermediates that had stayed in the tricarboxylic acid cycle for more than one turn.     

Assimilation of methane and inorganic carbon by microbial communities mediating the anaerobic oxidation of methane

The anaerobic oxidation of methane (AOM) is a major sink for methane on Earth and is performed by consortia of methanotrophic archaea (ANME) and sulfate-reducing bacteria (SRB). Here we present a comparative study using in vitro stable isotope probing to examine methane and carbon dioxide assimilation into microbial biomass. Three sediment types comprising different methane-oxidizing communities (ANME-1 and -2 mixture from the Black Sea, ANME-2a from Hydrate Ridge and ANME-2c from the Gullfaks oil field) were incubated in replicate flow-through systems with methane-enriched anaerobic seawater medium for 5–6 months amended with either ¹³CH₄ or H¹³CO₃^-. In all three sediment types methane was anaerobically oxidized in a 1:1 stoichiometric ratio compared with sulfate reduction. Similar amounts of ¹³CH₄ or ¹³CO₂ were assimilated into characteristic archaeal lipids, indicating a direct assimilation of both carbon sources into ANME biomass. Specific bacterial fatty acids assigned to the partner SRB were almost exclusively labelled by ¹³CO₂, but only in the presence of methane as energy source and not during control incubations without methane. This indicates an autotrophic growth of the ANME-associated SRB and supports previous hypotheses of an electron shuttle between the consortium partners. Carbon assimilation efficiencies of the methanotrophic consortia were low, with only 0.25–1.3 mol% of the methane oxidized.     

Combined monitoring of changes in δ13CH4 and archaeal community structure during mesophilic methanization of municipal solid waste

Reconstituted municipal solid waste (MSW) with varying contents of putrescible and cellulosic waste was incubated anaerobically under mesophilic conditions. Standard physicochemical parameters were monitored, together with stable isotopic signatures of produced CH₄ and CO₂. δ¹³C values for CH₄ indicated a change of methanogenic metabolism with time. CH₄ was predominantly produced from H₂/CO₂ at the beginning of the incubations. This period was associated with important shifts in archaeal communities monitored by automated ribosomal intergenic spacer analysis (ARISA) and FISH of oligonucleotidic probes targeting specifically 16S rRNA gene of various methanogenic groups. The onset of the active methane generation phase was characterized by an increase of CH₄δ¹³C, indicating a progressive shift toward an aceticlastic metabolism. When the methane production levelled off, a decrease in the isotopic signature was observed toward values characteristics of hydrogenotrophic metabolism. ARISA profiles were, however, found to be stable from the beginning of the active methane generation phase until the end of the experiment. FISH observation indicated that members of the family Methanosarcinaceae were predominant in the archaeal community during this period, suggesting that these methanogens might exhibit a high metabolic versatility during methanization of waste.     

Metabolic Precursors and Compartmentation of Cerebral GABA in Vigabatrin-Treated Rats

Abstract: The metabolic precursors and cerebral compartmentation of the augmented GABA pool induced by vigabatrin, an irreversible inhibitor of GABA transaminase, have been investigated by ¹³C NMR. Adult rats receiving rat chow ad libitum were given either drinking water only or drinking water containing 2.5 g/L vigabatrin for 7 days. Both groups of animals were infused either with [1,2-¹³C₂]acetate (15 µmol/min/100 g body weight), an exclusive precursor of GABA formation through the glial glutamine pathway, or with [1,2-¹³C₂]glucose (15 µmol/min/100 g body weight), a substrate that can produce GABA through the glial glutamine pathway or by direct metabolism in the neurons. The brains were frozen in situ, extracted with perchloric acid, and analyzed by ¹³C NMR. In vigabatrin-treated animals [¹³C]glutamine, a common intermediate for [¹³C]GABA synthesis from glucose or acetate, was accumulated to similar amounts during infusions with [1,2-¹³C₂]glucose or [1,2-¹³C₂]acetate. However, [¹³C]GABA accumulation was sevenfold higher during [1,2-¹³C₂]glucose infusions or twofold higher during [1,2-¹³C₂]acetate infusions. These results show that the direct pathway of GABA formation by neuronal metabolism of glucose predominates over the alternative pathway through glial glutamine. Near-equilibrium relationships of the aminotransferases of GABA and aspartate imply that the observed [¹³C]GABA accumulation occurs initially in the neuronal compartment.     

Protonmotive force-driven synthesis of ATP during methane formation from molecular hydrogen and formaldehyde or carbon dioxide in Methanosarcina barkeri

Abstract Methane formation from formaldehyde and H₂ or from carbon dioxide and H₂, as performed by cell suspensions of Methanosarcina barkeri , was coupled to ATP synthesis. In correspondence with this, methane formation was inhibited by N , N '-dicyclohexylcarbodiimide (DCCD), which at the same time, caused a decrease of the intracellular ATP concentration but only a slow decrease of the membrane potential. Addition of the uncoupler tetrachlorosalicylanilide (TCS) led to a relief of the inhibition of methane formation from CH₂O + H₂, but not from CO₂+ H₂.     

PYRUVATE METABOLISM IN THE LOBSTER NERVE AS AFFECTED BY THE PARTIAL PRESSURE OF CARBON DIOXIDE: OBSERVATIONS ON THE SYNTHESIS OF ACETYLCHOLINE AND ON METABOLIC COMPARTMENTATION

Abstract— In the lobster nerve the fixation of CO, at various levels of _pCO2 was studied by the incorporation of [l-¹⁴C]pyruvate. Incorporation of ¹⁴C was solely dependent on CO₂ fixation since the C-1 was decarboxylated in the formation of acetyl-CoA. Paired-nerve studies with [2-¹⁴C]pyruvate afforded a study of pyruvate metabolism in the lobster nerve. [I¹⁴C]Pyruvate was incorporated to nearly the same extent at all levels of pCO₂ including zero pCO₂, a finding that suggested metabolic recycling of CO₂. The magnitude of the metabolic recycling of C-1 of pyruvate or pyruvate dismutation was estimated to be nearly 20 per cent of total CO₂ fixation. Re-evaluation of the relative contributions of the CO₂ fixation. and acetyl-CoA pathways on the basis of more extensive data gave a ratio of 2:3.
The pCO₂ affected synthesis of ACh and the level of citrate. With increasing pCO₂, the specific radioactivity of ACh decreased much more than the content of ACh. The decrease in the specific radioactivity of ACh but not that of citrate further suggested metabolic compartmentation. The implication of these findings is discussed.
Alanine functioned as a metabolic sink for the incorporated pyruvate. Pyruvate levels were estimated to be approximately 0.1 nmol/mg of protein.     

Heavy metal inhibition of methanogenesis by Methanospirillum hungatei GP1

Abstract Washed whole cells of Methanospirillum hungatei incubated in TES buffer retained methanogenic activity in the absence of any reducing agents. Washed cells grown with 80% H₂-20% CO₂ and acetate produced methane from H₂/CO₂ and 50 mM formate at 1.1 to 1.8 and 15 μmol methane · h⁻¹· mg⁻¹ protein, respectively. Cadmium at a concentration of 15 μM and 50 μM mercury, copper or zinc completely inhibited methane production from H₂/CO₂ by M. hungatei . The chelating agent, EDTA, protected the cells from inhibition by cadmium but acetate and citrate did not. The activity of formate dehydrogenase and hydrogenase remaining in cells after incubation with copper, mercury, zinc or cadmium was reduced with formate dehydrogenase being the more sensitive.     

Competition between reductive acetogenesis and methanogenesis in the pig large-intestinal flora

Washed bacterial suspensions obtained from the pig hindgut were incubated under ¹³CO₂ in a buffer containing NaH¹³CO₃ and carbohydrates. Incorporation of ¹³C into short chain fatty acids was assayed by quantitative nuclear magnetic resonance. The effects of different levels of H₂ added to the gas phase (0, 20 and 80% v/v) and of the specific methanogenesis inhibitor 2-bromoethane-sulphonic acid (BES) were determined. In control incubations increasing the concentration of H₂ markedly increased methane production. Single- and double-labelled acetate and butyrate were formed in all incubations. In the absence of BES, increasing H₂ significantly increased the incorporation of ¹³CO² into butyrate and the proportion of double-labelled acetate in total labelled acetate. The addition of BES proved to be very successful as a methane inhibitor and greatly enhanced the amount of mono- and double-labelled acetate, especially at the highest H₂ partial pressure. The results suggest that methanogenesis inhibited both routes of reductive acetogenesis, i.e. the homoacetate fermentation of hexose (represented for the most part by single labelling) and the synthesis of acetate from external CO₂ and H₂ (represented mostly by double labelling). A highly significant interaction between BES and H₂ concentration was observed. At the highest pH2 BES increased the proportion of labelled acetate in total acetate from 17.1% for the control to 50.9%. It was concluded that although acetogenesis and methanogenesis can occur simultaneously in the pig hindgut, reductive acetogenesis may become a significant pathway of acetate formation in the absence of methanogenesis.     

Elevated pyruvate,orthophosphate dikinase (PPDK) activity alters carbon metabolism in C3 transgenic potatoes with a C4 maize PPDK gene

Changes in carbon metabolism and δ¹³C value of transgenic potato plants with a maize pyruvate,orthophosphate dikinase (PPDK; EC 2.7.9.1) gene are reported. PPDK catalyzes the formation of phospho enol pyruvate (PEP), the initial acceptor of CO₂ in the C₄ photosynthetic pathway. PPDK activities in the leases of transgenic potatoes were up to 5.4‐fold higher than those of control potato plants (wild‐type and treated control plants). In the transgenic potato plants, PPDK activity in leaves was negatively correlated with pyruvate content (r²= 0.81), and was positively correlated with malate content (r²= 0.88). A significant increase in the δ¹³C value was observed in the transgenic potato plants, suggesting a certain contribution of PEP carboxylase as the initial acceptor of atmospheric CO₂. These data suggest that elevated PPDK activity may alter carbon metabolism and lead to a partial operation of C₄‐type carbon metabolism. However, since parameters associated with CO₂ gas exchange were not affected, the altered carbon metabolism had only a small effect on the total photosynthetic characteristics of the transgenic plants.     

Glucose fermentation to acetate and alanine in resting cell suspensions of Pyrococcus furiosus: Proposal of a novel glycolytic pathway based on 13C labelling data and enzyme activities

Abstract Suspensions of maltose-grown cells of the hyperthermophilic archaeon Pyrococcus furiosus , when incubated at 90°C with 35 mM [1-¹³C]glucose or [3-¹³C]glucose, consumed glucose at a rate of about 10 nmol min⁻¹ (mg protein)⁻¹. Acetate (10 mM), alanine (3 mM), CO₂ and H₂ were the fermentation products. The ¹³C-labelling pattern in alamine and acetate were analyzed. With [1-¹³C]glucose the methyl group of both alanine and acetate was labelled; with [3-¹³C]glucose only the carboxyl group of alanine was labelled whereas acetate was unlabelled. Extracts of maltose-grown cells contained glucose isomerase (12.8 U mg⁻¹, 100°C), ketohexokinase (0.23 U mg⁻¹, 100°C), and fructose 1-phosphate aldolase (0.06 U mg^{−1, 100°C). Enzymes catalyzing the formation of fructose 1,6-bisphosphate from fructose 1-phosphate or fructose 6-phosphate could not be detected. As publihed previously by our group and other authors P. furiosus also contains enzymes of glyceraldehyde conversion to 2-phosphoglycerate according to a non-phosphorylated Entner-Doudoroff pathway, of dihydroxyacetone phosphate conversion to 2-phosphoglycerate according to the Embden-Meyerhof pathway, and of 2-phosphoglycerate conversion - via pyruvate - to acetate and alanine. Based on the enzyme activities in P. furiosus , the following pathway for glucose degradation to alanine and acetate in cell suspensions is proposed which can explain the [13}C]glucose labelling data: glucose→ fructose → fructose 1- phosphate → dihydroxyacetone phosphate + glyceraldehyde and further conversion of both trioses to alanine and acetate via pyruvate.     

Ontogeny and Cellular Localization of the Pyruvate Recycling System in Rat Brain

Abstract: The ontogeny of the cerebral pyruvate recycling pathway and the cellular localization of associated enzymes, malic enzyme (ME) and phosphoenolpyruvate carboxykinase (PEPCK), have been investigated using a combination of ¹³C NMR spectroscopy, enzymatic analysis, and molecular biology approaches. Activity of the pathway, using [1,2-¹³C₂]acetate as a substrate, was detected by ¹³C NMR in brain extracts 3 weeks after birth, increasing progressively up to the third month of age. In whole-brain homogenates, ME activity increased to adult levels with the same time course as the recycling pathway. PEPCK activity was low during the first 2 weeks of life and decreased further toward adulthood. ME and PEPCK activity were found in primary cultures of astrocytes and in synaptosomal fractions of adult brain. Primary cultures of cortical neurons showed PEPCK activity but no detectable ME activity. The cytosolic ME gene was expressed in primary cultures of neurons and in astrocytes as well as in the neonatal and adult brain. The PEPCK gene was expressed both in primary cultures of cortical neurons and in astrocytes, but the level of its expression in the neonatal and adult brain was undetectable.     

Carbon metabolism of Listeria monocytogenes growing inside macrophages

The intracellular metabolism of Listeria monocytogenes was studied by ¹³C-isotopologue profiling using murine J774A.1 macrophages as host cells. Six hours after infection, bacteria were separated from the macrophages and hydrolyzed. Amino acids were converted into tert-butyl-dimethylsilyl derivatives and subjected to gas chromatography/mass spectrometry. When the macrophages were supplied with [U-¹³C₆]glucose prior to infection, but not during infection, label was detected only in Ala, Asp and Glu of the macrophage and bacterial protein with equal isotope distribution. When [U-¹³C₆]glucose was provided during the infection period, ¹³C label was found again in Ala, Asp and Glu from host and bacterial protein, but also in Ser, Gly, Thr and Val from the bacterial fraction. Mutants of L. monocytogenes defective in the uptake and catabolism of the C₃-metabolites, glycerol and/or dihydroxyacetone, showed reduced incorporation of [U-¹³C₆]glucose into bacterial amino acids under the same experimental settings. The ¹³C pattern suggests that (i) significant fractions (50–100%) of bacterial amino acids were provided by the host cell, (ii) a C₃-metabolite can serve as carbon source for L. monocytogenes under intracellular conditions and (iii) bacterial biosynthesis of Asp, Thr and Glu proceeds via oxaloacetate by carboxylation of pyruvate.     

Effect of plant succession, ploughing, and fertilization on the microbiological oxidation of atmospheric methane in a heathland soil

Abstract: The effect of plant succession on methane uptake was measured on intact soil cores collected from seven heathland sites. Six of the sites had undergone either secondary succession with grass or oak, ammonium fertilization or ploughing, while the seventh site was located in the native heathland. There was a positive relationship between methane uptake rate and time elapsed since the plant invasion had taken place in the native heathland. The native heathland site showed an insignificant atmospheric methane uptake of 0.01 mg CH₄ m⁻² d⁻¹, whereas the established oak brushwood (70 years old) and the grass invaded heathland (13 years old) showed rates of 1.36 mg CH₄ m⁻² d⁻¹ and 0.73 mg CH₄ m⁻² d⁻¹, respectively. In the fertilized heathland plot (112 kg N ha⁻¹ six years prior to this study) grass had become the dominating species and showed a methane oxidation rate of 0.28 mg CH₄ m⁻² d⁻¹. Ploughing of the heathland resulted in methane oxidation rates seven times the rates measured in the native heathland. The results suggested that an increased future atmospheric nitrogen deposition in heathlands and other nutrient poor ecosystems may have a stimulating effect on the soil sink for atmospheric methane.     

Inhibition of Methanosarcina barkeri strain 227 by cadmium

Abstract The effect of cadmium (Cd) on methane formation from methanol and/or H₂–CO₂ by Methanosarcina barkeri was examined in a defined growth medium and in a simplified buffer system containing 50 mM Tes with or without 2 mM dithiothreitol (DTT). No inhibition of methanogenesis by high concentrations of cadmium was observed in growth medium. Similarly, little inhibition of methanogenesis by whole cells in the Tes buffer system was observed in the presence of 430 μM Cd or 370 μM mercury (Hg) with 2 mM DTT. When the concentration of DTT was reduced to 0.4 mM, almost complete inhibition of methanogenesis from H₂–CO₂ and methanol by 600 μM Cd was observed. In the absence of DTT, 150 μM Cd inhibited methanogenesis from H₂–CO₂ completely and from methanol by 97%. Methanogenesis from H₂–CO₂ was more sensitive to Cd than that from methanol.     

Temperature profile of oxygen activation and defense against oxygen toxicity in a psychrophilic member of the Bacteroidaceae

Abstract The temperature profiles have been determined for O₂ reduction by activating substrates for whole cells and cell extracts of the psychrophilic, obligately anaerobic bacterium, strain B6, belonging to the Bacteroidaceae. The profiles were similar whether the cells were grown at 15 or 1°C, and also for cells harvested in the exponential or stationary phase. The H₂O producing pyruvate oxidase displayed in cell-free extracts a considerably higher activity than the H₂O₂ producing NADH and NADPH oxidases at all temperatures in the range 30–1°C, and characteristically makes up a larger proportion of the total O₂ reduction capacity the lower the temperature. It thus seems that the O₂ scavenging property of the pyruvate oxidase, postulated to be utilized in a defense mechanism against the detrimental effects of the H₂O₂ producing pyridine nucleotide oxidases, is particularly well adapted to function at the low temperatures of the Barents Sea, from which this obligately anaerobic organism originates.     

Glial Formation of Pyruvate and Lactate from TCA Cycle Intermediates: Implications for the Inactivation of Transmitter Amino Acids?

Abstract: Cerebral formation of lactate via the tricarboxylic acid (TCA) cycle was investigated through the labeling of lactate from [2-¹³C]acetate and [1-¹³C]glucose as shown by ¹³C NMR spectroscopy. In fasted mice that had received [2-¹³C]acetate intravenously, brain lactate C-2 and C-3 were labeled at 5, 15, and 30 min, reflecting formation of pyruvate and hence lactate from TCA cycle intermediates. In contrast, [1-¹³C]glucose strongly labeled lactate C-3, reflecting glycolysis, whereas lactate C-2 was weakly labeled only at 15 min. These data show that formation of pyruvate, and hence lactate, from TCA cycle intermediates took place predominantly in the acetate-metabolizing compartment, i.e., glia. The enrichment of total brain lactate from [2-¹³C]acetate reached ∼1% in both the C-2 and the C-3 position in fasted mice. It was calculated that this could account for 20% of the lactate formed in the glial compartment. In fasted mice, there was no significant difference between the labeling of lactate C-2 and C-3 from [2-¹³C]acetate, whereas in fed mice, lactate C-3 was more highly labeled than the C-2, reflecting adaptive metabolic changes in glia in response to the nutritional state of the animal. It is hypothesized that conversion of TCA cycle intermediates into pyruvate and lactate may be operative in the glial metabolism of extracellular glutamate and GABA in vivo. Given the vasodilating effect of lactate on cerebral vessels, which are ensheathed by astrocytic processes, conversion of glutamate and GABA into lactate could be one mechanism mediating increases in cerebral blood flow during nervous activity.     

Metabolism of Cysteine in Astroglial Cells: Synthesis of Hypotaurine and Taurine

Abstract: The synthesis of hypotaurine and taurine was investigated in astroglia-rich primary cultures obtained from brains of neonatal Wistar rats using ¹H and ¹³C nuclear magnetic resonance (NMR) spectroscopy. Cell extracts of astroglial cultures analyzed by ¹H NMR spectroscopy show prominent signals of hypotaurine. To identify cysteine as precursor for hypotaurine and taurine synthesis in astroglial cells, primary cultures were incubated with [3-¹³C]cysteine for 24 or 72 h. Cell extracts and incubation media were then analyzed with ¹³C NMR spectroscopy. Labeled hypotaurine, taurine, glutathione, and lactate were identified in the cell extracts. Within 72 h, 35.0% of the total intracellular hypotaurine and 22.5% of taurine were newly synthesized from [3-¹³C]cysteine. The presence of [1-¹³C]hypotaurine and [1-¹³C]taurine in the incubation medium proves the release of those products of cysteine metabolism into the medium. Minor amounts of the [3-¹³C]cysteine were used for the synthesis of glutathione in astroglial cells or metabolized to [3-¹³C]lactate, which was found in cell extracts and media. These results indicate that the formation of hypotaurine and taurine is a major pathway of cysteine metabolism in astroglial cells.     

Glucose Metabolism in the Developing Cerebral Cortex as Detected by 1H{13C} Nuclear Magnetic Resonance Spectroscopy Ex Vivo

Abstract: Metabolism of [1-¹³C]glucose was monitored in superfused cerebral cortex slice preparations from 1-, 2-, and 5-week-old rats using ¹H-observed/¹³C-edited (¹H{¹³C}) NMR spectroscopy. The rate of label incorporation into glutamate C-4 did not differ among the three age groups: 0.52–0.67% of total ¹H NMR-detected glutamate/min. This was rather unexpected, as oxygen uptake proceeded at 1.1 ± 0.1, 1.9 ± 0.1, and 2.0 ± 0.1 µmol/min/g wet weight in brain slices prepared from 1-, 2-, and 5-week-old animals, respectively. Steady-state glutamate C-4 fractional enrichments in the slice preparations were ∼23% in all age groups. In the acid extracts of slices glutamate C-4 enrichments were smaller, however, in 1- and 2-week-old (17.8 ± 1.7 and 16.8 ± 0.8%, respectively) than in 5-week-old rats (22.7 ± 0.7%) after 75 min of incubation with 5 m M [1-¹³C]glucose. We add a new assignment to the ¹H{¹³C} NMR spectroscopy, as acetate C-2 was detected in slice preparations from 5-week-old animals. In the acid extracts of slice preparations acetate C-2 was labeled by ∼30% in 5-week-old rats but by 15% in both 1- and 2-week-old animals, showing that the turnover rate was increased in 5-week-old animals. In the extracts 3–4% of the C-6 of N -acetyl-aspartate (NAA; CH₃ of the acetyl group) contained label as determined by both NMR and mass spectrometry, which indicated that there was no significant labeling to other carbons in NAA. NAA accumulated label from [1-¹³C]glucose but not from [2-¹³C]acetate, and the rate of label incorporation increased by threefold on cerebral maturation.     

Carbon dioxide assimilation during postharvest removal of astringency from persimmon fruit

Application of anaerobic conditions with CO₂ or N₂ atmospheres to remove astringency from harvested persimmon fruit ( Diospryros kaki L. cv. Triumph), caused production of more acetaldehyde under CO₂ than under N₂, ¹⁴CO₂ applied in a 100% CO₂ atmosphere, for 48 h to astringent persimmon fruits was incorporated mainly into malate and very little into other metabolites, such as carbohydrate or amino acids. Application of malate or pyruvate to pulp discs of astringent persimmons caused an immediate rise in acetaldehyde production. The higher levels of acetaldehyde produced by whole fruits held in a CO₂ atmosphere, than by fruits held in a N₂ atmosphere, can be explained through fixation of atmospheric CO₂ into malate, leading to acetaldehyde production.     

Specificity of Muscarinic Acetylcholine Receptor Regulation by Receptor Activity

Abstract— Regulation of muscarinic acetylcholine receptor concentration by receptor activity in neuron-like NG108-15 hybrid cells is a highly specific process. Receptor levels, monitored by binding of [³H]quinuclidinyl benzilate ([³H]QNB), decreased 50-75% following 24-h incubation of cells with muscarinic agonists, but none of the following cellular processes was altered by this chronic receptor stimulation: (1) glycolytic energy metabolism, measured by [³H]deoxy- d -glucose ([³H]DG) uptake and retention; (2) rate of cell division; (3) transport, measured by [³H]valine and [³H]uridine uptake; (4) RNA biosynthesis, measured by [³H]uridine incorporation; (5) protein biosynthesis, measured by [³H]valine and [³⁵S]methionine incorporation into total protein and into protein fractions obtained by polyacrylamide gel electrophoresis. In contrast, chronic stimulation did cause a threefold decrease in the capacity of carbachol to stimulate phosphatidylinositol (PI) turnover, a receptor-mediated response. In addition to cholinomimetics, the neuroeffector adenosine (1 m m for 24 h) also caused a decrease in [³H]QNB binding levels, but chronic stimulation of α -adrenergic, opiate, prostaglandin E₁, and prostaglandin F_2α receptors found on NG108-15 cells caused no changes. The data indicate that loss of muscarinic receptors caused by receptor stimulation is not a consequence of fundamental changes evoked in overall cellular physiology but reflects a specific regulation of cholinoceptive cell responsiveness.