Mechanism of Action of Acamprosate. Part II. Ethanol Dependence Modifies Effects of Acamprosate on NMDA Receptor Binding in Membranes from Rat Cerebral Cortex

Acamprosate is a putative anticraving drug used to maintain abstinence in alcohol-dependent patients. Its mechanism of action is uncertain, but the drug is thought to interact with neuronal NMDA receptors and calcium channels, and these proteins are implicated in the induction of alcohol dependence. In these experiments, the effects of acamprosate were studied on the binding of the NMDA receptor ligand [³H]dizocilpine to rat brain membranes under nonequilibrium conditions; 10 μM glutamate and 1 μM glycine were present in the binding assays to partially activate the receptor. At clinically relevant concentrations (in the micromolar range), acamprosate significantly enhanced [³H]dizocilpine binding to cortical membranes from control animals (suggesting that acamprosate may increase the rate of association of the radioligand), whereas at higher concentrations binding was inhibited. This effect is consistent with a partial agonist effect of acamprosate on the NMDA receptor protein. However, when rats were made dependent on ethanol (exposure to the drug for 10 days by inhalation) and cortical membranes were prepared from these animals, acamprosate in vitro no longer produced any enhancement of [³H]dizocilpine binding. Similar results were obtained when membranes were used from rats that had received 400 mg/kg/day of acamprosate in their drinking water with or without concurrent ethanol inhalation for 10 days. Thus, in brain membranes from all these treatment groups, acamprosate in vitro caused inhibition of [³H]dizocilpine binding only. The results suggest that acamprosate may have excitatory or inhibitory effects on NMDA receptors, depending on the experimental conditions. The effects of the drug on this system appear to be shifted toward inhibition in alcohol dependence, and this finding may be important to its clinical mechanism.     

Effects of Chronic Ethanol on the Mobilization of Arachidonate and Docosahexaenoate Stimulated by the Type 2A Serotonin Receptor Agonist (±)-2,5-Dimethoxy-4-iodoamphetamine Hydrochloride in C6 Glioma Cells

We studied the effects of chronic ethanol exposure on the mobilization of polyunsaturated fatty acids stimulated by activation of the type 2A serotonin receptor in C6 glioma cells. In our in vitro model, we prelabeled cells with [³H]arachidonate and [¹⁴C]docosahexaenoate and subsequently stimulated with the type 2A serotonin receptor agonist (±)-2,5-dimethoxy-4-iodoamphetamine hydrochloride. In as early as 10 days of exposure to 20 or 50 mM ethanol, the (±)-2,5-dimethoxy-4-iodoamphetamine hydrochloride-stimulated mobilization of [³H]arachidonic acid ([³H]AA) and [¹⁴C]docosahexaenoic acid ([¹⁴C]DHA) was significantly inhibited, and this inhibition was accompanied by decreased mobilization of intracellular [Ca²⁺]_i. Exposure to ethanol did not alter significantly the release of [³H]AA and [¹⁴C]DHA stimulated by the calcium ionophore A23187 nor the incorporation of [³H]AA and [¹⁴C]DHA into cellular lipids. Decreased mobilization of polyunsaturated fatty acids and calcium in astroglia may contribute to neurotoxicity caused by chronic ethanol exposure.     

Regional Differences in the Effects of Chronic Ethanol Administration on [3H]Zolpidem Binding in Rat Brain

A strong association has been observed between [³H]zolpidem binding and the presence of γ-aminobutyric acid (GABA_A) receptor mRNA for α₁-, β₂-, and γ₂-subunits in specific brain regions. This correlates with observed sensitivity of individual neurons to zolpidem and ethanol in these same regions. Previous studies using homogenate binding approaches showed small alterations in [³H] zolpidem binding levels after chronic ethanol exposure. This study was undertaken to ascertain if there is regional specificity of the effects of chronic ethanol administration on [³H] zolpidem binding levels. Chronic ethanol administration induced small, but significant alterations in [³H] zolpidem (5 nM) binding in the Inferior colliculus, substantia nigra, and the medial septum. [³H]Zolpidem binding was increased in the inferior colliculus and substantia nigra, and decreased in the medial septum. No significant differences in [³H] zolpidem binding were noted in any other brain area analyzed, including the cortex and cerebellum. These findings show that chronic ethanol administration has small effects on [³H] zolpidem binding, although they occur in a site-specific and bidirectional manner. Moreover, there is no correlation between changes in [³H] zolpidem binding and alterations In GABA_A receptor subunit expression.     

Effects of Prenatal and Early Postnatal Ethanol Exposure on [3H]MK-801 Binding in Rat Cortex and Hippocampus

The effects of prenatal and/or early postnatal exposure to ethanol at high concentrations on N-methyl-D-aspartate (NMDA) receptor number and functioning in the weanling rat were examined. The bingelike exposure protocol was used in an animal model of acute ethanol effects at two critical periods of development. [³H]MK-801 binding parameters for the internal channel phencyclidine site were assessed in the presence of 10 μM glutamate and 10 μ M glycine activation. Four treatment groups were included: (1) animals exposed to ethanol both prenatal and postnatal; (2) animals exposed only prenatal; (3) animals exposed early postnatal only; and (4) control animals with no exposure to ethanol. The results of the [³H]MK-801 binding experiments showed that both prenatal and postnatal exposure to ethanol resulted in a significant decrease in the density of NMDA receptors. In addition, data indicated an apparent increase in the percentage of high-affinity state (open channel state) relative to low-affinity state (close channel state) receptors in the ethanoltreated groups. These results show that both prenatal and postnatal ethanol exposure decrease NMDA receptor density in the cortex and hippocampus. The findings are consistent with previous observations by our laboratory and others that NMDA-mediated calcium influx is reduced in these regions, as well as in whole brain by prenatal ethanol exposure. It is suggested that after ethanol exposure, the remaining functional NMDA receptors might have altered sensitiviity to coagonist activation with an increased probability of channel opening.     

Equilibrative Adenosine Transport in Rat Hepatocytes after Chronic Ethanol Feeding

Acute treatment of cells with ethanol in vitro inhibits adenosine uptake via equilibrative nucleoside transporters. After longer periods of exposure to ethanol in culture, rechallenge with ethanol no longer inhibits adenosine uptake. Herein, we have investigated the long-term effects of ethanol consumption in vivo on equilibrative nucleoside transport. Rats were fed a liquid diet containing 35% of calories as ethanol (ethanol-fed). Control rats were pair-fed a liquid diet that isocalorically substituted maltose dextrins for ethanol. After 4 weeks of ethanol consumption, nucleoside transport was measured in isolated hepatocytes. Uptake of [³H]adenosine was lower in ethanol-fed rats compared with control. Influx of the nonmetabolizable nucleoside analog, [³H]formycin B, was also decreased after ethanol feeding. However, neither the number of nitrobenzylthioinosine (NBMPR) binding sites or inhibition of adenosine uptake by NBMPR were affected by ethanol feeding. In controls, acute treatment of isolated hepatocytes with 100 itim ethanol inhibited [³H]adenosine uptake by 30–40%. However, in ethanol-fed rats, acute challenge with ethanol did not inhibit [³H]adenosine uptake. These data demonstrate that long-term ethanol feeding decreases equilibrative nucleoside transport in hepatocytes independent of a change in the number of nucleoside transporters and renders adenosine uptake insensitive to inhibition by ethanol.     

Long-Term Effects of Chronic Ethanol on Muscarinic Receptor Binding in Rat Brain

Effects of chronic ethanol treatment (CET) on muscarinic acetylcholine receptor (mAChR) binding properties were investigated via quantitative autoradiography in rats maintained on an ethanol-containing liquid diet for 28 weeks and withdrawn from ethanol for 8 weeks before harvesting of tissues. Controls received an identical diet in which sucrose was substituted isocalorically for ethanol. Maximal binding of the radiolabeled mAChR antagonist quinuclidinyl benzilate ([³H]QNB) was not reduced in hippocampal area CA1, dentate gyrus, neocortex, striatum, or thalamus, suggesting that CET results in no significant mAChR loss in these regions. Binding affinities of the cholinergic agonist carbachol to mAChRs were unaffected by CET in each of these regions, as determined by competitive displacement of [³H]QNB labeling. These results suggest that CET-induced functional deficits in brain cholinergic responses are not due to direct effects of CET on mAChR binding properties.     

Sensitivity to Ethanol Across Development in Rats: Comparison to [3H]Zolpidem Binding

Previous research has suggested that rats tested at 28 to 30 days of age show a marked subsensitivity to the sedative effects of ethanol. In the present study, rats of different ages were tested for aerial righting following acute ethanol (3 g/kg) treatment. These results were compared with the effects of the atypical benzodiazepine zolpidem (3 and 5 mg/kg) and pentobarbital (10 and 15 mg/kg). Animals tested at 25,28, or 35 days of age were significantly less impaired by ethanol than preweanling rats (age 20 days) or older rats (age 65 to 75 days), whereas animals tested at 25 or 28 days of age were less impaired by the higher dose of zolpidem. With pentobarbital, the most distinct age-related trend was greater impairment in 20-day-old rats. Because ethanol may be active at the same type I GABA_A receptor site selectively labeled by [³H]zolpidem, levels of [³H]zolpi-dem binding were determined for rats of different ages. Although some brain regions showed progressive increases in binding of [³H]zolpidem across development, other regions demonstrated increased binding from day 12 or 17 to day, then a plateau of binding levels across days 25, and 28, with further increases occurring by day 36 or day 60. This pattern was observed in the cingulate cortex, medial septal nucleus, globus pallidus, inferior colliculus, red nucleus, and cerebellum. Overall, the results indicate that the period of subsensitivity to the sedative effects of ethanol is coincident with a change in the developmental pattern of GABA_A receptor sites targeted by [³H]zolpidem.     

Effects of Ethanol on Cultured Fetal Astroglia

This study investigated the effects of a 4-day ethanol exposure on cultured rhombencephalic astroglia. The contents of astroglial protein and DNA, and astroglial uptake of serotonin (5-HT) were determined. Fetal rhombencephalic astroglia were examined because of this laboratory's evidence that in utero ethanol exposure markedly impairs the development of serotonergic neurons, which are located in this fetal brain area, and because of the recently demonstrated importance of local support glia in neuronal development.
The results of these experiments demonstrated that protein was significantly reduced in astroglia cultured in ethanol at either 150 or 300 mg/dl. In addition, these astroglia exhibited decreased [³H]5-HT uptake per well. However, no significant ethanol-associated differences were detected when [³H]5-HT uptake was expressed per mg protein rather than per well. In contrast to the effects of a 4-day ethanol exposure, the acute ethanol exposure did not significantly alter astroglial uptake of [³H]5-HT/well. In addition, the 4-day exposure to 50 to 300 mg/dl of ethanol did not significantly alter astroglial DNA content.
In summary, it appears that a 4-day exposure of cultured fetal rhombencephalic astroglia to 150 to 300 mg/dl of ethanol reduces astroglial protein content and astroglial 5-HT uptake. A reduction in total astroglial proteins, potentially including those that act as essential growth factors, could contribute to some of the ethanolassociated alterations in central nervous system development.     

Effects of Chronic Ethanol Exposure on GABA Receptors and GABAB Receptor Modulation of 3H-GABA Release in the Hippocampus

Chronic ethanol treatment (CET), sufficient for decreasing long-term potentiation (LTP) in rats, also enhances ³H-GABA release from hippocampal slices in these same animals. The mechanism for an increase in GABA release may involve changes in presynaptic receptors. Therefore, we characterized presynaptic autoreceptor modulation of ³H-GABA release in hippocampal slices from control and CET rats. The effects of a GABA_B receptor agonist (baclofen) and antagonist [2-hydroxy (OH)-saclofen] were tested for their ability to modulate electrically stimulated ³H-GABA release from superfused hippocampal slices. Baclofen decreased stimulated release in a dose-dependent manner and 2-OH-saclofen increased release consistent with the existence of presynaptic GABA_B autoreceptors in hippocampus. The GABA_A antagonist bicuculline did not significantly modulate basal or stimulated release. When the effects of baclofen and 2-OH-saclofen were measured in animals 48 hr after withdrawal from CET, presynaptic modulation of release by baclofen and 2-OH-saclofen was decreased. In addition, we examined the density of ³H-baclofen and ³H-bicuculline binding in the hippocampal formation using quantitative autoradiographic techniques. We found that the density of ³H-baclofen binding sites was not affected by CET, whereas the density of ³H-bicuculline binding sites was increased by 28% in ethanol-treated rats. These data may explain how CET increases presynaptic regulation of GABA release from hippocampus that may contribute to the decrease in LTP seen in rats after CET.     

First-Pass Metabolism of Ethanol Is Negligible in Rat Gastric Mucosa

Controversy exists concerning whether first-pass metabolism of imbibed ethanol occurs in the gastric mucosa or liver. We assessed ethanol metabolism in rat gastric mucosa by determining to what extent intact [¹⁴C]ethanol in body water plus hepatic metabolism could account for [¹⁴C]ethanol absorbed from the pylorus-ligated stomach. Intact [¹⁴C]ethanol in systemic body water accounted for 84 ± 1.9% of the [¹⁴C]ethanol absorbed from the stomach over a 30-min period. Assuming a 15 ml/min hepatic blood flow, the predicted hepatic metabolism of [¹⁴C]ethanol over the 30 min of the study was 18% of the dose. The sum of intact [¹⁴C]ethanol and predicted hepatic metabolism accounted for 100% of the ethanol absorbed from the stomach. We conclude that negligible metabolism of ethanol occurred in the gastric mucosa.     

Characterization of P2x1 purinoreceptors on rat platelets: effect of clopidogrel

This study aimed to determine the binding characteristics of [³H]α,β-Me-ATP, a specific ligand of the P2x1 receptors to rat platelets, and to investigate the effect of clopidogrel, a thienopyridine compound which has been found to selectively inhibit ADP-induced platelet aggregation and adenylyl cyclase ex vivo . Binding of [³H]α,β-Me-ATP to rat platelets was time-dependent and saturable. Scatchard analysis of the saturation binding data indicated that [³H]α,β-Me-ATP bound to one population of specific binding sites with high affinity ( K _D  =  23.6 ± 1.6 n m ; B _max = 690 ± 24 fmole/10⁸cells) ( n= 3). Unlabelled α,β-Me-ATP as well as 2-MeS-ADP and ADP competitively inhibited the specific binding of [³H]α,β-Me-ATP with IC₅₀ values of 19.0 ± 6.6, 103 ± 20 and 1120 ± 80 n m respectively ( n= 3). Other nucleotide analogues such as ATP, ATP-γS, UTP and GTP also antagonized [³H]α,β-Me-ATP binding. When administered orally (10 mg/kg, p.o.), clopidogrel inhibited ADP- or 2-MeS-ADP-induced platelet aggregation but did not affect the binding of [³H]α,β-Me-ATP to rat platelets ex vivo. In vitro , α,β-Me-ATP did not induce the aggregation or shape change of rat platelets and did not interfere with ADP-induced platelet aggregation.     

Dopamine D2 Receptor Gene Expression in Rat Lines Selected for Differences in Voluntary Alcohol Consumption

A selective breeding program has led to the establishment of the alcohol-preferring AA (Alko, Alcohol) and alcohol-avoiding ANA (Alko, Nonalcohol) rat lines. To reveal putative baseline differences in dopamine receptor gene expression and dopamine receptor binding profile in the AA and ANA rat lines, we assessed striatal D₂ mRNA levels in these two rat lines. Autoradiographical studies on dopamine D₁ and D₂ receptors in the striatum and nucleus accumbens were also performed with [³H]SCH 23390 and [¹²⁵I]iodosulpiride/[³H]spiperone, respectively. The baseline differences in D₁ or D₂ receptor binding and D₂ receptor gene expression between AA and ANA rat lines are marginal, and are not likely to play a role in the genetic background of the differential alcohol drinking behavior of these rat lines.     

Differences in NMDA Receptor Antagonist-Induced Locomotor Activity and [3H]MK-801 Binding Sites in Short-Sleep and Long-Sleep Mice

Short-Sleep (SS) and Long-Sleep (LS) mice differ in initial sensitivity to ethanol. Ethanol acts as an antagonist at N-methyl d -aspartate receptors (NMDARs). Therefore, we tested whether SS and LS mice also differ in initial sensitivity to NMDAR antagonists. Systemic injection (intraperitoneal) of either the noncompetitive NMDAR antagonist MK-801 (dizocilpine) or the competitive NMDAR antagonist 2-carboxypiperazin-4-yl-propyl-1-phosphonic acid (CPP) produced similar results. At lower drug doses, SS mice showed greater loco-motor activation than LS mice; and at higher doses, SS mice continued to be activated whereas LS mice became sedated. Brain levels of [³H]MK-801 were 40% higher in SS, compared with LS, mice. However, blood levels of [³H]MK-801 and [³H]CPP and brain levels of [³H]CPP were similar in the two lines. NMDARs were measured using quantitative autoradiographic analysis of in vitro [³H]MK-801 binding to SS and LS mouse brains. Significantly higher (20 to 30%) receptor densities were observed in the hippocampus and cerebral cortex of SS mice. Our results support the hypothesis that SS and LS mice differ in initial sensitivity to NMDAR antagonists and suggest that the line differences in the dose-response relationships for MK-801- and CPP-induced locomotor activity are qualitatively similar to those reported for ethanol. Differences in pharmacokinetics and number of NMDARs may contribute to, but are unlikely to entirely account for, the differential behavioral responsiveness of SS and LS mice to MK-801 and CPP.     

Ethanol Suppresses Growth Hormone-Mediated Cellular Responses in Liver Slices

Previous studies indicate that both acute and chronic ethanol administration inhibit protein synthesis and decrease the secretion of insulin-like growth factor-1 (IGF-1). Although IGF-1 synthesis and secretion are regulated by growth hormone secretion from the pituitary gland, we assessed whether ethanol inhibits tissue response to growth hormone. Liver slices from male Sprague-Dawley rats were prepared, placed into F-12 media, and incubated at 37°C with [³H]leucine, and either 0.25 or 1 nM rat growth hormone and 0, 37 (physiological levels), or 175 mM (toxic levels) ethanol. Tissues were removed at 0,15,30, and 60 min. Protein synthesis increased linearly during this time period, and administration of growth hormone (1 nM) significantly increased protein synthetic rate by 48% ( p < 0.01), whereas addition of 37 or 175 mM ethanol attenuated the effects of growth hormone ( p < 0.01). Analysis of IGF-1 mRNA indicated a 2-fold increase in response to growth hormone ( p < 0.01), whereas ethanol administration decreased the growth hormone-induced rise of IGF-1 mRNA. Ethanol (175 mM) inhibited the release of IGF-1 into the media ( p < 0.05). Ethanol did not alter growth hormone receptor binding, and exposure of tissue slices to ethanol did not influence the number of growth hormone receptors or the affinity of growth hormone for its receptor.
Our results demonstrate that (1) growth hormone is a potent acute regulator of IGF-1 mRNA and IGF-1 peptide release, (2) ethanol inhibits growth hormone-induced protein synthesis and induction of IGF-1 gene expression, and (3) the inhibitory effects of ethanol on growth hormone occur without changing growth hormone receptor number or binding characteristics. We conclude that ethanol suppresses growth hormone-induced signal transduction, resulting in a decrease in IGF-1 gene expression.     

Uptake of Tritiated Folates by Human Bone Marrow Cells in Vitro

S ummary . Using incubation periods up to 4 hr, it was demonstrated that uptake of tritiated pteroylglutamic acid ([³H]PteGlu) by human bone marrow cells in vitro was in the range of six-fold greater than uptake by reticulocytes. Uptake was temperature-dependent, increasing during 4 hr at 37°C but not at 4°C; a similar temperature dependence was found for the uptake of [³H]methyltetrahydrofolate ([³H-CH₃]H₄PteGlu). The pH optimum for [³H]PteGlu uptake was in the range of 7.4. Percentage uptake decreased as concentration of [³H]PteGlu increased. Preincubation with unlabelled PteGlu reduced uptake of subsequently added [³H]PteGlu by twice as much as did preincubation with methotrexate, suggesting that methotrexate may only partly share the uptake mechanism for [³H]PteGlu. [³H]PteGlu uptake was not affected by preincubation with diphenylhydantoin or a sulphydryl inhibitor. Uptake of [³H-CH₃]H₄PteGlu by human bone marrow cells appeared to be approximately twice the uptake of [³H]PteGlu. The findings support the concept of two mechanisms for folate uptake by human reticulocytes and bone marrow cells: an energy-dependent, active carrier mechanism probably of primary physiologic significance, and a seemingly passive diffusion-like mechanism, probably primarily of pharmacologic significance.     

Effect of Chronic Ethanol Exposure on Myocardial Phosphoinositide Turnover

The effect of chronic ethanol consumption (2 months) on atrial contractility and the myocardial phosphoinositide signaling system was examined in rat heart. Two months of ethanol consumption was not associated with changes in heart weight-to-body weight ratios; however, developed twitch tension was significantly lower in the ethanol atria compared with the control atria. Cytosolic and membrane-associated phospholipase C activity in atrial and ventricular tissue was measured and ethanol consumption was only associated with changes in ventricular cytosolic phospholipase C activity. When examining α₁-adrenergic stimulated phosphoinositide turnover in [³H]inositol radiolabeled left atria, no differences in phenylephrine (10 μM)-stimulated inositol monophosphate, inositol bisphosphate, inositol trisphosphate, and inositol tetrakisphosphate were found between groups before or at various times after the addition of phenylephrine. It is concluded that short-term ethanol consumption is associated with depressed contractile function, but not the development of hypertrophy or changes in α₁-adrenoreceptor-stimulated phosphoinositide turnover.     

Serotonin2C Receptors and Serotonin2C Receptor-Mediated Phosphoinositide Hydrolysis in the Brain of Alcohol-Preferring and Alcohol-Nonpreferring Rats

To examine the role of serotonin_2C (5HT_2C) receptors in alcohol drinking behavior, the binding indices of 5HT_2C receptors were determined in various brain regions of alcohol-preferring (P) and alcohol-nonpreferring (NP) rats. 5HT_2C receptor-mediated phosphoinositide hydrolysis in the choroid plexus of P and NP rats was also determined. It was observed that the densities of 5HT_2C receptors are significantly higher in the hippocampus, the amygdala, and the choroid plexus, but not in the cortex of P rats compared with NP rats. The K _d values of [³H]mesulergine binding to 5HT_2C receptors were not different in these brain regions of P rats compared with NP rats. It was also observed that 5HT-stimulated [³H]inositol 1-phosphate formation was significantly higher in the choroid plexus of P rats compared with NP rats. The results of this study indicate that the numbers of 5HT_2C receptors are higher in the hippocampus, the amygdala, and the choroid plexus, and that 5HT_2C receptor-mediated phosphoinositide hydrolysis is more elevated in the choroid plexus of P rats compared with NP rats. Thus, it seems from these results that increased 5HT_2C receptors may be involved in the genetic vulnerability to alcohol drinking behavior.     

Effects of Prenatal Ethanol Exposure on Hippocampal Ionotropic-Quisqualate and Kainate Receptors

Previous studies from our laboratories have shown that the consumption of moderate quantities of ethanol by rat dams during pregnancy reduces N-methyl-D-aspartate (NMDA) agonist receptor binding and NMDA-mediated electrophysiological responses in the hippocampal formation of adult offspring. We hypothesized that prenatal ethanol exposure would produce similar effects on receptor number and agonist-mediated responses of two so-called "non-NMDA" subtypes of glutamate receptors, the ionotropic-quisqualate (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)-sensitive and the kainate-sensitive receptors. Sprague-Dawley rats were fed either a liquid diet containing 3.35% ethanol, an isocalorically matched liquid diet, or lab chow ad libitum throughout gestation. No significant differences between offspring from these three groups in the agonist concentration-response curves for either AMPA-induced or kainate-induced depolarization of hippocampal CA1 pyramidal neurons were observed. Furthermore, no significant differences in the density of [3H]-AMPA or [3H]-vinylidene kainic acid binding sites in any of the apical dendritic field regions of dorsal or ventral hippocampal formation were observed between the groups. These results indicate that the ionotropic quisqualate and kainate receptors, located in the apical dendritic field regions of the principal hippocampal neurons, are not affected by the same degree of prenatal ethanol exposure, which is known to reduce NMDA receptor binding and function in these same regions.     

A Vitamin B12 Binder with Transcobalamin I Characteristics Synthesized and Released by Human Granulocytes in Vitro

S ummary . In-vitro synthesis and release of a vitamin-B₁₂ binding protein by peripheral leucocytes derived from healthy subjects and patients with chronic myeloid leukaemia (CML), acute promyelocytic leukaemia (APL) and acute myeloblastic leukaemia (AML) were investigated.
Myeloid cells, incubated in a nutrient medium containing [³H]leucine, incorporated labelled amino-acid into the vitamin-B₁₂ binding protein with an increase of the unsaturated vitamin-B₁₂ binding capacity (UBBC) of the cells.
The increase in the UBBC of the medium was due to the presence of the newly produced leucocyte binder which was released into the medium by the cells during incubation. On chromatography the newly synthesized binder was similar to transcobalamin I.
The incorporation of [³H]leucine into the leucocyte binder by mature granulo-cytes and undifferentiated myeloblasts was very limited and reached a maximum after 60 min of incubation. Cells from CML and APL patients showed a higher rate of incorporation of the labelled amino-acid which reached a plateau after 40 hr. The increase in the UBBC of the leucocyte binder was paralleled by the incorporation of [³H]leucine.
The percentage of immature myeloid cells in the incubation mixture (promyelocytes to stab forms) was correlated with both the incorporation of [³H]leucine and with the increase in UBBC.