Control of normal differentiation of myeloid leukemic cells. XII. Isolation of normal myeloid colony-forming cells from bone marrow and the sequence of differentiation to mature granulocytes in normal and D+ myeloid leukemic cells

An enriched population of early myeloid cells has been obtained from normal mouse bone marrow by injection of mice with sodium caseinate and the removal of cells with C3 (EAC) rosettes by Ficoll-Hypaque density centrifugation. This enriched population had no EAC or Fc (EA) rosettes and contained 87% early myeloid cells stained for myeloperoxidase and/or AS-D-chloroacetate esterase, 7% cells in later stages (ring forms) of myeloid differentiation and 6% unstained cells, 2% of which were small lymphocytes. After seeding in agar with the macrophage and granulocyte inducer MGI, the enriched population showed a cloning efficiency of 14% when removed from the animal and of 24% after one day in mass culture. Both the enriched and the unfractionated bone marrow cells gave the same proportion of macrophage and granulocyte colonies. The normal early myeloid cells were induced to differentiate by MGI in mass culture in liquid medium to mature granulocytes and macrophages. The sequence of granulocyte differentiation was the formation of EA and EAC rosettes followed by the synthesis and secretion of lysozyme and morphological differentiation to mature cells. D⁺ myeloid leukemic cells with no EA or EAC rosettes had a similar morphology to normal early myeloid cells and showed the same sequence of differentiation. The induction of EA and EAC rosettes occurred at the same time in both the normal and D⁺ leukemic cells, but lysozyme synthesis and the formation of mature granulocytes was induced later in the leukemic than in the normal cells. The results indicate that selection for non-rosette-forming normal early myeloid cells also selected for myeloid colony forming cells, that these normal early myeloid cells can form colonies with differentiation to macrophages and granulocytes, that normal and D⁺ myeloid leukemic cells have a similar sequence of differentiation and that the normal cells had a greater sensitivity for the formation of mature cells by MGI.     

Regulation of actin and other proteins in the differentiation of myeloid leukemic cells.

The regulation of cytoplasmic proteins in mutants of mouse myeloid leukemic cells, differing in their competence to be induced to differentiate by the normal macrophage- and granulocyte-inducing protein (MGI) and the steroid inducer dexamethasone, was analyzed using SDS-polyacrylamide gel electrophoresis of ³⁵S-methionine-labeled proteins. Before induction, no consistent differences in the pattern of cytoplasmic proteins were found between clones with different capabilities to differentiate.Four MGI⁺D⁺ clones, which are induced by MGI for Fc and C3 rosettes, the synthesis and secretion of lysozyme, and the formation of mature macrophages and granulocytes, all showed the same nine prominent changes in cytoplasmic proteins after induction. Five of these changes were either an increase or a decrease in proteins present in uninduced cells; four proteins appeared to be newly synthesized. One of the proteins that increased after induction was identified as actin. The pattern of cytoplasmic proteins from MGI-induced MGI⁺D⁺ clones more closely resembled that of normal peritoneal macrophages and granulocytes than the pattern of the uninduced clones. The relationship of these protein changes to cell differentiation was further substantiated by the finding that MGI⁺D^? cells, which can be induced by MGI for Fc and C3 rosettes and lysozyme, but not for mature cells, showed only four cytoplasmic protein changes which were quantitatively less than those found for MGI⁺D⁺ clones. An MGI^?D^? clone which was not inducible for any differentiation-associated properties by MGI showed no alteration in protein synthesis. Thus in all the clones studied, there was a correlation between the number and extent of protein changes and the degree of MGI-induced differentiation.In MGI⁺D⁺ clones, some of the differentiation-associated properties induced by MGI can be induced by the steroid hormone dexamethasone. Of the nine protein changes induced by MGI, six were also induced by dexamethasone, and no changes were induced by dexamethasone which were not also induced by MGI. These results, which were also shown by two-dimensional polyacrylamide gel electrophoresis, indicate that in cells which can respond to both MGI and dexamethasone, the proteins induced by dexamethasone were a subset of those induced by MGI.     

Control of normal differentiation of myeloid leukemic cells. XI. Induction of a specific requirement for cell viability and growth during the differentiation of myeloid leukemic cells

Normal hematopoietic cells require the presence of a protein (MGI) in the appropriate conditioned medium (CM) for cell viability and growth and for differentiation to mature macrophages and granulocytes. Clones of myeloid leukemic cells have been established in culture (D⁺ clones) which require CM with this protein for differentiation, but not for cell viability and growth. It has been shown that these leukemic cells can be induced by CM to again require, like normal cells, the presence of CM for cell viability and growth. Induction of this requirement, which will be referred to as RVG, occurred before the D⁺ cells differentiated to mature granulocytes. Clones of myeloid leukemic cells (D^? clones) that could not be induced to differentiate to mature cells, did not show the induction of RVG. The steroid hormones prednisolone and dexamethasone can induce some, but not all the changes associated with differentiation of D⁺ cells. Incubation with these steroids did not result in the induction of a requirement for these steroids for cell growth and viability. Studies with CM from different sources have shown, that all batches that induced RVG also induced differentiation of D⁺ cells and that both activities were inhibited after treating the CM with trypsin. It is suggested that the same protein (MGI) may be involved in both activities. Incubation of D⁺ cells with CM resulted in an increase in agglutinability by concanavalin A and this increase was maintained even in the absence of CM. This suggests, that the induction of RVG in D⁺ myeloid leukemic cells is associated with a change in the cell surface membrane.     

Control of normal differentiation of myeloid leukemic cells. IV. Induction of differentiation by serum from endotoxin treated mice

Sera from different strains of mice injected with endotoxin induced clones (D⁺) from a cultured line of myeloid leukemic cells to undergo normal differentiation to mature granulocytes and macrophages. Other clones (D^?) derived from the same cell line were not inducible by these sera to undergo normal cell differentiation. Sera from the same strains of mice that had not been injected with endotoxin, increased the cloning efficiency of D⁺ and D ^? clones but did not induce differentiation. Endotoxin serum induced differentiation in D⁺ cells at dilutions up to 1:64, but increased the cloning efficiency of these cells at dilutions up to 1:2048. The end point of the dilution of endotoxin serum that induced differentiation in D⁺ cells, was also the end point that induced the formation of colonies with differentiation from normal bone marrow cells. The results indicate that serum from endotoxin treated animals can serve as a good in vivo source to induce normal differentiation in D⁺ myeloid leukemic cells; that the progeny of a single leukemic cell was induced to undergo differentiation to both macrophages and granulocytes; that endotoxin serum contained two activities, one that increased cloning efficiency and the other that induced cell differentiation; and that the same material in endotoxin serum induced cell differentiation in normal and leukemic cells.     

Suppression of the in vivo malignancy and in vitro cell multiplication of myeloid leukemic cells by hybridization with normal macrophages.

Mouse myeloid leukemic cells that multiplied in vitro and were malignant in vivo were hybridized with non-malignant non-multiplying normal mouse or human macrophages. Both the hybrids with mouse and with human macrophages were non-malignant and non-multiplying. The suppression of malignancy and cell multiplication in these hybrids was associated with the expression of eight other properties expressed in normal macrophages but not in the myeloid leukemic cells. These properties were C3 and Fc rosettes and immune phagocytosis, rosettes and phagocytosis of uncoated erythrocytes, synthesis and secretion of lysozyme and a high frequency of cap formation by concanavalin A (ConA). The hybrids also expressed two properties of the myeloid leukemic cells, a lack of cell attachment to the surface of a Petri dish and staining for myeloperoxidase. The use of specific antibodies has shown that the lysozyme produced by hybrids between human macrophages and mouse myeloid leukemic cells was human lysozyme. The results indicate that the in vivo malignancy and in vitro cell multiplication of myeloid leukemic cells was suppressed in hybrids with normal macrophages, and that his suppression can be dissociated from the normal macrophage property of cell attachment.     

Control of normal differentiation of myeloid leukemic cells. X. Glucose utilization,cellular ATP and associated membrane changes in D+ and D− cells

Glucose utilization, energy metabolism and associated membrane changes, have been studied in D⁺ myeloid leukemic cells that can be induced to undergo cell differentiation to mature granulocytes by incubation with the appropriate conditioned medium (CM) and in D^? myeloid leukemic cells that cannot be induced to differentiate to mature cells. Before incubation with CM, glycolysis and the glycolytic production of ATP were lower and the activity of the pentose cycle was higher in D⁺ than in D^? cells. ATP depletion induced a higher degree of agglutination by concanavalin A in D^? than in D⁺ cells, indicating a difference in their surface membrane. There were no detectable differences in the transport of glucose and the synthesis of sterols and fatty acids. After incubation with CM, the D⁺ cells, like normal granulocytes, showed a higher glycolysis, produced their ATP more through glycolysis than oxidative phosphorylation, became less dependent on the exogenous supply of glucose and oxygen and had a lower rate of sterol and fatty acid synthesis. The differentiating D⁺ cells also showed a change in their surface membrane resulting in an increased agglutinability without a change in ATP content and a stimulation of the pentose cycle by concanavalin A. These properties, which were not acquired by D^? cells, were found before most of the D⁺ cells had differentiated to mature granulocytes. The data indicate, that the block in the ability of the D^? cells to differentiate and the acquisition of the metabolic properties of normal granulocytes by differentiating D⁺ cells, were associated with differences in the organization of the cell surface membrane.     

Role of Vitamin D3 Receptor in the Synergistic Differentiation of WEHI-3B Leukemia Cells by Vitamin D3 and Retinoic Acid

WEHI-3B D⁻ cells differentiate in response to 1,25-dihydroxyvitamin D₃ (1,25-(OH)₂D₃) but not to all-trans-retinoic acid (RA) or other inducing agents. Combinations of RA with 1,25-(OH)₂D₃ interact to produce synergistic differentiation of WEHI-3B D⁻ cells. To determine factors involved in the synergistic interaction, expression of the 1,25-(OH)₂D₃ receptor (VDR) and retinoid receptors, RARα and RXRα, was measured. No VDR was detected in untreated WEHI-3B D⁻ cells; however, RA and 1,25-(OH)₂D₃ when used as single agents caused a slight induction of the VDR and in combination produced a marked increase in the VDR. In contrast, no changes in RARα and RXRα were initiated by these compounds. An RAR-selective agonist combined with 1,25-(OH)₂D₃ produced synergistic differentiation of WEHI-3B D⁻ cells, whereas an RXR-selective agonist did not. To gain information on the role of the VDR in the synergistic interaction, the VDR gene was transferred into WEHI-3B D⁺ cells, in which no VDR was detected and no synergism was produced. Expression of the VDR conferred differentiation responsiveness to 1,25-(OH)₂D₃ in WEHI-3B D⁺ cells. These findings suggest that (a) induction of VDR expression is a key component in the synergistic differentiation induced by 1,25-(OH)₂D₃ and RA and (b) RAR and not RXR must be activated for enhanced induction of the VDR and for the synergistic differentiation produced by RA and 1,25-(OH)₂D₃.     

The effect of amphotericin B on lectin-induced aggregation of cell surface receptors

The mobility of concanavalin A (ConA) and ricin receptors from NS20 neuroblastoma and C₆ glioma cells was studied using an electrophoretic technique. Cells attached to a solid support were exposed to an electrical field (12V cm⁻¹) at room temperature. The distribution of lectin receptors on the cell surface was revealed by fluorescent conjugates of lectins and microscopic observation of the fixed cells. This technique allowed the estimation of the mobilities of lectin receptors either in free or liganded form, depending on the time at which the cells are labeled with lectins (either after or before electrophoresis). In line with previous observations [1] it is shown that in their free form ConA and ricin receptors are mobile all over the cell surface. Ligand binding induced an apparent receptor immobilization. Immobilization of ricin receptors from C₆ glioma cells could be induced either by the multivalent or the monovalent form of the lectin indicating that cross-linking of receptors by the ligand did not play a predominant role in the process of receptor immobilization. Amphotericin B but not ionophores like valinomycin or gramicidin blocked ligand-induced receptor immobilization. It is concluded from this observation that the effect of amphotericin B is not related to its ionophoretic properties but more likely to its capacity to interact with membrane cholesterol. When cells were incubated at 37 °C extensive patching of lectin receptors could be observed. This process was also inhibited by amphotericin B. A model is proposed to account for a role of cholesterol in ligand-induced receptor immobilization and patching.     

Lysis of halophilic Vibrio alginolyticus and Vibrio costicolus induced by chaotropic anions

High concentration (1.0 M) of KSCN, but not of NaSCN, induced lysis of slightly halophilic Vibrio alginolyticus and moderately halophilic Vibrio costicolus, and the decrease in absorbance of the cell suspension was complete after 30 min at 25°C. Replacement of K⁺ with Na⁺ effectively prevented the lysis by SCN⁻. K⁺ salts of NO₃⁻, Br⁻, however, induced no significant lysis. In electron micrographs, a prolonged exposure of the cells of V. alginolyticus to 1.0 M KSCN displaced the nucleoplasm to maintain close contact with the cell membranes. After 40 min of interaction, 50% of the cellular protein, 96% of RNA and 94% of DNA were recovered in the lysed cells. In contrast to lysis in hypotonic conditions, the lysis induced by KSCN is due mainly to a partial release of protein from the cells. V. costicolus was more susceptible to SCN⁻ than V. alginolyticus, whereas nonhalophilic Escherichia coli was resistant to 1.0 M KSCN. Thus, lysis by SCN⁻ is characteristic of halophilic bacteria and cell membranes of more halophilic bacteria are more susceptible to chaotropic anions. The protective effect of Na⁺ observed here was considered to be manifested by specific interactions of Na⁺ with components of cell membranes, thereby rendering their structures resistant to the action of chaotropic anions.     

Molecular dissection of differentiation in normal and leukemic myeloblasts: Separately programmed pathways of gene expression

The regulation of gene expression in leukemic and normal myeloblasts induced to differentiate by the normal macrophage and granulocyte inducing protein MGI was studied by analysis of protein changes using two-dimensional polyacrylamide gel electrophoresis. During the 6-day period of differentiation from myeloblasts to mature cells, there was a programmed sequential change in the rate of synthesis of 217 of the 450 proteins detected in a MGI⁺D⁺ leukemic clone. The developmental program was initiated with a decrease in the synthesis of many proteins within the first hour, whereas the synthesis of new proteins occurred later, mostly between the second and fourth days. The mature cells showed a specialization associated with a changeover and increased synthesis of the major protein species. Both the MGI⁺D⁺ leukemic and the normal myeloblasts showed a similar sequence of protein changes during differentiation. The normal developmental program was thus maintained in the MGI⁺D⁺ leukemic cells. Cell mutants which differ in their competence to be induced to differentiate by MGI were used to dissect the developmental program of differentiation. Sixty-six protein changes were induced by MGI in partially differentiatable MGI⁺D^? clones, whereas only 12 or 16 protein changes were induced in different MGI^?D^? clones which had not been induced for any previously known differentiation-associated property. In these mutant clone types, the induced protein changes were subsets of those induced by MGI in the MGI⁺D⁺ leukemic and normal myeloblasts. These subsets spanned the whole 6-day period of differentiation and had the same developmental sequence as in the fully differentiatable MGI⁺D⁺ cells. These data indicate that the protein changes during differentiation are not organized as one sequence, but rather as multiple, parallel sequences which can be separately induced. MGI induced some, but not all, of these sequences in the mutant clones. It is concluded that differentiation consists of multiple, parallel, separately programmed pathways of gene expression. Analysis of the initial differences between the proteins synthesized in untreated normal and leukemic myeloblasts has shown that all the leukemic clones, when compared to normal myeloblasts, constitutively expressed the MGI-induced state for a common subset of 14 proteins. In addition, the MGI⁺D^? and MGI^?D^? clones, compared to MGI⁺D⁺, constitutively expressed the differentiated state for other subsets of proteins. The size of these constitutively expressed subsets was larger in MGI^?D^? than in MGI⁺D^? clones. It is, therefore, suggested that the constitutive expression of some pathways of gene expression results in leukemia, whereas the constitutive expression of other pathways results in a decreased competence for the induction of differentiation.     

Capping of concanavalin a receptors and their association with microfilaments in monolayer grown human fibroblastoid cells

Summary The distribution of ConA binding sites on the surface of normal human fibroblastoid cells grown in monolayer culture was carefully examined. Low concentrations of ConA (between 0.5–5.0 g/ml) were found to induce the ConA receptors to form a single, large cap structure. High concentrations of ConA (between 50–100 g/ml) inhibit cap formation at temperatures above 20° C. Pretreatment of the cells in the cold or with colchicine allows cap formation to occur with high concentrations of ConA. The ConA caps appear to be preferentially localized near the nucleus. Using a double immunofluorescence technique, we have observed actin and myosin molecules concentrated underneath the surface receptor cap in the perinuclear region of the cells. These findings suggest that the binding of ConA to fibroblastoid cells may trigger the transmembrane association of cytoplasmic microfilaments with surface membrane receptors as previously proposed for lymphocytes and other round cells grown in suspension culture.Abbreviations IgG immunoglobulin - Fl fluorescein - Rh rhodamine - PBS 0.1 M phosphate buffer (pH:7.4) plus 0.15 M NaCl - ConA Concanavalin A This work was supported by U.S. Public Health Service Grants AI 15258-01 and CA 22453     

Clusterin over-expression modulates proapoptotic and antiproliferative effects of 1,25(OH)2D3 in prostate cancer cells in vitro

Prostate cancer is the most commonly diagnosed cancer in the majority of western countries. Due to their antiproliferative and proapoptotic activity, vitamin D analogues have been introduced recently as an experimental therapy for prostate cancer. Clusterin (CLU) is a glycoprotein that has two known isoforms generated in human cells. A nuclear form of CLU protein (nCLU) is pro-apoptotic, and a secretory form (sCLU) is pro-survival. In this study, we analyzed whether proapoptotic and antiproliferative effects of 1,25(OH)₂D₃ on LNCaP prostate cancer cells are modulated by expression of sCLU. Using colony forming assay, we studied the effect of treatment with different doses of 1,25(OH)₂D₃ (10⁻⁶, 10⁻⁷, 10⁻¹⁰ M) on proliferation of LNCaP cells that were stable transfected and over-express sCLU (LNT-1) as compared to empty vector-transfected cells (LN/C). We also measured apoptosis using TUNEL assay. sCLU over-expression protected against both antiproliferative (30%) and proapoptotic (15%) effects of 1,25(OH)₂D₃, although this effect was statistically not significant. In conclusion, our findings demonstrate that expression of sCLU modulates growth regulatory effects of 1,25(OH)₂D₃ in prostate cancer indicating that CLU interferes with vitamin D signalling pathways.     

Inhibition of sodium-plus-potassium-stimulated adenosine triphosphatase (Na-K-ATPase) by protein kinase C activators in the gills of Atlantic cod (Gadus morhua)

Exogenous (phorbol ester) and endogenous (diacylglycerol) activators of protein kinase C (PKC) inhibited sodium efflux across the gills of Atlantic cod Gadus morhua and inhibited sodium-plus-potassium-stimulated adenosine triphosphatase (Na⁺-K⁺-ATPase) in isolated chloride cells. The branchial sodium efflux measured in a perfused whole-body preparation was inhibited by 47% on administration of 10⁻⁶ mol.L⁻¹ phorbol 12, 13-dibutyrate (PDB). The branchial perfusion pressure was increased by 46% by 10⁻⁶ mol.L⁻¹ PDB. In contrast the synthetic diacylglycerol, 1-oleoyl-2-acetyl gycerol (OAG) did not alter significantly perfusion pressure but did reduce sodium efflux by 13% at a concentration of 4 × 10⁻⁶ mol.L⁻¹. The effects of these agents on Na⁺-K⁺-ATPase activity were determined in isolated chloride cells with a control activity of 30.9 ± 1.9 μmol Pi mg protein⁻¹ hour⁻¹. PDB and OAG both inhibited enzyme activity in a dose-dependent manner, with 10⁻⁵ mol.L⁻¹ causing 45% and 26% inhibition, respectively. These results suggest that PKC is involved in regulating sodium efflux in the gills of cod by modulating Na⁺-K⁺ATPase activity.     

Conformational change of core particles studied by quasielastic light scattering

The diffusion translational coefficient D_T of core particles in monodisperse solutions has been measured by the quasielastic light scattering method in a large scale of salinities over the range 6.10⁻⁴ to 2M Na⁺ or K⁺. The observed values of D_T are independent of particle concentration in the range 0.1–2 mg/ml and do not vary with the scattering vector q corresponding to scattering angles between 40°–120°. When the salinity is progressively raised an increase of D_T from 1.9.10⁻⁷ cm²s⁻¹ to 3.2.10⁻⁷ cm²s⁻¹ was observed at about 2.10⁻³ M NaCl followed by a decrease of D_T beyond 0.6 M NaCl.The various possible causes of the changes of D_T such as interactions between particles or between particles and salt ions are discussed. We show that the single low ionic strength change is due to a conformational transition of the core particles, while the second variation of D_T accompanies the disorganization of the core particles.     

Differences in membrane unsaturated fatty acids and electron spin resonance in different types of myeloid leukemia cells

The fatty acid composition and some physical properties of intact cells and isolated plasma membranes of two types of mouse myeloid leukemia cell clone grown in culture have been examined. One clone type, MGI⁺D⁺, can be induced by the macrophage and granulocyte-inducing protein (MGI) to differentiate into mature macrophages and granulocytes. The other clone type, MGI⁺D^?, could not be induced to differentiate into mature cells. A two-fold increase in the ratio of saturated fatty acid to unsaturated fatty acid was found in the MGI⁺D^? compared to the MGI⁺D⁺ clones. The MGI⁺D^? clones produced an unusual polyunsaturated C_20:5 fatty acid at 28°C, whereas the MGI⁺D⁺ clones did not grow at this temperature. The cells and their isolated plasma membranes were studied by electron spin resonance. The motion of the 5-nitroxide stearate spin label was found to be higher in the intact cells and in the membranes of MGI⁺D^? clones than of the MGI⁺D⁺ clones. The cells of MGI⁺D⁺ clones showed a similar freedom of motion to normal myeloblasts from the bone marrow. The results indicate that myeloid leukemia cells which differ in their competence to be induced to differentiate into mature cells have different physical properties of their plasma membranes and that this is correlated with their fatty acid acyl chain composition.     

Subpopulations of human peripheral blood lymphocytes.

Multiple staining protocols have been developed for the classification of subpopulations of human peripheral blood lymphocytes. Of the non-T (E^?) cells, roughly half (10–20% PBL) have receptors for complement components as detected with complement-coated zymosan particles, but do not show Fc receptors as detected with Ripley IgG-coated human RBC. The other half are C^?, Fc⁺, with a small percentage possessing both receptors. The C⁺, Fc^? cells can be subdivided into cells which are IgM⁺ (75%) or IgM^?. Cells with Fc receptors detected with aggregated IgG were IgM⁺.     

The role of hERG1 K channels and a functional link between hERG1 K channels and SDF-1 in acute leukemic cell migration

Stromal cell-derived factor-1 (SDF-1) and its unique receptor, CXCR4, regulate stem/progenitor cell migration and retention in the bone marrow and are required for hematopoiesis. Recent studies found that hERG1 K⁺ channels were important regulators of tumor cell migration. In this study, we investigated whether SDF-1 induced acute leukemic cell migration associated with hERG1 K⁺ channels. Our results showed that E-4031, a specific hERG1 K⁺ channels inhibitor, significantly blocked SDF-1-induced migration of leukemic cell lines, primary acute leukemic cells, leukemic stem cells and HEK293T cells transfected with herg-pEGFP. The migration of phenotypically recognizable subsets gave the indication that lymphoblastic leukemic cells were inhibited more than myeloid cells while in the presence of E-4031 which maybe associated with herg expression. SDF-1 increased hERG1 K⁺ current expressed in oocytes and HEK293T cells transfected with herg-pEGFP. There were no significant changes of CXCR4 expression on both HL-60 cells and primary leukemic cells regardless if untreated or treated with E-4031 for 24 h (P > 0.05). The hERG1 K⁺ current increased by SDF-1 might contribute to the mechanism of SDF-1-induced leukemic cell migration. The data suggested that hERG1 K⁺ channels functionally linked to cell migration induced by SDF-1.     

The acetylcholine receptor: Isolation of a brain nicotinic receptor and its preliminary characterization in lipid bilayer membranes

The technique of affinity chromatography with the curarizing neurotoxins of Naja naja venom has been employed to extract nicotinic acetylcholine receptors from the brain tissues of mouse and hog. Both carbochol and hexamethonium were used as linear or step gradients to elute the receptor and its properties were investigated in lipid bilayer membranes. Of particular interest is the observation that discrete quanta of conductance could be observed across an NaCl gradient of 1.0:0.1 M. By switching the voltage-clamp across the bilayer between a positive and negative 80 mV, the separate Na⁺ and Cl⁻ conductances of these quanta could be estimated and the following conductances of the smallest discrete quanta were observed: 3.7 · 10⁻¹¹ Ω⁻¹ (Na⁺) and 5.9 · 10⁻¹¹ Ω⁻¹ (Cl⁻) for mouse brain receptors; 3.8 · 10⁻¹¹ Ω⁻¹ (Na⁺) and 4.7 · 10⁻¹¹ Ω⁻¹ (Cl⁻) for hog brain receptors. Large aggregates of receptors appeared to activate and deactivate as multiples of a basic conductance size, although there is evidence that they may not represent the actual gating of ion channels. A “background noise” that is not within the temporal capability of the recording system is also present at an intensity that seems to parallel the number of activated receptors, and in view of recent electrophysiological evidence that the relaxation lifetime of the open channel state is of a millisecond duration, it may be that this “noise” actually represent the channel gating.