Intact Brain Cells Research Articles

Agonist-induced desensitization of muscarinic acetylcholine receptor in rat brain

Intact brain cell aggregates were dissociated from adult rat brains without cerebellum using a sieving technique. This preparation was used to elucidate the binding characteristics of agonist to muscarinic acetylcholine receptors (mAchR) in brain. Incubation of cells with carbamylcholine (carbachol) was shown agonist-induced receptor down-regulation depending on the concentration of agonist, not depending on the incubation time. This effect of carbachol was due to a reduction in the maximal binding capacity (B max ) to the mAchR without decreasing the affinity of the remaining receptors in incubation at 37 °C but was not apparent in incubation at 15 °C. In addition, it was abolished when the receptors were blocked by atropine. The decline in (3H)N-methylscopolamine((3H)NMS) binding induced by agonist was reflected as a significant reduction in the receptor density with no change in receptor affinity, suggesting that ‘tru’ receptor down-regulation takes place. Moreover, when the receptors were labeled with the lipophilic antagonist (3)NMS, the magnitude of the observed receptor down-regulation was significantly lower in case of the former than the latter. This suggests that exposure of intact brain cells to muscarinic agonists might induce a slight degree of accumulation of receptors in intracellular sites before the receptors are actually degraded.

Adenosine uptake and [ 3H]nitrobenzylthioinosine binding in developing rat brain

The ontogenesis of adenosine tranport sites as labelled with [ 3H]nitrobenzylthioinosine ([ 3H]NBI) was examined using radioligand binding and membrane preparations from whole brain and 4 brain regions of rats between the postnatal ages of one day through to adulthood. In whole brain, cerebral cortex and cerebellum, [ 3H]NBI binding was two-fold higher in 6-day-old than in 50-day-old rats. In contrast, [ 3H]NBI binding was higher in adults than in one-day-old rats by 4-fold in hypothalamus and 8-fold in superior colliculus. In cortex and hypothalamus, the levels of [ 3H]NBI binding in newborn and adult rats were reflected by changes in B max and not K d values. As a measure of the utility of [ 3H]NBI as a probe for identifying functional adenosine transport sites, we examined [ 3H]NBI binding to and [ 3Hadenosine accumulation by intact brain cells prepared from adult and newborn rats. For [ 3H]NBI binding to brain cells from adult rats, the values of K nd were 0.092 nM and of K max were 274 fmol/mg protein. For newborns, slightly higher K d and B max values were observed: 0.2 nM and 395 fmol/mg protein, respectively. [ 3H]Adenosine accumulation was higher in brain cells from one-day-old from adult rat brains. Kinetically this uptake was best described by a two-component model: the V max values for the high low-affinity uptake, and the K m value for the high-affinity component in one-day-old rats were greater than in adults. These results are consistnt with the increased demand for purines in rapidly dividing cells and the concept that [ 3H]NBI, at least in some tissues and for some purposes,is a useful marker substance for adenosine uptake sites.

45Ca2+ uptake in rat brain neurons: absence of sensitivity to the Ca2+ channel ligands nitrendipine and Bay K 8644

K+-stimulated 45Ca2+ uptake into intact rat brain cells was biphasic, consisting of a fast first phase and a slow second phase; the latter was Na+ dependent. Cobalt and cadmium at 10(-4) and 10(-3) M produced 19-97% block of first phase 45Ca2+ uptake, but nitrendipine (to 10(-6) M) and Bay K 8644 (to 10(-6) M) were without effect on uptake and were similarly without effect in cells prepared in the presence of ATP, cAMP, Mg2+, and protease inhibitors. The second phase of K+-stimulated 45Ca2+ uptake was inhibited by 3,4-dichlorobenzamil (IC50, 29.6 microM). Depolarization-induced 45Ca2+ uptake into intact rat brain cells occurs by at least two different mechanisms. The first phase probably represents uptake through 1,4-dihydropyridine-insensitive Ca2+ channels, while the second phase is probably due to Na+-Ca2+ exchange.

Agonist-induced muscarinic acetylcholine receptor down-regulation in intact rat brain cells

Intact brain cells were prepared by dissociating whole adult rat brains without the cerebellum using a sieving technique. It has been found that preincubation of these cells with the muscarinic acetylcholine receptor agonist, carbamylcholine, results in a significant reduction in the specific binding of [ 3H]N-methylscopolamine to the receptors after the agonist was washed away. This agonist-mediated receptor down-regulation increased with prolongation of the exposure period to the agonist, and a steady state was achieved after 3 h at 37°C. This effect of agonist was concentration-dependent, reaching a 30–35% decline in subsequent ligand binding upon preincubation with 1 mM carbamylcholine for 3 h. Carbamylcholine-induced receptor down-regulation was not apparent when exposure to the agonist was performed at 15°C. In addition, it was abolished when the receptors were blocked by atropine. The decline in [ 3H]N-methylscopolamine binding induced by agonist was reflected as a significant reduction in the receptor density with no change in receptor affinity, suggesting that ‘true’ receptor down-regulation takes place. Moreover, when the receptors were labeled with the lipophilic antagonist [ 3H]quinuclidinyl benzilate instead of the hydrophilic ligand [ 3H]N-methylscopolamine, the magnitude of the observed receptor down-regulation was significantly lower in case of the former than the latter. This suggests that exposure of intact brain cells to muscarinic agonists might induce a slight degree of accumulation of receptors in intracellular sites before the receptors are actually degraded. These results are discussed in relation to previous findings regarding muscarinic receptor regulation in clonal cell lines.

Morphine-induced opioid receptor down-regulation detected in intact adult rat brain cells.

Intact brain cells dissociated from the brains of adult rats were used to study the regulation of opioid receptors by in vivo chronic morphine treatment. When the specific binding of [3H]naloxone in a physiological iso-osmotic buffer was compared in cells obtained from sham-operated rats and in those treated for 6 days with four (2 X 2) 75 mg morphine pellets, it was found that morphine treatment resulted in a significant reduction in the density of [3H]naloxone binding sites. This receptor down-regulation was not accompanied by a change in the receptor affinity for the ligand. This effect of morphine was reversed upon the removal of morphine pellets for 18 h after tolerance induction. When similar experiments were performed using brain homogenates prepared and assayed in the same physiological buffer, there was an increase in the number of [3H]naloxone binding sites in morphine-treated animals compared to controls. On the other hand, when the binding experiments were conducted in 50 mM Tris-HCl buffer, no difference in ligand binding was apparent between control and morphine-treated groups. The present results demonstrate opioid receptor down-regulation by chronic morphine treatment measured in intact brain cells, and suggest that different conclusions may be reached when other tissue preparations are used to assess the receptor density.

Comparison of Ca2+ -dependent phosphorylation in viable dispersed brain cells with calmodulin-dependent protein kinase activity in cell-free preparations of rat brain.

Using two depolarizing agents, veratrine and high concentrations of extracellular KCl, we studied depolarization-stimulated phosphorylations in 32P-labelled dispersed brain tissue in order to identify phosphoprotein substrates for Ca2+ - and calmodulin-dependent protein kinase activity at the cellular level, for comparison with findings in cell-free preparations. In intact brain cells, the only prominent depolarization-stimulated phosphorylation was a 77 kDa protein separated on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. This phosphorylation was dependent on external Ca2+, since chelation of Ca2+ in media with 6 mM-EGTA or the presence of verapamil (a Ca2+ -channel blocker) in the incubation media inhibited depolarization-stimulated phosphorylation of the 77 kDa protein. Phosphorylation of the 77 kDa protein also appeared to be dependent on calmodulin, because depolarization-stimulated phosphorylation was significantly decreased (P less than 0.05) when 100 microM-trifluoperazine was present in the incubation media. Polymyxin B, an inhibitor of Ca2+- and phospholipid-dependent phosphorylation, and 12-O-tetradecanoylphorbol 13-acetate, the phorbol ester enhancing Ca2+- and phospholipid-dependent phosphorylation, had no effect on the phosphorylation of the 77 kDa protein. The 77 kDa phosphoprotein was identified as a protein previously named synapsin I [Ueda, Maeno & Greengard (1973) J. Biol. Chem 248, 8295-8305] on the basis of similar migration of native and proteolytic fragments of the 77 kDa protein with those of authentic synapsin I on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Whereas several studies with cell-free preparations showed that 57 kDa and 54 kDa endogenous phosphoproteins were the most prominent species phosphorylated in a Ca2+ and calmodulin-dependent manner, these results indicate that synapsin is the most prominent Ca2+-and calmodulin-dependent phosphorylation in intact cells. The phosphorylations of 54 kDa and 57 kDa proteins may not be as important in vivo, but instead occur as a result of the disruption of cellular integrity inherent in preparation of cell-free subfractions of brain tissue.

Formula omitted] binding to muscarinic receptors in intact adult rat brain cell aggregates

Intact brain cell aggregates were dissociated from adult rat brains, by a simple sieving technique, and were used to study the binding characteristics of [ 3 H]N- methylscopolamine to muscarinic acetylcholine receptors. The magnitude of binding of this ligand was related linearly to the amount of cell protein in the binding assay, with a high ratio of total to nonspecific binding. In addition, specific binding showed saturability and high affinity. Muscarinic receptor antagonists displaced specific [ 3 H]N- methylscopolamine binding according to the law of mass-action, while it was possible to resolve displacement curves using receptor agonists into high- and low-affinity components. The results are discussed in terms of the usefulness of dissociated intact rat brain cells in studying muscarinic acetylcholine receptors in the central nervous system.

Expression of monoclonal antibody-defined cell surface antigens during rat brain development

Using single-cell suspensions of mechanically dissociated, prenatal BDIX-rat brain cells (13th, 15th, and 21st days after fertilization) for immunization, we have established a collection of 37 monoclonal antibodies (Mabs) directed against neural cell surface determinants. The developmental-stage-dependent expression of cell-surface antigens recognized by these Mabs was analyzed both on plasma membranes isolated from whole brains of BDIX rats (prenatal days 13–22 and adults) using an indirect 125I so-lid-phase radioimmunoassay, and on intact BDIX-rat brain cells (prenatal days 13-22) using a fluorescence-activated cell sorter. Different types of developmental stagedependent profiles of Mab binding were found, these being indicative of the presence of neural cell surface determinants whose expression increases, decreases, or does not change with brain development. Some of the Mab-binding profiles showed transient changes as a function of developmental stage. These Mabs are currently being used for the characterization, reproducible identification, and isolation of neural cell subpopulations of the developing rat brain, with the aim of investigating the cell type dependence and developmental (differentiation) stage dependence of malignant transformation following pulse exposure to the carcinogen N-ethyl-N-nitrosourea at defined stages of brain development.

Use of intact rat brain cells as a model to study regulation of muscarinic acetylcholine receptors

Intact rat brain cells were dissociated and used to study the regulation of muscarinic acetylcholine receptors upon exposure to muscarinic receptor agonists. Incubation of cells with carbamylcholine resulted in a time-dependent decrease in subsequent [ 3H]N-methylscopolamine specific binding, an effect which reached a steady state after 3 hr at 37°C. This effect of carbamylcholine was dependent on the concentration of the agonist in the incubation medium and was due to a reduction in the maximal binding capacity of the receptor with no decrease in the affinity of the remaining receptors. This preparation might be useful in future studies to elucidate the mechanisms underlying the regulation of muscarinic acetylcholine receptors in the central nervous system.

Intact brain cells: A novel model system for studying opioid receptor binding

This paper presents the use of a novel tissue preparation to study opioid receptor binding in viable intact cells derived from whole brains of adult rats. Mechanically dissociated and sieved cells, which were not homogenized at any stage of the experimental protocol, and iso-osmotic physiological buffer were used in these experiments. This system was adapted in order to avoid mechanical and chemical disruption of cell membranes, cytoskeletal ultrastructure or receptor topography by homogenization or by the use of nonphysiological buffers, and to mimic in vivo binding conditions as much as possible. Using [ 3H]naloxone as the radioligand, our studies showed saturable and stereospecific high-affinity binding of this opioid antagonist in intact cells, which in turn showed consistently high viability. [ 3H]Naloxone binding was also linear over a wide range of tissue concentrations. This technique provides a very promising model for future studies of the binding of opioids and of many other classes of drugs to brain tissue receptors in a more physiologically relevant system than those commonly used to date.

Pharmacological specificity of brain histamine H2-receptors differs in intact cells and cell-free preparations.

Despite increasing evidence that histamine may be a neuro-transmitter in brain, the functions of putative histaminergic neurones are just beginning to be known1,2. Both H1- and H2-receptors3 seem to be present in brain4–7 and histaminergic neurones might control arousal mechanisms or carbohydrate metabolism, via H1-receptors8–11. The actions mediated by cerebral H2-receptors are less clear mainly because available agonists and antagonists3,12–15 do not cross the ‘blood–brain barrier’. However, on a histamine-sensitive adenylate cyclase from guinea pig brain homogenates with pharmacological properties similar to those of H2-receptors16,17 compounds like tricyclic antidepressants or promethazine were extremely potent antagonists18,19 suggesting that this might represent the molecular basis of clinical antidepressant activity19. Although electrophysiological20 and behavioural21 studies partially confirmed that one of these compounds, amitryptiline, could block cerebral histamine receptors in the living animal, their potency as H2-antihistamines has never been precisely established in intact brain cell preparations. Histamine strongly stimulates cyclic AMP synthesis in brain slices but, in contrast with homogenates, both H1- and H2-receptors are involved4,5,22. As compounds like promethazine are potent H1-antihistamines7,8, we have developed a test specific for H2-receptors consisting of the stimulation of cyclic AMP accumulation by impromidine, a potent and highly selective H2-receptor agonist14, in slices from guinea pig hippocampus and using it find that H2-receptors differ considerably in their drug discriminatory properties from those mediating the stimulation of adenylate cyclase activity in homogenates.

Fatal encephalitis due to Herpesvirus hominis: use of intact brain cells for isolation of virus.

A specimen of brain from a patient with fatal encephalitis was prepared for inoculation into tissue-culture monolayer systems by dispersal of cells with trypsin diluted in saline and by homogenization with mortar and pestle. In primary isolations and in reisolations from the original materials, Herpesvirus hominis (HVH) was recovered from 24 of 31 cell cultures inoculated with trypsindispersed brain cells and from none of 32 cultures inoculated with brain homogenate. Inoculation of tissue-culture systems with intact, infected cells appears to be a more sensitive method of recovering the agent from patients with HVH encephalitis than inoculation of homogenized tissue; thus this method should be evaluated further.

Cell-free protein synthesis by mouse brain during early development.

Abstract—Cell‐free homogenates were employed to study the nature of the mechanism that is responsible for the rapid decrement in protein synthesis during early neural development. There was a progressive loss of polypeptide synthesis in post‐mitochondrial fractions that were isolated from increasingly older tissue. By the time the animals were approximately 17 days old, the rate of amino acid incorporation had decreased to the rate that was measured in adult brain preparations. This decrement in synthetic activity was similar to that previously measured in developing intact brain cells. The loss in protein synthesis was demonstrated to be independent of cellular membrane permeability and under the influence of intracellular control mechanisms. Although the nature of the control mechanism is still not clear, a lack of template RNA to direct protein synthesis was not the limiting factor in the decreased synthesis of the older brain preparations.