Dopamine Receptors in Subcellular Fractions from Bovine Caudate: Enrichment of [3H]Spiperone Binding in a Postsynaptic Membrane Fraction

Abstract: Specific binding of tritiated dopamine, spiperone, and N-propylnorapomorphine was examined in subcellular fractions from bovine caudate nucleus. All fractions contained at least two sets of specific binding sites for [³H]spiperone (K_{D 1}^aPP= 0.2 nM, K_{D 2}^aPP= 2.2 nM), the higher affinity sites accounting for one-third to one-eighth of the total. [³H]Spiperone binding was slightly enriched over the total particulate fraction in P₂, P₃, SPM, and a crude fraction of synaptic mitochondria. A microsomal subfraction (P₃B₂) exhibited the highest specific binding capacity obtained, representing a fourfold enrichment over the total particulate fraction. [³H]Dopamine exhibited apparent binding to a single class of high-affinity sites in all fractions examined (K_D^aPP= 4.0 nM). A greater than twofold enrichment was observed in all fractions except myelin and P₃, with a fivefold enrichment in SPM and P₃B₂. At least two classes of receptors were labeled by [³H]-N-propylnorapomorphine (K_{D 1}^aPP= 0.55 nM, K_{D 2}^aPP= 20 nM), using 50 nM-spiperone together with 100 nM-dopamine to define nonspecific binding. Although binding to the higher affinity site was displaced by spiperone, and lower affinity binding by dopamine, comparison of receptor densities with values obtained by using [³H]spiperone and [³H]dopamine directly suggested that [³H]-N-propylnorapomorphine labeled additional sites. We have also examined a postsynaptic membrane (PSM) fraction obtained from SPM by successive extraction with salt and EGTA followed by sonication and separation on a density gradient. [³H]Spiperone binding in PSM was enriched two- to threefold over unfractionated SPM with a concomitant decrease in [³H]dopamine binding. The enrichment in spiperone receptors was almost entirely due to an increase in the number of lower affinity binding sites, suggesting that these sites may be associated with the postsynaptic membrane.     

Regulation of Calmodulin- and Dopamine-Stimulated Adenylate Cyclase Activities by Light in Bovine Retina

Abstract: Neural retina from most species contains 3,4-dihydroxyphenylethylamine (dopamine) receptors coupled to stimulation of adenylate cyclase activity. It has been demonstrated that release of dopamine from its neurons and subsequent occupation of dopamine receptors is increased by light. In this study, we have shown that adenylate cyclase activity in bovine retina is highly responsive to the endogenous Ca²⁺-binding protein, cal-modulin, and that calmodulin can increase dopamine-sen-sitive adenylate cyclase activity in bovine retina. We further demonstrate that both dopamine- and calmodulin-stimulated adenylate cyclase activities can be regulated by alterations in light. Bovine retinas were dissected from the eye under a low-intensity red safety light, defined as dark conditions, and incubated for 20 min in an oxygenated Krebs Henseleit buffer under either dark or light conditions. The retinas were then homogenized and adenylate cyclase activity measured in a paniculate fraction washed to deplete it of endogenous Ca²⁺ and calmodulin. Activation of adenylate cyclase activity by calmodulin, dopamine, and the nonhydrolyzable GTP analog, gua-nosine-5′-(β,γ-imido)triphosphate (GppNHp), was significantly (60%) greater in paniculate fractions from retinas that had been incubated under dark conditions as compared to those incubated under light conditions. Basal, Mn²⁺-, and GTP-stimulated adenylate cyclase activities were not altered by changes in lighting conditions. Calmodulin could increase the maximum stimulation of adenylate cyclase by dopamine in retinas incubated under either dark or light conditions, but the degree of its effect was greater in retinas incubated under light conditions. Activation of adenylate cyclase by calmodulin, dopamine, and GppNHp in paniculate fractions from retinas incubated under light conditions was indistinguishable from the activation obtained when retinas were incubated in the dark in the presence of exogenous dopamine. These results suggest that an increased release of dopamine occurs in light. The decreased response of adenylate cyclase to exogenous dopamine can then be explained by a subsequent down-regulation of dopamine receptor activity. The down-regulation of dopamine receptor activity can also regulate activation of adenylate cyclase by GppNHp and calmodulin. The results suggest that dopamine, calmodulin, and GppNHp are modulators of a common component of adenylate cyclase activity, and this component is regulated by light.     

Partial purification of dopamine D2 receptors using lectin affinity columns

Dopamine D₂ receptors , detected by [³H]spiperone Dopamine D₂ receptors , detected by [³H]spiperone binding, were solubilized from bovine caudate nucleus by cholate/sodium chloride and were found to bind to wheat germ agglutinin immobilized on agarose. Specific elution could be achieved with N-acetylglucosamine whereas other sugars tested were inactive in this regard . The eluted preparation was enriched in solubilized receptors about sevenfold. The pharmaco-logical properties of the preparation were essentially unchanged by the lectin affinity purification procedure. The D₂ dopamine receptor is therefore a glycoprotein. binding, were solubilized from bovine caudate nucJeus by cholate/sodium chloride and were found to bind to wheat germ agglutinin immobilized on agarose. Specific elution could be achieved with N-acetylglucosamine whereas other sugars tested were inactive in this regard . The eluted preparation was enriched in solubilized receptors about sevenfold. The pharmacological properties of the preparation were essentially unchanged by the lectin affinity purification procedure. The D₂ dopamine receptor is therefore a glycoprotein.     

Glial uptake of monoamines in primary cultures of rat median raphe nucleus and cerebellum

Summary Glial uptake of serotonin and dopamine was studied in primary cultures of the median raphe nucleus and cerebellum by using consecutive demonstration of monoamine fluorescence and glial fibrillary acidic protein immunofluorescence. Most of the glial cells taking up monoamines were glial fibrillary acidic protein positive. Astrocytes with a strong immunoreactivity exhibited monoamine fluorescence only occasionally, although such cells did take up L-dopa readily. Glial fibrillary acidic protein negative cells — morphologically identified as astrocytes — were seen to exhibit monoamine fluorescence after exposure. Glial uptake of serotonin at a concentration of 10^–4 M was detected in cerebellar cultures but not in cultures from the median raphe nucleus. When the concentration was 10^–3 M uptake of serotonin took place in both the areas but was weaker in cultures from the median raphe nucleus. At concentrations greater than 10^–5 M glial uptake of dopamine was detected in cultures from both the regions studied. No region dependent differences in glial uptake of dopamine was demonstrated, however. Based on these observations astrocytes and astrocyte-like glial cells take up dopamine and serotonin. Also glial cells with a remarkably high content of the glial fibrillary acidic protein are more resistant to monoamine uptake than cells exhibiting less intense or no glial fibrillary acidic protein immunofluorescence. The existence of regional differences in uptake of serotonin between the median raphe nucleus and cerebellum suggests that glial uptake of monoamines is not an entirely passive mechanism but may be actively controlled by glial cells in a region dependent manner.     

Subcellular distribution of prostaglandin and gonadotrop in receptors in bovine corpora lutea.

The subcellular distribution of prostaglandin (PG) E₁, F₂α and gonadotropin receptors in bovine corpora lutea was critically examined by preparing various subcellular fractions, assaying for various marker enzymes to assess the purity and examining ³H-PGE₁, ³H-PGF₂α and ¹²⁵I-human lutropin (hLH) specific binding. The marker enzyme data suggested that subcellular fractions were relatively pure with little or no cross contamination. The binding of ³H-PGs and ¹²⁵I-hLH was markedly enriched in plasma membranes with respect to homogenate. The other subcellular fractions also exhibited binding despite very little or no detectable 5′-nucleotidase activity. If 5′-nucleotidase was assumed to lack sensitivity and reliability to detect minor contamination with plasma membranes and ³H-PGs or ¹²⁵I-hLH binding were used as sensitive plasma membrane markers, it was still difficult to explain binding in other fractions based on plasma membrane contamination. Therefore, these results lead to the inevitable conclusion that plasma membranes were primary (or one of the primary) but not exclusive sites for PGE₁, PGF₂α and gonadotropin receptors.     

Direct binding of 3H-lisuride to adrenergic and serotonergic receptors

The characteristics of the specific binding of ³H-lisuride hydrogen maleate (³H-LHM) to homogenates of rat striatum and bovine frontal cortex tissue were investigated. In rat striatum 50% of ³H-LHM binding was inhibited potently by spiperone and haloperidol indicating a component of ³H-LHM binding to D₂ dopamine receptors. Specific ³H-LHM binding was detected in rat striatum after selective blockade of the D₂ dopamine component indicating specific ³H-LHM binding to other striatal sites. In bovine frontal cortex clonidine and serotonin competition curves for specific ³H-LHM binding included high affinity phases indicating alpha₂ adrenergic and high affinity serotonergic components of binding. Blockade of the adrenergic component by 10^?7M clonidine resulted in the specific ³H-LHM binding exhibiting distinctly serotonergic characteristics. Conversely, blockade of the serotonergic component by 2 × 10^?7M serotonin resulted in the specific ³H-LHM binding exhibiting distinct alpha₂ receptor characteristics. These data demonstrate the specific binding of ³H-LHM to alpha₂ adrenergic receptors, to a high affinity serotonin site, and to D₂ dopamine receptors.     

Astroglial Cells Express Large Amounts of GABAA Receptor Proteins in Mature Brain

Abstract: GABA_A receptors were characterized in cellular fractions isolated from adult bovine brain. The fraction enriched in cortical astrocytes is very rich in high-affinity binding sites for [³H]flunitrazepam and other "central-type" benzodiazepine ligands. The amount of specific [³H]flunitrazepam binding was more than five times higher in the glial fraction than in synaptosomal and perikaryal fractions. [³H]Flunitrazepam was displaced by low concentrations of clonazepam and other specific ligands for central GABA_A receptors. Specific binding sites for GABA, flunitrazepam, barbiturates, and picrotoxin-like convulsants were characterized. Allosteric interactions between the different sites were typical of central-type GABA_A receptors. The presence of α-subunit(s), as revealed by [³H]flunitrazepam photoaffinity labeling, was demonstrated in all brain fractions at molecular mass 51–53 kDa. Photoaffinity labeling was highest in the glial fraction. However, in primary cultured astrocytes from neonate rat cortex, no photoaffinity labeling was detected. Information obtained from astrocytes in culture should thus be taken with caution when extrapolated to differentiated astroglial cells. Our results actually show that, in mature brain, most of the fully pharmacologically active GABA_A receptors are extrasynaptic and expressed in astroglia.     

D2 Receptor Antagonists Do Not Modify Guanine Nucleotide-Sensitive Interactions Between Dopamine and D1 Dopamine Receptors Under In Vitro Conditions

Abstract: This study investigated possible D₁/D₂ interactions in rat and bovine striatal tissue by examining the effects of D₂ antagonists on the action of dopamine at D₁ dopamine receptors. In addition, the extent to which D₂ antagonists may induce an agonist low-affinity state of the D₁ receptor was evaluated in comparison with the effects of the guanine nucleotide analogue 5′-guanylylimidodiphosphate [Gpp(NH)p]. In saturation experiments dopamine caused a dose-dependent decrease in rat striatal and bovine caudate D₁ receptor density. This effect of dopamine, which has been shown to be sensitive to Gpp(NH)p, was not altered by pretreatment with either of the selective D₂ antagonists eticlopride (200 nM) or domperidone (200 nM). Results from displacement experiments show that the affinity of dopamine for D₁ receptors and the proportion of receptors in an agonist high-affinity state, are reduced by Gpp(NH)p (100 µM) but not by eticlopride. A molar excess of dopamine (100 µM) promotes the dissociation of (±)-8-chloro-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-3-benzazepine-7-ol ([³H]SCH 23390) from rat striatal D₁ receptors at a rate that is significantly slower than when dissociation is initiated using 1 µM piflutixol. After pretreatment with Gpp(NH)p, [³H]SCH 23390 dissociation induced by dopamine occurred at an even slower rate. Pretreatment with eticlopride had no effect on the dopamine-induced rate of [³H]SCH 23390 dissociation. These results indicate that all experimental approaches detected dopamine effects at D₁ receptors that are Gpp(NH)p sensitive and D₂ antagonist insensitive and provide no evidence to support a D₁/D₂ link operating at the receptor level.     

Dopamine receptors in canine caudate nucleus

Summary Physiological, pharmacological, histochemical and biochemical studies indicate that dopamine receptors are heterogenous in the, central nervous system with each individual functions. This review describes pharmacological and biochemical characteristics of dopamine receptors, particularly in canine caudate nucleus, which have been studied in our laboratory with a brief comparison to the current studies by other workers in similar research fields.Two distinct dopamine receptors have been characterized by means of [³H]dopamine binding to the synaptic membranes from canine caudate nucleus. One of the receptors with a Kd of about 3 M for dopamine may be associated with adenylate cyclase and referred to as D, receptor. The other receptor with a Kd of about 10 nM for dopamine is independent of adenylate cyclase and referred to as D₂. A photochemical irreversible association of [³H]dopamine with the membraneous receptors makes it possible to separate D₁ and D₂ receptors from one another by gel filtration on a Sephadex G-200 column after solubilization with Lubrol PX. On the basis of selective inhibition of [³H]dopamine binding to D₁ and D₂ receptors, dopamine antagonists can be classified into three classes: D₁-selective (YM-09151-2), D₂-selective (sulpiride) and nonselective (haloperidol, chlorpromazine). Effects of these typical antagonists on the metabolism of rat brain dopamine suggest that D₁ receptor is more closely associated with the neuroleptic-induced increase in dopamine turnover. Studies with 28 benzamide derivatives and some classical neuroleptics reveal that apomorphine-induced stereotypy displays a greater association with D₁ than with D₂ receptors.Dopamine-sensitive adenylate cyclase in canine caudate nucleus can be solubilized with Lubrol PX in a sensitive form to either dopamine, Gpp(NH)p or fluoride. Sephadex G-200 gel filtration separates adenylate cyclase from D₁ receptors with a concomitant loss of dopamine sensitivity. Addition of the D₁ receptor fraction to the adenylate cyclase restores the responsiveness to dopamine. The solubilized dopamine-unresponsive adenylate cyclase can be further separated into two distinct fractions by a batch-wise treatment with GTP-sepharose: a catalytic unit which does not respond to fluoride, and a guanine nucleotide regulatory protein. The regulatory protein confers distinct responsiveness to Gpp(NH)p and fluoride upon adenylate cyclase. These results indicate that dopamine-sensitive adenylate cyclase is composed of at least three distinct units; D₁ receptor, guanine nucleotide regulatory protein and adenylate cyclase.     

Binding of3H-ADTN,a dopamine agonist,to membranes of the bovine retina

The binding of³H-ADTN, a potent dopamine receptor agonist, to crude membrane preparations of bovine retina was studied, using a filtration method to isolate membrane-bound ligand. Specific binding was found to be saturable and occurred at a single binding site with an affinity constant of 7.3 nM. Binding was sodium-independent, slightly enhanced by Triton X-100 treatment, but drastically reduced by both trypsin and sodium laurylsulphate. The binding sites demonstrated a high degree of pharmacological specificity, with dopamine, apomorphine, and epinine being potent displacers of³H-ADTN. A higher degree of³H-ADTN binding was associated with subcellular fractions enriched with conventional synaptosomes rather than with fractions enriched with photoreceptor synaptosomes.     

Evaluation of positron-emitting SCH 23390 analogs as tracers for CNS dopamine D1 receptors

Two positron-emitting analogs of SCH 23390, one labelled with ⁷⁵Br (or ⁷⁶Br) and another with ¹¹C, were evaluated as potential PET tracers for central dopamine D1 receptors. In vivo studies were performed to assess the time course of the biodistribution of these tracers in mice and to determine whether dopamine receptors mediated their uptake in the brains of these animals. Results show that indeed cerebral uptake was consistent with dopamine receptor innervation, i.e. uptake and clearance was regionally consistent with the target receptors and that specific uptake was saturable. Because of the relatively rapid pharmacokinetics of this drug, ¹¹C-labelled SCH 23390 would be best suited for PET imaging although the metabolism of this compound needs to be further examined.     

Dopamine receptors in the goldfish retina: 3H-spiroperidol and 3H-domperidone binding; and dopamine-stimulated adenylate cyclase activity

Dopamine receptors in the goldfish retina have been examined by binding studies using ³H-spiroperidol and ³H-domperidone as specific ligands, and by measuring retinal adenylate cyclase activities in the presence and absence of dopamine. Our results indicate that washed membranes from goldfish retinal homogenate bind a variety of dopamine agonists and antagonists with high affinities and with characteristics similar to those reported for the brain, with the exception that in this retina there is virtually no binding of the specific D₂ receptor antagonist, ³H-domperidone. In addition, there is a very low basal activity of adenylate cyclase which can be greatly stimulated by dopamine, possibly reflecting a high degree of coupling between this enzyme and the dopamine receptor. Taken together, our findings indicate that the goldfish retina contains a high density of D₁ type dopamine receptors and few, if any, D₂ type receptors.     

Chemical lesion and drug induced supersentivity and subsensitivity of caudate dopamine receptors

In order to elucidate the mechanism of denervation supersensitivity, the effects of 6-hydroxydopamine lesion, placed in the substantia nigra, were examined on rat brain caudate adenylate cyclase and ³H-haloperidol binding to membrane dopamine receptors. In addition, the effects of chronic administration of L-DOPA, bromocriptine and piribedil were also investigated on ³H-haloperidol binding and dopamine, K⁺ isoproterenol (IPNE) and 2-Cl-adenosine stimulated formation of cyclic AMP in caudate slices. 6-Hydroxydopamine lesions resulted in significantly greater stimulation of adenylate cyclase by dopamine at various concentrations tested. The haloperidol binding sites were increased by 28% on lesioned side caudate without changes in dissociation constants (K_D). Three weeks after treatment with L-DOPA, bromocriptine or piribedil, the ³H-haloperidol binding sites were decreased by 40% with no change in K_D. The stimulatory effect of dopamine on cyclic AMP formation was also abolished, although there was no change in IPNE, K⁺, or 2-Cl-adenosine stimulated cyclic AMP formation in caudate slices, suggesting a specific effect of dopamine agonists on dopamine receptors. The results of these studies suggest a close relationship between at least some populations of dopamine receptors and adenylate cyclase in the caudate nucleus.     

In vivo receptor binding: attempts to improve specific/non-specific ratios.

$\text{In vivo}$ receptor binding was examined using ³H-spiperone and ³H-pimozide for dopamine receptors and ³H-LSD for serotonin receptors. Two strategies for improving total: nonspecific binding ratios were tested. The first was to deplete endogenous ligands by various pharmacological treatments prior to ³H-ligand administration in an attempt to increase specific receptor binding; the second was to perfuse the brain with ice-cold saline after ³H-ligand administration in an attempt to reduce nonspecific binding. Alteration of dopamine and serotonin by administering d-amphetamine, reserpine, alpha-methyl-paratyrosine or parachlorophenylalanine did not significantly elevate striatal: cerebellar or cortical: cerebellar (measures of total: nonspecific) bonding ratios. However, perfusion with ice-cold saline significantly improved the ratios for both dopamine and serotonin receptors. Thus, cold saline perfusion may be of value in reducing blank values in autoradiographic and other studies requiring $\text{in}$$\text{vivo}$ labelling of receptors.     

Guanine nucleotides do not inhibit tritiated dopamine agonist binding to bovine striatal membranes

The addition of GTP (50 M), MnCl₂ (1 mM) or EDTA (2 mM) had no effect on the affinity or capacity of bovine striatal plasma membranes for [³H]spiperone. However, GTP caused a decrease in the potency of dopamine as an inhibitor of [³H]spiperone binding under all conditions tested. Manganese enhanced the potency of dopamine both in the presence and absence of GTP, but NaCl (100 mM) had no effect. Neither manganese nor GTP caused any change in the affinity or capacity of bovine striatal membranes for the tritiated agonists dopamine, apomorphine or ADTN. GPPNHP, a nonhydrolyzable analog of GTP, was also ineffective. However, in identical experiments using rat striatal membranes, 50 M GTP caused a decrease in affinity for all three tritiated agonists and this effect was observed both in the presence and absence of manganese (1 mM). In addition, binding capacities for [³H]dopamine and [³H]ADTN were doubled when manganese was present. In light of this and other reports that GTP inhibits tritiated agonist binding in rat striatum, it is suggested that the absence of such inhibition in bovine striatal membranes may reflect a fundamental difference between the two species with regard to their receptors for dopamine agonists.     

Atypical SCH23390 Binding Sites Are Present on Bovine Adrenal Medullary Membranes

D1-selective dopamine receptor agonists inhibit secretagogue-stimulated catecholamine secretion from bovine adrenal chromaffin cells. The purpose of the studies reported here was to use the radiolabeled D1-selective dopamine receptor antagonist, SCH23390, to characterize putative D1-like dopamine receptors responsible for this effect. Characterization of SCH23390 binding sites demonstrated an unusual pharmacological profile inconsistent with classical D1-like receptors. [¹²⁵I]SCH23390 bound to adrenal medullary membranes was competed for by non-radioactive iodo-SCH23390 (Kd = 490 ± 50 nM), but not by (+)butaclamol. Other classical D1 antagonists had little, if any, effect. Competition with dopamine receptor agonists demonstrated a relative rank order of potency profile characteristic of D1-like dopamine receptors, however, K_is were higher than those found in other tissues. The K_is for competition of [¹²⁵I]SCH23390 binding by C1-APB and SKF38393 (16 and 118 M, respectively) are nearly identical to the IC₅₀s previously observed for inhibition of secretion (9 and 100 M, respectively). Combined these data suggest that adrenal medullary membranes contain a novel SCH23390 binding site involved in the inhibition of secretion by D1-selective agonists.     

Dopamine receptor-coupled modulation of the K+-depolarized overflow of 3H-acetylcholine from rat striatal slices: alteration after chronic haloperidol and alpha-methyl-p-tyrosine pretreatment

The effect of dopamine on the K⁺-depolarized overflow of ³H-acetylcholine from rat striatal slices was investigated to determine whether drug-induced changes in neuronal sensitivity to dopamine might be manifested in changes in striatal cholinergic activity. Dopamine was found to produce a dose-dependent inhibition of the K⁺-evoked release of ³H-Ach. This inhibition could be blocked by prior exposure of the slices to haloperidol, a dopamine receptor blocker. Dopamine receptors localized on striatal cholinergic axon terminals and possibly postsynaptic dopamine receptors on cholinergic perikarya and dendrites may mediate the DA inhibition of ³H-Ach release induced by high K⁺. Chronic pretreatment with haloperidol followed by alpha-methyl-p-tyrosine resulted in a significant shift to the left in the dose-dependent inhibition of K⁺-stimulated overflow of ³H-Ach by dopamine. This shift to the left in the dose-response curve may be the result of an increase in the number of striatal dopamine receptors produced by chronic dopamine receptor blockade and inhibition of dopamine synthesis.     

Subclasses of Adenosine Receptors in Brain Membranes from Adult Tissue and from Primary Cultures of Chick Embryo

Abstract: Membranes from adult chicken brain have high-affinity binding sites for N⁶-cyclohexyl[³H]adenosine (CHA) (K_D= 4 nM, Bmax = 0.6 pmol/mg protein). This CHA binding could be attributed to adenosine receptors of the A₁ type, since substituted adenosine analogs, e.g. N⁶-(l -2-phenylisopropyl)adeno sine (IC50 = 60 nM), were very potent displacers. Binding sites for 1,3-diethyl- 8-[³H]phenylxanthine (DPX) in adult brain membranes have a moderate affinity (K_D= 50 nM, Bmax = 1.5 pmol/mg). The association of DPX with these sites could be completely displaced by 8-phenyltheophylline (IC₅₀= 300 nM) and other xanthines, but only 45% of specific DPX binding could be displaced by phenylisopropyladenosine. This suggests that about half of DPX sites are putative A₁ receptors and the other half are of the A₂ type. Primary cultures of pure glial and neuronal cells from chick embryo brain were also examined for adenosine receptors. Specific binding of CHA could not be detected in these preparations, but both glial and neuronal membranes have specific sites for DPX. At a [³H]DPX concentration of 20 nM, specific binding was 50% higher (per mg protein) in glial than in neuronal membranes. The maximum binding of DPX to glial membranes (B_max= 1.6 pmol/mg) was comparable to values for adult brain, but the glial affinity (K_D= 90 nM) was somewhat less. Phenylisopropyladenosine was able to displace less than 20% of the total glial sites for DPX. This finding was in accord with the lack of CHA sites and demonstrates that A₁ receptors make little contribution to DPX binding in glial membranes. In decreasing order of potency, 8-phenyltheophylline, CHA, theophylline, caffeine, and 3-isobutyl-I-methylxanthine completely displace DPX association with glia. DPX binding to glial membranes thus appears due to a single class of receptors, which may prove to be of the A₂ type.     

INCORPORATION IN VIVO OF RADIOACTIVE LEUCINE INTO NEURONAL AND GOAL NUCLEAR PROTEINS OF RAT BRAIN

Purified neuronal and glial nuclei were separated from rat brain cells. The fraction rich in neuronal nuclei contained 68 ± 9 per cent neuronal nuclei and the fraction rich in glial nuclei contained 89 ± 6 per cent glial nuclei. The fraction rich in neuronal nuclei isolated from cells of adult rat brain incorporated l -[4,5-³H]leucine into TCA-insoluble material at a rate comparable to those of the microsomal and the soluble fractions of the brain, and at a much higher rate than the fraction rich in glial nuclei. The proteins soluble in buffered-saline, the acid-soluble deoxyribonucleoproteins, and the residual proteins of the neuronal nuclei are apparently the proteins which account for the higher specific activity of neuronal proteins compared with glial nuclear proteins. In liver and kidney, the incorporation of [³H]leucine into nuclear proteins was lower than into other subcellular fractions from the same organs.     

Simultaneous Isolation of Glial and Neuronal Fractions from Rat Brain Homogenates: Comparison of High-Affinity L-Glutamate Transport Properties

An improved three-step Percoll density gradient centrifugation technique is described for simultaneous isolation of glial plasmalemmal vesicles (GPV) and synaptosomal vesicles (SYN) from a rat brain homogenate. While electron microscopy revealed that fractions contained intact vesicles with markedly distinct morphological features, measures of high-affinity [³H]choline uptake, glutamine synthetase and carbonic anhydrase activities, as well as Western blot analyses for glial fibrillary acidic protein and neuron specific enolase, served to confirm the low level of neuronal contamination in GPV fractions as well as the low level of glial contamination in SYN fractions. In addition, GPV and SYN fractions were used to characterize the kinetic and pharmacological properties of sodium-dependent [³H]L-glutamate transport. In conclusion, these results demonstrate the usefulness of this method for obtaining highly-enriched, functionally viable populations of glial and neuronal elements which are suitable for studies of their respective cell functions in vitro.