High Affinity Stereospecific Binding of [3H] Cocaine in Striatum and its Relationship to the Dopamine Transporter

A high affinity (K_D 35 nM) binding site for [³H]cocaine is detected in rat brain Striatum present at 2–3 pmol/mg protein of synaptic membranes. This binding is displaced by cocaine analogues with the same rank order as their inhibition of [³H]dopamine ([³H]DA) uptake into striatal synaptosomes (r = 0.99), paralleling the order of their central stimulant activity. The potent DA uptake inhibitors nomifensine, mazindol, and benztropine are more potent inhibitors of this high affinity [³H]cocaine binding than desipramine and imipramine. Cathinone and amphetamine, which are more potent central stimulants than cocaine, displace the high affinity [³H] cocaine binding stereos-pecifically, but with lower potency (IC₅₀ ～ 1μM) than does cocaine. It is suggested that the DA transporter in Striatum is the putative “cocaine receptor.

Binding of [³H] cocaine, measured in 10 mM Na₂HPO₄-0.32 M sucrose, pH 7.4 buffer, is inhibited by physiologic concentrations of Na⁺ and K⁺ and by biogenic amines. DA and Na⁺ reduce the affinity of the putative “cocaine receptor” for [³H]cocaine without changing the B_max, suggesting that inhibition may be competitive. However, TRIS reduces [³H]cocaine binding non-competitively while Na⁺ potentiates it in TRIS buffer. Binding of [³H]mazindol is inhibited competitively by cocaine. In phosphate-sucrose buffer, cocaine and mazindol are equally potent in inhibiting [³H]mazindol binding, but in TRIS-NaCl buffer cocaine has 10 times lower potency. It is suggested that the cocaine receptor in the striatum may be an allosteric protein with mazindol and cocaine binding to overlapping sites, while Na⁺ and DA are allosteric modulators, which stabilize a lower affinity state for cocaine.     

The uptake of (3H)dopamine by homogenates of rat corpus striatum: effects of cations

The uptake of [³H]dopamine was studied with a synaptosomal preparation of the corpus striatum. The accumulation of dopamine was found to be temperature-dependent and very rapid, but linear over time for at least 5 min. at 37°C with characteristics of saturable kinetics. The optimum concentrations for Na⁺ and K⁺ were 150–160 m$\text{M}$ and 2.5–4.8 m$\text{M}$, respectively, while uptake was progressively inhibited at concentrations of K⁺ greater than 5 m$\text{M}$. Rubidium was capable of substituting for potassium whereas cesium was a much less effective replacement. The uptake of DA was blocked by the antibiotics, valinomycin and gramicidin-D which bind K⁺ or both Na⁺ and K⁺, respectively, and thereby might interfere with the transport of cations across neuronal membranes. Similarly, ouabain which blocks the active transport of Na⁺ markedly antagonized the accumulation of DA into striatal homogenates. In contrast, tetrodotoxin which does not prevent the active transport of Na⁺, had no effect. Uptake appeared not to require Ca⁺⁺ and it was not inhibited by increasing total osmolarity to 400 mos$\text{M}$. In general, the cationic requirements for DA-uptake in striatal tissue and its responses to several inhibition of ionic transport, do not appear to be greatly different from those reported for NE with synaptosomes prepared from whole brain.     

Dopamine Uptake is Differentially Regulated in Rat Striatum and Nucleus Accumbens

Active uptake of 3,4-dihydroxyphenylethylamine (dopamine) is sodium- and temperature-dependent, strongly inhibited by benztropine and nomifensine, and present in corpus striatum and nucleus accumbens. In rat striatum dopamine uptake is related to a receptor that is specifically labelled by [3H]cocaine in the presence of Na+ and is located on dopaminergic terminals. The dopamine uptake is differentially affected in the two areas by single or repeated injections of cocaine. Cocaine inhibits dopamine uptake in slices of corpus striatum. Moreover Na+-dependent [3H]cocaine binding is not detectable in nucleus accumbens. Nomifensine inhibits [3H]dopamine uptake by interacting with low- and high-affinity sites in corpus striatum, but shows only low affinity for dopamine uptake in nucleus accumbens. The present data indicate that different mechanisms are involved in the regulation of dopamine uptake in corpus striatum and nucleus accumbens.     

Modeling of the Interaction of Na+ and K+ with the Binding of the Cocaine Analogue 3β-(4-[125I]Iodophenyl)tropane-2β-Carboxylic Acid Isopropyl Ester to the Dopamine Transporter

Abstract: The present study examines the interaction of Na⁺ and K⁺ with the binding of the cocaine analogue 3β-(4-[¹²⁵I]iodophenyl)tropane-2β-carboxylic acid isopropyl ester to dopamine transporters (DATs) in rat striatal synaptosomal membranes at 37°C. The binding increases with [Na⁺] from 10 to 100 mM and decreases with higher [Na⁺]. The presence of K⁺ reduces the maximal stimulatory effect of Na⁺ and causes a nonlinear EC₅₀ shift for Na⁺. K⁺ strongly inhibits the binding at low [Na⁺]. Increasing [Na⁺] produces a linear IC₅₀ shift for K⁺. Saturation analysis indicates a single binding site changing its affinity for the radioligand depending on [K⁺]/[Na⁺] ratio in the assay buffer. A reduced B_max was observed in the presence of 10 mM Na⁺ and 30 mM K⁺. Both high [Na⁺] and high [K⁺] accelerate the dissociation of the binding, and K⁺-induced acceleration was abolished by increasing [Na⁺]. Least squares model fitting of equilibrium data and kinetic analysis of dissociation rates reveal competitive interactions between Na⁺ and K⁺ at two sites allosterically linked on the DAT: One site mediates the stimulatory effect of Na⁺, and the other site involves the radioligand binding and the inhibitory effect of cations on the binding. Various uptake blockers and substrates, dopamine in particular, display reduced potency in inhibiting the binding at a higher [K⁺]/[Na⁺] ratio.     

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.     

[3H]Threo-(±)-Methylphenidate Binding to 3,4-Dihydroxyphenylethylamine Uptake Sites in Corpus Striatum: Correlation with the Stimulant Properties of Ritalinic Acid Esters

Saturable and stereoselective binding sites for [3H]threo-(+/-)-methylphenidate were characterized in rat brain membranes. The highest density of [3H]threo-(+/-)-methylphenidate binding sites was found in the synaptosomal fraction of corpus striatum. Scatchard analysis revealed a single class of noninteracting binding sites with an apparent dissociation constant (KD) of 235 nM and a maximum number of binding sites (Bmax) of 13.4 pmol/mg protein. Saturable, high-affinity binding of [3H]threo-(+/-)-methylphenidate to striatal synaptosomal membranes was dependent on the presence of sodium ions. A good correlation (r = 0.88; p less than 0.001) was observed between the potencies of various psychotropic drugs in displacing [3H]threo-(+/-)-methylphenidate from these sites and their potencies as inhibitors of [3H]3,4-dihydroxyphenylethylamine ( [3H]dopamine) uptake into striatal synaptosomes. A good correlation (r = 0.85; p less than 0.001) was also observed between the potencies of a series of ritalinic acid esters in inhibiting [3H]threo-(+/-)-methylphenidate binding to striatal synaptosomal membranes and their potencies as motor stimulants in mice. These observations suggest that the binding sites for [3H]threo-(+/-)-methylphenidate described here are associated with a dopamine uptake or transport complex, and that these sites may mediate the motor stimulant properties of ritalinic acid esters such as methylphenidate.     

Isotopic labeling of synaptosomes that accumulate choline and the effect of narcotic drugs

Subcellular studies of choline uptake of rat striatum indicated a correspondence between the Na⁺-dependent uptake and choline acetyltransferase (ChAc), whereas there was a lack of correspondence between the Na⁺-independent uptake and ChAc. Subcellular studies also showed a correspondence between the Na⁺-dependent uptake and hemicholinium-3 inhibition, and more important, particles that accumulate choline were shown to consist of at least two subcellular populations. A comparison was made of kinetic data from three areas of the rat brain: corpus striatum, cerebral cortex, and hypothalamus. Taken together, our data on choline uptake give added support to the idea that the Na⁺-dependent choline transport is concentrated in the striatum and specifically related to cholinergic nerve endings. Morphine and methadone in vitro inhibited the Na⁺-dependent choline uptake. In vivo morphine induced a significant lowering of theV _max in the rat cerebral cortex, but not in the striatum. This finding is consistent with the known action of morphine on acetylcholine turnover.Preliminary reports of this work were presented at the Fifth Meeting of the American Society for Neurochemistry in New Orleans, March 1974, and the Fall ASPET Meeting in Montreal, August 1974 (1,2).     

Biochemical and Pharmacological Characterization of [3H]GBR 12935 Binding In Vitro to Rat Striatal Membranes: Labeling of the Dopamine Uptake Complex

Abstract: Binding of the selective dopamine (DA) uptake inhibitor [³H]GBR 12935 to rat striatal membranes was characterized biochemically and pharmacologically. [³H]-GBR 12935 binding at 0°C was reversible and saturable and Scatchard analysis indicated a single binding site with a K_D of 5.5 nM and a B_max of 760 pmol/mg tissue. [³H]GBR 12935 labeled two binding sites. One binding site was identified as the classic DA uptake site, since methylphenidate, cocaine, diclofensine, and Lu 19–005 potently inhibited [³H]GBR 12935 binding to it. Binding to the second site was inhibited by high concentrations of the above compounds. IC₅₀ values for inhibition of [³H]GBR 12935 binding to the DA uptake site were proportional to IC50 values for inhibition of DA uptake. However, substrates of DA uptake, e.g., DA and 1-methyl-4-phenylpyridine, and DA releasers, e.g., the amphetamines, inhibited [³H]GBR 12935 binding less than DA uptake. Rate experiments excluded the possibility that these “weak” inhibitors affected the binding by alloste-ric coupled binding sites. The second binding site was not a noradrenergic, serotonergic, or GABAergic uptake site. Neither was it a dopaminergic, acetylcholinergic, histaminic, serotonergic, or adrenergic receptor. However, [³H]GBR 12935 was potently displaced from it by disubstituted piper-azine derivatives, i.e., flupentixol and piflutixol. DA uptake and the DA uptake binding site of [³H]GBR 12935 were located primarily in the striatum, but the piperazine acceptor site was distributed uniformly throughout the brain. Also only the DA uptake binding site was destroyed by 6-OH-DA. Thus, [³H]GBR 12935 labels the classic DA uptake site in rat striatum and also a piperazine acceptor site. Substrates for DA uptake and releasers of DA inhibited [³H]GBR 12935 binding with low potency, but did not alter the rate constants for [³H]GBR 12935 binding. Therefore inhibitors of DA uptake label the carrier site and prevent the carrier process.     

Regional Characteristics of Histamine Uptake into Neonatal Rat Astrocytes

Histaminergic signalling constitutes an attractive target for treatment of neuropsychiatric disorders. One obstacle to developing new pharmacological options has been failure to identify putative specific histamine transporter responsible for histamine clearance. Although high-affinity histamine uptake was detected in neonatal cortical astrocytes, its existence in other brain regions remains largely unexplored. We investigated whether cerebellar and striatal astrocytes participate in histamine clearance and evaluated the role of organic cation transporters (OCT) in astroglial histamine transport. Kinetic and pharmacological characteristics of histamine transport were determined in cultured astrocytes derived from neonatal rat cerebellum, striatum and cerebral cortex. As well as astrocytes of cortical origin, cultured striatal and cerebellar astrocytes displayed temperature-sensitive, high-affinity histamine uptake. Exposure to ouabain or Na⁺-free medium, supplemented with choline chloride markedly depressed histamine transport in cortical astrocytes. Conversely, histamine uptake in striatal and cortical astrocytes was ouabain-resistant and was only partially diminished during incubation in the absence of Na⁺. Also, histamine uptake remained unaltered upon exposure to OCT inhibitor corticosterone, although OCTs were expressed in cultured astrocytes. Finally, histamine transport in cerebellar and striatal astrocytes was not sensitive to antidepressants. Despite common characteristics, cerebellar astrocytes had lower affinity, but markedly higher transport capacity for histamine compared to striatal astrocytes. Collectively, we provide evidence to suggest that cerebellar, striatal as well as cortical astrocytes possess saturable histamine uptake systems, which are not operated by OCTs. In addition, our data indicate that Na⁺-independent histamine carrier predominates in cerebellar and striatal astrocytes, whereas Na⁺-dependent transporter underlies histamine uptake in cortical astrocytes. Our findings implicate a role for histamine transporters in regulation of extracellular histamine concentration in cerebellum and striatum. Inhibition of histamine uptake might represent a viable option to modulate histaminergic neurotransmission.     

The binding of phloridzin to the isolated luminal membrane of the renal proximal tubule

The binding of [³H]ploridzin by isolated luminal membranes of the rabbit proximal tubule and by slices of rabbit kidney cortex was studied.Kinetic analyses of the relationship between the concentration of phloridizin in the incubation medium and the binding of phloridzin to the membrane indicated two distinct classes of receptors sites. One class, comprising high affinity sites, reached saturation at 20–25 μM phloridzin, had a K(phloridzin) of 8 μM, and 8·10⁺² nmoles interacted with 1 mg of brush border protein. The other class, comprising low affinity sites, had a K(phloridzin) of 2.5 mM, and the number of binding sites was 1.25 nmoles/mg Na⁺ was required for the binding of phloridzin at the high affinity sites. Na⁺ decreased the apparent K_i for phloridzin; the apparent V of binding was not altered. Binding at the low affinity sites was independent of Na⁺. Ca²⁺ was necessary for maximal binding at the high affinity sites. Binding of phloridzin at high affinity sites was more sensitive to N-ethylmalcimide and mersalyl than was binding at low affinity sites. Binding at high affinity sites, but not at low affinity sites, was temperature dependent.d-Glucose was a competitive inhibitor of the high affinity binding of phloridzin. The apparent K₁ was 1 mM. D-Glucoe inhibited non-competitively at the low affinity sites. l-Glucose had no influence on phloridzin binding. Phloretin was a competitive inhibitor of high affinity phloridzin binding with an apparent K_i of 16 μM. Phloretin inhibited low affinity bindings of phloridizin non-competitively. Binding of phloridzin at high affinity sites was completely reversible. Binding at low affinity sites was only partially reversed. Phloridzin bound at high affinity sites on the brush border was displaced by phloridzin and phloretin but not by d-glucose.The mechanism of the high affinity binding of phloridzin was distinguished from that of the initial interaction of d-glucose with the membrane. Binding of phloridzin required Na⁺, whereas the interaction of d-glucose with the membranes had a prominent Na⁺-independent component.Intact renal cells in cortical slices accumulated phloridzin. The uptake did not saturate, was Na⁺ independent, and was not competitively inhibited by sugars. These characteristics resemble those for the low affinity binding of phloridzin by isolated membranes. It is suggested that low affinity binding may represent an initial binding followed by uptake of the glycoside into membrane vesicles.     

Rat mesencephalic neuronal cells cultured for different periods as a model of dopamine transporter ontogenesis

Ventral mesencephalic neurons contained only low-affinity and sodium-independent binding sites of [³H]WIN 35,428 (marker of dopamine transporter) during the first 10d in primary cultures. These sites were present in cytosol, and they are not very probably related to dopamine transporter. After 12 d in culture, membrane-bound, high-affinity, and sodium-dependent [³H]WIN 35,428 binding sites were detected. In membranes prepared from cells 14 d in culture, cocaine displaced [³H]WIN 35,428 binding with similar potency to that in striatal membranes of adult rat brain. The high-affinity [³H]WIN 35,428 binding sites in mesencephalic neuronal cell cultures are very probably related to dopamine transporter. The development of high-affinity [³H]WIN 35,428 binding sites in neurons cultured for different time periods could be a useful model of dopamine transporter ontogenesis.     

Uptake of selenate and selenite by isolated intestinal brush border membrane vesicles from pig,sheep, and rat

Selenate and selenite uptakes by isolated intestinal brush border membrane vesicles (BBMV) from pig, sheep, and rat were investigated. Selenate uptake into jejunal and ileal, but not duodenal, BBMV from pig was stimulated by an inwardly directed transmembrane Na⁺ gradient (Na _out ⁺ >Na _in ⁺ ). Selenate transport into rat ileal and sheep jejunal BBMV was also enhanced in the presence of a Na⁺ gradient. Unlike selenate uptake, selenite uptake was not Na⁺ dependent, neither in pig small intestine nor in sheep jejunum and rat ileum. Uptake of selenate represented real uptake into the vesicular lumen, whereas selenite uptake was a result of an extensive binding of⁷⁵Se to the membranes. Thiosulfate at a 250-fold concentration of selenate completely inhibited Na⁺-dependent selenate uptake into pig jejunal BBMV. Furthermore, Na⁺-dependent sulfate uptake was totally inhibited in the presence of a 250-fold selenate concentration. The results clearly show that selenate transport across the BBM of pig jejunum and ileum, sheep jejunum, and rat ileum is partially energized by a transmembrane Na⁺ gradient. Moreover, it is concluded from the results that there exists a common transport mechanism for sulfate and selenate in the BBM. The extensive binding of⁷⁵Se from⁷⁵Se-labeled selenite to the membranes could be from a spontaneous reaction of selenite with membrane-associated SH groups.     

3H-dopamine binding to rat striatal D-2 and D-3 sites: Enhancement by magnesium and inhibition by guanine nucleotides and sodium

Previous studies have demonstrated high affinity ³H-dopamine binding sites on mammalian striatal membranes. These putative dopamine receptors of unknown physiological significance have been termed D-3 sites. Such studies have failed, however, to demonstrate high affinity ³H-dopamine binding to D-2 sites, which can be labeled by ³H-butyrophenones, and which represent the putative dopamine receptors most stronly implicated in the behavioral correlates of dopaminergic CNS activity. We now report that preincubation of membrane homogenates with Mg⁺⁺ and inclusion of Mg⁺⁺ (1–10mM) or other divalent metal cations during binding allows high affinity D-2 specific ³H-dopamine binding in rat striatal membranes, and that these ions also increase the Bmax of D-3 specific ³H-dopamine binding. GTP, GDP, and GppNHp can completely abolish all D-2 specific ³H-dopamine binding, while only a magnesium-dependent portion of D-3 sites appears to be GTP sensitive. These data are consistent with the hypothesis that the striatal D-2 receptor exists in two agonist affinity states whose interconversion is effected by guanine nucleotides and divalent metal cations. The GTP sensitive/magnesium dependent nature of ³H-dopamine binding to so-called D-3 sites suggests that some such sites may in fact represent a high agonist-affinity state of the D-1 adenylate cyclase stimulating dopamine receptor also found in this tissue.     

A Multisubstrate Kinetic Mechanism of Dopamine Transport in the Nucleus Accumbens and Its Inhibition by Cocaine

Abstract: Kinetic studies of dopamine transport into suspensions of nucleus accumbens (NAcc) and effects of Na⁺ and Cl^? as cosubstrates were performed using rotating disk electrode voltammetry. To mimic chemical neurotransmission, dopamine was added as a rapid pulse, and transporter-mediated clearance of dopamine was evaluated kinetically. This paradigm was shown to approximate a zero trans entry transport experiment. Dopamine was taken up with apparent K_m and V_max values of 1.3 µM and 375 pmol/s/g wet weight, respectively. Transport exhibited apparent trans acceleration. Substitution of Na⁺ with choline or Cl^? with isethionate reduced dopamine transport with reaction orders of two and unity, respectively, accompanied by reductions in V_max with no changes in K_m. Apparent K_Na and K_Cl values were 70.0 and 92.1 mM, respectively. Dopamine transport in NAcc was found to follow a partially random, sequential mechanism in which dopamine and Na⁺ bind randomly to the transporter followed by binding of Cl^? before transport. Cocaine inhibited dopamine transport and the influences of the other substrates allosterically with an overall K_i of 0.30 µM. Thus, the general kinetic mechanism of the transport of dopamine in the NAcc is identical to that previously reported by this laboratory for dopamine transport in the striatum. However, the dopamine transporter in the NAcc is more tightly regulated by Na⁺, possesses a higher kinetic turnover rate, is four times more sensitive to cocaine than the striatal transporter, and exhibits cocaine inhibition independent of [substrate]. These findings suggest that cocaine modulates chemical signaling in NAcc differently than in striatum, providing down-regulation of function irrespective of [substrate], thereby enhancing dopaminergic signaling more robustly in the NAcc than in the striatum.     

Characterization of Sodium-Dependent [3H]GBR-12935 Binding in Brain: A Radioligand for Selective Labelling of the Dopamine Transport Complex

High-affinity and saturable binding sites for the diphenyl-substituted piperazine derivative [3H]GBR-12935 have been characterized in crude synaptosomal membranes prepared from rat brain. The specific binding of [3H]GBR-12935 is sodium-dependent and is unevenly distributed among various brain regions, with the highest concentration of binding sites being found in the corpus striatum and nucleus accumbens. Sodium-dependent [3H]GBR-12935 binding in all other brain areas was 10% or less of the binding found in the striatum. The affinity of [3H]GBR-12935 for binding sites in the striatum is increased in the presence of Na+ but other cations, including K+, Ca2+, or Mg2+, inhibit specific binding. There is an excellent correlation (r = 0.96, p less than 0.01) between the potencies of a series of drugs in inhibiting [3H]GBR-12935 binding to striatal membranes and their potencies in inhibiting [3H]3,4-dihydroxyphenylethylamine ([3H]dopamine) uptake in synaptosomes. Agonists and antagonists of other neurotransmitter receptor or drug recognition sites have little or no effect on specific [3H]GBR-12935 binding to striatal membranes. In addition, prior intracerebroventricular administration of 6-hydroxydopamine results in a decrease in the number of specific [3H]GBR-12935 binding sites in the striatum. These data indicate that [3H]GBR-12935 is a selective radioligand of the presynaptic dopamine transport complex in brain.     

Age and Brain Structural Related Effects of Glutaric and 3-Hydroxyglutaric Acids on Glutamate Binding to Plasma Membranes During Rat Brain Development

(1) In the present study we determined the effects of glutaric (GA, 0.01–1 mM) and 3-hydroxyglutaric (3-OHGA, 1.0–100 μM) acids, the major metabolites accumulating in glutaric acidemia type I (GA I), on Na⁺-independent and Na⁺-dependent [³H]glutamate binding to synaptic plasma membranes from cerebral cortex and striatum of rats aged 7, 15 and 60 days. (2) GA selectively inhibited Na⁺-independent [³H]glutamate binding (binding to receptors) in cerebral cortex and striatum of rats aged 7 and 15 days, but not aged 60 days. In contrast, GA did not alter Na⁺-dependent glutamate binding (binding to transporters) to synaptic membranes from brain structures of rats at all studied ages. Furthermore, experiments using the glutamatergic antagonist CNQX indicated that GA probably binds to non-NMDA receptors. In addition, GA markedly inhibited [³H]kainate binding to synaptic plasma membranes in cerebral cortex of 15-day-old rats, indicating that this effect was probably directed towards kainate receptors. On the other hand, experiments performed with 3-OHGA revealed that this organic acid did not change Na⁺-independent [³H]glutamate binding to synaptic membranes from cerebral cortex and striatum of rats from all ages, but inhibited Na⁺-dependent [³H]glutamate binding to membranes in striatum of 7-day-old rats, but not in striatum of 15- and 60-day-old rats and in cerebral cortex of rats from all studied ages. We also provided some evidence that 3-OHGA competes with the glutamate transporter inhibitor L-trans-pyrrolidine-2,4-dicarboxylate, suggesting a possible interaction of 3-OHGA with glutamate transporters on synaptic membranes. (3) These results indicate that glutamate binding to receptors and transporters can be inhibited by GA and 3-OHGA in cerebral cortex and striatum in a developmentally regulated manner. It is postulated that a disturbance of glutamatergic neurotransmission caused by the major metabolites accumulating in GA I at early development may possibly explain, at least in part, the window of vulnerability of striatum and cerebral cortex to injury in patients affected by this disorder.     

The Transport Systems for Selenomethionine/Methionine and Selenocystine/Cystine in Escherichia Coli K-12 Appear to be Cooperative

Dopamine transporters of bovine and rat striata were identified by their specific [³H]cocaine binding and cocaine-sensitive [³H]dopamine ([³H]DA) uptake. Both binding and uptake functions of bovine striatal transporters were potentiated by lectins. Concanavalin A (Con A) increased the velocity but did not change the affinity of the transporter for DA; however, it increased its affinity for cocaine without changing the number of binding sites. This suggests that the DA transporter is a glycoprotein and that Con A action on it produces conformational changes

Inorganic and organic mercury reagents inhibited both [³H]DA uptake and [³H]cocaine binding, though they were all more potent inhibitors of the former, n- Ethylmaleimide inhibited [³H]DA uptake totally but [³H]cocaine binding only partially. Also, n-pyrene maleimide had differential effects on uptake and binding, inhibiting uptake and potentiating binding. [³H]DA uptake was not affected by mercaptoethanol up to 100 mM, whereas [³H]cocaine binding was inhibited by concentrations above 10 mM. On the other hand, both uptake and binding were fairly sensitive to dimercaprol (< 1 mM). The effects of all these sulfhydryl reagents suggest that the DA transporter has one or more thiol group(s) important for both binding and uptake activities. The Ellman reagent and dithiopyridine were effective inhibitors of uptake and binding only at fairly high concentration (>10 mM). Loss of activity after treatment with the dithio reagents may be a result of reduction of a disulfide bond, which may affect the transporter conformation     

Solubilization and Characterization of the A2-Adenosine Receptor

Abstract

Binding of [³H]CGS 21680, an agonist radioligand selective for A₂-adenosine receptors (A₂AR), to membranes and solubilized preparations from bovine brain striatum revealed labelling of a single high affinity binding state. In membranes, guanine nucleotides per se were ineffective in modulating agonist binding whereas cations, Na⁺ and Mg⁺⁺, had distinct effects. The addition of NaCl (200 mM) as well as the Mg⁺⁺-free preparation of membranes led to a significant decrease in binding affinity and the number of binding sites. Moreover, the presence of Na⁺ was required for the demonstration of a guanine nucleotide effect, i.e. a decrease in maximal binding. Following solubilization, agonist-A AR interactions were sensitive to guanine nucleotides even in the absence of Na⁺²; guanine nucleotides and Na⁺ had additive effects in reducing the number of binding sites. Moreover, the effect of GTP was reversible, i.e. binding returned to control levels upon removal of the nucleotide. This suggests the A₂AR and its G protein (presumably G_S) are solubilized as a functional unit and may not dissociate even in the presence of GTP following solubilization. We, therefore, believe that a “tight” association exists between receptor and G protein (G_S), and that guanine nucleotides and sodium act at different sites on the R–G complex. Drawing an analogy with similar observations on the avian β-adrenergic receptor (Hertel et al, J.Biol.Chem. 265:17988–94, 1990; Parker & Ross, J.Biol.Chem. 266:9987–96, 1991) we postulate that the regulatory features of the A₂AR can be attributed to a distinct receptor domain that interacts with cellular regulatory elements.     

Stimulation of sodium and calcium uptake by scorpion toxin in chick embryo heart cells

Scorpion toxins, the basic miniprotiens of scorpion venom, stimulated the passive uptake of Na⁺ and Ca²⁺ in chick ermbryo heart cells. Half-maximum stimulation was obtained for 20–30 nM Na⁺ and 40–50 nM Ca²⁺. Scorpion toxin-activated Na⁺ and Ca²⁺ uptakes were fully inhibited by tetrodotoxin, a specific inhibitor of the action potential Na⁺ ionophore in excitable membranes. Half-maximum inhibition was obtained with the same concentration of tetrodotoxin (10 nm) for both Na⁺ and Ca²⁺. Scorpion toxin-stimulated Ca²⁺ uptake was dependent on extracellular Na⁺ concentration and was not inhibited by Ca²⁺ channel blocking drugs which are inactive on heart cell action potential. Thus, in heart cells scorpion toxin affects the passive Ca²⁺ transport, which is coupled to passive Na⁺ ionphore. Other results suggest that (1) tetrodotoxin and scorpion toxin bind to different sites of the sarcolemma and (2) binding of scorpion toxin to its specific sites may unmask latent tetrodotoxin — sensitive fast channels.     

High affinity stereospecific binding of [3H] cocaine in striatum and its relationship to the dopamine transporter

A high affinity (KD 35 nM) binding site for [3H]cocaine is detected in rat brain striatum present at 2-3 pmol/mg protein of synaptic membranes. This binding is displaced by cocaine analogues with the same rank order as their inhibition of [3H]dopamine ([3H]DA) uptake into striatal synaptosomes (r = 0.99), paralleling the order of their central stimulant activity. The potent DA uptake inhibitors nomifensine, mazindol, and benztropine are more potent inhibitors of this high affinity [3H]cocaine binding than desipramine and imipramine. Cathinone and amphetamine, which are more potent central stimulants than cocaine, displace the high affinity [3H]cocaine binding stereospecifically, but with lower potency (IC50 approximately equal to 1 microM) than does cocaine. It is suggested that the DA transporter in striatum is the putative "cocaine receptor." Binding of [3H]cocaine, measured in 10 mM Na2HPO4-0.32 M sucrose, pH 7.4 buffer, is inhibited by physiologic concentrations of Na+ and K+ and by biogenic amines. DA and Na+ reduce the affinity of the putative "cocaine receptor" for [3H]cocaine without changing the Bmax, suggesting that inhibition may be competitive. However, TRIS reduces [3H]cocaine binding noncompetitively while Na+ potentiates it in TRIS buffer. Binding of [3H]mazindol is inhibited competitively by cocaine. In phosphate-sucrose buffer, cocaine and mazindol are equally potent in inhibiting [3H]mazindol binding, but in TRIS-NaCl buffer cocaine has 10 times lower potency. It is suggested that the cocaine receptor in the striatum may be an allosteric protein with mazindol and cocaine binding to overlapping sites, while Na+ and DA are allosteric modulators, which stabilize a lower affinity state for cocaine.