Lori L. Coffey

Regulation of the functional activity of the human dopamine transporter by protein kinase C

The role of protein kinase C (PKC) was examined in the regulation of dopamine transport in C6 glioma cells stably expressing the human dopamine transporter. The PKC activating phorbol esters phorbol 12-myristate 13-acetate (PMA) and 4 beta-12,13-dibutyrate phorbol-ester (PDBu) inhibited [3H]dopamine uptake concentration dependently. These effects were attenuated by the PKC inhibitor staurosporine but were unaltered by another inhibitor, chelerythrine, or the phosphatase inhibitor okadaic acid. The potency of PMA in inhibiting [3H]dopamine uptake was similar to that in inhibiting the binding of 2 beta-carbomethoxy-3 beta-(4-fluorophenyl)tropane ([3H]WIN 35,428), and again staurosporine, but not chelerythrine, weakened the effect of PMA. The reduction in dopamine transporter activity by PMA was caused by a decrease in the Vmax value of [3H]dopamine uptake, opposed by a smaller reduction in the Km value, whereas the effect of PMA on [3H]WIN 35,428 binding was caused by a reduction in the Bmax value without a change in the Kd value. The lower Km value in the presence of PMA was accompanied by a higher IC50 of dopamine in inhibiting [3H]WIN 35,428 binding; the latter effect was attenuated by the co-presence of staurosporine. The results are discussed in the context of transporter loss from the cell surface, or a model with phosphorylation affecting the shared dopamine and WIN 35,428 binding domain on the transporter as well as affecting a part of the dopamine binding domain lying outside that for WIN 35,428.

Cationic and Anionic Requirements for the Binding of 2β-Carbomethoxy-3β-(4-Fluorophenyl)[3H]tropane to the Dopamine Uptake Carrier

Abstract: The present study reports the ion dependency of 2β‐carbomethoxy‐3β‐(4‐fluorophenyl)[3H]tropane ([3H]‐ CFT) binding to the dopamine transporter in the rat striaturn. The results indicate that [3H]CFT binding to synaptosomal P2 membranes requires low concentrations of Na+ (peak binding between 20 and 50 mM Na+), is stimulated by phosphate anion or l‐, but is unaffected or only slightly affected by F‐, Cl‐, Br‐, NO3‐, or SO42‐, Concentrations of Na+ of >50 mM become inhibitory except in the presence of l‐, which shifts peak binding levels toward higher Na+ concentrations and also elevates the peak binding level. K+ strongly decreased [3H]CFT binding with a shallow inhibition curve, and Na+ could not overcome this effect. Saturation analysis of [3H]CFT binding revealed a single binding site changing its affinity for CFT depending on the concentration of sodium phosphate buffer (6, 10, 30, 50, 130, or 200 mM; 1 mM plus 49 mM NaCIversus 10 mM plus 40 mM NaCI; or 1 mM plus 129 mM Nal versus 10 mM plus 120 mM Nal). No differences were observed in the density of CFT binding sites between any of the conditions examined.

Amygdala Kindling of Forebrain Seizures and the Occurrence of Brainstem Seizures in Genetically Epilepsy‐Prone Rats

Forebrain seizures were kindled in rats by daily electrical stimulation of the amygdala. Genetically epilepsy‐prone rats scoring 9 (GEPR‐9s) on the seizure severity scale during audiogenic seizure (AGS) screening (“brainstem seizure‐experienced”) required fewer stimulations to achieve fully kindled seizures (forelimb clonus with rearing and falling) than control rats. AGS‐naive GEPR‐9s required an intermediate number of stimulations, indicating a role for both genetic predisposition and previous acoustically evoked brainstem seizure experience. Other forebrain kindling indices such as afterdischarge thresholdlduration and seizure latencylduration also involved genetic as well as phenotypic (previous seizure experience) factors. In most GEPR‐9s in both groups, severe brainstem seizures occurred after forebrain stimulation. The occurrence of brainstem seizures had a random nature and was not related to the sequence of kindling‐dependent forebrain seizure progression. The lack of a difference in the occurrence of brainstem seizures between seizure‐experienced and AGS‐naïve GEPR‐9s suggest that genetic predisposition is the major factor in forebrain seizure‐induced activation of brainstem seizure circuitry. This brainstem seizure activity appears to model pertinent aspects of secondary generalization observed in human partial seizures.

Translocation of dopamine and binding of 2β-carbomethoxy-3 β-(4-fluorophenyl) tropane (WIN 35,428) measured under identical conditions in rat striatal synaptosomal preparations: Inhibition by various blockers

Translocation of [3H]dopamine and binding of 2 beta-carbomethoxy-3 beta-(4-fluorophenyl)[3H]-tropane ([3H]WIN 35,428) were measured in crude synaptosomal preparations from rat striatum under identical conditions of assay buffer (phosphate-Krebs) and temperature (25 degrees). [3H]Dopamine uptake as a function of time was close to linear for at least 8 min, whereas [3H]WIN 35,428 binding had reached equilibrium within 1 min and remained at its plateau value for at least 20 min. The following inhibitors were tested in uptake and binding assays run in parallel with the same synaptosomal preparation: cocaine, WIN 35,428, benztropine, nomifensine, mazindol, methylphenidate, N-[1-(2-benzo[b]-thiophenyl)cyclohexyl]piperidine (BTCP), Lu 19-005 (Indatraline), 1-(2-(di(4-fluorophenyl)-methoxy)-ethyl)-4-(3-phenyl-2-propyl)piperazine (GBR 12909), 1-(2-(diphenylmethoxy)-ethyl)-4-(3-phenyl-2-propyl)piperazine (GBR 12935) and 7-trifluoromethyl-4-(4-methyl-1-piperazinyl)-pyrrolo [1,2-a]quinoxaline (CGS 12066B). When present together with [3H]dopamine or [3H]WIN 35,428 for 8 min, the observed binding IC50 values were generally higher (average 1.4-fold) than the uptake IC50 values, with a significant y-axis intercept in linear regression analysis of binding on uptake IC50. For slowly equilibrating inhibitors, estimates of uptake IC50 values were overestimates, and relatively lower values were obtained by monitoring [3H]dopamine uptake for 1 min only during the last minute of the 8-min presence of inhibitor; under these conditions, binding over uptake IC50 ratios were on the average 2.3. Kinetic calculations, taking into account both radioligand and inhibitor equilibration kinetics, indicated that the latter comparison between binding and uptake measurements was most relevant, and suggested the involvement of complexities beyond simple competitive inhibition of dopamine transport, such as different binding domains for substrate and blocker recognition, or spare receptors for blockers. The present data indicate that binding over uptake IC50 ratios should be interpreted with caution, depending on the experimental conditions used to measure these ratios.

[3H]WIN 35,428 binding to the dopamine uptake carrier. I. Effect of tonicity and buffer composition

In the present study, the dopamine transporter on rat striatal membranes was labeled with [3H]WIN 35,428 (2 beta-carbomethoxy-3 beta-(4-fluorophenyl)-tropane), and its binding state or form was manipulated by changing tonicity and buffer composition. Binding to P2 membranes was enhanced by the presence of sucrose in the assay. This effect was not due solely to factors relating to tonicity because creation of isotonicity by dextrose or N-methyl-D-glucamine was less effective, and an increase in binding by sucrose was also observed in assays that were already isotonic by a mixture of sodium phosphate and NaCl. Under the latter conditions, fructose and mannose were equally effective as sucrose. Other important factors were the presence of sodium phosphate in the homogenizing buffer and the presence of sucrose during resuspension of the membranes. When P2 membranes were prepared from homogenates in 0.32 M sucrose, the effect of sucrose in the polytronning step or in the binding assay was restricted to a decrease in the Kd of the main binding component.

GBR 12909 and 12935 block dopamine uptake into brain synaptic vesicles as well as nerve endings

GBR 12909 and 12935, commonly used as potent neuronal dopamine uptake blockers, also inhibit dopamine uptake into brain synaptic vesicles. The concentrations required for the latter activity (34-45 nM) are one order of magnitude higher than those required for inhibiting neuronal uptake of dopamine (1-6 nM). In contrast, the two activities differ by three orders of magnitude for cocaine (137 microM versus 0.35 microM). We propose that the vesicular effect of GBR-type dopamine uptake blockers should be taken into account when interpreting in vivo experiments.

Binding domains for blockers and substrates on the cloned human dopamine transporter studied by protection against N-Ethylmaleimide-induced reduction of 2β-carbomethoxy-3β-(4-fluorophenyl)[3H]tropane ([3H]WIN 35,428) binding

Binding sites for 2 beta-carbomethoxy-3 beta-(4-fluorophenyl)[3H]tropane ([3H]WIN 35,428) on the human dopamine transporter expressed in C6 glioma cells were alkylated with N-ethylmaleimide (NEM), and the protective potency of the blockers cocaine, N[1-(2-benzo[b]thiophenyl) cyclohexyl]piperidine (BTCP), and benztropine, and of the substrates dopamine, d-amphetamine, and norepinephrine was measured. In general, the protective potency was lower (at least 4-5 times) than the potency in inhibiting [3H]WIN 35,428 binding with the compounds present under the same experimental conditions used for the NEM alkylation. However, the disparity was substantially greater for all substrates tested (23- to 44-fold) than for the blockers (4- to 11-fold), especially cocaine (5-fold) and BTCP (4-fold). Benztropine took an intermediate place (11-fold) between cocaine (5-fold) and BTCP (4-fold), on the one hand, and dopamine (23-fold), on the other hand. [3H]WIN 35,428 binding was best described by a one-site model under the present conditions. The results are discussed in terms of models involving blocker-induced conformational changes and overlapping nonidentical binding domains for blockers and substrates.

Cations affect [3H]mazindol and [3H]WIN 35,428 binding to the human dopamine transporter in a similar fashion.

Abstract: The present study addresses the possibility that there are different cocaine‐related and mazindol‐related binding domains on the dopamine transporter (DAT) that show differential sensitivity to cations. The effects of Zn2+, Mg2+, Hg2+, Li+, K+, and Na+ were assessed on the binding of [3H]mazindol and [3H]WIN 35,428 to the human (h) DAT expressed in C6 glioma cells under identical conditions for intact cell and membrane assays. The latter were performed at both 0 and 21°C. Zn2+ (30–100 µM) stimulated binding of both radioligands to membranes, with a relatively smaller effect for [3H]mazindol; Mg2+ (0.1–100 µM) had no effect; Hg2+ at ∼3 µM stimulated binding to membranes, with a relatively smaller effect for [3H]mazindol than [3H]WIN 35,428 at 0°C, and at 30–100 µM inhibited both intact cell and membrane binding; Li+ and K+ substitution (30–100 mM) inhibited binding to membranes more severely than to intact cells; and Na+ substitution was strongly stimulatory. With only a few exceptions, the patterns of ion effects were remarkably similar for both radioligands at both 0 and 21°C, suggesting the involvement of common binding domains on the hDAT impacted similarly by cations. Therefore, if there are different binding domains for WIN 35,428 and mazindol, these are not affected differentially by the cations studied in the present experiments, except for the stimulatory effect of Zn2+ at 0 and 21°C and Hg2+ at 0°C.

[3H]WIN 35,428 [2β-Carbomethoxy-3β-(4-fluorophenyl)tropane] binding to rat brain membranes: Comparing dopamine cell body areas with nerve terminal regions

Abstract Potential differences between somatodendritic and axonal dopamine transporters were examined by comparing the binding constants of [ 3 H]WIN 35,428 [2β-carbomethoxy-3β-(4-fluorophenyl)tropane] binding to membranes prepared from the rat ventral mesencephalon, containing A9 and A10 dopamine cell bodies, and from the nucleus accumbens. Saturation analysis of [ 3 H]WIN 35,428 binding, in the presence of compounds to occlude norepinephrine and serotonin transporters, was performed by both the “unlabeled” method (varying unlabeled ligand) and “labeled” method (varying radioligand). The density of binding was substantially lower in the ventral mesencephalon than in the nucleus accumbens, but the binding affinity was only slightly different. Likewise, the differences between the two regions in the inhibitory potency of cocaine and GBR 12909 [1-(2-(di(4-fluorophenyl)-methoxy)-ethyl)-4-(3-phenylpropyl)piperazine] were not substantial. The results suggest that somatodendritic and axonal dopamine transporters in the ventral mesencephalon and nucleus accumbens are not very different as far as their binding domains for uptake blockers such as cocaine and GBR 12909 are concerned.