Effect of CH3HgCl and several transition metals on the dopamine neuronal carrier; peculiar behaviour of Zn2+.

CH3Hg+ and metal ions inhibited the specific binding of (1-[2-(diphenylmethoxy)ethyl]-4-(3-phenyl-2-[1-3H]propenyl) piperazine) ([3H]GBR 12783) to the dopamine neuronal carrier present in membranes from rat striatum with a general rank order of potency CH3Hg+ > Cu2+ > Cd2+ > Zn2+ > Ni2+ = Mn2+ = Co2+, suggesting that -SH groups are chiefly involved in this inhibition. Five millimolar dithiothreitol reversed the rather stable block of the specific binding produced by Cd2+ or Zn2+. An increase in the concentration of Na+, or addition of either K+ or Ca2+ reduced the inhibitory effects of metal cations, except Cu2+. Zn2+ (3 microM) reduced the inhibitory potency of Cd2+ on the binding but was ineffective against CH3Hg+ and Cu2+. Zn2+ at 0.3 to 10 microM significantly enhanced the specific binding of [3H]GBR 12783 and [3H]cocaine by 42 to 146%. Zn2+ (3 microM) increased the affinity of all pure uptake inhibitors tested and of the majority of the substrates for the [3H]GBR 12783 binding site. Dissociation experiments revealed that Zn2+ both inhibited and enhanced the [3H]GBR 12783 binding by recognizing amino acids located close to or in the radioligand binding site. Micromolar concentrations of Zn2+ noncompetitively blocked the [3H]dopamine uptake but they did not modify the block of the transport provoked by pure uptake inhibitors. These findings suggest that Na+, K+, Ca2+ and metal ions could recognize some -SH groups located in the [3H]GBR 12783 binding site; low concentrations of Zn2+ could allow a protection of these -SH groups.

Effects of several cations on the neuronal uptake of dopamine and the specific binding of [3H]GBR 12783: attempts to characterize the Na+ dependence of the neuronal transport of dopamine.

We have studied the effects of several cations on (1) the neuronal uptake of [3H]dopamine ([3H]DA) and (2) the specific binding of 1-[2-(diphenylmethoxy)ethyl]-4-(3-phenyl-2-[1-3H]propenyl)piperazi ne ([3H]GBR 12783) to a site associated with the neuronal carrier of DA, in preparations obtained from rat striatum. When studied under the same experimental conditions, both the uptake of [3H]DA and the binding of [3H]GBR 12783 were similarly impaired by the gradual replacement of NaCl by sucrose. In both processes, no convenient substitute for Na+ was found. Furthermore, potential substitutes of Na+ acted as inhibitors of the uptake with a rank order of potency as follows: K+ = Li+ > or = Cs+ > or = Rb+ > choline+ > Tris+ > sucrose, which was somewhat different from that observed in binding studies, i.e., Cs+ > Rb+ > choline+ > or = K+ > Li+ > Tris+ > sucrose. In the presence of either 36 mM or 136 mM Na+, [3H]DA uptake was optimal with 2 mM Mg2+, 1 mM K+, or 1 mM Ca2+. In contrast, higher concentrations of divalent cations competitively blocked the uptake process. K+ concentrations > 50 mM impaired the specific binding, whereas in the millimolar range of concentrations, K+ noncompetitively inhibited the uptake. Decreasing the Na+ concentration increased the inhibitory effect of K+, Ca2+, and Mg2+ on the specific uptake. An increase in NaCl concentration from 0 to 120 mM elicited a significant decline in the affinity of some substrates for the [3H]GBR 12783 binding site. An uptake study performed using optimal experimental conditions defined in the present study revealed that decreasing Na+ concentration reduces the affinity of DA for the neuronal transport. We propose a hypothetical model for the neuronal transport of DA in which both Na+ and K+ membrane gradients are involved.

Kinetic analysis of the chloride dependence of the neuronal uptake of dopamine and effect of anions on the ability of substrates to compete with the binding of the dopamine uptake inhibitor GBR 12783.

The specific binding of [3H]1-[2-(diphenyl-methoxy)ethyl]-4-(3-phenyl-2-propenyl)piperazine ([3H]GBR 12783) to the dopamine (DA) neuronal carrier present in membranes prepared from rat striatum was not affected when Cl- was replaced by either Br- or NO3-. In media containing Cl-, Br-, or NO3-, d-amphetamine and DA competed with the radioligand in a monophasic manner with Hill coefficients of close to 1 (0.94-1.12). Replacement of Cl- by Br- impaired the ability of some substrates (d-amphetamine, DA, p-hydroxyamphetamine, and m-tyramine) to compete with [3H]GBR 12783. The potency of Br- to decrease the affinity of substrates for the specific binding site was significantly correlated (t = 7.07, p less than 0.001) with their affinity for this binding site. These results suggest that the various substrates tested could bind to recognition sites in which Cl- is differently involved; as a consequence, substrates could bind to the neuronal carrier by means of partly different links. In experiments dealing with the specific uptake of [3H]DA, F-, NO3-, isethionate-, or acetate- was unable to substitute for Cl-, whereas Br- was quite a total substitute. Replacement of Cl- by equimolar concentrations of either NO3- or isethionate- resulted in inhibition curves of DA specific uptake with Hill coefficients of close to 1 (0.77 and 1.04 respectively); this indicates that both NO3- and isethionate- are devoid of inhibitory effects on neuronal uptake and are quite ineffective substitutes for Cl-.(ABSTRACT TRUNCATED AT 250 WORDS)