Margaret Elizabeth Smith

Dopamine transporter phosphorylation site threonine 53 is stimulated by amphetamines and regulates dopamine transport, efflux, and cocaine analog binding

The dopamine transporter (DAT) controls the spatial and temporal dynamics of dopamine neurotransmission through reuptake of extracellular transmitter and is a target for addictive compounds such as cocaine, amphetamine (AMPH), and methamphetamine (METH). Reuptake is regulated by kinase pathways and drug exposure, allowing for fine-tuning of clearance in response to specific conditions, and here we examine the impact of transporter ligands on DAT residue Thr-53, a proline-directed phosphorylation site previously implicated in AMPH-stimulated efflux mechanisms. Our findings show that Thr-53 phosphorylation is stimulated in a transporter-dependent manner by AMPH and METH in model cells and rat striatal synaptosomes, and in striatum of rats given subcutaneous injection of METH. Rotating disc electrode voltammetry revealed that initial rates of uptake and AMPH-induced efflux were elevated in phosphorylation-null T53A DAT relative to WT and charge-substituted T53D DATs, consistent with functions related to charge or polarity. These effects occurred without alterations of surface transporter levels, and mutants also showed reduced cocaine analog binding affinity that was not rescued by Zn2+. Together these findings support a role for Thr-53 phosphorylation in regulation of transporter kinetic properties that could impact DAT responses to amphetamines and cocaine.

III. Vitamin A Deficiency on Concentration of Sugar, Alkaline Reserve, and Glycogen Content of the Liver.∗

We have employed 24 animals, 29 to 55 days of age, that were transferred from our stock diet No. 2 1 to vitamin A deficient rations. The experimental basal ration used in this work had the following composition: Casein (hot-alcohol extracted) 20; Northwestern yeast 10; salts No. 185, 2 4; lard 2; dextrin 64, irradiated for 30 minutes to insure an adequacy of vitamin D. In a number of experiments the lard in the ration was replaced by 1 to 2% of butter fat, in order to prolong the experimental period, so that the animals would be suffering from an insufficiency of vitamin A rather than from complete depletion of this dietary factor. This was done to prevent sudden death from pneumonia which occurs when all traces of vitamin A are removed from purified diets. The period of experimentation ranged from 80 to 150 days. The methods used for the determination of blood constituents have been reported in previous publications. 3 , 4 The same technique was employed in our studies of vitamins D and G deficiencies, the results of which follow in the subsequent articles. In this study, as well as in those that follow, daily records were kept of food and water intake in the case of all animals, and the blood sugars were determined twice weekly. In addition, determinations of specific gravity were made at each bleeding, in order to obtain information on blood concentration. Our results show that in various stages of vitamin A deficiency characterized by the severity of eye lesions, there are no significant changes in the concentration of true blood sugar. The figures approximate those found in animals on satisfactory diets. 3 In a good many instances the concentration of apparent sugar is considerably higher in the pathological animals than in the controls.

Vitamin Requirements of Nursing Young. IX. Effect of Vitamin B Deficiency on Glycogen Content of Liver of Nursing Young of Albino Rat.

In this study we have employed 50 nurslings, 29 control and 21 pathological. The animals were sacrificed by cutting both carotid arteries with a sharp scissors. The vagus nerves were also severed and death occurred almost instantaneously. The chemical method used for determination of glycogen of the livers was one kindly furnished us by Dr. C. F. Cori, which is a modification of the Pflueger method 1 used by Cori and Cori in their studies on “The Fate of Sugar in the Animal Body.” 2 In the case of normal animals and those animals whose livers contained appreciable amounts of glycogen, the Folin-Wu method 3 of determining apparent sugars was used. In cases where the livers contained very small amounts of glycogen we substituted the Folin micro-ferricyanide method. 4 The glycogen content of the livers was expressed in terms of glucose, i. e., total reducing sugars. It was found that the glycogen content of the livers of the nurslings on maternal stock diet No. 1, 5 which is the control ration, varied from 1300 to 1600 mg. of glucose per 100 gm. of liver. On the other hand, the glycogen content of the livers of the polyneuritic nursing young 6 showed a range of 20 to 110 mg. of glucose per 100 gm. of liver. Expressed per 100 gm. of body weight, the glycogen content of the control animals showed a range of 45 to 56 mg. of glucose; whereas, the pathological group showed a range of 0.8 to 6.2 mg. of glucose. The marked decrease in the glycogen content of the liver is the most pronounced chemical change we have found to date in nursing young suffering from uncomplicated vitamin B deficiency.