The dopamine transporter constitutively internalizes and recycles in a protein kinase C-regulated manner in stably transfected PC12 cell lines

The dopamine transporter (DAT) removes dopamine from the extracellular milieu and is potently inhibited by number of psychoactive drugs, including cocaine, amphetamines, and methylphenidate (Ritalin). Multiple lines of evidence demonstrate that protein kinase C (PKC) down-regulates dopamine transport, primarily by redistributing DAT from the plasma membrane to endosomal compartments, although the mechanisms facilitating transporter sequestration are not defined. Here, we demonstrate that DAT constitutively internalizes and recycles in rat pheochromocytoma (PC12) cells. Temperature blockades demonstrated basal internalization and reliance on recycling to maintain DAT cell surface levels. In contrast, recycling blockade with bafilomycin A1 significantly decreased transferrin receptor (TfR) surface expression but had no effect on DAT surface levels, suggesting that DAT and TfR traffic via distinct endosomal mechanisms. Kinetic analyses reveal robust constitutive DAT cycling to and from the plasma membrane, independent of transporter expression levels. In contrast, phorbol ester-mediated PKC activation accelerated DAT endocytosis and attenuated transporter recycling in a manner sensitive to DAT expression levels. These data demonstrate constitutive DAT trafficking and that PKC-mediated DAT sequestration is achieved by a combination of accelerated internalization and reduced recycling. Additionally, the differential sensitivity to expression level exhibited by constitutive and regulated DAT trafficking suggests that these two processes are mediated by independent cellular mechanisms

Brief Psychotherapy for Management of Primary Headaches: a Clinical Grounded Approach

This research explores the potentialities of psychotherapy for the management of chronic pain. The model used is brief therapy of systemic orientation and the chronic pain managed is primary headaches (namely, migraines and tension-type headaches). In order to produce clinically relevant material, this research is carried out within an alternative research paradigm. The raw data are the audio-recordings of two cases: one with a man suffering from migraines; the other with a woman suffering from chronic tension-type headaches (aggravated by migraine episodes). These were selected from a pool of cases because they illustrate the phenomena under study and both completed a follow-up which confirmed an acceptable headache management outcome. The recordings were transcribed in order to be studied using discourse analysis of social constructionist orientation (DA hereafter). The research questions explored are: How were the headache problems, the therapeutic aims and the resources for managing them constructed during therapy? What did the participants do with these constructions? How was this particular type of talk interaction helpful in changing the way these two people managed their primary headaches? DA reveals that: (1) the headache problems are entangled in many vicious cycles, Catch-22 situations and even double-binds, and that these patterns have the tendency to perpetuate the problems; (2) the meaning of the headaches vary from one patient to the other, being greatly influenced by their personal experiences, family histories and interaction with health professionals; (3) these meanings influence the co-construction of the therapeutic aims, with management (rather than a cure) emerging as a more achievable goal, with additional auxiliary aims also becoming very important; (4) specific interventions for managing the headaches and for achieving the auxiliary aims lead to concrete changes; (5) these changes are sometimes generalized for other situations, and therapy is seen as a useful resource. Thus, this study shows some of the potentialities of brief therapy of systemic orientation to manage primary headaches, producing concrete suggestions that can be applied in clinical work

Measuring Plasma Membrane Protein Endocytic Rates by Reversible Biotinylation

Plasma membrane proteins are a large, diverse group of proteins comprised of receptors, ion channels, transporters and pumps. Activity of these proteins is responsible for a variety of key cellular events, including nutrient delivery, cellular excitability, and chemical signaling. Many plasma membrane proteins are dynamically regulated by endocytic trafficking, which modulates protein function by altering protein surface expression. The mechanisms that facilitate protein endocytosis are complex and are not fully understood for many membrane proteins. In order to fully understand the mechanisms that control the endocytic trafficking of a given protein, it is critical that the protein s endocytic rate be precisely measured. For many receptors, direct endocytic rate measurements are frequently achieved utilizing labeled receptor ligands. However, for many classes of membrane proteins, such as transporters, pumps and ion channels, there is no convenient ligand that can be used to measure the endocytic rate. In the present report, we describe a reversible biotinylation method that we employ to measure the dopamine transporter (DAT) endocytic rate. This method provides a straightforward approach to measuring internalization rates, and can be easily employed for trafficking studies of most membrane proteins