Whether amphetamine acts principally at the plasma membrane or at synaptic vesicles is controversial. We find that d-amphetamine injection into the Planorbis giant dopamine neuron causes robust dopamine release, demonstrating that specific amphetamine uptake is not required. Arguing for action at vesicles, whole-cell capillary electrophoresis of single Planorbis dopamine neurons shows that amphetamine reduces vesicular dopamine, while amphetamine reduces quantal dopamine release from PC12 cells by > 50% per vesicle. Intracellular injection of dopamine into the Planorbis dopamine neuron produces rapid nomifensine-sensitive release, showing that an increased substrate concentration gradient is sufficient to induce release. These experiments indicate that amphetamine acts at the vesicular level where it redistributes dopamine to the cytosol, promoting reverse transport, and dopamine release.

Quantification of vesicular transmitter contents is important for studying the mechanisms of neurotransmission and malfunction in disease and yet it is incredibly difficult to measure the small contents of neurotransmitters in the attoliter volume of a single vesicle, especially in the cell environment. We introduce a novel method, intracellular vesicle electrochemical cytometry. A nano-tip conical carbon fiber microelectrode is used to electrochemically measure the total contents of electroactive neurotransmitters from individual nanoscale vesicles in single PC12 cells as these vesicles lyse on the electrode inside the living cell. The results demonstrate that only a fraction of quantal contents of neurotransmitter is released during exocytosis. These data support the intriguing hypothesis that the vesicle does not open all the way during the normal exocytosis process, resulting in incomplete expulsion of the vesicular contents.

Biological membrane fusion is crucial to numerous cellular events, including sexual reproduction and exocytosis. Here, mass spectrometry images demonstrate that the low-curvature lipid phosphatidylcholine is diminished in the membrane regions between fusing Tetrahymena, where a multitude of highly curved fusion pores exist. Additionally, mass spectra and principal component analysis indicate that the fusion region contains elevated amounts of 2-aminoethylphosphonolipid, a high-curvature lipid. This evidence suggests that biological fusion involves and might in fact be driven by a heterogeneous redistribution of lipids at the fusion site.During membrane fusion, two adjacent lipid bilayers merge and a channel (a fusion pore) forms, which joins the aqueous volumes initially enclosed within the membranes. The protozoan Tetrahymena thermophila (Fig. 1A) is an attractive cell system for membrane fusion studies because it is possible to induce the simultaneous formation of hundreds of fusion pores within a well-defined membrane region of about 8 μm (1,2). During mating, or conjugation (Fig. 1B), the membranes of two complementary Tetrahymena join at the anterior end and 100-to 200-nm-sized fusion pores form (Fig. 1C) to allow the migration of micronuclei between the cells. Conjugation depends on de novo lipid synthesis (3), and Tetrahymena can readily modify the lipid composition of the cell membrane via intracellular lipid exchange (4). Thus, it seems likely that certain types of lipid are required to allow the mass formation of fusion pores, and that these biophysically relevant lipids may be synthesized or redirected to the fusion site, called the conjugation junction. Additionally, in preparation for conjugation, these cells actively modify their pattern of protein synthesis, and the anterior ends of the cells transform from pointed to blunt in shape and from ciliated and ridged to smooth in texture (2). The dependence of conjugation on lipid synthesis, the membrane morphological changes, and the excess of fusion pores might suggest that there are substantial spatial alterations in the chemistry of the membrane bilayer in the conjugation junction.The cellular machinery and thermodynamic driving forces behind biological fusion are a bit of an enigma, although it is widely believed that the machinery involves an intricate cooperation between membrane proteins, the cytoskeletal framework, and lipids (5,6). The interaction of complementary membrane proteins might dictate the location of fusion and regulate the fusion events (5), and the cell cytoskeleton might play a role by confining fusogenic proteins to domains where fusion occurs in the membrane (6). The existence of biophysically functional lipid domains, or rafts, which are membrane regions concentrated in a particular type of lipid, is well documented (7-10). Lipid movement through the membrane can be restricted and create a heterogeneous distribution of lipids. These structures appear to involve longer-term or semipermanent formations and may drive biol...

Evidence has been obtained that catecholamines and their metabolites are present in single lymphocytes and extracts of T-and B-ceUl clones by use of capiUlary electrophoresis with electrochemical detection. Pharmacological inhibition of tyrosine hydroxylase reduces observed catecholamine levels, suggesting catecholamine synthesis by lymphocytes. Intracelular dopamine levels are shown to be increased by extracellular dopamine, suggesting a cellular-uptake mechanism. Furthermore, incubation with either dopamine or L-dihydroxyphenylalanine, a precursor of dopamine, results in a dosedependent inhibition of lymphocyte proliferation and differentiation. Together, these results suggest the presence of an autocrine loop whereby lymphocytes down-regulate their own activity.