Mass Spectrometry Imaging Suggests That Cisplatin Affects Exocytotic Release by Alteration of Cell Membrane Lipids

We used time-of-flight secondary ion mass spectrometry (TOF-SIMS) imaging to investigate the effect of cisplatin, the first member of the platinum-based anticancer drugs, on the membrane lipid composition of model cells to see if lipid changes might be involved in the changes in exocytosis observed. Platinum-based anticancer drugs have been reported to affect neurotransmitter release resulting in what is called the “chemobrain”; however, the mechanism for the influence is not yet understood. TOF-SIMS imaging was carried out using a high energy 40 keV (CO₂)₆₀₀₀⁺ gas cluster ion beam with improved sensitivity for intact lipids in biological samples. Principal components analysis showed that cisplatin treatment of PC12 cells significantly affects the abundance of different lipids and their derivatives, particularly phosphatidylcholine and cholesterol, which are diminished. Treatment of cells with 2 μM and 100 μM cisplatin showed similar effects on induced lipid changes. Lipid content alterations caused by cisplatin treatment at the cell surface are associated with the molecular and bimolecular signaling pathways of cisplatin-induced apoptosis of cells. We suggest that lipid alterations measured by TOF-SIMS are involved, at least in part, in the regulation of exocytosis by cisplatin

Single-Cell Lipidomics: Characterizing and Imaging Lipids on the Surface of Individual Aplysia californica Neurons with Cluster Secondary Ion Mass Spectrometry

Neurons isolated from Aplysia californica, an organism with a well-defined neural network, were imaged with secondary ion mass spectrometry, C₆₀-SIMS. A major lipid component of the neuronal membrane was identified as 1-hexadecyl-2-octadecenoyl-<i>sn</i>-glycero-3-phosphocholine [PC­(16:0e/18:1)] using tandem mass spectrometry (MS/MS). The assignment was made directly off the sample surface using a C₆₀-QSTAR instrument, a prototype instrument that combines an ion source with a commercial electrospray ionization/matrix-assisted laser desorption ionization (ESI/MALDI) mass spectrometer. Normal phase liquid chromatography mass spectrometry (NP-LC–MS) was used to confirm the assignment. Cholesterol and vitamin E were also identified with in situ tandem MS analyses that were compared to reference spectra obtained from purified compounds. In order to improve sensitivity on the single-cell level, the tandem MS spectrum of vitamin E reference material was used to extract and compile all the vitamin E related peaks from the cell image. The mass spectrometry images reveal heterogeneous distributions of intact lipid species, PC­(16:0e/18:1), vitamin E, and cholesterol on the surface of a single neuron. The ability to detect these molecules and determine their relative distribution on the single-cell level shows that the C₆₀-QSTAR is a potential platform for studying important biochemical processes, such as neuron degeneration

Mechanistic Aspects of Vesicle Opening during Analysis with Vesicle Impact Electrochemical Cytometry

Vesicle impact electrochemical cytometry (VIEC) has been used to quantify the vesicular transmitter content in mammalian vesicles. In the present study, we studied the mechanism of VIEC by quantifying the catecholamine content in single vesicles isolated from pheochromocytoma (PC12) cells. These vesicles contain about one tenth of the catecholamine compared with adrenal chromaffin vesicles. The existence of a prespike foot for many events suggests the formation of an initial transiently stable pore at the beginning of vesicle rupture. Increasing the detection temperature from 6 to 30 °C increases the possibility of vesicle rupture on the electrode, implying that there is a temperature-dependent process that facilitates electroporation. Natively larger vesicles are shown to rupture earlier and more frequently than smaller ones in VIEC. Likewise, manipulating vesicle content and size with drugs leads to similar trends. These data support the hypothesis that electroporation is the primary force for pore opening in VIEC. We further hypothesize that a critical step for initiating vesicle opening by electroporation is diffusion of membrane proteins away from the membrane region of contact with the electrode to allow closer contact, increasing the lateral potential field and thus facilitating electroporation

Distribution of the decay time in (A) PACAP and (B) L-DOPA treated PC12 cells.

<p>Histograms indicate the existence of two populations of spikes that can be well fitted with two Gaussian functions. PACAP reduces the proportion of rapid spikes of the decay time. Moreover, PACAP shortens the decay time of both fast and slow spikes. In contrast, distributions of decay time of both fast and slow spikes are shifted to the right by the treatment of L-DOPA.</p

Histograms of the cube root of quantal size, amplitude and half-width for control and PACAP-treated PC12 cells (n = 482 events for control cells, n = 1436 events for PACAP-treated cells).

<p>Distribution of cube root transforms of the quantal sizes can be fitted with one Gaussian function in both control and PACAP-treated cells.</p

Representative amperometric foot current transients (A) and summary of foot duration, foot quantal size and mean catecholamine flux (B).

<p>Flux was computed as foot area divided by duration. Error bars represent mean ± SEM (control, 93 events; PACAP, 146 events and L-DOPA, 52 events). *** p<0.001 and ** p<0.01 vs. control, respectively (ANOVA test).</p

Representative TEM images of (A) control and (B) PACAP-treated cells.

<p>Large dense core vesicles are distributed near and far from the plasma membranes. A portion of the nucleus can be seen in the cells. Scale bars = 200 nm. (C) Mean vesicle sizes of control and PACAP-treated cells (n = 17 cells from control group, n = 24 cells for PACAP-treated group; **<i>p</i><0.01 vs. control cells, <i>t-</i>test).</p

Summary of the shape characteristics of individual release events from control, PACAP-treated, and L-DOPA-treated cells.

<p>*<i>p</i><0.05 vs. control,</p><p>**<i>p</i><0.01 vs. control.</p

Summary of rise time and decay time in PACAP and L-DOPA treated cells.

<p>Error bars represent mean ± SEM (control, 1767 events; PACAP, 1436 events and L-DOPA, 2246 events). Significance: *** p<0.001 vs. control, respectively (ANOVA test).</p

Capillary Electrophoresis–Mass Spectrometry-Based Detection of Drugs and Neurotransmitters in Drosophila Brain

Capillary electrophoresis coupled to mass spectrometry has been used to determine the in vivo concentrations of the neuroactive drug, methylphenidate, and a metabolite in the heads of the fruit fly, Drosophila melanogaster. These concentrations, evaluated at the site of action, the brain, have been correlated with orally administrated methylphenidate. D. melanogaster has a relatively simple nervous system but possesses high-order brain functions similar to humans; thus, it has been used as a common model system in biological and genetics research. Methylphenidate has been used to mediate cocaine addiction due to its lower pharmacokinetics, which results in fewer addictive and reinforcing effects than cocaine; the effects of the drug on the nervous system, however, have not been fully understood. In addition to measurements of drug concentration, the method has been used to examine drug-dose dependence on the levels of several primary biogenic amines. Higher in vivo concentration of methylphenidate is observed with increasing feeding doses up to 25 mM methylphenidate. Furthermore, administrated methylphenidate increases the drug metabolism activity and the neurotransmitter levels; however, this increase appears to saturate at a feeding dose of 20 mM. The method developed for the fruit fly provides a new tool to evaluate the concentration of administered drug at the site of action and provides information concerning the effect of methylphenidate on the nervous system