Secretory vesicle rebound hyperacidification and increased quantal size resulting from prolonged methamphetamine exposure

Abstract

Acute exposure to amphetamines (AMPHs) collapses secretory vesicle pH gradients, which increases cytosolic catecholamine levels while decreasing the quantal size of catecholamine release during fusion events. AMPH and methamphetamine (METH), however, are retained in tissues over long durations. We used optical and electron microscopic probes to measure the effects of long-term METH exposure on secretory vesicle pH, and amperometry and intracellular patch electrochemistry to observe the effects on neurosecretion and cytosolic catecholamines in cultured rat chromaffin cells. In contrast to acute METH effects, exposure to the drug for 6-48 h at 10 microM and higher concentrations produced a concentration-dependent rebound hyperacidification of secretory vesicles. At 5-10 microM levels, prolonged METH increased the quantal size and reinstated exocytotic catecholamine release, although very high (> 100 microM) levels of the drug, while continuing to produce rebound hyperacidification, did not increase quantal size. Secretory vesicle rebound hyperacidification was temperature dependent with optimal response at approximately 37 degrees C, was not blocked by the transcription inhibitor, puromycin, and appears to be a general compensatory response to prolonged exposure with membranophilic weak bases, including AMPHs, methylphenidate, cocaine, and ammonia. Thus, under some conditions of prolonged exposure, AMPHs and other weak bases can enhance, rather than deplete, the vesicular release of catecholamines via a compensatory response resulting in vesicle acidification.

Fig. 1

Time course of METH-induced vesicle acidification. Time course of vesicle pH changes assessed by the retention of AO in cells treated with 10 μM and 100 μM METH. The upper bar shows the time scale of two consecutive METH additions (solid arrows) and a drug washout (open arrow). AO was added for 10 min before each measurement. Data point represent mean fluorescence intensities ± SEM from N = 30 – 40 cells. Dotted line and shadowed box represent the mean ± SEM in control cells at time zero.

Fig. 2

In situ measurement of vesicular pH in chromaffin secretory vesicles using LSYB. (a) Maximal projection two-photon images of LSYB-stained chromaffin cell. Two emission channels 445 nm (Blue) and 510 nm (Yellow) were pseudo-colored as green and red, correspondingly. Note the punctate LSYB staining with negligible signal from the cytosol. Scale bar is 10 μm. (b) Examples of LSYB epifluorescence images taken at 510 nm (Y) and 445 nm (B) from untreated cells and cells exposed to 100 μM METH for 4 min or 24 h; λ_ex = 365 nm. Scale bar is 10 μm. (c) Calibration curve for ratiometric measurement of vesicular pH. (d) Time course of vesicular pH changes in chromaffin cells acutely treated with 100 μM METH, 100 μM chloroquine and 10 mM ammonium chloride. N = 10 cells at each experimental group. (e) Vesicular proton concentration and pH in cells treated with different METH concentrations for 24 h. All treatment groups were significantly different from untreated cells (p < 0.005 by one-way ANOVA). Dotted line and shadowed box represent the mean ± SEM in untreated cells.

Fig. 3

Dependence of vesicle acidity on weak-base properties of various drugs. (a) Vesicle pH in chromaffin cells treated for 24 h with drugs at 10 μm (grey bars) and 100 μM (black bars) concentrations. N = 30 cells in each group, * - p < 0.05 compared to untreated cells. (b) Dependence of vesicular pH on pKa values of the drugs. All weak bases were used at 100 μM concentrations for 24 h. Acidity of vesicles in cells treated with 50 μM puromycin (pKa = 7.2) for 6 h is shown as the leftmost point. The values of pKa for other drugs were form the online Hazardous Substances Data Bank (http://toxnet.nlm.nih.gov/cgi-bin/sis/htmlgen?HSDB): AMPH – 10.1, METH – 9.9, MPh – 8.8, cocaine – 8.6. (c) Dose-dependent accumulation of AO in vesicles of chromaffin cell treated with ammonium chloride for 24 h. Dotted line and shadowed box represent the mean ± SEM in cells treated with 100 µM METH for 24 h.

Fig. 4

Immunoelectron microscopy of chromaffin granules in METH-treated cells. (a) Representative micrographs of DAMP-stained untreated cells and cells treated with 100 μM METH for 1 h or 24 h. The scale bar is 500 nm. Insets show lysosomes at the same scale. (b) Quantification of vesicular and lysosomal pH (see Materials and Methods). * - p<0.05 from untreated cells. (c) Cumulative distribution of pH values of individual secretory vesicles in untreated and METH-treated chromaffin cells (N = 212 vesicles in control, 146 in 1h METH, and 224 in 24h METH). All curves are different from each other with p<0.001 by Kolmogorov-Smirnov test.

Fig. 5

Involvement of enzymatic component in METH-induced vesicle hyperacidification. (a) Effect of translation inhibitor, puromycin, on METH-induced hyperacidification of chromaffin secretory vesicles. Cells were treated with 100 μM METH for 6 h with or without 50 μM puromycin, followed by pH measurements with LSYB. N = 30 – 40 cells, * - p < 0.005 compared to untreated cells. (b) Temperature dependence of vesicle pH in control cells and those treated with 100 –M METH for 6 hours. N = 40 cells in each group. (c) Temperature dependence of METH-induced vesicle hyperacidification calculated as the difference between the two curves on (b).

Fig. 6

METH concentration dependences of vesicular and cytosolic catecholamine contents. (a) Quantal size vs. METH concentration in cells treated with the drug for 24 h. Data are presented as the means ± SEM of the median Q_N values from individual cells (N = 97 cells in control and 11 – 43 in METH-treated groups). (b) Cytosolic catecholamine concentrations in cells incubated with METH for 24 h (N = 22 – 46 cells). * - p < 0.05 compared to untreated cells. Dotted line and shadowed box represent the mean ± SEM in untreated cells.

Fig. 7

Effects of 10 μM METH on exocytotic quantal size in chromaffin cells. (a) Representative amperometric recordings of quantal catecholamine release from untreated cells and cells exposed to 10 μM METH for 1 h or 24 h. (b) Time dependence of quantal sizes in cells incubated with 10 μM METH. After 24 h of treatment, the cells were washed with drug-free media and incubated for additional 6 h before the amperometric recordings (grey bar). Data presented as percentages of changes relative to matched untreated sister cultures at the same day. * - p < 0.05 compared to untreated cells (see also Table 1). (c) Normal probability plots of the Log-transformed Q_N from untreated cells (N = 6,522 events) and cells treated with 10 μM METH for 1 h (N = 963 events), 24 h (N = 3,045) or 48 h (N = 955).

Fig. 8

Relationship between the square of the vesicle proton concentration and quantal size. The values for Q_N are the averages of cell medians from untreated cells and those treated with 10 μM METH for 24 and 48 h. The square of the vesicular proton concentration, which has been found in empirical studies to be directly related to vesicular catecholamine concentration (see Methods), was calculated from ratiometric pH measurements as: [H]² = (10^−pH)².