Reserpine As A Fluorescent Probe For Catecholaminergic Vesicle Tracking

Lindsey van Gemeren, Peter Sidaway, Keith Brain. University of Birmingham, Birmingham, West Midlands, UK.

Reserpine was one of the earliest clinical antihypertensives (Achor et al., 1955), acting through the depletion of vesicular catecholamines, and is receiving contemporary support for its reintroduction as an antihypertensive (Slim et al., 2011). It is a vesicular monoamine transporter (VMAT) inhibitor (Erickson et al., 1992). Reserpine fluorescence has been noted in analytical chemistry (Calatayud and Benito, 1991), 0however this property has not exploited in biological research. The fluorescence of reserpine and related alkaloids could be a useful indicator of catecholamine-containing vesicles.

Reserpine (10 µM) was incubated with cultured PC-12 cells, a widely used model for catecholamine storage due to their large dense core vesicles (LDCVs). Reserpine location was monitored using conventional fluorescence microscopy (405 nm LED light source). Male BALB/c mice (8-12 weeks of age) were humanely sacrificed in accordance with the European Communities Council Directives (86/609/EEC, 24 November 1986). Isolated murine vasa deferentia were supported in an oxygenated Kreb’s solution and exposed to 10 µM reserpine. The uptake was imaged by laser scanning confocal microscopy using a 405nm laser.

Fluorescence excitation and emission spectra showed that reserpine has a maximum excitation at 396nm and maximum emission at 504nm. Reserpine accumulated within PC-12 cells in the low micromolar range (n = 4), with visible punctuate regions, attributed to the LDCVs. Punctate fluorescence increased linearly over 90 minutes, whilst the mean cellular fluorescence remained constant after 30 minutes (F test, n = 5, P < 0.05). Pre-incubation with tetrabenazine (TBZ; 10 µM), a non-competitive VMAT inhibitor, did not significantly affect the rate of accumulation of punctate fluorescence (from an arbitrary rate of 3.13 min-1, R2 = 0.89, to 3.67 min-1, R2 = 0.91, unpaired t-test, P = NS), arguing against an active mechanism of uptake. Reserpine was relatively stable under continuous excitation, with a 30% loss of signal over 2.5 minutes of continuous exposure (n = 4). In reserpine loaded isolated murine vasa deferentia, strings of puncta were visible, consistent with the well-characterised sympathetic nerve terminal structures found in this tissue.

Reserpine is presented as a potential fluorescent probe for cellular imaging. It could have particular uses in vesicle tracking during imaging of neural and endocrine systems. It can be used at 10μM for cellular imaging, and chemical or photochemical oxidation to 3,4-dehydroreserpine will increase the sensitivity. The insignificant effect of the VMAT inhibitor TBZ on the vesicular uptake argues against transport of reserpine through VMAT, but does not exclude binding or lipophilic interactions. Intravesicular pH may induce oxidation and hence lead to the apparent localization. Future work would aim to further characterise the mode of vesicular and cellular accumulation.

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