Ageladine A, a pyrrole–imidazole alkaloid from marine sponges, is a pH sensitive membrane permeable dye

Abstract

a b s t r a c tThe alkaloid ageladine A, a pyrrole-imidazole alkaloid isolated from marine Agelas sponges shows fluorescence in the blue-green range during excitation with UV light with the highest absorption at 370 nm. The fluorescence of this alkaloid is pH dependent. Highest fluorescence is observed at pH 4, lowest at pH 9 with the largest fluorescence changes between pH 6 and 7. Ageladine A is brominated, which facilitates membrane permeation and therefore allows for easy staining of living cells and even whole transparent animal staining. To calculate the exact pH in solutions, cells, and tissues, the actual concentration of the alkaloid has to be known. A ratiometric measurement at the commonly used excitation wavelengths at 340/380 nm allows pH measurements in living tissues with an attenuated influence of the ageladine A concentration on calculated values. The fluorescence changes report small intracellular pH changes induced by extracellular acidification and alkalization as well as intracellular alkalization induced by ammonium chloride.Ó 2008 Elsevier Inc. All rights reserved.The pyrrole-imidazole alkaloid ageladine A was first isolated and described by Fujita et al.[1] using bioassay guided fractionation of extracts of the marine sponge Agelas nakamurai.The alkaloid showed biological effects such as the inhibition of matrix metallo-proteinases and the inhibition of cell migration of bovine endothelial cells. In 2006 the total synthesis of ageladine A was completed by the groups of Weinreb and Karuso [2,3] and later optimized [4,5].Brominated pyrrole-imidazole alkaloids are known to be fish feeding deterrent against the reef fish Thalassoma bifasciatum [6,7] and demonstrate antibiotic activity [8] even against pathogens like Helicobacter pylori [9]. Especially, the degree of bromination and the guanidine moieties have shown to be important for the alkaloids efficacy to disturb cellular calcium ion entry via voltage operated channels in neuroendocrine cells [10][11][12], which possess mainly L-and N-type calcium channels common in neuronal cells. During these fluorescence measurements of cellular effects by pyrrole-imidazole alkaloids, ageladine A was noticed to show fluorescence during UV excitation [12], which was also described earlier by Fujita et al. [1]. We demonstrate here other surprising properties of ageladine A such as its sensitivity to pH changes covering a wide range and because of its high membrane permeability an easy whole animal pH sensitive staining. Material and methodsCulture methods. PC12 cells from the DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany) were kept in culture medium containing RPMI 1640, 10% fetal calf serum, 5% horse serum, and 100 U penicillin/streptomycin per milliliter. Cells were cultivated in an incubator at 37°C, 90% humidity and 5% CO 2 with regular medium changes twice a week or when additionally necessary. Cells grew in culture flasks and 1-2 days prior to the experiments were seeded into petri dishes coated with coll...

The physiological response of the marine platyhelminthMacrostomum lignanoto different environmental oxygen concentrations

“…For Ageladine-A (Fujita et al, 2003), this was done following a previously described method (Bickmeyer et al, 2008;Bickmeyer, 2012) in which the average values obtained for each of the treatments are expressed as a proportion of the control values, and by assuming that non-specialized cells under these control conditions would present a putative pH i of 7.3-7.4. In contrast, as BCECF fluorescence intensity at 490nm linearly increases between pH6.4 and 7.8 (Silver, 2003), BCECF results were converted to pH i by constructing a calibration curve using the nigericin technique (nigericin sodium salt, 5mg, N7143, Sigma-Aldrich, St Louis, MO, USA) described previously (Thomas et al, 1979).…”

Tracking of fast moving neuronal vesicles with ageladine A

“…Ageladine A has been reported to be a moderate inhibitor of matrix metalloproteases and to be intensely fluorescent [13]. This fluorescence is pH dependent as it is stronger under acidic conditions and barely detectable in alkaline solutions [15]. The synthesis of ageladine A has been described by several groups [16,17,18,19], which provides a ready source of this compound and a viable alternative to isolation from natural sources.…”

“…The synthesis of ageladine A has been described by several groups [16,17,18,19], which provides a ready source of this compound and a viable alternative to isolation from natural sources. As ageladine A is pH sensitive and a useful dye for isolated cells as well as transparent whole animals [15] we postulated that the dye might be useful for staining acidic vesicles inside cells.…”

Inhibition of multidrug/xenobiotic resistance transporter by MK571 improves dye (Fura 2) accumulation in crustacean tissues from lobster, shrimp, and isopod

“…These small compounds in prey and food may be secondary metabolites produced in algae or sponges which show in part feeding deterrence (Pawlik et al, 1995;Assmann et al, 2000) as well as disturbance of neuronal and hormonal signalling in mammalian endocrine cells (Bickmeyer et al, 2004;Bickmeyer, 2005;Hassenklöver et al, 2006). Some of these natural products seem to be not expelled from living tissues and cells and are therefore under suspicion to inhibit-beside other effects-xenobiotic transporters (Bickmeyer et al, 2008b). In the investigated midgut glands only H. gammarus showed increased dye accumulation in the presence of MK571.…”