The protonephridium of platyhelminths including Schistosoma mansoni plays a pivotal role in their survival by excretion of metabolic wastes as well as xenobiotics, and can be revealed in the living adult parasite by certain fluorescent compounds which are concentrated in excretory tubules and collecting ducts. To determine the presence of the multidrug resistance-associated protein (MRP) as a possible transporter in protonephridial epithelium, adult schistosomes were exposed to a fluorescent Ca2+ indicator, fluo-3 acetyloxymethyl ester, which is a potential substrate of mammalian MRP. Specific fluorescence related to fluo-3/Ca2+ chelate delineated the whole length of the protonephridial system. Simultaneously, a fluorescent substance was accumulated in the posterior part of collecting ducts and the excretory bladder. Similarly, when other fluorogenic substrates for mammalian MRP such as monoclorobimane, fluorescein diacetate, and 5(6)-carboxyfluorescein diacetate were applied to adult schistosomes, these fluorescent markers were observed in the excretory tubules through to the excretory bladder. The excretory system of mechanically-transformed schistosomula was not labelled with any of these 4 fluorescent markers. These findings suggest that the protonephridial epithelium of adult schistosomes, but not schistosomula, might express the homologue of the mammalian MRP transporting organic anionic conjugates with glutathione, glucuronate or sulphate as well as unconjugated amphiphilic organic anions.

A confocal attachment (Odyssey) to an inverted microscope was modified to better study living cultured epithelial cells stained with fluorescent dyes. Improvements to the instrument included elimination of light leaks, improved electronic shielding, reduction of thermal effects, and use of low dark current detectors. In addition, rapid changes in illumination wavelength and power were accomplished by replacing the original mechanical filter changer by an acousto-optic tunable filter attached to the argon laser light source. The addition of a liquid crystal tunable filter to one of the two photomultiplier detectors also permitted rapid spectral scanning of the fluorescence emission. High-resolution, differential interference contrast transmitted light images were formed simultaneously by replacement of the photodiode-based transmitted light detector with a photomultiplier tube and dichroic mirror assembly. An illumination intensity of only 40 μW/cm2 at the back focal plane of the microscope objective allowed high-quality fluorescence and transmitted light images of living cells at video rates with minimal bleaching and photodynamic damage. Both excitation ratio imaging and emission spectral scanning of living epithelial cells were accomplished. The system performance was evaluated by optical sections of fluorescent beads and thin films.