Co-expression studies of the orphan carrier protein Slc10a4 and the vesicular carriers VAChT and VMAT2 in the rat central and peripheral nervous system.

The orphan carrier protein Slc10a4 represents a novel member of the so-called "sodium-bile acid co-transporter family," SLC10. Slc10a4 has a close phylogenetic relationship with the liver bile acid carrier Ntcp (Slc10a1), but has no transport activity for bile acids. In a previous study Slc10a4 proved to be predominantly expressed in the rat brain, where it was localized within cholinergic neurons. However, whether this cholinergic expression pattern was exclusive for Slc10a4 and whether this protein might also be expressed in the peripheral nervous system or other peripheral organs, remained unclear. Therefore, in the present study we analyzed the expression of Slc10a4 in neuronal and non-neuronal rat tissues more systematically, employing immunofluorescence co-localization studies of the vesicular acetylcholine transporter VAChT and the vesicular monoamine transporter VMAT2. The Slc10a4 protein was found to be widely expressed throughout structures of the CNS and peripheral nervous system. In addition to cholinergic neurons in the CNS, the retina, the neuromuscular junction and parasympathetic innervations, Slc10a4 was also localized in certain monoaminergic neurons and nerve fibers in the substantia nigra, the spinal cord and sympathetic innervations. Slc10a4 expression was also detected in granules of rat peritoneal and tissue mast cells using immunofluorescence and electron microscopy. Western blot and immunoprecipitation experiments with rat brain vesicle preparations revealed that the Slc10a4 protein was expressed in synaptic vesicles where it co-localized with synaptophysin, VAChT and VMAT2. This vesicular expression pattern was also shown in the rat adrenal pheochromocytoma cell line PC12 by immunofluorescence. Based on the findings of the present study we can speculate about the function of Slc10a4 as follows: (I) Slc10a4 could be a novel vesicular transporter for cholinergic and/or various monoaminergic neurotransmitters in the central and peripheral nervous system or (II) may be involved in the regulation of the synaptic vesicle sorting or exocytosis process.

Comparative pharmacology of chemically distinct NADPH oxidase inhibitors.

BACKGROUND AND PURPOSE: Oxidative stress [i.e. increased levels of reactive oxygen species (ROS)] has been suggested as a pathomechanism of different diseases, although the disease-relevant sources of ROS remain to be identified. One of these sources may be NADPH oxidases. However, due to increasing concerns about the specificity of the compounds commonly used as NADPH oxidase inhibitors, data obtained with these compounds may have to be re-interpreted. EXPERIMENTAL APPROACH: We compared the pharmacological profiles of the commonly used NADPH oxidase inhibitors, diphenylene iodonium (DPI), apocynin and 4-(2-amino-ethyl)-benzolsulphonyl-fluoride (AEBSF), as well as the novel triazolo pyrimidine VAS3947. We used several assays for detecting cellular and tissue ROS, as none of them is specific and artefact free. KEY RESULTS: DPI abolished NADPH oxidase-mediated ROS formation, but also inhibited other flavo-enzymes such as NO synthase (NOS) and xanthine oxidase (XOD). Apocynin interfered with ROS detection and varied considerably in efficacy and potency, as did AEBSF. Conversely, the novel NADPH oxidase inhibitor, VAS3947, consistently inhibited NADPH oxidase activity in low micromolar concentrations, and interfered neither with ROS detection nor with XOD or eNOS activities. VAS3947 attenuated ROS formation in aortas of spontaneously hypertensive rats (SHRs), where NOS or XOD inhibitors were without effect. CONCLUSIONS AND IMPLICATIONS: Our data suggest that triazolo pyrimidines such as VAS3947 are specific NADPH oxidase inhibitors, while DPI and apocynin can no longer be recommended. Based on the effects of VAS3947, NADPH oxidases appear to be a major source of ROS in aortas of SHRs.

Localization of carbohydrate determinants common to Biomphalaria glabrata as well as to sporocysts and miracidia of Schistosoma mansoni.

The presence of antigenic carbohydrate epitopes shared by Biomphalaria glabrata as well as by the sporocysts and miracidia representing snail-pathogenic larval stages of Schistosoma mansoni was assayed by immunohistochemical staining of paraformaldehyde-fixed tissues. To this end, both polyclonal rabbit antiserum raised against soluble egg antigens (SEA) of S. mansoni and monoclonal antibodies recognizing the carbohydrate epitopes LDN [GalNAc(beta1-4)GlcNAc(beta1-)], F-LDN [Fuc(alpha1-3)GalNAc(beta1-4)GlcNAc(beta1-)], LDN-F [GalNAc(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-)], LDN-DF [GalNAc(beta1-4)[Fuc(alpha1-2)Fuc(alpha1-3)]GlcNAc(beta1-)] and Lewis X [Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-)] were used. Intriguingly, anti-SEA serum as well as anti-F-LDN antibodies displayed significant binding in the foot region, anterior tissue and the hepatopancreas of uninfected snails, whereas the Lewis X epitope was only weakly detectable in the latter tissue. In contrast, increased binding of antibodies recognizing LDN, LDN-F and LDN-DF was observed in infected snail tissue, in particular in regions involved in sporocystogenesis, in addition to an enhanced binding of anti-SEA serum and antibodies reacting with F-LDN. A pronounced expression of most of these carbohydrate antigens was also observed at the surface of miracidia. Hence, the detection of shared carbohydrate determinants in uninfected snail tissue, sporocysts and miracidia may support the hypothesis of carbohydrate-based molecular mimicry as a survival strategy of S. mansoni.

Immunohistochemical localization of cardio-active neuropeptides in the heart of a living fossil, Nautilus pompilius L. (Cephalopoda, Tetrabranchiata).

Neuropeptides play an important role in modulating the effects of neurotransmitters such as acetylcholine and noradrenaline in the heart and the vascular system of vertebrates and invertebrates. Various neuropeptides, including substance P (SP), vasoactive intestinal polypeptide (VIP) and FMRFamide, have been localized in the brain in cephalopods and the neurosecretory system of the vena cava. Previous studies involving cephalopods have mainly focussed on the modern, coleoid cephalopods, whereas little attention was paid to the living fossil Nautilus. In this study, the distributions of the peptides related to tachykinins (TKs) and the high affinity receptor for the best characterized TK substance P (tachykinin NK-1), VIP, as well as FMRFamide were investigated in the heart of Nautilus pompilius L. by immunohistochemistry. TK-like immunoreactivity (TK-LI) was seen associated to a sub-population of hemocytes, VIP-LI glial cells in larger nerves entering the heart, whereas FMRFamide immunoreactivity was distributed throughout the entire heart, including the semilunar atrioventricular valves. The pattern of FMRFamide immunoreactivity matched that of Bodian silver staining for nervous tissue. The NK-1-LI receptor was located on endothelial cells, which were also positive for endothelial nitric oxide synthase-LI (eNOS). The results indicate that neuropeptides may be involved in the regulation of the Nautilus heart via different mechanisms, (1) by direct interaction with myocardial receptors (FMRFamide), (2) by interacting with the nervus cardiacus (VIP-related peptides) and (3) indirectly by stimulating eNOS in the endothelium throughout the heart (TK-related peptides).

Hemocyanin re-uptake in the renal and branchial heart appendages of the coleoid cephalopod Sepia officinalis.

The renal and branchial heart appendages of Sepia officinalis L. were investigated in order to elucidate a possible involvement of their excretory epithelia in hemocyanin metabolism. Immunocytochemical findings and tracer experiments indicate that after passing the barrier of ultrafiltration the hemocyanin molecules are taken up by the epithelial cells of the renal and branchial heart appendages and are subsequently carried back to the circulatory system, suggesting a mechanism of hemocyanin recycling. Apart from a function in maintaining constant hemocyanin levels, the present study indicates that the renal and branchial heart appendages are also sites of temporary hemocyanin storage.

Tracer studies of food absorption in the digestive tract of Nautilus pompilius (Cephalopoda, Tetrabranchiata).

In Nautilus pompilius, tracer experiments with 14C-labelled food show that the midgut gland, caecum and crop are involved in absorption of nutrients. According to liquid scintillation and light- and electron-microscopic autoradiography, the midgut gland exhibits the highest activity, followed by the caecum and crop. The density of silver precipitates is highest in the terminal alveoli of the midgut gland. Precipitates are also seen in the main cells of the caecal epithelium. Few precipitates are found in the lamina epithelialis mucosae of the crop, indicating that, in addition to food storage, digestive processes begin in this organ. These results have been confirmed by injection of the protein ferritin into the buccal cavity. The largest amount of ferritin is seen in the dense bodies of the main cells of the midgut gland, whereas those of the main cells of the caecum and crop contain less ferritin.

Cytobiological studies on hemocyanin metabolism in the branchial heart complex of the common cuttlefish Sepia officinalis (Cephalopoda, Dibranchiata).

The present study confirms previous investigations that demonstrated a high copper content in the branchial heart and its appendage, and that gave the first indication that this organ complex might be involved in hemocyanin metabolism in Sepia officinalis L. Immunocytochemical localization of hemocyanin molecules within the endocytotic lysosomal system of the ovoid cells and tracer experiments with 125I-labeled Sepia hemocyanin suggest its endocytotic uptake. Energy-dispersive X-ray microanalysis and histochemical methods reveal a high copper content within the ovoid cells of the branchial heart. In view of the turnover of the respiratory pigment in the branchial heart of Sepia officinalis L., we believe that the ovoid cells are a site of hemocyanin catabolism.

Cytomorphological aspects on the response of the branchial heart complex of Sepia officinalis L. (cephalopoda) to xenobiotics and bacterial infection.

The ovoid cells of the branchial heart complex of Sepia officinalis L. were investigated with respect to their role in detoxification processes. The electron microscopical localization of in situ injected ferritin in the endocytotic-lysosomal system, and the fluorescence microscopical localization of protein-bound Evans blue in the ovoid cells of in vivo incubated animals, indicate that foreign materials are eliminated from the hemolymph by the branchial heart tissue. In addition to the non-circulating ovoid cells of the branchial heart, hemocytes in the circulating blood and in the wall of the branchial heart are also involved in the incorporation of allogeneic substances and bacteria, or their debris. Based on these observations, we propose that the ovoid cells, together with circulating and adhesive hemocytes in the branchial hearts, are an important component of a more comprehensive defence and detoxification system in dibranchiate cephalopods that prevents contamination of the whole organism by endocytotic removal of noxious substances from the hemolymph.