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Expression and immunolocalization of aquaporin-7 in rat gastrointestinal tract.

Background information. In the gastrointestinal tract of mammals, water can either be secreted with digestive juices or absorbed by the small and large intestine. Transcellular water movement can be mediated by the transmembrane protein family of AQPs (aquaporins), as has also been recently identified in the gastrointestinal tract. However, the localization, expression and functioning of AQPs in the gastrointestinal tract have not been completely characterized. For the present study, we investigated: (1) the expression of AQP7 in some portions of rat gastrointestinal tract by semiquantitative reverse transcriptase—PCR and by immunoblotting and (2) the cellular and subcellular localization of AQP7 by immunohistochemistry.

Epidermal growth factor induces intracellular Ca2+ oscillations in microvascular endothelial cells

An increase in intracellular Ca2+ concentration ([Ca2+]i) may play a role in the proliferative effect of several growth factors. In this study, the changes in [Ca2+]i elicited by epidermal growth factor (EGF) in rat cardiac microvascular endothelial cells (CMEC) have been investigated by using fura‐2 conventional and confocal microscopy. A large heterogeneity in the latency and in the pattern of the Ca2+ response was found at each dose of EGF (2.5–100 ng/ml), whereas some cells displayed a non‐oscillatory behavior and others exhibited a variable number of Ca2+ oscillations. On average, the fraction of responsive cells, the total number of oscillations and the duration of the Ca2+ signal were higher at around 10 ng/ml EGF, while there was no dose‐dependence in the lag time and in the amplitude of the [Ca2+]i increase. EGF‐induced Ca2+ spikes were abolished by the tyrosine kinase inhibitor genistein, but not by its inactive analogue daidzein, and by the phospholipase C blocker NCDC. Only 1–2 transients could be elicited in Ca2+‐free solution, while re‐addition of extracellular Ca2+ recovered the spiking activity. The oscillatory signal was prevented by the SERCA inhibitor thapsigargin and abolished by the calcium entry blockers Ni2+ and La3+. Moreover, EGF‐induced Ca2+ transients were abolished by the InsP3 receptor blocker caffeine, while ryanodine was without effect. Confocal imaging microscopy showed that the Ca2+ response to EGF was localized both in the cytoplasm and in the nucleus. We suggest that EGF‐induced [Ca2+]i increase may play a role in the proliferative action of EGF on endothelial cells.

Aquaporin-6 is expressed along the rat gastrointestinal tract and upregulated by feeding in the small intestine

BackgroundSeveral aquaporins (a family of integral membrane proteins) have been recently identified in the mammalian gastrointestinal tract, and their involvement in the movement of fluid and small solutes has been suggested. In this direction we investigated, in some regions of the rat gastrointestinal tract, the presence and localization of aquaporin-6, given its peculiar function as an ion selective channel.ResultsRT-PCR and immunoblotting experiments showed that aquaporin-6 was expressed in all the investigated portions of the rat gastrointestinal tract. The RT-PCR experiments showed that aquaporin-6 transcript was highly expressed in small intestine and rectum, and less in stomach, caecum and colon. In addition, jejunal mRNA expression was specifically stimulated by feeding.Immunoblotting analysis showed a major band with a molecular weight of about 55 kDa corresponding to the aquaporin-6 protein dimer; this band was stronger in the stomach and large intestine than in the small intestine. Immunoblotting analysis of brush border membrane vesicle preparations showed an intense signal for aquaporin-6 protein.The results of in situ hybridization experiments demonstrate that aquaporin-6 transcript is present in the isthmus, neck and basal regions of the stomach lining, and throughout the crypt-villus axis in both small and large intestine. In the latter regions, immunohistochemistry revealed strong aquaporin-6 labelling in the apical membrane of the surface epithelial cells, while weak or no labelling was observed in the crypt cells. In the stomach, an intense staining was observed in mucous neck cells and lower signal in principal cells and some parietal cells.ConclusionThe results indicate that aquaporin-6 is distributed throughout the gastrointestinal tract. Aquaporin-6 localization at the apical pole of the superficial epithelial cells and its upregulation by feeding suggest that it may be involved in movements of water and anions through the epithelium of the villi.

Osmotic water permeability of rat intestinal brush border membrane vesicles: involvement of aquaporin-7 and aquaporin-8 and effect of metal ions

Water channels AQP7 and AQP8 may be involved in transcellular water movement in the small intestine. We show that both AQP7 and AQP8 mRNA are expressed in rat small intestine. Immunoblot and immunohistochemistry experiments demonstrate that AQP7 and AQP8 proteins are present in the apical brush border membrane of intestinal epithelial cells. We investigated the effect of several metals and pH on the osmotic water permeability (Pf) of brush border membrane vesicles (BBMVs) and of AQP7 and AQP8 expressed in a cell line. Hg2+, Cu2+, and Zn2+ caused a significant decrease in the BBMV Pf, whereas Ni2+ and Li+ had no effect. AQP8-transfected cells showed a reduction in Pf in the presence of Hg2+ and Cu2+, whereas AQP7-transfected cells were insensitive to all tested metals. The Pf of both BBMVs and cells transfected with AQP7 and AQP8 was not affected by pH changes within the physiological range, and the Pf of BBMVs alone was not affected by phlorizin or amiloride. Our results indicate that AQP7 and AQP8 may play a role in water movement via the apical domain of small intestine epithelial cells. AQP8 may contribute to the water-imbalance-related clinical symptoms apparent after ingestion of high doses of Hg2+ and Cu2+.

Calyx and dimorphic neurons of mouse Scarpa's ganglion express histamine H3 receptors

BackgroundHistamine-related drugs are commonly used in the treatment of vertigo and related vestibular disorders. The site of action of these drugs however has not been elucidated yet. Recent works on amphibians showed that histamine H3 receptor antagonists, e.g. betahistine, inhibit the afferent discharge recorded from the vestibular nerve. To assess the expression of H3 histamine receptors in vestibular neurons, we performed mRNA RT-PCR and immunofluorescence experiments in mouse Scarpas ganglia.ResultsRT-PCR analysis showed the presence of H3 receptor mRNA in mouse ganglia tissue. H3 protein expression was found in vestibular neurons characterized by large and roundish soma, which labeled for calretinin and calbindin.ConclusionThe present results are consistent with calyx and dimorphic, but not bouton, afferent vestibular neurons expressing H3 receptors. This study provides a molecular substrate for the effects of histamine-related antivertigo drugs acting on (or binding to) H3 receptors, and suggest a potential target for the treatment of vestibular disorders of peripheral origin.

Histamine H1 receptors are expressed in mouse and frog semicircular canal sensory epithelia.

Histamine-related drugs are commonly used in the treatment of vertigo and related vestibular disorders. Their site and mechanism of action, however, are still poorly understood. To increase our knowledge of the histaminergic system in the vestibular organs, we have investigated the expression of H1 and H3 histamine receptors in the frog and mouse semicircular canal sensory epithelia. Analysis was performed by mRNA reverse transcriptase-PCR, immunoblotting and immunocytochemistry experiments. Our data show that both frog and mouse vestibular epithelia express H1 receptors. Conversely no clear evidence for H3 receptors expression was found.

Activation of the CREB/c-Fos Pathway during Long-Term Synaptic Plasticity in the Cerebellum Granular Layer

The induction of long-term potentiation and depression (LTP and LTD) is thought to trigger gene expression and protein synthesis, leading to consolidation of synaptic and neuronal changes. However, while LTP and LTD have been proposed to play important roles for sensori-motor learning in the cerebellum granular layer, their association with these mechanisms remained unclear. Here, we have investigated phosphorylation of the cAMP-responsive element binding protein (CREB) and activation of the immediate early gene c-Fos pathway following the induction of synaptic plasticity by theta-burst stimulation (TBS) in acute cerebellar slices. LTP and LTD were localized using voltage-sensitive dye imaging (VSDi). At two time points following TBS (15 min and 120 min), corresponding to the early and late phases of plasticity, slices were fixed and processed to evaluate CREB phosphorylation (P-CREB) and c-FOS protein levels, as well as Creb and c-Fos mRNA expression. High levels of P-CREB and Creb/c-Fos were detected before those of c-FOS, as expected if CREB phosphorylation triggered gene expression followed by protein synthesis. No differences between control slices and slices stimulated with TBS were observed in the presence of an N-methyl-D-aspartate receptor (NMDAR) antagonist. Interestingly, activation of the CREB/c-Fos system showed a relevant degree of colocalization with long-term synaptic plasticity. These results show that NMDAR-dependent plasticity at the cerebellum input stage bears about transcriptional and post-transcriptional processes potentially contributing to cerebellar learning and memory consolidation.

Expression and localization of ryanodine receptors in the frog semicircular canal.

Several experiments suggest an important role for store-released Ca2+ in hair cell organs: drugs targeting IP3 and ryanodine (RyRs) receptors affect release from hair cells, and stores are thought to be involved in vesicle recycling at ribbon synapses. In this work we investigated the semicircular canal distribution of RyRs by immunofluorescence, using slice preparations of the sensory epithelium (to distinguish cell types) and flat mounts of the simpler nonsensory regions. RyRs were present in hair cells, mostly in supranuclear spots, but not in supporting cells; as regards nonsensory regions, they were also localized in dark cells and cells from the ductus. No labeling was found in nerve terminals, although nerve branches could be observed in proximity to hair cell RyR spots. The differential expression of RyR isoforms was studied by RT-PCR and immunoblotting, showing the presence of RyRα in both ampulla and canal arm and RyRβ in the ampulla only.

Glutamic acid decarboxylase 67 expression by a distinct population of mouse vestibular supporting cells.

The function of the enzyme glutamate decarboxylase (GAD) is to convert glutamate in γ-aminobutyric acid (GABA). Glutamate decarboxylase exists as two major isoforms, termed GAD65 and GAD67, that are usually expressed in GABA-containing neurons in the central nervous system. GAD65 has been proposed to be associated with GABA exocytosis whereas GAD67 with GABA metabolism. In the present immunofluorescence study, we have investigated the presence of the two GAD isoforms in the semicircular canal cristae of wild type and GAD67-GFP knock-in mice. While no evidence for GAD65 expression was found, GAD67 was detected in a distinct population of peripherally-located supporting cells, but not in hair cells or in centrally-located supporting cells. GABA, on the other hand, was found in all supporting cells. The present result indicate that only a discrete population of supporting cells use GAD67 to synthesize GABA. This is the first report of a marker that allows to distinguish two populations of supporting cells in the vestibular epithelium. On the other hand, the lack of GABA and GAD enzymes in hair cells excludes its involvement in afferent transmission.

Immediate early genes expression in the cerebellar cortex correlates with LTP and LTD induction

The consolidation of changes following activity-dependent neural plasticity are believed to involve specific patterns of gene expression. In the hippocampus, immediate early genes are thought to contribute to long-term synaptic plasticity (LTP and LTD); this phenomenon may occur also in the cerebellum, in which the transcription factors c-Fos and P-CREB have been identified. The cerebellum granular layer (GL) can manifest both LTP and LTD following a Theta Burst Stimulus (TBS) delivered to the mossy fibers. We have employed VSD imaging in rat cerebellar slices (P18-24) in order to map the spatial distribution of LTP and LTD in the cerebellum GL. Fluorescence changes were correlated to LTP or LTD in two different post-TBS time ranges (15 and 120 min). Slices were then fixed and processed for immunohistochemistry in order to identify levels of c-Fos and P-CREB expression. The induction of long-term plasticity increased the average level of P-CREB both at 15 min (+39±4.9, p<0.01%) and 120 min (+24±7.2, p<0.05%) after TBS. The level of c-Fos was unaltered at 15 min, while it significantly increased at 120 min (+37±8.9, p<0.05%). By spatially correlating longterm synaptic plasticity with the corresponding variation of P-CREB and c-Fos, we observed that regions showing LTP well correlated (p<0.05) with positive variations of P-CREB and c-Fos. Conversely, areas showing LTD correlated exclusively (p<0.05) with negative variations of P-CREB. Slices were also evaluated by in situ hybridization and a similar analysis was performed. The levels of fos and CREB mRNA expression and their spatial correlation with the sign of long-term synaptic plasticity corresponded with the immunohistochemical results. As a further test, VSD recordings showed that the induction of granular layer LTP and LTD could be prevented by applying 50 mM D-APV, a selective NMDA receptor blocker. Moreover, in situ hybridization and immunohistochemistry analysis evidenced that in these conditions both mRNA and protein expression levels of c-fos and CREB were unchanged, confirming the involvement of these two transcription factors in cerebellar granular layer plasticity.