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

Background: Several 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. Results: RT-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. Conclusion: The 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

Aquaporin-7 (A), and -10 (B) protein expression in human adipocytes plasma membranes.

<p>Blots representative of four were shown. Lanes were loaded with 35 µg of proteins, probed with anti-AQP7 rabbit polyclonal antibody (A left) and processed as described in Materials and Methods. The same blots were stripped and re-probed with anti-AQP10 rabbit polyclonal antibody (B left), and anti-β-actin antibody as housekeeping (C). A major band of about 34 kDa and two bands of about 30 kDa (monomer) and 60 kDa (dimer) were observed when the blots were probed with anti-AQP7 and anti-AQP10 antibodies respectively. No bands were detected when preadsorbed anti-aquaporin-7 or -10 antibodies were used (A and B right). AM, adipocytes plasma membranes. D, duodenal crude homogenate.</p

Water and glycerol permeability of human adipocyte plasma membrane vesicles.

<p>(A) Representative light scattering curves were obtained by exposing the isolated adipocytes to a 150 mOsm osmotic gradient in two different conditions: normal untreated cells (Control) and cells treated for 15 min with 0.5 M DMSO (DMSO). (B) Bars represent water permeability of isolated adipocytes, expressed as relative k. Values are means ± SEM of at least 15 single shots for each of five different adipocyte preparations. *, P<0.05 vs Control (Student <i>t</i> test for pair data). (C) Representative light scattering curves were obtained by exposing the adipocytes plasma membrane vesicles to a 150 mOsm osmotic gradient in three different conditions: normal untreated vesicles (Control), vesicles treated for 10 min with 1 mM HgCl₂, vesicles treated with 1 mM HgCl₂ followed by 15 min treatment with 15 mM β-mercaptoethanol (β-ME). (D) Bars represent water permeability of adipocyte plasma membrane vesicles, expressed as relative k. Values are means ± SEM of at least 8 single shots for each of five different preparations. *, P<0.05 vs Control and β-ME (repeated measure ANOVA, followed by Newman-Keuls’s <i>Q</i> test). (E) Representative light scattering curves were obtained by exposing the adipocytes plasma membrane vesicles to a 150 mM inwardly directed gradient of glycerol. The initial increase in light scattering results from osmotic water efflux caused by vesicle shrinkage (before the dashed line), while the subsequent slower decrease is caused by glycerol entry (after the dashed line). (F) Water (Pf) and glycerol (Pgly) permeability coefficients were calculated as described in Materials and Methods.</p

Effect of insulin and isoproterenol on subcellular localization of aquaporin-7 (AQP7) and -10 (AQP10) in human cultured differentiated adipocytes.

<p>The aquaporin localization was detected in basal conditions (control: A, B), after insulin (C, D) or isoproterenol (E, F) stimulation. Control adipocytes show an intracellular AQP7 and AQP10 green labeling, particularly evident around small lipid droplets. Green labeling indicated the presence of AQP-7-10, while nuclei were counterstained by DAPI (blue). Insulin treatment increased the AQP7 and AQP10 staining around the lipid droplets while that of isoproterenol reduced the lipid droplets labeling, greatly increasing the plasma membrane staining. Negative controls gave a faint or negligible signal (H). Accumulation of lipid droplets in adipocytes was demonstrated by Oil Red O staining (G).</p

Aquaporin-10 Represents an Alternative Pathway for Glycerol Efflux from Human Adipocytes

<div><h3>Background</h3><p>Glycerol outflow from adipocytes has been considered for a decade to be mediated by aquaporin-7, an aquaglyceroporin highly expressed in the adipose tissue. Its involvement in glycerol metabolism has been widely studied also in humans. Recent studies in different aquaporin-7 KO mice models pose two different questions 1) the exact localization of aquaporin-7 in human white adipose tissue; 2) the existence of other aquaglyceroporins that work with aquaporin-7 to guarantee glycerol efflux and thus a normal adiposity in humans. To this purpose we investigated the expression, the localization and the functioning of aquaglyceroporin-10 in subcutaneous white adipose tissue, in isolated and cultured differentiated adipocytes.</p> <h3>Methodology/Principal Findings</h3><p>Aquaporin-7 and -10 were expressed in the white adipose tissue both at mRNA and at protein level. Immunofluorescence revealed aquaporin-7 and -10 labelling in the human adipose tissue both to the plasma membrane and to a thin rim of cytoplasm of adipocytes. Aquaporin-7, but not aquaporin-10, colocalized with the endothelial marker CD34. Human cultured differentiated adipocytes showed an aquaporin-7 and -10 labelling mainly in the cytoplasm and in the lipid droplets with insulin reinforcing the lipid droplets staining and isoproterenol inducing its translocation to the plasma membrane compartment. Water and glycerol permeability measurements using adipocytes and adipose membrane vesicles confirmed the presence of functioning aquaglyceroporins. Aquaporin-10 silencing in human differentiated adipocytes resulted in a 50% decrease of glycerol and osmotic water permeability.</p> <h3>Conclusions/Significance</h3><p>The results indicate that aquaporin-7, differently from mice, is present in both adipocyte and capillary plasma membranes of human adipose tissue. Aquaporin-10, on the contrary, is expressed exclusively in the adipocytes. The expression of two aquaglyceroporins in human adipose tissue is particularly important for the maintenance of normal or low glycerol contents inside the adipocyte, thus protecting humans from obesity.</p> </div

Representative immunofluorescence confocal microscopical images of aquaporin-7 and -10 localization in human subcutaneous adipose tissue.

<p>Green labeling indicates the presence of aquaporin-7 (A) and -10 (B) at the plasma membrane and at the cytoplasm of the human adipocytes. No or faint staining was observed when anti-aquaporin-7 and anti-aquaporin-10 preadsorbed antibodies were used (C-D). Nuclei were counterstained by DAPI (blue). Colocalization of aquaporin-7 or -10 and the endothelial cell marker CD34 in human subcutaneous adipose tissue (E-F) was performed as detailed in “Materials and methods”. Green labeling indicated the presence of aquaporin-7 (E) or -10 (F), red labeling the vessels, while nuclei were counterstained by DAPI (blue). Merged images showed strong colocalization signal of aquaporin-7 and CD34 (yellow labelling) in the capillary, even though aquaporin-7 was also expressed in the adipocytes (E). On the contrary, aquaporin 10 and CD34 did not colocalized (F).</p

Aquaporin-7 (AQP7) and -10 (AQP10) mRNA expression in human subcutaneous adipose tissue (AT) and in isolated adipocytes (A).

<p>Upper panels, mRNA levels were measured by real-time RT-PCR relative to the β-actin internal standard (see Materials and Methods section) and the values obtained were reported as ΔCt. Bars represent the mean ± SEM of at least 4 different experiments each from different RNA extracts. *P<0.05 versus A (Student’s <i>t</i> test). Lower panels, Gel electrophoresis of the PCR products. Specific PCR products for AQP7 (139 bp band), AQP10 (115 bp band) and β-actin (146 bp) were observed in both AT and A.</p

Aquaporin-10 (AQP10) silencing in human differentiated adipocytes.

<p>AQP10 short interfering RNA (siRNA) and scrambled siRNA (Ctr) were transfected in differentiated adipocytes as described in Materials and methods. A, AQP10 mRNA levels were measured by real-time RT-PCR relative to the β-actin internal standard and the values obtained were reported as fold change (see Materials and Methods section). Bars represent the mean ± SEM of at least 4 different experiments each from different RNA extracts. AQP10 transcript was reduced in silenced differentiated adipocytes compared to controls; *P<0.006 versus Ctr (Student’s <i>t</i> test). B, Western blot and densitometry demonstrate that undifferentiated adipose stem cells (ASC) had no AQP10 expression, while AQP10 protein was reduced in silenced differentiated adipocytes (siRNA) compared to controls (scrambled; Ctr)(*, P<0.003; Student’s t test). Blots representative of three were shown (B, lower pannel). The same blots were stripped and re-probed with anti-β-actin antibody. Bands of the expected molecular weights were shown and acquired with the Image Master VDS (GE Healthcare Life Sciences, Italy). Densitometric analysis of the bands was performed by Total Lab V 1.11 computer program (GE Healthcare Life Sciences, Italy) and the results were normalized to the corresponding β-actin (B, upper panel). C, Water (Pf) and glycerol (Pgly) permeability coefficients were calculated as described in Materials and Methods. Both Pf and Pgly were significantly reduced in siRNA compared to controls by 46% and 51%, respectively (*, P<0.002; Student’s t test).</p

Cardiac microvascular endothelial cells express a functional Ca2+-sensing receptor

The mechanism whereby extracellular Ca(2+) exerts the endothelium-dependent control of vascular tone is still unclear. In this study, we assessed whether cardiac microvascular endothelial cells (CMEC) express a functional extracellular Ca(2+)-sensing receptor (CaSR) using a variety of techniques. CaSR mRNA was detected using RT-PCR, and CaSR protein was identified by immunocytochemical analysis. In order to assess the functionality of the receptor, CMEC were loaded with the Ca(2+)-sensitive fluorochrome, Fura-2/AM. A number of CaSR agonists, such as spermine, Gd(3+), La(3+) and neomycin, elicited a heterogeneous intracellular Ca(2+) signal, which was abolished by disruption of inositol 1,4,5-trisphosphate (InsP(3)) signaling and by depletion of intracellular stores with cyclopiazonic acid. The inhibition of the Na(+)/Ca(2+) exchanger upon substitution of extracellular Na(+) unmasked the Ca(2+) signal triggered by an increase in extracellular Ca(2+) levels. Finally, aromatic amino acids, which function as allosteric activators of CaSR, potentiated the Ca(2+) response to the CaSR agonist La(3+). These data provide evidence that CMEC express CaSR, which is able to respond to physiological agonists by mobilizing Ca(2+) from intracellular InsP(3)-sensitive stores