Diane Gingras

In situ localization of P-glycoprotein (ABCB1) in human and rat brain.

Transport of several xenobiotics including pharmacological agents into or out of the central nervous system (CNS) involves the expression of ATP-dependent, membrane-bound efflux transport proteins such as P-glycoprotein (P-gp) at the blood-brain barrier (BBB). Previous studies have documented gene and protein expression of P-gp in brain microvessel endothelial cells. However, the exact localization of P-gp, particularly at the abluminal side of the BBB, remains controversial. In the present study we examined the cellular/subcellular distribution of P-gp in situ in rat and human brain tissues using immunogold cytochemistry at the electron microscope level. P-gp localizes to both the luminal and abluminal membranes of capillary endothelial cells as well as to adjacent pericytes and astrocytes. Subcellulary, P-gp is distributed along the nuclear envelope, in caveolae, cytoplasmic vesicles, Golgi complex, and rough endoplasmic reticulum (RER). These results provide evidence for the expression of P-gp in human and rodent brain capillary along their plasma membranes as well as at sites of protein synthesis, glycosylation, and membrane trafficking. In addition, its presence at the luminal and abluminal poles of the BBB, including pericytes and astrocyte plasma membranes, suggests that this glycoprotein may regulate drug transport processes in the entire CNS BBB at both the cellular and subcellular level.

Cellular localization and functional expression of P‐glycoprotein in rat astrocyte cultures

We investigated the cellular/subcellular localization and functional expression of P‐glycoprotein, an ATP‐dependent membrane‐associated efflux transporter, in astrocytes, a brain parenchyma compartment that is poorly characterized for the expression of membrane drug transporters. Analyses were carried out on primary cultures of astrocytes isolated from the cerebral cortex of neonatal Wistar rats and CTX TNA2, an immortalized rat astrocyte cell line. Both cell cultures display morphological features typical of type I astrocytes. RT‐PCR analysis revealed mdr1a and mdr1b mRNA in primary cultures of astrocytes and in CTX TNA2 cells. Western blot analysis using the P‐glycoprotein monoclonal C219 antibody detected a single band of appropriate size in both cell systems. Immunocytochemical analysis using the monoclonal antibodies C219 and MRK16 labeled P‐glycoprotein along the plasma membrane, caveolae, coated vesicles and nuclear envelope. Immunoprecipitation studies using the caveolin‐1 polyclonal H‐97 antibody demonstrated that P‐glycoprotein is physically associated with caveolin‐1 in both cell culture systems. The accumulation of [3H]digoxin (an established P‐glycoprotein substrate) by the astrocyte cultures was significantly enhanced in the presence of standard P‐glycoprotein inhibitors and an ATP depleting agent. These results demonstrate the cellular/subcellular location and functional expression of P‐glycoprotein in rat astrocytes and suggest that this glial compartment may play an important role in the regulation of drug transport in the CNS.

Endocrine and Exocrine Secretion of Leptin by the Gastric Mucosa

Leptin is a hormone that plays important roles in nutritional status and in obesity. By means of immunocytochemistry, two populations of leptin-secreting cells were found in the lower half of the gastric mucosa. One consists of numerous large cells located around the gastric pits, the Chief epithelial cells, whereas the second refers to much smaller cells, strongly stained, few in number, and scattered between the gastric pits, the endocrine cells. By double immunostaining, leptin and pepsinogen were colocalized in the Chief cells, whereas the endocrine cells were positive only for leptin. Immunoelectron microscopy showed that leptin is present along the rough endoplasmic reticulum–Golgi-granules secretory pathways of the Chief and endocrine cells. On the other hand, leptin-receptor (long and short forms) immunolabelings, although absent in the gastric epithelial cell plasma membranes, were present in enterocytes at the level of their apical and basolateral membranes. Duodenal, jejunal, and ileal enterocytes displayed similar labelings for the leptin receptor. Thus, exocrine and endocrine secretions of leptin together with the presence of leptin receptors on enterocyte plasma membranes constitute a gastroenteric axis that coordinates the role played by leptin in the digestive tract.

Control of glycogen synthase through ADIPOR1-AMPK pathway in renal distal tubules of normal and diabetic rats

Diabetic nephropathies are characterized by glycogen accumulation in distal tubular cells, which eventually leads to their apoptosis. The present study aims to determine whether adiponectin and AMPK are involved in the regulation of glycogen synthase (GS) in these structures. Western blots of isolated distal tubules revealed the presence of adiponectin receptor ADIPOR1, catalytic AMPK subunits alpha(1) and alpha(2), their phosphorylated active forms, and the glycogen-binding AMPK subunit beta(2). ADIPOR2 was not detected. Expression levels of ADIPOR1, AMPKalpha(1), AMPKalpha(2), and AMPKbeta(2) were increased in streptozotocin-treated diabetic rats, whereas phosphorylated active AMPK levels were strongly decreased. Immunohistochemistry revealed the presence of ADIPOR1 on the luminal portion of distal tubules and thick ascending limb cells. Catalytic subunits alpha(1) and alpha(2), their phosphorylated active forms, and the glycogen-binding subunit beta(2) were also found in the same cells, confirming immunoblot results. In vitro, 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR; 2 mM) and globular adiponectin (10 mug/ml) activated catalytic AMPK in distal tubules isolated from kidneys of normal rats but much more weakly in those from diabetic rats. GS inhibition paralleled AMPK activation in both groups of animals: active GS levels were low in control animals and elevated in diabetic ones. Finally, glucose-6-phosphate, an allosteric activator of GS, was also increased in diabetic rats. These results demonstrate that in distal tubular cells, adiponectin through luminal ADIPOR1 activates AMPK, leading to the inhibition of GS. During hyperglycemia, this regulation is altered, which may explain, at least in part, the accumulation of large glycogen deposits.

Apoptosis of Tubular Epithelial Cells in Glycogen Nephrosis During Diabetes

The important problem of the fate of glycogen-accumulating clear cells in glycogen nephrosis is still unsettled. In this study, we examine whether apoptosis plays a relevant role in the development of diabetic glycogen nephrosis and explore the involvement of the Fas/Fas-L system and the activation of the caspase cascade. Diabetes was induced in rats by streptozotocin injection. Glycogen-accumulating clear cells were identified in renal tissues of hyperglycemic rats. They were found to be concentrated in the thick ascending limbs and distal tubules. Large cellular glycogen accumulations were confirmed by biochemical assays and enzyme-gold cytochemistry. Clear cells displayed apoptotic features such as Annexin V binding, nuclear TUNEL (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling), and the simultaneous occurrence of Fas, Annexin V, and TUNEL positivity. Western blot analysis demonstrated enhanced expression of Fas receptor/ligand and the activation of the caspase cascade in these cells because cleaved forms of the caspase-3, -8, and -9 were detected. Furthermore, active caspase-3 was located in nuclei by immunoelectron microscopy. Our results indicate that epithelial cells in thick ascending limbs and distal tubules that develop glycogen nephrosis in response to hyperglycemia undergo Fas/Fas-L mediated cell death. Thus, apoptosis could be playing a significant role in renal epithelial cell deletion during diabetes.

The 20S proteasome core, active within apoptotic exosome-like vesicles, induces autoantibody production and accelerates rejection

Exosome-like vesicles containing an active 20S proteasome core contribute to autoimmunity and vascular allograft inflammation. Friendly fire from organ failure Despite advances in organ transplantation, rejection still poses a substantial risk. Autoantibodies contribute to rejection, but how these autoantibodies are generated remains unknown. Dieudé et al. found that injection of apoptotic exosome-like vesicles apoExo stimulated autoantibody production in mice, which led to increased graft rejection after transplant. The apoExo contained active 20S proteasome core complexes, and inhibition of proteasome activity decreased the immunogenicity of apoExo and graft rejection in transplanted mice. Circulating apoExo and increased anti-autoantibody titers were also observed in mouse models of ischemia-reperfusion injury, suggesting that the same organ failure that necessitates the transplant might increase the risk of rejection. Therefore, proteasome inhibitors could provide a new therapeutic avenue for graft rejection. Autoantibodies to components of apoptotic cells, such as anti-perlecan antibodies, contribute to rejection in organ transplant recipients. However, mechanisms of immunization to apoptotic components remain largely uncharacterized. We used large-scale proteomics, with validation by electron microscopy and biochemical methods, to compare the protein profiles of apoptotic bodies and apoptotic exosome-like vesicles, smaller extracellular vesicles released by endothelial cells downstream of caspase-3 activation. We identified apoptotic exosome-like vesicles as a central trigger for production of anti-perlecan antibodies and acceleration of rejection. Unlike apoptotic bodies, apoptotic exosome-like vesicles triggered the production of anti-perlecan antibodies in naïve mice and enhanced anti-perlecan antibody production and allograft inflammation in mice transplanted with an MHC (major histocompatibility complex)–incompatible aortic graft. The 20S proteasome core was active within apoptotic exosome-like vesicles and controlled their immunogenic activity. Finally, we showed that proteasome activity in circulating exosome-like vesicles increased after vascular injury in mice. These findings open new avenues for predicting and controlling maladaptive humoral responses to apoptotic cell components that enhance the risk of rejection after transplantation.

Internalization and Transcytosis of Pancreatic Enzymes by the Intestinal Mucosa

As early as the beginning of the twentieth century some data indicated that macromolecules are able to cross the intestinal mucosa to reach the blood. Further evidence was added over the years; however, pathways for this transport still remain to be established. We report here the transfer of two pancreatic enzymes, amylase and lipase, from the intestinal lumen to the blood. Both are present in higher concentrations in the intestinal mucosa and in blood of fed rats. Upon cholinergic stimulation of pancreatic secretion, there was not only an increase in blood enzyme concentrations, but evidence for internalization by duodenal enterocytes was obtained. Following insertion of fluorochrome-tagged amylase and lipase into the duodenal lumen of fasting rats, blood and intestinal tissues were sampled at different time points. Serum activities for both enzymes clearly increased with time. Light microscopy established internalization of both proteins by duodenal enterocytes, and immunogold outlined the pathway taken by both proteins across the enterocytes. From the intestinal lumen, enzymes are channeled through the endosomal compartment to the Golgi apparatus and to the basolateral membrane reaching the interstitial space and blood circulation. Transcytosis through the intestinal mucosa thereby represents an access route for pancreatic enzymes to reach blood circulation. (J Histochem Cytochem 54:781-794, 2006)

Cellular and Subcellular Expression of Golf/Gs and Gq/G11 α-Subunits in Rat Pancreatic Endocrine Cells

We studied the cellular and subcellular localization of G α-subunits in pancreas by immunocytochemistry. Golfα and G11α were specifically localized in islet insulin B-cells and glucagon A-cells, respectively. Gs α and Gqα labeling was more abundant in B-cells. The presence of Golfα in B-cells was confirmed by in situ hybridization. In B-cells, Golfα and Gsα were found in the Golgi apparatus, plasma membrane (PM) and, remarkably, in mature and immature insulin secretory granules, mainly at the periphery of the insulin grains. Gqα was detected on the rough endoplasmic reticulum (RER) near the Golgi apparatus. In A-cells, the Gα-subunits were mostly within the glucagon granules: G11 α gave the strongest signal, Gsα less strong, Gq was scarce, and Golf was practically absent. Gqα and Gsα immunoreactivity was detected in acinar cells, although it was much weaker than that in islet cells. The cell-dependent distribution of the Gα-subunits indicates that the stimulatory pathways for pancreatic function differ in acinar and in islet B- and A-cells. Furthermore, the G-protein subunits in islet cell secretory granules might be functional and participate in granule trafficking and hormone secretion.

EphA4 is localized in clathrin―coated and synaptic vesicles in adult mouse brain

J. Neurochem. (2010) 10.1111/j.1471‐4159.2010.06582.x

Differences in secretory granule content in pancreatic acinar cells from peri-insular and tele-insular regions.

By applying morphometrical and quantitative double immunocytochemical techniques, differences in size and in amylase and chymotrypsinogen contents were found among pancreatic zymogen granules. These differences were present in granules of peri-insular and tele-insular acinar cells, the peri-insular ones displaying higher numbers of granules of smaller sizes. No correlation was found among enzyme contents in individual granules, nor was there a correlation between enzyme content and granule size. The results suggest that each individual secretory granule is formed in an independent way and that each enzyme is processed and packaged into granules independently. The differences among granules may be associated with nonparallel secretion, since this phenomenon has been reported in the intracellular processing of secretory enzymes. This hypothesis, however, remains to be demonstrated.