Search

Previously we have demonstrated that the GJ protein connexin 30.2 (Cx30.2) is expressed in pancreatic beta cells and endothelial cells (ECs) of the islet. In the present study, we address whether Cx30.2 is expressed in the exocrine pancreas, including its vascular system. For this, adult mouse pancreatic sections were double labeled with specific antibodies against Cx30.2 and CD31, an endothelial cell marker, or with anti-α-actin smooth muscle, a smooth muscle cell (SMC) marker or anti-mucin-1, a marker of epithelial ductal cells, using immunofluorescence (IF) studies. Cx30.2-IF hot spots were found at junctional membranes of exocrine ECs and SMCs of blood vessels. Furthermore, Cx30.2 was localized in mucin-1 positive cells or epithelial ductal cells. Using immunohistochemistry (IHC) studies, it was found that in vessels and ducts of different diameters, Cx30.2 was also expressed in these cell types. In addition, it was found that Cx30.2 is already expressed in these cell types in pancreatic sections of 3, 14 and 21 days postpartum. Moreover, this cell specific pattern of expression was also found in the adult rat, hamster and guinea pig pancreas. Expression of Cx30.2 mRNA and protein in the pancreas of all these species was confirmed by RT-PCR and Western blot studies. Overall, our results suggest that intercellular coupling mediated by Cx30.2 intercellular channels may synchronize the functional activity of ECs and SMCs of vascular cells, as well as of epithelial ductal cells after birth.

Previous studies have shown that following peripheral nerve injury there was a downregulation of the gap junction protein connexin 36 (Cx36) in the spinal cord; however, it is not known whether Cx36 protein is expressed in the dorsal root ganglia (DRGs), nor if its levels are altered following peripheral nerve injuries. Here we address these aspects in the adult rat lumbar DRG. Cx36 mRNA was detected using qRT-PCR, and Cx36 protein was identified in DRG sections using immunohistochemistry (IHC) and immunofluorescence (IF). Double staining revealed that Cx36 co-localizes with both anti-β-III tubulin, a neuronal marker, and anti-glutamine synthetase, a satellite glial cell (SGC) marker. In neurons, Cx36 staining was mostly uniform in somata and fibers of all sizes and its intensity increased at the cell membranes. This labeling pattern was in contrast with Cx36 IF dots mainly found at junctional membranes in islet beta cells used as a control tissue. Co-staining with anti-Cx43 and anti-Cx36 showed that whereas mostly uniform staining of Cx36 was found throughout neurons and SGCs, Cx43 IF puncta were localized to SGCs. Cx36 mRNA was expressed in normal lumbar DRG, and it was significantly down-regulated in L4 DRG of rats that underwent sciatic nerve injury resulting in persistent hypersensitivity. Collectively, these findings demonstrated that neurons and SGCs express Cx36 protein in normal DRG, and suggested that perturbation of Cx36 levels may contribute to chronic neuropathic pain resulting from a peripheral nerve injury.

Nowadays, connexin (Cx) 36 is considered the sole gap junction protein expressed in pancreatic beta cells. In the present research we investigated the expression of Cx30.2 mRNA and protein in mouse pancreatic islets. Cx30.2 mRNA and protein were identified in isolated islet preparations by qRT-PCR and Western blot, respectively. Immunohistochemical analysis showed that insulin-positive cells were stained for Cx30.2. Confocal images from double-labeled pancreatic sections revealed that Cx30.2 and Cx36 fluorescence co-localize at junctional membranes in islets from most pancreases. Abundant Cx30.2 tiny reactive spots were also found in cell cytoplasms. In beta cells cultured with stimulatory glucose concentrations, Cx30.2 was localized in both cytoplasms and cell membranes. In addition, Cx30.2 reactivity was localized at junctional membranes of endothelial or cluster of differentiation 31 (CD31) positive cells. Moreover, a significant reduction of Cx30.2 mRNA was found in islets preparations incubated for 24h in 22mM as compared with 3.3mM glucose. Therefore, it is concluded that Cx30.2 is expressed in beta and vascular endothelial cells of mouse pancreatic islets.

To identify when during fetal development connexins (Cxs) 26 (Cx26) 32 (Cx32), and 36 (Cx36) begin to be expressed, as well as to characterize their spatial distribution, real time polymerase chain reaction and immunolabeling studies were performed. Total RNA from mouse pancreases at 13 and 18 days postcoitum (dpc) and 3 days postpartum (dpp) was analyzed. In addition, pancreatic sections of mouse at 13, 14, 15, 16, 18 dpc and 3 dpp and of rat at term were double labeled with either anti-insulin or anti-α-amylase and anti-Cx26 or -Cx32 or -Cx36 antibodies and studied with confocal microscopy. From day 13 dpc, Cxs 26, 32, and 36 transcripts were identified and their levels increased with age. At 13-14 dpc, Cxs 26 and 32 were localized in few acinar cells, whereas Cx36 was distributed in small beta cell clumps. From day 14 dpc onwards, the number of labeled cells and relative immunofluorescent reactivity of all three Cxs at junctional membranes of the respective cell types increased. Cxs 26 and 32 colocalized in fetal acinar cells. In rat pancreas at term, a similar connexin distribution was found. Relative Cxs levels evaluated by immunoblotting also increased (two-fold) in pancreas homogenates from day 18 dpc to 3 dpp. The early cell specific, wide distribution, and age dependent expression of Cxs 26, 32, and 36 during fetal pancreas ontogeny suggests their possible involvement in pancreas differentiation and prenatal maturation.