Immunolocalization of AQP5 in resting and stimulated normal labial glands and in Sjögren's syndrome.

OBJECTIVE: In our current work, in vivo examination of AQP5 distribution in labial salivary glands following stimulation of secretion has been carried out in normal individuals and in patients with Sjögren's syndrome. SUBJECTS AND METHODS: For this study, we selected five patients with primary Sjögren's syndrome (mean age 62.4 ± 10.6 s.d. years) diagnosed in accordance with the European Cooperative Community classification criteria. There were five patients (mean age 27 ± 2.5 s.d. years) in the control group. The subcellular distribution of AQP5 in human labial gland biopsies was determined with light and immunoelectron microscopy before and 30 min after administration of oral pilocarpine. RESULTS: In unstimulated control and Sjögren's labial glands, AQP5 is about 90% localized in the apical plasma membrane, with only rarely associated gold particles with intracellular membrane structures. We have found no evidence of pilocarpine-induced changes in localization of AQP5 in either healthy individuals or patients with Sjögren's syndrome. CONCLUSIONS: Our studies indicate that neither Sjögren's syndrome itself, nor muscarinic cholinergic stimulation in vivo caused any significant changes in the distribution of AQP5 in the labial salivary gland cells.

Immunolocalization of electroneutral Na(+)-HCO cotransporters in human and rat salivary glands.

Patterns of salivary HCO secretion vary widely among species and among individual glands. In particular, virtually nothing is known about the molecular identity of the HCO transporters involved in human salivary secretion. We have therefore examined the distribution of several known members of the Na(+)-HCO cotransporter (NBC) family in the parotid and submandibular glands. By use of a combination of RT-PCR and immunoblotting analyses, the electroneutral cotransporters NBC3 and NBCn1 mRNA and protein expression were detected in both human and rat tissues. Immunohistochemistry demonstrated that NBC3 was present at the apical membranes of acinar and duct cells in both human and rat parotid and submandibular glands. NBCn1 was strongly expressed at the basolateral membrane of striated duct cells but not in the acinar cells in the human salivary glands, whereas little or no NBCn1 labeling was observed in the rat salivary glands. The presence of NBCn1 at the basolateral membrane of human striated duct cells suggests that it may contribute to ductal HCO secretion. In contrast, the expression of NBC3 at the apical membranes of acinar and duct cells in both human and rat salivary glands indicates a possible role of this isoform in HCO salvage under resting conditions.

Immunolocalization of aquaporin-8 in rat kidney, gastrointestinal tract, testis, and airways.

The purpose of this study was to determine the cellular and subcellular localization of aquaporin-8 (AQP8) in rat kidney and other organs by RT-PCR analyses and by immunoblotting and immunohistochemistry using peptide-derived rabbit antibodies to rat AQP8. RT-PCR and Southern blotting revealed the presence of AQP8 mRNA in all kidney zones. LLC-PK(1) cells transfected with a rat AQP8 construct exhibited strong labeling with the affinity-purified antibodies, whereas controls using cells transfected with the vector, but without the insert, were negative. The labeling was almost exclusively associated with intracellular vesicles. Immunoblotting of kidney membrane fractions revealed a predominant single band of 26-28 kDa. AQP8 immunoreactivity was mainly present in the cortex and outer stripe of the outer medulla. Sequential ultracentrifugation of rat kidney membrane revealed that AQP8 resides predominantly in intracellular vesicular fractions. Immunocytochemistry revealed modest labeling of proximal tubules and weak labeling of collecting ducts in cortex and medulla of rat kidney. The labeling was confined to cytoplasmic areas with no labeling of the brush border. Immunoblotting and RT-PCR/Southern blotting also revealed the presence of AQP8 protein and mRNA in rat liver, testis, epididymis, duodenum, jejunum, colon, and bronchi/trachea. Consistent with this, immunohistochemistry revealed AQP8 labeling in the hepatocytes and spermatogenic cells in testis and in the basal cells in ductus epididymis, trachea, and bronchial epithelia. Moreover, AQP8 labeling was observed in the myoepithelial cells in salivary, bronchial, and tracheal glands with no labeling of acini or ductal epithelial cells. AQP8 is also present in the surface epithelial cells in duodenum, jejunum, and colon. In conclusion, AQP8 is expressed at low levels in rat kidney proximal tubules and collecting ducts, and it is present in distinct cell types in liver, testis, epididymis, duodenum, jejunum, colon, trachea, and principal bronchi as well as in multiple glands, including salivary glands.

Identification and localization of aquaporin water channels in human salivary glands.

Aquaporin (AQP) water channels are expressed in a variety of fluid-transporting epithelia and are likely to play a significant role in salivary secretion. Our aim was to identify and localize the aquaporins expressed in human salivary glands. Total RNA was extracted from human parotid, submandibular, sublingual, and labial glands and from human brain. Expression of aquaporin mRNA was assessed by RT-PCR using specific primers for human AQP1, AQP3, AQP4, and AQP5. All four aquaporins were detected by RT-PCR in all of the glands, and the sequences were confirmed after further amplification with nested primers. Cleaned PCR products were then used as (32)P-labeled cDNA probes in a semiquantitative Northern blot analysis using glyceraldehyde-3-phosphate dehydrogenase as reference. Only AQP1, AQP3, and AQP5 mRNAs were present at significant levels. AQP localization was determined by immunohistochemistry on paraffin sections using affinity-purified primary antibodies and peroxidase-linked secondary antibodies. Each salivary gland type showed a broadly similar staining pattern: AQP1 was localized to the capillary endothelium and myoepithelial cells; AQP3 was present in the basolateral membranes of both mucous and serous acinar cells; AQP4 was not detected; and AQP5 was expressed in the luminal and canalicular membranes of both types of acinar cell. We conclude that AQP3 and AQP5 together may provide a pathway for transcellular osmotic water flow in the formation of the primary saliva.

Expression of aquaporin 1 (AQP1) water channels in human labial salivary glands.

Aquaporin (AQP) water channels are widely expressed in the membranes of fluid-transporting epithelia. Despite the fact that salivary glands are the site of considerable water movement, relatively little is known about the role of aquaporins in human salivary glands. We have examined the expression of AQP1 in human parotid, sublingual and labial salivary glands. Total RNA was extracted from glandular tissue obtained from surgery or biopsy. The presence of AQP1 mRNA was demonstrated in each of the three glands by RT-PCR using primers specifically designed for human AQP1. The PCR product from the labial gland RNA was further amplified with nested primers and the sequence confirmed by automated fluorescent DNA sequencing. The cleaned first PCR product from these glands was then used as a 32P-labelled hybridization probe in a Northern analysis which confirmed the presence of significant amounts of AQP1 transcript in all three glands. AQP1 expression was also demonstrated in cryosections of human labial glands by immunohistochemistry using peroxidase-linked antibodies. Antibody labelling was most prominent in the capillaries but was also evident in the basal regions of the labial gland acini, and may therefore be associated with the serous demilunes which are believed to be a significant site of fluid movement.