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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.

Both the acinar and ductal cells of the pancreas secrete a near-isotonic fluid and may thus be sites of aquaporin (AQP) water channel expression. Northern blot analysis of mRNA from whole rat pancreas revealed high levels of AQP1 and AQP8 expression, whereas lower levels of AQP4 and AQP5 expression were just detectable by RT-PCR Southern blot analysis. Immunohistochemistry showed that AQP1 is localized in the microvasculature, whereas AQP8 is confined to the apical pole of the acinar cells. No labeling of acinar, ductal, or vascular tissue was detected with antibodies to AQP2-7. With immunoelectron microscopy, AQP8 labeling was observed not only at the apical membrane of the acinar cells but also among small intracellular vesicles in the subapical cytoplasm, suggesting that there may be regulated trafficking of AQP8 to the apical plasma membrane. To evaluate the contribution of AQPs to the membrane water permeability, video microscopy was used to measure the swelling of acinar cells in response to hypotonic stress. Osmotic water permeability was reduced by 90% following exposure to Hg(2+). Since AQP8 is confined to the apical membrane, the marked effect of Hg(2+) suggests that other water channels may be expressed in the basolateral membrane.

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1 We have cloned, expressed and pharmacologically characterized the Human 5-HT 5A receptor.

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