Objectives: 

To study the effect of aquaporin 5 (AQP5) on embryonic glandular morphology and cell volume regulation.

Methods: 

Mouse E13.5 submandibular salivary glands were placed in DMEM/F12 medium and cultured for 24h with 500 nM AQP5 or fibronectin (control) siRNA and INTERFERin transfection medium, and were photographed at 1 and 24 h. AQP5 expression was analysed with qPCR. Some glands were dissociated with 100 U/ml collagenase IV and cells were exposed to iso- (320 mOsm) and hyposmotic (220 mOsm) solutions. Cell volume changes, gland areas, and bud development were measured.

Results: 

AQP5 and fibronectin siRNA knock down functioned in about 50% of the experiments (2 Ct-values decrease). AQP5 knock down did not affect glandular growth or budding. Fibronectin knock down showed 35% decrease in budding (P<0.001; control for siRNA uptake). Normal regulatory volume decreases (RVD) following hyposmotically induced swelling varied among glands. About 50% of the control glands showed a 60% RVD, while about 25% of the AQP5 knock down glands showed a similar RVD. When both regulating and non-regulating glands were grouped, control cells showed rapid swelling (max swelling within 1 min after hyposmotic exposure, then the cell volumes stabilized; i.e., no decrease). The AQP5 knock down cells swelled less rapidly and showed a gradual swelling throughout the experiment.

Conclusion: 

A significant mRNA knock down can be achieved using siRNA techniques with whole embryonic mouse salivary glands. AQP5 is an important aquaporin in salivary glands, but decreasing its mRNA level does not seem to affect glandular morphology. However, osmotic properties of the cells are affected by an AQP5 mRNA decrease. Thus, AQP5 apparently has a more important role in osmosis than in normal glandular development.