Anil G. Menon

The mechanisms for provisioning maternal resources to offspring in placental mammals involve complex interactions between maternally regulated and fetally regulated gene networks in the placenta, a tissue that is derived from the zygote and therefore of fetal origin. Here we describe a novel use of an embryo transfer system in mice to identify gene networks in the placenta that are regulated by the mother. Mouse embryos from the same strain of inbred mice were transferred into a surrogate mother either of the same strain or from a different strain, allowing maternal and fetal effects on the placenta to be separated. After correction for sex and litter size, maternal strain overrode fetal strain as the key determinant of fetal weight (P < 0.0001). Computational filtering of the placental transcriptome revealed a group of 81 genes whose expression was solely dependent on the maternal strain [P < 0.05, false discovery rate (FDR) < 0.10]. Network analysis of this group of genes yielded highest statistical significance for pathways involved in the regulation of cell growth (such as insulin-like growth factors) as well as those involved in regulating lipid metabolism [such as the low-density lipoprotein receptor-related protein 1 (LRP1), LDL, and HDL], both of which are known to play a role in fetal development. This novel technique may be generally applied to identify regulatory networks involved in maternal-fetal interaction and eventually help identify molecular targets in disorders of fetal growth.

Hg²⁺ is commonly used as an inhibitor of many aquaporins during measurements of transcellular water transport. To investigate whether it could also act on the paracellular water transport pathway, we asked whether addition of Hg²⁺ affected transport of radiolabeled probes through tight junctions of a salivary epithelial cell monolayer. Inclusion of 1 mM Hg²⁺ decreased transepithelial electrical resistance by 8-fold and augmented mannitol and raffinose flux by 13-fold, which translated into an estimated 44% increase in pore radius at the tight junction. These Hg²⁺-induced effects could be partially blocked by the protein kinase A (PKA) inhibitor N-[2-((p-bromocinnamyl) amino) ethyl]-5-isoquinolinesulfonamide, 2HCl (H89), suggesting that both-PKA dependent and PKA-independent mechanisms contribute to tight junction regulation. Western blot analyses showed a 2-fold decrease in tight junction-associated occludin after Hg²⁺ treatment and the presence of a novel hyperphosphorylated form of occludin in the cytoplasmic fraction. These findings were corroborated by confocal imaging. The results from this study reveal a novel contribution of the PKA pathway in Hg²⁺-induced regulation of tight junction permeability in the salivary epithelial barrier. Therapeutically, this could be explored for pharmacological intervention in the treatment of dry mouth, Sjögren9s syndrome, and possibly other disorders of fluid transport.

To investigate potential physiological interactions between the transcellular and paracellular pathways of water transport, we asked whether targeted deletion of Aquaporin 5 (AQP5), the major transcellular water transporter in salivary acinar cells, affected paracellular transport of 4-kDa FITC-labeled dextran (FITC-D), which is transported through the paracellular but not the transcellular route. After i.v. injection of FITC-D into either AQP5 wild-type or AQP5−/− mice and saliva collection for fixed time intervals, we show that the relative amount of FITC-D transported in the saliva of AQP5−/− mice is half that in matched AQP5+/+ mice, indicating a 2-fold decrease in permeability of the paracellular barrier in mice lacking AQP5. We also found a significant difference in the proportion of transcellular vs. paracellular transport between male and female mice. Freeze-fracture electron microscopy revealed an increase in the number of tight junction strands of both AQP5+/+ and AQP5−/− male mice after pilocarpine stimulation but no change in strand number in female mice. Average acinar cell volume was increased by ≈1.4-fold in glands from AQP5−/− mice, suggesting an alteration in the volume-sensing machinery of the cell. Western blots revealed that expression of Claudin-7, Claudin-3, and Occludin, critical proteins that regulate the permeability of the tight junction barrier, were significantly decreased in AQP5−/− compared with AQP5+/+ salivary glands. These findings reveal the existence of a gender-influenced molecular mechanism involving AQP5 that allows transcellular and paracellular routes of water transport to act in conjunction.

Although aquaporin 5 (AQP5) is the major water channel expressed in alveolar type I cells in the lung, its actual role in the lung is a matter of considerable speculation. By using immunohistochemical staining, we show that AQP5 expression in mouse lung is not restricted to type I cells, but is also detected in alveolar type II cells, and in tracheal and bronchial epithelium. Aqp5 knockout ( Aqp5 ^−/− ) mice were used to analyze AQP5 function in pulmonary physiology. Compared with Aqp5 ^+/+ mice, Aqp5 ^−/− mice show a significantly increased concentration-dependent bronchoconstriction to intravenously administered Ach, as shown by an increase in total lung resistance and a decrease in dynamic lung compliance ( P < 0.05). Likewise, Penh, a measure of bronchoconstriction, was significantly enhanced in Aqp5 ^−/− mice challenged with aerosolized methacholine ( P < 0.05). The hyperreactivity to bronchoconstriction observed in the Aqp5 ^−/− mice was not due to differences in tracheal smooth muscle contractility in isolated preparations or to altered levels of surfactant protein B. These data suggest a novel pathway by which AQP5 influences bronchoconstriction. This observation is of special interest because studies to identify genetic loci involved in airway hyperresponsiveness associated with asthma bracket genetic intervals on human chromosome 12q and mouse chromosome 15, which contain the Aqp5 gene.